Life and death under salt stress: same players, different timing?
Journal of Experimental Botany
Life and death under salt stress: same players, different timing?
Ahmed Ismail 2
Shin Takeda 1
Peter Nick 0
0 Molecular Cell Biology, Botanical Institute, Karlsruhe Institute of Technology (KIT) , Germany
1 Bioscience and Biotechnology Center, Nagoya University, Chikusa , Nagoya 464-8601 , Japan
2 Department of Horticulture, Faculty of Agriculture, Damanhour University , Damanhour , Egypt
Salinity does not only stress plants but also challenges human life and the economy by posing severe constraints upon agriculture. To understand salt adaptation strategies of plants, it is central to extend agricultural production to salt-affected soils. Despite high impact and intensive research, it has been difficult to dissect the plant responses to salt stress and to define the decisive key factors for the outcome of salinity signalling. To connect the rapidly accumulating data from different systems, treatments, and organization levels (whole-plant, cellular, and molecular), and to identify the appropriate correlations among them, a clear conceptual framework is required. Similar to other stress responses, the molecular nature of the signals evoked after the onset of salt stress seems to be general, as with that observed in response to many other stimuli, and should not be considered to confer specificity per se. The focus of the current review is therefore on the temporal patterns of signals conveyed by molecules such as Ca2+, H+, reactive oxygen species, abscisic acid, and jasmonate. We propose that the outcome of the salinity response (adaptation versus cell death) depends on the timing with which these signals appear and disappear. In this context, the oftenneglected non-selective cation channels are relevant. We also propose that constraining a given signal is as important as its induction, as it is the temporal competence of signalling (signal on demand) that confers specificity.
ABA; adaptaion; calcium; cell death; cross-talk; jasmonate; salinity; signal on demand; proton influx; ROS
stress-dependent losses of crop yield are estimated to range
between 65 and 87%
(Buchanan et al., 2002)
. Hunger and
malnutrition still represent the primary health risk, exceeding
the impact of AIDS, malaria, and tuberculosis, and leaving 1
billion people in the world without enough food to be healthy
. This underlines the importance of plant stress
for society. Salinity, in particular, has been a threat to
agriculture in some areas in the world for more than 3000 years
; it affects more than 80 million ha of arable
(reviewed by Munns and Tester, 2008)
estimated annual global costs equivalent to US$11 000
million in 2011
. High salinity is commonly caused
by high concentrations of sodium (Na+) and chloride (Cl–)
ions in the soluble fraction of the soil resulting in both
hyperionic and hyperosmotic conditions, which in turn impair the
ability of plants to take up water and micronutrients. This not
only leads to increased concentration of ions to levels that are
toxic to plants but also causes degraded soil structure. Plants
have acquired different adaptive mechanisms to control the
negative impacts of salinity and are classified into two groups
with respect to their adaptability to salinity: halophytes are
efficient in adaptation and therefore are able to inhabit saline
environments, whereas glycophytes cannot cope with saline
soils and therefore are excluded from saline habitats
by Flowers et al., 1977; Hasegawa et al., 2000)
. In this review,
we present and discuss recent advances in our understanding
of the mechanisms by which plants respond and adapt to salt
stress. In particular, we focus on the temporal pattern of
signals that are crucial for adaptation and the role of these
temporal patterns for the orchestration of cross-talks between
signalling pathways. We propose that the correct timing of
these cross-talks decides which salinity-triggered signalling
will culminate in successful adaptation.
Salinity can trigger two qualitatively different modes of cellular response
Salinity can challenge plants to a degree that may even lead to
cell death. Salt stress causes membrane disorganization,
metabolic toxicity, formation of reactive oxygen species (ROS),
inhibition of photosynthesis, and reduced nutrient
(reviewed by Hasegawa et al., 2000; Tuteja, 2007)
stress intensity reaches non-permissive levels, these processes
can culminate in cell death. Growth responses to salinity are
comprised of two phases
. Rapid and often
transient changes in growth occur within minutes and are
attributed to the osmotic effects of salt ions in the rhizosphere.
Hormonal signals originating from the roots are assumed to
regulate the growth reduction during this phase. The second
phase of growth reduction is the result of a salt-specific effect
and needs some time (days, weeks, or months) to develop.
This second phase is not a mere consequence of water stress
. Cellular damage in this second phase
is due to salt accumulation in transpiring leaves, leading to
levels that exceed the ability of the cells to sequester salts into
(Munns, 2002; reviewed by Läuchli and Grattan,
2007; Munns, 1993, 2005)
. It should be kept in mind that the
resulting cell death may be deleterious for the individual cell
but adaptive for the plant as a whole, as plants can dump
ions to toxic levels in their older leaves and then remove the
salt by simple abscission
(Munns and Tester, 2008)
the ionic effects of salinity stress occur later than the osmotic
effects, influx of Na+ has been observed from very early time
points after the onset of salt stress. For maize—an extreme
glycophyte—Na+ ions accumulate in chloroplasts within 4 h,
even preceding any changes of water potential in the
(De Costa et al., 2007; Zörb et al., 2009)
. As we
will discuss in this review, this early influx of Na+ might act as
a signal to trigger salinity adaptation and thus would not be
a mere manifestation of cellular toxicity leading to cell death.
Na+ might play a dual role: as an early signal triggering
‘salinity signalling’ (which can result in successful adaptation), and
as a late noxious factor that, upon accumulation, will lead to
cell death. It is thus possible to order the complex
salinitytriggered events in terms of a two-mode model for the cellular
response. As we will elaborate in the following section, the
relationship between (adaptive) salt signalling and
(destructive) salt accumulation depends on the timing of the events
triggered by salinity stress.
Salinity signals: same inputs, different outputs
Conceptual framework: temporal signatures define response quality
Plants respond to salinity challenges at the level of both cells
and the whole organism. It is important to identify factors
responsible for the adaptability to stress and to understand
the underlying mechanisms connecting these factors to
cellular signalling pathways in order to improve plant growth
and productivity under stressful conditions. However, to
dissect the biological function of the individual stress signals is
difficult; the events involved in stress adaptation overlap, at
least partially, with those accompanying stress-dependent
cellular damage. In addition, in the case of salinity stress, plants
experience two stress qualities at a similar time: osmotic
and ionic stresses. What determines the fate of a cell under
salinity stress? How can virtually the same signalling
molecules cause adaptation in one plant but trigger cell death in
another? Below, we propose and elaborate a model where not
the molecular nature of signals but also their temporal
signatures define the cellular response to salinity. The level of
explanation will be explicitly cellular; for the sake of scientific
reduction we do not consider the systemic level, although it is
evident that the cellular events described below are integrated
into interactions of different tissues and organs
referred to the comprehensive review by Munns and Tester,
The central idea of this model is that the two response
modes (adaptation versus cell death) depend on the relative
timing of two signal chains: one triggered by calcium and the
other triggered by oxidative burst in the apoplast (Fig. 1).
A delay in generation and dissipation of a salinity-triggered
calcium-dependent signal relative to a signal conveyed by
ROS will lead in the unconstrained activation of jasmonate
(JA) signalling culminating in cell death. In contrast, the same
molecular signal carrier (calcium) can, if properly timed,
initiate adaptive processes such as sequestration and extrusion
of sodium, and induce efficient constraint of JA signalling
through the activation of abscisic acid (ABA) signalling.
