title: Evidence for phosphorylation of the major seed storage protein of the common bean and its phosphorylation-dependent degradation during germination.
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.
authors.affiliation: Department of Genetics, University of Santiago de Compostela, 15782, Santiago de Compostela, Spain.
authors.forename: María
authors.initials: M
authors.lastname: López-Pedrouso
authors.forename: Jana
authors.initials: J
authors.lastname: Alonso
authors.forename: Carlos
authors.initials: C
authors.lastname: Zapata
mesh_terms: D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D018525:Germination; D008969:Molecular Sequence Data; D027805:Phaseolus / Q000378:metabolism*; D010766:Phosphorylation; D010940:Plant Proteins / Q000032:analysis* / Q000378:metabolism; D020033:Protein Isoforms / Q000032:analysis / Q000378:metabolism; D059748:Proteolysis; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D012639:Seeds / Q000254:growth & development / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D010940:Plant Proteins; D020033:Protein Isoforms; D020543:Proteome; C048826:phaseolin protein, Phaseolus vulgaris