Journal of Experimental Botany, Page 1 of 16doi:10.1093/jxb/err460
REVIEW PAPER
Drought, salt, and temperature stress-induced metabolicrearrangements and regulatory networks
Julia Krasensky and Claudia Jonak*
GMI–Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030 Vienna, Austria
* To whom correspondence should be addressed. E-mail: [email protected]
Received 7 October 2011; Revised 21 December 2011; Accepted 22 December 2011
Abstract
Plants regularly face adverse growth conditions, such as drought, salinity, chilling, freezing, and high temperatures.
These stresses can delay growth and development, reduce productivity, and, in extreme cases, cause plant death.
Plant stress responses are dynamic and involve complex cross-talk between different regulatory levels, including
adjustment of metabolism and gene expression for physiological and morphological adaptation. In this review, infor-
mation about metabolic regulation in response to drought, extreme temperature, and salinity stress is summarized and
the signalling events involved in mediating stress-induced metabolic changes are presented.
Key words: Abiotic stress, metabolism, protein kinase, signal transduction, transcription factor.
Plants in adverse environments
Plants frequently encounter unfavourable growth conditions.Climatic factors, such as extreme temperatures (heat, cold,
freezing), drought (deficient precipitation, drying winds), and
contamination of soils by high salt concentration, are major
abiotic environmental stressors that limit plant growth and
development, and thus agronomical yield, and play a major
role in determining the geographic distribution of plant
species. These different adverse but not necessarily lethal
conditions are generally known as stress.Environmental stress can disrupt cellular structures and
impair key physiological functions (Larcher, 2003). Drought,
salinity, and low temperature stress impose an osmotic stress
that can lead to turgor loss. Membranes may become dis-
organized, proteins may undergo loss of activity or be de-
natured, and often excess levels of reactive oxygen species
(ROS) are produced leading to oxidative damage. As a con-
sequence, inhibition of photosynthesis, metabolic dysfunc-tion, and damage of cellular structures contribute to growth
perturbances, reduced fertility, and premature senescence.
Different plant species are highly variable with respect to
their optimum environments, and a harsh environmental
condition, which is harmful for one plant species, might not
be stressful for another (Larcher, 2003; Munns and Tester,
2008). This is also reflected in the multitude of different
stress-response mechanisms. Two major strategies can bedistinguished: stress avoidance and stress tolerance (Levitt,
1980). Stress avoidance includes a variety of protective
mechanisms that delay or prevent the negative impact of a
stress factor on a plant. For example, cacti have constitu-
tively adapted their morphology, physiology, and metabo-
lism to hot and arid climates. Adaptation is stable and
inherited. On the other hand, stress tolerance is the potential
of a plant to acclimate to a stressful condition. For example,in summer, trees and herbaceous plants in northern latitudes
cannot withstand freezing. Exposure to chilling temperatures,
however, induces hardening and acclimated plants survive
winter temperatures far below freezing. Plants can increase
their resistance to various stresses including heat, saline, and
drought conditions in response to a period of gradual
exposure to these constraints. Acclimation is plastic and
reversible. The physiological modifications induced duringacclimation are diverse and are usually lost when the adverse
environmental condition does not persist.
Responses to environmental stresses occur at all levels of
organization. Cellular responses to stress include adjustments
of the membrane system, modifications of the cell wall
architecture, and changes in cell cycle and cell division. In
addition, plants alter metabolism in various ways, including
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production of compatible solutes (e.g. proline, raffinose, and
glycine betaine) that are able to stabilize proteins and cellular
structures and/or to maintain cell turgor by osmotic adjust-
ment, and redox metabolism to remove excess levels of ROS
and re-establish the cellular redox balance (Bartels and
Sunkar, 2005; Valliyodan and Nguyen, 2006; Munns and
Tester, 2008; Janska et al.; 2010). At the molecular level, gene
expression is modified upon stress (Chinnusamy et al., 2007;Shinozaki and Yamaguchi-Shinozaki, 2007) and epigenetic
regulation plays an important role in the regulation of gene
expression in response to environmental stress (Hauser et al.,
2011; Khraiwesh et al., 2011). Stress-inducible genes comprise
genes involved in direct protection from stress, including the
synthesis of osmoprotectants, detoxifying enzymes, and tran-
sporters, as well as genes that encode regulatory proteins such
as transcription factors, protein kinases, and phosphatases.
In this review, the focus is on metabolic adjustments in
response to high salt stress, drought, and extreme temper-
atures and our current knowledge of signal transduction
components that regulate metabolite levels under thesestress conditions will be summarized. Figures 1 and 2 give
an overview of the biosynthesis and degradation pathways
of important metabolites connected with stress tolerance
and will guide you through the next paragraphs.
Fig. 1. Schematic overview of amino acid, polyamine, and glycine betaine metabolism. Plants with enhanced or reduced activity of the
indicated enzymes show altered tolerance to abiotic stress. ApGSMT, Aphanothece halophytica glycine sarcosine methyl transferase (EC
2.1.1.156); ApDMT, Aphanothece halophytica dimethylglycine methyltransferase (EC 2.1.1.157); codA, choline oxidase (EC 1.1.3.17);
betA, choline dehydrogenase (EC 1.1.99.1); betB, betaine aldehyde dehydrogenase (EC 1.2.1.8); GAD, glutamate decarboxylase (EC
4.1.1.15); GABA-T, 4-aminobutyrate aminotransferase (EC 2.6.1.19); SSADH, succinic semialdehyde dehydrogenase (EC 1.2.1.16);
P5CS, 1-pyrroline-5-carboxylate synthetase (EC 2.7.2.11 and EC 1.2.1.41); P5CR, pyrroline-5-carboxylate reductase (EC 1.5.1.2);
ProDH, proline dehydrogenase (EC 1.5.99.8), P5CDH, 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12); d-OAT, ornithine
d-aminotransferase (EC 2.6.1.13); ADC, arginine decarboxylase (EC 4.1.1.19); ODC, ornithine decarboxylase (EC 4.1.1.17); SPDS,
spermidine synthase (EC 2.5.1.16); SPMS, spermine synthase (EC 2.1.5.22); PAO, polyamine oxidase (EC 1.5.3.11); DAO, diamine
oxidase (EC 1.4.3.22).
