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Postharvest Ripening
Physiology of Crops
Edited by
Sunil Pareek
Postharvest Ripening
Physiology of Crops
Series Editor
Sunil Pareek
Department of Agriculture and Environmental Sciences
National Institute of Food Technology Entrepreneurship and Management
Kundli, Sonepat, Haryana, India
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Contents
DEDICATION XXI
SERIES PREFACE XXVII
FOREWORD XXIX
PREFACE XXXI
ACKNOWLEDGMENTS XXXIII
EDITOR XXXV
CONTRIBUTORS XXXVII
v
Co ntents
vi
Co ntents
3.8 Temperature 99
3.9 Handling of Cut Flowers 102
3.10 Potted Plants 103
3.11 Conclusions and Future Perspectives 104
Acknowledgment 105
References 105
vii
Co ntents
viii
Co ntents
ix
Co ntents
x
Co ntents
xi
Co ntents
xii
Co ntents
xiii
Co ntents
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Co ntents
xv
Co ntents
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Co ntents
xvii
Co ntents
xviii
Co ntents
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Co ntents
xx
Dedication
During his more than 35 years at the University of California, Davis (UC
Davis), Professor Adel Kader maintained an extremely active teaching,
research, and extension program in postharvest biology and technology of
horticultural crops. His prodigious output includes more than 230 technical
manuscripts, many book chapters, and numerous extension publications. He
was the most cited author by postharvest colleagues. Kader was the undis-
puted leader of a research team that has reached high levels of knowledge
in the different disciplines; he was a reference for researchers, enterprises,
and international institutes. His research efforts were initially focused on
improving the more obvious aspects of the postharvest quality of fruits and
vegetables. He worked with a large number of fruits and vegetables during
his career in his endeavor to elucidate the physiological and biochemical
bases for quality maintenance. His papers have been milestones for others
to follow. Over time, he became increasingly interested in the less obvious
characteristics of flavor and nutritional quality. He realized that an improve-
ment in human diets through the consumption of more fruits and vegetables
would occur only when the appearance quality of fruits and vegetables was
matched by an increase in their flavor and nutritive quality. He became a
vocal proponent for coupling the traditional studies for improved appear-
ance quality with studies of flavor and nutritional quality changes during
harvest, storage, transport, and marketing. All this because he recognized
the importance of bringing to the end user a product that not only looks
great but also tastes wonderful and is at its optimal nutritive value.
xxi
D ed ic ati o n
xxii
D ed ic ati o n
xxiii
D ed ic ati o n
xxiv
D ed ic ati o n
died, he was coordinating the writing of the fourth edition, his signature
book. He also served as author and editor of many publications, including
the popular Small-Scale Postharvest Handling Practices: A Manual for
Horticultural Crops, which he coauthored with Lisa Kitinoja and was trans-
lated into 11 additional languages. He was also associate editor of the book
Dates: Postharvest Science, Processing Technology and Health Benefits, which
was published after his death. Adel led the development of the Postharvest
Technology website (www.postharvest.ucdavis.edu), which has become
the premier place to find postharvest information and receives several mil-
lion page views annually.
In teaching and research, Adel was a wonderful colleague. He and
his students were particularly focused on understanding the physiology,
biochemistry, and technology of controlled and modified atmosphere stor-
age of fruits and vegetables. He held the highest ethical, professional, and
research standards for both himself and others, which challenged everyone
with whom he worked to perform to their highest possible level. In addition
to hundreds of peer-reviewed papers and popular articles describing his
research, Adel, with photographer Don Edwards, also produced hundreds
of high-quality slides demonstrating his research findings. These slides are
still an essential component of many of the presentations made by members
of the UC Davis postharvest team. We would like to remember his intellec-
tual integrity and his way of discussing and interacting with colleagues and
students. He was happy to share his knowledge, he had no secrets, and he
believed research and information should be shared by and with everyone.
He was happy to give a bibliographic reference, photos, PowerPoint presen-
tation, publications, and so on.
Adel was convinced that improved postharvest practices would not
only raise the quality, taste, and nutrition of fruits and vegetables in the
United States, but also improve food supply and farmers’ incomes in the
developing world. From the start of his career, he was constantly involved
in development activities. As a key player in the Agriculture Development
Strategy (ADS) project, which sought to bring the expertise of U.S. hor-
ticulturists to Egypt, he made postharvest handling a central theme. The
subsequent flourishing of horticulture and horticultural exports from his
home country can, at least in part, be attributed to his efforts.
After retirement, Professor Kader maintained a very active interest in
postharvest programs worldwide and frequently participated in seminars at
UC Davis and many international meetings, chaired the California Citrus
Quality Council and the research advisory board of the Produce for Better
Health Foundation, and was board director of the Postharvest Education
Foundation. He continued to do some consulting to raise funds for the UC
Davis Postharvest Endowment. For the past several years, Adel served as a
key player in the Global Horticulture Assessment, which laid the foundation
for the development of the Horticultural Collaborative Research Support
xxv
D ed ic ati o n
xxvi
Series Preface
xxvii
Foreword
Horticultural crops have myriad uses in societies worldwide; not only are
fruits and vegetables critical components of the diet, but together with flow-
ers and ornamentals, they are pleasing aesthetic products that contribute
to human well-being. Edible products are a source of antioxidant vitamins
(A, C, and E), phenolics, carotenoids, phytonutrients, and dietary fiber that
are important health-promoting compounds in the human diet. The con-
tribution that these products make to human health has become increas-
ingly recognized and includes reduction of the incidences of degenerative
diseases as well as cardiovascular disease, hypertension, and cancers.
However, no amount of information about the “goodness” of any fruit or
vegetable is useful if it is not visually appealing or flavorful or does not meet
the quality expected by the consumer.
Access to horticultural products can vary greatly, with scarcity in
some areas and surplus in others. Estimates for losses of horticultural
crops vary widely, but it is interesting from survey research that losses of
fruits and vegetables are similar in the developing and developed worlds.
Yet, where those losses occur, between the “farm and fork,” can be mark-
edly different. In the developing world, losses are largely a result of fac-
tors such as lack of harvest, storage, and transport infrastructure and poor
marketing systems. In the developed world, losses are greater after harvest
because of stringent quality standards and factors such as excess produc-
tion, loss of product quality after harvest, and “plate waste” (unconsumed
food after purchase by consumers). Interestingly, the locavore movement,
or seeking locally grown food, often organic, in countries such as the United
States, can sometimes resemble the situation in developing countries, with
xxix
Fo re wo rd
Christopher B. Watkins
xxx
Preface
Ripening is an important aspect of the production and shelf life of fresh pro-
duce. Timing and stage of ripening affect the buying behavior of produce,
shelf life, quality, and nutraceuticals. Ripening physiology is complicated
and affected simultaneously by many factors. Looking at these facts, this
book provides information on the postharvest physiology, biochemistry, and
molecular biology of ripening. Quality, physiology, and molecular biology
of flower senescence are also discussed. Detailed information on advances
in respiration measurement, stomatal relations in postharvest, and factors
controlling postharvest water loss has been provided. Lysophospholipids
research is gaining popularity and provides information on ripening and
extending shelf life of horticultural produce. A detailed account on posthar-
vest quality in relation to lysophospholipids is also included.
This book is a comprehensive interdisciplinary reference source for
the various aspects of fruit ripening and postharvest behavior. It focuses on
the postharvest physiology and ripening overview of fruits and vegetables.
Flower senescence is equally important for the postharvest horticulture
industry, and chapters are included on the postharvest quality of ornamen-
tal plants and molecular biology of flower senescence. The share of colored
fruit in the total fruit production is quite significant, with the largest con-
tributing several billion dollars annually. This has encouraged scientists
to study the pigmentation mechanism in fruits. Various developments that
have taken place in the last decade with respect to identifying and altering
the function of ripening-related genes are described in this book. Taking
clues from studies in grape as a model fruit, we review a few case studies,
xxxi
Preface
Sunil Pareek
xxxii
Acknowledgments
xxxiii
Ac k n o w l e d g m e n t s
acknowledge my better half, Dr. Shilpi, and son, Mr. Sabhya, who under-
went all sorts of hardships and sufferings to support my spirit and endeavor
at every step.
xxxiv
Editor
xxxv
Ed i t o r
xxxvi
Contributors
xxxvii
Co ntributo rs
xxxviii
Co ntributo rs
xxxix
Co ntributo rs
xl
Chapter 1
Ripening Physiology:
An Overview
Sunil Pareek 1,2
1Maharana Pratap University of Agriculture and
Technology, Udaipur, Rajasthan, India
2National Institute of Food Technology Entrepreneurship
and Management, Ministry of Food Processing
Industries, Kundli, Sonepat, Haryana
Abstract 2
1.1 Introduction 2
1.2 Climacteric Phenomenon 4
1.3 Physicochemical and Metabolic Changes 12
1.3.1 Color Changes 13
1.3.2 Sugar Changes 14
1.3.3 Organic Acid Changes 16
1.3.4 Flavor and Aroma Changes 18
1.3.5 Cell Wall and Textural Changes 22
1.3.6 Physiological Changes 28
1.4 Conclusions and Future Perspectives 33
References 33
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
Abstract
Ripening is an important event in the fruit life cycle and from the consum-
ers’ point of view. Mature fruit undergo ripening, which is a coordinated
process typically involving many changes, such as pigmentation, flavor,
aroma, respiration, ethylene, texture, softening, sugar, and organic acid.
Physiologists typically classified fruits into climacteric and nonclimacteric
categories on the basis of their respiration peaks and internal ethylene
concentrations. This chapter reviews the climacteric and nonclimacteric
ripening of fruits and physiological and metabolic changes during ripen-
ing. In climacteric fruit, components responsible for the production of cli-
macteric ethylene have been identified. Less progress has been made on
nonclimacteric fruit; still, knowledge is poor to classify many fruits into
climacteric or nonclimacteric. A comprehensive climacteric classification
of fruits is given in this chapter. Textural changes and the role of enzymes
are also reviewed.
1.1 Introduction
Ripening is the composite of the processes that occur from the latter stages
of growth and development through the early stages of senescence and that
result in characteristic aesthetic and food quality, as evidenced by changes
in composition, color, texture, or other sensory attributes. Fruit ripening
is a complex process that undergoes dramatic changes mainly involving
flavor, color, and texture. It is a controlled process where the cellular com-
munication is important, as well as the influence of plant growth regulators
and environmental signals (Cruz-Hernandez and Paredes-Lopez, 2012).
Ripening could also be defined as a physiological process that is geneti-
cally programmed and comprises several physical, chemical, and biochemi-
cal changes that render fruit attractive and palatable (Lelievre et al., 1997;
Giovannoni, 2001).
One early view of ripening was that it is due to entirely to a series
of catabolic reactions associated with changes in membrane permeabil-
ity and a decrease in the structural integrity of the cell, resulting in the
release or activation of hydrolytic enzymes. This view is now clearly
untenable since genetic studies suggest that the expression of specific
genes is also required for ripening. It is supported by biochemical evi-
dence that shows there are changes in specific mRNAs and the de novo
synthesis of protein during ripening. Changes in gene expression are
both positive and negative. They involve genes in the nucleus and plas-
tids, and expression of at least some of them is confined to ripening fruit
tissues. Thus, fruit ripening is a highly controlled and programmed
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Ripen in g P hy s i o l o g y : A n Ov e r v i e w
3
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Climacteric peak
Carbon dioxide production
Post-climacteric
decline
Climacteric
rise
Pre-climacteric
minimum
Non-climacteric pattern
Time
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Ripen in g P hy s i o l o g y : A n Ov e r v i e w
Climacteric fruit
180 Breadfruit
Cherimoya
160
140
ml O2 or CO2/Kg-Hr
120
100
Mango
80
60
Fig
40
20 Tomato
Apple
0 2 4 6 8 10 12 14 16 18
Time units
5
P OST H AR V EST RI P ENING P H Y SIOLOG Y
not show any increase in respiration and ethylene synthesis during ripen-
ing (Figure 1.3). In fact, nonclimacteric fruits show decline in their respira-
tion rate and ethylene production throughout the ripening process.
Climacteric events are mainly regulated by the gaseous phytohor-
mone ethylene, which is also involved in the decrease in flesh firmness
typical of many economically relevant crops, such as tomato and peach
(Prinsi et al., 2011). On the other hand, ripening of nonclimacteric fruits
such as pepper, citrus, and strawberry is ethylene independent, although
similar major visual, texture, flavor, and metabolic changes occur, as in cli-
macteric fruits. Many of the changes have been mainly characterized in
climacteric ripening fruits, whereas nonclimacteric fruit ripening is still
poorly understood. A list of climacteric and nonclimacteric fruit updated
from Biale and Young (1981), Kays (1991), and Watkins (2002) is shown
in Table 1.2. Interestingly, this physiological behavior is not linked to
Nonclimacteric fruit
30
Strawberry
ml O2 or CO2/Kg-Hr
Grape
20
Pineapple
Cherry
10
Lemon
0
0 1 2 3 4 5 6 7 8 9 10
Time units
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Ripen in g P hy s i o l o g y : A n Ov e r v i e w
(Continued )
7
P OST H AR V EST RI P ENING P H Y SIOLOG Y
8
Ripen in g P hy s i o l o g y : A n Ov e r v i e w
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
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Ripen in g P hy s i o l o g y : A n Ov e r v i e w
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
12
Ripen in g P hy s i o l o g y : A n Ov e r v i e w
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
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Ripen in g P hy s i o l o g y : A n Ov e r v i e w
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
(Fait et al., 2008), and peach (Lo Bianco and Rieger, 2002) is evident dur-
ing ripening. The main sugar at the fruit ripening stage depends on the
plant species. Glucose is the major sugar in table grape, whereas fructose
is the predominant sugar in berries, mango, and citrus species. Those
fruits that accumulate fructose and glucose show very low concentrations
of sucrose. However, apricot, plum, nectarine, and peach have sucrose as
the main sugar, which is accumulated during stage 3 as a result of a rise
in the activity of sucrose synthase (Morandi et al., 2008). The proportions
of fructose, glucose, and sucrose are important in the perception of taste
since fructose is 80% sweeter than sucrose, whereas glucose is only 60%
sweeter than sucrose (Yamaguchi et al., 1970). A regular increase in the
level of sugars in pear throughout the period of fruit development has been
observed due to translocation of photosynthates from leaves to the young
fruit, which are partly used for the synthesis of pectic substances and other
cell wall materials and partly converted to the usual storage product, the
starch. With the advancement of maturity, the accumulated starch is hydro-
lyzed into sugars, which is known as a characteristic event for fruit ripening
(Hulme, 1958). The starch-degrading enzymes in fruits and their mode of
starch mobilization are given in Table 1.6. Further breakdown of sucrose
into glucose and fructose is probably mediated by the action of invertase.
Low levels of invertase activity have been linked to the sucrose accumula-
tion trait in tomatoes (Yelle et al., 1991).
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Ripen in g P hy s i o l o g y : A n Ov e r v i e w
Pyruvate GLYOXYSOME
PDH
CoA
NAD-ME acetylCoA CoA
acetylCoA citrate citrate
CS ACO
OAA CS citrate OAA
OAA
ACO glyoxylate
NAD-MDH
cycle isocitrate isocitrate
malate NAD-MDH
cis-aconitate
malate malate ICL
ACO
TCA Cycle
fumarate MS
isocitrate glyoxylate
succinate
NAD-IDH CoA
succinate CoA NADP-IDH
2-oxoglutarate
succinyl-CoA
citrate ATP-CL acetylCoA
MITOCHONDRION OAA metabolism
GABA shunt
ACO acetylCoA
NADP-ME isocitrate
Pyruvate malate
NADP-IDH flavonoids/
2-oxoglutarate isoprenoids
PPDK NAD-MDH
gluconeogenesis
PEPCK glutamate
glucose PEP OAA
glutamine
PEPC GABA
glucolysis
CYTOSOL
succinate
Figure 1.4 Citrate and malate metabolic pathways in fruit mesocarp cells.
The probable direction of reversible reactions is indicated by the large
arrow. ACO, aconitase; ATP-CL, ATP-citrate lyase; CS, citrate synthase; ICL,
isocitrate lyase; MS, malate synthase; NAD-MDH, NAD-malate dehydro-
genase; NAD-ME, NAD-malic enzyme; NAD-IDH, NAD-isocitrate dehy-
drogenase; NADP-ME, NADP-malic enzyme; NADP-IDH, NADP-isocitrate
dehydrogenase; PDH, pyruvate dehydrogenase; PEPC, phosphoenolpyru-
vate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; PPDK, pyru-
vate orthophosphate dikinase. (Adapted from Etienne, A. et al., Journal of
Experimental Botany, doi:10.1093/jxb/ert0352013.)
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
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Ripen in g P hy s i o l o g y : A n Ov e r v i e w
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
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Ripen in g P hy s i o l o g y : A n Ov e r v i e w
Table 1.8 Common Polymers of Primary Cell Wall and Their Structures
Polymer Molecular Structure
Cellulose (1→4)β-D-Glucan chains held together with
hydrogen bonding, forming very long crystalline
microfibrils
Cross section contains 36 glucan chains
Glucomannan Backbone contains regions of (1→4)β-D-glucan and
(1→4)β-D-mannan in nearly similar amounts
galactomannan
Occasionally terminal; has a side chain of single
units of α-D-galactose
Glucuronoarabinoxylan Backbone of (1→4)β-D-xylan
Side chains of single unit of nonreducing terminal
α-L-arabinose and α-D-glucoronic acid
Homogalacturonan Made of long chains of (1→4)α-D-galacturonic acid
Initially highly methyl esterified
Rhamnogalacturonan I Made of alternating α-D-rhamnose and α-D-
(RG-I) galacturonic acid residues; long side chains of
either unbranched (1→4)β-D-galactan or branched
α-L-arabinans or type I arabinogalactans attached
to the rhamnose residues
Rhamnogalacturonan II Backbone made of (1→4)α-D-galacturonic acid like
(RG-II) homogalacturohan; complex side chains of
different types of neutral sugar
A minor cell wall component
RG-II monomers can domimerize together as boron
diesters and may affect the cell wall porosity
Structural proteins Four different types including expansin; some are
heavily glycosylated
Xyloglucan Backbone similar to cellulose, i.e., (1→4)β-D-glucan
Regular substitution on three out of four consecutive
glucose residue with α-D-xylose
Xylose occasionally extended with β-D-galactosyl-α-L-
fucose or α-L-arabinose in some species
The reducing end of unsubstituted glucose residues
is susceptible to cleavage by Trichoderma endo-
(1→4)β-D-glucanase (EGases) producing similar
amounts of heptasaccharide (G1c4.Xy13) and
nonasaccharide (G1c4.Xy13.Gal.Fuc) xyloglucan
subunit oligosaccharides
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
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Ripen in g P hy s i o l o g y : A n Ov e r v i e w
These six changes have been observed in a wide range of fruit types,
although the extent and relative timing vary somewhat between species.
Such observations indicate that pectin solubilization and cell wall swelling
are important events in the control of softening in kiwifruit and probably
most other species with melting texture.
Graham Seymour and colleagues at HRI–Wellesbourne are investi-
gating the biochemistry of fruit texture in order to improve the palatabil-
ity and shelf life of produce (Marin-Rodriguez et al., 2002; Seymour et al.,
2002). Marin-Rodriguez et al. (2002) reviewed the role of pectate lyases in
fruit softening. Pectate lyases (PELs) catalyze the Ca 2+ -dependent cleavage
of deesterified pectin, which is a major component in the primary cell walls
of many higher plants. Initially, it was thought that these enzymes were
produced solely by plant pathogens to macerate plant tissues. As a result of
both plant genome sequencing and EST programs, however, it has become
clear that these enzymes are encoded by large gene families in plants and
expressed throughout the plant, including ripening fruit. Tomato, straw-
berry, grape, and banana fruits all express PELs, where they may play a
significant role in fruit softening. Other enzymes involved in modifying
25
P OST H AR V EST RI P ENING P H Y SIOLOG Y
80
Pectin solubilization
Fruit firmness (%)
60 Phase 1
initiation
Soluble pectin depolymerization
galactose loss
40
Aroma production
respiratory climacteric
Phase 2 pectin depolymerization
20 rapid softening
Loss of middle lamella
Phase 3
eating window Phase 4
over-ripe
Time
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Ripen in g P hy s i o l o g y : A n Ov e r v i e w
27
P OST H AR V EST RI P ENING P H Y SIOLOG Y
(Continued )
28
Ripen in g P hy s i o l o g y : A n Ov e r v i e w
29
P OST H AR V EST RI P ENING P H Y SIOLOG Y
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48
Chapter 2
Postharvest
Physiology of Fruits
and Vegetables
Peter M.A. Toivonen
Agriculture and Agri-Food Canada
Summerland, British Columbia, Canada
Abstract 50
2.1 Introduction 51
2.2 Classification of Fruits and Vegetables Based on
Physiological Characteristics 51
2.2.1 Ethylene Biology 51
2.2.2 Respiratory Characteristics: Climacteric and
Nonclimacteric 56
2.2.3 Developmental or Maturity Stage at Harvest 56
2.2.4 Tolerance to Low Temperatures 58
2.3 Factors Affecting Physiology of Fruits and Vegetables in
Postharvest Systems 60
2.3.1 Temperature 60
2.3.2 Humidity 62
2.3.3 Atmospheric Modification 64
2.3.4 Abiotic Stresses 65
49
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Abstract
This chapter covers a wide range of issues pertaining to the many aspects
of fruit and vegetable tissue physiology and changes that occur during
postharvest handling and storage. Understanding the specific ethyl-
ene biology, respiration behavior, and developmental or maturity stage
at harvest can provide great insight to postharvest quality retention
characteristics of a fruit or vegetable. Ethylene itself is one of the most
important factors determining quality retention, with different fruit and
vegetable classes having differing production rates and sensitivity to the
levels of ethylene present. Numerous disorders and quality defects can
be attributed to exposures to ethylene in postharvest systems, and these
are discussed. Respiration behaviors can be separated into two classes:
(1) climacteric and (2) nonclimacteric. The classification of respira-
tory behavior will have an impact on postharvest approaches required
for optimal handling of a particular fruit or vegetable in question. The
anatomical structure and developmental maturity of a fruit or vegetable
can range from an immature vegetable tissue all the way to a ripe fruit
tissue, and this will also determine which postharvest procedures will
be successful for handling and storage life potential. The importance of
temperature, humidity, and atmosphere management on quality reten-
tion is discussed in a mechanistic manner to explain the physiological
responses to these postharvest interventions. Finally, endogenous and
exogenously applied phytohormones are important to signaling meta-
bolic shifts that influence quality retention, and these implications are
explored. The discussion in this chapter is intended to provide a basic
and mechanistic understanding of how and why quality changes occur
in postharvest handling of fruits and vegetables that have contrasting
physiological characteristics. The discussion culminates in discussion
on improving quality retention of fruits and vegetables through multi-
disciplinary cooperation of plant breeders, physiologists, biochemists,
and molecular biologists, leading to more robust solutions to postharvest
problems.
50
P o s t h a r v e s t P hy s i o l o g y o f F r u i t s a n d V e g e t a b l e s
2.1 Introduction
Development of successful harvest, handling, storage, and distribution
protocols for fruits and vegetables has relied on the understanding of the
physiology of each type of produce and how that physiology responds to
the postharvest environment to which it is exposed (Toivonen and Hodges,
2011). Because of limitations in produce handling systems, many different
types of fruits and vegetables are held in common rooms or containers,
and this has led to issues of incompatibility of produce having different
physiologies. This incompatibility is expressed as shorter storage or shelf
life, development of off-flavors, and accumulation of bitter compounds
(Thompson et al., 1996). Another consequence of common storage areas
is that some fruits and vegetables have differing tolerances to low tempera-
tures, and therefore some fruits or vegetables held in common-use, low-
temperature storage might suffer chilling injuries (Thompson et al., 1996;
Cantwell, 2002). This phenomenon continues to exist and can still today
be visualized in retail produce displays as pitting with associated decay in
cantaloupes and summer squash or as sheet pitting in green peppers (per-
sonal observation). The challenge facing postharvest science and technol-
ogy today is the development of innovative strategies to resolve problems
associated with differing fruit and vegetable physiologies in the real-world
harvest, storage, distribution, and retail systems.
This chapter is devoted to providing an overview of the fundamen-
tal aspects of fruit and vegetable postharvest physiologies to allow a better
appreciation for challenges faced in their handling. Understanding of the
fundamental principles will provide grounded principles that will hopefully
be the basis of new, innovative strategies for harvest, storage, handing, and
distribution of these living and very perishable products.
51
P OST H AR V EST RI P ENING P H Y SIOLOG Y
5’-Methylthioribose-
Methylthioribose kinase 1-phosphate
α-Keto-γ-methylthio-
5’-Methylthioribose
butyric acid
Methylthioadenosin Methionine
nucleosidase Transaminase
Cycle
5'-Methylthioadenosin Methionine
1-Amino-cyclopropane- CO2
C
AC
C fera
Low emperat rs
high l scaveng
radic
C
A an s
thy high
tr Ethylene receptor
e
oxyg
t
a
sites
ou ing,
obalt
u
e
Fru
52
P o s t h a r v e s t P hy s i o l o g y o f F r u i t s a n d V e g e t a b l e s
membrane is associated with the cell wall (Mattoo and Lieberman, 1977).
This understanding of the localization of production may explain the
observations that ethylene production is transient during ripening and
senescence or in response to wounding (Figure 2.2). The latter stages of
senescence involve cell wall breakdown and cell membrane disintegration
(Hurkman and Kennedy, 1975), which would imply that the association
of membrane with cell wall is lost. Also in wounding, cell membranes are
known to lose their functionality (Toivonen and DeEll, 2002), again imply-
ing the loss of cell wall membrane associations that exist in intact cells.
Transient ethylene production occurs only at the onset of climacteric ripen-
ing and senescence (Biale et al., 1954; Aharoni and Lieberman, 1979) or
early in the wound response (Toivonen and DeEll, 2002), which is consis-
tent with this hypothesis.
Fruits and vegetables have varying sensitivities to ethylene, and
responses to ethylene are manifested in many different ways. Table 2.1
shows the relative sensitivity of many fruits and vegetables commonly mar-
keted in North America. It is also important to note that many of the sensi-
tive fruits and vegetables can also produce significant amounts of ethylene,
because ethylene production is linked to the ripening of that fruit. Table 2.2
lists the responses that are evoked with exposure to ethylene by various
Climacteric peak
Carbon dioxide production
Post-climacteric
decline
Climacteric
rise
Pre-climacteric
minimum
Non-climacteric pattern
Time
53
P OST H AR V EST RI P ENING P H Y SIOLOG Y
(Continued )
54
P o s t h a r v e s t P hy s i o l o g y o f F r u i t s a n d V e g e t a b l e s
55
P OST H AR V EST RI P ENING P H Y SIOLOG Y
56
P o s t h a r v e s t P hy s i o l o g y o f F r u i t s a n d V e g e t a b l e s
often harvests at a “breaker” stage to ensure that the fruit will respond to
ethylene and ripen after harvest.
The rate of developmental change is also important to understand in
handling fruits and vegetables. In general, those fruits and vegetables har-
vested at the vegetative growth stage to the physiological maturity stage
of the development continuum will have the most rapid metabolism and
hence shortest shelf potential (Toivonen, 2010). In addition, there is usu-
ally a higher respiratory rate, and it is well known that shelf life is consid-
ered to be inversely proportional to respiration rate (Saltveit, 2004). For
fruits and vegetables harvested at the ripening phase of the developmental
continuum, if the correct timing is chosen, respiration rates can be quite
low; for example, if potatoes are allowed to grow to maturity and are cured
before storage, they have much lower respiration rates than if they are
harvested as immature potatoes (Toivonen, 2010). Also, climacteric fruit,
which can exhibit high respiration rates in the latter stages of ripening,
57
P OST H AR V EST RI P ENING P H Y SIOLOG Y
when harvested at the early climacteric stage and properly stored, have
relatively low respiration rates and can be stored for long periods of time
(Saltveit, 2004; Watkins et al., 2004).
Because timing of harvest is an important factor in determining
storage life, various practical indices have been developed to aid in mak-
ing optimal harvest decisions, ranging from visual and tactile indices to
precise instrumental measures (Reid, 2002). The approach in each case
depends on monitoring visual, tactile, or compositional changes that occur
in the fruit or vegetable, leading to the horticultural optimal maturity for
harvest. In the case of lettuce, this means assessing the solidity of the head;
in apples, this means measuring the loss of starch with iodine staining,
and in persimmons the tannin content (Reid, 2002). Clearly, each fruit or
vegetable has a distinct approach to assessing maturity for harvest. In addi-
tion, there continues to be challenges for fruits and vegetables or new culti-
vars emerging into the export markets, where practical and reliable harvest
maturity indices have yet to be developed.
58
P o s t h a r v e s t P hy s i o l o g y o f F r u i t s a n d V e g e t a b l e s
Chilling temperature
Primary response
Phase transition of membrane
from liquid crystalline to solid gel
Secondary responses
ROS accumulations
Ethylene production
Increased respiration
Decoupling of electron transport from
energy production
Alteration of cellular structure
Alterations in metabolic pathways
Recovery
Short exposure
to pre-stressed
–processes reversible
functionality
Prolonged exposure
Symptoms of injury
Discoloration
Surface pitting
Internal breakdown
Loss of capacity to ripen
Wilting
Decay
Aroma Loss
59
P OST H AR V EST RI P ENING P H Y SIOLOG Y
2.3.1 Temperature
Temperature influences the rate of all metabolic processes, including res-
piration. Metabolic processes all rely on enzymatic activity, which can be
further generalized as a chemical reaction. As such, effects of temperature
can be described by an Arrhenius plot, which is derived as
An enzymatic reaction can be generalized as
V = k × [Substrate A] × [Substrate B]
60
P o s t h a r v e s t P hy s i o l o g y o f F r u i t s a n d V e g e t a b l e s
Vt +10
Q10 =
Vt
where Vt is the rate measured at a temperature (t) measured in degrees
Celsius and Vt+10 is the rate at 10°C higher than t.
The Q10 has been widely used in the context of respiratory behavior
cold storage and to explain the effect of low temperatures on enhancing
the storage life of produce (Nunes and Emond, 2003; Bron et al., 2005).
However, it has to be noted that not all enzymes involved in respiratory
and nonrespiratory pathways have identical Q10 values in the tempera-
ture ranges studied (0°C–20°C). Pollock and ap Rees (1975a) demon-
strated that enzymes involved with sugar metabolism have wide ranges in
Q10 values, between 2°C and 10°C, and this consequently explained some
of the changes associated with low temperature sweetening in potatoes
(Pollock and ap Rees, 1975b). It has also been shown that enzyme systems
can become acclimatized to low temperatures, becoming less sensitive
to temperature change over time, and thus having lower Q10 values after
time in storage (Atkin and Tjoelker, 2003). Rhodes et al. (1981) demon-
strated differential changes in enzymes involved with phenolic metabo-
lism in response to temperature and differential temporal changes in
these enzyme activities with time at low temperature storage. This work
clearly emphasizes the importance of temperature for imposing shifts in
metabolic pathways, and doing so in such a manner that relative contents
of phenolic constituents are modified, and this relationship also shifts
over time as some of the enzymes acclimate, and others do not, to low
temperatures.
While Q10 principles have been applied to enzymatic processes in
fruits and vegetables, temperature also has effects on physical move-
ments into, out of, and within fruit tissue; that is, rates of gaseous and
liquid diffusion in tissues can be influenced by temperature (Lammertyn
et al., 2001). This finding emphasizes how little is known about the com-
ponents of apparent Q10 values developed for postharvest analysis (Bron
et al., 2005).
The importance of low temperatures to controlling quality decline in
fruits and vegetables is well known (Nunes and Emond, 2003). However, a
scan of the literature indicates that there are limited reports on differen-
tial effects of low temperature on fruit and vegetable synthetic, catabolic,
and respiratory pathways. The lack of good tools to measure a broad range
of proteins (enzymes) and metabolic substrates and products has likely
limited work in this area. However, with new tools and data processing
61
P OST H AR V EST RI P ENING P H Y SIOLOG Y
2.3.2 Humidity
Humidity is the second most important factor, after temperature, for mod-
ulating changes in physiology and the quality of fruits and vegetables in
postharvest systems. The humidity of the air surrounding a fruit or veg-
etable exerts some vapor demand on the produce if the humidity is below
100%, which is normally the case in postharvest systems. Water loss can be
calculated as
J=
(P − P ) × A
i a f
( RD × T ) × r
where J is the water loss in weight of water per unit time per surface area
of the fruit, Pi and Pa are the steady-state vapor pressures of water in the
intercellular spaces of the fruit and in the atmosphere, respectively, A f is
the surface area of the fruit, R D is the gas constant, T is the absolute tem-
perature in degrees Kelvin, and r is the specific resistance of the fruit to
water loss. While this model provides precise estimates of water loss, it is
essential to determine the surface area of the fruit and the specific resis-
tance of that fruit to water loss. In practice, it is more important to get an
estimate of the Pi − Pa component of the water loss model so that relative
changes in driving force for water loss (i.e., vapor pressure deficit) can
be quantified, allowing evaluation of the effect of changes in postharvest
handling protocols on relative water loss from the fruit.
In order to calculate vapor pressure deficit, the saturation vapor pres-
sure of the fruit or vegetable and the surrounding air must be calculated.
Saturation vapor pressure (VPs ) can be calculated as
17.27 × T
VPs = 0.6108 × e T + 237.3
where T is the temperature of the produce or the surrounding air in degrees
Celsius.
The intercellular spaces of the fruit or vegetable are considered to be
at saturation vapor pressure (Ben-Yehoshua and Rodov, 2003). However,
the air vapor pressure (VPa ) can be calculated as
62
P o s t h a r v e s t P hy s i o l o g y o f F r u i t s a n d V e g e t a b l e s
RH a
VPa = × VPsa
100
where RHa is the relative humidity of the air in percent and VPsa is the calcu-
lated saturation vapor pressure of the air at that temperature.
Water vapor deficit (VPD) is the driving force for water loss from
fruits or vegetables and is calculated as
where VPp is the calculated saturation vapor pressure of the fruit or veg-
etable and VPa is the calculated vapor pressure of the surrounding air based
on its temperature and relative humidity.
It is a straightforward task to calculate vapor pressure deficits for
many postharvest handling scenarios to demonstrate the need for rapid pre-
cooling compared to room cooling. It can be demonstrated that warm fruit
will have higher integral vapor pressure deficits if room cooled, and hence
greater water loss than fruit that is rapidly forced-air cooled (Toivonen
and Hodges, 2011). Clearly, it is important to cool fruits and vegetables to
temperatures as close to that of the storage room or transport container as
soon as possible in order to minimize water loss. In addition, it is impor-
tant to manage the relative humidity in a storage room or container such
that it is maintained as high as is suitable for a particular fruit or vegetable
(Cantwell, 2002).
As implied earlier, there are a number of factors that determine the
resistance of produce to water loss—the composition and thickness of
the epidermis; the presence of surface structures on the epidermis, such
as hairs; the number or presence of stomata (or lenticels); and the mor-
phological structure (a bulky organ with a low surface area-to-volume
ratio vs. a leafy green with a high surface area-to-volume ratio). The
resistance to water loss can be modulated with wax coatings or applica-
tion of packaging to control humidity around the produce (Toivonen and
Hodges, 2011).
If water loss is not controlled, there is certainly a loss of turgor leading
to wilting, but there are also a few other metabolic changes that can occur
(Toivonen and Hodges, 2011). The enzyme activities of polygalacturonase
and pectinesterase increase, leading to loss of cell wall structure and con-
comitant increases in soluble sugars in tomato (Inari et al., 2002). Similar
results have been found with cucumbers, in which water stress caused an
upregulation of polygalacturonase activity, leading to the hypothesis that
the physical loss of water (i.e., loss of turgor in the tissue) was not the only
factor in causing softening of stressed fruit (Kubo et al., 2000). Water stress
also induces increased ethylene production (Kubo et al., 2000), and this
63
P OST H AR V EST RI P ENING P H Y SIOLOG Y
64
P o s t h a r v e s t P hy s i o l o g y o f F r u i t s a n d V e g e t a b l e s
65
P OST H AR V EST RI P ENING P H Y SIOLOG Y
66
P o s t h a r v e s t P hy s i o l o g y o f F r u i t s a n d V e g e t a b l e s
of energy homeostasis has emerged from this work (Figure 2.4), which
is congruent with earlier understanding in regard to the maintenance of
cellular organization and function. Clearly there is significant understand-
ing yet to be had in regard to the maintenance metabolism of tissues in
storage.
Stress (senescence)
+ −
Sucrose
ABA ATP Deficit
SnRK Signaling
Pathway
ROS
Fruit Quality
Deterioration
67
P OST H AR V EST RI P ENING P H Y SIOLOG Y
68
P o s t h a r v e s t P hy s i o l o g y o f F r u i t s a n d V e g e t a b l e s
69
P OST H AR V EST RI P ENING P H Y SIOLOG Y
70
P o s t h a r v e s t P hy s i o l o g y o f F r u i t s a n d V e g e t a b l e s
71
P OST H AR V EST RI P ENING P H Y SIOLOG Y
groups providing the most comprehensive results are those who have truly
multidisciplinary collaborators, including crop production physiologists,
postharvest physiologists, chemists, and molecular b iologists. This new
era of postharvest physiology requires new ways of working together and a
multidisciplinary approach to provide both perspective on the physiological
problem and a detailed understanding of the molecular mechanisms that
will allow us to better predict postharvest quality outcomes from treatment
applications and for directed breeding programs.
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Aharoni, N., and Lieberman, M. 1979. Patterns of ethylene production in
senescing leaves. Plant Physiology 64, 796–800.
Aharoni, A., Keizer, L.C.P, Van Den Broeck, H.C., Blanco-Portales, R., Muñoz-
Blanco, J., Bois, G., Smit, P., De Vos, R.C.H., and O’Connell, A.P. 2002.
Novel insight into vascular, stress, and auxin-dependent and -independent
gene expression programs in strawberry, a non-climacteric fruit. Plant
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79
Chapter 3
Postharvest Quality
of Ornamental Plants
Fernando L. Finger, Tania P. Silva, Fernanda
F. Araujo, and Jose G. Barbosa
Federal University of Viçosa, Viçosa, Minas Gerais, Brazil
Abstract 82
3.1 Introduction 82
3.2 Quality Attributes in Ornamental Plants 83
3.3 Influence of Water Relations on Ornamental Longevity 85
3.4 Action of Ethylene on Ornamental Plant Quality 88
3.4.1 Inhibition of Ethylene Synthesis 91
3.4.2 Inhibition of Ethylene Action 93
3.4.3 Ethylene Absorbers 95
3.5 Role of Abscisic Acid, Gibberellins, and Cytokinins 96
3.6 Role of Calcium on Flower Senescence 97
3.7 Respiration 98
3.8 Temperature 99
3.9 Handling of Cut Flowers 102
3.10 Potted Plants 103
3.11 Conclusions and Future Perspectives 104
Acknowledgment 105
References 105
81
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Abstract
In this chapter, the behavior of cut flowers and potted ornamental plants is
studied regarding the physiological and environmental factors that affect
the rate of senescence and longevity. Water uptake by cut flowers, carbohy-
drate supply, and response of flowers to ethylene interact alone or together
to affect the length of the vase life. Depending on whether ethylene is the
main cause of flower senescence, particular handling is required to extend
vase life. Periodically, recuts of the flower stem or pulsing with sucrose
improves the water uptake and floret opening of the bird-of-paradise. Any
flower species or potted plants with high sensitivity to ethylene have a
much better shelf life when treated with inhibitors of ethylene action such
as 1-methylcyclopropene (1-MCP) or silver thiosulfate (STS). The orchid
Epidendrum ibaguense had a positive response by reducing flower abscis-
sion when treated with the aminoethoxyvivylglycine (AVG) inhibitor of
ethylene synthesis. But in those flowers insensitive to ethylene presence,
water status and carbohydrate supply play a very important role in the
length of the vase life. Thus, pulsing solutions containing sugar or STS
may be effective in postponing earlier senescence in many flower species.
Based on the rate of leaf yellowing and abscission of fruits and leaves, treat-
ment of potted ornamental peppers with 1-MCP prolongs postproduction
life in indoor conditions. However, ethylene partially reversed the inhibition
of 1-MCP on leaf yellowing and abscission.
3.1 Introduction
Ornamental plants, in general, have limited vase or postproduction life;
therefore, the use of adequate postharvest practices of handling is essential
to maintain the quality and prolong the usefulness of the potted plant or
flower. The applied techniques should allow for transportation, and for the
majority of ornamental species, relatively short-term storage—at wholesale
or retail stores—is required before reaching the final consumer.
From the consumer’s point of view, quality is associated with the length
of the shelf life, either at home or in display areas. But the shelf life varies
from ornamental plant to plant, as well as the initial quality of the product
and previous treatments applied to extend the postharvest life. Particularly
in underdeveloped and developing countries, the production areas of orna-
mental plants are not far from the selling commercial centers, and even with-
out modern postharvest handling, the final consumer still receives products
of reasonable quality. However, with the rapid increase in urban population,
the production areas have moved farther away. Under such a scenario, the
use of new techniques for preservation is required, which must guarantee
82
Postharvest Quality of Ornamental Pl ants
quality to the florist and be satisfactory to the consumer. Among the several
techniques available for ornamental plant storage, temperature, relative
humidity, and composition of the atmosphere must be addressed. In addi-
tion, florists have to incorporate treatments to reduce the water and tem-
perature stresses, avoid the deleterious effects of ethylene, and minimize
the influence of low-level radiation in the storage and display areas.
After harvesting, the cut flowers or the potted plants at the postpro-
duction phase are subjected to a series of abiotic stresses, including water
stresses, intense transpiration, exposure to ethylene, and development of
physiological disorders. Usually, leafy ornamental plants are more resis-
tant to senescence than flowers. Flowers, on the other hand, due to their
ephemeral nature and the reduced supply of organic substances, find that
the catabolic metabolism accelerates quickly, thus altering important phys-
iological processes, such as reduction of the water uptake rate, depletion
of respiratory substrates, and an increase of ethylene production and sen-
sitivity. Despite the important influence of ethylene in reducing the vase
life of many ornamental crops, ornamental plants may respond to other
hormones, such as abscisic acid, gibberellins, and cytokinins, to extend of
their postharvest life.
Like any fresh product, ornamental plants, once harvested or trans-
ferred to the display areas, maintain intense respiratory and transpiratory
activities. Respiration will deplete the reserves of organic substrates, which
are already limited, whereas intense transpiration will result in the fast wilt-
ing of leaves and flowers.
Production of carbon dioxide by the ornamental plants can be dimin-
ished by reducing the storage temperature, usually as low as possible, but
above the freezing point of the cells. Many ornamental plants, however,
originate from tropical and subtropical climates and are susceptible to
chilling injury, which restricts the length of shipping and storage at low
temperatures.
In this chapter, we address the different aspects involved in the pres-
ervation of potted ornamental plants and cut flowers, with special attention
to postharvest treatments available to diminish the rate of leaf and flower
senescence.
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
Regarding the flower itself, the most important attributes are the size of the
bud, stage of development, and lack of wilted or abscised petals.
Two important criteria must be achieved at harvest for any ornamen-
tal plant: a presence quality compatible with the consumer’s wishes and an
extended shelf life after harvest or postproduction of potted plants, which
will allow enough time for transportation, eventual storage, and final dis-
play. In order to reach these goals, ornamental plants have to be harvested
at a specific stage of development, as presented in Table 3.1.
The establishment of the developmental stage for harvesting a
particular flower is based on the ability of its bud flower to open after har-
vest, its response to abiotic stresses, and the length of its shelf life.
The standard quality for potted plants depends on the plant s pecies.
For instance, for chrysanthemum and ornamental peppers, the plant canopy
84
Postharvest Quality of Ornamental Pl ants
should cover the whole surface area of the pot. Also, shorter plants with
a compact canopy have a better appearance than taller ones. But in pots
containing orchids and African violets, the presence of flowers in different
stages of opening and the absence of senescent flowers or leaves are more
important than the shape of the plant canopy.
85
P OST H AR V EST RI P ENING P H Y SIOLOG Y
105
100
Fresh weight (%)
95
90
85
80
0 20 40 60 80 100 120
Hours after harvest
Figure 3.1 Behavior of fresh weight in Zinnia elegans cut flowers recut ( )
and uncut ( ) at base of the stem every 12 hours. (From Carneiro, T.F. et al.,
Pesquisa Agropecuaria Brasileira 37, 1065–1070, 2002.)
86
Postharvest Quality of Ornamental Pl ants
96
94
Relative water contet (%)
92
90
88
86
80
0 2 4 6 8 10
Days
and suberin (Van Doorn and Vaslier, 2002). But when inhibitors for either
enzyme were applied to vase water, a delayed blockage of the xylem vessels
was observed, resulting in a longer time until leaf wilting for the cut chry-
santhemum flower.
In roses, the vascular occlusion in the xylem is primarily associated
with the presence of bacteria in the cut at the base of the stem and by the
growing of bacteria in the water of the vase, blocking the water flow to the
flower due to the presence of extracellular polysaccharides and other degra-
dation products that originated from the dead bacteria (Van Doorn, 1997). In
such condition, the roses will develop several symptoms of water deficiency,
including lack of petal opening, bending of the stem neck below the flower,
and leaf wilting (Bleeksma and Van Doorn, 2003). But when roses were main-
tained in the vase in a solution containing 200 mg L −1 8-hydroxyquinoline
sulfate alone or mixed with 30 g L −1 sucrose, the hydraulic conductance of
the stem was kept close to the initial level at harvest, and as consequence,
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Table 3.2 Symptoms of Ethylene Action in Ornamental Cut and Potted Plants
Plant Symptoms
Alstroemeria Darkening, petal abscission
Asiatic lily Abscission of flower, inhibition of flower opening
Carnation Flower wilting
Chrysanthemum Accelerates flower, leaf senescence
Delphinium ajacis Flower abscission
Gypsophila Flower wilting
paniculata
Iris Accelerates flower senescence
Orchids Flower abscission, senescence, epinasty, reddish petal
color
Ornamental Leaf yellowing; abscission of fruits, leaves, and flowers
peppers
Poinsettia Leaf and flower abscission, leaf epinasty
Roses Accelerates flower senescence, induces bud and petal
abscission, interruption of bud flower opening
Snapdragon Floret abscission
88
Postharvest Quality of Ornamental Pl ants
89
P OST H AR V EST RI P ENING P H Y SIOLOG Y
90
Postharvest Quality of Ornamental Pl ants
25 8
20
Abscission (flower day −1)
Longevity (days)
15
10
2
0 200 400 600 800 1000
Ethephon (mg l−1)
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values followed by the same uppercase and lowercase letters are not signifi-
cantly different at the 5% level.
