Review Article

Relevance of Proteomic Investigationsin Plant Abiotic Stress Physiology

Khalid Rehman Hakeem,1 Ruby Chandna,1 Parvaiz Ahmad,2 Muhammad Iqbal,1 and Munir Ozturk3

Abstract

Plant growth and productivity are influenced by various abiotic stresses. Stressful conditions may lead to delaysin seed germination, reduced seedling growth, and decreased crop yields. Plants respond to environmentalstresses via differential expression of a subset of genes, which results in changes in omic compositions, such astranscriptome, proteome, and metabolome. Since the development of modern biotechnology, various researchprojects have been carried out to understand the approaches that plants have adopted to overcome environ-mental stresses. Advancements in omics have made functional genomics easy to understand. Since the funda-mentals of classical genomics were unable to clear up confusion related to the functional aspects of the metabolicprocesses taking place during stress conditions, new fields have been designed and are known as omics.Proteomics, the analysis of genomic complements of proteins, has caused a flurry of activity in the past fewyears. It defines protein functions in cells and explains how those protein functions respond to changingenvironmental conditions. The ability of crop plants to cope up with the variety of environmental stressesdepends on a number of changes in their proteins, which may be up- and downregulated as a result of alteredgene expression. Most of these molecules display an essential function, either in the regulation of the response(e.g., components of the signal transduction pathway), or in the adaptation process (e.g., enzymes involved instress repair and degradation of damaged cellular contents), allowing plants to recover and survive the stress.Many of these proteins are constitutively expressed under normal conditions, but when under stress, theyundergo a modification of their expression levels. This review will explain how proteomics can help in eluci-dating important plant processes in response to various abiotic stresses.

Introduction

The potentially adverse abiotic stress conditionscommonly encountered by plants include extreme tem-

peratures, low water availability (drought), waterlogging(flooding), and high salinity and mineral deficiency, whichresult in significant reductions in the yield of economicallyimportant crops (Ahmad and Prasad, 2012a, 2012b; Ahmadand Umar, 2011). These unfavorable conditions bring aboutalterations in plant metabolism, growth, and development,ultimately leading to plant death (Ahmad et al., 2010a, 2012a;Ashraf, 2010; Manavalan et al., 2009; Tester and Langridge,2010; Tran and Mochida, 2010a). Being sessile in nature,plants lack mechanisms to escape from adverse conditions. Anotable feature of plants to adapt to abiotic stresses is theactivation of multiple responses involving complex networksthat are interconnected at many levels (Sarwat et al., 2012;Shinozaki and Yamaguchi-Shinozaki, 2007; Tran et al., 2007a,

2007b, 2010b). These complex responses initiated by the signaltransduction pathways, via which plants perceive and re-spond to environmental stresses, are not well understood. Inmany cases, several types of abiotic stress collectively chal-lenge plants (Hadiarto and Tran, 2011; Manavalan et al.,2009; Shinozaki and Yamaguchi-Shinozaki, 2007; Tran andMochida, 2010a).

Proteomics has appeared as an important tool in the field ofplant science, enabling us to interpret the stress responsesoccurring in plants. The vast range of applications of pro-teomics in biological fields has greatly increased its use overthe last decade (Bindschedler et al., 2008; Chen et al., 2011;Evers et al., 2012; Kaufmann et al., 2011; Nanjo et al., 2011;Thelen and Peck, 2007; Yang et al., 2011; Yokthongwattanaet al., 2012; Zhang et al., 2012; Zheng et al., 2012). The term‘‘proteome’’ (PROTEins expressed by genOME) represents thesurvey of the expression of all proteins in a given time andcondition. Proteomics is also described as the study of the

1Molecular Ecology Laboratory, Department of Botany, Jamia Hamdard, New Delhi, India.2Department of Botany, Amar Singh College, University of Kashmir, Srinagar, India.3Department of Botany, Ege University, Bornova, Izmir, Turkey.

OMICS A Journal of Integrative BiologyVolume 16, Number 11, 2012ª Mary Ann Liebert, Inc.DOI: 10.1089/omi.2012.0041

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quantitative totality of proteins of a cell, tissue, or organism,which helps in understanding the structural and develop-mental as well as functional aspects of the plant (Bindschedlerand Cramer, 2011; Boisvert et al., 2010). Proteomic researchaims at the identification of new proteins, revealing theirfunctions and also unravelling the regulatory networks thatcontrol their expression (Acero et al., 2011).

Proteomics is now gaining momentum in three major as-pects of plant science: in cellular and subcellular, structuraland developmental, and physiological and genetic studies.Hossain and colleagues (2012) summarized information onorganelle proteomes relevant to the plant tolerance mecha-nism to react to abiotic stress at the protein level. Recently,studies of proteomics have established that it has become auseful tool for studying the protein variations of the physio-logical events occurring in different plant organs (Kosovaet al., 2011; Tran and Mochida, 2010a; Timperio et al., 2008;Yan et al. 2006). Adverse environmental conditions altervarious physiological processes in plants, which are directlycontrolled by genes and functionalized by different proteins.Proteomics together with transcriptomics and metabolomicshave enabled us to carry out large-scale analyses of genes,transcripts, proteins, and metabolites, leading to a better un-derstanding of biological processes (Fig. 1). The long-termchallenges in proteomic research include the identificationand quantification of the structural and functional aspects ofcomplete complements of proteins. Functional genomics isuseful in the critical step of tackling the sets of clone genera-tion that are representative of each protein of a proteome, andthen analyzing these sets of clones on a genome-wide basis.

Principal Techniques of Global Protein Analysis

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the most commonly used technique for proteinseparation and for obtaining quantitative data (Dooki et al.,2006). High-resolution 2D-PAGE has important applicationsin the resolution of a large number of proteins (Aghaei et al.,2009), and has been used to study the gene products inducedin different plant species exposed to different abiotic stresses(Debnath et al., 2011), including salt treatments (Manaa et al.,2011a, 2011b; Shi et al., 2011; Sobhanian et al., 2011; Yan et al.,2006), dehydration/drought (Ge et al., 2012; Ladrera et al.,2007), high and low temperatures (Ahsan et al., 2010; Carrasco