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Amino acids
Accumulation of amino acids has been observed in many
studies on plants exposed to abiotic stress (Barnett and
Naylor, 1966; Draper, 1972; Handa et al., 1983; Rhodes
et al., 1986; Fougere et al., 1991; Kaplan et al., 2004;
Brosche et al., 2005; Zuther et al., 2007; Kempa et al.,
2008; Sanchez et al., 2008; Usadel et al., 2008; Luganet al., 2010). This increase might stem from amino acid
production and/or from enhanced stress-induced protein
breakdown. While the overall accumulation of amino
acids upon stress might indicate cell damage in some
species (Widodo et al., 2009), increased levels of specific
amino acids have a beneficial effect during stress acclima-
tion.
Proline
For many years, the capacity to accumulate proline has
been correlated with stress tolerance (Barnett and Naylor,
1966; Singh et al., 1972; Stewart and Lee, 1974). Proline is
considered to act as an osmolyte, a ROS scavenger, and
a molecular chaperone stabilizing the structure of proteins,
thereby protecting cells from damage caused by stress (Hare
and Cress, 1997; Rhodes et al., 1986; Verbruggen and
Hermans, 2008; Szabados and Savoure, 2010). Proline
accumulates in many plant species in response to different
environmental stresses including drought, high salinity, and
heavy metals. Interestingly, heat stress did not lead to
proline accumulation in tobacco and Arabidopsis plants
and induced proline accumulation rendered plants more
Fig. 2. Schematic overview of starch, fructan, sugar, and polyol metabolism. Plants with enhanced or reduced activity of the indicated
enzymes show altered tolerance to abiotic stress. SEX1, a-glucan water dikinase (EC 2.7.9.4); St. phos, starch phosphorylase (2.4.1.1);
BMY, b-amylase (EC 3.2.1.2); DPE2, glucanotransferase (EC 2.4.1.25); 1-SST, sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99); 6-
SFT, sucrose:fructan 6-fructosyltransferase (EC 2.4.1.10); FBF, fructan beta-fructosidase (EC 3.2.1.80); mtlD, mannitol-1-phosphate
dehydrogenase (EC 1.1.1.17); S6PDH, sorbitol-6-phosphate dehydrogenase (EC 1.1.1.200); SDH, sorbitol dehydrogenase (EC
1.1.1.14); Pase, unspecific phosphatase; MIPS, inositol-1-phosphate synthase (EC 5.5.1.4); IMP, inositol-1-phosphate phosphatase (EC
3.1.3.25); IMT, inositol methyltransferase (EC 2.1.1.40); GolS, galactinol synthase (EC 2.4.1.123); RS, raffinose synthase (EC 2.4.1.82),
StS, stachyose synthase (EC 2.4.1.67); TPS, trehalose-6-phosphate synthase (EC 2.4.1.15); TPP, trehalose-6-phosphate phosphatase
(EC 3.1.3.12).
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sensitive to heat (Rizhsky et al., 2004; Dobra et al., 2010;
Lv et al., 2011). Proline levels are determined by the
balance between biosynthesis and catabolism (Szabados
and Savoure, 2010). Proline is produced in the cytosol or
chloroplasts from glutamate, which is reduced to gluta-
mate-semialdehyde (GSA) by D-1-pyrroline-5-carboxylatesynthetase (P5CS). GSA can spontaneously convert to
pyrroline-5-carboxylate (P5C), which is then further re-duced by P5C reductase (P5CR) to proline. Proline is
degraded in mitochondria by proline dehydrogenase
(ProDH) and P5C dehydrogenase (P5CDH) to glutamate.
Stress conditions stimulate proline synthesis while proline
catabolism is enhanced during recovery from stress. Over-
expression of P5CS in tobacco and petunia led to increased
proline accumulation and enhanced salt and drought
tolerance (Kishor et al., 1995; Hong et al., 2000; Yamadaet al., 2005), whereas Arabidopsis P5CS1 knock-out plants
were impaired in stress-induced proline synthesis and were
hypersensitive to salinity (Szekely et al., 2008). Consis-
tently, ProDH antisense Arabidopsis accumulated more
proline and showed enhanced tolerance to freezing and
high salinity (Nanjo et al., 1999). It is discussed that, in an
alternative pathway, mitochondrial P5C can be produced
by d-ornithine aminotransferase (d-OAT) from ornithine(Miller et al., 2009). Over-expression of Arabidopsis d-OAT
has been shown to enhance proline levels and to increase
the stress tolerance of rice and tobacco (Roosens et al.,
2002; Qu et al., 2005) even though Arabidopsis plants
deficient in d-OAT accumulated proline in response to
stress and showed a salt stress tolerance similar to the wild
type (Funck et al., 2008).
GABA
The non-protein amino acid c-aminobutyric acid (GABA)
rapidly accumulates to high levels under different adverseenvironmental conditions (Shelp et al., 1999; Rhodes et al.,
1986; Kinnersley and Turano, 2000; Kaplan and Guy, 2004;
Kempa et al., 2008; Renault et al., 2010).
