Soybean Proteomics for Unraveling Abiotic Stress ... - ACS Publications

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Soybean Proteomics for Unraveling Abiotic Stress Response Mechanism Zahed Hossain,*,† Amana Khatoon,‡ and Setsuko Komatsu*,§ †

Plant Stress Biology Lab, Department of Botany, West Bengal State University, Kolkata 700126, West Bengal, India Department of Plant Sciences, Kohat University of Science and Technology, Kohat 26000, Khyber Pakhtunkhwa, Pakistan § National Institute of Crop Science, National Agriculture and Food Research Organization, Tsukuba 305-8518, Japan ‡

ABSTRACT: Plant response to abiotic stresses depends upon the fast activation of molecular cascades involving stress perception, signal transduction, changes in gene and protein expression and post-translational modification of stress-induced proteins. Legumes are extremely sensitive to flooding, drought, salinity and heavy metal stresses, and soybean is not an exception of that. Invention of immobilized pH gradient strips followed by advancement in mass spectrometry has made proteomics a fast, sensitive and reliable technique for separation, identification and characterization of stress-induced proteins. As the functional translated portion of the genome plays an essential role in plant stress response, proteomic studies provide us a finer picture of protein networks and metabolic pathways primarily involved in stress tolerance mechanism. Identifying master regulator proteins that play key roles in the abiotic stress response pathway is fundamental in providing opportunities for developing genetically engineered stress-tolerant crop plants. This review highlights recent contributions in the field of soybean biology to comprehend the complex mechanism of abiotic stress acclimation. Furthermore, strengths and weaknesses of different proteomic methodologies of extracting complete proteome and challenges and future prospects of soybean proteome study both at organ and whole plant levels are discussed in detail to get new insights into the plant abiotic stress response mechanism.

KEYWORDS: soybean, proteomics, abiotic stresses, methodology, review

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INTRODUCTION

fundamental step in developing genetically engineered stresstolerant plants. Soybean, the world’s most widely grown seed legume, provides an inexpensive source of protein and vegetable oil for human consumption. This important legume crop is adapted to grow in a wide range of climatic conditions; however, growth, development and yield of soybean are greatly affected by several abiotic stressors, such as flooding,4,5 drought,6 salinity7 and heavy metal cadmium.8 Although these harsh environmental conditions affect soybean plant growth throughout the different developmental stages starting from seed germination to flowering, seedling stage is more prone to the abiotic stresses, in particular flooding and drought. In addition to morphological and physiological responses of soybean, several molecular studies elucidating stress-induced changes in gene expressions and metabolites have been documented.9 However, genomic studies only highlight

Ever-changing environmental conditions impair cell metabolism, which in turn affects plant growth and productivity. Climate model forecast has predicted drastic changes in global surface temperature leading to changes in rainfall patterns that in turn would impose serious threat to plant vegetation worldwide.1 Of the total loss in yield potential annually, 70% has been counted due to imbalance in physiochemical environments.2 Plants have developed sophisticated defense mechanisms to perceive and respond to various abiotic stresses so that they can cope with the harsh environmental conditions.3 To elucidate the stress response mechanism, the slightest changes in gene and protein expression need to be studied with precision. High-throughput genome-based OMICS techniques have been used extensively to dissect plants’ stress responsive pathways. Nevertheless, due to lack of correlation between the expression of mRNAs and the abundance of their corresponding proteins, proteomic techniques provide one of the best options for the functional analysis of translated regions of the genome. Identifying master regulator proteins that play essential roles in plant stress regulatory pathways is thus a © XXXX American Chemical Society

Special Issue: Agricultural and Environmental Proteomics Received: June 25, 2013

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soybean proteome study both at organ and whole plant levels are discussed in detail to comprehend the underlying mechanism of abiotic stress acclimation.

mRNA level, which may not be necessarily translated into the final gene products, i.e., proteins. There is a loose correlation between mRNA and protein levels, especially for organelle genes. Moreover, several proteins undergo post-translational modifications (PTM) such as removal of signal peptides, phosphorylation and glycosylation that are extremely important for protein function. Thus, genomic data does not always represent a true picture of the plant stress adaptation process. Recent advancements in the field of proteomics offer a better opportunity for dissecting quantitative traits in a more comprehensive and meaningful way. Proteomics provide a powerful tool for the analysis of molecular mechanisms of plant responses against stresses, and it provides a path for linking gene expression to cell metabolism. In soybean, various methods have been developed for extracting and identifying the proteins from various tissues and organelles.10−14 These methods have overcome various shortcomings in extraction and purification of proteins free of carbohydrates and phenolic compounds that render the purification process more complicated. Stress response of soybean organs depends upon the severity, duration and type of stress that lead to various changes at the proteome level. The nature and intensity of responses may vary depending on the stress; however, few common defense mechanisms are found to be activated irrespective of the type of stresses. For instance, higher abundances of antioxidant proteins have been documented irrespective of type of abiotic stresses, which indicates stress-induced higher reactive oxygen species (ROS) production with concomitant oxidative burst.15 Plants are equipped with a robust antioxidative defense system that scavenges excess ROS, thus protecting vital cellular components from oxidative damage.3 Furthermore, abundance and oxidation state of redox-sensitive multiple proteins across the range of biochemical pathways, including redox systems, carbon metabolism, photosynthesis, signaling and homeostasis systems, and amino acid metabolism, were found to be altered to cope with the stress. The applications of proteomic techniques in dissecting molecular mechanism have been validated at both cellular and subcellular levels against various abiotic stresses.3,4 The information gathered from recent proteomics research has covered all aspects of plant response ranging from seed germination,16 seedling stage5,8,15 and later stages of growth.17 They inferred that the benefits derived from all these versatile research approaches can be optimized by integrating function and interactions of proteins and the understanding from this research might be a key for the generalized application of response mechanism in soybean to other plants as well. Proteomic approaches can be utilized to ascertain target enzymes and proteins responsible for important crop traits that could be utilized to enhance the natural tolerance against abiotic stress in different crop varieties. The functional analysis of stressresponsive proteins provides a clear insight into the complex mechanisms involved in the response of plants to stress. Keeping in view all these facts, a comprehensive account of this research is needed as a baseline strategy for developing stresstolerant plants. This review summarizes this targeted research on soybean with the aim to broaden the application spectra of this research to other plants to provide a clear insight into the complex mechanisms involved in the stress response mechanisms of plants to abiotic stresses. Furthermore, strengths and weaknesses of different proteomic methodologies of extracting complete proteome and challenges and future prospects of

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SAMPLE PREPARATION AND SEPARATION TECHNIQUE Extraction of proteins from various organs is one of the most crucial and challenging steps to have a clear picture of complete proteome profile or any minute changes in protein abundance in response to abiotic stresses, as the amount and quality of the extracted proteins ultimately determine the protein spot number, resolution and intensity. The method for protein extraction is highly dependent on the type of the plant species, tissues, organs, and the nature of desired proteins to be extracted (Table 1). Various interfering substances, such as phenolic compounds, proteolytic and oxidative enzymes, terpenes, organic acids, and carbohydrates offer more complications in the process of protein extraction, resulting in inferior results such as proteolytic breakdown, streaking and charge heterogeneity.32 A successful extraction method from a given sample would be capable of reproducibly capturing and solubilizing the full complement of proteins. In addition, it should meet the challenges offered by postextraction artifact formation, proteolytic degradation, and nonproteinaceous contaminants.33 The different protocols of protein extraction from diverse soybean tissues highlighted in this review mostly used trichloroacetic acid (TCA)/acetone or phenol-based method for analysis of stress responsive proteins. Nevertheless, different solubilization/lysis buffers with varying chemical compositions and concentrations have been used. Rapid and uniform homogenizations of pant tissue and protein pellets in lysis buffer are important steps in every proteomic study. Usually, the initial tissue disruption and final pellet homogenization are based on manual, laborious, and uncontrolled tissue grinding with an extraction buffer/liquid nitrogen or pellet homogenization using a suitable lysis buffer. In recent time, introduction of a high-performance, single-tube sample preparation device has enabled noncontact tissue disruption and protein pellet homogenization, thereby avoiding contamination and degradation. Toorchi et al.34 compared this advanced acoustic technology using Covaris device with the conventional mortar/pestle and vortex/ultrasonic methods for high-performance disruption and extraction of proteins from different tissue samples of soybean and rice using two types of lysis buffer, O- and T-buffer. With respect to the total numbers of detected protein spots on 2-DE gels, significant differences between these three methods were recorded. Despite a greater number of protein spots, the Covaris instrument results in a clearer protein pattern than the other conventional methods. This technology performed far better than water bath sonication by producing high-quality 2-DE gels and minimizing the processing time required for high-throughput proteomics research. As compared to O-buffer [8 M urea, 2% Nonidet P-40, 2% ampholine (pH 3.5−10.0), 5% 2-ME, and 5% polyvinylpyrrolidone (PVP)-40], T-buffer [7 M urea, 0.2 M thiourea, 0.2 mM tributylphosphine (TBP), 0.4% CHAPS, 0.2% ampholytes (pH 3−10), and 5% PVP-40] was found to be more effective in producing high-quality 2-DE pattern particularly for pure protein extracts such as plasma membrane purified protein in soybean.34 It has been reported that TBP enhances the protein solubility during IEF, resulting in better focusing and resolution.35 B

