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Available online at www.sciencedirect.com

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Comparative proteomic and physiological analyses reveal the protective effect of exogenous calcium on the germinating soybean response to salt stress Yongqi Yina,b , Runqiang Yanga , Yongbin Hana , Zhenxin Gua,⁎ a

College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China College of Food Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225127, PR China

b

AR TIC LE I N FO

ABS TR ACT

Article history:

Calcium enhances salt stress tolerance of soybeans. Nevertheless, the molecular

Received 12 March 2014

mechanism of calcium's involvement in resistance to salt stress is unclear. A comparative

Accepted 26 September 2014

proteomic approach was used to investigate protein profiles in germinating soybeans under NaCl-CaCl2 and NaCl-LaCl3 treatments. A total of 80 proteins affected by calcium in 4-day-old germinating soybean cotyledons and 71 in embryos were confidently identified.

Keywords:

The clustering analysis showed proteins were subdivided into 5 and 6 clusters in cotyledon

Soybean

and embryo, respectively. Among them, proteins involved in signal transduction and

Proteomic

energy pathways, in transportation, and in protein biosynthesis were largely enriched while

Calcium

those involved in proteolysis were decreased. Abundance of nucleoside diphosphate kinase

Salt

and three antioxidant enzymes were visibly increased by calcium. Accumulation of

Germination

gamma-aminobutyric acid and polyamines was also detected after application of

Gamma-aminobutyric acid

exogenous calcium. This was consistent with proteomic results, which showed that proteins involved in the glutamate and methionine metabolism were mediated by calcium. Calcium could increase the salt stress tolerance of germinating soybeans via enriching signal transduction, energy pathway and transportation, promoting protein biosynthesis, inhibiting proteolysis, redistributing storage proteins, regulating protein processing in endoplasmic reticulum, enriching antioxidant enzymes and activating their activities, accumulating secondary metabolites and osmolytes, and other adaptive responses. Biological significance Soybean (Glycine max L.), as a traditional edible legume, is being targeted for designing functional foods. During soybean germination under stressful conditions especially salt stress, newly discovered functional components such as gamma-aminobutyric acid (GABA) are rapidly accumulated. However, soybean plants are relatively salt-sensitive and the growth, development and biomass of germinating soybeans are significantly suppressed under salt stress condition. According to previous studies, exogenous calcium counters the harmful effect of salt stress and increases the biomass and GABA content of germinating soybeans. Nevertheless, the precise molecular mechanism underlying the role of calcium in resistance to salt stress is still unknown. This paper is the first study employing comparative proteomic and physiological analyses to reveal the protective effect of

⁎ Corresponding author. Tel./fax: +86 25 84396293. E-mail address: [email protected] (Z. Gu).

http://dx.doi.org/10.1016/j.jprot.2014.09.023 1874-3919/© 2014 Elsevier B.V. All rights reserved.

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exogenous calcium in the germinating soybean response to salt stress. Our study links the biological events with proteomic information and provides detailed peptide information on all identified proteins. The functions of those significantly changed proteins are also analyzed. The physiological and comparative proteomic analyses revealed the putative molecular mechanism of exogenous calcium treatment induced salt stress responses. The findings from this paper are beneficial to high GABA-rich germinating soybean biomass. Additionally, these findings also might be applicable to the genetic engineering of soybean plants to improve stress tolerance. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Soybean (Glycine max L.) is a traditional edible legume. It is very popular in China, Korea, Japan, and other Southeast Asian countries. Large amounts of protein, minerals, vitamins, and functional compounds such as isoflavone, saponins and phosphatidylcholine exist in its seeds [1]. However, its nutritional value is limited by anti-nutritional factors like phytic acid, lectins, and tannins [2]. Studies have demonstrated that seed germination can improve nutritional composition and lower the levels of antinutritional components in legumes [3–5]. Newly discovered functional components such as gammaaminobutyric acid (GABA) are generated after germination. GABA, a neurotransmitter in the mammalian brain and spinal cord, can prevent certain forms of cancer and reduce the risk of cardiovascular disease [6]. For this reason, GABA-enriched germinating soybeans have become popular as a source of therapeutic and salubrious functional ingredient. Germination under abiotic stress such as hypoxia and salt stress are the most common and effective ways of causing GABA to accumulate in soybeans [7–9]. However, the growth, development and biomass of germinating soybeans were found here to be significantly suppressed under stressful germination conditions, although the GABA content increased markedly. Calcium has been widely examined for its protective role in most abiotic stresses: drought, cold, heat, heavy metals, oxidative stresses, and especially salt stress [10–13]. Although the in vivo role of calcium in plants subjected to abiotic stress or transgenic overexpression has been investigated in recent studies, the traditional and classical approach is the use of exogenous calcium [14–16]. Application of exogenous calcium can improve the inhibition of growth and development and maintain the integrity of cell function and structure of plants under abiotic stress [17,18]. Similarly, according to our previous studies, exogenous calcium countered the harmful effect of salt stress and increased the biomass and GABA content in germinating soybean plants [9]. All of the studies cited above indicate that calcium might be an ideal biotechnological target for improvement of GABA-enriched germinating soybeans grown under stressful conditions [9]. Nevertheless, the precise molecular mechanism underlying the role of calcium in resistance to salt stress is still unclear. Proteomics is the best molecular tool available for describing the proteome profile and dissecting the complex molecular mechanism underlying plant physiology [19]. Shi et al. identified 36 differentially regulated proteins in Bermuda grass and proposed that polyamines could activate electron transport and energy pathways that facilitated the adaptation of Bermuda grass to salt and drought [20]. Hossain et al.

suggested that β-aminobutyric acid pretreatment helped the soybean to combat cadmium stress by modulating the defense mechanism in plants and activating their cellular detoxification system [21]. Another previous study demonstrated that exogenous calcium increased the abundance of proteins involved in fermentative metabolism, the TCA cycle, glycolysis, defense against reactive oxygen species, and in nitrogen metabolism in cucumber roots under hypoxic conditions [22]. It was also found that the impact of salinization on metabolism, ripening process, and on inducing salt tolerance was limited by calcium in tomato fruit [23]. However, only a limited number of studies have investigated the effects of exogenous calcium on the proteome of soybean germinating in the dark and under salt stress. The present study was to derive new insight into the molecular mechanism of calcium mediated salt stress response using exogenous calcium and the inhibitor of Ca2+ channels on soybean plants germinating in the dark. This might shed light on the role of calcium in physiological response of plants to salt stress in vivo. Comparative proteomic analysis was performed via two-dimensional electrophoresis (2-DE), and MALDI-TOF/ TOF-MS was performed to identify differentially displayed proteins affected by calcium. The effect of exogenous calcium and the inhibitor of Ca2+ channels on protein level changes in the cotyledons and embryos of germinating soybeans were also compared. The results of physiological assays and comparative proteomic analyses might also provide information regarding the physiological and molecular mechanisms of calcium in the response of germinating soybean plants to salt stress.

2. Materials and methods 2.1. Plant materials and growth conditions Dry soybean seeds (Glycine max L. cultivar Yunhe) were sterilized with 10 mM sodium hypochlorite for 30 min, washed, and steeped with distilled water at 30 ± 1 °C for 4 h. The soaked seeds were then placed in a soybean sprouting machine and germinated in a dark incubator at 30 ± 1 °C for 4 d with culture solution containing different additives: (a) NaCl: 50 mM NaCl; (b) NaCl-CaCl2: 50 mM NaCl + 6 mM CaCl2; (c) NaCl-LaCl3: 50 mM NaCl + 5 mM LaCl3. Based on a previous study and on pilot experiments, 50 mM NaCl, 6 mM CaCl2, and 5 mM LaCl3 were selected as effective concentrations [9]. During the 4 days of experiment, the culture solution was replaced daily. Three independent biological experiments were performed, and 4 d after treatment, the cotyledons and embryos were carefully collected for identification of differentially displayed proteins using the proteomic approach.

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Soybean materials were collected during germination for an assay of physiological parameters.