GABA is mainly synthesized from glutamate in the
cytosol by glutamate decarboxylase (GAD) and then
transported to the mitochondria. GABA transaminase
(GABA-T) and succinic semialdehyde dehydrogenase
(SSADH) convert GABA into succinate that feeds intothe TCA-cycle (Shelp et al., 1999; Fait et al., 2008). GABA
metabolism has been associated with carbon–nitrogen
balance and ROS scavenging (Bouche and Fromm, 2004;
Song et al., 2010; Liu et al., 2011). A functional GABA
shunt is important for stress tolerance. Salt stress enhances
the activity of enzymes involved in GABA metabolism
(Renault et al., 2010). Arabidopsis mutants defective in
GABA-T were hypersensitive to ionic stress and showedincreased levels of amino acid (including GABA), while
carbohydrate levels were decreased (Renault et al., 2010).
Disruption of the SSADH gene led to the accumulation of
ROS associated with dwarfism and hypersensitivity to UV-B
and heat stress (Bouche et al., 2003).
Amines
Polyamines
Polyamines (PA) are small aliphatic molecules positively
charged at cellular pH. Various stresses, such as drought,
salinity and cold, modulate PA levels, and high PA levels
have been positively correlated with stress tolerance (Yang
et al., 2007; Cuevas et al., 2008; Groppa and Benavides,
2008; Usadel et al., 2008; Kovacs et al., 2010; Quinet et al.,2010; Alcazar et al., 2011).
Putrescine, spermidine, and spermine are the most com-
mon PAs in higher plants. Putrescine is produced from either
ornithine or arginine by ornithine decarboxylase (ODC) and
arginine decarboxylase (ADC), respectively. Putrescine is
converted to spermidine by spermidine synthase (SPDS) and
then to spermine by spermine synthase (SPMS). Spermidine
and spermine are substrates of polyamine-oxidases (PAOs),which catalyse the back-conversion to putrescine.
PAs have been implicated in protecting membranes and
alleviating oxidative stress (Groppa and Benavides, 2008;
Alcazar et al., 2011; Hussain et al., 2011) but their specific
function in stress tolerance is not well understood. Analyses
of transgenic plants and of mutants involved in PA meta-
bolism clearly showed a positive role of PAs in stress
tolerance. Plants deficient in ADC1 or ADC2 had reducedputrescine levels and were hypersensitive to stress (Urano
et al., 2004; Cuevas et al., 2008), whereas constitutive or
stress-induced over-expression of ADC led to higher putres-
cine levels and enhanced drought and freezing tolerance
(Capell et al., 2004; Alcazar et al., 2010; Alet et al., 2011).
Arabidopsis plants over-expressing SPDS produced higher
amounts spermidine and were more resistant to drought, salt,
and cold stress (Kasukabe et al., 2004). Plants deficient inSPMS were unable to synthesize spermine and are hypersen-
sitive to salinity (Yamaguchi et al., 2006). Furthermore, the
introduction of ornithine decarboxylase (ODC) from mouse
enhanced polyamine levels and the tolerance of tobacco to
salt stress (Kumriaa and Rajam, 2002).
Glycine betaine
Glycine betaine (GB) is a quaternary ammonium compound
that occurs in a wide variety of plants, but its distribution
among plants is sporadic (Cromwell and Rennie, 1953).
Arabidopsis and many crop species do not accumulate GB.In plants that produce GB naturally, abiotic stress, such as
cold, drought, and salt stress, enhances GB accumulation
(Rhodes and Hanson, 1993; Chen and Murata, 2011).
GB can be synthesized from choline and glycine (Chen
and Murata, 2011). Introduction of the GB biosynthesis
pathway genes into non-accumulators improved their abil-
ity to tolerate abiotic stress conditions (Hayashi et al., 1997;
Alia et al., 1998; Sakamoto et al., 1998, 2000; Holmstromet al., 2000; Park et al., 2004; Waditee et al., 2005; Bansal
et al., 2011), pointing to the beneficial role of GB in stress
tolerance. Targeting GB production to the chloroplasts led
to a higher tolerance of plants against stress than in the
cytosol (Park et al., 2007). In salt-tolerant plant species, GB
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can also accumulate to osmotically significant levels (Rhodes
and Hanson, 1993). It has been suggested that GB protects
photosystem II, stabilizes membranes, and mitigates oxida-
tive damage (Chen and Murata, 2011).
Carbohydrates
Fructans
Plants accumulate carbohydrates such as starch and fruc-
tans as storage substances that can be mobilized during
periods of limited energy supply or enhanced energetic
demands. While most plant species use starch as their main
storage carbohydrate, several angiosperms, mainly from
regions with seasonal cold and dry periods, accumulatefructans (Hendry, 1993). Accumulation of fructans might
be advantageous, due to their high water solubility, their
resistance to crystallization at freezing temperatures, and
the fact that fructan synthesis functions normally under
low temperatures (Vijn and Smeekens, 1999; Livingston
et al., 2009). Furthermore, fructans can stabilize mem-
branes (Valluru and Van den Ende, 2008) and might
indirectly contribute to osmotic adjustment upon freezingand dehydration by the release of hexose sugars (Spollen
and Nelson, 1994; Olien and Clark, 1995).
Fructans are branched fructose polymers that are syn-
thesized in the vacuole by fructosyltransferases, including
1-SST and 6-SFT, which transfer fructose from sucrose to
the growing fructan chain (Vijn and Smeekens, 1999;
Livingston et al., 2009). The introduction of fructosyltrans-
ferases to fructan non-accumulating tobacco and rice plantsstimulated fructan production associated with enhanced
tolerance to low-temperature stress and drought (Pilonsmits
et al., 1995; Li et al., 2007; Kawakami et al., 2008).