et al., 2011), heavy-metal stress (Durand et al., 2010), andherbicides (Castro et al., 2005). Differential proteomics is usedto compare distinct proteomes (e.g., normal versus stressedcells and normal versus treated cells), differing both in proteinquality and quantity (Ahsan et al., 2010). Thus proteomeanalysis connects gene expression to cell metabolism. Withthe growing collection of technologies available for the ex-traction and identification of proteins and for studying theirinteractions, proteomics permits clarification of the mecha-nisms that are expressed in the responses of cells to abioticstresses. Different staining techniques are used to visualize theseparated proteins. The color density and size of the detectedspots quantifies the protein expressed. However, the accuracyof staining methods is limited due to low dynamic range. Thefluorescent dyes available for proteins have overcome thislimitation to a significant degree (Timms and Cramer, 2008).Mass spectroscopy (MS) for peptide mass fingerprinting(PMF) is gaining importance because of their sensitivity withpartial sequencing of proteins and affordability (Wong andCagney, 2010). High-performance liquid chromatography(HPLC)-based separation methods have also entered the fieldof proteomics as an alternative to 2D-PAGE. They help inidentifying and functionally characterizing the recombinantproteins encoded by differentially expressed cDNA clones(Stoevesandt et al., 2009). High-throughput proteomics re-search would be incomplete without image-analysis software(Palmblad et al., 2007). These programs help in removing thebackground patterns and by quantification of spots, matchingimages from relative gels and comparing the intensities ofrelative spots (http://www.lsbi.mafes.msstate.edu/p_data.htm). Processes involved in the identification of differentially-expressed proteins under stress conditions are shown dia-grammatically in Figure 2. The challenge lies in identifying

FIG. 1. Proteomics is gaining momentum in all three levelsof plant science. These three levels are concerned withstudying the proteins at (1) subcellular and cellular levels, (2)structural and developmental levels, and (3) physiologicaland genetic levels.

FIG. 2. Overview of the currently available disciplines forlarge-scale analyses of genes, transcripts, proteins, and me-tabolites (PTM, post-translational modification).

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proteins in the typically complex and large plant genomes.Proteome information is a direct link of genome sequencewith biological activity; therefore proteome analyses relyon well-annotated sequence databases (Bindschedler andCramer, 2011). Various bioinformatics tools are also availablefor plant proteome analysis (Table 1). The completion of thefirst plant genome in 2000 for the model plant Arabidopsisthaliana, and later for rice in 2002, also yielded informationabout sequencing for plant genomes like Lotus, Populus, to-mato, Medicago, and maize. This amazing applicability of thetool has added information related to the functional aspects ofmany genes simultaneously in the gene data bank (Bind-schedler and Cramer, 2011; International Rice GenomeSequence Project, 2005; Rensink and Buell, 2005; Schneideret al., 2009; Swarbreck et al., 2008; The Arabidopsis GenomeInitiative, 2000; Vij and Tyagi, 2007). Increasing sequencingspeed, and improvements in gene annotation and modeling,have helped to complete well-annotated plant genomes. TheNational Center for Biotechnology Information (NCBI)website (http://www.ncbi.nlm.nih.gov/genomes/PLANTS/PlantList.html) provides genomics data for the soybean,Medicago truncatula, the wine grape, maize, poplar, and sor-ghum. The UniprotKB and SwissProt protein databases arealso available for plant data. However, only a small propor-tion of the reviewed proteins are available in these manuallyannotated reference databases. For example, the protein en-tries for Arabidopsis, rice, maize, and other ‘‘viridiplantae’’organisms in Uniprot were 12,522, 10,643, 950, and approxi-mately 8500, respectively (http://www.uniprot.org/uniprot/;accessed February 2012). However, an investigation of theArabidopsis proteome has led to the discovery of 778 novelproteins associated with unpredicted open reading frames(ORFs), suggesting that the proteogenomic approach cangreatly improve and validate the annotation of the genes(Castellana et al., 2008). Therefore, the genome-proteome-widestrategy can be used to gain insights into stress-responsivegenes, thus adding to our understanding of the mechanismsinvolved in stress tolerance.

Proteomics of Plant Physiology Under Abiotic Stress

Under a number of abiotic stress conditions, variousphysiological and biochemical changes occur in plants

(Ahmad and Prasad, 2012a, 2012b; Ahmad and Umar,2011; Hadiarto and Tran, 2011; Shinozaki and Yamaguchi-Shinozaki, 2007; Tran and Mochida, 2010a). Crop plants oftencome into contact with various abiotic stresses, includingdrought, high and low temperatures, salinity, heavy metals,low soil fertility, and mechanical wounding. It is reported thatthese stresses can reduce the crop yield to less than 60%(Hadiarto and Tran, 2011; Tester and Langridge, 2010; Tranand Mochida, 2010a). A basic step toward understanding themolecular mechanisms via which plants cope with stressconditions is gene or protein identification. The first questionis the response of the plant to its changing environment, andthis is dependent on the ability of the plant to detect stimuli.This leads to signal transduction within the plant cell thatbrings about activation of the related genetic programs(Ashraf, 2010; Hadiarto and Tran, 2011; Shinozaki andYamaguchi-Shinozaki, 2007). Protein modification is also animportant mechanism initiated in the response to stress. Thereare many signalling pathways that lead to these various de-fense responses. Signalling pathways are interconnected withone another at many levels. Only proteomics tools can help inunderstanding the interactions between the different signallingpathways. Several researchers have reported that proteins re-spond to stress in different plants differently (Komatsu et al.,2009; Kosova et al., 2011; Witzel et al., 2009). These findings arerelated to the different stress enzymes expressed in plant cellsin response to abiotic stress. The complete sequencing of thegenomes of Arabidopsis (Kaul et al., 2000), and rice (Tanakaet al., 2008), has enabled plant breeders to access large numbersof plant genes. The identification of genes or proteins functionalin the plant’s response to stress requires a range of geneticinformation and molecular tools (Rossignol et al., 2006).

2D-PAGE is used to identify the specific proteins whoseaccumulation is altered under abiotic stresses (Table 2). Pro-tein expression in stress-tolerant and stress-sensitive plantscan also be evaluated with the help of proteomics ( Jorrın et al.,2007). Using proteomics, the effects of environmental stress onthe proteins at the subcellular, cellular, and organ levels, suchas mitochondria of Pisum sativum (Baginsky, 2009; Tayloret al., 2005), the nucleus of Arabidopsis ( Jain et al., 2006), theanther of rice (Imin et al., 2004), and poplar leaves (Renautet al., 2004), have also been successfully explored. Experi-ments by Boudsocq and associates (2007), using proteomics to

Table 1. Proteome Database Used in the Protein Identification of Some Important Plants

Crop Proteome database Reference

Rice http://oryzapg.iab.keio.ac.jp/ Helmy et al., 2011http://gene64.dna.affrc.go.jp/RPD/ Komatsu et al., 2004