In the following sections, we will consider, step by step, the
details of the individual signalling events (Fig. 1). For each
step, we will try to define: (i) by which events the signal is
generated (referred to as the ‘on’ state), (ii) by which events
the signal is dissipated (referred to as the ‘off ’ state), (iii) what
is the appropriate target of the signal, and (iv) what is the
inappropriate target of this signal in case of a delayed time
Non-selective cation channels (NSCCs): the earliest players but often overlooked
Ions are selectively conducted through channels in the
plasma-membrane channels depending on electrochemical
potential. In plants, these ion fluxes are tightly controlled
by the gating of these channels
(reviewed by Yeo, 1998)
However, in saline conditions, a rapid influx of Na+ from the
soluble phase of the soil into the cortical cytoplasm of plant
roots occurs through NSCCs, and, later, through the
highaffinity K+ transporter (HKT1)
(Essah et al., 2003; reviewed
by Tester and Davenport, 2003)
NSCCs have been classified according to their voltage
dependence or to their responsiveness to certain ligands
and physical stimuli, and their physiological roles under
salinity have been described previously
Demidchik and Maathuis, 2007; Kronzucker and Britto,
. Hyperpolarization-activated (HA)-NSCCs, which
are activated later and weakly selective for monovalent
(Davenport and Tester 2000; reviewed by Demidchik
et al., 2002)
, might be the predominant type of channel in
plasma membranes of sensitive species. In contrast, more
efficient NSCCs [depolarization-activated (DA)-NSCCs
and voltage-insensitive (VI)-NSCCs] might take the lead
in tolerant species. This idea was tested for cells of
grapevine, where otherwise very similar pairs of species can be
compared, such as the salt-tolerant Vitis rupestris and
the salt-sensitive Vitis riparia
(Ismail et al., 2012)
rupestris inhabits rocky, sunny slopes, and therefore has
evolved a considerable osmotic tolerance. In contrast,
V. riparia occurs in alluvial woods and performs poorly
under osmotic stress. In the drought-sensitive V. riparia,
sodium influx was observed to be slow, consistent with
the hypothesis that HA-NSCCs might be the predominant
type of channel
(Ismail et al., 2014)
. In contrast, the
vigorous and rapid influx observed in V. rupestris indicates that
the more efficient DA-NSCCs and/or VI-NSCCs are the
major types of channel. Interestingly, the role of NSCCs
and their kinetic activities not only determine the pattern
of Na+ influx but also modulate the cytoplasmic signatures
of two crucial signalling elements, Ca2+ and H+. In the
sensitive V. riparia, gradual and low kinetics of Na+ influx
were observed, while in the tolerant V. rupestris, NSCCs
catalysed a fast and strong Na+ influx reaching its
maximal amplitude after only 2 min (the first time point
measured). This initial and rapid Na+ uptake could serve as an
calcium-dependent signalling from processes leading to ion sequestration/
extrusion towards processes constraining the ABA status, such that the
parallel activation of the JA status will be released from ABA-dependent
control. (This figure is available in colour at JXB online.)
efficient signal to activate adaptation to the osmotic part of
(Fig. 2A, ① ; reviewed by Munns and Tester 2008)
Furthermore, the elevated intra- and extracellular Na+
are partially able to inhibit the K+ outward rectifiers and
thereby prevent the loss of cellular K+, maintaining cellular
(Shabala et al., 2006)
. Thus, in the
tolerant line, rapid influx of Na+ by the more effective NSCCs
has advantageous effects, at least during the first phase of
the salt stress response. The slower influx in sensitive cells
will be less efficient in adjusting the water potential such
that cells will lose water (Fig. 3, ①). These differences in
sodium content are very short lived and therefore their
connection with differential responses at the later stages must
be conveyed by signals of adifferent molecular nature.
Cells cannot tolerate the harmful effects of Na+ accumula- sodium entry is later and does not contribute to protective
tion in the cytoplasm due to impaired enzyme activities and signalling. Conversely, in the Arabidopsis mutants hkt1-1 and
cytotoxicity (ion-specific effects). To circumvent such toxicity, hkt1-2, Na+ entry was suppressed to some extent, but these
cells exclude Na+ from the cytosol and/or sequester sodium mutants were found to be endowed with enhanced salinity
inside the vacuole. To block additional influx of sodium, tolerance
(Rus et al., 2001; reviewed by Schroeder et al., 2013)
VI-NSCCs are rapidly deactivated by cAMP or cGMP pro- To arrest further influx of sodium is not sufficient,
howposed to be generated by turgor-sensitive membrane-located ever; ions have to be removed from the cytoplasm. The roles
cyclases (Fig. 2B, ③), which results in improved plant salinity of the salt overly sensitive (SOS) pathway in expelling Na+
tolerance (Maathuis and Sanders, 2001). A second route of out of the cell or caging it inside the vacuole via an SOS/NHX
entry is the high-affinity K+ transporter HKT1. However, this interaction (Fig. 2B, ③) seems to be the central mechanism
for Na+ exclusion
(reviewed by Ji et al., 2013)
, although the
role of the SOS pathway for plant salt tolerance has been
discussed controversially, as reviewed by Kronzucker and
Britto (2011). It has been questioned whether extrusion of
sodium through the plasma membrane by the SOS1 exporter
represents an effective strategy, as Na+ ions will re-enter the
cell though the NSCCs (if these were not deactivated by
cAMP/cGMP) and HKT1 channels, respectively, and the
accumulation of sodium in the apoplast would generate a
more negative water potential such that the cell would lose
(reviewed by Shavrukov, 2013)
. A further
caveat was found when the role of SOS1 in stress tolerance
was examined at a whole-plant level: In Arabidopsis thaliana,
SOS1 is expressed preferentially in the rhizodermis near to
the root tip, i.e. in cells that are still weakly vacuolated, and
in xylem parenchyma cells, from which Na+ is uploaded to
xylem vessels for long-distance transport from the root to
(Shi et al., 2002; reviewed by Munns and Tester,
. Mutants impaired in the expression of sos1 in both
Arabidopsis (Shi et al., 2002) and tomato
(Olías et al., 2009)
accumulate more sodium in the shoots under salinity stress.
Therefore, Na+ exclusion by SOS1 seems to be important as a
mechanism by which cells get rid of excess Na+. However, if
not accompanied by other adaptive responses, mere
enhancement of SOS1 activity (e.g. by overexpression) may not be
sufficient to improve salt tolerance
(Oh et al., 2010; reviewed
by Kronzucker and Britto, 2011;,Ji et al., 2013)
. As will be
given in detail in the section on ‘Ca2+ ions’, SOS1 is activated
via a calcium-dependent pathway. Simultaneously, the very
long cytoplasmic tail of this transporter has been proposed
to sense Na+ directly
(reviewed by Zhu, 2002)
However, the SOS system integrates a second strategy for
sodium dissipation: sequestration into the vacuole. As will be
outlined in more detail in the following section on calcium,
SOS2 and -3 will activate the NHX1 transporter that
pumping sodium from the cytoplasm into the vacuole (Fig. 2B, ③).
Compared with sodium extrusion, this strategy is efficient in
removing noxious sodium from the cytoplasm but at the same
time lowers the water potential of the entire protoplast, such
that additional water loss to the environment is prevented,
and not only the ionic but also the osmotic component of
salinity stress is encountered. It should be mentioned that
NHX1 is not the only transporter able to sequester sodium
into the vacuole. The tonoplast harbours slow-activating and
fast-activating channels that can facilitate sodium uptake to
the vacuole and improve salinity tolerance in leaves of quinoa
(Bonales-Alatorre et al., 2013)
. In the case of a non-tolerant
cell, the SOS system is activated in only a sluggish manner
(the reason is linked to the reduced activity of NSCCs, as will
be pointed out in the subsequent section) such that sodium
will remain in the cytoplasm and accumulate there (Fig. 3B)
to toxic levels.
