**2. Analyses of soybean proteomics**

### **2.1. Physiological and biological changes in the soybean proteome**

### *2.1.1. Whole plant organs*

The various tissues of soybean have specific groups of associated proteins at each develop‐ mental stage. While leaves at various developmental stages showed 26 differentially ex‐ pressed proteins, the first trifoliate stage manifested the greatest increase in protein types of the outer/inner envelope of choloroplast membrane and also of the protein transport machi‐ neries. Young leaves showed abundant chaperonin-60, while HSP 70 and TP-synthase b were present in all the tissues analyzed. Age dependent correlation was observed in net photosynthesis rate, chlorophyll content and carbon assimilation. During the flowering stage, flower tissue expressed 29 proteins that were exclusively involved in protein trans‐ port and assembly of mitochondria, secondary metabolism and pollen tube growth (Ahsan and Komatsu., 2009 [76]. Soybean peroxisomal adenine nucleotide carrier (GmPNC1) is as‐ sociated with the peroxisomal membrane and facilitates ATP and ADP importing activities. The proteins At PNC1 and At PNC2 are arabidopsis orthologs of Gm PNC1. Under constant darkness, Gm PNC1 increased in cotyledons up to 5 days post germination and the levels were rapidly reduced when the seedlings were exposed to light. RNA interference studies on arabidopsis At PNC1 and At PNC2 suggests that PNC1 assists with transport of ATP/ADP in the peroxisomal fatty acid-b oxidation pathway post germination (Arai et al., 2008 [77]. This probably helps the seedling establish vigour for future growth.

In order to establish if xylem proteins and the apoplast conduit are involved in long distance signalling in autoregulation of nodulation (AON) in the soybean-*B. japonicum* symbiosis, xy‐ lem and apoplast fluids were collected from hypocotyl, epicotyl and stem tissues. In addi‐ tion, proteins from imbibing seeds were evaluated to determine possible relationships of these proteins with the xylem and apoplast proteins, especially during the seed to seedling stage transition. The proteins secreted from imbibing seeds were different from the set of xy‐ lem-related proteins. Hypocotyl, epicotyl and stem xylem proteins were generally similar. Comparison of wild type and nts1007 plants showed no difference in xylem protein profiles, suggesting that xylem proteins were not involved in AON. However, a lipid transfer protein and Kunitz trypsin inhibitor, both known to have roles in plant signalling, were identified within the xylem proteins [78].

Proteomic studies on chasmogamous (CH) CH cv. Toyosuzu and cleistogamous (CL) CL cv. Karafuto-1 flowerbuds using 2D gel revealed differential protein levels of β-galactosidase and protein disulfide isomerase. Cleistogamy occurs in plants under diverse stress condi‐ tions, such as drought and cold, and can also vary with temperature and light [79]. Soybean cv Maverick was used to study proteomics during seed filling stages, at 2, 3, 4, 5 and 6 weeks after flowering, using 2D and MALDI-TOF-MS. Storage proteins, proteins involved in metabolism and metabolite transport and defense related proteins were the most abun‐ dant, along with cysteine and methionine biosynthesis proteins, lipoxygenases and 14-3-3 like proteins [80,81].

Based on these findings, it is clear that the plant partitions its proteomics based on ontogeny and this specificity probably plays a crucial role in organ maturation and transition from one stage to another in the plants life cycle. Understanding this is of fundamental impor‐ tance in agriculture, global food production, biofuel production and issues such as plant re‐ sponses to climate change.

### *2.1.2. Seeds*

ciation (CID). This procedure allows for the fragmenting of the peptides to produce a prod‐ uct ion spectrum, the MS/MS spectrum. In addition, the fragmentation occurs preferentially at the amide bonds, to generate N-terminal fragments (b ions) and C-terminal fragments (y ions) at specific m/z ratios, providing structural information about the amino acid sequence and sites of modification. The b ion and y ion patterns are matched to a peptide sequence in a translated genomic database to help identify the proteins present in the sample [72-75]. A variety of database searching and compiling algorithms are used to interpret the data ob‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

