Plant Cell Tiss Organ Cult DOI 10.

1007/s11240-013-0340-0

ORIGINAL PAPER

Proteomic analysis of cotyledonary explants during shoot organogenesis in Vigna radiata

Suparna Ghosh Amita Pal

Received: 26 March 2013 / Accepted: 30 May 2013 Springer Science+Business Media Dordrecht 2013

Abstract Vigna radiata or mungbean belongs to the legume family of plants. Mature mungbean seeds are rich source of dietary proteins for human nutrition. The present study was aimed to analyze the comparative protein proles of two cotyledon types, Cot and Cot E, prior to and during early time points of shoot morphogenic induction to understand the unique differential regeneration response in these two explant types which was reported earlier. These explants were grown separately in vitro on the shoot induction medium (SIM) containing Gamborgs B5 basal nutrient composition supplemented with 15 lM N6-benzyladenine. Isolation and characterization of the proteomes from Cot and Cot E explants at different time points, during early events of shoot differentiation, were performed using two dimensional gel electrophoresis following matrix assisted laser desorption-ionization tandem mass spectrometry. A total of 112 differentially identied proteins were classied according to their putative biological function. The differential control of protein synthesis between these explants under control condition, i.e. before in vitro culture, was also noted. In Cot E explants SIM induced prompt acquisition of competence for direct shoot morphogenesis probably through fast phytohormone signaling. Over accumulated proteins in Cot E indicated stimulation of several metabolic and associated pathways earlier than Cot explants. Abundance of stress and defense related proteins in Cot E explants was presumably to cope up with stressful
Electronic supplementary material The online version of this article (doi:10.1007/s11240-013-0340-0) contains supplementary material, which is available to authorized users.
S. Ghosh A. Pal (&) Division of Plant Biology, Bose Institute, Kolkata 700054, India e-mail: amita@bic.boseinst.ernet.in; amita_pal@yahoo.com; amita@mail.jcbose.ac.in

cultural condition. Enhanced accumulation of foldingassisted proteins involved in organogenesis mediated cellular reprogramming in Cot E explants contributed further in rapid and efcient regeneration responsiveness. Keywords Cytokinin MALDI-TOF-TOF Proteomics Shoot regeneration Vigna radiata Abbreviations SIM 2-D GE BA BSA Cv CV DTT IEF MALDI TOF MS

MEV MOWSE PMSF RT-PCR SDS PAGE

Shoot induction medium Two-dimensional gel electrophoresis N6-benzyladenine Bovine serum albumin Cultivar Coefcient of variation Dithiothreitol Isoelectric focusing Matrix assisted laser desorptionionization time-of-ight mass spectrometry MultiExperiment viewer Molecular weight search Phenylmethylsulfonyl uoride Reverse transcription polymerase chain reaction Sodium dodecyl sulphate polyacrylamide gel electrophoresis

Introduction Vigna radiata or mungbean is a widely cultivated species of genus Vigna and a rich source of dietary proteins, vitamins and minerals. Like other legumes it improves the nitrogen status of the soil by xing atmospheric nitrogen.

123

Plant Cell Tiss Organ Cult

The productivity of the crop, however, remained inadequate for last few decades mainly due to its intolerance to abiotic stresses like salinity, drought and susceptibility to several fungal, bacterial or viral pathogens and insects. Attempts made to improve resistance against disease and insects by classical breeding methods have yielded poor results due to unavailability of compatible breeding partners and a narrow genetic pool to introduce desirable characteristics. Agricultural biotechnology can complement the classical breeding methods that involve the technique of genetic engineering. A reliable and efcient in vitro regeneration system is an essential prerequisite for successful genetic transformation. Legumes, particularly V. radiata, however, are generally considered to be recalcitrant in nature where regeneration is often slow and the frequency of transformation is often low (Somers et al. 2003). In our laboratory, cotyledons of V. radiata were used along with various other explants, in order to establish efcient regeneration and transformation protocols under cultural condition. During such attempts differential regeneration response between two cotyledon types (referred as Cot and Cot E) was observed. The two cotyledons of a mature mungbean seed were identied separately as Cot that is easily separated from the embryonal axis and Cot E, which remains rmly attached to the axis following germination. Differential regeneration responses both in mode and efciency of regeneration were observed in these two cotyledonary explants when cultured separately (Chandra and Pal 1995). Regeneration efciency of Cot E explants was signicantly high in nine Cvs tested in three different concentrations of three cytokinins. Histological studies revealed meristematic region initiated in Cot E explants from 3rd day onwards followed by formation of shoot apical meristem and leaf primordia on 6th day and is visible by naked eye from Cot E at 6th day when grown on N6-benzyladenine (BA) supplemented Gamborgs B5 medium (Das and Pal 2004). In contrast callus formation was noted in Cot on 6th day onwards and meristematic activity initiated from 9th day, suggesting dedifferentiation (i.e. retrieval of meristematic activity and competence of shoot development) occurs in Cot E explants at an early time point than Cot explants in presence of suitable shoot induction medium (SIM). This unique phenomenon led us to probe differential proteomic proles in Cot E with respect to Cot explants at different time points during in vitro shoot induction to interpret differential regeneration response in the two cotyledonary explants of V. radiata at the molecular level. The new tools and techniques of proteomics have been shown to facilitate understanding of qualitative and quantitative changes of proteins at different stages of development precisely