To summarize the main points on sodium as a salinity
1. The ‘on’ state is generated by the rapid influx of sodium
(along with calcium ions and protons) through DA-NSCCs
and/or VI-NSCCs (Fig. 2A, ①). This rapid sodium/
calcium peak represents the first signal that allows the
discrimination of salinity stress from mere osmotic stress
(e.g. as a consequence of drought), i.e. here is the point
where the ionic component in salinity signalling bifurcates
from osmotic signalling. In parallel, the osmotic challenge
will result in activation of mechanosensitive calcium
channels yielding additional influx of calcium ions and protons
(Fig. 2A, ②).
2. The rapid elevation in the cytoplasmic concentration of
sodium/calcium activates a signalling (which is explained
in detail in the subsequent sections) that will initiate a
rapid dissipation of the sodium signal (Fig. 2B, ③): to
achieve the ‘off ’ state, on the one hand, additional influx
of sodium ions is prevented by deactivation of the NCCS
through cAMP/cGMP-dependent signals (Fig. 2B, ③), as
well as deactivation of the slower HKAT channel. On the
other hand, sodium ions are removed from the cytoplasm
either by extrusion (SOS1), or by sequestration into the
vacuole (SOS2/3 and NHX1).
3. The appropriate target for the sodium signal is actually the
concomitant influx of calcium (Fig. 2A, ②), which will
carry on signalling even after the sodium signal has been
dissipated by the mechanisms given in (2).
4. In the case of a delayed sodium/calcium influx through the
HA-NCCS (Fig. 3A, ①), activation of the SOS system as
well as the block of sustained sodium influx will not be
efficient. This not only results in accumulation of
cytoplasmic sodium to toxic levels (Fig. 3B), but will also lead to
sustained accumulation of calcium that, as pointed out in
the subsequent section, will go astray and channel towards
overactivation of the JA pathway.
Ca2+ ions: promiscuous but choosy
Calcium ions (Ca2+) are considered the most prominent
ubiquitous second messenger in cells ranging from bacteria
and plants up to specialized neurons
(reviewed by Clapham,
. The normal cytoplasmic Ca2+ (Ca2+cyt) level is ~100–
200 nM, while in membrane-enclosed organelles it is ~1–2 mM
(reviewed by White, 2000)
. Ca2+cyt signals are shaped by influx
or efflux of ions from the extracellular space (cell wall or
apoplast in plants) through a couple of different channels in the
plasma membrane, some of which seem to be
mechanosensitive, whereas others are voltage gated and might be
identical to the NCCS
(for a recent review, see Swarbreck et al.,
. Different channels are localized at the surface of
intracellular compartments (such as vacuoles, chloroplasts,
or mitochondria). Slow vacuolar channels, such as TPC1,
are targets of different signalling molecules including Ca2+,
calmodulin (CaM), and nucleotides, and play a crucial role in
raising cytosolic Ca2+ under a wide range of environmental
and developmental cues
(Pottosin et al., 2009; reviewed by
Hedrich and Martena, 2011; Peiter, 2011)
. The spatial
pattern of Ca2+ signals (e.g. cytosol, nucleus, organelles, or other
specific regions of the cell), the temporal propagation of
Ca2+ levels, the amplitude of the signal, and the frequency of
Ca2+ oscillations are all informative aspects of Ca2+ signals,
which are perceived by adaptor proteins or Ca2+-modulated
proteins that regulate downstream signalling events (reviewed
by Bouché et al., 2005;
Kudla et al., 2010
). Interestingly, Ca2+
signals participate in virtually all developmental, hormonal,
and stress cues
(reviewed by Reddy et al., 2011)
apparent ambiguity of this signal is even amplified by the fact that
nitric oxide (NO), a small, uncharged, short-lived, water- and
lipid-soluble, highly diffusible, ubiquitous, volatile, highly
reactive free radical, can act as a Ca2+-mobilizing
(reviewed by Neill et al., 2003; Besson-Bard et al., 2008;
Siddiqui et al., 2011)
Under salinity, the earliest cellular response seems to
be a rapid increase in free cytosolic Ca2+ within 1–5 s via
influx through either NSCCs or a mechanosensitive calcium
channel in the plasma membrane, which can be amplified
through release from internal stores, especially the vacuole
(Knight et al., 1997; Donaldson et al., 2004)
cytosolic Ca2+, in turn, activates the plasma-membrane
ATPases mediated by Ca2+/CaM-dependent protein kinases,
restoring membrane voltage after Na+-induced
depolarization, maintaining membrane integrity and ionic
homeostasis, promoting H+ influx, and inhibiting both K+ and H+
(Klobus and Janicka-Russak, 2004; Shabala et al.,
2006; reviewed by Wolf et al., 2012)
. Moreover, cytosolic
Ca2+ activates salt overly sensitive 3 (SOS3), a member
of the calcineurin B-like (CBL) family known as CBL4,
to interact with SOS2 (a CBL-interacting protein kinase,
CIPK24). The SOS3/SOS2 complex, in turn, activates SOS1
(a plasma-membrane Na+/H+ antiporter) through its
phosphorylation (Fig. 2B, ③). The activated SOS1 extrudes Na+
from the cell, thus reducing its harmful effects on cellular
(reviewed by Zhu, 2002; Harper et al., 2004;
Munns and Tester, 2008)
. SOS1 directly signals to a putative
K+ transporter by-passing SOS2 and SOS3 and therefore
was proposed to be necessary for safeguarding the K+
permeability of the plasma membrane during salinity stress
and Spalding, 2004; Shabala et al., 2005)
. In addition, the
Na+/H+ exchanger (NHX) class of transporters (Fig. 2B,
③)—the plant homologue of the yeast Na+/H+ exchanger
(Apse et al., 1999; Gaxiola et al., 1999)
that catalyses the
electroneutral exchange of Na+ or K+ with H+,
maintaining intracellular pH and Na+ and K+ homeostasis in all
(reviewed by Martinoia et al., 2012)
interconnected to Ca2+ signals via SOS/NHX interaction (Qiu
et al., 2004). To date, six members of the NHX gene family
have been identified in A. thaliana and classified according
to their intracellular localization into vacuolar (NHX1–4)
and endosomal (NHX5 and -6) compartments
(Bassil et al.,
. The generation of salt-resistant tomato by
overexpression of NHX1 was considered one of the milestones
of green genetic engineering
(Zhang and Blumwald, 2001)
Despite a long history of biotechnological application, the
actual reason for salinity tolerance conferred by NHX1 is
still under debate. Overexpression of Arabidopsis NHX1 or
tomato NHX2 in tomato did not yield a consistent elevation
of vacuolar Na+
(Rodriguez-Rosales et al., 2008; Leidi et al.,
and the protective effect was attributed to improved
potassium partitioning to the vacuole. On the other hand,
the Arabidopsis double mutant nhx1 nhx2 showed a similar
salinity sensitivity to the wild type but a reduced vacuolar
pool of K+ at simultaneously elevated sequestration of Na+.
