Influence of abiotic stresses on plant proteome and metabolome changes

19 Pages·2013·0.33 MB·English

ActaPhysiolPlant(2014)36:1–19
DOI10.1007/s11738-013-1402-y
REVIEW
Influence of abiotic stresses on plant proteome and metabolome
changes
Paweł Rodziewicz • Barbara Swarcewicz •
Klaudia Chmielewska • Anna Wojakowska •
Maciej Stobiecki
Received:14August2013/Revised:25September2013/Accepted:2October2013/Publishedonline:18October2013
(cid:2)TheAuthor(s)2013.ThisarticleispublishedwithopenaccessatSpringerlink.com
Abstract Plant responses to abiotic stresses are very concernedtotheinfluenceofabioticstressesonchangesin
complex phenomena with individual characteristics for thelevelofdifferentproteingroupsandmetaboliteclasses.
various species. Abiotic stresses (e.g. drought, salinity, Basic information about physicochemical methods applied
flooding, cold, heat, UV radiation, heavy metals, etc.) toqualitativeandquantitativeanalysesofbiopolymersand
strongly affect plant growth and development. It is esti- natural products is also briefly presented.
mated that they are the cause of more than 50 % of crop
yield losses. Abiotic stresses are known to activate a Keywords Abiotic stress (cid:2) Crop plants (cid:2) Enzymes (cid:2)
multigene response resulting in the changes in various Mass spectrometry (cid:2) Metabolites (cid:2) Metabolome (cid:2)
proteins and primary and secondary metabolite accumula- Proteins (cid:2) Proteome
tion. Therefore, proteomic and metabolomic approaches
are becoming very important and powerful tools used in
studying plants’ reaction to various stimuli. Precise ana- Introduction
lysis of proteome and metabolome is essential for under-
standing the fundamentals of stress physiology and Abiotic stresses are the major factors which negatively
biochemistry. In this review, we focus on recent reports influenceplantdevelopmentandproductivity.Theyarethe
main cause of extensive agricultural production losses
worldwide (Barnabas et al. 2008; Athar and Ashraf 2009).
Among abiotic stresses, drought, salinity and extreme
temperatures are the major environmental constraints that
CommunicatedbyP.Wojtaszek. modern agriculturehas tocope with. It has been estimated
thattheymayberesponsibleforover50 %yieldreduction
P.Rodziewicz,K.Chmielewska,B.SwarcewiczandA.Wojakowska
in major crop plants. However, severity of losses depends
contributedequallytothiswork.
on the plant development stage at which the stress occurs,
P.Rodziewicz(cid:2)B.Swarcewicz(cid:2)K.Chmielewska(cid:2) its intensity and duration (Bray et al. 2000; Ashraf et al.
A.Wojakowska(cid:2)M.Stobiecki(&)
2008; Atteya 2003; Monneveux et al. 2006; Lafitte et al.
InstituteofBioorganicChemistry,PolishAcademyofSciences,
2007).
Noskowskiego12/14,61-704Poznan,Poland
e-mail:[email protected] According to the United Nations Food and Agriculture
Organization (FAO), up to 26 % of arable land is sub-
P.Rodziewicz
e-mail:[email protected] jected to drought and over 20 % of the irrigated land is
salt-affected (Rehman et al. 2005; Pitman and Lauchli
B.Swarcewicz
e-mail:[email protected] 2002). Thus, as the climatic conditions are getting worse,
new resistant crop varieties are needed. It is possible to
K.Chmielewska
e-mail:[email protected] obtain them through the selection of cross-bred lines or
by the means of genetic engineering. However, better
A.Wojakowska
e-mail:[email protected] understanding of the mechanisms involved in plant stress
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2 ActaPhysiolPlant(2014)36:1–19
responses is necessary to reach that goal. Incorporation of biopolymersandnaturalproductsinplantmaterialarealso
modern knowledge into traditional breeding strategy is presented.
necessary for development of new varieties with
improved adaptation to non-optimal environmental
conditions. Instrumentation applied in proteomic
Plant responses towards abiotic stresses have been the and metabolomic studies
subject of various researches since decades. Initially
focused on the model plants, now moved to various crop Identification of proteins and metabolites is mainly based
plants like wheat, barley, rice, maize, and other economi- on application of mass spectrometric techniques hyphen-
cally important species. The development of the ‘‘omics’’ ated to chromatographic instrumentation and electropho-
technologies (e.g. genomics, transcriptomics, proteomics, retic techniques. Proper choice of ionization method and
metabolomics) has revolutionized plant science research analyzer type used in mass spectrometer in protein and/or
and has enabled more holistic study of interactions among metabolite analysis is crucial. Both ‘‘omics’’ need rather
biological components using models and/or networks to different configuration of mass spectrometric (MS) instru-
integrategenes,metabolites,proteins,regulatoryelements, mentation (Glinski and Weckwerth 2006).
fluxes and other, this methodology is defined as systems During analysis with mass spectrometry ionized mole-
biology (Yuan et al. 2008). Recent research permitted to cules are measured. The values of mass to charge ratio
explainfunctionsofmanykeygenes,proteins,metabolites (m/z, m-mass and z-charge) of created ions are estimated
and molecular networks involved in plant responses to after separation in the MS analyzer with accuracy of one
salinity,drought,heat,cold,heavymetalsandotherabiotic massunit(atomicmassunit—amu)ortothefourthdecimal
stresses. point, low- and high-resolution mass spectra, respectively.
