Roshchina2014 PDF

Victoria V.
Roshchina
Model Systems
to Study the
Excretory
Function of
Higher Plants
Model Systems to Study the Excretory
Function of Higher Plants
Victoria V. Roshchina
Model Systems to Study

the Excretory Function
of Higher Plants
Victoria V. Roshchina
Institute of Cell Biophysics
Russian Academy of Sciences
Pushchino
Russia
ISBN 978-94-017-8785-7 ISBN 978-94-017-8786-4 (eBook)

DOI 10.1007/978-94-017-8786-4
Springer Dordrecht Heidelberg New York London
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Abstract
This monograph presents plant model systems suitable for vital microscopic analy-
sis of excretory function that have been studied by the author during the last 15
years. The approaches to modeling and the screening of similar models that are
described may be of interest to the wider ring of biologists working in the fields of
cell biology, ecology, medicine, and pharmacology. Without vivisection and
fixation, a researcher can observe the processes of secretion and the cellular reac-
tions to exometabolites and can analyze the mechanisms of action. Special models
are recommended for studies of cell–cell contacts. Some of the model systems may
be used in express-diagnostics for biotechnology, ecological monitoring, and phar-
macology instead of animal models.
v
Preface
Modeling is widely used in biology, in areas such as in genetics, physiology, and

pharmacology, where biological, physicochemical, and mathematical models of
processes are considered. Biological models are simpler living systems that are suit-
able for experimental studies. Laboratory animals, plants, and various strains of
microorganisms can serve as biological models. To explain the causes of various
biological phenomena their mechanisms also can be modeled. Modeling of the life
conditions at the levels of individuals, populations, and ecosystems is possible too.
Choosing similar models allows the analysis of characteristics, features, and laws of
biological processes occurring in real complex organisms. In such models, research-
ers can reproduce certain conditions that permits the mechanisms of an event or
process to be studied. A model process may be demonstrated in artificial systems
using genetic disorders or mutants or in natural ones by changes in temperature,
light regimes, poisons, etc.
Since the 1970s, there have been attempts to model excretory function at differ-
ent levels of organization: molecular, subcellular, cellular, tissue, organ, organism,
and population levels that described in monographs (Roshchina VD and
Roshchina VV 1989, Roshchina VV and Roshchina VD 1993). Currently, there is a
need to attract researchers to modeling as one approach to the study of excretory
function involving cell donors and cell acceptors of secretion, biosensors that per-
ceive and react to the components of secretion in the form of a physiological
response. The aim of this type of modeling is to understand the mechanisms of
intercellular signaling and regulation through excretions in the chemical communi-
cation of organisms. This monograph is focused on cellular models to study plant
excretions in vital conditions by microscopic and spectral methods. The objects
should be clearly seen under various microscopes and have intensively colored and/
or fluorescent secretory products.
Pushchino, Russia Victoria V. Roshchina
vii
Acknowledgement
I am grateful to my colleague Dr. Valerii Yashin and to the engineers Nadezhda

Prizova, Lyubov Khaibulaeva, Nikolai Shvirst, and Andrei Kuchin for their constant
cooperation in experimental work.
ix
Contents
1 Approaches to Choice of Model Systems for Microscopic Studies . . . . 1

1.1 Color and Absorbance in Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.1.1 Usual Microscopy and Stereomicroscopy . . . . . . . . . . . . . . . 3
1.1.2 Microspectrophotometry and Spectrophotometry . . . . . . . . . 7
1.2 Fluorescence in Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.2.1 Autofluorescence of Secretory Components . . . . . . . . . . . . . 9
1.2.2 Special Fluorescent Probes or Markers . . . . . . . . . . . . . . . . . 12
1.2.3 Fluorescence-Related Technique . . . . . . . . . . . . . . . . . . . . . . 12
1.3 Cellular Observation of Secretory Process . . . . . . . . . . . . . . . . . . . . 19
1.3.1 Secretion Transport and Excretion . . . . . . . . . . . . . . . . . . . . . 20
1.3.2 Unicellular Models Suitable for Microspectral Analysis. . . . 21
1.4 Model Process Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
1.4.1 Secretion of Donor Models . . . . . . . . . . . . . . . . . . . . . . . . . . 26
1.4.2 Reactions of Acceptor Models Sensitive
to Secretory Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2 Intact Secretory Cells as Donor Models of Secretions . . . . . . . . . . . . . . 35
2.1 Intracellular Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.1.1 Models of Secretion into Vacuole . . . . . . . . . . . . . . . . . . . . . 36
2.2 Intratissual Secretory Systems I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.2.1 Laticifers as Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.2.2 Idioblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.3 External Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2.3.1 Secretion into Free Space. . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2.3.2 Models of External Secretion. . . . . . . . . . . . . . . . . . . . . . . . . 50
3 Acceptor Models of Secretions and Their Reactions
on Exometabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.1 Biosensors and Their Sensitive Reactions . . . . . . . . . . . . . . . . . . . . . 65
3.1.1 Exometabolites as Chemosignals and Regulators . . . . . . . . . 68
3.1.2 Sensory Systems in Acceptor Models . . . . . . . . . . . . . . . . . . 76
xi
xii Contents
3.2 Sensitive Reactions of Models to Exometabolites . . . . . . . . . . . . . . . 77

3.2.1 Microspores as Biosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3.2.2 Multicellular Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
4 Modeling of Cell–Cell Contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.1 Modeling of Cell–Cell Contacts Based
on Microscopic Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.1.1 Pollen–Pistil Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4.1.2 Pollen–Pollen Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.1.3 Direct Contacts in Plant–Animal
or Plant–Microorganism Interactions. . . . . . . . . . . . . . . . . . . 110
4.2 Models to Study Pollen Allelopathy . . . . . . . . . . . . . . . . . . . . . . . . . 119
4.2.1 Modeling of Interaction Between Pollen Donors
and Pollen Acceptors In Vivo and In Vitro . . . . . . . . . . . . . . 120
4.2.2 Modeling with Pollen Excretions. . . . . . . . . . . . . . . . . . . . . . 124
4.3 Modeling with Individual Components of Excretions as
Markers and Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
4.3.1 Colored and Fluorescing Compounds . . . . . . . . . . . . . . . . . . 129
4.3.2 Fluorescing Colorless Compounds . . . . . . . . . . . . . . . . . . . . 135
5 Application of Models in Pharmacology, Medicine, and Ecology . . . . 141
5.1 Unicellular Models in the Analysis of Effects
of Pharmaceuticals and Allelochemicals . . . . . . . . . . . . . . . . . . . . . . 143
5.1.1 Testing on Microspores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
5.1.2 Staining of Cells with Extracts . . . . . . . . . . . . . . . . . . . . . . . 147
5.2 Cellular Targets and Mechanisms of Action of Drugs
and Allelochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
5.2.1 The Surface Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
5.2.2 Targets Within Cell Model . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
5.3 Application of Models in the Environmental Monitoring . . . . . . . . . 153
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Latin Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Introduction
The importance of suitable model systems in plant biology is demonstrated by its

wide scientific audience (Mandoli and Olmstead 2000). In fundamental studies of
plant excretory function, various model systems also may be used. Models are sig-
nificant not only for understanding fundamental secretory process; the modeling of
processes is also one of the experimental approaches to study the mechanisms of
intercellular signaling in the chemical communication of organisms (Roshchina VD
and Roshchina VV 1989, Roshchina VV and Roshchina VD 1993, 2012).
First of all, model systems are necessary for ecological monitoring to analyze the
chemical interactions via excretions between living organisms. The screening of
secreting cellular models depends on the aim of the study. The secretory cells of
medicinal plants, in which the biosynthesis of physiologically active secondary
compounds occurs, seem to be of interest for medical and pharmacological prac-
tices. Different cultivation methods (prolonged cultivation, stress factors, and the
use of the precursors (predecessors) of the end products biosynthesis) allow their
application in the analysis of secretions. Cellular models could be represented
mainly as pollen grains, especially in allergology, where pollen excretion induces a
reactive immune response in humans.
Model systems may be distinguished as shown in the scheme in Fig. 1. The three
main fields of model studies are (1) biochemical investigations (analysis of the
composition of secretory products and the chemical nature of the individual compo-
nents of the secretions, the key biochemical pathways of their synthesis, and the
modes of their regulation); (2) direct observation of the secretory process with vari-
ous quantitative and qualitative methods, from single secreting cells to secretory
organs of whole organisms; and (3) genetic manipulation to create mutants with
variations in secretory products or secretory organs and enzymes of their synthesis
or genetically modifying systems (like green-fluorescing protein included systems)
that are used for secretory traffic within a cell. According to the scheme in Fig. 1,
researchers can use any suitable model to study changes in composition of secre-
tions, sensitivity of contacting organisms to certain secretions, effects of external
factors on secretory processes, etc.
xiii
xiv Introduction
Model systems
to study plant excretory function
1. Biochemical studies 2. Visualization

of secretory process
a. Composition of secretion
Models enriched in Models suitable for the
essential oils, observation of secretory
phenols, amines, process in vivo and in vitro
etc (Mentha piperita, Ovena sativa,
Ruta graviolens)
At the organism level At cellular level
b.
Sensitivity to secretions
Vital models suitable
3. Genetic studies
for microscopic research
Models sensitive
Models genetically
to secretory products
modified and mutants
of suitable objects
(Arabidopsis thaliana) c.
Effects of external factors
on secretory process
Models for analysis

of external factors’ effects
on secretory function
Fig. 1 Modeling in the study of excretory function
Objects characterized as models may be related to whole plants with secretory

organs or to individual secretory cells. The modern tendency is to prefer genetic
manipulations. Arabidopsis thaliana (The Arabidopsis Book 2010–2012) is the most
multifunctional genetic model for studying secretory organization, including the loca-
tion of most of the biochemical products, mainly enzymes, in the secretory structures.
The wide approach to studies with the Arabidopsis model dealt with the biochemical
characterization of the components of the secretions (nectars, essential oils, resins,
phenolic products, etc.) and the genetics of the secretory products.
Today, the most well-studied model is Arabidopsis in terms of the secretions to
pollen exine (Dobritsa et al. 2011). The oldest studies with the Arabidopsis model
began from anatomical research of secretory cells and today they have been fol-
lowed (a) to the analysis of the biological activity of known secretions or their
components on suitable sensitive systems – from microorganisms to plant and ani-
mals, and (b) to experiments with the actions of various external factors on the
secretory process/internal factors influencing the secretory processes, which are
also thought to be an important problem, although not yet studied. A promising
example of the use of this model is in the genetic analysis of the structures and
composition of nectar and nectaries (Kram et al. 2009). Particularly important are
genetic studies that address methods of nectar biosynthesis in the model system of
Arabidopsis thaliana, where the exploration of the expression of different genes in
mutants is convenient (Kram and Carter 2009). Sets of genes encoding components
Introduction xv
of the floral nectar called transcriptosomes were found (Kram et al. 2009). Another
suitable model for similar analysis is the nectaries of tobacco (Nicotiana tabacum)
that express genes encoding the synthesis of specific proteins, nectarines; this pro-
cess is regulated by numerous promoters (Carter and Thornburg, 2000, 2003).
Secretory products also are tested in the Arabidopsis model, for example, low
molecular weight phospholipase A2 regulates cell elongation and shoot gravitrop-
ism (Lee et al. 2002). Endocytic and secretory traffic in Arabidopsis is often ana-
lyzed (Viotti et al. 2010). To analyze the mutants for various purposes, one could
study the biochemical pathway of secretory products, for example, in mucilage
transport. The epidermal cells of the Arabidopsis seed coat undergo a complex dif-
ferentiation process in which there is sequential synthesis of pectinaceous mucilage
followed by secondary cell wall production (Arsovski et al. 2010). The release of
mucilage from secretory cells is a model to study the processes and regulation of
cell wall production and modification during plant development. In addition, the
polar secretion of pectin during mucilage production also makes this system a good
model with which to dissect the mechanisms of targeted secretion in plants, and is
used in the T.L. Western laboratory (Harpaz-Saad et al. 2011).
The biochemical pathways of essential oils (Dudareva and Pichersky 2006;
Gershenzon and Dudareva 2007) and genetic engineering of medically valuable
monoterpenes are studied in the leaves and flowers of Mentha piperita as a model
(Turner and Croteau 2004; Wildung and Croteau 2005; Turner et al. 2012). Similar
models for analyses of the composition of secretions, for example, the assortment
and variability of essential oils in the flowers and leaves of Hypericum perforatum
L. are valuable (Radusiene et al. 2005). Tissue root and leaf cultures inoculated by
various microbial agents in collections of 30 species of plants belonging to 17
families are also used as such models in the studies of secretory alkaloids; for
example, Peganum harmala L. for analysis of β-carbolinic alkaloids (Berlin et al.
1994; Kuzovkina et al. 2004).
The consideration of secretory cells as individual systems led to particular atten-
tion on the problem (Wagner 1991) and to the elaboration of methods for the isola-
tion of secretory structures that could be applied for chemical analysis (Gershenzon
et al. 1987a, b; Yerger et al. 1992; Goodger et al. 2010). This approach to the model-
ing of plant excretory function based on isolated secretory cells (Gershenzon et al.
1987a, b) allows the direct study of the biosynthesis of plant natural products in
isolated secretory cells. In particular, such model may be intact subdermal secretory
cavities (relatively large and rich in essential oils) from Eucalyptus leaves with cavi-
ties (Goodger et al. 2010). Isolated secretory root hairs are also good models for the
study of the biosynthesis of lipid resorcinols and benzoquinones (Dayan et al.
2007). A new method that combined hollow fiber liquid-phase microextraction with
in situ derivatization combined with gas chromatography–mass spectrometry was
also applied to the analysis of the root exudate of Capsicum annuum L. (Sun and
Wang 2013).
Special attention is paid to sensitive unicellular models used for the analysis of
effects of exometabolites, independently on the plant or microorganism is studied.
For example, compounds evoked from cultures of yeast Saccharomyces cerevisiae
xvi Introduction
(Raghuraman and Brewer 2010; Oleskin et al. 2013) may influence other organ-
isms. In this aspect, the invasion and defense mechanisms related to the truncation
of chitinases from Arabidopsis by secreted fungal proteases can be analyzed
(Naumann and Price 2012). In earlier publications, pollen grains were chosen as
suitable models for the analysis of cell wall and exine formation (Sheldon and
Dickinson 1986; Takahashi and Skvarla 1991) or male sterility (van der Veen and
Wirtz 1968).
Researchers also need to understand the complex mechanisms of the effects of
exometabolites in biocenosis and the cellular mechanisms of interspecies relations
with respect to the problem of invasive plants in various ecosystems (see the book
edited by Kohli et al. 2009) or allelopathy (Inderjit et al. 1999; Narwal et al. 1999a,
b). Among different natural models, there are mathematical models, for instance,
for the study of nectar secretion in animal-pollinated plants (Sakai, 1993) as well as
for the analysis of the biological response to allelochemicals (An et al. 1993).
The toxicity of various products excreted by plants seems to be testable with
various models such as two mammalian cell lines, Neuro 2A and Vero, in the green
alga Chlamydomonas reinhardtii and the bacteria Vibrio fischeri (Perreault et al.
2012). In addition, this is possible to do on animal unicellular models that are easily
cultured in the laboratory, in particular, Ciliates, e.g., Tetrahymena and Paramecium
(Beisson et al. 2010). The unicellular model system is also useful for investigations
of the multigenerational effects of hormonal imprinting (Kőhidai et al. 2012; Csaba
2012), and for observation of chemotaxis and cellular differentiation, such as in
Chlamydomonas cells (Ermilova 2013). Plant excretions and individual compo-
nents of secretions may also be tested in animal multicellular models, such as
Planaria serving as a model system analogous (similar with) Mammalia because of
specific behavioral patterns that are analogous to mammalian stereotypes in
response to drugs acting on acetylcholine or dopamine transmission (Buttarelli
et al. 2000). Planaria as a model organism is uniquely poised to be used to investi-
gate the mechanisms of tissue regeneration, stem cell regulation, tissue turnover,
pharmacological action of diverse drugs, cancer, and aging (Oviedo et al. 2008). It
is also amenable to molecular genetic techniques aimed at understanding complex
biological tasks.
Various models may be used for practical application. Of particular interest is the
topic of plant models for the study of mechanisms in plant and human pathogenesis
because plants, microorganisms, and humans have common means of secretion and
genetic regulation (Lugtenberg et al. 2002; Guttman 2004). All type of cells share
many virulence factors, such as extracellular polysaccharides and some type-
secreted effectors, and have common virulence mechanisms. Common principles
and mechanisms play roles in the interactions of microbial pathogens, biofertilizers,
phytostimulators, rhizoremediators, and biocontrol agents with plants (Lugtenberg
et al. 2002). Special emphasis is given to colonization, phase variation, two-
component systems, quorum sensing, complex regulation of the syntheses of extra-
cellular enzymes and secondary metabolites, etc. The study of the composition of
secretions and their effects in model systems, such as plant–nematode, provides
information that is useful for phytopathologists (Vanholme et al. 2004).
Introduction xvii
Seeds from some economic plants released volatile metabolites that serve as pro-
tector compounds against parasites are of especial interest in phytopathology for
modeling plant-parasite relations (Roshchina and Roshchina 1993, 2012), but it
should keep in mind that simultaneously the excretions are the sole carbon and
energy source for some bacteria (Sidorenko and Buzoleva 2012). Relations “plant-
insect” may be studied on special plant models which floral scent with the com-
pounds attracts insects-pollinators that is necessary for succesful pollination in plant
fertilization (Dobson and Heidi 2006).
The recent tendency to understand natural events in comparison with technology
includes two criteria proposed to characterize the diverse relations between nano-
technology and Nature (Schiemann 2005). Assuming that nature is not produced by
human action, the first criteria endorses the difference between natural and artificial
objects in nanotechnology and the second criteria allows the discussion of potential
nanotechnological modifications in nature. The selection of vital models sensitive to
small concentrations of certain chemicals may serve as part of a similar
technology.
In this book, widely known models are not described, referring readers to special
literature. Instead we concentrated on cellular models (not much known) that can be
used in vital regimes without fixation and vivisection. The focus of this book is on
new cellular models analyzed with in vivo microscopy that permit estimation of the
accumulation and release of secretions and their biological effects, including signal-
ing and contacts with other cells.
Chapter 1
Approaches to Choice of Model Systems
for Microscopic Studies
Modeling with secretions and receipting systems usually includes the analysis of
such reactions as composition of the secretions and their spectral characteristics
(absorbance and fluorescence), the germination, and the growth and development.
In this case, the donor model of the secretion and the acceptor model of the secre-
tions should be distinguished. Modeling processes involve donor cell secretion
(model system 1) and acceptor cells-biosensors (model system 2) that perceive and
react to the components of this secretion in the form of a physiological response,
which is one of the experimental approaches to study the mechanisms of intercel-
lular signaling in the chemical communication of organisms (Fig. 1.1). Donors may
be considered not only as plants because excretions of other organisms enriched in
similar secretory products, for example, known neurotransmitters acetylcholine and
biogenic amines as well as related enzymes of their synthesis and catabolism, are
observed in animals and microorganisms (Roshchina 2010). Plant cells also serve as
acceptor models of the external chemicals independently on the organism which
adds the secretion (microorganism, animal, or other plant species). As shown in
Fig. 1.1, the contact of models 1 and 2 in turn may serve as model 3 when both
contacted models changed their parameters in the form of physiological replies.
To choose model systems suitable for microscopic observation is a necessary
step in the studies. The objects used as intact model system can be well seen under
various microscopes, having intensively colored or fluorescent secretory products.
In many cases, they fast germinated or developed during the short time of visualiza-
tion. The researcher also observes the localization of secretory products and their
accumulation in secretory structures on the different stages of plant development as
well as express identification of the prevailing component. The technique for analy-
sis may be suitable for the investigation of mechanisms when one cell (a donor cell)
releases a molecule that is received by another cell (an acceptor cell) belonging to a
different species.
The success of such investigations requires models, mainly pigmented and/or
fluorescent cells suitable for visual manipulation under the microscope and, in some
V.V. Roshchina, Model Systems to Study the Excretory Function of Higher Plants, 1
DOI 10.1007/978-94-017-8786-4_1, © Springer Science+Business Media Dordrecht 2014
2 1 Approaches to Choice of Model Systems for Microscopic Studies
Model system 1 Model system 2
Cells-donors of secretions Cells-acceptors of secretions
secretion
sensitivity to secretion
secretion released
Parameters of analysis Parameters of analysis
localization of secretions in cells and extracellular reception of

secretory organs secretory products as chemosignals
express-indentification of secreting sensor systems to individual

components components of secretions
pernetration of secretory products
development of secretory systems into cell
excretions of secretory products physiological response of model
into water medium system
Model system 3
Contacts of cell-donors and cells-acceptors of secretion

in biocenosis
physiological responses of contacted model systems 1 and 2
Fig. 1.1 Model systems which could be used for microspectral analysis
cases, measurable as the absorbance or fluorescence parameters. As a whole, the

search is concerned mainly with biologically (allelopathically) active plant species
which also possess medicinal features. Multicellular or unicellular objects may be
chosen as models depending on the purpose.
It is possible to observe cellular models by microscopic methods: (a) the transport
of the compounds analyzed into an acceptor cell selected as a model (biosensor), (b)
changes in the autofluorescence or color of model cells, (c) the subcellular location
of secretion in models, (d) interaction of exometabolites from a donor cell with
certain compartments of the acceptor cell, and (e) changes in growth and develop-
ment of the acceptor cell as an integral response of the biosensor to secretions from
a donor cell.
The success of such investigations requires models, mainly pigmented and/or
fluorescent cells suitable for visual manipulation under the microscope and, in some
cases, measurable as the absorbance or fluorescence parameters.
Cellular models are possible to be observed by microscopic methods: (a) the
transport of the compounds analyzed into an acceptor cell selected as a model
(biosensor), (b) changes in the autofluorescence or color of cells, and (c) the subcel-
lular location where the chemical acted within the acceptor cell (model) and inter-
acted with certain compartments.
1.1 Color and Absorbance in Analysis 3
The use of the cellular models is possible to observe by microscopic methods for
the analysis of cell–cell contacts. In this case, one could see the changes in the state
of both the donor cell of the secretion and the acceptor cell of the secretion. Among
the processes tested may be the following: (a) the transport of the compounds ana-
lyzed into an acceptor cell selected as a model (biosensor), (b) changes in the auto-
fluorescence or color of donor cells or acceptors of the secretion, and (c) the
subcellular location where the chemical acted within the acceptor cell (model) and
interacted with certain compartments. Images of contacting surfaces with secretions
may be observed by usual microscopy, stereomicroscopy, and various modifications
of luminescence microscopy, including laser-scanning confocal microscopy and
microspectrofluorimetry.
In this chapter, the various approaches of secretory process testing with micro-
spectral and usual spectral techniques that could be used for various model systems
are considered.
1.1 Color and Absorbance in Analysis
Images of contacting surfaces with secretions may be observed by usual micros-

copy, stereomicroscopy, and various modifications of luminescence microscopy,
including laser-scanning confocal microscopy and microspectrofluorimetry. In
some cases, the choice of the objects recommended as a model could be connected
with the possibility to receive spectral data with microspectroscopy or microspec-
trofluorimetry as well as by using special apparatuses related to laser-scanning
confocal microscopy. Visual observation and spectral measuring of colored sam-
ples may occur under the usual microscope or stereomicroscope and various spec-
trophotometers (Table 1.1). The luminescence technique includes various
luminescence microscopes and their modifications such as microspectrofluorime-
ters (combination of the luminescence microscope with a multiphotometer for the
emission spectra registration) and laser-scanning confocal microscopes (a tech-
nique which permits to do optical slices of the object and to register the emission
spectra from different parts of the sample). Optical coherent images (registered by
optical coherence microscopy) could also be received for secretory structures
impregnated with calcium or siliceous salts, in particular idioblasts including oxa-
lates of calcium or tips of stinging emergences (Kutis et al. 2005; Roshchina et al.
2007a).
1.1.1 Usual Microscopy and Stereomicroscopy
Usual microscopy is useful when the samples contain pigmented secretions well
seen, as can be observed in particular for the leaf oil ducts and secretory cells of
Hypericum perforatum (Fig. 1.2). The main pigment of oil ducts colored in red is
Table 1.1 Technique for the study of colored plant samples

Material for Advantages of the
Apparatus Aim of the study the study methods
Usual microscope with Visual and photographic All parts of Simplicity and
transmitting light analysis of the plants noninvasive
secretion of intact observation
secretory cells
Stereomicroscope Visual volume and All parts of Simplicity and
photographic analysis plants noninvasive
of intact secretory cells observation
Microspectrophotometer Detailed spectral analysis All thin parts Noninvasive observation
for the spectrum of the secretions of of plants and receiving of
recorder intact and fixed spectral quantitative
secretory cells and qualitative
characteristics of the
sample
Usual spectrophotometer Spectral analysis of the All parts of Noninvasive observation
with different absorbance of intact plants and receiving of
cuvettes, glasses, and secretory cells spectral quantitative
glass cameras and qualitative
sample
known to be anthraquinone hypericin (Brockmann et al. 1950) and used as medical

drug (Karioti and Bilia 2010). The absorbance spectrum of hypericin has maxima at
590 and 595 nm (Roshchina 2008). Colorless secretory cells do not include the pig-
ment. Other examples may be the leaf phenol-containing glands of Lysimachia
nummularia (Fig. 1.3). Some secretory cells of the genus Lysimachia include both
phenols and their quinones (Fig. 1.4), for example, benzodilactones and quinones in
glands of Lysimachia fordiana (Huang et al. 2009).
In practice, the accumulation of vacuolar pigments is analyzed. In vacuoles,
anthocyanins are usually concentrated (Conn et al. 2010), although instead of these
flavonoids, two major types of betalains, the red-purple betacyanins and the yellow-
orange betaxanthins, are met (Harris et al. 2012). The color of many flower petals
and sometimes non-generative parts of plants permits the observer to see internal
secretory structures as vacuoles simply under the usual microscope with water or oil
immersion.
Stereomicroscopy is used mainly for multicellular objects to be seen in bright
light. Modern stereomicroscopes are equipped with a zoom lens system or a rotating
drum (containing Galilean telescopes) that are utilized to increase and decrease
overall magnification (achieved by objective and eyepiece magnifications, plus that
contributed by any intermediate or external auxiliary magnifying lens systems).
Stereomicroscopes are used for the observation of secretory structures and the
natural secretions on the surface of allelopathic plants, in particular essential
1 2 3
4
OH O OH
HO OH
HO OH
OH O OH
Fig. 1.2 Leaf oil ducts and secretory cells of Hypericum perforatum seen in transmitting light of
the usual microscope. (1) Leaf with dark and light spots of secretory structures, bar = 2 mm; (2)
oil duct with red pigment hypericin, bar = 500 μm; (3) glandular cell with light secretion, bar = 200
μm; (4) formulae of hypericin, main component of the secretion in oil duct
oil-containing structures on leaves and flowers (Gerbach 2002; Pacek and

Stpiczynska 2007) or roots (Hazak et al. 2010), as well as for the collection of secre-
tory structures necessary for chemical analysis of the secretions (Huang et al. 2009).
A researcher could see the different components of the secretion from the plant
donor or the changes in the images of plant acceptors undergoing the treatment with
the secretion.
By stereomicroscopy, flower capitate glandular trichomes of Rubus odoratus are
seen as multicellular structures with a transparent head filled with oleoresin and
red-colored stalk (Fig. 1.5). Oil secretion is accumulated on the bottom of the head.
By stereomicroscopy, one may also see the secreting hairs of the pistil of Tulipa sp.
or Hippeastrum hybridum, where there was a difference in the components of
secretions on the cell surface (Roshchina et al. 2013a).
Stereomicroscopy permits to observe vital changes in the secretion accumulation
with minimal damage of the secretory structures and to take photos of the model
selected.
1 2
Fig. 1.3 Phenol-containing leaf glands of Lysimachia nummularia with red phenolic pigments in
transmitting light (1) and under UV light (arrows show gland location) of the luminescence micro-
scope Leica DM6000 (2). Bar = 200 μm
O O
H3CO O H3CO O
O HO
H3C(H2C)4
O H3C(H2C)6 O
(CH2)6CH3 (CH2)6CH3
H O O
Fordianin A Fordianin B
(CH2)6CH3 O
(CH2)6CH3 O
O HO OH
HO
H3CO O (CH2)6CH3
H3CO O (CH2)6CH3
O
Fordianaquinone A Fordianaquinone B
Fig. 1.4 Phenols and quinones found in leaf gland of Lysimachia genus (Huang et al. 2009)
a b c
calyx
calyx
surface
hair
Fig. 1.5 The images of secretory structures such as calycinal glandular resin-containing hairs
from the flower of Rubus odoratus. (a) Common view of a flower (image a) with hairs shown with
arrows; (b, c) secretory hairs seen on stereomicroscope. On image c, one can observe a drop of
resinous secretion in the base of the head in secretory hair. Bar = 500 μm
1.1.2 Microspectrophotometry and Spectrophotometry
Microspectrophotometry and spectrophotometry with special holders are tech-

niques that permit to measure the absorbance of pigmented cells and register their
absorbance spectra (Karnaukhov 1978, 2001; Merzlyak et al. 2008). Secretion to
the vacuole of Allium cepa may be observed by this apparatus (Weisenböck et al.
1987). In the vacuolar region, the absorbance spectra of the object had maxima at
265 and 325 nm, whereas fluorescence spectra had a maximum at 520 nm.
Microspectrophotometric measurements of the absorbance spectra of guard cells of
Commelina communis and Allium cepa indicate a high absorbance in the blue region
of the spectrum with a maximum at 340 nm that is related to vacuoles containing
phenols and anthocyanins (Donkin and Martin 1981). The small maxima at 440–
445 and 470 nm were also seen in alkaline medium guard cells of Commelina com-
munis that had a maximum at 440 nm. The contribution, for example of flavonoids
and chalcones in the spectral characteristics was possible in the range of 336–
370 nm. Vacuolar fluorescence in onion Allium cepa with a maximum at 520 nm
was also observed and related to flavin or flavoprotein as possible blue light receptor
in tonoplast (Zeiger and Hepler 1979). Measurements of absorbance after plasmoly-
sis and deplasmolysis of the cells indicated that maximum blue light absorption
occurred within the cytoplasmic or vacuolar contents. Chromatographic analysis of
epidermal pigments indicated the presence of considerable quantities of flavonoids
which absorb light in the UV and blue region of the spectrum. Anthocyanin absorp-
tion maxima in vivo, as estimated with microspectrophotometry of vacuoles in
leaves (in the leaf cross sections) of Norway maple (Acer platanoides L.), cotoneas-
ter (Cotoneaster alaunica Golite), hazel (Corylus avellana L.), Siberian dogwood
(Cornus alba L.), and Virginia creeper (Parthenocissus quinquefolia (L.) Planch.),
were found between 537 and 542 nm (Merzlyak et al. 2008). In non-senescent
leaves, strong anthocyanin absorption was found between 500 and 600 nm (with a
70–80 nm apparent bandwidth). The objects were studied when they are differing in
pigment composition and at different stages of ontogenesis. Vacuolar absorbance

spectra were also measured for Hydrangea macrophylla by a microspectrophotom-
eter combined with a proton-selective microelectrode (Yoshida et al. 2003).
Using a microspectrophotometer, light absorption of colored vacuole was exam-
ined on intact cells of 20 plant species containing various kinds of pigments (Hayashi
1983). Based on their spectral curves, colored cells were classified into six groups
according to the type of curves, the location of absorption maxima, and the number
of peaks. There are group I (no absorption peak in the visible region as seen on vacu-
oles of all white flowers like in cacti flowers, dahlia yellow flowers, and broad bean
dark brown eye in petals, showing the presence of flavonols, chalcones plus aurones,
and melanins, respectively) and group II (1 spectral peak appearing on vacuoles of
red and red-purple organs in the region, 534–552 nm, which were due to anthocya-
nins like it is in stock rose flower or betacyanins in common four o’clock flower). In
group III, there are 2 peaks on the vacuoles of yellow, orange, or red organs, in
which betaxanthins alone (flowers of common portulaca) or both betacyanins and
betaxanthins (flowers of cacti) are contained. In group IV, 3 or more spectral peaks
appeared on the vacuoles of blue day flower petals (Commelina communis L.) and
orange tritonia flowers (Tritonia crocosmaeflora Lemoine). The former blue is due
to the metal or copigment complex of anthocyanin, while the latter is due to both
water-soluble carotenoid and anthocyanin. In group V, 2 or more peaks appeared on
the plastids of yellow, red, or green organs, showing the presence of carotenoids (red
pepper fruit) or chlorophylls (amaranth leaf), but none in the vacuoles. In group VI,
2 or more peaks appeared on the plastid and 1 on the vacuole of red flowering cactus
(Brasilicactus haselbergii Backbg.). Among these peaks, those of plastids were due
to carotenoids, and another peak of vacuole was due to betacyanin. Accordingly, it
becomes evident that the characteristics of absorption curves of intact cells are suf-
ficiently indicative of a series of major pigment components in colored plant organs.
The usual spectrophotometer also may be used for vacuolar estimation of pig-
mented flower components such as pollen and petals (Fig. 1.6). Anthocyanins from
various plant species have certain maxima in the absorbance spectra depending on
the pH – in acid medium at 525–540 nm or in base medium at 575–630 nm (Conn
et al. 2010). Pollen grains in some plant species may include the pigments in the
sporopollenin of their cover (which is well seen from the absorbance spectrum of
the Petunia hybrida microspore) and easily released out after moistening (Stanley
and Linskens 1974). Vacuolar pigments in petals of Hibiscus rosa-sinensis and
Epiphyllum hybridum show maxima at 440 and 540 in the absorbance spectra that
correlates with the anthocyanin contents.
1.2 Fluorescence in Analysis
In the use of fluorescence of models selected, one could see both autofluorescence
(natural emission of the analyzed cells) and fluorescence of fluorescent markers or
probes. Depending on the purpose, the parameters are studied by special lumines-
cence technique (Sect. 1.2.3).
1.2 Fluorescence in Analysis 9
Anthocyanins from pollen Intact pollen

Petunia hybrida Petunia hybrida
0.3 597 nm 0.03

537 nm
625 nm
Absorbance (units of optical density)
0.2
0.1
0
Papaver orientale 400 500 600 700
Wavelength (nm)
0.3 535 nm
600 nm
Intact surface of petals
0.2
2.4
Hibiscus
0.1
rosa-sinensis
0
2.2
Tulipa sp 527 nm Epiphyllum
0.3
hybridum
620 nm
0.2 2.0
0.1
1.8
0
25 21 17 13
400 500 600 700
Wavelength x 103(m-1) Wavelength (nm)
Fig. 1.6 The absorbance spectra of the individual anthocyanins and anthocyanin-containing pol-
len and petals
1.2.1 Autofluorescence of Secretory Components
Autofluorescence is the natural emission of cellular components due to a contribu-

tion of fluorescent compounds located in different cellular compartments. Plant
secretory cells often contain fluorescing products, and it permits to study their natu-
ral emission (Roshchina et al. 1997b; Roshchina 2003, 2008).
The visible (400–700 nm) fluorescence of intact living cells (usually called auto-
fluorescence) excited by ultraviolet, violet, or blue light is often observed under the
luminescence microscope. Autofluorescence of living cells may serve as biosensor
and bioindicator reaction for various studies of plants – from the taxonomic position
of plant or pollen viability to the chemosignaling processes. This reaction is ana-
lyzed by the following ways: (1) on undamaged structures, if their surface is com-
posed of fluorescent components or interior of the structures filled with fluorescent
secretion, and (2) modeling with individual cellular components showing the pic-
ture of the fluorescing cellular structure observed. The autofluorescent structure
images are studied depending on the purpose, either for anatomic characteristics
applying to taxonomic sign in plant systematics or for a comparison of samples in
the control variants and those that are exposed under some factor. Moreover, optical
slices made with laser-scanning confocal microscopy permit to show a location of
fluorescing secondary metabolite naturally synthesized within the cell or exome-

tabolite interacted with the cell at cell–cell communications or at experimental addi-
tion of the chemical agent. Besides images, the fluorescence spectra of emitted
structures may be recorded with microspectrofluorimetry or certain laser-scanning
confocal microscopes. Measuring the absorbance and fluorescence spectra, there is
a possibility to estimate the quantitative yield of fluorescence not only for individual
fluorophores but also for certain cells, such as flower petals.
Autofluorescence of the cellular compounds may serve (1) as markers for cyto-
diagnostics and (2) as a natural indicator of cellular state because the emission often
shows changes in cellular metabolism and responses to the external and internal
signals. This is true for the plant secretory structures analyzed under the lumines-
cence microscope (the structures are not seen in the usual microscope without spe-
cial histochemical staining) or laser-scanning confocal microscope (Roshchina
2008, 2012). Due to special microspectrofluorimeters or confocal microscopes (see
below), the fluorescence spectra of objects studied and the emission intensity can
also be measured.
The main natural fluorophores met in living cells of various organisms are shown
in Fig. 1.7. Their fluorescence makes visible not only plant cells but also microbial
(mainly fungal) or animal (insect) cells. In general, blue and green emission reflects
the participation of universal cellular fluorophores NAD(P)H (max. 460 nm), pter-
ins (max. 450 nm), and flavins (max. 520 nm), although in dry state, weak blue fluo-
rescence (420–450 nm) is also peculiar to isolated nucleotides, RNA, and DNA as
well as some proteins and seen in the films under the luminescence microscope, but
water quenches the emission. Besides, there are characteristic fluorescent com-
pounds in various organisms (Pöhlker et al. 2011, 2012). In animal cells, chitin
(max. 450–460 and 520 nm), collagen, and elastin (max. 400–430, 465, 495, and
520 nm) or cellulose (max. 420–430 nm) in plants and fungi can also contribute to
blue fluorescence (Pöhlker et al. 2011, 2012). Orange-red fluorescence depends on
the organism. In plant cells, the main known fluorophore used for environmental
monitoring is chlorophyll with maxima at 675–680 nm (Agati 1998; Andersson
et al. 1998), in photosynthesizing bacteria – bacteriochlorophyll (Pöhlker et al.
2011, 2012). In flowers and fruits, within secretory organelles and vacuoles, flavo-
noids such as anthocyanins are met that, depending on the chemical structure, pH of
medium, and metal complex, may fluoresce with different maxima – from 435 nm
up to 665 nm (Figueiredo et al. 1990). As plant vacuolar fluorophores in certain
families within the order Caryophyllales are also betaxanthins emitted in green
unlike betacyanins (Gandía-Herrero et al. 2005a, b, 2009). Uroporphyrinogen and
hemoglobin contribute to red lightening of mammalians, while green-red range
emission may reflect a contribution of sea animal fluorescent proteins used for the
genetic experiments as vector for the study of cellular components (Karnaukhov
1978; Roshchina 2008; Pöhlker et al. 2011, 2012). Terpenes, some alkaloids (col-
chicine), and flavonoids in plants emit in the blue or in the blue-green (470–525 nm)
region of the visible spectrum, whereas polyacetylenes, isoquinoline, and acridone
alkaloids emit in the yellow and orange region (Roshchina 2008). Red fluorescence
of plant cells may also be due to anthocyanins and azulenes (600–630 nm)
Fig. 1.7 Main wavelength (nm)

fluorophores met in living
organisms 400 500 600 700
NAD(P)H
phycoerythrins
bacteriochlorophyII
flavins
phycobillins
flavoproteins
Bacteria
Non-photosynthesing
(Pseudomonas fluorescens, etc)
Photosynthesing
(cyanobacteria
Rhodopseudomonas, etc).
NAD(P)H
Fungi
flavins
flavoproteins
NAD(P)H
flavins
flavoproteins
Plants
sesquiterpene lactones
isoquinaline alkaloids
acridone alkaloids
Monoterpenes
polyacetylenes
azulenes
anthocyanins
chlorophyII
colchicine
tannins
coumarins
flavonoids
NAD(P)H
flavins
flavoproteins
Animals
Insects
Ticks
Spiders and spider web
Green
red
yellow
Sea organisms
(medusa, etc)
fluorescent Proteins
Urophorphirin
Haemoglobin
Vertebrata
(mammalian)
(Roshchina 2008). At ageing or, if the object being undergone ozone and relative
reactive oxygen species, fluorescent pigments lipofuscins are formed that may emit
in wider spectral range – from blue (450–480 nm) to red (>600 nm) depending on
the nature of the organism and duration of the exposure under an unfavorable factor
(Merzlyak 1988; Roshchina and Karnaukhov 1999; Roshchina 2003). Especially it
is clearly seen after the influence of ozone and reactive oxygen species (Roshchina
and Roshchina 2003).
Some secretory products may themselves serve as fluorescent markers and dyes
that are especially valuable in the analysis of the cell–cell contacts (Roshchina
2005a, c, 2012; Roshchina et al. 2011a).
1.2.2 Special Fluorescent Probes or Markers
Special fluorescent probes or markers are often used for the analysis of intracellular
and extracellular traffic as well as reception and membranous penetration of exog-
enous chemicals. Most used compounds are summed in a special book of Haugland
(1999). Among similar molecular probes, protein transport could be monitored with
fluorescent markers such as Lucifer yellow CH (L-453). Alkaloid berberine has
been used for exocytosis of secretory granules in animal mast cells. Luciferin was
analyzed in the ATP secretion experiments. Hypericin as an inhibitor of protein
kinases is also used in fluorescence imaging (Haugland 1999). Recently, some col-
ored and fluorescing components from natural excretions (anthocyanin pelargoni-
din, sesquiterpene lactone azulene, and alkaloid rutacridone) were represented for
the study of the allelochemical penetration into the cell (Roshchina 2005a, 2012;
Roshchina et al. 2011a).
1.2.3 Fluorescence-Related Technique
Fluorescence may be analyzed by various types of techniques (Table 1.2).

Luminescence microscopy permits to study both multicellular and unicellular mod-
els. The light emission of living plant cells is the subject of attention in plant physi-
ology (Gilroy 1997). Fluorescence from intact secreting plant cells was observed
and photographed under the usual luminescence microscope (Roshchina and
Melnikova 1995, 1999; Roshchina et al. 1998a) or its derivatives: laser-scanning
confocal microscope (Roshchina et al. 2004, 2007a, 2008, 2009a, 2009b; Roshchina
2008) and microspectrofluorimeter (Roshchina and Melnikova 1995, 1999;
Roshchina et al. 1998a, 2002; Roshchina 2003, 2007b, 2008). In the luminescence
microscope, light from the ultraviolet source (UV source) excites the fluorescence
of the object; the luminescent image might be caught directly by the eye of an
observer. Microspectrofluorimetry as a modification of luminescent microscopy
permits to receive fluorescence spectra from the individual cells or to measure the
fluorescence intensity at one or double wavelengths. Unlike usual luminescence
microscopy, the confocal microscope has a special confocal aperture (pinhole),
from which a fluorescence of the object excited with a laser beam with a certain
wavelength passes and multiplies by the photomultiplier before the eye visualiza-
tion. The construction of the aperture permits to focus the light beam on the
Table 1.2 Technique for the study of fluorescent plant samples

Material for Advantages of the
Apparatus Aim of the study the study methods
Luminescence microscope Visual and All parts of Simplicity and
(or in combination with photographic plants noninvasive
transmitting microscope) analysis of the observation
emission of intact
secretory cells
Laser-scanning confocal Detailed analysis of Thin lamellar Noninvasive observation
microscope the emission of surface of on the deep layers of
intact and fixed plant studied cells and
secretory cells objects cellular compartments
with optical slices
Microspectrofluorimeter for Spectral analysis of All parts of Noninvasive observation
the spectrum recorder the emission of plants and receiving of
intact secretory spectral quantitative
cells and qualitative
sample
Double-wavelength Comparative analysis All parts of Noninvasive observation
microspectrofluorimeter of the emission plants and receiving of
intensity of intact qualitative
secretory cells in characteristics of the
two different sample fluorescence
wavelengths
simultaneously
Spectrofluorimeter for the Spectral analysis of Extracts from Chemical analysis of the
liquid and rigid samples the emission from plant allelochemical
extracts and samples presence
pollens
Fluorimeters Analysis of liquid Extracts from Dynamics of certain
samples at one plant allelochemicals
wavelength samples
different depths of the object. A well-seen image of the cellular structure and its
optical slices permit to show changes induced by many experimental factors.
1.2.3.1 Luminescence Microscopy
Fluorescing secretory cells are well seen among nonsecretory or secretory cells with
other non-fluorescing products. Microscopy of donor cells (secretory cells) enriched
in fluorescent allelochemicals was specially considered earlier in some publications
(Roshchina 2003, 2008; Roshchina et al. 1997b). Photos, which are represented
paper of Roshchina with co-authors (2013a), demonstrated the examples of the light
emission that is seen under the luminescent microscope, in particular for the rosette
obtained from leaf secretory glandular hairs of Hibiscus rosa-sinensis (blue-green-
ish emission, especially bright for crystals of released secretion, mainly due to
terpenes and phenols). Microscopy of donor cells (secretory cells) enriched in
fluorescent allelochemicalsvwas specially considered earlier in some special publi-

cations (Roshchina 2003, 2008; Roshchina et al. 1997b).
To select objects as modeling systems for the analysis of chemosignaling repre-
sents an important methodological task. Among the advantages of using the secretion
of plant cells as models for cell biology is their ability to brightly fluoresce when
excited by ultraviolet or violet light. The nature and intensity of autofluorescence cells
are markedly changed in development or under the influence of oxidants, some trans-
mitters, as well as various proteins. This ability can be the basis for developing appro-
priate tests, using conventional fluorescent and scanning confocal microscopes.
1.2.3.2 Microspectrofluorimetry
The recording microspectrofluorimetry is also applied for cellular biology as one of

the noninvasive methods used for cellular diagnostics. The luminescence of microob-
jects excited by shortwave radiation of an arc lamp and after spectral decomposition
is registered by detectors–photomultipliers. With the apparatuses one can detect
fluorescence from individual cells and even from a cell wall, large organelles, and
secretions in the periplasmic space (space between the plasmalemma and cell wall),
as well as from the drops secreted by secretory cells remaining on the cellular sur-
face. This method permits the investigation of physiological activity of a secretory
process in vivo (Roshchina et al. 2007a, b, 2011a; Roshchina 2008).
Microspectrofluorimeters, having a detector with optical probes of various diam-
eters up to 2 μm (the changed areas or probe holes composed with the system of
mirrors), have been constructed in the Institute of Cell Biophysics of Russian
Academy of Sciences (Karnaukhov et al. 1982, 1983, 1987; Karnaukhova et al.
2010) and may record the fluorescent spectra or measure the emission intensities or
the fluorescence intensity at two separate wavelengths (Karnaukhov et al. 1982,
1987; Karnaukhova et al. 2010). Microspectrofluorimeters can receive a magnitude
fluorescence image of a certain area of the specimen that appears on a spherical mir-
ror. The emission data may be written in a form of the fluorescence spectra by the
help of an X-Y recorder (could be coupled with a computer). The fluorescence
spectra of secretory cells were registered recently with microspectrofluorimetric
technique (Roshchina et al. 1995, 1996, 1997a, b, c, 1998a, 2002; Roshchina and
Melnikova 1995, 1996, 1999). The examples of the fluorescence spectra of some
secretory cells from allelopathically active species (in which excretions may regu-
late the growth and development of the surrounding inhabitants of biocenosis) are
shown in Table 1.3. At excitement by UV (360–380 nm) or violet (420–430 nm),
certain models that have autofluorescence may be applied to the study of the accu-
mulation of secretory product into cell. In particular, bract gland (named lupulin) or
idioblasts of the ovule from hop Humulus lupulus are a clear example shown in
Fig. 1.8. Emission from glandular cells during development demonstrates a maxi-
mum in the blue region (460 nm) which is absent in a matured structure. It appears
to show the appearance of bitter compounds responsible for the characteristic
hop smell instead of phenolic components that prevailed at the beginning of gland
development. Moreover, the difference between secretory (no chlorophyll) and
Table 1.3 Fluorescence of secretory cells located in some allelopathically active species
Color of Fluorescence
Species Secretory cells fluorescence maxima, nm Fluorescent components
Betula verrucosa Secretory cells Bright blue 460–470 Flavonoids quercetin,
Ehrh., birch of woody (exudate) kaempferol, etc.
bud scales Blue-green 500, 520
(secretory
hair)
Blue-green 480–495, 520,
(secretory 680
cell)
Solanum Leaf secretory Blue-green 465. 550 or Alkaloids noscapine,
tuberosum L., hairs 475-, 540 β-solamarine
potato
Campanula Glandular Blue-green 475, 560 Acids phenol carbonic,
persicifolia L., surface of vanillic, caffeic,
bellflower pistil ferulic, chlorogenic,
stigma and flavonoid
diosmetin
Ruta graveolens Root tip Orange (root 500, 590 Flavonoids rutin and
L., rue surface, tip) quercetin, alkaloid
idioblasts Idioblast 585–590 rutacridone
Yellow-orange 480, 575
(root
secretory
hair)
Yellow (leaf 565
secretory
cell)
Sources: Roshchina and Roshchina (1993), Roshchina (1999a, b, 2001b, 2008)
nonsecretory (chlorophyll maximum at 680 nm) cells is well seen. The fluorescence
spectra were also measured by Leitz MPV-SP microspectrophotometer, in particu-
lar for the analysis of the flavonoid accumulation in Fabaceae at the nodule forma-
tion (Mathesius et al. 1998).
1.2.3.3 Confocal Microscopy
Unlike the usual luminescence microscope, in confocal microscopy before catching

by the photomultiplier, the fluorescence beam from the sample studied passes
through a confocal aperture named pinhole (Pawley and Pawley 2006). Changing
the diameter of the aperture, the pinhole limits a scattered light from the object
points which are out of a focal plane and contrasts the volume image. The construc-
tion of a laser-scanning confocal microscope permits to observe cellular structures
by the mode of a regulation of the depth of an object slide (LSCM images). The
advantages of the technique are (1) to receive common complicated interference
image of the object, (2) a possibility to have the increased depth penetration for
receiving 20 visual slices (optical sections) or the complete volume (the information
a gland (lupulin) c
25 Developing gland
bract Non-secretory cell
Fluorescence (relative units)

b
0
Matured gland
25
0
400 500 600 700
Wavelength (nm)
Fig. 1.8 Autofluorescence of the gland in bract of hop Humulus lupulus (sources: Roshchina
(2008) and unpublished data). (a) Common view of bract with gland named lupulin; (b) fluores-
cence of the gland surface, excitation 405–420 nm, bar = 500 μm; (c) the fluorescence spectra of
glandular cells measured by a microspectrofluorimeter MSF-1, excitation 360–380 nm
must also be quantitatively extracted), (3) pattern analysis of the structure, and (4)
registration and analysis of the fluorescence spectra of some part of a cell. Confocal
microscopy offers several advantages over conventional optical microscopy, includ-
ing controllable depth of field, the elimination of image degrading out-of-focus
information, and the ability to collect serial optical sections from thick specimens.
The key to the confocal approach is the use of spatial filtering to eliminate out-of-
focus light or flare in specimens that are thicker than the plane of focus. Cells of
algae and nonsecretory cells of some higher plants were studied by the method,
mainly labeling with immunofluorescent probes or fluorescent dyes (Wymer et al.
1999). Confocal imaging (LSCM) of secreting plant cells was studied for pollen and
vegetative microspore analysis (Salih et al. 1997; Roshchina et al. 2004; Roshchina
2008). Scanning of the object studied along the Z-coordinate with an interval of
1.0 μm was seen for the slices and stack of the slices of the leaf secretory hairs on
Solidago virgaurea or on Solidago canadensis L., species enriched in terpenoids
(Roshchina et al. 2007b; Roshchina 2008). The slices can be collected by the help
of a special computer program. Some examples of LSCM images of secretory cells
registered by this technique (Roshchina et al. 2013a) will be represented below.
Figure 1.9a–d analyzed the LSCM images of oleoresin-evolving calycinal secretory
hair from the flower of fragrant thimbleberry (purple-flowering raspberry) Rubus
odoratus. Their fluorescence spectra were measured from parts of the secretory
structures in rings (Fig. 1.9e, f 1–4). A laser beam of sufficiently high power, in the
short time of observation, does not prevent the normal development of some plant
a b 1
c d
4
3
Fig. 1.9 The LSCM images (left a–d) and the fluorescence spectra (right e, f, see p. 18) of calyci-
nal glandular resin-containing hairs from the flower of Rubus odoratus with resinous secretion
(Adapted from Roshchina et al. (2013a)) Images seen under laser-scanning confocal microscope
Leica TSC SP 5: (a) Optical slice of the hair. Bar = 250 μm. (b) Stack of the image of the hair head
and stalk. Bar = 100 μm. Laser excitation 405 nm. Blue and blue-green fluorescence seen appears
to be due to various flavonoids and terpenes. Red-fluorescing chloroplasts are seen. (c) Head of the
trichome covered by evolved secretion (bar = 50 μm). Oil drops of blue fluorescent secretion were
seen. (d) Oil resin drops (bar = 20 μm). Numerals of rings mean the parts of the structures from
which the fluorescence spectra e and f were measured
cells. Oil in the trichome head evolved along the capitate hair has one and the same
emission maximum at 520 nm peculiar to terpenes and phenols. The stalk fluoresces
in red due to chlorophyll in chloroplasts. After the 10 min extraction from calyx by
water (1:10 W/v) and then the same volume of ethanol, the head was liberated from
e
240
200
Viewer pos
2
160
120
80
1
Fluorescence intensity (relative units)
40
0
f
3 4
0.80
0.72
Viewer pos
0.56
0.39
0.23
0.06
450 500 550 600 650 700 750 [nm]
Wavelength
Fig. 1.9 (continued)
oleoresinous secretion, and chloroplasts became visible here. The stalk fluoresces in
red due to chlorophyll in chloroplasts.
1.2.3.4 Images and the Fluorescence Spectra of Cells in Cytodiagnostics
The search for secretory cells among the nonsecretory ones basing on the difference
in their vital fluorescence has perspective for the selection of model systems. In
particular, there are the images seen under usual luminescence or confocal micro-
scopes and the fluorescence spectra measured by microspectrofluorimeters or some
1.3 Cellular Observation of Secretory Process 19
OMe
O + R2 O
MeO N
(+) O
HO HO N
O
OH
H Berberine
O
O
R5
H OH HO O
+ N
B H
R7 O O OH
Brefeldin A A C R3
HO R6 O
Anthocyanin Betacyanin
R5
glc (common formulae)
Fig. 1.10 Molecular probes for the study of secretory process
types of confocal microscopes. The research of fluorescence maxima in secretory

cells showed the correlations and differences in the emission spectra excited by UV
light among the plant species studied and their specialized structures (Roshchina
2003, 2008). For example, nectaries mainly fluoresce in the blue region, while spo-
rangia of fungi, mosses, horsetails, and ferns fluoresce in the green-yellow region
(500–550 nm). Except fungi, the structures also have a maximum at 680 nm in red,
peculiar to chlorophyll located in chloroplasts. Crystals of secretions emit brighter
than liquids. The secretion peaks were compared with the maxima of individual
components of the same secretions in solutions or films (Roshchina et al. 2011a).
Interference of various fluorophores in total emission may take place, but diagnos-
tics of the prevailing component of the secretion is real.
Changes in the fluorescing compounds may be seen on model systems repre-
sented by big plant cells that are suitable for observation both in vivo (such as in
nature) and in vitro (in cultivated systems). Among similar objects are generative
cells of flowers – pollen (male gametophyte) and pistil (female gametophyte) – and
non-generative cells, such as plant vegetative microspores and special secretory
cells (see Sect. 1.3). Endogenous idioblasts are single secretory cells found among
tissual nonsecretory cells and are also met in tissue cultures of undifferentiated
cells. Idioblasts differ from other cells, mainly due to the fact that there are usually
only specialized cells, producing a secretion. Phenol-containing idioblasts, in par-
ticular of Humulus lupulus bracts, are well seen in blue on the tissue slices under the
luminescence microscope at the excitation with easily 420–430 nm or 530–550 nm
(see Fig. 2.6). On the bract gland slice, there are also peltate glands (Srečec et al.
2010) which brightly fluoresce (see Fig. 1.8).
1.3 Cellular Observation of Secretory Process
At the cellular level, one could observe secretion release and interactions with con-
tacted cells on model systems with special molecular probes added into the medium
or in vivo basing on the natural color or fluorescence of the model system selected.
To select the model system shown above, it is necessary to choose (1) big cells well
seen under the microscope, (2) objects stored or those which live in a vital state dur-
ing the whole time of the experiment, and (3) plant species universal and easily in a
reproduction in laboratory or in the field conditions.
1.3.1 Secretion Transport and Excretion
Secretion traffic from organelles to vacuoles or out of the cell occurs via the trans-
port of secretory vesicles. In most cases, exocytosis is associated with intracellular
membranes and can be regulated by contractile proteins, mainly actin that forms
coats and rings promoting a regulated type of exocytosis (Nightingale et al. 2012),
Golgi apparatus, and the eventual loss of Golgi apparatus morphology (Haugland
1999, c. 280). Plants contain Rho-like small G proteins (small GTPases) called
RACs or ROPs that, like fungal and metazoan Rhos, are regulators of cell polarity.
The function of Rho proteins requires their association with discrete domains in the
plasma membrane where they orchestrate cytoskeleton organization vesicle traf-
ficking (Hazak et al. 2010) as well as local gradients of calcium ions and reactive
oxygen species (ROS). Exocytosis in this case may be controlled by receiving
mutants and by the genetic inclusion of green fluorescent protein (GFP) to the root
of transgenic Arabidopsis serving also as a model for the studies (Hazak et al. 2010).
To study the transport of proteins and their cellular secretion, use special block-
ers, such as brefeldin A that inhibits the transport of proteins from the endoplasmic
network in the Golgi apparatus and the reverse traffic of Golgi apparatus protein in
the endoplasmic reticulum (Satiat-Jeunemaitre et al. 1996; Nebenfuhr et al. 2002).
The proteins accumulate inside the endoplasmic reticulum. The target of the
brefeldin action is believed to be a stretch of GTP exchange containing the GTPase,
which is included in the transport vesicle protein to the outer membrane.
Brefeldin A, fungal compound isolated mainly from Penicillium brefeldianum
and Eupenicillium brefeldianum (Fig. 1.10), is now used to study protein transport
within a cell. Exposing cells to the molecular probe causes a distortion in intracellular
protein traffic from the endoplasmic reticulum to the Golgi apparatus and the even-
tual loss of Golgi apparatus morphology (Haugland 1999). Brefeldin A also alters
the morphology of endosomes and lysosomes. Brefeldin action could be monitored
with fluorescent markers such as Lucifer yellow CH (L-453). Exposing cells to
brefeldin A as a model of fungal compound effect may cause a distortion in intracel-
lular protein traffic from the endoplasmic reticulum to the Golgi apparatus and the
eventual loss of Golgi apparatus morphology (Haugland 1999).
As for phenols, flavonoids such as anthocyanins were studied as color markers
for the pigment traffic. It was shown on Arabidopsis thaliana (Poustka et al. 2007),
benefiting from the unique fluorescent properties of anthocyanins and using green
fluorescent protein involved by genetic engineering. One route for anthocyanin
transport to the vacuole involves vesicle-like structures shared with components of
the secretory pathway. By colocalizing the red fluorescence of the anthocyanins
with green fluorescent protein markers of the endomembrane system in Arabidopsis

seedlings, anthocyanins are shown to be also sequestered to the endoplasmic reticu-
lum and to endoplasmic reticulum-derived vesicle-like structures targeted directly
to the protein storage vacuole in a Golgi-independent manner.
But red coloration of pigments (Fig. 1.10) such as anthocyanin (Markham et al.
2000; Nielsen et al. 2003; Sato et al. 2011), betacyanin, betalain (Harris et al. 2012),
and hypericin (Karioti and Bilia 2010) could also be used to study both the secretion
into the vacuole and exocytosis when the pigments are liberated out from the cell.
Berberine (Fig. 1.10) has been used for exocytosis of secretory granules in ani-
mal mast cells (Haugland 1999). Luciferin was analyzed in the ATP secretion
experiments (Haugland 1999). Hypericin as an inhibitor of protein kinases is also
used in fluorescence imaging (Haugland 1999). The fluorescing alkaloid capsaicin
is also applied to study the plasmalemma permeability (Roshchina et al. 1986).
Intracellular secretion is mainly observed in the vacuole (which contains com-
pounds which are spared and may be included in cell metabolism again if it would
be necessary) and the cell space using self-coloration or autofluorescence of com-
pounds of vacuolar sap. Colored and fluorescent vacuolar products are mainly
anthocyanins and betacyanins (Fig. 1.10) which are also considered as vital cellular
markers.
1.3.2 Unicellular Models Suitable for Microspectral Analysis
In a search of model systems suitable for microspectral analysis of unicellular and

multicellular objects should be chosen as candidates. Unicellular model systems
may be considered as free-living (spores) or parts of extratissual and intratissual
secretory systems. Microspores of plants are known as vegetative
(in spore-bearing species) or generative (in seed-bearing species). Moreover, uni-
cellular objects are idioblasts within certain plant tissues. Single cells of glandular
structures (e.g., hairs, glands) are also potential models but today are far from real
modeling yet.
1.3.2.1 Vegetative Microspores of Spore-Bearing Plants
Vegetative microspores of spore-bearing (cryptogams) plants, in particular of horse-

tail Equisetum arvense L., are unicellular objects suitable for use, due to their sizes
for the microscopic observations in cell biology (Roshchina et al. 2002; Roshchina
2003, 2004a, d, 2006a, b, 2007a, b, 2008). The cells are visible even at minimal
objective × 10 of common usual and luminescence microscopes. The advantages of
the microspores as object are as follows: (1) faster division after moistening for
1–2 h, not more than 24 h; (2) good germination in artificial nutrient medium or in
water; (3) changes in cell interior and cell wall that are visible under the usual and
the luminescent microscopes; (4) sensitivity to natural substances; and (5)
Luminescence microscopy
Microspectrofluorimetry
1 2 3
registered MSF - 1 duable-wavelength MSF - 2