Arabidopsis http://ppdb.tc.cornell.edu/ Sun et al., 2009http://bioinf.scri.ac.uk/cgi-bin/atnopdb/home Leung et al., 2003

Soybean http://proteome.dc.affrc.go.jp/Soybean Sakata et al., 2009http://www.oilseedproteomics.missouri.edu/soybean.php Hajduch et al., 2005

Maize http://ppdb.tc.cornell.edu Sun et al., 2009Wheat http://www.proteome.ir. Hajheidari et al., 2005

http://www.gramene.org/protein Ware et al., 2002Chickpea http://bioinfo.noble.org/manuscript-support/legumedb Lei et al., 2011Pea http://bioinfo.noble.org/manuscript-support/legumedb Lei et al., 2011Alfalfa http://bioinfo.noble.org/manuscript-support/legumedb Lei et al., 2011Barley http://www.gramene.org/protein Ware et al., 2002Sorghum http://www.gramene.org/protein/ Ware et al., 2002

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study the effects of various abiotic stresses on the levels of heatshock proteins and a dnaK-type molecular chaperone, haverevealed a reduced expression of these elements, whereas a26S proteosome regulatory subunit was responsive only toosmotic stress. Regulation of gene expression is one of thestrategies used by plants to fight stress. Brechenmacher andcolleagues (2009) have exploited the full potential of pro-teomics for studying the functional genomics of soybeans. Aprotein reference map of soybean root-hair cells was gener-ated, which showed a total of 1492 different proteins. Theseobservations improved our understanding of protein func-tions in plant water and nutrient uptake. Also, Ahsan andKomatsu (2009) did proteome analysis at various develop-mental stages of soybean leaves and flowers and reported 500and 600 proteins for the leaf and flower tissues, respectively.

They found differential expression of 26 proteins in the leavesat various developmental stages, whereas in buds and flowersthe proteome profile revealed 29 differentially expressedproteins. A total of 478 non-redundant proteins were identi-fied using both 2-PAGE and multidimensional protein iden-tification technology (MudPIT) in proteome profiling of thesoybean during seed filling. The genetic nature of the over-all environmental stress mechanism in plants is shown inFigure 3.

Salinity stress

Salt is a major environmental stress that limits the cropyield, especially in arid and semiarid regions. Soil salinity isknown to affect about 800 million hectares of agricultural land

Table 2. Proteomics Studies That Explore the Response to Abiotic Stress Factors in Plants

Differentially expressed proteins in response to stress

Abiotic stresses Upregulated Downregulated Reference

Drought J-type co-chaperone Hsc20,a putative ABC transporter,ATP-binding protein,NtrX, HslU

Homeobox-leucine zipperprotein, AP2/EREBPtranscription factor

Hajheidari et al., 2005Bhushan et al., 2007Pandey et al., 2008Xu et al., 2009

Osmotic (salt) 31-kDa glycoprotein, b-conglycinin,protease inhibitor transporterproteins (vacuolar protonATPase, ABC transporters,exocyst subunits)

Carbohydrate metabolism,(GAPDH, fructokinase 2),CO2 assimilation (RubiscoLSU and SSU, PGK)

Abbasi and Komatsu,2004 Kim et al., 2005Caruso et al., 2008Sobhanian et al., 2010Manaa et al., 2011a

Chilling(low temperature)

Cysteine synthase,S-adenosylmethioninesynthetase, glutaminesynthetase, enolase

HSP90; saccharide anabolism(formation of UDP-glucose)

Yan et al. 2006Lee et al., 2009Degand et al., 2009Cheng et al., 2010

Anoxia Glycolytic (ENO1, GAPDH)and fermentation (PDC,ADH1) enzymes, cytokininmetabolism (b-D-glucosidase),cytoskeleton (actin)

Proteosynthesis (eIF-4A, eEF-2,mitochondrial EF Tu)

Chang et al., 2000Huang et al., 2005

Nutritional deficiency(nitrogen, iron,boron, manganese,potassium)

Ribose 5-phosphate isomeraseprecursor, malatedehydrogenase, ATPsynthase, protease Do-like 1,methionine synthase

Carbon assimilation(Rubisco-binding protein,Rubisco activase, PRK),components of Mn clusterin OEC (OEC23), b6 subunitof proteasome, PR proteins

Bahrman et al., 2004Alves et al., 2006Brumbarova et al.,2008 Kim et al., 2009

Heavy metals (arsenic,cadmium, copper,mercury, cobalt, zinc)

Protein kinase C inhibitor,ATP sulfurylase, glycinehydroxymethyltransferase,trehalose-6-phosphatephosphatase, glutathioneS transferases, glyoxalase I,peroxyredoxin, aldosereductase

Photosynthetic proteins(Rubisco LSU, OEE 1 andOEE 2, Fe–S componentsof cytochrome b6–f complex,chloroplast ferredoxin-NADP+oxidoreductase)

Requejo and Tena, 2005Roth et al., 2006Ahsan et al., 2007; 2008Duquesnoy et al., 2009

Ultraviolet radiationand ozone stress

Phytoalexins, pathogenesis-related(PR) genes, (OsPR1b, OsPR5,and OsPR10a)

CRT, LOX, glucose- andsucrose-binding proteins;signalling proteins(x subunit of 14-3-3protein)

Renaut et al., 2009Bohler et al., 2010Du et al., 2011Tripathi et al., 2011

Herbicide Antioxidative enzymes,ATP-dependent efflux pumpsCDR1 and CDR2, ABC(ATP-binding cassette)transporters, demethylationinhibitors (DMI) resistance

Photosynthetic enzymes(Rubisco)