Subsequent development of dense chromosome maps Application of high-resolution mass analyzers permits to
enabled identification of corresponding quantitative trait conclude aboutelementalcompositionofthedetected ions
loci (QTLs), which became a key molecular tool used in presented in mass spectra. This information is important
plant breeding programs, defined as marker-assisted during investigation on structural characterization of
selection (Tuberosa and Salvi 2004). However, despite studied compounds with MS techniques. First of all, it is
newer and more complex results, there are still lots of possible to estimate molecular mass and elemental
unknown in molecular processes occurring in plants while composition of the molecules from registered m/z values
coping with a stress. for protonated [M?H]? or deprotonated molecules [M-
Proteome and metabolome analyses have become H]-.FragmentorproductionsregisteredintheMSspectra
powerful tools to monitor changes in response to various bring additional data about the structure. Unambiguous
environmental stimuli. The results of such studies give identificationofcompoundsisinhighdegreedependenton
insight into the functioning of plants under specified con- the mass spectrometric system applied. Various strategies
ditions and are an indispensable part in revealing the of mass spectrometric identification and quantification of
molecular mechanisms underlying responses to abiotic proteins were established in the past two decades. MS
stresses(SetiaandSetia2008;WeckwerthandKahl2013). instruments equippedwith electrospray ionization(ESI) or
The knowledge of the key proteins and metabolites matrix-assisted laser desorption/ionization (MALDI)
involved in plant growth, development and response to source may be used. The MALDI ionization may be cou-
various stress conditions and proper interpretation of the pledtooneortwocombinedtimeofflightanalyzers(TOF
data enables the identification of potential biomarkers or TOF/TOF). The electrospray ionization (ESI) source
linked to enhanced tolerance to adverse environmental works well with quadrupole (Q), ion trap (IT), time of
factors and is critical for the introduction of desired bio- flight (TOF) analyzers and combination of them, quadru-
logical features to crop plant (Salekdeh and Komatsu pole/time of flight (qTOF) for instance. The highest reso-
2007). Appropriate analytical methods capable of identi- lutioninmassanalyzercanbeachievedwithioncyclotron
fying and monitoring changes in protein and metabolite resonance with Fourier transformation instruments (FT
accumulation are crucial. Nowadays mass spectrometry ICRMS),whenESIisusedasionizationmethod.Tandem
combined with various chromatographic orelectrophoretic mass spectrometric analyses (MS/MS), which enable the
techniques plays a central role in proteome and metabo- fragmentation of proteins or peptides performed by colli-
lome analysis. In this article we have reviewed main pro- sion induced dissociation (CID), electron transfer dissoci-
teingroupsandmetaboliteclasses,whichwereidentifiedin ation (ETD) or electron capture dissociation (ECD) are
the recent reports concerning proteomic and metabolomic widely used.
analyses of plants subjected to various abiotic stresses. Identification of proteins may be performed after sepa-
Some instrumental methods directed to analysis of ration of proteins present in the sample by electrophoretic
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ActaPhysiolPlant(2014)36:1–19 3
methods on polyacrylamide gels developed in one or two peakretentiontimealignmentbetweenindividualanalyses.
dimensions. Excised gel fragments containing proteins are After utilization of this procedure various statistical cal-
subjected to enzymatic digestion (proteolysis with trypsin culations may be performed in order to quantify the
or other proteases). Arisen peptides are then analyzed by amounts of metabolites, which enables determining the
mass spectrometry and molecular masses of peptides are changes of a particular compound in defined conditions
recorded as protonated molecules [M?H]?. In this (van den Berg et al. 2006; Goodacre et al. 2007; Korman
approach, peptide mass spectra of peptides mixture are et al. 2012; Weckwerth and Kahl 2013).
recorded and identification of proteins is realized on the The number of primary and secondary metabolites may
basis of consecutive peptide maps created from registered vary from a few thousands to tens of thousands in a single
mass spectra after sample proteolysis. The second tech- organism and their concentrations differ in several orders
nology is called shotgun proteomics. This approach relies of magnitude. On other hand, the dynamic range of mass
on direct digestion of proteins present in the extract sam- spectrometersappliedasdetectorsinmetabolomicanalyses
ple. The obtained peptide mixture is further separated by usually does not exceed five orders of magnitude. In these
1D or 2D chromatographic system hyphenated to mass circumstances, detection and identification of all com-
spectrometer. In the first method, proteins are identified pounds present in a sample originating from single
after MS analysis on the basis of the registered [M?H]? organism is not possible due to the separation power of
ions originating from the peptides present in the sample chromatographic systems and sensitivity range of mass
after proteolysis (peptides maps). The second method spectrometers. Several strategies for analysis of metabo-
relays on deciphering of amino acid sequence from the lites have been developed: metabolite profiling, metabolic
registered CID MS/MS spectra of the peptides ions sub- fingerprinting, metabolite target analysis and metabonom-
jected to fragmentation (bottom-up approach). This appli- ics (Dunn and Ellis 2005).
cation allows also proteins quantification (Swanson and
Washburn 2005). In the top-down method of protein
identification utilization of mass spectrometer instruments Proteomics
withveryhighresolutionisrequired(above100,000atfull
width at half maximum—FWHM). Orbitrap or ion cyclo- Activation of stress-responsive pathways on different
tron resonance mass spectrometers (FT ICR) enable ana- molecular levels due to environmental adverse conditions
lysis of intact proteins and their sequencing (Yates et al. causes significant changes in plant proteome. Proteins
2009). fulfill a vast diversity of functions. They act as enzymes,
InthecaseofmetabolomicsstudiesseveraldifferentMS transcriptional factors, interact with other molecules, have
analyzer configurations with low- and high-resolution protective functions, are involved in energy transfer or
combined to gas or liquid chromatographs are used. The radicals scavenging and take part in signaling pathways
choice ofthe system stronglydependson physicochemical and others. Specific composition of proteins present in
properties of studied compounds. Molecular masses and cells in the defined environmental conditions reflects the
polarity of the components present in the samples should true biochemical outcome of genetic information and
be taken into account while choosing a proper identifica- indicates the biochemical pathways that may be involved.