The various tissues of soybean have specific groups of associated proteins at each develop‐ mental stage. While leaves at various developmental stages showed 26 differentially ex‐ pressed proteins, the first trifoliate stage manifested the greatest increase in protein types of the outer/inner envelope of choloroplast membrane and also of the protein transport machi‐ neries. Young leaves showed abundant chaperonin-60, while HSP 70 and TP-synthase b were present in all the tissues analyzed. Age dependent correlation was observed in net photosynthesis rate, chlorophyll content and carbon assimilation. During the flowering stage, flower tissue expressed 29 proteins that were exclusively involved in protein trans‐ port and assembly of mitochondria, secondary metabolism and pollen tube growth (Ahsan and Komatsu., 2009 [76]. Soybean peroxisomal adenine nucleotide carrier (GmPNC1) is as‐ sociated with the peroxisomal membrane and facilitates ATP and ADP importing activities. The proteins At PNC1 and At PNC2 are arabidopsis orthologs of Gm PNC1. Under constant darkness, Gm PNC1 increased in cotyledons up to 5 days post germination and the levels were rapidly reduced when the seedlings were exposed to light. RNA interference studies on arabidopsis At PNC1 and At PNC2 suggests that PNC1 assists with transport of ATP/ADP in the peroxisomal fatty acid-b oxidation pathway post germination (Arai et al.,

tained for structure and function of the identified proteins.

**2.1. Physiological and biological changes in the soybean proteome**

2008 [77]. This probably helps the seedling establish vigour for future growth.

In order to establish if xylem proteins and the apoplast conduit are involved in long distance signalling in autoregulation of nodulation (AON) in the soybean-*B. japonicum* symbiosis, xy‐ lem and apoplast fluids were collected from hypocotyl, epicotyl and stem tissues. In addi‐ tion, proteins from imbibing seeds were evaluated to determine possible relationships of these proteins with the xylem and apoplast proteins, especially during the seed to seedling stage transition. The proteins secreted from imbibing seeds were different from the set of xy‐ lem-related proteins. Hypocotyl, epicotyl and stem xylem proteins were generally similar. Comparison of wild type and nts1007 plants showed no difference in xylem protein profiles, suggesting that xylem proteins were not involved in AON. However, a lipid transfer protein

**2. Analyses of soybean proteomics**

*2.1.1. Whole plant organs*

Relationships

8

Both 2D gel and peptide mass fingerprinting techniques (MALDI-TOF-MS) were used to study the proteins of mature and dry soybean (cv. Jefferson) seeds. Sucrose binding pro‐ teins, alcohol dehydrogenase and seed maturation proteins were some of the key proteins identified (Mooney and Thelen 2004 [82]. A comparison of four methods for protein isola‐ tion and purification from soybean seed was one of the first reports on soybean proteomics; thiourea/urea and TCA protocols were found to be the best. Proteins extracted with these two methods and further characterized by MALDI-TOF-MS and LC-MS helped identify pro‐ teins such as β-conglycinin, glycinin, Kunitz trypsin inhibitor, alcohol dehydrogenase, Gm Bd 28K allergen and sugar binding proteins in seeds [83]. The two major soybean storage proteins are α-conglycinin and glycinin. While the α-conglycinin subunits separated well in the pH range 3.0-10.0, glycinin polypeptides could be separated in pH ranges 4.0-7.0 and 6.0-11.0. Apart from these major storage proteins, this combined proteomic approach (2D-PAGE and immobilized pH gradient strips) also identified 44 storage proteins in wild soy‐ bean (*G. soja*) and 34 additional storage proteins in its cultivated counterpart (*G. max*) [84]. A comparative proteome analysis of soybean seed and seedling tissue suggested that there were dramatic changes in the protein profiles during seed germination and during seedling growth. The seed storage proteins β-conglycinin and glycinin were seen to degrade rapidly and their degradation products were either accumulated or degraded further as the seeds germinated. This degradation of the storage proteins indicates that the proteolysis process provides amino acids and energy for the growing seedlings, and gives access to new detail regarding these processes [85].

Synthesis of soybean glycinin and conglycinin, was suppressed by RNA interference. The storage protein knockdown (SP2) seeds were very similar to the wild type during develop‐ ment and at maturity. Proteomic analysis of the SP2 soybean genotypes and next-generation

transcript sequencing (RNA-Seq) suggested that the seeds could rebalance their transcrip‐ tome and metabolome in the face of at least some alterations. GFP quantification for glycinin allele mimics further revealed that glycinin was not involved in proteome rebalance and that seeds are capable of compensating through increases in other storage proteins, to main‐ tain normal protein content, even if the major storage proteins were not available [86].