Zhang and Riechers,2004 Castro et al.,2005 Acero et al., 2011

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worldwide (Munns and Tester, 2008). This is more than 6% ofthe world’s total land area. Due to land clearing or irrigation,salinity has affected a significant part of cultivated agricul-tural land. Excessive salinity of soil or water is a limitation ofagriculture production in some semi-arid regions where ag-riculture is dependent on irrigation (Munns and Tester, 2008).Increases in various salt concentrations, especially of Na + andCl - , and also Ca2 + , K + , (CO3)2 - , (NO3 ) - , and (SO4 )2 - , de-creases the water retentive potential of soil, which in turndecreases water uptake by roots. Therefore, elevated levels ofNaCl cause osmotic stress and disturb the ion homeostasis ofthe cell. During osmotic stress, there is accumulation of sev-eral low-molecular-weight osmolytes, such as glycine betaine(GB) and proline- and raffinose-derived oligosaccharides,as well as of high-molecular-weight hydrophilic proteinsfrom the late embryogenesis-abundant (LEA) superfamily(Ahmad, 2010; Ahmad and Sharma, 2010; Ahmad et al.,2010b, 2012b; Azooz et al., 2011; Cheng et al., 2009; John et al.,2009a). Also, a number of adverse processes are known tooccur in plants during salinity stress, including membranedisorganization, increases in the amounts of toxic metabolites,the generation of reactive oxygen species (ROS), reducednutrient uptake, inhibited photosynthesis, and finally thedeath of the cell and plant (Ahmad and Prasad, 2012a, 2012b;Ahmad and Umar, 2011; Ahmad et al., 2008, 2009, 2010a,2010b, 2011a; John et al., 2009b; Witzel et al., 2009). Yan andassociates (2006) observed 1100 spots in the proteome of riceroots that were seen upon exposure to high salt concentrations(150 mmol/L) on 2D gels, with 34 protein spots upregulatedand 20 spots downregulated. Abbasi and Komatsu (2004)showed that salt stress reduces plant growth. This was alsoreported by Yan and associates (2006), who showed reduc-tions in the levels of glutamate synthetase (GS), a key enzymefor nitrogen assimilation, during salt stress. It is interesting tonote that many proteins respond to salt stress and also to otherabiotic stresses. For instance, two proteins (peroxidase andputative nascent polypeptide-associated complex a-chain)respond to rice plants under salt stress, and also show asimilar response to sugar beet leaves (Beta vulgaris) underdrought stress (Hajheidari et al., 2005). Dooki and colleagues(2006) observed that 13 proteins in young rice panicles sig-

nificantly changed their expression levels under salt stress.MS analysis of highly abundant proteins of the panicle led tothe identification of proteins that are involved in several salt-responsive mechanisms. These proteins may help the plant toadapt to salt stress by upregulation of antioxidants and theproteins involved in translation, transcription, signal trans-duction, and ATP generation. Abbasi and Komatsu (2004)showed that photosystem II (PII), an oxygen-evolving en-hancer protein precursor, was upregulated in the rice leafsheath under salt stress. In wheat, ferredoxin NADP + oxi-doreductase was upregulated during salt stress (Caruso et al.,2008). In the roots of soybean seedlings, metabolism-relatedproteins are mainly downregulated under salt stress. Adecrease in some glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase; GAPDH), and several proteinsinvolved in CO2 assimilation, were also observed as a result ofsalinity stress in crops (Sobhanian et al., 2010). Proteins withimportant functions, like membrane stabilization, proton ho-meostasis, and signal transduction, always show increasedexpression. Cheng and associates (2009) reported a new saltresponding to leucine-rich-repeat type receptor-like proteinkinase, OsRPK1. Wang and co-workers (2008) found someinteresting results while studying the responses of the com-mon Chinese wheat cultivar Jinan 177 and its hybrid undersalt stress with a salt-tolerant Thinopyrum ponticum. Sig-nificant changes in the expression of proteins involved insignal transduction (small G proteins, ethylene signalling, andthe MAP kinase cascade) were observed in both genotypes. Anumber of transcription and translation factors, such asthe putative transcription factor BTF3 from the nascentpolypeptide-associated NACA protein family, and DEADbox RNA helicase involved in the modulation of stress-inducible CBF/DREB transcriptional activators, were upre-gulated at high salt concentrations. Upregulation of severaltransporter proteins (vacuolar proton ATPase subunit E in-volved in Na + /H + antiport, ABC transporters involved in thetransport of secondary metabolites, and the exocyst subunitsSEC1a and EXO70), and several proteins with protectivefunctions (HSP70 and other chaperones), was observed undersalt stress. Additionally, an increase in the level of transcrip-tion factor proteins by genetic engineering, such as DREBs

FIG. 3. A simple model of the environmental stress responses in crop plants (ROS, reactive oxygen species).

PROTEOMICS AND PLANT ABIOTIC STRESS 625

and NACs, significantly enhanced the tolerance of both themodel and crop plants to a wide range of environmentalstresses, including drought, salt, and cold, even in field con-ditions (Ahmad and Prasad, 2012a, 2012b; Ahmad and Umar,2011; Ahmad et al., 2010a; Hu et al., 2006; Nakashima et al.,2007; Qin et al., 2008; Tran et al., 2004, 2007b). Aghaei andcolleagues (2009) reported that some proteases make theirway to the cytosol from injured vacuoles under salt stress inthe hypocotyls and roots of the soybean, and were found toneutralize the deleterious effects of salt on cell proteins. Theexpression levels of proteins also showed significant changesin grapevines under salt stress ( Jelloulia et al., 2008). A total of48 proteins showed differential expression, of which 32showed upregulation, 9 showed downregulation, and 7 newprotein spots were observed. Thus adaptation to salt stress isthe result of modifications of gene expression that alter thecellular machinery. Moreover, when evaluating plant re-sponses to salt stress, quantitative analysis of gene expressionat the protein level is essential. Expression profiling at theprotein level of plants under stress reflects the importance ofthe proteomic approach (Aghaei et al., 2009). Therefore, mo-lecular tools are required for the identification of salt-stress-responsive genes, and proteomics is the most reproducibletool to determine the functionality of the identified genes(Afroz et al., 2011; Aghaei et al., 2009; Kosova et al., 2011;Sobhanian et al., 2011; Zhang et al., 2012). Evers and associates(2012) concluded that during cold and salt stresses in thepotato the majority of photosynthesis-related genes weredownregulated, whereas cell rescue and transcription factor-related genes were mostly upregulated.

Temperature stress

Temperature stress can greatly affect plant metabolism(Suzuki and Mittler, 2006). High and low (chilling andfreezing) temperatures are known stressors that decrease theproduction of many important crops such as rice, wheat, andmaize. In temperate regions low temperatures (0–12�C) arecommon during the growing season, and are known to de-crease crop production, since chilling temperatures signifi-cantly alter plant metabolism and physiology (Foyer andNoctor, 2005). To study and understand the changes occur-ring inside plants due to cold stress, a proteomics approach isa wonderful tool for deciphering these changes (Afroz et al.,2011; Evers et al., 2012; Kosova et al., 2011; Zheng et al., 2012).Experiments by Yan and co-workers (2006) showed that theprotein-expression pattern in rice leaf changes in response tocold stress. In this study, over 1000 protein spots were re-solved when leaf proteins were separated by 2D-PAGE, andamong them 31 proteins showed downregulation and 65proteins showed upregulation. Yan and associates (2006)showed that the majority of the spots that were differentiallyexpressed were consistently upregulated or downregu-lated until 24 h of recovery from temperature stress. Thesedifferentially-expressed proteins were further analyzed byPMF or matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF/MS) and functionally classified. An-other study by Kosmala and colleagues (2009) examined thefunctional distribution of differentially-expressed proteins inFestuca pratensis under cold stress. The functional group thatwas most affected by cold stress was photosynthesis, with35.3% of cold-responsive proteins residing in this category