Starch, mono- and disaccharides
Starch is composed of glucose polymers arranged intoosmotically inert granules. It serves as the main carbohy-
drate store in most plants and can be rapidly mobilized to
provide soluble sugars. Its metabolism is very sensitive to
changes in the environment. In addition to diurnal fluctua-
tions in starch levels, salt and drought stress generally leads
to a depletion of starch content and to the accumulation of
soluble sugars in leaves (Todaka et al., 2000; Kaplan and
Guy, 2004; Basu et al., 2007; Kempa et al., 2008). Sugarsthat accumulate in response to stress can function as
osmolytes to maintain cell turgor and have the ability to
protect membranes and proteins from stress damage
(Madden et al., 1985; Kaplan and Guy, 2004).
Starch is produced in plastids from excess sugars during
photosynthesis and involves ADP-glucose pyrophosphory-
lase (AGPase), starch synthases, branching enzymes, and
debranching enzymes. Phosphorylation of starch granulesby glucan-water dikinase (GWD) and phosphoglucan-water
dikinase (PWD) stimulates starch degradation. b-amylases
produce maltose from glucans. In the cytosol, maltose is
converted to glucose and, subsequently, fructose and sucrose
are formed (Tetlow et al., 2004; Kotting et al., 2010).
Hydrolysis of starch by the b-amolytic pathway repre-
sents the predominant pathway of starch degradation in
leaves under normal growth conditions and may also be
involved in stress-induced starch hydrolysis. Arabidopsis sex1
(starch excess 1) mutants, that are impaired in GWD activity,
were compromised in cold-induced malto-oligosaccharide,
glucose, and fructose accumulation during the early stages
of cold acclimation and sex1 plants that have been brieflypre-exposed to cold showed a reduced freezing tolerance
(Yano et al., 2005). Osmotic stress was shown to enhance
total b-amylase activity and to reduce light-stimulated
starch accumulation in wild-type Arabidopsis but not in
bam1 (bmy7) mutants, which appeared to be hypersensitive
to osmotic stress (Valerio et al., 2011). Similarly, BMY8
(BAM3) antisense plants accumulated high starch levels,
were impaired in cold-induced maltose, glucose, fructose,and sucrose accumulation, and showed a reduced tolerance
of photosystem II to low temperature stress (Kaplan and
Guy, 2005). Data by Zeeman et al. (2004) also suggest a role
of the phosphorolytic starch degradation pathway during
stress. Arabidopsis plants deficient in plastidial a-glucanphosphorylase showed enhanced formation of lesions sur-
rounded by cells with high starch levels after exposure to
low air humidity or salt stress.
Trehalose
The non-reducing disaccharide trehalose accumulates to high
amounts in some desiccation-tolerant plants, for example,Myrothamnus flabellifolius (Bianchi et al., 1993; Drennan
et al., 1993). At sufficient levels, trehalose can function as an
osmolyte and stabilize proteins and membranes (Paul et al.,
2008). In most angiosperms however, trehalose is present in
trace amounts and abiotic stress increases the levels of
trehalose only moderately (Fougere et al., 1991; Garg et al.,
2002; Kaplan et al., 2004; Panikulangara et al., 2004;
Rizhsky et al., 2004; Guy et al., 2008; Kempa et al., 2008).In plants, trehalose is synthesized in a two-step process
(Paul et al., 2008). Trehalose-6-phosphate synthase (TPS)
generates trehalose-6-phosphate (T6P) from UDP-glucose
and glucose-6-phosphate followed by dephosphorylation
to trehalose by trehalose-6-phosphate phosphatase (TPP).
Transgenic expression of trehalose biosynthesis genes showed
that enhanced trehalose metabolism can positively regulate
tolerance to abiotic stress, even though only a limited increasein trehalose content could be observed, excluding a direct
protective role of trehalose in these plants. Heterologous
expression of genes involved in the trehalose pathway from
E. coli or Saccharomyces cerevisiae enhanced tolerance to
drought, salt, and low temperature stress in several plant
species (Iordachescu and Imai, 2008). Over-expression of
different isoforms of TPS from rice conferred enhanced
resistance to salinity, cold, and/or drought (Li et al., 2011).Arabidopsis plants constitutively over-expressing AtTPS1
showed a small increase in trehalose and T6P levels and were
more tolerant to drought (Avonce et al., 2004). Consistently,
loss of TPS5 function, a TPS with a TPP domain, reduced
the basal thermotolerance of Arabidopsis (Suzuki et al., 2008).
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TPP activity and trehalose levels transiently increased in
response to cold stress in rice (Panikulangara et al., 2004) and
over-expression of OsTPP1 rendered rice more tolerant to
salinity and cold even though no increase in the trehalose
content could be observed (Ge et al., 2008). These genetically
engineered plants provide evidence that enhanced trehalose
metabolism can positively regulate stress tolerance, but the
precise role of T6P and trehalose during abiotic stressremains to be further elucidated.
Raffinose family oligosaccharides
Raffinose family oligosaccharides (RFO) such as raffinose,
stachyose, and verbascose accumulate in various plant
species during seed desiccation (Peterbauer and Richter,
2001) and in leaves of plants experiencing environmental
stress like cold, heat, drought or high salinity (Castonguay
and Nadeau, 1998; Gilmour et al., 2000; Taji et al., 2002;Cook et al., 2004; Kaplan et al., 2004; Peters et al., 2007;
Kempa et al., 2008; Usadel et al., 2008). RFOs have been
implicated in membrane protection and radical scavenging
(Hincha, 2003; Nishizawa et al., 2008). Biosynthesis of RFO
is initiated by the formation of galactinol from myo-inositol
and UDP-galactose by galactinol synthase (GolS). Sequen-
tial addition of galactose units, provided by galactinol, to
sucrose leads to the formation of raffinose and higher orderRFO (Peterbauer and Richter, 2001).