Transgenic soybean seeds have higher amounts of malondialdehyde, ascorbate peroxidase, glutathione reductase, and catalase (29.8, 30.6, 71.4, and 35.3%, respectively) than non-trans‐ genic seeds. Precursors of glycinin, allergen Gly m Bd 28k, actin and sucrose binding pro‐ teins were the other proteins identified [87,88]. High protein accessions of soybean (with 45 % or more protein in seeds) were compared with soybean cultivar Williams 82. 2-DE-MAL‐ DI-TOF-MS followed by Delta2D image analysis showed huge differences in 11S storage globulins amongst the accessions. In addition, the trait for high protein from PI407788A was moved to experimental line LG99-469 and was stable upon transformation [89,90].

### *2.1.3. Roots, root hairs and nodules*

Since the root apical meristem (RAM) is responsible for the growth of the plant root system and root architecture plays and important role in determining the performance of crop plants, a proteome reference map of the soybean root apex and the differentiated root zone was established. The root apex samples comprised of 1 mm of the root apex, encasing the RAM, the quiescent center and the root cap. The predominant proteins in the root belonged to those of stress response, glycolysis, redox homeostasis and protein processing machinery. The root apex contained key proteins, such as those involved in redox homeostasis and fla‐ vonoid biosynthesis, but was underrepresented in glycolysis, stress response and TCA cycle related proteins [91]. Analysis of the proteome of isolated soybean root hair cells using 2-D gel and shotgun proteomics approaches identified proteins involved in basic cell metabo‐ lism, those whose functions are specific to root hair cell activities, including water and nu‐ trient uptake, vesicle trafficking, and hormone and secondary metabolism [92, 93]. Proteomic studies of soybean roots and root hairs after *B. japonicum* inoculation explains the importance of initial plant-bacteria symbiotic interaction. A 2-D, MALDI-TOF, MS based ap‐ proach shows that enzymes such as chitinase and phosphoenolpyruvate carboxylase are dif‐ ferentially expressed in root hairs. As well as peroxidase and phenylalanine-ammonia lyase, found to be expressed during rhizhobial inoculation, other novel proteins such as phospho‐ lipase D and phosphoglucomutase were found to be expressed [94]. Nodule cytosol proteins from soybean cv. Williams 82 were found to be 28% related to carbon metabolism, 12% relat‐ ed to nitrogen metabolism, 12% related to reactive oxygen metabolism and 11% related to vesicular trafficking proteins. The vesicular trafficking proteins could be involved in the ex‐ change of micro- and macro-molecules during the process of nodulation, while carbon, ni‐ trogen and reactive oxygen species are related to physiological functions during nitrogen fixation [95]. The peribacteroid membrane (PBM) of the soybean symbiosome contains chap‐ eronins such as HSP60, BiP (HSP70) and PDI, and serine and thiol protease, all of which are involved in protein translocation, folding, maturation and degradation of proteins related to the symbiosomes. Nodulin proteins 53b and 26B, associated with the PBM, were also present, although their function is not clear [96].

### **2.2. Soybean proteomics under stress conditions**

Like all plants, soybean also encounters various stressors during its life cycle. Work related to flooding, drought, salt, heat, biotic stressors, metal toxicity, ozone, phosphorous deficien‐ cy and seed protein allergens are reviewed here.

### *2.2.1. Flooding stress*

transcript sequencing (RNA-Seq) suggested that the seeds could rebalance their transcrip‐ tome and metabolome in the face of at least some alterations. GFP quantification for glycinin allele mimics further revealed that glycinin was not involved in proteome rebalance and that seeds are capable of compensating through increases in other storage proteins, to main‐ tain normal protein content, even if the major storage proteins were not available [86].

