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Unravelling chemical priming machinery in plants: the
role of reactive oxygennitrogensulfur species in
abiotic stress tolerance enhancement
Chrystalla Antoniou1,3, Andreas Savvides1,3, Anastasis Christou2
and Vasileios Fotopoulos1
Abiotic stresses severely limit crop yield and their detrimental
effects are aggravated by climate change. Chemical priming is
an emerging field in crop stress management. The exogenous
application of specific chemical agents before stress events
results in tolerance enhancement and reduction of stress
impacts on plant physiology and growth. However, the
molecular mechanisms underlying the remarkable effects of
chemical priming on plant physiology remain to be elucidated.
Reactive oxygen, nitrogen and sulfur species (RONSS) are
molecules playing a vital role in the stress acclimation of plants.
When applied as priming agents, RONSS improve stress
tolerance. This review summarizes the recent knowledge on the
role of RONSS in cell signalling and gene regulation
contributing to abiotic stress tolerance enhancement.
Addresses
1
Department of Agricultural Sciences, Biotechnology and Food Science,
Cyprus University of Technology, Lemesos, Cyprus
2
Agricultural Research Institute, Ministry of Agriculture, Rural
Development and Environment, Nicosia, Cyprus
Corresponding author: Fotopoulos, Vasileios
(vassilis.fotopoulos@cut.ac.cy)
3
These authors contributed equally to this work.
Current Opinion in Plant Biology 2016, 33:101107
This review comes from a themed issue on Cell signalling and gene
regulation
Edited by Kimberley Snowden and Dirk Inze

http://dx.doi.org/10.1016/j.pbi.2016.06.020
1369-5266/# 2016 Elsevier Ltd. All rights reserved.

Introduction
Abiotic stresses severely limit crop yield and their detrimental effects are aggravated by climate change worldwide [1]. Accordingly, to maintain global food security,
stress tolerance improvement in plants is attracting much
attention during the last decades. Different methodologies have been employed to enhance stress tolerance;
some are particularly time-consuming (e.g. conventional
breeding) and others are currently unacceptable in many
countries around the world (e.g. plant genetic modification) [2]. Chemical priming is a rapidly emerging field in
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plant stress physiology and crop stress management [3].


Plants treated with certain natural or synthetic compounds (i.e. chemical agents) prior stress events show
enhanced tolerance when exposed to sub-optimum conditions (e.g. drought, heat, salinity, heavy metals; [3]).
Stress impacts on plant growth and yield in pre-treated
(i.e. primed) plants are remarkably reduced in comparison
with non pre-treated (i.e. non-primed) plants (Figure 1;
[3]). In addition, chemical priming may be considered as
a cost-effective (i.e. low cost chemical compounds used at
low concentrations) methodology [3], suggesting a low
cost-benefit ratio if applied in crop stress management.
The critical time period between the exogenous application of a chemical agent and the time point at which the
primed plant is optimally prepared to face an abiotic stress
event is called the primed state (PS; Figure 1). The
enhanced tolerance in primed plants under stress is
obviously a result of biological procedures triggered during the PS, which are not well-understood yet (Figure 1).
Unravelling the PS events preceding enhanced tolerance
will eventually unlock the secrets underlying chemical
priming machinery.
Reactive oxygen (e.g. H2O2), nitrogen (e.g. NO) and
sulfur (e.g. H2S) species (RONSS) are molecules involved
in cell signalling and gene regulation during stress and
play a pivotal role in the stress acclimation of plants [4,5].
Previous studies focused on elucidating their signalling
role but also the cross-talk between them when plants are
exposed to biotic and abiotic stresses [4,6]. Over the last
years, a growing number of studies indicates that these
molecules or their donors (e.g. sodium nitroprusside
[SNP] for NO and sodium hydrosulfide [NaHS] for
H2S), when applied at low concentrations (mainly in
the range of 11000 mM), can be effectively used as
priming agents for empowering abiotic stress tolerance
mechanisms (Figure 1; [3]). This remarkable function
strongly suggests that RONSS act as signalling molecules
that trigger the biological procedures during the PS
necessary to achieve enhanced tolerance later under
abiotic stress conditions.
The pre-treatment of plants with chemical agents can
initiate a mild stress-like cue, similar to an acclimation
response [3]. Primed plants are better prepared to face
an abiotic challenge, while non-primed plants are usually
Current Opinion in Plant Biology 2016, 33:101107

