Plant Molecular Biology

, Volume 84, Issue 4–5, pp 415–428 | Cite as

Evidence for phosphorylation of the major seed storage protein of the common bean and its phosphorylation-dependent degradation during germination

  • María López-Pedrouso
  • Jana Alonso
  • Carlos Zapata


Phaseolin is the major seed storage protein of common bean, Phaseolus vulgaris L., accounting for up to 50 % of the total seed proteome. The regulatory mechanisms responsible for the synthesis, accumulation and degradation of phaseolin in the common bean seed are not yet sufficiently known. Here, we report on a systematic study in dormant and 4-day germinating bean seeds from cultivars Sanilac (S) and Tendergreen (T) to explore the presence and dynamics of phosphorylated phaseolin isoforms. High-resolution two-dimensional electrophoresis in combination with the phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescent stain and chemical dephosphorylation by hydrogen fluoride–pyridine enabled us to identify differentially phosphorylated phaseolin polypeptides in dormant and germinating seeds from cultivars S and T. Phosphorylated forms of the two subunits of type α and β that compose the phaseolin were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MALDI-TOF/TOF tandem MS. In addition, we found that the levels of phosphorylation of the phaseolin changed remarkably in the seed transition from dormancy to early germination stage. Temporal changes in the extent of phosphorylation in response to physiological and metabolic variations suggest that phosphorylated phaseolin isoforms have functional significance. In particular, this prospective study supports the hypothesis that mobilization of the phaseolin in germinating seeds occurs through the degradation of highly phosphorylated isoforms. Taken together, our results indicate that post-translational phaseolin modifications through phosphorylations need to be taken into consideration for a better understanding of the molecular mechanisms underlying its regulation.


Phaseolin Phaseolusvulgaris Phosphoproteins Post-translational protein modification Seed phosphoproteome Phosphorylation-dependent regulation 



Two-dimensional electrophoresis


Abscisic acid


Gibberellic acid


Hydrofluoric acid


Relative molecular mass


Matrix-assisted laser desorption/ionization time-of-flight


Mass spectrometry




β-Phaseolin gene


Isoelectric point


Phosphorylation rate


Pro-Q Diamond phosphoprotein stain


Post-translational protein modification




Seed storage protein





This research was supported in part by Grant 10PXIB262008PR (Xunta de Galicia, Spain).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11103_2013_141_MOESM1_ESM.doc (1.8 mb)
Supplementary material 1 (DOC 1861 kb)