tion system (Villas-Boˆas et al. 2004). It is also very It is now accepted that explaining proteome changes is
important to design the experiments according to the critical for understanding of rules with which cells work
standards of Metabolomic Standard Initiative (Sumner and adapt towards various stimuli. Much attention has
et al. 2007; Fiehn et al. 2007), which recommends defined been paid to proteomic studies on crop plants in recent
procedures for the proper biological material preparation, years. Examples of researches on crop plants include,
metabolite extraction procedures and analytical protocols. among others, barley (Ashoub et al. 2013), maize (Pe-
According to the published rules, suitable number of chanova et al. 2013) rice (Lee et al. 2009), wheat (Peng
sample repetitions (biological and technical), plant growth et al. 2009) and soybean (Cheng et al. 2010) subjected to
conditionshavetobemonitoredanddescribed,alsocontrol various abiotic stresses. Especially, comparative proteo-
of MS parameters during mass spectra registration is mic studies based on analyzing contrasting plant geno-
required. This information provides conditions for appro- types in the stressed and non-stressed conditions are very
priate identification of compounds and further quantitation informative and give insights into the plant reactions to
of metabolites and reliable statistical calculation (Sansone environmental stimuli. Depending on the tolerance level
et al. 2007). of the studied genotype, plants may accumulate or
Interpretation of big data sets requires appropriate and enhance expression of particular proteins, which exhibit
advancedstatisticalmethods.Preliminaryanalysisincludes the protective functions. Various classes of molecules
usage of mathematical computing for chromatographic may be considered, for example heat-shock proteins,
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4 ActaPhysiolPlant(2014)36:1–19
proteins involved in osmolyte biosynthesis or belonging transcriptionalfactors(Hsf)inresponsetodifferentabiotic
to ROS-scavenging systems and others. stresseswasrecentlyinvestigated.Itwasrevealedthatmost
Correlation of results obtained on the proteome level HsfsandHSPshadsimilarresponseandregulationpatterns
with other data, such as genetic, transcriptomic, meta- under different stresses; however, some of those genes
bolomic and physiological parameters, may contribute to showed a highly specific response to distinct factors (Hu
discovery of biomarkers linked to enhanced tolerance to et al. 2009). In the study, in which proteomic response of
the defined stress. This information may be further incor- riceleavestohightemperatureswasexamined,18outofa
porated into breeding programs. total of 48 identified proteins belonged to HSPs. Among
them,membersofHSP70,HSP100,DnaK-type,molecular
Heat shock proteins chaperone BiP were identified and showed up-regulation.
Such a result may reflect a rapid adaptation to changing
Abiotic stresses cause protein dysfunction, thus maintain- environmental conditions. Seven induced sHSPs were also
ingproteinsattheproperconformationbypreventingthem found (Lee et al. 2007). In the reports concerning barley
from aggregation and incorrect folding is crucial for plant (Su¨le et al. 2004) and wheat (Majoul et al. 2003), the
survivalundersevereconditions.Heatshockproteins(HSP upregulation and induction of HSPs were also observed.
or chaperones) are a large family of proteins playing roles Plantsthatareexposedtolowtemperaturesalsosynthesize
inkeepingcellularhomeostasisbothunderoptimalgrowth HSPs (Miura and Furumoto 2013). In particular, the
conditions and under stress. They are responsible for cor- upregulation of HSP70, HSP100 and sHSPs is commonly
rect folding, translocation and degradation of proteins. reported (Cui et al. 2005; Hashimoto and Komatsu 2007).
They also prevent proteins from aggregation. The role of The comparative analysis of barley leaves subjected to
HSPs in abiotic stresses has been extensively reviewed drought stress revealed the upregulation of HSP100,
(Wangetal.2004;Timperioetal.2008;Al-Whaibi2011). HSP90 in both sensitive and tolerant genotype. A member
In plants, similarly to prokaryotes and other eukaryotes, of HSP70 was found to be down-regulated only in the
five major families of HSPs are recognized. They are sensitive genotype (Ashoub et al. 2013). In the study
classified on the basis of their approximate molecular conducted by Kausar and co-workers, HSP70 was also
weight and according to their amino acid sequence down-regulated in drought-sensitive barley (Kausar et al.
homologies and functions: (1) HSP100 family; (2) HSP90 2013). Three HSP70 proteins were down-regulated during
family; (3) HSP70 family; (4) HSP60 family; (5) small the analysis of sugar beet leaves (Hajheidari et al. 2005).
HSP family (sHSP; 15–40 kDa) (Kotak et al. 2007; Gupta HSP90 and HSP70 were found to be required for stomata
etal.2010).TheHSP100/Clptakespartintheremovaland closure and modulation of transcriptional responses to
reactivation of aggregated, misfolded, non-functional abscisic acid (Cle`ment et al. 2011); thus, they may be
polypeptides (Agarwal et al. 2001). Proteins belonging to crucial for enhanced drought-tolerance. Tobacco plants
HSP90classplayaroleinproteinfoldingandregulationof overexpressing NtHSP70-1 demonstrated higher tolerance
signal transduction (Buchner 1999; Pratt and Toft 2003). towaterdeficiency(ChoandHong2006).Intheanalysisof
The HSP70 family prevents newly synthesized proteins thechangesintheproteomeofgrasspeaexposedtosalinity
from aggregation and helps them to fold correctly. They and drought, HSP 70 was found to be up-regulated in the
are also involved in transport and proteolytic degradation plants exposed to increased salinity and drought (Chatto-
of unstable proteins (Su and Li 2008). Proteins of HSP60 padhyay et al. 2011). HSP70 was noted to be up-regulated
family are crucial to achieve native forms by newly syn- in both conditions. HSP70 proteins were also evaluated as
thesized proteins. For example, they assist in the proper potential markers of heavy metal, osmotic and heat stress
foldingofRUBISCO(Wangetal.2004).AmongallHSPs/ (Ireland 2004).
chaperonins in plants, small HSPs represent the most pre-
valent and diverse family with respect to their sequence Late embryogenesis abundant proteins
homology, functions and cellular location (Waters et al.
1996). They cannot refold non-native proteins; however, Late embryogenesis abundant proteins (LEA) were origi-
they bind to partially folded or denatured proteins and nally discovered more than three decades ago in cotton
prevent aggregation or non-functional folding. They are seed,wheretheyweresynthesizedandaccumulatedduring
also capable of degrading misfolded proteins (Sun et al. late embryo development (Dure et al. 1981). In plants,
2002). HSPs are known to be inducible by various abiotic LEA proteins and their mRNAs accumulate to high con-
stresses, including drought, salinity, high and low temper- centrations in the embryo tissue during the last stages of
atures, light, ozone and metal stress (Wang et al. 2003; seeddevelopment(Bakeretal.1988).Dehydrinsconstitute
Sabehat et al. 1998; Lee et al. 2000). The comparison of one of the groups of LEA proteins (Roberts et al. 1993).