2.2. Protein extraction and 2-DE Total protein extraction was performed using trichloroacetic acid (TCA)/acetone precipitation with some modifications [24], and the protein content was determined according to the Bradford method [25]. To minimize errors, ten cotyledons or embryos from ten germinated soybeans were pooled for each biological repeat sample. Briefly, sample was ground in liquid nitrogen, and then protein powder was suspended in 10 wt.%/vol. TCA in acetone containing 1 mM dithiothreitol (DTT), and held at − 20 °C for 2 h. After centrifugation and rinse, the pellet was air dried and dissolved at 4 °C in lysis buffer [7 M urea, 2 M thiourea, 4 wt.%/vol. of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate, 40 mM DTT and 2% (v/v) pharmalyte]. The 2-DE was performed as previously described with minor modification [22]. Briefly, 1 mg of cotyledon protein was applied onto a linear gradient immobilized pH gradient (IPG) strip (24 cm, pH 4–7, GE Healthcare, Buckinghamshire, UK), and was subjected to isoelectric focus (IEF) in the Protein IEF system (GE Healthcare) at 20 °C for a total of 94,850 V · h. Similarly, 0.8 mg embryo protein was applied onto a linear gradient IPG strip (18 cm, pH 4–7, GE Healthcare), and was subjected to IEF for a total of 90,850 V · h. After IEF, the strips were equilibrated and the second electrophoresis dimension was performed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and run on the Ettan Six vertical set (GE Healthcare) in electrophoresis buffer at 15 °C with a cooling device (GE Healthcare). Then the gels were stained with Coomassie brilliant blue (CBB) G-250.

re-suspended in 5 mg/mL a-cyano-4-hydroxy-trans-cinnamic acid containing 0.1% TFA and 50% ACN.[26] MALDI-TOF/TOF-MS analyses were conducted by an ABI 4800 Plus MALDI-TOF/TOF™ analyzer (Applied Biosystems, California, USA). Both the MS and MS/MS data were integrated and performed using the GPS Explorer™ software V3.6 (Applied Biosystems). The obtained peptide mass spectra were searched using an in-house licensed MASCOT search engine V3.5 (Matrix Science, London, UK), and compared to the soybean genome sequence database which was downloaded from the phytozome database (version 8.0, http://www. phytozome.net/soybean). The following parameters were used in the search: trypsin as the digestion enzyme; one missed cleavage allowed; cysteine carbamidomethylation as a fixed modification and methionine oxidization as a variable modification; no fixed modifications; 50 ppm for peptide ion tolerance and 0.2 Da for fragment ion tolerance. Only significant hits, as defined by the MASCOT probability analysis ( p < 0.05) or individual ion score > 38, were accepted. Identified proteins were annotated to their biological function according to KEGG (http://www.kegg.jp/kegg/pathway. html) and the literature. Information on these proteins was obtained from Universal Protein Resource (http://www.uniprot. org/). Moreover, WOLF PSORT prediction (http://wolfpsort.org/) and ESLpred (http://www.imtech.res.in/raghava/eslpred/) were used to predict subcellular localization of all identified proteins.

2.4. Determination of calcium content Calcium content was determined by inductively coupled plasmaoptical emission spectroscopy (ICP-OES, Optima 2100 DV, Perkin Elmer, USA) following the method of Larrea-Marin et al. [27].

2.5. Determination of malondialdehyde (MDA) content 2.3. Gel image analysis and protein spot identification by MALDI-TOF/TOF-MS The stained 2-DE gels were digitalized with a gel scanner (Imagescanner III, GE Healthcare), and analyzed with PDQuest™ Software (Version 7.2.0, Bio-Rad, California, USA). Spots were detected, matched, and normalized on the basis of total quantity of valid spots with the parameter of percent volume according to the software guide. For each protein spot, triplicate gels were used for each sample and the mean relative volume was used to designate the significant and differentially displayed proteins (changed more than 1.5 folds or less than 0.66 fold and statistically significant as calculated by one-way ANOVA, p < 0.05). The differentially displayed protein spots were excised from gels, washed with double-distilled water three times, destained with 25 mM NH4HCO3 for CBB G-250 staining spots, dehydrated with 50% (v/v) acetonitrile (ACN) in 25 mM NH4HCO3, reduced with 10 mM DTT in 50 mM NH4HCO3 for 1 h at 56 °C, alkylated with 55 mM iodoacetamide in 50 mM NH4HCO3 for 1 h at 25 °C, dried twice with 100% ACN, and digested overnight at 37 °C with sequencing grade modified trypsin (Promega, Madison, WI, USA) in 50 mM NH4HCO3. The peptides were extracted twice by washing the gel pieces with 0.1% trifluoroacetic acid (TFA) in 67% ACN. Extracts were pooled and lyophilized. The lyophilized tryptic peptides were

The extent of lipid peroxidation in terms of MDA formation was measured following the method of Madhava and Sresty [28].

2.6. Determination of H2O2 content and antioxidant enzyme activities The H2O2 content was determined using a H2O2 Assay Kit (A064, Nanjing Jiancheng Bioengineering Institute, China). The superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), and peroxidase (POD, EC 1.11.1.7) activities were determined using an SOD Assay Kit (A001, Nanjing Jiancheng Bioengineering Institute, China), a CAT Assay Kit (A007, Nanjing Jiancheng Bioengineering Institute, China), a Plant POD Assay Kit (A084-3, Nanjing Jiancheng Bioengineering Institute, China), respectively. The glutathione peroxidase (GPX, EC 1.11.1.9) activity was measured following the method of Drotar et al. [29].

2.7. Measurements of proline content, GABA content and free polyamines (PAs) content The amount of proline was measured according to Li et al. [18]. The soluble sugar was determined according to Shi et al. [20] Free PAs content was analyzed by HPLC as described by Xing et al. [8]. GABA content was determined according to Yang et al. [30].

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2.8. Statistical analyses All experiments in this study were repeated at least three times in independent experiments. Average values and standard deviations were computed according to the experimental data. One way analysis of variance (ANOVA) with Tukey's test was conducted on the data, and a p value at 0.05 was considered significant.

3. Results 3.1. Changes of calcium content in germinating soybeans The calcium content was significantly higher (p < 0.05) in both organs of germinating soybeans exposed to CaCl2 than in NaCl-stressed control and LaCl3 treatment (Fig. 1). After germinating for 2 and 4 d under CaCl2 treatment, the calcium contents were 1.75- and 2.82-fold of the NaCl-stressed control in cotyledon, and 2.96- and 5.14-fold in embryo, respectively (Fig. 1). The calcium content in the cotyledons and embryos of 2-day germinating soybeans treated with LaCl3 was not significantly different from that in the plants subjected to NaCl stress alone, but the 4-day germinating soybeans showed significantly less calcium content than plants subjected to NaCl stress alone (Fig. 1). The above facts indicated that calcium content was significantly affected by CaCl2 and LaCl3 in soybeans during germination.

3.2. Changing patterns in protein abundance in germinating soybeans

Calcium content (mg/100g DW)

Based on 2-DE and MALDI-TOF/TOF-MS, comparative analysis of cotyledon proteins and embryo proteins from the NaClstressed, germinating soybeans treated with NaCl-CaCl2 and NaCl-LaCl3 showed differential abundance of some proteins (Fig. 2). Three independent experiments were performed

400

a

NaCl NaCl+CaCl2 NaCl+LaCl3

300

a

200

a a

100

b

b

b

c

b

b b

c

SC-4d

SE-4d

0 SC-2d

SE-2d

Fig. 1 – Calcium content affected by exogenous calcium treatment under salt stress in germinating soybeans. SC-2d: 2-day germinated soybean cotyledons; SE-2d: 2-day germinated soybean embryos; SC-4d: 4-day germinated soybean cotyledons; SE-4d: 4-day germinated soybean embryos. The results shown are the means ± SE of three independent experiments.