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Transgenic soybean seeds have higher amounts of malondialdehyde, ascorbate peroxidase, glutathione reductase, and catalase (29.8, 30.6, 71.4, and 35.3%, respectively) than non-trans‐ genic seeds. Precursors of glycinin, allergen Gly m Bd 28k, actin and sucrose binding pro‐ teins were the other proteins identified [87,88]. High protein accessions of soybean (with 45 % or more protein in seeds) were compared with soybean cultivar Williams 82. 2-DE-MAL‐ DI-TOF-MS followed by Delta2D image analysis showed huge differences in 11S storage globulins amongst the accessions. In addition, the trait for high protein from PI407788A was

Since the root apical meristem (RAM) is responsible for the growth of the plant root system and root architecture plays and important role in determining the performance of crop plants, a proteome reference map of the soybean root apex and the differentiated root zone was established. The root apex samples comprised of 1 mm of the root apex, encasing the RAM, the quiescent center and the root cap. The predominant proteins in the root belonged to those of stress response, glycolysis, redox homeostasis and protein processing machinery. The root apex contained key proteins, such as those involved in redox homeostasis and fla‐ vonoid biosynthesis, but was underrepresented in glycolysis, stress response and TCA cycle related proteins [91]. Analysis of the proteome of isolated soybean root hair cells using 2-D gel and shotgun proteomics approaches identified proteins involved in basic cell metabo‐ lism, those whose functions are specific to root hair cell activities, including water and nu‐ trient uptake, vesicle trafficking, and hormone and secondary metabolism [92, 93]. Proteomic studies of soybean roots and root hairs after *B. japonicum* inoculation explains the importance of initial plant-bacteria symbiotic interaction. A 2-D, MALDI-TOF, MS based ap‐ proach shows that enzymes such as chitinase and phosphoenolpyruvate carboxylase are dif‐ ferentially expressed in root hairs. As well as peroxidase and phenylalanine-ammonia lyase, found to be expressed during rhizhobial inoculation, other novel proteins such as phospho‐ lipase D and phosphoglucomutase were found to be expressed [94]. Nodule cytosol proteins from soybean cv. Williams 82 were found to be 28% related to carbon metabolism, 12% relat‐ ed to nitrogen metabolism, 12% related to reactive oxygen metabolism and 11% related to vesicular trafficking proteins. The vesicular trafficking proteins could be involved in the ex‐ change of micro- and macro-molecules during the process of nodulation, while carbon, ni‐ trogen and reactive oxygen species are related to physiological functions during nitrogen fixation [95]. The peribacteroid membrane (PBM) of the soybean symbiosome contains chap‐ eronins such as HSP60, BiP (HSP70) and PDI, and serine and thiol protease, all of which are involved in protein translocation, folding, maturation and degradation of proteins related to

moved to experimental line LG99-469 and was stable upon transformation [89,90].

*2.1.3. Roots, root hairs and nodules*

Relationships

10

Plasma membrane proteins from the root and hypocotyl of soybean seedlings were purified and subjected to 2-D gel electrophoresis, followed by MS and protein sequencing, and also using nanoliquid chromatography followed by nano-LC-MS/MS based proteomics. The two techniques were used to compare the proteins present, and this indicated that during flood‐ ing stress proteins typically found in the cell wall were up-regulated in the plasma mem‐ brane. Also, the anti-oxidative proteins were up-regulated to protect the cells from oxidative damage, heat shock proteins to protect protein degradation and signaling proteins to regu‐ late ion homeostasis [97]. MS based proteomics applied to root tips of two-day-old seedlings flooded for 1 day showed increased levels of proteins involved in energy production. Pro‐ teins involved in cell structure maintenance and protein folding were negatively affected, as was their phosphorylation status [98].

Two-day-old germinated soybean seeds were subjected to water logging for 12 h and total RNA and proteins were analyzed from the root and hypocotyl. At the transcriptional level, the expression of genes for alcohol fermentation, ethylene biosynthesis, pathogen defense, and cell wall loosening were all significantly up-regulated, while scavengers and chaperons of reactive oxygen species were seen to change only at the translational level. Transcription‐ al and translational level changes were observed for hemoglobin, acid phosphatase, and Ku‐ nitz trypsin protease inhibitors. This adaptive strategy might be for both hypoxia and more direct damage of cells by excessive water [99]). Proteins from 2-day-old soybean seedlings flooded for 12 h were analyzed using 2-D gel MS, 2-D fluorescence difference gel electro‐ phoresis, and nanoliquid chromatography. Early responses to flooding involved proteins re‐ lated to glycolysis and fermentation, and inducers of heat shock proteins. Glucose degradation and sucrose accumulation increased due to activation of glycolysis and downregulation of sucrose degrading enzymes, in addition the methylglyoxal pathway, a detoxi‐ fication system linked to glycolysis, was up-regulated. 2-D gel based phosphoproteomic analysis showed that proteins involved in protein synthesis and folding were dephosphory‐ lated under flooding conditions [100]. Water logging stress imposed on very early soybean seedlings (V2 stage) resulted in a gradual increase of lipid peroxidation and *in vivo* H2O2 production. Proteomic studies of the roots using 2-D gel, MALDI-TOF-MS or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis, identified 14 up-regulated and 5 down-regulated proteins. Five newly discovered proteins were associated with water log‐ ging, a known anaerobic stress. The proteins included those associated with signal transduc‐ tion, programmed cell death, RNA processing, redox homeostasis and energy metabolism.