global expression of genes coding HSP and their Theseproteinsplayasignificantroleinplantresponseand
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ActaPhysiolPlant(2014)36:1–19 5
adaptation to abiotic stresses. The synthesis of dehydrins 2009).Inanotherstudyitwasshownthattheexpressionof
may be induced in the vegetative tissue by various stress the durum wheat dehydrin DHN5 in A. thaliana led to an
factors.TheroleofLEAproteinsinabioticstresstolerance increase in tolerance toward salt and osmotic stress in
was widely documented and may suggest that in general transgenic plants (Brini et al. 2007). Likewise, increased
these proteins are active to adaptation to various abiotic tolerance to drought and salinity was also observed in
stresses,suchasdrought,salinityandlow-temperature,but tobacco expressing Rab16A LEA gene from salt-tolerant
their precise function remains still unclear (Bray 1993). It rice (RoyChoudhury et al. 2007). Cheng and coworkers
seems that these proteins play a critical role in cellular showed thatoverexpressionofthewheatPMA80dehydrin
protectionduringtheosmoticshock.Theystabilizethecell in rice enhanced its tolerance to drought and salinity
and protect the tissue from water loss. In the research (Cheng et al. 2002). It seems that LEA proteins, in par-
conducted by Ford and co-workers, three wheat cultivars ticular dehydrins, can be used as molecular markers for
with different tolerance level have been evaluated under stress tolerance. However, the obtained results are not
droughtconditions.DehydrinCOR410wasup-regulatedin always clear and easy for interpretation and sometime are
allstudiedgenotypesincomparisonwiththecontrolplants. contradictory.
However, the most significant change in the expression
levelofthatproteinwasobservedintheintolerantcultivar Osmolyte biosynthetic enzymes
(Ford et al. 2011). Lopez and co-workers during their
research found a correlation between the accumulation of Compatible solutes like glycine betaine (GB) or proline
dehydrin in seven winter wheat cultivars during the expo- play an important role in enhancing plant resistance of
suretowaterdeficit.Threeofthetestedcultivarsshoweda abioticstresses,includingdrought,salinityandtemperature
significantly enhanced expression of 24 kDa dehydrin in stress. Osmolytes exhibit protective functions on enzymes
comparison with other genotypes (Lopez et al. 2003). In and membrane integrity and take part in regulation of
control plants of other analyzed genotypes, the expression osmotic pressure. During abiotic stresses their synthesis
of these proteins was not detected. The enhanced accu- usually increases in plants (Quan et al. 2004a; Chen and
mulation of dehydrin was related to the acquisition of Murata2011).Betainealdehydedehydrogenase(BADH)is
drought tolerance, which was characterized by a greater an enzyme involved in the synthesis of glycine betaine
growthofshootsinthreemosttolerantcultivars.Although through the conversion of betaine aldehyde. Studies of
theroleofdehydrinremains unknown,theauthorssuggest transgenic plants overexpressing BADH suggest higher
thatLEAproteinsmightbeusedtoimprovetheadaptation accumulation of glycine betaine and were correlated with
in drought conditions. In another study, seedlings of two increased tolerance to abiotic stresses, including water
barley cultivars with contrasting tolerance [differentiation deficit and salinity (Chen and Murata 2011; Turan et al.
based on the Relative Water Content (RWC) analysis] to 2012). Accumulation of betaine glycine in the transgenic
water deficit were subjected to proteomic analysis. line of wheat with overexpressing BADH gene showed an
IncreasedaccumulationofdehydrinDHN3andDHN4was enhanced tolerance to cold (Zhang et al. 2010). The
observedinthetolerantvariety,butnotinthesensitiveone. transgenic wheat containing gene encoding BADH cloned
Similar results were obtained with mature plants. The from Atriplex hortensis showed increased tolerance to
authorsalsosuggestthatthecorrelationbetweenDhn3and drought and heat stress. It was also found that overpro-
Dhn4 transcript accumulation and several traits associated duction of betaine glycine enhances photosynthesis and
withdroughttolerance(RWCanddroughtyieldindex)can antioxidant activity in wheat (Wang et al. 2010). Tobacco
serve as potential markers to determine drought-tolerant overexpressingBADHgenewasfoundtobemoretolerant
genotypes (Park et al. 2006). Transgenic wheat expressing to salinity than its wild counterpart. It also showed
the barley LEA gene HVA1 showed increased desiccation enhanced CO assimilation and increased activity of the
2
tolerance, which was correlated with biomass productivity enzymes involved in photosynthesis (Yang et al. 2008).
(Sivamanietal.2000).Quantitativedifferencesindehydrin The key enzyme involved in the metabolic pathway of
gene expression and dehydrin protein accumulation under proline is delta-1-pyrroline-5-carboxylate synthetase
extreme temperatures have been studied in wheat. The (P5CS)(VerbruggenandHermans2008).Prolineisoneof
correlation between an increase of WCS120 LEA protein the most abundant osmolytes synthesized in response to
synthesisandthelevelofacquiredfrosttolerancehasbeen environmental stresses. Research indicates that high accu-
shown (Houde et al. 1992). Two barley cultivars with mulation of proline in plants correlates with an increased
contrasting responses to salinity were evaluated by Wiztel tolerancetowardwaterdeficit(NayyarandWalia2003).A
and co-workers. The identified LEA proteins did not show study performed by Zhu and coworkers on transgenic rice
any differences in the expression pattern of tested geno- overexpressing P5CS showed an enhanced tolerance to
types between stressed and control plants (Witzel et al. osmotic stress caused by salinity and drought. Transgenic
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plants demonstrated an increase of biomass under salt and concerning drought responses in barley, the following
water stress in comparison with non-transformed plants proteins were considered: RUBISCO-binding protein,
(Zhu et al. 1998). It was also demonstrated that proline RUBISCO activase and Photosystem I Reaction Centre II.
overproduction in transgenic tobacco led to lower reduc- These proteins were decreased in the sensitive genotypes.