102 Cell signalling and gene regulation

Figure 1

Non-primed
cell

Abiotic
stress

Normal
tolerance

Impact

Primed cell under stress

RONSS exogenous
application
Abiotic
stress

Primed
cell

Pronounced cell signalling & gene


regulation

Enhanced
tolerance
Impact

Osmoprotection

Primed cell

ROS
detoxification

SNP

Ionic homeostasis
Protein homeostasis

NO
NaHS

Cross-talk
H2O2

H2O2

H2 S

Abiotic
stress

Enhanced tolerance

Physiological
homeostasis

Enhanced
growth

Cell signalling & gene regulation

Stress-responsive
genes

PTMs

e.g. antioxidant, redox cell


homeostasis enzymes,
osmoprotectant biosynthetic
enzymes, polyamine
Gene
metabolic enzymes

e.g. tyrosine nitration,


S-nitrosylation,
carbonylation,
S-sulfhydration

regulatory
elements

e.g. transcription factors,


miRNAs, kinases
Current Opinion in Plant Biology

RONSS exogenous application enhances stress tolerance of plants later exposed to abiotic stress and ameliorates stress impacts. Exogenous
application of either H2O2 or SNP (NO donor) or NaHS (H2S donor) initially results in an endogenous accumulation of the chemical compound and
in changes of the endogenous levels of the other RONSS (i.e. cross-talk). A signalling stream is triggered resulting in cell signalling (e.g. posttranslational modifications; PTMs) and gene regulation of stress responsive genes and gene regulatory elements. After exposure to abiotic stress,
pronounced cell signalling and gene regulation events occur leading to enhanced tolerance mechanisms, such as osmoprotection, ROS
detoxification, ionic homeostasis as well as protein homeostasis. Primed plants have increased physiological homeostasis and enhanced growth
when compared with non-primed plants under stress.

initiating their protective responses simultaneously or


sometime after the abiotic stress occurrence resulting
in fatal costs (Figure 1). The events taking place during
the PS in RONSS-primed plants may not be identical to
those occurring during abiotic stress in non-primed plants
[7]. It is, therefore, necessary to elucidate the events
occurring during the PS but also during stress in primed
plants.
This review aims at bridging the recent knowledge on
RONSS signalling and interactions in the context of
chemical priming and opening new directions in research
for further unravelling the chemical priming machinery in
plants.
Current Opinion in Plant Biology 2016, 33:101107

Gene regulation and protein modifications


underlying the enhanced tolerance after
RONSS application
The survival advantage of RONSS-primed plants after
their exposure to stress compared with non-primed plants
has been mainly attributed to the signalling feature of these
molecules [4,6]. Exogenous application of NO, H2S as well
as H2O2 enhances plant tolerance by regulating the expression of several defence-related genes (listed in Suppl.
Table 1) before abiotic stress imposition, but also by more
pronounced gene expression levels when under stress
(Figure 1; [8,9]). This pool of transcripts could correspond
to genes encoding proteins directly involved in plant stress
protection, gene regulatory elements, such as transcription
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Priming plants with RONSS against abiotic stress Antoniou et al. 103

factors (TFs), and genes implicated in signal transduction


pathways (Figure 1; [10,11,12,13]). The level of gene
regulation and expression mainly depends on the priming
agent concentration [12,14], the duration between the
application and stress imposition [13,15], the part of the
plant on which the application took place [8,16], the plant
organ [8,17], and also the type of stress itself [18,19].

experimental evidence suggests a regulatory mechanism


with many similarities. Universal elements of priming
could be genes-coding antioxidant enzymes, TFs as well
as genes implicated in signal transduction networks (e.g.
kinases), redox homeostasis, and osmoprotectants biosynthesis (e.g. proline), which are regulated after the application of any of the three compounds.