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(Supplementary Figs. S1 and S2). A total of 80 proteins were found to be affected by CaCl2 and LaCl3 in germinating soybean cotyledons and 71 in embryos (Tables 1, 2, and Fig. 3). A hierarchical clustering analysis was performed with the abundance data of proteins in germinating soybean cotyledons and embryos from the NaCl, NaCl-CaCl2 and NaCl-LaCl3 samples to determine the nature of any trends. The clustering analysis led to the division of the 80 cotyledon proteins into 5 prominent categories while 71 embryo proteins were subdivided into 6 clusters (Tables 1, 2, and Fig. 4). The abundance of proteins in germinating soybean cotyledons belonging to clusters i and ii gradually decreased under the NaCl-LaCl3 treatment compared with NaCl treatment. Under NaCl-CaCl2 treatment, the abundance of cotyledon proteins belonging to cluster iii was lower than that in NaCl-stressed controls while higher than that in samples treated with NaCl-LaCl3. However, the abundance change tendency of cotyledon proteins belonging to clusters iii and iv was quite the opposite. The intensity of cluster v proteins exposed to LaCl3 showed higher than that of NaCl-treated and CaCl2treated samples. In germinating soybean embryos, the abundance of proteins under NaCl and NaCl-CaCl2 treatment belonging to clusters i and ii decreased while that belonging to clusters iv and v increased compared with NaCl-LaCl3 treatment. Among the 80 proteins identified in germinating soybean cotyledons, 26 showed increased abundance under CaCl2 treatment and 20 under LaCl3 treatment. A total of 53 proteins showed decreased abundance in cotyledons exposed to CaCl2 treatment and 47 proteins showed decreased abundance in those exposed to LaCl3 (Supplementary Fig. S3, Supplementary Table S1). Functional class analysis showed that most of the cotyledon proteins were seed storage proteins (SSPs), and the remainder was divided into 5 functional classes, i.e. metabolism, cell growth/division, proteolysis, transportation and disease/defense (Fig. 5). All identified cotyledon proteins were analyzed with WoLF PSORT and ESLpred to determine their subcellular localization. More than 50% of the differentially displayed proteins identified were predicted to be chloroplast proteins, and most of the remaining proteins were predicted to localize in the cytoplasm and nucleus (Fig. 5). Out of 71 embryo proteins found to be affected by the treatments, 38 showed increased abundance under CaCl2 treatment and 14 under LaCl3 treatment, and 26 proteins showed decreased abundance under CaCl2 and 40 under LaCl3 treatment (Supplementary Fig. S4, Supplementary Table S1). These embryo proteins were divided into 10 functional classes, i.e. metabolism, energy, disease/defense, and protein synthesis. The majority of the identified embryo proteins were localized in the cytoplasm and chloroplasts (Fig. 5). Twelve of the embryo proteins were uncharacterized. Homologs of these proteins were located using BLAST (http://www. ncbi.nih.gov/BLAST/). Five of them showed more than 65% positive matches at the amino acid level, indicating that they might have functions similar to those of the logged proteins (Supplementary Table S3). In cotyledons, 64 proteins were found to be coregulated by both CaCl2 and LaCl3, and 41 in embryos, indicating that these proteins might play important roles in calcium-mediated stress responses (Supplementary Fig. S3 and Fig. S4).

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Fig. 2 – 2-DE pattern of proteins in 4-day germinated soybean cotyledons and embryos subjected to NaCl + CaCl2 or NaCl + LaCl3 treatment. NaCl-treated soybean plants germinated for 4 days served as a control. Proteins extracted from (A) cotyledons and (B) embryos were separated using 2-DE. In cotyledons, production of most of the SSPs increased under CaCl2 treatment and that of proteolysis-related proteins decreased (Fig. 5A). All of the energy-related, proteolysis-related, cell-growth/division-related, transportation-related, and signaltransduction-related embryo proteins and most of the metabolism-related and secondary-metabolism-related embryo proteins increased significantly under CaCl2 treatment, but all transportation-related, signal-transduction-related, and secondary-metabolism-related embryo proteins decreased under LaCl3 treatment (Fig. 5E and F). Three antioxidant enzymes (E47, POD precursor; E48, MnSOD; E51, phospholipid hydroperoxide GPX) were also significantly affected by CaCl2 and LaCl3, indicating that ROS metabolism and the underlying antioxidant enzymes might be affected by calcium (Fig. 3 and Table 2).

3.3. Calcium and accumulation of reactive oxygen species (ROS) and antioxidant enzyme activity in germinating soybeans The concentrations of the leading indicators of stress-triggered oxidative damage and ROS level, MDA and H2O2, were measured

in germinating soybeans under CaCl2 and LaCl3 treatment. The MDA and H2O2 concentrations were significantly lower in both organs of the 4-day-old plants exposed to CaCl2 than those in untreated, NaCl-stressed controls, but higher in the plants treated with LaCl3 (Fig. 6A–C). Furthermore, after 4 days of germination, the NaCl-CaCl2-treated germinating soybeans clearly showed more growth than NaCl-stressed germinating soybeans. The NaCl-LaCl3-treated plants showed markedly less growth (Supplementary Fig. S5A). Under NaCl stress conditions, the CaCl2 treated plants showed significantly greater sprout length, higher respiratory rate and heavier fresh weight than the untreated plants, while the sprout length, respiratory rate and fresh weight of LaCl3 treated plants decreased significantly ( p < 0.05), compared with the NaCl-stressed plants (Supplementary Fig. S5B–D). These findings showed that exogenous application of CaCl2 could improve NaCl stress tolerance in germinating soybeans. Proteomic analysis indicated that three antioxidant enzymes were affected by CaCl2 and LaCl3 treatment, therefore antioxidant enzyme activities were further assayed. Under

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Table 1 – List of changed proteins in cotyledon of germinated soybeans under NaCl-CaCl2 and NaCl-LaCl3 treatments. No. a

Homolog protein

b

Accession no. c

Plant species

TMr/TpI d

EMr/EpI

e

Score f M.P. g SC h% Local i

Metabolism j C1 UTP—glucose-1-phosphate uridylyltransferase C2 Lactoylglutathione lyase

XP_003544964.1

Glycine max

51.55/5.20

55.89/5.28

534

8

24

Cyto

XP_003534533.1

Glycine max

33.56/6.14

33.78/5.81

185

3

14

Cyto

Cell growth/division C3 LEA protein C4 Seed biotinylated protein 68 kDa C5 35 kDa seed maturation protein C6 Embryonic protein DC-8 C7 Embryonic protein DC-8 C8 Calreticulin-1 precursor C9 Expansin-like B1

AAC61783.1 ACS49840.1 AAD01431.1 XP_003535256.1 XP_003535256.1 NP_001236351.1 XP_003549946.1

Glycine Glycine Glycine Glycine Glycine Glycine Glycine

max max max max max max max

67.89/6.10 67.96/6.18 35.32/5.96 48.77/6.12 48.77/6.12 48.31/4.43 28.16/6.30

67.14/6.38 66.25/6.37 39.73/6.04 54.06/6.22 49.85/6.38 52.09/4.65 25.65/6.01

782 755 372 446 634 298 469

7 8 8 8 10 4 8

18 18 28 22 31 11 32

Mito Mito Nucl Nucl Nucl Mito Chlo

Proteolysis C10 Bd 30 K C11 Cysteine proteinase RD21a C12 Bd 30 K C13 Bd 30 K C14 Inhibitor Tia, trypsin C15 Bowman–Birk inhibitor C16 Kunitz trypsin protease inhibitor C17 Chain A, soybean trypsin inhibitor

BAA25899.1 XP_003545327.1 ACG59282.1 BAA25899.1 1108235A AAD09816.1 P01071.1 1BA7

Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine

max max max max max max max max

43.13/5.63 55.56/5.70 43.13/5.65 43.13/5.65 20.31/4.61 15.09/6.67 20.25/4.66 20.31/4.61

29.56/4.46 39.67/4.69 32.59/4.59 33.86/4.67 14.86/5.19 18.71/4.54 21.67/4.78 20.69/4.84

132 411 166 166 411 142 136 862

2 5 2 2 4 2 2 9

4 19 4 4 30 59 21 58

Nucl Chlo Vacu Vacu Cyto Chlo Cyto Cyto

BAA74452.2 AAO45103.1 BAA23360.2 AAO45103.1 XP_003556052.1 BAA74452.2 BAA74452.2 BAA74452.2 BAA74452.2 BAA74452.2 BAA74452.2 BAA74452.2 XP_003556051.1 XP_003556051.1 XP_003556051.1 P13916.2 P13916.2 P13916.2 P13916.2 BAA23360.2 BAA23360.2 BAA23360.2 BAA23360.2 BAA23360.2 P13916.2 P13916.2 P13916.2 P13916.2 1IPJ|A

Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine

max max max max max max max max max max max max max max max max max max max max max max max max max max max max max

