title: Analysis of tyrosine phosphorylation and phosphotyrosine-binding proteins in germinating seeds from Scots pine.
abstract: Protein tyrosine phosphorylation in angiosperms has been implicated in various physiological processes, including seed development and germination. In conifers, the role of tyrosine phosphorylation and the mechanisms of its regulation are yet to be investigated. In this study, we examined the profile of protein tyrosine phosphorylation in Scots pine seeds at different stages of germination. We detected extensive protein tyrosine phosphorylation in extracts from Scots pine (Pinus sylvestris L.) dormant seeds. In addition, the pattern of tyrosine phosphorylation was found to change significantly during seed germination, especially at earlier stages of post-imbibition which coincides with the initiation of cell division, and during the period of intensive elongation of hypocotyls. To better understand the molecular mechanisms of phosphotyrosine signaling, we employed affinity purification and mass spectrometry for the identification of pTyr-binding proteins from the extracts of Scots pine seedlings. Using this approach, we purified two proteins of 10 and 43 kDa, which interacted specifically with pTyr-Sepharose and were identified by mass spectrometry as P. sylvestris defensin 1 (PsDef1) and aldose 1-epimerase (EC:5.1.3.3), respectively. Additionally, we demonstrated that both endogenous and recombinant PsDef1 specifically interact with pTyr-Sepharose, but not Tyr-beads. As the affinity purification approach did not reveal the presence of proteins with known pTyr binding domains (SH2, PTB and C2), we suggest that plants may have evolved a different mode of pTyr recognition, which yet remains to be uncovered.
authors.affiliation: Ukrainian National Forestry University, Chuprynka St., 103, Lviv, Ukraine.
authors.forename: Valentina
authors.initials: V
authors.lastname: Kovaleva
authors.affiliation: Department of Chemistry, University of Reading, Whiteknights, Reading RG6 6AD, UK.
authors.forename: Rainer
authors.initials: R
authors.lastname: Cramer
authors.affiliation: Ukrainian National Forestry University, Chuprynka St., 103, Lviv, Ukraine.
authors.forename: Hryhoriy
authors.initials: H
authors.lastname: Krynytskyy
authors.affiliation: Department of Structural and Molecular Biology, Institute of Structural and Molecular Biology, University College London, London WC1E 6BT, UK.
authors.forename: Ivan
authors.initials: I
authors.lastname: Gout
authors.affiliation: Ukrainian National Forestry University, Chuprynka St., 103, Lviv, Ukraine. Electronic address: rgoutmollab@ukr.net.
authors.forename: Roman
authors.initials: R
authors.lastname: Gout
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D018525:Germination; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry*; D041605:Pinus sylvestris / Q000378:metabolism*; D012639:Seeds / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D019000:Phosphotyrosine; D014443:Tyrosine
keywords: Pinus sylvestris; Seed germination; Tyrosine phosphorylation; pTyr-binding proteins