Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 115, Issue 1, pp 55–68 | Cite as

Proteomic analysis of cotyledonary explants during shoot organogenesis in Vigna radiata

  • Suparna Ghosh
  • Amita PalEmail author
Original Paper


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 profiles 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 Gamborg’s B5 basal nutrient composition supplemented with 15 μM 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 identified proteins were classified 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 cultural condition. Enhanced accumulation of folding-assisted proteins involved in organogenesis mediated cellular reprogramming in Cot E explants contributed further in rapid and efficient regeneration responsiveness.


Cytokinin MALDI-TOF-TOF Proteomics Shoot regeneration Vigna radiata 



Shoot induction medium

2-D GE

Two-dimensional gel electrophoresis




Bovine serum albumin




Coefficient of variation




Isoelectric focusing


Matrix assisted laser desorption-ionization time-of-flight mass spectrometry


MultiExperiment viewer


Molecular weight search


Phenylmethylsulfonyl fluoride


Reverse transcription polymerase chain reaction


Sodium dodecyl sulphate polyacrylamide gel electrophoresis



Authors are thankful to the Department of Science and Technology, Government of India (DST Sanction no. SR/SO/PS-58/05) for constant financial 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.

Supplementary material

11240_2013_340_MOESM1_ESM.jpg (418 kb)
Supplementary Fig. 1. Involvement of the identified proteins in the Glycolysis/Gluconeogenesis pathway assigned by KEGG database in Blast2go software. (JPEG 417 kb)
11240_2013_340_MOESM2_ESM.jpg (355 kb)
Supplementary Fig. 2. Involvement of the identified protein in the tricarboxylic acid cycle pathway assigned by KEGG database in Blast2go software. (JPEG 354 kb)
11240_2013_340_MOESM3_ESM.doc (200 kb)
Supplementary material 3 (DOC 199 kb)
11240_2013_340_MOESM4_ESM.doc (34 kb)
Supplementary material 4 (DOC 34 kb)