65.16/5.23 44.99/5.62 63.18/4.92 44.99/5.62 50.47/5.88 65.16/5.23 65.16/5.23 65.16/5.23 65.16/5.23 65.16/5.23 65.16/5.23 65.16/5.23 70.55/5.12 70.55/5.12 70.55/5.12 70.53/5.07 70.53/5.07 70.53/5.07 70.53/5.07 63.18/4.92 63.18/4.92 63.18/4.92 63.18/4.92 63.18/4.92 70.54/5.07 70.54/5.07 70.54/5.07 70.54/5.07 47.88/5.67

27.88/4.35 11.47/4.37 25.82/4.55 21.23/5.12 20.88/5.41 47.65/4.96 49.78/5.01 43.69/5.16 51.75/5.21 49.15/5.13 67.89/5.20 57.98/5.29 67.35/5.19 57.09/5.15 56.96/5.19 66.68/5.02 50.12/5.03 41.89/5.31 39,34/5.17 31.59/5.21 39.57/5.24 56.43/5.13 58.83/5.24 59.45/5.45 63.23/5.13 36.83/5.45 33.67/5.48 37.46/4.89 28.34/5.66

334 62 257 380 270 428 553 614 710 748 607 717 365 598 579 657 692 661 402 375 332 126 440 649 460 532 706 325 454

6 3 4 7 4 8 8 8 9 9 8 9 4 9 9 10 9 8 7 6 5 4 8 8 8 6 9 5 6

12 6 8 17 14 15 16 18 22 20 20 20 10 17 17 20 20 17 12 11 9 6 16 18 17 14 18 11 19

Chlo Cyto Cyto Cyto Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Cyto Cyto Cyto Cyto Cyto Chlo Chlo Chlo Chlo Mito

1IPJ|A

Glycine max

47.88/5.67

23.76/4.91

396

5

15

Mito

1IPJ|A

Glycine max

47.88/5.67

24.45/5.01

283

4

12

Mito

1309256A 1309256A

Glycine max Glycine max

56.28/5.78 56.30/5.89

12.54/4.94 12.65/5.05

192 149

3 3

7 6

Chlo Chlo

Storage C18 Alpha′ subunit of beta-conglycinin C19 Alpha′ subunit of beta-conglycinin C20 Alpha′ subunit of beta-conglycinin C21 Beta-conglycinin alpha′ subunit C22 Beta-conglycinin alpha′ subunit C23 Alpha′ subunit of beta-conglycinin C24 Alpha′ subunit of beta-conglycinin C25 Alpha′ subunit of beta-conglycinin C26 Alpha′ subunit of beta-conglycinin C27 Alpha′ subunit of beta-conglycinin C28 Alpha′ subunit of beta-conglycinin C29 Alpha′ subunit of beta-conglycinin C30 Beta-conglycinin, alpha chain C31 Beta-conglycinin, alpha chain C32 Beta-conglycinin, alpha chain C33 Beta-conglycinin, alpha chain C34 Beta-conglycinin, alpha chain C35 Beta-conglycinin, alpha chain C36 Beta-conglycinin, alpha chain C37 Alpha subunit of beta conglycinin C38 Alpha subunit of beta conglycinin C39 Alpha subunit of beta conglycinin C40 Alpha subunit of beta conglycinin C41 Alpha subunit of beta conglycinin C42 Beta-conglycinin, alpha chain C43 Beta-conglycinin, alpha chain C44 Beta-conglycinin, alpha chain C45 Beta-conglycinin, alpha chain C46 Soybean Beta-conglycinin beta homotrimers complexes with N-acetyl-D-glucosamine C47 Soybean beta-conglycinin beta homotrimers complexes with N-acetyl-D-glucosamine C48 Soybean beta-conglycinin beta homotrimers complexes with N-acetyl-D-glucosamine C49 Glycinin G1 C50 Glycinin G1

(continued on next page)

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Table 1 (continued) No. a

Accession no. c

Plant species

TMr/TpI d

EMr/EpI

1309256A ACT53400.1 CAB57802.1 CAB57802.1 1309256A 1309256A P04776.2 P04776.2 P04776.2 P04776.2 P04776.2 NP_001235810.1 NP_001235810.1 NP_001235810.1 NP_001235810.1 NP_001235810.1 NP_001235810.1 AAB23212.1 AAB23212.1 CAA55977.1 NP_001238008.1 AAB71140.1

Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Soybeans Glycine soja Glycine max Glycine max

56.30/5.89 44.07/5.51 24.35/4.46 24.35/4.46 56.28/5.78 56.28/5.78 56.29/5.89 56.29/5.89 56.29/5.89 56.29/5.89 56.29/5.89 54.36/5.46 54.36/5.46 54.93/5.46 54.93/5.46 54.93/5.46 58.12/5.78 64.09/5.38 64.10/5.38 58.61/5.52 64.21/5.17 19.02/5.20

16.32/6.81 17.43/5.41 24.73/4.34 24.79/4.39 40.42/5.22 44.74/5.19 41.82/5.44 40.62/5.53 59.29/5.57 41.99/5.59 22.89/6.70 41.97/5.16 42.39/5.28 36.87/5.49 36.74/5.64 29.86/5.79 24.86/6.87 41.61/5.03 15,85/5.89 44.85/5.23 40.12/5.03 18.21/5.03

207 352 796 589 400 394 725 340 596 630 464 741 780 694 689 259 482 600 289 979 899 150

4 6 7 4 9 4 9 5 8 9 9 10 8 9 9 6 6 7 3 8 8 6

9 16 43 32 24 16 27 15 26 24 21 25 23 22 22 15 19 17 8 21 23 13

Chlo Chlo Nucl Nucl Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Chlo Mito

Transportation C74 Maturation polypeptide C75 Maturation polypeptide C76 Sucrose-binding protein-like C77 Coatomer subunit beta′-2-like

NP_001238546.1 NP_001238546.1 XP_003536630.1 XP_003546337.1

Glycine Glycine Glycine Glycine

max max max max

50.61/6.33 50.61/6.33 58.35/6.08 35.38/5.77

58.33/6.43 57.82/6.76 18.09/5.68 42.89/5.96

370 777 154 432

7 10 4 10

25 28 9 33

Nucl Nucl Cyto Cyto

Disease/defense C78 Nad-dependent formate dehydrogenase C79 Endoplasmin homolog C80 Acidic chitinase

BAA77337.1 XP_006596543.1 BAA77676.1

Oryza sativa Glycine max Glycine max

41.45/6.87 93.49/4.83 32.18/5.01

20.47/6.37 66.68/4.91 33.86/4.77

244 79 312

3 5 6

11 7 12

Cyto Nucl Chlo

C51 C52 C54 C55 C56 C57 C58 C59 C60 C61 C62 C63 C64 C65 C66 C67 C68 C69 C70 C71 C72 C73

Homolog protein

b

Glycinin G1 Mutant glycinin subunit A1aB1b Glycinin Glycinin Glycinin A1aBx Glycinin A1aBx Glycinin A1a subunit Glycinin A1a subunit Glycinin A1a subunit Glycinin A1a subunit Glycinin A1a subunit Glycinin G2 precursor Glycinin G2 precursor Glycinin G2 precursor Glycinin G2 precursor Glycinin G2 precursor Glycinin A3B4 subunit Glycinin G4 subunit Glycinin G4 subunit Gy5 Glycinin A5A4B3 precursor 2S albumin precursor

e

Score f M.P. g SC h% Local i

a

Spot no., spot numbering corresponds to the 2-DE gel in Fig. 2. Homolog protein name and species obtained via the MASCOT software from the database. c Accession no., accession number according to the database. d TMr, theoretical molecular weight. TpI, theoretical isoelectric point. e EMr, experimental molecular weight. EpI, experimental isoelectric point. f Score, MOWSE score probability for the entire protein. g M.P., number of query matched peptides. h SC, sequence coverage. i Local, localization category using local classification, Chlo, chloroplast; Mito, mitochondria; Nucl, nuclear; Cyto, cytoplasm; Cysk, cytoplasm skeleton; E.R., endoplasmic reticulum; Vacu, vacuole. b

NaCl stress conditions, the CaCl2-treated germinating soybeans exhibited significantly higher activity of four major antioxidant enzymes (SOD, POD, CAT and GPX) in both cotyledons and embryos after 4 days of treatment than the untreated germinating soybeans (Fig. 7C–F). The antioxidant activity of these four enzymes was significantly lower under LaCl3 treatment than under NaCl stress alone (Fig. 7C–F). These results indicated that treatment with exogenous CaCl2 might increase antioxidant enzyme activity, which in turn might lead to less ROS accumulation in germinating soybeans.

those in the plants subjected to NaCl stress alone (Fig. 7A). As expected, the GABA content was markedly higher in both organs in CaCl2-treated germinating soybeans than in the untreated germinating soybeans (Fig. 7B). The concentration of endogenous free PAs was markedly higher in both CaCl2treated and LaCl3-treated germinating soybean plants (Fig. 7C). Calcium might positively modulate the concentration of proline, GABA and free PAs under salt stress, which in turn regulated plant stress tolerance.