tion of leaf osmotic potentials during drought, when The carbon-metabolism-related proteins such as malate
comparing with the wild plants. Reduction of proline dehydrogenase and triosephosphate isomerase were up-
synthesis,hypersensitivitytohighconcentrationofsaltand regulated in tolerant species or showed no significant
accumulation of reactive oxygen species were observed in change, while in sensitive one they were down-regulated
the research conducted on A. thaliana P5CS knockout (Kausar et al. 2013). However, in a study conducted by
mutants (Sze´kely et al. 2008). These results suggest that Wendelboe-NelsonandMorrislargesubunitsofRUBISCO
proline accumulation may be involved in the tolerance to and other photosynthetic enzymes were generally found to
osmotic stress, caused by other adverse factors such as bedown-regulatedinatolerantvariety(Wendelboe-Nelson
drought or salinity. andMorris2012).Theeffect ofdroughtandsalinity stress
studied on a somatic hybrid of wheat and its parent also
Photosynthesis and carbon metabolism revealed degradation of the RUBISCO enzyme. However,
greater amounts of the fragmented RUBISCO subunits
Abiotic stresses disturb cell homeostasis and affect pho- were observed in parental genotype, it was also in agree-
tosyntheticmetabolism.Therate ofphotosynthesisusually ment with the measured rate of photosynthesis and chlo-
decreases during exposure to various stresses in higher rophyll activity, which were higher for the hybrid. The
plants (Chaves et al. 2009). The main reason is stomata capacity to sustain photosynthesis under stress by the
closure, which leads to decrease in internal CO concen- somatic hybrids may be the result of effective removal of
2
tration (Cornic 2000). However, as the stress progresses, ROS and more robust homeostasis (Peng et al. 2009). In
the metabolic impairment of the photosynthetic apparatus salt-stressed plants of wheat (Triticum durum L.), small
starts to be an important issue. Drought, salinity, high/low subunits of RUBISCO were found to be down-regulated.
temperatures, ultraviolet radiation and excessive light Two different fragments of the large RUBISCO subunit
generate additional oxidative stress caused by enhanced wereup-regulated,suggestingdegradationoccurringunder
production of reactive oxygen species, which further that stress condition and decreased synthesis of the
damages the photosynthetic machinery (Mittler 2002). enzyme. On the other hand, two molecular chaperones,
Ribulose-1,5-bisphosphate carboxylase/oxygenase RUBISCO activase and RUBISCO-binding protein, were
(RUBISCO)isanenzymeinvolvedinthemajorstepinthe up-regulated during stress treatment. It may suggest a role
CO incorporation into organic compound and the com- in protecting proteins against the denaturation of RUBI-
2
peting photorespiratory carbon oxidation (Spreitzer and SCO under stress conditions. Phosphoglycerate kinase,
Salvucci 2002). A decrease in RUBISCO activity was which catalyzes the first reaction in the reduction phase in
identified as one of the non-stomatal reasons for lowering the Calvin cycle, was decreased in salt-stressed wheat. It
the photosynthesis rate (Flexas et al. 2006). However, the may be connected to a decrease of CO fixation caused by
2
reductionofRUBISCO activityunderdroughtstressisnot the reduction of stomata conductance and degradation of
primarily caused by lower CO concentrations, but rather RUBISCO.Otherenzymesinvolvedinmetabolicpathways
2
due to the presence of tight-bound inhibitors, for example such as pentose phosphate pathway (phosphoribulokinase)
phosphorylated carbohydrates (Parry et al. 2002). The andglycolysis(fructose1,6-bisphosphatealdolase)showed
effect of drought on the activity of RUBISCO may vary a decreased abundance, in comparison with control plants.
fromstrongtonone,dependingontheanalyzedspecies,its However, enzymes involved in the regulation of carbon
tolerance to the stress and differences in experimental metabolism (phosphopyruvate hydratase, triose phosphate
design (Parry et al. 2002). In the study conducted by As- isomerase, glucose-6-phosphate dehydrogenase) were up-
houb and coworkers, it was found that RUBISCO was regulated in salt-treated plants (Caruso et al. 2008). An
more down-regulated in the drought-sensitive barley increased number of RUBISCO proteolytic fragments was
genotype than in the tolerant one. Transketolase, an also reported in metal-stressed rice (Hajduch et al. 2001).
enzyme involved in the Calvin cycle, showed a greater In the seedlings subjected to the cold, RUBISCO large
decrease in the accumulation profile in the sensitive culti- subunits and RUBISCO-binding protein were also down-
var. Glyoxysomal malate dehydrogenase and alanine gly- regulated, which may suggest the chloroplast damage.
oxylate aminotransferase, which are enzymes involved in Enzymesrelatedtoenergyproductionwereshowntobe
photorespiration were increased, especially in the tolerant activatedunderlowtemperatures(HashimotoandKomatsu
genotype suggesting a higher rate of photorespiration 2007). During a study conducted on spring wheat exposed
(Ashoub et al. 2013; Aubry et al. 2011). In another study to prolonged cold stress, a massive down-regulation of
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ActaPhysiolPlant(2014)36:1–19 7
photosynthetic proteins was observed. Identified enzymes (Pfannschmidt 2003), mitochondria (electron transport
from the Calvin and Krebs cycles were also down-regu- chain) (Rasmusson et al. 2004) or peroxisomes (photores-
lated. Oxygen-evolving enhancer protein, which stabilizes piration, b-oxidation) (del R´ıo et al. 2002). Although the
themanganesecluster,theprimarysiteofwatersplittingin ROS are considered to be very toxic for the plant, espe-
Photosystem II and ferredoxin NADPH oxidoreductase, cially at high concentrations, they also play a role in sig-
which participates in energy and electron transfer, were naling and are involved in the activation of defense
both down-regulated, suggesting disturbances in electron responses and in acclimation to changing environmental
flow throughthe photosynthetic machinery. Fragmentation conditions (Dat et al. 2000). Various stresses, both abiotic
of RUBISCO was also evidenced, with a greater degrada- and biotic, cause excessive production of ROS (Polle
tion of small subunits. It was suggested that RUBISCO 2001). In the case of abiotic stresses, diminished concen-
proteolysis is caused by an increased ROS concentration trationsofinternalCO duetostomataclosingleadstothe
2
and sugar accumulation during cold exposure. However, leakage of electrons to O and is one of the main reasons
2
RUBISCOactivase,whichremovestight-boundinhibitors, fortheexcessiveROSproduction.IfthegenerationofROS
wasup-regulated, which mayberelated tothe reactivation exceeds the plant scavenging capacity, it will cause dis-
of RUBISCO to fix remaining CO (Rinalducci et al. turbances in cell homeostasis and, as a further conse-
2
2011). Analysis of rice leaves subjected to high tempera- quence, may activate processes leading to cell death (Dat
tures (42 (cid:3)C) for 24 h also revealed down-regulation of et al. 2000). Plants developed various antioxidant mecha-
photosynthesis-related proteins, including RUBISCO (Lee nisms, both enzymatic and non-enzymatic, which very
et al. 2007). Similar findings were reported by Gammulla often form a common metabolic pathway. The ascorbate–
and coworkers. They subjected rice seedlings to various glutathione cycle is one of the major ROS scavenging
high and low temperatures for 72 h. Most of the proteins pathways. The enzymatic components of this cycle are
involved in the light-harvesting complex were responsive ascorbate peroxidase (APX), monodehydroascorbate
to either high- or low-temperature stress or both. The reductase (MDHAR), dehydroascorbate reductase
identifiedRUBISCOlargesubunitdecreaseditsabundance (DHAR), glutathione reductase, utilize ascorbic acid and
at different temperatures: 44/36, 12/5 and 20/12 (cid:3)C (day/ glutathione to scavenge H O (Jimenez et al. 1997). Other
2 2
night), with the most significant decrease shown at significant ROS removal enzymes are superoxide dismu-
20/12 (cid:3)C (day/night) (Gammulla et al. 2011). In the study tase(SOD),whichisoneofthemosteffectiveantioxidants,
concernedwithcoldacclimationofFestucapratensis,half which catalyzes the removal of O(cid:2)- by its dismutation to
2
ofallselectedproteinsthatweredifferentiallyaccumulated H O and O . SODs are found in various locations within
2 2 2
before and during cold acclimation in plants of different the cell. They are classified by the metal cofactor they
levels of frost tolerance were directly involved in the require:manganese(Mn-SOD),iron(Fe-SOD)andcopper/
process of photosynthesis (oxygen-evolving protein, cyto- zinc (Cu/Zn-SOD) (Mittler 2002). Catalases (CAT) found
chrome b /f and Rieske Fe–S protein). One of the differ- in the peroxisomes are very efficient enzymes capable of
6
ences observed between the sensitive and tolerant dismutatingH O toH OandO (PolidorosandScandalio
2 2 2 2
genotypes was the down-regulation of RUBISCO in the 1999). Other enzymes involved in antioxidant defense
low frost tolerant species. These results may suggest an include glutathione-S-transferases (GST) (Dixon et al.