  1. Ahn YJ, Chen GQ (2008) In Vitro regeneration of castor (Ricinus communis L.) using cotyledon explants. Hort Sci 43:209–215Google Scholar
  2. 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 21:739–746PubMedGoogle Scholar
  3. Baba A, Nogueira F, Pinheiro C, Brasil J, Jereissati E, Jucá T (2008) Proteome analysis of secondary somatic embryogenesis in cassava (Manihot esculenta). Plant Sci 175:717–723CrossRefGoogle Scholar
  4. Bian F, Zheng C, Qu F, Gong X, You C (2010) Proteomic analysis of somatic embryogenesis in Cyclamen persicum Mill. Plant Mol Biol Rep 28:22–31CrossRefGoogle Scholar
  5. 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 64:145–157CrossRefGoogle Scholar
  6. Chandra M, Pal A (1995) Differential response of the two cotyledons of Vigna radiata in vitro. Plant Cell Rep 15:248–253Google Scholar
  7. Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K (1993) Plant chitinases. Plant J 3:31–40PubMedCrossRefGoogle Scholar
  8. Das S, Pal A (2003) Differential DNA endoreduplication and protein profile during cotyledon ontogeny of Vigna radiata. J Plant Biochem Biotech 12:11–18CrossRefGoogle Scholar
  9. Das S, Pal A (2004) Differential regeneration response in two cotyledons of Vigna radiata: histomorphological analysis and effect of ß- arabinogalactan. J Plant Biochem Biotech 13:101–106CrossRefGoogle Scholar
  10. Das S, Sengupta DN, Pal A (2006) Differential protein pattern of two cotyledons of Vigna radiata during induced in vitro differentiation: probable implication in the conundrum of differential regeneration response. J Plant Biochem Biotech 15:123–129CrossRefGoogle Scholar
  11. Fujimoto Y, Nagata R, Fukasawa H, Yano K, Azuma M, Iida A, Sugimoto S, Shudo K, Hashimoto Y (1998) Purification and cDNA cloning of cytokinin-specific binding protein from mung bean (Vigna radiata). Eur J Biochem 258:794–802PubMedCrossRefGoogle Scholar
  12. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158PubMedCrossRefGoogle Scholar
  13. Ghosh S, Pal A (2012) Identification of differential proteins of mungbean cotyledons during seed germination: a proteomic approach. Acta Physiol Plant 34:2379–2391CrossRefGoogle Scholar
  14. Greenbaum D, Colangelo C, Williams K, Gerstein M (2003) Comparing protein abundance and mRNA expression levels on a genomic scale. Genome Biol 4:117PubMedCrossRefGoogle Scholar
  15. Gulati A, Jaiwal PK (1990) Culture conditions effecting plant regeneration from cotyledons of Vigna radiata (L.) Wilczek. Plant Cell Tissue Org Cult 23:1–7CrossRefGoogle Scholar
  16. Han JS, Oh DG, Mok IG, Park HG, Kim CK (2004) Efficient plant regeneration from cotyledon explants of bottle gourd (Lagenaria siceraria Standl). Plant Cell Rep 23:291–296PubMedCrossRefGoogle Scholar
  17. 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 4:1883–1896PubMedCrossRefGoogle Scholar
  18. Ko TS, Lee S, Schaefer SC, Korban SS (2003) Characterization of a tissue-specific and developmentally regulated β-1,3-glucanase gene family in Prunus persica. Plant Physiol Biochem 41:955–963CrossRefGoogle Scholar
  19. Konieczny ML, Konieczny R, Suro′wka E, S′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 110:123–131CrossRefGoogle Scholar
  20. 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 74:337–349PubMedCrossRefGoogle Scholar
  21. Kundu S, Chakraborty D, Das K, Pal A (2013a) An efficient in-gel digestion protocol for mass spectral analysis by MALDI-TOF-MS and MS/MS and its use for proteomic analysis of Vigna mungo leaves. Plant Mol Biol Rep 31:47–54CrossRefGoogle Scholar
  22. 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 11:15PubMedCrossRefGoogle Scholar
  23. 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 267:737–745PubMedCrossRefGoogle Scholar
  24. 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 Google Scholar
  25. 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 107:417–425CrossRefGoogle Scholar
  26. Mangat BS, Pelekis M, Cassells AC (1990) Changes in the starch content during erganogenesis in in vitro cultured Begonia rex stem explants. Physiol Plant 79:267–274CrossRefGoogle Scholar
  27. 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 25:385–388CrossRefGoogle Scholar
  28. Milena M, Marcella B, Luca E, Bhakti P, Alfredo N, Candida V (2008) Proteomic analysis of somatic embryogenesis in Vitis vinifera. Plant Cell Rep 27:347–356CrossRefGoogle Scholar
  29. Mitrovic A, Janosevic D, Budimir S, Bogdanovic Pristov J (2012) Changes in antioxidative enzymes activities during Tacitus bellus direct shoot organogenesis. Biol Plant 56:357–361CrossRefGoogle Scholar