through time course analyses. Several proteomics studies have been undertaken during the last couple of years to investigate the changes in protein proles during in vitro plant embryogenesis (Baba et al. 2008; Bian et al. 2010; Nogueira et al. 2007; Zhang et al. 2009); while changes in protein proles during earlier stages of shoot differentiation from organogenic callus cultures of Vanilla planifolia (Palama et al. 2010), in cell suspension cultures of Boesenbergia rotunda (Tan et al. 2012) were reported. Activities of globulin and cysteine proteinases during germination and seedling growth of Vicia sativa (Schlereth et al. 2000); and modulation of protein proles in cotyledons of V. radiata during seed germination (Ghosh and Pal 2012) were also reported. Among these studies, the workers have identied proteins such as, b-tubulin and annexin acting as molecular markers for the developmental stages of somatic embryos of Manihot esculenta (Baba et al. 2008), proteins viz. triosephosphate isomerase, proteasome complex, NADP-dependent glyceraldehyde-3phosphate dehydrogenase supposed to be involved in the regulation of somatic embryogenesis of Cyclamen persicum Mill (Bian et al. 2010), protein expression patterns and role of several proteins including pathogenesis related protein 4 (PR4) and PR10 associated with embryogenic cell suspension culture of Vigna unguiculata (Nogueira et al. 2007), stress related proteins exclusively expressed and thereby different stress response pathways activated in embryogenic callus culture of Vitis vinifera (Zhang et al. 2009). In other studies proteins involved in amino acid and protein metabolism and photosynthetic activity were signicantly expressed at earlier stages of shoot differentiation from callus cultures of Vanilla planifolia (Palama et al. 2010) and proteins involved in the avonoid and phenylpropanoid pathways to generate important bioactive compounds were identied (Tan et al. 2012). Mobilization of storage proteins in cotyledons of V. sativa during germination and seedling growth (Schlereth et al. 2000) and changes in cotyledonary proteins during seed gemination of V. radiata and proteins associated with starch and sucrose metabolism (Ghosh and Pal 2012) were investigated through proteomics approach. In this study we have analyzed the comparative protein proles of Cot and Cot E before exposing them to in vitro culture and during early stages of shoot induction. Differentially regulated proteins were identied employing twodimensional gel electrophoresis (2-D GE) followed by mass spectrometric analyses. The presence study portrayed participation of different proteins involved in various pathways during shoot differentiation. The perception of altered regulations of an array of proteins for efcient shoot regeneration in Cot E explants of V. radiata might help to understand recalcitrant nature of Vigna as well as other grain legumes.

123

Plant Cell Tiss Organ Cult

Materials and methods Plant material Seeds of V. radiata (L) Cv B-1 were collected from Berhampore Pulse and Oil Seed Research Station, West Bengal, India, and multiplied at the Madhyamgram Experimental Farm of Bose Institute. The seeds were treated with 0.1 % mercuric chloride solution and washed thoroughly 34 times with sterile double distilled water. The seeds were then imbibed for 22 h in sterilized water and kept at 30 C. The cotyledons were excised after removal of the seed coat. These two cotyledons (Cot and Cot E) were used as separate explants and placed in Gamborgs B5 basal nutrient medium (Gamborg et al. 1968) supplemented with 2 % sucrose and 15 lM BA. The pH of the medium was adjusted to 5.8 prior to autoclaving and was solidied with 0.8 % agar. Cultures were maintained under photoperiodic condition (16/8 h day night cycles) of cool-white uorescent light providing a quantum ux density of 27 lmol m-2 s-1 and were incubated at 25 1 C. Cultures were harvested on 1st, 2nd, 3rd, 4th, 5th and 6th days of culture. Cotyledons excised from seeds after 22 h imbibitions were considered as 0 day explants. Protein extraction and two-dimensional gel electrophoresis (2-D GE) Proteins were extracted from cotyledonary explants according to the method described by Vasconcelos et al. (2005) with minor modications (Ghosh and Pal, 2012) and two-dimensional gel electrophoresis (2-D GE) was performed according to the manufacturers recommendation (Bio-Rad). Image analysis of 2D-PAGE gel Coomassie stained 2-D gels were digitalized using VersaDocTM (Model 4000) Imaging System (Bio-Rad) and analyzed with PDQuest AdvancedTM 2-D Analysis software (version 8.0.1, Bio-Rad) following Kundu et al. (2011). Each set of experiments was carried out with minimum of three replicates. Spots in the gels were detected automatically by the Spot Detection Parameter Wizard using the Gaussian model with background sensitivity 4. To compare spot quantities across gels accurately, non-expression-related variations in spot intensity was compensated through normalization by local regression model. Only spots present in the replicate groups of each experiment were considered for subsequent analysis. Quantitative analysis was performed with the replicate groups to determine the average change of their protein spots in quantity. Comparison of proteome between the two

cotyledons (Cot and Cot E) isolated prior to inoculation in medium was done in order to nd out differences in the protein pattern of Cot and Cot E under control condition. To elucidate differentially regulated proteins during in vitro shoot induction protein proles of Cot and Cot E at 1st to 6th each consecutive days during in vitro differentiation were analyzed. Gradual qualitative and quantitative changes of the differential spots on each of the time points under study were determined. The quantitative changes of the spots were estimated between limit (two-fold) and outside limits ([?two-fold or \-two-fold) and expressed as ratio of average spot quantity between two sample sets. Spots showing a minimum of 1.5 fold change and a p value of 0.05 or less were considered to be expressed differentially in signicant amount. Two integrated gel images, one for Cot and the other for Cot E, were created combining sets of gels for different time point experiments through PDQuest software containing all the protein spots resolved in. These gels were individually prepared from individual protein samples. Selected protein spots were subjected to in-gel digestion for identication by MALDI TOF MS and MS/MS. MALDI TOF MS and MS/MS analysis and database search Selected protein spots were excised from the gel using EXQuest Spot Cutter (Bio-Rad). In gel digestion was performed following the method of Kundu et al. (2013a). Identied proteins were assigned to functional categories by Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/kegg2.html) and UniProt database. Cluster analysis of the differential proteins at different time points (including 0 day) was performed taking the log transformed fold change value of the protein expression level using MultiExperiment Viewer (MEV) software 4.8.1. Semi-quantitative RT-PCR Total RNA was extracted with Trizol reagent (Invitrogen) from approximately 200 mg of tissues (both Cot and Cot E) harvested at 0 day and on 1st, 2nd, 3rd, 4th, 5th and 6th days after culturing on SIM. The RNA was then quantied using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, USA). A two-step reverse transcription polymerase chain reaction (RT-PCR) was performed using GeNeiTM RT-PCR Kit with ve pairs of gene specic primers; actin primer pair was used to amplify actin as an internal control (Table 1). 1.2 lg of total RNA samples were used to synthesize the rst strand cDNA using AMV reverse transcriptase at 42 C for 60 min. The cDNA was then diluted and subsequently used for PCR

123

Plant Cell Tiss Organ Cult

amplication. The thermal cycling program performed was as follows: initial denaturation at 94 C for 3 min followed by 27 cycles comprised of denaturation at 94 C for 45 s, primer annealing at 60 C for 30 s, primer extension at 72 C for 45 s and a nal extension at 72 C for 5 min. The RT-PCR products were analyzed by electrophoresis in a 1.5 % (w/v) agarose gel, stained with ethidium bromide and photographed using a gel imaging system (VersaDocTM, Bio-Rad). Band intensities were quantied using Quantity One densitometric software (Bio Rad). Ratios of band intensities were calculated for Cot E over Cot after normalization with actin. Statistical analyses Coefcient of variation (CV) was computed for the matched spot quantities in each set of experiments with the replicate groups. Analysis of variance (ANOVA) at the 95 % signicance level was employed to evaluate differential protein spots statistically.