3.4. Calcium and accumulation of osmolytes under salt stress

4. Discussion

The proline content in the cotyledons of the 4-day germinating soybeans treated with CaCl2 had shown no significant differences from that in the plants subjected to NaCl stress alone, but the embryos showed significantly less proline than

Previous studies clearly indicated that soybean plants were relatively salt-sensitive and that salt stress had a number of deleterious effects, especially on soybean growth and development [31,32]. However, the concentration of GABA, a new

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Table 2 – List of changed proteins in embryo of germinated soybean under NaCl-CaCl2 and NaCl-LaCl3 treatments. No.

Homolog protein

Accession no.

Plant species

TMr/TpI

EMr/EpI Score M.P. SC % Local

Metabolism E1 UTP—glucose-1-phosphate uridylyltransferase E2 Glutamine synthetase PR-2 E3 Phospholipase D alpha 1 E4 Aminoaldehyde dehydrogenase E5 Glutamine synthetase precursor E6 S-adenosylmethionine synthetase E7 S-adenosylmethionine synthase E9 Linoleate 9S-lipoxygenase-4 E10 Methionine synthase E11 UDP-N-acetylglucosamine pyrophosphorylase E12 Ubiquitin-conjugating enzyme E2 variant 1D E13 Nucleoside diphosphate kinase E14 Regulator of ribonuclease

XP_003544964.1 P24099.1 XP_003531710.1 KC478661.1 XP_006593484.1 XP_003546550.1 XP_003554569.1 P38417.1 AAQ08403.1 XP_003531103.1 XP_006588855.1 AAN77501.1 XP_003563400.1

Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Glycine max Brachypodium distachyon

51.55/5.20 39.29/5.32 92.06/5.42 55.60/5.23 47.91/8.34 43.42/5.50 43.37/5.74 96.64/5.71 84.40/5.93 54.69/5.88 16.71/6.20 16.33/6.30 18.30/5.60

56.72/5.28 40.69/5.40 95.45/5.78 70.69/5.62 49.39/5.68 48.65/5.82 56.85/6.04 98.64/6.26 88.26/6.50 60.45/6.38 18.26/6.72 16.95/6.61 18.38/5.13

535 129 465 395 342 606 555 528 703 231 366 302 64

8 3 9 3 7 8 8 10 10 6 9 6 2

23 13 17 6 24 32 25 13 20 12 56 40 11

Cyto Chlo Cysk Chlo Chlo Cyto Cyto Cyto Cyto Cyto Cyto Cyto Cyto

Energy E15 ATP synthase 24 kDa subunit E16 NADP-dependent isocitrate dehydrogenase E17 NADP-dependent malic enzyme E18 NADP-dependent malic enzyme E19 ATPase subunit 1 E20 V-type proton ATPase subunit E

XP_003525085.1 AAC64182.1 XP_003547744.1 XP_003516804.1 AFR34317.1 XP_003533059.1

Glycine Glycine Glycine Glycine Glycine Glycine

max max max max max max

27.51/6.09 47.11/8.67 65.43/5.75 65.32/5.83 55.58/6.23 26.98/5.87

29.56/5.41 53.79/6.19 72.54/5.94 15.34/6.15 58.57/6.48 31.67/6.26

339 75 272 332 658 240

4 2 9 10 8 7

13 5 18 17 23 31

Mito Cyto Cyto Cyto Mito Mito

Cell growth/division E21 Calreticulin-1 precursor E22 cell division cycle protein 48 E23 Ripening related protein E24 Embryonic protein DC-8

BAF36056.1 P54774.1 AAD50376.1 XP_003535256.1

Glycine Glycine Glycine Glycine

max max max max

48.31/4.43 90.51/5.18 17.72/5.38 48.77/6.12

63.81/4.43 97.67/5.45 16.70/6.51 50.87/6.54

297 414 67 644

4 7 2 10

11 34 17 25

Mito Cyto Cyto Nucl

Protein synthesis E25 Eukaryotic translation initiation factor 5A3 E26 Elongation factor 2

ACJ76773.1 XP_003531498.1

Glycine max Glycine max

17.68/5.60 18.30/6.00 94.95/5.80 96.69/5.68

457 412

7 10

42 20

Cyto Cyto

Protein destination and storage E27 Kunitz trypsin protease inhibitor E28 Ihibitor Tia,trypsin E29 Trypsin inhibitor p20 E30 Peptidyl-prolyl isomerase FKBP12

ACA23205.1 1108235A BAA82254.1 XP_003593250.1

23.92/5.17 20.31/4.61 22.85/5.39 12.26/7.77

23.41/4.52 21.23/4.59 20.34/5.63 13.85/6.24

573 641 416 150

8 9 7 5

50 58 45 23

Cyto Cyto Chlo Cyto

E31 E32 E33 E34

31kD glycoprotein 31kD glycoprotein Subtilisin-like protease Peptidyl-prolyl cis-trans isomerase CYP20-2

P10743.1 P10743.1 XP_003547763.1 XP_003556583.1

Glycine max Glycine max Glycine max Medicago truncatula Glycine max Glycine max Glycine max Glycine max

29.43/6.72 29.43/6.72 82.53/6.24 27.37/9.10

32.05/6.29 32.42/6.53 84.59/6.15 22.97/6.89

572 517 378 184

9 8 9 3

34 34 17 16

Cyto Cyto Cyto Chlo

Transportation E35 Nuclear transport factor 2 E36 nascent polypeptide-associated complex subunit alpha

XP_003524901.1 XP_003523308.1

Glycine max Glycine max

13.72/5.86 14.16/5.96 23.72/4.43 33.06/4.31

358 134

4 3

45 19

Cyto Cyto

NP_001240182.1 XP_003528531.2

Glycine max Glycine max

22.00/6.95 24.10/6.78 22.04/6.95 22.01/6.98

73 103

2 2

12 22

Cyto Cyto

XP_003545770.1 XP_003543105.1 BAA77676.1 XP_003532006.1 XP_003545030.1 XP_003544594.1 NP_001243073.1

Glycine Glycine Glycine Glycine Glycine Glycine Glycine

17.99/5.14 19.32/4.54 32.18/5.01 64.97/4.86 93.62/4.83 80.42/4.98 41.76/5.93

16.83/4.48 18.97/4.34 32.69/4.35 78.07/4.94 95.27/4.95 84.70/5.11 43.36/6.39

376 537 312 631 79 369 475

5 7 6 8 5 9 7

45 69 12 19 7 16 30

Vacu Vacu Chlo Cyto Nucl Cyto Cyto

XP_003552370.1 AAD11481.1

Glycine max Glycine max

53.79/5.67 65.10/6.00 39.23/6.85 45.72/6.94

467 319

9 6

26 23

Cyto Cyto

Signal transduction E37 Auxin-binding protein ABP19a E38 Auxin-binding protein ABP19a Disease/defense E39 Pathogenesis-related protein 1 E40 pathogenesis-related protein 1 E41 Acidic chitinase E42 Nucleoredoxin 1 E43 Endoplasmin E44 Heat shock cognate protein 80 E45 3′-hydroxy-N-methyl-(S)-coclaurine 4′-O-methyltransferase E46 Selenium-binding protein 1 E47 Peroxidase precursor, partial

max max max max max max max

(continued on next page)

118

J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 1 1 0 – 12 6

Table 2 (continued) No. E48 E49 E50 E51

Homolog protein

Plant species

TMr/TpI

EMr/EpI Score M.P. SC % Local

ABQ52658.1 BAH01715.1 XP_003554704.1 XP_003532707.1

Glycine Glycine Glycine Glycine

max max max max

26.69/8.56 26.81/6.26 23.31/5.55 25.29/9.14

27.15/6.97 23.84/6.67 21.05/6.52 19.17/6.33

250 332 469 410

6 6 5 4

29 41 33 36

Cyto Chlo Chlo Mito

XP_003537997.1 XP_003552695.1 XP_003530453.1 AAB86942.1

Glycine Glycine Glycine Glycine

max max max max

71.43/5.11 71.19/5.10 73.66/5.08 73.82/5.15

78.98/5.23 81.00/5.15 83.89/5.18 83.67/5.23

487 217 747 717

7 7 10 10

15 10 23 19

Cyto Chlo E.R. E.R.