Additionally to steering the SOS pathway, calcium can
activate gene expression. For instance, the CaM-binding
transcription activators CAMTA1–4, and CAMTA6 are all
(Yang and Poovaiah, 2002)
. In addition, a
specific CaM isoform in soybean (GmCaM4) interacts directly
with a MYB2 transcription factor, enhancing the
transcription rate of MYB2-dependent genes, such as P5CS1,
conferring salt adaptation to Arabidopsis overexpressing GmCaM4
(Yoo et al., 2005). However, calcium ions can also act directly
without the need for a protein adaptor: the Arabidopsis salt
stress-responsive gene 1 (AtNIG1), a basic helix–loop–helix
(bHLH)-type transcription factor, is the first known
Ca2+binding transcription factor involved in the plant response to
(Kim and Kim, 2006)
. Interestingly, Ca2+ can also
stimulate the NADPH oxidase (Fig. 2B, ⑥), a primary source
of stress-related oxidative burst in plants, and thus generates
a further important stress signal
(Dubiella et al., 2013)
Similar to sodium, the protective function of calcium
signalling can turn deleterious when cytosolic calcium
levels remain high over a longer period. Under these
circumstances, Ca2+ can activate degradative processes or cell death
by precipitating phosphate (depleting, among others, ATP
as ‘cellular currency’), cause aggregation of proteins and
nucleic acids, and impair the integrity of lipid membranes
(reviewed by Clapham, 1995; Case et al., 2007)
. For example,
ROS-activated sustained Ca2+ influx (feedback stimulated
by threshold levels of H2O2) was followed by programmed
cell death in soybean (Levine et al., 1996). Therefore, plants
have adopted different strategies to restore Ca2+cyt levels
after the completion of Ca2+ signalling, and the balance
between reactions that cause elevated Ca2+cyt (‘on’ reaction)
and reactions through which the Ca2+ signal is damped by
buffering, pumping, and exchanging machineries (‘off ’
reactions) determines the intracellular Ca2+ levels at any time
(reviewed by Berridge et al., 2003; Bouché et al., 2005;
Clapham, 2007; Bose et al., 2011)
. The central plant
vacuoles (equivalent to lysosomes of animal cells with regard to
their degradation and autophagy functions) represent the
major Ca2+ store in a mature plant cell. High-capacity
vacuolar Ca2+ exchangers (CAXs) play crucial roles in ion
homeostasis and signal transduction
. In the resting
state, these CAX pumps are complemented by auto inhibited
Ca2+-ATPase pumps of the PIIB-type. However, upon Ca2+cyt
elevation, Ca2+/CaM will bind to the N-terminal
autoinhibitory domain, releasing them from autoinhibition and
restoring Ca2+cyt by a feedback regulation (Fig. 2B, ④) that is an
(reviewed by Pittman, 2011)
A. thaliana, there are six members of CAX (CAX1–6) that
seem to function specifically with regard to different cues. For
instance, the cax1 mutant displayed enhanced freezing
tolerance, while cax3 resulted in higher salinity sensitivity
et al., 2003; Zhao et al., 2008; reviewed by Bose et al., 2011)
Interestingly, CAX1 is also interconnected to the SOS
pathway and activated via SOS2, restoring Na+/Ca2+
homeostasis, whereas elevated expression of deregulated CAX1 caused
(Cheng et al., 2004)
Failure to dissipate a cytosolic calcium signal in a timely
fashion will strongly interfere with the signalling status of
important stress hormones such as JA and ABA (Fig. 3C).
Details on the function of these hormones are given in the
sections below—however, here their regulation by calcium
is considered. Calcium released from the vacuole through
the TPC1 channel can activate JA synthesis
(Fig. 3B, ③;
Bonaventure et al., 2007)
, whereas CBL proteins impede both
the synthesis and the signal transduction of ABA (Fig. 3C,
④; Pandey et al., 2004).
To summarize the main points on calcium as a salinity
1. The ‘on’ state is generated by the rapid influx of calcium
through different channels, probably including the NCCS
carrying sodium ions (Fig. 2A, ①), and a
mechanosensitive calcium channel triggered by membrane load caused
by osmotic water loss (Fig. 2A, ②).
2. The ‘off ’ state is restored by rapid dissipation of calcium
by either binding to SOS3 (Fig. 2B, ③) or by
sequestration by the CAX transporters that are controlled through
SOS2, which means that calcium activates its own removal
from the cytoplasm (Fig. 2B, ④).
3. The appropriate target for the calcium signal is on the one
hand the SOS system driving the elimination of sodium
ions from the cytoplasm (Fig. 2B, ③), and the CAX
transporters (Fig. 2B, ④) that will contribute to the shut-off
of the calcium signal. Calcium-triggered activation of the
NADPH oxidase will relay the signal to the next player,
apoplastic ROS (Fig. 2B, ⑥).
4. In case of a delayed sodium/calcium influx (Fig. 3A, ①),
activation of the SOS system as well as the block of
sustained sodium influx will not be efficient. As a result, the
sequestration of calcium into the vacuole will be slowed
down, and the calcium signal is conveyed to other
calciumadaptor proteins, such as CBL9 (Fig. 3B, ④). This will
impede the ABA ‘status’ as a dynamic product of
synthesis and signalling (Fig. 3C, ④).This situation might even
become accentuated by sustained calcium released from
the vacuole through the slowly activated TPC1 channels
(Fig. 3B, ③).
Proton influx: a signal enhancer?
Protons (H+) play crucial roles for cell signalling either
directly or in cross-talk with phytohormones or Ca2+
et al., 2004a)
. In addition, protons directly regulate enzymatic
conformations and thus metabolic activities
(Roberts et al.,
. However, intracellular pH can also act as a second
messenger for several signalling pathways. For instance, a
cytoplasmic alkalinization is able to convey methyl-JA (MeJA)
and ABA signalling during stomatal closure of A. thaliana
(Suhita et al., 2004)
, and is also involved in plant responses to
salinity and drought stresses, indole-3-acetic acid, and gravity
(Gao et al., 2004a; Fasano et al., 2001; reviewed by Kurkdjian
and Guern, 1989)
. Proton influx can occur concomitantly
with calcium, and the resulting apoplastic alkalinization
has been used extensively as a robust reporter for the rapid
activation of calcium influx channels by elicitors
(Felix et al.,
or abiotic stresses including salinity stress (Ismail
et al., 2012, 2014;
Geilfuß and Mühling, 2013
). With respect
to the downstream signals, it should be noted that pH
controls the ratio between the active and the inactive enantiomer
of the bioactive JA conjugate JA-Ile
(Fonseca et al., 2009)
A comparison of two Vitis cell lines differing in salt
tolerance (see ‘Conceptual framework’ section) showed that
efficient adaptation in V. rupestris correlated with a more rapid
and more persistent apoplastic alkalinization compared with
the salt-susceptible V. riparia
(Ismail et al., 2014)
alkalinization, in turn, might promote adaptive events such
as activation of wall-consolidating enzymes such as pectin
methyl esterase, or, on the other hand, inhibition of expansins
that render the wall softer
(reviewed by Wolf et al., 2012)
should also be considered that depletion of protons in the
apoplast will release anionic binding sites to complex sodium
ions. In addition, the elevated steady-state level of apoplastic
superoxide as a further relevant signal (see following section)
will be enhanced if the level of protons is low. Furthermore,
stress-induced pH changes in the xylem sap might act as a
root signal through ABA anions that redistribute and
accumulate due to the low membrane-permeability of the charged
anion, promoting stomatal closure (Taiz and Zeiger, 2010).