30 520 Histogrames
Spectrum 20
0.13 ± 0.006
Amount of cells
20 10
elaters
0
680
elaters cell elaters 20
3.15 ± 0.4
10
0 0
400 500 600 700 2 4
Wavelength (nm)
Laser-scanning confocal microscopy
4 5 6 7 200
3
1
(relative units)
Fluorescence
100 2
Dry spore 2 h of moistening 3 h of moistening
3
(optical slice) (Stacks of optical slices)
1
2 3 2
Development of microspore 1 0
400 500 600 700
Wavelength (nm)
Optical slice and emission
spectra of various parts
Fig. 1.11 The images and fluorescence spectra of the vegetative microspores of horsetail
Equisetum arvense (sources: Roshchina et al. (2007a, b, 2011b); Roshchina (2008, 2012) and non-
published data of author). Bar = 20 μm. Upper side: luminescence microscopy (left). Views (1–3)
of fluorescing microspores under UV light of the luminescence microscope Leica DM6000. Blue-
green fluorescence of surface with elaters is seen as well as red emission of chloroplasts.
Microspectrofluorimetry. The fluorescence spectrum (middle) and the emission intensity distribu-
tion in the form of numerical data or histogram (right). The fluorescence intensity (I) on histo-
grams measured by dual-wavelength microspectrofluorimeter MSF-2 shows as I520 and I680,
respectively, the emission intensity in the green region with a maximum at 520 nm and red region
with a maximum at 680 nm. Lower side: laser-scanning confocal microscopy. Images 4–7 – under
laser-scanning confocal microscopes LSM 510 NLO “Carl Zeiss” and Leica TCS SP-5 (4 and 7,
dry microspores; 5, moistened spore; 6, developing spore that put off the blue-fluoresced rigid
cover envelope and then divided, respectively). On the optical image slice (image 7), numbers
mean cellular parts, from which the appropriate fluorescence spectra have been received: exine (1),
its inner layer intine with plasmic membrane (2), and chloroplast (3) are marked. Excitation for the
luminescence microscope or microspectrofluorimeter was 420 nm and for laser-scanning confocal
microscopy, 405 nm laser
significant autofluorescence recorded after 1–2 min or 1–2 h of moistening in con-

trol experiments.
The spores are collected from the meadows by the end of April or in the begin-
ning of May and stored in dry conditions. They are analyzed under ordinary and
luminescent microscopes with photocamera (even one microscope with lumines-
cent and transparent regimes) and, if possible, microspectrofluorimeters or laser-
scanning confocal microscopes (Fig. 1.11). The objects are observed on slides under
the microscope, and if needs to having germinated spores used Petri dishes with
paper filters moisten with nutrient medium (Roshchina et al. 2002, 2004, 2007a, b).
Vegetative microspores of horsetail Equisetum arvense fluoresce and germinate on
the object glasses (slides) after moistening with artificial medium or simple water
(Roshchina et al. 2002, 2003; Roshchina 2004a, b). The number of germinated veg-
etative microspores (red fluorescing) was counted using objective × 10, 20, or 40
under the luminescent microscope. When vegetative microspores from Equisetum
arvense develop, new molecules of chlorophyll are formed, which fluoresce in the
red spectral region (fluorescing vegetative microspores could be photographed).
Hence, the blue fluorescence of non-germinated spores (when the rigid cover is on
the microspore cell) changes to red fluorescence of germinated ones (after the cover
has been put off, the cell starts to divide, and the content of chloroplasts is increased),
2–24 h after moistening (Fig. 1.11 images 5 and 6). The number of germinated
vegetative microspores (red fluorescing) was counted using the luminescent micro-
scope. The changes in the germination rates of vegetative microspores of Equisetum
arvense are considered as possible biosensor reactions to study mechanisms of the
secretion action on the other cells, including those of other plants, animals, and
microorganisms (Roshchina 2003, 2004a, 2006a, b, 2007a, b).
Observation of each test sample in vegetative microspores of Equisetum arvense
lasts from 2 h (for fresh microspores, <1 month after collection) to 6–24 h (for
microspores stored >1 month). The observer fixes the start of development as a
missing of rigid blue-fluorescing cover and elaters and after 2–24 h may count the
number of red-fluorescing microspores that increase in the red chlorophyll fluores-
cence in comparison to undeveloped (fluoresce in blue) and see the divided cells
(Fig. 1.13). Under the ordinary microscope, after 2–3 days, each microspore may
form rhizoid cells, which have less amount of chloroplasts than prothallium (first
divided cells) and thallus cells giving gametophyte. One hundred microspores were
analyzed per slide.
Figure 1.11 shows examples of fluorescent analysis of vegetative microspores of
horsetail Equisetum arvense using various techniques. Under luminescence micro-
scope, green fluorescence of cell wall and elaters (shell employees to attach micro-
spore to the substrate) is visible. Through the rather transparent cover, bright red
fluorescence of chloroplasts, located inside the cell among non-lightening cyto-
plasms, is also observed. The total spectra of fluorescence from cells measured by a
microspectrofluorimeter depended on the presence of appropriate pigments and had
the three peaks – in blue with maxima at 450–460 nm (azulenes, phenols), in green
with maxima at 540–550 nm (carotenoids), as well as in the most intense red emis-
sion with maxima at 675–680 nm (chlorophyll). Cell cover liberated during the
microspore development does not have a maximum in red spectral region (Roshchina
et al. 2002). The average fluorescence intensity of 100 cells measured on one sub-
ject slide by dual-beam microspectrofluorimeter in green (520 nm) and red (640–
680 nm) differed many times as seen from numerical data of summary emission
shown in the histogram area (3.15 relative units in red against 0.13 relative units in
green). Red emission prevails. Summed distribution histograms of green and red
fluorescence in Fig. 1.11 demonstrate significant variability among the cells just in
red emission – number cells with the fluorescence intensity from 2 up to 4 relative
units. During the microspore development, the red fluorescence intensity increases
in comparison with dry rest (non-developed) cells. If the division of the spore cell
occurs (and duplication of chloroplasts takes place), the two times increase in red
fluorescence is the indicator of cellular division because amounts of chloroplasts
and fluorescing chlorophyll are doubled.
Unlike microspectrofluorimetry, a more sensitive laser-scanning confocal
microscopy made it possible to see images of optical slices of intact microspore
cells at different depths and register the spectra of fluorescence from individual
parts (marked as numerals in Fig. 1.11 image 7), viz., cell wall, including its outer
layer exine (1), its inner layer intine with plasma membrane (2), and chloroplast
(3). Spectra of fluorescence measured with confocal microscope in this case differ
from the total spectrum of cell measured by microspectrofluorimetry that depends
on just the optical slice position in the spore structure. Exine mainly fluoresces in
blue and green spectral regions with maxima at 450 and 500 nm peculiar to phe-
nols, while intine with close-fitting plasmic membrane fluoresces in the blue-green
region with maxima at 465 and 520–530 nm (phenol and flavine luminesce),
whereas plastids fluoresce in the red region (chlorophyll maxima at
675–680 nm).
Fluorescence and related germination of the vegetative microspores may serve as
test reactions to study effects of plant secretions at the cellular level in vivo (see
Chaps. 1 and 3). The spores collected in May retained the ability to germinate and
developed up to the formation of gametophyte with sexual organs (archegonia and
antheridia) in 2 months. But the cells can be divided and formed multicellular thal-
lus several years after collection.
1.3.2.2 Pollen of Seed-Bearing Plants
Pollens are generative microspores or the male gametophyte of seed-breeding plants

(gymnosperms and angiosperms). They are also unicellular objects with hard cover
that contains polymers of phenols or carotenoids. Unlike vegetative microspores,
pollen grains produce male gametes – spermia with haploid genome. They have no
elaters and cannot develop as autotrophic cells because they lack chloroplasts.
Pollen development may be observed either on the pistil stigma (in vivo) or on the
artificial medium (in vitro). After moistening (by secretion of flower pistil stigma or
artificial medium), this cell forms the male gametes called spermia. When pollen is
developed on a flower pistil, spermia moves within a forming pollen tube to its tip
growing to the ovule. Then one spermia is copulated with the egg cell, both nuclei
are combined, and the fertilization occurs.
Pollens release the secretions in the first seconds of moistening by pistil stigma
liquid in vivo or by nutrient medium in vitro experiments (Stanley and Linskens
1974). Figure 1.12 shows the drops of fluorescing secretions recording by laser-
scanning confocal microscopy.
1.4 Model Process Testing 25
1 2
Fig. 1.12 LSCM images (stacks) of fluorescing pollen of Cichorium intybus L. (1) Dry pollen; (2)
after moistening (drops of released green-fluorescing secretion are seen). Laser excitation 488 nm.
Bar = 10 μm. Confocal microscope LSM 510 NLO “Carl Zeiss.” (Adapted from Roshchina et al.
(2011c))
Pollens of knight’s star Hippeastrum hybridum L. (Herb.), clivia Clivia hybrida,

and mock orange Philadelphus grandiflora Willd. easily forming pollen tube were
visible even at minimal objective ×10 of the microscope. Nucleus in pollen, sper-
mia, and pollen tubes could be observed, respectively, in light and luminescent
microscopes, by using objective ×10, 20, and 40 (Fig. 1.13).
1.4 Model Process Testing
Among known model process testing, we could consider such reactions as compo-
sition of the secretions and their spectral characteristics (cellular absorbance and
autofluorescence), the germination, and the growth and development. In this case,
the donor model of the secretion and the acceptor model of the secretions should be
distinguished. Donors may be considered not only as plants because excretions of
other organisms enriched in similar secretory products, for example, known neu-
rotransmitters acetylcholine and biogenic amines as well as related enzymes of
their synthesis and catabolism, are observed in animals and microorganisms
(Roshchina 2010). Plant cells serve also as acceptor models of the external chemi-
cals independently on the organism which adds the secretion. This chapter consid-
ers the types of model process testing that could be used for various model systems.
Modeling processes involving donor cell secretion and acceptor cells, biosensors
that perceive and react to the components of this secretion in the form of a physio-
logical response, is one of the experimental approaches to study mechanisms of
intercellular signaling in the chemical communication of organisms (Roshchina
and Roshchina 1989, 1993, 2012).
1 4 8
Fig. 1.13 The images of dry (1–3, bar = 50 μm) and germinated pollen (4–9, bar = 200 μm) of
Hippeastrum hybridum under the usual light microscope (1–2, 4, 6, 8) and luminescence (excita-
tion by UV light 360–380 nm) microscope (3, 5, 7, 9). Big nucleus is seen in the center of pollen
under transmitting light
1.4.1 Secretion of Donor Models
The choice of modeling processes in donor cells is based on the analysis of the
secretion from the objects. In the liquid secretions, it was possible to analyze the
number of pigments, fluorescing compounds, proteins, and enzyme activity by
spectral methods.
1.4.1.1 Secretion of Pigments and Other Low-Molecular Metabolites
Objects being pigmented contain phenols such as anthocyanins, anthraquinones,

betacyanins, and betalains in the vacuole of cells from flowering plant species
or in the cell walls of such spores as pollen of seed-bearing plants or vegetative
microspores of spore-bearing plants (Stanley and Linskens 1974). In cell walls

and free space of some species and spores, azulenes have been found (Roshchina
et al. 1995, Roshchina 2008). Under the influence of external chemosignal
(passing from the plasmalemma to organelles through the cytoplasm), secretory
products can go outside or into vacuoles. On such plant objects, one can see
visually and highlight pigments from cells and quantitatively analyze the output
of the products and spectral characteristics of these compounds. It can be
expected to consider changes in absorption and fluorescence of intact samples
in situ and their secrets in solution under the influence of various factors.
Pigmented cells are proposed to be models that excrete pigments, and by this
mode, one permits to observe and estimate the process. The quantity of the secretion
pigment released appears to be analyzed by spectral methods. Among those models
are cells capable of excreting colored products: (1) from cell wall and/or free cel-
lular space (between cell wall and plasmalemma) or (2) from secretory vesicles
located in the cytoplasm and vacuole.
Colored objects able to excrete pigments are (1) unicellular system – vegeta-
tive spores and generative spores (pollen) – and (2) multicellular systems of
plants and animals including flower petal cells. The spectra of the extracts may
show the presence of certain compounds. Microspores contain colored products,
mainly phenol anthocyanins, carotenoids, and azulenes in the cell wall that well
seen under the microscope. Pollens from Petunia hybrida, Hemerocallis fulva,
Philadelphus grandiflorus, Zephyranthes grandiflora, and other species release
the pigments into water or ethanol (sometimes in oil medium, like carotenoids of
Lilium pollen) which is a natural medium of plant tissues (Roshchina et al.
2009a, b). Multicellular objects were analyzed for the abovementioned aim
(Roshchina et al. 2013b). Among them were those containing pigments in vacu-
oles from petal cells of cacti Epiphyllum hybridum (L.), Hibiscus rosa-sinensis
(L.), Saintpaulia ionantha Wendl, bracts of milkweed Euphorbia milii Desmoul,
scales of red onion Allium cepa L., leaf oil gland and ducts as St. John’s wort
Hypericum perforatum L., and bracts of hop Humulus lupulus L. From animal
objects, some perspective was for planarian Dugesia tigrina easily cultivated in
a mixture of tap and distilled water 2:1.
Cellular mechanisms of excretions in allelopathic relations may also include the
breakup products (products of a cleavage) of some exometabolites. In particular,
neurotransmitters acetylcholine and histamine are found in pollen (Marquardt and
Vogg 1952) and in other parts of plants. (Marquardt and Falk 1957), including bac-
terial microflora (Marquardt and Spitznagel 1959) as well.
Secretions of the cells have been obtained by extraction with an aqueous
solution without nitrate salts containing in μg/l of potassium phosphate, cal-
cium chloride, sodium chloride, and magnesium chloride during a short
period – from 20 min up to 60 min (Roshchina et al. 2009a, b, 2013). Protein
secretions and their enzymatic activity were determined (Roshchina 2009a, b;
Roshchina and Yashin 2013). The absorption and fluorescence of the cellular
extracts as well as their effects on acceptor models (see Chaps. 2 and 3) were
analyzed.
1.4.1.2 Secretion of Proteins and Their Enzymatic Activity
Secretion of proteins from the cells is often accompanied with their various enzy-
matic activities (esterase, peroxidase, superoxide oxidase, superoxide dismutase,
etc.). Cells from various species release oxidases or reductases (monoaminooxi-
dases, cytochromes, methemoglobin) and hydrolases (cholinesterase, amylase, pep-
sin, trypsin, etc.), the examples of which are demonstrated in Fig. 1.14 where a
similar activity is shown for the secretion from pollen, vegetative microspores, and
pistil.
Many proteins were found (Roshchina 2009a, b) in the excretions from
Hippeastrum hybridum pollen and pistils as well as from vegetative microspores of
horsetail Equisetum arvense (Fig. 1.14). Main fractions from all studied extracts
contained high-molecular proteins (molecular mass near 1,000–2,000 kDa) pos-
sessing cholinesterase (ChE) activity, and some proteins with lower molecular
masses had peroxidase (PO) activity, but superoxide dismutase (SOD) activity was
found only in some minor proteins. Electrophoresis of pollen proteins without SDS
confirmed that the presence of protein with molecular mass higher than 224 kDa
(Roshchina 2001a) had cholinesterase activity. Besides, three protein bands with Mr
60, 55, and 43 kDa were also seen. In the electrophoretic analysis with DDS, the
high-molecular complexes were broken into lower subunits and 90 kDa proteins in
molecular masses identical to plant cholinesterases (Roshchina 2001a); other pro-
teins with Mr < 35 kDa may belong to proteins with 28 kDa similar to olfactory
mucilage proteins with 28 kDa (Roshchina et al. 1998c). Of special interest is cho-
linesterase, an enzyme found in all living organisms and released via plasmatic
membrane (Roshchina 2001a, 2010).
Cholinesterase of living cells is a hydrolytic enzyme, which hydrolyzes acetyl-
choline and is sensitive to artificial and natural toxins (Augustinsson 1963; Gupta
ana Gupta 1997; Budantsev 2005; Budantsev A Yu and Roshchina VV 2005). It
serves as biomarker for toxicants in environments and for acetylcholine (Jemec
et al. 2009). Higher concentrations of acetylcholine and cholinesterases may serve
as biomarkers in models of stress (Yamamota et al. 2011). The enzyme is actively
excreted by animal cells, including parasites (Ros-Moreno et al. 2002) that may be
observed by biochemical Ellman method, using 5,5″-dithio-bis(p-nitrobenzoic
acid) which interacts with thiocholine, forming a yellow product with a maximum
of absorbance at 412 nm, or by immunoblot assays. One of the examples is for
flower bract glands from hop Humulus lupulus L. (Table 1.4). The identity of the
activity was with specific inhibitors of the animal cholinesterase – neostigmine and
physostigmine which decreased the hydrolysis of the substrate acetylthiocholine.
Cholinesterase may be identified in vital model systems by histochemical meth-
ods. In particular, after the staining with red Ellman analog on the thiol groups
2,2-dithio-bis-(p-phenyleneazo)-bis-(1-oxy-8-chlorine-3,6)-disulfur, the blue color
appears (Roshchina 2001a, b, 2007a). Blue color ring concentrated on the plasma-
lemma of pollen, whereas other parts have red color (Roshchina et al. 1994). Many
allelochemicals, especially pharmaceutical alkaloids, demonstrate the pesticidal
characteristics, affecting cholinesterases of plants and animals (Narahashi 1979;
pollen of Hippeastrum
kDa
ChE activity SOD activity PO activity

protein (mg/ml)
10
kDa 2000 kDa 67 kDa 12
0
5 10 15 20 25 30 35
vegetative microspores of Equisetum

protein (mg/ml)
10
ChE activity SOD activity PO activity
0
5 10 15 20 25 30 35
pistil of Hippeastrum
15
ChE activity SOD activity

protein (mg/ml)
10 PO activity
0
5 10 15 20 25 30 35
number of fraction
Fig. 1.14 The chromatography on Toyopearl HW-75 of the extracts from pollen, pistils of
Hippeastrum hybridum, and vegetative microspores of Equisetum arvense (Adapted from
Roshchina (2009a, b)). ChE, SOD, and PO activity means activities of cholinesterase, superoxide
dismutase, and peroxidase, respectively
Ulrichova et al. 1983; Atta-ur-Rahman et al. 2001; Budantsev and Roshchina

2004). The phenomenon is also of interest to understand the allelopathic mecha-
nisms of growth regulation, in particular exogenous acetylcholinesterase stimu-
lates the growth reactions of animals (Sternfeld et al. 1998) and an inhibitor of
cholinesterase – plant alkaloid physostigmine – increases the saliva excretions
Table 1.4 Hydrolysis of acetylthiocholine by water extracts (1:40 w/v) from bracts with glands
named lupulins and individual lupulins of hop Humulus lupulus L. calculated in ×10−8 М/g weight
per min (1) or × 10−8 М/mg protein per min (2)
Bracts with glands Lupulins
Variant 1 2 1 2
Control 4.2 ± 0.06 14 ± 0.10 0.032 ± 0.0003 0.82 ± 0.006
+ Neostigmine 10−5 М 2.1 ± 0.04 7 ± 0.04 0.0096 ± 0.0001 0.24 ± 0.003
+ Physostigmine 10−5 М 2.5 ± 0.03 8.1 ± 0.04 0.0148 ± 0.0010 0.37 ± 0.002
Source: Roshchina et al. (2013b)
(Khosravani et al. 2007). These effects may be analyzed using the biotests based on
the preparation from either animal or plant cells as well as from pure commercial
enzyme cholinesterase (Budantsev and Roshchina 2007). Cholinesterase is found
in excretions of glands, in particular lupulins of hop Humulus lupulus (Table 1.4).
Histochemical staining may permit to observe cholinesterase released in various
plant cells and tissues (Bednarska and Tretyn 1989; Bednarska 1992; Momonoki
and Momonoki 1993b; Roshchina 1999b; Roshchina 2001a, 2007a).
The presence of cholinesterases in pollen, pistils, and vegetative microspores of
cryptogams, perhaps, is a common feature in breeding processes. Cholinesterase,
especially acetylcholinesterase (EC 3.1.1.7.), is also present in many pollen grains
from various species (Bednarska 1992; Roshchina et al. 1994; Roshchina 2001a, b)
as well as in anthers (Semenova and Roshchina 1993; Roshchina and Semenova
1995), and this activity is low in pollen from self-compatible clones of Petunia hyb-
rida (Kovaleva and Roshchina 1997). The pollen from self-incompatible clone of
Petunia has a low concentration of enzyme (Kovaleva and Roshchina 1997). The
pollen tube elongation in pistils of Lilium longiflorum cv. Hinomoto after self-
incompatible pollination was stimulated by acetylcholine and other choline deriva-
tives or neostigmine, an inhibitor of acetylcholinesterase (Tezuka et al. 2007a, b).
Activities of this enzyme and choline acetyltransferase (acetylcholine-forming
enzyme) (EC 2.3.1.6.) in pistils were associated with self-incompatibility. Genetic
defects of acetylcholine signaling promote the protein degradation in muscle cells
(Szewczyk et al. 2000).
Other enzymes are excreted by microspores and pistils (Fig. 1.14), mainly super-
oxide dismutase and peroxidase (Roshchina 2009a, b). The small concentration of
superoxide dismutase in pollen is correlated to the higher concentration of superox-
ide anion radical in the pollen or vegetative microspore surfaces (Roshchina and
Roshchina 2003; Roshchina et al. 2003). Peroxidase activity in all samples belonged
to some fractions, and that confirms earlier information (Stanley and Linskens
1974). The acid phosphatase, ribonuclease, esterase, amylase, and protease activity
as well as other proteins are present in the pollen walls of 50 angiosperms and pines
and also in the spore wall of Equisetum sp. (Knox and Heslop-Harrison 1969, 1970).
In the pollen of Pinus sylvestris, extracellular nuclease with Mr 25–25.7 kDa was
found (Matoušek and Turkova 1987), but no immunochemical relationships were
observed between the pine nuclease and extracellular proteins from vegetative
spores of Equisetum arvense.
1.4.1.3 Autofluorescence as Biosignal or Marker
The search of the donor cell of secretions may be based on the autofluorescence of
secretory products – secondary metabolites (Roshchina 2003, 2008, 2012). The
fluorescence spectra and the emission intensity of secretory cells may be studied in
the comparison of the secretion accumulation at normal conditions and under the
treatment of various factors.
New look on autofluorescence as a biosignal for insects appears with several
works dealing with the search of signal for pollinators which prefer flowers of certain
plant species (Iriel and Lagoio 2010a, b). Frey-Wyssling and Agthe (1950) have dis-
covered fluorescence of flower nectar, as a possible signal for insect pollinators, and
today the phenomenon has been demonstrated for various floral and extrafloral nec-
taries (Roshchina 2008). Moreover, some secondary metabolites served as the defense
agents against parasitic invasion, for example, phenols emit under the excitation of
UV light, and their autofluorescence should mark their location in cells and tissues.
A quantitative evaluation of the light emerging from intact petals of Rhododendron
indicum flowers of different colors was performed based on the measurement of
reflectance and fluorescence emission (Iriel and Lagoio 2010a, b). Emitted photons
as fluorescence were compared with reflected photons. The fluorescence quantum
yielded values (Φf) varying from 7.6 × 10−5 to 6.3 × 10−4 for the emission in the blue
region of the electromagnetic spectrum and from 2.4 × 10−5 to 1.9 × 10−4 for the
emission in the red one. (Φf) was calculated for flowers of Bellis perennis (white,
yellow, pink, and purple), Ornithogalum thyrsoides (petals and ovaries), Limonium
sinuatum (white and yellow), Lampranthus productus (yellow), Petunia nyctagini-
flora (white), Bougainvillea spectabilis (white and yellow), Antirrhinum majus
(white and yellow), Eustoma grandiflorum (white and blue), Citrus aurantium (pet-
als and stigma), and Portulaca grandiflora (yellow). The highest values were
obtained for the ovaries of O. thyrsoides (Φf = 0.030) and for Citrus aurantium pet-
als (Φf = 0.014) and pistil stigma (Φf = 0.013). The fluorescence emission as an opti-
cal signal in biocommunication was negligible if compared to the light reflected by
the petals. Nevertheless, the calculation of quantum catches for each studied flower
species described the visual sensitization of insect eye photoreceptors.
The fluorescence spectra and the emission intensity of secretory cell models
included fluorescent drugs that are important for medicinal and simultaneously alle-
lopathic plant species because this may characterize the state of pharmaceutically
valuable material (Roshchina and Karnaukhov 2010).
1.4.2 Reactions of Acceptor Models Sensitive to Secretory

Products
Models which are used as test systems to analyze the effects of secretions released
by other organisms may be chosen from unicellular (microspores) and multicellular
sensitive objects. Analyzed reactions in every case should select individually for
each model.
Among known model process testing, we have considered such reactions as cel-
lular spectral characteristics, the germination, and the growth and development.
Moreover, the amounts and rate of excretion of secretory products from cells, like
in Saintpaulia petals (see Chap. 3), could be measured.
Among potential models, there are systems having autofluorescence changed
under various factors. Changes in autofluorescence usually are valuable as a natural
indicator of cellular state because the emission often shows changes in cellular
metabolism and responses to the external and internal signals. The intensity and
spectral composition of the emission differ depending on, viz., (1) the nature of the
organism or (2) cell analyzed, (3) taxonomic position of the organism tested, and (4)
environmental and experimental conditions (actinic light, temperature, humidity,
physiological state of a cell as a whole and phase of development, influence of
neighbor cells or organisms, including parasites). Autofluorescence could be used
in the diagnostics of cellular damage and in the analysis of cell–cell interactions
(Roshchina and Roshchina 2003; Roshchina 2003, 2008; Roshchina et al. 2008,
2009a, b). Perhaps, it is a resource for the nondestructive vital microscopic analysis
of natural organisms. It already has practical application in various diagnostic pro-
cedures, for cell biology analysis, ecology, medicine, and pharmacology.
Similar biosensors based on petal fluorescence in flowers such as Mirabilis
jalapa may also be applied to the analysis of chemosignaling dealing with certain
pigment fluorophores (Gandía-Herrero et al. 2005a, b). The visible fluorescence
emitted by one pigment, a yellow betaxanthin, is absorbed by another, a violet beta-
cyanin, to create a contrasting fluorescent pattern on the flower’s petals for pollina-
tors. The analysis of free-radical processes with the participation of the pigments
opened new horizons to search biosensors among pigmented plant cells.
Perspective models today appear to be mycorrhizal roots of plants sensitive to a
colonization of arbuscular mycorrhizal fungal structures (such as hyphae, vesicles,
and spores) that brightly autofluoresce among root tissues under ultraviolet, blue, or
green light excitation (Ames et al. 1982; Dreyer et al. 2006; Dreyer 2009). Whole
roots of lucerne Medicago sativa may be suitable for the aim.
The microspore (both vegetative from spore-bearing species and generative ones
such as pollens of seed-bearing species) is a unique unicellular object for the study
of mechanisms of the plant excreta action on the growth processes in the germina-
tion of a pollen in a favorable medium. Their germination is fast, and the rate of the
process is often about some millimeters per hour (Esau 1977). For this purpose,
plant vegetative microspores of horsetail Equisetum arvense and the pollen of
knight’s star Hippeastrum hybridum that have physiological responses to
neurotransmitters seen as changes in own fluorescence of cells and growth of reac-
tion are represented (Roshchina 2004a, 2006a, b, 2007a, b; Roshchina et al. 2009a).
Other pollens from various species such as Vallota speciosa, Plantago major, and
Haemanthus katherinae are suitable for the cellular studies too.
Multicellular models studied are pistils of intact plant such as knight’s stars
Hippeastrum hybridum (Roshchina and Melnikova 1998a, b, 1999; Roshchina
2008) or petals of Saintpaulia ionantha, Hibiscus rosa-sinensis, and Epiphyllum
hybridum (Roshchina et al. 2013b). These models will be considered in Chap. 3.
The fluorescence and germination of plant microspores, both in generative

(pollen or male gametophyte of gymnosperms and angiosperms) and in vegetative
(in cryptogams), may be used as test reactions on the secretory product addition.
The microspores serve for sexual and vegetative breeding, respectively, and may
germinate on artificial media.
Some enzymatic reactions (e.g., the cholinesterase activity) are also tested in the
microspores. The autofluorescence of pollen and pistils as biosensors can be used in
the study of various processes (Roshchina 2006a, b, 2008) from the damages by
ozone and other reactive oxygen species to chemosignaling (Roshchina and
Melnikova 2001; Roshchina and Roshchina 2003).
Among the reactions sensitive to plant excretions earlier, the chloroplast move-
ment on Elodea canadensis cells (Roshchina 1965, 1975; Roshchina et al. 1979)
was considered as the test reaction that is also recommended today (Budantsev
2012).
Growth and development of multicellular models such as leaves of Lemna minor
or of Chara corallina as well as root tips of seedlings of Raphanus sativus are also
considered in Chap. 3.
Chapter 2
Intact Secretory Cells as Donor Models
of Secretions
Models of secretion may be considered at the cellular level such as secretion into
organelles and out of the cell or at tissual and organ levels. In multicellular systems,
it is necessary to distinguish the secretory systems that release secretion within a
cell (intracellular secretion) or tissues (intratissual secretion) and those that liberate
secretory product out from the organism (external secretion). Besides the main divi-
sions, models on which secretions are seen in the extracellular space or within cell
are also of interest to the researchers.
The purpose of this chapter is to show objects with clear images received by vari-
ous microscopic methods and suitable for the investigation of intact secretory cells
which could be donors or acceptors of secretions or simultaneously donors and
acceptors depending on the aims of the experiments.
2.1 Intracellular Secretion
Substances that could disturb cell homeostasis are removed from the sphere of
active metabolism. The intracellular secretion is an evolutionarily determined path-
way of elimination of excessive metabolites. This pathway appeared, probably,
when terrestrial forms of life arose, in particular, giant forms of plants, which lim-
ited the ability of cells to excrete metabolites to the environment.
The main physiological functions of the cell occur in the cytoplasm with included
organelles. This active part of the cell is separated from the cell wall by the plasma-
lemma and from the vacuole by an internal membrane, the tonoplast. An excess of
metabolites which can cause disturbances in normal reactions of the organism is
evacuated by diffusion or via the active mechanism of transport through the
plasmatic membrane or the tonoplast. In the former case, the secreted substance is
accumulated in the free cell space occurring out of the plasmalemma-limited volume
and in the latter case in the vacuole.
36 2 Intact Secretory Cells as Donor Models of Secretions
Metabolites are evacuated by diffusion or via the active mechanism of transport

through the plasmatic membrane or the tonoplast of vacuole:
Free cellular space ¬ Secretion in secretory vesicles of cytoplasm ® Vacuole
Secretions in the free space should be considered as a part of external secretion;

therefore, related model systems will be discussed in Sect. 2.3.1.
2.1.1 Models of Secretion into Vacuole
A completely developed plant cell has a large central vacuole which can occupy up
to 90 % of the cell volume. The vacuole is the seat of accumulation of water-soluble
compounds which come from the cytoplasm. The membrane surrounding the vacu-
ole possesses selective permeability, so it admits to the vacuole substances of a
particular type. Physiologically, the substances accumulated in the vacuole belong
to two different categories.
Substances of the first category are useful for the plant: sugars, amino acids,
organic acids, etc., which are retained in the cell and can be further involved in the
metabolism. The vacuole can contain up to 100 types of proteins. Among them are
many hydrolases: acidic proteinase, acidic phosphatase, mannosidase, and galacto-
sidase (Kenyon and Black 1986). Oxidoreductases are represented by the main iso-
forms of peroxidases (Schloβ et al. 1987). Like all proteins, the enzymes are
synthesized in the cytoplasm on ribosomes and are then transported across the tono-
plast to the vacuole.
On the other hand, the vacuolar sap contains secondary metabolic products, fla-
vonoids, alkaloids, etc., whose accumulation is caused by a limited excretion capac-
ity of the cell. Thus, the central vacuole of the cell presents a kind of secretory
system where substances of various chemical origins are isolated from the sphere of
active transformations. The composition of the material secreted by the cytoplasm
to the vacuole varies strongly depending on plant species, the phase of development,
and the organ (root, leaf, etc.) where the cell is located. The vacuole has a number
of functions (osmoregulation, turgor maintenance, storage of assimilates, and secre-
tion). The secretory function becomes prevalent in the mature cell when non-utilized
metabolites are accumulated in the vacuole (Matile 1987). The total concentration
of substances as well as of particular compounds in the cell sap is higher than in the
cytoplasm, so the movement of molecules is directed against the concentration gra-
dient. As a rule, the transport of secreted substances across the tonoplast is carried
out by active mechanisms: exocytosis and pinocytosis (Fineran 1971); in some
cases, however, a passive process is possible (Buzuk and Lovkova 1986). The mech-
anism of transporting secondary substances across the tonoplast is poorly studied,
except for phenols and alkaloids. Today, there are three hypotheses applied to the
accumulation of secondary metabolites (mainly based on experiments with alka-
loids) in vacuoles: (1) ion-trap mechanisms (Matile 1976), (2) transport with
2.1 Intracellular Secretion 37
specific carriers with energetic expenditures (Deus-Neumann and Zenk 1984,

1986), and (3) a directly energized transporter in the tonoplast, the ATP-binding
cassette transporter for many secondary metabolites (Klein et al. 2000).
Phenolic compounds may also be synthesized in both the vacuole and chloro-
plasts. In the vacuole, chalcone synthase, chalcone isomerase, and flavonoid glyco-
side transferase were found, while in the chloroplasts – phenylalanine ammonia-lyase,
cinnamate 2-hydroxylase, hydroxycinnamoyl ~ CoA: quinate hydroxycinnamoyl
transferase, and chalcone synthase were found (Hrazdina and Wagner 1985). Under
permanent illumination, chloroplasts arose whose formation was accompanied by a
significant increase in the amount of total soluble phenolic compounds and by
appearance among them of flavones along with catechins and proanthocyanidins
(Zaprometov 1985). Some elements of the endoplasmic reticulum, probably, par-
ticipate in the transport of phenols into the vacuole (Charriere-Ladreix 1977). In this
case, phenolic substances being transported within secretory vesicles penetrate
through the tonoplast into the vacuole by exocytosis. It is also possible that the
movement of compounds across the vacuolar membrane proceeds via pinocytosis.
Such type of absorption in the central vacuoles of parenchymatous cells has been
shown by Fineran (1971). According to his scheme of the event, a particle or a mol-
ecule is absorbed on the tonoplast that initiates invagination of the membrane result-
ing in the formation of a vesicle which is found in the vacuole. After the membrane
of the vesicle has disappeared, the adsorbed substance appears in the vacuolar sap.
Anthocyanin transport to the vacuole studied as color markers (by colocalizing the
red fluorescence of the anthocyanins with green fluorescent protein markers of the
endomembrane system) for the pigment traffic on Arabidopsis thaliana involves
vesicle-like structures shared with components of the secretory pathway (Poustka
et al. 2007). Anthocyanins are also sequestered to the endoplasmic reticulum and to
endoplasmic reticulum-derived vesicle-like structures targeted directly to the pro-
tein storage vacuole in a Golgi-independent manner.
Besides phenols, alkaloids, and terpenes, some hydrocarbons synthesized in
chloroplasts and leucoplasts can also be accumulated then in the vacuole. Some
studies have been devoted to the accumulation of alkaloids in the vacuole (Müntz
1984; Deus-Neumann and Zenk 1984, 1986; Buzuk and Lovkova 1986). The syn-
thesis of alkaloids takes place in the cytoplasm (Deus-Neumann and Zenk 1984) or
can sometimes occur in plastids or in some individual vesicles (Wink 1985;
Hartmann 1985).
2.1.1.1 Models with Pigments in Vacuoles
For modeling, pigmented objects are of interest, especially in the studies of pathways
of intracellular transport into and out of vacuoles (Marty 1999). Figure 2.1 shows
the main known pigments met in vacuoles. As a whole, this is a red- or blue-colored
(depending on the pH of the vacuolar sap) anthocyanin. At pH < 7, red color pre-
vails, while blue color prevails at pH > 7. Certain families within the order
Caryophyllales produce pigments called betalains instead of anthocyanins. Betalains
Pelargonidin Cyanidin Betacyanin

O+ R2O
HO O+ OH
OH HO O
OH (+)
OH HO N
OH OH
OH OH
Delphinidin Malvidin
O+ OH O+ O Me
HO HO
OH OH
OH O Me HO O
OH OH N
OH OH H
O OH
Betaxanthin Dopaxanthin Indicaxanthin

HO
R1
O O (+) O
N HO N N
OH OH OH
HO O HO O HO O
N N N
H H H
O O OH O OH
OH
Fig. 2.1 Formulae of pigments occurred in vacuoles
are divided into two classes: the betaxanthins and betacyanins, which produce yel-
low to orange or violet colors, respectively (Gandía-Herrero et al. 2005a, b).
Betaxanthins have a maximum at 480 nm, while betacyanins have a maximum at
536 nm in the absorbance spectra. Betacyanin at pH < 7 has a peak of 535 nm and
red color, but at pH > 7, this maximum disappeared.
Anthocyanins fluoresce in blue and in red and can emit not only in blue with
maxima at 450–470 nm, as well as in green (at 510 and 515 nm), but also in yellow
orange and even red (555, 570–585, 610 nm) at excitation by light 360–380 nm
(Roshchina and Melnikova 1996; Melnikova et al. 1997). The anthocyanin fluores-
cence depends on the formation of complexes or conjugates with metals or other
cellular components. Poustka et al. (2007), benefiting from the unique fluorescent
properties of anthocyanins, showed anthocyanin transport to the vacuole that
involves vesicle-like structures in Arabidopsis thaliana. Unlike anthocyanins, green
autofluorescence is characteristic of betaxanthins (Gandía-Herrero et al. 2005a, b,
2009; Harris et al. 2012). Betacyanin fluorescence is observed in the range of 580–
660 nm (Rabasovica et al. 2009). Basing on fluorescent betaxanthins, the flower of
common purslane Portulaca oleracea serves as a model for the analysis of tyrosi-
nase activity characteristics (Gandía-Herrero et al. 2009).
1 3
0.15
555
Absorbance (units opt.density)

585
0.10
2 0.05
0
500 550 600 650
Wavelength (nm)
Fig. 2.2 The common view (1) of bracts from Euphorbia milii, their cells with vacuoles (2), and
the absorbance spectra of their extracted pigments purified by thin-layer chromatography on silica
gel (3). Bars = 50 μm. In the spectra, broken line, pigments in acid medium (pH < 6), and unbroken
line at pH > 7
As a rule, the occurrence of betalains and anthocyanins is mutually exclusive. In

some cases, through a combination of genetic modification and substrate feeding
(by L-dihydroxyphenylalanine), anthocyanin-producing species are able to form
betalains in cell cultures of Solanum tuberosum (potato) and petals of Antirrhinum
majus as well (Harris et al. 2012).
Anthocyanosomes, bodies containing anthocyanins and coated with membrane
within vacuoles, are studied on various model objects (Yasuda and Shinoda 1985;
Markham et al. 2000; Conn et al. 2010). Some of the interesting models for the obser-
vation of pigments (anthocyanin accumulation) in vacuoles are shown in Figs. 2.2 and
2.3. These are scarlet bracts of crown of thorns Euphorbia milii Des Moul. and blue
petals from flowers of Saintpaulia ionantha Wendl. In their vacuoles, anthocyanins
are concentrated in anthocyanosomes. Arrows of Figs. 2.2 and 2.3 show the position
of anthocyanosomes. E. milii produces various red anthocyanin pigments in the cells
and in the suspension cultures (Yamamoto et al. 1989; Hamade et al. 1994). There are
maxima at 555 and 585 nm in the absorbance spectra of sum of the pigments (Fig. 2.2
image 3). Unlike crown of thorns, on the visible absorption spectral curves of fresh
violet-blue petals and in their crude extracts in pH 5.0 buffer solution, there are two
characteristic absorption maxima at 547 and 577 nm, with a shoulder near 620 nm
(Roshchina and Yashin 2013). Acylated anthocyanidin 3-rutinoside-5-glucosides were
isolated from the violet-blue flowers of Saintpaulia which have a blue color (Tatsuzawa
et al. 2012). The anthocyanins were determined by chemical and spectroscopic
Intact petal
1 3
2.25
Absorbance (units of opt.density)

2.20
1.75
Anthocyanins
0.4
0.2
2 0
400 500 600 700
Wavelength (nm)
5 6
a
a
7
b 200
b a
100
B
c
c
c 0
400 500 600 700
Wavelength (nm)
Fig. 2.3 The flower petals from Saintpaulia ionantha. Common view of petal (1), their cells with
vacuoles (2), the absorbance spectra of their intact cells and pigment anthocyanins extracted and
purified by thin-layer chromatography on silica gel (3), fluorescing surface of the petals with secre-
tory hairs (4), secretory hair in transmitted light of usual microscope (5), in laser light 405 nm of
confocal microscope (6), and the emission spectra from different parts (7). Bars = 50 μm (2), 100 μm
(4), and 25 μm (6). In 3 broken lines, pigments in acid medium (pH < 6), and unbroken line at pH > 7.
In 5, 6 head (a), stalk (b), base (c)
blue-green
1. 2. 3. 4.
green-yellow emitted cell