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C

Osmotic stress

Enrei Harosoy Naviko

100 μM CdCl2, 3 d

50 μM Al3+, 24−72 h 37, 58, 116 ppb BaXi 10 Pioneer 93B15

Harosoy Fukuyutaka

100 μM CdCl2, 3 d

Cadmium

Aluminum Ozone

Aldana

Cold: +10 °C/water, osmotic: +25 °C /−0.2 MPa, cold +osmotic: +10 °C/−0.2 MPa

Osmotic, Cold, Osmotic + cold stress

3−10 μM Cd2+, 24, 48, 72 h

Enrei

0, 5, 10, 20% PEG 1, 4 d

Osmotic stress

Enrei

Taegwang

Withholding water, 5 d, rewatering, 4 d 10% PEG, 1, 2, 3, 4 d

Enrei

1d

Enrei

Enrei

12−48 h

Stop watering 10% PEG, 4 d

Asoagari

3, 7 d

Taegwang

Enrei

1−4 d

Flooded with 100 mM NaCl, 2 d

Enrei

2d

Enrei

Enrei

2d

3, 6 d

Enrei

cultivar

5d

treatment time and dose

Flooding Low oxygen Flooding Salinity Drought

Flooding

stress

Root microsome Cell Suspension Root Leaf Root

Leaf Root

Root

Hypocotyl Root PM Root

Root

Hypocotyl Root Leaf Hypocotyl Root

Root

Hypocotyl Root PM

Hypocotyl Root

Hypocotyl Root Root

Hypocotyl Root MT Hypocotyl Root cell wall

Leaf Hypocotyl Root

organ/ organelle

IPG, 2-DE, MALDI-TOF MS IEF gel strip, SDS-PAGE, nanoLC−MS/MS

IPG, SDS-PAGE, nanoLC− MS/MS SDS-PAGE/Q-TOF MS

IPG, 2-DE, nanoLC−MS/MS, MALDI-TOF MS

IPG, SDS-PAGE, MALDI-TOF MS IEF tube gel, SDS-PAGE, LC− MS/MS, nanoLC−MS/MS IEF tube gel, SDS-PAGE, MALDI-TOF MS, protein sequencing IPG, 2-DE, LC/nanoESI-MS

IPG, SDS-PAGE, BN-PAGE, nanoLC−MS/MS IEF, SDS-PAGE, MALDI-TOF MS, nanoLC−MS/MS, protein sequencing IEF/IPG, SDS-PAGE, MALDITOF MS, protein sequencing IPG, SDS-PAGE, MALDI-TOF MS, ESI-MS/MS IEF/IPG tube gel, 2-DE, MALDI-TOF MS, nanoLC− MS/MS, protein sequencing IEF tube gel, 2-DE, MALDITOF MS, nanoLC−MS/MS, protein sequencing IEF, SDS-PAGE, MALDI-TOF MS, nanoLC−MS/MS IPG, SDS-PAGE, MALDI-TOF MS IPG, SDS-PAGE, nanoLC− MS/MS

IEF, SDS-PAGE, nanoLC−MS/ MS

proteomic methodologies

Table 1. Summary of Soybean Proteome Analyses in Response to Abiotic Stressesa

Flavonoid biosynthesis; antioxidant defense Energy (glycolysis, TCA cycle, oxidative phos phorylation); photosynthesis; antioxidant defense; flavonoid biosynthesis; amino acid and nucleotide metabolism

Energy (glycolysis, TCA cycle); antioxidant defense; glycoprotein biosynthesis; amino acid synthesis Energy (glycolysis, TCA cycle, oxidative phos phorylation); antioxidant defense; fatty acid metabolism; antioxidant defense; amino acid and nucleotide metabolism Energy (glycolysis, TCA cycle, oxidative phosphorylation); photosynthesis; antioxidant defense; glycoprotein biosynthesis; amino acid synthesis Energy (fermentation); antioxidant defense; amino acid synthesis Amino acid synthesis; flavonoid biosynthesis

Energy (glycolysis, pyruvate decarboxylation, fermentation) Energy (glycolysis, fermentation); photosynthesis; glycoprotein biosynthesis Energy (glycolysis, TCA cycle, oxidative phos phorylation); photosynthesis; antioxidant defense; glycoprotein biosynthesis; amino acid synthesis Antioxidant defense; amino acid synthesis; flavonoid biosynthesis Antioxidant defense; glycoprotein biosynthesis

Antioxidant defense

Energy (glycolysis, fermentation); glycoprotein biosynthesis Energy (glycolysis, fermentation); amino acid synthesis Energy (glycolysis, pyruvate decarboxylation, fermentation); antioxidant defense

Energy (glycolysis, pyruvate decarboxylation, TCA cycle, oxidative phosphorylation) Lignin biosynthesis; amino acid synthesis

Energy (glycolysis, pyruvate decarboxylation, TCA cycle); amino acid synthesis; flavonoid biosynthesis

stress-induced modulation of major metabolic pathways

Ene, DisDef, Sgnl, Trans Ene, Met

DisDef

Met, Ene

Met, Ene, DisDef, ProtDesSt

GrDev, TrStr, Ene, DisDef, Secmet, Trans

Met, Ene, Sgnl, DisDef, CellSt Sgnl, Met, ProtSyn, DisDef, Trans DisDef, Ene, ProtDesSt, Met, CellSt, Secmet

ProtDesSt, ProtSyn, DisDef, CellDiv, Trans, Pmet, Ene, Secmet, Sgnl Met, Ene, ProtDesSt, Sgnl, ProtSyn, DisDef Met, Ene, ProtDesSt, DisDef Met, Ene, ProtSyn, DisDef

Ene, DisDef, Pmet, CellSt, Secmet, Sgnl

ProtDesSt, DisDef, Ene, Pmet, CellSt, Trans Met, Ene, DisDef, ProtSyn

Met, ProtDesSt, DisDef

Ene, DisDef

Met, Ene, ProtDesSt, DisDef, ProtSyn

function

18 9

10

− − −

21

−

24 25 26

− −

23

− −

8

16

Chlo, Cyto, Mito, Vacu

−

22

17

−

−

6

Chlo, Cyto, Nucl, Mito

20

19

12

−

Cyto, Chlo, Nucl −

11

13

5

ref.

Sec

Mito, Nucl, Cyto, Extr, ER, Cysk, PM Mito, Chlo

localization

differentially expressed protein classification

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Abbreviations: IPG, immobilized pH gradient; IEF, isoelectric focusing; LC, liquid chromatography; MS, mass spectrometry; PM, plasma membrane; TCA, trichloroacetic acid. Functional classification: Met, metabolism; Ene, energy; ProtDesSt, protein destination/storage; ProtSyn, protein synthesis; DisDef, disease/defence; CellDiv, cell division; Trans, transporter; Pmet, primary metabolism; Secmet, secondary metabolism; CellSt, cell structure; Sgnl, signal transduction; GrDev, growth and development; TrStr, translocation and storage. Subcellular localization: Chlo, chloroplast; Mito, mitochondria; PM, plasma membrane; Nucl, nuclear; Cyto, cytoplasm; Extr, extracellular matrix; ER, endoplasmic reticulum; Cysk, cytoskeleton; Vacu, vacuolar; Sec, secretory pathway.