The impact of proton activity on the enzymes or other
proteins in the cytoplasm is very critical, where Ca2+-induced
H+ influx might feedback on Ca2+ signalling by affecting
Ca2+ affinity for CaM
(reviewed by Busa and Nuccitelli,
. Moreover, the activities of the important
transporters NHX1, CAX1, and CAX2 are inhibited by cytosolic
(Pittman et al., 2005; reviewed by Padan et al., 2001)
Thus, the influx of protons would promote the temporary
accumulation of the concomitant calcium and sodium
signals. However, this fluctuation of cytoplasmic pH will remain
transient, because protons are rapidly extruded by powerful
proton ATPases at the plasma membrane and especially the
V-ATPase at the tonoplast (Fig. 2B, ⑤), and at the same time
are complexed by the high buffering capacity of the cytosol
(reviewed by Kurkdjian and Guern, 1989)
In summary, although proton influx does not act as an
independent signal, it can act as an enhancer of early sodium
and calcium signals:
1. The ‘on’ state is generated by influx together with calcium
through the NSCC (Fig. 2A, ①) and the mechanosensitive
calcium channels (Fig. 2A, ②).
2. The ‘off ’ state is restored by rapid buffering of protons
in the cytosol and active extrusion through the proton
ATPases at the plasma membrane, and, most importantly,
by vacuolar sequestering by the V-ATPase (Fig. 2B, ⑤).
3. The appropriate target is the inhibition of NHX1 and
CAX activities acting as an amplifier of the initial sodium
and calcium signal (Fig. 2B, ③, ④). At the same time, the
depletion of protons from the apoplast will increase the
lifetime of ROS (Fig. 2B, ⑥), and improve the matrix
buffering for sodium ions.
4. In the case of a delayed sodium/calcium influx (Fig. 3A,
①), apoplastic protons will be available for quenching
ROS, thus dampening a further important signal (Fig. 3B,
⑤). In the cytosol, the initial sodium/calcium signal would
not be enhanced, and later acidification might even
interfere negatively with hormonal signalling.
ROS: bifunctional in the response to salinity stress
ROS are continuously produced in plant compartments
such as mitochondria, chloroplasts, and peroxisomes as
unavoidable by-products of aerobic metabolism such as
photosynthesis, photorespiration, and respiration
by Abogadallah, 2010; Apel and Hirt, 2004)
. As aerobic
metabolism is based on electron flow across membranes, even
mild damage of mitochondrial or plastidic membranes will
result in uncontrolled intracellular oxidative burst (Fig. 3C,
⑦).The term ROS comprises both free radical (O2•–,
superoxide radicals; OH•, hydroxyl radical; HO2•, perhydroxy radical;
and RO•, alkoxy radical), and non-radical (molecular) forms
(H2O2, hydrogen peroxide; and 1O2, singlet oxygen) (reviewed
by Gill and Tuteja, 2010). The different ROS vary not only in
their chemical nature but also in their toxicity. The superoxide
O2•– is considered the earliest ROS, while OH• is among the
most highly reactive ROS known. The accumulation of ROS
causes oxidative damage to DNA, proteins, carbohydrates,
and lipids. However, they also could function as signalling
molecules regulating responses of development and various
aspects of stress. Therefore, they must be closely regulated by
(reviewed by Miller et al., 2010)
different stimuli, the elevated levels of ROS are sensed at the
plasma membrane, for instance by two-component signalling
systems (membrane-localized histidine kinases) that, in turn,
activate the mitogen-activated protein kinase (MAPK)
signalling cascades. Under salinity challenge, different MAPK
elements are activated such as MAPK4, MAPK6, and
(reviewed by Taj et al., 2010)
overexpressors for AtMAPKK2 exhibited constitutive MAPK4
and MAPK6 activity, constitutively unregulated expression
of stress-induced marker genes, and increased freezing and
salt tolerance. Transcriptomic analysis of this mutant showed
altered expression of 152 genes involved in transcriptional
regulation [such as STZ (slt tolerance zinc finger protein),
WRKY and MYB], signal transduction (such as a
MAPKK5related protein and a putative calmodulin), cellular defence
(such as lipoxygenase and the ACC synthase AtACS-6), and
stress metabolism (including a flavonol synthase and P5CS,
a gene encoding a key enzyme of proline biosynthesis)
et al., 2004)
. Although MAPK4 regulates the cross-talk
between SA and JA, supporting the JA/ethylene signalling
(Brodersen et al., 2006)
, growth assays and northern
blot analysis of transcripts did not detect differences between
the mpk4 Arabidopsis mutant compared with the wild type
under salinity, cold, or heat shock, although differences were
noted for pathogen challenge
(Petersen et al., 2000)
indicating that, in the context of abiotic stress, the alternative
MKK2/MAPK6 cascade is relevant. However, MAPK
signalling can also act as an antagonist for abiotic stress
signalling—for instance, AtMAPK1 negatively regulates a putative
Na+/H+ antiporter, leading to salinity sensitivity
Chinnusamy et al., 2006)
. In addition to the MAPK
pathways, ROS can modulate gene expression by modifying
(reviewed by Apel and Hirt, 2004)
. A third
mechanism is the reversible oxidation of critical thiols in key
(reviewed by Forman and Torres, 2002)
However, ROS production needs to be tightly controlled to
act as a signal, otherwise an excessive oxidative burst would
result in cell death. In fact, ROS are a hallmark of
plant-specific forms of programmed cell death, so called necroptosis
(reviewed by Coll et al., 2011)
. Interestingly, animals and plants
share common apoptosis signal transduction pathways
triggered by oxidative stress, where H2O2-induced lipoxygenase
activities that are able to introduce molecular oxygen into the
fatty acid moieties of phospholipids lead to increasing
mitochondrial membrane lipid peroxidation and, subsequently,
cytochrome c release (reviewed by Maccarrone et al., 2001).
Singlet oxygen, on the other hand, is used as a substrate of
lipoxygenases triggering a metabolic pathway that will
generate a further important stress signal, JA
(Fig. 3C, ⑥; Farmer
and Mueller, 2013)
. Also, ABA synthesis is activated by ROS
(Xiong and Zhu, 2003)
. Salinity- or drought-stressed plants
close their tomata, which in turn limits water loss (favourable
effect) and the influx of CO2 (unfavourable effect)
. Consequently, carbon reduction and
photosynthetic NADPH consumption by the Calvin cycle decrease,
resulting in electron leakage from photosystem I to O2 as an
alternative electron acceptor, initiating the Mehler reaction
(reviewed by Türkan and Demiral, 2009)
. The resultant O2•–
is considered the earliest ROS that consequently gives rise to
other ROS, including the most noxious OH•. Additionally,
the peroxisomal glycolate oxidase during photorespiration,
plasma-membrane located NADPH oxidases, amine
oxidases, and cell-wall-bound peroxidases are important sources
of ROS that are active to a certain extent even under normal
conditions but are activated in response to stress (reviewed
by Mittler, 2002). Plants must strictly maintain ROS
homeostasis to mitigate the toxicity of ROS. Therefore, plants have
employed different scavenging machineries that tightly
control ROS levels, both enzymatic and non-enzymatic. Plant
enzymatic antioxidant mechanisms include superoxide
dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX),
glutathione reductase (GR), monodehydroascorbate
reductase (MDHAR), dehydroascorbate reductase (DHAR),
glutathione peroxidase (GPX), guaicol peroxidase (GOPX), and
glutathione S-transferase (GST). The metalloenzyme SOD
is the most effective intracellular enzymatic antioxidant and
acts by dismutating superoxide to H2O2, which in turn can be
detoxified by APX, GPX, and CAT. The non-enzymatic
antioxidants comprise ascorbic acid (ASH), glutathione (GSH),
phenolic compounds, alkaloids, non-proteinogenic amino
acids, and α-tocopherols
(reviewed by Apel and Hirt, 2004;
Gill and Tuteja, 2010)
The quelling of ROS accumulation can also be achieved
by other signals, such as NO. NO has the ability to
neutralize Fenton-type oxidative damage by scavenging superoxide,
therefore preventing the formation of oxidants (such as O2•–,
H2O2, and alkyl peroxides), which makes it easier to recover
a redox homeostasis
(Lamattina et al., 2003)
. For example,
pre-treatment with 1 mM sodium nitroprusside, a NO donor,
results in enhancement of the antioxidant defence and
methylglyoxal detoxification systems in salt-stressed wheat seedlings
(Hasanuzzaman et al., 2011)
. In addition, NO is considered
a redox regulator of the NPR1/TGA1 system, a key
redoxcontrolled regulators in plant systemic acquired resistance in
(Lindermayr et al., 2010)
. As an additional regulator,
hydrogen sulfide (H₂S) has emerged as a signalling molecule
in plants that increases GSH levels, alters enzyme activities,
and interacts with NO and ROS metabolism
Paul and Snyder, 2012; Lisjak et al., 2013)
. As NO is
acting as a secondary messenger of ABA signalling
by Hancock et al., 2011)
, this molecule provides cross-talk
between oxidative and phytohormonal signalling. This
crosstalk is even bilayered, because also ROS deriving from the
activity of the NADPH oxidase in the plasma membrane are
essential for ABA induced signalling (Kwak et al., 2003).