50 blue-green green-yellow emitted cell
emitted cell emitted cell
25
blue
emitted cell
0
400 500 600 700 400 500 600 700 400 500 600 700 400 500 600 700
Chloroform extract Chloroform extract Chloroform extract Chloroform extract
600 600
600 600
400 400
400 400
200 200 200 200
0 0 0 0
400 500 600 700 400 500 600 700 400 500 600 700 400 500 600 700
Wavelength (nm) Wavelength (nm) Wavelength (nm) Wavelength (nm)
Fig. 2.4 The common views (upper side) and the fluorescence spectra of petal secretory cells
from various colored flowers of Achillea millefolium L. registered by recorded microspectrofluo-
rimeter MSF-1 (lower side). Excitation 360–380 nm. Secretory cells (unbroken lines) and nonse-
cretory cells (broken lines) (Source: Roshchina et al. (2011c))
methods to be 3-O-[6-O-(4-O-(acetyl)-α-rhamnopyranosyl)-β-glucopyranoside]-
5-O-(β-glucopyranoside)s of malvidin, peonidin, and pelargonidin. HPLC analysis
revealed that malvidin 3-O-acetylrutinoside-5-O-glucoside existed as a dominant pig-
ment in the violet-blue flowers.
Anthocyanin-containing cells are considered as models for the analysis of the
anthocyanosomes and membranous damages under the action of various compo-
nents of plant secretions (Roshchina et al. 2013b; Roshchina and Yashin 2013).
Secreting cells with pigments are also applied as models to study the role of acetyl-
choline in signaling and communications (Roshchina et al. 2013). Some examples
are described in Chap. 3.
Betacyanins also serve as markers of membrane permeability on model systems
from beet root disks (Roshchina and Roshchina 1970, 1989; Roshchina 1972, 1974;
Jesudian and Bose 1983).
Besides, isolated vacuoles of Beta vulgaris var. rubra (where red betacyanins are
found) are also used for the analysis of participants of intracellular signaling, in
particular the accumulation of cyclic AMP and adenylate cyclase (Rykun 2013).
Secretory cells of milfoil Achillea millefolium L. (Asteraceae) may serve as a
model too (Roshchina et al. 2011c). Among allelopathically active and medicinal
plants, there is the fluorescence from petal secretory cells of Fig. 2.4 that demon-
strates how the changes in the color of petals from the flowers are reflected in the
fluorescence spectra of their secretory cells. Pigmentation of flowers is varied
depending on the amount of anthocyanidines and their composition. Blue (maximum
at 460 nm)- and blue-green (maximum at 500 nm)-emitted secretory cells in white
Table 2.1 The fluorescence of extracts from the petals of Achillea millefolium
Sample 3 (rose
petal with weak Sample 4 (petal with
Sample 1 (white Sample 2 anthocyanin red-rose anthocyanin
Solution petal) (white-rose petal) pigmentation pigmentation)
Water No emission No emission No emission No emission
Ethanol No emission Weak emission Small maximum Maximum at
at 590–600 nm 590–600 nm
Chloroform One maximum at One maximum at One maximum at One maximum at
403–405 nm 403–405 nm 403–405 nm 403–405 nm
Excitation 360 nm
petals, green-yellow (maximum or shoulder at 450 and maximum at 550 nm)-

emitted secretory cells in rose-colored petals, and brightly fluorescing blue-green
(maximum at 450 nm and shoulder at 550 nm) secretory cells in red-colored petals
are seen. In most cases, nonsecretory cells had no significant emission in the blue-
orange spectral region, and only red fluorescence with a maximum at 680 nm,
peculiar to chlorophyll, was seen. The fluorescence maxima at 430–460 nm may
relate to azulenes, whereas emission at 550–600 nm may relate to anthocyanins.
The abovementioned changes in the emission of secretory cells may be described
by the alterations in the metabolism of milfoil Achillea genus that depends on the
various factors. As shown by Bashirova et al. (2003) on the example of Achillea
asiatica, there is a correlation between anthocyanin and azulene synthesis. If the
anthocyanin content in the flowers was high, the accumulation of azulenes was also
observed up to 300 % of control.
The 10 min extracts from the above-seen petal samples (Table 2.1) showed
weak or no emission after extraction with water or 80 % ethanol. The significant
fluorescence was seen only after the extraction with chloroform in the blue-vio-
let region at 403–406 nm that reflects the presence of lipophilic components in
secretory structures, possibly azulenes in the Achillea genus (Kaloshina et al.
1992). Only anthocyanin-pigmented samples may give the emission in the
orange spectral region. The changes in the composition of allelochemicals such
as azulenes and phenolic compounds anthocyanins are well seen in the
experiments.
Anthocyanins as phenols may undergo oxidation within vacuoles due to the for-
mation of hydrogen peroxide and the vacuolar peroxidase activity. Class III peroxi-
dases are plant enzymes capable of using H2O2 to oxidize a range of plant secondary
metabolites, in particular phenolic compounds. It has been shown on isolated leaf
vacuoles from Catharanthus roseus where more than 90 % of total leaf peroxidase
activity was localized (Ferreres et al. 2011). There were three caffeoylquinic acids
and four flavonoids in this organelle that serve as substrates for the
peroxidase-mediated oxidation. These phenols or similar compounds were shown
to be good substrates for the peroxidases.
2.1.1.2 Models for Analysis of Alkaloid Accumulation in Isolated Vacuoles
Initially, the conceptions of the accumulation of alkaloids in vacuoles were based on

the fact that the vacuolar sap has an acidic reaction (low pH). According to this
model, alkaloids can quickly penetrate through the tonoplast by diffusion in lipo-
philic layers. In the acidic medium of the vacuole, alkaloids are protonated and,
hence, captured as cations for which the tonoplast is either poorly permeable or abso-
lutely impermeable (Matile 1984). Moreover, phenols and other components of the
cell sap can immobilize alkaloids by forming complicated complexes with them.
This mechanism known as “ion trap” has been evidenced by classical observations
with neutral red whose absorption mechanism is believed to be analogous to that of
alkaloids (Matile 1984). However, this phenomenon is considered to be not a wide-
spread one, which follows from the experiments with isolated vacuoles (Deus-
Neumann and Zenk 1986). It has been shown that isolated vacuoles accumulate only
alkaloids specific for a given plant. The vacuoles of plant species that do not synthe-
size these alkaloids are unable to accumulate them. Mende and Wink (1987) studied
the uptake of alkaloids lupinine (1-oxymethylquinolisidine) and atropine by proto-
plasts and isolated vacuoles from tissue culture cells of Lupinus polyphyllus, Spinacia,
Galium, and Atropa belladonna. Lupinine was taken up only by vacuoles of Lupinus,
whereas atropine was taken up only by analogous organelles of Atropa. The vacuoles
of other studied plants absorbed neither lupinine nor atropine. Some specificity in
alkaloid uptake by isolated latex vacuoles from Chelidonium majus and Papaver
somniferum was also observed in later works (Hauser and Wink 1990; Roberts et al.
1991). Based on similar experiments, a concept of high-taxon-specific carriers of
alkaloids which function with the expense of metabolic energy and are localized in
the tonoplast has been created (Deus-Neumann and Zenk 1986). However, there is
evidence that the specificity in the alkaloid uptake by vacuoles depends not only on
the taxonomy of plants which synthesize the substances. In particular, Sato et al.
(1990) have demonstrated the absorption of alkaloid berberine (yellow color permits
to see the transport) by protoplasts and vacuoles isolated from cells of tissue culture
of Coptis japonica and Thalictrum flavum which practically do not synthesize or
poorly synthesize this compound. But when being absorbed by protoplasts, this alka-
loid was not accumulated in the vacuole. In another case, cells in tissue culture of
Thalictrum minus excreted berberine and were unable to take up the exogenous alka-
loid (Sato et al. 1990). Probably, the absorption by vacuoles was due to not only the
taxonomic specificity but also the ability to excrete this alkaloid. Participation of
intact vacuoles and a tonoplast vesicle system in the berberine transport is shown by
scientists of Sato Laboratory (Otani et al. 2005). The alkaloid uptake was stimulated
by Mg/ATP, as well as GTP, CTP, UTP, and Mg/pyrophosphate. This input of berber-
ine was strongly inhibited by NH4+ and bafilomycin A1, while vanadate (inhibitor of
ATP-binding cassette transporters) had only a slight effect on the tonoplast unlike the
plasma membrane of this plant cell, where the ATP-binding cassette transporter is
involved in berberine transport. Model experiments with liposomes demonstrated
that an ion-trap mechanism was hardly implicated in berberine transport.
Some authors (Buzuk and Lovkova 1986; Roberts et al. 1991), using alkaloids of
different structures, have shown that the rate of penetration of these substances into
vacuoles varies strongly. Matern in his review (1987) believes that the sterical
parameters of a molecule to be transported are essential for the transport of second-
ary metabolites into the vacuole and their fixation.
The advantage of the stereoisomeric model is that it explains the penetration into
the vacuole of basic, acidic, or neutral substances and their selective accumulation.
The evidence for the validity of this model could be the isolation of conformation-
and configuration-specific carriers from the tonoplast.
There is a tonoplast vesicle system of Catharanthus roseus as a model from one of
the most studied medicinal plants containing dimeric terpenoid indole alkaloids vin-
blastine and vincristine and being applied in cancer chemotherapy (Carqueijeiro et al.
2013). It has been shown that the alkaloids are actively taken up by leaf mesophyll
vacuoles through a specific H+ antiport system and not by an ion-trap mechanism or
ABC transporters. Vindoline uptake was ATP dependent. Studied alkaloids were able
to dissipate H+ gradient preestablished across the tonoplast. The initial rates of H+
gradient dissipation followed the Michaelis–Menten kinetics, suggesting the involve-
ment of mediated transport, and this activity was species and alkaloid specific.
The rate of the alkaloid uptake by vacuoles is actually high (0.03 mg/mg of vacu-
olar protein) as shown in the experiment of Deus-Neumann and Zenk (1986). It may
be due to both active and passive mechanisms of transport through the tonoplast.
The participation of both mechanisms has been shown by using specific blockers
(Buzuk and Lovkova 1986). Some factors point to the participation of the proton
mechanism of H-ATPase in the active transport of alkaloids. In particular, vesicles
isolated from the latex of Papaver somniferum were unable to absorb morphine
from the external solution (Pham et al. 1989) if ATP and Mg were absent in external
media or did it poorly (Roberts et al. 1991). The absorption of lupinine by Lupinus
vacuoles was activated 30-fold by Mg, ATP, and KCl and inhibited by DCCD (dicy-
clohexylcarbodiimide) which usually blocks the activity of H-ATPase (Mende and
Wink 1987). Hauser and Wink (1990) have shown that the protoberberine and ben-
zophenanthridine alkaloids, naturally accumulated by latex vacuoles of Chelidonium
majus, penetrate into these isolated organelles via diffusion rather than with the
participation of carriers. The kinetics of the uptake is rather characteristic of a sim-
ple diffusion. But among the seven studied heterologous alkaloids of various struc-
tures (nicotine, vinblastine, 9, 10-dihydroergocryptine, strychnine, colchicine,
lupanine, 13-hydroxylupanine), dihydroergocryptine and vinblastine whose move-
ment is stimulated by ATP had the highest rate of their accumulation by isolated
vacuoles. Thus, except for the case of the two latter alkaloids, ATP had no influence
on this process or decreased it slightly although the alkaloid transport occurred
against the concentration gradient. It is believed to be due to chelidonic acid whose
concentration in vacuoles achieves 661 mM, whereas its concentration in latex ves-
icles achieves only 58 mM. Chelidonic acid forms complexes with these alkaloids
and by this way can prevent their back diffusion from the vacuolar compartment.
This is a possibility for the alkaloids to be accumulated within vacuoles via a pas-
sive mechanism.
2.2 Intratissual Secretory Systems I 45
The isolated latex vacuoles from Papaver somniferum took up alkaloids mor-
phine, codeine, thebaine, nicotine, noscapine, papaverine, and caffeine. The
rate of the process is dependent on the maintenance of tonoplast pH and on
ATPase which generates this pH (Roberts et al. 1991). Exogenous ATP stimu-
lated the morphine accumulation. Sequestration of alkaloids taken up appears to
involve protonation and anion–cation stabilization with the participation of
meconate and sulfate, important acids of vacuoles. The specificity of alkaloid
uptake had no clear correlation with pK or lipophilicity. A possible channel
mechanism of alkaloid influx more related to alkaloid shape is suggested.
According to Roberts et al. (1991), it is more real than the mechanism of protein
carriers.
Among alkaloids to be secreted, there are physiologically active and sometimes
toxic substances. It remains unclear why the cells or organelles are resistant to
them. Thus, it has been proposed (Roos and Luckner 1986) that it is due to an
asymmetrical architecture of the membranes which provides that their external
and internal sides possess different sensitivities to specific metabolites. This was
demonstrated in experiments with vacuoles from the latex of Chelidonium majus
where binding of isoquinoline alkaloids, sanguinarine and chelerythrine, was
observed. Both compounds induced lysis of isolated vacuoles if their concentra-
tion was higher than the organelles could be capable to accumulate. The vacuolar
membrane was more stable if the alkaloids were accumulated only in the vacuole
but was damaged when the same substances were accumulated outside the
vacuole.
Penetration of other secondary metabolites through the tonoplast has been poorly
studied. In order to gain some insight into the physiological role of the secondary
metabolites taken up by the vacuole, it is essential to know that they are accumu-
lated irregularly and only in particular plant species and that they can be released
from the cell only after damage of the latter.
Although most of the secondary metabolites are stored in nonplasmatic com-
partments of living cells, the lipophilic substances can form lipid drops inside
the cytoplasm. Among the compounds are carotenoids, resins, and essential
oils. In these drops, other lipophilic substances such as alkaloids can be dis-
solved as well (Roos and Luckner 1986). Similar drops are also found in
vacuoles.
Prior to being excreted from the cytoplasm, the metabolites overcome the cyto-
plasmic membranes: the plasma membrane when the substance is transported to the
free space of the cell or the tonoplast when it is secreted to the vacuole.
2.2 Intratissual Secretory Systems I
Modeling with intratissual secretory systems is not considered yet, and potential
models may be fluorescent laticifers and idioblasts in which emission changes dur-
ing their development or plant development as a whole.
2.2.1 Laticifers as Models
Suitable models for the study of intratissual secretion may be laticifers due to their
fluorescent characteristics. The reservoirs with latex called laticifers are living cells.
The presence of laticifers in natural conditions can be easily recognized by the latex
efflux from damaged plant tissue. Two types of laticifers are known: articulated and
non-articulated ones. Articulated laticifers originate from many neighboring cells
whose cell walls have been dissolved and the cell contents combined into a continuous
branched system. A laticifer cell becomes secretory just after the arising. The duration
of functioning of laticifers varies from species to species, being, as a rule, shorter than
the life of the plant itself (Roshchina and Roshchina 1993). The protoplasm and other
organelles in a laticifer cell remain alive. The cytoplasm is localized near the walls,
whereas the rest of the space is occupied by the vacuolar sap. Further development of
this cell is characterized by degradation of cellular organelle. The isolated latex vacu-
oles from Papaver somniferum filled with alkaloids may serve as a model for the study
of the accumulation of these secondary metabolites (Roberts et al 1991).
A non-articulated laticifer is a giant cell which when arising in the embryo is not
divided more but grows continuously, elongates, and branches. Such laticifers per-
meate all organs of the plants belonging to family Euphorbiaceae. A model for the
luminescence studies is laticifers of Euphorbia milii bract (Fig. 2.5). Due to the
content of phenols and alkaloids in latex, we can see fluorescent laticifers on the
slice. The emission spectra of laticifers fluoresced in blue show maxima at 475 nm
(differ from cell wall and parenchyma with a maximum at 675–680 nm) at the laser
excitation of 405 nm, while other nonsecretory parts of the bract fluoresce in the red
region of the spectrum characteristic of chlorophyll. Some parts of laticifers contain
different amounts of fluorescing latex that are seen from the height of peak 475 nm
in the spectra that are peculiar to phenol and/or alkaloid accumulation. The laticifer
fluorescence permits to observe the accumulation of latex during the development
of the plant. The laticifers emit in blue at UV light excitation of 360–380 nm and in
green at violet light excitation of 430 nm (Table 2.2). The emission is clear in UV
light. The latex drop may have undergone the influence of the air oxygen and, as
seen in Table 2.2, fluoresces more brightly if compared with undamaged laticifer.
Latex of this model is of interest for the study of antiparasitics (including the mol-
luscicidal activity, carrier of dangerous infection from trematode Schistosoma man-
soni) as shown in some publications (Souza et al 1997; Bakry and Mohamed 2011).
Perhaps, the active matter is lectin euphorbin (Dias-Baruffi et al. 2000).
2.2.2 Idioblasts
Idioblasts, cells filled with secretion, are met within the tissue. These cells are single
secretory systems. The evolution of secretory structures is supposed (Fahn 1979) to
go from scattered cells, idioblasts, to organized intratissual canals, ducts, and cavi-
ties covered by epithelial cells and to end on the glands located on the plant surface.
2.2 Intratissual Secretory Systems I 47
0.975
0.922
0.869
0.816
0.763
3
0.71
2
0.657
0.604 2
0.551
0.498 1
0.446
0.393
0.34
0.287
0.234
1 3
0.181
0.128
0.075
0.022
450 500 550 600 650 700 750
Wavelength (nm)
Fig. 2.5 The LSCM images of fluorescent petal laticifers with latex (blue parts on the longitudinal
slice, shown with arrows) from flower bracts of Euphorbia milii and their fluorescence spectra
marked with green color (1). (2 and 3) The emission spectra from parenchyma (yellow line) and
cell wall (violet line), relatively. Bar = 300 μm (Courtesy of Dr. Yashin VA)
Table 2.2 The fluorescence intensity (I) of laticifers and latex in Euphorbia milii measured by
microspectrofluorimeter
Excitation 360–380 nm Excitation 430 nm
Part of plant I460 I680 I520 I680
Flower bract (part without 0.06 ± 0.003 1.46 ± 0.080 0.03 ± 0.003 1.05 ± 0.009
laticifers)
Laticifers 0.09 ± 0.003 1.25 ± 0.007 0.08 ± 0.003 0.40 ± 0.030
Latex drop 0.52 ± 0.004 1.09 ± 0.030 0.03 ± 0.003 0.03 ± 0.003
Stem (part without laticifers) 0.02 ± 0.003 1.17 ± 0.020 0.01 ± 0.003 1.25 ± 0.040
Laticifers 0.07 ± 0.002 0.40 ± 0.007 0.07 ± 0.002 0.34 ± 0.030
Latex drop 0.16 ± 0.004 1.09 ± 0.030 0.03 ± 0.003 0.03 ± 0.003
Idioblasts may include salts of calcium and silicium, phenols, and alkaloids. As
model system Khaphagi (2007) represented cell suspension culture of Peganum
harmala enriched in idioblasts with β-carboline alkaloids and serotonin, suitable for
the observation of the culture development species, also could serve as models for
phenol-storing idioblasts in cytochemical manipulations with chemical analysis by
HPLC (Beil and Rauwald 1993). In Aloe barbadensis and A. succotrina, acetogenic
a b c
Fig. 2.6 Fluorescence of idioblasts (shown by arrows) containing phenols, seen under lumines-
cence microscope Leica DM6000 from transverse slice (Done by courtesy of Dr. Likhanov) of
flower ovule bag from hop Humulus lupulus. Excitation by light 360–410 nm (a), 420–450 nm (b),
and 560 nm (c). Bar = 100 μm
Table 2.3 The maxima (λ) in the fluorescence spectra of intact cells on the Ruta graveolens roots,
extracts from the root tissue (1 h of soaking), and known individual compounds of the tissue
Pure substance from
Root intact cells λ (nm) secretion λ (nm)
Nonsecretory cell 480 Rutacridone 600–605
Idioblasts (fluoresced in yellow and red 475, 550, or 610 Rutacridone glycoside 595
orange)
Crystals Quercetin 450, 610
1. Orange 610
2. Yellow 530
3. Green yellow 490,520
Tip of root (young root) 590–595 Rutin 610
Adapted from Roshchina (2007b)
anthranoid and 2-alkylchromones were found in isolated aloin cells. Aloins A and
B as well as their derivatives were identified. If the cells contain fluorescent com-
pounds, their location within tissues and development appear under the lumines-
cence microscope (Eilert et al. 1985, 1986; Kuzovkina et al. 1975; Roshchina 2002,
2005a, 2008).
Clear autofluorescence is observed for some species (Fig. 2.6). A similar model
of idioblast appears to be ovule bag of hop Humulus lupulus where idioblasts are
well seen as yellow structures at excitation by violet light or as red cells under
orange light among the dark surface parenchyma (Fig. 2.6). The main components
of the secretory cells are phenols, perhaps flavonoids.
Earlier as analogous fluorescing model for the study secretions in idioblasts root
cells of Ruta graveolens was considered where emission in spectral region 570–
610 nm was changed during the cell development in tissue and tissual culture
(Roshchina 2008). Among the fluorescent components in idioblast secretions of this
species are furanocoumarins and acridone alkaloids. Acridone alkaloids occurring
in root idioblasts of Ruta graveolens brightly fluoresce at 580–600 nm among
weakly blue-emitted cells (Kuzovkina et al. 1975; Roshchina 2008). Their forma-
tion could be observed during plant development. In Table 2.3, the maxima in the
2.3 External Secretion 49
fluorescence spectra of idioblasts and root extracts by certain solvents. The

composition of nonsecretory cells (maxima in blue) and idioblasts (maxima in yel-
low orange) containing rutacridone differs significantly, although similar with the
tip of the root. Besides alkaloid rutacridone, flavonoids quercetin and rutin may
contribute to orange emission. The secretory products are released on the surface of
the model object and form various crystals. Furanocoumarins have also autofluores-
cence in orange (Zobel and March 1993).
2.3 External Secretion
External secretion is accomplished by specialized secretory cells which are located

on the surface of aboveground parts of the plant (stem, leaves, bud scales, flowers).
Among them are glandular hairs or trichomes, glands such as salt glands and glands
of carnivorous plants, nectaries, osmophores, and hydathodes. Glandular hairs and
glands perform the biosynthesis of many substances such as essential oils, terpenoids,
flavonoids, fatty acids, and slimes and often excrete their mixtures (Wiermann 1981).
2.3.1 Secretion into Free Space
Secretion, before releasing out the secretory cell, enters into free space (Evert and
Esau 2006). The free space of every secretory cell usually means the cell part that is
outside the plasmalemma (Esau 1977; Vasilyev 1977). Since the cell wall and the
plasmalemma are considered as a single complex which can be separated with great
difficulty, secretion via the plasma membrane must be virtually the secretion into
the free space of the cell wall. Some space between the cell wall and the plasma
membrane (periplasmic space) arises really only as a result of secretory activity of
the cell when the inclusion of the Golgi vesicle is rejected outside the plasma mem-
brane, and in this case, the plasma membrane may be separated from the cell wall
by a rather great space filled with the secretion.
There are less data about model systems where the secretion is being studied
directly. Phenols are known to be secreted into the free space of cell. The model sys-
tem may be swede Brassica napus where the secretory products appear in just the first
stage of embryogenesis (36 h after the beginning of germination) either in the cyto-
plasm (in small vacuoles) or outside the plasmalemma. In the latter case, the secretion
of phenolic substances serves as a barrier preserving from infection (Zobel 1989).
Special attention has been paid to the synthesis of cell wall elements and their involve-
ment in the biogenesis of the cell wall (Harpaz-Saad et al. 2011). Secretion into the
free space of the cell is particularly observed in specialized secretory cells, being the
first stage of excretion from the cell. This process, however, is intrinsic in any plant
cell, in particular dividing meristemic cells form the cell wall from the components
secreted to the free space. Major constituents of the cell wall are an amorphous matrix
with high water content and the fibrillar system of the skeleton made of cellulose. The
matrix elements hemicelluloses (essentially ureides) and pectins are synthesized in
the Golgi apparatus and then excreted through the plasmalemma by exocytosis. The
fibrillar system of the skeleton consists of cellulose (β-1,4-polyglucosan) and galac-
tan (β-1,3-polyglucosan) which are synthesized outside the plasma membrane in the
newly formed matrix of the cell wall. These elements are synthesized by a complex
of enzymes localized in the plasmalemma (Frey-Wyssling 1973). Recently, it was
shown that phospholipase A secreted into free space also participates in cell elonga-
tion and shoot gravitropism in the genetic model of Arabidopsis (Lee et al. 2003). As
model system, Arabidopsis seed coat mucilage is used for genetic analysis of plant
cell wall structure and function (Haughn and Western 2012).
Special bodies – exosomes – may participate in the multivesicular body-mediated
secretion into free space (Denzer et al. 2000; Lopez-Verrilli and Court 2013).
Multivesicular bodies (spherical endosomal organelles containing small vesicles
formed by inward budding of the limiting membrane into the endosomal lumen in
order to deliver them into lysosomal/vacuolar compartments for degradation) appear
to fuse with the plasma membrane in an exocytic manner, leading to release their
contents including internal vesicles into the extracellular space. These released ves-
icles are termed exosomes. The bodies and paramural vesicles proliferate when cell
wall appositions are rapidly deposited beneath fungal penetration attempts or during
plugging of plasmodesmata between hypersensitive cells and their intact neighbor-
ing cells. For example, there are multivesicular compartments in intact cells in bar-
ley leaves attacked by the barley powdery mildew fungus, and there are intravacuolar
multivesicular bodies with double limiting membranes in an intact epidermal cell
adjacent to a hypersensitive epidermal part (Lopez-Verrilli and Court 2013).
Endosomes play an important role in the traffic of the products in the cell that is
shown on the genetic model of Arabidopsis (Contento and Bassham 2012).
2.3.2 Models of External Secretion
Modeling of external secretions may be based on the observation of the color and/
or fluorescence of secretory products in vital conditions. The excretions from the
cells are also suitable to analyze with special dyes for histochemical analysis that
permits to identify the presence of certain metabolites in their content and within the
cells. Modeling may occur in both unicellular and multicellular systems. Since
color and fluorescent histochemical analyses of secretions for the main groups of
secondary metabolites, viz., phenols, lipids, and alkaloids, are described in the set
of books and reviews (Gilroy 1997; Roshchina 2003, 2008), fluorescent microscopy
of living plant cells in this section is the main focus of attention that is devoted to
new materials dealing with the participation of excretions in biocenotic relations.
Components of their secretions are involved in cell recognition processes and the
regulation of cellular development. Model systems where secretions are well seen
and in which accumulation may be analyzed are preferable.
2.3.2.1 Models of External Secretions
Some of the secretory components in models chosen are colored and/or fluoresce.
Their autofluorescence is well seen within and out of cells (see Sects. 1.2, 1.3, and
1.4). In ultraviolet light, most phenols with aromatic rings – stilbenes, catechols,
indoles, anthraquinones, and flavonoids, except some compounds as galangin and
small concentrations of kaempferol – fluoresce in blue (419–440 nm) or in blue
green (500–520 nm). Anthocyanins also have peaks of the emission at 510, 515,
555, 570–585, and 610 nm. Products of lipid peroxidation formed in the reaction
between malonic dialdehyde and free amino groups fluoresce in blue (430–470 nm)
too. For non-fluorescing compounds, one could apply histochemical staining with
molecular probes or markers. Histochemical methods alone or in combination with
biochemical assays also have perspective in express analysis.
2.3.2.2 Single Cell Models
Single cells, in particular plant microspores (vegetative microspores of spore-

breeding species and pollens) and stinging hairs (tip cells of complex trichome) of
representatives from family Urticaceae, can serve as unicellular donor models of
secretions. The stinging hair or emergence, part of complex stinging hair as object,
was suitable for the study of the accumulation of the secretion fluoresced under UV
light of the luminescence microscope during the development of Urtica dioica as
well as Urtica urens (Roshchina 2008).
Secretions of single cells are very variable, and their compositions depend on
genetic characteristics and conditions of plant inhabitation as well as on factors
related to environmental pollution. At the beginning of the twenty-first century,
special attention was paid to the analysis of natural accumulation of some secretory
metabolites found in medicinal and allelopathically active species. A similar model
as the stinging cell of Urtica dioica or Urtica urens is of interest because it contains
compounds named animal neurotransmitters – acetylcholine and biogenic amines
(Roshchina 2010). They play a basic role in chemosignaling between different cells
(see Sect. 3.1).
The main information about secretions from plant microspores relates to pollen,
although basic characteristic is peculiar to vegetative microspores as well. Slime-like
pollen secretion contains polysaccharides, proteins (lectins, enzymes such as peroxi-
dases, esterases), and lipids with complexes of lipophilic compounds (Stanley and
Linskens 1974). Growth regulators (flavonoids and biomediators, acetylcholine and
histamine) were also found in pollens of many species (Marquardt and Vogg 1952;
Bednarska 1992; Roshchina 2001a, b). Lipophilic components of pollen excretions
such as carotenoids, phytosteroids including hormone of the insect metamorphosis
ecdysone and sex hormones testosterone and rostenedione (Šaden-Krehula et al.
1971, 1983), and sesquiterpene lactones (azulenes and proazulenes) are also found
in pollens (Heftmann 1975; Roshchina et al. 1995; Roshchina 2008). Besides liq-
uids, pollens also release volatile compounds (geranyl acetate, geraniol, geranial,
neral, and others – aldehydes and alcohols) (Dobson et al. 1987; 1996; Roshchina
and Roshchina 1993), and male flowers release terpenoids 3-carene, limonene, and
myrcene (Roshchina and Roshchina 1993). All these compounds can influence the
pollen germination. Volatile compounds such as linalool inhibited the pollen devel-
opment in lower concentrations 10−8–10−9 M, while citral (geraniol + neral) and
cymol stimulated the process (Roshchina 2007a). Sesquiterpene lactones may stimu-
late (azulene) or inhibit (austricine or gaillardine) the pollen germination (Roshchina
2004a, 2007a, b). Phenols may also be inhibitors (Murphy 1999a, b) or stimulators
(Waser and Fugate 1986) of pollen germination.
Enzymatic activity of secretions is also very important in order to recognize the
low-molecular compounds released from competing pollen or pistil stigma (Stanley
and Linskens 1974; Waser 1983; Waser and Fugate 1986); in particular, cholinester-
ase decomposes acetylcholine (Roshchina 2001a, b). This activity is concentrated in
intine, pores, and colpus of pollens from many species and vegetative spores of
Equisetum genus (Knox and Heslop-Harrison 1970). Cholinesterase is an enzyme
which catalyzes the hydrolysis of acetylcholine and is a marker for the presence of
acetylcholine in the cells that are found in both vegetative microspores and pollen
grains (Bednarska 1992; Roshchina et al. 1994; Roshchina 2001a, b, 2007a).
Pollen and anther fructokinase and hexokinase regulate the pollen germination,
possibly by providing fructose-6-phosphate for glycolysis or through conversion to
UDP glucose to support the biosynthesis of cell wall material for pollen tube growth
(Karni and Aloni 2002). Moreover, the microspore surface includes or forms the
reactive oxygen species in the atmosphere ozone (Roshchina and Roshchina 2003)
that also may react with antioxidants of secretions and both low-molecular and
high-molecular enzymes. The secretion is functionally similar to olfactory muci-
lage, which helps in chemosignal transduction and its recognition by the acceptor
cell (Roshchina et al. 1998b). Mechanisms of chemosignaling that influences the
cell growth reactions are not well known in pollen allelopathy, but such information
is important. The possible mechanisms of microspore germination in pollen alle-
lopathy compared to other spore-forming systems of cryptogams, far in evolution
from phanerogams, reflect the similar natural technology. Here compounds in spore
secretions may play the role of growth regulators, and due to the high biological
activity of microspores (Roshchina 2005a, b, 2006a, b, 2007a), they are already
used in medicine as the pollen loads collected by bees or to determine the activity
of allelochemicals, many of which are potential drugs. In other words, biomimetic
nanotechnology copying nature on a molecular level may be the origin for new
improved plant growth regulators and/or pharmaceuticals; therefore, our knowledge
of cellular mechanisms in allelopathic relations is necessary to achieve this aim.
Vegetative microspores and pollen of various species may have bright autofluo-
rescence due to secretory products. The first drop of secretion after moistening of the
cells may contain the metabolites, in particular flavonoids emitted in blue or blue
green (Roshchina 2008) or cholinesterase whose presence is observed after staining
with the red analog of Ellman reagent (Roshchina 2001a). For example, a researcher
could see induced fluorescence showing the occurrence of biogenic amines such as
catecholamine (dopamine) and histamine (also known as neurotransmitters in animal
organisms having neural system) within and out of the cell. The components of the
secretions may play a role in chemosignaling in biocenosis (see Chaps. 3 and 4).
Secretions from the plant microspores and some stinging hairs (emergences)
may contain nitrogen-containing physiologically active compounds known as neu-
rotransmitters in animal physiology (Roshchina 1991, 2001a, b, 2010). Among the
compounds contained in many plant species are acetylcholine and biogenic amines
such as catecholamines (dopamine, noradrenaline, adrenaline), serotonin, and hista-
mine (see references in monograph by Roshchina 2001a). Acetylcholine was inves-
tigated then in many plants by the methods of biotests, paper and thin-layer
chromatography, electrophoresis, gas–liquid chromatography and its combination
with mass spectroscopy, as well as nuclear magnetic resonance (NMR) spectros-
copy. Highest concentrations of acetylcholine and histamine were found in stinging
hairs of species belonging to Urticaceae, in particular in common nettle Urtica dio-
ica and U. urens (Emmelin and Feldberg 1947; see also monographs of Roshchina
1991, 2001a). Histamine is also found in pollen, mainly of wind-pollinated species
Agrostis alba, Alopecurus pratensis, Bromus erectus, Corylus avellana, Cynosurus
cristatus, Dactylis glomerata, Gelsemium sempervirens, Lolium perenne, Phleum
pratense, Poa pratensis, Secale cereale, and Zea mays, as well as of some insect-
pollinated species Syringa vulgaris, Tilia platyphyllos, and T. cordata (Marquardt
and Vogg 1952). Histamine found in pollen may be the agent of allergic reaction in
humans (Stanley and Linskens, 1974). Under stress reactions, a sharp increase in
histamine is observed in plants, like in animals. In plants, the rise in histamine is
found during drought, for instance, for sunflower seeds (Korobova et al. 1988).
Histochemical approaches are used to study the cell secretion of these com-
pounds primarily called neurotransmitters in animal physiology (Roshchina 2010).
Among them, one could apply glyoxylic acid as reagent for catecholamines such as
dopamine, noradrenaline, and adrenaline (Björklund et al. 1972; Axelsson et al.
1973; Lindvall et al. 1974; Lerke and Bell 1976) and o-phthalic aldehyde as reagent
for histamine (Cross et al. 1971; Kuruma et al. 1994; Ekici and Coşkun 2002, 2006).
Glyoxylic acid is used mainly for animal tissue research, but recently this reagent
was also applied for plant microspores (Roshchina et al. 2011b). Phthalic aldehyde
is known mainly as reagent for fluorescent assay of histamine in animal organisms
(Cross et al. 1971), and its application for other organisms is at the beginning
(Barwell 1989; Ekici and Coşkun 2002). Glyoxylic acid (glyoxylate) and phthalates
are found in plant tissues as stated in the monograph by Roshchina V. V. and
Roshchina V. D. (2012); therefore, the reagents may also be considered as natural
metabolites.
Glyoxylate, reacting with catecholamines, forms a fluorescent product (emission
maximum 520 nm) that is a derivative of isoquinolines (Fig. 2.7). The complex fluo-
resces mainly in blue green. Under the influence of glyoxylic acid (Fig. 2.7), blue or
green-yellow fluorescence significantly increases in vegetative microspores of
Equisetum arvense or in the pollen of Hippeastrum hybridum and their excretions
from the cells (Table 2.4). Red emission inside the vegetative microspore, on the
contrary, is diminished by the newly formed fluorophores – derived from isoquino-
lines (Fig. 2.7).
R
HO – H2O – CO2
+ 2 HOOC–CHO R
HO
HO
glyoxylic acid ⊕
NH2
HO N – CH3 COO
catecholamine
R = H dopamine 2–carboxymethyl–3,4–dihydroisoquinoline
R = OH noradrenaline derivative (strongly fluoresced)
Equisetum arvense Hippeastrum hybridum
control + glyoxylic control + glyoxylic

acid acid
Fig. 2.7 The staining of vegetative microspores of Equisetum arvense and pollen of Hippeastrum
hybridum with glyoxylic acid, reagent for the determination of catecholamines. Upper side: sche-
matic chemical reaction of catecholamine dopamine with the reagent with the formation of fluo-
rescent isoquinolines. Lower side: common view of microspores before (control) and after the
treatment with glyoxylic acid under UV light (360–405 nm) of luminescent microscope. Blue fluo-
rescence of secretion is seen as the holo of E. arvense (bar = 20 μm) and green-yellow emission, for
H. hybridum (bar = 50 μm)
Table 2.4 Autofluorescence of Equisetum arvense microspores and their fluorescence after
histochemical reaction with glyoxylic acid (glyoxylate) on catecholamine (determined as the
intensity of fluorescence of cells and their secretions)
Green fluorescence at 520 nm (520) (I520)
Variant Cells Excretions Away from cell
Background controls (without additives) 0.06 ± 0.003 No emission 0.02 ± 0.002
Control + (glyoxylate) 0.29 ± 0.030 0.23 ± 0.026 0.04 ± 0.005
Brefeldin А 20 μg μmol/ml 0.08 ± 0.005 No emission 0.02 ± 0.001
Brefeldin А220 μg μmol/ml + glyoxylate 0.35 ± 0.013 0.20 ± 0.003 0.04 ± 0.001
Adapted from Roshchina et al. (2011b)
The treatment with brefeldin B, the secretion inhibitor acting on protein transfer
in secretory vesicles, slightly reduces the intensity of the green emission out of cells
and strengthens it within the cell. All this indicates both the presence of catechol-
amines in the secretory vesicles inside the cells and the ability of these neurotrans-
mitters to release outward. Thus, Equisetum arvense microspores contain both
acetylcholine (as shown with its marker cholinesterase; also see Chap. 1) and cate-
cholamines that may be involved in intercellular communication with cells of other
organisms. Cells of E. arvense are sensitive to redox changes in the environment, in
particular to ozone (Roshchina and Roshchina 2003). Table 2.5 also demonstrates
that even small concentrations of strong oxidants such as ozone increased emission
(twofold) and deals with catecholamines (Roshchina and Yashin 2014).
Stinging hairs of Urtica dioica on leaves and stems have demonstrated high
emission at 520 nm and decrease red fluorescence of chlorophyll at 680 nm after the
Table 2.5 Effects of ozone on the accumulation of catecholamines (green fluorescence intensity
at 520 nm) in Equisetum arvense microspores staining with glyoxylic acid. Excitation
360–380 nm
(I520)
Variant Cells Excretions
Control (stained with glyoxylic acid) 0.26 ± 0.001 No emission
+ Ozone 0.009 ppm per 5 h exposition 0.53 ± 0.039 0.44 ± 0.040
+ Ozone 0.009 ppm per 15 h exposition 0.64 ± 0.030 0.77 ± 0.057
Table 2.6 The emission of catecholamines after the treatment of secretory hairs on common nettle
Urtica dioica with glyoxylate
Green fluorescence at 520 nm (I520) Red fluorescence at 680 nm (I680)
Control (without Control (without
Variant treatment) + Glyoxylate treatment) + Glyoxylate
Leaf hairs 0.38 ± 0.011 3.73 ± 0.066 0.52 ± 0.016 2.76 ± 0.069
Stem hairs 0.53 ± 0.016 1.25 ± 0.026 0.59 ± 0.018 1.94 ± 0.050
treatment with glyoxylate due to catecholamines contained in secretions (Table 2.6).

Stem emergences contain lesser neurotransmitters than the leaf ones. Stinging nettle
leaf hairs show increase (approximately ten times) in the characteristic of green
fluorescence with a maximum at 520 nm. Besides, red fluorescence increased five
times only. In hairs of stems, green emission was about 2.5 times higher after the
treatment with glyoxylate than in control.
Histamine may also be determined in secretions by histochemical method.
Phthalic aldehyde is known as a reagent for fluorescent assay of histamine in organ-
isms (Cross et al. 1971). The reaction with histamine leads to the formation of fluo-
rescent product (Fig. 2.8) that appears to be complex, including derivatives of
imidazolyl ethylamine. The complex fluorescence is inhibited in acidic medium
and emits in alkaline medium, although unstable (Douabalé et al. 2003). At small
concentrations of 10−8–10−7 M, the emission of animal tissue slices under UV light
of the luminescence microscope in the reaction is blue, while at higher concentra-
tions, the complex of fluorescing products emits in green and in maximum in yel-
low. Nuclei of fibroblasts fluoresce as well as elastic net fibrils (Zhirnova et al.
2007). Quantification of the method was applied to the measurement of histamine
in human blood and urine (Kuruma et al. 1994), and its detection limit was 2–5 pg.
The method is also used for food (including plants) analysis in order to select toxic
samples that as a rule relates to bacterial pollution of the products (Ekici and
Coşkun 2002). The ability of bacterial microflora to produce histamine as well is a
problem for human health. In fundamental studies, histamine was determined by
this method in marine algae, in particular in thallus Furcellaria lumbricalis
(Barwell 1989). In this object, mean values varied from 60 to 500 μg g−1 fresh
weight.
In our experiments with unicellular models after the treatment with o-phthalic
aldehyde, bright blue or even green-yellow (at concentrations of histamine higher
HO H
N HOC N – H2O O
NH 2 + 2 N N
NH HOC N
NH H OH
NH HO OH
histamine o-phathalic aldehyde Fluorescing
products
Equisetum arvense
+ o-phthalic + o-phthalic
control aldehyde
aldehyde
Urtica dioica
control
+ +
o-phthalic o-phthalic
aldehyde aldehyde
Fig. 2.8 The staining of vegetative microspores of Equisetum arvense (bar = 20 μm) and stinging
hair (emergence bar = 50 μm) of Urtica dioica with phthalic aldehyde, reagent for the determina-
tion of histamine. Upper side: schematic chemical reaction of histamine with the reagent with
phthalic aldehyde when the fluorescent products are formed. Lower side: common view of micro-
spores before (control) and after the treatment with phthalic aldehyde under UV light (360–
405 nm) of luminescence microscope. Blue fluorescence of secretion is seen as holo of E. arvense
and green-yellow emission, for Urtica dioica (bar = 50 μm)
than 10−6 M) fluorescence excited by ultraviolet light (360–405 nm) was peculiar
both for the cell and their secretions seen as holo (Fig. 2.8) and may be measured by
microspectrofluorimeters (Tables 2.6 and 2.7). It was especially clear for vegetative
microspores of Equisetum arvense and green-yellow emission for Urtica dioica.
Blue fluorescence of secretion is seen as holo of E. arvense and green-yellow emis-
sion for Urtica dioica surface of stinging hair (Fig. 2.8). In the first model system,
the chloroplasts and nucleus appear as blue organelles (Roshchina et al. 2014). As
seen in Table 2.7, the blue emission intensity in maximum at 450–460 nm (mea-
sured by dual-wavelength microspectrofluorimeter) increased twofold after the
staining with o-phthalic aldehyde and red fluorescence at 680 nm, peculiar to chlo-
rophylls. Usually, blue emission is measured at 430 nm too.
Like in animals, stress conditions may increase the secretion of histamine as well
as in plants during drought in sunflower seeds (Korobova et al. 1988). Truly salina-
tion stimulated the emission related to histamine in microspores of E. arvense
Table 2.7 Autofluorescence of Equisetum arvense microspores and their fluorescence after
histochemical reaction with O-phthalic aldehyde on histamine (determined as the intensity of
fluorescence of cells and their secretions)
Red fluorescence at
Blue fluorescence at 460 nm (I460) 680 nm (I680)
Away from
Variant Cells Excretions cell Cell
Background controls 0.10 ± 0.001 No emission 0.02 ± 0.002 0.74 ± 0.05
(without additives)
+ O-phthalic aldehyde 0.21 ± 0.020 0.07 ± 0.001 0.03 ± 0.005 0.03 ± 0.005
Table 2.8 Influence of 24-h exposure of the germination of Equisetum arvense microspores after
salination by Na2SO4 solutions moistening on their fluorescence after histochemical reaction with
O-phthalic aldehyde
Blue fluorescence at 460 nm Red fluorescence at 680 nm
(I460) (I680)
Variant Cells Excretions Cells
Controls (without additives) 0.13 ± 0.009 0.03 ± 0.003 0.74 ± 0.010
+ 0.1 % Na2SO4 0.15 ± 0.030 0.05 ± 0.020 0.92 ± 0.020
+ 0.5 % Na2SO4 0.17 ± 0.005 0.39 ± 0.030 0.41 ± 0.060
+ 1 % Na2SO4 0.08 ± 0.013 0.61 ± 0.09 0.37 ± 0.030
Average error experience for n = 4 (count 100 cells in each of the four subject slides)
(Table 2.8). Moreover, the emission progressed in excretions with the growth of
concentration of salt in the medium that accompanied depression in chlorophyll
formation (red fluorescence).
After the treatment of stinging hairs from Urtica dioica (Table 2.9) with
o-phthalic aldehyde, one can see the increase in the fluorescence related to hista-
mine. From the data the possible concentration of the neurotransmitter within cells
may be estimated. It is highest in leaf base of hair where the secretion is
concentrated.
Table 2.10 includes results of the fluorescence measurement from pollen stained
with both histochemical probes – glyoxylic acid and phthalic aldehyde. Among the
species, high concentration (about 10−3 M) of histamine was shown for pollen of
Populus balsamifera, Taraxacum officinale, and Tussilago farfara. Most wind-
pollinated species studied have no or smaller amounts of catecholamines (10−7–
10−6 M). Completely neither catecholamines nor histamine were for pollen from
Phleum pratense and Betula verrucosa. Knowledge of the accumulation of cate-
cholamines and histamine in pollen is an important problem for medicine because
the neurotransmitters play a role as growth regulators, demencion agents, and com-
ponents of allergic reactions. Moreover, it is unknown for us whether the com-
pounds participate in plant fertilization.
Table 2.9 The fluorescence intensity (I) of emergences from Urtica dioica stained with o-phthalic
aldehyde for histamine determination. Excitation 360–380 nm for I430–450 and 405–430 nm for I520
Common view of
stinging hair (bar Concentration of
200 μm) Leaf hair I430–450 I520 histamine (M)
Control 0.03 ± 0.001 0.11 ± 0.003
+ Phthalic aldehyde 0.50 ± 0.007 0.55 ± 0.030 10-3 M
(near sting tip of
hair)
Base (thick part)of hair 0.68 ± 0.050 0.74 ± 0.030 10-2 M
Stem hair
Control 0.01 ± 0.003 0.01 ± 0.003
+ Phthalic aldehyde 0.20 ± 0.002 0.56 ± 0.010 >10-5 M
(near tip of hair)
2.3.2.3 Multicellular Models
Today, among multicellular models having secretory cells, quantitative and qualita-
tive results deal with the measurement of autofluorescence (Roshchina 2008) or
some histochemical procedures with reactions on the presence of terpenes, lipids,
and phenols in secretions (Ascensao and Pais 1987; Ascensão et al. 1997; Muravnik
2008; Marin et al. 2010).
Salt glands are inherent in many plant species adapted to living on saline soils, in
particular in the representatives of Plumbaginaceae, Tamaricaceae, Verbenaceae,
Gramineae, and other families. A great amount of salts is constantly released on the
surface of the plant leaves and then is blown away by wind or washed off with rain.
Salt glands originate from the epidermis and are located on the surface of both sides
of the leaf and young stems. Generally, they are embedded into the epidermis.
Similar leaf glandular structures may be illustrated on the example of salt glands
that eliminate salts into vacuoles and may store or secrete them outside the secretory
cells.
The glandular structure of leaves is also peculiar to salt-accumulated plants
that belonged to Chenopodiaceae such as in the allelopathically active genus
Chenopodium (Alam and Shaikh 2007). The structure of the similar gland in both
Chenopodium and Atriplex genera is a one-celled or (seldom) two–three-celled
Table 2.10 Fluorescence of pollen from various species stained with reagents on catecholamines
and histamine
Concentration of neurotrans-
Variant I450 I530 mitter, M
Wind-pollinated species
Betula verrucosa Ehrh.
Control 0.03 ± 0.002 0.02 ± 0.002
+ Glyoxylate 0.05 ± 0.003 0.07 ± 0.001 Traces
+ Phthalic aldehyde 0.07 ± 0.007 0.12 ± 0.010 Traces
Corylus avellana L.
Control 0.06 ± 0.005 0.05 ± 0.006
+ Glyoxylate 0.41 ± 0.010 0.14 ± 0.010 Cat 10−6
+ Phthalic aldehyde 0.03 ± 0.006 0.13 ± 0.010 No
Larix decidua Mill.
Control 0.29 ± 0.016 0.08 ± 0.001
+ Glyoxylate 0.46 ± 0.033 0.12 ± 0.023 Cat 5 × 10−7
Phleum pratense L.
Control 0.56 ± 0.006 0.58 ± 0.001
+ Glyoxylate 0.40 ± 0.030 0.04± 0.020 No
Populus balsamifera L.
Control 0.02 ± 0.004 0.03 ± 0.003
+ Glyoxylate 0.10 ± 0.006 0.39 ± 0.017 Cat 10−6
+ Phthalic aldehyde 0.19 ± 0.001 0.79 ± 0.047 Hist > 10-3 M
Quercus robur L.
Control 0.04 ± 0.003 0.05 ± 0.002
+ Glyoxylate 0.13 ± 0.008 0.08 ± 0.004 Cat 10−8-10-7
Insect-pollinated species
Acer platanoides L.
Control 0.05 ± 0.001 0.05 ± 0.002
+ Glyoxylate 0.15 ± 0.007 0.47 ± 0.004 Cat 10−6
+ Phthalic aldehyde 0.22 ± 0.007 0.43 ± 0.006 Hist > 10−3
Aloe vera
Control 0.06 ± 0.001 0.12 ± 0.020
+ Glyoxylate 0.18 ± 0.010 0.20 ± 0.022 Cat 10−7
+ Phthalic aldehyde 0.18 ± 0.010 0.21 ± 0.010 Hist 10−8
Anemone ranunculoides L.
Control 0.12 ± 0.005 0.12 ± 0.001
+ Glyoxylate 0.05 ± 0.004 0.33 ± 0.015 Cat 10−6
Anthriscus sylvestris L.
Control 0.02 ± 0.002 0.04 ± 0.001
Epiphyllum hybridum L.
(continued)
Table 2.10 (continued)

Control 0.34 ± 0.008 1.49 ± 0.028
+ Glyoxylate 0.35 ± 0.004 1.3 ± 0.010 No
Hibiscus rosa-sinensis L.
Control 0.21 ± 0.05 0.11 ± 0.020
+ Glyoxylate 0.45 ± 0.008 0.29 ± 0.023 Cat 5 × 10−7
Hippeastrum hybridum
Control 0.02 ± 0.003 0.12 ± 0.001
+ Glyoxylate 0.03 ± 0.005 0.02 ± 0.004 No
Narcissus pseudonarcissus L.
Control 0.36 ± 0.030 0.14 ± 0.012
+ Glyoxylate 0.59 ± 0.073 0.29 ± 0.020 Cat 10−6 M
+ Phthalic aldehyde 0.66 ± 0.060 0.14 ± 0.073 Hist > 10−5 M
Picea excelsior (excelsa) L.
Control 0.12 ± 0.012 0.34 ± 0.030
+ Glyoxylate 0.26 ± 0.013 0.43 ± 0.020 Cat 5 × 10−7 − 10−6 M
Pinus sylvestris L.
Control 0.10 ± 0.011 0.24 ± 0.020
+ Glyoxylate 0.17 ± 0.010 0.26 ± 0.010 Cat 10−7
Philadelphus grandiflorus
Willd.
Control 0.09 ± 0.009 0.20 ± 0.020
+ Glyoxylate 0.11 ± 0.010 0.24 ± 0.010 No
+ Phthalic aldehyde 0.21 ± 0.020 0.40 ± 0.020 Hist 5 × 10−6
Plantago major L.
Control 0.22 ± 0.002 0.49 ± 0.004
+ Glyoxylate 0.09 ± 0.010 0.17 ± 0.0080 No
Rubus odoratus L.
Control 0.30 ± 0.008 0.31 ± 0.004 No
+ Glyoxylate 0.17 ± 0.010 0.17 ± 0.008 No
+ Phthalic aldehyde 0.14 ± 0.003 0.09 ± 0.010
Syringa vulgaris L.
Control 0.30 ± 0.001 0.31 ± 0.001
+ Phthalic aldehyde 0.85 ± 0.070 0.80 ± 0.060 Hist > 10-2
Taraxacum officinale L.
Control 0.04 ± 0.001 0.13 ± 0.070
+ Glyoxylate 0.35 ± 0.080 0.45 ± 0.080 Cat 10-6
Table 2.10 (continued)

Tilia cordata Mill.
Control 0.05 ± 0.009 0.46 ± 0.040
+ Glyoxylate 0.03 ± 0.001 0.30 ± 0.090 No
Tussilago farfara L.
Control 0.41 ± 0.010 0.35 ± 0.070
+ Glyoxylate 0.98 ± 0.080 0.26 ± 0.040 Cat 5 × 10−6
Tulipa sp.
Control 0.20 ± 0.010 0.35 ± 0.070
+ Glyoxylate 0.47 ± 0.009 0.40 ± 0.040 Cat 10−6
Urtica dioica L.
Control 0.22 ± 0.033 0.38 ± 0.070
+ Glyoxylate 0.11 ± 0.020 0.21 ± 0.010 No
Excitation 360–380 nm for I430 and 405–430 nm for I520 (Сat catecholamines, Hist histamine)
Source: Roshchina and Yashin (2014)
Chenopodium album Solidago virgaurea
Fig. 2.9 Fluorescence of salt-containing gland of Chenopodium album L. on the lower side of the
leaf. Left. The view of fluorescing gland with crystallic salt in the center under luminescent micro-
scope. The rigid secretion includes the fluorescing phenols (positive coloration with FeCl3).
Excitation 360–380 nm. Bar = 200 μм. Right. Terpenoid-enriched petal glandular hairs of Solidago
virgaurea seen under laser-scanning confocal microscope. Laser 405 nm. Bar = 50 μm. Secretion
released is seen on the surface of one cell
Table 2.11 The fluorescence intensity (I) of salt glands on the lower leaf side of Chenopodium
album
Cell Green emission (I520) Red emission (I680)
Salt gland with crystal 0.19 ± 0.018 1.95 ± 0.10
Surrounding nonsecretory cells 0.02 ± 0.003 3.02 ± 0.40
I520 and I680 – the emission intensity at 520 and 680 nm, respectively, at excitation 430 nm
Table 2.12 The fluorescence

intensity of secretory cells of Cell I520–540 I640–680
medicinal plant Solidago Leaf oil reservoir 0.26 ± 0.06 7.24 ± 0.9
virgaurea L. measured by Nonsecretory cells 0.07 ± 0.037 7.48 ± 1.0
double-beam Flower oil cell 0.26 ± 0.01 0.44 ± 0.03
microspectrofluorimeter
Nonsecretory cells 0 0.20 ± 0.03
MSF-2
Root tip secretion 0.32 ± 0.01 0.57 ± 0.09
Root nonsecretory cells 0.11 ± 0.01 0.17 ± 0.02
Excitation 420–430 nm
stalk bearing a distended head, the bladder cell (Fahn 1979). Being excited by
light 360–380 nm, the salt-containing leaf glands of lamb’s quarter Chenopodium
album L. (Chenopodiaceae) fluoresced in green or green yellow (Fig. 2.9).
The yellow fluorescence of salt glands may be due to the presence of flavonoids,
which impregnated the calcium crystals in the glandular cell. Moreover, sometimes
its blue fluorescence is seen. Calcium sulfates and silicates usually have no fluores-
cence, but perhaps when the crystalline salts are impregnated with phenols, the
blue-green emission appears (Roshchina 2008). The fluorescence color (greenish
yellow) and intensity of the salt glands differ from the surrounding nonsecretory
cells emitted in red (Table 2.11). As shown in Table 2.11, the emission intensity in
leaf salt glands is higher (seven to ten times), whereas red fluorescence is lower (two
to three times) than in the surrounding nonsecretory cells.
Perhaps, phenolic compounds contribute to the autofluorescence of the salt
glands. The green-yellow fluorescing crystals of salts are also observed on the sur-
face of seeds and cotyledons of the seedlings in the salt-enriched plant Chenopodium
album. They are formed presumably on the medium containing sulfates (Roshchina
2008). Allelopathic features of the leaf extract of nettle leaf goosefoot Chenopodium
murale depressed the seedling growth of rice (Alam and Shaikh 2007). Chenopodium
genus is rich in phenolics identified as protocatechuic, ferulic, p-coumaric, and
syringic acids that may be allelochemicals (Batish et al. 2007). These components
have light emission (Roshchina 2008). Alkaloid trigonelline and ferulic acid of the
plant species (Golovkin et al. 2001) may contribute to the visible fluorescence too.
Many phenolic allelochemicals may exist in such structure and affect the plants
grown around them, when the salt crystals fall outside leaves and fruits. Images of
leaf salt glands impregnated with phenolic compounds have been observed earlier
by the LSCM (Roshchina et al. 2007a, b). Big crystalline bands are seen on one of
the slices. Small crystals of phenols are also seen on the stack of the slices (sum
image) as yellow-green lightening spots, brighter than other surrounding structures.
In the optical slice type of the gland, it is seen in the confocal microscope as deposits
of salt on the edges of the layered structure (Roshchina et al. 2007a, b). The maxima
in the fluorescence spectra of glands and leaf extracts by various solvents are domi-
nated in blue (420–430 nm) and yellow-green (530–560 nm) spectral regions. In the
glandular cells with salt, it falls along with transpiration of the sap of xylem endings
of spending bunches. Their movement is carried out mainly by apoplast.
Terpene-enriched oil cells of goldenrod Solidago virgaurea (such as secretory hairs

shown in Fig. 2.9) fluoresce in green three to four times more intensive than nonsecre-
tory cells (Table 2.12). In red spectral region leaf cells, both secretory or not, has the
same values of the intensity.
However, at 640–680 nm, secretory cells of flowers and roots emit two to five
times stronger than nonsecretory ones. The green fluorescence may be related to the
terpenoids mixed with phenols in oil cells (Roshchina and Roshchina 1993). Green
fluorescent compounds appear to be flavonoids rutin, quercetin, kaempferol, isorh-
amnetin, and astragalin; chlorogenic and caffeic acids; and coumarin esculin found
in leaves, flowers, and herbs of the Solidago virgaurea (Golovkin et al. 2001). The
plant contains saponins that are antifungal and act specifically against the Candida
fungus as well as phenolic glycosides which are anti-inflammatory. The leaves and
flowering tops are anthelmintic, anti-inflammatory, antiseptic, aromatic, astringent,
carminative, diaphoretic, mildly diuretic, febrifuge, and stimulant. A good vulner-
ary herb, it has also proven of value when used internally in the treatment of urinary
infections, chronic catarrh, skin diseases, influenza, etc.
Chapter 3
Acceptor Models of Secretions
and Their Reactions on Exometabolites
Model systems are also necessary in the analysis of cellular mechanisms in response
to secretions released by own cells or those of foreign organisms. It is valuable for
understanding of contacts between organisms as a whole. Modeling processes
involve a search of suitable acceptor cells, biosensors that react with the compo-
nents of the secretions and form a physiological response. In studies, some exome-
tabolites are considered as chemosignals.
Biosensors are usually known as the analytical systems, which contain sensitive
biological elements and detectors. Plant intact cells as possible biosensors have a
natural structure that determines their high activity and stability. The search for
similar systems among plant species suitable for determining the mechanisms of
action of biologically active substances as external factors of the environment is a
real problem (Roshchina and Roshchina 2003; Roshchina 2004a). In this chapter
we shall pay attention to acceptor cells as sensitive models-biosensors, considering
possible exometabolites–chemosignals, sensory systems and reactions of cell stud-
ied on the secretions and secretory products.
3.1 Biosensors and Their Sensitive Reactions
The criteria in the screening of plant cells as biosensors in modeling effects of

secretions and their individual components should be as follows: (1) reaction is fast
based on the time of response, (2) reaction is sensitive to small doses of analyzed
compounds or their mixtures, and (3) methods of detection such as biochemical,
histochemical, and biophysical (in particular, spectral changes in absorbance or
fluorescence) are easy in the laboratory and in the field.
Now the search for natural biosensors is aimed at cells with most sensitive
reactions to biologically active compounds. Such reactions are important because
they show changes in the process of recognition during the chemical interactions
of plant cells, including pollen–pistil contacts at the fertilization of seed-bearing
plants (Roshchina 1999a, b, 2001a, b), germination of vegetative microspores
66 3 Acceptor Models of Secretions and Their Reactions on Exometabolites
in cryptogams (Roshchina 2004a, b, c, 2005a, b), or interaction of the trap cell

of insect as in carnivorous plant species (Muravnik and Ivanova 2002). Test
objects may be cells, which serve in sexual (pollen, male gametophyte, and pistil,
female gametophyte, in flower) and vegetative breeding (vegetative microspores),
respectively.
We shall consider the following biosensors for secretions and their individual
components: (1) vegetative microspores of spore-bearing plants, (2) pollen as gen-
erative (male) microspores of seed-bearing plants, (3) pistil stigma (female game-
tophyte) of seed-bearing plants, and (4) secretory cells of some plants (Roshchina
1999a, b, 2001a, b, 2004a, b, c), mainly, in (5) multicellular objects such as the
pigmented ones (Roshchina et al. 2013b).
Due to their spectral features and ability to develop, making them suitable for
the study, plant intact cells may serve as biosensors and bioindicators because they
have a natural structure that determines their high activity and stability (Roshchina
2012). The criteria in the screening of biosensors for biologically active compounds
should be as follows: (1) the object is easy to use in the laboratory and in the field
and (2) changes in absorbance and autofluorescence should be one of the physiolog-
ical responses to external chemicals because they reflect fast biochemical changes.
Some acceptor models (vegetative microspores and pollen) could develop in vitro,
which also is useful.
The test reactions of biosensors are summarized in Table 3.1 and will be consid-
ered in special sections with concrete examples. Some model systems – biosensors
used in the studies of intercellular contacts – will be described in Chap. 4.
The abovementioned test reactions on biosensors could also serve for some
biologically active excretions having drugs’ characteristics from plants known as
weeds and medicinal or poisonous plant species (Roshchina 2004a, b, c, 2007a, b,
2008, 2012). These plant biosensors are more suitable for similar testing than that
of animals, which needs procedures of vivisection, tissue isolation, and fixation
(Roshchina 2006a, b; Roshchina and Karnaukhov 2010). Autofluorescence, changes
of membrane potential, and secretion of digestive slime of insectivorous species are
fast responses to the external chemical factor (Table 3.1). The germination of micro-
spores and seeds has longer reactions.
Multicellular biosensors (Table 3.1) such as leaves of Lemna minor or Chara cor-
allina (water-living species) as well as seeds and seedlings of radish Raphanus sati-
vus were analyzed for fluorescence of chlorophyll or cell wall phenols. Perspectives
for new similar biosensors have trichomes in traps bubbles are represented by ten
insectivorous species having receptors, after which irritation by insects digestive
glands excrete trapping slime internal structure is secreting the protein animal
cages. They secrete ribonucleases, lipases, esterases, acidic phosphatase, and a huge
number of proteases (Fahn 1979, 2002). Hydrolytic enzymes, including cholines-
terase (Roshchina and Semenova 1995; Roshchina 2001a), appear on the surface of
leaves only after receiving signal from insects, not after raindrops, for instance. The
signaling occurs via the changes of membrane potential (Volkov et al. 2009, 2011).
Among known model process testing, we could consider such reactions as cellular
spectral characteristics (color, adsorbance, and fluorescence), the germination,
3.1 Biosensors and Their Sensitive Reactions 67
Table 3.1 Possible biosensors and sensor reactions