(Kosmala et al., 2009). Lee and associates (2009) identifiedsome novel proteins known to be involved in energy produc-tion and metabolism, vesicular trafficking, and detoxification(e.g., acetyl transferase, phosphogluconate dehydrogenase,fructokinase, NADP-specific isocitrate dehydrogenase, puta-tive alpha-soluble N-ethylmaleimide-sensitive factor [NSF]attachment protein, and glyoxalase 1), and found that theyshowed higher sensitivity to cold stress in rice. Hashimotoand Komatsu (2007) observed downregulation of certainproteins at the organ level under cold stress. For example, 5-methyletetrahydropteroyl triglutamate-homocysteine S-methyltransferase was downregulated not only in the roots, butalso in leaf sheaths, suggesting that common cold-stress-responsive pathways exist for the regulation of some proteinsin roots and leaf sheaths. Studies carried out by Kamal andassociates (2010) on wheat under cold stress revealed thatsome cold stress-related proteins, such as cold acclimationproteins (9000–22,000), cold shock proteins (16,000–38,000),ABA-inducible proteins (10,000–41,000), cyclophilin (13,000–18,000), low-temperature-regulated proteins (7000–14,000),kinase-like protein (6000–74,000), nitrogen-activated proteins(40,000–80,000), transcriptional adaptors (7000–29,000), andtranslation initiation proteins (12,000–17,000), were inducedin wheat. In the winter wheat variety Cheyenne, Rinalducciand co-workers (2011) showed that out of 1000 protein spotsthat were reproducibly detected on each gel, 31 protein spotswere downregulated and 65 were upregulated. MS analysis ofthese proteins showed 85 differentially-expressed proteins,including the well known and novel cold-responsive proteins,like RNA-binding proteins, lectin protein, and peptide me-thionine sulfoxide reductase (PMSR). PMSR is known to cat-alyze the reduction of protein-bound methionine sulfoxidegroups to methionine, which protects the proteins from in-activation and degradation by ROS. During chilling stressmany proteins, especially proteins related to photosyntheticactivity, (e.g., Rubisco large subunit, of which 19 fragmentswere detected), showed degradation.

High-temperature stress is prevalent in plants because ofthe emission of greenhouse gases. A large part of crop loss isdue to increases in temperature worldwide, especially whencombined with drought or a variety of other stressors. Heatstress is responsible for improper folding of proteins and leadsto the denaturation of intracellular proteins and membranecomplexes. Heat stress increases the expression of proteinswith chaperon functions, such as heat-shock proteins (HSPs),and so-called small HSPs (sHSPs). Peng and associates (2004)observed that rice grain yields decreased by 10% for each 1�Cincrease in the minimum temperature during the growingseason. Lee and colleagues (2007) identified novel high tem-perature stress-responsive genes and determined their ex-pression patterns. Heat stress is also responsible for oxidativedamage to biomolecules. Also, several enzymatic and non-enzymatic antioxidants are upregulated and aid in redoxhomeostasis. These enzymes include dehydroascorbate re-ductase (DHAR), glutathione-S-transferase (GST), thioredox-in h-type (Trx h), and chloroplast precursors of superoxidedismutase (SOD). Dong and colleagues (2007) also used aproteomics approach to analyzing heat-responsive proteins inrice leaves. This study showed that heat stress affects proteins,namely sHSPs. Zhang and co-workers (2010) also showedthat heat stress induces profound changes in cytoskeletoncomposition, indicating its reorganization. In addition, an

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increased accumulation of some eukaryotic translation initi-ation factors (eIF4F and eIF5A-3) was observed, indicatingprofound cellular organization leading to programmed celldeath (PCD) under heat stress. Ahsan and associates (2010)reported enhanced accumulation of several other proteinswith chaperone functions (chaperonin 60 b subunit CPN60-b,HS90, chaperonin CPN10, and chloroplast chaperonin) insoybean seedlings under heat stress.

Heavy metal stress

Intensive industrial activity over the past 100 years hasresulted in massive releases of heavy metals into the envi-ronment. Many heavy metals such as nickel (Ni), chromium(Cr), lead (Pb), cadmium (Cd), mercury (Hg), and others, areimportant health-threatening pollutants. Major sources ofheavy metal pollution include power stations, cement facto-ries, zinc smelting, coal burning, paint factories, and the use ofphosphate fertilizers. Ions of these heavy metals are known tobe mobile in the geosphere, and their accumulation in thehuman food chain is a cause of concern for health specialists.The main entry pathway of heavy metals into human andanimal foods is through uptake by crop plants (Ahmad et al.,2011a; John et al., 2009a, 2009b). There are different ways thatplants respond to heavy metal toxicity, such as immobiliza-tion, exclusion, chelation, and compartmentalization of metalions (Ahsan et al., 2008). The formation of peptide metal-binding ligand phyto-chelatins (PCs) and metallothioneins(MTs), as well as more general stress-response mechanisms,are among the ways plants combat stress (Ahsan et al., 2009;Hradilova et al., 2010). Impairments in growth and develop-ment are among the effects of metal ions in plants. The gen-eration of ROS through chain reactions characterized by anexcess of free metals causes damage at a molecular level(Kieffer et al., 2009). It is known that increased ROS produc-tion induces the expression of HSPs and chaperones, andprotects the cell from oxidative damage. If these HSPs areconsidered stress markers, they mark the morphologicalchanges seen due to stress in the aerial plant parts. A study byHradilova and colleagues (2010) showed that HSPs were ableto indicate stress symptoms up to day 14, and that othertoxicity symptoms were not apparent. Nearly 20% of thestressed plants had necrotic spots that were seen mostly inyoung leaves. Cheng and associates (2009) reported that thelarge number of stress proteins induced by heavy metal stresshave a molecular mass of 10,000–70,000 Da in plants. There-fore proteome analysis of the proteins involved in the re-sponse to heavy metals can deliver more accurate andcomprehensive information for better understanding of theplant response to heavy metal stress. Labra and co-workers(2006) reported the upregulation of proteins involved in dif-ferent cellular compartments and metabolic pathways in re-sponse to the protein changes caused by potassiumdichromate treatments in Zea mays. They suggested that theactivation of oxidative stress mechanisms affects sugar me-tabolism and ATP synthesis. Additionally, patterns of proteinexpression of maize roots under arsenic stress was examinedby Requejo and Tena (2005), who reported that about 10% ofall detected maize root proteins were upregulated or down-regulated by arsenic. Twenty proteins showed reproducibleeffects of the metal, and were selected for further analysis byMALDI-TOF/MS. Out of these 20 proteins, 11 were identified