Leaf Stem Root Enrei Heat

40 °C, 6−24 h

Hypocotyl Root Enrei 50−200 mM NaCl, 1−4 d

20−80 mM NaCl, 7 d

Efforts have been made to standardize sample preparation protocols to get optimized yield. Instead of having physicochemical limitations of each and every protocol, the TCA/acetone precipitation and phenol extraction methanol/ammonium acetate precipitation methods are most used as standard methods for removing interfering substances to obtain high quality gels.36 Isolation of both abundant and less abundant proteins from soybean seeds using conventional extraction methods is more challenging, as the seeds contain large proportion of abundant storage proteins that account for about 70−80% of the total protein. Presence of abundant storage proteins, such as β-conglycinin and glycinin, often hinders the isolation and characterization of less abundant seed proteins. Sample fractionation technique has proved to be an efficient strategy for successful removal of such high-abundance storage proteins. With the simple addition of 10 mM calcium chloride to the salt soluble soybean seed protein extract in low ionic strength buffer, the α, α′, and β subunits of β-conglycinin and the acidic and basic subunits of glycinin were found to be reduced significantly from the total protein extract.37 This simple, fast and inexpensive method effectively removed 87% of the highly abundant seed proteins from the extract, allowing easy detection of 541 previously inconspicuous proteins present in soybean seed after SYPRO Ruby in-gel fluorescent protein staining. Furthermore, this fractionation technique allowed detection of 63 new phosphorylated protein spots and enhanced the visibility of 15 phosphorylated protein spots, using 2-DE separation and ProQ Diamond in-gel phosphorylated protein staining. Similarly, isopropanol extraction was found to be an efficient method that facilitates resolution of lowabundance proteins.38 Analysis of 2-DE revealed that proteins that were less abundant or found absent in the conventional extraction procedure were clearly visible in the isopropanol extract. The authors claim that extraction of soybean seed powder with 40% isopropanol enriches lower abundance proteins of legume seeds and is a suitable method for 2-DE separation and identification. Earlier, the same group39 compared four different protein extraction/solubilization methods, urea, thiourea/urea, phenol, and a modified TCA/acetone, to determine their effectiveness in separating soybean seed proteins by 2-DE. The thiourea/urea and TCA methods were found to be more suitable in resolving less abundant and high molecular weight proteins. In addition, these two methods exhibited higher protein resolution and spot intensity as compared to rest of the methods. Later, Ahsan and Komatsu40 evaluated three different protein extraction protocols, TCA precipitation,34 phenol extraction method41 with modifications and direct tissue homogenizing in suitable protein solubilization buffers, to compare soybean leaf and flower proteomes at different developmental stages. To optimize protein pellet solubilization buffer, A-buffer containing 8 M urea, 2% Nonidet P-40, 2% ampholine (pH 3.5−10), 5% 2-mercaptoethanol, and 5% PVP- 40; B-buffer34 containing 7 M urea, 0.2 M thiourea, 0.2 mM TBP, 0.4% CHAPS, 5% PVP-40, and 2% ampholine (pH 3−10); and C-buffer containing 8.5 M urea, 2.5 M thiourea, 5% CHAPS, 1% dithiothreitol (DTT), 1% Triton X-100, and 0.5% ampholines (pH 3−10 and 5−8) were tested. Combination of the phenol-based method with C-solubilization buffer generated high quality proteome maps in terms of wellseparated resolved spots, spot intensity, and the number of proteins in the 2-DE gels with no horizontal streaking and high background noise levels. The authors claim that this optimized protein extraction protocol is equally efficient for all other

a

31 − DisDef, Met, Ene, ProtSyn, Secmet Energy (glycolysis, TCA cycle, fermentation); photosynthesis; antioxidant defense; flavonoid biosynthesis; amino acid metabolism

30 − ProtDesSt, DisDef

7 −

Energy (glycolysis); photosynthesis; antioxidant defense; glycoprotein biosynthesis; amino acid synthesis Glycoprotein biosynthesis; antioxidant defense

Met, Ene, ProtDesSt, ProtSyn, DisDef

29 − Energy (glycolysis); antioxidant defense

IPG, SDS-PAGE, MALDI-TOF MS IEF tube gel, SDS-PAGE, MALDI-TOF MS, protein sequencing IEF tube gel, SDS-PAGE, ESI-Q/ TOF-MS/MS, protein sequencing IPG, 2-DE, MALDI-TOF MS, nanoLC−MS/MS 100 mM NaCl, 12 d Salinity

Germinating seed Leaf Hypocotyl Root

Clark: Standard, magenta Lee 68 N2899 Enrei Natural levels of UV−B UV−B

Leaf

Met, Ene, DisDef

28 − Photosynthesis; Energy (glycolysis); flavonoid biosynthesis; antioxidant defense

Ene, Met, DisDef

27

Reviews

Chlo

localization function

Met, Ene, ProtSyn, DisDef Photosynthesis; energy (glycolysis, TCA cycle); antioxidant defense; amino acid synthesis

IEF tube gel, SDS-PAGE, MALDI-TOF MS, protein sequencing IPG, SDS-PAGE, MALDI-TOF MS Leaf chloroplast Enrei 120 ppb, 3 d

treatment time and dose stress

Table 1. continued

cultivar

organ/ organelle

proteomic methodologies

stress-induced modulation of major metabolic pathways

differentially expressed protein classification

ref.

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Figure 1. Differential modulations of various cellular pathways under abiotic stresses. The scheme is based on the published proteomic works on different abiotic stress mediated changes in soybean proteome. Up- and down-regulation of proteins are represented by respective contrasting colored boxes. Abbreviations: ABP, ATP binding protein; ADH; alcohol dehydrogenase; AH, aconitate hydratase; ALD, aldolase; APX, ascorbate peroxidase; CaM, calmodulin; CAT, catalase; CIS, citrate synthase; CS, cysteine synthase; DHAR, dehydroascorbate reductase; ENO, enolase; FR, fumarase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; G6PI, glucose-6-phosphate isomerase; Gly-I, glyoxalase I; GPx, glutathione peroxidase; GR, glutathione reductase; GS, glutamine synthetase; GSH, reduced glutathione; GST, glutathione-S-transferase; HM, heavy metal; Hsp, heat shock proteins; IDH, isocitrate dehydrogenase; JA, jasmonic acid; LOX, lipoxygenase; LSU, large subunit; MDAR, monodehydroascorbate reductase; MD, malate dehydrogenase; MTs, metallothioneins; MS, methionine synthase; MG, methylglyoxal; OEE, oxygen-evolving enhancer protein; PR, pathogenesis-related; PC, phytochelatin; PD, pyruvate dehydrogenase; PDC, pyruvate decarboxylase; PFK, phosphofructokinase; PGK, phosphoglycerate kinase; PHGDH, 3-phosphoglycerate dehydrogenase; PHP, 3-phosphohydroxypyruvate; PK, pyruvate kinase; POD, peroxidase; PS, proteasome subunit; PS I, photosystem I; Prx, peroxidoxin; ROS, reactive oxygen species; RuBisCO, ribulose-1,5-bisphosphate carboxylase oxygenase; SAMS, S-adenosylmethionine synthetase; SAM, S-adenosylmethionine; SD, succinate dehydrogenase; SSU, small subunit; TPI, triosephosphate isomerase; Trx, thioredoxin.