Biosensor Species Sensitive reaction Targets
Unicellular systems
Vegetative Equisetum arvense Autofluorescence Chloroplasts,
microspores cell wall
Secretion Plasmalemma,
cholinesterase
Germination Cholinoreceptors, ion
channels
Development Chloroplasts, nucleus
Pollen (generative Hippeastrum hybridum Autofluorescence Cover components
microspores)
Hippeastrum hybridum Germination Ca2+ channels
Vallota speciosa Germination Ion channels
Haemanthus katherinae Germination Ion channels
Multicellular systems
Flower cells Mirabilis jalapa Autofluorescence Vacuolar betalains
Flower cells Saintpaulia ionantha Secretion Cholinoreceptors
Flower cells Hibiscus rosa-sinensis Secretion Cholinoreceptors
Red bulb cells Allium cepa Secretion Cholinoreceptors
Algae cells Chara corallina Autofluorescence Chloroplasts, cell wall
Leaves Lemna minor Autofluorescence Chloroplasts, cell wall
Seeds and seedlings Raphanus sativus Autofluorescence Chloroplasts, cell wall
Cells of Pinguicula vulgaris, Secretion and changes Receptors
insectivorous Drosera, Aldrovanda in membrane
plants vesiculosa potential
Source: Muravnik (2000), Roshchina (2004a, b, c, 2007a, 2008, 2009a, b), Roshchina et al. (2004,
2009a, b, 2010, 2011a, b, c, d, 2013), Gandía-Herrero et al. (2005a, b), Volkov (2006), Volkov
et al. (2009, 2011)
growth, and development on the examples of the various types of model biosensors.
In choosing modeling processes, one may also base on the analysis of the secretion
from the objects. In liquid secretions it was possible to analyze the number of pig-
ments, fluorescing compounds, proteins, and enzyme activity by spectral methods.
If the secretory compound has color, the excretion may be observed visually and,
perhaps, its absorbance could be measured by spectrophotometer or microspec-
trophotometer (Chap. 1). Fluorescent substances in the excretions are seen under
luminescence microscope or measured by spectrofluorometer and microspectroflu-
orometer as described in Chap. 1. If the excretions have neither color nor emission
under actinic light, special dyes should be applied for the studies.
Changes in autofluorescence usually are valuable as natural indicator of cellu-
lar state because the emission often shows alterations in cellular metabolism and
responses to the external and internal signals. The intensity and spectral composi-
tion of the emission differ depending on, viz., (1) the nature of the organism or (2)
cell analyzed, (3) taxonomic position of the organism tested, and (4) environmen-
tal and experimental conditions (actinic light, temperature, humidity, physiological
state of a cell as a whole and phase of development, influence of neighbor cells
or organisms, including parasites, etc.). Autofluorescence could be used in diag-

nostics of cellular damage and in analysis of cell–cell interactions (Roshchina and
Roshchina 2003; Roshchina 2003, 2008; Roshchina et al. 2008; 2009a, b, 2012).
Perhaps, it is a resource for the nondestructive vital microscopic analysis of natural
organisms. It has already practical application in various diagnostic procedures, for
cell biology analysis, ecology, medicine, and pharmacology.
3.1.1 Exometabolites as Chemosignals and Regulators
Considering the plant life one could see their sensitive reactions to exometabo-
lites which serve both as chemosignals and regulators. Plant “living” environment
includes microorganisms and animals as well as other plant beings. Many compo-
nents of the secretions may be served as chemosignals in these relations: plant–
animal, plant–plant, and plant–microorganism. The final physiological response to
exometabolites is usually in the changes of growth and development, although some
earlier responses may be observed too.
A lot of compounds are released by living organisms, and among the chemicals
both universal (peculiar to every biological system) and specific ones are found.
Exometabolites of secretions may be distinguished in two groups: (1) those which
are found in all living organisms and (2) those found only in plants. To the first
group one should relate reactive oxygen species and some nitrogen-containing
compounds. The second group of molecules includes both the growth regulators
common for plants as a whole (indolyl acetic acid, gibberellins, abscisic acid, cyto-
kinins, and various phenols) and taxon-specific substances.
Among the universal signaling molecules are amine-containing compounds act-
ing in any cells primarily known as neurotransmitters in animals – acetylcholine
and biogenic amines (Roshchina 1991, 2001a, 2010) and flavonoids as redox agents
(Peer and Murphy 2006). Besides the compounds, among secondary metabolites
(alkaloids and terpenoids), blockers and inhibitors of the sensory systems sensi-
tive to the neurotransmitters are also found (Roshchina and Roshchina 1989, 1993,
2012). They may be released and also act on the model for biosensor reactions.
Relations between all of them occur via the irritation events. The mechanism of irri-
tability appears to have a common base in the form of chemical signals, chemicals
which are uniform for every cell.
3.1.1.1 Amine-Containing Compounds Common for all Organisms
Signaling compounds likely to be amine-containing compounds such as acetylcho-

line, dopamine, noradrenaline (norepinephrine), adrenaline (epinephrine), sero-
tonin, and histamine known as neurotransmitters were found not only in animals
(Wessler et al. 2001; Wessler and Kirkpatrick 2001; Boron and Boulpaep 2005)
but also in plants (Roshchina 1991, 2001a; Murch 2006) and microorganisms
Fig. 3.1 Neurotransmitters Acetycholine
(biomediators) found in plant

excretions O
N Serotonin
HO
O
Dopamine NH2
HO NH2 NH
HO
Noradrenaline
Histamine
HO NH2
N
OH
HO
Adrenaline
+ NH2
HO NH 2 CH3 NH
OH
HO
(Hsu et al. 1986; Strakhovskaya et al. 1991; Lyte and Ernst 1992; Oleskin et al.
1998a, b; Tsavkelova et al. 2006; Freestone and Lyte 2008; Freestone et al. 1999,
2000, 2007, 2008). Today, we have more and more evidence that neurotransmitters,
which participate in synaptic neurotransmission, are multifunctional substances
participating in developmental processes in microorganisms, plants, and animals.
Moreover, their universal role as signal and regulatory compounds is proclaimed and
supported (Roshchina 1991, 2001a; Baluška et al. 2005, 2006; Brenner et al. 2006).
Any organism may release neurotransmitters, and due to the excretions the “living
environment” influences every other inhibitor of biocenosis, determining relation-
ships between organisms such as microorganism–microorganism, microorganism–
plant, microorganism–animal, plant–animal, plant–plant, and animal–animal.
The universal character of the compounds’ occurrence and similarity of their
functions at the cellular level should convince scientists to have doubt in naming
acetylcholine and biogenic amines as “neurotransmitters” (Fig. 3.1). Later the com-
mon term “biomediators” has been applied for any living cell, not only organisms
with nervous systems (Roshchina 1989, 1991, 2001a).
This phenomenon permits us to imagine evolutionary picture, where the sub-
stances were participators of different cellular processes, concentrating mainly
on non-synaptic systems of microorganisms and plants (Oleskin et al. 1998a, b,
2011; Roshchina 2010). Nonnervous functions of neurotransmitters, named as
“biomediators” (Roshchina 1989, 1990) because they have arisen before objects
with nervous system, are analyzed and compared in different kingdoms (Wessler
et al. 2001; Roshchina 2010; Oleskin et al. 2010; Oleskin 2012). The compounds
are easily formed in every living organism from ordinary amino acids. The concen-
tration range of the neurotransmitter compounds is similar for all three kingdoms
of the living organisms, although some organs and specialized cells of multicellular
organisms may be enriched in the compounds (Roshchina 2010).
Acetylcholine
Acetylcholine, one of the most exemplary neurotransmitters, has been detected in

bacteria, algae, protozoa, tubullaria, and primitive plants, suggesting an extremely
early appearance in the evolutionary process and a wide expression in nonneuro-
nal cells (Roshchina 2001a, 2010; Wessler et al. 2001). The compound and/or the
synthesizing enzyme choline acetyltransferase has been demonstrated practically
in all types of animal cells, including the protozoa Paramecium primaurelia. In
the plant kingdom, acetylcholine was found in 65 species of 33 families (Fluck
and Jaffe 1974; Hartmann and Gupta 1989; Tretyn and Kendrick 1991; Roshchina
1991, 2001a, 2010; Wessler et al. 2001; Murch 2006). It was synthesized not only
in free but in conjugated form as well, in particular as conjugates of cholinic esters
with auxins (Fluck et al. 2000). The compound is particularly abundant in secretory
cells of common nettle stinging hairs, where its concentration reaches 10−1 M or
120–180 nmol g−1 of fresh mass (Roshchina 1991, 2001a). Acetylcholine was also
identified as a component of bacteria (see review of Roshchina 2010 and mono-
graphs of Oleskin et al. 2010; Oleskin 2012).
Today it is known that the amine-containing compounds can be regulators of
growth processes. In particular, acetylcholine (1 nM) promotes the elongation of
lateral roots of Raphanus sativus (Suquiama and Tezuka 2011) and causes rooting
in vitro explants of tomato Lycopersicon esculentum Miller seedlings (Bamel et al.
2007). Another example had shown the stimulation of the plant microspores’ germi-
nation (Roshchina 2004a) by biogenic amines dopamine and serotonin.
In plants acetylcholine has many functions – from regulation of membrane per-
meability to growth regulation (Fluck and Jaffe 1976; Hartmann and Gupta 1989;
Tretyn and Kendrick 1991; Roshchina 1991, 2001a, b, 2010). The ubiquitous
expression of acetylcholine and acetylcholine-synthesizing activities among life
forms without nervous systems has been demonstrated and supports the notion that
acetylcholine has been expressed and may be active as a local mediator and modu-
lator of physiological functions since the beginning of life (Wessler et al. 2001;
Roshchina 1991, 2001a, b, 2010). Acetylcholine may serve as signal molecules in
environment, and cholinergic system participates in the connections between differ-
ent organisms (Roshchina and Melnikova 1998a, b; Roshchina 2001a, b, 2010). The
enzyme cholinesterase catalyzed the hydrolysis of the compounds. Protein serves
as a marker of the acetylcholine occurrence in cells and tissues. In seedlings the
acetylcholinesterase activity is sharply increased during heat stress, and Ca2+ is a
trigger for acetylcholine release (Momonoki et al. 1997).
Nonneuronal acetylcholine can be involved in the regulation of basic cell func-
tions like gene expression, proliferation, differentiation, cytoskeletal organization,
cell–cell contact (tight and gap junctions, desmosomes), locomotion, migration,
ciliary activity, electrical activity, secretion, and absorption (Wessler et al. 2001;
Wessler and Kirkpatrick 2008). In plants, the compound is involved in the regula-
tion of membrane potential, water resorption and photosynthesis (Hartmann and
Gupta 1989; Roshchina 1989; 1990, 1991, 2001a, b), plant growth and development
(Tretyn and Kendrick 1991), etc. (more information in the monograph of Roshchina
2001a). The widespread expression of nonneuronal acetylcholine is accompanied

by the ubiquitous expression of cholinesterase and acetylcholine-sensitive receptors
(nicotinic, muscarinic). Both receptor populations interact with more or less all cel-
lular signaling pathways. Nonneuronal acetylcholine also plays a role in the control
of unspecific and specific immune functions in animals (Wessler et al. 2013).
Biogenic Amines
In every organism biogenic amines catecholamines (dopamine, noradrenaline,

adrenaline), serotonin, and histamine are synthesized (Roshchina 2010). In nonneu-
ronal systems they play role of exometabolites and extracellular chemosignals. In
plants catecholamines have been found in 28 species of 18 plant families (Roshchina
1991, 2001a; Kuklin and Conger 1995; Kulma and Szopa 2007). The amount of
dopamine found varies during plant development (Kamo and Mahlberg 1984) and
sharply increased during stress (Świędrych et al. 2004). Especially it needs to be
noted that increased amounts (1–4 mg/g fresh mass) of dopamine are found in flow-
ers and fruits, in particular in Araceae species (Ponchet et al. 1982) which shows
the important role of the neurotransmitter in fertilization as well as in fruit and
seed development. Recently catecholamines norepinephrine and dopamine have
been identified in microorganisms by high-performance liquid chromatography by
Tsavkelova with coauthors (2000). Dopamine concentrations of 0.45–2.13 μmol/l
and noradrenaline of 0.21–1.87 μmol/l were found in the biomass of many bacteria
as well as in fungi such as Saccharomyces cerevisiae (0.21 μmol/l) and Penicillium
chrysogenum (21.1 μmol/l) (Oleskin et al. 2010; Roshchina 2010; Oleskin 2012).
As a whole, the content of catecholamines in microorganisms is higher than in
animals, for example, in human blood norepinephrine is ~0.04 μmol/l (Kruk and
Pycock 1990).
Besides catecholamines, serotonin is found in 42 plant species of 20 families
(Roshchina 1991, 2001a; Rayne 2010; Ramakrishna et al. 2011). The compound
may be excreted by stinging hairs from representatives of genus Urtica (Regula
and Devide 1980) and from banana fruits at the maturing of seeds (Rayne 2010;
Ramakrishna et al. 2011). Besides free serotonin, conjugated serotonins such as
N-feruloylserotonin, N-(p-coumaroyl) serotonin, and N-(p-coumaroyl) serotonin
mono-β-D-glucopyranoside have been isolated from safflower Carthamus tinctorius
L. seeds (Roshchina 2001a). In 1998 Oleskin et al. also established the presence of
serotonin in the phototrophic bacterium Rhodospirillum rubrum (1 μg/billion of cells
~3–12,500 μg/g of fresh mass) as well as in non-phototrophic bacteria Streptococcus
faecalis and Escherichia coli (50 and 3.3 μg/billion of cells, relatively).
Bacteria, in particular Bacillus subtilis, may release norepinephrine and dopa-
mine from the cell and, perhaps, by this way participate in intercellular communi-
cation both in microorganism–microorganism and bacteria–host contacts (Oleskin
2012). In practice of the ecologists, it is necessary to know the content of these com-
pounds in insects. The salivary glands of insects contain stimulators of plant secre-
tions and may contain neurotransmitters. Exogenous neurotransmitters serotonin
and dopamine (10−7 − 6 × 10−7 M) stimulate similar secretion in model experiments

on salivary glands of cockroach Periplaneta americana (Just and Walz 1996).
Unlike catecholamines and serotonin, histamine was first found in ergot fungi
Claviceps purpurea and subsequently in many bacterial and plant cells by Werle
and coauthors (1948, 1949). Significant amounts of histamine have also been
observed in higher plants, initially by Werle and Raub in 1948, and subsequently
described for 49 plant species belonging to 28 families from basidiomycetes to
angiosperms (Roshchina 1991, 2001a). Especially high levels are observed in plant
species belonging to the family Urticaceae that could be one of the taxonomic clas-
sification signs. In plants, besides histamine itself, its derivatives N-acetylhistamine,
N,N-dimethylhistamine, and feruloylhistamine are also found in plants (Roshchina
2001a). The Brazilian stinging shrub Jatropha urens (family Euphorbiaceae) con-
tains 1,250 μg of histamine per 1,000 hairs. The presence of histamine in sting-
ing hairs serves in order to frighten off predatory animals by inducing burns, pain,
and allergic reactions. Under stress conditions a sharp increase of histamine is
observed in plants (Korobova et al. 1988), like in animals. The maximum value
(74.91 mg kg−1) was obtained from a sample of hot pepper pickles. Now it is
observed in many types of foods as the result of microbial activity, in particular bac-
teria Morganella morganii, Proteus sp, and Klebsiella sp. (Ekici and Coşkun 2002;
Ekici et al. 2006). Histamine was determined in the intact tissues by luminescence
method after the treatment of the sample with O-phthalic aldehyde (see Chap. 2).
It is one of the biogenic amines formed mainly by microbial decarboxylation of
amino acids in numerous foods such as fish, cheese, wine, and fermented products.
A number of microorganisms can produce histamine.
The amount of histamine varies according to the phase of plant development.
For example, in the marine red algae Furcellaria lumbricalis (Huds.) Lamour, the
occurrence of histamine was from 60 to 500 μg g−1 fresh mass observed in both
non-fertile fronds and sexual-expressed parts, in all regions of the thallus of male,
female, and tetrasporophyte (Barwell 1979, 1989). The amount of histamine (in μg
g−1 fresh mass) in the male plant was 90–490 (sometimes up to 1,100), in female
plant 60–120, and in asexual tetrasporophyte 100–500. Especially enriched were
the neurotransmitter cells of male plants, as the ramuli were approximately five
times higher in histamine than female and asexual plants. Histamine was also found
in the seawater surrounding these host algae. The host algae, for example, red alga
Delisea pulchra and Ecklonia radiata, inhabited on the larvae of the Australian
sea urchin Holopneustes purpurascens and induced to settle and metamorphose
(termed settlement herein) by the excretion of histamine (0.9–4.5 μM) as shown by
Swanson et al. (2004).
3.1.1.2 Specific Compounds Excreted by Plants
Compounds specific for plants (many alkaloids, phenols, and terpenes as well as
certain proteins) are accumulated in plant secretory cells and organs and serve as
chemosignals for useful or parasitic insects (Fig. 3.2). The compounds play the role
OH
CH3-OOC
E–β-farnesene D–cadinene β–caryophyllene Yohimbine

H
H H
NH
MeO N
+ Me
d–Tubocurarine N
Me
H
OH NH
CH2 Me
O O
Me
Me
O
OMe Atropine
Fig. 3.2 Biologically active terpenes and alkaloids in plant excretions
of repellents or attractants too. In the process of fertilization, attractants for insect

and bird pollinators have a decisive significance, valuable not only for biocenotic
relations (Ashman et al. 2000; Kanaoka et al. 2011; Carter and Thornburg 2000,
2003, 2004a, b), but also for agriculture and forestry (Inderjit et al. 1999; Narwal
1999a, b; Murphy 2002; Inderjit and Mukerji 2006: Jacob et al. 2011). Often, the
accumulation of phenols, terpenes, and alkaloids also appears to be markers of any
damage (Roshchina and Roshchina 1993, 2012). The accumulation of alkaloids and
terpenoids protects against herbal (e.g., weeds in allelopathy), microbial, or animal
invasion. Phenol functions are similar. Moreover, pigmented phenols also signal
about the attractiveness or dangers from other habitants of biocenosis. Lipophilic
components of excretions such as all terpenoids and some alkaloids and phenols
with unsaturated binds may interact with membranous lipids. For example, alkaloid
capsaicin contained in secretory cavities of fruits of red pepper Capsicum annuum
induces efflux of some proteins from animal membranes (Roshchina et al. 1986).
Known alkaloids, antagonists of neurotransmitters, such as d-tubocurarine, atropine,
and yohimbine found in plant excretions, are also drugs used in modern medicine.
Special interest is to terpenes in signaling in biocenosis. Monoterpenes prevailing
in any flower fragrance (Knudsen et al. 1993) may act on pollen germination and,
therefore, on successful fertilization (Dobson et al. 1987, 1996). Pollen excretions
in turn influence the larvae of flower-specialist bee (Dobson and Peng 1997).
Exudation of trichomes in genus Solanum (Solanum tuberosum, S. berthaultii)
contains sesquiterpenes to which insects are very sensitive (Gregory et al. 1986).
Among them, E-β-farnesene, β-caryophyllene, β-cubebene, and D-cadinene are
most active. Perhaps, all of them act on lipids of membranes, inducing block in a
digestive function of insects. Sesquiterpene lactones appear to be chemosignals.
Some of them with specific smell also may be considered as chemosignals. For
example, similar odor peculiar to common wormwood is prochamazulene absinthin
isolated from the herb Artemisia absinthium L. (Herout et al. 1956) and its chemical
structure has been established (Zhang et al. 2005). The compound has both ecologi-
cal (anthelmintic) and medicinal significances (Lachenmeier 2007). Azulenes may
act as protecting and signaling compounds in some plant species (Roshchina 2008;
Roshchina et al. 2011b).
Flavonoid anthocyanins were also studied as color markers for chemosignaling
with pigment accumulation as very important for insect attraction or repelling. But
red coloration of anthocyanin (Markham et al. 2000; Nielsen et al. 2003; Sato et al.
2011), betacyanin, betalain (Harris et al. 2012), and hypericin (Karioti and Bilia
2010) could be also used to study both the secretion into the vacuole and exocy-
tosis when the pigments are liberated from the cell. Anthocyanin pelargonidin in
the concentration of 10 mg/ml is red in color only in damaged pollen grains, while
the undamaged ones did not have any coloration (Roshchina et al. 2011a). At the
concentrations of 5 mg/ml only the nucleus was blue colored. Benefiting from the
unique fluorescent properties of anthocyanins is demonstrated on Arabidopsis thali-
ana (Poustka et al. 2007). An interaction of anthocyanidins released from plants
with other components of secretions is not known yet, but inhibition of monoamine
oxidase by anthocyanins, for example, decreases the dopamine decomposition that
prevents human dementia.
Proteins present in the composition of many secretions also play various roles
in communication between organisms. They may act as enzymes such as the many
esterases in the pollen and participate in the germination process (Rejơn et al.
2012). Among the compounds are carboxylesterases, acetylesterases, acetylcholin-
esterase, and lipases. Moreover, trypsin and pepsin found in animals also are present
in pollen excretions (Stanley and Linskens 1974). Recently special attention was
paid to protector proteins phylloplanins (from the English word phylloplane = leaf
surface). Such proteins are found on the surface of leaf trichomes near to or directly
on the plant genus Nicotiana trichomes (Shepherd et al. 2005). Phylloplanins are
involved in plant–insect interactions and serve to protect the leaves from being eaten
by animals.
3.1.1.3 Reactive Oxygen Species
Reactive oxygen species released by plants may serve as chemosignals. The ozone
and its derivatives of reactive oxygen (hydrogen peroxide, hydroxyl radical, super-
oxide anion radical, and other free radicals) are powerful oxidizers. They are formed
and enter into a variety of reactions with living cells. Detailed material on this sub-
ject is contained in the monograph Roshchina and Roshchina (2003).
Conventional air oxygen under the influence of UV radiation (320–200 nm) in
the process of dissociation is initially formed into atomic oxygen, which in associa-
tion with diatomic oxygen with the presence of any particle (M) transforms into
ozone (O3):
UV radiation → O 2 → O + O → [ O ] + O 2 + M → O3
O3 may occur in any processes where atomic oxygen occurs, including in biolog-
ical systems. Recently ozone formation on light has been found in photosynthesiz-
ing sea algae Polysiphonia and Phyllophora nervosa (Dzhabiev and Kurkina 2007).
In the presence of the catalyst manganese, chloroplasts were able to form ozone,
because of oxidation processes which often formed atomic oxygen (Dzhabiev et al.
2005). Apparently this process involves photosystem II. Excretions of plants (par-
ticularly concentrated in forests) may also participate in the formation of ozone,
which is formed when electricity from lightning strikes the atomic oxygen. Thus,
plants can produce and evolve ozone, though in small amounts as compared to the
concentrations in air polluted with emissions from industry and vehicles (Roshchina
and Roshchina 2003).
Besides ozonation of many compounds and water, reactive oxygen species in
plant excretions are formed in many free-radical reactions as well as in reactions
of oxidation and peroxidation, including lipid peroxidation that is considered as
a protector in a cascade of reactions (Aver’yanov et al. 1987; Aver’yanov and
Lapikova 1988). The events occur outside the cell and in different parts within the
cell (Bhattacharjee 2011). The reactions (with oxygen radicals and oxidative burst)
may be related to plant defense against parasitic invasion and as early plant response
to pathogen infection (Tzeng and DeVay 1996; Wojtaszek 1997). Reactive oxy-
gen species play many roles in plants (Kovalchuk 2011). In small concentrations,
reactive oxygen species serve as external chemosignals (Sarkar and Sharma 2011)
which possibly switches on works of intracellular system of secondary messengers
(cyclic AMP and GMP, inositols, Ca2+, etc.). They also control the activity of genes
coding antioxidant synthesis (Mylona and Polidoros 2011). Finally, the chemosig-
nals can regulate the growth and development of acceptor cells and plants. In higher
concentrations the accumulation of reactive oxygen species leads to synthesis of
antioxidants (Locato et al. 2011) and/or, at last, to programmed cell death (Gechev
et al. 2011). Oxidant and antioxidant relations, both within excretions outside of the
cells and within the acceptor cell, define the final response of contacting organisms.
Therefore, in signaling processes, it is necessary to consider the participation of
antioxidants as well.
These antioxidant compounds are proteins and substances related to reactive
oxygen species (oxidants and antioxidants) found in pollens and vegetative micro-
spores and demonstrate biological activity, in particular on pollen or vegetative
microspore germination (Roshchina 2009a, b). Both types of microspores con-
tained high-molecular-weight (<2,000 kDa and subunits with SDS near 90–94 kDa)
and low-molecular-weight (<30 kDa) proteins having cholinesterase, peroxidase,
and superoxide dismutase activity (Roshchina 2009a, b). The effects of pollen pro-
teins (monoamine oxidase, peroxidase, hexokinase, α-amylase, pepsin, trypsin,
cytochrome C, ferritin, hemoglobin, cholinesterase, and lysozyme) were tested on
microspore development (Roshchina 2009a, b). Among the proteins, cytochrome
C, ferritin, hemoglobin, subtilisin, and trypsin strongly inhibited pollen germina-
tion, whereas the inhibition in vegetative microspores was observed with pepsin
and amylase. The other analyzed peptides either weakly stimulated or had no effect
on spore’s germination. Reactive oxygen species (peroxides or superoxide) formed
anion radical from the catecholamines (dopamine and noradrenaline) and transam-
ine and iprazide (inhibitors of monoamine oxidases) and stimulated the germination
(20–100 % increase) of both spore types. On the contrary, antioxidants, such as
dithionite, mannitol, ascorbate, peroxidase, superoxide dismutase, ecdysone, caf-
feic and chlorogenic acids, austricine, gaillardine, and grosshemine, inhibited the
germination. However, high concentration of ozone (0.2 μl/l) inhibited the reaction
and damaged the cells. Therefore the role of reactive oxygen species for growth
enhancement and antioxidant retardation may be important in pollen–pollen and
pollen–pistil interactions (Roshchina 2001b). Possible mechanisms of cell–cell
interactions with identified compounds will be discussed in Chap. 4.
Besides the abovementioned features of reactive oxygen species, one should
keep in mind the changes in the characteristics of responses on exometabolites at
the ozonation in nature or indoors (Roshchina 2001b, 2004a, b, c). Pre-fumigation
of ozone led to the shifts in the effects of serotonin on the germination of pollen
from Hippeastrum hybridum (Roshchina 2004a). In small doses the stimulation of
the process is enhanced, while in higher doses even the depression in a comparison
with a control was observed.
3.1.2 Sensory Systems in Acceptor Models
Today, the mechanism of action of plant excreta on subcellular and molecular levels
is studied intensively. The main targets of the action of normal and stress compo-
nents of plant excreta in cells of animals, plants, and microorganisms are the cellular
membranes, composed basically of lipids and protein complexes, which form ion
channels and (or) receptors, the transport of K/Na ATPases, ATPases of coupling
membranes, and other enzymatic systems. Many reactions occur on the plant sur-
face at the contact with exometabolite and shortly one could imagine it as follows:
1. Exometabolite → enzymatic and redox reactions on the cellular surface where
sensors are enzymes and cell wall components
2. Exometabolite → sensor systems of plasmalemma (receptors, ion channels,
enzyme sensors)
The concept of membrane receptors is well known for animals and can be
applied to the theory of the action of neurotransmitters and hormones and today
has been applied to explain the action of plant hormones (Venis 1985) and
neurotransmitters such as acetylcholine, dopamine, noradrenaline, adrenaline,
serotonin, and histamine which are found in plants as well (Roshchina 1991,
2001a, b, 2010). The concept could be useful for the understanding of mecha-
nisms of action of plant excreta (Roshchina and Roshchina 1989; Roshchina
and Roshchina 1993, 2012). Prevailing hypothesis for acceptor cell reaction to
exometabolites is the existence of special receptors in membranes. Discreteness
of membranous structure permits to indicate sites where one or another chemical
compound is specifically binding.
3.2 Sensitive Reactions of Models to Exometabolites 77
Protein components of membranes are most sensitive to components of

excretions. Some of them function mainly as catalysts, i.e., as enzymes par-
ticipating in metabolic and energetic reactions. Other proteins have function of
“recognition”, i.e., are receptors of low-molecular-weight hormones and media-
tors. Often enzymatic and receptor systems are connected structurally and func-
tionally. Among enzymes of plasmatic membranes are ATPases, in particular
calcium ATPases, components of receptor-mediated signaling in plant immune
responses and development (Frey et al. 2012). The binding of the released sub-
stances with similar receptors serves as chemical signal to change either the
cascade of enzymatic processes or membrane permeability for certain ions –
Na+, K+, Ca2+, etc. Ion channels are mediated by receptors or can form receptor
itself, in particular Na+ channel for cholinoreceptor sensitive to acetylcholine
(Changeux et al. 1984). Protein subunits of ion channels can have enzymatic
function as well (Eisenberg 1990).
All receptors are proteins which can have a high affinity in specific binding (non-
covalently) with some low-molecular-weight agents. The best model to understand
the reception work in all systems is the cholinergic system of animals which, on the
cellular level, is analogous to the system in plants (Roshchina 1991, 2001a, 2010).
The cholinergic system includes the mediator acetylcholine and cholinoreceptor
sensitive to acetylcholine, as well as choline acetyltransferase, the enzyme for syn-
thesis, and cholinesterase, the enzyme for hydrolysis. The mechanism of the action
of such a system is as follows. Acetylcholine synthesized with the participation of
choline transferase binds with a cholinoreceptor that changes the conformation of
the receptor. As a result, ion channel opens and ion permeability is increased, which
induces the arising of electric potential known as action potential which spreads
along the membrane as a result of irritation. There are receptors not only for com-
pounds, called neurotransmitters, but also for biomediators of organisms without a
nervous system. Many volatile plant compounds sensed by animals are perceived
through special olfactory receptors which can be very sensitive to smells and odors,
in particular for camphor in concentrations of 10−10 M. Besides neurotransmitters
and hormones, plant receptors may be linked with redox systems of the cells, for
example, abscisic acid receptors through cascade reactions regulate K+ and Cl−
channels through reactive oxygen species (Wang et al. 2013).
Intercellular signaling occurs via a cascade of reactions linked with external
receptors via the formation of action potential or systems of secondary messengers
(calcium ions, cyclic AMP or GMP, inositol triphospate, etc.) and participation of
enzymes such as kinases, phosphoesterases, phospholipases, etc.
3.2 Sensitive Reactions of Models to Exometabolites
In this section our experiments with various model systems are represented includ-
ing microspores and multicellular objects. Their reactions were determined using
microscopic and spectral methods.
3.2.1 Microspores as Biosensors
The fluorescence and germination of plant microspores, both in generative (pol-

len or male gametophyte of gymnosperms and angiosperms) and in vegetative (in
cryptogams), may be used as test for reactions on secretory products (see Chap.
1). In some cases secretions from microspores may be sensitive to exometabolites.
Enzymatic reactions (e.g., the cholinesterase activity) are also tested in the micro-
spores. In artificial medium during the first 1–5 s, pollen releases proteins with
enzymatic activity, while grass pollen on the pistil shows lipid drop in excretion
also for few seconds (Stanley and Linskens 1974). The drop is rich in various low-
molecular-weight biologically active compounds such as phenols and terpenoids
(Anaya et al. 1992; Murphy 2007). Low-molecular-weight and high-molecular-
weight components of mixture could participate in redox reactions occurring on
the contacted surfaces (Roshchina and Roshchina 2003). The similar phenomenon
of protein–lipid drop releasing was observed in horsetail vegetative microspores
(Roshchina 2008). Slime-like pollen secretion contains polysaccharides, proteins
(lectins, enzymes such as peroxidases and esterases), and lipids with complexes of
lipophilic compound (Stanley and Linskens 1974). Growth regulators (flavonoids
and biomediators acetylcholine and histamine) were also found in pollens of many
species (Marquardt and Vogg 1952; Stanley and Linskens 1974; Roshchina 2001a,
b). The compounds excreted from plants may act on receptors or other sensors
located at the plasmalemma and be recognized by the proteins.
3.2.1.1 Effects of Complex Excretions
The experiments with natural complex excretions (both with volatiles and liquids)
were carried out on plant microspores (Roshchina 2004a, b, c, 2005b, 2007a).
Table 3.2 demonstrates the results of the microspores’ treatment with volatile
excretions from lavender (Lavandula sp.) oil, disrupted fruits of red pepper
(Capsicum annuum), and cut bulb of garlic (Allium sativum) as well as the water
extracts from the same samples (Roshchina 2007a). Lavender oil or cut bulb of
garlic was put in a Petri dish, in small vessels near the object glass with micro-
spores being studied. The volatile products from the materials are influenced
by air. Besides, water (nutrient medium for microspores) extracts from fruits of
red pepper and garlic bulbs (1:1,000 w/v) were prepared and added directly to
the microspores on the object glasses. Lavender oil tested from fresh blossom-
ing lavender contained linalyl acetate (up to 40 %) among 100 components such
as linalool, lavandulol, lavandulyl acetate, terpineol, cineol, limonene, ocimene,
caryophyllene, etc. (Roshchina and Roshchina 1993; Golovkin et al. 2001). Fruits
of red pepper release biologically active alkaloid capsaicin which may be in
volatile and soluble form and acts on animal special receptors (Roshchina and
Roshchina 1993). Garlic volatile excretions contain thiocyanates alliin and alli-
cin, whereas their water extracts from bulbs contain diallyl disulfide (Roshchina
and Roshchina 1993). The effects on germination of microspores depend on the
Table 3.2 The effects of volatile plant excretions and water extracts (1: 1,000) w/v) from
allelopathic species with pesticidal features on the germination of horsetail vegetative microspores
Germination of microspores (% of control)
Water extract
from the same
Excretion Near slide 2.5 cm distance probe (1:1,000)
Vegetative microspores
of Equisetum arvense
Lavender (Lavandula vera) oil 64.5 ± 6 98.5 ± 6 –
Disrupted fruits (79 mg) 139 ± 9 125 ± 10 88 ± 14
of Capsicum annuum
Cut bulb of garlic Allium 154 ± 10 132 ± 10 104 ± 6
sativum L. (350 mg)
Pollen Hippeastrum hybridum
Lavender (Lavandula vera) oil 0±2 38 ± 6 –
Disrupted fruits (79 mg) 55 ± 10 108 ± 11 26.5 ± 8
of Capsicum annuum
Cut bulb of garlic Allium 98 ± 8 132 ± 10 73 ± 5
sativum L. (350 mg)
Adapted from Roshchina (2007a)
concentration and the distance from the object glass with microspores moistened
with nutrient medium lavender oil (active matter) which may or may not inhibit
the process, while the volatile excretions from other plant species have no inhibi-
tory, but stimulatory, effects. Only the water extract from red pepper demonstrates
weak activity.
In experiments with liquid excretions on germination of vegetative micro-
spores of Equisetum arvense shown as effects with water and ethanol extracts from
sesquiterpene-enriched plants Achillea millefolium and Gaillardia pulchella were
given in paper of Roshchina (2007a). The inhibitory effects were dependent on the
solvents and the concentration of plant material. The strongest depressing effects
were observed, mainly, for extracts from leaves and flowers, while root extractions
often stimulated the germination of the microspores.
3.2.1.2 Effects of Amine-Containing Compounds
Models of microspores demonstrated a significant response (in their fluorescence

and germination) undergoing additions of some amine-containing compounds
which served as neurotransmitters in animals (Roshchina 2004a, b, c). The infor-
mation on similar lines was earlier summed in a special monograph (Roshchina
2001a). But the possibility that plant excretions contained acetylcholine and bio-
genic amines, for example, in common nettle Urtica dioica, wood nettle Laportea
moroides, and other species from Urticaceae (Roshchina 2001a), to regulate func-
tions of animals and microorganisms was not analyzed yet.
Acetylcholine affected pollen germination depending on the plant species
(Roshchina 2001a). It has weak effect on microspore germination in the models chosen.
Pollen Vegetative microspores

260 Hippeastrum hybridum Equisetum arvense
2
240
220
200
Germination (% of control)
180 3
160
1
140
120 1
100 2
3
80
60
–8 –7 –6 –5 –4 –8 –7 –6 –5 –4
Concentration (Lg M) Concentration (Lg M)
Fig. 3.3 The effects of biogenic amines as regulators on the germination of plant microspores –
models 1 dopamine, 2 noradrenaline, 3 serotonin (Adapted from Roshchina (2004a))
The stimulation of plant microspore germination (both of vegetative microspores of

Equisetum arvense and pollen of Hippeastrum hybridum) by the biogenic amines
dopamine and serotonin was shown in Fig. 3.3. The most effective were dopamine and
serotonin, while noradrenaline has lesser activity. Earlier the phenomenon of the stim-
ulation at the concentrations 10−7–10−5 M was demonstrated for histamine on pollen
of Hippeastrum hybridum too (Roshchina and Melnikova 1998a, b; Roshchina et al.
1998a). Dopamine, noradrenaline, and serotonin are known to increase the rate of the
seed germination in such species as Raphanus sativus (Roshchina 1992, 1994).
Autofluorescence as first reaction on the exometabolites was also sensitive to
some neurotransmitters as shown on vegetative microspores studied (Roshchina
et al. 2011c).
Certain mechanisms of the response dealt with the reception were also demon-
strated. In vegetative microspores of Equisetum arvense, the stimulation of their
fluorescence and germination undergoing additions of acetylcholine and biogenic
amines was observed (Fig. 3.4). Effects of the secreting neurotransmitters and their
antagonists (alkaloids, which also are found in plant excretions) on the autofluores-
cence have been studied (Roshchina et al. 2011a, b, c). The examined compounds
added into the medium affect the amount of division and accordingly stronger or
weaker fluorescent cells compared to control. During the microspore development,
the red fluorescence intensity increases in comparison with dry rest (non-developed)
cells. If the division of the spore cell occurs (and duplication of chloroplasts takes
place), the twofold increase in red fluorescence is the indicator of cellular division
because the amounts of chloroplasts and fluorescing chlorophyll are doubled.
Control Acetylcholine Dopamine Histamine

20
Cell number
10
0
d-Tubocurarine Yohimbine Azulene
0 2 4 20
Fluorescence,
relative units 10
0
0 2 4 0 2 4 0 2 4
Fluorescence, relative units
Fig. 3.4 Examples of histograms for the fluorescence intensity (at maximum 680 nm) distribution
among 100 cells of vegetative microspores of horsetail Equisetum arvense (measured at 640–
680 nm) after the treatment with neurotransmitters (10−5 M). Cells with the emission intensity >2
germinate (Source: Roshchina et al. (2011b))
Table 3.3 The summed intensity of red emission from microspores in the presence of
neurotransmitters and their antagonists (10−5 M) in relative units
Variant I680 Variant I680
Control 3.28. ± 0.32
+ acetylcholine 3.25 ± 0.30 + d-tubocurarine 1.20 ± 0.30
+ dopamine 4.05 ± 0.21 + yohimbine 2.54. ± 0.22
+ histamine 4.15 ± 0.24 + azulene 0.95 ± 0.19
In the presence of the neurotransmitters acetylcholine, dopamine, histamine

(10−6−10−5 M), and their natural antagonists (alkaloids d-tubocurarine, yohim-
bine, and sesquiterpene lactone azulene) often found in excretions, variations in
red fluorescence intensity, rather than in green, were observed (Fig. 3.2). Summed
intensity of red emission and influence of the compounds abovementioned is shown
in Table 3.3. In a population of 100 cells, red emission intensity increases more
than twofold, which indicates the presence of germinated (dividing) microspores.
Reliable stimulation of fluorescence (20–30 % of the control) and the cell division
according to the microspore germination has been mainly undergone the action of
dopamine and histamine (Fig. 3.2).
Alkaloid d-tubocurarine, acetylcholine antagonist, and alkaloid yohimbine, cate-
cholamines antagonist, as well as an antihistaminic agent sesquiterpene lactone azu-
lene significantly (up to 60–70 %) reduce the intensity of fluorescence (Table 3.3)
and the growth of a culture of microspores indicating the presence of cholino- and
adrenoreceptors as well as, perhaps, histaminic receptors. According to the infor-
mation received at different depths from optical slices by laser-scanning confocal
microscopy, as shown for yohimbine in Fig. 3.5 (similar phenomenon was also
observed for d-tubocurarine), the main antagonist binding occurred on the surface
of the microspore. As can be seen from the fluorescence spectra of single sections of
exine (1) and intine (2a) with adjacent plasma membrane (2b), originally emitting in
Fluorescence,
relative units 10 Solution of
yohimbine
5
1
0
400 500 600 700 200
Fluorescence, relative units

Wavelength, nm 2a
Scheme of optical slice 2b
1 2a 100
2b 3 3
3
0
400 500 600 700
Wavelength, nm
Fig. 3.5 Laser-scanning confocal microscopy of vegetative microspores from Equisetum arvense
after addition of catecholamines’ antagonist yohimbine (10−5 M). 405 nm laser light excitation. Top
left fluorescence spectrum of the yohimbine solution in a 1 cm cuvette, bottom left diagram of the
optical slice of the microspore under the microscope, right fluorescence spectra from single parts
of the microspore. The parts of microspore on optical diagram slice and the fluorescence spectra
under confocal microscope are marked by numerals: exine (1), its inner layer intine (2a), the
plasma membrane (2b), and chloroplast (3) (Source: Roshchina et al. (2011b))
blue area, spectrum solutions of antagonists are located just in protoplasmic mem-
brane where there are relevant receptors (the membrane has no emission in the vari-
ant without the treatment on Fig. 3.5, and the fluorescence is observed only after the
addition of the antagonist). The maxima of the fluorescence from part 2b shift to a
shorter region of the emission spectrum – 460–465 and 500–520 nm.
Unlike the other antagonists studied, azulene quickly bleached under laser beam
of confocal microscope; so it is easier to see its interaction with the microspores
under normal luminescence microscope from Leica DM6000 lens ×40. This micro-
scope allows us to make optical slices, using sources of actinic light, causing not so
much damage to the cells. In contrast with d-tubocurarine and yohimbine, azulene
passed the inside of the cell and bounded DNA-containing organelles; the green
emission of nuclei and chloroplasts (Roshchina et al. 2011a) observed explained the
general decline in red fluorescence of cells. Azulene is known as a drug, a medici-
nal antihistaminic agent (Roshchina and Karnaukhov 2010), because it diminished
the stimulation of microspore germination by histamine. Its mechanism of action
is probably due to its extreme lipophilicity, resulting in easier permeability through
the plasma membrane.
The analysis of the effects of histamine and antihistaminic compounds acting as
antagonists of animal histaminic receptors H1 type (Table 3.4) has shown the exis-
tence of similar histaminic receptors or their analogs in the plasmalemma of both
Table 3.4 Effects of antihistaminic compounds on the germination of plant microspores as

biosensors. Index of germination = amount germinated cells/total amount of cells studied
Index of germination
Variant of Equisetum arvense Pollen of Hippeastrum hybridum
24 h 2h 24 h
Control 0.36 ± 0.04 0.49 ± 0.02 0.49 ± 0.02
Suprastin 0.09 ± 0.01 0.0 ± 0.0 0.11 ± 0.01
Diazoline 0.08 ± 0.01 0.10 ± 0.01 0.10 ± 0.01
Dimedrol 0.04 ± 0.01 0.0 ± 0.0 0.03 ± 0.01
Blockers of H1 –histaminic receptors: suprastin = chloropyramine = N-p-chlorobenzyl-N’. N’-
dimethyl-N-{2-pyridyl]ethylenediamine hydrochloride; diazoline = mebhydrolin napadisyl-
ate = 3-methyl-9-benzyl-1,2,3,4,4-tetrahydrocarbolinenaphthaline-1,5-disulfonol;
dimedrol = Benadryl = dimedrol = diphenhydramine = β-dimethylaminoethyl ester benzhydrol
hydrochloride
models – vegetative and generative (pollen) microspores. All the chosen blockers of
histaminic receptors inhibited their germination.
The mechanisms of the abovementioned effects with acetylcholine are related
to the influence on ion channels that may or may not be included in appropriate
receptors. The chemosignaling may also occur through the systems of second-
ary messengers (cyclic AMP and GMP, inositol phosphates, Ca2+, etc.). In cases
of biogenic amines, the main effects appear to link with the switch on the cas-
cades of alternative ways of intracellular signaling in secondary messengers. The
participation of ion channels and contractile proteins in chemosignaling in plant
microspores as models using specific blockers or anticontractile agents to treat
microspores has been established (Roshchina and Vikhlyantsev 2009). The germi-
nation of microspores was determined, and experiments lasted up to 24 h (short-
time experiments) or up to 1–1.5 months (longtime experiments). To treat ion
channels, α-bungarotoxin (binding Na+ channels and nicotinic cholinoreceptors),
tetraethylammonium (blocking K+ channel and also binding nicotine cholinorecep-
tors), and verapamil (blocking Ca2+ channels) were chosen. The blockers decreased
the growth rate of microspores to different degrees depending on concentration and
type of microspores (Fig. 3.6). The depressive effects on pollen formation (reduc-
tion in fruits and lack of seeds) were observed not only in vitro (Fig. 3.6), but also
in vivo, after the treatment of pistil stigma and following pollination (Table 3.5).
In vivo, the abnormal fruits were produced in variants with 2 min moistening of
pistil stigma by solutions of the ion channel blockers. Similar picture was observed
earlier for d-tubocurarine, antagonist of nicotinic-type cholinoreceptor, which binds
with subunits of Na+ channels (Roshchina 2004a, b, c). The normal pollen develop-
ment is likely depressed in this case at earliest time of recognition and reception of
chemosignal, although pleiotropic effects related to female gametophyte (pistil) are
also possible.
The mechanism of acetylcholine action on ion channels may be also linked
with the contractile protein actin having contacts or included into Na+ channels in
Pollen of Hippeastrum hybridum

Ion channel blockage Anticontractile agents
100 100
80 Clofelin
% of control
80
Papaverine
TEA 60
60
40
40 Bung Colchicine
20
20 Ver Cytochalasin B
0 0
–9 –8 –7 –6 –5 –4 –8 –7 –6 –5 –4
Vegetative microspores of Equisetum arvense
100 100 Papaverine

80 Bung 80 Clofelin
% of control
Colchicine
60 TEA 60
Ver 40
40 Cytochalasin B
20 20
0 0
–9 –8 –7 –6 –5 –4 –9 –8 –7 –6 –5 –4
Concentration (Lg M) Concentration (Lg M)
Fig. 3.6 The effects of blockers of ion channels and anticontractile agents on the germination of
pollen of knight’s star (Hippeastrum hybridum) and vegetative microspores of horsetail (Equisetum
arvense) during 24 h. Bung α-bungarotoxin, TEA tetraethylammonium, Ver verapamil (Source:
Roshchina and Vikhlyantsev (2009))
animal cell (Cantiello 1997) or into K+ channels of plant cells (Hwang et al. 1997).
In experiments on plant microspores, similar possibility (Table 3.5) was demon-
strated with cytochalasin B, a cell-permeable fungal toxin from Helminthosporium
dematiodeum, which inhibits cell division by blocking the actin polymerization and
formation of contractile actomyosin microfilaments (Roshchina and Vikhlyantsev
2009). This drug inhibited the germination of microspores (Roshchina 2005a). In
some cases the fluorescence of actin filaments in cells after the staining with cyto-
chalasin B occurs and isolated actin fluoresces, forming a complex with this com-
pound (Roshchina and Vikhlyantsev 2008).
3.2.1.3 Effects of Alkaloids
Alkaloids from allelopathically active species were also tested on the germination
of vegetative and generative microspores (Roshchina 2004a, b, c, 2007a). They are
known as medicinal drugs. Figure 3.7 shows that inhibition in most cases (30–80 %)
was observed. Isochinolinic alkaloids (berberine, glaucine, and sanguinarine) inhib-
ited the germination of vegetative microspores, but glaucine did not affect pollen
germination. Berberine inhibited the reactions only at low concentrations. The
differences may be due to the different chemical structures – the number of five
carbon heterocycles and the position of benzene rings. Colchicine and casuarine
Table 3.5 The development of microspores in long-duration experiments (28 days after moistening
with nutrient medium) under blockers of ion channels and the anticontractile agents of 10−5 M
Gametophyte developed from
Variant Pollen germination in vivo vegetative microspores
Fluorescence at
640–680 nm
Fruits Seeds (relative units) Antheridia
Control Yes Yes 3.14 ± 0.42 Yes
α-Bungarotoxin No No 0 No
Tetraethylammonium No No 0.25 ± 0.2 No
Verapamil Reduced No or reduced 0.27 ± 0.2 No
Cytochalasin B No No 0.90 ± 0.12 No
Colchicine Yes Yes 0.38 ± 0.10 No
Papaverine No No 0.87 ± 0.08 Yes
Clofelin Yes Yes 0 No
The formation of fruits and seeds of Hippeastrum hybridum after pollination as well as fluores-
cence of the gametophyte (chlorophyll accumulation) and formation of the antheridia from vegeta-
tive microspores from Equisetum arvense were observed
have different chemical structures, but both compounds depressed the germination
of the microspores. Colchicine is secreted from the species of Colchicium genus,
which binds tubulin and prevents mitosis (Wolff et al. 1991). Moreover, the com-
plex of colchicine–tubulin fluoresces (Croteau and Leblanc 1978; Bhattacharyya
et al. 1986). One mechanism of action of some allelochemicals in excretions from
plants and fungi that depress the plant growth occurs through the inhibition of cel-
lular motility.
3.2.1.4 Effects of Monoterpenes and Sesquiterpene Lactones
The effects of the terpenoids found in secretions of many allelopathically active

plants were studied on plant microspores (Roshchina 2001b; 2004a, b, c, 2007a).
The results of experiments were dependent on the concentration used. Volatile
monoterpenes of the flower aroma from various plants such as linalool, cymol, and
citral (mixture of geranial and neral) in very small concentrations stimulated pollen
germination (Fig. 3.7). The effect of sesquiterpene lactone absinthin found in excre-
tions from flowers and herb of wormwood Artemisia absinthium (Lachenmeier
2007) was similar at higher amounts (Fig. 3.8). The compound is known as a pun-
gent and exceptionally bitter essential oil (0.2–1.5 %) that is dark green to brown or
blue in color. Unlike the abovementioned sesquiterpene lactones related to proazu-
lenes, absinthin showed strong inhibitory effect on arising pollen tube. The possible
common mechanism of action in human is in binding with taste receptors, but it is
unknown whether similar receptors occur in plants.
Other sesquiterpene lactones, known as proazulenes gaillardine, grosshemine,
and austricine excreted by many flowers, stimulated the germination of pollen
Hippeastrum hybridum in concentrations up to 10−7–10−6 M and depressed in higher
amounts (Roshchina et al. 1998c). Artemisinin, inulicine, deacetylinulicine, and
Germination (% of control) Equisetum arvense Hippeastrum hybridum