by comparing their peptide mass fingerprints to a protein andexpressed sequence tag database. Their study found thatmaize root proteins highly responsive to arsenic exposureincluded a major homogenous group of seven enzymes thatare involved in cellular homeostasis for redox perturbation(e.g., three superoxide dismutases, two glutathione peroxi-dases, one peroxiredoxin, and one p-benzoquinone reduc-tase), in addition to four additional functionally heterogenousproteins (e.g., ATP synthase, succinyl-CoA synthetase, cyto-chrome P450, and guanine nucleotide-binding protein bsubunit). These findings strongly suggest that the induction ofoxidative stress is the main process underlying heavy metaltoxicity in plants.

According to Lee and associates (2010), upregulation ofenzymes cooperating with reduced glutathione (GSH) inplants was observed with increasing cadmium concentration.During the ROS-quenching process, approximately half of theupregulated proteins in the roots of rice belonged to the cat-egory involved in the oxidative stress response and GSHmetabolism, including peroxidases, putative ferredoxin:NADP(H) oxidoreductase, and APX1. Accumulation of pro-teins like copper chaperone (CCH), ROS-scavenging en-zymes, and those involved in the biosynthesis of GSH such asGS-like 1, are protective in function and are induced by ele-vated levels of cadmium. The activity of Rubisco, such asRubisco LSU-binding proteins, Rubisco activase 2, a chloro-plast precursor of Rubisco activase, ribulose-phosphate 3-epimerase, and carbonic anhydrase, decreased significantly inresponse to cadmium stress. A number of studies have alsobeen conducted on elevated concentrations of arsenic (Ahsanet al., 2008), copper (Ahsan et al., 2007), nickel (Ingle et al.,2005), mercury (Isarankura-Na-Ayudhya et al., 2009), andzinc (Isarankura-Na-Ayudhya et al., 2009). These studies in-dicated the formation of necrotic spots associated with adrastic degradation of Rubisco, especially the Rubisco LSU.Cysteine synthase (CS), GST, GST-tau, and tyrosine-specificprotein phosphatase proteins (TSPP) were markedly upre-gulated under heavy metal stress.

Thus the proteomics approach enables us to reveal mole-cules that play important roles in plants during exposure toheavy metals, and generates data about stress-related re-sponses in different tissues. This information aids in the de-sign and selection of plants that resist deleterious metalaccumulations in the food chain. This may also help solveproblems with essential metal deficiencies in humans.

Osmotic and water-deficit stress

Among the abiotic stresses, drought is responsible forwidespread decreased crop production worldwide. Variousmechanisms, including physiological and molecular mecha-nisms, are involved in the plant adaptation to drought. Inplants, drought-induced proteins are known to be involved inphysiological adaptations to water stress. Proteomics hasbeen found to be a useful tool for the identification ofthe mechanisms involved in drought stress and tolerance(Hajheidari et al., 2005). A proteomics approach has been usedby several groups to study drought responsiveness in manyplant species, such as poplar (Durand et al., 2011), maize (Zhuet al., 2007), rice (Liu and Bennet, 2011), soybean (Yamaguchiet al., 2010), and oak trees (Sergeant et al., 2011). Zang andKomatsu (2007), and Joshep and Jini (2010), gave mannitol

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treatments to rice plants to evaluate the mechanisms of theresponses of plants to osmotic stress using a proteomic ap-proach. The observations of Joshep and Jini led to the identi-fication of 327 drought-responsive proteins. Among them thelevels of 12 proteins were upregulated and 3 proteins weredownregulated with increases in the duration of mannitoltreatment. Xu and associates (2009) observed the damageoccurring in Hippophae rhamnoides (seabuckthorn) withdownregulation of HsIU (a heat shock protein belonging tothe Hsp100/Clp family, with ATPase and unfoldase activity),and a putative ABC transporter ATP-binding protein, due tothe effect of drought stress. They also observed an increase in[Fe–S] cluster assembly that is known to occur under drought.This was implied by the accumulation of J-type co-chaperoneHsc20. From this result, they concluded that iron–sulfur [Fe–S]proteins that are commonly found in all types of organismsplay important roles in processes such as redox and non-redox catalysis and signalling processes in seabuckthorn.Yamaguchi and colleagues (2010) studied the effect ofdrought on soybean primary roots using a proteomics ap-proach. They observed that several enzymes and proteinsrelated to isoflavonoid biosynthesis had increased expressionlevels. This may contribute to growth maintenance of rootsunder stressful conditions. Contrary to this, caffeoyl-CoA O-methyltransferase, an enzyme playing role in lignin synthesis,was highly upregulated. This might be associated with theenhanced accumulation of lignin, which is related to the in-hibition of growth. Several proteins were increased in abun-dance in water-stressed roots of the soybean, and played arole in protection from oxidative damage (Yamaguchi et al.,2010). These authors were able to detect 35 proteins that ex-hibited significant changes in their expression profiles indrought-affected soybean primary roots. These differentialexpressions were observed in at least one region of water-stressed roots, compared with well-watered controls. Manaaet al. (2011a) found that during osmotic stresses, levels ofABA, phospholipid signalling, and mitogen-activated proteinkinases (MAPKs) were increased in tomato. Different sig-nalling processes were integrated to work together againstosmotic stress (e.g., ABA and phospholipid moleculesappeared to function upstream of the osmotic stress-activatedprotein kinases; Manaa et al., 2011a). Drought stressesare known to induce Ca2 + -binding proteins that serveas transducers of Ca2 + signals (Kosova et al., 2011; Li et al.,2010; Sarwat et al., 2012). Ca2 + -binding proteins such ascalmodulin have been identified in plants (Sarwat et al., 2012).Ca2 + -dependent protein kinases, calcineurin B–like proteins,and SOS3 are involved in ABA-dependent stress responses inplants. Still, the functions of many proteins induced in higherplants are unknown, and their functions can only be inferredfrom information available for other organisms. Proteomicscan be used to analyze water-deficit-responsive proteins invarious plants (Ge et al., 2012). It may also help in identifyingthe functions of proteins that have a significant role in theplant response to drought stress.