Increases in glycolysis and fermentation pathway associated proteins were indicative of adaptation of the plant to this alternate energy provision pathway. Other novel proteins, such as a translation initiation factor, apyrase, auxin-amidohydrolase and coproporphyrino‐ gen oxidase, were also identified [101]. Mitochondrial proteomics from 2-day-flooded 4-dayold soybean seedlings identified increases in the levels of proteins and metabolites associated with TCA cycle and the γ-amino butyrate shunt. Increases in NADH and NAD and a decrease in ATP during the stress suggest that the electron transport chain is disrupt‐ ed, although NADH production increases through TCA cycle activity [102].

Soybean seeds germinated for 48 h were subjected to water logging stress for 6-48 h. In addi‐ tion to general stress responses due to increases in reactive oxygen species scavengers, sev‐ eral glycolytic enzymes were up-regulated, suggesting changes in energy generation [103].

### *2.2.2. Water stress – Drought*

Soybean root activities are affected during water stress. The root can be partitioned into zones 1 (apical 4 mm zone) and 2 (4-8 mm zone), based on maximum elongation during well watered conditions. Soluble proteins from these regions, studied under both well-watered and water deficit stress conditions, revealed region-specific regulation of the phenylpropa‐ noid pathway. Zone 1 of roots manifested increases in isoflavanoid biosynthesis related en‐ zymes and proteins that contribute to growth and maintenance of the roots under water stress conditions. However, zone 2 of water stressed roots manifested up-regulation of caf‐ feoyl-CoA *O*-methyltransferase (a protein involved in lignin biosynthesis), protective pro‐ teins related to oxidative damage, ferritin proteins that sequester iron, and 20S proteasome α-subunit A. Increases in lignin accumulation and ferritin proteins preventing availability of free iron in this zone were suggested to be the factors affecting root growth during water stress [104]. An investigation of the soybean plasma membrane proteome, under osmotic stress, was conducted using 2-day-old seedlings subjected to 10% PEG for 2 days; both geland nano-LC MS/MS-based proteomics methods were utilized to analyze the samples. Out of the 86 proteins identified by nano-LC MS/MS approach, 11 were up-regulated and 75 pro‐ teins down-regulated under PEG mediated stress. Three homologues of plasma membrane transporter proteins H1-ATPase and calnexin were prominent [105]. Similarly, 3-day-old soybean seedlings were subjected to 10% PEG treatment or water withdrawal and samples collected from roots, hypocotyl and leaves, 4-days after treatment, for proteome analysis. The root was the most responsive and affected organ for both drought stress induction methods. The leaves showed increases in metabolism-related proteins, while the energy pro‐ duction and protein synthesis machineries were negatively affected. HSP70, actin isoform B and ascorbate peroxidase were up-regulated in all the tissues analyzed. Importantly, me‐ thionine synthase, a drought response protein, decreased, suggesting negative effects of drought stress on these seedlings [106].

#### *2.2.3. High temperature stress*

Tissue specific proteomics under high temperature stress revealed 54, 35 and 61 differential‐ ly expressed proteins in the leaves, stems and roots, respectively. Heat shock proteins and those involved in antioxidant defense were up-regulated while proteins for photosynthesis, amino acid and protein synthesis and secondary metabolism were down- regulated. HSP70 and other low molecular weight HSPs were seen in all the tissues analyzed. ChsHSP and CPN-60 were tissue specific and the sHSPs were found only in tissues under heat stress, and were not induced by other stresses such as cold or hydrogen peroxide exposure [107].