Glaucine
100 * 100 * *
*
*
Berberine
* *
* *
*
* Glaucine
50 * Casuarine 50 Casuarine
Berberine Colchicine
Colchicine Sanguinarine
Sanguinarine
0 0
0 –8 –7 –6 –5 –4 0 –8 –7 –6 –5 –4
Concentration (lg M) Concentration (lg M)
Fig. 3.7 The effects of alkaloids–allelochemicals on the germination of vegetative microspores

from Equisetum arvense and generative microspores (pollen) from Hippeastrum hybridum
(Adapted from Roshchina (2007a))
Cymol Linalool Citral Absinthin
Geranial Neral
HO
HO
CH3 O O HH
200 CH3
H H H
HO
H H H
O
O
O
100
O
Fig. 3.8 Effects of volatile monoterpenes and sesquiterpene lactone absinthin on the germination
of pollen from Hippeastrum hybridum in concentrations of 10−10, 10−8, and 10−5 M. Concentrations
of cymol, linalool, and citral were 10−10, 10−8, and 10−5 M, while those for absinthin were 10−5, 10−4,
and 10−3 M (Adapted from Roshchina (2001b) and unpublished data)
ledol increased the rate of the pollen germination only in concentrations of 10−6–
10−4 M (Roshchina et al. 1998c; Roshchina and Roshchina 2012).
Monoterpenes act on animal odor receptors. Proazulenes can penetrate into plant
cells via lipid parts of plasmalemma and bind with organelles, for example, gail-
lardine is bounded with nucleus (Roshchina 2004a, b, c, 2005c).
3.2.1.5 Effects of Proteins
In model systems of generative microspores (pollen) from Hippeastrum hybridum

and vegetative microspores of horsetail Equisetum arvense, the effects of pollen
proteins (monoamine oxidase, peroxidase, hexokinase, α-amylase, pepsin, trypsin,
cytochrome C, ferritin, hemoglobin, cholinesterase, and lysozyme) were tested

on microspore development (Roshchina 2009a, b). Some of the compounds were
found in pollens and vegetative microspores themselves, and other compounds in
natural biocenosis (from cells of animals, microorganisms and plants belonging to
another species) may be either excreted or liberated as a result of destruction of
the cells. Both types of microspores themselves contained high-molecular-weight
(<2,000 kDa and subunits with SDS near 90–94 kDa) and low-molecular-weight
(<30 kDa) proteins (Roshchina et al. 1998c; Roshchina 2009a, b) having cholines-
terase, peroxidase, and superoxide dismutase activity. Among the proteins studied
(Roshchina 2009a, b), cytochrome C, ferritin, hemoglobin, subtilisin, and trypsin
strongly inhibited pollen germination, whereas inhibition in vegetative microspores
was observed with pepsin and amylase (Fig. 3.9). The other analyzed peptides either
weakly stimulated or had no effect on spore germination. Strong inhibition in both
types of microspores was observed for hydrolytic enzymes (pepsin and trypsin) as
well as bacterial protease subtilisin, and inhibition was greater in pollen germina-
tion. The effect of hydrolytic enzymes α-amylase (hydrolysis of glucan bonds) on
pollen germination was not significant, while 50 % of inhibition occurred in horse-
tail vegetative microspores, perhaps due to the accumulation of excessive sugars
after germination that stopped photosynthesis. Lysozyme, which breaks down the
bacterial cell wall and hexokinase (transferase of hexoses), stimulated microspore
germination. Hexokinase participating in sugar metabolism either has no influence
or caused slight stimulation. Hemoglobin (blood iron-containing protein and analog
of plant lethaemoglobin), cytochrome c (iron-containing protein), and ferritin (the
reserve protein participating in iron metabolism and oxidation) reduced the ger-
mination in both types of microspores. Cholinesterase from pea leaves had weak
effects on pollen germination in vitro; nevertheless, the self-incompatible clones of
Petunia hybrida pollen have smaller amounts of the enzyme in a comparison with
self-compatible clones (Kovaleva and Roshchina 1997). Therefore, cholinesterase
genetically linked with the incompatibility phenomenon. Ferritin, iron storage pro-
tein, and structural mitochondrial protein cytochrome C may serve as antioxidants,
because they can reduce oxidized compounds (see following section). Their inhibi-
tory effects on microspore germination reduced the stimulation by oxidants. Like
some of the abovementioned proteins, glycoproteins also stimulate the pollen tube
growth (Wu et al. 1995).
The cytoskeleton proteins released from plants and animals via exocytosis or
as a result of damages in plasmatic membranes (in plants after fungal invasion)
have biological activity, besides their function in cellular motility. Bioactivity of
the cytoskeleton proteins such as actin, myosin, and titin extracted from the rabbit
muscle was analyzed on plant microspores (vegetative microspores from horsetail
Equisetum arvense and pollen of knight’s star Hippeastrum hybridum) as unicellular
biosensors (Roshchina and Vikhlyantsev 2012). The fibrillar forms of the proteins
stimulated the germination of both types of microspores. In special experiments
on the study of mechanisms of the effects, contractile proteins tested were able to
reverse the blockade of ion channels by drugs d-tubocurarine and α-bungarotoxin,
acting on cholinoreceptors linked with Na+/K+ ion channels, or verapamil, regulat-
ing Ca2+ channels. The addition of the exogenous proteins prevents negative effects
Iysozyme
150
Pepsin
Albumin
Hexokinase
Cholinesterase
Amylase
Cytochrome C
100
Hemoglobin
Ferritin
Trypsin
Subtilisin
50
Vegetative microsporse of Equisetum arvense

Iysozyme
Hexokinase
150
Subtilisin
Cholinesterase
Cytochrome C
Albumin
Trypsin
100
Amylase
Pepsin
Hemoglobin
Ferritin
50
Fig. 3.9 The effects of various proteins (1 mg/ml) from the microspore excretions on the germina-
tion of pollen of Hippeastrum hybridum and vegetative microspores of horsetail Equisetum arvense
(Source: Roshchina (Roshchina et al. 2009a, b))
of blockers of ionic channels. Exogenous actin and myosin from the rabbit muscles
being perfused into the algae Chara corallina cells were shown to cause the genera-
tion of the cytoplasm movement (Nothnagel et al. 1982). Being added into the exter-
nal medium, actin filaments from animal tissues modulated the opening of stomata
and K+ ionic channels in the guard cells of Vicia faba L. (Hwang et al. 1997) as well
as the opening of the sodium channels in animal cellular culture (Cantiello 1997).
In the experiments potential participation of the actin domains in the work of ionic
channels was examined. The cytoskeleton proteins being excreted or liberated from
the cells after destruction of tissues may play certain role in chemosignaling and
regulating of growth processes.
3.2.1.6 Effects of Natural Oxidants and Antioxidants
Effects of redox systems (oxidants and antioxidants) on microspores’ germination

were also observed on plant microspores (Roshchina 2009a, b). Ozone, hydrogen
peroxide, dopamine, and other compounds play the role of oxidants, while some
phenols, terpenoids, and enzymes play the role of antioxidants. Figure 3.10 showed
some experiments on redox reactions on the pollen surface. Similar effects were also
seen in vegetative microspores (Roshchina and Roshchina 2003; Roshchina et al.
2003). The oxidants – peroxides (generators of superoxide anion radical dopamine
and noradrenaline) and transamine or iprazide (inhibitors of monoamine oxidases
that resulted in the accumulation of the catecholamines) – stimulated the pollen
germination of Hippeastrum. The mechanisms of stimulation by superoxide anion
radical and inhibitors of monoamine oxidases can also be related to the formation
of hydrogen peroxide in the following reactions:
Superoxide dismutase
1. ( O’ −2 ) superoxide anion radical + H2O ⇒ ⇒ ⇒ H2O2
Monoamine oxidase
2. Catecholamine + H2O + O2 ⇒ ⇒ ⇒ R–COOH + H2O2 + NH3
Inhibition of monoamine oxidase leads to the accumulation of catecholamines,
which formed superoxide anion radical being used in reaction 1, with formation
of hydrogen peroxide. Photochemical smog produces ozone in high concentration
(>0.01 μl/l) that may induce the metabolic processes, like it is seen in ageing poly-
mers (Roshchina and Roshchina 2003). Depending on the concentration, ozone and
its derivatives may stimulate or depress microspore germination and even shift the
fluorescence maxima of the cell (Roshchina and Melnikova 2001; Roshchina and
Roshchina 2003; Roshchina et al. 2003). At high concentration, the ozone gas inhib-
ited spore germination. Ozonated (oxidized) compounds differed in their effects
from the non-ozonated ones, like those seen in some phenols (Roshchina 2001b).
But the microspore surfaces as well as pistil stigma contain many compounds
having double bonds, and therefore they may act as antioxidants, in particular fla-
vonoids such as kaempferol (Vogt and Taylor 1995). The levels of phytoecdyster-
oids vary significantly between the different plant species (Heftmann 1975) and
ecdysone found in pollen (Šaden-Krehula et al. 1971, 1983). The opposite effects
were observed for antioxidants: 10−4 M reductant dithionite Na2S2O4 and 10−4 M
mannitol. Reactive oxygen species (peroxides or superoxide) formed anion radical
Pollen of Hippeastrum hybridum Inhibitors of

monoaminooxidase
Transamine
Reactive Generators Antioxidants
oxygen of superoxide
species radical
200
Chlorogenic acid 10-6M
Iprazide
Dopamine
peroxide
Noradrenaline
Hydrogen
peroxide
Butvl
150
Grosshemine
Mannitol
Dithionite
Ozone
Ascorbate
100
Caffeic acid
Austricine
Ecdysone
Peroxidase
Gaillardine
50
Transamine
peroxide
Vegetative microspores of Equisetum arvense

Butvl
200 Iprazide
Dopamine
Noradrenaline
Hydrogen
peroxide
150
Austricine
Mannitol
Ascorbate
Grosshemine
100
Gaillardine
Ecdysone
Ozone
Dithionite
Chlorogenic acid
Caffeic acid
Peroxidase
50
Fig. 3.10 The effects of reactive oxygen species, ozone, and antioxidants on germination of
Hippeastrum hybridum pollen and vegetative microspores of Equisetum arvense (Source:
Roshchina (2009a, b)). The concentration of proteins was 0.6 mg/ml; ozone 0.2 μl/l; dopamine and
noradrenaline; hydrogen peroxide and butyl peroxide 10−5 M; ecdysone 10−5 M; chlorogenic and
caffeic acids 10−6 M; ascorbate, austricine, gaillardine, and grosshemine 10−5 M; dithionite
Na2S2O4; and mannitol10−4 M
from catecholamines such as dopamine and noradrenaline and transamine and ipra-
zide (inhibitors of monoamine oxidases) and stimulated the germination (20–100 %
increase) of both spore types, while antioxidants (dithionite, mannitol, ascorbate,
peroxidase, superoxide dismutase, ecdysone, caffeic and chlorogenic acids, aus-
tricine, gaillardine, and grosshemine) inhibited the germination. However, high
concentration of ozone (0.2 μl/l) inhibited the reaction and damaged the cells.
Therefore, the role of reactive oxygen species for growth enhancement and antioxi-
dant retardation may be important in pollen–pollen and pollen–pistil interactions.
Possible mechanisms of cell–cell interactions with identified compounds have been
discussed.
3.2.1.7 Effects of Exometabolites on the Secretions from Microspores
Microspores, mainly spores of some species, contain colored products such as phe-
nols anthocyanins, carotenoids, and azulenes in the cell wall (Stanley and Linskens
1974; Roshchina 2009a, b). Pollens from varieties of Petunia hybrida, Hemerocallis,
Philadelphus, Zephyranthes, and others release the pigments into water or ethanol –
natural media of plant tissues. The spectra of the compounds in the extracts may
show certain compounds. It can be expected to consider changes in absorption and
fluorescence of intact samples of their own and their secretions in solution under
the influence of various factors. The pollens may be unicellular pigmented mod-
els in some cases to study excretions (Roshchina 2006a). Many pollen grains also
released proteins.
The vegetative microspores of horsetail Equisetum arvense and pollen of
Hippeastrum hybridum excreted proteins (see Sect. 3.2.1.5). The technique of
cultivating the spores on slides in Petri dishes, as well as statistical processing, is
described in the literature (Roshchina 2004a, b, c, 2007a, b, 2009a, b).
Among the proteins, cholinesterase is a significant enzyme in catabolism of ace-
tylcholine as a chemosignal. Cholinesterase, an enzyme that catalyzes the acetyl-
choline hydrolysis, was found in many plant species (Fluck and Jaffe 1974; Gupta
and Maheshwari 1980; Gupta et al. 1998, 2001; Roshchina 1991, 2001a). The cho-
linesterase activity may be considered as an indicator of sensitivity to excretory
products which stimulate or depress the enzyme activity. The enzyme also serves
as the indicator of plant stress (Momonoki and Momonoki 1992, 1993a, b). The
protein enzymatic activity occurs in secretions of all living organisms. First, it was
found in animal secretions and later in those of plants (pollen and pistil excretions)
and microorganisms. The review looks on the universality in some publications
(Roshchina 2001a, 2010). The enzyme may be maximally active to acetylcho-
line, in this case called acetylcholinesterase, while if it hydrolyzes other cholinic
esters at a similar rate, it is simply called cholinesterase or pseudocholinesterase
(Augustinsson 1963).
Cholinesterase plays many roles in organisms. Today the attention is paid to its
function in cell–cell contacts between cells of one and the same organism (in rela-
tion to pollen–pistils) or between different organisms, in particular plant–animal or
Table 3.6 The rate (V) of the acetylthiocholine hydrolysis by water extracts (1:10 weight/volume)
Equisetum arvense in the presence of allelochemicals – alkaloids and ozone
Compound V × 10−3 M kg−1 fr.ws−1 % of control
Controls without alkaloids 1.2 ± 0.03 100 ± 4.2
Neostigmine 10−6 M 0.99 ± 0.03 83 ± 4.2
10−5 M 0.97 ± 0.04 81 ± 4.0
Physostigmine10−6 M 0.95 ± 0.05 79 ± 0.4
10−5 M 0.92 ± 0.05 78 ± 0.4
Berberine 10−6 M 0.99 ± 0.04 82.6 ± 5
10−5 M 0.97 ± 0.04 81 ± 3.9
10−4 M 1.03 ± 0.08 86 ± 7.2
Glaucine 10−7 M 0.30 ± 0.03 25 ± 0.3
10−6 M 0.40 ± 0.009 33 ± 0.1
10−5 M 0.40 ± 0.0009 33 ± 0.1
10−4 M 0.38 ± 0.05 30 ± 5.0
Sanguinarine 10−7 M 0.85 ± 0.03 70 ± 3.0
10−6 M 0.77 ± 0.04 64 ± 4.0
10−5 M 0.53 ± 0.04 44 ± 4.0
10−4 M 0.83 ± 0.06 69.6 ± 6.5
Ozone 0.8 х10−9 M 1.2 ± 0.06 100 ± 6.5
2 х 10−9 M 0.67 ± 0.06 57.5 ± 6.5
4 х 10−9 M 0.97 ± 0.06 78 ± 6.5
8 х 10−9 M 0.99 ± 0.06 80 ± 1
Source: Budantsev and Roshchina (2004, 2007)
plant–microorganism. Secretions contained cholinesterase released by Arthrobacter

ilicis, which is associated with the marine sponge (Mohapatra and Bapuji 1998).
Cholinesterase is formed and released by the microorganism cultures in the medium
(Imshenetskii et al. 1974, 1976a, b).
In any cases the enzyme is sensitive to components of plant excretions (Budantsev
and Roshchina 2004, 2007). The cholinesterase activity of unicellular models was
determined in the presence of alkaloids or reactive oxygen species ozone and per-
oxides (Table 3.6). The experiments with water extract from horsetail microspores
used alkaloids present in plant excretions (Glaucium, Berberis, etc.) and inhibited
acetylthiocholine hydrolysis. Although animal cholinesterase is more sensitive to
alkaloids (Budantsev and Roshchina 2007), but unlike acetylcholinesterase of eel,
maximal inhibition was observed at concentrations of 10−7–10−5 M for variants with
glaucine (70–75 %), lesser with sanguinarine (30–50 %), whereas that with ber-
berine, physostigmine, and neostigmine was not higher (20–30 %). Since about
20–30 % of the rate of acetylthiocholine hydrolysis was inhibited by the known
inhibitors of the animal cholinesterase such as physostigmine and neostigmine
(Augustinsson 1963), therefore, the pure cholinesterase activity of microspores
was also approximately 20–30 %. In the case of more deep inhibition, in particular
by glaucine and sanguinarine, the activity of nonspecific esterase also (Roshchina
2003) decreased the rate of the acetylthiocholine hydrolysis by 20–30 %. Ozone can
be formed and released by plants as shown for some algae (Dzhabiev et al. 2005)
and the gas depressed cholinesterase activity in the plant microspores studied.
Table 3.7 I50 for inhibition Inhibitor I50, M

of cholinesterase from
Vegetative microspores Equisetum arvense
microspores by biologically
active compounds used +neostigmine 0.9 × 10−5
+flacoside 6 × 10−4
+tert-butyl hydroperoxide 10−4 M 1.1 × 10−4
+neostigmine 5 × 10−6
+physostigmine 10−4
Source: Budantsev and Roshchina (2004, 2007)
Flacoside = 7-β-D-glucopyranoside-8 (3-methylbut-2-enyl)
-4′,5,7 –trioxyflavanone from Phellodendron amurense Rupr.
(family Rutaceae)
Table 3.8 The action of acetylcholine and nicotine on protein secretion after 5 min wetting of
vegetative microspores of Equisetum arvense (10 mg microspores + 2 ml), in % of controls, and
cholinesterase activity of secretion, in μmol/min. mg protein
Cholinesterase activity
Concentration Acetylcholine Nicotine at the presence acetylcholine
10−8 М 150 ± 3 102 ± 4 4.5 ± 0.1
10−7 М 291 ± 10 152 ± 11 1.0 ± 0.2
Control (0.1 mg protein/ml)
The sensitivity of the cholinesterase of the microspores to alkaloids analyzed

may be also estimated from the comparison of I50 (a concentration of the inhibi-
tor, which decreases the rate of the enzyme synthesis by 50 % of the control). As
seen from Table 3.7, among the studied compounds the highest sensitivity to neo-
stigmine was observed. Rutin-like phenolic compound flacoside, from Amur cork
tree Phellodendron amurense, is also able to inhibit the cholinesterase activity, but
in higher concentrations. Organic peroxide tert-butyl hydroperoxide and alkaloid
physostigmine were equally effective in similar concentrations. On comparing the
cholinesterase sensitivity of the models studied, pollen grains from Hippeastrum
hybridum were most sensitive than vegetative microspores of Equisetum arvense,
because the effect of inhibitor is observed at lower concentrations.
Cholinesterase activity may be the test not only for plant analysis in the experi-
ments with excretions but also for the research of intercellular contacts of differ-
ent organisms. For example, slime which is a secretion of planarians demonstrated
cholinesterase activity sensitive to alkaloid eserine (Li 2008). Insecticidal activity
of many exometabolites has potential to be tested on various insects, in particular
on aphid cholinesterase (Blackman and Eastop 1984). Changes in esterases released
are also observed in response to blast infection (Muarlidharan et al. 1996).
The mechanisms of the action of some alkaloids on the secretion of proteins,
including cholinesterase, from plant microspores were also studied (Roshchina
et al. 2013b). Table 3.8 shows the effects of small concentrations of acetylcholine
and alkaloid nicotine (agonist of acetylcholine binding with the cholinoreceptor in
the same site) on the excretions of the vegetative microspores which induced the
protein output, including cholinesterase. The effect of nicotine on protein excretion
Table 3.9 The effect of Plant biosensor (acceptor cells) Serotonin Histamine
biogenic amines (10−5 M) on
Chara corallina 192 ± 20 95 ± 4
the fluorescence intensity (%
of control) at 640–680 nm on Lemna minor 90 ± 10 236 ± 25
the thallus meristem of Chara Source: Roshchina et al. (2009b)
corallina and leaf of Lemna
minor measured with
microspectrofluorometer
MSF–2
as a whole was not as strong as that of acetylcholine. Cholinesterase activity deter-

mined in the proteinous extracts dropped with the presence of 10−7 M acetylcholine,
perhaps due to the increased, exogenous neuromediator that inhibits the enzyme
activity (Augustinsson 1963). Acetylcholine and nicotine acted on cholinorecep-
tor which regulates the membrane permeability and the ion channel state, while
d-tubocurarine may the proteinous extracts dropped with the presence of 10−7 M
acetylcholine.
3.2.2 Multicellular Biosensors
Multicellular biosensors react with secretions changing autofluorescence, mem-

brane potential, or secretion in external medium. Slower reactions are related to
growth and development.
3.2.2.1 Changes in Fluorescence
Multicellular biosensors such as leaves of Lemna minor or leaves of Chara coral-

lina as well as root tips of seedlings of Raphanus sativus were analyzed with the
addition of dopamine, noradrenaline, serotonin, and histamine – physiologically
active aminous compounds that also are found in plant excretions (Roshchina et al.
2009b). The changes in the fluorescence treated with the individual components
of plant secretions could be used as a response to the chemical tested, in particular
biogenic amines serotonin and histamine (Table 3.9) or dopamine (Table 3.10).
Microspectrofluorometry permits measurement of the changes in fluorescence in
the cells. Similar changes in the fluorescence intensity of the chlorophyll in leaves
of L. minor and of C. corallina in red spectral region were analyzed with the addi-
tion of the serotonin and histamine that also are allelochemicals (Roshchina et al.
2009b). The changes in the fluorescence treated with biogenic amines serotonin and
histamine or dopamine (found in plant secretions – see Sect. 3.1.1) are responses
to the compounds tested (Table 3.9). Serotonin and histamine stimulated the chlo-
rophyll fluorescence in the leaves of L. minor and C. corallina, whereas dopamine
enhanced the green emission in seedlings of R. sativus that is related to the dopa-
mine oxidation (Table 3.10). Dopamine may be released from L. minor (Roshchina
2001a) and roots of various plants and undergo the oxidation on the air oxygen
Table 3.10 The effect of Part of seedling Primary root meristem Root hairs
biogenic amine dopamine
Control 0.11 ± 0.01 0.02 ± 0.03
(10−5 M) on the fluorescence
intensity (relative units) at +dopamine 0.29 ± 0.02 0.23 ± 0.03
530 nm on the Raphanus Source: Roshchina et al. (2009b, 2011c)
sativus seedlings measured
with microspectrofluorometer
MSF–2
(Roshchina et al. 2011a). The water-living species could be suitable for modeling of
allelopathic relations like marine algae and other representatives of phytoplankton
(Solé et al. 2005; Roy et al. 2007) because it demonstrates possible interactions
between exometabolites – allelochemicals excreted by donor and acceptor cells.
Dopamine is known to be excreted from green algae Ulvaria obscura (van Alstyne
et al. 2006) and contained in Lemna sp. (Roshchina 2001a). Histamine occurs in the
thallus of algae Furcellaria lumbricalis, predominantly in the male and tetrasporo-
phyte cells (Barwell 1989), and may be released into the surrounding water with
other living inhabitants.
3.2.2.2 Secretion from Cells
Sensitivity of plant secretory process to exometabolites that originated from differ-

ent organisms may be studied on appropriate model sensors. The stimulatory effects
of exogenous biogenic amines serotonin and dopamine on salivary secretion of
cockroach Periplaneta americana are known (Just and Walz 1996). Serotonin stim-
ulates secretion of proteins, whereas dopamine only stimulates the nonproteinous
components of the saliva. Alkaloid atropine found in Atropa belladonna, on the
contrary, blocks the secretion in the digestion system of mammalian (Mashkovskii
2005).
Objects chosen as acceptor models often contain pigment phenols (anthocya-
nins, anthraquinones, betacyanins, betalains) in the vacuole of cells from flowering
plant species. Under the influence of external chemosignal (passing from plasma-
lemma to organelles through the cytoplasm), secretory products can go out of the
cell and from the vacuoles. On such plant objects one can see highlighted pigments
from cells and quantitatively analyze the output of the products and spectral charac-
teristics of these compounds.
Pigmented cells are proposed to be models that excrete pigments and by this
mode one permits to observe and estimate the process. The quantity of the secretion
pigment released appears to be analyzed by spectral methods. Among those models
are cells capable to excrete colored products from (1) cell wall and/or free cellu-
lar space (between cell wall and plasmalemma) or (2) from vacuole and secretory
vesicles located in cytoplasm and originated from vacuole.
Recently, studies on multicellular pigmented secretory systems of plants and ani-
mals have started (Roshchina et al. 2013b). The objects included secreting flower
petal cells of cacti (Epiphyllum hybridum (L.)), Chinese hibiscus (Hibiscus rosa-
sinensis L.), common African violet (Saintpaulia ionantha Wendl), and bracts of
1 2
(units opt.density) 0.020
Allium d–Tubocurarine
Absorbance
0.015 Saintpaulia 10–6 M

Hypericum
0.010 Euphorbia
Hibiscus
0.005 Epiphyllum
0
–8 –7 –6 –5
Concentration of
acetylcholine (Lg M)
Fig. 3.11 Effects of acetylcholine (1) and d-tubocurarine (2) on the output of red pigmented secre-
tions from flower petals and leaves of various species (Source: Roshchina et al. (2013)). Epiph
Epiphyllum hybridum (30 ml solution on 360 mg of petal), Hib Hibiscus rosa-sinensis (40 ml
solution on 500 mg of petal), Saint Saintpaulia ionantha (3 ml solution on 20 mg of petal), Euph
Euphorbia milii (3 ml solution on 20 mg of bracts), Al Allium cepa (onion) (3 ml solution on
100 mg of bulb skin), Hyper Hypericum perforatum (3 ml solution on 20 mg of leaves). The absor-
bance was estimated at a maximum of 530 нм (Hib, Epiph, Al, Euph), 594 нм (Hyper), 635 нм
(Saint)
milkweed (Euphorbia milii Des Moul), as well as bulbs of onion (Allium cepa L. var.
“Excellent”) grown in the greenhouse, leaves of common St. John’s wort (Hypericum
perforatum L.), and bracts of common hop (Humulus lupulus L.). The cell responses
based on their absorption, fluorescence, and the secretion release through 0.5–1 h
after the addition of reagents. Secretions of cells obtained by extraction with an aque-
ous solution without nitrate salts contain potassium phosphate, calcium chloride,
sodium chloride, and magnesium chloride (Roshchina and Yashin 2013). Protein
secretions and their cholinesterase activity were observed in all samples determined.
Figure 3.11 demonstrates the pigment excretions from the objects. In untreated (con-
trol) samples there was no pigment release up to 20 h of exposure. The output of
anthocyanins (as well as anthraquinones for Hypericum perforatum) was seen only
under the addition of acetylcholine into the medium and was determined accord-
ing to the absorbance and fluorescence spectra of the 30–60 min. extracts by water.
Increase in optical density, typical for anthocyanin, was at 520–530 or 590–630 nm
depending on the color of petals – red (characteristic for acid pH of cellular sap) or
blue (peculiar to base pH). Changes in fluorescence of the extracts at 465 nm secre-
tion were also measured. Antagonist of acetylcholine d-tubocurarine in different
degrees inhibited the pigment output (except H. perforatum) that indicated possible
participation of cholinoreceptors in the secretory process.
More details in the signaling process with possible participation of cholino-
receptors have been studied on intact petal cells of Saintpaulia ionantha Wendl
(Roshchina and Yashin 2013). The petal cells of blossoming plants released antho-
cyanins and proteins (including the enzyme cholinesterase) to outer environment
after the treatment with acetylcholine and its agonists nicotine (binds with nicotinic
Arecoline
10–7
0.02 Acetylcholine 0.02
10–6
10–6 10–5–10–4
0.01
10–8 – 10–7
Absorbance (opt. density)
Control Control
0 0
400 500 600 700 400 500 600 700
Wavelength, nm Wavelength, nm
0.020
Nicotine
Arecoline
0.015 Acetylcholine
d-Tubocurarine
0.010
Atropine Muscarine
0.005
0
–8 –7 –6 –5 –4 –8 –7 –6 –5 –4
Concentration, Lg M Concentration, Lg M
Fig. 3.12 Output of anthocyanins from petal secretory cells of Saintpaulia ionantha after treat-
ment with the acetylcholine agonists and antagonists (Source: Roshchina and Yashin (2013))
type of animal cholinoreceptors), muscarine, arecoline, and quinuclidinyl ben-

zilate (all three compounds bind with muscarinic type of animal cholinoreceptor).
Maximal stimulation of the output of anthocyanins from petals was observed at a
concentration of 10−7 M. The anthocyanin release by the agents is shown in Fig. 3.12
Among agonists the highest effect is shown for nicotine. The secretion was largely
decreased by the antagonists of acetylcholine d-tubocurarine (binds with nicotinic
type of animal cholinoreceptors), tetraethylammonium (blocker of K+ channels
linked with nicotinic type of animal cholinoreceptors), atropine, and platyphylline
(acting on muscarinic cholinoreceptors) that indicates the participation of mixed-
type cholinoreceptors (both nicotinic and muscarinic) in the secretory process.
The mechanisms of intercellular signaling on the model were also studied using
forskolin (stimulator adenylate cyclase) and theophylline (inhibitor of phosphodi-
esterase), and agents affected the synthesis and catabolism of secondary messen-
ger cyclic AMP, relatively (Roshchina and Yashin 2013). Both compounds which
induced the cAMP accumulation within a cell stimulated the output of secretory
products. This appears to be a way of intracellular signaling with secondary mes-
senger cyclic AMP from plasmalemma to the vacuoles containing anthocyanins. An
alternative way of intracellular signaling may be related to the formation of action
potential spreading within cells from plasmalemma by the increase of the perme-
ability for Na+ and K+ as observed for Chara cells (Kisnieriene et al. 2012).
The sensitivity of the petal cells to acetylcholine as well as to its agonists and
antagonists permitted the use of Saintpaulia ionantha as a model for studies of
participation of plant cholinergic system in the secretory process.
The release of another red pigment –betacyanin – from root cells of Beta vul-
garis var rubra under acetylcholine treatment was also demonstrated parallel to the
changes in Na+/K+ output/input (Roshchina 2001a). This model system earlier was
used for analysis of effects of plant excretions (Roshchina and Roshchina 1970) and
individual compounds such as organic acid and phenols on the cell permeability
(Roshchina 1972, 1975). Today it is also a prospect for investigations.
Chapter 4
Modeling of Cell–Cell Contacts
Cell–cell communication is based on chemical interactions between organisms,

including relations between cells of the same species or cells of different species
(allelopathic interactions). Under normal conditions, molecular mechanism of the
interactions consists of excretion of active substance on the surface of donor cell
and the recognition of this chemical signal by the surface of receiver cell named
acceptor cell. Many secretory components may serve as allelochemicals, pharma-
ceuticals (drugs), and natural pesticides. To test their biological activity and espe-
cially their mechanisms of action on the cellular level, special model systems are
used as shown in previous chapters. Germination and the changes in fluorescence
are main physiological responses of the objects (plants, animals, or microorgan-
isms) to chemical signaling with components of plant secretions. Among known
model process testing, one could use such reactions as the cellular autofluorescence,
composition of the secretions and their spectral characteristics, germination, and
growth and development. Models of cell–cell contacts may represent pollen–pistil,
pollen–pollen, host cell–parasitic cell, plant cell–animal cell, and other interactions.
Below, we shall consider the various types of the models useful to study cell–cell
contacts where components of secretions play an essential role in a physiological
reply.
4.1 Modeling of Cell–Cell Contacts Based on Microscopic

Observation
Modeling of cell–cell contacts is possible by several modes. Direct contacts

observed based on the fluorescence alterations were studied in pollen mixtures
(Roshchina and Melnikova 1996, 1999; Roshchina et al. 2008, 2009a), in the
interactions between pollen and pistil (Roshchina et al. 1996; Roshchina 2008), or
in the relations between plant and parasitic invader (Roshchina 2012). Another
mode of modeling includes the interaction of acceptor cell biosensor with
100 4 Modeling of Cell–Cell Contacts
compounds (allelochemical) from excretion of donor cell. For this purpose,

multicellular and unicellular biosensors are suitable (see Table 3.1, Sect. 3.1). Cell
donor of chemosignal (signaling substance in its excretion) and cell acceptor of
the same chemosignal (model has recognizing systems in cell surface) interact via
their excretions. Changes in color or fluorescence in the model system could be
registered (Roshchina 2004a, b, c, 2005a, b, c; Roshchina et al. 2011a, b, c). The
absorbance and fluorescence of both contacting cells may be altered. If the secre-
tory product of cell donor has color or emission under light of luminescence
microscope, we can see how the secretion penetrates into the acceptor cell or not
(Roshchina et al. 2011a, b, c; Roshchina 2012). Development and growth of the
cell acceptors simulated or depressed after the contact with secretory products of
donor cells are also used as test reactions in the analysis of cell–cell contacts
(Roshchina et al. 2009a).
Approaches and examples of the studies will be based on the visualization of direct
cell–cell contacts in plants when acceptor cell with the help of its own excretion and
sensor systems receives a chemosignal from a secretion excreted by another cell, a
donor cell. The interactions are better observed on large well-seen cells. The secretory
products are located in surface (0.01–0.02 μm in diameter) channels of the cell wall,
for example, in the cuticle of nectary cells (Koteeva 2005) or in pollen exine (Rowley
et al. 1959) as well as in the secretory ducts of pistil stigma (Fahn 1979). The recogni-
tion of the chemosignal derived from secretions and the response to the signaling was
shown for such contacts as pollen–pistil or pollen–pollen (Knox et al. 1976; Knox
1984; Murphy 1992). Secretions play an essential role as chemical signals and/or as
medium for the reception of the signals (Roshchina 1999a, b, 2001a, b).
4.1.1 Pollen–Pistil Interactions
At plant fertilization, generative male microspore named pollen is added on the

surface of pistil by the wind, insects, or human hand in the selection practice.
Foreign pollen, which can be carried by wind or insect on the surface of pistil
stigma, interacted with pollen of own sp. that leads to the antagonistic or favorable
effects on the pollen germination.
The first attempt to analyze direct pollen–pistil interaction was when the spread-
ing of the excitation wave (electric impulse) along the pistil stigma was measured
with electrodes (Sinyukhin 1973). Thus, the action potential was first registered in
the reproductive organs of Ipomoea flower, when pollen was added on pistil stigma
(Sinyukhin and Britikov 1967). Primary reactions of contacting cells, both pistil
and pollen, at fertilization may be changes not only in electric potential but also
in their autofluorescence that reflects the sexual recognition of “own-self species.”
Chemical relations between different species in a competition between pollen of
own and foreign species usually take place, when they are added on the one and
same pistil. In the latter cases, “pollen–pistil” and “pollen–pollen” interactions
4.1 Modeling of Cell–Cell Contacts Based on Microscopic Observation 101
will be analyzed in connection with a fertility and pollen allelopathy. Pollen–pollen

contacts shall be described in Sect. 4.1.2. Below, we shall consider modeling in
system pollen–pistil.
At plant fertilization, generative male microspore named pollen is added on the
surface of pistil by the wind, insects, or human hand in the selection practice.
Foreign pollen, which can be carried by wind or insect on the surface of pistil
stigma, interacted with pollen of own sp. that may lead to the antagonistic or favor-
able effects on the pollen germination. Pollen allelopathy, known as pollen–pollen
competition on the pistil surface, plays a major role in normal plant fertilization in
biocenosis (reviews of Murphy 1992, 1999a, b; Murphy et al. 2009a, b; Chauhan
et al. 2005; Gaur et al. 2007; El-Ayeb et al. 2009). Studies reported the stimulatory
or inhibitory effect of pollens from different species modeling their germination in
mixtures in vitro (Branscheidt 1930; Grümmer 1955; Char 1977).
Especially modeling of cell–cell contacts that occurred on the pistil stigma
between own and foreign pollen grains was demonstrated by the observation of the
fluorescence changes at fertilization of Hippeastrum hybridum as suitable model
(Roshchina et al. 1996, 1997b; Roshchina 2008). Secretions released either by pistil
surface or by pollen affect their contacts which could be analyzed directly (by
microscopy, microspectroscopy, or microspectrofluorimetry) or indirectly (by the
observation of the formation of fruits and seeds). Today, there are only few exam-
ples of similar studies described for the model of Hippeastrum hybridum as a direct
visualization of pollen–pistil reactions at their surface contact (Roshchina et al.
2008, 2009a).
4.1.1.1 Visualization of Pistil Stigma Reaction on Pollination
Cell–cell interactions of pollen with a cell of the pistil stigma of knight’s star
Hippeastrum hybridum as model system at their contact result in the changes in the
fluorescence spectra of cells contacted (Fig. 4.1). The fluorescence of both contact-
ing cells is changed if the cells belong to one and the same plant species (Roshchina
et al. 1996, 1997b; Roshchina and Melnikova 1999). In opposite case, there were
no changes. It should mark that before the pollen addition only green-yellow emis-
sion of the pistil stigma papillae was seen. Visual increase in blue fluorescence of
the stigma papillae, when pollen was put down on the pistil surface, was demon-
strated. The events occurring upon cross- and self-pollination seem to be more
complicated. The pollen grains of diverse plant species are transferred to stigma
surface, but only the pollen of own species is capable of germination. Probably, the
liquid excreted by stigma contains inhibitors and stimulators of pollen germination,
including plant-specific alkaloids, phenols, terpenoids, and other fluorescent sub-
stances. These compounds may also be involved in free-radical reactions occurring
on both contacting surfaces; thus, they may rapidly trigger pollen germination to
participate in germination as a component of the signaling system (Roshchina
2001b).
Stigma Pollen grain

1 1
2 2
0 0
2
4 2
4
2 2
0 0
400 500 600 700 400 500 600 700
Wavelength (nm) Wavelength (nm)
Fig. 4.1 Common view under stereomicroscope (left) and the fluorescence spectra of pollen and
pistil stigmas (stigmata) of Hippeastrum hybridum upon pollination measured by microspectro-
fluorimetry (right) (Adapted from Roshchina et al. (1996, 1997b, 2013a)). The fluorescence spec-
tra of the pistil stigma of Hippeastrum hybridum without (−−−) and with (___) pollen grain added.
1 Foreign pollen of Dactylis glomerata; 2 self-pollen of Hippeastrum hybridum. Right: the fluores-
cence spectra of pollen with (__) and without (−−) interaction with the pistil stigma of Hippeastrum
hybridum. 1 Foreign pollen of Dactylis glomerata; 2 self-pollen of Hippeastrum hybridum. All
changes are observed only at the contact of pistil with self-pollen – pollen of the same plant
species
By the help of fluorescent cytodiagnostics, a researcher can know whether cells

recognize which is “own” or “foreign.” Figure 4.1 shows the changes in fluores-
cence of pistil with pollen from different species, which are added on the stigma. So
the pistil refuses to receive “foreign” pollen and does not permit pollen to germi-
nate. Moreover, pollen can germinate only on the pistil of the same plant species.
Possible contacts between “own” and “foreign” pollen grains also demonstrated
out pistils (in pollen mixtures), modelling their interactions on the pistil stigma
(Roshchina and Melnikova 1996, 1999). This is interpreted (Roshchina 2008) as
models of allelopathic relations between pollens of different plant species that lead
to the inhibition or the stimulation of their ability to grow on the pistil stigma
(Roshchina 2001b). This is necessary for the specialists in the plant selection and
the genetic practice to know. As shown in Fig. 4.1, the fluorescence of foreign pol-
len from Dactylis glomerata leads to the small changes of the pistil stigma fluores-
cence if any, while the addition of own pollen induces the appearance of the
response.
Modeling of the pistil secretion effects on pollen development is also possible.
The germination of pollen grains was determined in the experiments that lasted up
to 24 h (short-time experiments) or up to 1–1.5 months (long-time experiments
in vivo to study the fertilization and the formation of fruits and viable seeds). Pollen
develops in vitro on the nutrition medium (10 % sucrose solution and tested
compounds dissolved in 10 % sucrose; boron is added) and forms pollen tube
(Roshchina 2007a, b). Using light microscope, the author determined the micro-
spore germination (number of developed pollen tubes was counted) 2–24 h after
moistening. In nature, pollen germinates on the pistil stigma, i.e., in vivo, when
microspores are aided by the wind, insects, or human hand. If mechanisms of the
pollen secretion on pistil stigma are studied, test solutions of reagents were added
on the pistil stigma before the hand pollination. After 1 month, the formation of
fruits and seeds indicated successful or unsuccessful fertilization from the pollen
germination in vivo.
4.1.1.2 Individual Components in the Pollen–Pistil Contact
At the contacts, “pollen–pistil” recognition of specific signal stimuli in plant excre-

tions by acceptor cell takes place. Pistil or pollen secretion is a primary medium for
a chemosignal spreading from pollen surface to plasmalemma (Roshchina et al.
1998b). Secretion of the recipient pollen cover may serve as a recognizing and
transporting liquid like olfactory slime in animals for chemosignal.
The mechanisms of the changes in fluorescence or growth and development reac-
tions at pollination could be studied by the analysis of the effects observed after the
addition of the individual compounds occurring in the secretions of either pollen or
pistil stigma. Similar chemosignals may be acetylcholine and biogenic amines
(dopamine, noradrenaline, serotonin, and histamine) known in animal cells as neu-
rotransmitters and also found in plant cells, in particular in pollen (Roshchina
2001a, b). Another group of chemosignals is proposed as reactive oxygen species –
ozone, free radicals, and peroxides or antioxidants (Roshchina and Roshchina 2003;
Roshchina et al. 2003). Phenols, in particular flavonoids (Peer and Murphy 2006),
also may serve as chemosignals and growth regulators.
Acetylcholine and Biogenic Amines
Acetylcholine and cholinesterase, enzyme that hydrolyzes this compound, are found
in plant secretions, in particular from those as pollen and pistil of some species
(Bednarska and Tretyn 1989; Roshchina et al. 1994; Roshchina and Semenova
1995; Roshchina 1999a, b; Roshchina 2001a, b, 2007a). In male cells of animals
(spermatozoa), acetylcholine (Nelson 1978), catecholamines, and serotonin (Young
and Laing 1990) are also found and participate in fertilization as hormone. The
secretions of pollen contain neurotransmitters – acetylcholine and histamine
(Marquardt and Vogg 1952), catecholamines (Roshchina and Melnikova 1999), as
well as enzyme cholinesterase, hydrolyzing acetylcholine (Bednarska 1992;
Roshchina et al. 1994). Cholinesterase also serves as marker on the presence of
acetylcholine.
The treatment of the pistil stigma with acetylcholine and histamine as well as
their agonists (imitators) and antagonists (binding with membrane receptors in one

5 + Pollen 5 5
Histamine
+ Acetylcholine
C
C
0 0 0

400 500 600 700 3 d-tubocurarine 2
Wavelength (nm) Tavegil
Acetylcholine O 0
N+
Tavegil + Histamine
0 3
d-tubocurarine + Acetylcholine
O 2
Histamine
N
0 0
400 500 600 700 400 500 600 700
H2 N Wavelength (nm) Wavelength (nm)
NH
Fig. 4.2 The effects of 10−5 M neurotransmitters acetylcholine, histamine, and their antagonists on
the fluorescence spectra of the pistil stigma in Hippeastrum hybridum flower (Sources: Roshchina
(2001a, 2008))
and the same part of the membrane and regulating their activity) also induced the
shifts in the fluorescence spectra and intensity (Roshchina et al. 1998a, b; Roshchina
2001a, b). The addition of acetylcholine on the pistil stigma (Fig. 4.2) almost dou-
bled the fluorescence intensity in a maximum at 475 nm and shoulder at 530 nm.
However, after the preliminary treatment with its antagonist d-tubocurarine, which
blocks animal nicotinic cholinoreceptor, the effects were not observed. Similar pic-
ture was for antagonist atropine (Roshchina 2001a, b). It showed the presence of the
receptors of acetylcholine on the stigma surface. Histamine also stimulated the
autofluorescence of pistil stigma (Fig. 3.12) and, unlike acetylcholine, induced a
maximum at 510 nm and two shoulders at 450 and 560 nm. The fluorescence inten-
sity was also stimulated but after preliminary treatment with Tavegyl (clemastine),
antagonist of histamine, to a small extent compared with histamine alone. This
showed the presence of structures like histamine receptors on the stigma surface.
Another surface sensor on the pistil is enzyme cholinesterase, present in the stigma
excretions (Roshchina and Semenova 1995). The treatment of the pistil stigma with
neostigmine, inhibitor of cholinesterase, also makes smooth the fluorescence spec-
tra of the structure (Roshchina 2008). Therefore, it was shown that acetylcholine
and histamine induced the changes in the pistil stigma fluorescence, as observed at
pollen addition (Roshchina 2001a). Moreover, this response was absent if the pistil
stigma was preliminarily treated with antagonists of acetylcholine or histamine such
as d-tubocurarine or clemastine (Tavegyl) that links receptors on the cellular sur-
face. Thus, autofluorescence of the cells at contacts of pollen and pistil appears to
be a biosensor reaction for neurotransmitters and the antitransmitter substances.
Mechanisms of the effects of the amine-containing secretory products were also
studied (Roshchina and Vikhlyantsev 2009). Modeling appears to be applied for the
+ BUNG
+ TEA
Control + VER
Cytochalasin B Colchicine Papavernie
Fig. 4.3 The effects of blockers of ion channels and anticontractile agents on knight’s star
(Hippeastrum hybridum) the fruit formation after 2 min preliminary treatment of pistil stigma in
flower before pollination. Bung α-bungarotoxin, TEA tetraethylammonium, and Ver verapamil
search of receptors and contractile proteins which participated in the mechanisms of

recognition of either own pollen or own pistil. For the purpose of special inhibitors
of natural and artificial origins which affect receptors, ion channels and contractile
systems in both animal and plant cells could be used (Roshchina 2007a, b; Roshchina
and Vikhlyantsev 2009). The following substances were used as reagents: (1) antag-
onist of acetylcholine acting as the blocker of Na+ channels and nicotinic type of
cholinoreceptor α-bungarotoxin; (2) tetraethylammonium that blocks the K+ chan-
nel binding with nicotinic type of cholinoreceptor; (3) verapamil, calcium channel
blocker; (4) cytochalasin B, inhibitor of actin polymerization; (5) colchicine, inhibi-
tor of tubulin polymerization; and (6) alkaloid papaverine, antismazmodic drug
relaxing contractile systems. The depressive effects on pollen formation (reduction
in fruits and lack of seeds) were also observed in the studies of mechanisms of
reception and sensitivity to some components of secretions (Roshchina 2007a, b;
Roshchina and Vikhlyantsev 2009) not only in vitro but also in vivo (Fig. 4.3), after
the treatment of pistil stigma and following pollination. At 10−6–10−4 M
concentrations, the blockers of ion channels and cholinoreceptors retarded or inhib-

ited fruit and seed formation. Figure 4.3 compared the effects of ion channel block-
ers with those induced by anticontractile agents such as actin-binding cytochalasin
B (fungal metabolite), tubulin-binding colchicines (alkaloids), muscle relaxant, and
known allelochemical (species of family Papaveraceae) papaverine. Papaverine was
selected for analysis based on its effects as blocker of ion channels because it affects
the development of amphibian embryos, perhaps inhibiting the calcium fluxes.
Figure 4.3 illustrates the images of abnormal fruits produced in variants with 2 min
moistening of pistil stigma by solutions of the ion channel blockers. Similar picture
was observed for d-tubocurarine, antagonist of nicotinic type of cholinoreceptor,
which binds with subunits of Na+ channels (see also Table 3.5, Chap. 3). Blockage
of the cholinoreceptor and/or ion channels leads to ugly fruits or lack of the fruits
with seeds at all. The activity of ion channels may be linked with contractile pro-
teins (Cantiello 1997; Hwang et al. 1997). Moreover, some domains of ion channel
subunits are also common for proteins of cytoskeleton. Cytochalasin B, fungal
metabolite, acting on the actin depolarization, as well as alkaloid papaverine, relax-
ing muscle contractile elements, depressed the fruit formation unlike alkaloid col-
chicine from Colchicum spp. which blocks mitosis due to binding with contractile
protein tubulin. The normal pollen development is likely depressed in this case at
earliest time of recognition and reception of chemosignal, although pleiotropic
effects related to female gametophyte (pistil) are also possible.
Oxidants and Antioxidants
Reactive oxygen species such as ozone and its derivatives – free radicals and perox-
ides – are constantly formed as oxidants in atmosphere and water (Baird 1995) as
well as in living cells (Roshchina and Roshchina 2003; Dzhabiev et al. 2005;
Dzhabiev and Kurkina 2007). Similar species such as free radicals, in particular
superoxide anion radical and peroxides, arise in both pollen and pistil (Roshchina
et al. 2003; Roshchina and Roshchina 2003). The hydrogen peroxide solution itself
has weak emission in blue. The fluorescence spectra of the pistil stigma are changed
during the first 22–300 s after the addition of oxidants and antioxidants – superoxide
dismutase or peroxidase (Roshchina et al. 1998a). It was shown (Roshchina 2008)
that the light emission increased after the addition of noradrenaline that generates
superoxide anion radical O & . The shift of maximum from 500 to 530 nm and shoul-
2
der at 530 nm occurs after the treatment with hydrogen peroxide (this reagent has a
weak fluorescence), but water has no effect. Another character of the fluorescence
spectra was observed under the treatments with antioxidants, also constantly found
on the cellular surface. Low-molecular antioxidant ascorbate forms shoulder at
600 nm in the emission spectrum, which is transformed to a maximum at 560–
600 nm. Antioxidant enzyme superoxide dismutase (catalyzer of the superoxide
radical dismutation that leads to the formation of hydrogen peroxide) induces the
arising of new maximum at 580 nm, and peroxidase (catalyzer of the peroxides
decomposition) maxima at 530 and 600 nm (Fig. 4.4). A maximum at 530 nm may
Oxidants Antioxidants
3
5
2
Peroxidase Superoxide dismutase
H2O2
0
400 500 600 700 400 500 600 700 400 500 600 700
Wavelength (nm) Wavelength (nm) Wavelength (nm)
Fig. 4.4 The effects of oxidants and antioxidants on the fluorescence of the pistil stigma in
Hippeastrum hybridum (Adapted from Roshchina et al. (1998a)). 1 Without treatment, 2 and 3
300 s after the addition of noradrenaline at 10−4 M and hydrogen peroxide at 10−5 M. Enzymes
(0.6 mg/ml) peroxidase and superoxide dismutase. H2O2 – emission of hydrogen peroxide out of
cell. Excitation 360–380 nm
be related to oxidized flavines, and longer wavelength maxima – to flavonoids or

azulenes in a complex with cell wall (Roshchina 2008).
As antioxidants on the contacting surface of pollen and pistil, some phenolic and
terpenoid compounds may occur including pigments such as phenols anthocyanins,
carotenoids, and others (Stanley and Linskens 1974). Blue pigment of azulene pres-
ent in pollen exine (Roshchina et al. 1995; Roshchina 2008) is also antioxidant and
also can be the origin of free radical (Barriero et al. 1992). If it is added on the pistil
stigma, the fluorescence of the cellular surface increases, and smooth maxima at
500 and 600 nm become clear and 2.5-fold higher during 22–110 s, and then the
emission achieves a control level.
4.1.2 Pollen–Pollen Interactions
The first attempt to modeling with pollen–pollen contacts via their secretions has
been made in the 1930s of 20 century by Branscheidt (1930) and Zanoni (1930).
The results were summed and described in the monograph of Grümmer (1955) and
became a base for the study of allelopathic relations estimating the germination of
pollen from different species in various mixtures (Char 1977; Murphy 1992;
Murphy 2007; Murphy et al. 2009a, b; Chauhan et al. 2005; Gaur et al. 2007).
Today, due to the autofluorescence of pollen acceptors and pollen donor of the
secretions, a researcher could see direct interactions of pollens from various species
by the help of laser-scanning confocal microscopy that permits to receive LSCM
images (Roshchina et al. 2008, 2009a, b).
Matricaria
Oenothera
1. 2.
Artemisia Knautia
3. 4. Artemisia
Fig. 4.5 Visualization of mixture that contained fluorescent pollen grains on white background
under laser-scanning microscope LSM 510 NLO “Carl Zeiss” (laser 488 nm) (Sources: Roshchina
et al. (2008, 2009, 2013a)). 1 and 2 Frontal view and optical slice of pollen from Oenothera bien-
nis (single large microspore) interacting with pollen grains from Matricaria chamomilla (small
microspores). 3 and 4 Frontal view and optical slice of pollen from Knautia arvensis (single large
microspore) interacting with pollen from Artemisia absinthium (small microspores). Orange/red-
fluorescing emission from A. absinthium pollen is seen on the blue-green-fluorescing surface of
pollen K. arvensis. Bars = 100 μm
4.1.2.1 LSCM Images of Fluorescence in Pollen–Pollen Interactions
LSCM images of fluorescent pollens in their mixtures showed how secretions are
released and contacted (Roshchina et al. 2008, 2009a). It made possible to directly
observe, viz., (1) the contact of intact cell, (2) changes in their emission, and (3)
location of contacting secretions. The experiments in this line were carried out on
pollen from various species. Figure 4.5 demonstrates the direct contacts in pollen
mixtures under laser-scanning confocal microscope (LSCM images) on examples
of pollen grains Oenothera biennis, Matricaria chamomilla, Knautia arvensis, and