The role of the RFO pathway in acquiring stress
tolerance is not yet fully understood. Arabidopsis plants
over-expressing Arabidopsis GolS1 or GolS2 accumulated
high levels of galactinol and raffinose and were more
tolerant to drought and salinity stress (Taji et al., 2002;
Nishizawa et al., 2008). Similarly, constitutive and stress-
inducible expression of GolS1 from the resurrection plantBoea hygrometrica in tobacco conferred enhanced drought
tolerance (Wang et al., 2009). By contrast, increased raffinose
content in Arabidopsis plants constitutively expressing cu-
cumber GolS did not enhance freezing tolerance (Zuther
et al., 2004) and neither gols1 knock-out plants nor knock-
out mutants in the raffinose synthase gene, that were both
impaired in raffinose accumulation, showed any obvious
increase in sensitivity to heat or freezing stress, respectively(Panikulangara et al., 2004; Zuther et al., 2004).
Polyols
Polyols are implicated in stabilizing macromolecules and in
scavenging hydroxyl radicals, thereby preventing oxidative
damage of membranes and enzymes (Smirnoff and Cumbes,
1989; Shen et al., 1997). Accumulation of the straight-chain
polyols, mannitol and sorbitol, has been correlated with
stress tolerance in several plants species (Stoop et al., 1996).
Expression of mannitol-1-phosphate dehydrogenase (mtlD)from E. coli, which catalyses the reversible conversion of
fructose-6-phosphate to mannitol-1-phosphate in Arabidopsis,
tobacco, poplar, and wheat induced mannitol accumula-
tion and enhanced tolerance to salinity and/or water deficit
(Tarczynski et al., 1993; Thomas et al., 1995; Abebe et al.,
2003; Chen et al., 2005). Similarly, photosystem II was less
affected by salinity in persimmon trees that accumulated
sorbitol by over-expression of sorbitol-6-phosphate dehy-
drogenase (S6PDH) from apple (Gao et al., 2001).
The cyclic polyols myo-inositol, and its methylated
derivatives D-ononitol and D-pinitol, accumulate upon salt
stress in several halotolerant plant species (Adams et al.,
1992; Vernon and Bohnert, 1992; Ishitani et al., 1996;Sengupta et al., 2008). L-myo-inositol-1-phosphate synthase
(MIPS) forms myo-inositol-1-P from glucose-6-P, which is
dephosphorylated by myo-inositol 1-phosphate phosphatase
(IMP) to form myo-inositol. Inositol O-methyltransferase
(IMT) methylates inositol to form D-ononitol, which is
epimerized to D-pinitol. In line with a stress-protective role,
over-expression of MIPS and IMT from halotolerant plants
increased cyclic polyols levels and salt stress tolerance oftobacco (Sheveleva et al., 1997; Majee et al., 2004; Patra
et al., 2010).
Complexity of the metabolic response
Metabolic adjustments in response to unfavourable con-
ditions are dynamic and multifaceted and not only depend
on the type and strength of the stress, but also on the
cultivar and the plant species. Traditionally, metabolic
studies focused on single metabolites or groups of metabo-
lites. Recent advances in metabolic profiling allow increas-
ingly comprehensive metabolite analyses, illustrating thecomplexity of metabolic adjustments to stress. The meta-
bolic profiles of different plant species, including rice,
poplar, Vitis vinifera, Thellungiella halophila, and Arabidopsis
thaliana, have been analysed after exposure to drought
(Rizhsky et al., 2004; Cramer et al., 2007; Urano et al.,
2009), salinity (Gong et al., 2005; Cramer et al., 2007;
Gagneul et al., 2007; Kempa et al., 2008; Sanchez et al.,
2008; Janz et al., 2010; Lugan et al., 2010), and temperaturestress (Cook et al., 2004; Kaplan et al., 2004, 2007; Rizhsky
et al., 2004; Usadel et al., 2008; Espinoza et al., 2010;
Caldana et al., 2011).
Plants respond to stress by a progressive adjustment of
their metabolism with sustained, transient, early- and late-
responsive metabolic alterations. For example, raffinose and
proline accumulate to high levels over the course of several
days of salt exposure, drought, or cold, whereas centralcarbohydrate metabolism changes rapidly in a complex,
time-dependent manner.
Some metabolic changes are common to salt, drought,
and temperature stress, whereas others are specific. For
example, levels of amino acids, sugars, and sugar alcohols
typically increase in response to different stress conditions.
Notably, proline accumulates upon drought, salt, and low
temperature but not upon high temperature stress (Kaplanet al., 2004; Rizhsky et al., 2004; Gong et al., 2005; Cramer
et al., 2007; Gagneul et al., 2007; Kempa et al., 2008;
Sanchez et al., 2008; Usadel et al., 2008; Urano et al., 2009;
Lugan et al., 2010;). In most studies, organic acids and
TCA-cycle intermediates decreased in glycophytes after salt
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stress (Gong et al., 2005; Gagneul et al., 2007; Zuther et al.,
2007; Sanchez et al., 2008), but increased in response to
temperature or drought stress (Kaplan et al., 2004; Usadel
et al., 2008; Urano et al., 2009). A direct comparison of the
metabolic profile of Arabidopsis thaliana acclimated to
either low or high temperatures showed a significant over-
lap, but also major differences in metabolic adjustments
(Kaplan and Guy, 2004). A greater number of metabolitelevels changed specifically in response to cold than to heat,
pointing towards a strong impact of cold on plant metabo-
lism. Similarly, metabolic rearrangements after drought
were more profound than after heat stress and the
metabolic profile of Arabidopsis plants exposed to a combi-
nation of drought and heat was more similar to that of
drought-stressed than heat-stressed plants. Exposure of
drought-stressed plants to heat also induced unique meta-bolic responses highlighting the complexity of metabolic
adjustments in natural environments (Rizhsky et al., 2004).