buffer, reduced IEF running temperatures, and replacement of fresh wicks during IEF are some of the key steps that allow construction of high resolution soybean protein map. Total leaf protein extraction from the young soybean seedlings and subsequent separation by 2-DE exploiting this modified method revealed a remarkable difference in the quality of separation and in the number of individual proteins present in the gels. As compared to the traditional method, significantly higher, well resolved and focused protein spots were detected on 2-DE gel, which renders this modified method as common protein extraction protocol for different organs/tissues of soybean.42

tissues and organs of soybean, including the hypocotyl, leaf, petiole, stem, flower bud, and flower. By the inclusion of many small changes in the traditional phenol-based protein extraction protocol, Sarma et al.42 succeeded in developing a modified method that not only improved the resolution and reproducibility of 2-DE but also shortened the time of analysis. Introduction of DTT at all steps in the procedure, use of phenol rather than TCA/acetone for protein extraction, application of acid-washed sand to disrupt the plant tissue, precipitation of the phenol extracts with methanol containing ammonium acetate for only 2 to 3 h but at −80 °C, further washing of the methanol/ammonium acetate pellets with 90% ethanol, use of a mixture of detergents at high concentrations and sonication to increase resolubilization of the protein, combination of DTT and 2-HED (2-hydroxyethyl disulfide) for thiol stabilization, addition of glycerol to IEF

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FLOODING-INDUCED CHANGES IN SOYBEAN PROTEOME COMPOSITION Soil flooding is an ever existing environmental constrain that negatively affects plant productivity of arable farmland. E

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Legumes are often sensitive to flooding, and soybean is not an exception of this trait. Progressive decreases in soil oxygen concentration and redox potential are major physiological consequences of submergence.43 To adapt oxygen-poor conditions, plants generally activate anaerobic pathways, generate ATP through glycolysis, and regenerate NAD+ through ethanol fermentation as initial responses by selectively synthesizing flooding-inducible proteins involved in sucrose breakdown, glycolysis, and fermentation.44 Protein ubiquitination, a critical post-translational regulatory mechanism, plays an important role in plant tolerance against various biotic and abiotic stresses including flooding.45 Ubiquitination of target proteins requires the sequential action of three different enzymes, namely, Ub-activating enzyme (E1), Ub-conjugating enzyme (E2 or UBC), and Ub ligase (E3).46 Usually, the target substrate protein is polyubiquitinated before being recognized and degraded by the 26S proteasome. It is believed that the Ub/proteasome-mediated proteolysis of enzymes involved in glycolysis and fermentation pathways may be negatively controlled under the hypoxic condition caused by flooding stress in soybean.4 Since the ring-type E3 ligase GmRFP1 is up-regulated by ABA treatment,47 it is likely that the negative regulator of Adh might be polyubiquitinated by GmRFP1 followed by its degradation by 26S proteasome. Gelbased and gel-free proteomic studies have also revealed differential regulation of 20S proteasome subunits in flooded soybean.48 Altered expression of each 20S proteasome subunit in response to flooding stress may thus affect the amount as well as the activity of the 26S proteasome, thereby altering flooding tolerance. Among the cell organelles, mitochondrion has been a target for subcellular proteomic study, as most of the abiotic stresses primarily impair mitochondrial electron transport chain resulting excess ROS generation. Proteomic techniques have been employed to elucidate the mitochondrial responses to environmental stresses such as salinity,49 drought,50 and chilling.51 Changes in the expression of mitochondrial genes and proteins have been studied in soybeans under flooding stress using proteome analyses.13 The expression of processing peptidase was found to be increased as a result of flooding, while ATPase beta subunit decreased. However, there is limited understanding of the role of mitochondrial proteins in sensing and initiating signaling in response to flooding. In separate proteomic-based flooding experiments, mitochondrial matrix and membrane proteins were separated by 2-DE and blue native-polyacrylamide gel electrophoresis (BN-PAGE), respectively, and differentially expressed proteins and metabolites of roots and hypocotyls of soybean experiencing flooding stress were identified using MS.13 Proteins and metabolites related to the tricarboxylic acid cycle and GABA shunt were found to be increased by flooding stress, while inner membrane carrier proteins and proteins related to complexes III, IV, and V of the electron transport chains were decreased. The authors observed that the amounts of NADH and NAD were increased; however, ATP was significantly decreased under flooding stress. These findings led them conclude that flooding stress directly impairs electron transport chains, although NADH production increases in the mitochondria through the tricarboxylic acid cycle. Plant cell wall plays essential role in stress sensing and signal transduction between the apoplast and symplast. Hence, in recent time cell wall proteome science has been the subject of intense research. Analysis of CaCl2-extracted cell wall proteins, purified from the roots during the early stages of soybean

seedling development, revealed that 16 proteins responded to flooding stress.11 Out of the differentially expressed proteins, two lipoxygenases, four germin-like protein precursors, three stem glycoprotein precursors, and one Cu−Zn superoxide dismutase (SOD) were reported to be decreased (Figure 1). In combination, these results suggest that under flooding stress, the roots-of soybean respond through signaling cascades that result in down-regulation of lignification caused by a decline in the reduction of hydrogen peroxide and ascorbate, or by the reduction in linking of polysaccharides. Change at the protein level is one of the main cellular responses required for perception of a stress signal and its transduction into the cell that mostly happens in the plasma membrane. It plays vital role in cell communication, as it acts as a primary interface between the cellular cytoplasm and the extracellular environment. To explore the alterations in the plasma membrane proteins of soybean exposed to flooding stress, plasma membrane was first purified from root and hypocotyl of soybean seedlings using an aqueous two-phase partitioning method and then analyzed using gel-based and gelfree proteomics techniques.10 It is presumed to be the first report of identifying flood-induced plasma membrane proteins in soybean exploring 2-DE MS/sequencer-based proteomics and nanoLC−MS/MS-based proteomics techniques. A total of 35 stress-induced novel proteins were identified, mostly associated with plants’ defense system. Among them, SOD was found to be remarkably up-regulated, suggesting that the antioxidative system may play a crucial role in protecting cells from oxidative damage following exposure to flooding stress. The SOD family of proteins are essential enzymes of ascorbate glutathione cycle and act as the first level of defense against ROS as it directly controls the concentrations of two important ROS, namely, superoxide radicals and hydrogen peroxide. The induction of SOD by anaerobic stress in plants has been previously reported.43 Furthermore, flood-induced higher accumulation of heat shock cognate 70 kDa protein might protect proteins from denaturation and degradation during flooding stress. One of the early responses to flooding stress appears to involve closing of stomata to avoid water loss, with a subsequent down-regulation of the photosynthetic machinery.52 Floodinduced down-regulation of proteins involved in Calvin cycle and photosynthetic electron transport chain markedly influence photosynthetic efficiency. Leaf proteome analysis of young soybean seedlings revealed that most of the chloroplastic and metabolism associated proteins were decreased. In addition, low abundances of proteins associated with plant defense (SOD, CAT) were documented in flooded soybean (Figure 1).19 Among the reduced metabolism-related proteins, isoflavone reductase, involved in the biosynthesis of plant defense metabolites such as lignins and isoflavonoids, plays an important role in mitigating oxidative injuries.53 Low expression of chlorophyll a-b binding protein might be another decisive factor for reduced photosynthetic activity. Overall, decreased synthesis of photosynthetic assimilates with a concomitant breakdown of effective ROS scavenging system lead to suppression of seedling growth under flooding. In plants, the root is the immediate responding organ where stress signals of flooding are perceived and transmitted, thereafter leading to various cellular and molecular level changes. Proteomics of soybean root under flooding stress have been well analyzed with great focus.5,11−15,19,48 Marked changes in protein abundance have been reported in soybean roots even F