As with most stress signals, ROS are ambiguous,
switching between activation of adaptive events and causing
oxidative damage. Again, it depends on timing and regulation as to
whether they act as a ‘signal on demand’ or go wild as cellular
1. The ‘on’ state is generated by metabolic disbalance of
oxidative processes such as respiration or photosynthesis, but
in the context of signalling, calcium-triggered activation
of the membrane-bound NADPH oxidase seems to be
central(Fig. 2, ⑥). The elevated steady-state level of the
resulting apoplastic ROS is increased due to the apoplastic
depletion of protons, such that these ROS can enter the
cytoplasm, probably through aquaporins.
2. The ‘off ’ state is on the one hand provided by enzymatic
and non-enzymatic antioxidants (Fig. 2C, ⑥), and on the
other by dissipation into stress signalling (e.g. MAPK
cascades) and activation of phytohormone synthesis.
3. The appropriate targets are signal cascades such as the
MAPK pathway leading to the activation of adaptive
genes but also the activation of JA and ABA synthesis
(Fig. 2C, ⑦).
4. In the case where ROS accumulate at later stages (Fig. 3B,
⑤), or are formed as a consequence of mitochondrial
damage (Fig. 3C, ⑦) this will result not only in a pertinent
hyperactivation of JA synthesis due to excessive lipid
peroxidation but also in autocatalytic oxidative burst (Fig. 3C,
⑥), as the membrane damage will impair the functionality
of electron transport in both mitochondria and plastids.
ABA: commitment for adaptive responses?
The phytohormone ABA, synthesized via the terpenoid
pathway, regulates numerous plant biological processes ranging
from development, including the inhibition of
growth/germination and bud dormancy, to adaptive stress responses,
such as drought, salt, ozone, and pathogen infection, and
therefore is seen as a stress-related hormone
Xiong and Zhu, 2003)
. The perception and signalling
pathways of ABA have been studied extensively in A. thaliana
and other species using biochemical and molecular genetic
(Ishibashi et al., 2012; reviewed by Cutler et al.,
2010; Raghavendra et al., 2010)
. In 2009, the long search
for the ABA receptor succeeded with the identification of
pyrabactin resistance 1 (PYR1), a member of the PYR/
PYR1-like (PYL)/regulatory component of the ABA
receptor (RCAR) group of proteins. This novel ABA-binding
protein was demonstrated as a soluble ABA receptor by two
independent research groups, considered as a breakthrough
for the understanding of ABA signalling
(Ma et al., 2009;
Park et al., 2009)
. These receptors, now termed PYR/PYL/
RCAR, represent a family of soluble proteins of about 150–
200 aa that share a conserved START domain. The
ABAfree ‘open-lid’ conformation of PYR1 is converted to a more
compact and symmetric closed-lid dimer upon binding to
(Nishimura et al., 2009, 2010)
. Plants constrain ABA
signalling through clade A protein phosphatases 2C (PP2C)
[mainly ABI1, ABI2, and HOMOLOGY TO ABI (HAB1
and HAB2), which negatively regulate (dephosphorylate)
downstream kinases. However, in response to
environmental or developmental signals, ABA is synthesized and bound
to PYR1, and this receptor, in turn, binds to PP2Cs
inducing a conformational change resulting in its inhibition, and
thus terminating the inhibition of the downstream
ABAactivated kinases (OST1/SnRK2.6/SRK2E, SnRK2.2, and
SnRK2.3). The released SnRK2s are able to phosphorylate
downstream factors, such as the majority of osmotic
stressresponsive genes harbouring ABA-responsive promoter
elements/complexes (ABREs) and bZIP transcription
factors (such as ABI5), ion channels (SLAC1, KAT1), and the
NADPH oxidase AtrbohF
(reviewed by Hubbard et al., 2010;
Umezawa et al., 2010; Joshi-Saha et al., 2011)
. ABA activates
genes that encode enzymes for the biosynthesis of
(as shown for water-stress-induced betaine
in pear leaves; Gao et al., 2004b)
(Strizhov et al.,
, and cellular chaperones (dehydrins and LEA-like
proteins) that protect proteins and membranes under stress
et al., 2013; reviewed by Hasegawa et al., 2000, Shinozaki
and Yamaguchi-Shinozaki, 2007)
. In addition, ABA causes
induction of Ca2+cyt via ROS or IP3 recruitment
et al., 2001; Taiz and Zeiger, 2010)
. Furthermore, ABA and
JA play pivotal roles in controlling stomatal closure, which is
considered a fast response in stressed plants, although plants
cannot keep stomata closed over a long period as they need
to fix CO2 for survive. Interestingly, ABA and JA
transduction pathways leading to stomatal closure share overlapping
signalling elements. Several ABA mutants with NCED (the
key regulatory gene in ABA biosynthesis) overexpression
showed better drought adaptation, while ABA-deficient aba
mutants of Arabidopsis perform poorly under drought or salt
stress or even die
(reviewed by Zhu 2002; Bartels and Sunkar,
. Direct comparison of two genetically similar
grapevine cell lines differing in their osmotic sensitivity under salt
stress revealed that salt susceptibility was accompanied by a
delayed accumulation of ABA (Ismail et al., 2014). By
keeping in mind that some osmotic stress-responsive genes are
ABA independent and are activated via JA signalling
including MYC and MYB elements
(Ishitani et al., 1997; reviewed
by Bartels and Sunkar, 2005)
, although both rd22BP1/
AtMYC2 and AtMYB2 proteins were firstly identified as
ABA-inducible transcriptional activators under drought
(Abe et al., 2003)
, ABA was concluded not to be the only
adaptive signal, as will be discussed in the next section.