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Cut flowers of Consolida ajacis treated with 1-MCP had their longevity
extended significantly, even when the flowers were exposed to 100 mg L −1
ethephon after they had been exposed to 0.5 g m−3 SmartFresh for 6 h at room
temperature. When ethephon was applied 2 h before the use of 1-MCP, lower
longevity was observed than with 1-MCP alone or when 1-MCP was applied
after the flowers had been treated with ethephon. But for ethephon-treated
flower, a longer vase life was observed than for control fl
owers (Table 3.5).
Thus, the reduction in flower longevity when ethephon was applied before
1-MCP suggests that the latter did not occupy the entire available receptor
sites for ethylene.
In another work, Cameron and Reid (2001) found that 1-MCP had
a transitory effect to block the abscission of Pelargonium peltatum petals
induced by ethylene, and a second fumigation was needed to prevent flower
abscission. Thus, the reduction of flower longevity when ethephon was
applied before 1-MCP in Consolida ajacis and the transient effect observed
in P. peltatum flowers suggest that binding sites for ethylene were not com-
pletely occupied by 1-MCP, or new receptors sites were available by the
presence of exogenous ethylene. It is well known that the presence of ethyl-
ene is able to make the flowers more sensitive to its action.
In addition to 1-MCP, other similar cyclopropene compounds have
shown high effectiveness in blocking ethylene action. 1-Hexylcyclopropene
(1-HCP) and 1-octylcyclopropene (1-OCP) more effectively blocked the del-
eterious effects of ethylene in Kalanchoë blossfeldiana cv. ‘Alexandra’ flow-
ers than 1-MCP (Kebenei et al., 2003). The authors found that pretreatment
with 1-OCP was more effective than that with 1-MCP in delaying flower
senescence at a concentration of 200 nl L −1 for 6 hours, followed by exposi-
tion to 2 µl L −1 ethylene, while the 1-HCP pretreatment had an effect similar
to that of 1-MCP, but at a 5 or 10 times higher concentration.
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When cv. ‘Kiss’ roses were sprayed with 10 or 20 mM calcium sulfate mixed
with 0.01% Tween 20 until the solution ran off, applied 24 hours before har-
vest, postharvest infection with Botrytis cinerea was decreased during
display, thus increasing the vase life by at least 30% at room temperature
(Capdeville et al., 2005). In a similar work, Capdeville et al. (2003) found
that pulsing the same rose cultivar with 50 mM calcium sulfate for 15 hours
reduced the severity of gray mold caused by Botrytis cinerea by 88% and
increase the vase life.
3.7 Respiration
In general, flowers have high respiration rates compared to other horticul-
tural products, which might lead to carbohydrate depletion because flowers
are not adapted as a long-term storage organ. In addition, other deleterious
effects develop under high respiratory activity, including higher transpira-
tion rates and elevated ethylene production and action. The Q10 factor for
most of vegetables and fruits is close to 2, but in flowers, it might reach
values up to 8 (Wills et al., 1998). In cut Narcissus flowers, the respiration
rate in two commercial cultivars was exponentially increased when the
temperature was increased from 0°C to 12.5°C, given a Q10 close to 3.5.
The elevation of the temperature resulted in a negative linear relationship
between the respiratory activity and the vase life (Cevallos and Reid, 2000).
Similar respiration and vase life behavior was observed for cut flowers of
gerbera and sunflower when kept under temperatures ranging from 0°C to
20°C (Çelikel and Reid, 2002). Thus, for these flowers, at the temperature
conditions studied, the respiration can be used as an excellent index to pre-
dict the longevity of the vase life.
The respiration of flowers with high sensitivity to ethylene can be
reduced by applying inhibitors of its action, in particular STS, which acts
as a persistent inhibitor of ethylene action. Altman and Solomos (1995)
found that carnation flowers had no response to exogenous ethylene
regarding the development of petal wilting or an increase in respiration
when the vase solution contained 0.2 mM STS. The senescence of the
flowers in Consolida ajacis is associated with the increase in ethylene pro-
duction and respiration (Finger et al., 2004). When cut flowers of C. ajacis
were pulsed with 1 mM STS or 1 mM STS + 5% sucrose, the increase
of respiration was inhibited, thus prolonging the vase life of the flower
(Figure 3.4). However, by loading the flowers with sucrose alone, no influ-
ence on the longevity was detected, probably due to the weak effect of the
carbohydrate in lowering the respiration compared to the STS (Finger
et al., 2004). Similarly to sucrose pulsing, spraying the flowers with 2 mM
AOA or 20 mM ASA had no effect on the respiration or, as a consequence,
the flower longevity (Figure 3.4).
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240 Control
5% sucrose
1mM STS
200 1 mM STS + 5% sucrose
AOA
ASA
160
Respiration (μl/g/h)
120
80
40
0
0 2 4 6 8 10 12 14 16
Days after harvest
Figure 3.4 Influence of pulsing with sucrose and STS and of spray with
AOA or ASA on respiration of Consolida ajacis flowers. (From Finger, F.L.
et al., Pesquisa Agropecuaria Brasileira 39, 533–537, 2004.)
3.8 Temperature
Temperature is the most important postharvest factor influencing the quality
and longevity of cut flowers and potted ornamental plants. Similar to any
fresh horticultural product, ornamental plants maintain high respiratory and
transpiratory activities to maintain their vital cell reactions. The intermedi-
ate organic molecules are used for the synthesis of new compounds and to
generate energy in the form of ATP. But respiration also produces heat as
a by-product, which increases when the temperature rises and accelerates
the aging process. The rate of senescence is dramatically reduced by cooling
the ornamental plants immediately after harvest. By far, the storage of cut
flowers at low temperatures has been used as the most adequate technique
for long-distance transport and, in many instances, for short-term storage
by the retail florists. The major effects of temperature on ornamental plant
storability and their posterior display are determined by the rate of respira-
tory substrate depletion and by the intensity of the transpiration rate and how
much production and action of ethylene will develop in the plants submitted
to temperature stress. Furthermore, all the previous effects of undesirable
storage temperatures will favor the development of postharvest diseases.
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Postharvest Quality of Ornamental Pl ants
Table 3.6 Vase Life of Bird-of-Paradise Flowers after Pulsing with 40%
Sucrose for 24 Hours and 7, 14, 21, and 28 Days of Storage at 10°C
Vase Life (Days)
Treatments/Days of Storage
at 10°C 7 14 21 28
Distilled water 8.3 a 6.7 b 3.3 a 1.6 a
Pulsed before cold storage 5.6 b 4.2 c 2.0 b 0.7 b
Pulsed after cold storage 8.6 a 8.0 a 4.0 a 2.0 a
Source: Finger, F.L. et al., Acta Horticulturae 628, 863–867, 2003.
Note: Mean separation within columns by Tukey’s test; values followed by a
letter in common are not significantly different at the 5.0% level.
life was observed (Table 3.6). Furthermore, by pulsing the flowers with
40% sucrose for 24 h after the 10°C storage, a higher number of open
florets was obtained, at least after 7 and 10 days of storage (Table 3.7).
The reduced vase life after 28 days of storage was associated with the
development of chilling symptoms, characterized by discoloration and
Penicillium spp. contamination in the flowers.
When the storage temperature for a particular ornamental species
is not known, determine the optimum temperature of storage by placing
the flowers at different temperatures, usually ranging from 0°C up to 15°C.
Most of the temperate ornamental crops are chilling insensitive and can be
stored between 0°C and 2°C. However, for plants that originate from tropi-
cal and subtropical regions, it is always important to consider their sensitiv-
ity to chilling injury. In the majority of cases, the symptoms of injury will
develop under the chilling-induced temperatures; if not, the plants have to
be moved to a higher temperature in order to show any disturbance.
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Postharvest Quality of Ornamental Pl ants
14. Clear flame: Make sure that any burner close to the storage room
has a clear flame because a yellow-flame burn releases high
ethylene-concentrated fumes.
15. Stability: Avoid excessive transit in the cold storage room due to
rapid changes in the temperature and relative humidity.
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plants for control of fruit ripening and senescence of vegetables, cut flowers,
and ornamental potted plants (Blankenship and Dole, 2003).
Treatment with 1 g m−3 EthylBloc (0.14% of 1-MCP) for 6 h at a tem-
perature of 20°C to 25°C inhibited the action of ethylene on the abscission
of leaves of the ornamental pepper cultivar ‘Calypso’ (Segatto et al., 2013).
The application of 1-MCP extended the vase life of plants and completely
blocked the sites of ethylene action. Thus, once sprayed with 1-MCP, plants
do not exhibit abscission of leaves when exposed to ethylene (Figure 3.5).
There was no degradation of chlorophyll or leaf abscission in plants of the
cultivar ‘Calypso’, which were pretreated for 6 hours with 1 g m−3 EthylBloc
and subsequently exposed to an atmosphere containing 10 ml L −1 ethylene
at 48 h (Figure 3.5).
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Postharvest Quality of Ornamental Pl ants
of ethylene action prolong vase and shelf life by reducing flower wilting
and abscission of leaves, flowers, and fruits. Furthermore, inhibitors of
ethylene synthesis seem to be less effective than inhibitors of its action
in delaying flower and leaf senescence. Additional data are required to
establish the critical levels of ethylene for the senescence of many orna-
mental plants. The role of abscisic acid, gibberellins, and cytokinins in
the senescence of flowers must be better understood before they can be
of any practical use.
Acknowledgment
FAPEMIG and CNPq provided scholarship and financial support.
References
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of carnations pulsed or continuously treated with silver thiosulfate.
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Bleeksma, H.C., and Van Doorn, W.G. 2003. Embolism in rose stems as
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Brecheisen, S., Haas, H.P., and Röber, R. 1995. Influence of water quality and
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Cameron, A.C., and Reid, M.S. 2001. 1-MCP blocks ethylene-induced
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Campanha, M.M., Finger, F.L., Cecon, P.R., and Barbosa, J.G. 1997. Water
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Capdeville, G. de, Maffia, L.A., Finger, F.L., and Batista, U.G. 2003. Gray mold
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Capdeville, G.D., Maffia, L.A., Finger, F.L., and Batista, U.G. 2005. Pre-harvest
calcium sulfate applications affect vase life and severity of gray mold in
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Carneiro, T.F., Finger, F.L., Santos, V.R., Neves, L.L.M., and Barbosa, J.G.
2002. Influência da sacarose e da base da haste na longevidade de
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Moraes, P.J., Finger F.L., Mapeli A.M., Cecon P.R. and Barbosa J.G. 2009.
Growth and Flower Development of Epidendrum ibaguense Orchid.
Acta Horticulturae 813, 565–570.
Newman, J.P., Dodge, L.L., and Reid, M.S. 1998. Evaluation of ethylene
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Panavas, T., Walker, E.L., and Rubinstein, B. 1998. Possible involvement of
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Paull, R.E., and Chantrachit, T. 2001. Benzyladenine and the vase life of tropi-
cal ornamentals. Postharvest Biology and Technology 22, 303–310.
Pompodakis, N.E., Joyce, D.C., Terry, L.A., and Lydakis, D.E. 2004.
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Porat, R., Borochov, A., Halevy, A.H., and O’Neil, S.D. 1994b. Pollination-
induced senescence of Phalaenopsis petals. Plant Growth Regulation 15,
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Santos, V.R., Finger, F.L., Barbosa, J.G., and Barros, R.S. 2005. Influência do
etileno e do 1-MCP na senescência e longevidade de inflorescências de
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(Capsicum annuum L.). Acta Horticulturae 1000, 217–222.
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and senescence of Grevillia ‘Sylvia’ inflorescences, flowers and flower
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ment in tulip tepal abscission. Physiologia Plantarum 108, 321–329.
Shaul, O., Elad, Y., and Zieslin, N. 1995. Suppression of Botrytis blight in
cut roses flowers with gibberellic acid: Effects of postharvest timing
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108
Chapter 4
Physiology and
Molecular Biology of
Flower Senescence
Kenichi Shibuya and Kazuo Ichimura
National Agriculture and Food Research
Organization (NARO), Fujimoto, Tsukuba, Japan
Abstract 110
4.1 Introduction 110
4.2 Ethylene and Senescence of Cut Flowers 111
4.2.1 Ethylene Response of Cut Flowers 111
4.2.2 Types of Senescence in Cut Flowers with High
Ethylene Sensitivity 112
4.2.3 Ethylene in Petal-Wilting-Type Flowers 113
4.2.4 Ethylene in Petal-Abscission-Type Flowers 114
4.2.5 Ethylene Biosynthesis 114
4.2.6 Ethylene Signal Transduction 115
4.2.7 Acceleration of Flower Senescence by
Pollination 116
4.2.8 Acceleration of Flower Senescence by
Wounding 119
4.2.9 Effect of Temperature on Ethylene Production
and Perception 119
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Abstract
Flower longevity is one of the most important traits of cut flowers. An under-
standing of the postharvest biology of flowers is needed to efficiently develop
postharvest technology. Ethylene is a key factor that regulates flower senes-
cence in many plant species. Ethylene biosynthetic and signaling pathways
have been well characterized, and it is now possible to improve the vase life
of some cut flowers by controlling the effects of ethylene. In contrast, regula-
tory mechanisms of senescence in flowers that show ethylene-independent
senescence remain largely unknown. Petal senescence is a highly regulated
developmental process and is considered to be a type of programmed cell
death, which is an actively regulated process. To identify key regulators of
programmed cell death during petal senescence, many studies have been
conducted. In this chapter, we outline studies on the postharvest physiology
and molecular biology of flowers, focusing on regulatory mechanisms for
petal senescence by ethylene and programmed cell death.
4.1 Introduction
Several postharvest techniques are applied to improve the vase life of many
cut flowers. These techniques have been developed based on knowledge
from basic studies of plant hormones, sugar, and water relations. Ethylene
plays a very important role in the senescence of many cut flowers, and this
plant hormone has been of primary interest in the postharvest biology of
flowers. Besides ethylene, other plant hormones play an important role in
flower senescence and the biochemical functions of biomembranes during
senescence. After the 1990s, with the advances in molecular b iology, genes
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The last two reactions are catalyzed by ACC synthase and ACC
oxidase.
In vegetative tissues of plants, ACC synthase is considered to be a
rate-limiting step in ethylene biosynthesis. However, activities of not only
ACC synthase but also ACC oxidase rise during petal senescence of car-
nation, suggesting that both enzymes are responsible for the increase in
ethylene production (Woodson et al., 1992). In the gynoecium of carnation
flowers, on the other hand, ACC oxidase activity is constantly high during
senescence, and thus the increase in ethylene production in this tissue is
likely due to a rise in ACC synthase activity (Yangkhamman et al., 2007).
ACC synthase and ACC oxidase are encoded by multigene families,
and genes encoding these enzymes have been isolated in many ornamental
plant species. Three ACC synthase genes have been isolated from carna-
tion (Park et al., 1992; Henskens et al., 1994; Jones and Woodson, 1999a),
four from rose (Wang et al., 2004), three from snapdragon (Woltering et al.,
2005), two from geranium (Wang and Arteca, 1995), and two from petunia
(Lindstrom et al., 1999). ACC synthase genes are differentially regulated
in a tissue-specific manner. For example, among the three carnation ACC
synthase genes, DcACS1 is most abundant in petals, while DcACS2 and
DcACS3 are preferentially expressed in styles (Jones and Woodson, 1999a).
For ACC oxidase, two genes have been isolated from carnation
(Wang and Woodson, 1991; Norikoshi et al., 2008), one from rose (Xue
et al., 2008), one from snapdragon (Woltering et al., 2005), one from
Phalaenopsis (Nadeau et al., 1993), five from tulip (Momonoi et al., 2007),
four from petunia (Tang et al., 1993), and three from geranium (Clark
et al., 1997). ACC oxidase genes are regulated in a spatial- and temporal-
specific m anner. In carnation, DcACO1 is clearly upregulated in petals dur-
ing senescence, while DcACO2 is constantly expressed in the gynoecium
(Norikoshi et al., unpublished data). Differential expression of ACC oxi-
dase genes has also been reported in petunia (Tang et al., 1994).
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
responses (Hua and Meyerowitz, 1998). The receptors act through CTR1
(CONSTITUTIVE TRIPLE RESPONSE1), which is homologous to the
Raf family of Ser/Thr kinases and negatively regulates ethylene signaling
(Kieber et al., 1993; Huang et al., 2003). Downstream of the receptor–CTR1
complex is EIN2 (ETHYLENE INSENSITIVE2), which has homology to
Nramp metal transporters and positively regulates signaling (Alonso et al.,
1999). EIN2 undergoes proteasome-mediated turnover (Qiao et al., 2009),
and ethylene stimulates phosphorylation-dependent cleavage and nuclear
movement of a carboxyl-terminal EIN2 fragment (Qiao et al., 2012). Toward
the end of the signaling pathway, ethylene responses are mediated by tran-
scription factors including EIN3 (ETHYLENE INSENSITIVE3) and ERF1
(ETHYLENE RESPONSE FACTOR1) (Chao et al., 1997; Solano et al.,
1998). Regulation of the stability of EIN3 protein is a key aspect of the ethyl-
ene response (Guo and Ecker, 2003; Yanagisawa et al., 2003).
Genes encoding ethylene receptors have been isolated in several
ornamental plant species, for example, three in carnation (Shibuya et al.,
2002), five in rose (Müller et al., 2000a, 2000b), one in chrysanthemum
(Narumi et al., 2005), two in Delphinium (Kuroda et al., 2003; Tanase and
Ichimura, 2006), four in petunia (Wang and Kumar, 2007), two in geranium
(Dervinis et al., 2000), and two in gladiolus (Arora et al., 2006). Genes
have been reported that encode CTR1 in rose (Müller et al., 2002) and
Delphinium (Kuroda et al., 2004), EIN2 in petunia (Shibuya et al., 2004),
and EIN3 in carnation (Waki et al., 2001; Iordachescu and Verlinden, 2005),
rose (Müller et al., 2003), and petunia (Shibuya and Clark, 2006).
Ethylene receptors are negative regulators of ethylene signaling, and
depletion of the receptors is considered to lead to an increase in ethylene
sensitivity (Hua and Meyerowitz, 1998). However, it has been reported that
the transcript levels of ethylene receptors in rose are higher in cultivars with
higher ethylene sensitivity in rose (Müller et al., 2000a). The inconsistency of
the relationship between levels of transcript for ethylene receptors and ethyl-
ene sensitivity might be explained by posttranslational regulation of ethylene
receptor proteins. Kevany et al. (2007) showed that protein levels for ethylene
receptors in tomato are not correlated with corresponding transcript levels
during fruit development and that the receptors are rapidly degraded in the
presence of ethylene. Thus, the level of ethylene receptor proteins seems
to be responsible for ethylene sensitivity. Similar regulation of the ethylene
response is expected in flower senescence. Analysis of ethylene receptors at
the protein level is necessary in the study of flower senescence.
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and Dijkman, 1967; Porat et al., 1994), petunia (Gilissen and Hoekstra,
1984; Whitehead et al., 1984), Eustoma (Ichimura et al., 1998; Ichimura
and Goto, 2000), carnation (Larsen et al., 1995; Jones and Woodson, 1997),
Campanula (Kato et al., 2002), Delphinium (Okamoto and Ichimura, 2010),
and Torenia (Goto et al., 1999). In particular, flower longevity of orchid spe-
cies is dramatically shortened by pollination. In daffodil flowers, which
show relatively low ethylene sensitivity, pollination also accelerates flower
senescence (Hunter et al., 2004b).
Pollination raises ethylene production from flowers, and treatment
with inhibitors of ethylene biosynthesis or ethylene perception cancels
the acceleration of flower senescence in many plant species (Stead, 1992).
Furthermore, transgenic petunia plants with reduced ethylene sensitiv-
ity due to suppressed expression of PhEIN2 exhibit significant delays in
petal senescence after pollination (Figure 4.1) (Shibuya et al., 2004). These
observations suggest that ethylene is a primary signal for pollination-
accelerated flower senescence.
In Eustoma (Shimizu-Yumoto and Ichimura, 2006b), petunia (Gilissen,
1977), and Digitalis (Stead and Moore, 1979), a greater amount of pollen
pollinating the stigma results in faster progression of flower senescence.
In this case, a greater concentration of ethylene inhibitors is needed to
counter the effect of pollination in flower senescence.
Compatibility between pollen and gynoecium involves the accel-
eration of flower senescence by pollination. In self-incompatible petunia,
compatible pollination accelerates the second peak of ethylene synthesis,
leading to hastened petal senescence, while incompatible pollination does
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not accelerate the second peak of ethylene synthesis and does not lead to
hastened petal senescence (Singh et al., 1992). Similar phenomena have
been observed in carnation (Larsen et al., 1995).
In pollinated orchid flowers, ethylene sensitivity rises prior to an
increase in ethylene production. This rise in ethylene sensitivity is not
suppressed by an ethylene synthesis inhibitor, AOA, suggesting that the
increase in ethylene sensitivity is regulated independently from e thylene
synthesis (Porat et al., 1994, 1995a). In addition, treatment with STS, an eth-
ylene perception inhibitor, suppresses the increase in ethylene production.
These observations suggest that the dramatic rise in ethylene production
after pollination in orchid flowers may be caused by the perception of a rela-
tively low level of ethylene as a result of an increase in ethylene sensitivity.
Detailed analyses of ethylene production patterns after pollination
have been conducted in several flower species. Petunia flowers produce
ethylene in styles within 3 h and then in petals at 2–3 days after pollination
(Hoekstra and Weges, 1986; Singh et al., 1992). In carnation, an increase
in ethylene production is observed at around 12 hours in the stigma and at
24 hours in ovaries, and then at 36 hours in petals after pollination (Jones
and Woodson, 1999b). In Phalaenopsis, ethylene increases at 12 hours in the
stigma and column, at 24 hours in the labellum, and after 48 hours in the
perianth after pollination (O’neill et al., 1993).
It has been reported that in pollinated Phalaenopsis flowers, the accu-
mulation of mRNA for ACC synthase and ACC oxidase increases in the
gynoecium, while mRNA for ACC oxidase is not detectable in the perianth
(O’Neill et al., 1993). Based on this observation, the following model has
been proposed: in the perianth, ACC is translocated from other flower parts,
such as the gynoecium, and is converted to ethylene (O’Neill et al., 1993).
However, mRNA for ACC synthase has since been detected in the perianth
using real-time reverse transcription polymerase chain reaction (RT-PCR),
a more sensitive technique, and the level of PhalACS1 mRNA increases in
the perianth after pollination (Ichimura and Niki, unpublished data).
In petunia, when the style is removed within several hours of pol-
lination, petal senescence is not accelerated by pollination (Gilissen and
Hoekstra, 1984). This observation suggests the existence of mobile signals
that induce petal senescence after pollination. Candidates for the mobile
signals are ACC and ethylene. When radiolabeled ACC is applied to the
stigmas of carnation flowers, radioactive ethylene is produced both by the
gynoecia and by the petals, suggesting that ACC may be a signaling com-
pound transported from the stigmas to the petals (Reid et al., 1984). In con-
trast, radiolabeled ACC applied to the stigma is largely immobile in flowers
of Cymbidium (Woltering et al., 1995) and petunia (Woltering et al., 1997).
Furthermore, in petunia, eluates collected from the styles were able to
induce petal senescence, but ACC was not detected in the eluates (Gilissen
and Hoekstra, 1984). These observations suggest that ACC is not a mobile
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4.3.1 Auxin
Auxin at relatively high concentrations induces ethylene synthesis. In
carnation, treatment with indoleacetic acid or 2,4-dichlorophenoxyacetic
acid, a synthetic auxin, induces ethylene production, leading to hastened
petal senescence (Sacalis and Nichols, 1980; Wulster et al., 1982). Auxin
at high concentrations seems to induce ACC synthase activity, resulting in
an increase in ethylene production. In contrast, application of 1-naphtha
leneacetic acid, another synthetic auxin, suppresses abscission of bougain-
villea flowers (Chang and Chen, 2001).
4.3.2 Gibberellin
Gibberellin treatment prolongs flower longevity by suppressing ethylene
synthesis in carnation (Saks et al., 1992). It also prolongs the vase life of
daffodil, which has relatively low ethylene sensitivity (Ichimura and Goto,
2002). Since it is difficult to accurately quantify the gibberellin content in
plants, little is known of the distribution and movement of gibberellins in
cut flowers.
4.3.3 Cytokinin
Cytokinin generally delays senescence in plants. In rose (Mayak and
Halevy, 1970) and carnation (van Staden et al., 1987), cytokinin-like activity
decreases in senescent flowers. Application of benzyladenine, a synthetic
cytokinin, to presenescent cut carnation flowers delays petal senescence
(Mayak and Dilley, 1976a; Eisinger, 1977; Cook et al., 1985).
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the petals senesce (Yamada et al., 2009). DAD1 (defender against apoptotic
cell death) is a protein that suppresses the progression of PCD in animal
cells. The mRNA level of a DAD1 homolog starts to decrease at early stages
of petal senescence in Alstroemeria (Breeze et al., 2004), while it decreases
at later stages in carnation (Hoeberichts et al., 2007), gladiolus (Yamada
et al., 2004), and Iris (Van Doorn et al., 2003). However, the role of DAD1 in
flower senescence is unknown.
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137
Chapter 5
Respiratory
Metabolism
Mikal E. Saltveit
University of California, Davis, California
Abstract 140
5.1 Introduction 140
5.2 Why Measure Respiration? 141
5.3 Major Components of Respiration 142
5.3.1 Glycolysis 142
5.3.2 Pentose-Phosphate Shunt 142
5.3.3 Anaerobic Diversion 143
5.3.4 Tricarboxylic Acid Cycle 143
5.3.5 Electron Transport (Chemiosmotic
Phosphorylation) 145
5.4 Measurement of Respiration 146
5.4.1 Loss of Substrate, Heat Production,
and Water 146
5.4.2 Consumption of Oxygen and Production
of Carbon Dioxide 147
5.4.2.1 Static System 147
5.4.2.2 Flow-Through or Dynamic System 149
5.5 Sampling and Analyzing 151
5.6 Instruments and Techniques 152
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Abstract
The three groups of catabolic reactions comprising respiration provide the
energy, the reducing power and carbon fragments used in subsequent ana-
bolic metabolic reactions. Horticultural crops are harvested either at the
optimal stage of maturity and quality (e.g., cucumber, lettuce, zucchini) or
at a stage of maturity that requires subsequent changes to reach maximal
quality (e.g., avocadoes, bananas, tomatoes). In the first group, respiratory
metabolism is suppressed in the postharvest environment to levels only
needed to maintain product quality. In the latter group, the often profound
changes that accompany ripening (e.g., pigment destruction and synthesis,
synthesis of characteristic aroma and flavors, tissue softening, and textural
changes) require significant inputs of energy and substrates derived from
respiration. Measurements of respiration (e.g., carbon dioxide production,
oxygen consumption) give a good indication of the metabolic activity of
the tissue, and thereby the rate of changes in product quality. The rate of
respiration and metabolic activity is tightly coupled to tissue temperature.
Both are reduced by lower product temperatures and increased by elevated
product temperatures. Some commodities (especially those of tropical and
subtropical origin, e.g., avocado, banana, bean, and squash) suffer physi-
ological damage (i.e., chilling injury) if exposed to temperatures below
10°C (50°F) for commodity-specific inductive periods. Respiratory and
metabolic activity can also be reduced by limiting the availability of oxygen
to the tissue. However, too little oxygen induces anaerobic respiration with
production of uncharacteristic compounds that can reduce product quality.
5.1 Introduction
The function of respiratory metabolism is threefold: to extract energy
stored in the chemical bonds of substrate molecules (e.g., glucose, fatty
acids, proteins) and convert it into high-energy phosphate bonds (i.e., ATP),
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Respir ato ry Me ta bo lism
141
P OST H AR V EST RI P ENING P H Y SIOLOG Y
inversely related to the shelf life of the commodity. The higher the rate of
respiration, the more quickly there will be a decrease in the quality, nutri-
tion, or taste of the commodity. Since we cannot easily measure changes in
these parameters of shelf life, we use measurements of the rate of respira-
tion as their surrogate.
5.3.1 Glycolysis
As its name implies, the 10 enzymatic reactions in glycolysis break (or lyse)
the 6-carbon glucose molecule into two 3-carbon pyruvate molecules. The
reactions of glycolysis take place in the cytoplasm. Glucose is first phosphory-
lated in a series of reactions by two ATPs to produce fructose-1,6-diphosphate.
Living cells are homeostatic over short periods of time and tend to maintain
the status quo by carefully regulating the concentration of ATP. A key enzyme
in glycolysis is phosphofructokinase (PFK), a multicomponent enzyme that
converts fructose-6-phosphate into fructose-1,6-diphosphate, thereby pre-
paring the hexose for cleavage into two dissimilar phosphate-containing
3-carbon molecules (i.e., dihydroxyacetone phosphate and glyceraldehyde-
3-phosphate [G3P]), which are interconvertible. The rate of glycolysis is reg-
ulated primarily through controlling the activity of PFK, an enzyme that is
inhibited by ATP, one product of respiration. However, almost every enzyme
in the respiratory pathway is under some sort of metabolic control, indicating
the importance of careful regulation of respiration and metabolism.
Each molecule of G3P undergoes a series of reactions during which
one molecule of NADH and two molecules of ATP are produced. The result-
ing two molecules of the 3-carbon pyruvate contain all six carbons that were
in the original molecule of glucose. Two ATPs were needed to initiate the
reaction, but four ATPs and two NADHs were produced, for a net gain of
two ATPs and two NADHs. So while there are the same numbers of carbon
atoms in the two pyruvate molecules as there were in the original glucose
molecule, the pyruvate molecules have lost about 20% of the extractable
energy originally present in the glucose molecule. The remaining 80% is
extracted by the two subsequent reactions of respiration. Notice that no oxy-
gen was consumed, and no carbon dioxide was produced during glycolysis.
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Respir ato ry Me ta bo lism
143
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Polysaccharides
Proteins Fats
Simple sugar
(e.g., glucose)
Amino acids Fatty acids
glycerol
Glycolysis
-oxidation
CO2
Pyruvate
Acetyl CoA
Citric
acid CO2
cycle
CO2
Reducing Power
3 O2 (NADH, FADH2)
Electron transport
ATP ADP + Pi
3 H2O Mitochondria Cytoplasm
completing the cycle. During the cycle, three NADHs, one FADH2, and one
ATP are produced from each pyruvate molecule (Figure 5.2).
High-energy molecules are not the only product of the TCA cycle. The
intermediates within the cycle can be substrates for subsequent synthetic
reactions. For example, pyruvate is used in the synthesis of acetaldehyde,
ethanol, lactic acid, and alanine; acetyl CoA is used in the synthesis of fatty
acids, cuticular compounds, isoprenoids, carotenoids, sterols, terpenes,
and aromatic amino acids; α-ketoglutarate is used in the synthesis of glu-
tamic acid, other amino acids, chlorophyll, cytochromes, and phytochrome;
144
Respir ato ry Me ta bo lism
Pyruvic acid
COOH Pyruvic
C=O decarboxylase CO2
NADH
CH3
Lactate Acetaldehyde
dehydrogenase Pyruvic H-C=O
2H+ oxidase CH3 NADH
+
NAD
Alcohol
CO2 dehydrogenase
COOH S-CoA 2H+
COOH C=O +
NAD
H-C-OH CH3 H
CH3 H-C-OH
Acetyl-CoA CH3
Lactic acid
Ethanol
To TCA cycle
and oxaloacetic acid is used in the synthesis of aspartic acid, alkaloids, and
nucleic acids.
Notice that in the TCA cycle, all the carbon from the glucose molecule
was released as CO2, but no oxygen has yet been consumed.
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
membrane. This ATPase uses the energy in the returning protons to pro-
duce ATP from ADP and Pi (inorganic phosphate), a process called chemi-
osmotic phosphorylation.
146
Respir ato ry Me ta bo lism
147
P OST H AR V EST RI P ENING P H Y SIOLOG Y
0.4
O2 CO2
Carbon dioxide %
0.3
0.2
0.1
0
0 1 2 3 4 5
Time
(a) (b)
may strongly affect the tissue and its respiration rate. As can be seen in the
graph, the accumulation of CO2 becomes nonlinear at around 0.2%.
Problems with the accumulation of physiologically active concen-
trations of CO2 and C 2H4 can be overcome by including KOH to absorb
CO2 and KMnO 4 to oxidize C 2H4 . Indeed, some respirometers ( jargon for
a machine that measures respiration) measure the pressure (or volume)
change resulting from absorption of respired CO2 . The most notable of
these, the Warburg respirometer, was instrumental in determining the
pathway of glycolysis and has driven many postharvest physiologists to
strong drink because of the technical difficulties often encountered in
its use.
To calculate the rate of respiration in a static system, you need to know
the following: volume of container, weight of tissue, initial carbon dioxide
concentration, length of time, and final carbon dioxide concentration.
For example, assume that a 280 g apple is enclosed in a 1560 ml
container for 15 min. During that time, the CO2 concentration in the jar
increased from 0.03% to 0.23%, or the CO2 concentration increased 0.2%.
Converting the weight and time units to kilograms and hours gives a
weight of 0.28 kg and a sampling period of 15/60 = 0.25 h. The simplest
way to calculate the amount of CO2 produced during the sampling period
would be to multiply the difference in CO2 concentrations between the
beginning and end of the sampling period (i.e., 0.2%) by the total volume of
the container (0.2% × 1560 ml = 3.12 ml). Dividing that volume of CO2 by
148
Respir ato ry Me ta bo lism
the weight (in kg) and the sampling period (in hours) would give the rate
of CO2 production (i.e., 3.12 ml/(0.28 kg × 0.25 h) = 44.6 ml CO2/kg-h).
However, this calculation could be criticized because many assump-
tions were made that can significantly alter the answer. For example, it did
not take into account the fact that the volume occupied by the apple reduced
the total gas volume in the jar; i.e., the density of the tissue could deviate
from 1 and affect its volume, and the solubility of the gas being measured
in the cellular solution. The density of an apple is around 0.8 g/ml, so the
volume occupied by the apple would be 280/0.8 = 350 ml. The liquid in the
apple fruit will absorb some of the CO2 produced. The solubility of 100% CO2
in water at 20°C is 0.878 ml CO2/ml water (and increases with decreasing
temperatures). Taking all these caveats into consideration, we can conclude
that under normal physiological conditions, the rate of CO2 production can
be calculated to within about 5% of its true value by ignoring the volume
changes introduced by the tissue. The volume of CO2 produced can be cal-
culated by multiplying the change in concentration by the entire void vol-
ume of the container.
The significantly lower solubility of oxygen and ethylene in the
aqueous solution of most commodities requires that the volume of the
tissue be subtracted from the container volume when calculating their
production rate.
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
Gas in
0.15
O2 CO2
Carbon dioxide %
0.10
0.05
0.00
0 2 4 6 8 10
(a) (b) Time
Gas out
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Respir ato ry Me ta bo lism
151
P OST H AR V EST RI P ENING P H Y SIOLOG Y
152
Respir ato ry Me ta bo lism
153
P OST H AR V EST RI P ENING P H Y SIOLOG Y
than predicted; that is, the rate of respiration falls abnormally rapidly. In
some cases, the respiration rate may even increase dramatically at lower
temperatures, possibly associated with increased glycolysis to compensate
for loss of mitochondrial oxidation. These events are symptoms of the onset
of chilling injury, an economically important low-temperature phenomenon.
Another important respiratory effect of exposure of sensitive com-
modities to chilling temperatures is abnormally high respiration rate (sus-
tained if the tissue has been irreversibly damaged) when the commodity is
returned to normal temperatures. This enhanced respiration presumably
reflects the cells’ efforts to detoxify metabolic intermediates that may have
accumulated during the chilling exposure, as well as to repair damage to
membranes and other subcellular structures.
As the temperature rises beyond the physiological range, the rate
of increase in respiration falls. It becomes negative as the tissue nears its
thermal death point, when metabolism is disorderly and enzyme proteins
are denatured. Many tissues can tolerate high temperatures for short times
(e.g., minutes), and this property is used to advantage in killing some surface
fungi in stone fruits and papaya. Continued exposure to high temperatures
causes phytotoxic symptoms and then tissue collapse. However, condition-
ing and heat shocks (i.e., short exposures to potentially injurious tempera-
tures) can modify the tissue’s responses to subsequent harmful stresses.
154
Respir ato ry Me ta bo lism
signal that migrates from the site of injury and induces a wide range of
physiological changes in adjacent, nonwounded tissue. Some of the more
important changes include enhanced respiration, ethylene production,
phenolic metabolism, and wound healing. Most wound responses are det-
rimental to quality (e.g., rapid softening, browning, toughening), but some
(e.g., wound healing in potatoes) are necessary for their long-term storage.
5.7.4.1 Genotype
Some fruits are more perishable than others. You would not, for example,
expect a strawberry to last as long as apples in your fruit bowl. This relative
perishability is usually reflected in the respiration of the different commodi-
ties. The respiration of strawberries is much higher than that of apples.
Some short-lived apple and melon fruit are from cultivars with higher rates
of respiration than long-lived cultivars.
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References
Kader, A.A. 1992. Postharvest Technology of Horticultural Crops. 2nd ed.
Division of Agriculture and Natural Resources, University of California,
Oakland.
Kader, A.A., and Saltveit, M.E. 2003. Respiration and gas exchange. In
Postharvest Physiology and Pathology of Vegetables, ed. J.A. Bartz and J.K.
Brecht. Marcel Dekker, New York, pp. 7–30.
Kays, S.J., and Paull, R.E. 2004. Postharvest Biology. Exon Press, Athens, GA.
Wills, R.B.H., McGlasson, B., Graham, D., and Joyce, D. 1998. Postharvest:
An Introduction to the Physiology and Handling of Fruit, Vegetables and
Ornamentals. Wallingford, Oxon, UK: CAB International.
156
Chapter 6
Stomata and
Postharvest Physiology
Uulke van Meeteren1 and Sasan Aliniaeifard 2
1Wageningen University, Wageningen, the Netherlands
2University of Tehran, Tehran, Iran
Abstract 158
6.1 Introduction 158
6.2 Stomata 159
6.2.1 Role of Stomata in Plants 159
6.2.2 Mechanism of Stomatal Closure and Opening 161
6.2.3 Signal Transduction Pathways in Guard Cells
for Stomatal Closure 164
6.3 Role of Stomata in the Postharvest Phase 166
6.3.1 Stomata in Relation to Vase Life of Cut Flowers 166
6.3.2 Stomata in Relation to Quality of Vegetables 169
6.3.3 Stomata in Relation to Quality of Fruits 172
6.4 Preharvest Conditions Leading to Postharvest
Problems via Stomata 174
6.4.1 Relative Humidity and Stomata Control 175
6.4.1.1 How Does Preharvest Low VPD Affect
Postharvest Stomata Control? 176
6.4.1.2 Induction of Stomata Morphological
Changes by Low VPD 179
157
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Abstract
Stomata are pores in the gastight waxy cuticula that covers the outer surface
of aerial parts of plants. They make uptake of CO2 possible, which is needed for
photosynthesis. At the same time, water vapor will leave the plant via the sto-
mata. To optimize photosynthesis, while at the same time preventing excess
water loss, stomata control their opening by a signaling network of pathways
that respond to environmental conditions such as light and darkness, water
vapor pressure deficit (VPD), temperature, CO2, and ethylene. Water loss is
one of the most obvious changes in harvested vegetables, often limiting mar-
keting life, and a negative water balance (uptake of water is insufficient to
compensate transpiration) is one of the most important reasons for the end of
the vase life of cut flowers. Also, the postharvest quality of some fruits is nega-
tively affected by water loss. In leafy vegetables, stomata are portals that make
invasion of bacteria into the inner tissue possible and protect in that way bac-
teria for sanitizers in washing solutions with the risk of food-borne bacterial
diseases. This chapter discusses how several environmental factors, during
preharvest cultivation as well as postharvest storage, influence stomata clos-
ing control in harvested cut flowers, vegetables, and fruits. Also, the role of the
number of stomata and their variability between genotypes and due to cultiva-
tion conditions are discussed in relation to postharvest life. One of the most
striking factors is low VPD (high humidity) during the growth of plants: after
exposure of several days to low VPD, the control of stomata closure is largely
disturbed; stomata do not respond anymore to stimuli that normally induce
closure. This malfunctioning is very persistent and results in high water loss
afterward in the harvested products. Fast cooling of produce can close the
stomata of some crops, while in others, the stomata stay open until wilting.
6.1 Introduction
A waxy cuticle covers the outer surface of the epidermal cells of the a erial
parts of plants. This cuticular layer protects plants from drying via its
airtight properties. However, a plant needs gas exchange (intake of CO2
158
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and O2) for photosynthesis and respiration. Therefore, there are pores in the
cuticle, called stomata, of which the opening can be controlled by surround-
ing guard cells. Stomata are the main openings connecting the internal
leaf space to the outside environment. The intercellular air spaces in plant
tissues are saturated with water vapor, which will exit the tissue through
these stomata. Water loss and respiration are two of the most important
processes involved in postharvest physiology of freshly harvested products;
therefore, understanding the control of the opening of the stomata in the
postharvest phase is of importance to control the postharvest behavior of
plant produce.
Besides gas exchange, stomata also offer opportunities for fungi
and bacteria to enter plant tissues. The infection can take place during the
preharvest growth period as a latent infection that results in postharvest
losses, but contamination with bacteria can also take place during handling
after harvest. Especially when human pathogenic bacteria enter plant tis-
sue via the stomata, they are a serious threat to human health.
The closing and opening behavior of stomata is instantly affected by
many environmental cues. Above this, exposure for some time to specific
environmental conditions can influence the stomatal closing control after-
wards. As a consequence, climatic conditions during cultivation can affect
the stomatal closing control during the postharvest phase. In this chapter,
we describe the control mechanism of stomata control, how this control can
be affected by previous environmental conditions, and the specific possible
consequences for quality changes during the postharvest phase of orna-
mentals, vegetables, and fruits.
6.2 Stomata
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(a)
(b)
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162
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Stom ata a n d P ostharvest P hy s i o l o g y
protein kinases are positive regulators of ABA signaling (Fujii et al., 2009;
Fujii and Zhu, 2009; Ma et al., 2009; Park et al., 2009; Umezawa et al., 2009;
Vlad et al., 2009; Lee and Luan, 2012). Downstream of ABA receptors,
PP2Cs, and SnRKs are ion channels that control stomatal movements (Fujii
et al., 2009; Geiger et al., 2009; Lee et al., 2009). The guard cell slow-type
anion channel (SLAC1) may represent an essential component for stomatal
closure induced by ABA or other signals (Negi et al., 2008; Vahisalu et al.,
ABA
ABA Receptors
Calcium-independent ABA signaling
OST1 CDPKs
Ion channels
(e.g., SLAC1)
Stomatal
closure
Figure 6.2 Simplified ABA signal transduction pathway in guard cells for
closure of the stomata. In a calcium-independent ABA signaling pathway,
perception of ABA by receptors leads to inactivation of type 2C protein
phosphatases (PP2C). As a result, S-type anion channels (SLAC1) will be
activated by a SnRK2-type protein kinase (OST1). Consequently, stomatal
closure occurs. In the calcium-dependent ABA signaling pathway, calcium-
dependent protein kinases (CDPKs) can induce stomatal closure via activa-
tion of SLAC1 (Data from Hubbard, K.E. et al., Genes and Development
24, 1695–1708, 2010; Kim, T.H. et al., Annual Review of Plant Biology
61, 561–591, 2010; Antoni, R. et al., Current Opinion in Plant Biology 14,
547–553, 2011; Sreenivasulu, N. et al., Gene 506, 265–273, 2012; Lee,
S.C. et al., Molecular Plant 6, 528–538, 2013).
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2008, 2010; Geiger et al., 2009). SLAC1 acts as a substrate for and is acti-
vated by OST1 (Geiger et al., 2009; Lee et al., 2009, 2013).
Calcium-dependent protein kinases (CDPKs) function as essential
elements of the calcium-dependent ABA signaling (Zhu et al., 2007). It has
been shown that SLAC1 can be activated by CDPKs, which leads to stoma-
tal closure (Mori et al., 2006; Geiger et al., 2010) (Figure 6.2).
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The decrease in water potential can cause cavitations, which will further
enlarge the imbalance between water uptake and loss.
The negative effect of a decreased stem hydraulic conductance on the
water balance of a cut flower will be postponed or less severe when the tran-
spiration rate of the cut flower is low. As an example, it has been shown that
the vase life of cut roses can be prolonged by various ways that lower their
transpiration: cooling, high air humidity, and leaf removal (Zieslin, 1989).
Mayak and Halevy (1974) showed that the difference in vase life between a
short-lived cultivar and two long-lived cultivars of rose was large, especially
under conditions promoting high transpiration rates, and was narrowed when
either flowers were held in mild conditions or the leaves were stripped off.
Most cut flowers include, besides one or more flowers, a stem that
often bears leaves. The main parts of a flower consist of a vegetative part,
containing sepals and petals, and a reproductive part, containing stamens
and a gynoecium. Because a waxy cuticle covers the outer surface of the
epidermal cells of leaves, sepals, petals, and the gynoecium, most of the
transpiration of cut flowers is via the stomata. Therefore, it can be expected
that the number and the closing and opening behavior of stomata play a role
in vase life. Stomata are usually present in green epidermal tissues such as
leaves, sepals, and green (parts of) petals (Chimona et al., 2012), but also in
some nongreen petals, stomata can be found (Effmert et al., 2005; Chimona
et al., 2012). Stomata can also be completely absent in petals (Mayak and
Halevy, 1974; Tahir and Rajput, 2010). In petals of commercially important
crops like roses (Rosa hybrida), carnations (Dianthus caryophyllus), and ger-
bera (Gerbera jamesonii), stomata were not found (Van Doorn, 1997). As
shown in the overview by Van Doorn (1997), numbers of stomata in petals
vary between 0 and 45 per cm 2, which is very low compared with the num-
bers of stomata found in leaves, which varies between 5 and 1000 per mm 2,
depending on species and growth conditions (Table 6.1) (Hetherington and
Woodward, 2003). When present, stomata in petals are not always func-
tional, as shown for several orchids (Hew et al., 1980). An exceptional flower
in this context is tulip; tulip petals can contain 5–49 stomata per mm 2, and
a temperature-dependent stomatal movement in tulip petals controls water
transpiration during flower opening and closing (Azad et al., 2007). In con-
clusion, for most cut flowers, the main transpiration is via the stomata in the
leaves of the cut flowering stem.