Comparative analysis of proteome patterns of Cot and Cot E during in vitro differentiation The total soluble protein content was found to be higher in Cot E than Cot explants in all the time points (Fig. 2A). 2D gel proles of Cot and Cot E were compared under the same physical and chemical milieu at each consecutive day (1st6th) and control condition as well, revealing temporal differences in protein synthesis, accumulation and utilization. Differential pattern of total protein composition was also reected by the number of resolved spots on the gel. Image analysis by PD Quest software revealed an average of 400480 spots at different time points. Quantitation of spot intensity was regarded as the abundance level of the corresponding protein. Quantitative analysis showed a total of 158 signicantly modulated protein spots. Among these, 112 spots were identied by MALDI TOF MS and MS/MS. Different physical parameters of these proteins are given in Supplementary Table 1. The classication of proteins according to their putatively assigned functional categories is shown in Fig. 2B. The spots were represented in the integrated gel image created from gel images at seven different time points (Fig. 3). Functional category of a large portion of the differentially regulated proteins was metabolism related followed by protein processing, stress, storage etc. Proteins that did not show homology with any other protein in the database, that were designated as unknown. Several identied proteins appeared in multiple spots representing more than one isoforms with different isoelectric point (pI) and molecular weight (Mw). 78 differential proteins present under control condition were in higher and 33 were in lower abundance in Cot E explants than Cot. Highly abundant proteins include ADP glucose pyrophosphorylase (S1, fold change 2.9), two isoforms of seed albumin (S4 and S7, fold change 2 and 10.1, respectively), protein disulphide isomerase (S9, fold change 14.6), dihydropicolinate synthase (S107, fold change 4.8). Some representative, differentially abundant protein spots under metabolism, signal transduction, stress and protein processing category showing in-gel changes in spot volume are represented in Fig. 4.

Results Differential regeneration response in Cot and Cot E explants Swelling of the cotyledonary explants was observed within 3 days upon incubation in SIM. The colour of the cotyledons initially changed from pale yellow to light green and gradually became dark green with the progression of time. Direct shoot-bud regeneration from the proximal end of the cotyledons of the Cot E explants was observed within 6 days during in vitro differentiation (Fig. 1). The regenerated shoot-buds grew further followed by basal callus formation as observed on 9th day. Multiple shoots regenerated from Cot E explants upon further incubation in SIM. Subsequent shoot-buds regenerated from the close proximity of the rst adventitious shoot. In contrast, callus differentiated after 3rd day onwards from the proximal end of Cot explants. The callus increased in size in the progressive days followed by shoot-bud initiation from 12th day onwards.
Table 1 Primers used in semi-quantitative RT-PCR Gene Aldehyde reductase Ascorbate peroxidase Cyclophilin HSP70 PR10 Actin NCBI accession no. gi|5852202| gi|187962069| gi|18146785| gi|45331280| gi|60418923| gi|6934187| Forward primer

Reverse primer 50 -TTCCGGGTCAGAATACCAAG-30 50 -GGCCTTCCTTTTCACCACTCA-30 50 -CTCCTTCACCACGTTCAGTC-30 50 -AGAGTCCTCTTCGCCCTCTC-30 50 -AGCCTTGAGCTCATCTTCGT-30 50 -AGCCTTCGCAATCCACATCTG-30

50 -TCCTGGCTCGTCAAGTTTCT-30 50 -CTTTGACGTTAGCACGAAGACC-30 50 -GACCTTCATTTCCCGTCTCT-30 5 -TGGTTACTGTCCCCGCTTAC-3 5 -TGGCTGTTTTCACATTCGAC-3

0 0 0 0

50 -ATGACTCAGATCATGTTTGAG-30

123

Plant Cell Tiss Organ Cult

Fig. 1 Cot and Cot E explants after 22 h imbibation (0 day) and showing differential mode of in vitro differentiation at 3rd, 6th, 9th and 12th day grown in SIM. Panel A Callus differentiation from the proximal end of Cot explants from 3 day onwards. The callus increased in size on 6th and 9th day followed by callus mediated

shoot initiation on 12th day. Panel B Direct shoot initiation from the proximal end of the abaxial side of Cot E explants on 6th day. The regenerated shoots grew further followed by basal callus formation on 9th day. Multiple shoots regenerated from Cot E explants on 12th day

Fig. 2 A Total soluble protein contents in Cot and Cot E explants on 0 day (before inoculation in the culture medium) and on consecutive 7 days during in vitro differentiation. B Functional classes of

identied proteins. Proteins are assigned with putative biological functions using KEGG (http://www.genome.jp/kegg/kegg2. html) and UniProt database

A comprehensive overview on the time dependent change in abundance of the identied proteins was achieved through cluster analysis. It was revealed from the heat map that majority of proteins under the class metabolism were present in increased abundance in Cot E during most of the time points under study as indicated in Fig. 5. Several metabolism related proteins were found to be over expressed in Cot E throughout the study, for example, fructose bisphosphate aldolase isoform S22, malate dehydrogenase isoforms S50, S73 and S99, glutamine synthetase isoform S97, phosphoglycerate mutase and aspartate aminotransferase. Whereas, malate dehydrogenase isoforms S35, S39, enolase, hexokinase, fructokinase, starch phosphorylase, shikimate kinase,UDP glycosyl transferase and glucose-6-phosphate dehydrogenase present initially with insignicant differences between these two explants

but started to synthesize more in Cot E from 3rd to 6th day during initiation of shoot differentiation. Varied patterns of differential abundance amongst proteins related to protein processing were observed. Increased abundance of proteins, for instance, cyclophilin isoforms S6, S41 and S47 and 20S proteasome was observed in Cot E maximum time periods and protein disulphide isomerase and DnaK-type molecular chaperone was over expressed in Cot E throughout the entire time points. Cysteine protease isoforms S57 and S36 were present in higher abundance on advanced days, from 4th to 5th day, respectively. The relative expressions of protein namely 17.7 kDa small HSP were insignicant in Cot E with respect to Cot during most time points examined. Amongst rest of the proteins, cyclophilin isoform S52 and 20 kDa chaperonin were upregulated in Cot at the initial phase of callus differentiation

123

Plant Cell Tiss Organ Cult

Fig. 3 Representative 2D PAGE gel image of A Cot E and B Cot spots representing identied protein spots in an integrated gel image of seven different time points