Gene regulation before stress

Primary or secondary oxidative damage is caused by all


abiotic stresses, as a result of an accumulation of reactive
oxygen species (ROS) in the cell [22]. Primed plants have
the ability to better ameliorate the deleterious effects of
ROS by regulating the expression of genes encoding
proteins involved in redox cell homeostasis (i.e. ASCGSH cycle) and antioxidant machinery [9,23]. Transcriptional analysis of primed plants under non-stress and/or
stress conditions showed regulation of antioxidants genes
such as superoxide dismutase isoforms (Cu/Zn-SOD, MnSOD, Fe-SOD), xanthine oxidoreductase (XOR), alternative oxidase (AOX), catalase (CAT), ascorbate peroxidase
(APX) and glutathione reductase (GR) [8,9,15,23,24,25].
Regarding redox cell homeostasis, experimental evidence
indicated an up-regulation of ASC-GSH cycle biosynthetic genes (GDH, GCS and GS) in primed (with NaHS, SNP
or H2O2) strawberry plants under non-stress and stress
(salinity and heat) conditions [9,20,21]. Interestingly,
more pronounced gene up-regulation was recorded in
primed salt stressed compared with primed non
stressed plants [9]. This suggests that the exposure of
primed plants to stress amplifies the level of gene regulation already initiated in PS.

Several reports exist indicating whole genome transcriptional changes that happen shortly (i.e. within hours or
even minutes) after NO or H2O2 application. SNP infiltration in Arabidopsis leaves, revealed significant regulation
of genes encoding proteins implicated in stress response,
photosynthesis, cellular transport, disease resistance and
basic metabolism, within 10 minutes, 1 h and 3 h after
treatment [13]. Remarkably, the majority of these genes
(38%) was rapidly transcriptionally activated within
10 minutes, but the activation was short-lasting, whereas
8.5% and 29.6% of the transcripts showed increased levels
1 h and 3 h after treatment, respectively [13]. In another
study where SNP was applied in Arabidopsis roots, transcriptional analysis on the rosette revealed regulation of
several genes with diverse functions, such as transcripts
encoding proteins related to plant defence response, amelioration of ROS, iron homeostasis and cellular detoxification (e.g. ABC transporter, glutathione S-transferase; GST).
Interestingly, part of the regulated transcripts were genes
encoding TFs and genes implicated in signal transduction
pathways (e.g. mitogen activated protein (MAP) kinases;
[12]). Additionally, in the same study, the transcriptional
response of two SNP concentrations (0.1 mM and 1 mM)
was assessed and 162 genes showed a dose-dependent
expression increase [12]. In a similar report, transcriptional analysis of H2O2 pre-treated plants showed an induction of stress-responsive genes, TFs (e.g. CBFs, DDF1)
and detoxification related genes (e.g. cytochrome P450), but
also a suppression of some genes involved in plant growth
and development [11]. It is proposed that H2O2 is a global
regulator of transcriptional expression [11]. In regard with
H2S, which is the latest player to enter the field, the
available information is limited on the expression profile
of specific genes (i.e. stress-related genes). Application of
NaHS on Arabidopsis and strawberry plants before a
multitude of stress factors affected the expression of
stress-responsive genes, such as heat shock proteins (HSPs;
[20]), Salt Overly Sensitive (SOS; [21]) components,
DREBs[21], CBFs, NCED3 and ABA2 [10]. The massive
transcriptional changes observed in previous studies during
the PS suggests that the exogenous application of RONSS
prepares plants to respond faster, stronger or both when
encountering an abiotic stress pressure.
Genes regulated by the three compounds under
different stresses

The mode of action of these priming agents as signalling molecules is not completely elucidated; however,
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Osmoprotective molecules (e.g. proline and oligosaccharides) and polyamines (e.g. putrescine, spermidine and
spermine) are metabolites with pivotal role in osmotic
adjustment and maintenance of membrane stability
[26,27]. Several reports have shown an up-regulation of
genes-coding biosynthetic enzymes implicated in the
biosynthesis of osmoprotectants in primed plants under
non-stress and stress conditions. For instance, induction
of the genes P5CS (delta 1-pyrroline-5-carboxylate
synthase), which encodes a rate-limiting enzyme in proline formation, and SPS (sucrose-phosphate synthase)
were observed after SNP and H2O2 application in Oryza
sativa plants [28]. A comprehensive gene expression
analysis on the biosynthetic (e.g. ADC) and catabolic
enzymes of polyamines pathway (e.g. PAO, DAO) after
treatment of mature and senescing Medicago truncatula
plants with SNP demonstrated more pronounced gene
expression levels in senescing plants [29].
TFs, kinases and miRNAs are also an important part of
the RONSS-regulated genes pool. They can act downstream by regulating the expression of several genes
involved in many physiological and stress tolerancerelated processes during the PS and after the exposure
to stress. SNP and H2O2 primed plants revealed an
Current Opinion in Plant Biology 2016, 33:101107