The role of stomata in vase life was elegantly demonstrated by Halevy
et al. (1974) in an experiment about the effects of application of ABA on
the vase life of cut roses. Application of ABA can induce stomatal closure,
reduce water loss, and extend the vase life of cut rose flower shoots bear-
ing leaves. Its effect was very pronounced when the flowers were exposed
to conditions that promote transpiration (28°C and 45% RH). However, in
a stomata-less system (leafless flower shoots) or in leafy shoots held in
darkness when all stomata were closed, ABA shortened vase life because it
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increased the aging of the flowers and some biochemical processes associ-
ated with it (RNase activity and reduction in protein content).
Elibox and Umaharan (2008) evaluated the vase life of 26 anthurium
(Anthurium andraeanum Hort.) cultivars. Among the tested cultivars, there
were large differences in vase life, varying from 14 to 49 days. Anthurium cut
flowers do not hold leaves on their cut stem and have a low transpiration rate
and long vase life. Still, symptoms associated with the end of the vase life in
anthurium are typical of water stress (Paull, 1982). Anthurium cut flowers
have a brightly colored modified leaf, the spathe, which subtends a spadix
with more than 300 spirally arranged minute flowers. This spathe contains
stomata, mostly on the abaxial side, of which the density varies between
the cultivars from 1.8 to 25.7 per mm 2. Although the rate of water loss of
anthurium cut flowers is relatively low, of 12 morphophysiological character-
istics, only spathe color and abaxial stomatal density were able to accurately
predict vase life (Elibox and Umaharan, 2008). In their study, Elibox and
Umaharan (2008) also showed that changes in vase life of cultivars over
season were mediated through changes in stomatal density. However, culti-
var differences in stomata density of the spathe explained only a small pro-
portion of cultivar variation in vase life. In a later study, the same authors
showed that the balance between factors affecting water uptake, which is
determined by the timing, extent, and duration of vascular occlusion, and
those affecting water loss (e.g., stomatal density and regulation) may con-
tribute to cut flower senescence in anthurium (Elibox and Umaharan, 2010).
In an evaluation of snapdragon (Antirrhinum majus) cultivars, that
differed in length of vase life, leaf stomatal numbers and postharvest water
loss were shown to be important factors in vase life. Cut flowers of a geno-
type with 9 days longer vase life had 53% fewer leaf stomata (Schroeder
and Stimart, 2005). However, long vase life was especially associated with
an early reduction in transpiration followed by low, steady transpiration,
while short-lived genotypes had a linear transpiration pattern over the vase
period. This indicates that differences in stomatal control (opening and
closing) of water loss between cultivars are likely to be a more important
factor than the number of stomata (Schroeder and Stimart, 2005). This
agrees with a previous paper of Rutland et al. (1987), in which they showed
that stomatal density did not correlate with vase life and transpiration of 10
different snapdragon cultivars.
Number of stomata (stomatal density) is not only dependent on geno-
type (cultivar), but also affected by the environmental conditions during
the cultivation of the cut flowers. Four of five rose cultivars tested showed
a significantly higher number of stomata per cut flower at higher cultiva-
tion temperatures (Pandey et al., 2007). However, the vase life of the cut
flowers was not affected by the preharvest temperature conditions (Pandey
et al., 2010). This strengthens the conclusion that stomata number per se
is not the determining factor for vase life. High air humidity (RH) during
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cultivation greatly reduced the vase life of cut roses, which was accompa-
nied by high transpiration rates (Torre, 1999; Mortensen and Gislerod,
2000) and symptoms that are typically related to water stress, including
premature flower and leaf wilting, as well as pedicel bending (Mortensen
and Gislerod, 2000; Torre and Fjeld, 2001; In et al., 2007). Roses grown at
high RH (90–95%) showed a significantly higher number of stomata and
longer stomata, as well as wider stomatal apertures, than roses grown at
a moderate RH of 60%–70% (Torre et al., 2003; Fanourakis et al., 2013a).
However, in rose leaflets subjected to desiccation, the enhanced stomatal
conductance in high-RH- as opposed to moderate-RH-grown plants was
mostly due to poor stomatal closure and, to a lesser extent, the combined
result of higher stomatal density and longer pore length (Fanourakis et al.,
2013a). This confirms that the reduced degree and, especially, the reduced
rate of stomatal closure are the primary causes of the large genotypic varia-
tion in the control of water loss in high-RH-grown rose plants. It is known
that stomata respond rapidly to air humidity, resulting in higher stomatal
conductance at high RH (Hall et al., 1975; Morison and Gifford, 1983).
Growth of rose plants at high RH, however, resulted in stomata that are
less responsive to desiccation. As a result, transpiration of the cut flowers
remains also high when the water potential in the cut flower decreases. This
implies that cultivation of cut flowers at high RH makes the flowers more
vulnerable to dry storage or transport, and to a hampered water uptake at
the consumer’s, caused by a decreased hydraulic stem conductance of the
cut flower. A limited exposure period to high RH of a few days can already
disturb the stomata closure response to desiccation (Rezaei Nejad and van
Meeteren, 2008; van Meeteren et al., 2009; Aliniaeifard et al., 2014). This
suggests that a period of some days of high RH during the preharvest stage
can have a large impact on the postharvest quality of cut flowers.
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cucumber, okra, and several legumes, such as pea and bean, are used in an
immature stage as a vegetable. The stomata number on pickle cucumbers
varies from 10 to 100 mm−2 (Smith et al., 1979), what is comparable to some
leaves (see before). The stomatal density decreases when the cucumber
fruit size increases, because stomata are differentiated in an early stage of
fruit development. So, the total number of stomata per fruit does not change
during growth of the fruit. In the legume plants, pots are also rich in sto-
mata. In the pot of broad bean or pea, stomata in the exocarp are 25% of the
stomatal density on the leaf abaxial surface and 50% of the stomatal density
on the adaxial side of the leaf (Willmer and Johnston, 1976; Atkins et al.,
1977; Blanke and Lenz, 1989). The stomatal density in broad bean is around
19 stomata mm−2, and in pea is around 40 stomata mm−2. It has been shown
that 12,000–14,000 stomata exist on one pea pod. The maximum density of
stomata in the pea can be observed after anthesis (Blanke and Lenz, 1989).
Water loss is one of the most obvious changes in harvested vegetables,
often being the factor that limits marketing life (Ben-Yehoshua and Rodov,
2003; Shamaila, 2005). Therefore, it is surprising that the effects of prehar-
vest growth conditions and postharvest handling practices on the stoma-
tal aperture of harvested vegetables have received little attention. Water
loss during storage can be controlled with proper and rapid precooling and
storage humidification. In chilling-sensitive vegetables, however, control of
water loss is more difficult, since they can only be stored at warmer tem-
peratures. Also, the environmental conditions in different links of a supply
chain are not always under strict control, which makes the role of stomata
of even more importance. In a study by Thomson (2005), observations
were made on stomatal apertures in the leaves of Japanese mustard spin-
ach (Brassica rapa var. perviridis ‘Komatsuna’) and a large India mustard
(Brassica juncea ‘Red Giant’) in response to cooling and holding treatments
representing commercial procedures. When the leaves were held in air at
20°C, the stomatal aperture of both Brassicas progressively reduced after
harvest; after 15 min, closure was largely complete in B. juncea, while in
B. rapa, stomata closure took about 30 min. Stomata of B. juncea also closed
at 4°C; however, 4°C inhibited stomatal closure of B. rapa. Besides cool-
ing by air, following harvest, B. rapa leaves were also dipped for 20 min in
iced water and in water at 20°C. Neither water dips interfered with stoma-
tal closure. However, after these dips and a further 25 min in air at 20°C,
stomata remained largely closed for iced leaves, but had begun to open on
leaves dipped in the 20°C water.
Reducing water loss in B. juncea not only prevented wilting, but
also reduced leaf yellowing; it increased the sweetness and retarded pro-
tein degradation and the loss of vitamin C (Lazan et al., 1987a, 1987b).
Also, in lettuce, the retention of water played an important role in main-
taining ascorbic acid content (Agüero et al., 2011). In general, the nutri-
tional value of vegetables is very much affected by water loss (Paull, 1999;
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Ben-Yehoshua and Rodov, 2003; Shamaila, 2005). However, there has only
been limited research evaluating the impact of water loss on the nutritive
value of vegetables. Ezell and Wilcox (1959, 1962) found that water loss was
associated with significant losses of both vitamin C content and carotene in
a wide range of vegetables (kale, collards, turnip greens, spinach, cabbage,
and snap beans). Diminishing water loss of broccoli by misting resulted in
significant retention of ascorbic acid (Barth et al., 1992). Also, preventing
water loss of strawberries (by wrapping) reduced the loss of ascorbic acid.
As modifications of O2 and CO2 levels were only minimal in the wrapped
treatments, the effect was not due to their modification (Nunes et al., 1998).
Water loss is clearly an important factor in the retention of quality, includ-
ing nutritional quality, in vegetables. As discussed in Section 6.4, the culti-
vation of plants at low VPD can influence the closing ability of stomata. In
that way, the VPD during the preharvest phase of vegetables influences the
postharvest life and nutritional quality of leafy vegetables via the stomata. A
higher rate of water loss after harvest of plant leaves was observed in basil
(Ocimum basilicum L.) and lemon balm (Melissa officinalis L.) as a result
of exposure during cultivation to low VPD than in plants grown at higher
VPDs (Islam et al., 2010). Postharvest life was negatively correlated to the
rate of water loss via stomata after harvest of the plants (Islam et al., 2010).
Whether VPD during the growth of legume plants also affects the stomatal
closing ability of the pots is unknown.
Some recent outbreaks of bacteria-related diseases after consumption
of fresh vegetables have increased the interest in food-borne pathogens and
especially in bacteria–plant interactions. Since stomata are the portals into
the plant for many bacterial species that cause plant diseases, there is more
and more interest in the role that stomata play in food safety, especially
in leafy greens. Scanning electron microscopy studies of lettuce leaves
showed that bacteria can infiltrate stomata and in that way are protected
from chemicals such as sodium hypochlorite during the washing of the
leaves (López-Gálvez et al., 2010). Therefore, washing can be ineffective in
eliminating Escherichia coli cells on leaf surfaces, and such is also the case
for sanitizers used in the washing solution. The main function of sanitizers
in the washing of fresh-cut products is to avoid cross-contamination.
Incubation of GFP-tagged Salmonella enterica with iceberg lettuce
leaves in the light resulted in aggregation of bacteria near open stomata
and invasion into the inner leaf tissue (Kroupitski et al., 2009). In contrast,
when stomata were closed due to darkness, incubation resulted in a scat-
tered attachment pattern and very poor stomatal internalization. However,
Salmonella internalization was not enhanced by forcing stomatal opening
in the dark by the use of fusicoccin. These results imply not only that light
was necessary for stomata opening, but also that the pathogen is attracted
to nutrients produced de novo by photosynthetically active cells. Some plant
pathogens force stomata to open after they have closed as part of the plant’s
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between 10 and 20 stomata mm−2 around the petal fall (Blanke, 1986; Blanke
and Lenz, 1989). Then after 1 week, it decreases to 6–8 stomata mm−2 , and
thereafter, when the expansion of the cuticle occurs, it decreases further to
less than 1 mm−2 (Schwertfeger and Buchloh, 1968; Blanke, 1986; Blanke
and Lenz, 1989). It is reported that an apple fruit with 10 mm diameter
would have around 600–6000 stomata per fruit (Table 6.2) (Blanke and
Lenz, 1989).
Also in Valencia oranges, the stomatal density is at its maximum
(160 mm−2) when the fruit is small. Fruit growth expands the space
between the stomata; in large fruits, the number of stomata is decreased
to 20–50 mm−2 (Moreshet and Green, 1980). In navel oranges, the stoma-
tal density (10–16 mm−2) is lower than that in the fruit of Valencia orange
(Turrell and Klotz, 1940). It seems that in all fruits, even when they are ripe,
stomata exist on them, but they are covered by a wax layer.
In banana fruit, contrary to the leaf, the stomata are not distributed in
a parallel way. The number of stomata is between 3 and 11 mm−2 in imma-
ture fruits and 3 and 6 mm−2 in full and mature fruits (Johnson and Brun,
1966). The stomatal density in the leaf (Table 6.1) of banana (1700 mm−2) is
considerably higher than their numbers on the fruits (Table 6.2) (Wardlaw,
1936). Although many studies reported nonfunctional stomata in banana
fruit, it has been shown that just before the harvest, they are functional in
response to light and CO2 and capable of gas exchange with the environ-
ment (Moreshet and Green, 1980; Burg, 2004).
Stomatal density in tomato fruits is very low, and in some studies,
even no stomata were reported on the tomato fruit, while the number of sto-
mata in cucumber is relatively high (Table 6.2). Higher stomatal densities
correlate positively with higher water loss from the fruits. Measuring the
amount of water loss immediately after harvest showed a 20 times higher
water loss for cucumber fruits than for tomato fruits (Adams and Ho, 1995).
In ABA-deficient orange plants, an increase in stomatal opening on the fruit
caused higher fruit water loss than in wild-type oranges (Romero et al.,
2013). This also demonstrated the role of stomata in the water loss of fruits.
Therefore, fast handling after harvest is important in order to restrict water
loss and quality loss in fruits with higher stomatal densities.
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Moran et al., 2009; Islam et al., 2010; Fanourakis et al., 2011; Burchi and
Prisa, 2013; Tudela et al., 2013). During growth of the plants, the manage-
ment of environmental conditions such as relative humidity (RH), light, and
temperature can considerably influence postharvest performance of horti-
cultural products. Some of these conditions affect postharvest performance
via effects on the stomata (number, morphology, and closing ability).
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exposed to high VPDs (Rezaei Nejad and van Meeteren, 2005; Rezaei
Nejad et al., 2006; Aliniaeifard and van Meeteren, 2013; Fanourakis et al.,
2013a; Aliniaeifard, 2014; Aliniaeifard et al., 2014). Such a transfer from
low to high VPD is common after vegetative propagation by leafy cuttings,
after in vitro propagation, and at the end of the cultivation period of orna-
mentals when plants or cut flowers are transferred to domestic conditions.
Although plants can be cultured in vitro on a large scale under low-VPD
conditions, in vitro–produced plants are usually susceptible to wilting
upon transfer to normal atmospheric VPDs (Brainerd and Fuchigami,
1982; Ghashghaie et al., 1992; Santamaria et al., 1993; Aguilar et al., 2000;
Hronková et al., 2003; Hazarika, 2006; Aracama et al., 2008; Khan et al.,
2010). This is also because of malfunctioning of the stomata in response to
a wide range of closing stimuli, such as darkness, abscisic acid (ABA), and
elevated calcium (Ca 2+) levels (Brainerd and Fuchigami, 1982; Ziv et al.,
1987; Santamaria et al., 1993). It has been shown that higher rates of water
loss after desiccation (postharvest phase) in the plants grown at low-VPD
conditions are mainly caused by the stomata, compared to the role of the
cuticle (Ziv et al., 1987; Santamaria and Kerstiens, 1994; Aliniaeifard and
van Meeteren, 2013; Fanourakis et al., 2013a).
As discussed in specific sections before, cultivation at low VPD not
only influences the vase life and visual appearance of cut flowers, but also
can influence the postharvest life and nutritional quality of leafy vegetables
via the stomata. Leaves of plants that lose their moisture more easily are
more vulnerable to losing vitamin than plants that are resistant to wilting
(Ezell and Wilcox, 1959; Lee and Kader, 2000).
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
(Xu et al., 2002; Priest et al., 2006). ABA-GE is readily reversible but
not easily permeable to biomembranes and may function as a realizable
and transportable form of ABA (Dietz et al., 2000; Sauter et al., 2002).
When ABA is needed, ABA-GE is hydroxylated through β-glucosidase
to increase the active form of ABA (Lee et al., 2006). The activity of
β-glucosidase was decreased in rose plants that had been grown in low
VPD compared with its activity in moderate-VPD-grown plants. This
resulted in a higher ratio of ABA-GE to ABA (Arve et al., 2012). However,
it is still not clear whether the lower ABA level after long-term exposure
to low VPD is due to lower production or the higher catabolism of ABA.
It has been shown that exposure to different VPDs influences ABA in
the leaves of plants. In many plant species, such as spinach (Spinacia olera-
cea) (Zeevaart, 1974), rose (Arve et al., 2012; Giday et al., 2013a), Arabidopsis
(Okamoto et al., 2009), and spiderwort (Tradescantia virginiana) (Rezaei
Nejad and van Meeteren, 2007, 2008), the foliar [ABA] decreases as a result
of exposure to low-VPD conditions. In Tradescantia, 1 day after transfer-
ring moderate-VPD-grown plants to a low-VPD condition, the foliar [ABA]
decreased to the ABA level found in plants grown at low VPD. Reciprocal
transfer of moderate-VPD-grown plants back from low to moderate VPD
increased the foliar [ABA] again to its original level in moderate-VPD-
grown plants (Rezaei Nejad and van Meeteren, 2008). In rose plants, con-
trary to moderate-VPD-grown plants, exposure of low-VPD-grown plants
to darkness did not result in elevation of the foliar [ABA] (Arve et al., 2012;
Giday et al., 2013a). In Arabidopsis, the [ABA] decreased sharply even 1 h
after exposure to a low-VPD condition (Okamoto et al., 2009). Moreover,
in vitro–propagated plants that were produced under low-VPD conditions
were deficient in ABA (Hronková et al., 2003). Therefore, in the absence of
stresses, the foliar [ABA] depends on the VPD as well; decreasing the VPD
will result in a decrease in [ABA].
A foliar threshold level for ABA has been suggested in order to keep
the stomata responsive to closing stimuli. Since the foliar ABA level in mod-
erate-VPD-exposed plants is always higher than this threshold, their sto-
mata are always responsive. In different rose cultivars and also Arabidopsis
accessions, higher ABA levels than the threshold were still present after
low-VPD exposure; these genotypes maintained responsiveness to closing
stimuli in the postharvest phase (Giday et al., 2013a; Aliniaeifard and van
Meeteren, 2014). In genotypes that showed malfunctioning of stomata after
a 4-day exposure to low VPD, a daily spray of ABA during the 4-day expo-
sure to low VPD resulted in maintaining the stomatal closing response to
closing stimuli in Arabidopsis and bean (Aliniaeifard and van Meeteren,
2013, 2014; Aliniaeifard, 2014; Aliniaeifard et al., 2014). This confirmed that
a long-term low ABA level results in loss of stomatal closing ability after-
wards. Changes in the signaling pathway inside the guard cells of stomata
due to long-term exposure to low-VPD conditions are reviewed in detail by
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Stom ata a n d P ostharvest P hy s i o l o g y
L M
100 μm 100 μm
Figure 6.3 Stomatal size and density in low (L)- and moderate (M)-VPD-
grown Vicia faba plants.
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the other hand, smaller stomata are usually associated with higher stoma-
tal density per leaf area (Hetherington and Woodward, 2003). To optimize
the trade-off between carbon gain and transpirational water loss, these
characteristics (smaller stomata and higher stomatal density) allow the
leaf to attain high stomatal conductance under favorable conditions, and
to promptly reduce stomatal conductance when conditions are unfavor-
able, which help the plant cope with stress conditions (Xu and Zhou, 2008;
Doheny-Adams et al., 2012). Using five closely related species of the genus
Banksia, it has been demonstrated that the rate of stomatal response was
negatively correlated with stomatal size (Drake et al., 2013). It has been
indicated that smaller stomata require less leaf drying to close and that
plants’ stomatal size underlays much of the variation in the regulation of
transpiration upon desiccation (postharvest phase) after growth of the
plants in low and moderate VPDs (Franks and Farquhar, 2007; Doheny-
Adams et al., 2012; Drake et al., 2013; Giday et al., 2013b).
In cut rose flowers, although changes in stomatal length had no
influence on stomatal functionality, anatomical features per se represent a
significant and direct contribution to the increased water loss in the post-
harvest phase of the roses (Fanourakis et al., 2013a). Higher stomatal den-
sities enlarge the stomatal pore area per leaf area, leading to an increase
in the transpiration rate in low-VPD-grown plants. However, in faba bean,
stomatal density decreased as a result of growing them at low VPD. On
the other hand, in faba bean, stomata of low-VPD-grown plants were
larger for all guard cell dimensions than those of moderate-VPD-grown
plants (Aliniaeifard et al., 2014). Therefore, high stomatal conductance
in low-VPD-grown plants can be attributed to their larger stomata with
wider pore area in comparison with the stomata in m oderate-VPD-grown
plants. In the previous studies, investigating the impact of stomatal mor-
phological traits on stomatal functionality, the stomatal responsiveness
of different species was investigated or plants from one or several geno-
types were exposed to contrasting environments, which usually induced
changes in both stomatal morphology and responsiveness (Hetherington
and Woodward, 2003; Torre et al., 2003; Franks and Farquhar, 2007; Drake
et al., 2013; Fanourakis et al., 2013a; Giday et al., 2013b). Analyzing the
importance of the stomatal morphological traits on the stomatal function-
ality, not only in moderate and low-VPD-grown plants, but also in plants
that had developed their leaves in moderate VPD and thereafter trans-
ferred for 1–4 days to low VPD, confirmed that stomata morphological
traits are not always determinant of stomatal functionality: the morphol-
ogy of stomata in plants that had developed their leaves at moderate VPD
and were then transferred for 4 days to low VPD was more similar to that
of moderate-VPD-grown plants, while their response to ABA and des-
iccation was similar to the stomatal response of low-VPD-grown plants
(Aliniaeifard et al., 2014).
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Stom ata a n d P ostharvest P hy s i o l o g y
6.4.2 Temperature
As discussed in Section 6.2.1, stomata are involved in controlling plant
t emperature through the transpiration rate. Therefore, it could be
expected that stomata will open more with increasing temperatures to
prevent overheating of the plant. However, the response of stomata to tem-
perature is not constant for different temperatures, and usually it is species
dependent. There are contradictory reports regarding stomatal response
to different temperatures. Both stomatal closure and stomatal opening
have been reported as a result of an increase in temperature (Schulze
et al., 1973; Wilkinson et al., 2001). Likely this is because stomata opening
responds to many factors; among these factors are the CO2 concentration
in the stomatal cavity and the water potential of the leaves. These param-
eters are respectively affected by the rate of photosynthesis and by water
uptake and water loss. These processes are also largely influenced by
temperature. Schulze et al. (1973) showed for five species (Zygophyllum
dumosum, Artemisia herba-alba, Hamada scoparia, Reaumurian egevensis,
Prunus armeniaca) that stomata opened more in response to a gradual
increase in temperature (from 31.6°C to 39.8°C) at low water stress. This
response was independent of VPD. At high plant water stress, however,
the stomatal response was reversed; that is, the stomata closed when the
temperature was gradually increased. In leaf disks of legumes floating on
water, stomata opened in darkness by increasing the temperature from
20°C to 45°C (Feller, 2006; Reynolds-Henne et al., 2010), even when ABA
was applied. By using epidermal strips floating on water, the direct effect
of temperature on guard cells was studied, excluding the effects of pho-
tosynthesis and water loss (Rogers et al., 1979). They showed that stoma-
tal aperture increased by an increase of temperature from 10°C to 40°C,
while at 45°C the aperture was less than those obtained at 35°C and 40°C.
In the experiments mentioned, there was a fast recovery of stomata to its
original apertures when temperature was decreased again. This indicates
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
6.4.3 Light
Greenhouse crop production frequently makes use of supplementary light-
ing to improve plant productivity in periods of the year when natural irradi-
ance is low; on some occasions, continuous lighting is used (Mortensen
and Fjeld, 1998; Dodd et al., 2005; Pettersen et al., 2006; Velez-Ramirez
182
Stom ata a n d P ostharvest P hy s i o l o g y
et al., 2011). For example, in pot roses, extending the lighting period from
18 to 24 h per day shortened the time to flowering by 12% and the num-
ber of flowers increased by 34% (Pettersen et al., 2007). Although grow-
ing plants under continuous light has several advantages (e.g., reduction
of powdery mildew), it adversely influences the postharvest life of the cut
flowers (Mortensen and Fjeld, 1998; Mortensen and Gislerød, 1999, 2011)
due to poor stomatal closure under conditions of decreasing leaf water
potential and turgor (Slootweg and van Meeteren, 1991; Arve et al., 2012).
The vase life of the roses decreased by using supplemental lighting. The
vase life depends on the duration of exposure to light. The longer the light-
ing period during growth, the shorter the vase life afterwards (Mortensen
and Fjeld, 1998; Pettersen et al., 2006; Mortensen and Gislerød, 1999, 2011;
Arve et al., 2012). Extending the lighting period from 16 to 20 h decreased
the postharvest life in lemon balm, while it is not affected in basil; however,
24 h exposure to light resulted in decreased postharvest life in both herbs
(Islam et al., 2010). It has been shown that by decreasing the RH in the
growing units when plants are grown under continuous light, it is possible
to reduce the disability of stomata to close and, as a result, increase the vase
life of cut flowers (Mortensen and Fjeld, 1998; Mortensen and Gislerød,
1999, 2011; Pettersen et al., 2006; Arve et al., 2012). However, even in mod-
erate RH conditions, exposure to continuous light results in decreased vase
life of cut roses (Mortensen and Fjeld, 1998).
Exposure to light not only influences the sugar content through pho-
tosynthesis, but also can induce sugar accumulation because of osmotic
adjustment in the cells. For example, higher sugar levels have been found
in the rinds of mandarin fruits positioned outside of the tree canopy fac-
ing toward sunlight. Sugar accumulated (osmotic adjustment) in the rinds
of sunlight-exposed fruits because of dehydration of the rind’s cells due
to high transpiration via stomata of sun-exposed fruit surfaces (Yakushiji
et al., 1998; Magwaza et al., 2013). Accumulation of sugar and other bio-
chemicals in the rinds of sun-exposed fruit during the growing season
improves their postharvest performance and decreases susceptibility to
rind breakdown (Magwaza et al., 2013).
The amount of healthy compounds of a plant can be influenced by
the lighting period and light intensity. Although light has no direct role
in the production of ascorbic acid (vitamin C), the duration and intensity
of light can influence the production of ascorbic acid in the leaf (Lee and
Kader, 2000). In general, the amount of ascorbic acid increases by light
(intensity and duration) and decreases in the dark period (Lee and Kader,
2000). On the other hand, plants that have been produced in a prolonged
lighting period could lose their vitamin C more easily after harvest because
of higher water loss via stomata after harvest; therefore, there should be
a balance for the lighting period for producing vitamin C before harvest
and preventing loss of vitamin C after harvest of leafy vegetables. However,
183
P OST H AR V EST RI P ENING P H Y SIOLOG Y
184
Stom ata a n d P ostharvest P hy s i o l o g y
the presence of more stomata and a larger stomatal aperture area in the fruit
surface could increase the susceptibility to bacteria. Wang et al. (2011a)
found that under the same experimental conditions, ‘Meiwa’ kumquat has a
lower stomatal density and smaller stomatal openings than ‘Newhall’ navel
orange. These stomatal characteristics of ‘Meiwa’ make this cultivar better
resistant to canker than ‘Newhall’. ‘Pinalate’ (Citrus sinensis L. Osbeck) is a
yellow ABA-deficient mutant derived from the orange ‘Navelate’; therefore,
its stomata will close less. ‘Pinalate’ has a higher transpiration rate, and due
to the impaired stomatal functioning, it is more susceptible to pathogenic
infections (Alférez et al., 2005).
Since stomata are the entrance site of many bacterial and fungal
diseases into leaves or fruits of horticultural products, the immunity of a
product to pathogen virulence in many cases depends on the function of
the stomata (Zeng et al., 2010). Many postharvest treatments are efficient
when they restrict the entrance of pathogens into the products. For exam-
ple, heat treatments such as hot water dips, vapor heat, short hot water
rinse, and brushing, causing melting of the wax layer in the surface of the
fruits, and therefore covering of the stomata, provide a mechanical barrier
for the pathogens, restricting the invasion of pathogens into the internal
tissues (Porat et al., 2000; Schirra et al., 2000; Waller et al., 2001; Mulas
and Schirra, 2007). On some occasions, exogenous wax is applied in order
to extend the quality of the fruits. The amount of wax applied on the fruit is
important to prevent pathogen infection. Low-coating loads result in some
uncoated surface areas with bare stomata, while sufficient wax loads cover
all the stomata and restrict pathogen infections (Njombolwana et al., 2013).
Salicylic acid is one of the plant hormones known to be involved in sensing
the pathogenic invasion. Salicylic acid–induced stomatal closure is one of
the cellular pathways that has been suggested after infection by pathogens
(Lu and Chen, 2005; Melotto et al., 2006; Khokon et al., 2011). It has been
shown that inducing stomatal closure by salicylic acid improves the plant
capacity to cope with diseases. For example, in lily, salicylic acid treatments
lead to induction of stomatal closure, causing more tolerance of lily plants to
Botrytis elliptica (Lu and Chen, 2005).
In some products, monitoring stomatal conductance can be used
as a nondestructive index for storability and quality status. Controlling
water loss from the leaves (especially from the stomata) can be consid-
ered an important way to minimize quality problems and extend the
postharvest life in crops such as rose cut flowers and kalanchoe cuttings
(Bredmose and Nielsen, 2009; Fanourakis et al., 2013a, 2013b). Stomatal
density in tomato fruits is very low, whereas the number of stomata in
cucumber is relatively high (Table 6.2). Higher stomatal densities posi-
tively correlate with higher water loss from the fruits. Measuring the
amount of water loss immediately after harvest showed 20 times higher
185
P OST H AR V EST RI P ENING P H Y SIOLOG Y
water loss for cucumber fruits than tomato fruits (Adams and Ho, 1995).
Therefore, fast handling after harvest is important in order to restrict
water loss and quality loss in crops with higher stomatal densities. High
humidity during growth decreases movement of calcium to the leaves in
cucumber and tomato plants. However, due to the higher stomatal den-
sity in the fruits of cucumber than in tomato fruits, a higher level of cal-
cium accumulates in the cucumber fruits (Adams and Ho, 1995). This
confirmed that transpiration by fruits plays a pivotal role in determining
calcium levels in the fruits and susceptibility to physiological disorders
related to calcium deficiency.
186
Stom ata a n d P ostharvest P hy s i o l o g y
187
P OST H AR V EST RI P ENING P H Y SIOLOG Y
188
Stom ata a n d P ostharvest P hy s i o l o g y
Sisler and Wood, 1988). The dual effect of high CO2 during storage is pre-
sented in Figure 6.4. At atmospheric pressure, closure of stomata occurs
during storage and transport in darkness, but using low-pressure (LP)
storage encourages stomatal opening even in darkness (Kirk and Skytt
Anderson, 1985; Kirk et al., 1986; Veierskov and Kirk, 1986). This low sto-
matal resistance at LP results in improved gas diffusion (such as ethylene)
and considerably improves intercellular ventilation. In Valencia orange with
open stomata, the transpirational resistance is 13.5 scm−1 (Moreshet and
Green, 1980). In the fruits, the stomatal resistance to ethylene transport is
23 scm−1. Due to stomatal closure during harvest, the resistance to ethyl-
ene transport increases to 6886 scm−1 in the orange fruits (Ben Yehoshua
et al., 1979). Moreover, fruit’s cuticular resistance to ethylene transport is
approximately 10-fold higher than its diffusive resistance to ethylene of
the stomata. Under the situation of closed stomata, the internal ethylene
concentration largely increases. Therefore, in order to prevent detrimental
effects of ethylene, it is vital to decrease the internal ethylene concentration
by some means, such as vacuum extraction (Burg, 2004).
Detaching organs from the mother plants and placing them in dark-
ness can promote leaf senescence in many plant species. A relationship
between stomatal aperture and senescence has been suggested (Thimann
Stomatal closure
After
High internal harvest
ethylene level
Accelerated
senescence
(quality loss)
189
P OST H AR V EST RI P ENING P H Y SIOLOG Y
190
Stom ata a n d P ostharvest P hy s i o l o g y
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Zhang, K., Xia, X., Zhang, Y., and Gan, S.S. 2012. An ABA-regulated and Golgi-
localized protein phosphatase controls water loss during leaf senes-
cence in Arabidopsis. Plant Journal 69, 667–678.
215
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Zhang, X., Takemiya, A., Kinoshita, T., and Shimazaki, K.I. 2007. Nitric oxide
inhibits blue light-specific stomatal opening via abscisic acid signaling
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Zhang, X., Wang, H., Takemiya, A., Song, C.P., Kinoshita, T., and Shimazaki,
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through hydrogen peroxide-induced dephosphorylation of the plasma
membrane H+ -ATPase in guard cell protoplasts. Plant Physiology 136,
4150–4158.
Zhao, X., Qiao, X.R., Yuan, J., Ma, X.F., and Zhang, X. 2012. Nitric oxide inhib-
its blue light-induced stomatal opening by regulating the K+ influx in
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calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic
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Zieslin, N. 1989. Postharvest control of vase life and senescence of rose flow-
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216
Chapter 7
Abstract 218
7.1 Importance of Water Loss 219
7.2 Properties of Water 220
7.3 Psychrometrics: Behavior of Water in Air 221
7.4 Transpiration: Diffusion of Water Vapor 224
7.5 Resistance to Diffusion of Water Vapor 226
7.6 Measurement of Transpiration 226
7.6.1 Weight Loss 227
7.6.2 Direct Measurement 227
7.6.3 Diffusion Porometer 227
7.7 Factors Affecting Water Loss 227
7.7.1 Commodity Factors 227
7.7.1.1 Surface-to-Volume Ratio 228
7.7.1.2 Routes of Water Loss 228
7.7.1.3 Anatomy of the Evaporating Surface 229
7.7.1.4 Physiological State of the Commodity 230
217
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Abstract
Water loss from harvested horticultural commodities will continue to be
a problem as long as warm products need to be cooled and kept cold
during storage and marketing. Even a small amount of water loss can
adversely affect product quality, marketability, and storability. Apart
from the loss of fresh weight that accompanies water loss, a few percent
water loss can stimulate physiological changes that hasten senescence.
The physics of heat transfer in a mechanical refrigeration system neces-
sitates that the air in a cold room will have a vapor pressure less than
that of the commodity. This inherent difference in the vapor pressure
between water near the surface of fresh fruits, vegetables, and ornamen-
tals and the ambient cooling air is the prominent force that determines
the rate of water loss. Increasing the relative humidity of the surround-
ing air or the resistance of the surface of the commodity to the diffu-
sion of water vapor will decrease the rate of water loss. This can be done
by applying a wax or edible coating to the surface of whole or fresh-cut
commodities, by enclosing them in a package that raises the ambient
humidity, and by using harvest and postharvest practices that reduce
injuries (e.g., bruises, wounds, abrasions, etc.) that decrease the ability
of the surface to retard water loss. Preharvest factors (e.g., cultivar selec-
tion, growing environment, cultural practices, and maturity at harvest)
can have significant effects on rates of water loss. Familiarity with the
218
WATER LOSS
219
P OST H AR V EST RI P ENING P H Y SIOLOG Y
220
WATER LOSS
away from the tissue. The epidermis of all terrestrial plants is covered with
a waxy cuticle that evolved to significantly reduce diffusion of water vapor
from the tissue. However, this coating also reduces the diffusive exchange
of O2 and CO2 with the external environment. Specialized cells in the epi-
dermis of photosynthetically active leaves (i.e., guard cells) surround a
pore (i.e., the stoma) that breaches the epidermal barrier. Guard cells can
actively change their shape and size to alter the size of the stomatal open-
ing to balance the import of CO2 against the loss of water vapor. In other
organs, static ventilation systems such as lenticels allow diffusion of CO2
and O2 across the epidermis while minimizing water loss. The cuticle may
be modified during growth, development, and ripening to alter its water
diffusion characteristics. The rate of water loss, that is, transpiration, is
therefore controlled by active (i.e., stomata) and passive (e.g., lenticels)
routes of water loss. Transpiration in actively growing plants also provides
the driving force for water uptake by the roots and movement through the
xylem to the leaves.
221
P OST H AR V EST RI P ENING P H Y SIOLOG Y
3.5
25
)
°C
e(
3.0
ur
rat
2.0
15
1.5
10
5 1.0
0
100
Relative 80 0.5
60
humidity 40
(%) 20
0 0.0
0 5 10 15 20 25 30 35 40 45
Dry bulb temperature (°C)
222
WATER LOSS
Let’s use a few examples to see how we can use the psychrometric chart
(Figure 7.1).
The relative humidity (RH) can be determined by finding the inter-
section of the line extending upward from the dry bulb temperature with
the downward-sloping line from the wet bulb temperature. For example,
air with a dry bulb temperature of 25°C and a wet bulb temperature of 18°C
has a RH of 50% (point A on Figure 7.2). The vapor pressure (VP) of that
50% RH air can be determined by following the horizontal line to its inter-
section with the vertical axis at 1.6 kPa (point B on Figure 7.2). The dew
point temperature of 14°C can be determined by following the horizontal
3.5
)
°C
25
e(
3.0
ur
rat
20 2.5
lb
bu
18
50
et
2.0
W
15 A B
C 14
1.6
1.5
10
5 1.0
0
100
Relative 80 0.5
60
humidity 40
(%) 20
0 0.0
0 5 10 15 20 25 30 35 40 45
Dry bulb temperature (°C)
Figure 7.2 Example of how the psychrometric chart can be used to find the
vapor pressure and dew point from measurements of the wet and dry bulb
temperatures (refer to text for detailed description).
223
P OST H AR V EST RI P ENING P H Y SIOLOG Y
line left to its intersection with the wet bulb temperature curve (point C on
Figure 7.2).
In another example, the RH of air with a dry bulb temperature of 15°C
and a wet bulb temperature of 12°C will be 70%, with a vapor pressure of
1.2 kPa and a dew point of 10°C.
If a sample of warm moist air is cooled (move left along a horizontal
line), its relative humidity increases until it reaches saturation (at the left-
most line, the wet bulb temperature curve). The cooled air is now at its dew
point. Further removal of heat will result in condensation of water from the
air onto the cool surface. In contrast, the RH of air rapidly decreases with
increasing temperature (move right along the horizontal line from the wet
bulb temperature curve). This means that at warm temperatures, air can
hold much more water vapor than at cool temperatures. The water holding
capacity of air just about doubles with every 11°C rise in temperature.
If a cold commodity is placed in a warm room, the air in contact with
the commodity will become cooler, and depending on the initial RH of the
air, water may condense on the surface of the fruit. In contrast, if a warm
commodity is placed in a cool atmosphere, the air in contact with the fruit
will become warmer (move to the right along the horizontal lines) and its
RH will decrease, thereby facilitating water evaporation from the tissue.
224
WATER LOSS
equation (these calculations are only correct when other conditions, such
as barometric pressure, nature of the commodity’s surface, and air velocity
past the product remain constant).
J = K × VPD
where J = rate of water loss (g/h, % FW/day, etc.), K = proportionality
constant (a function of many features of the commodity that will be
discussed later), and VPD = vapor pressure difference, or deficit (kPa,
mmHg, mbar, etc.).
For example, consider a harvested head of lettuce at 25°C that loses
1% of its fresh weight when held for 1 hour (i.e., J = 1%/h) in a room at 25°C
and 80% RH. What will be its initial and final rates of weight loss during
cooling to 4°C in a store at 80% RH (Figure 7.3)?
From the psychrometric chart we can determined that at 25°C, the
saturation VP is 3.2 kPa (point A on Figure 7.3). The VP of the 25°C air
3.5
)
(°C
25
A
re
3.0
tu
ra
20 2.5
lb
bu
et
2.0
15
1.5
10
5 1.0
0 B
100
Relative 80 0.5
humidity
60
40
VPD = 0.8 – 0.6 = 0.2
(%) 20
0 0.0
0 5 10 15 20 25 30 35 40 45
Dry bulb temperature (°C)
Figure 7.3 Example of how the psychrometric chart can be used to calcu-
late the vapor pressure deficit (VPD) experienced by a head of lettuce placed
into a cold room as it cools from 25°C to 4°C (refer to text for detailed
description).
225
P OST H AR V EST RI P ENING P H Y SIOLOG Y
at 80% RH is 80% of the saturation VP or about 2.5 kPa, and the VPD is
the difference of the two values, or about 0.7 kPa (i.e., 3.2 − 2.5 = 0.7).
Substituting in the equation J = K × VPD, we can calculate the proportional-
ity constant K [K = (1%/h)/(0.7 kPa) = 1.4% FW/kPa-h].
The VP of 100% RH air at 4°C is 0.8 kPa, while 80% RH air at the
same temperature has a VP of 0.6 kPa (0.8 × 0.80) (point B of Figure 7.3).
When the 25°C lettuce is initially put in the 80% RH 4°C cold room, the VPD
between the tissue and the air will be 2.6 kPa (i.e., 3.2 − 0.6). The initial rate
of water loss will therefore be 2.6 kPa × 1.4% FW/kPa-h = 3.6% FW/h. This
initial high rate of water loss is one reason why cooling to the correct stor-
age temperature should be done as quickly as possible. Once the product
is cooled to 4°C, the VP of the lettuce decreases to 0.8 kPa and the VPD
between the product and the air decreases to 0.2 kPa (0.8 − 0.6). The new
rate of water loss is 0.2 kPa × 1.4% FW/kPa-h = 0.28% FW/h. This amounts
to a 92% reduction in the rate of water loss.
226
WATER LOSS
227
P OST H AR V EST RI P ENING P H Y SIOLOG Y
can have markedly different rates of water loss depending on where and
when it was grown and how it was handled during and after harvest.
Table 7.2 Equations for the Calculation of the Area and Volume for a Sphere
(Tomato), a Cylinder with Spherical Ends (Cucumber), and A right Circular
Cone with One Hemispherical End (Carrot).
Sphere Cylinder Cone
Area 4πr2 2πLr + 4πr 2 πr'r2 + L2 + 2πr2
Volume 4/3πr 3 πLr2 + 4/3πr3 1/3πLr2 + 2/3πr3
228
WATER LOSS
tubers. Water losses through the epidermal cells occur by a relatively com-
plex process whereby water molecules must dissolve into the epidermal cov-
ering, diffuse through it, and then evaporate into the outside atmosphere.
The rate at which evaporation occurs is often limited by the solubility of
water in the outer surface coating.
The surface coating is very effective in retarding water loss. For exam-
ple, evaporation from the cells inside the leaf is 10-fold higher than from
cells on the leaf surface. In very heavily cutinized or suberized organs, very
little water escapes by direct evaporation from the surface cells. Instead,
most water is lost in the vapor phase through apertures in the surface of
the organ, such as stomata, lenticels, and wounds. The rate of evaporation
is affected not only by the route of evaporation, but also by the architecture
of the evaporating surface.
229
P OST H AR V EST RI P ENING P H Y SIOLOG Y
7.7.1.5 Cultivar
The many structural and chemical differences between different cultivars
can substantially affect weight loss.
230
WATER LOSS
7.7.2.1 Humidity
The importance of the water vapor content of the air on water loss has
been emphasized already. Lower-humidity environments also frequently
adversely affect the quality of stored commodities. Because of the natural
cycling of temperatures in cold storage rooms, a compromise must be made
between the danger of condensation of water on cold fruit during the warm
part of the cycle in rooms with very high RH, and unacceptable levels of
water loss if the RH is maintained at levels safe for the entire cycle.
231
P OST H AR V EST RI P ENING P H Y SIOLOG Y
7.7.2.3 Temperature
The close interdependence of temperature, VP, and RH is implicit in the
psychrometric chart, and the importance of temperature in the VPD-driven
loss of water from perishable commodities cannot be overemphasized.
Some studies have shown a substantial effect of temperature on water loss,
even allowing for the changes in VPD. Enhanced respiration and changes
in the composition of the cuticle at warmer temperatures may account for
these observations.
232
WATER LOSS
7.8.3 Packaging
Placing commodities in packages of any sort obviously changes their geom-
etry with respect to weight loss. The larger the package, the smaller is its
surface area in relation to its volume. This is because the surface area
increases as a function of the dimensions of the package squared, while
the volume increases as a function of the dimensions cubed. In effect, the
package reduces the VPD within the package because the air inside the
package will be at high RH. To further reduce water loss, some products
are packed in polyethylene or other plastic liners, usually ventilated to avoid
anaerobic or high CO2 conditions in the package. Airflow through holes in
233
P OST H AR V EST RI P ENING P H Y SIOLOG Y
the package to maintain the desired product temperature and gas concen-
trations will nullify this benefit.
Certain containers, such as wooden or plain fiberboard boxes, can
absorb a substantial amount of water, thereby decreasing the initial VP in
the package. It is not feasible to equilibrate the containers with the storage
atmosphere, and waxed fiberboard cartons may be too expensive for many
commodities. Under these circumstances, waxed paper or polyethylene lin-
ers can substantially reduce weight loss for little extra cost.
7.8.4 Waxing
An alternative route to those noted above for reducing water loss is to raise
the resistance of the fruit surface to diffusion of water vapor by the applica-
tion of wax. Waxing is widely used as a treatment for fruits, frequently for
cosmetic reasons rather than to reduce weight loss. The waxes employed
are proprietary formulations and probably comprise largely waxes derived
from petrochemicals. Remember that although waxing can slightly reduce
the rate of water loss, temperature (even allowing for differences in the
VPD) can have a far greater effect than waxing. Waxing can also modify
the internal atmosphere of perishable commodities. There is a danger
that the use of surface coatings can produce toxic internal levels of CO2,
particularly when the commodity is returned to ambient temperatures after
transportation or storage.