#### *2.2.4. Salt stress*

Increases in glycolysis and fermentation pathway associated proteins were indicative of adaptation of the plant to this alternate energy provision pathway. Other novel proteins, such as a translation initiation factor, apyrase, auxin-amidohydrolase and coproporphyrino‐ gen oxidase, were also identified [101]. Mitochondrial proteomics from 2-day-flooded 4-dayold soybean seedlings identified increases in the levels of proteins and metabolites associated with TCA cycle and the γ-amino butyrate shunt. Increases in NADH and NAD and a decrease in ATP during the stress suggest that the electron transport chain is disrupt‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Soybean seeds germinated for 48 h were subjected to water logging stress for 6-48 h. In addi‐ tion to general stress responses due to increases in reactive oxygen species scavengers, sev‐ eral glycolytic enzymes were up-regulated, suggesting changes in energy generation [103].

Soybean root activities are affected during water stress. The root can be partitioned into zones 1 (apical 4 mm zone) and 2 (4-8 mm zone), based on maximum elongation during well watered conditions. Soluble proteins from these regions, studied under both well-watered and water deficit stress conditions, revealed region-specific regulation of the phenylpropa‐ noid pathway. Zone 1 of roots manifested increases in isoflavanoid biosynthesis related en‐ zymes and proteins that contribute to growth and maintenance of the roots under water stress conditions. However, zone 2 of water stressed roots manifested up-regulation of caf‐ feoyl-CoA *O*-methyltransferase (a protein involved in lignin biosynthesis), protective pro‐ teins related to oxidative damage, ferritin proteins that sequester iron, and 20S proteasome α-subunit A. Increases in lignin accumulation and ferritin proteins preventing availability of free iron in this zone were suggested to be the factors affecting root growth during water stress [104]. An investigation of the soybean plasma membrane proteome, under osmotic stress, was conducted using 2-day-old seedlings subjected to 10% PEG for 2 days; both geland nano-LC MS/MS-based proteomics methods were utilized to analyze the samples. Out of the 86 proteins identified by nano-LC MS/MS approach, 11 were up-regulated and 75 pro‐ teins down-regulated under PEG mediated stress. Three homologues of plasma membrane transporter proteins H1-ATPase and calnexin were prominent [105]. Similarly, 3-day-old soybean seedlings were subjected to 10% PEG treatment or water withdrawal and samples collected from roots, hypocotyl and leaves, 4-days after treatment, for proteome analysis. The root was the most responsive and affected organ for both drought stress induction methods. The leaves showed increases in metabolism-related proteins, while the energy pro‐ duction and protein synthesis machineries were negatively affected. HSP70, actin isoform B and ascorbate peroxidase were up-regulated in all the tissues analyzed. Importantly, me‐ thionine synthase, a drought response protein, decreased, suggesting negative effects of

Tissue specific proteomics under high temperature stress revealed 54, 35 and 61 differential‐ ly expressed proteins in the leaves, stems and roots, respectively. Heat shock proteins and

ed, although NADH production increases through TCA cycle activity [102].

*2.2.2. Water stress – Drought*

Relationships

12

drought stress on these seedlings [106].

*2.2.3. High temperature stress*

Salt stress is also an important abiotic stressor that affects crop growth and productivity. Of the 20% of agricultural land available globally, 50% of the cropland is estimated by the Unit‐ ed Nations Environment Program (The UNEP) to be salt-stressed [108]. As the plant grows under salt stresses conditions, depending on the severity of the stress, the plants can experi‐ ence reduced photosynthesis, protein and energy production, and changes in lipid metabo‐ lism [109,110]. As soil salinity increase, the effects on seed germination and germinating seedlings are profound. Responses to salinity and drought stress are similar; they affect the osmotic activity of the root system, thereby affecting the movement of water and nutrients into the plants. In Canadian soils, salinity varies between spring and fall and the most saline conditions are seen at the soil surface just after spring thaw. In the Canadian prairies, the dominant salts of saline seeps include calcium (Ca), magnesium (Mg) and sodium (Na) cati‐ ons, and sulphate (SO4 - ) anions [111]. Soybean is very sensitive to Cl- , but not greatly affect‐ ed by Na+ , because of its ability to restrict movement of Na+ to leaves [112].