Comparison of the metabolome of stress-tolerant acces-
sions, cultivars, or species, with that of stress-sensitive ones,
further show that, the role of metabolism in natural stress
tolerance is multifaceted and diverse (Gong et al., 2005;
Hannah et al., 2006; Zuther et al., 2007; Janz et al., 2010;
Korn et al., 2010; Lugan et al., 2010). Generally, stresstolerant plants have higher levels of stress-related metabo-
lites under normal growth conditions and/or accumulate
larger amounts of protective metabolites, such as proline
and soluble sugars, under unfavourable conditions, indicat-
ing that their metabolism is prepared for adverse growth
conditions. For example, analyses of the metabolic profile
of Arabidopsis accessions with different freezing tolerances
point to a crucial role of compatible solutes, including pro-line and raffinose, in freezing tolerance (Hannah et al.,
2006; Korn et al., 2010). Similarly, comparison of the meta-
bolome of cultivars or species with different salt tolerance
indicates a beneficial role of compatible solutes under
salinity conditions (Gong et al., 2005; Zuther et al., 2007;
Janz et al., 2010; Lugan et al., 2010). Interestingly, meta-
bolism of salt-tolerant plants appeared to be pre-adapted to
saline environments by constitutively high levels of someprotective metabolites such as proline and raffinose. Con-
sistent with classical studies, these recent metabolomic
analyses illustrate that plants have developed a whole range
of strategies to adapt their metabolism to unfavourable
growth conditions and that enhanced stress resistance is not
restricted to a single compound or mechanism. Different
plant species accumulate different metabolites (e.g. treha-
lose, proline, glycine betaine) and there is no absolute req-uirement for the accumulation of a specific metabolite for
acclimation to stress. In some cases, the flux through a meta-
bolic pathway, rather than the accumulation of a specific
metabolite per se, might contribute to stress tolerance.
Several metabolites/metabolic pathways that contribute
to stress acclimation also play a role in development.
Appropriate proline levels have been shown to be important
for embryo and flower development (Nanjo et al., 1999;Samach et al., 2000; Mattioli et al., 2008, 2009; Szekely
et al., 2008). GABA regulates pollen tube growth and
guidance (Wilhelmi and Preuss, 1996; Palanivelu et al.,
2003). Polyamines regulate several developmental processes
including embryogenesis, meristem, flower, and gameto-
phyte development (Hanzawa et al., 2000; Imai et al., 2004;
Alcazar et al., 2005; Gupta and Kaur, 2005; Deeb et al.,
2010; Zhang et al., 2011b). Trehalose metabolism is essential
for proper embryo maturation as well as for vegetative and
inflorescence development (Eastmond et al., 2002; vanDijken et al., 2004; Satoh-Nagasawa et al., 2006).
Signal transduction and transcriptionalcontrol involved in stress-induced metabolicchanges
Acclimation of plants to changes in their environment
requires a new state of cellular homeostasis achieved by a
delicate balance between multiple pathways. Hormones,secondary messengers, phosphatases, and protein kinases
are crucial components within the stress-induced signalling
network that regulates a multitude of biochemical and
physiological processes (Hirayama and Shinozaki, 2010).
As highlighted in the previous paragraphs, metabolic
adjustments to stress are vital for acquiring stress tolerance.
Transcriptional analyses of stress signalling mutants showed
that, in numerous mutants with altered stress tolerance,genes involved in the synthesis of stress-associated metabo-
lites are altered, and thus might be directly or indirectly
involved in metabolic adjustment upon stress. However, due
to post-transcriptional and post-translational modifications,
compartmentalization, metabolite stability, substrate avail-
ability, etc. changes in the abundance of transcripts are
not necessarily translated into changes in metabolite levels.
A start has been made to link signal transduction with themetabolite response upon drought, salt, and temperature
stress conditions including targeted analyses of metabolites
and metabolic profiling of mutants in signalling compo-
nents.
ABA and metabolic adjustments
Abscisic acid (ABA) is an integral regulator of abiotic stress
signalling (Cutler et al., 2010; Hubbard et al., 2010;
Raghavendra et al., 2010; Umezawa et al., 2010). ABA
quickly accumulates in response to different environmentalstress conditions and ABA-deficient plants have an altered
stress response. ABA promotes stomatal closure, inhibits
stomatal opening to reduce water loss by transpiration,
induces the expression of numerous stress-related genes, and
recent studies indicate a role in regulation of stress-induced
metabolic adjustments.
Comparison of the metabolic profile of Arabidopsis plants
treated with ABA, or exposed to high soil salinity, revealedABA-induced and ABA-independent steps of salt stress-
induced metabolic rearrangements (Kempa et al., 2008).