7.8.6 Curing
In some commodities, the formation of new or heavier deposits of suber-
ized cells on the surface will substantially increase their resistance to water
loss. Onions, potatoes, and sweet potatoes are frequently cured to improve
234
WATER LOSS
their postharvest life and reduce water loss. High temperatures and RH
often assist in the suberization process that occurs during curing.
References
Kader, A.A. 2002. Postharvest Technology of Horticultural Crops. 3rd ed.
Division of Agriculture and Natural Resources, University of California,
Oakland.
Kader, A.A., and Saltveit, M.E. 2003. Respiration and gas exchange. In Bartz,
J.A., and Brecht, J.K. (eds.), Postharvest Physiology and Pathology of
Vegetables. Marcel Dekker, New York, pp. 7–30.
Kays, S.J., and Paull, R.E. (eds.). 2004. Postharvest Biology. Exon Press,
Athens, GA.
Wills, R.B.H., McGlasson, B., Graham, D., and Joyce, D. (eds.). 2007.
Postharvest: An Introduction to the Physiology and Handling of Fruit,
Vegetables and Ornamentals. CAB International, Wallingford, UK.
Woods, J.L. 1990. Moisture loss from fruits and vegetables. Postharvest News
and Information 1, 195–199.
235
Chapter 8
Lysophospholipids and
Postharvest Quality
of Fruits, Vegetables,
and Cut Flowers
Domingos P.F. Almeida
Universidade de Lisboa, Lisbon, Portugal
Abstract 238
8.1 Introduction 238
8.2 Lipids as Signal Molecules in Plant Senescence and
Stress Response 239
8.2.1 Chemistry 239
8.2.2 Metabolism 240
8.3 Modulation of Postharvest Quality by Lysophospholipids 241
8.3.1 Effects on Color 242
8.3.2 Effects on Texture 242
8.3.3 Other Effects on Postharvest Quality-Related
Features 246
8.4 LPE Treatment Condition 247
8.5 Improvement of Postharvest Quality by
Lysophospholipids: Potential and Limitations 248
8.6 Conclusions and Future Perspectives 249
References 249
237
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Abstract
Regulatory lipid molecules are involved in plant responses to environmental
stresses and developmental cues. Phosphatidic acid is one of the best-
known signaling lipids in plants, but other lipidic molecules are involved in
signal transduction pathways. Therefore, the potential exists for the tech-
nological or biotechnological use of signaling lipids as tools to modulate the
features of fresh produce with postharvest relevance. One attempt has been
made with the exogenous application of lysophosphatidylethanolamine
(LPE) to crops and produce. Modulation of postharvest quality by lyso-
phospholipids has been documented in dozens of horticultural commodi-
ties. Reported effects include delayed senescence and improved color and
texture, among other postharvest quality-related features. Despite the evi-
dence for the regulatory effects of lysophospholipids, two decades of trials
of LPE applications to horticultural produce have not yet been conclusive
regarding the development of lipids as plant growth regulators to improve
postharvest quality. Different approaches can be envisioned to harness the
potential for quality modulation by regulatory lipids: genetic engineering
to alter endogenous levels of regulatory lipids, pharmacological treatments
to target the biosynthesis of the signal transduction pathways of regulatory
lipids, and exogenous application of formulations containing signaling or
regulatory lipids. The improvement of our fundamental understanding of
lipid-mediated signaling and metabolic regulation is likely to result in novel
technological applications aimed at modulated produce quality.
8.1 Introduction
The importance of membranes on the postharvest quality of perishable
plant organs has long been recognized for their structural role, cellu-
lar compartmentation, and site of intense metabolic activity (Marangoni
et al., 1996). However, in addition to these functions, lipids have emerged
as an important class of regulatory and signaling molecules, and their role
in responses to environmental stresses and developmental cues is now
evident in plants and mammalians. Many regulatory lipids are related to
membrane phospholipids, as anabolic precursors or degradation products.
Phosphatidic acid is one of the best-known signaling lipids in plants (Wang
et al., 2006), but other lipidic molecules are involved in signal transduction
pathways. Therefore, the potential exists for the technological or biotech-
nological use of signaling lipids as tools to modulate the features of fresh
produce with postharvest relevance. One attempt has been made with the
exogenous application of lysophosphatidylethanolamine (LPE) to crops
and produce.
238
L Y SO P H OS P H OLI P I D S AN D P OST H AR V EST
8.2.1 Chemistry
Phospholipids are structural components of cell membranes. In addition,
phospholipids and their catabolic products are important messengers in
the regulation of plant growth, development, and response to stress. These
molecules are chemically derived from a glycerol backbone esterified to
fatty acids in the sn-1 and sn-2 positions and phosphorylated at the sn-3 posi-
tion (Figure 8.1). The diversity of phospholipids is made possible by the
nature of the fatty acids and the head groups in the structure. The total
phospholipid composition of Arabidopsis leaves can be taken as represen-
tative of plant cells. The head groups most common in plants are choline,
ethanolamine, and glycerol (Miquel and Browse, 1992; Li et al., 2006), with
lower quantities of inositol and serine (Li et al., 2006). The fatty acids most
PLA1
PAT-PLA
H O
H C O C R1
O
R2 C O C H
O
PLA2
H C O P O X
H O
PLC PLD
Figure 8.1 Structure of a generic phospholipid with the acyl chains R1 and
R2 and the head group X, indicating the covalent bond hydrolyzed by PLA,
PLC, and PLD.
239
P OST H AR V EST RI P ENING P H Y SIOLOG Y
commonly found in plant phospholipids are 16:0, 16:3, 18:2, and 18:3, with
a lower proportion of 16:1, 16:2, 18:0, and 18:1 (Miquel and Browse, 1992).
The sn-2 carbon is usually esterified to an unsaturated acyl chain.
8.2.2 Metabolism
Plant membrane phospholipids are hydrolyzed by phospholipases, a large
and diverse group of enzymes belonging to three major classes: phospholi-
pases A, C, and D. Phospholipase A (PLA) hydrolyzes the acyl chain from
the glycerol backbone at the sn-1 or sn-2 positions or both, releasing free
fatty acids and lysophospholipids as reaction products. Three subtypes
of PLA are defined based on their positional specificity. PLA 1 and PLA 2
specifically hydrolyze the ester bond at the sn-1 and sn-2 positions, respec-
tively, whereas patatin-like PLA (PAT-PLA) catalyzes the hydrolysis at
both positions. Phospholipase C (PLC) cleaves the linkage between the
phosphate and the glycerol, releasing a phosphoryl head group and adiacyl
glycerol. Phospholipase D (PLD) hydrolyzes the terminal phosphodiester
bond between the head group and the phosphate group, releasing phospha-
tidic acid. Each of these classes of phospholipases is encoded by a large
gene family and contains several isoforms that can be grouped according
to their sequence similarities and substrate specificities (Qin and Wang,
2002; Meijer and Munnik, 2003; Li et al., 2007, 2009; Tasma et al., 2008;
Chen et al., 2011). The hydrolytic products of phospholipase activity have
a wide range of roles in plant metabolism, including responses to abiotic
stresses (drought, salinity, freezing, wounding, ultraviolet radiation), dis-
ease responses, hormone effects (absicic acid and auxin), jasmonic acid
biosynthesis, nutrition, including nitrogen assimilation and response to
phosphorus starvation, cell growth and elongation, shoot gravitropism,
onset of senescence, intracellular trafficking, stomatal function, and cyto-
skeletal organization (Wang et al., 2006; Xue et al., 2009; Chen et al., 2011;
Boss and Im, 2012; Dong et al., 2012).
Considering the broad range of physiological processes that can be
mediated by phospholipid-derived molecules, technological applications of
these molecules to modulate postharvest quality attributes in fruits, veg-
etables, and ornamentals can be foreseen. Arguably, the most immediate
technology is the direct application of exogenous lipidic molecules to crops
or harvested produce. This approach has been attempted with LPE.
LPE is the lysophospholipid resulting from the hydrolysis of phospha-
tidylethanolamine (PE) at the sn-2 position catalyzed by PLA 2. LPE is natu-
rally present in small concentrations in plant tissues (Li et al., 2006). The
chemical nature of LPE depends on the acyl chain present at the sn-1 posi-
tion of the glycerol backbone. LPE containing a 14:0 acyl chain is seldom
240
L Y SO P H OS P H OLI P I D S AN D P OST H AR V EST
present in plant tissues (Ryu et al., 1997), but the 16:0, 18:0, and 18:1 acyl
chains exist in plants.
241
P OST H AR V EST RI P ENING P H Y SIOLOG Y
242
Table 8.1 Summary of LPE Effects on Horticultural Commodities
Unaffected or
Species Increased Decreased or Delayed Inconsistent References
Fruits
Apple Firmness, color Respiration rate Farag and Palta (1991c)
uniformity, peel
anthocyanin content,
ethylene production
Banana Shelf life, fruit diameter, Senescence, respiration Fresh weight loss, Workmaster and Palta
marketable fruits, rate, soluble solids ethylene production (1996), Ahmed and
firmness content, electrolyte Palta (2010, 2011a)
leakage
Cranberry Fruit set, anthocyanin Chlorothalonil toxicity, Özgen and Palta (1999,
content, color ethylene production 2003, 2004), Farag and
uniformity, firmness Palta (1991c), Ryu et al.
L Y SO P H OS P H OLI P I D S
(1997)
Cantaloupe Aldehyde production Volatile alcohols and Amaro et al. (2012)
(fresh cut) esters
AN D
(Continued )
P OST H AR V EST
243
244
Table 8.1 Summary of LPE Effects on Horticultural Commodities
Unaffected or
Species Increased Decreased or Delayed Inconsistent References
Pepper Ripening, ethylene Ethephon-induced injury, Kang et al. (2003), Hong
production chilling injury and Chung (2006)
P OST H AR V EST
Tomato Ripening, firmness, Ethylene production and Farag and Palta (1991b,
ethylene production respiration rates and 1991d, 1993a, 1993b),
(mature-green fruit), ACC oxidase activity in Mangat and Palta
respiration rate (mature ripening fruit, electrolyte (1995), Özgen et al.
green), ACC oxidase leakage, decay, (2000), Hong et al.
activity (mature green) polygalacturonase (2001, 2002), Altwies
activity, injury by et al. (2002), Hong
RI P ENING
Leaves and
cotyledons
Arabidopsis Freezing tolerance Rajashekar et al. (2006)
thaliana
Philodendron PAL or insoluble acid Hong et al. (2009a)
cordatum invertase, ABA-induced
increase in
malonaldehyde,
chlorophyll
Potato Chlorophyll content Özgen et al. (2005)
(in vitro)
Radish Acid invertase, Polyphenoloxidase and Soluble acid invertase Cowan et al. (2006),
phenylalanine ammonia 3-hydroxy-3- activity, glucose and Hong et al. (2009b)
lyase, endo β-1,3(4)- methylglutaryl sucrose levels
glucanase and coenzyme A reductase
peroxidase (transient), activity
PAL and insoluble acid
invertase activity
L Y SO P H OS P H OLI P I D S
AN D
P OST H AR V EST
245
P OST H AR V EST RI P ENING P H Y SIOLOG Y
246
L Y SO P H OS P H OLI P I D S AN D P OST H AR V EST
247
P OST H AR V EST RI P ENING P H Y SIOLOG Y
egg, which is the 18:0-LPE (Cowan, 2009). However, the 18:1-LPE has stron-
ger biochemical effects on ethylene and phospholipase D (Ryu et al., 1997).
248
L Y SO P H OS P H OLI P I D S AN D P OST H AR V EST
of the reported effects attributed to LPE. Ethanol, per se, can delay
ripening-related changes and reduce decay in fruits (Margosan et al.,
1997; Chervin et al., 2005; Pesis, 2005).
References
Ahmed, Z.F.R., and Palta, J.P. 2010. Lysophosphatidylethanolamine, a natu-
ral phospholipid, may retard senescence and improve the shelf life of
banana fruit. HortScience 45, 66. (Abstr.).
Ahmed, Z.F.R., and Palta, J.P. 2011a. A natural lipid, lysophosphatidyletha-
nolamine, may promote ripening while reducing senescence in banana
fruit. HortScience 46, 273.
Ahmed, Z.F.R., and Palta, J.P. 2011b. Hormone-like effect of a natural lipid,
lysophosphatidylethanolamine, can mitigate calcium deficiency injury
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254
Chapter 9
Abstract 256
9.1 Introduction 256
9.2 Economics of Color Fruit 258
9.3 Carotenoid Pigments in Fruits 259
9.4 Anthocyanin Pigments in Fruits 260
9.5 Grape as a Model System for Pigmentation Studies
in Fruit Crops 270
9.6 Case Studies 278
9.6.1 Apple 278
9.6.2 Strawberry 279
255
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Abstract
The color of fruit is an important phase in its life cycle and results from the
presence of carotenoid and anthocyanin pigments. During coloration, sev-
eral physiological and biochemical changes take place through differential
expression of various genes that are developmentally regulated. Expression
and suppression of these genes contribute to various changes in the fruit
that make it visually attractive and edible. Pigments and surface topography
selectively absorb and refract incident visible light to produce a reflectance
spectrum characteristic of a particular fruit skin. The share of colored fruit
in the total fruit production is quite significant, which is contributing sev-
eral billion dollars annually. contributing several billion dollars annually.
This has encouraged scientists to study the pigmentation mechanism in
fruits. Pigmentation evokes several responses during coloration through
a signaling cascade, and thousands of genes are involved, with each gene
being a member of a multigene family. The transcripts are abundant in the
skins of cultivars with red skin, but rarely in skins of cultivars with other
fruit colors, suggesting that these genes have major roles in the determina-
tion of fruit skin color. In this chapter, various developments that have taken
place in the last decade with respect to identifying and altering the function
of ripening-related genes have been described. Taking clues from the stud-
ies in grape as a model fruit, a few case studies are reviewed.
9.1 Introduction
Fruits are an important part of the human supplementary diet, with the
quality of fruit being determined by a wide range of desirable character-
istics, such as food value, flavor, shelf life, and fruit color. Carotenoid and
anthocyanin are the most important pigments, being responsible for a
256
ROLE O F P IGMENTS D URING F RUIT RI P ENING
257
P OST H AR V EST RI P ENING P H Y SIOLOG Y
carotenoids, metal complexes, vacuolar pH, and cell shape, have been
known to influence the final pigmentation phenotype. The genetics and
biochemistry of the a nthocyanin biosynthetic pathway, a major branch
of flavonoid metabolism, have been well characterized in maize, petunia,
snapdragon, and more recently, Arabidopsis (Holton and Cornish, 1995;
Winkel-Shirley, 2001; Kim et al., 2003; Martens et al., 2010). The regula-
tion of anthocyanin biosynthesis has also been studied thoroughly and is
comprised of basic helix–loop–helix (bHLH) transcription factors, inter-
acting with R2R3 MYB transcription factors to activate either all or part of
the anthocyanin genes (Allan et al., 2008). Many studies have generated
transgenic plants with either increased concentration or altered composi-
tion of anthocyanin in flowers, or fruit by manipulating the expression of
the anthocyanin r egulatory genes (Quattrocchio et al., 1998; Davies et al.,
2003; Ben Zvi et al., 2008; Butelli et al., 2008; Tanaka and Ohmiya, 2008;
Oren-Shamir, 2009).
Fruit color is one of the characteristics that are important for both the
commercial and the aesthetic value of fruit. The mechanism of color forma-
tion in the fruit has not been studied extensively at the molecular level.
Color variation has a genetic basis that is species specific, with different
genes in the pathway having been found to be responsible for color varia-
tion in different species (Arakawa et al., 1999; Awad et al., 2000; Jaakola
et al., 2002; Ben-Yehudah et al., 2005). In this chapter, we review various
developments that have taken place in the last decade with respect to iden-
tification and alteration of functions of genes involved in fruit pigmentation
during fruit ripening.
258
ROLE O F P IGMENTS D URING F RUIT RI P ENING
Mangoes, mangosteens,
and guavas
Grapes
Figure 9.1 Percent share of major color fruit out of the total world p
roduction
in metric tons.
259
P OST H AR V EST RI P ENING P H Y SIOLOG Y
260
Table 9.1 Carotenoid Contents (mg/100 g fresh weight) of Some Common Fruits
Family Genus Species Name α-Carotene β-Carotene Lycopene References
ROLE
(Continued )
RI P ENING
261
262
Table 9.1 Carotenoid Contents (mg/100 g fresh weight) of Some Common Fruits
Family Genus Species Name α-Carotene β-Carotene Lycopene References
Actinidiaceae Spondias dulcis L. Hog plum — 0.201 0.364 Hulshof et al.
Actinidia (1997)
deliciosa Kiwifruit ND 0.074 ND Yano et al.
P OST H AR V EST
ND 0.035 ND
ND 0.027 ND
[0.005]
0.006 0.023 —
Moraceae Artocarpus heterophyllus Jackfruit ND 0.026–0.36 0.037 Charoensiri
Lam. et al. (2009)
D URING
0.058 0.058 ND
[0.007] [0.006]
(Continued )
RI P ENING
263
264
Table 9.1 Carotenoid Contents (mg/100 g fresh weight) of Some Common Fruits
Family Genus Species Name α-Carotene β-Carotene Lycopene References
paradisiaca Banana — 0.097 0.114 Setiawan et al.
L. (2001)
var. Ambon
P OST H AR V EST
bright (0.006)
var. May glo — 0.058 — Gil et al. (2002)
(0.005)
var. — 0.131 — Gil et al. (2002)
F RUIT
September (0.023)
red
(Continued )
RI P ENING
265
266
Table 9.1 Carotenoid Contents (mg/100 g fresh weight) of Some Common Fruits
Family Genus Species Name α-Carotene β-Carotene Lycopene References
persica (L.) Peach 0.001 0.097 — Holden et al.
Batsch [0.001] [0.013] (1999)
var. Summer — 0.04 (0.01) — Gil et al. (2002)
P OST H AR V EST
sweet
var. Snow — 0.008 — Gil et al. (2002)
king (0.002)
var. Santa — 0.049 — Gil et al. (2002)
Rosa (0.012)
var. — 0.057 — Gil et al. (2002)
Angeleno (0.009)
RI P ENING
var. ND 0.218 ND
Ponteroza [0.019]
var. Soldam ND 0.439 ND
[0.029]
Rosaceae Prunus spp. Cherry ND ND ND Lako et al.
— 0.14 (0.06) − (2007),
Bhaskarachary
et al. (1995)
P H Y SIOLOG Y
(1999)
Rubus sp. Raspberry 0.012 0.008 — Holden et al.
O F
(1999)
Rutaceae Citrus aurantium L. Orange — 0.17 (0.08) — Bhaskarachary
et al. (1995)
maxima Pummelo 0.014 0.32 — Holden et al.
Merr. (1999)
microcarpa Musk lime — 0.012 — Tee and Lim
P IGMENTS
Bunge (1991)
nobilis L. Orange — 0.025 — Tee and Lim
paradisiaca Grapefruit ND 0.452 1.869 (1991)
Macfad. var. [0.019] [0.654] Yano et al.
Star ruby (2005)
var. Pink 0.005 0.603 — Holden et al.
D URING
Osbeck (1999)
(Continued )
RI P ENING
267
268
Table 9.1 Carotenoid Contents (mg/100 g fresh weight) of Some Common Fruits
Family Genus Species Name α-Carotene β-Carotene Lycopene References
var. Navel 0.019 0.139 ND Yano et al.
[0.002] [0.014] (2005)
var. Valencia 0.015 0.051 ND Yano et al.
P OST H AR V EST
269
P OST H AR V EST RI P ENING P H Y SIOLOG Y
270
Table 9.3 Regulatory Genes Identified in Different Fruit Crops and Their Targeting Structural Genes Involved in Anthocyanin
Biosynthesis
Structural
ROLE
Fruit Species MYB bHLH Genes Fruit Tissue Accession No. References
Grapes VvMYB5a UFGT, C4H, Berry skin AY555190 Deluc et al. (2006)
O F
ANS, F3H
VvMYB5b MYCA1 VvLAR, VvANR Berry skin AY899404 Deluc et al. (2008)
VvMYBA1 CHI, CHS, Berry skin DQ886418 Walker et al. (2007)
4CL, DFR
VvMYBA2 UFGT, ANR DQ886419 Walker et al. (2007)
VlmybA1-1 UFGT AB073010 Kobayashi et al.
P IGMENTS
(2002)
VlmybA1-2 UFGT, OMT, AB073012 Kobayashi et al.
GST (2002)
VlmybA2 UFGT AB073013 Kobayashi et al.
(2002)
D URING
271
272
Table 9.3 Regulatory Genes Identified in Different Fruit Crops and Their Targeting Structural Genes Involved in Anthocyanin
Biosynthesis
Structural
Fruit Species MYB bHLH Genes Fruit Tissue Accession No. References
VvmybA2 UFGT AB097924 Kobayashi et al.
P OST H AR V EST
(2004)
VvmybA3 Unknown AB097925 Kobayashi et al.
(2004)
VvMybA1 UFGT DQ886417 Walker et al. (2007)
VvMybA2r UFGT DQ886419 Walker et al. (2007)
VvMybA2w Unknown DQ886420 Walker et al. (2007)
VvMybA3 Unknown DQ886421 Walker et al. (2007)
RI P ENING
273
P OST H AR V EST RI P ENING P H Y SIOLOG Y
anthocyanins in their skins (Pomar et al., 2005; Torres et al., 2010). Though
earlier studies were focused more on the molecular biology of fruit skin
color, genomics approaches have recently been used to reveal insights
into the control of primary coloration upstream of anthocyanin, coloration-
related signal transduction systems, and downstream metabolic networks.
Grape pleiotropic coloration mutations have added greatly to the under-
standing of fruit skin color. Genes of anthocyanin production and pigment
biosynthesis enzymes were among the first anthocyanin-responsive genes
isolated from fruits of grape, and anthocyanin biosynthesis in grape skin
has been extensively studied (Boss et al. 1996). In grapevine, anthocyanin
biosynthesis depends on Myb-related genes, which play a crucial role in
the different varieties that exhibit an assortment of biosynthesis modes. In
American hybrid grapevine, VlmybA1-2, VlmybA2, and VlmybA3 are located
in the pericarp, though the exact location is unknown, and also regulate
the anthocyanin biosynthetic gene cluster (dashed box in Figure 9.2). Gene
clusters can encode the production of the Myb protein, thus contributing to
the expression of the anthocyanin biosynthetic pathway in UFGT-turned
grape skin color. Grape color loci are mainly divided into three cases
(Figure 9.2):
274
ROLE O F P IGMENTS D URING F RUIT RI P ENING
Chr2
VvmybA2 VvmybA1
Mutation
I
Regulatory
function in red/
black pericarp
Chr2
Chr2
VvmybA2w VvmybA1a VvmybA2r VvmybA1c VvmybA1SUB
Chr2 VvmybA2w VvmybA1b
null null
VvmybA2w VvmybA1c II
Anthocyanin
Delphinidin Chr2
UFGT III VvmybA1-2 VvmybA1-3
Chr2
VvmybA2 VvmybA1-3
Anthocyanin OMT
Regulatory
function in red/
black pericarp
Figure 9.2 Schematic diagrams of the regulation of the skin color locus of
grapevine.
275
276
Table 9.4 Homolog Genes Involved in Anthocyanin Biosynthesis in Arabidopsis and Grape
Arabidopsis
Gene Gene ID Chromosome Start End Grape Gene Gene ID Chromosome Start End
AtPAL1 AT2G37040 2 15,557,376 15,560,363 VvPAL1 GSVIVP00013939001 chr16 700,746 703,220
AtPAL2 AT3G53260 3 19,744,051 19,746,780 VvPAL2 GSVIVP00013930001 chr16 648,662 651,184
AtPAL4 AT3G10340 3 3,204,013 3,208,022 VvPAL3 GSVIVP00013943001 chr16 724,118 727,239
P OST H AR V EST
277
P OST H AR V EST RI P ENING P H Y SIOLOG Y
9.6.1 Apple
Apple (Malus spp.) is one of the most popular fruit crops worldwide. There
are more than 7500 known cultivars of apples, with different cultivars
available for temperate and subtropical climates. Among the quality char-
acteristics of the cultivars, red fruit skin coloration is of major importance
and significantly determines the market value of the produce, because
consumers associate better-colored apples with better taste, ripeness, and
flavor (King and Cliff, 2002). Color is also an obvious characteristic that
facilitates product discrimination. For example, the apple cultivar ‘Cripps’
Pink’, commercially sold as Pink Lady, has been very successful due to
its distinct pink hue (Corrigan et al., 1997). The coloration of apple skin is
derived from anthocyanins belonging to a class of flavonoids (Gallus et al.,
2005; Liu et al., 2005; Rasmussen et al., 2005). Anthocyanin accumulation
positively correlates with the expression of at least five anthocyanin
biosynthetic genes (MdCHS, MdF3H, pDFR, MdANS, and pUFGluT ),
suggesting that the levels for the expression of the five genes basically cor-
respond to the degree of anthocyanin concentration (Honda et al., 2002;
Ubi et al., 2006).
Apple skin is also a rich source of compounds that are potent antioxi-
dants, such as anthocyanins, chitosans (CTs), and flavonols, whose intake
in the human diet has been correlated with lower incidences of cancer
and cardiac disease (Gallus et al., 2005; Liu et al., 2005; Rasmussen et al.,
2005). For these reasons, the antioxidative potential of apple skin may
become a new quality marker for different apple cultivars (Schirrmacher
and Schempp, 2003). Although the pigments that comprise fruit color vary
with fruits, the amount and composition of anthocyanins that belong to a
class of flavonoids are the major determinants of apple fruit skin redden-
ing. Anthocyanin biosynthesis in apple fruit is developmentally regulated
and occurs at two peaks. The first peak occurs at the fruitlet stage in both
red and nonred cultivars, and this peak is not economically important. The
second peak takes place later, at the ripening fruit stage, only in red cul-
tivars. The intensity and extent of reddening at the mature fruit stage are
most important, as they impact greatly the marketable value of the produce
and have direct bearing in the economics of apple production. The expres-
sion of the five anthocyanin biosynthetic genes, chalcone synthase (CHS),
flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocy-
anidin synthase (ANS), and UDP-glucose:flavonoid 3-O-glucosyltransferase
(UFGluT) during ripening in five early ripening apple cultivars, as well as
under UV-B and temperature treatments. The expression of CHS, ANS and
UFGluT was substantially depressed in fruit skin, whereas that of all five
278
ROLE O F P IGMENTS D URING F RUIT RI P ENING
genes including CHS, ANS and UFGluT was enhanced by UV-B and low
temperature treatments with the accumulation of anthocyanins. The accu-
mulation level of the main anthocyanin pigment, cyanidin 3-galactoside,
in the fruits under UV-B and low temperature treatment, was less than
that in the field-ripened fruits, while cyanidin 3-arabinoside was relatively
the same level. The UV-B and low temperature were important factors for
anthocyanin accumulation in apple fruit skin by inducing the expression
of the anthocyanin biosynthetic genes, especially CHS, ANS and UFGluT
genes. The recent studies at the molecular level have revealed that the
regulation of the expression of the biosynthetic genes is one of the causes
that determine the anthocyanin accumulation in apple skin.
9.6.2 Strawberry
Strawberry (Fragaria spp.) fruit is widely appreciated, not only for its
characteristic aroma, but also for its bright red color. Besides sweetness
and flavor, the development of red color is one of the most important char-
acteristics affecting the fruit quality of strawberry, as it contributes to
a consumer’s initial impression and decision to buy or not. These fruit
offer an interesting model to study fruit pigmentation by anthocyanin,
considering that the color of the white and red botanical forms reflects
the presence of such phytochemicals. In the Fragaria genus, fruit color
is determined by the accumulation of anthocyanin, the most abundant
flavonoid in strawberry fruits (Hannum, 2004). Pelargonidin 3-gluco-
side is the predominant anthocyanin in several varieties of red straw-
berries, usually followed by pelargonidin 3-rutinoside and cyanidin
3-glucoside (Gil et al., 1997; Kosar et al., 2004; Tulipani et al., 2008).
These three compounds represent more than 95% of the total anthocya-
nins in the cultivated strawberry (Lopes-da-Silva et al., 2007). Flavonoid
genes involved in anthocyanin biosynthesis exhibit upregulation dur-
ing ripening, leading to fruit pigmentation in strawberry and thereby
establishing a positive correlation between transcript levels of flavonoid
genes and anthocyanin accumulation (Manning, 1998; Almeida et al.,
2007; Carbone et al., 2009). The cDNA fragments of genes from phenyl-
propanoid (PAL, C4H, and 4CL) and the flavonoid biosynthetic pathway
(CHS, CHI, F3H, DFR, ANS, UFGT, LAR, and ANR) have been isolated
from the native Chilean strawberry, and the differential transcriptional
profiles of these genes analyzed in Fragaria chiloensis and Fragaria
patagonica through four different fruit developmental stages and tis-
sues by quantitative real-time polymerase chain reaction (qRT-PCR).
In addition, the accumulation of the two major anthocyanins, pelargo-
nidin 3-glucoside and cyanidin 3-glucoside, in these native strawberries
279
P OST H AR V EST RI P ENING P H Y SIOLOG Y
9.6.3 Tomato
Tomato (Lycopersicon esculentum) has dark green foliage and elevated
anthocyanin expression in the hypocotyls of seedlings and in the skin
and outer pericarp tissues of fruits. Tomato fruit is not usually reported
to contain anthocyanin (Jones et al., 2003), but Oregon State University
managed to develop a high-anthocyanin tomato cultivar using genes intro-
gressed from wild species. Cultivated tomatoes produce anthocyanin in
vegetative tissues, but only small amounts of other flavonoids, such as nar-
ingenin chalcone and flavonols, can be found in the fruit (Muir et al., 2001;
Torres et al., 2005; Bovy et al., 2007). As a consequence, tomato is consid-
ered an excellent candidate for an enhancement of the flavonoid and antho-
cyanin contents through transgenic approaches (Gonzali et al., 2009).
Recently, Butelli et al. (2008) expressed in tomato Delila and Rosea1, two
genes coding for transcription factors involved in anthocyanin produc-
tion in snapdragon. The tomato anthocyanin fruit (Aft) genotype is char-
acterized by purple color in the skin and outer pericarp of its fruits due
to higher levels of anthocyanins—flavonoid metabolites. The dominant
gene Aft (Anthocyanin fruit) was introgressed into domesticated tomato
plants by a cross with Solanum chilense (Jones et al., 2003). Aft triggers
anthocyanin accumulation in immature green fruit upon stimulation by
high light. Subsequently, pigments are produced continuously through-
out development (Mes et al., 2008). The Aft gene identity still has to be
revealed. Recent linkage analyses showed that the Aft locus cosegregates
with two different MYB transcription factor genes located on chromo-
some 10, SlAN2 (Mes et al., 2008; Boches et al., 2009) and anthocyanin
1 (SlANT1) (Sapir et al., 2008), both involved in anthocyanin synthesis
in tomato (Mathews et al., 2003; Mes et al., 2008). A recessive gene, Atv
(Atroviolacea), derived from the interspecific cross with Solanum chees-
maniae (L. Riley) Fosberg, has been shown to influence anthocyanin pig-
mentation in the entire tomato plant, particularly in the vegetative tissues
(Mes et al., 2008). The Atv gene has been located to chromosome 7 (Rick
et al., 1968), and previous studies indicated that its mutation may affect
phytochrome responses, since Atv plants exhibit an exaggerated response
to red light in terms of anthocyanin production (Kendrick et al., 1997). The
280
ROLE O F P IGMENTS D URING F RUIT RI P ENING
9.6.4 Litchi
Litchi (Litchi chinensis Sonn.) is a tropical and subtropical fruit that has
high commercial value due in part to its white and translucent aril and
attractive red color (Holcroft and Mitcham, 1996; Jiang et al., 2004).
The red color of the litchi fruit peel is mainly attributed to anthocyanin
(Prasad and Jha, 1978; Jiang et al., 2006). Litchi fruit pericarp contains
a large amount of pigments that are responsible for the red color (Lee
and Wicker, 1991). Cyanidin was identified as the major anthocyanin
present in pericarp tissues of fresh litchi fruit, followed by delphinidin
and pelargonidin (Oomah and Mazza, 1999; Jiang et al., 2004) and mal-
vidin (Prasad and Jha, 1978; Lee and Wicker, 1991). Pericarp browning
is a common and important defect of harvested litchi fruit, which results
in reduced market value (Huang and Scott, 1985) and has mainly been
attributed to the degradation of anthocyanin and the oxidation of pheno-
lics by polyphenol oxidase (PPO) (Jiang et al., 2004). Thus, postharvest
technologies have been developed to reduce the anthocyanin degradation
and maintain the red color of harvested litchi fruit (Jiang et al., 2006).
Lee and Wicker (1991) identified the red pigment in litchi as cyanidin
3-rutiside. Cyanindin 3-glucoside and malvidin 3-glucoside may also be
present in these anthocyanins (Zhang et al., 2000). Sarni-Manchado et al.
(2000) identified the anthocyanins as cyanidin 3-rutinoside, cyanidin glu-
coside, quercetin 3-rutinoside, and quercetin glucoside, while Zhang et al.
(2004) reported that the major anthocyanin of litchi fruit pericarp was
cyanidin 3-rutinoside. The differences in the reported identification of the
anthocyanins may be attributed to differences in the extraction and puri-
fication procedures (Jiang et al., 2004). Most of the red pigments in litchi
fruit pericarp present in the ethyl acetate fraction and the pigments are
identified as flavanols. Identification of structures of flavanols was needed
281
P OST H AR V EST RI P ENING P H Y SIOLOG Y
to explain the loss in the skin color of harvested litchi fruit, in relation to
their antioxidant activity.
Recent research has shown that pigments in postharvest fruits
exhibit a strong antioxidant activity (Einbond et al., 2004; Bao et al., 2005).
Antioxidative properties of pigments resulted from their high reactivity
as hydrogen or electron donors and from their ability to chelate transition
metal ions, that is, terminate the Fenton reaction (Rice-Evans et al., 1997;
Wada and Ou, 2002). It is well established that enzymatic browning of post-
harvest fruits is related to antioxidant activity (Martinez and Whitaker,
1995). Unfortunately, not much information on the antioxidant activ-
ity of skin pigments from harvested litchi fruit, as well as the molecular
mechanisms regulating this activity, is available.
282
ROLE O F P IGMENTS D URING F RUIT RI P ENING
9.6.7 Pear
Pear fruit pigmentation (red and green pear) is observed through tran-
scriptional and chemical approaches. The proportion of different anthoy-
anins is demonstrated to be characteristic of each botanical form, with
pelargonidin 3-glucoside being the most abundant in red fruit and cyani-
din 3-glucoside the major one in green fruit. Partial gene sequences of
the phenylpropanoid and flavonoid biosynthesis pathways are isolated
from the native Chilean strawberry fruits and used to design gene-spe-
cific primers in order to perform transcriptional analyses by qRT-PCR.
These genes show spatial, developmental, and genotypic-associated pat-
terns. The red fruit exhibited higher transcript levels of anthocyanin-
related genes and higher levels of anthocyanin than the green pigmented
fruit. The anthocyanin accumulation in red and green fruits is concomi-
tant with the particular progress of the transcriptional activity of genes
involved in the biosynthesis of flavonoid pigments. The differences in
anthocyanin contents, in terms of both type and quantity, between the two
283
P OST H AR V EST RI P ENING P H Y SIOLOG Y
9.6.8 Cherry
The cherry (Prunus cerasus L.), containing high levels of anthocyanins that
possess strong antioxidant and anti-inflammatory properties, has attracted
much attention. The evaluation and characterization of the in vitro–
produced pigments are compared to those of the anthocyanins found
in vivo in fruits of several cherry cultivars. Interestingly, the anthocyanin
profiles found in whole fruit extracts are similar in all tested genotypes.
The evaluation of antioxidant activity, performed by oxygen radical absor-
bance capacity (ORAC) and trolox equivalent antioxidant capacity (TEAC)
assays, revealed a relatively high antioxidant capacity for the fruit extracts
and a lower one for the callus extract. The structural gene DFR was deter-
mined from cherry where two MYB factors were responsible: PavMYB10
and PcrMYB10 (Wang et al., 2010).
9.6.9 Kiwifruit
The kiwifruit or Chinese gooseberry (often shortened to kiwi) is the edible
berry of a woody vine in the genus Actinidia, and it is native to northern
China. The anthocyanins responsible for the red color of red kiwifruit
were extracted in acidified ethanol and isolated by solid-phase extraction
(SPE), followed by preparative HPLC. Five anthocyanins were obtained
and subsequently identified as delphinidin 3-[2-(xylosyl)galactoside],
delphinidin 3-galactoside, cyanidin 3-[2-(xylosyl)galactoside], cyanidin
3-galactoside, and cyanidin 3-glucoside by a combination of liquid
chromatography–mass spectrometry (LC-MS)/ mass Spectrometry (MS),
Gas chromatography–mass spectrometry (GC-MS), and Two-dimensional
nuclear magnetic resonance spectroscopy (2D NMR).
284
ROLE O F P IGMENTS D URING F RUIT RI P ENING
285
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Table 9.5 U.S. Patents (Granted) on Transgenic Plants Developed for Fruit
Skin Color
Patent Named
No. Date Inventors Applicant Area
7678393 March 16, Bradford DB Laboratories Anthocyanin
2010 et al. LLC, Stites, mixture useful
Idaho for topical
and internal
applications
7682637 March 23, Gerald et al. Phenolics LLC, Compositions
2010 Omaha, enriched in
Nebraska total phenols
7727564 June 1, 2010 Kenneth et al. K2A LLC, Fruit-based
Springville, dietary
Utah supplements
7750211 July 6, 2010 Richard et al. Samuel Roberts Production of
Noble flavonoids and
Foundation, isoflavonoids
Ardmore,
Oklahoma
7767416 August 3, Spangenberg Ariculture Flavonoid
2010 et al. Victoria Service biosynthesis in
Pty. Ltd., plants
Altwood,
Australia, and
Agresearch
Ltd., Hamilton,
New Zealand
7855319 December Rommens J.R. Simplot Antioxidant-
21, 2010 et al. Company, containing
Boise, Idaho food
7880059 February 1, Richard et al. The Samuel Proanthocyanins
2011 Roberts Noble to improve
Foundation, forage quality
Ardmore
Oklahoma (US)
286
ROLE O F P IGMENTS D URING F RUIT RI P ENING
Table 9.6 U.S. Patent Applications on Transgenic Plants Developed for Fruit
Color
Named
Patent No. Date Inventors Applicant Area
20100016565 January 21, Connors Exelixis Plant Anthocyanin
2010 et al. Science, Inc. mutant (ANT1)
in tomato
20100015065 January 21, Matsumoto — Anthocyanin
2010 et al. composition in
food
20100021614 January 28, Nishijima — Bioabsorption of
2010 et al. flavonoid
20100041877 February Tamura — Purified
18, 2010 et al. anthocaynin
20100107277 April 29, Bruguera International Flavonoid
2010 et al. Flower 3,5-hydroxylase
Developments gene
Pty. Ltd.
20100216729 April 30, Gutierrez- — Cancer cell
2010 Uribe growth
et al. inhibition
20100199370 August 5, Levin et al. — Fruits with high
2010 levels of
anthocyanin
and flavonols
20110072539 March 24, Espley — Pigment
2011 et al. production in
plant
—, data not available.
287
P OST H AR V EST RI P ENING P H Y SIOLOG Y
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Chapter 10
Molecular Regulation
of Fruit Ripening
Ajay Arora
Indian Agricultural Research Institute, New Delhi, India
Abstract 300
10.1 Introduction 300
10.2 Transcriptional Control of Fruit Ripening 301
10.3 Hormonal and Transcriptional Regulation during
Ripening 305
10.3.1 Ethylene 306
10.3.2 Auxins 308
10.3.3 Gibberellins 308
10.3.4 Abscisic Acid 308
10.4 Epigenetic Regulation of Fruit Development
and Ripening 309
10.5 Ripening Pathways and Associated Fruit Quality 312
10.5.1 Sugar Accumulation 313
10.5.2 Cell Walls and Fruit Shelf Life 313
10.5.3 Color, Flavor, and Nutrition in Fruits 314
10.5.3.1 Chloroplast-to-Chromoplast
Conversion 314
10.5.3.2 Flavor and Aroma Production 315
10.5.3.3 Control of Color and Texture
Changes 316
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Abstract
Fruit ripening is a highly coordinated developmental process that coincides
with seed maturation. Regulated expression of thousands of genes controls
fruit softening as well as accumulation of pigments, sugars, acids, and
volatile compounds that increase attraction to animals. A combination of
molecular tools and ripening-affected mutants has permitted researchers
to establish a framework for the control of ripening. Key to crop improve-
ment is a deeper understanding of the processes underlying fruit ripening.
In tomato, mutations blocking the transition to ripe fruits have provided
insights into the role of ethylene and its associated molecular networks
involved in the control of ripening. However, the role of other plant hor-
mones is still poorly understood. Translation of information from tomato
to other fleshy-fruited species indicates that regulatory networks are con-
served across a wide spectrum of angiosperm fruit morphologies. In this
chapter, we describe how plant hormones, transcription factors, and epigen-
etic changes are intimately related to provide tight control of the ripening
process. These discoveries are likely to have a major impact on strategies
for crop improvement through genetic manipulation of ripening regulatory
genes in fruit-bearing species. The findings from comparative genomics
and system biology approaches are also discussed. Recent developments in
the sequencing of angiosperm genomes have provided the foundation for a
step change in crop improvement through the understanding and harness-
ing of genome-wide genetic and epigenetic variation.
10.1 Introduction
Ripening represents a summation of physiological and biochemical pro-
cesses of fleshy fruits, including, but not limited to, degreening and accu-
mulation of colored pigments for attraction, textural changes associated
with cell wall metabolism and cell turgor variation leading to softening,
and metabolic changes related to flavor and nutrient composition, generally
associated with accumulation of sugars, acids, and volatiles culminating
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in a diverse array of tastes and smells, varying among species (Klee and
Giovannoni, 2011). In climacteric fruit such as apple, tomato, banana, and
most stone fruits, the onset of ripening coincides with a burst of respira-
tion and ethylene, and ethylene is necessary to confer these physiological
changes. However, elevated respiration and ethylene are not required for
normal ripening in fruit classified as nonclimacteric, including important
fruit crops such as grape, citrus, and strawberry, indicating ethylene-
independent ripening events are important in many species, including
those undergoing climacteric ripening. Investigations into the molecular
and genetic regulation of ripening have focused largely on the model sys-
tem of tomato due to its dramatic ripening process, short life cycle, ame-
nability to genetic transformation, and the plethora of available molecular
and genetic resources, including its recently sequenced genome (Tomato
Genome Consortium, 2012). The agricultural importance and unique attri-
butes of many additional species have resulted in the characterization of
ripening physiology, metabolism, and molecular biology, which is high-
lighted in this chapter as well. The recent history of ripening molecular
biology can be divided into several distinct, though sometimes overlapping,
areas, including the elucidation of cell wall–modifying proteins and their
roles in textural modification, the characterization of ethylene synthesis
and signaling, the genes and pathways contributing to pigment accumula-
tion, the regulatory process associated with fruit plastid development, the
biochemistry of sensory attributes, and the overall genetic regulation of the
ripening process. We will attempt to highlight all areas, with emphasis on
primary regulators that are likely to be functional and conserved among
many fleshy fruit–producing species.
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nutritional value, flavor, processing qualities, and shelf life determine the
quality of fruits.
The main changes associated with ripening include color (loss of
green color and increase in nonphotosynthetic pigments that vary depend-
ing on species and cultivar), firmness (softening by cell wall–degrading
activities and alterations in cuticle properties), taste (increase in sugar
and decline in organic acids), and flavor (production of volatile compounds
providing the characteristic aroma).
The comprehensive molecular analyses of ethylene synthesis and sig-
nal transduction during ripening suggest the presence of additional regulatory
systems that coordinate the ethylene burst and production of this necessary
ripening hormone. A number of interesting tomato ripening mutants have
been collected by breeders and geneticists, and their subsequent physiologi-
cal characterization suggests that they may represent defects in ripening
regulatory systems. These mutations include the ripening-inhibitor (rin),
non-ripening (nor), and colorless non-ripening (Cnr) mutations that are non-
allelic but share common physiological characteristics in that all (1) develop
to the mature green stage, where the fruit is full size and the seeds are
mature, yet do not advance to ripening; (2) fail to undergo the climacteric
rise in respiration or produce ripening-associated ethylene; (3) do not ripen
in response to exogenous ethylene; and yet (4) respond to ethylene in other
tissues and fruit in which ethylene-responsive genes are induced (Eriksson
et al., 2004; Manning et al., 2006; Giovannoni, 2007).
Together, these characteristics suggest that all three mutations
impact central ripening phenomena necessary for the induction of ethyl-
ene, in addition to activities for which ethylene alone cannot compensate.
In this regard, it is a reasonable hypothesis that such genes might represent
conserved regulators impacting ripening even in nonclimacteric fruits.
All three genes have been isolated by positional cloning, and all encode
transcription factors. There is only one described rin mutation, and it also
displays a large sepal, or macrocalyx, phenotype. The rin locus encodes a
MADS-box transcription factor termed RIN-MADS, which is induced at the
onset of ripening (Vrebalov et al., 2002). MADS-box genes are developmen-
tal regulators conserved in eukaryotes and associated with floral develop-
ment in plants, including members of the ABC model of floral development
(Coen and Meyerowitz, 1991). RIN-MADS is a member of the SEPALLATA
clade associated in Arabidopsis with E function activities that determine the
growth and development of floral organs (Zahn et al., 2005).