  1. Aberlenc-Bertossi F, Chabrillange N, Duval Y, Tregear J (2008) Contrasting globulin and cysteine proteinase gene expression patterns reveal fundamental developmental differences between zygotic and somatic embryos of oil palm. Tree Physiol 28:1157–1167PubMedCrossRefGoogle Scholar
  2. Agrawal GK, Thelen JJ (2005) Development of a simplified, economical polyacrylamide gel staining protocol for phosphoproteins. Proteomics 5:4684–4688PubMedCrossRefGoogle Scholar
  3. Agrawal GK, Thelen JJ (2006) Large-scale identification and quantitative profiling of phosphoproteins expressed during seed filling in oilseed rape. Mol Cell Proteomics 5:2044–2059PubMedCrossRefGoogle Scholar
  4. Baud S, Boutin J, Miquel M, Lepiniec L, Rochat C (2002) An integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant Physiol Biochem 40:151–160CrossRefGoogle Scholar
  5. Bollini R, Vitale A, Chrispeels MJ (1983) In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps. J Cell Biol 96:999–1007PubMedCrossRefGoogle Scholar
  6. de la Fuente van Bentem S, Hirt H (2007) Using phosphoproteomics to reveal signalling dynamics in plants. Trends Plant Sci 9:404–411CrossRefGoogle Scholar
  7. de La Fuente M, Borrajo A, Bermúdez J, Lores M, Alonso J, López M, Santalla M, De Ron A, Zapata C, Alvarez G (2011) 2-DE-based proteomic analysis of common bean (Phaseolus vulgaris L.) seeds. J Proteomics 74:262–267CrossRefGoogle Scholar
  8. de La Fuente M, López-Pedrouso M, Alonso J, Santalla M, De Ron AM, Alvarez G, Zapata C (2012) In-depth characterization of the phaseolin protein diversity of common bean (Phaseolus vulgaris L.) based on two-dimensional electrophoresis and mass spectrometry. Food Technol Biotechnol 50:315–325Google Scholar
  9. Emani C, Hall TC (2008) Phaseolin: structure and evolution. Open Evol J 2:66–74Google Scholar
  10. Finch-Savage WE, Leubner-Metzger G (2006) Seed dormancy and the control of germination. New Phytol 171:501–523PubMedCrossRefGoogle Scholar
  11. Gepts P (1998) Phaseolin as an evolutionary marker. In: Gepts P (ed) Genetic resources of Phaseolus beans. Kluwer Academic Publishers, Dordrecht, pp 215–241Google Scholar
  12. Gepts P, Aragao FL, de Barros E, Blair MW, Brondani R, Broughton W, Galasso I, Hernandez G, Kami J, Lariguet P, McClean P, Melotto M, Miklas P, Pauls P, Pedrosa-Harand A, Porch T, Sánchez F, Sparvoli F, Yu K (2008) Genomics of Phaseolus beans, a major source of dietary protein and micronutrients in the tropics. In: Moore PH, Ming R (eds) Genomics of tropical crop plants. Springer, Philadelphia, pp 113–143CrossRefGoogle Scholar
  13. Ghelis T, Bolbach G, Clodic G, Habricot Y, Miginiac E, Sotta B, Jeannette E (2008) Protein tyrosine kinases and protein tyrosine phosphatases are involved in abscisic acid-dependent processes in Arabidopsis seeds and suspension cells. Plant Physiol 148:1668–1680PubMedCentralPubMedCrossRefGoogle Scholar
  14. Gutierrez L, Van Wuytswinkel O, Castelain M, Bellini C (2007) Combined networks regulating seed maturation. Trends Plant Sci 12:294–300PubMedCrossRefGoogle Scholar
  15. Hall TC, Chandrasekharan MB, Li G (1999) Phaseolin: its past, properties, regulation and future. In: Shewry PR, Casey R (eds) Seed proteins. Kluwer Academic Publishers, The Netherlands, pp 209–240CrossRefGoogle Scholar
  16. Hellmann H, Estelle M (2002) Plant development: regulation by protein degradation. Science 297:793–797PubMedCrossRefGoogle Scholar
  17. Hirayama T, Shinozaki K (2007) Perception and transduction of abscisic acid signals: keys to the function of the versatile plant hormone ABA. Trends Plant Sci 12:343–351PubMedCrossRefGoogle Scholar
  18. Irar S, Oliveira E, Pagès M, Goday A (2006) Towards the identification of late-embryogenic-abundant phosphoproteome in Arabidopsis by 2-DE and MS. Proteomics 6:S175–S185PubMedCrossRefGoogle Scholar
  19. Kami J, Gepts P (1994) Phaseolin nucleotide sequence diversity in Phaseolus. I. Intraspecific diversity in Phaseolus vulgaris. Genome 37:751–757PubMedCrossRefGoogle Scholar
  20. Karin M, Ben-Neriah Y (2000) Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol 18:621–663PubMedCrossRefGoogle Scholar
  21. Kersten B, Agrawal GK, Durek P, Neigenfind J, Schulze W, Walther D, Rakwal R (2009) Plant phosphoproteomics: an update. Proteomics 9:964–988PubMedCrossRefGoogle Scholar
  22. Kita K, Okumura N, Takao T, Watanabe M, Matsubara T, Nishimura O, Nagai K (2006) Evidence for phosphorylation of rat liver glucose-regulated protein 58, GRP58/ERp57/ER-60, induced by fasting and leptin. FEBS Lett 580:199–205PubMedCrossRefGoogle Scholar
  23. Kline-Jonakin KG, Barrett-Wilt GA, Sussman MR (2011) Quantitative plant phosphoproteomics. Curr Opin Plant Biol 14:507–511PubMedCrossRefGoogle Scholar