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after a very brief exposure (12 h) of flooding stress.10 Root proteome of 2-day-old soybeans exposed to waterlogging revealed that glycolysis-related proteins, including UDP-glucose pyrophosphorylase and fructose-bisphosphate aldolase, and disease/ defense-related proteins such as ROS scavengers, chaperones, hemoglobin, and/or acid phosphatase, were highly affected.10,48 Separate proteomic study of flooded soybean roots has shown a remarkable increase in the expression of alcohol dehydrogenase (Adh), the key enzyme of alcohol fermentation pathway, at an early growth stage (Figure 1).14 Nevertheless, significant expression of Adh2 gene was not observed under other abiotic stresses. Flood specific accumulation of Adh2 in soybean root strongly indicates activation of ethanolic fermentative pathway to withstand the hypoxic condition. Interestingly, growth inhibition of soybean seedlings caused by flooding stress was found to be reduced in transgenic soybean overexpressing Adh2 gene.54 Recent proteomic study has shown that gamma irradiated soybean mutants exhibit better root growth even under flooded condition.15 As compared to wild type, an increased number of fermentation-related proteins including different types of ADHs and pyruvate decarboxylase isozymes 1 and 2 were identified in the mutant on exposure to flooding stress. In addition, abundances of cell wall loosening-related proteins were not found to increase under flooding stress, thereby preserving the viability of the root tip and permitting rapid growth after withdraw of stress. This finding further supports the idea that ADH is strongly involved in flooding tolerance in soybean. Proteomic changes involved in plant recovery from flooding provide essential clues in better understanding the flooding injury. Salavati et al.55 reported that the proteins associated with cell wall modification and S-adenosylmethionine synthesis were involved in recovery mechanism. Response mechanism of soybean root against flooding stress at late growth stages has been studied in depth.18 On exposure to short-term submergence, root proteome profile of young seedlings exhibited higher expression of proteins primarily associated with glycolysis and fermentation pathways. This finding suggests that soybean seedlings respond similarly to flooding stress during early and late growth stages. An analysis of the carbohydrate contents and measurements of enzyme activities in flooded soybean confirmed that activation of glycolysis and decreased expression of sucrose-degrading enzymes caused an accumulation of sucrose in flooded seedlings.48 Expression of ROS scavenger proteins, peroxidases, cytosolic ascorbic peroxidase (cAPX) and SOD was reported to be decreased during flooding (Figure 1). Proteomic screening of six different soybean cultivars revealed significant down-regulation of (cAPX 2) protein under flooding condition.56 Northern-hybridization further confirmed that the abundance of cAPX 2 transcript decreased significantly after flooding, as did the enzymatic activity of APX. This finding indicates that APX plays a key role in the flood tolerance mechanism of soybean. In contrast, flooding-induced increase in APX activity was documented in other crops.43,57 This divergent behavior of APX implies crop-specific modulation of antioxidant enzymes in response to flooding. Most of the proteomic investigations done so far primarily highlighted the overall response of soybean root against flooding. There have been reports of predominant proteins involved in stress response, glycolysis, redox homeostasis, and protein processing located in differentiated root zones including root apex with different abundances.58 Root tip cell death with a concomitant suppression of root elongation has been reported

in different crops.59,60 Using gel-free MS-based quantitative proteomics and phosphoproteomics approaches, Nanjo et al.59 studied the altered protein expression profiles of soybean root tips under flooding stress. The protein categorization revealed that a majority of the proteins involved in glycolysis, fermentation, cell wall metabolism and nucleotide metabolism were up-regulated, while the expression of most of the proteins involved in amino acid metabolism and cell organization were down-regulated. On exposure to flooding, certain proteins like sucrose binding protein, phosphatidylinositol-4-phosphate 5-kinases, actins, and alpha-tubulins were reported to express specifically in root tip region. The higher abundance of sucrose binding protein might facilitate the active transport of sucrose in flooded soybean seedlings.59 Taken together, all these proteomic findings suggest that metabolic adjustment for the management of energy consumption was the key adaptive response in flooded roots and also that the regulation of defense systems is involved in adapting stressed conditions.

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MODULATION OF PROTEOME COMPOSITION IN SOYBEAN UNDER DROUGHT AND SALINITY Among the various abiotic stress factors, drought and salinity are the two adverse environmental conditions that negatively affect growth and productivity of major crops including soybean.3,7 The effects of drought stress on soybean have been investigated extensively exploiting physiological, biochemical and genomic approaches. Nevertheless, response of soybean to drought has not been well documented at the protein level. In recent time, Mohammadi et al.6 performed organ-specific proteomic analysis of drought-stressed soybean seedlings. In addition, response of soybean to PEG-induced osmotic stress was compared with drought at protein level. The functional categorization revealed that most of the droughtresponsive proteins were chiefly involved in redox regulation, oxidative stress response, signal transduction, protein folding, secondary metabolism, and photosynthesis. Root was found to be the most drought-responsive organ showing maximum changes in protein abundance in response to stress. Expressions of metabolism-related proteins were shown to be increased in leaves of both PEG-treated and drought-stressed seedlings, while proteins related to energy production and protein synthesis were decreased. In contrast to waterlogging stress, increased APX abundance was evident in drought-stressed soybeans (Figure 1). Proteome analysis of soybean roots exposed to short-term drought stress revealed differential protein abundances, mostly involved in various cellar functions including carbohydrate and nitrogen metabolism, cell wall modification, signal transduction, cell defense and programmed cell death.17 Moreover, ferritin and dehydrin were found to be newly induced under drought condition. Ferritin plays important role in sequestering highly reactive intracellular iron and thus reduces the formation of toxic hydroxyl radicals. Similarly, dehydrin has some positive function in abiotic stress tolerance by minimizing the negative effects of ROS.61 The authors claimed that novel accumulation of dehydrin and ferritin might play a crucial role in minimizing free iron and ROS level in drought-stressed soybean roots. Higher accumulation of H2O2 and increased TBARS levels in roots of drought-stressed soybean were also indicative of higher membrane damage. Interestingly, endogenous free proline level was found to be in accordance with stress level, while during the recovery period, proline content was decreased up to the G

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channels.68 Often, lack of specificity of transporter proteins that are primarily involved in uptake of essential elements allows the entry of HM ions.69 Within a cell, HMs disrupt normal cellular functions by a wide range of actions including binding of HM ions to protein sulfhydryl groups, replacement of essential cations from specific binding sites, leading to enzyme inactivation and ROS accumulation, which in turn results in oxidative damage to lipids, proteins and nucleic acids.70 To protect cells from the adverse affects of HMs, Plants have adopted coordinated complex detoxification mechanisms to regulate the uptake, mobilization, and intracellular concentration of HMs. One of the important strategies to detoxify HMs within the plant cell is to synthesize specific low molecular weight chelators to minimize the bindings of metal ions to functionally important proteins.71 In addition, plasma membrane exclusion method and synthesis of membrane transporters for vacuolar sequestration are the two common ways to protect the cell from the adverse effects of HMs. Mass spectrometric approach has been well exploited in addressing HMs stress response mechanism in soybean. Comparative proteomic studies of high and low Cd accumulating soybeans by our group have revealed enhanced expressions of glutamine synthetase (GS) and metabolites associated with glutathione biosynthesis (glutamine and glycine) under Cd stress (Figure 1).8,23,72 The enhanced expression of GS coupled with higher accumulation of glutamine and glycine lead to more GSH formation. Induction of GSH synthesis implies higher metal binding capacity as well as enhanced cellular defense mechanism against oxidative stress.71 Since GSH is the precursor of PC, enhanced expression of GS might help the Cd-stressed soybean cells to synthesize and accumulate more PC to detoxify cytosolic Cd2+. Our findings are in agreement with previous reports of Cd-induced upregulation GSH biosynthesis in other crops.73,74 It is an interesting finding that soybean cultivars exhibit different levels of cadmium tolerance; nevertheless, they share this common strategy to immediately synthesize GSH and PCs to minimize cadmium-induced cellular damages. Up-regulation of cysteine synthase (CS) and GSH pool involved in Al adaptation has also been documented in preoteomic studies of Al-stressed soybean.25 In addition, proteins involved in lignin biosynthesis were shown to be increased in soybean under Cd-stress. Proteomic findings suggest that translocation of Cd2+ from the root to the aerial parts might be prevented by the increased xylem lignifications.23 Furthermore, increased abundance of defense proteins for effective ROS scavenging and molecular chaperones help HM-stressed plants to maintain redox homeostasis. Molecular chaperones/heat-shock proteins (HSPs) are responsible for protein stabilization, proper folding, assembly and translocation under both optimum and adverse growth conditions.75 More than 2-fold increased abundance of HSP70 protein was detected in leaves of high Cd-accumulating soybean cultivar Harosoy, while low Cd-accumulating cv. Fukuyutaka exhibited decreased expression (Figure 1).8 Al-stress also is known to induce one LMW-HSP and three DnaJ-like proteins in soybean.25 DnaJ-like proteins are molecular chaperones that regulate Hsp70 adenosine triphosphatase (ATPase) activity in protein folding and assembly and disassembly of protein complexes. This higher accumulation of HSPs/DnaJ-like proteins might help the HM-stressed soybean cells in re-establishing normal protein conformation and hence maintain cellular homeostasis. The excess intracellular ROS level, an inevitable outcome of HM stress, alters protein structure by inducing oxidation of