Artemisia absinthium. In the mixtures, pollen of Oenothera biennis was considered
as pollen acceptor, whereas pollen of Matricaria chamomilla was considered as
pollen donor. For other mixtures, pollens from Knautia arvensis and Artemisia
absinthium served as acceptor and donor of the secretions, respectively. Red-
fluorescing secretion is characteristic for large pollen of Oenothera, while small
pollens of Matricaria emitted green (frontal view) and liberated green-fluorescing
secretions (image 1). Optical slices showed the interior of contacted pollen grains,
and green fluorescence was seen only on the surface of M. chamomilla spore, while
red emission increased. Red-fluorescing secretion is characteristic for large pollen
of Oenothera, while small pollens of Matricaria emitted green (frontal view) and
liberated green-fluorescing secretions. Optical slices showed the interior of con-
tacted pollen grains, and green fluorescence was seen only on the surface of M.
chamomilla spore, while red emission was also seen inside the pollen grain of O.
biennis (image 2). This picture explains why the red fluorescence of Matricaria pol-
lens increased after mixing with pollens of Oenothera. Besides, secretions from the
pollen acceptor enhanced the germination of M. chamomilla pollens (Roshchina
et al. 2009a, b). Similar findings have been done with the contacts of pollen grains
from Knautia arvensis and Solidago virgaurea (Roshchina et al. 2008). The interac-
tion of blue-green fluorescent pollen of Knautia arvensis and orange/red-fluorescing
secretion of pollen from Artemisia absinthium decreased the germination in both
contacting types of microspores. Orange/red-fluorescing secretion from A. absin-
thium was located on the green fluorescent surface of pollen of K. arvensis, and part
of the secretion was also seen within acceptor pollen grain (images 3 and 4).
Perhaps, the visible enhancement in the fluorescence is related to the energy loss
peculiar to nonviable pollens (Roshchina 2008).
Changes in the fluorescence intensity of both acceptor cell and donor cell in
green (maximum at 520 nm) and/or red (maximum at 680 nm) spectral regions
accompanied by the following alteration in the pollen germination were observed
for 42 plant species (Roshchina et al. 2009a, b). The experiments will be described
in Sect. 4.2 devoted to pollen allelopathy.
Among fluorescent compounds of pollen excretions could be phenols, aromatic
compounds which have hydroxyl groups bound with aromatic nucleus. A variety of
them include main groups able to fluoresce (Roshchina 2008): (1) flavonoids such
as anthocyanins, flavonols, and flavons; (2) quinones; (3) phenolic acids; and (4)
stilbenes (Roshchina 2008). Among emitted substances are especially known fluo-
rophores C6–C3 (acid), C6–C3 (coumarins), C6–C4 (naphthoquinones), C6–C3–C6
(flavonoids), C6–C3 of lignin in anther cell walls, C6–C3–C6 (condensed tannins), as
well as hydrolyzed tannins, in particular gallic and valoneic acids.
4.1.2.2 The Germination Reactions in Pollen Mixtures
Pollen–pollen contacts are the subject to study because it connects with the prob-
lems of breeding and ecosystem formation (El-Ayeb et al. 2009). Chemical interac-
tions between pollens of various species were first analyzed in the Grümmer
monograph (1955). In vitro, the stimulatory or inhibitory effects on germination of

the pollen grains in mixtures were observed (Branscheidt 1930; Golubinskii 1946;
Babadzhanyan 1947). These were the first model systems to study mixtures from
various species, in particular of grass lawn spp. (Lilium, Tulipa, and Narcissus) and
natural phytocenosis spp. (Corylus avellana, Linaria vulgaris, Papaver som-
niferum, etc.). Germination of pollen as test reaction as well as pollen tube growth
was also analyzed in experiments in vitro (Char 1977; Thomson et al. 1982; Ortega
et al. 1988; Murphy and Aaarssen 1989; Anaya et al. 1992). The abovementioned
studies were a base to determine the phenomenon named as pollen allelopathy
(Char 1977).
As seen today, pollen allelopathy, known as pollen–pollen competition on the pis-
til surface, plays a major role in normal plant fertilization in biocenosis (see reviews
of Murphy 1992, 1999a, b, 2000, 2002; Murphy and Aaarssen 1995a, b; Chauhan
et al. 2005; Gaur et al. 2005, 2007; Murphy et al. 2009a, b; El-Ayeb et al. 2009).
Pollen allelopathy is one consequence of heterospecific pollen transfer and occurs
only if sufficient numbers of grains containing exuded or surficial allelochemicals
alight on the stigma or other tissues of recipient individuals (Murphy et al. 2009a).
The main mechanisms elucidated have included pollen-to-pollen exudation and
absorption of chemicals, with some indication that a major mechanism is disruption
of mitosis involved in production of pollen tubes. Mutual effects were also analyzed
by the use of extracts enriched in individual compound of the pollen secretion or
pure components of the secretion (Ortega 1988; Anaya et al. 1992; Cruz-Ortega and
Anaya 2007; Murphy 2007).
Some models of pollen–pollen contact which could relate to pollen allelopathy
will be considered in Sect. 4.2.
4.1.3 Direct Contacts in Plant–Animal

or Plant–Microorganism Interactions
Secretion from plants is the primary means by which a cell interacts with its sur-
roundings, including animals, various foreign plant species, and microorganisms.
Approaches to analysis of the relations were out of the researchers’ attention yet.
Cytodiagnostics of the cell–cell interactions has perspectives also in the modeling
of chemical relations in the systems “plant–animal” and “plant–microorganism” in
biocenosis.
Theoretically, a base for similar studies could be the signal compounds common
for any organisms. Similar substances are proposed to be acetylcholine and biogenic
amines which are ancient chemosignals in evolution (Roshchina 1991, 2001a, 2010).
Never before was an investigation attributed on the problem of how plant excretions
containing the neurotransmitters acetylcholine and biogenic amines contact with
animals or how similar compounds in animal excretions contact with plant cells.
This appears to be a significant role of the above mentioned neurotransmitters
found in saliva and salivary glands of animals as described by Ribeiro (1987). The
secretions contain neurotransmitters, proteases, and other digestive proteins.
Moreover, exogenous neurotransmitters serotonin and dopamine (10−7 − 6 × 10−7 M)
stimulate similar secretion (Marg et al. 2004) that is shown earlier in model experi-
ments on salivary glands of cockroach Periplaneta americana (Just and Walz 1996).
A very important role belongs to histamine in secretions of various living organisms
and its influence on the cellular relations between different individuals and species.
The biogenic amine is found in salivary glands and venom of insects. It is organic
nitrogen compound of animals that involves in local immune responses (it triggers
the inflammatory response and allergy in human) as well as in regulating physiologi-
cal functions and acting as a neurotransmitter (Engel et al. 1988). It increases the
permeability of the capillaries to white blood cells and some proteins, to allow them
to engage pathogens in the infected tissues. But plant reactions on the insect hista-
mine are not studied yet.
Modeling of the plant–animal interactions via histamine as secretory product has
certain perspective. Only few publications are concerned with the direct experi-
ments on some models yet. In particular, such a model is Australian sea urchin
Holopneustes purpurascens which induced to settle and metamorphose (termed
settlement herein) by the excretion of histamine (0.9–4.5 μM) as shown by Swanson
et al. (2004). The possibility of plant excretions to regulate functions of animals and
microorganisms was not analyzed systematically yet. Bacteria also are capable of
producing histamine from amino acid histidine by the catalysis with the enzyme
histidine decarboxylase. The compound may be identified in plants and animals by
fluorescent methods with staining by glyoxylic acid (see Chap. 3). In some com-
mercial vegetables and foods, the bacterial pollution is analyzed based on the hista-
mine determination (Ekici and Coşkun 2002). A noninfectious form of foodborne
disease, scombroid poisoning, is due to histamine production by bacteria in spoiled
food, including the following: fermented foods and beverages naturally contain
small quantities of histamine due to a similar conversion performed by fermenting
bacteria or yeasts. Sake contains histamine in the 20–40 mg/L range; wines contain
it in the 2–10 mg/L range.
The examples of the possible models of contacted plant and animal or plant and
microorganisms may be the visualization of the insect attack (Lee et al. 2009) or
fungal invasion (Wu and Warren 1984a, b; Žižka and Gabriel 2006). Worms such as
planarians is also applied for the analysis effects of some plant excretions (Roshchina
et al. 2013b). Below, we shall illustrate examples of similar useful models.
4.1.3.1 Plant–Insect Contacts
Direct observation of similar contacts is based on the difference in the fluorescence

excited by ultraviolet light of luminescence microscope. Fungi or insects such as
ticks, spiders, or human skin fluoresce, mainly, in blue (460–470 nm) or blue green
(<520–530 nm), whereas muscle fluoresce in blue with maxima at 410 and 480 nm
a b c
Fig. 4.6 Fluorescent larva of aphis interacts with cellular complex of leaf gland of mint Mentha
piperita and hop Humulus lupulus under luminescence microscope Leica DM6000 (Adapted from
Roshchina (2012) and unpublished data 2012). (a) The common view of aphis larva out of the mint
gland, (b) view of larva on the mint gland, and (c) view of larva on the hop leaf surface. Excitation
by light at 360–410 nm. Bar = 100 μm
(Roshchina 2012). Insects fluoresce by all parts of their body, and it may be observed
separately on the plant cell surface. The blue emission intensity of fungal hyphen
and mycelium is much higher than in fluorescing secretory plant cells (Roshchina
2008) that permit to recognize fungal infection.
The example of parasitic invasion distinguished in the fluorescence is in Fig. 4.6
for leaf of medical plant mint Mentha piperita used by aphid for nutrition. On the
leaf, colorless microscopic larvae are plant louse not seen well up to the excitation
by actinic light of luminescence microscope. Blue-fluorescing gland of Mentha (if
excited by UV light) with maxima at 475, 540, and 675–680 nm in the fluorescence
spectrum (Roshchina 2008) is covered by weak blue-fluorescing aphis larva (chitin
emission, mainly at 450 nm). At excitation, fluoresced mint gland (surrounded by
red-fluorescing nonsecretory cells of leaf mesophyll due to chlorophyll presence)
mainly was seen through transparent larva body (non-visible in usual microscope)
larva body. In an analogous picture, one could observe on leaves of hop Humulus
lupulus that the aphid larvae arose. Excretions of defensive compounds from leaf
gland of Mentha piperita (e.g., monoterpenes) may kill the larvae, and we can see
the decomposed insect. Table 4.1 demonstrates the fluorescence intensity of the
larvae on the mentioned objects. Under ultraviolet light, larvae are well seen as blue
objects, and they cover the leaf surface decreasing red fluorescence, peculiar to
chlorophyll emission with a maximum at 675–680 nm. The excretions of mint kill
the larvae, and decomposed insect is also well seen. As shown in Humulus lupulus,
the insect releases pathogenic secretion that also weakly fluoresces in blue
(Table 4.1). Direct observation of the larvae of aphids under UV light of lumines-
cence microscope may help in phytopathology to recognize earlier invasion of aphid
when it is impossible for scientists to see larvae in the transmitted light of a usual
microscope. On this stage of aphid development, the preventive procedures, in par-
ticular the treatment with pesticides, for the plant normal growth are needed.
Table 4.1 The Plant species I460 I640–680

autofluorescence intensity (I)
Mentha piperita
of aphids on leaves of
Mentha piperita and Humulus Gland without larvae 0.54 ± 0.009 8.40 ± 0.5
lupulus measured by Larvae on the gland 0.11 ± 0.010 0.91 ± 0.05
double-wavelength Decomposed larvae on the leaf 0.09 ± 0.010 0.36 ± 0.05
microspectrofluorimeter Humulus lupulus
MSF-2. Excitation at Undamaged leaf surface 0.03 ± 0.003 3.56 ± 0.04
360–380 nm Larvae on the leaf 0.09 ± 0.009 1.26 ± 0.04
Excretion released by larvae 0.02 ± 0.05 0.25 ± 0.05
The aphid feeding widely varies depending on the plant species (Auclair 1963;
Leszczynski 1999). Mainly nitrogenous compounds prevail in its nutrition. The fact
that mint excretion (terpenes) is also attractive for aphid may be observed from
direct model of secretory leaf gland of mint and the insect (Roshchina 2012). The
modeling of the plant–aphid contact is useful to those persons who are interested in
host–plant resistance to aphids and virus transmission by aphids, as well as for a
discussion on possible nutritional functions of symbiotes. Homoptera, in particular
aphids, insert their stylets into plant tissues (mainly by the action of protractor and
retractor muscles attached on the enlarged bases of the stylets, probably aided by a
clamping action in the groove of the labium or might be propelled forward mainly
by successive short holds contrived by the joint pincer-like action of muscles in the
labium and labrum, the latter being also grooved). The saliva rapidly set into a gel
which covered the tip of the advancing stylets, eventually forming a salivary sheath
which remained within the plant tissue after withdrawal of the stylets. Such salivary
sheaths were reported as produced by most aphid species. Some compounds found
in plants may be excreted and affect the functions of contacted visitor. Leaf volatiles
reduce, in particular, moth oviposition (Allmann et al. 2013). Among the substances
are the neurotransmitter acetylcholine and biogenous amines that influence the
insect contacted (Roshchina 2001a, b, 2010). In particular, acetylcholine stimulated
the release of saliva by insects, although in many cases plants release repellents, in
particular diterpenes and their glycosides (Dinh et al. 2013).
4.1.3.2 Plant–Worm Contacts
Interactions between animals and plants may be observed on planarians as model

biosensor for components containing water plant excretions. Today, the object is
analyzed as universal model having characteristics similar with mammalians
(Oviedo et al. 2008). Exposure to biologically active compounds found in plant
excretions such as agonist of acetylcholine (alkaloid nicotine), antagonist of acetyl-
choline, blocker of muscarinic cholinoreceptors (alkaloid atropine), agonist of
dopamine (alkaloid apomorphine), or inhibitor of cholinesterase (alkaloid physo-
stigmine) induced various types of planarian hypokinesia (Buttarelli et al. 2000).
Planarians also excrete in their mucosa the enzyme cholinesterase (Li 2008;
Without treatment
a b
Histochemical staining
c d e f
g h i
Fig. 4.7 Modeling on the interaction of plant excretions with worm Girardia tigrina. (a, b) The
common views of the worm without any treatment under transmitted light and UV light (360–
410 nm) of luminescence microscope Leica DM6000, relatively; (c) staining on the cholinesterase
activity with red analog of Ellman reagent (Roshchina 2001a, b; Roshchina et al. 2013b) seen
under transmitted light; (d–f) treatment by hypericin as model of phenols–quinones released by
plants; (d, e) object seen in luminescence microscope; (f) view under laser-scanning confocal
microscope Leica TCS SP-5, laser 405 nm; (g–i) control or after the treatment with 10−6 M d-tubo-
curarine or yohimbine, respectively (Picture seen as fluorescence excited by light at 360–400 nm
of luminescence microscope Leica DM6000). Bar = 500 μm
Roshchina et al. 2013b) and, therefore, may be sensitive to acetylcholine and

biogenic amines excreted by water-inhabited plants. The enzyme cholinesterase,
marker of the acetylcholine’s presence and sensitivity, is found in similar species
such as those belonging to genera Nitella, Lemna, Chara (Roshchina 1991, 2001a),
and many Bryophyta spp. (Gupta et al. 1998, 2001). There is evidence of a trophic
action of acetylcholine in a planarian (Welsh 1946). Figure 4.7 shows histochemical
staining of the planarian Girardia tigrina with red analog of Ellman reagent on
cholinesterase (Roshchina 2001a), demonstrating characteristic blue color forma-
tion from red dye. Planarians treated with acid showed significant fluorescence due
to the porphyrin red emission (Macrae 1961). According to Macrae (1961), epider-
mal cells named rhabdites and subepidermal rhabdite-containing glandular cells
being treated with acid and fixed by ethanol look as red-colored cells in UV light
due to uroporphyrin. Unlike acid staining, in experiments without any treatments,
the object Girardia tigrina (Platywormhes, Turbellaria) shows weak blue fluores-
cence with maxima at 430–460 nm (Fig. 4.7, image b), perhaps related to NADH/
NADP of food particles (products of digestion). Slime released by the worm serves
as contacts with the surrounding living world. Among the water inhabitants are
plants containing and excreting phenols, quinones, amines, etc. In slime cover of the
worm, cholinesterase activity is concentrated (Fig. 4.7, image c), and the enzyme is
sensitive to acetylcholine as chemosignals evacuated from both plants and animals
of water basin (Roshchina 2010). Using the colored phenolic excretions from plants
such as red hypericin and pelargonidin, we could see the staining and the accumula-
tion of tested compound on the surfactive part of worm, mainly in the slime (Fig. 4.7,
images d–f). Confocal microscopy also permitted to see optical slice of the red-
emitted object (Fig. 4.7, image f). In this way, we also try to use blue pigment azu-
lene and yellow β-carotene which also are concentrated, mainly in planarian slime.
It may be one of the approaches to understand primary interaction between plant
excretion and animal (in particular worm). Images (d–h) also demonstrate experi-
ments on the treatment with alkaloids d-tubocurarine (acting on animal nicotinic
cholinoreceptor) and yohimbine (which binds with animal adrenoreceptors). In both
cases, blue fluorescence arises which is bright mainly on the periphery of the worm
body. Perhaps, the compounds are concentrated on slime and the surface receptors.
4.1.3.3 Relations Between Water-Grown Plants and Animals
As an antiherbivore defense, the temperate green alga Ulvaria obscura demon-

strates dopamine release and may serve as a sea model to study plant–animal inter-
actions (van Alstyne et al. 2006, 2013). The red color of the algae population is
concerned with the formation of red oxidized product of dopamine named dopach-
rome. To understand the mechanisms associated with dopamine release, van Alstyne
et al. (2013) experimentally determined whether light quantity and quality, desicca-
tion, temperature, exudates from conspecifics, and dissolved dopamine caused
dopamine release. Although dopamine released into seawater can reduce the sur-
vival or growth of potential competitors, its release is associated with significant
physiological stress and tissue mortality.
Among other possible models of plant–animal relations in water larvae of the sea
urchin, Holopneustes purpurascens were suitable objects that have undergone
secretions from a host alga containing histamine (Swanson et al. 2004, 2007). This
compound induced the settlement of larvae, and that is why the object may be a
model for the observation relations between plant and animal.
4.1.3.4 Plant–Microorganism Contacts
Most plant microscopic pathogens are extracellular, which demands a robust and
efficient host secretory system directed at the site of attack. Secretion is required not
only for the delivery of antimicrobial molecules but also for the biogenesis of cell
surface sensors to detect microbes (Doehlemann et al. 2009; Oleskin et al. 2010;
Wanga and Dongb 2011; Oleskin 2012). The extracellular material is important in
the defense at plant–bacteria or fungi–bacteria interactions against classical bacte-
rial pathogens (Doehlemann et al. 2009) as well as in the so-called “nonhost” resis-
tance. Representation of modern advances in the understanding of the molecular
details of the secretory pathway during plant–microbe interactions has been done
recently in a review (Wanga and Dongb 2011). Plant secretions play the role of a
chemical barrier for microorganisms like it is observed for trichomes of Lycopersicon
pennellii (Nonomura et al. 2009). Moreover, plant cell synthesizes and excretes
esterases in response to infection, for example, blast invasion in finger millet
Eleusine coracana as a means of defense (Muarlidharan et al. 1996).
Boosting the secretions as a response to invasion is for avoiding infection as well
as for achieving symbiosis, even though in the latter case the microbes are engulfed
in intracellular compartments. In many cases, a boosting of the proteinaceous secre-
tion capacity is vital for avoiding infection. The emerging evidence indicates that
secretion provides an essential interface between plant hosts and their associated
microbial partners. For example, secreted fluid of carnivorous Nepenthes plants
depresses microbial growth (Buch et al. 2013). But there are situations when plant
and fungi secretions allow beneficial interaction between the contacted organisms
(Camehl et al. 2013). In the last case, the model Arabidopsis thaliana may be
applied for the genetic analysis of the mechanism.
The parasitic invasion requires a specialized staining procedure for visualization,
except for the microorganisms that demonstrate autofluorescence. Confocal micros-
copy appears to be more suitable for plant–pathogen interaction in detail (Hardham
2012). Fungal fluorophores may depend on species and are not well studied yet.
Today, fungal infection is better registered observing the enhanced blue-green auto-
fluorescence of plant cell walls related to their phenols and sometimes auxin
exchange (Perrine-Walker et al. 2010). The autofluorescence properties of fungi
may be applied to practical diagnostics of pest. Perspectives to model contacts
between plant cell and pest cell are in a search of suitable objects.
Possible example is greenhouse-grown plant Callisia fragrans (Lindl.) Woodson
(family Commelinaceae). Figure 4.8 and Table 4.2 show results of the work with
fluorescing leaf surface of the plant species that have undergone fungal infection
seen as brown spots on leaves. Undamaged healthy leaf cells weakly fluoresce in
blue (430–460 nm) at the excitation by UV light (only chlorophyll emits in red
spectral region 640–680 nm). Fungal excretions fluoresce just on earlier stages of
the invasion when the observer was not able to see damaged cells in the usual micro-
scope. Excreting fungal hyphae are concentrated in cell walls and external spaces,
but even chloroplasts are seen as blue organelles. The latest phase of the invasion
characterizes as brightly blue fluorescence of excretions from both fungal and plant
a b
c d
Fig. 4.8 Leaf surface cells of plant Callisia fragrans undergone fungal infection on earlier stage
of pest invasion (a, b) and at visible damage (c, d). (a, c) Views under usual microscope in trans-
parent light, (b, d) fluorescence of the cells seen at UV light (360–380 nm) of luminescence micro-
scope Leica DM6000. Bar = 100 μm
Table 4.2 The autofluorescence intensity (I) at 460 and 640–680 nm (excitation UV light at 360–
380 nm) measured with microspectrofluorimeter MSF-2 from the leaf surface of Callisia fragrans
infected fungi forming brown necrotic spots
Cell surface I460 I640–680
Undamaged plant cell (red fluorescence 0.06 ± 0.005 4.48 ± 0.032
prevails)
Beginning of the invasion of fungi 0.14 ± 0.011 2.54 ± 0.034
Developed invasion 0.13 ± 0.008 0.68 ± 0.037
Complete invasion (blue fluorescence 0.02 ± 0.003 0.26 ± 0.051
prevails)
damaged cells. The fluorescence may be connected also with the formation of phe-
nolic components in the sites of invasion (Roshchina 2008). To our mind, this mod-
eling system or similar one appears to be useful for future investigations based on
the autofluorescence of contacting cells (Roshchina 2012).
Many pathogenic and saprophytic fungi fluoresce in blue green (in 390–490 nm –
spectral region) under actinic ultraviolet light (Wu and Warren 1984a, b; Žižka and
Gabriel 2006; Doehlemann et al. 2009). Moreover, the phenomenon was better seen
under stress, in particular after such unfavorable conditions as desiccation, heat, and
chemicals (Wu and Warren 1984a, b). Recently, blue-green autofluorescence of
fungi Ustilago maydis on maize and cell wall of this plant was shown as an indicator
of the invasion (Doehlemann et al. 2009). At the fungal infection, the value of auto-
fluorescence as a screening method for detecting fungi in tissues was compared with
conventional histochemical staining. Autofluorescence can be used as a rapid
screening method for identification of fungi in tissue sections as it does not require
any other specialized staining procedure. But fluorophores in fungal cell are weakly
studied.
Compositions of microbial excretions varied, mainly proteins prevail, in particu-
lar like antifungal glycolipid secreted by the yeast Sympodiomycopsis paphiopedili
(Kulakovskaya et al. 2004). Bacterial influence may be also analyzed based on the
animal smell. Mainly animal–bacteria interactions give characteristic smell due to
aromatic products of bacterial decomposition of animal excretions. In this case, the
human person is the best model of such contacts (see monograph of Inaba and Inaba
1992). Natural human odor can be a factor in human communication and is a signifi-
cant source of information involving body processes. At normal, gamma-lactones
form the scalp odor of hair; L-pyrroline, steroids of males, free fatty acids, and
compentandecalactone – the vaginal odor and genital odor; butyric and caproic
acids, acetic acid and palmitic acid, pelargonic acid, and stearic acid – the sweat
smell; and triglycerides, squalene, sebaceous esters, and cholesterol – the odor of
salivary glands. But the composition of the odor is changed at stress and disease, for
example, isovaleric acid and aldehydes derived from the acid or cis-undec-4-enol
arise from scent glands (deer or rabbit odors).
Some genetic models are also used today for the analysis of plant protection
against fungal parasitic attacks. In plant defense against fungal infection, the chitin-
ases ChitA and ChitB participate as shown in such suitable genetic models as mono-
cotyledon Zea mays and dicotyledon Arabidopsis thaliana (Naumann and Price
2012). In model Zea mays, the chitin-binding domain is removed by secreted fungal
proteases called fungalysins. Plant class IV chitinases have a small amino-terminal
chitin-binding domain and a larger chitinase domain and are involved in plant
defense. The effects of fungalysin-like proteases on four class IV chitinases from
the model Arabidopsis thaliana were analyzed, and four Arabidopsis chitinases
were heterologously expressed in fungi Pichia pastoris, purified and shown to have
chitinase activity against a chitohexaose (dp6) substrate. The incubation of these
four chitinases with Fv-cmp, a fungalysin protease secreted by fungi Fusarium ver-
ticillioides, resulted in the truncation of AtchitIV3 and AtchitIV5.
Recently, new models for the study of fungi–bacteria systems were represented
by Oleskin and colleagues (2013). Bacteria and fungi can synthesize norepinephrine
4.2 Models to Study Pollen Allelopathy 119
and dopamine and release them into the culture fluid (Shishov et al. 2009; Oleskin
et al. 2010; Oleskin 2012). The excretions participate in the relations between the
organisms. Mycelium of fungi Coprinus comatus being added into cultures of vari-
ous bacteria such as Bacillus subtilis, Arthrobacter globiformis, and Pseudomonas
fluorescens may be covered by bacterial films. At the contacts, biogenic amines
(known as neurotransmitters) play an important role because serotonin, dopamine,
or noradrenaline released by this fungal object inhibited the growth of similar bacte-
rial films that is confirmed by experiments with exogenous biogenic amines. The
authors proposed the participation of the compounds in other communications –
between yeast Saccharomyces cerevisiae and leaves of fruit plants (Malikina et al.
2010a, b). Biogenic amines catecholamines, serotonin, and histamine regulate the
state and growth of the yeast culture. Dopamine stimulated cell proliferation in the
eukaryote Saccharomyces cerevisiae, while norepinephrine did not significantly
influence this process (Malikina et al. 2010a, b).
Catecholamines stimulate biomass accumulation (estimated nephelometrically)
and cell proliferation (determined from colony-forming unit number increase) in
nonpathogenic E. coli K-12 (strain MC4100) as shown in Oleskin’s laboratory
(Anuchin et al. 2008). Dopamine stimulated proliferative activity and biomass accu-
mulation to a greater extent than norepinephrine (Anuchin et al. 2008). Moreover, in
human organism apart from cell proliferation, catecholamines stimulate the synthesis
of adhesins, toxins, and other virulence factors (Lyte et al. 1996; Freestone et al.
1999; 2000; 2007; 2008; Clarke et al. 2006). It has been shown that serotonin acceler-
ates the growth of the purple phototrophic bacterium Rhodospirillum rubrum (Oleskin
et al. 1998a, b), the soil myxobacterium Polyangium sp. (Oleskin et al. 1998), and the
yeasts Candida guilliermondii (Strakhovskaya et al. 1993) and S. cerevisiae (Malikina
et al. 2010a, b). Serotonin also exerts a stimulatory influence upon plants. For exam-
ple, it accelerates the germination of radish seeds (Roshchina 1991). Presumably,
serotonin produces its effects in plant systems because its chemical structure is simi-
lar to that of the plant hormone auxin, or 3-indoleacetic acid (IAA). However, studies
with bacteria Pseudomonas putida provided evidence that IAA is utilized by bacteria
only as a carbon, nitrogen, and energy source (Leveau and Lindow 2005).
Bacterial histamine in relation with plants plays significant role for food storage
as toxin (Roshchina 2010). Its high concentration in plant cell may be determined
by fluorescent method (see Chap. 2, Sect. 2.3.2) as a result of bacterial infection.
Micromolar histamine concentrations stimulate cell proliferation in S. cerevisiae
(Malikina et al. 2010a, b). In the future, the search of special models for the studies
is required.
4.2 Models to Study Pollen Allelopathy
Pollen allelopathy plays a major role in normal plant fertilization in biocenosis

(Char 1977; Sukhada and Jayachandra 1980a, b; Thomson et al. 1982; Ortega et al.
1988; Anaya et al. 1992; Murphy 1992, 1999a, b, 2007; Murphy and Aaarssen
1989, 1995a, b; Chauhan et al. 2005; Gaur et al. 2005, 2007; Roshchina 1999a, b,
2001a, b; El-Ayeb et al. 2009). The first studies of pollen mixtures composed of
different species, reported even during the years 1930–1940, showed the stimula-
tory or inhibitory effects of modeling events that occurred in nature for addition of
foreign pollen carried by the wind or insect (Branscheidt 1930; Zanoni 1930;
Golubinskii 1946; Babadzhanyan 1947; Grümmer 1955). Galen and Gregory (1989)
included interspecific pollen transfer as a mechanism of competition consequences
of foreign pollen contamination for seed set, in particular in the alpine wild flower
Polemonium viscosum. In the 1980s–1990s, allelopathic effects of pollen in mix-
tures of own and foreign pollens were studied on in vivo germination (Char 1977;
Sukhada and Jayachandra 1980a, b; Thomson et al. 1982; Anaya et al. 1992;
Murphy 1992, 1999a, b, 2007).
Pollen competition is supposed to be important phenomenon necessary for sta-
bile biocenosis. It is of special interest for ecology and economics due to occurrence
of competitive relations between grain crops and weeds (Murphy 1992; Chauhan
et al. 2005; Gaur et al. 2005, 2007; El-Ayeb et al. 2009). Pollen allelopathy may
delay the flowering in weeds to less favorable season or diurnal period to reduce the
weed pressure (Murphy and Aaarssen 1989, 1995a, b).
4.2.1 Modeling of Interaction Between Pollen Donors

and Pollen Acceptors In Vivo and In Vitro
To develop models for pollen–pollen interactions, the effects of allelochemicals

secreted by foreign pollen, i.e., donor pollens, were studied in vitro or in vivo on the
autofluorescence and germination of acceptor pollen (Roshchina 2004a, b, c, d;
Roshchina et al. 2008, 2009a, b). To model the pollen–pollen interactions occurring
in nature, one should study the effects of well-germinated pollen, which served as
pollen acceptor of allelochemicals released in secretions by the foreign pollen,
called as donor pollen. Both acceptor and donor pollens were chosen from the
indoor- (greenhouse) and outdoor-grown plants (Roshchina et al. 2009a, b).
Acceptor pollens had large size (20–50 μm), quicker germination on artificial nutri-
ent media, and the ability to fluoresce in ultraviolet light for observation with light
and luminescent microscopes (Roshchina 2003, 2007a, b, 2008). Pollen fluores-
cence in allelopathic response is also described by Murphy (2007).
If pollens from different species are mixed and simultaneously added to pollen
acceptor, their effects were often masked. It was needed to model the real situation
that occurs in nature. When foreign pollen appears on another plant’s stigma, its
secretion influences both the stigma reception of chemical signal and the reception
of same signal by own pollen which was added later. The first pollen that falls (or
added by insects) on pistil stigma suppressed all late-arriving pollens (which do not
germinate). Similar tendency was observed and able to determine if it was chemical
or physical interaction. If pollen (donor of secretion) is exposed for 20–30 min to
artificial nutrient medium (in vitro samples) or on the pistil stigma of such model
(% of control)
Germination
Control Hippeastrum+Echinopsis Hippeastrum+Lilium Knuatia+ Artemisia Knautia+ Artemisia Knautia + Centaurea

absinthium vulgaris
Fluorescence (% of control)
100
250 14.4± 7 26± 10 113± 13 0 0 33± 1.7 25± 1.5 97± 0.8 86± 1.8 129± 1.8
200
150
100
50
0
I blue – I red
green
Fig. 4.9 The interrelations of pollens in mixtures when the acceptors are pollen grains of green-
house species Hippeastrum hybridum or outdoor species Knautia arvensis, whereas donors are
pollen grains from greenhouse species Echinopsis bridgesii and Lilium martagon or outdoor
(meadow and weed species) species Artemisia absinthium, Artemisia vulgaris, and knapweed
Centaurea jacea L. (Asteraceae) (Adapted from Roshchina et al. (2009a, b)). Reactions are germi-
nation on artificial nutrient medium and autofluorescence excited by violet light at 420 nm and
measured in dual beams at 530 nm (green or blue-green emission) and at 640 nm (red emission).
Diagrams with additional middle light ring show the stimulation higher than control
system as Hippeastrum hybridum, significant effects were seen (Roshchina et al.

2009a, b).
One of the examples of similar experiments with acceptor models – pollen of
knight’s star Hippeastrum hybridum (greenhouse grown) and field scabious Knautia
arvensis (meadow grown) – is given in Fig. 4.9 where pollen mixtures with pollen
donors are analyzed. Both contacted pollen grains excreted biologically active com-
pounds, and mutual influence on either germination in vitro or autofluorescence as
test reactions was observed. Pollens from indoor-grown plant species Echinopsis
bridgesii and Lilium martagon being added to Hippeastrum pollen weakly germi-
nated or did not germinate at all. In the first case, pollen of knight’s star also retarded
the formation of pollen tubes. Similar pictures were seen for the field scabious
Knautia arvensis pollen as acceptor model. Pollen donor from common wormwood
Artemisia absinthium depressed the germination of the pollen, and in turn Knautia
pollen inhibited the pollen tube appearance in pollen donor. Unlike pollen donor
from common wormwood, the germination of pollen from mugwort Artemisia vul-
garis was not inhibited, although these microspores depressed the germination of
Knautia pollen. Knapweed Centaurea jacea pollen was weakly stimulated in the
germination and showed small percent inhibition of Knautia pollen. The shifts in
the rate of germination were accompanied with the alterations in autofluorescence
of contacted cells. As a rule, cellular damage is seen from the stimulated emission
in blue green (460–500 nm) or sometimes in red (640–680 nm).
The mutual effects of pollen species were compared in in vitro and in vivo exper-
iments (Table 4.3). Pollen from Acacia, which inhibited the Hippeastrum pollen
development in artificial conditions, also reduced the formation of fruits and seeds.
Pollen of Alstroemeria itself did not develop in vitro, and there was no influence on
Table 4.3 Interactions of foreign pollens with pistil stigma of Hippeastrum hybridum
Germination in vitro (percent of Fruit and seed formation of
germinated pollens in control as Hippeastrum after pollination with
100 %) mixture of own and foreign pollens
Foreign donor Hippeastrum Foreign Seeds, common
pollen pollen donor pollen Fruit weight (g) view
Out mixture 100 ± 3 7.6 ± 0.5 Dark brown,
(control) matured
Acacia dealbata 26 ± 10 150 ± 10 Non-matured, No
without seeds
Aloe vera 100 ± 3 100 ± 5 10 ± 0.3, Normal Dark brown,
matured
Alstroemeria 120 ± 7 0 7.6 ± 0.6, Normal Dark brown,
aurantiaca matured
Clivia hybrida 100 ± 10 100 ± 5 No No
Cyperus papyrus 92 ± 6 0 Abnormal reduced Non-matured,
with white seeds white color
Petunia hybrida 100 ± 8 125 ± 10 Non-matured No
Source: Roshchina et al. (2009a, b)
Hippeastrum in vivo because normal fruits and seeds were formed. Although Aloe
donor pollen itself germinated in vitro, there also were no effects of Aloe micro-
spores on Hippeastrum pollen in both in vitro and in vivo experiments. But data
in vitro for variants with pollen from Clivia and Petunia did not confirm; when the
pollen was added on the Hippeastrum pistil, no fruits and seeds were formed that
explains more complex interactions in vivo. In variant with Cyperus pollen, weak
effects on the Hippeastrum pollen development in vitro were observed, but there
were abnormal fruits and no seeds in experiments in vivo. Therefore, mechanisms
of pollen–pollen as well as pollen–pollen–pistil interactions may be different for
pollen from different species. Pollen of greenhouse plants (Clivia and Petunia)
stopped the development of microspores from Hippeastrum hybridum in vivo
(because there was no fertilization, hence, fruits with seeds were absent) during the
first stage of foreign pollen recognition. It is likely that non-mature fruits and seeds
were formed in variants with the addition of pollen from the Acacia or Cyperus on
the later stage of pollen tube development. The interaction with the pistil stigma
may also play a role in the effects of donor pollen.
As a whole in model systems “pollen–pollen” out pistil or “pollen–pollen sys-
tem” on pistil “(Roshchina et al. 2009a, b) various types of interactions were found
(Table 4.4). Microspores from Hippeastrum hybridum, Philadelphus grandiflorus,
Knautia arvensis, Plantago major, and Oenothera biennis were used as pollen
acceptors, while other pollen grains from 42 spp. were used as donor pollens. The
microspore germination inhibited or stimulated (in vitro and in vivo) and fertiliza-
tion (for Hippeastrum hybridum). The significant negative effects of donor pollen
were observed on three acceptor pollen: Hippeastrum hybridum, Philadelphus
grandiflorus, and Knautia arvensis. Their germination was inhibited by excretions
of foreign pollen grains from 18 spp. In mixtures of pollen from various species,
Table 4.4 Summary of responses induced in pollen–pollen interactions
Pollen acceptor Effects of donor pollens on germination of acceptor pollen spp.

plant spp. Stimulation Inhibition No effect
Hippeastrum Alstroemeria Acacia dealbata, Cyperus Aloe vera, Lilium
hybridum L aurantiaca papyrus, Echinopsis martagon
(Herb.) bridgesii, Petunia
hybrida, Pinus sylvestris,
Salix caprea,
Zephyranthes grandiflora
Knautia arvensis Solidago virgaurea, Artemisia absinthium, –
(L.) Coult. Tagetes patula Artemisia vulgaris,
Centaurea jacea,
Plantago major
Oenothera biennis Calendula – –
L officinalis,
Matricaria
chamomilla
Philadelphus Anthriscus Achillea millefolium, Alopecurus pratensis,
grandiflorus sylvestris Artemisia absinthium, Filipendula
Willd. Barbarea vulgaris, ulmaria, Hypericum
Chenopodium album, perforatum, Phleum
Dactylis glomerata, pratense
Knautia arvensis,
Ranunculus acer
Plantago major L. Calendula – –
officinalis
Tilia cordata Phleum pratense
four donor pollens (Alstroemeria aurantiaca, Anthriscus sylvestris, Solidago vir-

gaurea, and Tagetes patula) stimulated microspore germination, whereas micro-
spores from Aloe vera, Lilium martagon, Filipendula ulmaria, and Hypericum
perforatum showed no significant responses to foreign pollen. The rate of the pollen
tube formation was also changed in donor pollens themselves in the presence of
acceptor pollen grains. Colored excretions from donor pollen of Hypericum perfo-
ratum and Calendula officinalis stained the colorless pollens of Plantago major and
Oenothera biennis. Autofluorescence of acceptor and donor pollens in mixtures was
changed to blue-green and red spectral regions that may serve as the indicator
response on foreign microspores. Generally increased blue-green and occasionally
red emission indicated the decrease in rate of pollen germination. However, red
fluorescence was not related to germination and depended more on species used.
The germination of acceptor pollens was inhibited by many donor pollens from
both indoor- and outdoor-grown species. In mixtures of pollens from various spe-
cies, microspore germination was also stimulated by some donor pollens
(Alstroemeria aurantiaca, Anthriscus sylvestris, Solidago virgaurea, and Tagetes
patula), whereas different reactions were seen in the mixtures of the acceptor pol-
lens with the donor microspores of Aloe vera, Lilium martagon, Filipendula
ulmaria, and Hypericum perforatum. The capacity of Philadelphus or Lilium pollen

to decrease the germination of microspores from Malus domestica was known ear-
lier (Grümmer 1955), but there is no information for other species.
Autofluorescence of pollens from 48 species studied in mixtures was changed in
blue-green and red spectral regions; these indicate the pollen–pollen interactions
(Roshchina et al. 2009a, b). In most cases, the increase in blue-green fluorescence
was accompanied by decrease in pollen germination, while the change in red fluo-
rescence depended on pollen species. Fluorescent allelochemicals, mainly phenols
and alkaloids, are found in the secretions, whose individual components regulated
pollen development (Roshchina 2008). Murphy (2007) measured fluorescence
and received the emission spectra in pollens from hawkweed Hieracium canadense
and timothy Phleum pratense. There were big maximum at 520 nm, shoulder at
600 nm, and small maximum at 680 nm (perhaps chlorophyll remainders) in both
fluorescence spectra. A maximum at 520 nm was also seen when Phleum pollen
alights on pistil stigmas of Canadian species Elytrigia repens, Danthonia spicata,
and Danthonia compressa. This peak was due to the flavonoid rhamnetin extracted
from Phleum pollen. Flavonoid isorhamnetin was found as main allelopathic agent
of pollen grains from timothy Phleum pratense that inhibited the pollen tube forma-
tion in mixture with field sow thistle Sonchus arvensis and quack grass Elytrigia
repens (Murphy 2007). Only isorhamnetin depressed the germination of pollen
from Sonchus arvensis.
Some of the compounds in secretions may undergo the redox reactions in pollen
mixtures. Their oxidation leads to the formation of hydrogen peroxide, peroxides
(ozonides) of allelochemicals, and free radicals such as superoxide anion radical,
hydroxyl radical, and radicals of allelochemicals (Roshchina and Roshchina 2003).
Moreover, both superoxide anion radical and hydrogen peroxide are present on the
pollen surface (Roshchina et al. 2003). Peroxides stimulated the pollen germination
in Hippeastrum hybridum, whereas peroxidase or superoxide dismutase inhibited
the process (Roshchina and Melnikova 1998, 2001). These reactive oxygen species
can participate in allelopathic effects. Since low concentrations of studied allelo-
chemicals induced their effects, they may participate in chemosignaling on the con-
tacting plant surfaces. All these problems are new and need solutions through
further studies including modeling.
4.2.2 Modeling with Pollen Excretions
Composition of pollen excretions is different and depends on the species and condi-
tions of plant inhabitation. The pollen extracts by water and ethanol may contain
some biologically active compounds (including allelochemicals), and it needs to
compare their effects with data received for intact pollen. Besides, complete chemi-
cal analysis, for express diagnostic spectral analysis, may be applied in the studies
of pollen allelopathy (Roshchina et al. 2009a, b). One of the examples of the analy-
sis is shown in Fig. 4.10 and in Table 4.5.
Hippeastrum hybridum Knautia arvensis

1.0
(units of optical density)
1.0
Absorbance
Water extract
0.5 0.5
0 0
300 400 500 600 700 300 400 500 600
Fluorescence(relative units)
Ethanol extract
Water extract 10
10
Ethanol extract Water extract
Intact pollen 5
5
Intact pollen
0
0
450 500 600 700
450 500 600 700
Wavelength (nm)
Wavelength (nm)
(units of optical density)
1.0 Oenothera biennis Plantago major Philadelphus grandiflorus

1.0
Absorbance
Water extract Water extract

0.5 0.5 0.5 Water extract
0 0 0
300 400 300 400 300 400 500 600
Fluorescence(relative units)
Water extract
10 10 150 Water extract
Water extract
Ethanol extract
100 Ethanol
Ethanol extract intact
5 Intact pollen 5 extract
extract
Intact pollen 50
0 0 0
450 500 600 700 450 500 600 450 500 550 600 650
Wavelength (nm) Wavelength (nm) Wavelength (nm)
Fig. 4.10 The absorbance and fluorescence (excitation at 360–380 nm) spectra of the intact accep-
tor pollens and water or ethanol extracts from the microspores (Source: Roshchina et al. (2009a,
b))
The water extracts from pollen had large absorbance maxima at 260–280 or 350–
360 nm (flavonoids) and small absorbance maxima at 550 and 590 nm (anthocyanins),
while in their emission maxima blue (440–460 nm) or blue-green lightening (500 nm)
occurred in many flavonoids. The ethanol-soluble fractions showed the absorbance
maxima that have shorter wavelength at 430 nm (usually peculiar to terpenoids), but
their emission varied – maxima at 580–600 nm (peculiar to alkaloids or carotenoids)
and maxima at wavelengths longer than 600 nm may belong to azulenes and pro-
azulenes (Roshchina et al. 1995; Roshchina 2008). As for the absorbance maxima
at 580–590 nm of water samples, they appear to belong to some alkaloids or antho-
cyanins (as in Philadelphus, Hippeastrum, Knautia), but in Hypericum perforatum,
to red pigment anthraquinone hypericin (Brockmann et al. 1950). Ethanol extracts
lacked green emission but had expressed maxima at 592 nm (perhaps, flavonoids),
Table 4.5 The maxima (nm) in the absorbance and fluorescence spectra of donor pollens
Fluorescence maxima Fluorescence of intact
Plant spp. Absorbance maxima (nm) pollen excited by light at
pollen (nm) Excitation Emission 360–380 nm
Acacia dealbata
Water extract 297, 327, 334 310 398 430–460
Yellow 313, 346 310 430
ethanol
extract
Calendula officinalis
Water extract 260 360 405 460, 520, 580
Yellow 270, 307, 356, 419, 443, 360 402
ethanol 447, 457, 461, 469,
extract 485, 578, 582
Cyperus papyrus
Water extract 230 360 406 430–460
Yellow 226, 322 360 430
ethanol
extract
Hemerocallis fulva
Water extract 229, 279, 321, 328 320 No 550, shoulder
Yellow 315, 335, 362, 425, 434, 360 420 620
ethanol 445, 578, 582
extract
Matricaria chamomilla
Water extract No 350 380, 400 435, 465, 570, 20
Yellow 360, 465, 550, 590 350 420
ethanol
extract
Tagetes patula
Water extract No
Yellow 340–360, 445 340 382 480, 625
ethanol
extract
Zephyranthes
Water extract 265, 315 No
Yellow 258, 319, 327 310 398–400 490, 520
ethanol
extract
and a small peak of 640 nm that is characteristic for azulenes was found in the pollen
(Roshchina et al. 1995). Sesquiterpenoid compounds, peculiar to Artemisia absin-
thium, Artemisia vulgaris, Calendula officinalis, Solidago virgaurea, and Matricaria
chamomilla, contributed to the absorbance spectra with maxima in ultraviolet spectral
region and the maxima of fluorescence in violet and blue (Roshchina 2008). Yellow
pigments were found in the extracts from pollens of Hippeastrum hybridum and
Philadelphus grandiflorus, related to alkaloids and/or carotenoids.
The comparison of fluorescence maxima of extracts with the emission from

intact pollen showed that only part of microspore compounds was extracted by nat-
ural solvents like water and ethanol. But primary pollen drops secreted after moist-
ening also contain the lipid fraction that may be extracted only by more hydrophobic
solvents such as natural oils (Stanley and Linskens 1974). In Sect. 2.1 (LSCM
views), the secretions released by pollens contained very dense material like oil-
containing components. The interactions of some flavonoids and alkaloids (related
to allelochemicals) with oils may change the visible picture of fluorescence and
shift the emission maxima. The complex composition of secretion needs to be esti-
mated with extractions in different solvents.
To understand what components of secretion from pollen donor act as allelo-
chemical and possible mechanism of the effects on pollen acceptor, one needs to
study chemical composition of the excretions and to test individual compounds on
suitable models. Chemical and fluorescent analysis of pollen excretions in a form of
water and ethanol extracts was also studied in some papers (Ortega et al. 1988;
Anaya et al. 1992; Murphy 1992, 2007; Roshchina et al. 2009a, b). Anaya group
(Ortega et al. 1988; Anaya et al. 1992) first demonstrated that main allelochemical
of pollen from Zea mays is phenylacetic acid.
Flavonoid isorhamnetin as seen above was found as main allelopathic agent of
pollen grains from timothy Phleum pratense (Murphy 2007).
In stimulation activity, flavonols quercetin and kaempferol as well as phytohor-
mones indoleacetic acid, gibberellins, and others in concentrations of 10−12–10−9 M
were effective (Kovaleva et al. 2009). Potentially, pollen flavonoid anthocyanins are
also active pigments in the regulation of pollen development (Roshchina et al.
2011). Some components of pollen secretions are various proteins including
enzymes that may regulate pollen germination like peroxidase, catalase, and cholin-
esterase acting on redox and sensory reactions (Roshchina and Melnikova 1998;
Roshchina et al. 2003). Besides liquid secretions, volatiles (Egorov and Egofarova
1971; Dobson et al. 1987, 1996) excreted by pollen may influence pollen germina-
tion, in particular monoterpenes (Roshchina 2007a). Components of volatile excre-
tions such as alcohols and aldehydes linalool, geranial, and neral in concentrations
>10−9 M inhibited the germination of pollen from Hippeastrum hybridum (Roshchina
2007a). The volatiles are able to attract or repel insects (Dobson and Peng 1997).
Pollen from thorn apple, in particular Datura stramonium and Datura alba L.
(Solanaceae), and annual weeds can be allelopathically active due to alkaloids. Gaur
et al. (2005) quantified the allelopathic nature of pollen from weed Datura alba L.
in in vitro and in vivo pollen germination of four target crops, viz., Abelmoschus
esculentus L. (Malvaceae), Brassica juncea L. (Brassicaceae), Lycopersicon escul-
entum Mill., and Solanum melongena L. (Solanaceae). The pollen donor inhibited
the pollen germination in Solanum melongena, but stimulated the pollen germina-
tion in Brassica juncea, reduced the time taken for their germination, and increased
the pollen tube length.
The comparison of fluorescence maxima of extracts with the emission from
intact pollen showed that only part of microspore compounds was extracted by
natural solvents like water and ethanol. But primary pollen drops secreted after
moistening also contain the lipid fraction that may be extracted only by more
hydrophobic solvents such as natural oils (Stanley and Linskens 1974). LSCM
images analyzed with confocal microscopy showed that the secretions released
by pollens contained a very dense material like oil-containing components
(Roshchina et al. 2008, 2009a, b). The interactions of some flavonoids and alka-
loids (related to allelochemicals) with oils may change the visible picture of fluo-
rescence and shift the emission maxima (Roshchina 2008). The complex
composition of secretion needs to be estimated with extractions in different
solvents.
Besides pistil excretions and excretions from foreign pollens, pollen germina-
tion may be regulated by microorganisms inhabiting the cells. Murphy et al. (2009a)
tested for in situ pollen transfers and allelopathic impacts from the putatively pol-
len allelopathic species Alliaria petiolata L. and Hesperis matronalis L. to the sym-
patric, concurrently flowering species Alyssum alyssoides (L.) L., Cardamine
diphylla (Michx.) Willd, Lepidium campestre (L.) R. Br., and Sisymbrium altissi-
mum L. working in a sterilized environment. Authors have shown that fungi already
existing on pollen and stigmas began to grow. Proposing pollen allelopathic
impacts, it is still possible that the declines in pollen tube generation in species
receiving pollen from Alliaria petiolata or Hesperis matronalis may in fact be
caused by immediate exudation from fungal spores carried on pollen or stigmas
rather than pollens.
Modeling of cell–cell interactions under the microscope may consist of adding
the excretions (mainly water or ethanolic extracts enriched in color allelochemical)
or individual allelochemical to the acceptor cell (model) served as a biosensor and
an observation of behavior of cell receiving the chemosignal.
4.3 Modeling with Individual Components of Excretions

as Markers and Dyes
The presence of colored compounds in plant secretions made them potentially use-
ful to study the allelopathic interactions at a cellular level. Modeling of cell–cell
interactions by the use of pigmented and fluorescing components of secretions
released by the donor cells of allelopathic plant has been carried out on unicellular
biosensors – unicellular plant generative and vegetative microspores (Roshchina
et al. 2011a). Isolated cellular organelles and their components may be also tested
in the interactions with exometabolites. Steps of the observations were as
follows:
Step 1. Exometabolite → cell as a whole
Step 2. Exometabolite → isolated organelles
Step 3. Component of organelles (DNA, RNA, proteins, etc.)
According to this scheme, one can directly observe targets of their intracellular
binding and estimate possible mechanisms of action.
4.3 Modeling with Individual Components of Excretions as Markers and Dyes 129
Pelargonidin R Azulene Rutacridone

OH
+
HO O O OH
O
HO
OH Chamazulene N OH
R=H O CH3 O
Pelargonidin - RhamPhy - GluPyr OH
H3C
R = OH
HO HO O O
Cyanidin - RhamPhy - GluPyr OH
O
H3C OH
HO O OH
HO
HO
Fig. 4.11 Colored and fluorescent components of secretions used as dyes
4.3.1 Colored and Fluorescing Compounds
Fluorescent and/or pigmented secondary products released by the allelopathic

plants are suitable biomarkers for microscopic observations (Roshchina et al. 1997a,
b, 1998a, b, c; Roshchina 2004a, b, c, 2005a, b, c, 2008). The compounds may
stimulate or inhibit the germination of pollens, vegetative microspores, and seeds.
Pigmented secretions enriched in red anthocyanins (glucosides of pelargonidin
from Hippeastrum hybridum petals), blue azulenes (synthetic azulene and azulene
extracted from leaves of Artemisia absinthium), and yellow alkaloids (rutacridone
from Ruta graveolens roots) being added to the surface of contacted cells served as
tools to study the mechanisms of cell–cell interactions as possible natural markers
(Roshchina 2007a, 2008, 2012; Roshchina et al. 2011a). Formulae of the com-
pounds chosen are represented in Fig. 4.11. Pelargonidin was represented as a
hydrophilic allelochemical, while azulene and rutacridone are represented as lipo-
philic ones. The absorbance and fluorescence spectra of the compounds are given in
Fig. 4.12. Pelargonidin and azulene fluoresce in blue and red spectral regions, while
rutacridone glucoside which concentrated mainly in roots has bright emission with
maxima at 580–590 nm (Roshchina 2008). To analyze their mechanisms of action,
the experiments were carried out on intact cells, isolated organelles, and separated
components of the organelles (Roshchina et al. 2011a). Models for the testing were
vegetative microspores of Equisetum arvense and pollen grains from Hippeastrum
hybridum, Vallota speciosa, and Haemanthus katherinae (Figs. 4.13 and 4.14).
Treatments with the colored allelochemicals induced various changes in the accep-
tor cells. Either color or the fluorescence (Roshchina and Karnaukhov 2010) or both
color and fluorescence (Roshchina et al. 2011a) were changed after addition of
10−5 M of anthocyanin pelargonidin, sesquiterpene lactone azulene (for comparison,
Pelargonidin Chamazulene Chamazulene

-containing
0.4 535
650 extract
(units of opt.density)
Acidic
0.3
Absobance
590 590
Basic 0.09 605
0.2
Absobance
630
0.1 0.06
550
0
0.03
400 500 600 700
470 620
OH 0
5 +
500 600 700
HO O
(relative units)
Fluorescence
4
O
20 410
OH RhamPyr-GluPyr
455465
3
(relative units)
Fluorescence
485
2 Acidic
basic 10
1
0
400 500 600 700
0
Azulene 400 500
580
(units og opt. density)
Rutacridone
610 655 1.5 O OH

Absobance
Absobance
0.03
1.0 N O OH
CH3
H3C
0.5 HO O
O
HO
HO OH
0
0 400 500 600
500 600 700 150
(relative units)
Fluorescence
100
(relative units)
Fluorescence
10
420 50
5
0
0
500 400 500 600 700
400
Fig. 4.12 Absorbance and fluorescence spectra of colored allelochemicals (10−5 M) used. Acidic
form of anthocyanin pelargonidin, R = H, and basic form of cyanidin, R = OH
also chamazulene-enriched extract from Artemisia absinthium leaves was given), as

well as alkaloid rutacridone.
Staining of vegetative microspores from horsetail Equisetum arvense (as
chloroplast-containing models) with azulene induced blue (excitation 360–380 nm)
or blue-green (excitation 420–430 nm) fluorescence of nucleus and chloroplasts
(i.e., DNA-containing structures) instead of red fluorescence seen in the control
(Fig. 4.13).
The blue fluorescence intensity was also seen in the fluorescence spectra of pol-
len nuclei of H. hybridum and V. speciosa. Pure azulene- and chamazulene-
containing extracts were used in these experiments. The results were similar.
Vegetative microspores E. arvense

Untreated + Azulene + Rutacridone
Pollen H.hybridum Pollen V.speciosa
+ Azulene + Rutacridone + Azulene

200
3
(relative units)
100 150
Fluorescence
20 2 2 2
1 1 100
1
0 0 0
400 600 400 700 400 600
Wavelength (nm)
Fig. 4.13 The staining of the model cells with colored components of secretions. Views of
Equisetum arvense microspores (bar = 20 μm) and fluorescence spectra of pollens from
Hippeastrum hybridum and Vallota speciosa are seen. 1, 2, 3 cell without treatment, nucleus, and
cytoplasm, respectively
Treatment with rutacridone led to binding with nucleus of Hippeastrum hybridum.