  24. Kovaleva V, Cramer R, Krynytskyy H, Gout I, Gout R (2013) Analysis of tyrosine phosphorylation and phosphotyrosine-binding proteins in germinating seeds from Scots pine. Plant Physol Biochem 67:33–40CrossRefGoogle Scholar
  25. Kuyama H, Toda C, Watanabe M, Tanaka K, Nishimura O (2003) An efficient chemical method for dephosphorylation of phosphopeptides. Rapid Commun Mass Spectrom 17:1493–1496PubMedCrossRefGoogle Scholar
  26. Lee J, Feng J, Campbell KB, Scheffler BE, Garret WM, Thibivilliers S, Stacey G, Naiman DQ, Tucker ML, Pastor-Corrales MA, Cooper B (2009) Quantitative proteomic analysis of bean plants infected by a virulent and avirulent obligate rust fungus. Mol Cell Proteomics 8:19–31PubMedCrossRefGoogle Scholar
  27. Li G, Bishop KJ, Chandrasekharan MB, Hall TC (1999) Phaseolin gene activation is a two-step process: PvALF-facilitated chromatin modification followed by abscisic acid-mediated gene activation. Proc Natl Acad Sci USA 96:7104–7109PubMedCrossRefGoogle Scholar
  28. Lioi L, Bollini R (1984) Contribution of processing events to the molecular heterogeneity of four banding types of phaseolin, the major storage protein of Phaseolus vulgaris L. Plant Mol Biol 3:345–353PubMedCrossRefGoogle Scholar
  29. Marsolais F, Pajak A, Yin F, Taylor M, Gabriel M, Merino DM, Ma V, Kameka A, Vijayan P, Pham H, Huang S, Rivoal J, Bett K, Hernández-Sebastià C, Liu Q, Bertrand A, Chapman R (2010) Proteomic analysis of common bean seed with storage protein deficiency reveals up-regulation of sulfur-rich proteins and starch and raffinose metabolic enzymes and down-regulation of the secretory pathway. J Proteomics 73:1587–1600PubMedCrossRefGoogle Scholar
  30. Mensack M, Fitzgerald VK, Ryan EO, Lewis MR, Thompson HJ, Brick MA (2010) Evaluation of diversity among common beans (Phaseolus vulgaris L.) from two centers of domestication using ‘omics’ technologies. BMC Genomics 11:686PubMedCentralPubMedCrossRefGoogle Scholar
  31. Meyer LJ, Gao J, Xu D, Thelen JJ (2012) Phosphoproteomic analysis of seed maturation in Arabidopsis, rapeseed, and soybean. Plant Phys 159:517–528CrossRefGoogle Scholar
  32. Myernik JA, Hajduch M (2011) Seed proteomics. J Proteomics 74:389–400CrossRefGoogle Scholar
  33. Ng DW-K, Hall TC (2008) PvALF and FUS3 activate expression from the phaseolin promoter by different mechanisms. Plant Mol Biol 66:233–244CrossRefGoogle Scholar
  34. Saravanan RS, Rose JKC (2004) A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 4:2522–2532PubMedCrossRefGoogle Scholar
  35. Schubert P, Hoffman MD, Sniatynski MJ, Kast J (2006) Advances in the analysis of dynamic protein complexes by proteomics and data processing. Anal Bioanal Chem 386:482–493PubMedCrossRefGoogle Scholar
  36. Schwenke KD, Mothes R, Dudek S, Görtniz E (2000) Phosphorylation of the 12S globulin from rapeseed (Brassica napus L.) by phosphorous oxychloride: chemical and conformational aspects. J Agric Food Chem 48:708–715PubMedCrossRefGoogle Scholar
  37. Singh SP (2001) Broadening the genetic base of common bean cultivars: a review. Crop Sci 41:1659–1675CrossRefGoogle Scholar
  38. Slightom JL, Sun S, Hall TC (1983) Complete nucleotide sequence of a French bean storage protein gene: phaseolin. Proc Natl Acad Sci USA 80:1897–1901PubMedCrossRefGoogle Scholar
  39. Slightom JL, Drong RF, Klassy C, Hoffman LM (1985) Nucleotide sequences from phaseolin cDNA clones: the major storage proteins from Phaseolus vulgaris are encoded by two unique gene families. Nucleic Acids Res 13:6483–6498PubMedCentralPubMedCrossRefGoogle Scholar
  40. Spencer D (1984) The physiological role of storage proteins in seeds. Philos Trans R Soc Lond B 304:275–285CrossRefGoogle Scholar
  41. Sturm A, Van Kuik JA, Vliegenthart JFG, Chrispeels MJ (1987) Structure, position and biosynthesis of the high mannose and the complex oligosaccharide side chains of the bean storage protein phaseolin. J Biol Chem 262:13392–13403PubMedGoogle Scholar
  42. Vitale A, Bollini R (1995) Legume storage proteins. In: Kigel J, Galili G (eds) Seed development and germination. Marcel Dekker, New York, pp 73–102Google Scholar
  43. Wan L, Ross ARS, Yang J, Hegedus DD, Kermode AR (2007) Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds. Biochem J 404:247–256PubMedCrossRefGoogle Scholar
  44. Zhu K, Zhao J, Lubman DM (2005) Protein pI shifts due to posttranslational modifications in the separation and characterization of proteins. Anal Chem 77:2745–2755PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • María López-Pedrouso
    • 1
  • Jana Alonso
    • 2
  • Carlos Zapata
    • 1
  1. 1.Department of GeneticsUniversity of Santiago de CompostelaSantiago de CompostelaSpain
  2. 2.Proteomics LaboratoryCHUSSantiago de CompostelaSpain

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