control level. Increased accumulation of proline might facilitate soybean root to cope with drought stress by mediating osmotic adjustment. In summary, proteins associated with osmotic adjustment, defense signaling and programmed cell death play important roles in soybean for adaptation to drought. Change in protein profile of PEG-induced osmotic stressed soybean was monitored by using 2-DE gel-based proteomic approach in order to elucidate the underlying osmotic stress mechanism.22 Identifications of differentially expressed proteins using Edman sequencing and peptide-mass fingerprinting method revealed decrease of caffeoyl-CoA 3-O-methyltransferase, Hsp-70, S-adenosylmethionine synthetase with concomitant increase of defense associated proteins. Caffeoyl-CoA-O-methyltransferase is an enzyme involved in lignification, and its repression in soybean root under osmotic stress infers to the reduction of lignin content as an adaptive response to osmotic stress. Plasma membrane proteome study by Nouri and Komatsu21 revealed up-regulation of calnexin protein in 2-day-old soybean seedlings under PEG treatment. In addition, three homologues of plasma membrane H+-ATPase, acting as transporter proteins involved in ion efflux, were found to be up-regulated both at transcript and protein levels under osmotic stress. These observations imply that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux is accelerated by up-regulation of plasma membrane bound H+ATPase protein. Like drought, soil salinity also exerts osmotic stress.62 Genomic techniques are being successfully employed for screening and developing salt-tolerant crops.63,64 As compared to flooding, limited information about the changes in proteome composition under salt stress are available in soybean.7,29,30 Similar to drought stress, a positive correlation between proline content and salt stress tolerance was observed in a wide range of plant species.65 Proteome analysis of soybean hypocotyl and root under salt stress revealed concentration-dependent accumulation of free proline under NaCl stress.30 A separate proteomic study of salt response revealed higher accumulation of late embryogenesis-abundant (LEA) proteins and pathogenesis related proteins in soybean roots.66 In addition, a leucinezipper-like protein was found to be induced under salt stress in mature organs of soybean to counteract salt-triggered water potential changes.67 Low expression of metabolism-related proteins (Figure 1) coupled with decreased soybean plant growth indicate that metabolism of glucose through glycolysis is important to meet the required energy to overcome the salinity stress.7 Additionally, on exposure to salt stress, expressions of most of the photosynthesis-related proteins in soybean leaves were found to be decreased, suggesting that NaCl adversely affects photosynthesis and energy production, and consequently reduces plant growth. Moreover, concomitant decreases in the abundances of proteins involved in degradation of secondary metabolites and cell wall lignifications were recorded in salt-stressed roots,7 thus affecting overall soybean seedling growth. Overall findings indicate that soybean shares and exhibits different strategies to cope with the salinity stress.

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RESPONSES OF SOYBEAN TO HEAVY METAL STRESS Soil heavy metal (HM) toxicity has become a major environmental concern with regard to quality crop production. HM ions usually enter the root by specific/generic ion carriers or H

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both protein backbone and amino acid side chain residues.76 Among the Cd-induced defense-related proteins, enhanced expressions of antioxidant enzymes were evident in our proteomic investigation with soybean.8 Both APX and CAT enzymes are scavengers of peroxides and hence protecting cell membrane from hydroxyl radical induced lipid peroxidation.77 In addition to major antioxidant enzymes, plants are also equipped with some additional defense proteins, shown to be increased by HM stress. This group includes peroxiredoxin (Prx), thioredoxin (Trx), Trx-dependent peroxidase, NADP(H)-oxidoreductase, glyoxalase I (Gly I) (Figure 1). Apart from involving in PCs biosynthesis, cellular GSH pools also help in ROS scavenging through ascorbate glutathione cycle and play pivotal role in GSTs (glutathione S-transferases)-mediated decomposition of toxic compounds.78 Our proteomic study revealed that, among the contrasting Cd-accumulating soybean cultivars, abundances of antioxidant proteins were much higher in high Cd accumulator. In a separate experiment, GST mRNA level was found to be increased at early stress period (6 h) in soybean roots, suggesting that GST expression might be an early response to Al toxicity.25 Taken together, these increased expressions of defense-related proteins might help soybean seedlings to cope with the HM-induced oxidative stress. Interestingly, PR-10 protein, an important member of the pathogenesis-related (PR) protein family, often associated with systemic acquired resistance (SAR) against a wide range of pathogens,79 was reported to be highly up-regulated at the transcriptional and translational levels in Al-stressed soybean roots.25 Although the underlying precise molecular mechanism remains unclear, PR proteins are believed to play a key role in adaptation to stressful environments including HM toxicity.80 Kieffer et al.74 also documented marked increase in abundance of PR proteins endo-1,3-beta-glucanase, a class 2 PR protein, found to be induced in rice roots under short-term Cd-stress.81 Further proteomic investigations need to be carried out to understand the underlying molecular mechanism of PR proteins-mediated HM tolerance in soybean. Apart from synthesizing PCs, antioxidant molecules or molecular chaperones to alleviate the HMs induced stress effects, soybean plants also modulate vital metabolic pathways, primarily photosynthesis and mitochondrial respiration on exposure to HM stress. This controlled regulation of cellular pathways further helps to produce more reducing power to compensate high-energy demand of HM-challenged soybean cells. 2-DE-based proteomics approach was exploited for proteome determination in leaves of Cd-stressed soybean and revealed increased abundance of RuBisCO LSU-binding protein subunits alpha and beta, RuBisCO activase, oxygenevolving enhancer protein 1 and 2, NAD(P)H-dependent oxidoreductase, photosystem I and II related proteins (Figure 1).8 In contrary, low abundance of proteins involved in photosynthetic electron transport chain and Calvin cycle has been documented in Cd exposed Populus.73,74 Hajduch et al.82 also reported drastic reduction in abundance/fragmentation of large and small subunits of RuBisCO (LSU and SSU) in rice leaves exposed to HMs, suggesting complete disruption of photosynthetic machinery under HM stress. However, enhanced expressions of proteins involved in photosystem I, II and Calvin cycle as documented in our proteomic study might be an adaptive feature to overcome the Cd injury in soybean. In addition, overexpressions of glycolytic enzymes, phosphoglycerate mutase (PGM), glucose-6-phosphate isomerase (G6PI), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate

dehydrogenase (G3PDH), enolase (ENO), and pyruvate kinase (PK), have been reported in response to Cd.8,74 Like glycolysis, enzymes of tricarboxylic acid cycle cycle, citrate synthase (CS), succinate dehydrogenase (SD), malate dehydrogenase (MDH), aconitase (ACO), and aconitate hydratase (AH), were found to be up-regulated under Cd-stress (Figure 1).8,73 The 2-D-DIGE proteomic analysis of Al-tolerant and -sensitive soybean genotypes has also revealed similar kind of up-regulation of enzymes involved in citrate synthesis in Al-stressed soybean roots.83 In our opinion, contribution of high photosynthetic assimilates into respiration and up-regulation of glycolysis and tricarboxylic acid cycle might help soybean plants to yield more reducing power to compensate high-energy demand of HM challenged soybean cells.