Secondary metabolism E56 Polyphenol oxidase A1, chloroplastic E57 Caffeoyl-CoA O-methyltransferase E58 Isoflavone reductase homolog E59 Chalcone isomerase A

XP_006583808.1 XP_003518701.2 XP_006581203.1 AAK69432.1

Glycine Glycine Glycine Glycine

max max max max

69.13/5.88 37.17/6.57 34.19/6.10 23.31/6.23

79.84/5.72 35.14/5.58 39.32/6.12 27.99/6.57

474 546 450 724

9 8 8 9

16 31 26 47

Chlo Cyto Cyto Chlo

Uncharacterized E61 Uncharacterized E62 Uncharacterized E63 Uncharacterized E64 Uncharacterized E65 Uncharacterized E66 Uncharacterized E67 Uncharacterized E68 Uncharacterized E69 Uncharacterized

XP_003529661.1 NP_001239640.1 XP_003543859.2 NP_001237911.1 NP_001238655.1 NP_001241124.1 NP_001240189.1 NP_001237954.1 NP_001239731.1

Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine Glycine

max max max max max max max max max

14.56/4.05 13.07/4.00 46.29/4.80 16.52/4.68 16.70/4.80 17.15/5.10 38.16/5.32 21.79/5.92 38.56/6.01

27.89/4.05 32.65/4.05 65.82/4.68 16.71/4.66 17.64/4.81 17.03/5.02 44.24/5.30 22.01/6.14 40.73/6.90

165 164 460 124 113 180 304 369 497

2 2 8 2 3 3 6 4 6

36 41 24 24 24 19 31 40 25

Chlo Chlo Nucl Cyto Cyto Cyto Cyto Nucl Chlo

E52 E53 E54 E55

MnSOD PR-5b protein precursor Pro-hevein Phospholipid hydroperoxide glutathione peroxidase Hsc70 Heat shock cognate 70 kDa protein Luminal-binding protein 5 Endoplasmic reticulum HSC70

Accession no.

protein protein protein protein protein protein protein protein protein

LOC100788703 LOC100801797 At5g39570 LOC100305867 LOC100527097 LOC100783267 LOC100780391 LOC100499662 LOC100795412

functional component, was largely increased during soybean germination under stress, especially salt stress [8]. There is an urgent need for an effective way to alleviate the inhibition of soybean growth under salt stress and increase soybean biomass and GABA content. In the present study, the protective role of exogenous calcium in the response of germinating soybeans to salt stress was determined through exogenous application of CaCl2 and confirmed by an assay of growth performance, sprout length, respiratory rate, and the fresh weight of germinating soybeans (Supplementary Fig. S5). Meanwhile, the growth and development of germinating soybean plants was completely abolished through exogenous application of LaCl3. This is because LaCl3 is an inhibitor of Ca2+ channels. The increase in abiotic stress tolerance was consistent with the protective effect of calcium, which was observed using the exogenous application approach and genetic engineering in such plant species as Arabidopsis [15,33], Vigna [17], and tomato plants [34]. More importantly, the GABA content was significantly higher in germinating soybeans treated with exogenous CaCl2 (Fig. 7B). These facts suggested that calcium might be an ideal target for the improvement of GABA-enriched germinating soybeans under salt stress. However, the underlying molecular mechanism of calcium-mediated soybean salt stress responses needs to be discussed further.

4.1. Redistribution of storage proteins and regulation of protein synthesis and proteolysis In the present study, physiological and comparative proteomic analyses were performed to reveal the protective mechanism of calcium in response to salt stress. Through 2-DE and

MALDI-TOF/TOF-MS, a total of 80 proteins were found to be differentially displayed in germinating soybean cotyledons exposed to CaCl2 and LaCl3, and 71 in embryos. Among these 80 identified cotyledon proteins, more than 60% represented various SSPs, almost all of which were assigned to β-conglycinin subunits and glycinin. Β-conglycinin and glycinin were the products of hydrolysis of globulin and were mobilized to provide carbon and nitrogen for soybean germination and growth. GABA-enriched germinating soybeans were germinated in a dark artificial environment, so that organic compounds could not be synthesized via photosynthesis. These storage materials were the major source of nutrients for soybeans. The low levels of SSPs in cotyledons of soybeans subjected to CaCl2 treatment were general responses that increased plant growth. SSPs were mobilized but the composition and abundance of different types of subunits of β-conglycinin and glycinin showed different levels of enrichment in response to NaCl-CaCl2 treatment. Sales et al. [35] and Danchenko et al. [36] have suggested that SSPs may not only function as seed storage reserves but also play additional roles in plant defense. It remains to be seen whether the manner of these SSP changes in response to CaCl2 treatment could improve salt tolerance in germinating soybeans. Consistently, the abundance of all the synthesis-related proteins and six trypsin inhibitors (C11, C14, C16, C17, E27, and E28) was higher, indicating that the promotion of protein biosynthesis and inhibition of proteolysis were required for the germination in soybean growth and development. Komatsu et al. have suggested that calcium may play one role through heat shock protein 70 in the soybean cotyledon under flooding stress [37]. This research showed that a total of ten differentially displayed proteins were associated with protein processing in endoplasmic reticulum, including four heat shock proteins (E44, E52, E53

119

J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 1 1 0 – 12 6

A Relative intensity

0.4 a

a

a

a a a a a

0.1

0.75

C1

C2

a

a

a b b abb b

C3

b

b

b

C4

ab

b

C5

a

a a a

a

b

C6

b

C7

a b

b

a b b b

b

a

a

c

c

a a a a a

b

c

b b

b

b

b

a

a

b

b

C8 C10 C11 C13 C14 C15 C16 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C29 C31

a

a a

0.25

a

a

a

0.00

a

a a

b

b

b

b

b b

a

a a

a

a

1.0

a a

a b

b

b

0.50

a a

Relative intensity

a

0.2

0.0

Relative intensity

NaCl NaCl+CaCl2 NaCl+LaCl3

0.3

b

a bb

b

ab

c

c

bb

a

a

a

a

a

a

b

a a b

b b

b a b b

b b

a

a

a a b

b

b

a ba

b

a ab

b

C32 C34 C35 C36 C37 C38 C39 C40 C41 C42 C43 C45 C46 C47 C48 C49 C50 C51 C52 C54 C55 C56 C57 C58 C59 C60

0.8

a

a

a

a

a

ab

a

0.6

a

a

a

b

a

0.4

a a

a

0.2 a cb

0.0

b

a

a

b

cb

bb

c

a

a a bc

a

a

bb

bc

b

b

a a b c c b

b

b

b

b

bb

a

b

b

a

a a bb b b

a a

a a

b

b

c

b

c

c

c

C61 C62 C63 C65 C66 C67 C68 C69 C71 C72 C73 C74 C75 C76 C77 C78 C79 C80 C9 C12 C17 C28 C30 C33 C44 C64 C70

Spot number

B Relative intensity

0.60

NaCl NaCl+CaCl2 NaCl+LaCl3

0.45

a

a

a

a a

b

b ab

cb

0.00 E1

a a

b b a

E2

E3

E4

b

E5

E6

a

b b

b b

b

a ab

c

b b b

bab

E7

E9

a a

b b

ab

a a

b

a

b b b

a

b

b

a

a a

ab

a

0.15

a

a

ab

0.30

a

b b

a

b

b

b

b

b

aa

b c

E10 E11 E12 E14 E15 E16 E17 E18 E19 E21 E22 E23 E24 E25 E26 E29 E30

Relative intensity

0.60

a

a

0.45

a

a

a a

ab

0.30 b

b

b

0.15

b

b

ab

ab b

a a

b a b b b

b bb

a

bb

ab

a b b

a a

b b

a a b b ab

a a

a

b

a

c

a

b

a

a

a

a

ab

0.00

b

b

c

b

b

ab

b

a a

a

b

b

a

b

b b

c

b

c b

E33 E34 E35 E36 E37 E38 E39 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52 E53 E54 E55 E56 E57 E58 E59 E61