On the other hand, the ABA transient increase under
demanding conditions points to the importance not only of
the activation of signalling and biosynthesis but also the
suppression strategy. Indeed, ABA enhancement in an NCED
mutant resulted in accumulation of its catabolite, phaseic
acid, via (+)-ABA 8′-hydroxylase activation that catalyses the
first step in the oxidative degradation of ABA, in addition to
other catabolic pathways
(Qin and Zeevaart, 2002; reviewed
by Cutler and Krochko, 1999)
. Oxidative degradation might
be complemented by other ABA inactivation strategies, such
as ABA conjugation. As the phosphatase activity of ABI1
and ABI2 increases in response to ABA, dephosphorylation
of ABA signalling elements will constitute a
(Merlot et al., 2001)
. A further (negative) feedback
loop is provided by ABA-dependent inhibition of ABI5
degradation and simultaneous ABI-dependent promotion of the
RING E3 ABI3-INTERACTING PROTEIN 2 (AIP2) that
in turn suppresses ABI3, which interacts with ABI5,
enhancing its activity
. Both synthesis
(Kwak et al., 2003)
of the ABA
pathway are promoted by ROS, whereas calcium, through
calcineurin B, constrains both synthesis and signalling of
(Pandey et al., 2004)
ABA seems to be the first step that, by its molecular nature,
is committed to adaptation, as both calcium and ROS
modulate the ABA status (defined as dynamic equilibrium between
ABA content and signalling activity, Fig. 2C, ⑦), and this
point seems to be important for the decision between
adaptation and cell death:
1. The ‘on’ state is activated through both synthesis and
activation of signalling by ROS (Fig. 2C, ⑦).
2. The ‘off ’ state is achieved by the ABA signalling pathway
itself due to the induction of negative regulators (ABI
proteins) by ABA (Fig. 2C, ⑧).
3. The appropriate targets are adaptive genes harbouring
ABA-inducible adaptive genes that encode osmoprotectans
(such as the LEA proteins) but also signalling components
that adjust a sustainable ABA status as a balance between
constraint (through the ABIs) or promotion (through the
NADPH oxidase RboH generating ROS) (Fig. 2C, ⑥, ⑧).
4. In the case of delayed calcium signatures, calcium will,
through CBL proteins, impair the ROS-dependent
activation of the ABA pathway (Fig. 3C, ④). Due to this delay,
the concurrent JA pathway will become dominant,
culminating in cell death.
JAs: a dangerous switch
Jasmonic acid and related compounds, collectively named
jasmonates (JAs), are ubiquitously occurring lipid-derived
compounds, and function as a master switch in plant responses
to several abiotic and biotic stresses such as wounding
(mechanical stress), drought and salt stress, ozone and
pathogen infection, and insect attack
(reviewed by Wasternack,
2007; Wasternack and Hause, 2013)
. Similar to ABA, the
synthesis of JA is triggered by ROS, as the first committed
step of synthesis, the peroxidation of linoleic acid by
lipoxygenases, requires singlet oxygen
(Farmer and Mueller, 2013)
In contrast to the ABA pathway, which is negatively regulated
by CBL proteins, there is evidence that the JA pathway is
(reviewed by Hu et al., 2009)
. In addition to their
role as a general stress signal, JAs regulate many aspects of
plant development and growth such as seed germination, fruit
ripening, production of viable pollen, root growth, tendril
coiling, photomorphogenesis, leaf abscission, and senescence
(Creelman and Mullet, 1995, 1997a, b; Conconi et al., 1996;
Rao et al., 2000; Riemann et al., 2003, 2013; Haga and Iino,
2004; Ma et al., 2006; Robson et al., 2010; reviewed by Wang
et al., 2011)
. Among JA conjugates and derivatives,
(+)-7-isojasmonoyl-l -isoleucine (JA-Ile) formed by the enzyme JAR1
(Jasmonate-Resistant 1) was found to be an endogenous
bioactive form of JA
(Fonseca et al., 2009)
. Under non-stress
conditions, JA-Ile is maintained at low levels, and this allows
a multimeric protein complex to inactivate JA signalling in
plant cells. This machinery is composed of JAZ repressor
proteins that bind and repress the transcriptional activator
MYC2, via recruiting the Groucho/Tup1-type co-repressor
TOPLESS (TPL) and TPL-related proteins (TPRs) through
a transcriptional repressor called Novel Interactor of JAZ/
TIFY (NINJA) (Chini et al.,
2007; Thines et al., 2007
Pauwels et al., 2010; reviewed by
Kazan and Manners 2008
2012, 2013). In response to developmental or
environmental cues (including salinity), the levels of JA-Ile are elevated.
JA-Ile binds to COI1 and promotes the interaction of JAZ
proteins with COI1, leading to SCFCOI1-mediated
ubiquitination of the JAZ factors, followed by their degradation
via the 26S proteasome. This results in the release of
MYCtype transcription factors from repression by the JAZ factors
and thereby will induce transcription of early JA-responsive
genes including the JAZ genes themselves. In Arabidopsis,
derepression of AtMYC2 is induced under dehydration and
saline conditions. In addition, Arabidopsis plants in which
AtMYC2 is overexpressed exhibited less electrolyte leakage
under osmotic stress
(Abe et al., 2003)
. When treated with
MeJA, protective proteins against oxidative stress (which is
a true companion of many abiotic stresses including drought
or salt) accumulate in the wild type but at reduced levels in
a myc2 null mutant. These protective proteins include the
HSP20-like chaperone protein, the fibrillin precursor
protein, a luminal binding protein (BiP2), and GST
(Guo et al.,
. JA-dependent activation of OsbHLH148 upregulates
rice OsDREB1A, a functional orthologue of Arabidopsis
DREB1A (Fig. 2C, ⑨), which plays critical roles in
improving drought, salinity, and freezing tolerance but in an
(Dubouzet et al., 2003; Seo et al., 2011)
These results, at first sight, suggest a role for JA signalling in
conferring tolerance to drought and salinity, or oxidative stress.
However, a closer look reveals that JA signalling is tightly
controlled, as the transcription of JAZ genes is induced by JA.
The newly synthesized JAZ proteins interact with and restore
the repression of MYC2, which in turn deactivates the JA
signal transduction pathway (Chini et al.,
2007; Thines et al.,
; reviewed by Wager and Browse, 2012). This
negativefeedback loop and the resulting transient action of JA
indicate that unfavourable effects of overshooting JA signalling
have to be strictly avoided (Fig. 2C, ⑨). Indeed, MeJA was
found to induce cell death in Arabidopsis protoplasts
and Xing, 2008)
and Vitis cell suspensions
(Repka et al., 2004)
in a concentration- and time-dependent manner
. The MeJA-induced cell death correlates with ROS
production, alterations of mitochondrial dynamics, and
photosynthetic collapse (Fig. 3C, ⑥). The above-mentioned direct
comparison of the two salt-stressed grapevine cell lines showed
that the salt-sensitive V. riparia, where the accumulation of
ABA was delayed, accumulated tenfold higher levels of the
bioactive JA-Ile, whereas in the more salt-tolerant V. rupestris,
ABA accumulated earlier and strongly suppressed formation
of the JA-Ile signal
(Ismail et al., 2014)
. This more rapid and
more massive induction of JA-Ile was accompanied by a
pronounced oxidative burst in V. riparia accompanied by synthesis
of high amounts of δ-viniferin, a metabolic indicator for
ensuing programmed cell death
(Chang and Nick, 2012)
The importance of JA tuning is corroborated by analysis of
the rice mutant rice salt sensitive 3 (rss3), where root growth
is more severely inhibited under salinity compared with
the wild type
(Toda et al., 2013)
. This growth phenotype is
accompanied by elevated expression of JA-dependent genes.
RSS3 binds to JAZ and non-MYC-type bHLH transcription
factors, and has been proposed to repress an exaggerated
JA response in the root tip
(Toda et al., 2013)
these data suggest that fine-tuning JA signalling is important
for the growth and viability of plants under salinity stress.