The analysis of rutacridone interaction with acceptor cells (Roshchina et al. 2011a)
showed the shift of orange fluorescence with maxima at 580–586 nm to green emis-
sion in the region of nucleus in pollen of H. hybridum (Fig. 4.13) or the nuclei and
chloroplasts of vegetative microspores of Equisetum arvense. Results are similar
when this alkaloid was used to stain isolated nuclei from white petals of Phyladelphus
grandiflorus and Matricaria recutita (Roshchina et al. 2011a). The nuclei fluo-
resced in green and had two fluorescence spectrum maxima at 460–480 nm and
585–590 nm, whereas only one maximum at 585–590 nm was observed in the cyto-
plasm of cells or in the rutacridone solution. When the pollen of H. hybridum was
used as acceptor cells treated with anthocyanin pelargonidin, the nucleus of viable
cells (cells able to germinate and form a pollen tube) stained blue (Roshchina et al.
2011a) that is peculiar to the basic form of the allelochemical anthocyanidin cyani-
din (see Fig. 4.12) where the maximum color is in red at pH 6.0–6.5 with 520–
530 nm maxima and in blue at pH >7.0 with 590–600 nm maxima in the absorbance
spectra. Unlike viable pollen grains, nonviable ones did not change color under
transmitting light of microscope. The green fluorescence of same samples was seen
in luminescence microscope (Roshchina et al. 2011a). The optical slice of viable
pollen under confocal microscopes showed the location of emission only on the
0.951
0.882
0.813
0.743
0.674
0.604
0.535
0.466
0.396
0.327
0.257
0.188
a 0.119
0.049
Fluorescene (relative units) 0.977
0.857
0.737
0.617
0.497
0.377
0.257
b 0.137
450 500 550 600 650 700
Wavelength (nm)
Fig. 4.14 The staining of the model cells of pollen Haemanthus katherinae with pelargonidin.
Views LSCM images (left) and the fluorescence spectra (right) measured by Leica laser-scanning
confocal microscope. (a) View from surface and (b) optical slice after the treatment with pelargo-
nidin (1 mg/ml). Bar = 50 μm
surface that correlates with the lack of fluorescence of the basic form of pelargoni-
din (cyanidin). A red emission (peculiar to acidic form of anthocyanin) could be
seen in the center of nonviable pollen, whereas the surface fluoresced in green.
Figure 4.14 shows the staining of pollen Haemanthus katherinae with pelargoni-
din viewed under Leica laser-scanning confocal microscope. Common view of the
object is the fluorescence in blue green as well as in the cover of pollen. In the
center, blue-emitted nucleus is observed. The anthocyanin was bounded on the sur-
face and demonstrated blue-green fluorescence excited by laser at 405 nm (A), but
on optical slice mainly red fluorescence (B) is seen. The fluorescence spectrum
(excitation by laser at 405 nm) of the pollen surface stained with anthocyanin had a
maximum at 450 nm and a shoulder at 520 nm. Thus, pelargonidin was able to pen-
etrate into cell and color the nucleus.
The experiments were compared with the biological effects of anthocyanin
(Roshchina et al. 2011a). The germination of all microspores used as acceptor cells
from Plantago major, H. hybridum, and Equisetum arvense was stimulated by pel-
argonidin glucosides, which correlated with the results of cell staining for the
nucleus. The stimulation was very high both for P. major pollen tube growth (435 %
of control) and for vegetative microspores of E. arvense (342 % of control), while

the rate of pollen germination for P. major and H. hybridum the stimulation was
about 18 %. The stimulation of the microspore germination in the experiments con-
firms the hypothesis of the significant role of allelochemicals in plant–plant interac-
tions (Brooker 2010). In this aspect, colored compounds were used as histochemical
dyes to determine the cellular mechanisms during the allelopathic interactions
(Roshchina et al. 2011a).
Isolated chloroplasts or nuclei also were used as model systems to analyze mech-
anisms of the allelochemical action (Roshchina 2005b; Roshchina et al. 2011a).
Nuclei were isolated from white petals and well staining with colored exometabo-
lites (Roshchina 2005a, c). Isolated chloroplasts from red clover Trifolium pratense
were treated with the anthocyanin pelargonidin tested to determine whether there
was any interaction between this molecule and these organelles. A decrease in the
organelles’ emission at 640–680 nm was also observed. Isolated nuclei from white
petals of the abovementioned species showed that the changes in color of the model
system correlated with those effects seen on whole cells. The target of the anthocy-
anin action is proposed to be DNA (Sharma and Sharma 1999; Jaldappagari et al.
2008). Thus, the possible mechanism of the anthocyanin interaction with acceptor
cells involves the penetration into the cell and binding to the cell wall and DNA-
containing structures. Pelargonidin penetrates into the acceptor cells and is bounded
with DNA-containing organelles (nuclei and chloroplasts) that change the color or
fluorescence of cellular structures.
The mechanism of action of lipophilic component of secretions such as azulene
differed from hydrophilic anthocyanins (Roshchina et al. 2011a). Azulene modulates
the germination of plant microspores (Roshchina 2004a, b, c) and in particular binds
to the cell wall and the nucleus of pollen grains (Roshchina 2008). Isolated chloro-
plasts treated with azulene increased the blue fluorescence intensity and decreased
the red emission of organelles (Table 4.6). The extracts with chloroform from
chamazulene-containing species A. absinthium and Achillea millefolium that after
drying mixed with water staining nucleus by similar mode inhibited the germination
of pollen from H. hybridum and V. speciosa (Roshchina and Karnaukhov 2010). The
mechanism of action of the sesquiterpene lactones, such as azulenes and proazu-
lenes, with α-methylene-γ-lactone cycle is in their link with the sulfhydryl or thiol
groups of proteins via the reaction between group CH═C―C═O and SH groups of
enzymes (Konovalov 1995). The linkage leads to the inhibition of DNA polymerase,
thymidylate synthetase, and other enzymes and at the end to the blocking in the syn-
thesis of nucleic acid and proteins. Due to α, β-unsaturated γ-lactones, proazulenes
and azulenes, like other sesquiterpene lactones, demonstrate cytotoxic, antimicro-
bial, and phytotoxic activities as well as deterrents of insect feeding (Rodriguez et al.
1976; Konovalov 1995). Their mechanisms of action are associated with the pres-
ence of exocyclic methylene conjugated to a γ-lactone or with the presence of func-
tional groups such as epoxide, hydroxy, chlorohydrin, unsaturated ketone, or o-acyl
adjacent to the α-CH2 of γ-lactone. Azulenes have the protective effects against can-
cer (Racz-Kotilla et al. 1968); thus, it confirms the medicinal observation of antican-
cer features of the genus Artemisia, containing azulenes (Hartwell 1968).
Table 4.6 Fluorescence intensity of isolated chloroplasts from red clover T. pratense treated with
10−5 M of azulene (excitation at 360–380 nm)
Blue emission at 430–460 nm (relative Red emission at 640–680 nm (relative
Treatment units) units)
Control 0.01 ± 0.001 0.21 ± 0.010
+ azulene 0.02 ± 0.002 0.07 ± 0.014
Source: Roshchina et al. (2011a)
Table 4.7 Fluorescence intensity of isolated chloroplasts from red clover T. pratense treated with
10−5 M of rutacridone (excitation at 420–430 nm)
Green emission at 520 nm (relative Red emission at 640–680 nm (relative
Treatment units) units)
Control 0.01 ± 0.001 0.41 ± 0.020
+ rutacridone 0.02 ± 0.002 0.16 ± 0.010
Yellow lipophilic alkaloid rutacridone, active allelochemical of rue roots

(Baumert et al. 1982), may participate in root–root interspecies interactions and
inhibit the germination of seeds from Raphanus sativus (Roshchina 2008). The
staining of isolated chloroplasts with rutacridone decreased their red fluorescence
and enhanced the green emission (Table 4.7) that supports the hypothesis that such
allelochemicals can bind to chloroplasts, possibly at a DNA binding site (Roshchina
2002).
At the molecular level, the interaction of colored and fluorescent exometabolites
with the components of nucleic acids – nucleosides, i.e., pyrimidine and purine
bases bound to ribose or deoxyribose, thymidine, cytosine, adenosine, and guano-
sine as well as ribose (deoxyribose) itself – was studied using the films of com-
pounds on the subject glass (Roshchina et al. 2011a). Modeling with the films (their
solid systems fluoresce in a region at 380–410 nm) was due to several circum-
stances, viz., (a) in nature nucleic acids are in solid form and (b) in the solutions of
the nucleosides and sugars, their fluorescence was very small if any.
If the films of the abovementioned components of nucleic acids were stained
with pelargonidin at a short time exposure (<1 h) only in treatment with ribose
(deoxyribose), the anthocyanin blue fluorescence was quenched. This correlates
with data of whole cell staining, when the nucleus transformed fluorescent
pelargonidin to nonfluorescent cyanidin. The binding site of cyanidin is proposed to
be the phosphorus between two deoxyribose residues of the DNA near the contact
of ribose (deoxyribose) with nucleotides. Anthocyanin–DNA copigmentation com-
plex becomes the protection against oxidative damage (Sharma and Sharma 1999).
Unlike the results with pelargonidin, among the films, staining by azulene, only
guanosine increased the fluorescence at 420–430 nm, while other nucleosides did
not interact. DNA–azulene complex emits in blue green with 440 and 520 nm max-
ima (Roshchina 2008). Guanosine may be considered as a potential target for the
exometabolite. Another picture was after the staining by rutacridone. In this treat-
ment, blue-green emission was observed mainly for cytosine (maxima at 415 and
445 nm in the fluorescence spectra) and in small degree for ribose (smooth peak at
445 nm was seen), while solution of rutacridone had no maxima in the spectral
region. Green (or blue-green) fluorescence of nuclei and pure DNA (Roshchina
2004a, b, c, 2008) was also observed in the presence of rutacridone. Cytosine
appears to be one of the targets for rutacridone in the nucleic acid. Tested allelo-
chemicals may be considered as histochemical dyes and also used for indication of
DNA-containing cellular structures.
Modeling of cell–cell interactions using colored components of secretions and
unicellular models (biosensors) can be useful for the analysis of allelopathic mecha-
nisms at cellular level. Color and fluorescence of acceptor cells served as indicators
of cellular reactions after adding the allelochemicals from donor cells. The com-
pounds bind to the surface and/or penetrate and act on cellular organelles. As shown
with luminescence microscopic techniques, the anthocyanin pelargonidin and ses-
quiterpene lactone azulene reacted both with cellular surface (cell wall) and after
the penetration into the acceptor cell with nucleus and chloroplasts (DNA-containing
organelles). On the other hand, the alkaloid rutacridone reacted mainly with nucleus
and chloroplasts. DNA was the potential target for these allelochemicals, in particu-
lar as sugar ribose (deoxyribose) for pelargonidin, guanosine for azulene, and cyto-
sine for rutacridone. Therefore, these colored allelochemicals could be developed as
useful histochemical dyes to study the cellular mechanisms involved in allelopathic
interactions.
Some groups of colored compounds found in plant excretions such as yellow-
orange alkaloids berberine, sanguinarine, and chelerythrine were also tested as fluo-
rescent dyes and markers (Roshchina 2007b, 2008). Berberine fluoresced in yellow
with a maximum at 540 nm after the contacts with model systems – vegetative and
generative microspores – strengthened emission from cells in the range of
545–575 nm, while chelerythrine emitted in green yellow with maxima at 510 and
540 nm. Both alkaloids stained plasma membrane and cell wall. The target of the
compounds was also the enzyme cholinesterase. Sanguinarine acts in a similar
mode with the abovementioned compounds but fluoresces in orange with a maxi-
mum at 595 nm. Biological responses of the isochinolinic alkaloids were seen as
inhibition of the germination of vegetative and generative microspores – from
Equisetum arvense (I50 1 − 2 × 10−5 M for berberine and chelerythrine) and
Hippeastrum hybridum (I50 5 × 10−7 M for berberine). Inhibition by sanguinarine
was at concentrations <10−6 M. The differences may be due to the different chemical
structures – the number of five carbon heterocycles and the position of benzole
rings.
4.3.2 Fluorescing Colorless Compounds
Fluorescing colorless compounds of plant secretions also may be markers and dyes
in the analysis of the interactions of contacting cells (Roshchina 2008; Roshchina
et al. 2008). For this aim, compounds, which are nonfluorescent out of cell but
Table 4.8 Effects of components of secretions on the fluorescence and germination of microspores
Fluorescence (color/maxima in nm)
Cellular component
Component of In interaction with microspores target of fluorescent
plant secretion Pure compound E. arvense H. hybridum staining
Alkaloids
Atropine Blue/415 Blue/465–470 Blue/465–470 ChR in Plm
Casuarine Blue/475 Green/500 Blue/475 Nucleus, Glc
Colchicine Blue/440 Blue/475 Green/530 Tubulin in
microtubules
Glaucine Blue/440 Blue-green/440, Blue green/440, Nucleus, ChE, Plm,
510 500 CW
d-Tubocurarine Blue/415 Blue-green/440, Blue/465–475 ChR, Plm
510
Yohimbine Blue/455 Blue-green/440, Blue/475 AdR, Plm
510
Sesquiterpene lactones
Artemisinin Blue/440 Blue-green/450, Blue/440 Nucleus,
530 chloroplasts
Gaillardine Blue/420 Blue-green/450, Blue Nucleus
530 green/495–
500
Lactones from microorganisms
Brefeldin A Violet blue/405 Blue 405 Cytoplasm
Cytochalasin B Violet Blue 420 Cytoplasmic fibrils
blue/405–420
Plm plasmalemma, CW cell wall, ChE cholinesterase, ChR cholinoreceptor, AdR adrenoreceptor,
Glc glucosidase
become lightening after the contact with cell model, suit too. Table 4.8 includes
information about fluorescent individual components of plant secretions, mainly
alkaloids and sesquiterpene lactones which can bind with certain cellular compart-
ment. If the compounds (hydrophilic compounds) do not penetrate into cells, they
may bind cellular components on the surface. In particular, alkaloids atropine,
d-tubocurarine, and yohimbine link with receptors to acetylcholine or catechol-
amines in plasma membrane as we have seen in Chap. 3 (Sect. 3.1.1.1).
If the secretory component (lipophilic compound or with higher lipophilicity)
penetrates into cell, it binds with certain compartment or cytoplasmic structure.
Some alkaloids such as casuarine from plants of Casuarina genus or glaucine from
species of Glaucium genus may penetrate and fluoresce in different cellular com-
partments, mainly in nuclei (Roshchina 2005b, 2008). Among the lipophilic com-
pounds are blue-fluorescing sesquiterpene lactones artemisinin and gaillardine able
to be markers of nuclei. Possible targets appear to be nucleolar proteins. Moreover,
artemisinin may bind with DNA in both nuclei and chloroplasts as shown in vegeta-
tive microspores of Equisetum arvense (Roshchina 2005b, c, 2008). Besides, cer-
tain compounds demonstrate a specificity to bind cytoplasmic proteins, for example,
colchicine binds contractile protein of microtubules tubulin. Cytoskeleton may play

a role in the signaling and in responses on external stimuli such as nucleus migra-
tion and division and chromatin structures (Baluška and Barlow 1993).
Cholinesterase location in the cell acceptor may be also determined by staining
with glaucine (Table 4.8) or yellow berberine (Roshchina 2007b, 2008). The enzyme
activity serves as the test not only for plant analysis in the experiments with excre-
tions but also for the research of intercellular contacts of different organisms. For
example, slime which is a secretion of planarians demonstrated cholinesterase
activity sensitive to alkaloid eserine (Li 2008). Insecticidal activity of many exome-
tabolites has perspectives to be tested on various insects, in particular on aphid
cholinesterase (Blackman and Eastop 1984). Changes in esterases released are also
observed in response to blast infection (Muarlidharan et al. 1996).
One could compare the effects of binding with the biological response such as
germination of acceptor model. Alkaloids from allelopathically active species
known as medicinal drugs were also tested on the germination of vegetative and
generative microspores (Roshchina 2004a, b, c, 2007a, b). Inhibition in most cases
(30–80 %) was observed. Colchicine is secreted from the species of Colchicum
genus, which binds contractile protein tubulin and prevents the mitosis. Isochinolinic
alkaloid glaucine inhibited the germination of vegetative microspores but did not
affect pollen germination. Colchicine and casuarine, although having another chem-
ical structure, also depressed the germination of the microspores (Roshchina 2005b,
2008).
Objects of interest are also lactones from the excretions of microorganisms, in
particular of fungi that fluoresce in violet with a maximum at 405 nm or blue with a
maximum at 430–440 nm. Compounds being nonfluorescent or weak fluorescent in
a solution out of cell model and nevertheless emitting after contact with the cell
should be included in the problem too. For example, cytochalasin B, cell-permeable
fungal toxin from Wormhosporium dematiodeum, which inhibits cell division by
blocking the actin polymerization and formation of contractile actomyosin micro-
filaments, inhibits the germination of microspores (Roshchina 2005a). The com-
pound may fluoresce just as it penetrates into cell and marks the actin filaments
(Roshchina 2005a, b, c). Figure 4.15 shows the example of the interaction of actin
(globular and fibrillar forms) isolated from rabbit muscle with cytochalasin B
(Roshchina and Vikhlyantsev 2008). G- and F-actin as well as cytochalasin B itself
in solutions weakly fluoresced under the excitation of ultraviolet light. But in
mixtures of F-actin–cytochalasin (the cytochalasin concentrations >5 × 10−5 M), the
fluorescence with maxima at 410–415 and 430–435 nm increases in comparison
with the emission of the separate actin or cytochalasin. G-actin had no such reac-
tion. Other proteins, in particular bovine serum albumin, had no similar reaction
with cytochalasin B. This shows that in intact cells cytochalasin B binding with
fibrillar actin may be seen as fluorescence of some actin-bounded compartments –
actin filaments. The mechanism of the emission arising is unknown yet, and one of
the hypotheses proclaims the participation of redox reactions.
Alkaloids colchicines from plants that belonged to genera Colchicium, Merendra,
Ramond, and Gloriosa were able to bind and fluoresce in the cells. It is known that
The excitation spectra The emission spectra

(at 425 nm emission) (excitation 360 nm)
40
60
5 F-actin +
50 20
cytochalasin
3
40 Cytochalasin
5
0
30 –7 –6 –5 –4 –3
4 Cytochalasin B (Ig M)
3
20 4
2
10
1 2 1
0
270 310 350 390 380 400 420 440 460
Fig. 4.15 The fluorescence excitation (left) and emission (middle) spectra of actin treated with
cytochalasin B (Source: Roshchina and Vikhlyantsev (2008)). 1 and 2 G and F forms of actin
(0.4 mg/ml); 3 cytochalasin B 5 × 10−4 M; 4 and 5 F-actin + cytochalasin B 5 × 10−5 and 5 × 10−4 M.
Right the fluorescence intensity curves of the mixture F-actin with different concentrations of
cytochalasin B (fluorescence at 435 nm excited by UV light at 360 nm). G- and F-actin globular
and fibrillar forms
the colchicine solutions, if excited by UV light, fluoresce, and the light emission
increases at the contact with protein tubulin (Croteau and Leblanc 1978;
Bhattacharyya et al. 1986). The ability of colchicine to fluoresce in solutions with
purified tubulin, mainly in monomer form, has been described earlier (Bhattacharyya
et al. 1986). The promotion of the drug fluorescence at the colchicine–tubulin inter-
action appears to relate to the B ring of colchicine, a determinant recognized by the
binding site on tubulin. Especially N-acyl group of the B ring may participate. The
emitting ability of cytochalasin B is not clear yet. The emission of a pure compound
was ~300 times lesser. So this tubulin-binding agent, which blocks its polymerization
in microtubules, may bind with monomer forms of the contractile protein that are in
the environment of the microtubules.
The fluorescence spectra from the models – microspores of Equisetum arvense
and Hippeastrum hybridum treated with the anticontractile exometabolites – are
represented in Fig. 4.16. The ethanol (10−5 M) solutions of cytochalasin B or colchi-
cine fluoresce with a maximum at 430 or 460 nm, respectively. The dilution of the
stock solution with water medium up to a concentration of 10−5 M led to the shifts
in the maxima: two maxima at 448 and 479 nm for cytochalasin B and one maxi-
mum at 430–440 nm for colchicine were observed. But the diluted solutions (10−5–
10−7 M) had small emission on the object glasses, if any, as will be seen below.
These solutions were added to emission. Cytochalasin B-treated cells emit more
CH2
HO MeO
O O
HO A B N-Me
O
MeO
NH
MeO C
O
OMe
Cytochalasin B Colchicine
Out cell
15 30
10 20
5 20
0 0
400 450 500 550 400 450 500 550
Within cell
Equissetum arvense
5 5
0 0
Hippeastrum hybridum
4
5
3 3 2
2 1
1
0
400 500 600 700 400 500 600 700
Fig. 4.16 The fluorescent spectra of pure cytochalasin B and colchicine (unbroken line, ethanol,
and broken line, ethanol–water 1:1,000 solutions) and after their interaction with the 10−6 M solu-
tions with vegetative microspores from Equisetum arvense or pollen from Hippeastrum hybridum
(Adapted from Roshchina (2005b, 2006b)). Upper solutions outside the cell; unbroken line,
(10−5 M) in ethanol; broken line stock 10−3 M solution diluted by water to a concentration (10−5 M).
Lower (left) thin unbroken line, solution of anticontractile agent on the subject glass; broken line,
the untreated cell; thick unbroken line, the cell treated with 10−6 M of anticontractile agent.
Excitation at 360–380 nm
brightly in cytoplasm where actin filaments are concentrated. The enhanced fluores-
cence in colchicine-treated cells is seen well near nuclei and in the Equisetum
arvense microspores – also near the chloroplasts. At LSCM images (Roshchina
et al. 2007a, b), there were the brightly lightening nuclei, belonging to both vegeta-
tive cell of pollen and spermia (maximum of the emission 520–530 nm) in pollen
tube as well as the strands of cytoplasm along the plasma membrane, near the cell
wall (maximum at 475 nm and shoulder at 540 nm). This emission shows the sites
of the tubulin accumulation. The bright lightening is clearly seen on LSCM images
for some parts of the colchicine-treated pollen tube. Blue-green fluorescence
(excited by blue laser beam 458 nm) of the vegetative microspores took place. But
especially bright lightening was seen in elaters of vegetative microspores from
Equisetum arvense in the presence of colchicine. Elaters are motile, contractile
derivatives of the spore exine (the cover layer), which serve for the spore anchoring
to the substrate, in particular to soil. Thus, microtubules are not only near the cel-
lular surface of the spore but in the elaters too. Perhaps, the fluorescent sites are
microtubules. In particular, nuclei emitted intensively due to the fluorescing com-
plex “colchicine–tubulin” of the microtubules, which are located around these
organelles.
One mechanism of action of the abovementioned exometabolites from plants and
fungi when plant growth retardation appears is realized through the inhibition of
cellular motility. The plant microspores treated with cytochalasin B were depressed
in their germination and had the enhanced blue fluorescence at 420–460 nm,
expressing the possible binding of the inhibitor of the actin polymerization as
described in our earlier publication (Roshchina 2005a, b, c). Clear effect on pollen
was seen (Chen et al. 2001a, b). Cytochalasin B produced by Helminthosporium
dematioideum and Phoma exigua is found in tissues of potato gangrene (Scott et al.
1975). It was proposed that the gangrene and “silver scuff” formed by various spe-
cies of Helminthosporium and Phoma genera being a part of usual food induce
anencephaly and spina bifida in humans like Phytophthora infestans does. Scott
et al. (1975) supposed cytochalasin B to be a teratogen in vivo. Thus, there is a
bridge between plant, fungi, and animal organisms that are in mutual interactions.
Today, cell biologists proclaim these features of biocenosis (Wayne 2009).
Other mechanisms of action related to membranous agents may have only poten-
tial to be dyes and probes yet. For example, brefeldin A from fungi Penicillium
brefeldianum fluoresces in violet with a maximum at 405 nm and blocks protein
transport into the Golgi compartment, decomposing its structure. The target of
action is the domain containing GTPase (Satiat-Jeunemaitre et al. 1996). The field
of research of similar markers, perhaps, should include dyes within fungal organ-
isms in the future.
Chapter 5
Application of Models in Pharmacology,
Medicine, and Ecology
Models used for fundamental studies of plant excretory function (Chaps. 1, 2, 3, and 4)
may also be applied for express analysis in medicine and pharmacology as well as
toxicology linked with ecology, agriculture, and forestry. First of all plant secretions
are a base for natural drugs analyzed in pharmacology from ancient times. Today it
also has become a part of pharmacological botany. Botanical medicine, mainly, is
considered in microscopic analysis of actively secreting medicinal plant species (see
fundamental book Upton et al. 2011) and learned as the necessary discipline in
medicinal courses of universities (Murav’eva 1981; Samylina and Anosova 2010).
Handbooks dealt with the wide field of medicinal herbs, including analysis of their
biological activity, have been published (Golovkin et al. 2001; Duke 2002, 2003;
Polya 2003; Ebadi 2007). Studies in allelopathic relations in biocenosis become
linked with the pharmacology because most allelochemicals as secretory products
are drugs (Roshchina et al. 2011a, b). Moreover, secretions enriched in allelochemi-
cals may be biological controllers of pathogens in nature (Inderjit and Mukerji 2006).
Pharmacologists have started to use cellular models in animal physiology for
the testing of new drugs because the objects permit us to avoid vivisection and to
see the cellular development. Moreover, the main principles and modes of action
for many compounds, peculiar to mammalians such as acetylcholine and biogenic
amines, are also similar in simple, more primitive species, embryos, and even uni-
cellular organisms (Buznikov 1967, 1987, 1990; Wessler et al. 2001; Roshchina
1991, 2001a, 2010; Kawashima et al. 2007). In these systems substances earlier
known as neurotransmitters are met in every living organism and play roles of
regulators of various functions (Grando et al. 2012), especially well seen on ani-
mal embryonal cells and mammalian nonneuronal cells (Buznikov 1967, 1990,
2007; Buznikov et al. 1996; Wessler et al. 2001, 2013; Wessler and Kirkpatrick
2008; Kawashima et al. 2012). In other words, the presence of main neurotrans-
mitter compounds and sensitivity to the substances of nonneuronal animal cells as
well as plants and microorganisms (Roshchina 1991, 2001a, 2010; Lyte and Ernst
1992; Lyte and Bailey 1997; Lyte et al. 1997; Oleskin et al. 1998a, b, 2010; Oleskin
2012) may be a base for using simple cellular models. In this system, there are
main components necessary in the analysis of contacts between organisms and in
142 5 Application of Models in Pharmacology, Medicine, and Ecology
testing biologically active compounds. Similar models are applied on sea urchin
and larvae in toxicological and therapeutic aims (Buznikov 1983; Buznikov et al.
2003, 2007, 2013). Escherichia coli as a bacterial model was also represented (Lyte
and Ernst 1992, 1993). Recently, plants are believed to be models for the studies
of human pathogenesis because bacterial pathogens demonstrated many common
disease mechanisms in both plants and animals (Guttman 2004). Moreover, they use
common means of attachment, secretions (mainly extracellular polysaccharides and
special effectors), and genetic regulation.
Plant cell has a preference in this field because vivisection and complex bio-
chemical and expensive manipulations are not required, and, in most cases, the
material is received easily. Moreover, unlike small mammalian cells, plant cells are
so big that they can be seen using an ordinary microscope at objective magnitude
×10, 20, or 40. If the researcher studies plant secretions as an origin of natural
drugs, the fluorescent secretions are also well seen in luminescence microscope
(see Chaps. 1 and 2). Another purpose may be achieved if there is a need to test
the biological activity of certain drugs or raw material containing such a drug on
special cell biosensors (see Chaps. 3 and 4). The investigations are of interest for
ecologists too because natural biosensors such as insects–pollinators may recog-
nize that flowers differ in their sexual dimorphism, basing on blossom excretions
(Ashman et al. 2000) or preferable food (Dobson et al. 1987; Knudsen et al. 1993;
Dobson and Peng 1997). Moreover, pollen as a natural biosensor is sensitive to
the pollution, especially to ozone and reactive oxygen species (Roshchina and
Roshchina 2003), and pollen exine surface has antioxidant properties (Smirnova
et al. 2012). The mechanisms of the relations appear to be applied to environment
monitoring.
In cell biology selection and finding of new cell-based biosensors represents
an important problem of biophysics. Biosensors are analytical systems consisting
of sensitive biological detection systems and elements. Cellular sensors maintain
the natural surroundings for structures where the investigated reactions ensure
high performance and stability. The criteria for the selection of cellular models
as biosensors shall be as follows: (1) the reaction should occur rapidly (a time
of response within 1–2 h), (2) the reaction should be sensitive to small doses of
the test substance, and (3) method definitions should be readily available as bio-
chemical (such as changing the color or absorption) or biophysical test (change in
absorbance or fluorescence) of compounds. Finding of such cellular models should
include a simple and relatively cheap biosensor (change of absorption or fluores-
cence compounds, etc.) and sensitive and suitable test systems for laboratory and
field conditions. Unlike mammalians which have small cells and need vivisection,
fixation, and store of tissual samples, plant cell models overcome these things. To
develop biosensors based on cellular systems, you need to know which cells and
their reactions have the required sensitivity. Preferably one needs objects that can
be suitable for manipulation under a microscope. Special tasks require a simi-
lar model, capable of easily monitoring cultivation in the simplest conditions and
sometimes prolonged growth in artificial media at special conditions. When you
5.1 Unicellular Models in the Analysis of Effects of Pharmaceuticals 143
select the cell models, a preference should be given to the most sensitive to various
factors or especially to the factor analyzed.
Recently, special attention is paid to plant sites, which are sensory reactions
common to all living organisms. Such objects can replace costly laboratory animals
and, in addition, prevent difficulties from arising in vivisection. Good examples are
(1) green algae Chara australis and C. corallina as an alternative for the analysis of
ion channels and biosensor for pharmacologically important compounds instead of
oocyte from frog, Xenopus laevis, the use of which is linked to disruption of cells,
special storage conditions, and cultivation (Lühring 1986; Lühring and Witzemann
1995) and (2) cells of flowers of carnations and chrysanthemums (both minced and
intact tissue portions which convert biochemical substrates into volatile products)
in the manufacture of ammonia-sensitive electrodes, sensors in the presence of urea
and certain amino acids (Uchiyama and Rechnitz 1987). Among the single-cell sys-
tems for the same purposes are vegetative microspores of Equisetum arvense and
pollen of various species which are used for reproduction (see Chap. 3). Below we
shall represent some examples.
5.1 Unicellular Models in the Analysis of Effects

of Pharmaceuticals and Allelochemicals
Plant models could be applied to study the effects of raw pharmaceutical plant mate-
rial or individual components of plant excretions from the origin that often con-
tain allelochemicals which also may be drugs. Analysis of test reactions includes
the observation of cell development, enzyme (cholinesterase, in particular) activ-
ity, fluorescent analysis of drugs, and the observation of their penetration or non-
penetration into cells. In most cases plant microspores are suitable as unicellular
models. Some similar experiments were described in earlier publications (Roshchina
and Karnaukhov 2010; Roshchina et al. 2011a).
5.1.1 Testing on Microspores
Fluorescence as primary reaction and more distant integral reaction such as ger-
mination on the addition of the biologically active compounds are the simplest
approaches to know the toxic characteristics of the raw material tested and indi-
vidual drug/allelochemical. The fluorescence spectra and the emission intensity of
vegetative microspores of Equisetum arvense permit the estimation of such param-
eters as the rate of fluorescence and relative germination (see Chap. 1). Figure 5.1
represents data with the treatment of the spores with 0.5 ml of water or ethanol
extracts from medicinal plant species Symphytum officinale (10 g roots +20 ml of
solvent). Ratios of microspores and extract (w/v) added were 1:1, 1:10, and 1:100.
The effects were dependent on the solvent of extract and time of the exposure.
a b
Control
150
Fluorescence (% of control)
20
Water extract ethanolic extract
3.15 ± 0.4
100 10
Amount of cells
50 0
20 Gallic acid
0
1:1
1:1
1 : 10
1 : 10
1 : 100
1 : 100
1.2 ± 0.2
10
Concentration of extract (w/v)

0
0 2 4
I 680 (relative units)
Fig. 5.1 The effects of raw pharmacological material and individual drug on fluorescence of the
vegetative microspores from Equisetum arvense. (a) Plant extracts from roots of Symphytum offi-
cinale, (b) cell histograms of the emission intensity (I 680) at 680 nm after addition of gallic acid
10–5 M. Excitation 360–380 nm. Numerals are the total sum of the emission from 100 cells
(Source: Roshchina et al. (2011c) and unpublished data of Roshchina 2011)
Ethanol extracts demonstrated inhibitory activity, while water extracts are effective
only in small concentrations. The secretory cells of Symphytum officinale contain
alkaloids, among which is cyanoglossin – alkaloid, which fluoresces at 460–480 nm
(Roshchina and Melnikova 1995), and flavonoid rutin (Golovkin et al. 2001) also
can contribute in the emission. Besides alkaloids, the species contains saponins.
Moreover, slime secretions of the plant species contain tannins, which have a green
fluorescence with maximum at 520–530 nm. The impressive wound-healing prop-
erties of Symphytum are partially due to the presence of allantoin which promotes
cell proliferation (perhaps, this is a basis for some of its wound-healing properties).
All comfrey species contain hepatotoxic pyrrolizidine alkaloids (Betz et al. 1994).
These alkaloids, for instance, lasiocarpine, as well as chlorogenic and caffeic acids
found in Symphytum (Golovkin et al. 2001) may also give a contribution in the
secretion emission, and they also can inhibit the germination of the microspores.
Individual compounds such as drug tannin gallic acid which met in plants dam-
aged by certain insects and fluorescing in green also decreased amount of cells
fluoresced in red. It is due to binding of the compound on the cell wall (Roshchina
et al. 2011c). Histograms shift to left in a comparison with control (green emis-
sion prevails) and total sum of the emission decreased sharply. Tannins may also
be inhibitors (Table 5.1). In comparison the results dealt with the testing of some
natural and artificial drugs on the germination of vegetative microspores as biosen-
sors. One could see that tannins gallic and valoneic acids have inhibitory activity
due to their anti-inflammatory and viscosity features and the antitumor drugs unlike
Table 5.1 The effects of medicinal substances on the germination of the vegetative microspores
from Equisetum arvense
Natural drugs I50 or S 50 Artificial drugs Effect (%) or I50
Coumarin (S50) 10−7 Bisacodyl 10–5 M (I) 20–25 %
Gallic acid (I50) 8 × 10−7 Clofelin (I50) 5 × 10−6
Valoneic acid (I50) 10−6 Enalapril 10–5 M (I) 40 %
I – or S – concentrations induced inhibition or stimulation
Table 5.2 The fluorescence Variant I520–540 I640–680

intensity of the vegetative
Control 0.08 ± 0.005 3.23 ± 0.6
microspores of horsetail
Equisetum arvense after Root extract 0.23 ± 0.02 2.97 ± 0.3
treatment with (1: 10) water Petal extract 0.08 ± 0.008 1.51 ± 0.25
extracts from root and flower
petal secretory cells of
Tagetes patula L. (excitation
by the light 420 = 430 nm)
coumarins stimulated the reaction (Mashkovskii 2005). In groups of artificial drugs,

only clofelin which decreased arterial hypertension demonstrated similar inhibitory
effect in contrast with enalapril, also an antihypertensive drug, and bisacodyl, a
purgative (laxative) pharmaceutical.
Among allelochemicals of the genus Nicotiana, there are many alkaloids that
make the plants allelopathically active. The leachates from root and leaves of
Nicotiana alata inhibited both the water uptake and the germination of the micro-
spores (Roshchina 2008). This reaction may be due to binding of alkaloids that
acted on cholinoreceptors. The addition of acetylcholine ester did not change the
depression level (in variant with root leachate) or restore the rate of germination up
to a control level (in the case of leaf leachate). Therefore, irreversible and revers-
ible effects of acetylcholine demonstrate the possible site (cholinoreceptor related
to ion channels) of binding of the allelochemicals from leachates. Extracts from
many pharmacologically valuable plants may bind with dopamine receptors as well
(Luedtke et al. 2003).
Extracts from secretory cells of allelopathically active species may also change
the fluorescence of the tests such as the vegetative microspores of horsetail
Equisetum arvense. One of the examples is in Table 5.2 for water extracts from of
Aztec marigold Tagetes patula L. (Asteraceae). Extracts from the roots and flowers
of the species decreased the red fluorescence of the vegetative microspores as cell
acceptor at 640–680 nm. It appears to be because the oxidative processes related to
chlorophyll may occur. Only extract from root, perhaps due to inclusion of tannin
ellagic acid or flavonoids patuletin and luteolin (Golovkin et al. 2001), stimulated
the green fluorescence. Simultaneously, the root extract depressed the horsetail
development up to 30–50 % of control. The extracts include the secretions from root
secretory cells and petals of Tagetes patula. Among the cells are those that fluoresce
in green yellow and red. They also differ in the emission intensity measured by dou-
ble-beam microspectrofluorometer. The most intensive green fluorescence is seen
for green-fluorescing root cells, unlike the red- or brownish-fluorescing surrounding
Table 5.3 The sensitivity of horsetail Equisetum arvense microspores as biosensors to the antiviral
drugs from plant origins
Drug Chemical nature Plant drug origin I50 (М)
Alpisarin Flavonoid derivative Leaves of Mangifera sp. 10−6
(tetraoxyxanthone)
Flacoside Flavonoid Leaves of Phellodendron amurense No inhibition
I50 – concentration which inhibited the microspore germination by 50 %
OH
OH
OH OH
O OH O
HO O O
OH OH
OH
HO
100 HO O OH
HO
O
80 Alpisarin 20 Flacoside
60
40 10
20
0 0
400 500 600 400 500 600
Fig. 5.2 The fluorescence spectra of alpisarin and flacoside in water solutions. Excitation 380 nm
cells. The difference appears to be due to the different compositions of the emitted
cells. Green-yellow emission may be related with polyacetylenes and/or flavonoids
or tannin ellagic acid solved in oils (Golovkin et al. 2001). In the composition of
the allelopathically active and anthelmintic plant, there are polyacetylenes such as
α-tertienyl and flavonoids patuletin and luteolin (Golovkin et al. 2001). The com-
pounds may contribute in the visible oxidation in the root secretory cells.
Possible fields of application among antibacterial and antiviral drugs of plant ori-
gin on already investigated microspores of horsetail Equisetum arvense are shown
in Table 5.3. Here are examples of testing dealt with some antiviral drugs of plant
origin (Papuashvili et al. 2001; Mashkovskii 2005; Register of Medical Drugs of
Russia 2009) on germination vegetative spore of horsetail. Highest sensitivity of
vegetative microspores to the compounds tested (effect at smaller concentration)
was for the flavonoid alpisarin, while flavonoid flacoside has no inhibition activity
at all. But both drugs fluoresce (Fig. 5.2). Alpisarin has maxima 435 and 560 nm,
while flacoside 435 and 485 nm. Their input into pollen Hippeastrum hybridum
showed the green fluorescence in the nucleus which appear to be a target of the
drugs. Unlike these natural compounds, water solutions of hipporhamine (complex
of tannins from leaves of Hippophae sp.) and donelvine (complex quinones from
roots of Salvia sp.) emit in blue with maximum 480–485 nm, whereas eucalimine
(complex of terpenoids from leaves of Eucalyptus viminalis) is mainly in blue green
(Roshchina et al. 2011c). Staining of the pollen from H. hybridum demonstrated
Table 5.4 The germination Variant Index of germination

of the vegetative microspores
Control 0.220 ± 0.05
of horsetail Equisetum
arvense after treatment with +extract (1:1,000) 0.435 ± 0.05
water extracts (w/v) from (1:100) 0.335 ± 0.08
glands of Humulus lupulus (1:10) 0.250 ± 0.08
Table 5.5 Staining of the model acceptors with extracts from plants containing azulenes
Fluorescing
Donor plant Cell acceptor compartment Color of
species Extract from of plant species after staining emission
Artemisia Leaves (ethanol Pollen from Hippeastrum Nucleus Blue
absintium 1:10 W/v) hybridum L and Vallota
speciosa (L. f.)T. Durand
et Schinz
Artemisia Leaves (ethanol Vegetative microspores from Nucleus, Blue
absintium 1:10 W/v) Equisetum arvense L. chloroplasts
Achillea Leaves (ethanol Pollen from Vallota speciosa Nucleus Blue
millefolium 1:10 W/v)
Azulene Ethanol Pollen from Hippeastrum Nucleus Blue
(10–5 M) (10–5 M) hybridum
Azulene Ethanol Vegetative microspores from Nucleus, Blue
(10–5 M) (10–5 M) Equisetum arvense chloroplasts
enhanced (two- to four fold in a comparison with control) blue-green fluorescence,

concentrating in cell wall. All these drugs inhibited the germination of microspores
from Equisetum arvense.
Bitter acids and essential oils in water and ethanol extracts from glands (lupu-
lins) of Humulus lupulus are known as ancient natural sedative drug. In Table 5.4
there are experiments with the water extracts on the germination of microspores of
Equisetum arvense. Lupulin extracts stimulate the process that confirms favorable
action on living cells.
5.1.2 Staining of Cells with Extracts
Examples of staining of the unicellular biosensors (acceptors of exometabolites)

with the plant secretions including azulenes (Konovalov 1995; Roshchina et al.
1995; Roshchina 2008) as allelochemicals and natural drugs are represented in
this section. The blue pigments possess antibacterial and antihistaminic features
(see Chap. 3). On the basis of natural azulenes, new antiulcer pharmaceuticals are
made (Yanagisawa et al. 1990). Table 5.5 shows that, like pure azulene, 10 min
ethanol extracts from the compartments became blue fluorescent. Of special inter-
est are the changes of red emission of chloroplasts (680 nm maximum of chlo-
rophyll) to blue emission due to the staining with azulene (or even proazulenes
in the extracts). Sesquiterpene lactones such as azulenes have antibacterial and
antihistaminic effects, and the new azulene-containing preparations from leaves of
Solidago virgaurea or Picea pungens (Roshchina 1999a, b, 2001b) are perspective

for such analysis. Among sesquiterpene lactones, helenin, telekin, and artemisinin
suppressed cell growth in concentrations of 1–2 μg/ml, which exceed the cytotoxic-
ity of cyclophosphamide, an antineoplastic drug (Spiridonov et al. 2005).
After the treatment of cell acceptors as vegetative microspores of horsetail
Equisetum arvense) with the above mentioned antiviral and bactericidal drugs
used in medicine (Kryzhanovsky and Vititnova 2003; Register of Medical drugs
of Russia 2009) such as hipporhamine (complex of tannins from the leaves of
Hippophae rhamnoides L. (Elaeagnaceae)) and eucalimine (sum of triterpenoids
and terpenoid aldehydophenols from ethanolic extract leaves of Eucalyptus vimina-
lis Labill (Myrtaceae)) the green emission is concentrated on the surface of studied
cells. Histograms (Roshchina et al. 2011c) showed the shift to the left of the amount
of the cell acceptors emitted in red. From the experiments, we can see the changes
in the fluorescence of cell wall under treatment with tannins (possible interactions
with phenols on the surface of vegetative microspores), and the red fluorescence
(mainly related to chlorophyll) emitted by chloroplasts is also altered. Tannins may
mask the visible red fluorescence or affect the chloroplast function directly or indi-
rectly. Terpenoids of eucalimine (weakly lighten in the dimethyl sulfoxide solution)
stimulated both the green emission of the cell wall (the interaction with phenolic
constituents) and red emission of chloroplasts. Like tannins terpenoid drug eucali-
mine binds with cell walls too and stains it, inducing green fluorescence at the exci-
tation with UV light of 360–380 nm.
5.2 Cellular Targets and Mechanisms of Action of Drugs

and Allelochemicals
In order to know whether the exometabolite (allelochemical, drug) penetrates or

not into the cell and the possible mechanism of the action on the cellular level, one
could use the own fluorescence features of the compound tested (see Chap. 4, Sect.
4.3.1). Excretions may interact with plasmatic membranes. If they cannot penetrate
into the cell interior, their effects on cell acceptor may be related to the influence on
the sensors (some enzymes, e.g., cholinesterases), ion channels, or receptors located
in plasmalemma. If compound penetrates into cells, possible targets are intracellular
compartments.
5.2.1 The Surface Targets
Since compound does not penetrate into cells, possible targets are surface cel-
lular compartment cell wall, free space, and plasma membrane. Pharmaceuticals
act mainly on receptors, ion channels, and enzymes as sensors in plasmalemma of
model cells.
5.2 Cellular Targets and Mechanisms of Action of Drugs and Allelochemicals 149
5.2.1.1 Reception and Ion Channels
Main events occurred on the plasmalemma related to the opening of ion channels
and the binding with the receptors, which can direct the channel opening or are
related to the initiation of the chemosignal transfer into a cell via secondary mes-
sengers. Today, the theory of the reception is applied to such chemosignals known
as hormones and neuromediators such as acetylcholine and biogenic amines dopa-
mine, noradrenaline, and serotonin (Roshchina 2001a) as well as reactive oxygen
species such as free radicals and peroxides (Roshchina and Roshchina 2003). It
supposes the presence of sensitive receptors to the compounds. The receptions of
compounds on the cell surface may be studied by the use of pharmacological drugs
from plant origin, which can serve as agonists or antagonists of the hormones and
neuromediators. It is carried out by the estimation of the influence of the com-
pounds on the germination, water uptake, and autofluorescence of the plant cells as
well as on the fluorescence of these compounds within a cell (Chaps. 3 and 4). The
site of the compound binding is well seen in this case, and then their effects on the
microspores germination may be explained.
Opening of Na+/K+ channels may be regulated by the receptor of acetylcho-
line. Some antineurotransmitter compounds simultaneously may block the chan-
nels and cholinoreceptors of nicotinic type (Roshchina 2001a). As described
earlier (Roshchina 2006a), blockers of ion channels such as α-bungarotoxin and
d-tubocurarine (bind with nicotinic type of cholinoreceptors and block Na+/K+
channels), tetraethylammonium (blocker of K+ channels), and verapamil (blocker
of Ca2+ channels) depress the germination of vegetative and generative microspores.
Roshchina (2009b) showed that the water uptake by dry vegetative microspores of
horsetail Equisetum arvense also was inhibited. Similar picture was also observed
earlier for pollen of Hippeastrum hybridum (Roshchina 2001a).
Often exometabolites (drugs, allelochemicals) per se have, if any, weak fluores-
cence in visible spectral region and need to use some of their functional analogues
or fluorescent agonists and antagonists (Roshchina et al. 2003; Roshchina 2008).
Fluorescent exometabolite may be bound in the surface of cell if it binds with the
receptor of acetylcholine or biogenic amine. In contrast with the preliminary treat-
ment with the neurotransmitter, the fluorescence in the cellular compartment may
be small or even absent due to the binding in the same site. Acetylcholine and
biogenic amines may increase the germination of microspores and seeds due to
the first binding with special receptors of plasmalemma (Roshchina 2001a). Plant
toxins (antagonists and agonists of the compounds) can also bind in the same site
of the membrane, preventing the linkage of the ligands and so their effect. The
antineurotransmitter compounds are usually alkaloids, well-known medical drugs
d-tubocurarine found in Chondrodendron tomentosum Ruiz et Pav. and yohimbine
found in Corynanthe yohimbe K. Schum. Allelochemical d-tubocurarine, antagonist
of acetylcholine, forms blue-fluorescing ring on the cell models’ surface (Roshchina
2004a, b, c, 2007a, b) that shows its binding with surface receptors (see Chap. 3).
The same picture was observed when yohimbine, antagonist of noradrenaline, was
used (Fig. 3.5). Both acetylcholine and noradrenaline, previously being added to the
cells analyzed, prevent the surface binding of d-tubocurarine or yohimbine as seen

from the decrease of the fluorescence intensity.
5.2.1.2 Cholinesterase as Sensor
Among the surface enzymes’ main sensor is cholinesterase that hydrolyzes acetyl-
choline and so stops the irritation induced by the compound. Alkaloids contained in
many plant excretions (Atta-un-Rahman et al. 2001) may inhibit the enzyme, influ-
encing on the cholinergic mechanism of signaling (Roshchina 2001a). The cholin-
esterase activity of the excretions of cell acceptor is stimulated or inhibited by the
excretions or individual drug.
Cholinesterase activity was measured according to the modified biochemical
methods developed for crude preparations, using Ellman reagent 5,5″-dithio-bis(p-
nitrobenzoic acid) or its red analogue 2,2-dithio-bis-(p-phenyleneazo)-bis-(1-
oxy-8-chlorine-3,6)-disulfur acid in the form of sodium salt, which interact with
thiocholine (see method in Budantsev and Roshchina 2007). The rate of hydrolysis
of acetylthiocholine is measured based on the absorbance at 412 nm of yellow prod-
uct of the reaction of the substrate with Ellman reagent. Exposure with the enzyme
preparation is 45–60 min at 22 °C.
Anticholinesterase activity of exometabolites may also be analyzed histochemi-
cally with red analogue of Ellman reagent 2,2-dithio-bis-(p-phenyleneazo)-bis-(1-
oxy-8-chlorine-3,6)-disulfur acid in the form of sodium salt, which interacts with
thiocholine (see method in Roshchina 2001a). The red color of the dye in this case
converts to blue. Model pollen grains of Hippeastrum hybridum, Haemanthus kath-
erinae, and Vallota speciosa are more suitable for this aim.
Cholinesterases often blocked by some alkaloids, in particular physostigmine
from the roots of Physostigma venenosum Balf. from Fabaceae (Roshchina 2001a;
Budantsev and Roshchina 2007). Alkaloids sanguinarine and glaucine inhibited
the rate of the hydrolysis of acetylthiocholine (ATHCh) by the water extracts from
Equisetum arvense (Budantsev and Roshchina 2007) that is similar with the effects
of special inhibitors of cholinesterases neostigmine and physostigmine.
In these cases the enzyme activity of cell acceptor may be recommended to deter-
mine in the presence of the excretions from cell donor (Budantsev and Roshchina
2007) or histochemically (Roshchina 2001a, 2007a). The possible analysis of the
exometabolite effects on ion channels and receptors may be carried out for the
understanding of the mechanisms of action of active matter from the excretion
tested.
Vegetative microspores of horsetail Equisetum arvense as cell model with vis-
ible cholinesterase activity and high sensitivity to small concentrations of biologi-
cally active substances can be used in the analysis for the presence of pesticides,
toxicants, medical drugs such as alkaloids, and other compounds. For example,
among the alkaloids used in medicine, known drugs from plant origin alkaloids
berberine and glaucine demonstrated the highest anticholinesterase activity in
horsetail as biosensor (Budantsev and Roshchina 2004, 2007) and therefore may
5.2 Cellular Targets and Mechanisms of Action of Drugs and Allelochemicals 151
have application in pharmacology. The sensitivity of these models as potential

biosensors based on cholinesterase is comparable with similar goals of animal
blood products and even brain tissue (Roshchina 2001a, 2004b; Budantsev and
Roshchina 2004, 2005, 2007). New natural anticholinesterase preparations such
as containing alkaloids, coumarins, terpenoids and other compounds (Filho et al.
2006), may be tested on the cells.
Sensor system may include such enzymes as dopamine-β-hydroxylase which
inhibited by hypericins (I50 = 5 μmol/l) and flavonoids (quercetin, amentoflavone,
kaempferol) which I50 was more than 50 μmol/l (Denke et al. 2000). Hypericin
was bounded on the pollen surface, being not within cell (Roshchina et al. 2008;
Roshchina 2008).
5.2.2 Targets Within Cell Model
Within cell targets of exometabolites penetrated are intracellular compartments.