Phylogenetic analysis of the RIN gene and examination of the tomato
genome sequence (http://solgenomics.net) indicate two additional genes
compared to Arabidopsis, including RIN-MADS, with the additional gene
having no transcriptional support in the public tomato expressed sequence
tag (EST) collection. This suggests that it may have very limited expres-
sion or be a pseudogene. The RIN-MADS gene resides on c hromosome
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Transcription factors
Epigenetic dynamics
Senescence
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10.3.1 Ethylene
The role of ethylene in fruit ripening has been reviewed many times and
is therefore covered only briefly here. Fleshy fruits have historically been
divided into two classes: climacteric (e.g., tomato, apples, and banana)
and nonclimacteric (e.g., grape, strawberry, and citrus). Climacteric fruits
show a burst of respiration at the onset of ripening, along with a large rise
in ethylene production. Ripening in climacteric fruits can also be initiated
by exposure to exogenous ethylene. In nonclimacteric fruits, the respira-
tory increase is absent and ethylene does not appear to be critical for the
ripening process. Direct evidence that ethylene is critical for the induc-
tion of ripening in climacteric fruit came from work with tomato, where
antisense genes were used to suppress the expression of ACO1 and ACS2,
which encode ACC oxidase (ACO) and ACC synthase (ACS), respectively,
the major enzymes involved in ethylene biosynthesis (Grierson, 2013).
Autocatalytic ethylene biosynthesis (system 2 ethylene biosynthesis)
(Klee and Giovannoni, 2011) is operational during ripening; this involves
new forms of ACO and ACS that produce much higher levels of ethylene and
are not subject to autoinhibition. Ethylene is then perceived by a family of
copper binding membrane-associated receptors, several of which—tomato
ETHYLENE RESPONSE4 (LeETR4), LeETR6, and NR (which results in
the phenotype of the Never-ripe [Nr] tomato mutant)—show significantly
increased expression at the onset of ripening (Klee and Giovannoni, 2011).
The receptors interact with a Raf kinase–like protein, constitutive triple
response1 (CTR1), and repress ethylene responses. When the receptors
bind ethylene, this inhibition is relieved and a signaling process leads to
the ethylene response (Grierson, 2013; Klee and Giovannoni, 2011). This
involves activation of the ETHYLENE INSENSITIVE3 (EIN3) and EIN3-
like (EIL) transcription factors by EIN2. In the nonclimacteric pepper fruit,
EIL-like genes are induced during ripening (Lee et al., 2012), suggesting
common systems for downstream signal transduction in climacteric and
nonclimacteric fruits, which in the latter occurs in the absence of ethylene
biosynthesis. In climacteric fruit, at least, ethylene production enhances
the expression of some regulatory and structural genes. Ethylene does
not act to regulate ripening in isolation, but rather acts in concert with
other plant hormones. Lin et al. (2008) identified a tetratricopeptide (TPR)
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repeat protein, SlTPR1, from tomato that interacts with the ethylene recep-
tors LeETR1 and NR. Overexpression of SlTPR1 in tomato enhances
ethylene responses while causing reduced expression of the early auxin-
responsive gene IAA9.
In strawberry, which has emerged as a prime model of nonclimac-
teric fruit ripening, ethylene is relatively high in green fruits, decreases
in white fruits, and finally increases again at the red stage of ripening
(Perkins-Veazie et al., 1996; Iannetta et al., 2006). Interestingly, this last
increase is accompanied by an enhanced respiration rate that resembles
the one that occurs in climacteric fruits at the onset of ripening (Iannetta
et al., 2006). For better understanding of the function of ethylene during
strawberry ripening, different approaches have been used. External appli-
cation of ethylene caused the downregulation of several cell wall–related
genes, such as β-galactosidase, pectin methylesterase, or β-xylosidase
(Trainotti et al., 2001; Castillejo et al., 2004; Bustamante et al., 2009),
while the expression of other genes, such as the expansin FaEXP2 (Civello
et al., 1999), was ethylene insensitive. Recent studies at transcriptomic
and metabolomic levels in transgenic strawberry fruits with decreased
ethylene sensitivity indicate that ethylene action is required for normal
fruit development, acting differently in the two parts of strawberry fruit,
the achenes and receptacle. These results show that, although not as rel-
evant as in climacteric fruits, ethylene may nevertheless play a role in
strawberry fruit ripening.
Recent comparative transcriptome and metabolome studies dur-
ing the maturation processes of climacteric and nonclimacteric fruits
(tomato and pepper, respectively) suggest that both species have similar
ethylene-mediated signaling components. In pepper, the regulation of
these genes is, however, clearly different and may reflect altered ethylene
sensitivity or regulators other than ethylene in tomato (Osorio et al., 2012).
Unlike the situation described in tomato, the ethylene biosynthesis genes,
a minocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase,
are not induced in pepper. However, genes downstream of ethylene percep-
tion, such as cell wall–related genes, ethylene response factor 3 (ERF3),
and carotenoid biosynthesis genes, are upregulated during pepper fruit
ripening (Osorio et al., 2012). Other commonly regulated genes between
climacteric and nonclimacteric fruits have been described. In strawberry, a
SEPALLATA gene (SEP1/2; MADS-box) is needed for normal development
and ripening (Seymour et al., 2011). Similarly, in banana, which is classified
as a climacteric fruit, the MADS-box SEP3 gene also displays ripening-
related expression (Elitzur et al., 2010). In apple, MADS2 gene expression is
also associated with fruit firmness (Cevik et al., 2010), whereas in bilberry
fruit, the SQUAMOSA MADS-box ortholog of the TDR4 gene in tomato has
a role in regulation of anthocyanin biosynthesis (Jaakola et al., 2010).
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10.3.2 Auxins
Current knowledge about the role of hormones—other than ethylene—in
the development and ripening of climacteric and nonclimacteric fruits is
limited. In tomato, pepper, banana, muskmelon, and strawberry, the most
abundant free auxin, indole-3-acetic acid (IAA), has been reported to
decline prior to the onset of ripening; this reduction was accompanied by
an increase of its conjugated form (IAA-Asp) (Bottcher et al., 2010). The
conjugation reaction is catalyzed by the IAA-amino synthase gene (GH3).
In tomato, 15 members of the GH3 gene family have been described, but
only for 2 of them is the pattern of expression associated with ripening
(Kumar et al., 2012). Tomato fruits overexpressing the pepper GH3 gene
show anticipation of ripening (Liu et al., 2005), which is in agreement with
the view that the ratio between IAA and its AA-conjugated form, rather than
the level of IAA itself, may contribute to the temporal regulation of ripening
(Bottcher et al., 2010). In nonclimacteric fruits, no single growth regulator
appears to play a positive role analogous to that played by ethylene, but it
has been observed that auxin can negatively control the ripening of some
nonclimacteric fruits. In strawberry, it has been shown that the expression
of many ripening-specific genes can be downregulated by treatments with
an exogenous auxin. Also, in grape, auxin seems to play a negative role in
the regulation of ripening, with synthetic auxin treatments delaying the
expression of a number of ripening-related genes (Davies et al., 1997).
10.3.3 Gibberellins
As a consequence of the prominent role of auxin in the development and
ripening of some nonclimacteric fruits, little attention has been paid to pos-
sible roles of other plant hormones, such as gibberellins (GAs). However,
in strawberry, it has been reported that external application of GA 3 to
ripening fruits caused a significant delay in the development of the red
color (Martinez et al., 1996) and modified the expression of genes involved
in cell enlargement (de la Fuente et al., 2006) and cell wall disassembly
(Bustamante et al., 2009).
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the ripening process. For example, color change and fruit firmness are often
used as a surrogate for ripening, with other ripening characters completely
overlooked. It is becoming clear that some ripening traits are controlled inde-
pendently of each other (Johnston et al., 2009; Ireland et al., 2013). The use
of single physiological markers may hence lead to a misrepresentation of this
complex process. Here we have summarized the literature based on how dif-
ferent traits respond to hormones, rather than considering ripening as one
single process.
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Shelf life is important in tomato and other fruits. This trait is influ-
enced by many processes, including the rate of fruit softening. Modulation
of genes encoding two ripening-specific N-glycoprotein-modifying
enzymes—α-mannosidase and β-D-N acetyl hexosaminidase—reduces
the rate of softening and enhances fruit shelf life (Meli et al., 2010). More
than 50 cell wall structure-related genes show expression changes in
r ipening tomato fruits (Tomato Genome Consortium, 2012), and texture
involves complex quantitative trait loci (Chapman et al., 2012). Shelf life is
also related to water loss from the mature fruits.
Cuticle properties are critical for maintaining fruit integrity post-
harvest. Delayed Fruit Deterioration (DFD) mutant tomato fruits show
remarkably prolonged resistance to postharvest desiccation and remain
firm for many months after harvest (Saladie et al., 2007), and analyses of
cutin-deficient tomato mutants indicate the importance of waxes (Isaacson
et al., 2009).
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gallate. Cell, animal, and human studies have shown that green tea extract
and epicatechins prevent cancer development, particularly prostate can-
cer (Dixon and Ferreira, 2002). Similar effects may be gained by the
consumption of fleshy fruits such as cranberry, blueberry, and grape or by
the consumption of berry-juice products; these have relatively high levels
of catechins and epicatechins, which also protect against cardiovascular
disease (Dell’Agli et al., 2004).
Dietary intervention studies support the view that consumption
of fruit (especially when replacing calorie-dense food) helps maintain a
healthy weight and reduce the risk of a wide range of cancers (and car-
diovascular disease). Consequently, dietary improvement by increasing
fruit consumption or by improving fruit to contain higher levels of fiber or
phytonutrients should have a positive impact on the incidence and progres-
sion of chronic disease. Such improvement of fruit crops could be carried
out through genetic modification (Butelli et al., 2008) or marker-assisted
selection using existing variation.
Fresh fruits and their processed derivates represent important
sources of carotenoids, flavonoids, and anthocyanins, and the possibility
of improving content using bioengineering represents an opportunity to
improve access to healthy foods. The inherent resiliency of plant metab-
olism to maintain homeostasis has impaired efforts to facilitate changes
in some of these pathways, as has been especially well documented for
carotenoids (Fraser et al., 2009). An alternative approach is to focus on
regulatory rather than biosynthetic genes. Accumulation of carotenoids in
high-pigment fruit mutants has been reported to result from an increase in
the plastid number, larger plastid compartment size, increased plastid divi-
sion, and greater capacity for pigment storage (Galpaz et al., 2008; Wang
et al., 2009). These changes in pigmentation, plastid type, and metabolism
were associated with the elevation of transcripts from key carotenoid genes
such as phytoene synthetase 1 (Psy-1) and are not directly connected with
the ripening process (Fraser et al., 2007), thus allowing modifications
in carotenoid content without affecting other aspects of fruit ripening or
broader plant development. Moreover, this particular modification has
been reported to be stable in field tests for transgene stability and yield
performance (Giorio et al., 2007).
A MYB transcription factor from snapdragon was expressed using
the E8 promoter specifically in tomato fruit, causing elevated levels of
anthocyanins, compounds that have been shown to increase the longev-
ity of cancer-susceptible mice (Butelli et al., 2008). A homolog of this
transcription factor (MYB10) was previously cloned and characterized
from apple and was shown to positively regulate the anthocyanin pathway
(Espley et al., 2007). Further, an allelic rearrangement in the promoter of
MYB10 causes a novel autoregulatory motif, which is sufficient to account
for the increase in MYB10 levels and subsequent ectopic accumulation of
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that relate to fruit nutrient content (Cuevas et al., 2008), ripening, and
softening (Moreno et al., 2008). These tools make it possible to identify
potential candidate genes in model plants, like Arabidopsis, which could
lead to targeting important genes in crop species (Gorguet et al., 2008).
In one interesting example, tomatoes were biofortified with calcium by
the overexpression of an Arabidopsis calcium transporter (CA X1) (Park
et al., 2005). However, while the calcium levels were increased, the bio-
availability of the increased calcium was questioned. Similar experiments
were carried out, where the intake of calcium from transgenic carrots
overexpressing a CA X1 gene was monitored in both mice and humans
(Morris et al., 2008). Calcium absorption in bones increased for both
mice and humans eating CA X1-expressing carrots compared to controls,
demonstrating alternative means of fortifying fruits and vegetables with
bioavailable calcium.
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322
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323
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Chapter 11
Advances in Ethylene
Signal Transduction in
Fruits and Vegetables
Willis O. Owino1 and Jane Ambuko2
1Jomo Kenyatta University of Agriculture and
Technology, Nairobi, Kenya
2University of Nairobi, Nairobi, Kenya
Abstract 340
11.1 Introduction 340
11.2 Ethylene Physiology in Climacteric and
Nonclimacteric Fruits 341
11.3 Ethylene Signaling Components in Fruits 342
11.4 Analyses of Ethylene Signal Components in
Other Fruit Species 344
11.4.1 Climacteric Fruits 344
11.4.2 Nonclimacteric Fruits 348
11.5 Transcriptional Regulators of Fruit Ripening 349
11.6 Conclusions and Future Perspectives 352
References 353
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Abstract
Fruits and vegetables are a significant part of a healthy diet, as they are a
source of essential vitamins, minerals, fiber, and other health-promoting
compounds. The major challenges in improvement of these commodities is
the betterment of their health-promoting attributes while improving qual-
ity, postharvest shelf life, marketability, processing qualities, and consumer
appeal. To address these challenges, researchers have pursued strategies
of understanding key control points in global development regulation or
within specific processes in fruits and vegetables. This is informed by the
substantial insights into the mechanistic basis of ethylene biosynthesis,
perception, and signaling, and the identity of key regulators of ripening that
operate upstream of, or in concert with, regulatory pathways that mediate
the plant hormone ethylene. In this chapter, we highlight the contributions
made in our understanding of ethylene signaling, ethylene physiology,
and ethylene signaling components in both climacteric and nonclimac-
teric fruits. Tomato is the model system of choice for studying ethylene
signaling in fleshy fruit due to its commercial importance, a rich source of
genetic and biochemical information, relatively small genome, relative ease
of genetic transformation, and availability of developmental mutants that
are ripening impaired. Meanwhile, we look at how the translation of infor-
mation from tomato to other fleshy-fruited species indicates that ethylene
signaling and regulatory networks are conserved across a wide spectrum
of fruits and vegetables. We also discuss the ethylene additional regulatory
systems that coordinate the ethylene production. We finally summarize the
future perspectives of research in the ethylene signaling pathway.
11.1 Introduction
The phytohormone ethylene is a simple gaseous alkene that is a potent
modulator of proper plant development, growth, and survival (Bleecker
and Kende, 2000). The crucial role that ethylene plays in regulating mul-
tiple physiological and developmental processes in plants, such as leaf
and flower senescence, ripening, organ abscission, and growth transition,
and its involvement in the reactions of plants to abiotic and biotic stresses
are well documented (Chang and Bleecker, 2004; Seymour et al., 2013a).
Ethylene is physiologically active in trace amounts, and its effects are of
great commercial importance. To understand the roles of ethylene in plant
functions, it is important to know how this gaseous hormone is synthesized,
how its production is regulated, and how the signal is transduced.
Major breakthroughs in understanding how the ethylene signal is per-
ceived and transduced to mediate phenotypic responses have been made
340
ET H Y LENE SIGNAL TRANS D U C TION
341
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342
ET H Y LENE SIGNAL TRANS D U C TION
domains and a histidine kinase domain that lacks one or more elements that
are necessary for catalytic activity. The recently released tomato genome
sequence indicates that the seven ethylene receptors comprise the entire
inventory of tomato ethylene receptors.
The proteins encoded by these genes are structurally diverse and, at
worst, are less than 50% identical. The mRNA expression patterns of the
tomato ethylene receptors have been detected in all tissues analyzed, but
they present distinct expression patterns throughout development and in
response to differing environmental stimuli. The transcript levels of LeETR1
and LeETR2 are constitutive in all tomato tissues throughout development,
while NR, LeETR4, LeETR5, and LeETR6 exhibit increased levels in repro-
ductive tissues (flowers and fruit), with a significant increase of NR, LeETR4,
and LeETR6 in ripening fruits (Kevany et al., 2007). It is plausible that the
increased ethylene evolved at the onset of ripening in tomato is responsible
for the increased transcription of NR, LeETR4, and LeETR6 receptors, since
these three receptor genes are by far the most highly expressed in ripening
fruits. Together, these results indicate that subfamily II tomato receptors
may have far more important functions. The LeETR4, which has been dem-
onstrated to be a critical receptor for tomato fruit ripening, is multiply phos-
phorylated in vivo, and the phosphorylation level is dependent on ripening
stage and ethylene action (Kamiyoshihara et al., 2012).
The ethylene receptors transmit the signal to the downstream Raf
kinase–like protein, the constitutive triple response1 (CTR1) protein. The
subfamily I tomato receptors LeETR1, LeETR2, and NEVER-RIPE (NR)
can interact with multiple LeCTRs (1, 3, and 4) (Zhong et al., 2008). As
with the ethylene receptors, all tissues evaluated expressed CTR1 genes,
and their transcript differentially increased depending on tissue (Adams-
Phillips et al., 2004). The LeCTR1 is the highest expressed of the family
members in fruit, and its abundance is induced by exogenous ethylene appli-
cation and ripening. Genetic studies have illustrated that in the absence of
ethylene perception, the receptors repress ethylene responses by activat-
ing CTR1; binding of ethylene inactivates ethylene receptor signaling, and
CTR1 is consequently inactive, thereby leading to ethylene responses. The
molecular mechanism for how the receptors control CTR1 activity is yet to
be elucidated (Zhong and Chang, 2012).
The next component downstream of CTR1 is Ethylene Insensitive 2
(EIN2), a central positive regulator of ethylene responses residing in the
ER membrane (Bisson et al., 2009; Bisson and Groth, 2011). EIN2 encodes
a protein with similarity to the Nramp family of metal ion carriers and
for which loss-of-function mutations display ethylene insensitivity. EIN2
expression in tomato shows an increase at the onset of ripening (J. Wang
et al., 2007). The expression of LeEIN2 remains the same at different stages
of fruit development and is independent of ethylene. In addition to transcrip-
tional control of EIN2 mRNA accumulation, it is likely EIN2 is also a target
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
for protein turnover via the 26S proteosome. EIN2 is thought to play an
important role in the signal transduction of other hormones in addition to
ethylene, such as abscisic acid, auxin, cytokinin, and jasmonate, and thus
may represent a point of cross talk between multiple hormone signaling
pathways (Zhu et al., 2006; Cara and Giovannoni, 2008).
Downstream of EIN2, localized in the nucleus, is a family of short-lived
proteins that act as positive regulators of the ethylene signaling pathway
termed EIN3 and EIL (or EIN3-like) transcriptional regulators. Four EIL
genes have been cloned from tomato, and EIN3/EILs function as dimers,
and at least in the case of the tomato, EIL1, a mutation at a conserved phos-
phorylation site, disrupts fluorescence signal in a tobacco BiFC system, as
well as abolishes the activity of the respective trans-gene in tomato plants
(Li et al., 2012). EIN3/EILs are positive regulators of the ethylene signaling
pathway that act as trans-activating factors to trigger ethylene responses
mainly via the regulation of ethylene response factor (ERF) genes known
to be their downstream targets (Li et al., 2012).
The ethylene signaling cascade ends with a family of transcription
factors, the ERFs. ERF-type transcription factors are specific to plants and
belong to the APETALA2 (AP2)/ERF family. Proteins encoded by this
family have a highly conserved DNA binding domain known as the AP2
domain. The SlERF6 has been defined as a negative regulator of ethylene
and carotenoid biosynthesis in maturing tomato fruit (Lee et al., 2012).
Five tomato ERF genes (LeERF1–4 and LeERF3b) have been described to
date, and the transcript accumulation studies indicated a specific pattern of
expression for each gene, with LeERF2 (Sl-ERF2) and LeERF3b display-
ing ripening-associated transcript accumulation (Yang et al., 2010). While
LeERF2 expression increased during fruit ripening, LeERF3b accumu-
lated before and declined sharply after the onset of ripening. The LeERF2
transcripts were not detected in the tomato ripening mutants Nr, rin, and
nor. In contrast, LeERF3b mRNA is increased in low-ethylene tomato fruit
containing an ACC oxidase sense-suppression trans-gene and in Nr mutant
fruit (Chen et al., 2008).
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ET H Y LENE SIGNAL TRANS D U C TION
The melon fruit is second to tomato when it comes to research work car-
ried out on ethylene perception. Melon fruit is ideal for these studies due
to its development having three distinct stages, phases I–III. The flesh,
embryo, placenta, and seeds are well ordered; the fruit development can
be clearly divided into ethylene-insensitive and ethylene-sensitive stages;
and the developing fruit has a lower sensitivity to ethylene than does
the ripening fruit (reviewed in Ezura and Owino, 2007). In muskmelon,
Cm-ERS1 mRNA increased slightly in the pericarp of fruit during ripen-
ing, followed by the marked increase of Cm-ETR1 mRNA, which paral-
leled climacteric ethylene production. The increase of Cm-ERS1 mRNA
at a low concentration of ethylene before the increase of Cm-ETR1 mRNA
and ethylene production indicates that Cm-ERS1 may be sensitive to a
low concentration of ethylene, whereas Cm-ETR1 may be involved in
the response at a high concentration of ethylene. The melon receptor
Cm-ERS1 was determined to localize to the ER, and its topology is such
that it spans the membrane three times, with its N-terminus facing the
luminal space and the large C-terminal portion lying on the cytosolic side
of the ER membrane (Ma et al., 2006).
In mango MiETR1 mRNA level increased in both mesocarp and seed
tissues. However, in those same tissues, MiERS1 transcript accumulation
was barely detected (Ish-Shalom et al., 2011; Contreras-Vergara et al., 2012).
Three ethylene receptor genes, PeETR1, PeERS1, and PeERS2, have been
isolated from passion fruit. The PeETR1 and PeERS1 transcripts are more
elevated in arils than in seeds, while PeERS2 was detected only in the arils
of ripe purple fruit, suggesting that PeERS2 might play a role in repressing
ethylene responses at later developmental stages after fruit ripening has
been completed (Mita et al., 2002).
The avocado PA-ERS1 mRNA increased gradually from the day of har-
vest and did not change significantly until the climacteric peak, when it was
hyperinduced. 1-MCP, however, suppressed the accumulation of PA-ERS1
to basal levels, suggesting that the stimulated induction of PA-ERS at the
climacteric peak may be a mechanism by the avocado fruit to dissipate the
high levels of autocatalytic ethylene (Owino et al., 2002). The avocado seed
has been shown to be involved in the regulation of ethylene responsive-
ness during ripening and contributes to the delay of the climacteric peak in
mature seeded fruits (Hershkovitz et al., 2010). The seedless fruit ripened
more quickly, both at ambient temperature and in cold storage, than seeded
ones. The faster ripening was evident by an early onset of climacteric eth-
ylene production, and the expression of PaETR, PaERS1, and PaCTR1
increased in parallel with the onset of the ethylene burst in seedless fruit,
whereas PaETR increased predominantly in seeded ones. There were also
clear differences between seeded and seedless fruit in the mRNA accu-
mulation patterns of the PaCTR1 gene in response to endogenous or exog-
enous ethylene; PaCTR1 was dramatically induced by exogenous ethylene
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ET H Y LENE SIGNAL TRANS D U C TION
by cold and during ripening. Pc-ERS1a and Pc-ETR5 were less affected by
cold treatment, but all increased during post–cold treatment, ethylene-
dependent ripening (El-Sharkawy et al., 2003). A sharp peak of Pc-ETR1a
and Pc-ERS1a mRNA accumulation was observed during ripening in the
early season pears, in contrast to the gradual increase seen in ‘Passe-
Crassane’ (PC) fruit. A more pronounced difference between early cultivars
and PC fruit can be seen in the behavior of Pc-ETR5, a transcript accumula-
tion. Transcript levels for Pc-ETR5 diminish sharply before and during the
ethylene climacteric and ripening of early season pear fruit, whereas in PC
fruit they increase sharply. This suggests that a decrease in the expression
of a negative regulator could result in an increase in ethylene sensitivity
early in the ripening phase of early fruit development. However, given the
potential for redundancy in the ethylene receptor family, it remains to be
determined whether reduced levels of Pc-ETR5 affect the overall ethylene
sensitivity of early pear fruit (El-Sharkawy et al., 2003).
Three ethylene receptors, DkERS1, DkETR1, and DkETR2, have been
isolated and their expression determined during ripening of persimmon
fruit (Pang et al., 2007). The DkETR1 mRNA is constitutively expressed
during all stages of fruit ripening and is ethylene independent. Conversely,
DkERS1and DkETR1 mRNAs correlated with ethylene production during
fruit development and ripening and were induced by ethylene. The DkERS1
protein decreased gradually prior to fruit maturation and reached its lowest
level at the ripening stage, when ripening-related ethylene was produced,
suggesting the involvement of DkERS1 in ethylene perception during fruit
ripening.
Ethylene signaling elements have been isolated and characterized
in two Japanese plum cultivars, ‘Early Golden’ (EG), with normal cli-
macteric pattern, and ‘Shire’ (SH), with suppressed climacteric pattern
(El-Sharkawy et al., 2007, 2009). These included two receptors (ETR1-
like proteins Ps-ETR1 and Ps-ERS1), one CTR1-like protein (Ps-CTR1),
and an ethylene-responsive element binding factor (Ps-ERF). During the
early stages of development, all four ethylene components were downregu-
lated in an ethylene-independent manner. However, Ps-ETR1 and Ps-CTR1
transcripts accumulated during later stages in an ethylene-independent
manner. Ps-ETR1 was constitutively expressed during development, with
further increased expression during fruit ripening. Ps-ERS1 and Ps-ERF1
transcript levels increased sharply with the climacteric peak of EG fruit in
an ethylene-dependent manner. However, in the SH cultivar, the expression
was constitutive and low.
Five ethylene receptors (AdERS1a, AdERS1b, AdETR1, AdETR2, and
AdETR3), two CTR1-like genes (AdCTR1 and AdCTR2), and an EIN3-like
gene (AdEIL1) have been isolated from kiwifruit. The gene was found to be
fruit specific, and all were differentially expressed among various kiwifruit
tissues (Yin et al., 2008).
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
348
ET H Y LENE SIGNAL TRANS D U C TION
349
P OST H AR V EST RI P ENING P H Y SIOLOG Y
350
ET H Y LENE SIGNAL TRANS D U C TION
and SPBP transcription factors, respectively, and are necessary for ripen-
ing control (Manning et al., 2006).
Repression of TAGL1, MADS-RIN, CNR-SBP, or HB-1 is similar in
that all result in low-ethylene, nonripening fruit, suggesting that all four
genes operate upstream of ethylene biosynthesis and have some functions
that are ethylene independent. HB1 regulates climacteric ethylene through
direct regulation of ACO1, while TAGL1 regulates ethylene (either directly
or indirectly) through regulation of ACS2 with no effect on ACO1. CNR-SBP
mRNA accumulation is reduced in the rin mutant, suggesting that MADS-
RIN has a positive regulatory role on CNR-SBP expression (Vrebalov et al.,
2009). It is also noteworthy that (rin/Rin) hybrids form the basis for most
present-day production of slow-ripening, long-shelf-life, fresh-market toma-
toes demonstrating the commercial importance of the rin mutation.
Arlequin (Alq) is a semidominant T-DNA tomato mutant that exhibits
developmental changes affecting flower and fruit ripening. Genetic, molec-
ular, and functional analyses of the Alq T-DNA mutant indicated that it cor-
responds to the TAGL1 gene as a key MADS-box regulator of reproductive
development in tomato (Seymour et al., 2008; Giménez et al., 2010; Pineda
et al., 2010). Analysis of ALQ/TAGL1 demonstrated that it acts upstream
of ethylene-related genes, though independently to the ripening pathway
regulated by RIN, and regulates flower and fruit development, and there-
fore cannot be considered a specific fruit ripening gene. The ALQ/TAGL1
might act as a linking factor between flower development and fruit ripen-
ing networks, allowing the reproductive development to be successfully
completed.
Additional MADS-box genes have also been reported for other fruits;
however, most of them were suggested to be involved in early fruit devel-
opment. In peach, two MADS-box genes similar to TAG and TAGL1 have
been cloned, and their expression in tomato shows that they might be
responsible for fruit development (Tadiello et al., 2009). MADS-box genes
have also been isolated from Chinese pear, and there was no difference
in their expression in climacteric and nonclimacteric cultivars (Yamane
et al., 2007). Whether or not these genes are specifically involved in rip-
ening remains to be determined in these fruits. In ‘Grand Nain’ bananas,
MaMADS2 is the most likely candidate that serves as an upstream regu-
lator of ripening, since it is not affected by ethylene (Elitzur et al., 2010).
MuMADS1 and a ubiquitin-activating enzyme E1 gene fragment (MuUBA)
were co-regulated by ethylene, suggesting that it might play an important
role in postharvest banana fruit ripening (Liu et al., 2013).
An AGAMOUSMADS-box protein predominantly accumulated in
the climacteric banana fruit pulp and also in female flower ovary suggests
involvement in banana fruit ripening and floral reproductive organ devel-
opment (Roy Choudhury et al., 2012). A ripening-related homolog of the
tomato RIN gene has been isolated in strawberry, which increases neither
351
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352
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358
Chapter 12
Internal Atmosphere
of Fruits: Role and
Significance in
Ripening and
Storability
Vijay Paul and Rakesh Pandey
Indian Agricultural Research Institute,
New Delhi, India
Abstract 360
12.1 Introduction 361
12.2 Endogenous Volatiles in Fruits 363
12.3 Factors Affecting and Basis of Internal Atmosphere
of Harvested Fruit 363
12.4 Variability in the Internal Atmosphere of Fruits 366
12.5 Influence of the Internal Atmosphere on Ripening
and Related Aspects 367
12.5.1 Ripening 367
12.5.2 Flavor and Aroma 368
12.5.3 Fruit Decay 370
359
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Abstract
Ripening, storability, quality attributes, and postharvest losses in fruits
are interlinked with one another. The postharvest life of a fruit is primarily
determined by various physiological processes and associated metabolic
changes occurring in the fruit. The role of the external atmosphere in reg-
ulating the above processes and changes is relatively better understood.
However, little is known about the overall internal atmosphere of the fruit
and how it influences different aspects of ripening and storability. This
chapter looks into this emerging area: the basic and applied importance
of the internal atmosphere to postharvest physiology and food science and
technology. There are various gases and volatiles that make the internal
atmosphere of fruits. Their production and diffusion across the fruit tissues
360
INTERNAL ATMOS P H ERE O F F RUITS
12.1 Introduction
Postharvest physiology, shelf life, and losses in fruits are interlinked and
primarily governed by the last phase of fruit maturation, referred to as
ripening. Fruit ripening involves many physiological, biochemical, and
developmental changes occurring through a coordinated and genetically
regulated program (Stepanova and Alonso, 2005; Barry and Giovannoni,
2007; Bouzayen et al., 2010). Fruits, in general, show two distinctive respi-
ratory patterns during the course of ripening, and on this basis, fruits
are categorized into climacteric and nonclimacteric groups (Biale, 1964;
McMurchie et al., 1972; Biale and Young, 1981; Abeles et al., 1992; Paul
et al., 2012). Climacteric fruits show a dramatic increase in the rate of res-
piration during ripening, and this is referred to as climacteric rise. The
rise in respiration is simultaneous, or it is just followed after the rise in
the rate of ethylene production (Burg and Burg, 1962, 1965a; Lelievre
et al., 1997). The process of ripening can be triggered and also acceler-
ated by exogenous ethylene treatment in climacteric fruits (Tucker, 1993).
The plant hormone ethylene plays a major role in the ripening process
of climacteric fruits, and the presence of ethylene and its perception is
required for the expression of ripening-related genes, even at advance
stages of fruit ripening (Alexander and Grierson, 2002; Hoeberichts et al.,
2002). On the other hand, respiration rates increase at least temporarily
361
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362
INTERNAL ATMOS P H ERE O F F RUITS
363
P OST H AR V EST RI P ENING P H Y SIOLOG Y
There are three major routes through which gaseous exchange takes
place for a harvested commodity: the outermost layers (cuticle, cuticular
cracks, and periderm), apertures (stomata and lenticels), and stem scar
region (Solomos, 1987; Ben-Yehoshua and Rodov, 2003). In view of the pres-
ence of cracks on the cuticular layer, the exchange of gases and volatiles
was reported through the cuticular layer, and it is primarily determined
by the extent of cracks present on cuticular layer (Ehret et al., 1993). For
most of horticultural produce, the skin represents the major barrier to
gas exchange (Kader, 1987; Solomos, 1987). The diffusivity of gases for
the fruit flesh is 10–20 times higher than the diffusivity from the skin
(Solomos, 1987; Banks and Nicholson, 2000). In tomato, the stem scar (the
place where the pedicel, along with sepals, connects the fruits to the stem)
is the predominant site for gas exchange (Cameron and Yang, 1982). It was
also demonstrated that 85%–90% of ethylene exchange occurs through this
region of tomato fruit (de Vries et al., 1995). The release of volatiles from
the plant organs with lower rates of transpiration (such as bulky fruits with
well-developed diffusion barriers on the surface, peel, or pericarp) will
be less, and therefore the volatiles can accumulate in the tissues of such
organs. Keeping in view the varietal variation in diffusion barriers and their
characteristics for different plants and plant parts, the accumulation of vola-
tiles would be different, and thereby so would their effect on various physi-
ological processes (Paul et al., 2011; Paul and Pandey, 2014).
Gas diffusion in fruits follows Fick’s law (Burg and Burg, 1965b),
which states that the flux of a gas, diffusing normally through a barrier, is
dependent on the diffusion coefficient and concentration gradient. There
is a morphological and anatomical basis for the gaseous exchange across
the boundaries of harvested fruits (Kader and Morris, 1977; Solomos, 1987;
Kader and Saltveit, 2003a, 2003b). As per Kader and Morris (1977), anatom-
ical features, and not any alterations in biochemical pathways, are the rea-
sons for differences in the diffusion resistance. External (morphological)
and internal (anatomical) features were reported to determine the keep-
ing quality of grapes (Uys, 1974), storability of tomato berries (Niemirow-
Krizsai and Csillag, 1994; Mulas, 1994), and firmness or mechanical
resistance of tomato (Wann, 1996) and olive fruits (Mulas, 1994). In a
study by Bai et al. (2003), it was shown that ‘Granny Smith’ apples have
relatively few open pores (lenticels) in their peel surface, and therefore they
suffer from low rates of gas exchange; as a result, these fruits are prone
to develop off-flavors after they have been coated with waxes. In contrast,
‘Delicious’ apples have many open lenticels, and they retain sufficient gas
exchange even when coated with waxes. So, waxed ‘Delicious’ apples accu-
mulate only low concentrations of ethanol and off-flavors (Bai et al., 2003).
Likewise, it was reported that Murcott mandarins are much more sensi-
tive to anaerobic stress conditions than Star Ruby grapefruit (Shi et al.,
2005). Anatomical observations revealed that although the total thickness
364
INTERNAL ATMOS P H ERE O F F RUITS
of the peel (comprising the albedo, the white inner layer, and the flavedo
[the c olored outer layer]) was greater in grapefruit, it was more condensed
in mandarins. So, it was concluded that mandarins accumulate a larger
amount of acetaldehyde and ethanol after harvest than grapefruit because
of the higher activity of the enzyme alcohol dehydrogenase (ADH) in the
juice vesicles and lower permeability of their peel to gases (Shi et al., 2007).
The extent of diffusibility of gases across the fruit boundaries primar-
ily determines the internal atmosphere of the fruit in terms of the levels of
O2 and CO2 (Ben-Yehoshua et al., 1983; Nuevo et al., 1984). So, the composi-
tion of the internal atmosphere of the fruit is always different from that of the
external atmosphere in which it is kept (Dadzie et al., 1993). The exchange
of gases in fruits through the peel via diffusion from the openings (stomata
and lenticels) is proportional to the difference in the concentrations of gases
across the barrier, total area of the peel, solubility of a gas in peel, solid-
state diffusion coefficient, and total hole area available on the peel surface
(as contributed by openings of stomata and lenticels) (Hagenmaier, 2004,
2005). Gas transport in fruit tissue is governed by diffusion as well as by
permeation. The permeation is basically caused by an overall pressure gra-
dient of a given gas (Ho et al., 2006a). So, a permeation–diffusion–reaction
model was applied to study gas transport in an intact pear. Permeation was
found to be at a minimum across skin, and it gradually increased for cortex
and vasculature tissues of pear fruit (Ho et al., 2006a).
To a large extent, gas exchange depends on the arrangement pattern
of cells and intercellular spaces (Mendoza et al., 2007). For pear fruit, gas
diffusibility in the vertical axis was higher than that in the equatorial radius
axis (Ho et al., 2006b). In view of this, a very comprehensive model of gas
exchange of pear fruit was proposed for explaining the development of phys-
iological disorder, such as core breakdown and its role in long-term storage
of this fruit (Franck et al., 2007). Later on, Verboven et al. (2008) used high-
resolution phase tomography (making use of synchrotron radiation) to
explore the three-dimensional (3-D) structure and cellular arrangements
of pome fruit tissues in their natural state (i.e., with high water content) up
to a submicrometer resolution. For this study, pome fruits such as apple and
pear were selected because their gas exchange properties have been shown
to be very different and closely related to their storage lives (Schotsmans
et al., 2004; Ho et al., 2006a, 2006b; Franck et al., 2007; Verboven et al.,
2008; Ho et al., 2008). Results obtained (Verboven et al., 2008) revealed
very clearly that the apple fruit had more voids than the pear. The differ-
ences in void fraction (23% for apple cortex and only 5% for pear cortex),
along with the extent of network architectures of voids, explained the bet-
ter ability of tissues to facilitate the gas exchange in apple fruit. This lower
void volume in pear fruit than in apples (Verboven et al., 2008) was able to
explain the earlier findings where pear fruit was found to be more sensi-
tive to physiological disorder, such as internal browning and its relation to
365
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366
INTERNAL ATMOS P H ERE O F F RUITS
the growth and maturity of the fruit. The reason for this may be the changes
that occur in the anatomical properties and features of the fruit itself during
its development and maturation (Longhurst et al., 1994; Kader and Saltveit,
2003a; Bargel and Neinhuis, 2005; Paul et al., 2007; Ho et al., 2008).
Wide differences in the concentrations of volatiles were recorded
in different varieties of apricot (Guichad and Souty, 1988). The levels and
proportions of different volatiles were also found to be responsible for the
characteristic differences in flavor among the varieties of apricot (Guichad
and Souty, 1988). Similarly, Larsen and Poll (1990) found differences in fla-
vor among the 10 raspberry cultivars due to variation in the production of
aroma volatiles. In apple cultivars, wide differences in susceptibility to scald
were associated with the α-farnesene content (Huelin and Coggiola, 1968;
Watkins et al., 1993). Gran and Beaudry (1993) observed wide variability in
the threshold levels of oxygen required (at 0°C) for the induction of anaero-
bic respiration in three apple varieties: 0.7% for ‘Red Delicious’, 1.4% for ‘Red
Fuji’, and about 1.9% for ‘McIntosh’. In this way, variety and storage condi-
tion can influence the degree of accumulation of products of anaerobic res-
piration, such as acetaldehyde and ethanol in tissues. Likewise, out of two
varieties of raspberries (‘Meeker’ and ‘Qualicum’), when harvested at the
red-ripe stage and stored at 1°C with 90% relative humidity (RH) for 7 days,
the variety ‘Qualicum’ was found to be more susceptible to the accumulated
acetaldehyde, ethanol, and ethylacetate in the MAP, with high CO2 (i.e.,
10% CO2) with 5% O2 in comparison with other MAP conditions (6% CO2
with 10% O2 and 3% CO2 with 15% O2) (Toivonen et al., 1999). In tomato fruit,
differences in flavor among different varieties were in part due to the varia-
tion in production levels of aroma volatiles (Brauss et al., 1998). For mango
fruits, volatiles (in different varieties and at different maturity stages) have
been used as a marker for the identification of different maturity stages
in different varieties. So, volatiles can thereby be used in determining the
most optimum maturity stage for harvesting the mango fruits, as this can
result in attaining the best quality of harvested fruits on ripening (Lebrun
et al., 2008; Pandit et al., 2009). In another study, discrimination of 28 apri-
cot cultivars into four distinguishable aroma groups was achieved by ana-
lyzing their volatile constituents (Aubert and Chanforan, 2007).
12.5.1 Ripening
During the ripening of tomato fruits, a rise in the endogenous concentrations
of CO2 and ethylene was reported, along with a decrease in the concentration
of O2 (Lyons and Pratt, 1964). The significance of the stem scar region as
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
a major site for gaseous exchange of tomato fruit was pointed out by Calbo
et al. (1988) and Yang and Shewfelt (1999). The relationship between ripen-
ing behavior and the stem scar region of tomato fruit in different varieties
was studied by blocking the stem scar region either completely or to different
degrees (Paul et al., 2010b). It was made clear from this study that it is the
degree of blockage of the stem scar region that determines the extent to which
the rate of respiration and ripening are suppressed. In the above studies, the
basic change causing the delay in ripening or suppression of respiration was
primarily due to the lower levels of ethylene and the O2 -to-CO2 ratio within
the fruit. Toivonen (1997) had reviewed the accumulation of nonethylene and
nonrespiratory volatiles (alcohols, aldehydes, jasmonates, terpenes, carbox-
ylic acids, sulfur compounds, and ammonium) and discussed them in terms of
their biological activity in harvested fruits and vegetables. It was pointed out
that besides removing the ethylene, ethylene removing and absorbing agents
can also remove other organic volatiles from the storage or package atmo-
sphere (Toivonen, 1997). Therefore, at least some effects that were attributed
to the removal of ethylene may in fact be related to the removal of other vola-
tiles that have not been measured or identified. So, there is a strong reason
to evaluate the potential role and significance of volatiles other than ethylene
and their interaction with ethylene in postharvest situations and under differ-
ent storage conditions (Lougheed et al., 1987; Toivonen, 1997). Besides ethyl-
ene, the role of volatiles such as alcohols (mainly ethanol), aldehydes (mainly
acetaldehyde), and methanol has been demonstrated in the ripening of climac-
teric fruits such as tomato (Kelly and Saltveit, 1988; Saltveit and Sharaf, 1992;
McDonald et al., 1996) and apple (Pesis et al., 1994; Pesis, 1995). Besides this,
levels of volatiles are also found to be associated with storage disorders of
apple fruit, such as scald (Huelin and Coggiola, 1968) and internal browning
(Mendoza et al., 2007). The ripening and quality of nonclimacteric fruits such
as grapes, oranges, and strawberries were also influenced by these volatiles
(Saltveit and Ballinger, 1983; Ke and Kader, 1990; Ke et al., 1991).
368
INTERNAL ATMOS P H ERE O F F RUITS
(Continued )
369
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Sources: Salunkhe, D.K., and Do, J.Y., Critical Reviews in Food Science and
Nutrition 8, 161–190, 1977; Tandon, K.S. et al., Postharvest Biology
and Technology 20, 261–268, 2000; Tandon, K.S. et al., Proceedings
of the Florida State Horticulture Society 114, 142–144, 2001;
Lewinsohn, E. et al., Plant Physiology 127, 1256–1265, 2001; Baldwin,
E.A., in The Commercial Storage of Fruits, Vegetables and Florist and
Nursery Stocks, Gross, K.C. et al. (eds.), Agriculture Handbook Number
66, Agricultural Research Service, Beltsville, MD, 2004, 18. http://
usna.usda.gov/hb66/023flavor.pdf. Hui, Y.H. (ed.), Handbook of Fruit
and Vegetable Flavors, John Wiley & Sons, Hoboken, NJ, 2010.
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INTERNAL ATMOS P H ERE O F F RUITS
12.6.1 Tomato
Ethylene concentrations were unusually high in attached tomato fruits
(ranging from 2 to 13 µl L –1, depending on cultivars and seasonal condi-
tions), while in detached fruits the values drop to a level of 1.4 µl L –1 on an
average (Sawamura et al., 1978). Detachment of tomato fruit advances the
ripening and reduces the threshold value of endogenous ethylene required
to promote the ripening to a considerably lower level. This supports the
concept of a hypothetical labile ripening inhibitor that is translocated from
vegetative parts of the plant to the fruits, where it antagonizes the action on
ethylene (Sawamura et al., 1978). This concept might be true, but the large
decrease in the levels of ethylene in detached fruits may be explained by
more effective gaseous exchange from the stem scar region of harvested
tomato fruit as a result of detachment of the pedicel. This explanation is
supported by de Vries et al. (1995), who reported that 85%–90% of ethylene
is released by tomato fruit through this stem scar region.
There are various factors that affect the production and release of ethyl-
ene by fruit. In this regard, a theory of ethylene emission was developed and
used as the basis to develop the simulation model called ETHY by Genard
and Gouble (2005). This model was found to be highly sensitive to factors
such as the permeability of the fruit skin; internal concentrations of O2, CO2,
and ACC; changes in fruit growth and temperature; activities of ACC-oxidase
and ACC-synthase; concentration of the ethylene itself; and production sta-
tus of ATP. Similarly, a model has been proposed for gas exchange in pear
fruit (Ho et al., 2008). The role of surface morphology and the stem scar
region in determining the ripening behavior, rate of respiration, and water
371
P OST H AR V EST RI P ENING P H Y SIOLOG Y
loss by attached and detached tomato fruits was pointed out, along with a
variety-dependent increase in the density of lenticels with the progress of
ripening (Paul and Srivastava, 2006). Further, it is interesting to note that
the extent of the 1-methylcyclopropene (1-MCP)-mediated delay in ripening
depends on the endogenous level of ethylene in the tomato fruits (Zhang
et al., 2009; Paul et al., 2010a; Paul and Pandey, 2013).
12.6.2 Melons
Several cultivated cultivars and wild ecotypes of melons exhibit a climac-
teric pattern of ripening that is linked with the detachment of the fruit
from its parent plant (Abeles et al., 1992). There are variations in the mag-
nitude and duration of the respiratory rate, depending on the cultivars in
the harvested melon fruits. However, several melon cultivars that appar-
ently do not abscise show no burst in ethylene production and display a
long shelf life. These melon fruits emit little or no ethylene, and therefore
behave like nonclimacteric fruit (Shiomi et al., 1999). It has been reported
that the r ipening-associated increase in ethylene production is present,
but the respiratory climacteric is absent during ripening of melon fruit
attached to the plant, leading to the suggestion that climacteric respira-
tion is an artifact of harvest. To address the universality of this phenom-
enon, the ripening behavior in the melon cultivar ‘Charentais’ (Cucumis
melo cv. ‘Reticulatus F1 Alpha’) was investigated. The results, however,
showed that the respiratory climacteric occurs in fruit ripened both on
and off the plant (Hadfield et al., 1995). The sensitivity to ethylene in rela-
tion to the climacteric respiration is affected by detachment of the fruit
(Bower et al., 2002). In an another study, application of ethylene to anti-
sense ACC-oxidase melons stimulated O2 consumption only if they were
detached from the vine, showing that attachment to the plant inhibits the
effect of ethylene on respiration (Pech et al., 2008). This effect of detach-
ment on sensitivity to ethylene is known as the tree factor by posthar-
vest physiologists, as discussed previously in Section 12.6.1. Auxin has
been suggested to be a candidate, but experimental evidence is still lack-
ing (Pech et al., 2008). In muskmelon, the detached fruit also produced
a burst in respiration and ethylene, but attached fruits did not exhibit a
large increase in respiration in spite of the significant increase in the lev-
els of ethylene (Shellie and Saltveit, 1993).