important role of the chloroplast machinery in plant 2011) and various peroxiredoxins (PRX) (Dietz 2003), of
acclimation to cold (Kosmala et al. 2009). which some are implicated in the water/water cycle
(Rizhsky et al. 2003). The regulation of the expression of
Enzymatic reactive oxygen species scavengers the ROS-scavenging enzymes differs between the plant
species, depending on the tolerance level to the stress and
Partially oxidized oxygen particles, known as the reactive its intensity. The increased activities of APX and SOD
oxygen species (ROS), unlike atmospheric oxygen, have were found in all three analyzed varieties of beans in
the capability of oxidizing various molecules and cell responsetodrought,buttheCATactivitywasup-regulated
structures. Under normal environmental conditions, ROS only in two of them. Moreover, in the variety with the
are naturally present in plants; however, plants developed highestAPXandCATactivity,thelowestH O production
2 2
variousantioxidantsystemstobalancetheirproductionand and lipid peroxidation were observed (Zlatev et al. 2006).
quantity (Gill and Tuteja 2010a). Main forms of ROS that Shotgunproteomicanalysisofwheatcultivarssubjectedto
arise in plants are the superoxide radical (O(cid:2)-), hydrogen drought revealed that Cu/Zn SOD and CAT were highly
2
peroxide(H O ),singletoxygen(1O )andhydroxylradical up-regulatedinbothtolerantandsensitivegenotypes(Ford
2 2 2
(HO(cid:2)-) (Mittler 2002). They are by-products of biological et al. 2011). An increased activity of APX at mild drought
2
processes that take place in organelles with a high meta- was also reported in rice seedlings. However, the intensi-
bolic activity like chloroplasts (during photosynthesis) fication of the stress diminished its activity (Sharma and
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8 ActaPhysiolPlant(2014)36:1–19
Dubey 2005). In the comparative study of dehydration- Lignins, which are synthesized from phenylpropanoid
sensitive and -tolerant cultivars of chickpea, SOD, APX compoundsplayacrucialroleinconductingwaterinplant
and GST were found to be induced only in the resistant stem. Increased resistance to drought was observed in A.
genotype. Another enzyme, glyoxalase, involved in the thalianapal1pal2doublemutants,whichmighthavebeen
detoxification ofR-oxoaldehydes, wasalsoup-regulated in the effect of the reduced lignin synthesis as the mutants
the tolerant variety. The lack of induction of major ROS containedonly30 %oftheligninleveltypicalforthewild
catabolic enzymes may lead to hypersensitivity of plants type. The reduced lignin contents in the double mutants
during dehydration-induced oxidative stress (Subba et al. might cause a reduced efficiency of water transport in the
2013). The up-regulation of glyoxylate was also observed vascular tissue (Huang et al. 2010). The effects of drought
in rice root tissue in response to cold treatment (Lee et al. stressontheactivityofPALinmaizerevealedanincreased
2009). In another study concerning the analysis of cold- activity of this enzyme in leaves, while in the roots it
induced changes of proteins in the roots of rice seedlings, remained constant. The antioxidant activity in leaves was
APX was found to be up-regulated and also its phosphor- also enhanced. It may suggest that PAL is a responsive
ylation pattern was changed (Chen et al. 2012).According antioxidativeenzymeintheleaves,butnotinroots(Ashraf
to a recent review on maize proteome, APX and SOD 2011). In a study performed by Hura and co-workers,
belong to proteins whose accumulation is the most influ- drought-tolerant and -sensitive wheat genotypes were
enced by various abiotic stresses, which shows the selected,basedontheirfieldperformanceandbiochemical
importance of ROS catabolism during plant acclimation parameters.TheactivityofPALandphenoliccompounds’
(Pechanova et al. 2013). The effect of aluminum and synthesis level was correlated with crop yield and
cadmium on antioxidant enzyme activities in two barley according to these values, the level of wheat resistance to
cultivars revealed that the activity of SOD was higher in water deficit was described (Hura et al. 2007).
the resistant genotype (Guo et al. 2004). Among the cad- The protein synthesis elongation factor (EF-Tu protein)
mium-induced defense-related proteins between high- and playsaroleintheelongationphaseofproteinbiosynthesis.