Well-seen sites of binding are DNA-containing organelles and contractile elements
which are stained with colored or fluorescent exometabolites. In cytoplasm, actin
and tubulin are often stained by fluorescent secretions (see Sect. 4.3.2).
5.2.2.1 DNA-Containing Organelles
Some sesquiterpene lactones may mark fluorescent the DNA-containing structures,

mainly nuclei and chloroplasts (Roshchina 2004a, 2005a, b, 2008). One of the
examples is given on Fig. 5.3 for sesquiterpene lactone gaillardine from allelopathi-
cally active species Gaillardia pulchella Foug (Asteraceae). It inhibits the pollen
germination in vivo and in vitro as well as germination of seeds (Roshchina 2004a
and unpublished data of the author, 2004). Active part of the germination inhibition
is proposed as a five-chain ring shown on the picture formula that can interact with
SH groups of amino acids and amino acid residues in proteins (Rodriguez et al.
1976). Gaillardine increased the cellular fluorescence at 500 nm (Fig. 5.3); espe-
cially blue-green emission is seen in the nucleus of the vegetative and generative
(pollen) microspores studied. In vegetative microspores of Equisetum arvense, the
blue-green fluorescence, instead of red emission of chlorophyll, is seen in chloro-
plasts after the staining with gaillardine (Roshchina 2004a). The maxima of the
substance shifted in longer wavelength region in a comparison with pure allelo-
chemical. Nuclei and chloroplasts may be colored if the compound penetrates into
cells like azulene. Pollen tube is considered as a cellular model for the study of the
movement of organelles, in particular nuclei. Dry, non-developed pollen has one
nucleus (the so-called nucleus of vegetative cell). Then, during the germination,
the cell can form (within itself) two new cells named as gametes or spermia, which
include new nuclei. In developed pollens and vegetative microspores, we saw vari-
ous pictures of fluorescence changes.
15 Gaillardine
Me
CH3 Coo H
10 O
3
O
5 1 2 H
HO
Me CH2
0
400 500 600 700
Wavelength (nm)
Fig. 5.3 The fluorescence spectra of allelochemical gaillardine (10−6 M) staining nucleus in the
center of the Hippeastrum hybridum pollen as model. (1) Solution of pure gaillardine in ethanol;
(2 and 3) pollen without a treatment and after staining with the allelochemical. Broken line in the
formula shows the acting site for the germination triggering
The target of allelochemical action within cellular organelle could also be ana-
lyzed in the model system with isolated pure compounds, in particular from nucleus.
As seen from publication (Roshchina 2004a, 2008), gaillardine also changes the
fluorescence at the interaction with the components of nucleus – nucleic acids and
proteins. The effect is in the increase of the emission in blue spectral region (maxi-
mum 480 nm) in the mixture of lactone with DNA and albumin. Moreover, there
are no similar effects in variant with RNA. Unlike gaillardine, sesquiterpene lac-
tone azulene may also stimulate fluorescence in variants both with one DNA and
DNA + albumin (Roshchina 2004a). Therefore, the target of the drug action within
nucleus may be also analyzed. The interaction of such drugs as anthocyanin pelar-
gonidin, alkaloid rutacridone, and azulene with component of nucleic acid nucle-
osides and ribose or deoxyribose was considered (Roshchina et al. 2011a). The
information has been represented in Sect. 4.3.1.
5.2.2.2 Contractile Proteins
Among molecular probes of natural origin that inhibit cellular motility are fungal
products cytochalasins (from fungi Helminthosporium dematioideum) and antitu-
mor drugs (which block the mitosis in cancer cells) such as colchicines from plants
that belong to genera Colchicium and others (Haugland 2000). One of the targets
for the intracellular binding of the fluorescent compounds may be contractile pro-
teins – actin for cytochalasin B and tubulin for colchicines. The effects of the com-
pounds on the plant unicellular models have been described in Sect. 4.3.2. Both
exometabolites are the secretory products and may interact with sensitive living cells
inhibiting a polymerization of the contractile proteins. These substances, when it
has been released by cell donor, can interact with cell acceptor and demonstrate the
fluorescence in plant microspores, sensitive unicellular models (Roshchina 2005b, c,
5.3 Application of Models in the Environmental Monitoring 153
2006a, b). The promotion of the drug fluorescence at the colchicine–tubulin interac-
tion appears to relate to the B ring of colchicine, a determinant recognized by the
binding site on tubulin. Especially N-acyl group of the B ring may participate.
In the experiments with developed microspores, we have also received more
information about the connection between the spore fluorescence and the ability to
germinate after the binding of the anticontractile agents with the dry microspores
(Roshchina 2004b, 2005b, c). Cytochalasin B, which blocks the actin polym-
erization, and antitubulin agents colchicine and vinblastine in concentration of
10−7–10−5 M inhibited the germination of both microspores studied by 50–70 % of
control. The cell division was stopped in many vegetative microspores, and pollen
tubes of the pollen were rarely developed or did not develop at all. Pollen tube is
considered as a cellular model for the study of the non-muscle motility due to the
possibility of seeing the movement of organelles, in particular nuclei (Chen et al.
2001b; Vidali et al. 2001).
The analysis of drug effects on cellular contractile protein cytochemical visual-
ization is also possible when dopamine serves as exometabolite or drug (Shubina
et al. 2009, 2011; Parnyshkova et al. 2012). Concentrations of dopamine 10−4 M and
higher ones may induce the accumulation of globular actin preventing its polym-
erization in cancer cells. Actin of cancer cell is considered as therapeutic target for
dopamine. Moreover the antitumor effect of dopamine appears to connect with the
ability of dopamine to disaggregate amyloids.
5.3 Application of Models in the Environmental Monitoring
The role of biosensors in the monitoring of environment becomes significant in the

last years of global pollution and heating of atmosphere (see books eds by Greppi
and Mura 2011 or Preedy and Patel 2012). Similar model systems for the express
analysis are necessary for every laboratory. Especially valuable line is an application
of whole cells as biosensors (Buchinger et al. 2010; Buchinger and Reifferscheid
2012; Kiilerich-Pedersen and Rozlosnik 2012). In this aspect the use of plant veg-
etative spores and pollens as biosensors of different sensitivity to various chemicals
has perspectives. Among the ecological problems, a new direction dealts with trans-
genic plants, which are origins of biopharmaceuticals (Hefferon 2009) and insec-
ticidal proteins (Konrad et al. 2008) influencing all living organisms. For example,
there is hazard with crop plants that are pollinated by bees which bring the toxins to
nectar (precursor of honey) and transgenic elements in pollen (Konrad et al. 2008).
The toxins appear to be tested on the model biosensors.
As sensitive reactions, the germination of microspores, their autofluorescence,
and enzymatic (cholinesterase) activity were considered (Roshchina 2006a, 2007a,
b, 2008; Speranza et al. 2011). The mechanisms of the sensitivity in chemosignal
processes with the participation of biologically active compounds – neurotrans-
mitters (acetylcholine, dopamine, noradrenaline, serotonin, histamine) and natural
oxidants (ozone, free radicals and peroxides) – were discussed. Plant microspores
Table 5.6 Effects of pollutants on the development of plant microspores

Germination
Vegetative microspores Pollen of Hippeastrum
Pollutant of Equisetum arvense hybridum
Ozone 0.05–0.2 μl/l Weak stimulation Weak stimulation
(2 × 10−9 М − 8 × 10−9 М) or no effect
Ozone >0.2 μl/l (>8 × 10−9 М) Inhibition Inhibition
Hydrogen peroxide 10−6–10−4 М Stimulation Stimulation
Tert-butyl peroxide 10−6–10−4 М Stimulation Weak stimulation
may be used in laboratory practice as the objects of the study and in testing for the
determination of the components of sensory systems.
Components of environmental pollutions such as high concentrations of ozone, CO2,
NO, NO2, and other pollutants in air may be analyzed on the biosensors. Chichiricco
and Picozzi (2007) reported that fumigation of the gases induced reversible inhibition
of the pollen germination and stigma penetration in Crocus vernus. The increased con-
centration of allergens – pollens and spores of microorganisms in the environment –
also greatly changes the color and intensity of the fluorescence (Pöhlker et al. 2011,
2012; Saari et al. 2013) that can also be displayed in environmental monitoring.
The presence of pollen as allergen carriers as well as bacterial and fungal spores
in the atmosphere represents significant problem in both medicine and ecology. In
these aspects specific fluorescence of the cells serves as indicator of their occur-
rence (Knaus et al. 2013; Saari et al. 2013). Bacterial pollution leads to appearance
of toxic concentrations of histamine in food (Fernández et al. 2006).
Autofluorescence of the microspores allows analysis of the presence of reactive
oxygen species, such as ozone and peroxides (Roshchina and Karnaukhov 1999;
Roshchina and Melnikova 2001; Roshchina and Roshchina 2003; Roshchina et al.
2003). This effect is seen under a luminescence microscope after the microspore
fumigation by ozone in concentrations of 0.05–0.1 μl/l because it is easy to observe
how their fluorescence changes color. Vegetative microspores of Equisetum arvense
change red fluorescence to yellow (Roshchina and Roshchina 2003). Under chronic
exposures to ozone, pollen of Philadelphus grandiflorus, Hemerocallis fulva,
Papaver orientale, or Petunia hybrida enriched in carotenoids and/or anthocyanins
also changes the color of their fluorescence excited by UV light (Roshchina 2003;
Roshchina and Roshchina 2003). This has important implications for environmental
monitoring of ozone because the biosensors serve as biological indicators of ozone
pollution in the urban environment.
Forming in the atmosphere, ozone and other reactive forms of oxygen such as
peroxides (hydrogen peroxide, tert-butyl hydroperoxide, tert-butyl peroxide) in low
concentrations stimulate the germination of both types of microspores (Table 5.6).
Some of the low ozone concentrations can stimulate germination vegetative
microspores, but at high doses the gas usually acts as an inhibitor. The free radi-
cals, superoxide anion radical in particular, arise from the interaction of ozone with
cellular components and then quickly turn into peroxides. The peroxides cause a
stimulation of microspores’ germination.
5.3 Application of Models in the Environmental Monitoring 155
Table 5.7 The fluorescence % of germinated

intensity (I) at 640–680 nm Variant I640–680 pollens
and germination of the
Control 3.42 ± 0.260 100 ± 7.6
vegetative microspores of
horsetail Equisetum arvense +methylviologen
after treatment with 10−6 M 2.49 ± 0.184 72.8 ± 5.4
methylviologen (excitation 10−5 M 1.89 ± 0.115 55.2 ± 3.30
by the light 420 = 430 nm) 10−4 M 1.69 ± 0.205 49.4 ± 6.10
In high doses (>0.1 μl/l), ozone can cause and change the color of the pollen of
plants that are well marked in their fluorescence spectra. For example, there has
been a shift in the fluorescence maximum in yellow in pollen of Philadelphus gran-
diflorus, enriched with carotenoids, and in the blue region of the spectrum, as well
as the appearance of the orange fluorescence instead of green emission in pollen of
Hippeastrum hybridum (Roshchina and Roshchina 2003). In addition to ozone, the
fluorescence of the pollen of different plants undergoes changes by peroxides.
Ozonated compounds (0.1 μl/L ozone during 1 h) such as released allelochemical
esculetin and β-carotene (Roshchina 2001b) have various effects – from less inhibi-
tory effect for first compound to more depressive for a second one. Photochemical
smog produces ozone in high concentration (>0.01 μl/L) that may induce the changes
in metabolic processes, as seen at ageing of polymers. At the influence of ozone or
other factors that induced peroxidation of lipids in cells, fluorescent pigment lipofus-
cin is formed in both plant and animal cells. In pollen and vegetative microspores,
the changes in the autofluorescence related to the lipofuscin are well seen after ozone
fumigation (Roshchina and Karnaukhov 1999; Roshchina and Roshchina 2003).
In a dependence on concentration, ozone and its derivatives may stimulate or
depress the microspore germination and even shift the fluorescence maxima of the
cell (Roshchina and Roshchina 2003). Antioxidants found in the cellular excre-
tions – both low-molecular (phenols, carotenoids, azulenes) and high-molecular
(enzyme peroxidases) – also alter both germination and fluorescence.
Hydrogen peroxide and tert-butyl hydroperoxide caused stimulation of lumines-
cence in pollen of Hippeastrum hybridum, and antioxidant enzyme of peroxidase
reduced the intensity of the glow. In addition, under the action of hydrogen perox-
ide, the total fluorescence and fluorescence maximum in spectrum move to longer
wavelength region. This reaction is inhibited by addition of peroxidase. The influ-
ence of the enzyme superoxide dismutase and superoxide anion radical damage,
was low, although the radical formation from pollen.
Oxidants as pesticides also may play the roles of pollutants. One example is
methylviologen which inhibitory effects are demonstrated in Table 5.7. This herbi-
cide decreases both fluorescence of chlorophyll and the germination of Equisetum
arvense vegetative microspores as models. The mechanism of compound action as
pesticide consists in the strong oxidation of surface and intracellular components.
An important problem in medicine is the indication of pollutants such as aller-
gens or toxic matters in plant secretions. For example, pollen of allergenic plants
releases not only protein allergen (Puc 2003), but also histamine, an active factor
of an allergic reaction (Garcia et al. 1996; Cirkovic et al. 1999). We showed the
histamine occurrence in the pollen excretions (see Chap. 2, Sect. 2.3.2).

Determination of chemical components of food components subjected to stress, for
example, oil glands of oil-containing plants, is also important for human security
(Anderson et al. 1999). Poisonous plant-secreted toxicants are also particularly
important for preventive medicine (Frohne and Pfänder 2005).
Dopamine and noradrenaline, generators of reactive oxygen species, are exo-
metabolites, allelochemicals, and drugs that may regulate the seed or microspores
germination (Roshchina 2001a). An indicator of the oxidative processes in the pres-
ence of the compounds could be the fluorescence. It is also useful in the studies of
redox mechanism of the action (Cruz-Ortega and Anaya 2007) and pharmaceutical
(Spiridonov et al. 2005) effects on the living cells.
The surface of many pollen grains and vegetative microspores are also able
to form and release reactive oxygen species (Roshchina et al. 2003; Roshchina
and Roshchina 2003; Potocky et al. 2007). Reactive oxygen species produced by
NADPH oxidase are needed for normal pollen tube growth (Potocky et al. 2007).
Oxidants such as peroxides, generators of superoxide anion radical dopamine and
noradrenaline, and inhibitors of monoamine oxidases stimulated germination of
the pollen of Hippeastrum hybridum. Opposite effects were shown for antioxidants
(Roshchina 2009a, b): 10−4 M reductant dithionite Na2S2O4, 10−4 M mannitol (scav-
enger of hydroxyl radical), 10−5 M ascorbate (scavenger of superoxide anion radi-
cal), and enzymes (0.6 mg/ml) peroxidase (decomposes peroxides) and superoxide
dismutase (decomposes superoxide anion radical by dismutation).
The application of sensitive single-cell analytic systems (without vivisection
and, in some cases, labor-intensive biochemical tests) may be significant for the
development of the so-called basic environmental units (Rea 1994). Selective sensi-
tivity of plant cells to specific sensors can be used to develop new biosensors.
Conclusion
Plant model systems suitable for microscopic analysis of excretory function were
out of the attention of researchers up to now. Vital state of similar models and appro-
priate spectral and microscopic methods, including various modifications of lumi-
nescence microscopy, microspectrophotometry, and microspectrofluorimetry,
permits to estimate composition of secretions, state of secretory cells, and secretory
process using parameters of absorbance, fluorescence, and histochemical staining.
Due to microscopic and spectral methods, one could quickly analyze on various
models mechanisms of the interactions of cells belonging to different organisms,
biological activity of secretions, and sensor activity of secreting cells. In this book,
there is the experience of the author who for many years searched suitable models
for these purposes and who gives possible algorithms to select similar model sys-
tems in the future. Perhaps, it will be one of the branches of biotechnology in the
field of express-analysis. Nanotechnology might increasingly use the model of
nature as a point of orientation.
New tendency to use cellular models is realized in finding unicellular natural
objects – plant microspores. The possible mechanisms of microspore germina-
tion in pollen allelopathy compared to other spore-forming systems of crypto-
gams, far in evolution from phanerogams, reflect the similar natural technology.
Here, Compounds in the spore secretions may play the role as growth regulators.
Moreover, due to the high biological activity of microspores, their cells are
already used in medicine as the pollen loads collected by bees or to determine the
activity of allelochemicals, many of which are potential or known drugs. In other
words, biomimetic nanotechnology copying nature on a molecular level may be
the origin for new improved plant growth regulators and/or pharmaceuticals;
therefore, our knowledge on cellular mechanisms in allelopathic relations is nec-
essary to achieve this aim.
Allelopathy plays an important role in weed management (Narwal 1999a, b,
2007) and formation of cultural communities (Jacob et al. 2011). This concerned
not only with the interaction between plant and other plants, but between plant and
animal (mainly insects) or plant and microorganisms too. Similar relations may also
be named allelopathic for animal-animal contacts (Surinov et al. 2001). Formation
DOI 10.1007/978-94-017-8786-4, © Springer Science+Business Media Dordrecht 2014
158 Conclusion
of ecosystems has evolutionary history where allelochemical signals in secretions

influence the natural selection of allelopathically active plant species (Inderjit et al.
2011).
To select microscopic models for analysis, a researcher should choose objects in
dependence on the aims of the study. First of all distinguishing models as donors of
secretions and models as acceptors of the secretions is needed. Then the next step of
the investigation is to search both contacted objects that form model system which
contains two individuals interacted.
Both unicellular and multicellular objects may serve as cell-donors of secretions.
In this aspect, the model should be used for analysis in the accumulation of secre-
tory products (named exometabolites if they are released out of the cells) during
development of secretory structures or under the treatment by various factors (from
mineral elements and parasitic infection to pollutants and pesticides). At the cellular
level, the studies are at the beginning and only algorithms of the investigations have
been selected and proposed. The main line in the studies has been in the analysis of
the autofluorescence of secretions and express diagnostics of the individual compo-
nents that prevailed in the secretions (Roshchina 2003, 2008). Analyses of redox-
components of secretions as oxidants and antioxidants and their relations, perhaps,
are perspective in the future. Today, we have only information based on the effects
of ozone and some reactive species of oxygen on some model systems (Roshchina
and Roshchina 2003; Roshchina 2008). Use of the simple plant models permits to
spread the knowledge about the mechanisms of the ozone and other reactive oxygen
species’action to animal cells (these cells are not well seen under microscope unlike
plant ones). Similar oxidative stress inactivates the nicotinic type of cholinoreceptor
(Krishnaswamy and Cooper 2012) and appears to do by the same mode on vegeta-
tive microspores of Equisetum arvense, also sensitive to agonists and antagonists of
acetylcholine (Chap. 3).
Secretory cells of medicinal plants are enriched by pharmacologically useful
compounds, and their conditions determine the value of the raw materials or the
degree of the material preparedness or ready to use (Carr et al. 1970; Balandrin et al.
1985; Following 2002). Pharmacologists paid great attention up to now to the
analysis of morphological and anatomical structure of secretory cells of plants
described as botanical medicine, unlike microscopical express-analysis of secretory
products activity on testable model objects. The secretory products of some plants
might have some antibodies that are important to maintain the immunity of the ani-
mal organism (Wycoff 2005). It is also perspective to develop biotechnology for the
production of pharmaceutical preparations on the basis of secondary metabolites of
plants and microorganisms that also may serve as herbicides (Cutler 1999; Narwal
1999a, b).
Qualitative, semiquantitative, and quantitative methods may be used for the
study of the effects on models which serve as acceptors of exometabolites and
participate in intercellular contacts of different organisms. In similar cases, model
systems serve as biosensors of secretions released by other own cells or cells of
other organisms (animal, fungal, or microbial). Especially, it is of interest in the
field of understanding the role of biosensors for universal exometabolites – acetyl-
choline and biogenic amines which are found in every living organism. Plant
References 159
microspores and pigmented cells were recommended as such model – acceptors in

this book. The systems were sensitive to compounds named as neurotransmitters
acetylcholine and biogenic amines as well as to their agonists and antagonists
(mainly plant secretory alkaloids that show the presence of cholino- and adrenore-
ceptors in such cells). Cholinesterase activity (enzymatic hydrolysis of acetylcho-
line) was also shown in the systems. Own studies and practice of the author
demonstrate narrow stripe of the use of new models yet, and one could propose
their search in the following years.
Simple model systems are also needed for analysis of the disease and protectory
mechanisms (Di Carlo 2012), especially of interest are whole cell biosensors
(Buchinger and Reiffersscheid 2012; Buchinger et al. 2013). Acid and base trau-
matic proteins were found in drops of pollen secretions in hybrid Taxus (O’Leary
et al. 2007). Similar proteins are also proposed to participate in egg cell protection
against various damages. Therefore, there is a necessity to look for models of plant–
herbivore association: a singular case of oviposition-induced synomone for an egg
parasitoid (Conti et al. 2010) or of the analysis of protectory mechanisms in phyto-
pathogenicity (Bender 2003; González-Teuber et al. 2010). This aspect of modeling
and this significance for phytopathology are likely also of interest in the future.
Models of both types – donor and acceptors of secretions – are the base for new
model systems in the analysis of mutual interactions. It may be not only plant–plant
relations but also relations between plants and aphids or plants and planarians, as
examples which have been shown here. Most productive models of plant–plant con-
tacts appear to be pollen–pollen or pollen–pistil systems. Pollen allelopathy, medic-
inal models for natural drugs, and toxicant testing are ways for practical applications
of the models. This monograph represents some approaches and algorithms for
analysis of similar systems.
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Subject Index
A phenol carbonic, 15
Absinthin, 73–74, 85–86 phenylacetic, 127
Absorbance, 1–10, 25, 28, 38–40, 65–67, p-hydroxycinnamoyl, 37
96–97, 100, 125–126, 129–131, protocatechuic, 62
142, 150 syringic, 62
Absorbance spectra valoneic, 109, 144, 145
of petal, 8–9, 39–40, 131 Actin, 20, 83, 84, 87–89, 105–106, 137–138,
of pollen, 8, 126–127, 131 140, 151–153
Acetylcholine Actin microfilaments, 137
agonists, 93, 96–97, 103, 113, 149 Action potential, 77, 97, 100
antagonists, 80–83, 97, 103, 104, 106, Adenylate cyclase, 41, 97
113, 149 Adhesins, 119
effects, 96, 145 Adrenaline (epinephrine), 53, 68, 69, 71, 76
in stinging trichomes, 51, 53–54, 70–71 Adrenoreceptors, 81, 115, 136
Acetylcholinesterase Agonists
effects, 30 of acetylcholine, 93, 96–97, 103, 113
in pollen, 30 of biogenic amines, 103–104, 149
in traps, 66 of dopamine, 113
Acetylthiocholine, 28, 30, 92, 150 of histamine, 103
Acid of noradrenaline, 145, 149
abscisic, 68, 77 of serotonin, 145, 149
acetic, 118 Alkaloid-containing plants, 28–29, 36, 43–48,
butanoic (butyric), 118 62, 68, 72–73, 78, 80, 92–93, 95–96,
caffeic, 15, 63, 90, 91, 144 113–115, 135–138, 144–145
caffeoylquinic, 42 Alkaloid-containing structures, 44, 84–85
caproic, 118 Alkaloids
chelidonic, 44 acridone, 10, 11, 48
chlorogenic, 15, 63, 76, 90–91, 144 biological effects, 78, 137
coumaric, 62 carbolinic, xv, 47
ellagic, 145–146 fluorescence, 10–12, 15, 136, 143–144
ferulic, 15, 62 in glandular trichomes, 62
gallic, 109, 144, 145 isoquinaline, 11
indoleacetic, 119, 127 in latex, 43–45
isovaleric, 118 in pistil stigma exudates, 101–103, 124
palmitic, 118 pyrrolizidine, 144
pelargonic, 118 in vacuoles, 10, 21, 36–37, 43–45, 95
DOI 10.1007/978-94-017-8786-4, © Springer Science+Business Media Dordrecht 2014
188 Subject Index
Allantoin, 144 in cytodiagnostics, 18–19

Allelochemicals, 12–14, 28, 42, 52, 62, 85–86, of glands, 16, 58, 61–62, 66
92, 94–95, 99–100, 106, 110, 120, 124, of models, 2, 8
127–131, 133–135, 141, 143–156 at pest invasion, 116–117
Allelopathic relationship, 27, 52, 95, 102, of pollen, 33, 104, 107, 123–124
107, 141 of secretion and secretory cell, 9–12
Allelopathy, 52, 73, 101, 109–110, 119–128 of sporangia, 21–22
Allergens, 154–155 Auxins, 70
Allergic reactions, 53, 57, 72, 155 Azulenes
Allicin, 78 fluorescence, 27, 129–130, 134
Alliin, 78 in pollen, 27, 52, 74, 91, 108, 125–126,
Aloin, 48 130, 134, 153, 157
Alpisarin(e), 146 as probe, 12
Amines, 1, 25, 51–53, 68–72, 79–80, 83,
94–95, 103–106, 110, 114–115, 119,
136, 141, 149 B
Amylase, 28, 30, 75, 86–88 Bacteria, xvi, xvii, 10, 11, 55, 70–72, 87,
Antagonists 111, 116, 118–119, 142, 154
of acetylcholine, 81, 96, 97, 103–104, Bacteriochlorophyll, 10, 11
113, 149 Benzoquinone, xv, 4
of biogenic amines, 80, 83, 103–104, 149 Berberine, 12, 19, 21, 43, 84, 86, 92, 135,
of dopamine, 81 137, 150
of histamine, 81, 82, 103–104 Betacyanins, 4, 8, 10, 19, 21, 26, 37–38,
of noradrenaline, 149 41, 74, 95, 98
of serotonin, 149 Betalains, 4, 21, 26, 37–39, 67, 74, 95
Antheridia, 24, 85 Betaxanthins, 4, 8, 10, 32, 37–38
Anthocyanidines, 41 Biogenic amines, 1, 25, 51–53, 68–72, 80,
Anthocyanins 83, 94, 95, 103–106, 110, 111, 114,
absorbance, 7–9, 38–40, 96, 125, 129 119, 141, 149
effects, 132–133 Biomediators, 51, 69, 77, 78
fluorescence, 9 Biosensors, 1–3, 9, 23, 25, 32, 33, 65–78,
as markers, 12 83, 87, 94–100, 104, 113, 128, 135,
Anthocyanosomes, 39, 41 142–144, 146, 147, 150–151, 153,
Anthraquinones, 4, 51, 95–96, 126 154, 156
Antioxidants, 52, 75–76, 87, 89–91, 103, Biosensors cell-based, 142
106–107, 142, 155–156 Biotechnology, v
Antiviral agent, 146, 148 Bisacodyl, 145
Aphis (ds), 93, 112–113, 137 Boosting secretions, 116
Apomorphine, 113 Brefeldin A, 19, 20, 136, 140
Archegonia, 24 α-Bungarotoxin, 83–85, 87, 105, 149
Arecoline, 97
Artemisinin (e), 85, 136, 148
Ascorbate, 76, 90, 106, 156 C
Astragalin, 63 Cadinene, 73
ATP secretion, 12, 21 Caffeine, 45
Atropine, 43, 73, 85, 97, 104, 113, 136 Calcium, 3, 20, 27, 47, 62, 77, 96, 105, 106
Attractants, 73 crystals, 62
Aurones, 8 silicates, 62
Austricine, 52, 76, 85, 90 cAMP. See CyclicAMP (cAMP)
Autofluorescence Capsaicin, 21, 73, 78
at allelopathic relations, 52, 107 Carboxylesterase, 74
of biosignals, 31 Carene, 52
in cell-cell interactions, 32, 68 Carnivorous plants, 49
of crystals, 62 β-Carotene, 115, 155
Subject Index 189
Carotenoids, 8, 23, 24, 27, 45, 51, 91, 107, Colchicine(s), 11, 44, 84–86, 105, 106,
125, 126, 154, 155 136–140, 152, 153
β-Caryophyllene, 73 Collagen, 10
Casuarine, 84–86, 136, 137 Contacts
Catecholamines bacteria-plant, 116
effects, 55, 89 fungi-plant, 116
histochemical assay, 53 host-parasit, 99
redox-reactions, 89 microorganism-plant, 68, 69, 91–92,
in stinging trichomes, 53, 71 110, 116–119
Cell-cell communications, 10, 99 plant-animal, 68, 69, 110, 111, 115
Cell-cell contacts, 3, 12, 70, 91, 99–140 plant-insect, 74, 111–113
Cellular models, 2, 3, 141, 142, 151, 153 plant-worm, 113–115
Cellulose, 10, 50 pollen-pistil, 65, 76, 91, 99–107, 122
Cell wall fluorescence, 14, 23, 24, 66, pollen-pollen, 101, 107–110
116, 148 root-root, 134
cGMP. See Cyclic GMP (cGMP) Contractile elements and systems, 106, 151
Chalcones, 7, 8, 37 Contractile proteins, 20, 83–84, 87, 104–106,
Chamazulene, 129–130, 133 137, 138, 152–153
Channels for secretions, 77, 83, 89, 100, β-Cubebene, 73
105, 149 Cyanidin, 38, 129, 130, 132, 134
Chelerythrine, 45, 135 Cyanoglossin, 143–144
Chemosignaling, 2, 9, 14, 32, 33, 51–53, 65, Cyclic AMP (cAMP), 41, 77, 83, 97
68–76, 83, 89, 91, 95, 100, 103, 106, Cyclic GMP (cGMP), 75, 77, 83
110, 115, 124, 128, 149, 153 Cyclic nucleotides, 41, 75, 77, 83, 97
Chemotaxis, xvi Cytochalasin B, 84, 85, 105, 106, 136–140,
Chitin, 10, 112, 118 152, 153
Chitinase, xvi, 118 Cytochrome C, 75, 86–88
Chlorophyll Cytochromes, 28
fluorescence, 10, 17, 18, 23, 24, 42, Cytodiagnostics, 18–19, 102, 110
46, 54–55, 66, 80, 94, 112, 148, Cytokinins, 68
151, 155 Cytosine, 134–135
Chloroplasts, 17–19, 22–24, 33, 37, 56, 67,
75, 80, 82, 116, 130, 131, 133–136,
140, 147, 148, 151 D
Choline acetyltransferase, 30, 70, 77 Delphinidin, 38
Cholinesterase Digestive glands, 66
as biomarker, 28 Diosmetin, 15
determination, 92, 96 Diterpenes, 113
effects, 30, 87, 93, 150 Dithionite, 76, 89–91, 156
effects of inhibitors, 150 DNA, 10, 133–136
excretion, 28, 29, 92, 93, 150 DNA-containing organelles, 82, 133, 135,
histochemical staining, 30, 114–115 151–152
inhibitors, 28, 29, 93, 104, 113 DNA polymerase, 133
muscarinic, 97, 113 Donelvine, 146
nicotinic, 71, 83, 96–97, 104–106, 115, Dopachrome, 115
149 Dopamine
in pollen, 30, 87 effects, 80, 89, 95, 153
in pollen-pistil contacts, 91 in fungi and bacteria, 71
in self-incompatibility, 30 redox reactions, 89
in vegetative microspores, 28, 30, 52, 75, release, 115
87, 93, 150 Dopamine-β-hydroxylase, 151
Citral, 52, 85, 86 Dopaxanthin, 38
Clofelin, 84, 85, 144–145 d-Tubocurarine, 73, 81–83, 87, 94, 96, 97,
Codeine, 45 104, 106, 114, 115, 136, 149, 150
190 Subject Index
E Fluorescence spectra
Ecdysone, 51, 76, 89–91 of idioblasts, 14, 47–49
Elastin, 10, 55 of latex, 46, 47
Elaters, 22–24, 140 of laticifer, 46, 47
Electrophoresis of pollen proteins, 28 of pollen, 16, 101, 102, 104, 106, 124–126,
Emergence, 3, 31, 51, 53, 55, 56, 129–132, 139, 152, 155
58, 116 of trichomes, 17
Enalapril, 145 Fluorophores, 10, 11, 19, 32, 53, 116, 118
Eserine (physostigmine), 28–30, 92, 93, Fordianins, 6
137, 150 Forskolin, 97
Essential oils, xiv, xv, xvii, 4–5, 45, 49, Free radical reactions, 74, 75, 101, 103,
85, 147 106, 149
Esterase, 28, 30, 51, 66, 74, 78, 92, 93, 116, Free radicals, 32, 74, 103, 106, 107, 124,
127, 137 149, 153
Eucalimine, 146, 148 Fructokinase, 52
Euphorbin, 46 Fruit formation, 85, 101–103, 105, 106,
Exine of pollen, 100, 107, 142 121, 122
Exocytosis, 12, 20, 21, 36, 37, 50, 74, 87 Fungal invasion, 87, 111, 117
Exosomes, 50 Fungalysins, 118
Fungi
fluorescence, 10, 19, 85, 111, 116, 118,
F 137, 140, 152, 154
β-Farnesene, 73 Furanocoumarins, 48, 49
Ferritin, 75, 87, 88
Fertilization, 24, 57, 65, 71, 73, 100–103,
110, 119, 122 G
Flacosid (e), 93, 146 Gaillardine, 52, 76, 85, 90, 91, 136,
Flavins, 7, 10, 11, 24, 107 151, 152
Flavonoids Galactan, 50
absorbance, 4, 7, 10, 37 GEP. See Green-fluorescent protein (GEP)
fluorescence, 7, 10, 11, 15, 17, 20, 37, 48, Geranial, 51, 85, 86, 127
51, 52, 62, 63, 74, 78, 103, 107, 109, Geraniol, 51, 52
124, 125, 127, 144–146 Geranyl acetate, 51
as growth regulators, 51, 52, 68, Gibberellins, 68, 127
78, 103 Glands, 4–7, 13–17, 19, 21, 28, 30, 46, 49, 58,
in salt glands, 49, 62 61, 62, 72, 111, 113, 115, 118, 147
in vacuole, 4, 7, 10, 20, 37, 42, 74 Glandular trichomes (hairs), 5, 13, 17, 49, 61
Flavonols, 8, 109, 127 Glaucine, 84, 86, 92, 136, 137, 150
Flavons, 37, 109 Glyoxylic acid, 53–55, 57, 111
Flavoproteins, 7, 11 Green-fluorescent protein (GEP), 20, 21, 37
Fluorescence Grosshemine, 76, 85, 90, 91
aphid larvae, 112, 113 GTPase, 20, 140
fungi, 10, 11, 19, 85, 111, 116–119, 137, GTPbinding protein, 20
140, 152 Guanosine, 134, 135
insects, 10, 11, 31, 59, 66, 74, 111, 112,
133, 142, 144
of pistil, 19, 32, 78, 99, 101–104, H
106, 107 Helenine, 148
at pollen-pistil contact, 65, 99, 101, 103, Hemicellulose, 50
104, 106, 107 Hemoglobin, 10, 75, 87, 88
probes, 8, 12 Hexokinase, 52, 75, 86–88
proteins, 10, 11, 20, 21, 37 Hipporhamine, 146
technique, 1, 8, 12–19, 23 Histamine
Subject Index 191
in bacteria, 55, 71, 72, 111, 119, 154 Latex, 43–47

effects, 82, 103, 104 Laticifers, 45–47
in fungi, 71, 72, 119 Lectins, 46, 51, 78
histochemical assay, 53, 55, 57 Lethaemoglobin, 87
in pollen, 27, 51–53, 57, 78, 80, 103, Limonene, 52, 78
104, 154, 155 Linalool, 52, 78, 85, 86, 127
in stinging trichomes, 53, 56, 58, 72 Lipases, 66, 74
in vegetative microspores, 51, 52, 56, Lipofuscin, 11, 155
78, 80–83 LSCM. See Laser-scanning confocal
Histaminic receptor, 81–83 microscopy (LSCM)
Histidine, 111 Lucifer yellow, 12, 20
Histochemical dyes, 10, 30, 51, 114, 118, Luminescence microscopy, 3, 9, 10, 12–15,
133, 135 19, 21, 22, 51, 55, 56, 67, 82, 100,
Hydrogen peroxide, 42, 74, 89, 90, 106, 111, 112, 114, 131, 135, 142, 154
107, 124, 154, 155 Lupinine, 43, 44
Hydroxyl radical, 74, 124, 156 Lupulins, 14, 16, 30, 147
Hypericin, 4, 5, 12, 21, 74, 114, 115, Luteolin, 145, 146
125, 151 Lysozyme, 75, 87
Hypha(e), 32, 116
M
I Male sterility, xvi
Idioblasts Malondialdehyde, 51
fluorescence, 45, 48–49 Malvidin, 38, 41
Imidazolyl ethylamine, 55 Malvidin–diglucosides, 41
Inositol triphospate, 77 Mannitol, 76, 89–91
Insect feeding, 133 Melanins, 8
Insecticidal activity, 93, 137 Methylviologen (paraquate), 155
Insect-pollinated species, 53, 59 Microscopy
Insect-pollinator, 31 confocal, 10, 12, 15–19, 24, 25, 40, 62,
Insects, 10, 11, 31, 51, 53, 66, 71–74, 93, 100, 82, 115, 128, 131
101, 103, 111–113, 120, 127, 137, 144 laser-scanning confocal, 3, 9, 10, 12,
Inulicine, 85–86 15, 17, 22, 24, 61, 81, 82, 107, 108,
Ion channels 114, 132
blockers, 83, 84, 87–88, 105–106, 149 luminescence, 3, 9, 10, 12–15, 19, 21, 22,
Iprazide, 75–76, 89, 90 51, 55, 56, 67, 82, 100, 111, 112, 114,
Isolated secretory cells, xv, 20, 36, 131, 135, 142, 154
42–46, 128 optical coherence, 3
Isoquinolines, 10, 45, 53, 54 stereo, 3–7, 102
Isorhamnetin, 124, 127 usual, 3–7, 10, 40, 112, 117
Microspectrofluorimeter
double-beam, 23, 62, 145
K with X-Y recorder, 14
Kaempferol, 15, 51, 63, 89, 127, 151 Microspectrofluorimetry, 3, 10, 12, 14–15,
22, 24, 101
Microspectrophotometers, 8, 15
L Microspectrophotometry, 7–8
Larvae, 72, 73, 112, 115, 142 Models
Laser-scanning confocal microscopy (LSCM), amines, 1, 25, 149
3, 9, 10, 12, 15–17, 22, 24, 25, 47, 61, common scheme, xiii, 37, 128
62, 81, 82, 107–109, 114, 127, 128, enriched in, 1, 13, 14, 16, 25, 47, 69, 110,
132, 140 128, 129, 154
Lasiocarpine, 144 essential oils, 45, 49, 85, 147
192 Subject Index
Models (cont.) O
genetic, 50, 118 Odor, 73, 77, 86, 118
mathematical, vii, xvi Oil, 4, 5, 17, 45, 49, 63, 78, 79, 85, 127, 128,
multicellular, 2, 12, 32, 33, 58–63 146, 147
phenols, 4, 6, 7, 13, 17, 20, 23, 24, 26, ducts, 3, 5, 27, 46
31, 36, 37, 42, 43, 46–52, 58, 62, glands, 27, 156
63, 66, 68, 72, 73, 78, 89, 95, 98, reservoirs, 62
101, 103, 107, 109, 115, 116, 124, Oleoresin, 5, 16, 18
148, 155 o-Phthalic aldehyde, 53, 55–58, 72
pigmented, 1–3, 7, 26, 27, 37, 66, 91, Optical coherence microscopy, 3
128, 129 Oxidants, 14, 54, 75, 87, 89–91, 106–107,
pollen-interaction, 107–110 153, 155, 156
pollen-pollen interaction, 101, 107–110, Ozone, 11, 12, 33, 52, 54, 55, 74–76, 89–92,
120, 124 103, 106, 142, 153–155
secretion into vacuole, 36–45 Ozonides, 124
sensitive to secretions, xiv, 31–33 Ozonolysis, 45
suitable for microscopy, 1
suitable for visualization, 1, 2, 127
unicellular, 2, 12, 21–25, 27, 31, 51, 55, P
92, 143–148, 152 Papaverine, 45, 105, 106
vital, 5, 18, 20, 21, 28, 32, 50, 68, 116 Patuletin, 145, 146
Monoaminooxidase, 28, 90 Pectins, 50
Monoterpenes, xv, 73, 85–86, 112, 127 Pelargonidin, 41, 74, 115, 129–135, 152
Morphine, 44, 45, 113 Peonidin, 41
Mucilage, xv, 28, 50 Pepsin, 74, 75, 86, 87
Muscarine, 97 Peroxidase, 28–30, 36, 42, 75, 76, 78, 86, 87,
Muscarinic cholinoreceptors, 97, 113 91, 106, 107, 124, 127, 155, 156
Mutants, 20 Peroxides, 75, 89, 103, 106, 124, 149,
Myosin, 87, 88 153–156
Myrcene, 52 Pesticides, 99, 112, 150, 155
Pharmaceuticals (drugs), 28, 31, 52, 143–149,
153, 156
Phenol (s)
N effects, 89, 93, 98
NAD(P)H, 10, 11, 115, 156 in excretions, 49, 50, 73, 78, 98, 109, 114
Nanotechnology, xvii, 52 fluorescence, 48, 61, 66, 124
Naphthoquinones, 109 in latex, 46
Nectar, xiv, xv, xvi, 31, 153 at pest invasion, 117
Nectarins, xv in secretory cells, 4, 13, 36, 42, 48, 49, 58,
Nectary, 19, 49, 100 62, 63, 72
Neostigmine (proserine), 28, 30, 92, Phosphatase, 30, 36, 66
104, 150 Phosphodiesterase, 97
Neral, 52, 85, 127 Phospholipase, xv, 50, 77
Neurotransmitters, 1, 27, 32, 51–53, 55, 57, Photosynthesis, 10, 70, 75, 87
68–73, 76, 77, 79–81, 103, 104, 110, Phycobillin, 11
111, 113, 119, 141, 149 Phycoerythrin, 11
Nicotine, 44, 45, 83, 93, 94, 96, 97, 113 Phylloplanins, 74
Nicotinic cholinoreceptors, 83, 97, 104–106, Physostigmine (eserine), 28, 29, 92, 93, 150
115, 149 Pigment secretion, 27, 95
Noradrenaline (norepinephrine) Pinocytosis, 36, 37
effects, 80, 89–91, 106, 156 Pistil
in fungi, 71, 118 fluorescence, 102, 104, 107
Noscapine, 45 secretion, 102
Nucleus (ei), 24–26, 55, 56, 74, 82, 86, 109, stigma, 24, 31, 52, 66, 83, 89, 100–107,
130–137, 140, 146, 151–153 120, 122, 124
Subject Index 193
Pistil-pollen interaction, 76, 91, 100–102 S

Planaria (n), xvi, 27, 93, 111, 113–115, 137 Salivary glands, 71, 72, 111, 118
Platyphylline, 97 Salt-containing cells, 27
Pollen Salt glands, 49, 58, 61, 62
allelopathy, 101, 109, 110, 119–128 Sanguinarine, 45, 84, 92, 135, 150
allergens, 154, 155 Saponins, 63, 144
bee-collected (pollen load), 52 Secretion
as biosensors, 153 external, 35, 36, 49–63
development, 24, 52, 83, 102, 103, 106, intracellular, 21, 35–45
121, 122, 124, 127 intratissual, 21, 35, 45–49
fluorescence, 59, 108–109 transport, 20–21, 36
germination, 26, 32, 52, 73–76, 78, 79, Self-incompatibility, 30, 87
84–90, 100–103, 107, 109–110, Serotonin, 47, 53, 70–72, 76, 80, 94, 95, 103,
120–124, 127–129, 131, 133, 137, 111, 119, 149, 153
151, 156 Sesquiterpene lactones, 12, 51, 52, 73, 81,
loads, 52 85–56, 129, 133, 135, 136, 147, 148,
mixtures, 99, 102, 108–110, 120–124 151, 152
as model, 110, 119, 152 Slime, 49, 51, 66, 78, 93, 103, 115,
Pollen-pistil recognition, 52, 83, 102, 103, 137, 144
105, 106 SOD. See Superoxide dismutase (SOD)
Pollinators, 30–32, 53, 57, 73, 83, 85, β–Solamarine, 15
101–103, 105, 142, 153 Spectrofluorimetry, 3, 10, 12, 14–15, 22, 24,
Pollution, 51, 55, 75, 111, 142, 153–155 94, 101, 102
Polyacetylenes, 10, 145, 146 Spectrophotometry, 7–8
Polysaccharides XVI, 51, 78, 142 Spermia, 24, 140, 151
Proazulenes, 51, 85, 86, 133, 147 Squalene, 118
Protease, 30, 66, 87, 111, 118 Stilbenes, 51, 109
Protein digestive, 66, 95, 111 Stinging emergences (hairs), 51, 53, 54, 56,
Protein kinase, 12, 21 57, 70, 71
Protein phosphatase, 30, 36, 66 Strychnine, 44
Protein secretions, 27–30, 66, 67, 73, 93, Superoxide anion radical, 30, 74–75, 89, 106,
95, 96, 116, 127, 152 124, 154–156
Protoberberine, 44 Superoxide dismutase (SOD), 28–30,
Pterins, 10 75, 76, 87, 89, 91, 106, 107,
124, 155, 156
Q
Quercetin, 48, 49, 63, 127, 151 T
Quinones, 4, 6, 109, 115, 146 Tannins, 11, 109, 144–146, 148
Quinuclidinyl benzylate, 97 Tavegyl (clemastin), 104
Terpenoid-enriched structures, 16, 44, 49, 52,
61, 63, 68, 73, 78, 85, 89, 101, 107,
R 125, 146, 150, 153
Reactive oxygen species (ROS), 11, 12, 20, Terpenoids, 16, 44, 49, 52, 61, 63, 68, 73,
33, 52, 68, 74–76, 89–92, 103, 106, 78, 85, 89, 101, 107, 125–126, 146,
124, 142, 149, 154, 156 150, 153
Repellents, 73, 74, 113 tert-Butyl peroxide, 154, 156
Resin, 7, 17, 45 Testosterone, 51
Rhamnetin, 124 Tetraethylammonium, 83, 85, 97,
Ribonuclease, 30, 66 105, 149
Ribonucleic acid (RNA), 10, 128, 152 Theophylline, 97
Ribose, 36, 134, 135, 152 Titin, 87
RNA. See Ribonucleic acid (RNA) Trypsin, 28, 74–75, 86–88
ROS. See Reactive oxygen species (ROS) Tubulin, 85, 105–106, 137–138, 140,
Rutacridone, 12, 49, 129–131, 134, 135, 152 151–153
194 Subject Index
U Verapamil, 83–85, 87, 105, 149

Uroporphyrin, 115 Vinblastine, 44, 153
Uroporphyrinogen, 10 Vincristine, 44
Vindoline, 44
V
Vacuolar pigments, 4, 7–8, 20–21, 26–27,
W
37–42, 74, 95
Wind-pollinated plants, 53, 57, 59
Vacuolar transport, 20–21, 35–38, 44–45
Worm, 111, 113–115
Vacuole, 4, 7–8, 10, 20–21, 26–27, 35–45, 49,
58, 74, 95, 97
fluorescence, 22–24, 27, 81, 82, 85, 125, Y
131, 136, 138, 139, 144 Yeasts, 111, 118–119
germination, 65–66, 79, 83, 84, 86, 135, Yohimbine, 73, 81–82, 114–115, 136,
137, 144, 149 149–150
Latin Index
A Atriplex sp., 58
Abelmoschus esculentus, 127 Atropa belladonna, 43, 95
Acacia dealbata, 122, 123, 126
Acacia sp., 121
Acer platanoides L., 7, 59 B
Achillea asiatica, 42 Bacteria
Achillea millefolium, 41, 42, 79, 123, 133, 147 Arthrobacter globiformis, 119
Achillea sp., 42 Bacillus subtilis, 71, 119
Agrostis alba, 53 Escherichia coli, 71, 119, 142
Aldrovanda vesiculosa, 67 Klebsiella sp., 72
Alliaria petiolate, 128 Morganella morganii, 72
Allium cepa, 7, 27, 67, 96 Polyangium sp., 119
Allium sativum, 78, 79 Proteus sp., 72
Aloe barbadensis, 47 Pseudomonas fluorescens, 11
Aloe vera, 59, 122, 123 Pseudomonas putida, 119
Alopecurus pratensis, 53, 123 Rhodopseudomonas, 11
Alstroemeria aurantiaca, 122, 123 Rhodospirillum rubrum, 71, 119
Alstroemeria sp., 121 Streptococcus faecalis, 71
Alyssum alyssoides, 128 Vibrio fischeri, xvi
Anemone ranunculoides, 59 Barbarea vulgaris, 123
Animals Bellis perennis, 31
Digesia tigrina, 27 Berberis vulgaris, 123
Girardia tigrina, 114, 115 Beta vulgaris var rubra, 41, 98
Holopneustes purpurascens, 72, 111, 115 Betula verrucosa, 15, 57, 59
Paramecium, xvi Bougainvillea spectabilis, 31
Paramecium primaurelia, 70 Brasilicactus haselbergii, 8
Periplaneta americana, 72, 95, 111 Brassica juncea, 127
Tetrahymena, xvi Brassica napus, 49
Xenopus laevis, 143 Bromus erectus, 53
Anthriscus sylvestris, 59, 123
Antirrhinum majus, 31, 39
Arabidopsis thaliana, xiv, 20, 37, 38, 74, 116, 118 C
Artemisia absinthium, 74, 85, 108, 109, 121, Calendula officinalis, 123, 126
123, 126, 129, 130, 133 Callisia fragrans, 116, 117
Artemisia vulgaris, 121, 123, 126 Campanula persicifolia, 15
195
196 Latin Index
Capsicum annuum, xv, 73, 78, 79 Euphorbiaceae, 46, 72

Cardamine diphylla, 128 Euphorbia milii, 27, 39, 46, 47, 96
Carthamus tinctorius, 71 Eustoma grandiflorum, 31
Casuarina, 136
Catharanthus roseus, 42, 44
Centaurea jacea, 121, 123 F
Chara australis, 143 Filipendula ulmaria, 123–124
Chara corallina, 33, 66, 67, 88, 94, 143 Fungi
Chara sp., 114 Candida guillermondii, 119
Chelidonium majus, 43–45 Candida sp., 63
Chenopodiaceae, 58, 62 Claviceps purpurea, 72
Chenopodium album, 61, 62 Coprinus comatus, 119
Chenopodium murale, 62 Eleusine coracana, 116
Chlamydomonas reinhardtii, xvi Eupenicillium brefeldianum, 20
Chondodendron tomentosum, 149 Fusarium verticillioides, 118
Cichorium intybus, 25 Helminthosporium dematioideum,
Citrus aurantium, 31 140, 152
Clivia hybrida, 25, 122 Penicillium brefeldianum, 20, 140
Clivia sp., 122 Penicillum chrysogenum, 71
Colchicum sp., 106, 137 Phoma exigua, 140
Commelinaceae, 116 Phytophthora infestans, 140
Commelina communis, 7, 8 Pichia pastoris, 118
Coptis japonica, 43 Saccharomyces cerevisiae, xv, 71, 119
Cornus alba, 7 Sympodiomycopsis paphiopedili, 118
Corylus avellana, 7, 53, 59, 110 Ustilago maydis, 118
Corynanthe yohimbe, 149 Wormhosporium dematiodeum, 137
Cotoneaster alaunica, 7 Furcellaria lumbricalis, 55, 72, 95
Crocus vernus (vernalis), 154
Cynosurus cristatus, 53
Cyperus papyrus, 122, 123, 126 G
Gaillardia pulchella, 79, 151
Gaillardia sp., 79, 151
D Galium sp., 43
Dactylis glomerata, 53, 102, 123 Gelsemium sempervirens, 53
Danthonia compressa, 124 Glaucium sp., 92, 136
Danthonia spicata, 124 Gloriosa sp., 137
Datura alba, 127
Datura stramonium, 127
Delisea pulchra, 72 H
Drosera sp., 67 Haemanthus katherinae, 32, 67, 129,
132, 150
Hemerocallis fulva, 27, 126, 154
E Hesperis matronalis, 128
Echinopsis bridgesii, 121, 123 Hibiscus rosa-sinensis, 8, 9, 13, 27,
Ecklonia radiata, 72 32, 60, 67, 96
Elodea canadensis, 33 Hieracium canadense, 124
Elytrigia repens, 124 Hippeastrum hybridum, 5, 25, 26, 28, 29, 32,
Epiphyllum hybridum, 8, 9, 27, 32, 59, 95, 96 53, 54, 60, 67, 76, 79, 80, 83–88, 90,
Equisetum arvense, 21–23, 28–30, 32, 53–57, 91, 93, 101, 102, 104, 105, 107,
67, 79–88, 90–93, 129–133, 135, 136, 121–127, 129–133, 135, 136, 138, 139,
138–140, 143–151, 154, 155 146, 147, 149, 150, 152, 154–156
Equisetum sp., 52 Hippophae rhamnoides, 148
Eucalyptus, xv Humulus lupulus, 14, 16, 19, 27, 28, 30, 48,
Eucalyptus viminalis, 146, 148 96, 112, 113, 147
Latin Index 197
Hydrangea macrophylla, 8 Nepenthes sp., 116

Hypericum perforatum, xv, 3, 5, 27, 96, Nicotiana alata, 145
123–125 Nicotiana sp., 74, 145
Nicotiana tabacum, xv
Nitella sp., 114
I
Ipomoea sp., 100
O
Oenothera biennis, 108, 109, 122, 123, 125
J Ornithogalum thyrsoides, 31
Jatropha urens, 72 Ovena sativa, xiv
K P
Knautia arvensis, 108, 109, 121–123, 125 Papaver orientale, 9, 154
Papaver somniferum, 43–46, 110
Parthenocissus quinquefolia, 7
L Peganum harmala, xv, 47
Lampranthus productus, 31 Petunia hybrida, 8, 9, 27, 30, 87, 91,
Laportea moroides, 79 122, 123, 154
Larix decidua, 59 Petunia nyctaginiflora, 31
Lavandula sp., 78, 79 Phellodendron amurense, 93, 146
Lemna minor, 33, 66, 67, 94 Philadelphus grandiflorus, 25, 27, 60, 122,
Lemna sp., 95, 114 123, 125, 126, 131, 154, 155
Lepidium campestre, 128 Phleum pratense, 53, 57, 59, 123,
Lilium longiflorum, 30 124, 127
Lilium martagon, 121, 123 Phyllophora nervosa, 75
Lilium sp., 27, 110, 121, 124 Physostigma venenosum, 150
Limonium sinuatum, 31 Picea excelsa(sior), 60
Linaria vulgaris, 110 Picea pungens, 148
Lolium perenne, 53 Pinguicula vulgaris, 67
Lupinus polyphyllus, 43 Pinus sylvestris, 30, 60, 123
Lupinus sp., 43, 44 Plantago major, 32, 60, 122, 123,
Lycopersicon esculentum, 70, 127 125, 132, 133
Lycopersicon pennellii, 116 Poa pratensis, 53
Lysimachia fordiana, 4 Polemonium viscosum, 120
Lysimachia nummularia, 4, 6 Polysiphonia sp., 75
Lysimachia sp., 4, 6 Populus balsamifera, 57, 59
Portulaca grandiflora, 31
Portulaca oleracea, 38
M
Malus domestica, 124
Mangifera sp., 146 Q
Matricaria chamomilla, 108, 109, 123, 126 Quercus robur, 59
Matricaria recutita, 131
Medicago sativa, 32
Mentha piperita, xiv, xv, 112, 113 R
Merendra sp., 137 Ramond sp., 137
Mirabilis jalapa, 32, 67 Ranunculus acer, 123
Raphanus sativus, 33, 66, 67, 70, 80,
94, 95, 134
N Rhododendron indicum, 31
Narcissus pseudonarcissus, 60 Rubus odoratus, 5, 7, 16, 17, 60
Narcissus sp., 110 Ruta graveolens, xiv, 15, 48, 129
198 Latin Index
S Tilia cordata, 53, 61, 123

Saintpaulia ionantha, 27, 32, 39, 40, 67, Tilia platyphyllos, 53
95–97 Trifolium pratense, 133, 134
Saintpaulia sp., 32, 39, 96 Tritonia crocosmaeflora, 8
Salix caprea, 123 Tulipa sp., 5, 9, 61, 110
Salvia sp., 146 Tussilago farfara, 57, 61
Secale cereale, 53
Sisymbrium altissimum, 128
Solanum berthaultii, 73 U
Solanum melongena, 127 Ulvaria obscura, 95, 115
Solanum sp., 73 Urticaceae, 51, 53, 72, 79
Solanum tuberosum, 15, 39, 73 Urtica dioica, 51, 53–58, 61, 79
Solidago canadensis, 16 Urtica sp., 71
Solidago virgaurea, 16, 61–63, 109, Urtica urens, 51, 53
123, 126, 148
Sonchus arvensis, 124
Spinacea sp., 43 V
Symphytum officinale, 143, 144 Vallota speciosa, 32, 67, 129–131,
Syringa vulgaris, 53, 60 133, 147, 150
Vicia faba, 89
T
Tagetes patula, 123, 126, 145 Z
Taraxacum officinale, 57, 60 Zea mays, 53, 118, 127
Thalictrum flavum, 43 Zephyranthes grandiflora, 27, 123
Thalictrum minus, 43 Zephyranthes sp., 91, 126

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Victoria V.

Model Systems to Study

ISBN 978-94-017-8785-7 ISBN 978-94-017-8786-4 (eBook)

Library of Congress Control Number: 2014943518

© Springer Science+Business Media Dordrecht 2014

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Modeling is widely used in biology, in areas such as in genetics, physiology, and

Pushchino, Russia Victoria V. Roshchina

I am grateful to my colleague Dr. Valerii Yashin and to the engineers Nadezhda

1 Approaches to Choice of Model Systems for Microscopic Studies . . . . 1

3.2 Sensitive Reactions of Models to Exometabolites . . . . . . . . . . . . . . . 77

The importance of suitable model systems in plant biology is demonstrated by its

1. Biochemical studies 2. Visualization

Models for analysis

Fig. 1 Modeling in the study of excretory function

Objects characterized as models may be related to whole plants with secretory

Model system 1 Model system 2

Cells-donors of secretions Cells-acceptors of secretions

Parameters of analysis Parameters of analysis

localization of secretions in cells and extracellular reception of

express-indentification of secreting sensor systems to individual

Contacts of cell-donors and cells-acceptors of secretion

physiological responses of contacted model systems 1 and 2

cases, measurable as the absorbance or fluorescence parameters. As a whole, the

1.1 Color and Absorbance in Analysis

Images of contacting surfaces with secretions may be observed by usual micros-

1.1.1 Usual Microscopy and Stereomicroscopy

Table 1.1 Technique for the study of colored plant samples

known to be anthraquinone hypericin (Brockmann et al. 1950) and used as medical

oil-containing structures on leaves and flowers (Gerbach 2002; Pacek and

1.1.2 Microspectrophotometry and Spectrophotometry

Microspectrophotometry and spectrophotometry with special holders are tech-

pigment composition and at different stages of ontogenesis. Vacuolar absorbance

1.2 Fluorescence in Analysis

Anthocyanins from pollen Intact pollen

0.3 597 nm 0.03

1.2.1 Autofluorescence of Secretory Components

Autofluorescence is the natural emission of cellular components due to a contribu-

fluorescing secondary metabolite naturally synthesized within the cell or exome-

Fig. 1.7 Main wavelength (nm)

1.2.2 Special Fluorescent Probes or Markers

1.2.3 Fluorescence-Related Technique

Fluorescence may be analyzed by various types of techniques (Table 1.2).

Table 1.2 Technique for the study of fluorescent plant samples

1.2.3.1 Luminescence Microscopy

fluorescent allelochemicalsvwas specially considered earlier in some special publi-

The recording microspectrofluorimetry is also applied for cellular biology as one of

1.2.3.3 Confocal Microscopy

Unlike the usual luminescence microscope, in confocal microscopy before catching

bract Non-secretory cell

Fluorescence (relative units)

1.2.3.4 Images and the Fluorescence Spectra of Cells in Cytodiagnostics

Fig. 1.10 Molecular probes for the study of secretory process

types of confocal microscopes. The research of fluorescence maxima in secretory

1.3 Cellular Observation of Secretory Process

1.3.1 Secretion Transport and Excretion

with green fluorescent protein markers of the endomembrane system in Arabidopsis

1.3.2 Unicellular Models Suitable for Microspectral Analysis

In a search of model systems suitable for microspectral analysis of unicellular and

1.3.2.1 Vegetative Microspores of Spore-Bearing Plants

Vegetative microspores of spore-bearing (cryptogams) plants, in particular of horse-

Fluorescence (relative units)