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INTERNAL ATMOS P H ERE O F F RUITS
12.6.4 Saskatoon
Comparison of respiration and ethylene production rates at nine differ-
ent maturity stages of saskatoon (Amelanchier alnifolia Nutt.) fruits after
their harvest, with the fruits undergoing maturation and ripening on the
plant itself, indicated that the respiratory response was very different in
these two situations. The increase in respiration associated with ripening
was demonstrated only on a whole-fruit basis and only in attached fruits
(Rogiers and Knowles, 1999).
Based on the above information, it could be concluded that cultivar-
dependent variations in the ripening behavior of fruits can also be due to
the differences in their internal gaseous environment. It appears that there
are various factors that contribute differentially to determining the ripen-
ing behavior of attached and detached fruits. Such factors include differ-
ences in the production and release of ethylene, and thereby endogenous
levels of ethylene in the fruit; differences in the sensitivity of fruit to the
endogenous levels of ethylene; differences in the threshold level of ethylene
required to initiate the process of fruit ripening; and overall differences in
the endogenous gaseous microenvironment of fruits. In addition to this,
variability among varieties and cultivars in these factors could also contrib-
ute to the differences in ripening behavior. In view of these reasons, the role
of the above-listed factors was pointed out by Paul et al. (2012) in explaining
the deviations from the classical pattern of climacteric and nonclimacteric
ripening in different fruits.
12.7.1 Ethylene
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
many fruits. As stated earlier, this plant hormone plays a major role in
the r ipening process of climacteric fruits (Nagata et al., 1995; Klee, 2002;
Bouzayen et al., 2010; Paul et al., 2011; Paul and Pandey, 2013, 2014). The
production of aroma during the ripening of different fruits also depends
strongly on the production and action of ethylene (Golding et al., 1999;
Rupasinghe et al., 2000; Lurie et al., 2002; Flores et al., 2002; Dandekar
et al., 2004; Pech et al., 2008; Defilippi et al., 2009).
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INTERNAL ATMOS P H ERE O F F RUITS
Methionine
SAM-synthetase
S-Adenosyl methionine
_ +
ACC-synthase (ACS)
1-Aminocyclopropane-1-carboxylic acid
+ +
ACC-oxidase (ACO)
Ethylene
response is suppressed in
1-MCP _
presence of 1-MCP
Ethylene-mediated
Response
(Ripening and senescence, etc.)
375
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Table 12.2 Relative Activity of Ethylene and Its Analogs in Pea Straight
Growth Bioassay Test
ppm (µl L–1) in Gas Phase for
Gases Formula Half-Maximum Activity
Ethylene C2H4 0.1
Propylene C3H6 10
Vinyl chloride C2H3Cl 140
Carbon monoxide CO 270
Acetylene C2H2 280
Vinyl fluoride C2H3F 430
Methylacetylene (propyne) C3H4 800
Vinyl bromide C2H3Br 1,600
Allene (propadiene) C3H4 2,900
Vinyl methyl ether C3H6O 10,000
Ethylacetylene (1-butyne) C4H6 11,000
1-Butene C4H8 27,000
Vinyl ethyl ether C4H8O 30,000
Carbon dioxide CO2 30,000
1,3-Butadiene C4H6 500,000
Sources: Abeles, F.B. et al., Ethylene in Plant Biology, 2nd ed., vol. 15, Academic
Press, San Diego, 1992; Burg, S.P., and Burg, E.A., Physiologia Plantarum
18, 870–886, 1965; Abeles, F.B., and Gahagan, H.E., Plant Physiology
43, 1255–1258, 1968.
376
INTERNAL ATMOS P H ERE O F F RUITS
377
P OST H AR V EST RI P ENING P H Y SIOLOG Y
378
INTERNAL ATMOS P H ERE O F F RUITS
379
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Epidermis
Mesocarp
Seed cavity
7 22
6 21
5 20
% Carbon dioxide
4 19
% Oxygen
3 18
2 17
1 16
0
Distance
Figure 12.2 Cross section passing through a fruit showing how the con-
centration of O2 and CO2 can vary within different tissues of the fruit due
to respiration and external and internal barriers to gas diffusion. (From
Kader, A.A., and Saltveit, M.E., in Postharvest Physiology and Pathology of
Vegetables, Bartz, J.A., and Brecht, J.K. (eds.), New York, Marcel Dekker,
2003, 229–246.)
380
INTERNAL ATMOS P H ERE O F F RUITS
L-Methionine
SAM-synthetase
SAM
Low concentration of
+
SA/MSA/JAs, water stress
NO ACC-synthase
– > CO2, ethanol/acetaldehyde,
high concentration of SA/MSA/JAs
ACC
O2, < CO2, low concentration of
+
NO ACC-oxidase JAs, water stress
Ethylene, low
+
Auto-induced concentration of SA/MSA
NO Ethylene ethylene
production – > CO2, ethanol/acetaldehyde,
NO, high concentration of
SA/MSA/JAs
NO + Ethylene
< O2/CO2, hypoxia, anoxia, ethanol/
–
acetaldehyde (concentration dependent)
Figure 12.3 Different endogenous volatiles and other factors that play a
regulatory role in determining the production and response of ethylene. The
symbols + , − , , >, and < indicate inducers, suppressors, higher, lower,
and possible interactions, respectively. SAM, S-adenosyl-L-methionine; ACC,
1-amino-cyclopropane-1-carboxylic acid; SA, salicylic acid; MSA, methyl
salicylic acid; JAs, jasmonates; NO, nitric oxide.
switch to fermentative metabolism. This O2 level has been called the anaer-
obic compensation point (ACP) (Leshuk and Saltveit, 1990). The ACP may
vary for different fruits and for the same fruit at different maturities and at
different storage temperatures, besides being affected by different variet-
ies of a given fruit (Gran and Beaudry, 1993; Kubo et al., 1996; Borisjuk
et al., 2007).
The susceptibility of apple fruit to low O2 injury in CA storage was
found to be positively correlated with the resistance of the fruit to the dif-
fusion of gases. For a given strain or cultivar, resistance to gas diffusion
was found to be affected by the fruit’s maturity, duration of storage, and
whole-fruit volume (Park et al., 1993). It was noticed that the ‘Marshall’
strain of McIntosh apple had higher resistance, and thereby it showed more
susceptibility to low O2 injury, as this strain accumulated 10 times more
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
ethanol than ‘Rogers’ (another strain of McIntosh apple) (Park et al., 1993).
This demonstrates the extent of varietal variability among the fruits for
response toward their internal atmosphere, especially for the lower con-
centrations of O2 .
Modified atmospheres with low concentrations of O2 can slow down
the deterioration of fruits by decreasing respiration, ethylene production,
and tissue sensitivity to the ethylene (Kader et al., 1989). The extent of
decrease in ethylene production therefore depends not only on the O2 con-
centration that is present in the fruit’s internal atmosphere, but also on the
sensitivity of the ethylene production system under a prevailing concen-
tration of O2. Sanders and de Wild (2003) reported a lower partial pres-
sure of O2 (lower than the external partial pressure of O2) due to the rapid
consumption of O2 by the tomato fruit and higher resistance of fruit to the
diffusion of O2. Reduced O2 or elevated CO2 decreased the respiration rate
(Smith et al., 1987). In bulky storage organs such as fruits (where the length
of the diffusion path may be considerable), hypoxia conditions have been
demonstrated (Ho et al., 2008). So, low O2 stress may occur within the fruit
as it grows, and the resistance to the entry of O2 from the atmosphere into
the fruit (via the diffusion process through the skin and thickened cell lay-
ers of the cortex) becomes significant. In avocado fruit, synthesis of cellu-
lase and polygalacturonase was found to be directly related to the levels of
O2 (Knee, 1982; Kanellis et al., 1991).
382
INTERNAL ATMOS P H ERE O F F RUITS
383
P OST H AR V EST RI P ENING P H Y SIOLOG Y
384
INTERNAL ATMOS P H ERE O F F RUITS
of ethylene, but its action as well (Saltveit and Mencarelli, 1988; Ritenour
et al., 1997; Pesis, 2005; Asoda et al., 2009). Ethanol was also reported to
delay ripening as well as the production of CO2, loss of chlorophyll, and
synthesis of lycopene (Kelly and Saltveit, 1988; Yang and Shewfelt, 1999;
Podd et al., 2002).
It has been proposed that the ethanol-mediated delay in ripening is
basically caused by acetaldehyde. Acetaldehyde is produced by conver-
sion of ethanol to acetaldehyde via the reversible reaction catalyzed by the
enzyme ADH (Beaulieu et al., 1997; Podd et al., 2002). Both climacteric
and nonclimacteric fruits produce a lot of acetaldehyde and ethanol (Pesis,
2005). For a given fruit, genetic variability was also seen in the levels of
production of acetaldehyde and ethanol and in the ability to survive under
anaerobiosis (Pesis, 2005). Acetaldehyde inhibits the formation of ethylene
by preventing the action of ACC-synthase and action and synthesis of ACC-
oxidase (Pesis et al., 1998; Podd et al., 1998). Exogenous ethanol application
resulted in a marked increase in acetaldehyde levels, and this inhibited the
ethylene production and ripening of tomato fruits (Pesis and Marinansky,
1993). It was therefore suggested that acetaldehyde is the causal agent for
ethanol-induced inhibition of fruit ripening (Beaulieu et al., 1997). In light
of the above findings, it was concluded by Pesis (2005) that ethanol and
acetaldehyde are natural compounds that are essential in governing the
process of fruit ripening. These compounds are also associated with aroma
production and the removal of astringency. Various sites where acetalde-
hyde and ethanol can regulate the production and response of ethylene are
presented in Figure 12.3.
385
P OST H AR V EST RI P ENING P H Y SIOLOG Y
386
INTERNAL ATMOS P H ERE O F F RUITS
387
P OST H AR V EST RI P ENING P H Y SIOLOG Y
81 genes were found to show elevation (by more than threefold) in response
to MJ in wild-type tomato in comparison to nor mutant (Imanishi et al.,
2005). The results indicate a strong interaction of jasmonate and ethylene,
and in this way, the endogenous status of jasmonate could regulate the rip-
ening and ripening-related changes. In a study by Ziosi et al. (2007), exog-
enous application of JAs led to alterations in ethylene biosynthesis, ethylene
perception, and polyamine accumulation.
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INTERNAL ATMOS P H ERE O F F RUITS
predict the gaseous concentration in fruits because fruit tissue does not rep-
resent a continuum. Permeability therefore changes in a dynamic way with
metabolic activities of tissues, developmental stage, maturity, and storage
time. Besides this, the external factors, such as the temperature, relative
humidity, and gaseous composition of the immediate storage environment,
also affect the permeability within and across the fruit. Several researchers
have described different geometrical models with mathematical equations
that also take into the consideration the tissue microstructures, besides
other factors (Verboven et al., 2008; Ho et al., 2008, 2009, 2010; Steindel
et al., 2005).
Developments in the field of plant volatiles and their roles showed that
the quality and quantity of volatiles in plants and plant parts are linked to
various types of abiotic and biotic stresses (Steindel et al., 2005; Karl et al.,
2008; Loreto and Schnitzler, 2010). Some specific examples are hexanal,
trans-2-hexenal, and hexylacetate, which improve the safety of freshly sliced
apple (Lanciotti et al., 2003); trans-2-hexenal, which confers resistance to
anthracnose fruit decay in the highbush blueberry (Polashock et al., 2007);
hexanal, which reduces infection of tomatoes by Botrytis cinerea (Utto
et al., 2008); α-farnesene, which increases the susceptibility of apple fruits
to scald disorder (Watkins et al., 1993); different endogenous volatiles (alde-
hydes, alcohols, and esters) and trans-2-hexenal, which exhibit antifungal
properties against Colletotrichum acutatum, which causes anthracnose in
strawberry fruits (Arroyo et al., 2007); several terpenoids (sesquiterpenes
such as zingiberene and curcumene and monoterpenes such as p-cymene,
α-terpinene, and α-phellandrene), which strongly repel whiteflies that
infest tomatoes (Bleeker et al., 2009); and isoprene, which is reported to
provide thermotolerance in Arabidopsis (Sasaki et al., 2007)—however, its
role in fruits is yet to be studied. Strawberries treated with MJ alone or in
combination with ethanol showed higher antioxidant capacity, total pheno-
lics, and anthocyanins, along with longer postharvest life and better quality
and aromatic properties (Ayala-Zavala et al., 2005). These examples show
that at least some of the volatiles in the internal atmosphere of fruit can
provide protection against biotic and abiotic stresses during ripening and
storage and reflect potential practical applications in the overall posthar-
vest management of fruits.
Today, efforts are being made for the more effective use of some gases,
such as carbon monoxide (CO), nitrous oxide (N2O), nitrogen (N2), sulfur
dioxide (SO2), and chlorine dioxide (ClO2), in the storage environment
to reduce the microbial, insect, and pest infestation. Besides this, ozone
(O3) gas is also being exploited in delaying the ripening and improving the
quality of the stored fruits (Scully and Horsham, 2008; Hoehn et al., 2009;
Zambre et al., 2010; Rodoni et al., 2010). Ozone-treated fruits are reported
to show a delay in ripening and decay, the proper maintenance of quality,
suppression in microbial contamination, and a reduction of the levels of
389
P OST H AR V EST RI P ENING P H Y SIOLOG Y
ethylene in the storage environment and within the fruits as well (Xu, 1999;
Zambre et al., 2010; Rodoni et al., 2010). Exposure of O3 also reduced the
levels of pesticide in stored apples (Ong et al., 1996). Besides the practical
applicability of the above gases, required levels of O2, CO2, ethylene, humid-
ity, and condensation in the storage environment can also delay ripening
and improve the quality and storability of fruits (Mangaraj and Goswami,
2009; Hoehn et al., 2009; Yahia, 2009).
There are different endogenous gases and volatiles that play a
regulatory role in determining the production and response of ethylene
(Figure 12.3). Besides this, interactions of some of the volatiles, such as
C2H4, O2, CO2, SA/MeSA, JAs, and NO, on various components of the respi-
ratory metabolism were also reported (Ederli et al., 2006; Leakey et al.,
2009; Wang et al., 2010). This shows that different gases and volatiles can
affect the status and response of ethylene, along with a definite influence
on respiration, and this in turn is closely linked with the potential shelf life
of fruits after their harvest (Kader and Saltveit, 2003b; Paul and Srivastava,
2006; Paul et al., 2010b; Varoquaux and Ozdemir, 2005).
390
INTERNAL ATMOS P H ERE O F F RUITS
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Chapter 13
Proteomics of
Fruit Development
and Ripening
Aloysius Wong,1 Ludivine Thomas,1
Christoph Gehring,1 and Claudius Marondedze2
1King Abdullah University of Science and Technology,
Thuwal, Kingdom of Saudi Arabia
2University of Cambridge, Cambridge, United Kingdom
Abstract414
13.1 Introduction 414
13.2 Characteristics of Climacteric and Nonclimacteric Fruit
Ripening416
13.3 Proteomic Tools and Approaches for Fruit Ripening
Assessment417
13.3.1 Challenges in Proteomics Analysis 418
13.3.2 Protein Sample Preparation: Tissue Disruption,
Homogenization, and Solubilization 418
13.3.3 Protein Separation: Gel-Based and Gel-Free
Technologies421
13.4 Proteomes of Climacteric and Nonclimacteric Fruits 422
13.4.1 Overrepresented Functional Categories
during Development and Ripening 422
413
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Abstract
Comparative proteomics has emerged as a powerful tool used to study com-
plex biological processes in fruits throughout their development and ripen-
ing. This is greatly aided by the rapid growth of genomics, transcriptomics,
and expressed sequence tag (EST) databases, which allow for protein iden-
tification and pave the way for systems analyses and inference of molecular
data. Fruit development and ripening are complex developmental processes
that involve well-coordinated biological programs, the knowledge of which
has valuable economic ramifications centered on the agricultural industry.
Besides, fruit ripening is accompanied by numerous phenotypic and physi-
ological changes, such as skin and hypanthium color and increase in sugar
levels, which are regulated by environmental factors such as light and tem-
perature and internal factors such as hormonal and gene regulation. The lat-
ter is key in the classification of fruit species in their respective climacteric
and nonclimacteric categories. Comparative proteomics is therefore a useful
tool to gain information on the molecular events taking place during fruit
maturation, in addition to finding biotechnological strategies to improve hor-
ticultural traits such as fruit quality, shelf life, and yield. In this chapter, an
overview of methods utilized in fruit proteomics, as well as a global proteome
and systems biology analysis of fruits during ripening, is presented.
13.1 Introduction
Fruit development is a highly coordinated process that involves irreversible
cellular events and is accompanied by systematic molecular and physiological
414
Proteomics of Fruit Development and Ripening
changes (Bonghi and Manganaris, 2012). On the other hand, fruit ripening
correlates with a series of complex developmental changes, usually involving
various physiological processes, such as chlorophyll degradation, pigment
biosynthesis (carotenoid and xanthophyll) (Grassi et al., 2013), tissue soften-
ing (Payasi et al., 2009), starch degradation, simple sugar accumulation (Li
et al., 2012), and volatile compounds and organic acid production (Nergiz
and Ergönül, 2009; Etienne et al., 2013; Rambla et al., 2014). The final stage
of fruit development usually manifests apparent color, flavor, aroma, and
texture changes that are appealing to consumers and also to the broader
agricultural industry. In 2012, global banana (Musa acuminata) production
exceeded 100 million metric tons, while apples (Malus × domestica), oranges
(Citrus × sinensis), and grapes (Vitis vinifera) combined reached more than
200 million metric tons (FAOSTAT, 2013). Due to the growing economic
impact of fruit crops, understanding of events that occur during fruit mat-
uration has become of paramount importance, as this knowledge can be
exploited for breeding strategies to achieve desirable traits and improved
yield. While postharvest innovations such as fruit handling and storage strat-
egies have improved tremendously (Palma et al., 2011), physiological and
intracellular events, such as the expression and regulation of genes, signal-
ing mechanisms, and the biosynthesis and regulation of key molecules and
enzymes responsible for the fruit ripening process, have just recently gained
momentum. Better understanding of fruit maturation at the molecular level
is vital for further progress of postharvest technologies. In order to address
these challenges, advanced and high-throughput technologies have been
applied to discover the gene expression profile (Fei et al., 2004), the biosyn-
thesis of metabolites (Alba et al., 2005) and other molecules unique to fruit
development, and the proteome changes of various fruits (Sarry et al., 2004;
Rocco et al., 2006). The latter has recently seen a “burst” in the fruit science
field as reflected by more than 50 scientific literature outputs in the last 5
years. This is mainly due to the increasing availability of fruit crop genomes
and transcriptomes, from which plant biologists can extract functional infor-
mation. Since the fruit proteome is dynamic, elucidation of its profile at a
given development stage or in response to various signaling molecules or
stimuli is required to understand and capture key molecular events, which
are either not possible or less accurate with genome analyses alone. One
example is the occurrence and regulation of posttranslational modifications
(PTMs) that influence protein functions and interactions, and occur down-
stream and independently of the genetic code (Marondedze et al., 2014).
These PTMs render functional predictions from the genetic sequence inac-
curate. Additionally, recent advancements in proteomic approaches, such
as protein sample preparation, separation methods, and identification and
visualization methods (for review, see Orellana and Nilo, 2012), have aided
comparative analyses of the fruit proteome. This chapter highlights meth-
odological advances in fruit development and ripening proteomics; offers
415
P OST H AR V EST RI P ENING P H Y SIOLOG Y
416
Proteomics of Fruit Development and Ripening
because of its short life cycle and the availability of various tomato genomic
resources, such as microarrays, expressed sequence tags (ESTs), and the
recently completed tomato genome (Tomato Genome Consortium, 2012;
Kimura and Sinha, 2008). In addition to the development of effective and
stable tomato transformation systems (Ruf et al., 2001), numerous mutant
lines have also been generated and used for the characterization of ethyl-
ene biosynthesis and regulation (Saito et al., 2011). Several studies on key
genes involved in ethylene biosynthesis, including S-adenosylmethionine
decarboxylase, 1-aminocyclopropane-1-carboxylic acid synthase (ACS), and
1-aminocyclopropane-1-carboxylic acid oxidase (ACO), revealed that a reduc-
tion in ethylene synthesis in tomato fruit can delay fruit ripening, thus
increasing fruit shelf life (Hamilton et al., 1990; Oeller et al., 1991; Good
et al., 1994; Lokkamlue and Huehne, 2013). Further, transgenic tomato
lines with reduced ethylene perception, such as those involving the Never-
ripe and the Ethylene receptor (ETR) genes, also achieved delay in fruit rip-
ening (Wilkinson et al., 1997; Ciardi et al., 2000).
While limited, similar evidence in other fruits, such as apple, melon,
and papaya, has also been reported, of which some displayed encouraging
fruit ripening alterations (Flores et al., 2001; Dandekar et al., 2004; Lopez-
Gomez et al., 2009). In apple fruits, ACS or ACO genes were silenced and
fruits of the transgenic plants showed a firmer texture and increased shelf
life, although biochemical events such as sugar or acid accumulation were
unchanged (Dandekar et al., 2004). In ACO-silenced melon plants, the
chlorophyll and carotenoid pigments remained intact in the rind, and other
biochemical contents (sucrose and citric acid) were accumulated in the
pulp (Flores et al., 2001). Cosuppression of ACO in the tropical fruit papaya
also achieved delayed ripening and alterations in CO2 production and color
development (Lopez-Gomez et al., 2009). It is noted that the suppression
of ethylene perception approach, similar to the ETR suppression studies
in transgenic tomato plants, has yet to be reported in other fruits. As more
genetic resources and tools become available for other economically impor-
tant fruits, current discoveries on ripening from the tomato model system
can be translated to these fruits.
417
P OST H AR V EST RI P ENING P H Y SIOLOG Y
418
Proteomics of Fruit Development and Ripening
Tissues disruption
Fruit tissues
Homogenization
Protein extract
Quantitation
Gel-based (2-DE) Gel-free
Protein separation
pH gradient
IEF Alkylation, reduction
and digestion by trypsin
Alkylation, reduction and with/without labels
Small
MS and MS/MS
Spot excision and in-gel Data Analysis
tryptic digestion
water content and low protein concentration (Saravanan and Rose, 2004).
Protein extraction is further complicated by the presence of relatively
high amounts of contaminants, such as acids, lipids, polysaccharides, and
phenols, which interfere with downstream analyses. Taking into consid-
eration these challenges, the sample preparation strategy should include
419
P OST H AR V EST RI P ENING P H Y SIOLOG Y
420
Proteomics of Fruit Development and Ripening
421
P OST H AR V EST RI P ENING P H Y SIOLOG Y
422
Proteomics of Fruit Development and Ripening
2011) to obtain biological and molecular information that reflect the events
leading to fruit ripening. Based on our analysis, the major portion (21%)
of the differentially expressed proteins in climacteric species were plastid
proteins, while in nonclimacteric species, both plastid and plasma mem-
brane proteins shared a reduced majority of 17% (Figure 13.2A). The high
representation of proteins from plastids during fruit development and ripen-
ing is partly attributed to the biosynthesis of pigments such as carotenoids,
which give fruits their distinctive colors. While fruit plastids have reduced
photosynthetic capability in comparison to leaves, metabolic activities such
as the catabolism of starch and metabolites for the bioaccumulation of pig-
ments, lipids, and amino acids are high (Bouvier and Camara, 2006). Plasma
membrane proteins are also overrepresented in both climacteric (13%) and
nonclimacteric (17%) species since new membranes are being developed
to sequester pigments and lipid derivatives synthesized during ripening
(Camara et al., 1995). A remodeling of membrane protein composition and
organization takes place during the shift from photosynthetic green fruit to
less or nonphotosynthetic colored fruits during the transition from develop-
ment to ripening, and this possibly explains the overrepresentation of the
plasma membrane category. Key members of the membrane category, and
specifically in the plastid, include translocators, which function to shuttle
sugars and metabolites to and from the cytosol (Flügge, 1999). The phos-
phate translocators, involved in recruiting carbon source hexose phosphate
from the cytosol, and polyphenol oxidases, which are responsible for fruit
browning, are located at the plasma and thylakoid membranes and were
detected as overexpressed during fruit ripening (Flügge, 1999; Mayer,
2006). In climacteric fruits, overrepresented proteins from the cytosol rep-
resented 11% of total differentially expressed proteins. Contributions from
differentially expressed proteins from the extracellular region, mitochon-
dria, and nucleus were comparable between climacteric and nonclimacteric
species. However, a lower ratio of nucleus proteins was detected in noncli-
macteric (4%) than in climacteric (10%) species. Transcription factors, such
as the MADS box transcription factor ripening inhibitor (RIN), are known
to accumulate in the nucleus during ripening, and their absence has been
shown to halt fruit ripening (Ito et al., 2008). The higher representation of
nuclear proteins in climacteric species could be attributed to the necessity
for ethylene-related transcription factors, such as the ethylene-insensitive
(EIN) proteins, for the ripening of climacteric fruits (Chaves and de Mello-
Farias, 2006). Interestingly, Golgi apparatus proteins contributed to 5% of
overrepresented proteins in nonclimacteric species, although this category
was absent in climacteric species.
With regard to the molecular function, similar ratios were detected in
climacteric and nonclimacteric species for the nucleotide binding (31% and
30%, respectively), hydrolase activity (24% and 21%, respectively), transfer-
ase activity (18% and 15%, respectively), and protein binding (17% and 16%,
423
P OST H AR V EST RI P ENING P H Y SIOLOG Y
11 15
10 11
10
Nucleotide binding
10
7 Hydrolase activity
7 31 Transferase activity
17 30
5
Protein binding
B
Molecular function RNA binding
16 (%)
Structural molecule activity
Transporter activity
18 15 21
24
424
Proteomics of Fruit Development and Ripening
for enzymes to the gating of plant ion channels via cyclic nucleotides (Talke
et al., 2003; Zelman et al., 2012). Importantly, one group of nucleotide bind-
ing protein, the P-loop-containing cell wall hydrolases, is involved in fruit
softening (Fischer and Bennett, 1991; Thumdee et al., 2007), probably
explaining their overrepresentation during fruit ripening. The representa-
tion of the RNA binding category was, however, lower in nonclimacteric
species (5%) than in climacteric species (10%). Two molecular categories,
the structural molecular activity and transporter activity, contributed 7%
each and were present only in nonclimacteric species (Figure 13.2B).
In terms of biological process categories, both climacteric and non-
climacteric species showed overrepresentation of proteins involved in the
same 12 categories. The metabolic, cellular, and response to stimulus and
signaling categories were the most represented, contributing more than
60% to the responsive proteins (Figure 13.2C). No significant differences
of category ratios between climacteric and nonclimacteric species were
observed. In accordance with Molassiotis et al. (2013), the metabolism
category was the most represented in both climacteric and nonclimacteric
species. The authors also reported a large number of common proteins,
suggesting that similar regulatory mechanisms occur in climacteric and
nonclimacteric species (Molassiotis et al., 2013). Of interest is that meta-
bolic processes play a major role during fruit development, which is char-
acterized by massive cellular and physiological changes, including cell
division and expansion, which slows down with maturity and ripening onset
(Figure 13.3).
S MD NTR RIPE
425
P OST H AR V EST RI P ENING P H Y SIOLOG Y
426
Glycolysis/Gluconeogenesis Pyruvate metabolism TCA cycle
c nc c nc c nc
6 5 8 5 11 2
CO2
Starch and sucrose Phosphoenol- coA
metabolism Glucose Glu-6P 2 x Pyruvate Acetyl-coA
pyruvate Citrate
c nc NAD+ NADH Ribulose-1,5P
c nc Carbon fixation
4 8 10 CO2 c nc
Galactose Gly 3-P + Fru-6P Oxaloacetate Glycerate-3P
metabolism 2 6
c nc 2 16 2
H2O
1 4 5 Fructose/mannose PPP Amino acid metabolism
Amino and nuclotide c nc
metabolism
sugar metabolism c nc
7 13 9
1 7 4 Glyoxylate and dicarboxylate metabolism Galactose metabolism
His, Arg, Pro Ala, Asp and Glu Cys and Met Val, Leu and Isoleu Phenylalanine Gly, Ser and Thr
Phenylpropanoid
biosynthesis
Ethylene biosynthesis
4-Coumaroyl-CoA
Nucleotide metabolism Flavonoid biosynthesis
c nc c nc
8 9 3 4 3
Anthocyanidins and anthocyanins
427
Proteomics of Fruit Development and Ripening
STARCH AND SUCROSE METABOLISM
UDP-D-
Amino sugar and galacturonate Pectin Pectate 3.2.1.15 D-galacturonate
2.4.1.43 3.1.1.11
nucleotide sugar metabolism 3.2.1.67
428
5.1.3.6
D-Xylose 1,4β-D-Xylan β-D-Glucuroroside
Pentose and glucuronate
32.1.37 2.4.2.24 4.1.1.35 2.4.1.17 3.2.1.31 Ascorbate metabolism
interconversions
UDP-D-xylose UDP-D- D-Glucuronate
glucuronate
3.2.1.28
Sucrose-6P Retinol Trehalose D-Glucose
Sucrose
2.7.1.69 metabolism 24.1.245 2.4.1.64
(extracellular) Sucrose-6P
(2,6-β-D- 1.1.1.22 βD-Glucose-6P
2.4.1.14
Fructosyl)m+n (2,6-β-D-Fructosyl)n 5.4.2.6
3.2.1.26 3.2.1.65 2.4.1.10 3.1.3.12 βD-Glucose-1P
3.2.1.64 3.1.3.24 32.1.122 D-Glucose-6P
1.199.13
α-D-Glucose-6P 2.4.1.15
3.2.1.20 3-Ketosucrose Trehalose-6P 32.1.93
P OST H AR V EST
Dextrin
3.2.1.21 3.2.1.91 Cellulose
α-D-Glucose- 3.2.1.1
β-D-Glucose 1,4-β-D-Glucan 1P Glycogen; 2.4.1.25 3.2.1.20
3.6.1.21
Amylose 3.2.1.3 3.2.1.10 2.7.1.175
Amino sugar and
nucleotide sugar metabolism 2.7.7.33 2.4.1.18 3.2.1.3
CDP-glucose α-D-Glucose
3.2.1.33
2.7.1.41 2499.16
α-Maltose-1-P
2.7.1.10
Glycolysis/ 3.2.1.10
α-D-Glucose-1,6P2 2.7.1.106 5.4.2.2 Gluconegenesis 2.4.1.20
Isomaltose
3.1.3.9 α-D-Glucose-6P
Cellohexaose Cellotetraose
2.7.1.1 3.2.1.74 3.2.1.74 3.2.1.74 3.2.1.74 3.2.1.74
Celloheptaose Cellopentaose Cellotriose Cellobiose
α-D-Glucose 2.7.1.2 5.3.19
P H Y SIOLOG Y
β-D-Fructose-6P
(A)
Figure 13.5A Overview of the (A) starch and sucrose metabolisms, (B) fructose and mannose metabolisms, and (C)
galactose metabolism. These are some of the most represented metabolic pathways during fruit development and rip-
ening. The boxes highlight proteins with differential accumulation during fruit development and ripening. The symbols
highlight proteins whose expression significantly varies in the categories represented.
FRUCTOSE AND MANNOSE METABOLISM
L-Sorbose
D-Allose
1.19921
α-D-Glucose 1.1.2.2 2.7.1.55
Galactose
1.1.1.21 D-Sorbitol
metabolism 1.1.1.11 D-Mannitol-1P
1.1.1.67 3.1.3.22 D-Allose-6P
5.3.1.5 1.1.1.14 1.1.1.15 2.7.1.69
1.1.1.138 D-Mannitol
RpiB
D-Fructose-2,6P2
3.1.3.- 3.1.3.54
D-Fructose D-Fructose-2P 1.1.1.17 D-Allulose-6P
3.2.1.80 Fructan 3.1.3.46
5.3.1.7 2.7.1.1 2.7.1.4 2.7.1.105 5.1.3.-
2.7.1.69 β-D-Fructose-6P
Amino sugar and D-Mannose D-Mannose-6P
nucleotide sugar 2.7.1.1 5.3.1.8 Glycolysis
metabolism Amino sugar and
2.7.1.7
5.4.2.8 nucleotide sugar metabolism 1.1.1.140
ADP-Mannose D-Mannose-1P
3.6.1.21 GDP- D-Sorbitol-6P
3.2.1.78 3.2.1.77 3.2.1.137 D-mannuronate 4.2.2.3 Dinner
2.4.1.33
4.2.2.11 3.1.3.11 2.7.1.11 1.1.1.-
2.4.1.- 2.7.7.13 2.7.7.22 3.6.1.21 Alginate
Mannan 1.1.1.132 Mannosyl-
2.7.1.90 2.7.1.69
3-phosphoglycerate L-Sorbose-1P
2.4.1.- 2.41.217 3.1.3.70 Mannosyl-
1,4-β-Mannan GDP-D-mannose glycerate
D-Sorbitol 2.7.1.69
2.41.269
4.2.1.47
N-Glycan GDP-4-oxo-6-deoxy-
biosynthesis L-Sorbose
D-mannose
1.1.1.135
β-D-Fructose-1,6P2
GDP-6-deoxy-D-talcose
1.1.1.271
4.12.13
1.1.1.187
GDP-L-fucose
1.1.1.281 Glyceraldehycle-3P
GDP-D-rhamnose Glycolysis
2.7.7.30
L-Fucose
2.7.1.52 5.3.1.1
L-Fucose-1P
5.3.1.25
L-Fuculose-1P Glycerone-P
2.7.1.51 4.1.2.17 2.7.1.28 2.7.1.56
L-Fuculose
L-Lactate
Pyruvate
1.1.1.122 3.1.1.- 4.2.1.68 1.1.1.- 3.7.1.-
metabolism
L-Fucono- L-Fuconate 2-Dehydro-3-deoxy- 2,4-Diketo-3-deoxy-
lactone L-fuconate L-fuconate
L-Rhamno- D-Glycer-
furanose L-Rhamnonate
aldehyde
1.1.1.173 3.1.1.65 4.2.1.90 4.1.2.53
L-Rhamnono- L-Lactaldehyde
2-Dehydro-3-deoxy-
1,4-lactone L-rhamnonate
L-Rhamnose
5.3.1.14
L-Rhamnulose-1P
2.7.1.5 4.1.2.19
L-Rhamnulose
2.7.1.3
4.1.2.13
2.7.1.69 D-Fructose-1P
(B)
429
Proteomics of Fruit Development and Ripening
430
GALACTOSE METABOLISM
1.1.3.9
2-Dehydro-3-deoxy-
1.1.1.48 4.2.1.6 D-galactonate 4.1.2.55 D-Glyceraldehyde
3.1.1.25
1.1.1.120 D-Galactonate 4.2.1.140 4.1.2.51
D-Galctomono-1,4-lactone
1.1.1.360
2.7.1.58 2.7.1.178
1.1.1.359 D-Galactono-1,5-lactone
UDP-glucose 4.1.2.21 D-Glyceraldehyde-3P
Pentose and glucuronate Pentose phosphate
Amino sugar and nucleotide
interconversions 4.1.2.55 pathway
sugar metabolism 2-Dehydro-3-deoxy-
P OST H AR V EST
2.7.7.9 D-galactonate-6P
D-Galactose α-D-Galactose-1P
α-D-Glucose-1P
5.1.3.3 2.7.1.6 2.7.7.12 5.1.3.2 Glycerone-P
α-D-Galactose
2.7.7.10 2.7.7.64
4.1.2.40
UDP-galactose Galactinol
3.2.1.23 2.41.123
Galactan
5.4.99.9
3.2.1.23 Lactose 2.4.1.82
UDP-D- 5.4.2.2
2.4.1.22 Raffinose
3.2.1.108 galactofuranose
3.2.1.22 Sucrose
3.1.3.9 N-Acetyl-
RI P ENING
3.2.1.22
glycerol 5.3.1.26
D-Galactose-6P
Glycerol
Galactitol D-Tagatose
1.1.1.21 1.1.1.16 2.7.1.101 D-Tagatose-6P
(C)
2.7.1.69 1.1.1.251
Galactitol-1P
431
P OST H AR V EST RI P ENING P H Y SIOLOG Y
432
Glycolysis Gluconeogenesis
ATP
Glucose Pi
Glucose-6-phosphate (P) hexokinase Glucose-6-phosphatase
ADP H2O
NADP+ Glucose-6-P Glucose-6-
* dehydrogenase phosphate (P)
NADPH
6-phosphogluconolactone
H2O
* Gluconolactonase ATP Fructose-6-P H2O
H+ phospho-
H2O * * Fructose 1,6-bisphosphatase
fructokinase-1
6-phosphogluconate ADP
Oxidative phase
Fructose H2O
NADP+
* 6-phosphogluconate 1,6-bisP
* NADPH + CO2 dehydrogenae
*
Dihydroxyacetone P Dihydroxyacetone P
Ribulose-5-P
Ribulose-5-P isomerase Ribulose-5-P 3-epimerase
*
Ribulose-5-P Xylulose-5-P 2x Glyceraldehyde 3-P
Transketolase 2 (NAD++Pi) 2 (NAD++Pi)
* *
2 (NADH+H+) 2 (NADH+H+)
Glyceradehyde-3-P + Sedoheptulose-7-P
Non-oxidative phase
2x 1,3-bisphosphoglycerate
* Transaldolase 2x ADP 2x ADP
*
Fructose-6-P + Erythrose-4-P Xylulose-5-P 2x ATP 2x ATP
Transketolase 2x 3-phosphoglycerate
*
Glycolysis
Glyceraldehyde-3-P + Fructose-6-P 2x phosphoenol- 2x GDP
Climacteric only pyruvate PEP carboxykinase
non-Climacteric only 2x ADP 2x GTP
* both Climacteric and non-Climacteric Glycolysis * 2x oxaloacetate
fruit categories 2x ATP
2x ADP
(A) (B) 2x pyruvate pyruvate carboxylase
2x ATP
Figure 13.6 Overview of the (A) pentose phosphate pathway and (B) glycolysis and gluconeogenesis pathways. Common
and climacteric- and nonclimacteric-specific enzymes’ responsive proteins during fruit development and ripening are shown.
433
Proteomics of Fruit Development and Ripening
1.1.99.7
1.13.124
4.1.1.3 1.2.4.1 1.2.5.1
2-Hydroxy- 1.2.3.3
6.4.1.1 ThPP Acetyl-P
ethyl-ThPP 2.7.2.12
1.1.1.38 1.1.1.39
2.7.2.1
1.1.1.40 Lipoamide-E 1.2.4.1 1.2.7.1 1.2.3.6
2.3.1.54 S-Acetyl-
Formate
Oxaloacetate 1.8.1.4 dihydro- 3.6.1.7 4.1.2.36
lipoamide-E 2.3.1.8 EutD
Dihydro-
lipoamide-E 2.3.1.12
Glyoxylate
1.1.1.37 1.1.1.82
metabolism 3.1.2.1 Acetate
1.1.5.4 Acetyl-CoA
2.8.3.18
Propanoate 2.8.3.1
Glyoxylate 6.2.1.13
metabolism metabolism
6.2.1.1 6.2.1.1
(S)-Malate Acetyladenylate 1.2.1.3 1.2.1.-
4.2.1.2 1.3.5.4
Fumarate Succimate 1.2.99.3 1.2.99.6
2.3.3.9 1.2.1.10
Citrate cycle Acetaldehyde
2.3.3.13 Leucine biosynthesis
(R)-2-Ethylmate 2.3.3.6 3-Carboxy-3-hydroxy-
Acetoacetyl-CoA 4-methylpentanoate
Butanoate metabolism 2.3.1.9 2.3.3.14 Lysine biosynthesis
Homocitrate
Synthesis and degradation
of ketone bodies 4.1.3.- 6.4.1.2 Fatty acid biosynthesis
2-Propylmalate Malonyl-CoA
434
Proteomics of Fruit Development and Ripening
Phosphoenol-
41.1.32 pyruvate Glycolysis/
41.1.49 Gluconeogenesis
Fatty acid biosynthesis
1.1.5.4
(S)-Malate 1.1.1.42
Tyrosine 42.12
metabolism Oxalosuccinate 1.1.1.41 1.11286
Arginine and
proline metabolism Fumarate
1.1.1.42
135.4 135.1
ThPP 2-Oxo
6.2.1.4 Succinyl-CoA glutarate
6.2.1.5 23.1.61 1.2.42 1.2.42
Succinate 2.8.3.18 S-Succinyl- 3-Carboxyl-
dihydrolipomide-E hydroxypropyl-Thpp Ascorbate and aldarate
metabolism
1.8.1.4
Val, Leu & Ile Dihydro- Lipomide-E Alanine, aspartate and
degradation lipomide-E glutamate metabolism
1.2.7.3
D-Gln & D-Glu
metabolism
435
P OST H AR V EST RI P ENING P H Y SIOLOG Y
436
Proteomics of Fruit Development and Ripening
shown to increase during the véraison in grapes (Chervin et al., 2004; Sun
et al., 2010), at the onset of ripening in strawberries (Trainotti et al., 2005),
and in some cases, during ripening, such as in pineapple (Cazzonelli et al.,
1998), which are all nonclimacteric fruits. Even though a slight elevation in
ethylene has been reported in grape, strawberry, and capsicum ripening as a
result of the increase in ACO expression (Chervin et al., 2004; Iannetta et al.,
2006; Pham, 2007), the level of ethylene was very low compared to that in
climacteric fruits (Aizat et al., 2013). This low level of ethylene in nonclimac-
teric fruits might be compensated, at least partly, by the presence of other
hormones, such as brassinosteroids and abscisic acid, to facilitate ripening in
some fruits, as suggested in grape (Symons et al., 2006) and strawberry (Jia
et al., 2011), but further research is necessary.
437
P OST H AR V EST RI P ENING P H Y SIOLOG Y
(+)-Gallocatechin
non-climacteric only
438
Proteomics of Fruit Development and Ripening
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Chapter 14
Abstract 450
14.1 Introduction 451
14.2 Dormancy: General Considerations 451
14.2.1 Developmental Aspects 452
14.3 Genetics of Tuber Dormancy 453
14.4 Pre- and Postharvest Environmental Effects 453
14.5 Physiological Regulation of Tuber Dormancy 454
14.5.1 Auxin 455
14.5.2 Carotenoid-Derived Hormones: Abscisic
Acid and Strigolactone 456
14.5.3 Cytokinins 458
14.5.4 Ethylene 459
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14.5.5 Gibberellins 460
14.5.6 Summary and Conclusions 460
14.6 Transcriptional Regulation during Dormancy 461
14.6.1 Initiation of Tuber Dormancy 461
14.6.2 Dormancy Termination 462
14.6.3 Epigenetic Control of Tuber Dormancy 463
14.7 Control of Sprouting in Storage 463
14.7.1 Introduction and Importance 463
14.7.2 Dormancy, Cultivar Selection, and
Storage Temperature 464
14.7.3 Chemical Control 464
14.8 Conclusions and Future Perspectives 467
References 467
Abstract
Potato is the third most important food crop in the world and is an excel-
lent source of dietary calories, vitamins, and minerals. At harvest and for an
indeterminate period thereafter, potato tubers are in a state of physiological
dormancy and will not sprout. Tuber dormancy is lost during storage in a cul-
tivar- and environmentally dependent manner. The onset of sprouting, which
follows the loss of tuber dormancy, results in numerous biochemical changes
that are detrimental to the processing and nutritional qualities of potatoes.
The initiation of tuber dormancy is coincident with the initiation of
tuberization. The duration of tuber dormancy is affected by both genetic and
pre- and postharvest environmental factors. Endogenous plant hormones
are involved in the regulation of tuber dormancy progression. Current evi-
dence suggests that abscisic acid and ethylene are required for dormancy
initiation and maintenance, while cytokinins appear to regulate dormancy
exit. The exact roles of auxins and gibberellins in tuber dormancy control
have yet to be determined, but both are involved in the control of sprout
growth that follows dormancy termination.
Dormancy initiation and progression are associated with wholesale
changes in gene expression. Although the transcript abundances of many
genes change dramatically during tuber dormancy, in most cases, the bio-
logical significance of these changes has yet to be determined. Dormant
tuber meristems are arrested in the G1 position of the cell cycle, and the
onset of sprout growth following dormancy exit is accompanied by the
resumption of cell cycle progression and expression of genes encoding key
proteins involved in cell cycle control. Dormancy progression is also accom-
panied by changes in chromatin composition involving both DNA cytosine
methylation and covalent histone modification.
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14.1 Introduction
Together with rice and cereals, potatoes are among the top three crops
grown worldwide for human consumption. Global potato production
exceeds 300 million metric tons, with China, India, and the Russian
Federation leading in total production. In addition to its contribution to
basic total caloric intake, potatoes are excellent sources of minerals, vita-
mins, and fiber. A single 150 g potato contains roughly 45% of the minimum
daily requirement of vitamin C and more potassium than any commonly
consumed vegetable or fruit.
In the United States, potatoes are grown on more than 1.1 million
acres, and in 2012, more than 46.7 billion pounds of potatoes were har-
vested, with an estimated market value in excess of $3.9 billion (USDA/
NASS, 2013). More than 70% of the U.S. fall potato production is placed into
medium- to long-term storage to satisfy the yearlong demands of both the
processing industry and consumers. Unlike rice and cereals, potatoes are
stored in a fully hydrated and highly perishable form. Maintenance of the
potato market and nutritional qualities during storage is a critical aspect of
modern potato storage management practices.