This first report regarding soybean seedling proteomic responses to salt stress evaluated length and fresh weight of the hypocotyl and roots of soybean exposed to a series of NaCl concentrations. At 200 mM NaCl, the length and fresh weight of hypocotyl and roots were greatly reduced, with a simultaneous increase in proline content, suggesting activation of mechanisms for coping with salt stress. In addition, hypocotyl and root samples from 100 mM NaCl treated seedlings up-regulated seven key proteins, such as late embryogenesisabundant protein, b-conglycinin, elicitor peptide three precursor, and basic/helix-loop-helix protein. The same treatment caused down-regulation of protease inhibitor, lectin, and stem 31-kDa glycoprotein precursor. This combination of up- and down-regulated proteins indi‐ cates a metabolic shift and could represent a strategy used by soybean seedlings to enhance tolerance of, or adapt to, salt stress [113].

Sobhanian et al. [110,114] found that treatment of soybean seedlings with 80 mM NaCl ar‐ rests the growth and development of both hypocotyl and roots. This study assessed effects on leaf, hypocotyl and root proteomics of salt treated soybean seedlings and found that re‐ duction of glyceraldehyde-3-phospahte dehydrogenase was indicative of reduction in ATP production, and down-regulation of calreticulin was associated with disruption in the calci‐ um signalling pathway, both of which are associated with decreased plant growth. The lev‐ els of other proteins, such as kinesin motor protein, trypsin inhibitor, alcohol dehydrogenase and annexin, were also found to change, suggesting that these proteins might play different roles in soybean salt tolerance and adaptation [110,114].

Soybean cultivars Lee68 and N2899 are salt-tolerant and salt-sensitive, respectively. The per‐ centage germination was not affected when exposed to 100 mmol L-1 NaCl, however, the

mean germination time for Lee68 (0.3 days) and N2899 (1.0 day) was delayed, compared with control plants. Hormonal responses to salt stress differed between these cultivars. Both cultivars, increased abscisic acid levels and decreased giberrelic acid (GA 1, 3) and isopenty‐ ladenosine concentrations; auxin (IAA) increased in Lee68, but remained unchanged in N2899. 2-D gel electrophoresis, followed by MALDI-TOF-MS analysis, of the proteins from germinated seeds suggested increases in ferritin and the 20S proteasome subunit β-6 in both the cultivars. Glyceraldehyde 3-phosphate dehydrogenase, glutathione *S*-transferase (GST) 9, GST 10, and seed maturation protein PM36 were down-regulated in Lee68, but these pro‐ teins were naturally present in low concentrations in N2899 and were seen to up-regulate following exposure to salt stress [115].

### *2.2.5. Biotic stress*

The soybean-*Phytophthora soje* plant-oomycete interaction is of agriculture and economic im‐ portance, as this oomycete causes soybean root and stem rot, translating to an annual global loss of \$1-2 billion US. Twenty-six proteins were significantly affected in a resistant soybean cultivar (Yudou25) and 20 in a sensitive one (NG6255), as determined by 2-D gel analysis, followed by MALDI-TOF-MS. The distribution pattern of the affected proteins were - 26% energy regulation, 15% protein destination and storage, 11% defense against disease, 11% metabolism, 9% protein synthesis, 4% secondary metabolism, and 24% unknown/hypotheti‐ cal proteins [116].

Soybean mosaic virus (SBMV) causes one of the most serious viral infections of soybean; leaves of infected plants were studied at a series of time points using 2-D gel electrophore‐ sis, followed by MALDI-TOF-MS and tandem TOF/TOF-MS. Proteins expressed in the ino‐ culated leaves were identified and were seen to be involved in protein degradation, defense signalling, coping with changes in the levels of reactive oxygen species, cell wall reinforce‐ ment, and energy and metabolism regulation. Quantitative real time PCR was used to focus on gene expression related to some of these proteins. Photosynthesis and metabolism relat‐ ed genes were down-regulated at all the time points, while most of the energy related genes (respiration in this case) were up-regulated for at least five of the six time points studied [117]. At the time of this writing, this report is the only one addressing the proteomic ap‐ proach to molecular understanding of soybean-SBMV interaction.

### *2.2.6. Other miscellaneous stress related reports*

Aluminium toxicity is often observed in acidic soils and Baxi 10 (BX10) is an Al-resistant cul‐ tivar. One-week-old soybean seedlings treated with 50 mM AlCl3 for 24, 48 and 72 h were studied for characterization of root proteins in response to Al; and 2-D gel electrophoresis followed by MS revealed 39 proteins expressed differentially following Al treatment. Of these 21 were up-regulated (such as heat shock proteins, glutathione S-transferase, chalcone related synthetase, GTP-binding protein, ABC transporters and ATP binding proteins). Five proteins were also down-regulated and 15 newly induced proteins were present following AL treatment [118].