Relative intensity

1.5 a

1.2

a

a

a

ab ab b

0.9

b a

a

0.6 0.3 b

a c

a

a

a

0.0

a

a b

a

a a b

aa a b b b

ab b

a

a

a

b

aa

b

a a b

b bab

a

a

c

b b

b

b b

b

b c

b

c

bb

bb

E58 E59 E61 E62 E63 E64 E65 E67 E68 E69 E70 E71 E13 E20 E27 E28 E31 E32 E40 E41 E42 E66

Spot number

120

J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 1 1 0 – 12 6

A

N-Ca/N N-La/N N-Ca/N-La

Up Non

Down

Cluster C 14 C 19 C 22 C8 C 43 C 48 C 42 C7 C 13 C 80 C 46 C 11 C 12 C 25 C 35 C 38 C 36 C 26 C 40 C 61 C 39 C 41 C 18 C 20 C 10 C 16 C 78 C 21 C 51 C 37 C 30 C 50 C 47 C 52 C 56 C 60 C 72 C 59 C 76 C 67 C 71 C1 C5 C6 C 17 C 65 C2 C 75 C 45 C9 C 31 C 32 C 29 C3 C4 C 28 C 33 C 15 C 69 C 74 C 58 C 63 C 57 C 27 C 34 C 23 C 79 C 66 C 64 C 24 C 73 C 55 C 70 C 77 C 54 C 62 C 44 C 49 C 68

i

ii

iii

iv

v

B

N-Ca/N N-La/N N-Ca/N-La

Cluster E 16 E 44 E4 E 25 E 26 E9 E 10 E 48 E 34 E 13 E 28 E 17 E 56 E 19 E 46 E 38 E 43 E 39 E 22 E 29 E 55 E 65 E3 E5 E 54 E 52 E 53 E 41 E 42 E1 E 11 E 24 E 36 E 15 E 63 E 23 E 35 E 37 E 21 E 51 E 20 E6 E 59 E 14 E7 E 57 E 47 E 18 E 71 E 58 E 27 E 68 E 49 E 30 E 70 E 64 E 45 E 50 E 40 E 32 E 66 E 33 E2 E 62 E 69 E 12 E 67 E 61 E 31

I

II

III

IV

V

VI

Fig. 4 – Hierarchical clustering of proteins in 4-day germinated soybean cotyledons (A) and embryos (B) subjected to NaCl, NaCl + CaCl2 or NaCl + LaCl3 treatment. Hierarchical clustering based on the log2-transformed expression ratios of protein spots was performed using Gene Cluster 3.0 software with the Euclidean distance similarity metric and complete linkage method. The resulting clusters were visualized using JAVA TREEVIEW software. N: NaCl; N-Ca: NaCl + CaCl2; N-La: NaCl + LaCl3.

and E55), two endoplasmins (C79 and E43), two calreticulin-1 precursors (C8 and E21), cell division cycle protein (E22), and luminal-binding protein (E54), indicating that protein processing in endoplasmic reticulum might be an essential strategy for improving salt stress through calcium (Fig. 8).

4.2. Regulation of signal transduction Studies show calcium to be involved in signal transduction in plants [38,39]. It is not surprising that the signal transduction

pathway was largely enriched by CaCl2 but sharply suppressed by LaCl3 in this study. The calreticulin-1 precursor (C8 and E21) was detected and identified in both organs (cotyledon and embryo), but it was more intense in the organs treated with CaCl2 and less intense in those treated with LaCl3. This suggested that calcium could strengthen the calcium signaling pathway and other signal transduction pathways to enhance tolerance of germinating soybeans to salt stress. Similarly, under NaCl stress conditions, CaCl2 was found to increase the abundance of transporters but LaCl3 to decrease it.

Fig. 3 – Relative intensity of protein spots that are differentially displayed in germinating soybean cotyledons and embryos subjected to NaCl, NaCl + CaCl2, and NaCl + LaCl3 treatments. Staining intensities were quantified using PDQuest software. Results are presented as the mean ± SE of the relative protein intensity for gels from 3 biological replicates. The spot numbers are the same as in Table 1 and Table 2.

121

J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 1 1 0 – 12 6

A

B

Increase Decrease

Metabolism Cell growth/division Proteolysis Storage Transportation Disease/defense 0

15

30

45

60

C

0

15

30

45

60

0

12

24

36

48

0

4

8

12

16

10

20

30

40

D

Chloroplast Cytoplasm Nucleus Mitochondria Vacuole 0

12

24

36

48

E

F

Metabolism Energy Cell growth/division Protein synthesis Protein destination/storage Transportation Signal transduction Disease/defense Secondary metabolism Uncharacterized 0

4

8

12

16

G

H

Cytoplasm Chloroplast Cytoplasm skleton Mitochondria Nucleus Vacuole Endoplasmic reticulum 0

10

20

30

40

Number of proteins

0

Number of proteins

Fig. 5 – Classification of differentially displayed proteins in cotyledons and embryos of 4-day germinating soybean plants treated with NaCl + CaCl2 and NaCl + LaCl3. The functions of differentially expressed proteins in (A, B) cotyledons and (E, F) embryos treated with (left) NaCl + CaCl2 and (right) NaCl + LaCl3 were assigned according to KEGG and previous studies. The localization-based classification of proteins in (C, D) cotyledons and (G, H) embryos treated with (left) NaCl + CaCl2 and (right) NaCl + LaCl3 is shown in lower panels.

4.3. Maintenance of energy metabolism ATPases are membrane-bound enzymes. They combine ATP synthesis with the transportation of protons. ATP synthase 24 kDa subunit (E15), ATPase subunit 1 (E19), V-type proton ATPase subunit E (E20), NADP-dependent isocitrate dehydrogenase (E16), and two NADP-dependent malic enzymes (E17 and E18) are critical to glycolysis and the TCA cycle, which support plant growth and development. In the present

proteome analysis, application of exogenous CaCl2 increased the abundance of all the proteins identified above, which were associated with energy metabolism. ATP synthase 24 kDa subunit and two NADP-dependent malic enzymes significantly decreased and the remaining proteins showed no significant differences under LaCl3 treatment and NaCl stress alone. These results suggest that the energy pathway might play a pivotal role in increasing calcium tolerance to salt stress.

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H2O2 content (mM/g protein)

MDA content (mmol/g DW)

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POD activity (U/g protein)

SOD activity (U/g protein)

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GPX activity (U/g protein)

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CAT activity (U/g protein)

8

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a b 120

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SC-4d

SE-4d

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40

0

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c

a 80

c ba c

bb a SC-2d

SE-2d

SC-4d

SE-4d

Fig. 6 – ROS accumulation and antioxidant enzyme activity affected by exogenous calcium treatment during salt stress in germinating soybeans. (A) Quantifications of MDA content, (B) H2O2 content, (C) SOD activity, (D) POD activity, (E) CAT activity, and (F) GPX activity of germinating soybean plants subjected to different treatments at designated time intervals. SC-2d: 2-day germinated soybean cotyledons; SE-2d: 2-day germinated soybean embryos; SC-4d: 4-day germinated soybean cotyledons; SE-4d: 4-day germinated soybean embryos.

4.4. Disturbance of secondary metabolite process Secondary metabolites play an important role in osmotic adjustment in response to environmental stressors [40]. Here, increased abundance of caffeoyl-CoA-O-methyltransferase (CCoAOMT, E57), isoflavone reductase (IFR, spot E58), and chalcone isomerase A (E59) and decreased abundance of polyphenol oxidase A1 (E56) were detected in CaCl2-treated soybean embryos, indicating that the secondary metabolic pathways were affected. CCoAOMT plays a key role in the supply of substrates for the synthesis of lignin units [41]. The upregulation of CCoAOMT may be linked to the increased synthesis of monolignols, which may be due to calciuminduced growth acceleration [42]. In this study, IFR and chalcone isomerase were associated with isoflavonoid biosynthesis pathways. Isoflavonoids and polyphenol were potent antioxidants. They acted as scavengers of ROS under stressful conditions. The accumulation of isoflavones and polyphenol might play an essential role in regulating the growth response. These results indicated that the secondary metabolite synthesis pathways were the targets for exogenous

calcium response and that they promoted the growth and development of soybean embryos.