104 Cell signalling and gene regulation

up-regulation of TFs, such as ERFs, DREBs and CBFs,


but also genes involved in signal transduction, like
kinases (e.g. MAPKK; [11,12]). miRNAs are also an
important category of signalling molecules regulating the
expression of specific genes under stress conditions [30].
Interestingly, exogenous application of NaHS affected
the expression of drought associated miRNAs (e.g.
miRNA167, miRNA393, miRNA396, miRNA398) which
could regulate the expression of target genes and, therefore, improve drought tolerance [18].
Stress-specific gene regulation

Although, gene regulation phenomena before stress exposure share similarities between the three compounds,
there are also stress-specific transcriptional changes. For
example, application of H2O2 in soybean plants enhanced
the expression of genes implicated in oligosaccharides
biosynthesis, mainly 4 h and 6 h after treatment, such as
GmMIPS2 (i.e. myo-inositol synthase) and GmGolS (i.e.
galactinol synthase) under drought stress [16]. Furthermore, SNP application delayed salt-inducible leaf senescing by inducing the expression of genes-coding Na+/H+
antiporters (SOS1 and NHX1) and a cytokinin biosynthesis gene (IPT1), as well as by suppressing the expression
of genes implicated in ABA biosynthesis (NCED2,
NCED9) [31]. Another example is the maximum induction of five Cd-associated genes (MYB107, CAX3, POX1,
MT3 and PCS1) in Arabidopsis 3 h after NaHS pre-treatment [19].
Post-translational modifications

NO and H2O2 and to a lesser extent H2S, not only


regulate the expression of several genes, but can also
alter the activity of intracellular target enzymes by posttranslational modifications (PTMs). PTM phenomena
such as tyrosine nitration and S-nitrosylation, as well as
carbonylation and S-sulfhydration, have been identified
to occur in response to exogenous application of RONSS
[8,32]. It is well-known that these PTMs positively or
negatively affect the activity of enzymes which are crucial
for plant survival under sub-optimal conditions [33].
These modified proteins are usually involved in cell
homeostasis (e.g. antioxidant machinery) and photosynthesis-related procedures [8,34]. Interestingly, antioxidant enzymes which control ROS levels in the cell, such
as CAT, SOD, POX and ASC-GSH cycle-related
enzymes, are usually the main targets of NO-mediated
PTMs [35].
Self-propagation and organ-specific gene regulation

A number of reports exists building a hypothesis of selfpropagation of RONSS and translocation of the signal
from the compartment of the plant where application
takes place to other parts of the plant. This is quite
interesting, especially if, for instance, a priming event
occurred in the root and transcriptional differences could
be recorded in both roots and leaves suggesting a systemic
Current Opinion in Plant Biology 2016, 33:101107

effect [8,12,16]. Experimental evidence on H2O2 and


NO root-treated citrus plants demonstrated an organspecific transcriptional regulation of genes involved in
both H2O2 production and NO biosynthesis. An opposite
transcription pattern was observed between roots (gene
suppression) and leaves (gene induction) 8 days after salt
treatment, in both stressed and non-stressed primed
plants [8]. Additionally, in another study by the same
authors, application of NaHS in the roots regulated the
expression of genes encoding proteins implicated in NO
biosynthesis (e.g. NiR, NR) in the leaves under stress and
non-stress conditions [17]. These observations support
the notion of self-propagation of RONSS and organspecific regulation, which is another interesting feature
of RONSS priming.