Fig. 4 Differentially abundant selected protein spots under A metabolism B signal transduction C stress and D protein processing categories at different time points. Bar graphs represent the log transformed fold expression values of normalized spot quantity for Cot E over Cot

123

Plant Cell Tiss Organ Cult

and exceeded by the over-abumdance in Cot E from 3rd day onwards. Among the stress related proteins all the isoforms of ascorbate peroxidase were present in higher abundance in Cot E explants from 2nd to 5th day. Four isoforms of the antioxidant superoxide dismutase (SOD) were identied in this study. Two of them, S14 and S16, were present in relatively higher abundance from 2nd to 4th day and then decreased gradually in Cot E; whereas isoform S31 showed up-regulation in later periods, i.e. from 4th day onwards, while, isoform S15 displayed no signicant changes throughout the time period under study. Besides, the antioxidant enzymes, other stress related proteins like b-1, 3-glucanase and PR10 were highly abundant in Cot E from 1st and 2nd day, respectively until 5th day. Proteins implicated in signal transduction, cytoskeleton and cell wall modication, photosynthesis, secondary metabolism, meristem function etc. were also detected in differential abundance during in vitro shoot regeneration. Semi-quantitative RT-PCR Semi-quantitative RT-PCRs were performed for aldehyde reductase, ascorbate peroxidase, cyclophilin, HSP70 and PR10 (Fig. 6). Expression patterns of these transcripts were compared between Cot and Cot E explants at different time points during in vitro differentiation (Fig. 6A). Relative expression levels in terms of fold change in Cot E over Cot were represented graphically for both transcript and protein analyses (Fig. 6B). Proteins with multiple isoforms are represented by the average fold change value. Overall expression patterns of ve selected genes and corresponding proteins represent substantial correlation, except HSP70. While, the predominance of PR10 transcripts on 3rd day in Cot E was observed in the following day at the protein level.

commenced at an early time point in Cot E explants. This led us to detect differential proteomes in Cot E explants with respect to Cot from the induction of differentiation until shoot-bud development under in vitro condition, elucidating qualitative and quantitative changes in the differential proteins within this time period. Analyses of proteomes and changes in protein abundance during in vitro callus differentiation have been reported in Oryza sativa (Yin et al. 2007), shoot organogenesis in Vanilla plantifolia (Palama et al. 2010), somatic embryogenesis in Cyclamen persicum (Bian et al. 2010), Vitis vinifera (Milena et al. 2008), Citrus sinensis Osbeck (Pan et al. 2009), Crocus sativus (Shari et al. 2012), an inbred line of Zea mays (Sun et al. 2013); embryogenic cell cultures in Medicago trancatula (Imin et al. 2004) and in V. unguiculata (Nogueira et al. 2007). In all these studies protein proles retrieve on 2D-gel were only from the soluble fraction of the cell. Multiple spots identied as same proteins with different molecular weights and pI represent different protein species either originating from posttranslational modications or due to proteolytic degradation. The mean CV values for matched spot quantities in experiments of different time points were in a range of 2031 % (Supplementary Table 2). This acceptable range of CV values reected the reproducibility of the experiments as shown by Kundu et al. (2013b). In the present study, a total of 112 differentially expressed proteins were identied during in vitro shoot-bud differentiation. Several proteins could not be identied even with a good quality MS and/or MS/MS signals due to the unavailability of the specic protein sequences in public databases included in MASCOT search engine. This is most common especially in case of organisms without having complete genomic information. Possible biological roles of selected identied proteins during in vitro shoot regeneration are discussed below. Role of metabolism related proteins in shoot-bud differentiation of V. radiata In vitro shoot organogenesis involves extensive cell division over an extended period of time which eventually gives rise to shoot apical meristem development. Energy required for the enhanced cell division is provided by increased rate of metabolism within the cell. The higher abundance of proteins related to energy metabolism is therefore predominant in cell cultures to sustain the intense cell division activity in the developing organs. Carbohydrate metabolism Most of the enzymes of carbohydrate metabolism were differentially regulated in these two explant types. In vitro

Discussion Adventitious shoots differentiated only from the proximal end of the cotyledon. In fact the pioneer researchers (Gulati and Jaiwal 1990; Chandra and Pal 1995) reported that proximal tissues of mungbean cotyledon explants are more responsive to regeneration than the distal. Similar response was also reported in other species including castor (Ahn et al. 2008), soybean (Mante et al. 1989), squash (Ananthakrishnan et al. 2003) and bottle gourd (Han et al. 2004). In this study proteomic analyses has been undertaken in order to elucidate the underlying molecular mechanism of early and efcient morphogenic potentials of Cot E at the protein level. Previous histological study (Das and Pal 2004) indicated that the initiation of meristematic activity

123

Plant Cell Tiss Organ Cult

Fig. 5 Cluster analysis of expression proles of differentially abundant proteins belonging to functional category metabolism, protein processing, stress and storage during initiation of in vitro shoot differentiation. Protein abundance is expressed as the ratio (log

2 base transformed) of Cot E over Cot. The gradual change in colour in the above represented colour bar indicates change in expression from higher to lower in Cot E explants

123

Plant Cell Tiss Organ Cult

Fig. 6 A Semi-quantitative RT-PCR analysis of ve selected differential proteins using actin as an internal control. B Graphical representation of relative expression in terms of fold change in Cot E with respect to Cot for both transcript and protein expression level at different time points

cultures mainly depends on external carbon source as photosynthesis plays a trivial role in providing carbon skeletons and energy for metabolism associated with shootbud induction. Sucrose is considered as the most suitable source of carbon and energy for growth of plant tissue in culture medium (Ovono et al. 2009). Besides, Park et al. (2006) have demonstrated a shift in the stationary growth phase of Eschscholtzia californica culture to the logarithmic phase by adding sucrose in the culture medium. It was presumed that during in vitro differentiation sucrose is taken up by the cotyledonary explants from the medium to meet the high carbon demand of regenerating shoots. Genomic study has recently revealed that among the sucrose degrading enzymes, acid invertase plays a crucial role in determining the mode of regeneration of V. radiata cotyledons in presence of cytokinin (Maiti et al. lez (2004) reported that cytokinin 2011). Roitsch and Gonza upregulates invertase genes providing carbohydrates to the growing tissues. Thus, cytokinin triggers in vitro differentiation enhancing the ability of the cells to import carbohydrates in the form of hexose monomers, while hexoses provide necessary nutrient supply for the growing cells and act as metabolic signal to promote cell divisions. Continuous hydrolysis of sucrose takes place within cotyledonary explants and subsequently supplied to the adventitiously differentiating shoots by simple diffusion method (Maiti et al. 2011). Increased abundance of invertase in Cot E explants was noted up to 4th day indicating a positive correlation between invertase synthesis and adventitious