low-cadmium-accumulating cultivars of soybean, the This protein has been studied in several plants in response
enhanced expressions of antioxidant enzymes, APX, CAT toabioticstress,suchasextremetemperatures,salinityand
and Cu/Zn SOD, were evident in both genotypes (Hossain drought.EF-Tuactsasamolecularchaperoneandprotects
etal.2012).Analysisofthechangesintheproteomeofthe other proteins from thermal aggregation and degradation
roots of pea in response to salinity also revealed an (Rao et al. 2004). The first report regarding stress-induced
increased accumulation of SOD, but no other antioxidant expression and accumulation of the elongation factor (EF)
enzymes were identified (Kav et al. 2004). The study of showed such effect in maize. It was indicated that there
changes in the root proteome of barley genotypes with a was a correlation between the increased accumulation of
contrasting response towards salinity revealed that the EF inplastidsand the heat tolerance ofmaize lines (Ristic
MDAR, APX and CAT showed a higher constitutional et al. 1991). Similar results were obtained as an effect of
expressionlevelinthesensitivegenotypeandtwoofthem combined heat and drought stress applied to this plant
(MDAR, APX) were found to be more down-regulated (Ristic et al. 1999). The analysis of the proteome and
during the salinity treatment in the tolerant genotype. transcriptomeofmaizeindicatedthattheexpressionofEF-
However, two identified proteins involved in the glutathi- Tu factor was regulated differentially in heat-tolerant and
one-based detoxification of the reactive oxygen species sensitive cultivars. A significant increase in the accumu-
(ROS) were more abundant in the tolerant cultivar. The lation of both the EF-Tu transcript and EF-Tu protein was
higherconstitutiveexpressionoftheseproteinsinthemore observed in tolerant genotypes of maize. The authors
tolerant genotype could represent a pre-formed tolerance suggested that regulation of the expression of EF-Tu may
mechanism (Witzel et al. 2009). be different in heat-tolerant and sensitive genotypes
(Momcilovic and Ristic 2007).
Other proteins Transcription factors (TF) are the specific group of
proteins that play a regulatory role in gene control. TF
Anothergroupofproteinsrespondingtoabioticstressesare factors have the ability to control the expression of target
proteins involved in the secondary metabolism. Phenylal- genes through binding to the specific sequence in these
anine ammonia lyase (PAL) is the first enzyme of the genes. Large plant TF families play significant roles in
phenylpropanoid pathway, which catalyzes the conversion response to abiotic stress (Nakashima et al. 2009). In a
of phenylalanine to cinnamic acid. The phenylpropanoid studyoftheinfluenceoflong-termdroughtonwheatthere
pathway is crucial for the biosynthesis of lignins, phenolic was demonstrated enhanced expression of MYB transcript
acids and flavonoids, which play important roles in plant factor in a tolerant genotype in comparison with the sus-
defense mechanisms (Vogt 2010). ceptible genotype. Similar results have been obtained
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ActaPhysiolPlant(2014)36:1–19 9
during studies on salt stress, which may suggest that the ofthemostwidelydistributedosmolyte,thelevelofwhich
MYB transcription factor plays an important regulatory is elevated in different environmental stresses including
function in adaptation to abiotic stress in wheat (Rahaie drought,salinityandcoldstress(VerbruggenandHermans
et al. 2013). In another study, it was demonstrated that 2008; Szabados and Savoure 2010). This stress-responsive
overexpressionofthetranscriptionfactorGmbZIP1didnot amino acid is predominantly synthesized from glutamate
result in growth inhibition of wheat seedlings subjected to through pyrroline-5-carboxylate (P5C) by two reductions
drought stress, suggesting enhanced tolerance to water catalyzed by pyrroline-5-carboxylate synthetase (P5CS)
deficit (Gao et al. 2011). and pyrroline-5-carboxylate reductases (P5CR) (Hu et al.
1992). The important role of proline in osmotic stress was
confirmed in transgenic plants, e.g. by P5CS overexpres-
Metabolomics sion in tobacco, which led to an increased proline content
and a smaller decrease in osmotic potentials in the leaf of
Metabolomicsiscurrentlyanimportanttoolinvolvedinthe transgenic plants, compared with control plants after
selection process of plants resistant to changing climatic droughttreatment(KaviKishoretal.1995).Therefore,the
conditions. The most important abiotic stress factors, such increased levels of proline in plants in response to abiotic
as drought, salinity, soil flooding and extreme tempera- stresses were for many years regarded to be the stress-
tures, cause significant changes in the composition of the tolerance trait. Recently, the relationship between the
plant metabolome (Obata and Fernie 2012; Ruan et al. accumulation of osmolytes and stress tolerance has been
2013) The knowledge about the role played by low- discussed because of its questionable relevance to crop
molecular-weightprimaryandsecondarymetabolitesinthe yield (Serraj and Sinclair 2002). The increased content of
stress tolerance process is essential for crop species some amino acids, including proline, tryptophan, phenyl-
improvement. However, the function of secondary metab- alanine and histidine was reported in a research on maize
olites in the abiotic stress tolerance of plants is relatively hybrids subjected to drought stress (Witt et al. 2012).
least understood nowadays. Interestingly, very few metabolites were linked with
drought tolerance or drought susceptibility of the studied
Roleofprimarymetabolitesinresponsetoabioticstress hybrids. On the other hand, studies on drought response at
the metabolomic level in Andean potatoes revealed that
Plants developed various adaptive strategies to withstand plants with a phenotype indicating higher stress suscepti-
abiotic stresses, including alterations of metabolism in bility had increased proline content as compared with
different directions, to ensure their survival under adverse genotypes, which were more drought-resistant (Vasquez-
environmental conditions. One of the widely described Robinet et al. 2008). In studies on barley subjected to
plant responses to water deficit is osmotic adjustment, salinitystress,itwasfoundthatthecultivarwithasensitive
whichrequiresaccumulationofcompatiblesolutes,suchas phenotype had an elevated level of some amino acids,
aminoacids,carbohydrates,polyols,tertiarysulfoniumand including proline and GABA. It may suggest a higher
quaternary ammonium compounds (especially glycine susceptibility of these plants to this kind of stress. The
betaine). These molecules play an important role in authorsproposedthatthisaccumulationmightberelatedto
maintaining cell turgor, as well as stabilizing proteins and leaf necrosis and slower growth of the more sensitive
cell membranes. Other hypothesis indicates their contri- genotype(Widodoetal.2009).Furthermore,dataobtained
bution in re-establishing the redox balance by scavenging inresearchonArabidopsisshowthatprolinecanbeatoxic
reactive oxygen species, which could negatively affect compound and harmful for plant growth during heat stress
cellular structures and metabolism. In cold stress, the (Lv et al. 2011).
