Identification of phosphoproteins regulated by gibberellin in rice leaf sheath.
To identify the gibberellin (GA) signaling components involved in rice leaf sheath elongation process, protein phosphorylation changed by GA3 was analyzed. The protein kinase activities in rice leaf sheath were assessed in an in-gel kinase assay using SDS-polyacrylamide gel containing histone III-S as a substrate. The activity of a putative 54-kDa calcium dependent protein kinase (CDPK) in cytosolic fraction in rice leaf sheath increased significantly by GA3. Further, phosphorylation status of the proteins changed by GA3 in rice leaf sheath were detected by in vitro protein phosphorylation followed by two-dimensional polyacrylamide gel electrophoresis and the phosphoproteins were identified by mass spectrometry. Sixty phosphoproteins was detected after in vitro protein phosphorylation and the phosphorylation of 7 proteins was enhanced by GA3 treatment. The addition of GA3 treated cytosolic fraction of leaf sheath further increased the phosphorylation of 4 proteins, glyoxalase-I, cytoplasmic malate dehydrogenase, glyceraldehydes-3-phosphate dehydrogenase and another unknown protein. The protein kinase inhibitor, staurosporine inhibited the phosphorylation of these proteins in vitro. Other hormones, particularly, indole acetic acid, 6-benzylaminopurine and abscisic acid did not change the phosphorylation status of these proteins. The identified proteins did not show any change by GA3 treatment at transcription level. The abundance of glyoxalase-I and cytoplasmic malate dehydrogenase remained unchanged by GA3 treatment as detected on 2D-gel by silver staining, unlike for glyceraldehydes-3-phosphate dehydrogenase. Results suggest that the phosphoproteins, glyoxalase-I and cytoplasmic malate dehydrogenase in rice leaf sheath could be important signaling components of GA3, downstream to 54-kDa CDPK.
Khan
Md Monowar Karim
MM
Department of Molecular Genetics, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, 305-8602, Tsukuba, Japan.
Jan
Asad
A
Karibe
Hideji
H
Komatsu
Setsuko
S
D015152:Blotting, Northern; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005875:Gibberellins / Q000494:pharmacology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology; D007791:Lactoylglutathione Lyase / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism; D013058:Mass Spectrometry; D008970:Molecular Weight; D012275:Oryza / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010937:Plant Growth Regulators / Q000494:pharmacology; D018515:Plant Leaves / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018514:Plant Structures / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013379:Substrate Specificity; D013997:Time Factors
D002135:Calcium-Binding Proteins; D005875:Gibberellins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D010937:Plant Growth Regulators; D010940:Plant Proteins; D012333:RNA, Messenger; C015482:protein kinase modulator; D008291:Malate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011494:Protein Kinases; D007791:Lactoylglutathione Lyase; D019311:Staurosporine