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PROTEOMICS OF RADIATION-INDUCED RESPONSES IN SOYBEAN Light intensity and quality are the integral parts of the environment that influence growth and development of a plant.84 Excessive, deficient and inappropriate spectral distributions, e.g., more than required quantities of photosynthetically active radiation (PAR), increased absorption of UV radiation and ozone (O3) exert radiation stress in plants. Ozone-Induced Changes in Proteome Composition

Tropospheric O3 concentration higher than 40 ppb negatively affects soybean growth and yield with decreased shoot and pod biomass, fewer pods produced, and premature leaf senescence.85,86 Decrease in stomatal conductance under O3 stress reduces net carbon assimilation and photosynthetic activities that ultimately results in reduced seed yield.87 To elucidate the response mechanism of soybean to high O3 level, tissue-specific and subcellular proteome analyses have been carried out.27 Identification of differentially expressed proteins in O3-exposed leaves and chloroplasts revealed down-regulation of proteins involved in primary carbon assimilation and the Calvin cycle (Figure 1). Furthermore, a number of proteins associated with photosystem I/II and electron transport exhibited decreased abundances following exposure to O3. These results are in conflict with those obtained in total and redox proteome analyses of leaf and root tissues from soybean grown at SoyFACE under moderate O3 treatment.26 Increased expressions and/or oxidations of RuBisCO large and small subunits, RuBisCO activase, RuBisCO-associated protein, RuBisCO-binding proteins, chlorophyll α/β binding protein, ferredoxin reductase, and a chlorophyllase-like protein might help soybeans to maintain normal photosynthetic rate at early growth stage before the O3induced senescence started.26 Furthermore, significant decline in the starch content, with increase in sucrose concentration have been reported in the O3treated soybean leaves.27 Alternation of carbon catabolism/ metabolism-related enzymes and proteins might lead to the change in starch content during O3 stress (Figure 1). Thus, starch metabolism is required in order to feed the tricarboxylic acid cycle to cope with the energy demand. Ozone enters plant system through stomata, and thereafter it induces active ROS production. The subcellular localization of O3-induced H2O2 production has been well studied in Betula pendula leaf cells.88 Histochemical study with DAB and NBT staining is a powerful tool to detect invisible O3-induced lesions in leaves of O3-stressed plants. Ahsan et al.27 exploited this approach for early diagnosis of previsual O3 injury in soybean leaves. Presence of dark blue and deep brown spots on I

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at the proteome level. Ahsan et al.31 for the first time investigated the proteomic responses of leaves, stems, and roots of soybean seedlings to heat stress (40 °C) in order to identify the heat-induced, tissue-specific proteins and to demonstrate the tissue-specific defense strategy and thermoadaptive mechanisms. Two high molecular weight HSPs and several low molecular weight HSPs represented by several isoforms were common to all three tissues and showed a similar pattern of upregulation in response to heat stress. Small heat shock proteins (sHSPs), either newly synthesized or markedly up-regulated in response to heat stress, were common to all three tissues. These sHSPs mainly function as molecular chaperones to protect proteins from being denatured in extreme conditions. Proteomic analysis indicates that although different tissues share a common expression pattern of sHSPs and HSP70, some of the HSPs, e.g., CPN-60 α and β, HSP 90, ChsHSP, HSP 22.3, and HSP 17.6, exhibit tissue-specific expression. CPN-60 β was exclusively expressed in heat-stressed leaves, while subunit α was common between leaf and stem, but either less or no expression was observed in root. Along with the CPN-60β, ChsHSPs is uniquely expressed in green tissues, suggesting their probable involvement in protecting chloroplast proteins from heat stress-induced thermal aggregation and denaturation. In addition, on exposure to heat stress, glutamine synthase (GS) was found to be significantly down-regulated in all three tissues, while decreased abundances of cysteine synthase (CS) and glutamate dehydrogenase (GDH) were recorded in heatstressed leaves and roots, respectively. Notably, proteins involved in redox homeostasis, e.g., SOD [Cu−Zn] and cytosolic APX1, were increased significantly following exposure to heat (Figure 1), indicating their positive role in scavenging heatinduced excess ROS generation in soybean leaves. Taken together, these proteomic findings suggest that in course of time, tissue-specific defenses and adaptive mechanisms have been developed in soybean, along with the common defense strategies including up-regulation of several HSPs in all three tissues in order to defend each type of tissue against thermal stress.

O3-exposed leaves are clearly indicative of high H2O2 and superoxide accumulation under O3 stress. Furthermore, elevated levels of H2O2 and TBARS indicate severe oxidative burst prior to actual appearance of any visual symptoms that ultimately affect vital cellular pathways modulating plant response to O3 stress. Taken together, the authors suggest that carbon allocation is tightly programmed, and not only is the degradation of starch with concomitant increased sucrose level in response to short-term acute O3 exposure essential to feed the tricarboxylic acid cycle, but also the availability of sucrose may play a pivotal role in oxidative stress signaling and regulation pathways of antioxidative processes to withstand O3 stress. A recent proteomic study by Galant et al.26 also reveals higher expressions of enzymes associated with the reduction and regeneration phases of the Calvin cycle, glycolysis, and the citric acid cycle. These O3-induced changes in abundances and oxidation state of proteins are the part of adaptive responses that influence a wide range of metabolic processes, mitigating oxidative stress damages. Proteomic Changes under UV−B Radiation

Like O3 exposure, excessive UV−B radiation has deleterious effects on plant cell components, and it inhibits vital metabolic pathways.89 Plants possess an array of adaptive mechanisms to mitigate UV−B induced damages. 2-DE analysis of soybean leaf proteome revealed that abundances of metabolism, energy, protein destination/storage, disease/defense, transcription, protein synthesis, and secondary metabolism associated proteins were mostly changed in response to UV−B radiation.28 Findings indicate that presence of high levels of foliar flavonoids help soybean in mitigating UV−B stress more efficiently. Interestingly, all the responsive proteins related to photosystem (PS) were found to be enhanced by solar UV−B treatment. Nevertheless, Rubisco activase, Rubisco small subunit, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, phosphoribulokinase or other enzymes involved in primary carbon metabolism were decreased by UV−B radiation (Figure 1). Xiong and Day90 have reported that the inhibition of UV−B on photosynthesis is associated with enzymatic components, rather than PS II limitations. Unlike energy related proteins, only six defense responsive proteins exhibit changed abundances; e.g., catalase and peroxiredoxin were decreased, while ascorbate peroxidase, SOD and lectin were increased by UV−B. In summary, enhanced expressions of proteins related to PS and electron transport may lead to greater reducing power. With the concomitant down-regulation of enzymes involved in primary carbon metabolism, photosynthetic electron transport chain becomes over reduced, leading to the formation of ROS, which in turn cause oxidative damages to the UV-challenged soybean cells.

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PROSPECTS AND CHALLENGES Proteomics technique has validated its role in precise identification and characterization of target proteins that play essential roles in stress tolerance. However, the accuracy of this technique is highly dependent upon various steps, including isolation of full component of proteins, separation, visualization and their accurate identification. The different proteomic approaches that have been exploited extensively for quantification of stress-responsive proteins in soybean includes conventional 2-DE and 2D-fluorescence difference gel electrophoresis (DIGE), and MS-based methods that involve labelbased and label-free protein quantification. Instead of having limitations in identifying low-abundance and hydrophobic proteins, exceedingly large or small proteins, as well as basic proteins, 2-DE method has been used as a global technique to explore abiotic stress response in soybean.5−8,13,16,17,19,22,25 These inherent limitations of 2-DE approach can be overcome by the multidimensional protein identification technique (MudPIT), a nongel method of shotgun proteomics approach. In contrast to gel-based techniques where selected proteins are typically digested with trypsin after separation, MudPIT analysis involves digestion of all proteins in a sample into peptides before the separation steps. The separated peptides are then sequentially eluted into the mass spectrometer and

Modulation of Soybean Proteome under Heat Stress

When plants are exposed to a temperature that is five or more degrees above the optimal growth temperature, the synthesis of most normal proteins is repressed and a set of proteins called HSPs is induced.91 This rise in temperature level sufficient to cause irreversible damage to plant growth and development is considered to be a heat stress. A gradual increase in temperature also triggers HSPs synthesis that confers thermotolerance by preventing the denaturation of proteins during the heat stress and by facilitating the refolding of already misfolded/ unfolded proteins. Soybean is considered to be more sensitive to heat stress than the other legume crops.92 However, limited information is available on the heat-stress response of soybean J