Both ABA and salt stress led to a depletion of starch
and the accumulation of maltose. However, subsequent
carbon flux appears to be differentially regulated. While
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glucose-6-phosphate and fructose-6-phosphate levels, and
the glucose/fructose ratio, decreased under salt stress con-
ditions, glucose-6-phosphate and fructose-6-phosphate levels
and the glucose/fructose ratio increased in response to ABA
treatment. Interestingly, ABA did not induce galactinol and
raffinose accumulation (Kempa et al., 2008). In support of
an ABA-independent induction of these sugars, Arabidopsis
mutants deficient in stress-induced ABA accumulation wereable to induce galactinol and raffinose under drought con-
ditions (Urano et al., 2009). Similarly, stress-induced accu-
mulation of citrate, malate, succinate (TCA cycle), and
GABA is ABA-independent (Kempa et al., 2008; Urano
et al., 2009). In contrast, accumulation of many osmotic
stress-induced proteinogenic amino acids, including proline,
were induced and depended on ABA (Kempa et al., 2008;
Urano et al., 2009).
Signalling towards proline accumulation
Proline plays a mulifunctional role in stress defence. Several
protein kinases important for salt, drought, and/or cold
tolerance, have been shown to regulate proline accumula-
tion (Fig. 3). SNF-related protein kinases 2 (SnRK2s) areactivated by osmotic stress (Boudsocq et al., 2004). Analyses
of Arabidopsis mutants revealed that the ABA-responsive
SnRKs 2.2, 2.3, and 2.6 are important for osmotic stress-
and ABA-induced proline accumulation, whereas SnRK2.9
appears to play a negative role in ABA-induced proline
accumulation (Fujii et al., 2011).
SnRK3s, which interact with calcineurin B-like (CBL)
calcium binding proteins, and are thus also known as CBLinteracting kinases (CIPKs), are also involved in modulat-
ing proline levels. In rice, over-expression of OsCIPK03 and
OsCIPK12 enhanced tolerance to cold and drought, re-
spectively, and increased the levels of proline under stress
conditions. However, over-expression of the closely related
OsCIPK15 enhanced salt tolerance without any significant
influence on stress-induced proline content indicating that
only a subset of SnRK3s are involved in signalling towards
proline accumulation in response to stress (Xiang et al.,
2007).
In addition to the above-mentioned calcium-responsive
protein kinases, Arabidopsis calcium-dependent protein
kinase 6 (CDPK6) (Xu et al., 2010) and soybean calmodulin
GmCAM4 (Yoo et al., 2005) positively regulate stresstolerance and proline content in Arabidopsis, suggesting
a central role for intracellular calcium signals in proline
metabolism.
MAPK (mitogen activated protein kinase) cascades regu-
late numerous processes including abiotic stress responses.
Several MAPKs are activated by cold, salt, and drought in
monocot and dicot plants (Jonak et al., 1996; Hoyos and
Zhang, 2000; Ichimura et al., 2000; Xiong and Yang, 2003)and genetic manipulation of MAPK signalling alters plant
tolerance to these stresses (Xiong and Yang, 2003; Shou
et al., 2004a, b; Teige et al., 2004). Recent data indicate a
positive role for MAPK-based signalling in stress-induced pro-
line accumulation (Kong et al., 2011; Zhang et al., 2011a).
While ABA and several stress-related protein kinases can
stimulate proline accumulation, a maize protein phospha-
tase type 2C negatively regulated stress-induced prolineaccumulation and tolerance to hyperosmotic stress (Liu
et al., 2009). PP2C type phosphatases PP2Cs are involved in
regulating diverse processes including development and
responses to environmental stress and have been shown to
regulate stress-induced MAPK and SnRK2 protein kinases
negatively (Schweighofer et al., 2004; Cutler et al., 2010;
Hubbard et al., 2010; Raghavendra et al., 2010; Umezawa
et al., 2010).Phospholipid metabolism is involved in mediating
hyperosmotic-stress responses (Testerink and Munnik, 2011).
Based on a pharmacological approach PLC-based signalling
has been implicated in salt-induced proline accumulation
(Parre et al., 2007).
Protein kinases involved in stress-induced changes incarbon metabolism
Carbohydrate metabolism is quickly modulated in response
to adverse environments. Plant glycogen synthase kinase 3
(GSK-3)/shaggy-like kinases appear to connect stress signal-
ling and carbon metabolism (Kempa et al., 2007). TheMedicago sativa MsK4 is a positive regulator of salt stress
tolerance. Plants over-expressing MsK4 have elevated levels
of sugars including raffinose and galactinol, increased
amounts of putrescine and amino acids under normal growth
conditions. Remarkably, MsK4 associates with starch gran-
ules and over-expression of MsK4 in Arabidopsis increased
the levels of starch and of several soluble sugars under salt
stress conditions (Kempa et al., 2007). These data are alsointeresting from an evolutionary point of view as GSK-3 was
originally identified as a regulator of the animal storage
carbohydrate glycogen (Woodgett and Cohen, 1984).
OsCIPK03, OsCIPK12, and the MAPK kinase ZmMKK4,
which promote proline accumulation under stress conditions,
Fig. 3. Stress-related protein kinases mediating solute accumula-
tion. AtSnRK2.3, AtSnRK2.4, AtSnRK2.6, CDPK6, OsCIPK03,
OsCIPK12, GhMPK6, ZmMKK4, and MsK4 positively regulate
tolerance to high salinity, drought, and/or cold stress. Plants over-
expressing these protein kinases and/or knock-outs of these
kinases have altered proline and/or sugar levels. (This figure is
available in colour at JXB online.)
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also stimulated stress-induced increase in soluble sugar
content (Xiang et al., 2007; Kong et al., 2011).
Further to the protein kinases mentioned, the negative
regulator of drought tolerance, SAL1, has been associated
with sugar and starch metabolism (Wilson et al., 2009).