In addition to postharvest disease, uncontrolled sprouting results in
accelerated loss of tuber quality. At harvest, and for an indeterminate period
thereafter, potato tubers are dormant and will not sprout. During extended
postharvest storage, tuber dormancy is gradually lost and sprout growth
commences. Accompanying sprout growth is a host of biochemical changes
that severely impact both the nutritional and processing qualities of stored
tubers. In addition, the increased water loss from sprouting tubers predis-
poses the tubers to accelerated pathogen attack. For these reasons, control of
postharvest sprouting is an important aspect of potato storage management.
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14.5.1 Auxin
Despite being the first plant hormone described, there have been relatively few
detailed analyses of changes in endogenous auxins (notably Indole-3-acetic
acid (IAA) and its metabolites) during tuber dormancy progression, and as
such, their role in the regulation of potato tuber bud dormancy remains uncer-
tain. As determined by enzyme-linked immunosorbent assay (ELISA), endog-
enous IAA content in tuber apical buds remained essentially constant during 5
months of postharvest storage and declined markedly as sprouting commenced
(Sukhova et al., 1993). Subsequent studies using gas chromatography–mass
spectrometry (GC-MS) analysis of tuber apical bud extracts by Sorce et al.
(2000, 2009) provided conflicting data. In the first study, the content of IAA
increased dramatically during postharvest storage, peaking coincident with or
slightly after sprouting commenced. In the second study, using two cultivars
with differing lengths of innate dormancy, the endogenous content of IAA in
both cultivars was highest at harvest, declined during storage, and reached
a minimum as sprouting began. Consistent with the latter study, expression
of the auxin-inducible gene ARF6 was low in dormant buds and increased
as sprouting commenced (Faivre-Rampant et al., 2004), suggesting that an
increase in endogenous auxin content accompanies dormancy cessation.
Application of low concentrations of IAA to nondormant buds slightly
stimulates elongation but has no apparent effect on dormant buds (Hemberg,
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14.5.2 Carotenoid-Derived Hormones: Abscisic Acid
and Strigolactone
Prior to its isolation and chemical characterization, abscisic acid (ABA)
was known by two names, abscising and dormin, the latter arising from its
association with temperate perennial bud dormancy (Addicott and Carns,
1983). ABA was subsequently identified as a major bioactive component of
the β-inhibitor complex, whose levels (as judged by bioassay) were closely
correlated with several forms of meristem dormancy (Addicott and Carns,
1983; Hemberg, 1985). Studies using more selective and sensitive instru-
mental analyses have largely confirmed the early bioassay results and found
that tuber ABA content is highest in deeply dormant tuber tissues, declines
during postharvest storage, and is lowest at the time of sprouting (for
reviews, see Suttle, 2007; Sonnewald and Sonnewald, 2014). Unfortunately,
many of these studies used bulk tuber samples composed mainly of nonbud
tissue, thereby complicating interpretation of the data.
More recently, the effects of dormancy status on the ABA content
of isolated tuber tissues was determined using mass spectrometry. Using
GC-MS, Sorce et al. (1996) found the highest concentrations of ABA in
“subeye” tissues and also found a small increase in the ABA content of tuber
eyes as sprouting commenced. Subsequent LC-MS analyses examining
changes in the ABA content of microscopically excised tuber eyes during
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14.5.3 Cytokinins
Cytokinins are known to participate in the control of cell division and the
resumption of cell cycle activity following the termination of meristematic
quiescence (Mok and Mok, 2001; Kyozuka, 2007). As such, they have been
implicated in the reinitiation of growth following dormancy exit in peren-
nial tree buds and potato tubers.
Increases in isoprenoid cytokinins, including both trans- and cis-
zeatin derivatives, have been detected in potato tuber buds as they exit dor-
mancy and initiate sprout growth (for reviews, see Suttle, 2007; Sonnewald
and Sonnewald, 2014). Exogenous cytokinins are rapidly degraded to
adenine derivatives in potato tubers by the enzyme cytokinin oxidase
(Suttle, 2002). However, no significant changes in cytokinin metabolism or
expression of cytokinin oxidase genes were observed during natural and
chemically forced dormancy progression (Suttle et al., 2014). These results
suggest that metabolic control of tuber cytokinin content during dormancy
occurs at the level of biosynthesis. Although genes encoding isopentenyl-
transferases (the first committed step in cytokinin biosynthesis) have been
cloned and characterized from a number of plants (Sakakibara, 2006), the
effect of tuber dormancy status on their expression has not been reported.
After harvest and during storage, tubers pass through three stages
of dormancy. Initially, tubers are deeply dormant and will not sprout in
response to a variety of external treatments. Next, tubers remain dormant
but become responsive to external agents/treatments. Finally, tuber dor-
mancy ends and sprouting commences naturally. The endogenous content
of cytokinins is low in phases 1 and 2, but increases coincident with the
onset of sprout growth.
As was first reported by Hemberg (1970), once tubers become
responsive to external stimuli, exogenous cytokinins readily break tuber
dormancy. Many forms of naturally occurring cytokinins are active, includ-
ing free-base, nucleoside, and nucleotide forms of isopentenyl-, trans-, and
cis-zeatins (Suttle, 1998a; Suttle and Banowetz, 2000). Synthetic and meta-
bolically stable cytokinin derivatives are especially effective in breaking
tuber dormancy (Suttle, 2008). Although dormancy is effectively termi-
nated by cytokinin treatment, the resultant sprouts often remain quite
small and fail to elongate. However, simultaneous treatment with cytoki-
nin and gibberellin typically results in sustained sprout growth that more
closely resembles the situation following the natural dormancy break
(Hartmann et al., 2011).
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14.5.4 Ethylene
As is the case with other vegetative storage organs, potato tubers pro-
duce low (but detectable) amounts of ethylene during storage (Okazawa,
1974). The rate of ethylene production increases coincident with the onset
of sprouting either during the natural dormancy break or following treat-
ment with artificial dormancy-terminating agents (Okazawa, 1974; Suttle,
2009). Exogenous ethylene has a dual effect on tuber dormancy. Short-term
application of high concentrations of ethylene has been reported to hasten
subsequent tuber sprouting, while long-term or continuous ethylene treat-
ment suppresses sprout growth (Rylski et al., 1974). The latter effect was
the impetus for current efforts to commercialize the use of ethylene as a
sprout inhibitor in potato storages (Prange et al., 1998).
Despite these observations, no definitive role for endogenous eth-
ylene in tuber dormancy control was envisaged. This uncertainty was
addressed using an in vitro microtuber system and specific inhibitors of
ethylene action. In this system, ethylene production was highest during
the initial period of tuber development, and chemical inhibition of ethylene
action during the first week of tuber growth resulted in precocious sprout-
ing (Suttle, 1998b). These results suggested that endogenous ethylene
plays an essential role in the induction of microtuber dormancy.
It is surprising that genetic manipulation of ethylene synthesis or
action in potato tubers by sense or antisense approaches has seldom been
reported. Tubers developing on Russet Burbank plants transformed with
either sense or antisense constructs of the ethylene receptor ETR1 dis-
played a number of phenotypic alterations (Haines et al., 2003). When stored
at 20°C, no effects on dormancy duration were noted, but antisense tubers
stored at 4°C failed to sprout after 2 years. Whether this represents a direct
effect of decreased ETR1 expression on tuber dormancy progression or is
a reflection of impaired cold tolerance was not established. Transformation
of potato with antisense constructs of the polyamine biosynthesis gene
SAMDC resulted in increased ethylene evolution and altered tuber pheno-
type, but had no obvious effects on tuber dormancy (Kumar et al., 1996).
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14.5.5 Gibberellins
The endogenous gibberellins (GAs) of potato are members of the early
13-hydroxylation pathway, ultimately yielding the bioactive hormone GA 1
(Jones et al., 1988; Xu et al., 1998a). Gibberellins are potent inhibitors of tuber-
ization, and the initiation of tuber formation is preceded by a marked decline
in endogenous GA 1 content in subapical regions of the stolon (Xu et al., 1998a
As the processes of tuber initiation and dormancy inception are thought to
occur together, a decline in GA content may play a role in the initiation of
tuber bud dormancy. As a result of the antagonism between GA content and
tuber formation, the endogenous contents of all GAs are very low in tuber tis-
sues throughout growth, harvest, and early storage (Jones et al., 1988). The
effects of postharvest storage and dormancy progression on endogenous GA
levels in tuber apical bud tissues were determined using GC-MS with internal
standards (Suttle, 2004). The endogenous contents of GA 19, GA 20, and GA 1
declined slightly during early storage when tubers were deeply dormant,
remained at or below harvest levels as dormancy weakened and sprouting
commenced, and increased as sprout growth became more vigorous.
The ability of exogenous GA to prematurely terminate tuber dormancy
was first reported by Brian et al. (1955) and subsequently verified by oth-
ers (Hemberg, 1985). The dormancy-breaking efficacy of individual GAs in
the latter steps of the biosynthetic pathway was GA 19 < GA 20 < GA 1 (Suttle,
2004). As mentioned above, robust sprout growth typically requires the
simultaneous application of both GAs and cytokinin (Hartmann et al., 2011).
The effects of chemical and genetic manipulation of endogenous GA
content have also been reported (Suttle, 2004). Treatment of developing
microtubers with known inhibitors of GA biosynthesis did not extend the
dormant period. No differences in dormancy progression were observed in
studies comparing a wild-type and GA-deficient dwarf mutant of Solanum
andigena. Taken together, these studies suggest that endogenous GAs do
not directly participate in the regulation of tuber dormancy progression,
but may play a critical role in sprout growth following dormancy exit.
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has expanded greatly and continues to grow. Nevertheless, there are many
substantive questions that remain unresolved. First and foremost is the
need to acquire detailed quantitative data on the effects of dormancy status
on the hormonal content of tuber buds free from surrounding nongrowing
tissues. Second is the need to develop an understanding of the biochemi-
cal and molecular processes regulating tuber bud hormone biosynthesis
and metabolism during dormancy progression. Last, occurring coinciden-
tally with changes in hormone content are changes in hormone perception
and action. These processes are likely key to developing a more complete
understanding of the physiology of tuber dormancy control and have yet to
receive experimental scrutiny.
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relative to physiological aging and the potential to knock off the sprouts
during handling. The latter could cause an increase in stem numbers per
eye and may be an entry point for pathogen invasion.
The current common means to control or minimize sprouting include
cultivar selection, storage temperature, and chemical product application.
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will be dependent on cultivar, how the crop was grown, storage tempera-
ture, and the rate and timing of the product application. It also will vary
greatly between the type of sprout inhibitor and method of application.
Differences in cultivar dormancy length and sprout development charac-
teristics (Kleinkopf and Olsen, 2003) may warrant different application tim-
ings of a sprout inhibitor.
There are several commercialized products utilized for sprout sup-
pression. Not all listed are registered for use worldwide.
465
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466
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476
Chapter 15
Calcium Deficiency
Disorders in Plants
Sergio Tonetto de Freitas,1 Cassandro Vidal
Talamini do Amarante,2 and Elizabeth J. Mitcham3
1Brazilian Agricultural Research Corporation,
Embrapa Tropical Semi-arid, Petrolina, Pernambuco, Brazil
2Santa Catarina State University, Lages, Santa Catarina, Brazil
3 University of California, Davis, California
Abstract 478
15.1 History of Ca2+ Deficiency Disorders 479
15.2 Role of Ca2+ as an Essential Plant Macronutrient 479
15.3 Symptoms of Ca2+ Deficiency Disorders in Fruit 481
15.3.1 Apple 481
15.3.2 Tomato 484
15.3.3 Watermelon 484
15.3.4 Pepper 484
15.4 Symptoms of Ca2+ Deficiency Disorders in Leafy
Vegetables 485
15.4.1 Lettuce 485
15.4.2 Cauliflower 485
15.4.3 Artichoke 486
15.4.4 Celery 487
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
Abstract
Physiological disorders, such as bitter pit in apple; blossom-end rot in
tomato, watermelon, and pepper; tipburn in lettuce, cauliflower, artichoke;
and blackheart in celery are believed to be triggered by Ca 2+ deficiency and
can strongly reduce crop quality and yield. These disorders are character-
ized by dark brown lesions on distal young and fast-growing tissue. In leafy
vegetables, stunted growth and curling are also common symptoms. The
conserved symptoms and factors leading to Ca 2+ deficiency disorders sug-
gest the existence of conserved mechanisms regulating these disorders in
fruits and vegetables. Suggested mechanisms triggering these disorders
are involved in the inhibition of Ca 2+ accumulation or abnormal regulation of
cellular Ca 2+ partitioning in affected tissues. Interactions between Ca 2+ and
other nutrients in affected tissue have also been suggested to be involved.
Although recent ideas have suggested that oxidative stress may play an
important role in Ca 2+ deficiency disorder development, they remain to be
experimentally analyzed. Considering the complexity of Ca 2+ deficiency
478
C alci um D e f i c i e n cy D iso rd ers in Pl a nts
disorders, control strategies should first identify the genetic and environ-
mental factors triggering mechanisms leading to symptom development.
Based on the factors involved, specific approaches can be identified to
effectively inhibit Ca 2+ deficiency disorder development.
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
effect not only on cell wall strength, but also on cell wall pH due to its cation
effect on the cell wall medium (Demarty et al., 1984). In addition, Ca 2+ has
also been reported to affect the synthesis of cell wall polysaccharides, such
as 1,3-β-glucan (Kauss, 1987; Brett and Waldron, 1996).
The high binding capacity of Ca 2+ makes this ion an important signal-
ing molecule in the cytosol (Hepler and Wayne, 1985; White and Broadley,
2003; Batistic and Kudla, 2010). Indeed, Ca 2+ plays an important role in
cytosolic signal transduction pathways involved in cell responses to a wide
range of biotic and abiotic factors (Scrase-Field and Knight, 2003; White
and Broadley, 2003). In the resting or quiescent state, cytosolic Ca 2+ ranges
between 100 and 200 nM, reaching about 2 µM under stimulus (Rudd and
Franklin-Tong, 1999). Changes in cytosolic Ca 2+ concentrations may take the
form of single calcium transients (Knight et al., 1996), oscillations (McAinsh
et al., 1995), or repeated spikes (Ehrhardt et al., 1996). The spatial and tem-
poral characteristics of these stimuli-specific Ca 2+ transients have become
known as Ca 2+ signatures (Scrase-Field and Knight, 2003). Specific Ca 2+ sig-
natures have been suggested to encode information about the type and sever-
ity of the input stimulus (Dolmetsch et al., 1997), which is then decoded by
downstream components of the signaling pathway, eventually leading to the
specific and appropriate cellular response (Scrase-Field and Knight, 2003).
After raising cytosolic Ca 2+ levels, the resting state must be reestablished to
avoid Ca 2+ toxicity and, potentially, cell death. At high concentrations in the
cytosol, Ca 2+ can become toxic due to its precipitation with inorganic phos-
phate and other ionic substances, as well as its competition for binding sites
with other cations, such as Mg2+, that are required for enzyme activation and
proper cellular metabolism (Hepler and Wayne, 1985; Batistic and Kudla,
2010). For these reasons, cytosolic Ca 2+ must be under strict biochemical
and physiological control. After cytosolic oscillations, the resting state of
cytosolic Ca 2+ is reestablished by the activity of Ca-ATPases and H+/Ca 2+
exchangers, which are proteins that transport Ca 2+ from the cytosol into the
apoplast or into storage organelles in the cell (White and Broadley, 2003).
Calcium is needed at high concentrations inside cellular organelles
so it is available to be loaded into the cytosol during signaling responses,
and as a counterion to inorganic and organic anions (Jones and Bush, 1991;
White and Broadley, 2003). The vacuole is the largest pool of Ca 2+ in the cell,
with 90%–95% of the cell’s volume and 1–10 mM Ca 2+ (Bush, 1995; White
and Broadley, 2003). In the endoplasmic reticulum, Ca 2+ concentration can
range from 1 to 5 mM (Kendall et al., 1992). Chloroplasts and mitochondria
also contribute, with the Ca 2+ being stored inside these cellular organelles
at concentrations ranging between 0.1–10 µM and 0.2–1.2 µM, respectively
(Johnson et al., 1995; Logan and Knight, 2003). Inside these organelles,
Ca 2+ can be present as a free ion that can be used for cytosolic signaling
responses or as a countercation to inorganic and organic anions forming
complexes with substances such as organic acids, proteins, peptides, and
480
C alci um D e f i c i e n cy D iso rd ers in Pl a nts
other anions (Jones and Bush, 1991; White and Broadley, 2003). Studies
have suggested that free Ca 2+ present in each organelle is responsible for a
specific cellular response, and that all organelles act in concert to shape a
Ca 2+ signaling response (Batistic and Kudla, 2010).
The high affinity of Ca 2+ for anionic phosphate and carboxylate groups
of lipids and proteins at the membrane surface makes Ca 2+ an important
regulator of membrane structure and function (Hanson, 1960; Hauser et al.,
1976; Clarkson and Hanson, 1980; Legge et al., 1982; Kirby and Pilbeam,
1984; Picchioni et al., 1998; Jaiswal, 2001; Hirschi, 2004; Batistic and Kudla,
2010). Calcium binding reduces membrane fluidity by tightly packing the
membrane lipids and proteins, which reduces the passive flow of monova-
lent ions such as H+, Na+, and K + (Williams, 1970; Jaiswal, 2001; White and
Broadley, 2003; Plieth, 2005). Since cytosolic Ca 2+ must be maintained at
extremely low levels to avoid toxicity, apoplastic free Ca 2+ has been sug-
gested to be the most important pool to regulate proper membrane struc-
ture and function (Hanson, 1960; Steveninck, 1965; Wallace et al., 1966;
Lund, 1970; Kirby and Pilbeam, 1984; Picchioni et al., 1998).
15.3.1 Apple
The symptoms of Ca 2+ deficiency disorder in apple, known as bitter pit (BP),
start as water-soaked spots in the outer cortical flesh of the fruit, frequently
481
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Figure 15.1 Calcium deficiency disorders in fruit: (a, b) apple with BP initial
water-soaked symptoms that become dark brown, depressed on the fruit
surface; (c) blossom-end rot symptoms in tomato fruit; (d) watermelon; and
(e) bell pepper. (Panel c from Freitas and Mitcham, 2011a. Copyright ©
American Society of Plant Biologists, www.plantphysiol.org. Panel d from
Ontario Crop Integrated Pest Management (IPM), Blossom-end rot, 2009,
Queen’s Printer for Ontario, http://www.omafra.gov.on.ca/IPM/english/
cucurbits/diseases-and-disorders/blossom-end-rot.html. Copyright © 2015
Queen’s Printer for Ontario, image courtesy of John G. Strang, Department
of Horticulture, University of Kentucky. Panel e from Hochmuth, G.J., and
Hochmuth, R.C., Blossom-end rot in bell pepper: causes and prevention,
Institute of Food and Agricultural Sciences, University of Florida, Gainesville,
2012, http://edis.ifas.ufl.edu/ss497#FIGURE%203. Copyright © 2015
University of Florida, Institute of Food and Agricultural Sciences.)
just under the skin (Figure 15.1a, Table 15.1). Later, cell dehydration and
death result in the collapse of the outermost cells, causing small dark
brown depressed lesions (pits) on the surface (Figure 15.1b). BP lesions
have also been associated with vascular elements and in severe cases may
coalesce to form larger necrotic areas (MacArthur, 1940). Although pit-
ting of the flesh also occurs, symptoms are not always visible from the out-
side. The frequency of pitting is often greater toward the calyx end of the
482
Table 15.1 Calcium Deficiency Disorders in Fruits: Symptoms and Mechanisms Involved
Fruit Disorder Symptoms Mechanisms References
C alci um
Apple (A) BP Water-soaked spots 1. Reduction of total fruit Ca 2+ Bangerth, 1979; Ferguson and
(A) or tissue (T, W, P) concentration (A, T, W, P) Watkins, 1989; Saure, 1996;
that eventually 2. Reduction of Ca2+ White and Broadley, 2003;
becomes dark brown concentration at specific Freitas et al., 2010, 2013
Tomato (T) BER mainly (A) or always cellular compartments (A, T) Saure, 2001; White and
(T, W, P) at the distal 3. Interaction between Ca2+ Broadley, 2003; Taylor and
fruit tissue and other nutrients (N, K+, Locascio, 2004; Ho and White,
Mg2+) in the fruit (A) 2005; Saure, 2005; Freitas
D e f i c i e n cy
483
Pl a nts
P OST H AR V EST RI P ENING P H Y SIOLOG Y
fruitw(Ferguson and Watkins, 1989). Fruit pitting usually takes place after
harvest, but in severe cases, it can also develop before harvest (Ferguson
and Watkins, 1989).
15.3.2 Tomato
In tomato fruit, Ca 2+ deficiency disorder was named blossom-end rot
(BER) due to its appearance: decay at the blossom end of the fruit (Figure
15.1c, Table 15.1). During BER development, blossom end tissue shows
water-soaked symptoms that become dark brown on the fruit surface at
later stages. In severe cases, BER can expand from the blossom end tis-
sue toward the calyx end tissue, affecting the whole fruit (Ho and White,
2005; Freitas et al., 2011a). During BER development, death of fruit tissue
can favor pathogen infection, which also contributes to the rot symptoms.
The initial water-soaked symptoms are believed to be caused by high mem-
brane leakage, leading to cell dehydration and death that trigger phenol
oxidation and the dark brown color development (Suzuki et al., 2000, 2003;
Ho and White, 2005). In tomato, Ca 2+ deficiency symptoms usually occur
early during fruit growth and development, when limited Ca 2+ moves into
rapidly expanding fruit (Ho and White, 2005).
15.3.3 Watermelon
Calcium deficiency disorder in watermelon is also known as BER due to its
visual symptoms, as previously described for tomato. The symptoms are
characterized by softening and shriveling of the fruit blossom end tissue,
which eventually becomes dark brown, sunken, and leathery (Figure 15.1d,
Table 15.1) (Maynard and Hopkins, 1999). Genotypes with elongated fruit
have been reported to be more susceptible to BER than round fruit geno-
types (Hammouda, 1987).
15.3.4 Pepper
The symptoms of Ca 2+ deficiency disorder in pepper are quite similar to
those of tomato fruit, which is also known as BER (Figure 15.1e, Table 15.1).
The symptoms usually occur early during fruit growth and development,
starting as water-soaked tissue in the blossom end region, which becomes
brown, with necrotic areas at later stages (Marcelis and Ho, 1999). In severe
cases, BER can expand to the entire pepper fruit (Aktas et al., 2005). Fruit
tissue close to the BER symptoms tends to lose green color faster than the
rest of the pepper. In addition, BER favors tissue infection with saprophytic
484
C alci um D e f i c i e n cy D iso rd ers in Pl a nts
fungi and soft-rot bacteria species that enhance the rot-like appearance
(Hochmuth and Hochmuth, 2012).
15.4.1 Lettuce
Calcium deficiency disorder in lettuce, known as tipburn, is characterized
by dark brown lesions and necrosis on the margins of young developing
leaf tips (Figure 15.2a, Table 15.2) (Saure, 1998; Koike and Smith, 2010). In
some genotypes, tipburn is first seen on the small veins along the margin
of young leaves. Inner and younger leaves are more susceptible to tipburn
incidence than outer and older leaves (Koike and Smith, 2010).
15.4.2 Cauliflower
Calcium deficiency disorder in cauliflower, also called tipburn, is charac-
terized by a light brown coloration and tip necrosis of young inner wrapper
485
P OST H AR V EST RI P ENING P H Y SIOLOG Y
leaves that enclose the cauliflower head (Figure 15.2b, Table 15.2) (Rosen,
1990; Koike and Smith, 2010). In severe cases, Ca 2+ deficiency symptoms
can also result in curd discoloration due to secondary pathogen infection
(Maynard et al., 1981).
15.4.3 Artichoke
Calcium deficiency disorder in artichoke is also known as tipburn and is
characterized by brown discoloration and the death of leaf margins. In
addition, immature flower buds can also develop black lesions along the
486
C alci um D e f i c i e n cy D iso rd ers in Pl a nts
upper tips and edges of the flower bracts. These symptoms appear mainly
on bracts in the inner whorls of the bud (Figure 15.2c, Table 15.2) (Francois
et al., 1991; Koike and Smith, 2010). In severe cases, Ca 2+ -deficient bracts
can also be infected by pathogens (Francois et al., 1991).
15.4.4 Celery
The Ca 2+ deficiency disorder in celery is known as blackheart and is char-
acterized by light to dark brown speckling, lesions, and necrosis on the
margins of developing leaf tips deep within the central growing region
(Figure 15.2d, Table 15.2) (Koike and Smith, 2010). During plant growth
and development, the blackheart symptoms may turn black and expand
outward from the inner plant tissues (Koike and Smith, 2010).
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P OST H AR V EST RI P ENING P H Y SIOLOG Y
Environment
Genotype
Growth regulators
Ca2+ movement in
the fruit
Cellular Ca2+
distribution
Ca2+ deficiency
disorders
Reactive oxygen
species (ROS)
488
C alci um D e f i c i e n cy D iso rd ers in Pl a nts
Most of the root Ca 2+ uptake is believed to take place passively through the
apoplast at the root tip and lateral root growth regions where the Casparian
band is not present (White, 2001; Taylor and Locascio, 2004). In that case,
root growth enhances Ca 2+ uptake by increasing the number of root tips and
lateral roots that lead to higher apoplastic Ca 2+ movement into the root sys-
tem (White, 2001). Although root Ca 2+ uptake may also take place through
the symplastic pathway, the complex mechanisms that have evolved to
strictly regulate and maintain low levels of cytosolic Ca 2+ suggest other-
wise (White, 2001). In addition, root Ca 2+ uptake is also affected by uptake
competition between Ca 2+ and other ions available in the soil, as will be dis-
cussed later in this chapter on section 15.5.3. (Bangerth, 1979; Ferguson
and Watkins, 1989; Saure, 2005). Therefore, soil nutrient composition also
influences root Ca 2+ uptake and, consequently, leaf and fruit susceptibility
to Ca 2+ deficiency disorders.
After Ca 2+ is taken up by the roots, it moves in the xylem vessels
toward the leaves and fruit by mass flow in response to the water potential
gradient generated by leaf and fruit transpiration and growth (Saure, 1998;
Taylor and Locascio, 2004). Mature leaves have higher transpiration rates
than young leaves and fruit, which explain their higher Ca 2+ accumulation
(Saure, 1998; Ho and White, 2005; Freitas et al., 2011b). Accordingly, low rel-
ative humidity increases Ca 2+ uptake into mature high-transpiring leaves,
but reduces in low-transpiring fruit and the inner leaves of leafy vegetables
with a closed growing point (Olle and Bender, 2009). In addition, mature
leaves receive water exclusively from the xylem, whereas young leaves and
fruit receive water from both xylem and phloem vessels (Ho and White,
2005; Freitas et al., 2011b). Considering that Ca 2+ moves in the plant only
through the xylem, water uptake from the phloem further decreases the
capacity to uptake Ca 2+ through the xylem sap into young leaves and fruit,
compared to mature leaves. Similar behavior is also observed in other sink
organs, such as meristems that have low transpiration rates and receive
water from both the phloem and xylem, making sink organs highly sus-
ceptible to Ca 2+ deficiency disorders (Saure, 1998; Koike and Smith, 2010).
Studies have shown that increasing transpiration of sink organs, without
changing whole-plant transpiration, is more efficient to increase Ca 2+ con-
tent in these organs than increasing Ca 2+ availability in the soil (Tadesse
et al., 2001). In addition, reducing whole-plant transpiration either by
decreasing water vapor pressure deficit (WVPD) or by spraying the plant
with abscisic acid (ABA) can reduce xylem sap movement toward mature
leaves and increase xylemic flow toward low-transpiring fruit, which has
been shown to increase fruit Ca 2+ accumulation and decrease fruit suscep-
tibility to Ca 2+ deficiency disorders (Guichard et al., 2005; Freitas et al.,
2011b, 2014; Barickman et al., 2014).
Plant water stress has been widely reported to increase Ca 2+ defi-
ciency disorder development in both low-transpiring leaves and fruit
489
P OST H AR V EST RI P ENING P H Y SIOLOG Y
(Saure, 1998; Ho and White, 2005; Koike and Smith, 2010). Water stress,
triggered by low water availability or high salinity conditions, reduces
the water potential gradient between roots and low-transpiring leaves and
fruit, consequently decreasing Ca 2+ uptake and increasing tissue suscep-
tibility to Ca 2+ deficiency disorders (Taylor and Locascio, 2004; Freitas
and Mitcham, 2012). This effect is further enhanced by Ca 2+ immobility
through the phloem, ensuring that the high levels of Ca 2+ accumulated in
older leaves are not redistributed to low-transpiring Ca 2+ -deficient tissues
(White and Broadley, 2003).
Although transpiration and growth are important factors determin-
ing xylem sap flow and Ca 2+ uptake, the number of functional xylem vessels
and the hydrostatic gradient required for xylem sap movement can also
affect Ca 2+ content in sink organs (Ho et al., 1993; Tadesse et al., 2001;
Dražeta et al., 2004a; Bondada et al., 2005; Ho and White, 2005; Freitas
et al., 2011b). Accordingly, studies have shown that the number of func-
tional xylem vessels and Ca 2+ accumulation in the fruit decrease simulta-
neously during fruit growth and development (Nonami et al., 1995). High
numbers of functional xylem vessels have also been associated with high
levels of fruit Ca 2+ uptake and low fruit susceptibility to Ca 2+ deficiency
disorders (Freitas et al., 2011b). Besides a high number of functional xylem
vessels, the hydrostatic gradient in the xylem vessels may also be required
for xylem sap movement into distal fruit tissues (Bondada et al., 2005),
contributing to Ca 2+ accumulation and reducing susceptibility to Ca 2+ defi-
ciency disorders.
490
C alci um D e f i c i e n cy D iso rd ers in Pl a nts
Healthy fruit
491
P OST H AR V EST RI P ENING P H Y SIOLOG Y
15.5.3.1 Nitrogen
Nitrogen (N) is commonly applied at high rates in the soil to stimulate
plant growth and yield (Bramlage and Weis, 2004). However, excessive
N levels might increase fruit susceptibility to Ca 2+ deficiency disorders
(Figure 15.3) (Raese and Drake, 1997; Dris et al., 1998). Besides the total
amount of N, the form of N that is used can also influence fruit quality.
Soil fertilization with ammonium rather than nitrate nitrogen substantially
492
C alci um D e f i c i e n cy D iso rd ers in Pl a nts
493
P OST H AR V EST RI P ENING P H Y SIOLOG Y
15.5.3.3 Boron
Boron (B) is a micronutrient element that plays a key role in reproductive
processes such as pollen germination and pollen tube growth (Dickinson,
1978), and application of B increases fruit set and yield in B-deficient
apple and pear trees (Wojcik and Wojcik, 2003; Wojcik and Treder, 2006;
Wojcik et al., 2008). Boron has a synergistic effect with Ca 2+ and helps to
increase the Ca 2+ concentration in fruit (Dickson et al., 1973; Bramlage
et al., 1980; Wojcik et al., 1999; Wojcik and Wojcik, 2003; Bramlage and
Weis, 2004) and reduce BP incidence in apples (Faust and Shear, 1968;
Granelli and Ughini, 1990; Wojcik et al., 1999; Sen et al., 2010) and inter-
nal browning in pears (Wojcik and Wojcik, 2003; Mielke and Chaplin,
2008) (Figure 15.3). Boron might suppress the activity of indole-3-acetic
acid (IA A) oxidase (Marschner, 1995), leading to an increase in IA A
that promotes acropetal movement of Ca 2+ (Dela Fuente et al., 1986) and
stimulates xylem tissue differentiation in the pedicel of the fruit (Dražeta
et al., 2004b), thereby improving Ca 2+ uptake by fruits. Soil fertilization
with B also increased the dry weight of fine roots in apple trees (Wojcik
et al., 2008), where most of the Ca 2+ uptake is believed to take place
(White, 2000, 2001; Taylor and Locascio, 2004). This might also con-
tribute to increased Ca 2+ content of leaves and fruits in plants fertilized
with B. Boron also plays an important role in maintaining both the struc-
tural and functional properties of membranes (Cakmak and Römheld,
1977), thereby reducing internal browning disorders in pear fruit (Xuan
et al., 2001, 2005; Mielke and Chaplin, 2008).
15.5.3.4 Phosphorus
Mulder (1952) reported that BP in apple fruit was associated with low P con-
tent. However, several authors reported that BP was associated with high
P content (Brown, 1926; Oberly and Kenworthy, 1961; Sharples, 1964). The
high P content of the affected fruit is not surprising if one takes into account
the observation that mineral elements move into the pitted tissue (Chamel
and Bossy, 1981).
Apple trees supplied annually at bloom with P (with 20 g P per tree as
ammonium polyphosphate, and receiving adequate fertigation applications
of N, K+, and B) had fruit with reduced incidence of water core (also a Ca 2+
deficiency disorder) at harvest, higher resistance to browning of cut slices,
reduced membrane leakage, and elevated antioxidant content after cold-air
storage (Neilsen et al., 2008). This indicates a role for P in the maintenance
of apple fruit membrane stability and cellular energetics that might aid in
494
C alci um D e f i c i e n cy D iso rd ers in Pl a nts
495
P OST H AR V EST RI P ENING P H Y SIOLOG Y
496
C alci um D e f i c i e n cy D iso rd ers in Pl a nts
inhibitor, has been shown to maintain the number of functional xylem ves-
sels during fruit growth and development, which was highly correlated with
fruit Ca 2+ uptake and susceptibility to Ca 2+ deficiency disorders (Freitas
et al., 2012a). Treating tomato plants with ABA has also been shown to
maintain higher numbers of functional xylem vessels and higher xylem/
phloem ratios of water uptake into the fruit, which resulted in higher Ca 2+
uptake and lower fruit susceptibility to Ca 2+ deficiency disorders (Freitas
et al., 2011b, 2014; Barickman et al., 2014).
497
P OST H AR V EST RI P ENING P H Y SIOLOG Y
498
C alci um D e f i c i e n cy D iso rd ers in Pl a nts
499
P OST H AR V EST RI P ENING P H Y SIOLOG Y
500
C alci um D e f i c i e n cy D iso rd ers in Pl a nts
501
P OST H AR V EST RI P ENING P H Y SIOLOG Y
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Chapter 16
Abstract 514
16.1 Introduction 515
16.2 Aroma Composition in Fruits 516
16.2.1 Apple 516
16.2.2 Melon 517
16.2.3 Strawberry 517
16.2.4 Tomato 518
16.2.5 Citrus 518
16.2.6 Grape 519
16.2.7 Peach 519
16.2.8 Banana 520
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Abstract
Fruit consumers are not only looking for traditional quality attributes such
as sugar, acidity, firmness, and appearance. They also value other attri-
butes, including nutrient availability, antioxidants, and aroma, where each
of these attributes is the result of a complex interaction between intrinsic
and external variables.
Overall aroma is determined by a complex mixture of a large number
of odor-active volatile compounds, and differences in the concentrations
and thresholds of key volatiles ultimately determine the distinctive aroma
of a particular fruit species or cultivar. It is also the result of a dynamic
process, where during fruit development, there are many changes of
these m etabolites caused by their synthesis, transport, or degradation.
Finally, there are several pre- and postharvest factors affecting tree and
fruit p
hysiology, leading to important changes in fruit composition, such as
aroma-related volatiles.
Several studies have been performed identifying and characterizing
the most important genes and encoded enzymes involved in aroma-related
volatiles; however, research in the mechanisms of regulation or modulation
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16.1 Introduction
The quality of fresh fruit is related to many attributes, such as appearance,
texture, color, and flavor, with the last relying on a combination of taste
and aroma. In addition to its biological importance to fruit species survival,
aroma is a key contributor to fruit flavor perception and plays a primary role
in consumer acceptability.
A complex mixture of a large number of odor-active volatile compounds
determines the aroma characteristics (Dixon and Hewett, 2000), where dif-
ferences in the concentrations and thresholds of key volatiles ultimately
determine the distinctive aroma of a particular fruit species or cultivar
(Defilippi et al., 2009). With the advent of highly sensitive instrumental meth-
ods, a large number of volatile compounds have been identified in fruits, and
in combination with sensory analysis, these methods have made it possible
to categorize volatile compounds according to their organoleptic importance.
Research on fruit aroma has revealed that volatile compounds are made up
of diverse classes of chemical groups, including acids, aldehydes, ketones,
alcohols, esters, sulfur compounds, furans, phenols, terpenes, epoxides,
and lactones (Schwab et al., 2008), all of which are derived from different
biosynthetic pathways that are highly integrated with fruit development and
ripening. Volatile compounds are generally produced during ripening via the
metabolism of high-molecular-weight molecules such as proteins, carbohy-
drates, and fatty acids (Aharoni and Lewinsohn, 2010), processes that are
considered to be mediated by ethylene in climacteric fruit.
Significant advances have been made in recent years toward improv-
ing our knowledge of the biosynthetic pathways and regulation of aroma-
related volatiles. These advances have resulted in a better understanding
of the mechanisms involved in aroma production, as well as the influ-
ences of environmental factors, genetic background, and handling
procedures on fruit aroma. However, there are still many underlying
mechanisms of aroma volatile biosynthesis and modulation that remain
to be determined.
This chapter provides an overview of the critical metabolic pathways
and genes that control volatile biosynthesis in model fruits, with a special
emphasis on fruit ripening and the role of ethylene during this process.
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16.2.1 Apple
More than 350 volatile compounds, such as esters, alcohols, aldehydes,
ketones, terpenes, and ethers, have been reported as contributing to
the aroma profile of apples (Zhu et al., 2008; Dunemann et al., 2012). Of
these, esters, aldehydes, and alcohols are considered the most significant
contributors to the typical apple aroma (Plotto et al., 2000; Contreras and
Beaudry, 2013). However, which compounds are most important depends
on the developmental and ripening stage of the fruit. Esters are the most
abundant volatiles in apples, accounting for up to 80%–98% of total volatiles
(Altisent et al., 2009). Both straight- and branched-chain esters are found in
apples (Song and Forney, 2008), and among these, butyl acetate, 2-methyl
butyl acetate, and hexyl acetate are considered the major contributors to
the characteristic “red apple aroma” and “Cox-like aroma” in most apple
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cultivars (Holland et al., 2005; Zhu et al., 2005; Dunemann et al., 2009). The
alcohols 2-methyl propanol, (Z)-hexen-3-ol, 1-hexanol, and 1-octanol and
the aldehyde 1-hexanal are considered key volatile compounds in cultivars
such as ‘Golden Delicious’ and ‘Fuji’ (Altisent et al., 2009).
16.2.2 Melon
Of the approximately 240 volatile compounds identified in various melon
varieties (Shan et al., 2012), straight- and branched-chain esters, satu-
rated and unsaturated aldehydes, alcohols, and sulfur compounds are
likely to be the major contributors to the aroma of melon (Obando-Ulloa
et al., 2008; Verzera et al., 2011). As melon fruit contains climacteric and
nonclimacteric genotypes, the aroma profile depends on the ripening
behavior. Climacteric varieties, which are considered highly aromatic,
are composed predominantly of esters (Shalit et al., 2001), mainly in the
form of acetate derivatives, such as methyl butyl, ethyl, hexyl, nonenyl,
and benzyl acetates (Song and Forney, 2008). Among these, ethyl butano-
ate, methyl-2-methyl butanoate, and ethyl-2-methyl propanoate contribute
to the fruity and sweet aroma notes of muskmelon (Condurso et al., 2012).
In contrast, nonclimacteric varieties are normally less aromatic due to
the lack of volatile esters and their high levels of aldehydes and alcohols
(Gonda et al., 2010).
16.2.3 Strawberry
Strawberries possess one of the most complex fruit aromas, and therefore
their typical aroma has been extensively studied. Approximately 360 vola-
tile compounds contribute to the aroma of strawberries (Zorrilla-Fontanesi
et al., 2012), including esters, aldehydes, ketones, alcohols, terpenes, fura-
nones, and sulfur compounds (Vandendriessche et al., 2013). Straight esters
and furanones appear to be the most important aroma-active compounds
in strawberries (Blanch et al., 2011), with the esters being represented by
ethyl hexanoate, ethyl butanoate, methyl butanoate, and methyl hexanoate
(Jouquand and Chandler, 2008; Almenar et al., 2009), and the furanones pri-
marily represented by furaneol (2,5-dimethyl-4-hydroxy-3(2H)-furanone)
and mesifurane (2,5-dimethyl-4-methoxy-3(2H)-furanone). Esters contrib-
ute green and sweet fruity notes (Ménager et al., 2004), while furanones
contribute sweet caramel-like and floral notes (Jouquand and Chandler,
2008). Other compounds that contribute to strawberry aroma are terpenes
(linalool, nerolidol, terpineol, and α-pinene), which provide citrus and
spicy notes (Zorrilla-Fontanesi et al., 2012), and aldehydes (hexanal and
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16.2.4 Tomato
More than 400 aroma volatiles have been identified in intact fruit and
upon tissue disruption (Díaz de León-Sánchez et al., 2009; Bai et al., 2011).
These volatiles include hydrocarbons, alcohols, phenols, ethers, aldehydes,
ketones, carboxylic acids, esters, and lactones, among others (Zhu et al.,
2005). A combination of cis-3-hexenal, cis-3-hexenol, hexanal, 1-penten-
3-one, 3-ethyl butanal, trans-2-hexenal, 6-methyl-5-hepten-2-one, methyl
salicylate, 2-isobutylthiazole, and β-ionone, at appropriate concentrations,
provides the characteristic fresh ripe aroma of tomatoes (Baldwin et al.,
2000; Bai et al., 2011). Another important volatile compound that influences
flavor is the monoterpene S-linalool, which imparts a sweet, floral, alcoholic
note (Lewinsohn et al., 2001).
16.2.5 Citrus
The citrus aroma has been difficult to study because citrus juices are
l iquid–solid suspensions, and suspended solids can alter the headspace
concentrations and aroma thresholds of many volatiles (Rouseff et al.,
2009). There are three sources from which citrus volatiles are gener-
ated: the juice contained in the juice sacs, which provide volatile water-
soluble compounds; juice oil, which is present in globular bodies within
the juice sacs; and peel oil. Both juice oil and peel oil contribute oil-
soluble compounds (Cabral et al., 2010). The essential oil of orange juice
has been studied the most extensively, with approximately 220 volatile
compounds having been identified among its components. The oil con-
sists of up to 90% terpene hydrocarbons, with limonene being the most
abundant compound, and less than 2% aldehydes, alcohols, and esters
(Cabral et al., 2010). Flavor reconstitution experiments have reported
that the following combination of 22 volatiles, at appropriate concentra-
tions, reconstructed the aroma of orange very closely (Rouseff et al.,
2009): acetaldehyde, ethyl-2-methyl propanoate, (R)-R-pinene, ethyl
butanoate, (S)-ethyl-2-methyl butanoate, hexanal, (Z)-hex-3-enal,
myrcene, (R)-limonene, 3-methyl butanol, 2-methyl butanol, ethyl hex-
anoate, octanal, oct-1-en-3-one, nonanal, decanal, (S)-linalool, butanoic
acid, (R)-ethyl-3-hydroxyhexanoate, (E,E)-deca-2,4-dienal, trans-4,5-
epoxy-(E)-dec-2-enal, and vanillin (Buettner and Schieberle, 2001).
Research on fresh ‘Mor’ mandarins has shown that the compounds
hexanal, ethyl-2-methyl butanoate, limonene, linalool, and β-myrcene
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provide green, fruity, citrus, floral, and musty aromas, respectively, all
of which c ontribute to mandarin flavor (Tietel et al., 2011).
16.2.6 Grape
Several volatile compounds have been identified in Vitis vinifera berries, with
these compounds mainly being present in the skin. These volatiles include
terpenes, norisoprenoids, aliphatic and aromatic alcohols and aldehydes,
esters, and benzene derivatives (Ferrandino et al., 2012). Most of the infor-
mation available for this species has been obtained for wine varieties, leav-
ing a gap in the information available for table grape cultivars, especially
those consumed after a long-term storage period. Based on volatile com-
position, grape germplasm can be divided into three groups: Vitis labrusca
and its hybrids, which is characterized by abundant esters; Muscat cultivars
of V. vinifera, with high concentrations of terpenoids; and neutral grapes,
mainly from cultivars of V. vinifera. The aroma of this latter group is mainly
composed of very low concentrations of C6 alcohols and aldehydes, benzene
derivatives, esters, and nonglycosylated monoterpenes and sesquiterpenes
(Yang et al., 2011).
Monoterpenes, in the form of free volatiles and glycoside conjugates
of monoterpene alcohols, contribute to the grape and wine aromas of sev-
eral cultivars. Typical monoterpenol components of aroma-rich grape vari-
eties are S-linalool, geraniol, nerol, citronellol, and α-terpineol (Defilippi
et al., 2009). C6 compounds impart grassy and herbaceous notes. Aliphatic
aldehydes, such as octanal, decanal, and (Z)-2-heptenal, impart a citrus-
like aroma, whereas furfural and benzaldehyde impart an almond aroma
(Ferrandino et al., 2012). Some volatile thiols, such as 4-mercapto-
4-methylpentan-2-one, are considered to be impact odorants in Sauvignon
blanc wines, producing aromas that are reminiscent of box tree, passion
fruit, broom, and black current bud. Alternatively, 3-mercaptohexan-1-ol
and 3-mercaptohexyl acetate contribute to the aromas of passion fruit,
grapefruit, and citrus (Coetzee and du Toit, 2012).
16.2.7 Peach
More than 100 volatile compounds have been identified in peach, including
aldehydes, alcohols, alkanes, ketones, lactones, terpenes, and esters (Xi
et al., 2012). Among these compounds, lactones, in association with other
volatiles, such as C6 aldehydes and alcohols and terpenes, play important
roles in peach aroma (Aubert et al., 2003; Cano-Salazar et al., 2013). Lactones,
particularly γ- and δ-decalactones, and esters, such as hexyl acetate and
(Z)-3-hexenyl acetate, are responsible for fruity aromas (Xi et al., 2012).