The existence of a multimeric transcriptional co-repression
complex machinery to inactivate JA signalling (Chini et al.,
2007; Thines et al., 2007
; Pauwels et al., 2010), in addition to
JA-dependent repression of MYC2 via the MEK2/MAPK6
(Petersen et al., 2000)
, is evidence that suppression of
hazardous side effects of JA signalling is crucial for survival.
It should be kept in mind that ABA and JA signalling are
antagonistic on several levels—partially by mutual
competition for shared signalling factors such as MYC2
et al., 2004, Fig. 2C, ⑨)
The ambiguity of the ROS signal seems to be perpetuated
at the level of JA—early activation of this pathway seems to
be beneficial for salt adaptation, but sustained JA signalling is
clearly deleterious and culminates in cell death:
1. The ‘on’ state is triggered through oxidative cleavage
of membrane lipids and is therefore stimulated by ROS
(Fig. 2C, ⑦).
2. The ‘off ’ state is achieved by tight negative feedback of
the JA signal pathway itself with JAZ/TIFY proteins as
central players (Fig. 2C, ⑨).
3. Under adaptive conditions, the JA pathway will initially be
under tight constraint, probably triggered by a short
transient peak of ROS; some JA will be formed, but signalling
will be rapidly shut off, such that concurrent activation of
ABA signalling is ahead (Fig. 2A, ⑨).
4. In the case of delayed calcium signatures, impaired ABA
signalling (Fig. 3C, ④) in combination with
sodiumdependent mitochondrial membrane damage (Fig. 3C, ⑦)
will lead to excessive accumulation of JA accompanied by
accelerated oxidative burst, membrane damage, and
eventually cell death (Fig. 3C, ⑥).
The ubiquitin–26S proteasome system (UPS): executors of cross-talk?
In eukaryotes, such as plants, the UPS constitutes a tightly
regulated and highly specific machinery that is devoted to
(reviewed by Sullivan et al., 2003)
utilize the UPS to modulate almost all aspects of their
biology including growth, development, and stress responses
(Santner and Estelle, 2010)
. The crucial roles of UPS are
reflected in the number of genes encoding UPS components.
A genomic analysis of A. thaliana showed that over 1400
genes (or >5% of the proteome) code for UPS components
(Smalle and Vierstra, 2004)
. Interestingly, several enzymes in
the UPS are hormonal receptors. Moreover, the UPS controls
the levels of essential downstream signalling proteins in
hormonal signal transduction
(Santner et al., 2009)
. Thus, the
plant UPS not only removes abnormal proteins that arise due
to biosynthetic errors or normal proteins with the wrong
configuration (reviewed by Vierstra, 2009), but, in addition to this
canonical function, controls signal specificity by removal of
specific repressors. Furthermore, the UPS has been reported
to be critically involved in plant programmed cell death, as
the disruption of proteasome function by gene silencing of
the proteasome subunits activates programmed cell death in
(Kim et al., 2003)
As discussed above, salt-stressed plants increase the levels
of different hormones such as JA activating specific branches
of the UPS. The elevated levels of JA-Ile promote the
proteolytic degradation of JAZ proteins via UPS releasing JA
(reviewed by Wager and Browse, 2012;
Fig. 2C, ⑨)
. Conversely, in ABA signalling, the synthesis of
AIP2 is increased, which in turn suppresses the ABA
transcriptional regulator ABI3. However, ABA blocks
degradation of ABI5 (Fig. 2C, ⑧), a central regulator of ABA
signalling during post-germinative growth
It is possible to modulate stress tolerance through the
UPS. In fact, the functional ubiquitin-specific protease
UBP16 was found to increase salt tolerance by
stabilization of SERINE HYDROXYMETHYLTRANSFERASE1
(SHM1), which can reduce oxidative burst and therefore
repress cell death and at the same time positively
regulates plasma-membrane Na+/H+ antiporter activity
et al., 2012)
. Arabidopsis thaliana ABA insensitive RING
protein 3 (AtAIRP3/LOG2) is a positive regulator of the
ABA-mediated drought and salinity adaptation by
targeting RD21 (Responsive to Dehydration 21), which might
accelerate cell death progression during senescence and
(Kim and Kim, 2013)
. On the other hand,
two A. thaliana C3HC4 RING domain-containing proteins,
named DREB2A-INTERACTING PROTEIN1 (DRIP1)
and DRIP2, function as E3 ubiquitin ligases, and negatively
Life and death under salt stress: same players, different timing? | 2975
regulate drought-responsive gene expression by mediating
(Qin et al., 2008)
The convergence of different hormonal pathways on shared
elements of the UPS provides a molecular framework that
allows the integration of different signals, for instance the
relative status of JA versus ABA signalling (Fig. 2C, ⑧,⑨).
In other words, it might be this machinery that is dedicated to
specific destruction, where plant cells decode the ‘meaning’ of
different concurrent signal pathways.
To endow crops with enhanced stress tolerance, it is important
to understand the underlying mechanisms. The advances of
the last decade have revealed numerous molecular details of
salinity-triggered responses and mechanisms of adaptation.
In addition, complex and obviously precisely tuned cross-talk
between different pathways seems to be relevant. How is the
specificity of this cross-talk achieved? Both stress-tolerant
and -sensitive plants utilize the same signalling molecules.
However, it is important to conceptually discriminate signals
from the molecules that convey these signals. The central
message transported in this review is that the timing of stress
signals is decisive. Stress signals are activated transiently and they
are subsequently turned off. Whether a plant cell will adapt to
salt stress or whether it will yield to cell death appears to be
dependent on the correct timing of these transient signalling
events. Tolerant plants are tolerant because they can
orchestrate cross-talks between different signalling pathways (signal
on demand). This is likely to be achieved by controlling the
temporal signature and amplitude of the signalling. When
this temporal control turns loose, such that a signalling event
persists longer or initiates later, this will lead to inappropriate
cross-talk with downstream events of other pathways. These
downstream events would otherwise not be competent for the
respective signal, simply because they proceed at a time point
that is later. We have elaborated this heterochronous shift of
signalling for the interaction between calcium and JA versus
ABA signalling. A delay in the activation and, consequently,
also in the deactivation, of the calcium signature will
channel ROS-triggered signalling towards the JA pathway, which,
in consequence, will run out of control, culminating in cell
death. The same molecule (calcium), occurring at the right
time, will be recruited for the activation of sodium extrusion
and sequestering, such that ROS-triggered signalling will be
channelled to (protective) ABA signalling constraining the
JA pathway and thus leading to efficient adaptation.
Thus, a deeper understanding of the temporal patterns
in signalling will help us to dissect adaptive from
damagerelated events. But this conclusion also calls for a specific
experimental approach to stress physiology: to identify
temporal signatures in stress adaptation, we need approaches
that are both comparative and integrative—comparative in
the sense that systems that are biologically very similar but
differ in the outcome of their stress response are investigated
side by side under the same conditions, and integrative in
the sense that the different stages of the stress response are
studied in the same system in parallel with respect to their
time course. Knowledge of the molecular players of stress
adaptation is a necessary prerequisite but is t sufficient. We
need to consider and investigate molecular activities rather
than mere molecules.
AI was supported by the German Egyptian Research Long term Scholarship
ʿGERLSʾ program, which is jointly funded by the Ministry of Higher
Education and Scientific Research ‘MHESR’, Egypt and the Deutscher
Akademischer Austauschdienst ‘DAAD’, Bonn, Germany.
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