Transcription factors regulating metabolite levels
Salinity, drought, and temperature stress have a strong
impact on gene expression. Many genes coding for enzymes
involved in cellular metabolism are differentially expressed
upon stress, and modulation of some stress-related tran-
scription factors have been shown to induce changes instress-associated metabolite levels.
The CBF/DREB1 proteins (C-repeat binding factor or
dehydration responsive element binding proteins) are mem-
bers of the AP2/EREBP (Apetala2/ethylene-responsive
element binding protein) family of transcription factors.
CBF1/DREB1B, CBF2/DREB1C, and CBF3/DREB1A
play an important role in the transcriptional response to
osmotic stress (Shinozaki and Yamaguchi-Shinozaki, 2007;Thomashow, 2010). Over-expression of these transcription
factors in Arabidopsis improved tolerance to freezing,
drought, and/or salt stress (Liu et al., 1998; Kasuga et al.,
1999; Gilmour et al., 2000, 2004) and CBF/DREB1 over-
expressing plants accumulated higher levels of proline and
soluble sugars (glucose, fructose, sucrose, and raffinose)
when grown under normal growth conditions and during
cold acclimation (Gilmour et al., 2000, 2004; Cook et al.,2004; Achard et al., 2008). The overall metabolic profile of
CBF3/DREB1A over-expressers grown at normal growth
temperatures resembled that of cold-exposed plants (Cook
et al., 2004; Maruyama et al., 2009).
DREB2A (dehydration responsive element binding pro-
tein 2A) is activated by osmotic stress and over-expression
of a constitutively active version of DREB2A, lacking the
inhibitory domain, improved tolerance to dehydration butonly slightly to freezing temperatures (Sakuma et al., 2006a).
In line with this tolerance pattern, the metabolite profile of
plants over-expressing constitutively active DREB2A was
more similar to that of dehydration-exposed than that of
cold-treated plants (Maruyama et al., 2009). While the global
metabolic profile of plants over-expressing the constitutively
active DREB2A resembled that of stressed plants, levels of
galactinol, raffinose, and proline were not increased. Prolineaccumulates in response to drought, but not to heat or
a combination of drought and heat (Rizhsky et al., 2004).
Interestingly, DREB2A has also been shown to promote heat
stress tolerance (Sakuma et al., 2006b).
Heat shock transcription factors (HSF) are central
regulators of heat stress responsive genes. In plants, HSFs
are encoded by a large gene family with different expres-
sion patterns and functions (von Koskull-Doring et al.,2007). Over expression of HsFA2 led to the constitutive
accumulation of galactinol and raffinose and improved
the tolerance of Arabidopsis plants to different environ-
mental stresses (Nishizawa et al., 2006, 2008; Ogawa et al.,
2007). Similarly, over-expression of HSF3/HsfA1b enhanced
thermo tolerance (Prandl et al., 1998) and raffinose levels
under normal and heat stress conditions (Panikulangara
et al., 2004).
The NAC domain family of plant-specific transcrip-
tional regulators are involved in developmental processes,
as well as in hormonal control and stress defence (Olsen
et al., 2005). OsNAC5 is induced by osmotic stress and
ABA. Over-expression of OsNAC5 enhanced stress-induced proline and soluble sugar levels and tolerance to
cold, salt, and drought stress (Takasaki et al., 2010; Song
et al., 2011) whereas knock-down lines were impaired
in proline and soluble sugar accumulation upon stress
exposure and were more sensitive to stress (Song et al.,
2011).
Levels of soluble sugars (glucose, fructose, and sucrose)
and proline, as well as glycine betaine, were also elevatedunder normal and stress conditions in Arabidopsis plants
over-expressing the rice Myb transcription factor, OsMyb4.
Accumulation of these metabolites was concomitant with
enhanced drought and freezing tolerance (Mattana et al.,
2005).
Conclusion
During the last decade, our knowledge on the importance of
metabolic adjustments to unfavourable growth conditions
has increased considerably. Natural stress tolerance is a very
complex process involving numerous metabolites and meta-bolic pathways. Analyses of metabolic adjustments of
plants with different levels of stress tolerance and transgenic
approaches provide important complementing evidence for
better understanding the role of different metabolites in
adjusting to harsh environments. Despite these substantial
gains, several questions remain to be addressed. For example,
the developmental stage of a plant is important for its
potential to tolerate an adverse condition and cellular meta-bolism changes during development. Thus, it will be in-
teresting to analyze how the developmental stage influences
the metabolic adjustment to stress conditions.
Metabolic networks are highly dynamic. Metabolites can
move between different cellular compartments. Since meta-
bolic profiling only reveals the steady-state level of metabo-
lites, detailed kinetics and flux analyses will be instrumental
for a better understanding of metabolic fluctuations inresponse to stress. Metabolic analysis at the subcellular level
in specific tissues is a further challenge.
Stress-induced signalling networks are well studied.
However, the signalling processes required for homeostasis
of basic cellular and metabolic processes in adverse environ-
ments are just starting to emerge. Better understanding of
how environmental changes are communicated via cellular
signal transduction to induce a co-ordinated metabolic res-ponse, and how the function of metabolic enzymes are
adjusted by transcriptional and post-translational modifica-
tions, are of basic scientific interest and will contribute to
meet the goal of increased plant stress tolerance and
productivity in an ever-changing environment.
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Acknowledgements
Work in our laboratory is supported by grants from the
Austrian Science Fund P 20375-B03 and the EU FP7-ITN
‘COSI’. We apologize to authors whose papers have not
been included due to space limitation in this review.
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