shoot differentiation in V. radiata. Up regulation of a cytokinin signaling protein (histidine phosphotransfer protein) and the cytokinin biosynthesis protein (isopentenyl transferase) signies continuous cytokinin synthesis during the early time points in Cot E explants. This probably induced invertase expression providing energy for the shoot differentiation in Cot E explants. The higher availability of carbohydrate in Cot E explants was due to higher abundance of enzymes involved in glycolysis and TCA cycle. Over abundance of enzymes like hexokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase, enolase and malate dehydrogenase in Cot E during differentiation indicates enhanced rate of respiration in this explant (Supplementary Figs. 1 and 2). Presumably, the increased cellular respiration was due to rapid growth for meristematic tissue organization in Cot E explants. Starch synthesizing enzyme ADP glucose pyrophosphorylase (AGPase) was present in higher amount in Cot E explants before shoot-bud induction and during early stage of differentiation. Mangat et al. (1990) demonstrated starch accumulation prior to organ pimordia formation especially in sucrose containing media. The accumulated starch acts as an energy reservoir for the utilization in morphogenic processes as indicated by the gradual disappearance of AGPase enzyme with the progress of in vitro shoot regeneration. Presumably starch phosphorylase degrades the accumulated starch to provide nutrients for the growing shoot primordia. Another differential protein, UDP glycosyl transferase

123

Plant Cell Tiss Organ Cult

plays a central role as a glycosyl donor and regulates cellular metabolic pathways. One microsomal UDP-glycosyltransferase gene was over-expressed in Pisum sativum that was implicated in mitosis and the gene strongly induced in dividing cells (Woo et al. 1999). Several toxic aldehydes are produced during lipid peroxidation as a consequence of increased respiration. These toxic aldehydes need to be detoxied in order to protect membrane proteins, inactivate enzymes and receptors. Plant cells contain reductase group of enzymes, like alcohol dehydrogenase, aldehyde reductase, which reduce toxic acetaldehydes produced due to oxidative stress (Sunkar et al. 2003). Both of these enzymes were accumulated in comparatively higher amount in shoot regenerating Cot E explants from 4th and 5th day onwards, respectively. Presumably this kind of regulation salvages the cell from toxic effects under limiting oxygen condition. These enzymes use NADH as cofactor to generate NAD? which perhaps allows uninterrupted glycolysis to continue organogenic differentiation in Cot E explants under cultural condition. Nitrogen metabolism Glutamine synthetase, helps in the assimilation of ammonia generated by numerous plant processes and produces glutamine from the amino acid glutamate for the transportation purpose. The enzyme plays different roles based on its localization mainly in chloroplast or in cytosol (Teixeira et al. 2006). All of the three isoform of glutamine synthetase identied in this study were localized in chloroplast. The differential expression of this enzyme in two cotyledonary explants may be accounted for providing higher pool of transportable amino acids in Cot E that required as nutrient for the organ differentiation. The enzyme aspartate amino-transferase generates aspartate that acts as precursor for several other amino acids and participates in protein synthesis. This protein was up-regulated in Cot E explants from 4th to 6th day. The enzyme dihydropicolinate synthase is involved in biosynthesis of one of the essential amino acid lysine. Though it was present in higher amount during the onset of shoot differentiation, but subsequently disappeared from Cot E 5th day onwards, indicating its role in the synthesis of proteins essential for the induction of shoot primordium. Participation of storage proteins in shoot-bud differentiation of V. radiata Storage proteins serve as nitrogen reserves for embryonic development. Cotyledons of leguminous plants contain high concentrations of these proteins. Amino acids released by the turnover of storage proteins are either redirected to synthesize new proteins or utilized by the developing cells

for other metabolic purposes. Das et al. (2006) have shown that Cot E explants always contained higher amount of proteins than that of Cot. They have also shown using 35S radioisotope that de novo protein synthesis occurred much earlier in the former explants than the latter during in vitro differentiation. The present proteomic study also revealed higher abundance of storage protein in Cot E explants and during early days of in vitro culture. Perhaps the higher pool of storage protein was used up during shoot meristem differentiation in Cot E explants. An increase in endogenous protein content was also reported at the early time points during in vitro morphogenesis of Solanum carolinense (Reynolds 1989). Similarly, in vitro adventitious shoot-bud initiation in Torenia stem explant was also preceded by increase in protein content (Tanimoto and Harada 1983). Role of proteins related to protein processing in shootbud differentiation of V. radiata Cell proliferation during plant development is essentially dependent upon temporal and spatial regulation of the cell division. The regulation includes some cell cycle inhibitors at certain cell cycle stages that regulate the progression of the cell division depending upon positive and negative signals of intracellular or extracellular origin. These inhibitors are degraded rapidly and timely through proteolytic pathways involving small proteins called ubiquitin and a multi-subunit protease called the 26S proteasome. Plant organogenesis related role for 26S proteasome was demonstrated by Yanagawa et al. (2002) in rice where they found abundance of 26S proteasome in apical and marginal meristems of shoots and roots. Signicantly higher amount of 20S proteasome b-subunit was accumulated in Cot E throughout the time of in vitro culture under study. The asubunit was found to be up-regulated up to 4th day starting from the culture initiation indicating proteasome mediated cell cycle progression in highly dividing meristematic tissues of Cot E. Cysteine proteases are associated with storage protein hydrolysis, mobilization (Schlereth et al. 2000) and tissue differentiation (Ye et al. 1996). Differentially regulated cysteine proteases reported in this study suggest their putative role in shoot-bud differentiation by their presence in higher abundance in Cot E explants on later days of culture. Radical changes in overall gene expression occurred at the onset of organogenesis followed by massive alterations in the protein patterns including regulatory proteins. The reorientation in the protein pattern depends on chaperone like proteins which executes folding/unfolding of other proteins. Heat shock proteins (HSPs) are one such class of proteins which assist in proper folding of nascent and misfolded polypeptides by preventing their aggregation. It