Waterlogging and flooding stress

One of the most damaging abiotic stresses is waterloggingcaused by excessive water in the soil. Worldwide about half ofcrops (approximately 2 million hectares) is affected by floodingannually, causing a 25% reduction in yield (Valliyodan and

Nguyen, 2008). Waterlogging also restricts the availability ofoxygen to plant roots. Anaerobic metabolism occurs due to alack of oxygen and leads to glycolysis, followed by pyruvatefermentation. In higher plants the cell wall is the first organelleto respond to flood stress signals, and transmits it to the cell’sinterior, subsequently affecting the cell signalling cascade, andthe cell’s stress tolerance or intolerance (Komatsu et al., 2003,2010, 2012). Thus proteins in cell walls are involved in cell-wallsignal transduction, structure, metabolism, and cell enlarge-ment in response to the waterlogging stress. Numerous cell-wall proteins involved in stress tolerance have been identifiedin plants, such as soybean (Alam et al., 2010), chickpea(Bhushan et al., 2007), maize (Zhu et al., 2007), and rice (Choet al., 2009). Sakata and colleagues (2009) revealed relationshipsamong the 106 mRNAs, 51 proteins, and 89 metabolites thatshowed variations in the soybean under flooding stress. An-other study of roots and hypocotyls in soybean seedlings(Komatsu et al. 2009) identified several inducible genes andproteins. Within 12 h of stress, genes that were associated withethylene biosynthesis, alcohol fermentation, cell wall loosen-ing, and pathogen defense were significantly upregulated.Altered expression of trypsin protease inhibitor and acidphosphatase were also observed both at the transcriptional andthe proteome level. Several studies carried out on the soybean(Alam et al., 2010; Hashiguchi et al., 2009; Komatsu et al., 2010,2011; Nanjo et al., 2010) under flooding stress have confirmedthat proteins involved in glycolysis and fermentation path-ways, such as alcohol dehydrogenase, fructose-bisphosphatealdolase, UDP-glucose pyrophosphorylase, and GAPDH, ex-hibit upregulated expression. These results confirm thatflooding stress is also involved in stress due to oxygen star-vation. A comparative proteomic approach (Ahsan et al., 2007)in tomato leaves responding to waterlogging stress resulted inincreases in ion leakage and lipid peroxidation and increasedH2O2 content, whereas the chlorophyll content decreased.Using MALDI-TOF/MS, the authors identified 52 proteinspots that were differentially expressed in tomato. Many of theidentified proteins were involved in photosynthesis, energy,metabolism, and protein biosynthesis. A number of the pro-teins identified also play roles in disease resistance, and stressand defense mechanisms. It was also observed that stress leadsto disintegration of the fragments of large subunits of Rubisco.Proteome profiling by Alam and colleagues (2010) showed thatcontinuous waterlogging stress caused an increase in glycolyticflux to meet ATP requirements. Anaerobic energy metabolismwas upregulated to meet the objective. During flooding stress,the terminal electron acceptor (oxygen) of the electron trans-port chain (ETC) is unavailable, thus the intermediate electroncarriers become reduced, affecting the redox characteristics ofthe cell (i.e., the NADH/NAD + ratio). In this case, upregula-tion of GS in cells might help maintain the redox potential of thecell (Alam et al., 2010). Large protein sequence databases areavailable for Arabidopsis thaliana and rice, and they are thereforeconsidered to be model plants (Kosova et al., 2011). Thesestudies led to the identification of different pathways that areshared by different plant species under various stress condi-tions, as well as the pathways unique to a given stressor.

Hydrogen peroxide, ultraviolet rays, and ozone stress

ROS include singlet oxygen (O2), superoxide anions (O2 - ),hydrogen peroxide (H2O2), and hydroxyl radicals (HO$), are

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highly reactive and toxic, and lead to oxidative damage tobiomolecules (Ahmad and Prasad, 2012a, 2012b; Ahmad andUmar, 2011). Nonetheless, ROS also act as important regula-tors of many biological processes, such as cell growth anddevelopment, hormone signaling, and stress responses (Tri-pathi et al., 2011). ROS imbalance in the cell is closely linked toseveral types of oxidative destruction; therefore cellular redoxconditions should be well regulated. H2O2 is non-radical andcarries no net charge, and has a comparatively longer half-life,which makes it a more likely long-distance signalling mole-cule (Zhou et al., 2011). H2O2 acts as a physiological indicatorof stress intensity when plants are challenged with biotic and/or abiotic stresses. It can therefore be used to activate stress-responsive genes. Therefore, proteomic analysis of the H2O2

response may be of paramount importance for the under-standing of the plant network to environmental stresses(Bakalova et al., 2008). Detoxification of ROS by extracellularascorbic acid in the leaf apoplast has been identified as a po-tential tolerance mechanism toward H2O2 (Cheng et al., 2009).The apoplast consists of cell walls and the intercellular spaces.The root apoplast is important for a plant’s interactions withits environment. It can sense environmental changes andstress signals and then transfer them into the cell’s interior totrigger a cell-wide response. In addition to signal perceptionand transduction, apoplast proteins can also be used for cellwall modification and reconstruction, as well as for defenseresponses, although they comprise only 5–10% of the wall’sdry weight. Using a bioinformatics approach, we have dis-covered that there are more than 1000 different apoplasticproteins in Arabidopsis ( Jamet et al., 2008). Several reportshave appeared about leaf apoplast proteomes under variousabiotic stresses, such as salt in tobacco (Dani et al., 2005),dehydration in chickpea and rice (Bhushan et al., 2007), andboron deficiency in Lupinus albus (Alves et al., 2006). Thesestudies have broadened our understanding of the compli-cated regulation of leaf apoplast proteins. A few studies haveaddressed the dynamic changes in the proteome of the plantroot apoplast seen in response to oxidative stress, especially toH2O2, which serves as a physiological indicator of abioticstress intensity. High irradiance and elevated ozone (O3)concentrations are seen with UV-B exposure (315–280 nm)under field conditions. It is well known that atmospheric UV-Band O3 are important components affecting the global cli-mate, and the slightest change in their levels has adverseimpacts on physiological and biochemical growth character-istics, as well as on the productivity of many plants (Ahsanet al., 2010). Therefore it is important to study their interactiveeffects on plant systems. Experiments done by Kumari andassociates (2009) in Abelmoschus esculentus showed that im-portant target sites for the action of UV-B and O3 are cellmembranes, proteins, and DNA. The results of this study alsoindicated that UV-B and O3, individually as well as in com-bination, seem to have significant effects on the antioxidantsystem in plants. O3 uptake is an important phenomenoninside plant cells, and tends to be directly proportional tostomatal conductance (Tripathi et al., 2011). Therefore, onceinside the plant, O3 and UV-B are known to generate ROS incells, leading to oxidative damage. This leads to alterations inthe gene expression of proteins fundamentally important toall functions of cells, to produce different proteins to cope withthese stresses. Mechanisms for combating ROS depend on theinterrelated activities of several antioxidant enzymes, like