  30. 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 26:1333–1343PubMedCrossRefGoogle Scholar
  31. 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 99:55–59CrossRefGoogle Scholar
  32. 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 57:262–266CrossRefGoogle Scholar
  33. Palama TL, Menard P, Fock I, Choi YH, Bourdon E, Govinden-Soulange 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 10:82PubMedCrossRefGoogle Scholar
  34. Pan Z, Guan R, Zhu S, Deng X (2009) Proteomic analysis of somatic embryogenesis in Valencia sweet orange (Citrus sinensis Osbeck). Plant Cell Rep 28:281–289PubMedCrossRefGoogle Scholar
  35. Panigrahi J, Behera M, Maharana S, Mishra RR (2007) Biomolecular changes during in vitro organogenesis of Asteracantha longifolia (L.) Nees—A medicinal herb. Ind J Exp Biol 45:911–919Google Scholar
  36. 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 86:257–269CrossRefGoogle Scholar
  37. Petrov VD, Breusegem FV (2012) Hydrogen peroxide—a central hub for information flow in plant cells. AoB Plants. doi: 10.1093/aobpla/pls014 PubMedGoogle Scholar
  38. 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 65:77–85CrossRefGoogle Scholar
  39. Roitsch T, González MC (2004) Function and regulation of plant invertases: sweet sensations. Trends Plant Sci 9:606–613PubMedCrossRefGoogle Scholar
  40. Schlereth A, Becker C, Horstmann C, Tiedemann J, Müntz K (2000) 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 51:1423–1433PubMedCrossRefGoogle Scholar
  41. Sharifi G, Ebrahimzadeh H, Ghareyazie B, Gharechahi J, Vatankhah E (2012) Identification of differentially accumulated proteins associated with embryogenic and non-embryogenic calli in saffron (Crocus sativus L.). Proteome Sci 10:1–15CrossRefGoogle Scholar
  42. Smet ID, Voß U, Jürgens G, Beeckman T (2009) Receptor-like kinases shape the plant. Nat Cell Biol 11:1166–1173PubMedCrossRefGoogle Scholar
  43. Somers DA, Samac DA, Olhoft PM (2003) Recent advances in legume transformation. Plant Physiol 131:892–899PubMedCrossRefGoogle Scholar
  44. 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 113:103–119CrossRefGoogle Scholar
  45. Sunkar R, Bartels D, Kirch HH (2003) Overexpression of a stress-inducible aldehyde dehydrogenase gene from Arabidopsis thaliana in transgenic plants improves stress tolerance. Plant J 35:452–464PubMedCrossRefGoogle Scholar
  46. 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: identification of proteins involved in flavonoid and phenylpropanoid biosynthesis pathways. Plant Cell Tissue Org Cult 111:219–229CrossRefGoogle Scholar
  47. Tanimoto S, Harada H (1983) Protein synthesis during adventitious bud initiation in superficial cell layers of Torenia stem segments cultured in vitro. Biochem Physiol Pflanzen 178:391–400CrossRefGoogle Scholar
  48. Teixeira J, Pereira S, Queirós F, Fidalgo F (2006) Specific roles of potato glutamine synthetase isoenzymes in callus tissue grown under salinity: molecular and biochemical responses. Plant Cell Tissue Org Cult 87:1–7CrossRefGoogle Scholar
  49. 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 62:447–450CrossRefGoogle Scholar
  50. Waters ER, Lee GJ, Vierling E (1996) Evolution, structure and function of the small heat shock proteins in plants. J Exp Bot 47:325–338CrossRefGoogle Scholar
  51. Woo HH, Orbach MJ, Hirsch AM, Hawesa MC (1999) Meristem-localized inducible expression of a UDP-glycosyltransferase gene is essential for growth and development in Pea and Alfalfa. Plant Cell 11:2303–2315PubMedGoogle Scholar
  52. Yamasaki H, Sakihama Y, lkehara N (1997) Flavonoid-peroxidase reaction as a detoxification mechanism of plant cells against H202. Plant Physiol 115:1405–1412PubMedGoogle Scholar
  53. 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 43:604–613PubMedCrossRefGoogle Scholar
  54. Ye ZH, Varner JE (1996) Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans. Plant Mol Biol 30:1233–1246PubMedCrossRefGoogle Scholar
  55. Yin L, Tao Y, Zhao K, Shao J, Li X, Liu G, Liu S, Zhu L (2007) Proteomic and transcriptomic analysis of rice mature seed-derived callus differentiation. Proteomics 7:755–768PubMedCrossRefGoogle Scholar
  56. 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 Sauvignon—A proteomic approach. Plant Sci 177:103–113CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.Division of Plant BiologyBose InstituteKolkataIndia

Personalised recommendations