The process of nitrogen fixation demands large amounts of phosphorus [119]. When soy‐ bean plants are starved of phosphorus, 44 phosphate starvation proteins are expressed in soybean nodules [120].

mean germination time for Lee68 (0.3 days) and N2899 (1.0 day) was delayed, compared with control plants. Hormonal responses to salt stress differed between these cultivars. Both cultivars, increased abscisic acid levels and decreased giberrelic acid (GA 1, 3) and isopenty‐ ladenosine concentrations; auxin (IAA) increased in Lee68, but remained unchanged in N2899. 2-D gel electrophoresis, followed by MALDI-TOF-MS analysis, of the proteins from germinated seeds suggested increases in ferritin and the 20S proteasome subunit β-6 in both the cultivars. Glyceraldehyde 3-phosphate dehydrogenase, glutathione *S*-transferase (GST) 9, GST 10, and seed maturation protein PM36 were down-regulated in Lee68, but these pro‐ teins were naturally present in low concentrations in N2899 and were seen to up-regulate

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

The soybean-*Phytophthora soje* plant-oomycete interaction is of agriculture and economic im‐ portance, as this oomycete causes soybean root and stem rot, translating to an annual global loss of \$1-2 billion US. Twenty-six proteins were significantly affected in a resistant soybean cultivar (Yudou25) and 20 in a sensitive one (NG6255), as determined by 2-D gel analysis, followed by MALDI-TOF-MS. The distribution pattern of the affected proteins were - 26% energy regulation, 15% protein destination and storage, 11% defense against disease, 11% metabolism, 9% protein synthesis, 4% secondary metabolism, and 24% unknown/hypotheti‐

Soybean mosaic virus (SBMV) causes one of the most serious viral infections of soybean; leaves of infected plants were studied at a series of time points using 2-D gel electrophore‐ sis, followed by MALDI-TOF-MS and tandem TOF/TOF-MS. Proteins expressed in the ino‐ culated leaves were identified and were seen to be involved in protein degradation, defense signalling, coping with changes in the levels of reactive oxygen species, cell wall reinforce‐ ment, and energy and metabolism regulation. Quantitative real time PCR was used to focus on gene expression related to some of these proteins. Photosynthesis and metabolism relat‐ ed genes were down-regulated at all the time points, while most of the energy related genes (respiration in this case) were up-regulated for at least five of the six time points studied [117]. At the time of this writing, this report is the only one addressing the proteomic ap‐

Aluminium toxicity is often observed in acidic soils and Baxi 10 (BX10) is an Al-resistant cul‐ tivar. One-week-old soybean seedlings treated with 50 mM AlCl3 for 24, 48 and 72 h were studied for characterization of root proteins in response to Al; and 2-D gel electrophoresis followed by MS revealed 39 proteins expressed differentially following Al treatment. Of these 21 were up-regulated (such as heat shock proteins, glutathione S-transferase, chalcone related synthetase, GTP-binding protein, ABC transporters and ATP binding proteins). Five proteins were also down-regulated and 15 newly induced proteins were present following

proach to molecular understanding of soybean-SBMV interaction.

*2.2.6. Other miscellaneous stress related reports*

following exposure to salt stress [115].

*2.2.5. Biotic stress*

Relationships

14

cal proteins [116].

AL treatment [118].

Label free proteomics, coupled with multiple reaction monitoring (MRM) with synthetic iso‐ tope labelled peptides, was used to study 10 allergens from 20 non-genetically modified commercial varieties of soybean. The concentration of these allergens varied between 0.5-5.7 *μ*g mg-1 of soybean protein. At the time of this writing, this is the only proteomic report on soybean allergens [121].

The responses of soybean plants exposed to 116 ppb O3 involved significant changes to car‐ bon metabolism, photosynthesis, amino acid, flavanoid and isoprenoid biosynthesis, signal‐ ing, homeostasis, anti-oxidant and redox pathways [122], as indicated by shifts in expression of the relevant proteins.

More information regarding soybean functional genomics and proteomics is available at the publicly accessible Soybean Knowledgebase (SoyKB) http://soykb.org/ [123].