catalase (CAT), SOD, peroxidase (POD), ascorbate peroxidase(APX), and glutathione reductase (GR), and non-enzymaticantioxidants such as ascorbic acid, glutathione, and alpha-tocopherol (Ahmad and Umar, 2011; Ahmad et al., 2010a;Renaut et al., 2009). UV radiation may induce plants to gen-erate signal transduction intermediates, such as nitric oxide,ROS, and ethylene, followed by downregulation of photo-synthesis-related proteins, such as light-harvesting Chl a/b-binding protein, and upregulation of protective proteins, suchas pathogen-related protein-1 (PRP-1) and pigment-dispersingfactor 1.2 (PDF 1.2), at the mRNA level. Leaves are themain photosynthesizing organs, and they absorb nearly 90%of UV-B. A proteomic study by Du and co-workers (2011) ofrice leaf total proteins under early UV stress (at 8 h) found 22unique proteins by MALDI-TOF. This indicates how pro-teome analysis helps us discover the expression patterns ofUV-induced genes at the protein level. Recently, Tripathi andassociates (2011) observed several-fold reductions of majorleaf proteins, including Rubisco, in rice plants exposed to UV-B.For the O3 effect, Sarkar and Agrawal (2010) observed highprotein loss in rice plants grown in open-top chambers withelevated O3 levels. Feng and associates (2008) found a sig-nificant loss of photosynthetic proteins, including Rubisco, inrice plants exposed to O3. Besides the induction of stress-related proteins like APX and SOD, the induction of class 5proteins (PR5) and pathogenesis-related (PR) and PR10 pro-teins was also observed.

Phosphoproteomics

Protein phosphorylation comprises a major area of post-translational modifications, especially in plants that mobilizea high number of genes. Many crucial physiological andbiochemical functions, including responses to environmentalstimuli and diseases that contribute to the complexity of theproteome, comprise part of phosphoproteomics, and play arole in the identification of phosphoproteins, quantification ofphosphorylation sites, and the precise mapping of proteinsand their biological utility. Recently several plants have beenanalyzed using systemic phosphoproteomics, and this aidedin the optimization of in vitro and in vivo technologies to re-veal components of the phosphoproteome within the cell/organelle (Nakagami et al., 2010, 2012). The development of anew phosphoproteomic tool, the technically improved MS-based identification of phosphorylated residues in proteins, ishighly sensitive and accurate. Mass spectrometers in combi-nation with phosphopeptide enrichment methods, mainlyimmobilized metal affinity purification (IMAC) and titaniumdioxide (TiO2), are involved. This has led to the discovery ofnovel substrates that are specific for protein kinases. Thesemethods have enabled rapid identification of kinase sub-strates, such as kinase assays, using plant protein microarrays.Studies have shown that phosphorylation events are involvedin a wide range of biological processes taking place in plantsand other organisms (Sugiyama et al., 2008). To understandthe core regulatory systems, the characterization of conservedphosphoproteome features in plants is useful. Hence phos-phoproteomics can promote the improvement of agronomically-important plant species. Recent research projects inphosphoproteomics technology have led to the identifica-tion of thousands of phosphorylation sites in unfractionatedplant cells by simple enrichment methods. Sugiyama and

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colleagues (2008), using phosphoproteomics, reported morethan 2000 phosphorylation sites in Arabidopsis. Research onthe Medicago phosphoproteome has shed light on comparablephosphoproteomes from other plant species as well (Kerstenet al., 2009). Progress in the quantitative and dynamic analysisof mapped phosphorylation sites is also occurring. A newapproach, shortgun proteomics, is emerging for proteomeanalysis. Shortgun proteomics is useful for the identificationof thousands of proteins simultaneously from a complexsample (Nakagami et al., 2012). Continuing work on phos-phorylation sites in plants has prompted the creation of webresources for plant-specific phosphoproteomics data. In theera of computational biology, there is a need to significantlyimprove the sensitivity and specificity of the detection ofphosphorylation sites in plants (Grimsrud et al., 2010).

Conclusions

Tremendous progress in the field of plant proteomics hasbeen made in recent years. Studying the dynamic plant pro-teome has become comparatively simple and accurate withthe advancements seen in technology and bioinformatic tools.With these developments plant physiologists are now able tosolve mysteries about the physiological processes that takeplace inside plants. From seed germination to grain harvest,there has been tremendous growth in proteomics researcharound the world. Knowledge of the proteins involved innutritional and other plant processes have widened ourunderstanding of various metabolic processes using theproteomics approach. One remarkable achievement of pro-teomics is the discovery of the functions of various proteinsand their expression levels in plants undergoing various typesof stress. The proteomic strategy involved in the study ofprotein localization in cells is a necessary first step towardunderstanding protein functions in complex cellular net-works. These fascinating events taking place during themetabolism that governs various physiological processeshave been revealed by the dynamic nature of the proteome,and we believe that proteomics represents an importantadvance in the study of plant physiological processes.

Future Perspectives

Developments in proteomic technologies may aid re-searchers in understanding the plant engineering involved inimportant processes like nutrition, crop yields, and defense.Proteomics applications and advancements in technologysuch as multidimensional protein fractionation, isobaric tagsfor relative and absolute quantitation, label-free quantifica-tion mass spectrometry, and phosphoprotein and glycopro-tein enrichment and tagging, will enable the discovery ofproteins and novel regulatory mechanisms that occur duringsalt stress signalling and related metabolic pathways. Theintegration of proteomics with transcriptomics, metabo-lomics, and bioinformatics, has facilitated insights into themolecular networks underlying salt stress response and tol-erance. These data will be used to analyze how differentcomponents interact, and generate responses to control di-vergent metabolic pathways. Thus, the proteomics commu-nity needs to work in concert with those working intranscriptomics, metabolomics, and bioinformatics, to revealthe mechanisms affecting plant growth and the stressresponse.

Author Disclosure Statement

The authors declare that no conflicting financial interestsexist.

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Address correspondence to:Parvaiz Ahmad

Department of BotanyAmar Singh College

190008, University of KashmirSrinagar, India

E-mail: parvaizbot@yahoo.com

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