123

Plant Cell Tiss Organ Cult

is predictable that the initial up-regulation of DnaK-type molecular chaperone (one subfamily of HSP70) in Cot E explants was due to the reshufing of the protein pattern within the cell prior to shoot meristem differentiation and the later upsurge of HSP70 was probably to assist the folding of de novo synthesized polypeptides and translocation of precursor proteins during shoot-bud development. The chaperone like activity of HSP70 was assisted by co-chaperones, like chaperonins as evident by their over accumulation in Cot E explants during shoot-bud differentiation. Several isoforms of the protein acting as folding catalysts such as peptidyl prolyl cis trans isomerase or cyclophilin have been identied. All these isoforms and protein disulphide isomerase were found to maintain a higher abundance from the onset of shoot-bud induction in Cot E explants suggesting protein frame rearrangement takes place during shoot organogenesis. Calreticulin, a calcium binding protein, regulates the phytohormone mediated signaling cascade by modulating the intracellular calcium homeostasis (Li and Komatsu 2000). Nevertheless this protein was up-regulated in Cot E at later stages, i.e. from 4th to 6th day, suggesting its preferential chaperone like activity rather than phytohormone signaling; since, protein processing is considered to follow phytohormone signal acquisition. Possibilities of involvement of stress related proteins It is already established that oxidative stress is involved in in vitro regeneration and morphogenic processes (Konieczny et al. 2012). Wounding of the tissue occurs during explant preparation for the culture initiation, which is known to cause oxidative stress (Cassells and Curry 2001). Function of antioxidant enzymes in shoot organogenesis The role of antioxidant enzymes is crucial during organ differentiation (Panigrahi et al. 2007; Mitrovic et al. 2012). High hexose supply causes an enhanced rate of respiration by the actively growing cells. The enhanced oxidizing metabolic activity generates reactive oxygen species (ROS) such as superoxide radical (O2-) which is then converted to hydrogen peroxide (H2O2) by the action of superoxide dismutase (SOD). H2O2 generated by the activity of SOD acts as signaling molecule and most stable one among the ROS species. ROS and H2O2 mediated signaling is also implicated in plant growth and development (Reviewed by Petrov and Breusegem 2012). H2O2 activates the Ca?? channels of plasma membrane causing elevated level of cytosolic Ca?? concentration. Subsequently, a downstream cascade is activated via Ca?? binding proteins like

calmodulin and protein kinase eventually leading to the activation of transcription factors that regulate gene expression. The pattern of differential abundance of this antioxidant indicated a dual role of SOD i.e. providing competence to the regenerating tissues of Cot E explants to scavenge superoxide radicals generated due to enhanced rate of respiration and to generate signaling surge at the onset of meristemoid differentiation followed by shoot-bud differentiation. All isoforms of ascorbate peroxidase, the H2O2 detoxifying enzyme, was found in increased abundance from 2nd day onwards indicating that it acts alongside of SOD. One of the H2O2 inducible gene glutathione S-transferase (GST) has been identied in this study. GST acts as an antioxidant to maintain GSH/GSSG (ratio of reduced to oxidized glutathione) balance and maintains cellular redox level. It also conjugates and detoxies metabolites arising from the oxidative damage by lipid peroxidation. Glucose 6-phoshate dehydrogenase catalyses the rate limiting step in oxidative pentose phosphate pathway (OPPP) and serves as an antioxidant providing NADH? as reducing agent for the conversion of oxidized to reduced glutathione form that serves as a cofactor for glutathione peroxidase. Plant small HSP class of proteins is generally absent in normal vegetative tissue and is induced upon specialized cellular conditions like different developmental and stressed states (Waters et al. 1996). It acts as antioxidant and the antioxidative role of sHSPs has been assigned for their ability to decrease the level of cellular ROS. Comparatively higher expression of HSP17 in Cot E on 1st3rd day indicates its dynamic role in shoot meristems during active cell divisions. Other stress related proteins Among other stress related proteins identied, pathogenesis related protein 10 (PR10) from V. radiata has high sequence homology with cytokinin specic binding protein (Fujimoto et al. 1998), therefore it is assumed that PR10 binds with cytokinin to maintain an active cytokinin pool within the cell. Although PR10 protein is well known for its role in plant resistance against biotic and abiotic stresses (Liu et al. 2013), but the up-regulation of this protein in Cot E throughout 2nd5th day suggested its participation for higher meristematic activity in favor of morphogenesis by essentially maintaining a higher endogenous cytokinin level in this explant. On the other hand b-1, 3-glucanase has a role in cell wall loosening that facilitates cell elongation during plant development (Ko et al. 2003). It was up-regulated in Cot E up to 5th day. Class I chitinase is a class of plant endochitinases that implicated in defense processes but their additional role is generation of signal molecules that regulate plant morphogenesis (Collinge

123

Plant Cell Tiss Organ Cult

et al. 1993). It was up regulated in Cot E on 2nd and 3rd day and subsequently on 6th day corroborating with previous reports. Other notable proteins Amongst other proteins, the protein prolin was found to be initially up-regulated in Cot E indicating obvious cytoskeletal changes prior to shoot-bud induction. A leucine-rich repeat (LRR) domain containing receptor-like protein kinase has been identied in this study. Involvement of receptor-like kinases in the formation and maintenance of shoot apical meristem has been reported in Arabidopsis thaliana (Smet et al. 2009). Flavonoids are considered as a backup defense mechanism rather than a primary detoxication system in plants (Yamasaki et al. 1997). Identication of two enzymes related to avonoid biosynthesis (isoavone reductase and avonol 40 -sulfotransferase) suggested their radical scavenging activity in the process of shoot differentiation. Semi-quantitaive RT-PCR In order to validate the differential abundance of proteins as noted during proteomics study, transcript expression analyses were done using semi-quantitative RT-PCR. Five identied proteins, namely, aldehyde reductase, ascorbate peroxidase, HSP 70, cyclophilin and PR10 were randomly chosen for this study. The differential abundance/expression of these ve selected proteins as detected in 2-D GE complies with respective transcript level. Disagreement between the transcript level and protein level in few instances can be attributed to many complicated and varied post-transcriptional mechanisms involved in turning mRNA into protein. Different rates in transcription and translation may also account for the time lapse between transcription and translation process (Greenbaum et al. 2003).

also reported (Das and Pal 2003; Pal et al. 2004). The differential protein abundance regulates differential morphogenic development in these two explant types. The coordinated up-regulation of an array of proteins directed the Cot E explants towards shoot developmental pathway through well-timed acquisition of competence and phytohormone signal. Early stimulation of several metabolic and associated pathways induced shoot organogenic development in Cot E. Perhaps higher accumulation of stress and defense related proteins promptly tuned the potent explants to cope up with stressful in vitro environment thereby triggering cellular growth, changes in cytoskeleton and differentiation. Indispensable cellular reprogramming associated with morphogenesis is accomplished by synthesis of chaperone class of proteins appearing at an earlier time point in the Cot E explants. Thus, the present proteomics investigation portrayed the role of 112 identied proteins that actively participated in shoot organogenesis in Cot E explants of V. radiata under in vitro condition.
Acknowledgments Authors are thankful to the Department of Science and Technology, Government of India (DST Sanction no. SR/ SO/PS-58/05) for constant nancial support in this area of research; and to the Director, Bose Institute for providing all infrastructural facilities and a Senior Research Fellowship to SG. The proteomic facilities provided by DST through IRHPA project (IR/SO/LF02/ 2002) are thankfully acknowledged.