content of cryoprotective molecules, such as soluble sug-
ars, sugar alcohols and nitrogen-containing compounds is Polyamines
increased. This helps plant to cope with low temperatures
bypreventingiceadhesiontoplasmamembrane,whichcan Polyaminesarelow-molecular-weightnitrogencompounds
befollowedbycelldisruption(Ronteinetal.2002;Bartels with a positive charge at the cellular pH, which enables
and Sunkar 2005; Valliyodan and Nguyen 2006; Munns them to interact with negatively charged molecules like
and Tester 2008; Janska´ et al. 2010). nucleic acids, proteins and phospholipids. The most com-
monpolyaminesaretriaminespermidine(Spd),tetraamine
Amino acids spermine (Spm) and their diamine precursor putrescine
(Put). Due to their cationic nature, these commonly
Ithasbeendocumentedthatmanyaminoacidsaccumulate occurring compounds have been frequently related with
inplantsexposedtovariousabioticstresses.Prolineisone environmental stresses, including drought, salinity and
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10 ActaPhysiolPlant(2014)36:1–19
chilling stress, as well as UV-B and heavy metals (Kaur- aroleinadaptivemechanismstostress(Rameletal.2009).
Sawhneyetal.2003;GroppaandBenavides2008;Hussain Moreover,trehalose,arare,non-reducingdisaccharide,the
etal.2011).Polyamineshavebeenascribedtobeinvolved presence of which was multiple times related to stress
in the stabilization of membranes protecting them from tolerance in bacteria (Purvis et al. 2005) and fungi (Cao
denaturation under stress condition (Slocum et al. 1984), etal.2008),alsoaccumulatesinplantsandseemstoplaya
scavenging free radicals (Drolet et al. 1986), modulating protective role during abiotic stress; however, its specific
nucleic acid structures and also enzyme activities or functionisnotwellunderstood.Ingeneral,trehaloseisnot
function (Galston and Sawhney 1990). Moreover, it was accumulated at high levels and, except some resurrection
suggested that they might act as osmolytes because their plants, it is barely detectable in crop plants (Fernandez
functionissimilartothoseofprolineandothercompatible etal.2010).Nevertheless,itsmetabolismintheresponseof
solutes (Hussainet al. 2011). Additionally, several authors grapevine to chilling stress was studied recently (Fernan-
indicated that polyamines may act as a cellular signal dez et al. 2012). Interestingly, not only trehalose, but also
during stress response (Alca´zar et al. 2010a, b; Gill and trehalose-6-phosphate accumulated in grapevine tissue as
Tuteja 2010b). Polyamine levels change under environ- the effect of stress and their increase might be the reason
mental stress conditions. In some cases, their content for the resistance to this abiotic stress. The authors high-
increases as it was presented in the experiments on wheat, lightedthepossiblefunctionoftrehalose-6-phosphateasan
in which elevated content of Put and Spd was observed active compound in the stress response and suggested its
after cold treatment, as well as higher levels for Spm after link with the metabolism ofsucrose. Inaddition,Garg and
polyethyleneglycol-inducedosmoticstresswereregistered coworkers demonstrated that transgenic rice with the
(Kova´cs et al. 2010). In other cases, decreased levels of overexpression of trehalose biosynthetic genes was char-
polyamines were observed as a consequence of the stress. acterizedbytheenhancedtolerancetosalinityanddrought
Under long-term salt stress, a significant reduction of free stress (Garg et al. 2002). Transgenic lines shown in this
PutandSpmwasnotedininvitrogrownappleshoots(Liu study had improved physiological parameters, such as
et al. 2008). sustained plant growth, less photo-oxidative damage,
favorablemineralbalanceaswellasahigheraccumulation
Carbohydrates of trehalose, compared with nontransgenic controls. These
researchers suggested that trehalose might be involved in
It was widely reported that abiotic stresses lead to accu- sugar sensing and carbohydrate metabolism rather than
mulation of nonstructural carbohydrates like sucrose, being a compatible solute.
hexoses and polyhydric alcohols among many plant spe-
cies. Especially, there is a strong correlation between the Glycine betaine
carbohydrate accumulation and tolerance to osmotic
stresses,suchaswaterdeficitorsalinitystress(Bartelsand Another extensively studied osmoprotectant is glycine
Sunkar 2005). Soluble carbohydrates play an important betaine (N,N,N-trimethylglycine). Glycine betaine is a
role in plant metabolism as a source of carbon and energy quaternary ammonium compound, which is involved in
within a cell. Their level might be affected by different maintaining water balance, stabilizing macromolecules,
stresses, as the carbohydrate content is related to photo- protecting photosynthesis and detoxificating reactive oxy-
synthesis. Contents of some carbohydrates including gen radicals (Chen and Murata 2011). Many studies have
sucrose,raffinose,glucose,fructoseandmaltoseincreased, reported its important role in enhancement of plant toler-
whereas the myoinositol level decreased in water-stressed ance against abiotic stresses. However, not every crop
barleyroots(Sicheretal.2012).Solublesugarsfunctionas speciesaccumulatessignificantamountsofglycinebetaine
osmoprotectants during water deficit, reducing the detri- during stress. To this group belong wheat, maize and bar-
mental effects of osmotic stress, helping in maintaining ley. Additionally, some of the crops do not accumulate
turgor, stabilizing cell membranes and protecting plants glycine betaine at all, like rice (Giri 2011). Therefore,
from degradation (Basu et al. 2007). The increase in sugar attempts to achieve transgenic plants with enhanced tol-
content is mostly the effect of starch hydrolysis, which erance through glycine betaine biosynthesis have been
requires enzymes with a hydrolytic activity (Kaplan and made. One of the studies concerned transgenic maize
Guy 2004). Furthermore, soluble sugars like sucrose, raf- accumulatinghigherlevelsofglycinebetainethanthewild
finose, stachyose, trehalose and sugar alcohols, like sorbi- types. As a result of such a transformation, transgenic
tol, ribitol and inositol, act as cryoprotectants during cold maize better sustained chilling stress, as compared with
stress, protecting cell membranes against ice adhesion non-transformants (Quan et al. 2004b). Furthermore, other
(Janska´ et al. 2010). In addition, carbohydrates may act as transgenic crop plants were achieved through genetic
signalingmolecules(HansonandSmeekens2009)andplay engineering and tested against environmental stresses,
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