Crosstalk between RONSS


A considerable amount of research exists on the cross-talk
between RONSS when plants are exposed to an abiotic
challenge and the current knowledge is already incorporated in recent review articles [4,6,36]. However, some of
the main points of this cross-talk, such as the involvement
of H2O2, NO or H2S on the biosynthesis/removal, and
therefore, the accumulation of the other RONSS [8,37
39], are crucial to understand chemical priming effects on
stress tolerance.
For a molecule to act as a signal, its concentration should
be raised from a low to above a higher threshold level [40].
Exogenous application of either H2O2, NO or H2S
initially results in an endogenous accumulation of the
chemical compound (Figure 1; [8,21]). In addition,
exogenous application of either H2O2, NO or H2S regularly results in changes of the endogenous levels of the
other RONSS before the stress event (Figure 1). For
example, exogenous application of either H2O2 or SNP
resulted in endogenous accumulation of NO and H2O2,
respectively, in sour orange plants [8]. Additionally,
exogenous application of either SNP or NaHS resulted
in endogenous accumulation of H2S and NO, respectively, in bermudagrass and alfalfa plants [37,39]. In another
study, exogenous application of NaHS led to endogenous
accumulation of H2O2 in Arabidopsis thaliana [38]. The
use of specific RONSS scavengers and/or inhibitors
supports the involvement of RONSS accumulating in
response to the application of a RONSS priming agent
in tolerance enhancement to different abiotic stresses
[37,39]. Conclusively, it could be suggested that the
exogenous application of SNP, NaHS or H2O2 initially
triggers a signalling stream where RONSS play a pivotal
role during the PS in stress tolerance enhancement before
stress event (Figure 1).

Better together? The potential of multi-donor


compounds
The feed-forward/feedback complex relations between
RONSS during the PS and their important role in stress
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Priming plants with RONSS against abiotic stress Antoniou et al. 105

tolerance enhancement propose an interesting research


question. Could the exogenous application of a combination of RONSS act synergistically and further enhance
stress tolerance? Combinations of RONSS have already
been applied in previous studies. For instance, combination of NaHS and SNP did not reduce lipid peroxidation
further than NaHS and SNP alone in Cd-stressed alfalfa
plants [39] and bermudagrass [37]. Conversely, combination of SNP with H2O2 resulted in a concentration-dependent interaction between them regarding heat shock
protein expression under heat stress in Arabidopsis thaliana [14]. Therefore, the research question on whether a
combination of RONSS priming agents could act synergistically seems multifactorial and further research is
needed to be properly answered.

Known aspects of the mode of action of RONSS presented herein, including cell signalling and gene regulation during the PS, but also the triggered tolerance
mechanisms at stress exposure, strongly suggest that
RONSS may be effectively used as priming agents for
cross-protection against multiple/combined stresses.
However, further research is needed in this area. To
further understand the cellular transcriptional responses
caused by RONSS at PS and after exposure to stress,
comprehensive genomic analyses using detailed time
series with the use of mutants impaired in RONSS
biosynthesis should be incorporated in future studies.
This information is necessary to understand the gene
regulatory network build up by the signalling action of the
compounds.

Recently developed synthetic chemical compounds that


simultaneously donate more than one RONSS have
shown promising results as priming agents. For example,
NOSH-aspirin (NOSH-A; NBS-1120) is a novel hybrid
synthetic compound that was first established as a highpotency compound for its anti-inflammatory action [41].
NOSH-A is a donor of NO, H2S and aspirin (acetylsalicylic acid). Salicylates (e.g. salicylic acid) are produced
by plants as part of their defence systems against biotic
and abiotic stress conditions. It have been demonstrated
that NOSH-A significantly improves drought tolerance in
mature Medicago sativa plants when applied via foliar
spraying compared with well-watered plants, NOSHtreated (i.e. a synthetic compound donor of NO and
H2S) or aspirin-treated ones [42,43]. Further transcriptome analysis demonstrated a massive gene regulation in
NOSH or NOSH-A-primed plants, under drought conditions (Antoniou and Fotopoulos; unpublished data).
This implies that the combination of NO, H2S and aspirin
in a single donor-compound yields a potentially supreme
chemical agent for priming against drought and probably
also against other abiotic stresses that necessitates further
exploration and exploitation.

Acknowledgments

Concluding remarks
Exogenous application of RONSS results in massive
transcriptional regulation of genes during the PS. The
vast majority of these genes are defence-related genes,
such as enzymatic antioxidants and genes implicated in
the production of osmoprotectants and polyamines. The
similarities observed in the type of genes regulated by
either H2O2, NO or H2S application, the involvement of
each of them in the accumulation of the others during the
PS and pharmacological studies (the use of scavengers
and/or inhibitors) suggests a cross-talk network between
RONSS during the PS necessary for tolerance enhancement. The exogenous application of RONSS prepares
plants to respond faster, stronger or both when encountering an abiotic stress pressure. After stress imposition,
primed plants show more pronounced gene expression
levels compared with non-primed plants.
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CA would like to acknowledge financial support by the Alexander S.


Onassis Public Benefit Foundation.

Appendix A. Supplementary data


Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
pbi.2016.06.020.

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