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

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Abstract

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.

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Abbreviations

2-DE:

Two-dimensional electrophoresis

ABA:

Abscisic acid

GA:

Gibberellic acid

HF:

Hydrofluoric acid

M r :

Relative molecular mass

MALDI-TOF:

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

MS:

Mass spectrometry

P:

Pyridine

phas :

β-Phaseolin gene

pI :

Isoelectric point

PR :

Phosphorylation rate

Pro-Q DPS:

Pro-Q Diamond phosphoprotein stain

PTM:

Post-translational protein modification

S:

Sanilac

SSP:

Seed storage protein

T:

Tendergreen

References

  • 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 28:1157–1167

    Article  CAS  PubMed  Google Scholar 

  • Agrawal GK, Thelen JJ (2005) Development of a simplified, economical polyacrylamide gel staining protocol for phosphoproteins. Proteomics 5:4684–4688

    Article  CAS  PubMed  Google Scholar 

  • Agrawal GK, Thelen JJ (2006) Large-scale identification and quantitative profiling of phosphoproteins expressed during seed filling in oilseed rape. Mol Cell Proteomics 5:2044–2059

    Article  CAS  PubMed  Google Scholar 

  • 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 40:151–160

    Article  CAS  Google Scholar 

  • 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 96:999–1007

    Article  CAS  PubMed  Google Scholar 

  • de la Fuente van Bentem S, Hirt H (2007) Using phosphoproteomics to reveal signalling dynamics in plants. Trends Plant Sci 9:404–411

    Article  Google Scholar 

  • 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 74:262–267

    Article  Google Scholar 

  • 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 50:315–325

    Google Scholar 

  • Emani C, Hall TC (2008) Phaseolin: structure and evolution. Open Evol J 2:66–74

    CAS  Google Scholar 

  • Finch-Savage WE, Leubner-Metzger G (2006) Seed dormancy and the control of germination. New Phytol 171:501–523

    Article  CAS  PubMed  Google Scholar 

  • Gepts P (1998) Phaseolin as an evolutionary marker. In: Gepts P (ed) Genetic resources of Phaseolus beans. Kluwer Academic Publishers, Dordrecht, pp 215–241

    Google Scholar 

  • 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 113–143

    Chapter  Google Scholar 

  • 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 148:1668–1680

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gutierrez L, Van Wuytswinkel O, Castelain M, Bellini C (2007) Combined networks regulating seed maturation. Trends Plant Sci 12:294–300

    Article  CAS  PubMed  Google Scholar 

  • 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 209–240

    Chapter  Google Scholar 

  • Hellmann H, Estelle M (2002) Plant development: regulation by protein degradation. Science 297:793–797

    Article  CAS  PubMed  Google Scholar 

  • 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 12:343–351

    Article  CAS  PubMed  Google Scholar 

  • 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–S185

    Article  PubMed  Google Scholar 

  • Kami J, Gepts P (1994) Phaseolin nucleotide sequence diversity in Phaseolus. I. Intraspecific diversity in Phaseolus vulgaris. Genome 37:751–757

    Article  CAS  PubMed  Google Scholar 

  • Karin M, Ben-Neriah Y (2000) Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol 18:621–663

    Article  CAS  PubMed  Google Scholar 

  • Kersten B, Agrawal GK, Durek P, Neigenfind J, Schulze W, Walther D, Rakwal R (2009) Plant phosphoproteomics: an update. Proteomics 9:964–988

    Article  CAS  PubMed  Google Scholar 

  • 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 580:199–205

    Article  CAS  PubMed  Google Scholar 

  • Kline-Jonakin KG, Barrett-Wilt GA, Sussman MR (2011) Quantitative plant phosphoproteomics. Curr Opin Plant Biol 14:507–511

    Article  CAS  PubMed  Google Scholar 

  • 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 67:33–40

    Article  Google Scholar 

  • Kuyama H, Toda C, Watanabe M, Tanaka K, Nishimura O (2003) An efficient chemical method for dephosphorylation of phosphopeptides. Rapid Commun Mass Spectrom 17:1493–1496

    Article  CAS  PubMed  Google Scholar 

  • 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 8:19–31

    Article  CAS  PubMed  Google Scholar 

  • 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 96:7104–7109

    Article  CAS  PubMed  Google Scholar 

  • 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 3:345–353

    Article  CAS  PubMed  Google Scholar 

  • 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 73:1587–1600

    Article  CAS  PubMed  Google Scholar 

  • 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 11:686

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Meyer LJ, Gao J, Xu D, Thelen JJ (2012) Phosphoproteomic analysis of seed maturation in Arabidopsis, rapeseed, and soybean. Plant Phys 159:517–528

    Article  CAS  Google Scholar 

  • Myernik JA, Hajduch M (2011) Seed proteomics. J Proteomics 74:389–400

    Article  Google Scholar 

  • Ng DW-K, Hall TC (2008) PvALF and FUS3 activate expression from the phaseolin promoter by different mechanisms. Plant Mol Biol 66:233–244

    Article  CAS  Google Scholar 

  • Saravanan RS, Rose JKC (2004) A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 4:2522–2532

    Article  CAS  PubMed  Google Scholar 

  • 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 386:482–493

    Article  CAS  PubMed  Google Scholar 

  • 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 48:708–715

    Article  CAS  PubMed  Google Scholar 

  • Singh SP (2001) Broadening the genetic base of common bean cultivars: a review. Crop Sci 41:1659–1675

    Article  Google Scholar 

  • Slightom JL, Sun S, Hall TC (1983) Complete nucleotide sequence of a French bean storage protein gene: phaseolin. Proc Natl Acad Sci USA 80:1897–1901

    Article  CAS  PubMed  Google Scholar 

  • 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 13:6483–6498

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Spencer D (1984) The physiological role of storage proteins in seeds. Philos Trans R Soc Lond B 304:275–285

    Article  CAS  Google Scholar 

  • 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 262:13392–13403

    CAS  PubMed  Google Scholar 

  • Vitale A, Bollini R (1995) Legume storage proteins. In: Kigel J, Galili G (eds) Seed development and germination. Marcel Dekker, New York, pp 73–102

    Google Scholar 

  • 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 404:247–256

    Article  CAS  PubMed  Google Scholar 

  • Zhu K, Zhao J, Lubman DM (2005) Protein pI shifts due to posttranslational modifications in the separation and characterization of proteins. Anal Chem 77:2745–2755

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

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.

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Authors and Affiliations

  1. Department of Genetics, University of Santiago de Compostela, 15782, Santiago de Compostela, Spain

    María López-Pedrouso & Carlos Zapata

  2. Proteomics Laboratory, CHUS, Santiago de Compostela, Spain

    Jana Alonso

Authors
  1. María López-Pedrouso
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  2. Jana Alonso
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  3. Carlos Zapata
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Correspondence to Carlos Zapata.

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López-Pedrouso, M., Alonso, J. & Zapata, C. Evidence for phosphorylation of the major seed storage protein of the common bean and its phosphorylation-dependent degradation during germination. Plant Mol Biol 84, 415–428 (2014). https://doi.org/10.1007/s11103-013-0141-1

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