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16.2.8 Banana
More than 250 volatile compounds have been identified in banana culti-
vars (Imahori et al., 2013). A mixture of these compounds—mainly esters,
alcohols, and ketones—gives bananas their characteristic aroma (Brat
et al., 2004). Bananas mainly produce isoamyl and isobutyl esters, such as
3-methyl butyl and 2-methyl propyl esters of acetate and b utanoate. These
esters are considered to be the major contributors to the aroma of bananas
(Jayanty et al., 2002), providing the “fruity” top notes (Wendakoon et al.,
2006). The alcohols pentan-2-ol, 3-methyl b utanol, and 2-methyl p ropanol
also contribute to the succulent character of banana aroma, and the ketone
pentan-2-one, which is one of the major volatile compounds of bananas,
imparts the characteristic banana flavor (Brat et al., 2004).
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16.4.1.1 Genotype
Fruit aroma is largely determined by the fruit’s genetic background, and
accordingly, significant differences in the composition and concentra-
tions of volatile compounds have been observed among cultivars within
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Charentais cantaloupe melon, bred for longer shelf life, has much lower
concentrations of volatile compounds than short-shelf-life cultivars (Aubert
and Bourger, 2004).
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production, while an excess of radiation and heat could cause stress, either
by dehydration or by temperature increase in the plant, negatively affecting
aroma biosynthesis.
Cultural practices can also greatly influence fruit aroma, often reduc-
ing the quality of this attribute due to the use of crop management sys-
tems based almost exclusively on crop yield. Mattheis and Fellman (1999)
reviewed several studies reporting detrimental effects of high crop load and
the use of technologies for the control of diseases and pests on apple aroma.
As water status is a key factor in plant physiology, modulating pri-
mary and secondary metabolism, it is expected that irrigation might induce
changes in the biosynthesis of aroma volatiles. Veit-Köhler et al. (1999)
found that ‘Vanessa’ tomatoes produced a higher accumulation of C6 alde-
hydes, such as hexanal, (Z)-3-hexenal, and (E)-2-hexenal, under conditions
of lower water supply (without visible symptoms of water stress), resulting in
fruit with better quality without significant effects on fruit growth and yield.
The aroma potential and total content of C13 norisoprenoids were found to
increase in ‘Cabernet Sauvignon’ grapes grown with water deficit (Bindon
et al., 2007; Koundouras et al., 2009). An indirect effect of irrigation, particu-
larly on grape aroma, is its influence on shoot growth and leaf area, which
affect sunlight penetration and consequently aroma metabolism.
A practice that is often used to control soilborne diseases is g rafting.
It is known that yield and quality attributes of fruit can be affected by
grafting and the type of rootstock. For instance, grafting muskmelon onto
pumpkin has been found to reduce aromatic ester accumulation in this
fruit compared with melon grown on its own roots, as a result of decreased
activities of alcohol dehydrogenase and alcohol acyltransferase enzymes
(Chuan-qiang et al., 2011; Condurso et al., 2012).
Aminoethoxyvinylglycine (AVG), an ethylene biosynthesis inhibi-
tor, is commonly used to avoid premature fruit drop and extend the har-
vest period, mainly for apples, to which this inhibitor is applied 1 month
before harvest (Escalada and Archbold, 2009). A significant reduction in
total volatile compound production after storage has been reported for
several cultivars of AVG-treated apples, whose decline in aroma produc-
tion was enhanced as storage time proceeded, and especially in those
stored under controlled atmosphere conditions (Bangerth and Streif,
1987; Mir et al., 1999).
Fertilization is also considered to affect fruit aroma. Studies in which
the influences of nutrients at various concentrations have been examined
have shown that suitable levels of potassium and nitrogen induced ester
production in muskmelon and strawberry, respectively, improving their
aroma (Lin et al., 2004; Ojeda-Real et al., 2009). In contrast, either a defi-
cient or excessive use of fertilizers had negative effects on aroma produc-
tion in these fruits. However, extreme levels of nutrients might be beneficial
for some species. For instance, the contribution of the C13 norisoprenoid
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538
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Acknowledgments
The authors thank Conicyt/Fondecyt grants (1060179, 1100273, 1130107)
for funding their studies to further understand flavor metabolism on a pricot,
table grape, and avocado.
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551
Chapter 17
Abstract 554
17.1 Introduction 555
17.2 Exotic Fruits and Their Flavor Profiles 556
17.2.1 Lychee (Litchi chinensis) 556
17.2.2 Rambutan (Nephelium lappaceum L.) 556
17.2.3 Yellow Passion (Passiflora edulis) 558
17.2.4 Durian Fruit (Durio zibethinus) 559
17.2.5 Star Fruit or Carambola (Averrhoa
carambola L.) 560
17.2.6 Mangosteen (Garcinia mangostana) 560
553
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Abstract
The characteristic flavor of exotic tropical fruits is one of their most attrac-
tive attributes to consumers. In this chapter, the enormous diversity of
exotic fruit flavors is reviewed. Classifying some of the exotic fruits into two
classes on the basis of whether esters or terpenes predominate in the aroma
was also attempted. Indeed, as far as exotic tropical fruits are concerned,
the majority of fruits have terpenes predominating in their aroma profile.
554
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
Some of the fruits in this group are the Amazonian fruits, such as pitanga,
umbu-caja, camu-camu, garcinia, and bacuri. The ester group is made up
of rambutan, durians, star fruit, snake fruit, acerola, tamarind, sapodilla,
genipap, soursop, cashew, melon, jackfruit, and cupuacu. Also, this review
summarizes recent progress in the characterization of esters, terpenoids,
and sulfur volatile compounds’ biosynthetic genes and their spatiotemporal
expression patterns.
17.1 Introduction
Among the many attractive attributes that create demand for fruits from
the tropics and subtropics is the noticeable flavor characteristic. In addi-
tion, these fruits are often inexpensive and extremely rich in vitamins and
can be used in a wide range of food products. A great deal of attention has
been directed toward the volatile compositions of a wide variety of fruits
over the past several decades. In a few cases, individual “character-impact
compounds” have been pinpointed as being responsible for a characteris-
tic flavor, but most fruit flavors seem to be due to the integrated response
to a large number of contributing compounds (Marostica and Pastore,
2007). Indeed, as far as exotic tropical fruits are concerned, a scan of the
literature (Van Straten et al., 1988) shows that esters and terpenes are
their most prolific volatile components. It would also appear that a rough
classification of such fruit can be made on the basis of whether esters or
terpenes predominate in the aroma. In very rare cases, other classes of
compounds contribute significantly to the aroma notes of these exotic
fruits. For example, the attractive tropical flavor note of ripe yellow pas-
sion fruit has been shown to be associated with trace levels of sulfur vola-
tiles (Werkhoff et al., 1998). Volatile sulfur compounds are important trace
constituents of natural products and play an important role in the sensory
properties of food flavors.
The characteristic flavor of exotic fruits is one of the most attractive
attributes to consumers. The enormous diversity of exotic fruits represents
a promising area for research on aromas, with unusual sensory properties.
Nowadays, food industries are looking at how to use these volatiles to pro-
duce amazing new products that can accommodate this demand (Marostica
and Pastore, 2007). Accordingly, more research should be done in parallel
with the increasing demand of the food flavor industries for these exotic
fruits. The outcomes of this research may be necessary to stimulate the
production of accurate and natural flavor compounds for use in different
food systems. In view of the above, this work reviews the distinctive flavor
constituents of some exotic tropical fruits that are not well known in the
Western world.
555
P OST H AR V EST RI P ENING P H Y SIOLOG Y
556
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
O
cis-Hex-3-enyl Fruity, sweet
acetate O Mangosteen, star fruit
O
Ethyl butyrate O Fruity
Cupuacu, Costa Rican
O guava, durian,
camu-camu
Ethyl hexanoate O Fruity
Cupuacu, Costa Rican
guava, yellow passion
O
fruit, durian
Geraniol Roselike
OH
Lychee, star fruit,
mangaba fruit,
garcinia, evodia,
yellow passion
Geranyl acetol OH Sweet, fruity
Yellow passion fruit
(Continued )
557
P OST H AR V EST RI P ENING P H Y SIOLOG Y
cis trans
β-Pinene Woody-green
Garcinia, Costa Rican
guava, yellow passion,
pitanga, guabiroba,
araca-boi, umbu-caja,
camu-camu
β-Selinene Fruity
Costa Rican guava,
guabiroba, pitanga,
araca
558
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
beverages (Mussinan and Keelan, 1994). The analysis of the flavor com-
position of yellow passion fruit by four different isolation techniques,
namely, (1) vacuum headspace sampling (VHS), (2) the dynamic head-
space method, (3) simultaneous distillation and extraction at atmospheric
pressure, and (4) simultaneous distillation and extraction under reduced
pressure, produced approximately 180 flavor components (Werkhoff et al.,
1998). While the flavor of yellow passion fruit is determined by numer-
ous volatile components, the attractive tropical flavor note of ripe yellow
passion fruit is mainly attributed to the presence of trace levels of sulfur
volatiles in combination with other compounds. Compounds contribut-
ing significantly to the fruit aroma note are cis- and trans-γ-jasmine lac-
tone; δ-jasmine lactone, with a sweet, creamy, and fruity-flowery note;
trans-marmelolactone, with a strong fruity, floral, and quincelike aroma
(Tsuneya et al., 1980); cycloionone, with a fruity, sweet, floral, and woody
aroma (Nijssen et al., 1996); 4-hydroxy-2,5-dimethyl-3(2H)-furanone and
4-methoxy-2,5-dimethyl-3(2H)-furanone, with their characteristic fruity
notes; furaneol acetate; 3-mercaptohexyl pentanoate; 3-(methylthiol)pro-
pyl acetate, methionyl acetate, and geranyl acetol, with a sweet, fruity note;
and neryl acetol and (Z)-5-tangerinol, with sweet floral and woody notes,
respectively (Table 17.1).
559
P OST H AR V EST RI P ENING P H Y SIOLOG Y
560
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
Mangosteen Indian
(Garcinia subcontinent,
mangostana) Malaysia
(MacLeod
and Pieris,
1982)
(Continued )
561
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Mangaba Brazil
(Hancornia (Sampaio
speciosa and
Gomes) Nogueira,
2006)
(Continued )
562
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
Camu-camu Brazil
(Myrciaria (Franco and
dubia) Shibamoto,
2000)
Cupuacu Brazil
(Theobroma (Franco and
grandiflorum) Shibamoto,
2000)
Araca-boi Brazil
(Eugenia (Franco and
stipitata) Shibamoto,
2000)
Guabiju South
(Myrcianthes America
pungens) (Marin
et al., 2008)
(Continued )
563
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Guabiroba South
(Campomanesia America
xanthocarpa) (Marin
et al.,
2008)
(Continued )
564
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
565
P OST H AR V EST RI P ENING P H Y SIOLOG Y
are thought to contribute to the complexity of the Costa Rican guava flavor.
The fruity notes are ascribed to the many aliphatic esters, whereas floral
note is attributed to α-terpineol and linalool. The woody and spicy note is
ascribed to the (E)-β-caryophyllene and its epoxide, whereas the green
note is attributed to (Z)-3-hexenol and (Z)-3-hexenyl acetate.
566
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
567
P OST H AR V EST RI P ENING P H Y SIOLOG Y
568
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
pulp with a very attractive exotic flavor. Analysis of the volatile constitu-
ents of the fruit revealed linalool, 2-heptanone, and cis-3-hexenyl acetate
as the most potent flavor compounds (Alves and Jennings, 1979). However,
analysis of the free and bound flavor components of bacuri fruit with GC
and GC-MS using XAD-amberlite separation identified 75 compounds
(Boulanger et al., 1999). The compounds contributing significantly to the
aroma are the terpene alcohols, such as α-terpineol, hotrienol, nerol oxide,
nerol, geraniol, and linalool.
569
P OST H AR V EST RI P ENING P H Y SIOLOG Y
aqueous essence and the puree of fresh fruit using gas chromatography–
mass spectrometry (GC-MS) and gas chromatography–olfactometry
(GC-O) revealed 53 components in the essence and 38 in the fresh fruit,
respectively (Jordan et al., 2001). The compounds contributing significantly
to the melon aroma included 2-methyl-3-buten-2-ol, 2,3-butanediol, methyl-
3-phenyl propionate, ethyl-3-phenyl propionate, and ethyl-3-(methylthio)
propionate.
570
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
571
P OST H AR V EST RI P ENING P H Y SIOLOG Y
hexanoate and methyl hex-2-enoate were the two most abundant compo-
nents of soursop.
572
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
573
P OST H AR V EST RI P ENING P H Y SIOLOG Y
blend of volatiles that constitute the aroma. Often, a single fruit emits a
large spectrum of esters; for instance, more than 100 different esters have
been detected in ripe strawberry fruit (Zabetakis and Holden, 1997). Most
volatile esters have flavor characteristics described as fruity (Burdock,
2005). However, they not only are produced and emitted by fruit but also
are part of floral scents (Dudareva et al., 1996). Formation of volatile esters
is not restricted to the plant kingdom and is also common in microorgan-
isms such as yeast and fungi.
The metabolic pathways for volatile biosynthesis in fruits are not fully
understood. More than 80% of the volatiles produced by ripe strawberry
and melon are esters (Dirinck et al., 1989). Therefore, production of these
compounds has been the major focus of research in the past. Despite the
diversity of volatile compounds among these fruits, the basic biosynthetic
pathways may be similar. The metabolism of fatty acids and branched
amino acids may serve as a precursor for the biosynthesis of aroma vola-
tiles in fruits (Perez et al., 2002; Fellman et al., 2003). Fatty acids play a
major role in ester synthesis. According to Rowan et al. (1999), straight-
chain ester volatiles in whole fruit arise predominantly by β-oxidation of
fatty acids. It is widely assumed that lipoxygenase (LOX) may contribute
to the breakdown of long-chain fatty acids to C 6 aldehydes, which are con-
verted to alcohols by aldehyde dehydrogenase (Rowan et al., 1999; Fellman
et al., 2003). The alcohols are subsequently converted to hexyl and hexenyl
esters (Shalit et al., 2001; Perez et al., 2002).
Such a pathway has been demonstrated by Wang et al. (1996), who
altered the levels of fatty acids in transgenic tomato (Lycopersicon esculentum)
fruit, leading to dramatic changes in the aroma profile. Ripening-related
processes, such as changes in cell wall composition, might also contribute
intermediates to ester biogenesis. In tomato, evidence was provided that
the activity of pectin methyl esterase (which catalyzes demetoxylation of
pectin) regulates levels of methanol in the fruit (Frankel et al., 1998). The
accumulating methanol may then be utilized to generate methyl esters in
ripe fruit.
Branched amino acids are important substrates for branched-chain
volatiles such as 2-methyl butyl acetate and ethyl-2-methyl butanoate. This
pathway was shown to be present in both apple and strawberry (Perez et al.,
2002). For example, in strawberry, the amino acid alanine has been impli-
cated in the formation of ethyl esters during ripening (Perez et al., 1992).
However, the levels of amino acid precursors could not explain, in all cases,
the formation of the corresponding ester, and it has therefore been sug-
gested that the selectivity of enzymes preceding alcohol acyl transferase
(AAT) in the biosynthetic pathway also plays a role in determining ester
composition (Song and Forney, 2008). The enzyme that is responsible
for the final step of ester formation is AAT, which combines alcohols and
574
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
575
P OST H AR V EST RI P ENING P H Y SIOLOG Y
Cytosol / ER / Peroxisomes
Pyruvate
Acetyl-CoA +GA-3P
AACT DXS
AcAc-CoA DOXP
HMGS DXR
HMG-CoA MEP
MEP MCT
MVA HMGR PATHWAY
CDP-ME
PATHWAY Mevalonate (MVA)
CMK
MVK
CDP-ME2P Isoprene
Mevalonate-5-phosphate Plastid
MDS
PMK ISPS
ME-2,4cPP
Mevalonate-5-diphosphate
HDS
MVD HMBPP
IDI HDR HDR
IDI
DMAPP IPP IPP DMAPP
IDI
GGDS FDS
GDS IPP DMAPP GDS
FDS
GGPP
ria
Mono terpenes
Mi
Sesqui Sesqui
terpenes Sesquiterpenes terpenes
terpenes
576
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
IPP and DMAPP to produce geranyl diphosphate (GPP) (C10), the precur-
sor of monoterpenes.
577
P OST H AR V EST RI P ENING P H Y SIOLOG Y
H2 O
O NH2 O
alliinase S
S R OH S R Thiosulfinates
R COOH R S
S-alk(en)yl cysteine sulfoxides Sulfenic acid
(e.g. methiin, ethiin, butiin, alliin intermediates
isoalliin, propiin)
S O O
S
S R
R S
R
S R S R
S S R S
S R S R
S
C
Epithionitriles N
578
BIOS Y NT H ESIS O F F LA V OR AN D AROMA C OM P OUN D S
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Chapter 18
Impact of Postharvest
Technologies on the
Flavor of Fresh Fruits
and Vegetables
Charles F. Forney
Agriculture and Agri-Food Canada,
Kentville, Nova Scotia, Canada
Abstract 586
18.1 Introduction 587
18.2 Flavor of Fruits and Vegetables 588
18.2.1 Sensory Assessment 588
18.2.2 Chemical Flavor Constituents 589
18.3 Mechanism of Flavor Change 590
18.3.1 Metabolic Changes 591
18.3.2 Diffusional Changes 592
18.4 Impact of Postharvest Technologies 593
18.4.1 Ripening Manipulation 593
18.4.2 Temperature and Cold Storage 595
18.4.3 Controlled Atmosphere Storage 597
18.4.4 Packaging 599
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Abstract
The flavor of fruits and vegetables is complex and dynamic and is affected
by many pre- and postharvest factors. Desirable flavor is a critical factor
that can determine consumer satisfaction and thus sustainability of mar-
kets. While postharvest technologies are designed to prevent decay and
breakdown and maintain good appearance of fresh produce, their effect
on flavor is often neglected. Flavor is the human perception of a complex
combination of volatile, nonvolatile, and structural components contribut-
ing to appearance, aroma, taste, and texture. Volatile compounds contrib-
uting to flavor consist of diverse chemistries, including esters, terpenes,
alcohols, aldehydes, ketones, lactones, and sulfur compounds. Nonvolatile
compounds contributing to flavor include sugars, acids, and phenolics.
Changes in flavor can result from metabolic changes in flavor compounds,
diffusional loss of volatile flavor compounds, or gains of compounds from
the storage environment or treatments. Harvest maturity and posthar-
vest manipulation of ripening are important for optimizing flavor. The
postharvest environment, including temperature, atmosphere modifica-
tion through controlled atmosphere (CA) storage, modified atmosphere
packaging (MAP) or coatings, and the application of various postharvest
treatments, can preserve, enhance, or degrade product flavor. Reduced
temperatures can slow metabolic changes and diffusional loss of flavor
compounds, but also may inhibit normal ripening and cause flavor loss in
some chilling-sensitive commodities. Low oxygen atmospheres can inhibit
flavor synthesis or induce anaerobic atmospheres that can cause fermenta-
tion, which can result in off-flavors. Cutting fresh produce to add value and
consumer convenience can alter flavor by inducing the production of sec-
ondary flavor compounds, altering metabolism, and increasing diffusional
losses. A variety of postharvest treatments, such as heat, irradiation, ozone,
and chemical fumigation, are used to reduce decay, eliminate quarantine
pests, ensure product safety, and prolong storage life. These treatments
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18.1 Introduction
Flavor is an important characteristic determining quality and consumer
satisfaction of fresh fruits and vegetables. However, the flavor of fruits
and vegetables often does not meet the expectation of consumers. Initial
purchases are typically based on product appearance and firmness, while
repeat purchases depend on internal quality factors such as texture and
flavor (Baldwin, 2002). Loss of flavor that occurs during postharvest han-
dling, storage, and marketing of fresh fruits and vegetables often precedes
the loss of visual quality (Gorny, 2005). Therefore, ensuring good flavor
is critical to increase consumption and maintain and expand markets for
fresh produce.
A wide array of technologies have been developed to prolong the mar-
ket life of whole and fresh-cut produce. However, due to the diversity of
fruits and vegetables, postharvest technologies tend to be commodity spe-
cific in order to accommodate physiological and morphological differences.
Diversity among fresh produce is reflected in the variability of factors, such
as size, shape, firmness, cuticle thickness, transpiration rate, maturity, rip-
ening physiology, chilling sensitivity, metabolic activity, and susceptibility
to decay. These factors dictate how the product must be handled to opti-
mize market life and quality and will determine the appropriate postharvest
technology required. As a result, a wide range of strategies and technolo-
gies to prolong market life have been developed. Manipulation of product
maturity and ripeness can be achieved through harvest timing, as well as
a variety of postharvest treatments that can inhibit or stimulate ripening.
Proper precooling and postharvest temperature management slow meta-
bolic activity and product deterioration. Atmosphere modification through
controlled atmosphere (CA) technologies or modified atmosphere packag-
ing (MAP) during storage, transportation, and marketing can slow senes-
cence, inhibit decay, and reduce dehydration. Edible coatings can act as
diffusional barriers to gas exchange and have effects on produce market
life similar to those seen with CA and MAP. A variety of chemical and physi-
cal treatments have also been developed to reduce decay, inhibit microbial
growth, and slow deterioration.
Most of these postharvest technologies have been developed and opti-
mized to maintain visual quality and extend shelf life, while consideration
of organoleptic quality characteristics that are critical for consumer satis-
faction has often been neglected. Postharvest flavor change can be affected
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IM P A C T O F P OST H AR V EST TE C H NOLOGIES
responsible for flavor can generate a lot of data on sugars, acids, sugar/
acid ratios, and aroma volatiles, these data mean little if they are not related
to the human perception of flavor. Sensory tests are designed to answer a
variety of different questions, which include basic information about flavor,
differences between samples or products, magnitude of differences, and
consumer preference or “likeability.” Sensory tests can run from objective
to subjective (Baldwin et al., 2007).
Fundamental flavor information can be determined using a trained
panel to determine different flavor aspects and descriptors. Trained panels
can objectively rate the intensity of different flavor aspects and, in this way,
act like an instrument providing objective measures. Trained panels are
used to understand what comprises flavor, texture, and aroma in a quan-
titative manner (Meilgaard et al., 1999). Consumer panels, on the other
hand, are large, untrained panels that can rate the “likeability” of an attri-
bute or the “preference” of one product over another, or rank samples for a
particular attribute, such as sweetness (Baldwin et al., 2007). Flavor per-
ception is influenced by the socioeconomic, ethnic, and geographical back-
ground of the Panellists, further complicating sensory flavor e valuation
(Baldwin, 2002).
Sensory studies for fruits and vegetables have been used to iden-
tify optimal harvest maturity (Tandon et al., 2000), evaluate flavor qual-
ity in breeding material (Baldwin et al., 1998), determine optimal storage
(Maul et al., 2000) and handling conditions (Bett et al., 2001; Hagenmaier
and Shaw, 2002; Bai et al., 2003), assess effects of disinfestation or pre-
conditioning techniques on flavor quality (Bai et al., 2004; Plotto et al.,
2006), and measure flavor quality over the postharvest life of the product
(Baldwin, 2002).
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provide insights into how product flavor can be preserved and, in some
cases, enhanced during postharvest handling and marketing.
The two primary mechanisms of flavor change in fresh produce are
metabolic and diffusional (mass transfer) (Voilley and Souchon, 2006).
Both of these processes occur during development in the field as well as
postharvest. Metabolic changes involve the synthesis or catabolism of
flavor compounds or the synthesis of off-flavor compounds. Physiological
factors, such as maturity and ripeness and response to the growing and
postharvest environments, will dictate metabolic processes that affect both
volatile and nonvolatile flavor compounds. Preharvest factors affecting fla-
vor are diverse but include water availability (Baldwin et al., 2007), fertil-
ization (Randle and Lancaster, 2002), and chemical applications (Podoski
et al., 1997). The harvest maturity of fruits and vegetables has a large effect
on the postharvest flavor, with fruit harvested underripe having limited fla-
vor potential. For example, melon (Cucumis melo L.) maturity at harvest
affects the fruit flavor, with riper fruit having a sweeter aromatic taste than
underripe fruit even after postharvest ripening (Beaulieu et al., 2004).
Volatile compounds contributing to flavor are subject to diffusional changes
in which diffusion and mass transfer of volatile compounds into and out of
the commodity occur. Diffusion is determined by the volatility of the com-
pound, its concentration gradient, and diffusional barriers in the fruit or
vegetable or packaging materials. The role of each of these mechanisms
depends on the product, the environment in which it is held, and the treat-
ments to which it is subjected.
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ripe, but had a higher acid content. In addition, the postharvest ripened
fruit had higher total volatiles than fruit harvested ripe, with the highest
levels of monoterpenes, sesquiterpenes, and aromatic compounds, whereas
the ripe harvested fruit had the highest concentration of esters (Lalel et al.,
2003). These compositional differences suggest a difference in fruit flavor.
Climacteric fruits are responsive to the ripening hormone ethylene,
which stimulates ripening and flavor development when endogenously
produced or exogenously applied. Therefore, a common strategy is to har-
vest climacteric fruit, such as banana (Musa × paradisiaca L.) and tomato,
unripe and firm. Handling and shipping firm fruit reduces damage that can
occur when shipping fruit long distances. When fruit reaches its market,
ripening can then be stimulated with ethylene to achieve an acceptable fla-
vor, texture, and eating quality (Lèlievre et al., 1997). Commercial ripening
rooms that can control ethylene concentration, temperature, and humidity
are typically used for this purpose. The flavor quality reached under these
conditions is dependent on the harvest maturity and fruit species. Many
underripe fruits may not be able to obtain full flavor through postharvest
ripening. In tomato, fruit harvested immature green produced ripe fruit
with lower aroma volatile levels than mature-green harvested fruit, while
fruit harvested at the table-ripe stage had the highest intensity of flavor
components (Watada and Aulenbach, 1979). Ethylene treatment of toma-
toes was reported to speed ripening but did not affect flavor when compared
to fruit ripened without exogenous ethylene (Kader et al., 1978). The matu-
rity stage at which apple fruit are harvested also affects ester formation
and acid content during postharvest ripening (Fellman et al., 2000), thus
affecting fruit flavor. Flavor change during postharvest ripening of apples
during cold storage varies substantially among cultivars; therefore, the rate
and nature of flavor change can be cultivar specific (Corollaro et al., 2013).
Inhibition of ethylene action has been effective in prolonging the stor-
age life of apples and other fruits and vegetables. This has been achieved
through the application of 1-methylcyclopropene (1-MCP), which blocks
ethylene-induced ripening and other physiological processes (Blankenship
and Dole, 2003). Application of 1-MCP can be achieved through posthar-
vest fumigation (SmartFresh™ ) or, more recently, as a preharvest spray
(Harvista™ ). These treatments are effective in maintaining fruit firmness
and inhibiting acid loss, color change, and volatile synthesis (Blankenship
and Dole, 2003). In addition, they are effective in delaying senescence and
extending the storage life of apples and many other fruits and vegetables
(Watkins, 2006). The 1-MCP-induced reduction in volatile production
has been reported in many climacteric fruits, including banana (Golding
et al., 1998), plums (Prunus domestica) (Abdi et al., 1998), apples (Fan
and Mattheis, 1999; Mattheis et al., 2005), and pears (Pyrus communis L.)
(Argenta et al., 2003). However, in apples, this reduction in volatile produc-
tion by 1-MCP may not be perceived as negatively impacting consumer
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preference. ‘Anna’ and ‘Gala’ apple fruit treated with 1-MCP had reduced
ester production and perceived fruity aroma, but were preferred over con-
trols, possibly due to textural differences (Lurie et al., 2002; Maria et al.,
2007). Treatments with 1-MCP were also shown to inhibit ethylene-induced
bitterness in fresh carrots, which is caused by accumulation of isocouma-
rin-6-methyoxymellein (Fan and Mattheis, 2000; Song et al., 2003).
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failure to ripen normally, and loss of flavor (Saltveit and Morris, 1990).
When ripe tomato fruit were held for 2 days at the chilling temperatures
of 5°C, 10°C, and 12.5°C, their ripe aroma, sweetness, and tomato flavor
were rated by a sensory panel as significantly lower than those of tomatoes
held at 20°C (Maul et al., 2000). Similarly, when mature-green or light pink
tomatoes were held at 5°C for 7 days prior to ripening at 20°C, they were
found to be more acidic than similarly ripened fruit that were not held at 5°C
(Kader et al., 1978). These reductions in sensory ratings were accompanied
by reductions in volatile and nonvolatile flavor components. Some cultivars
of plums are chilling sensitive and will develop internal browning and poor
flavor if held at 1°C–5°C (de Kock and Taylor, 2014). However, to develop
optimum flavor, they must be cooled to –0.5°C for 3–10 days prior to ripen-
ing at 7.5°C. Similarly, fruit of chilling-sensitive peach cultivars that were
held at 5°C followed by 3 days at 20°C had a reduction in the lactones that
were shown to be aroma impact compounds of peach (Xi et al., 2012). This
chilling-reduced production of lactones was partially ameliorated by hold-
ing fruit at 0°C rather than 5°C, or by subjecting them to a low-temperature
conditioning treatment (8°C for 3 days followed by storage at 5°C).
In addition to the effects on metabolic activity, the volatility of volatile
flavor compounds increases as temperature increases. Therefore, elevated
temperatures can speed up both quantitative and qualitative changes in
flavor compounds, thus affecting flavor. To preserve flavor, it is thought
that the lowest acceptable storage temperature is generally best, espe-
cially when fruit are harvested at optimal eating quality. However, with the
dynamic and complex nature of flavor components, changes in temperature
can affect rates of both synthesis and loss of flavor compounds, and these
effects can be compound specific. In stored strawberry fruit, a change in
ester composition was found to be temperature dependent. When straw-
berry fruit were stored at 1°C, concentrations of the esters ethyl butanoate
and ethyl hexanoate increased, while those of methyl butanoate and methyl
hexanoate increased during storage at 15°C (Forney et al., 2000b). The tem-
perature-dependent effects on volatile composition could affect fruit fl avor
characteristics in strawberry as well as other fruits and deserve further
assessment.
Extended cold storage can also result in the development of off-
flavors, which are often described as old or stored. This may be the result
of the loss of natural flavor compounds or the migration of off-odor com-
pounds into the product from the storage environment. It is recognized
that flavor can be lost during the storage of fresh fruits and vegetables,
and that this may occur prior to a decline in product appearance (Baldwin
et al., 2007). The appearance of fresh strawberries was maintained for
7–9 days when stored at 5°C, but acceptable flavor was lost several days
sooner, depending on cultivar (Pelayo et al., 2003). Similar results were
reported for tomato (Maul et al., 2000) and mandarins (Tietel et al., 2012).
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18.4.4 Packaging
The use of MAP has increased in recent years with the increase in mar-
keting of fresh-cut fruits and vegetables (Forney and Yaganza, 2011).
Proper package design can maintain beneficial atmospheres and prolong
the market life of these highly perishable products by minimizing dehy-
dration, reducing the browning of cut surfaces, reducing decay and micro-
bial growth, and preventing product contamination (Forney et al., 2007).
Packaging can also affect product flavor through the effects of atmosphere
composition and volatile diffusion (Forney, 2008).
Atmosphere composition can affect fresh-cut produce flavor in a
manner similar to that seen in CA storage of whole fruits and vegetables.
However, due to their high perishability and the resulting short market life,
fresh-cut produce may tolerate more extreme atmospheres. Sliced ‘Gala’
apples packaged in microperforated bags that maintained a 16 kPa O2/6
kPa CO2 atmosphere had better flavor than slices packaged in a solid mul-
tilayered polyolefin film bag that maintained a 2–3 kPa O2/6 kPa CO2 atmo-
sphere (Cliff et al., 2010). The apple slices in the microperforated bags had
higher fruity aroma and flavor and perceived sweetness, which were asso-
ciated with higher volatile concentrations of estragole and straight-chain
esters. These flavor differences could be a result of the differences in O2
concentration in the package or interactions of the fruit volatiles with the
polymers in the packaging.
Since atmosphere composition is not actively controlled in MAP, pack-
ages run a greater risk of developing anaerobic atmospheres and objection-
able off-odors if exposed to temperature abuse (Forney and Yaganza, 2011).
Warming of packages increases product respiration rate, which can deplete
package O2 and create anaerobic conditions. The resulting of off-odor
development is dependent on product physiology. While broccoli produced
substantial levels of the malodorant methanethiol when held under anaero-
bic atmospheres, other fresh-cut Brassica vegetables varied substantially
in their production of methanethiol and other malodorants (Forney and
Jordan, 1999). Fresh-cut cauliflower (Botrytis group) florets, Chinese cab-
bage (Pekinensis group), and kohlrabi (Gongylodes group) tubers, while
producing ethanol, only produced trace amounts of methanethiol and other
malodorants. Therefore, these vegetables pose less of a risk of developing
objectionable odors if exposed to abusive temperatures and their result-
ing anaerobic atmospheres during marketing. Fresh-cut iceberg lettuce is
typically packaged in low O2 atmospheres to prevent cut-edge browning.
However, these atmospheres often induce fermentation, resulting in the
production of ethanol (Cameron et al., 1995; Smyth et al., 1998) and other
volatiles, including hexanal, cis-3-hexen-1-ol, β-elemene, ethyl acetate, and
dimethyl sulfide (Tudela et al., 2013). The resulting off-odor and -flavor,
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which are typically masked by salad dressing when consumed, have been
accepted by consumers. However, other technologies to prevent cut-lettuce
browning are needed to avoid this undesirable off-flavor and provide con-
sumers with a more desirable fresh flavor. Anaerobic off-odors associ-
ated with increased levels of ethanol, acetaldehyde, and ethyl esters are
typically found in fruit held in injurious atmospheres, as seen in packaged
pomegranate arils (Caleb et al., 2013).
To mitigate anaerobic effects, active MAP approaches have been tried
where initial atmospheres of super atmospheric O2 were established. When
‘Camarosa’ strawberry fruit was held at 2°C in MAP with an initial atmo-
sphere of 80 kPa O2, acceptable flavor was not maintained during 12 days of
storage (Allende et al., 2007). During this time, package O2 concentration
was maintained above 65 kPa, but CO2 concentrations increased to about
20 kPa, which appears to have induced anaerobic metabolism and develop-
ment of associated off-flavors.
Volatile flavor compounds in fresh produce and other food products
can interact with polymer packaging materials in several ways (Forney and
Yaganza, 2011). If the polymer acts as a barrier to the loss of flavor volatiles
from the package headspace, the resulting reduced concentration gradient
between the product and the surrounding atmosphere reduces diffusional
loss from the product. Most packaging materials provide a good barrier to
diffusional loss assuming the package is well sealed and leak-free. Fruity
esters in fresh-cut cantaloupe sealed in polyethylene terephthalate (PET)
bowls increased as much as 40% after 7 days of storage at 4°C (Beaulieu,
2006), whereas a rapid loss of these esters was observed under conditions
conducive to diffusional loss (Beaulieu, 2007).
Polymer packaging may also have a direct interaction with volatile
compounds in the headspace, resulting in sorption or permeation, which
removes the volatiles from the package atmosphere and ultimately the
packaged product (Brody, 2002; Forney and Yaganza, 2011). The widely
used packaging polymers polypropylene and polyethylene have a high
affinity for volatile compounds due to their nonpolar properties, whereas
polyesters, including the bio-based polymer polylactic acid (PLA), are
more polar and have a weaker affinity for flavor volatiles (Sajilata et al.,
2007). When diced onions were sealed in PLA containers, the onions
retained a fresh-cut onion aroma for 2 weeks, while those sealed in poly-
ethylene bags lost this fresh aroma within the first 2 days (Forney et al.,
2012a). This loss of aroma was associated with a rapid loss of sulfur-
containing aroma volatiles from the onions and the package headspace.
Fresh blueberry fruit stored in sealed PLA containers were found to have
better flavor than fruit held in vented PET containers (Almenar et al.,
2010). However, no significant effects on aroma volatiles were found
between the two package types; rather, greater water loss in the vented
PET packaged fruit appeared to be responsible for the poorer flavor.
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18.4.6.1 Cutting
The fresh-cut processing of whole fresh produce can cause both metabolic
and diffusional flavor changes. Secondary aroma volatiles are formed in a
number of fruits and vegetables as a result of cellular disruption. For exam-
ple, as described previously, the cutting of onions produces thiopropanal
S-oxide, prop(en)yl thiol, aldehydes, and a variety of sulfides as a result
of the reaction of S-alk(en)yl cysteine sulfoxides with alliinase that occurs
due to cellular rupture during cutting (Järvenpää et al., 1998). Fresh onion
aroma is maximized after about 30 min, but continues to change due to
further chemical reactions of the volatiles in the headspace. New aroma
volatiles are also formed as a result of lipid oxidation that can occur as a
result of cutting. When tomatoes are cut, an increase of many compounds,
including cis-3-hexenal, hexanal, and 1-penten-3-one, occurs that impacts
the product’s flavor (Baldwin et al., 2000).
In addition to the postharvest formation of flavor compounds follow-
ing cutting, flavor loss has been associated with fresh-cut processing. In
carrots, the loss of aroma was greater when cut with a dull blade than with
a sharp blade (Barry-Ryan and O’Beirne, 1998). Carrots cut with the dull
blade also developed off-odors more rapidly, which may have been a result
of injurious levels of CO2 that developed more rapidly in packages of dull-
blade cut carrots. Cantaloupe cut with a dull cork bore also developed more
off-odors and had a slightly higher level of ethanol than fruit cut with a
sharp cork bore, although both had a similar loss of aroma (Portela and
Cantwell, 2001). Lamikanra and Richard (2002) suggested that the rapid
loss of esters in cut melon tissue is a result of “stress induced enzymatic
hydrolysis of esters.” However, activity of esterase, an enzyme that breaks
down esters, decreased 24 h after cutting (Lamikanra and Watson, 2003),
and there was no relationship observed between ester loss and synthesis of
fatty acids, aldehydes, or alcohols, which are esterase products (Lamikanra
and Richard, 2002). Subjective aroma ratings of honeydew and cantaloupe
pieces were found to decline after 6 days when the cut fruit was held at 5°C
in permeable cheesecloth-covered jars (Portela and Cantwell, 1998, 2001).
Sensory tests also determined that fresh-cut orange slices stored at 4°C in
a closed plastic box that maintained ambient atmospheres lost flavor during
5 days of storage, which was associated with a loss of flavor components
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in the fruit (Rocha et al., 1995). While flavor loss is commonly associated
with cutting, definitive studies that determine the underlying mechanisms
responsible are limited.
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50% (Forney et al., 2011). In contrast, the ester content of ‘Cortland’ apples
subjected to the same treatments was reduced 30%–65%. The effects of heat
treatments on more mature fruit and following longer storage times tended
to decrease fruit ester content compared to untreated control fruit. Fruit
with the greatest loss of esters also developed skin and flesh browning. In
tomato fruit, a 5 min 52°C hot water dip of mature-green fruit resulted in
enhanced sensory profiles of both ‘TastiLee’ and ‘Florida-47’ fruit follow-
ing ripening at 20°C when compared to fruit dipped in 25°C water (Loayza
et al., 2012). Heat-treated fruit were considered soft with a complex sensory
profile, whereas fruit from the control were firm, sour, and bland. A 2 min
dip of sour cherries in 40°C water preserved color and texture during 17
days of storage at 4°C, but was detrimental to acceptable flavor (Ravanfar
et al., 2012). On the other hand, grapes that were subjected to a 5 min 55°C
water dip had no detrimental effect on flavor while slowing decay and prod-
uct deterioration (Sabir and Sabir, 2013).
Although many reports focus on the positive response of horticul-
tural commodities to heat treatment, high temperatures with long dura-
tions may cause tissue damage and increase decay development (Klein
and Lurie, 1992; Song et al., 2001; Fan et al., 2005a). Heat stress induces
fermentation, which may be transient in mild treatments or sustained in
more severe injurious treatments. This is associated with the production
of ethanol, acetaldehyde, and ethyl esters such as ethyl acetate. Exposure
of oranges for more than 200 min to 48.5°C forced air resulted in ethanol
synthesis that was associated with perceptible off-flavors (Obenland et al.,
1999). Similarly, treatments of mangoes at 46°C and 48°C forced air for
3–5 h resulted in acetaldehyde and ethanol accumulation (Mitcham and
McDonald, 1993). Hot water dips of 1–3 min at 52°C delayed yellowing
of broccoli held at 20°C (Forney, 1995). However, the 3 min dip caused a
“green floral” off-odor, which was associated with the production of ethanol,
cis-3-hexenol, and dimethyl trisulfide (Forney and Jordan, 1998).
18.4.6.3 Irradiation
Irradiation with ultraviolet (UV) and ionizing radiation has been used
to eliminate quarantine pests, reduce microbial load, and in some cases,
induce decay resistance. Exposure of thin slices of pineapple to UV light
caused a twofold increase of the sesquiterpenes copaene and ocimene,
which have antimicrobial activity (Lamikanra and Richard, 2004) and
woody and floral aroma notes. UV treatments also increased the produc-
tion of the terpenoids ß-ionone, ß-ionone epoxide, and dihydroactinidiolide
in fresh-cut cantaloupe, and it was suggested that these terpenoids could
be detrimental to fresh melon flavor (Beaulieu, 2007). Lamikanra et al.
(2005) reported fresh-cut cantaloupe treated with UV light (1180 mW/cm 2
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for 4 min) smelled less rancid and more fruity after 6 days of storage than
control fruit. The reduction in rancid smell was associated with a reduc-
tion in lipase activity in the treated fruit. Ionizing radiation treatments
have not been shown to have significant effects on fresh-cut product fla-
vor. Pluots (Prunus domestica × Prunus armeniaca) treated with up to 1.4
kGy of gamma irradiation were reported to have a quality similar to that
of untreated controls (Duvenhage et al., 2012). In strawberry fruit, treat-
ments of 2.0 and 2.5 kGy reduced fruit decay while maintaining acceptable
flavor (Silva et al., 2009). Romaine lettuce sealed in laminate PE bags and
irradiated with 0.15 or 0.35 kGy of gamma radiation developed off-odors
after 11 days of storage at 4°C, but the irradiation treatment had no effect
on off-odor induction (Pariasca et al., 2001).
18.4.6.4 Ozone
Ozone (O3) is a highly reactive form of oxygen with strong antimicrobial
properties. Antimicrobial treatments have been developed using ozone in
air or dissolved in water to reduce the decay of fresh produce and sanitize
water and equipment used in postharvest handling. Ozone and its reactive
products may induce many physiological changes in plant tissues, and under
high doses, cellular damage may occur, causing altered membrane perme-
ability, premature senescence, loss of pigments, and surface discoloration.
The use of ozone for postharvest sanitation and decay control of fresh fruits
and vegetables during handling and storage has been investigated for com-
mercial application on a wide variety of commodities (Forney, 2003). Ozone
has been shown to have some effect on the decay of carrots. Exposure of
carrots to 1 μl/L gaseous ozone at 10°C for 2–4 days induced resistance to
Botrytis cinerea (gray mold) but not to Sclerotinia sclerotiorum (white mold)
(Song et al., 2003; Forney et al., 2007). These treatments altered both volatile
and nonvolatile flavor compounds in the carrots. Concentrations of ethanol
and hexanal were 43 and 11 times greater, respectively, than controls imme-
diately after the 4-day exposure to 1 μl/L ozone. Terpenes, which contribute
to carrot flavor, also increased following treatment. However, these increases
in volatiles were transient, and concentration returned to control levels after
8 weeks of storage. The 4-day 1 μl/L ozone treatment also increased glucose
and fructose concentrations, reduced sucrose concentrations, and induced
the production of the phytoalexin isocoumarin-6-methoxymellien to concen-
trations that could cause bitterness in the carrots. Treatment of the carrots
with 1-MCP prior to ozone treatment prevented changes in sugars and
induction of isocoumarin-6-methoxymellien and Botrytis resistance, indicat-
ing that these effects were mediated by ozone-induced ethylene.
Ozone treatments can also affect the flavor of fruits. Ozone treatments
of 2.5 μl/L for 4 days followed by storage at 26°C for 12 days reduced the
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decay of papaya fruit while increasing the sensory ratings of sweetness and
texture above those of untreated controls (Ali et al., 2014). In tomato fruit
subject to 7 days of 0.15 mol/mol ozone enrichment, fruit were perceptibly
sweeter, retained their firmness, and were preferred over fruit subject to
traditional storage and transit practices (Tzortzakis et al., 2007). In straw-
berries, ozone treatments of 0.35 μl/L for 3 days at 2°C decreased the pro-
duction of esters by 35% compared to controls (Pérez et al., 1999). Ethanol
concentrations also decreased in strawberries following 2 and 4 additional
days in air at 20°C, and there was no effect on ethyl acetate or acetalde-
hyde concentrations, suggesting that ozone prevented the development of
off-flavors. Sucrose, glucose, and fructose were reduced by about 20% in
ozone-treated fruit, but after an additional 2 or 4 days in air at 20°C, these
differences were less apparent and the ozone-treated fruit actually had
higher concentrations of sucrose (Pérez et al., 1999). In cranberries, ozone
induced a faint but pleasant floral aroma (Norton et al., 1968). Treatments of
1.0 μl/L ozone for 24 h had no effect on the soluble solid content of peaches
(Ridley and Sims, 1967). Schomer and McColloch (1948) found that long-
term exposure of apples to 3.25 μl/L ozone resulted in a loss of flavor.
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FRUIT & VEGETABLE PRODUCTS
Postharvest Ripening
Physiology of Crops
Postharvest Ripening Physiology of Crops is a comprehensive interdisciplinary
reference source for the various aspects of fruit ripening and postharvest be-
havior. It focuses on the postharvest physiology, biochemistry, and molecular
biology of ripening and provides an overview of fruits and vegetables, including
chapters on the postharvest quality of ornamental plants and molecular biology
of flower senescence.
It describes various developments that have taken place in the last decade
with respect to identifying and altering the function of ripening-related genes.
Taking clues from studies in grape and tomato as model fruits, the book reviews
a few case studies and gives you a detailed account of molecular regulation of
fruit ripening, and signal transduction and internal atmospheres in relation to
fruit ripening. It also presents an overview of methods utilized in fruit proteomics,
as well as a global proteome and systems biology analysis of fruits during rip-
ening, and discusses the basics of dormancy, its molecular and physiological
basis, and methods to break the dormancy.
K24683
ISBN: 978-1-4987-0380-2
90000
9 781498 703802