References
Ahn YJ, Chen GQ (2008) In Vitro regeneration of castor (Ricinus communis L.) using cotyledon explants. Hort Sci 43:209215 Ananthakrishnan G, Xia X, Elman C, Singer S, Paris HS, Gal-On A, Gaba V (2003) Shoot production in squash (Cucurbita pepo) by in vitro organogenesis. Plant Cell Rep 21:739746 T (2008) Baba A, Nogueira F, Pinheiro C, Brasil J, Jereissati E, Juca Proteome analysis of secondary somatic embryogenesis in cassava (Manihot esculenta). Plant Sci 175:717723 Bian F, Zheng C, Qu F, Gong X, You C (2010) Proteomic analysis of somatic embryogenesis in Cyclamen persicum Mill. Plant Mol Biol Rep 28:2231 Cassells AC, Curry RF (2001) Oxidative stress and physiological, epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineers. Plant Cell, Tissue Org Cult 64:145157 Chandra M, Pal A (1995) Differential response of the two cotyledons of Vigna radiata in vitro. Plant Cell Rep 15:248253 Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K (1993) Plant chitinases. Plant J 3:3140 Das S, Pal A (2003) Differential DNA endoreduplication and protein prole during cotyledon ontogeny of Vigna radiata. J Plant Biochem Biotech 12:1118 Das S, Pal A (2004) Differential regeneration response in two cotyledons of Vigna radiata: histomorphological analysis and effect of - arabinogalactan. J Plant Biochem Biotech 13:101106 Das S, Sengupta DN, Pal A (2006) Differential protein pattern of two cotyledons of Vigna radiata during induced in vitro

Conclusion It is conrmed from the present proteomics information that there is differential regulation of proteins between the two explant types and that leads to differential regeneration responses. The qualitative and quantitative differences in total proteomes were also found under in vivo condition i.e. prior to cytokinin induced in vitro shoot differentiation. In fact, previous studies have shown differential control of gene expressions are the determinants of developmental switches. Variations in the degree of DNA endoreduplication and the pattern of storage protein accumulation during the various stages of cotyledon developmental were