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consequently analyzed by high-throughput automatic fashion.93 Typically, digested peptides are separated by two phase columns, strong cation exchange (SCX) and reversed-phase (RP) columns coupled with HPLC pump. However, analyzing complex biological mixtures often results in column clogging. To minimize this obstacle, split-phase columns where the sample is loaded only onto the SCX phase and washed extensively prior to connection to the RP column94 and RP-SCX-RP series columns have been designed to further improve the peptide separation capacity.95 Offline MudPIT approach is most applicable in the case of analyzing a large amount of peptides, while online MudPIT offers the advantage of minimal sample handling.93 Quantification in MudPIT analysis is usually performed through in vitro labeling techniques such as isotope tags for relative and absolute quantitation (iTRAQ) and isotope coupled affinity tags (ICAT). Though iTRAQ labeling requires expensive reagents and a mass spectrometer of relatively high resolution, the multiplexed reagents enable quantitative analyses of four or eight samples, rather than just two. The iTRAQ mass tagging strategy has wide application in elucidating phosphoproteome changes during the defense response mechanism, as documented in Arabidopsis thaliana−Pseudomonas syringae interaction.96 In the case of soybean, iTRAQ-based quantitative approach has been well exploited in proteomic analysis of elite cultivar exhibiting higher yield and enhanced resistance against abiotic stress over the parents. On the other hand, MS-based label-free quantification methodology allows differential expression analyses of multiple samples.97 Such label-free quantification has been extensively used in unraveling response mechanism of soybean seedlings against flooding and osmotic stress.9,21,98 In separate study, both 1D-PAGE-LC−MS/MS and MudPIT shotgun proteomics approaches and 2-DE were simultaneously explored in analyzing proteome of isolated soybean root hair cells.99 Construction of such root hair proteome reference map not only provides new insight into the metabolism and regulatory pathways, but also helps in deeper understanding of water stress tolerance mechanism at root hair proteome level. Similar quantitative label-free shotgun proteomic analysis has been carried out recently for analyzing longdistance drought signaling mechanism in rice grown in splitroot system.100 In the past decade, protein extraction protocols have been improved a lot to overcome the shortcomings in having full proteome profiles. Complete protein extraction from soybean leaf is indeed a challenging section of stress proteomics. Presence of extremely abundant photosynthetic CO2 fixation enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) not only limits the dynamic resolution and yield of low-abundance proteins of interest, but also masks other proteins or affects the electrophoretic migration of neighboring protein species.101 Different fractionation techniques based upon different physiological or biochemical principles have been proposed to deplete or reduce a substantial portion of RuBisCO from total leaf protein extract.102,103 In comparison to these complex, lengthy methods, Krishnan and Natarajan104 developed a fast and simple fractionation technique using 10 mM Ca2+ and 10 mM phytate to precipitate 85% of the RuBisCO from soybean leaf soluble protein extract. Using this method, the authors succeeded in easy detection and identification of previously inconspicuous protein spots using SYPRO Ruby fluorescent stain. Although this fractionation technique is an efficient way to remove RuBisCO, significant loss of

nontarget proteins is also reported. Hence, other methods such as immunoaffinity depletions need to be exploited further for specific removal of only the target proteins. Similarly, fractionation method based on 15% polyethylene glycol was found to be effective for elimination of RuBisCO.105 Hashimoto and Komatsu106 used anti-RuBisCO LSU antibody affinity column with protein A-Sepharose as a resin for the elimination of RuBisCO. Alam et al.107 recommended the re-extraction of PEG-fractionated samples with phenol that effectively eliminated interfering substances and results in optimal conductivity during separation in the first dimension of the isoelectric focusing. Despite recent advancement, more emphasis needs to be given on the accuracy of MS techniques for identification of very low-abundance proteins. Selected reaction monitoring (SRM), a highly sensitive MS technique, has become a potential tool for the reliable identification and accurate quantitation of very low-abundance proteins in complex biological mixtures and characterization of modified peptides.108 The outstanding multiplexing abilities, reproducibility, sensitivity and selectivity make SRM an invaluable tool in targeted proteomics for determining very subtle expression changes and thus facilitate protein network modeling. Similarly, improvements in multiple reaction monitoring (MRM) MS technique provide new insights into plant stress signaling pathways. Rainteau et al.109 used mass spectrometry in the MRM mode to analyze the fatty acid composition of the major glycerophospholipids in Arabidopsis suspension cells. The MRM-MS strategy was found to be a reliable method for determining the extract composition of glycerophospholipids in plants. These emerging, targeted and highly sensitive MS techniques could be exploited further for reliable identification and accurate quantitation of ultra-lowabundance stress responsive proteins for dissecting plant stress signaling pathways. Characterization of protein isoforms and protein species is another major challenge for quantitative soybean proteomics, as the soybean genome has undergone two rounds of whole genome duplication and many tandem duplications.110 Although a majority of the genes are present in multiple copies, many of these duplicated genes either become silenced or lost in due course of time. Due to higher gene duplication and recombination process, so many protein isoforms exist in soybean as compared to rice and Arabidopsis. The 2-DE based proteomic technique has significant contribution in identification and characterization of these different isoforms. Protein species occupy different positions on the 2-DE gel matrix based on their individual isoelectric point (pI) and relative molecular mass (Mr) but share the same identification. It has been documented that the occurrence of an increasing number of phosphorylation sites leads to a shift both in pI and Mr. Alternative to gel-based approach, bottom-up LC−MS/MS techniques offer more advantages in identifying protein species. In this method the unambiguous identification of a single protein species relies on the identification of at least one peptide sequence that is uniquely found in that protein species.111 Moreover, appropriate selection of the database used for searches is essential for identification of such protein species with accuracy.

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CONCLUDING REMARKS A comprehensive understanding of plants’ stress response mechanisms is important to elucidate the key factors affecting crop performance under adverse climate. The present review K

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*(S.K.) Tel: +81-29-838-8693. Fax: +81-29-838-8694. E-mail: skomatsu@affrc.go.jp.

outlines the response mechanisms of soybean to various abiotic stresses. Irrespective of the type and severity of the stress, soybean exhibits a tight control over the carbon metabolism (Figure 1, Table 1). Expressions of proteins involved in glycolysis and tricarboxylic acid cycle are highly modulated under stress to meet the cells’ required energy demand. Activation of anaerobic pathways to regenerate NAD+ is an additional strategy of flooded soybean to adapt the oxygen-poor condition. Both 2-DE and gel-free MS-based quantitative proteomic analyses of flooded soybean revealed up-regulation of proteins associated with the glycolysis and fermentation (Table 1). Moreover, up-regulation of photosynthesis-associated proteins under HM, salinity and radiation stresses contributes high photosynthetic assimilates into respiration to yield more energy needed to combat the stress. Another strategic management of stress mitigation involves activation of defense genes irrespective of type of stress. Differential modulations of stress-induced antioxidant proteins have been reported in most of the soybean proteomic studies (Figure 1). Taken together, these findings indicate presence of a robust antioxidative network in soybean to maintain the redox balance. In addition, enhanced expression of molecular chaperones might be another additional common defense mechanism of soybean for refolding of misfolded proteins and to stabilize protein structure and function, thus maintaining cellular homeostasis. Like other crops, soybean has also developed stress-specific defense mechanisms that get activated only under the concerned adverse condition. Activation proteins involved in HM detoxification pathways, namely, chelation and compartmentalization, help the HM-challenged soybean cells to maintain steady intracellular concentration of metal ions, thus alleviating the stress damages. In conclusion, despite its limitations and challenges, soybean proteomics has proved itself as a valuable tool for identifying stress responsive target proteins with a clear picture of translational and post-translational modifications. More attention needs to be paid to the research on protein−protein and protein−ligand interactions and interdisciplinary research in junction with metabolomics to establish networks of interaction between proteins and metabolites involved in abiotic stresstolerance mechanisms. The convergence of proteomic techniques with computational advancement is essential to overcome the limitations associated with the analytical variability of this technique. Apart from the technological advancement, more proteomic research works on organelle proteomes need be initiated for better understanding of the underlying molecular mechanism of how the cell modulates its protein signature to cope with a harsh environment. In depth studies on organelle proteomes would be of great contribution toward understanding the intracellular stress signaling pathways. Response of soybean to multiple stresses would be another interesting area of future proteomic research that could shed light on cross talk between different abiotic stress signal pathways with greater extant of accuracy. All these proteomic findings would further help us to move a step ahead in designing stresstolerant crops.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This review is an outcome of post-NICS, Japan, visit as DSTBOYSCAST fellow (Z.H.). The authors wish to acknowledge the support of the Department of Science and Technology, Government of India. This work is supported by the Grants from National Agriculture and Food Research Organization, Japan.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*(Z.H.) Tel: +91-33-2524-1975. Fax: +91-33-2524-1977. E-mail: [email protected]. L

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