4.5. Regulation of antioxidant system and antioxidant enzyme activities Other studies show that proteins that are differentially displayed in antioxidant defense pathways may play key roles in soybean adaptation to salt stress [43–45]. In the present study, three antioxidant enzymes, E47, E48, and E51, were detected among the calcium-mediated proteins. The abundance of all three antioxidant enzymes was much higher under CaCl2 treatment and much lower under LaCl3 treatment. The changes in abundance of lipoxygenase (E9), UTP-glucose-1-phosphate uridylyltransferase (C1 and E1), sucrose-binding protein (C76), and pathogenesis-related protein (E39, E40, and E49) indicated the involvement of exogenous calcium in jasmonic acid biosynthesis, glucose metabolism, sucrose metabolism, and the stress response. Generation of excess ROS in plant cells is an inevitable outcome of any abiotic stress, especially NaCl stress [46].

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A Proline content (µg/g DW)

1600

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NaCl NaCl+CaCl2 NaCl+LaCl3

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GABA content (mg/per soybean)

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determine the involvement of these antioxidant enzymes in calcium-mediated salt stress responses, the underlying antioxidant enzyme activities and level of ROS were assayed further. CaCl2 treatment was found to significantly increase MDA and H2O2 contents, and higher levels of activity of four antioxidant enzymes, SOD, POD, CAT, and GPX, were observed than in untreated germinating soybeans (Fig. 6). This conclusion was consistent with the results shown above in 2-D. However, the LaCl3-treated germinating soybeans exhibited significantly less activity of these antioxidants and higher concentrations of MDA and H2O2 than in untreated germinating soybeans at the same points in time (Fig. 6). Nucleoside diphosphate kinase (NDPK, E12 and E13) regulates the expression of antioxidant genes in plants. Recently, proteins from transgenic plants overexpressing AtNDPK2, such as potatoes [49], poplars [50], and sweet potatoes [51], were found to markedly enhance tolerance to multiple abiotic stresses that lowered the ROS level. These increases were effected through modulation of antioxidant enzyme activities. In this way, the increase in NDPK protein levels through exogenous calcium observed in the present study might contribute to the enhancement of salt tolerance in germinating soybean plants. The aforementioned changes in secondarymetabolism-related proteins might cause the accumulation of secondary metabolites such as lignins and isoflavonoids, which play an important role in the mitigation of oxidative injury. These results suggested that calcium might modulate stress-triggered ROS homeostasis and MDA by activating some antioxidant enzymes, including SOD, CAT, POD, and GPX, and by increasing the concentration of secondary metabolites.

4.5. Regulation of osmotic adjustment substances

800

200

123

aa a c SC-2d

SE-2d

ab

SC-4d

SE-4d

Fig. 7 – Proline, GABA, and free PAs levels affected by exogenous calcium treatment under salt stress in germinating soybeans. (A) Proline content, (B) GABA content, and (C) free PAs content of germinating soybean plants subjected to different treatments at designated time intervals. SC-2d: 2-day germinated soybean cotyledons; SE-2d: 2-day germinated soybean embryos; SC-4d: 4-day germinated soybean cotyledons; SE-4d: 4-day germinated soybean embryos.

These ROS cause membrane damage and attack proteins and nucleic acids [47]. In higher plants, the antioxidant system includes many antioxidant enzymes. It can scavenge these toxic compounds, alleviating the ROS-induced oxidative damage that occurs under stressful conditions [48]. In this study, three antioxidant enzymes (POD precursor, E47; MnSOD, E48; phospholipid hydroperoxide GPX, E51) were affected commonly by CaCl2 and LaCl3. Based on the proteomic analysis described above (Fig. 2 and Fig. 3). To

To counter abiotic stresses, plants increase the osmotic potential of their cells by synthesizing and accumulating proline, GABA, and PAs, which participate in osmotic adjustment. Many experiments have proved that application of exogenous proline, GABA, and PAs can improve salt stress tolerance in plants [52–54]. In the present study, in germinating soybeans treated with CaCl2, the GABA and PAs contents were much higher than those in the untreated controls, but the proline content was markedly lower in the embryo (Fig. 7). In plants, ornithine can be metabolized to proline and PAs through ornithine-δ-aminotransferase and ornithine decarboxylase, respectively [55,56]. Proline and GABA can also form through glutamate metabolic pathways [30,57]. PAs can be converted to γ-aminobutyraldehyde by diamine oxidase, and then γ-aminobutyraldehyde is converted to GABA by aminoaldehyde dehydrogenase [55]. All these findings suggest that proline, GABA, and PAs share some common substrates and that their metabolic pathways are complex networks. In the present proteomic analysis, the abundance of glutamine synthetase PR-2 (E2) and glutamine synthetase precursor (E5) decreased significantly under exogenous CaCl2 treatment. Glutamine synthetase (GS) catalyzed the assimilation of ammonium to glutamine using glutamate as its substrate. The decreased abundance of GS indicated that higher glutamate content might supply more substrates for proline and GABA biosynthesis. However, glutamate decarboxylase, which converted glutamate to GABA, was a Ca2 + binding protein.

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E52, E53, E55 E44

E54

C79, E43 C8, E21

E22

Fig. 8 – Differential regulation of proteins involved in protein processing in endoplasmic reticulum by calcium. The spot numbers are the same as in Tables 1 and 2.

The application of exogenous CaCl2 could enhance glutamate decarboxylase activity. Exogenous application of calcium might shift more substrates for GABA biosynthesis, especially under salt stress. Calcium ions lowered proline levels in NaCl-stressed plants [58]. Higher abundance of aminoaldehyde dehydrogenase (AMADH, E4), S-adenosylmethionine synthetase (E6 and E7), and methionine synthase (MS, E10) were observed in germinating soybean embryos exposed to CaCl2 treatment (Supplementary Fig. S6). S-adenosylmethionine synthetase and MS were involved in the polyamine biosynthesis pathway, so their levels of abundance increased markedly, which in turn increased the PAs concentration. Many of these PAs were converted to GABA through the polyamine degradation pathway. AMADH was indispensable for the polyamine degradation pathway and its abundance increased under CaCl2 treatment. Not only the abundance of proteins involved in Glu and PAs metabolism was affected by calcium, but also the abundance of AMADH decreased significantly under exogenous CaCl2 treatment in accumulating GABA. These results supported the conclusion that exogenous application of calcium might shift more substrates toward GABA and PAs biosynthesis instead of proline biosynthesis and that they might do so through modulation of amino acid metabolism and other related metabolic pathways, which would suppress the effects of salt stress.

Taken together, this study analyzed the protective role of calcium in germinating soybean responses to salt stress. Physiological and comparative proteomic analyses revealed that the putative molecular mechanism of exogenous calcium treatment induced salt stress responses. Collective observations here supported the putative roles of calcium in salt stress responses in germinating soybean plants (Supplementary Fig. S7). Exogenous calcium was found to redistribute storage proteins, regulate protein processing in the endoplasmic reticulum, enrich the signal transduction pathway, facilitate the energy pathway and transportation, promote protein biosynthesis, and inhibit proteolysis, and finally to increase the tolerance of germinating soybeans to salt stress. Calcium also raised the protein levels of NDPK and antioxidant enzymes and their activities, and caused the accumulation of the secondary metabolites, GABA, and PAs, all of which could alleviate cell damage and reduce the increase in osmotic pressure caused by salt stress. Calcium might also regulate other physiological changes and modulate global proteomic responses, including those associated with the remaining differently displayed proteins observed in the present study, and, in turn, might result in other adaptive responses that have yet to be fully understood. The present study provided evidence of the protective effect of exogenous calcium (6 mM) on the growth and metabolic activities of

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4-day-old germinating soybeans restrained under salt stress (50 mM). Further studies to investigate calcium-signaling components may contribute to clearer comprehension of the complex metabolic picture displayed during soybean germination under salt stress.

Transparency document The Transparency document associated with this article can be found, in the online version.

Acknowledgments This project was supported by the Natural Science Foundation of China (31401614), and was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2014.09.023.

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