123

Plant Cell Tiss Organ Cult differentiation: probable implication in the conundrum of differential regeneration response. J Plant Biochem Biotech 15:123129 Fujimoto Y, Nagata R, Fukasawa H, Yano K, Azuma M, Iida A, Sugimoto S, Shudo K, Hashimoto Y (1998) Purication and cDNA cloning of cytokinin-specic binding protein from mung bean (Vigna radiata). Eur J Biochem 258:794802 Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151158 Ghosh S, Pal A (2012) Identication of differential proteins of mungbean cotyledons during seed germination: a proteomic approach. Acta Physiol Plant 34:23792391 Greenbaum D, Colangelo C, Williams K, Gerstein M (2003) Comparing protein abundance and mRNA expression levels on a genomic scale. Genome Biol 4:117 Gulati A, Jaiwal PK (1990) Culture conditions effecting plant regeneration from cotyledons of Vigna radiata (L.) Wilczek. Plant Cell Tissue Org Cult 23:17 Han JS, Oh DG, Mok IG, Park HG, Kim CK (2004) Efcient plant regeneration from cotyledon explants of bottle gourd (Lagenaria siceraria Standl). Plant Cell Rep 23:291296 Imin N, Jong FD, Mathesius U, Noorden GV, Saeed NA, Wang XD, Rose RJ, Rolfe BG (2004) Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts. Proteomics 4:18831896 Ko TS, Lee S, Schaefer SC, Korban SS (2003) Characterization of a tissue-specic and developmentally regulated b-1,3-glucanase gene family in Prunus persica. Plant Physiol Biochem 41: 955963 Konieczny ML, Konieczny R, Suro0 wka E, S0 lesak I, Michalec Z, Rozpadek P, Miszalsk Z (2012) Pathways of ROS homeostasis regulation in Mesembryanthemum crystallinum L. calli exhibiting differences in rhizogenesis. Plant Cell, Tissue Organ Cult 110:123131 Kundu S, Chakraborty D, Pal A (2011) Proteomic analysis of salicylic acid induced resistance to Mungbean Yellow Mosaic India Virus in Vigna mungo. J Proteomics 74:337349 Kundu S, Chakraborty D, Das K, Pal A (2013a) An efcient in-gel digestion protocol for mass spectral analysis by MALDI-TOFMS and MS/MS and its use for proteomic analysis of Vigna mungo leaves. Plant Mol Biol Rep 31:4754 Kundu S, Chakraborty D, Kundu A, Pal A (2013b) Proteomics approach combined with biochemical attributes to elucidate compatible and incompatible plant-virus interactions between Vigna mungo and Mungbean Yellow Mosaic India Virus. Proteome Sci 11:15 Li Z, Komatsu S (2000) Molecular cloning and characterization of calreticulin, a calcium-binding protein involved in the regeneration of rice cultured suspension cells. Eur J Biochem 267: 737745 Liu JJ, Ekramoddoullah AKM, Hawkins B, Shah S (2013) Overexpression of a western white pine PR10 protein enhances cold tolerance in transgenic Arabidopsis. Plant Cell Tissue Org Cult. doi:10.1007/s11240-013-0317-z Maiti S, Kundu S, Chakraborty D, Paul S, Sengupta S, Das K, Pal A (2011) Developmentally regulated temporal expression and differential acid invertase activity in differentiating cotyledonary explants of mungbean [Vigna radiata (L.) Wilczek. Plant Cell Tissue Org Cult 107:417425 Mangat BS, Pelekis M, Cassells AC (1990) Changes in the starch content during erganogenesis in in vitro cultured Begonia rex stem explants. Physiol Plant 79:267274 Mante S, Scorza R, Cordts J (1989) A simple, rapid protocol for adventitious shoot development from mature cotyledons of Glycine max cv. Bragg. In Vitro Cell Dev Biol Plant 25:385388 Milena M, Marcella B, Luca E, Bhakti P, Alfredo N, Candida V (2008) Proteomic analysis of somatic embryogenesis in Vitis vinifera. Plant Cell Rep 27:347356 Mitrovic A, Janosevic D, Budimir S, Bogdanovic Pristov J (2012) Changes in antioxidative enzymes activities during Tacitus bellus direct shoot organogenesis. Biol Plant 56:357361 Nogueira FCS, Goncalves EF, Jereissati ES, Santos M, Costa JH, Oliveira-Neto OB, Soares AA, Domont GB, Campos FAP (2007) Proteome analysis of embryogenic cell suspensions of cowpea (Vigna unguiculata). Plant Cell Rep 26:13331343 Ovono PO, Kevers C, Dommes J (2009) Effects of reducing sugar concentration on in vitro tuber formation and sprouting in yam (Dioscorea cayenensisD. rotundata complex). Plant Cell Tissue Org Cult 99:5559 Pal A, Vrana J, Dolezel J (2004) Flow cytometric analysis of variation in the level of nuclear DNA endoreduplication in the cotyledons amongst Vigna radiata cultivars. Caryologia 57:262266 Palama TL, Menard P, Fock I, Choi YH, Bourdon E, GovindenSoulange J, Bahut M, Payet B, Verpoorte R, Kodja H (2010) Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage. BMC Plant Biol 10:82 Pan Z, Guan R, Zhu S, Deng X (2009) Proteomic analysis of somatic embryogenesis in Valencia sweet orange (Citrus sinensis Osbeck). Plant Cell Rep 28:281289 Panigrahi J, Behera M, Maharana S, Mishra RR (2007) Biomolecular changes during in vitro organogenesis of Asteracantha longifolia (L.) NeesA medicinal herb. Ind J Exp Biol 45:911919 Park JJ, Yoon SYH, Cho HY, Son SY, Rhee HS, Park JM (2006) Patterns of protein expression upon adding sugar and elicitor to the cell culture of Eschscholtzia californica. Plant Cell Tissue Org Cult 86:257269 Petrov VD, Breusegem FV (2012) Hydrogen peroxidea central hub for information ow in plant cells. AoB Plants. doi:10.1093/ aobpla/pls014 Reynolds TL (1989) Changes in RNA, protein, and translatable messenger RNA synthesis and accumulation during adventive organogenesis in somatic tissue cultures of Solanum carolinense. Plant Sci 65:7785 lez MC (2004) Function and regulation of plant Roitsch T, Gonza invertases: sweet sensations. Trends Plant Sci 9:606613 ntz K (2000) Schlereth A, Becker C, Horstmann C, Tiedemann J, Mu Comparison of globulin mobilization and cysteine proteinases in embryonic axes and cotyledons during germination and seedling growth of vetch (Vicia sativa L.). J Exp Bot 51:14231433 Shari G, Ebrahimzadeh H, Ghareyazie B, Gharechahi J, Vatankhah E (2012) Identication of differentially accumulated proteins associated with embryogenic and non-embryogenic calli in saffron (Crocus sativus L.). Proteome Sci 10:115 rgens G, Beeckman T (2009) Receptor-like Smet ID, Vo U, Ju kinases shape the plant. Nat Cell Biol 11:11661173 Somers DA, Samac DA, Olhoft PM (2003) Recent advances in legume transformation. Plant Physiol 131:892899 Sun L, Wu Y, Zou H, Su S, Li S, Shan X, Xi J, Yuan Y (2013) Comparative proteomic analysis of the H99 inbred maize (Zea mays L.) line in embryogenic and non-embryogenic callus during somatic embryogenesis. Plant Cell Tissue Org Cult 113:103119 Sunkar R, Bartels D, Kirch HH (2003) Overexpression of a stressinducible aldehyde dehydrogenase gene from Arabidopsis thaliana in transgenic plants improves stress tolerance. Plant J 35:452464 Tan EC, Karsani SA, Foo GT, Wong SM, Rahman NA, Khalid N, Othman S, Yusof R (2012) Proteomic analysis of cell suspension cultures of Boesenbergia rotunda induced by phenylalanine: identication of proteins involved in avonoid and

123

Plant Cell Tiss Organ Cult phenylpropanoid biosynthesis pathways. Plant Cell Tissue Org Cult 111:219229 Tanimoto S, Harada H (1983) Protein synthesis during adventitious bud initiation in supercial cell layers of Torenia stem segments cultured in vitro. Biochem Physiol Panzen 178:391400 s F, Fidalgo F (2006) Specic roles of Teixeira J, Pereira S, Queiro potato glutamine synthetase isoenzymes in callus tissue grown under salinity: molecular and biochemical responses. Plant Cell Tissue Org Cult 87:17 Vasconcelos EAR, Nogueira FCS, Abreu EFM, Goncalves EF, Souza PAS, Campos FAP (2005) Protein extraction from cowpea tissues for 2-D gel electrophoresis and MS analysis. Chromatographia 62:447450 Waters ER, Lee GJ, Vierling E (1996) Evolution, structure and function of the small heat shock proteins in plants. J Exp Bot 47:325338 Woo HH, Orbach MJ, Hirsch AM, Hawesa MC (1999) Meristemlocalized inducible expression of a UDP-glycosyltransferase gene is essential for growth and development in Pea and Alfalfa. Plant Cell 11:23032315 Yamasaki H, Sakihama Y, lkehara N (1997) Flavonoid-peroxidase reaction as a detoxication mechanism of plant cells against H202. Plant Physiol 115:14051412 Yanagawa Y, Hasezawa S, Kumagai F, Oka M, Fujimuro M, Naito T, Makino T, Yokosawa H, Tanaka K, Komamine A, Hashimoto J, Sato T, Nakagawa H (2002) Cell-cycle dependent dynamic change of 26S proteasome distribution in tobacco BY-2 cells. Plant Cell Physiol 43:604613 Ye ZH, Varner JE (1996) Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans. Plant Mol Biol 30: 12331246 Yin L, Tao Y, Zhao K, Shao J, Li X, Liu G, Liu S, Zhu L (2007) Proteomic and transcriptomic analysis of rice mature seedderived callus differentiation. Proteomics 7:755768 Zhang J, Mab H, Chen S, Ji M, Perl A, Kovacs L, Chen S (2009) Stress response proteins differential expression in embryogenic and non-embryogenic callus of Vitis vinifera L. cv. Cabernet SauvignonA proteomic approach. Plant Sci 177:103113

123