Role of protein phosphatases in the GA-dependent gene expression of Osamy-c in rice (Oryza sativa. L)

Cereal grain aleurone cells have been an attractive model in studying the GA-dependent gene expression. To understand the molecular basis of GA-dependent gene response, both of pharmacological and molecular biological approaches were used in my study. Three inhibitors, okadaic acid, microcystin-LR, and calyculin A, known to specifically inhibit Ser/Thr protein phosphatase 1 and 2A (PP1 and PP2A) were found to strongly inhibit the expression of Osamy-c as well as the germination of rice seeds.;To understand further the possible role of protein phosphatase 1 and 2A in the GA-dependent expression of Osamy-c, I cloned the cDNAs encoding the catalytic subunit of protein phosphatase 1 (OsPP1c ) and protein phosphatase 2A (OsPP2Ac). The deduced amino acid sequences of OsPP1c and OsPP2Ac are highly conserved (73% to 90% similarity) with the catalytic subunits of PP1 or PP2A from other organisms. Genomic Southern blot analysis indicated that there are only one or two copies of OsPP1c genes and more than two copies of OsPP2Ac genes in the rice genome. Northern blot analysis showed that OsPP1c and OsPP2Ac genes are expressed in several organs of rice and GA does not regulate the expression of either gene.;The enzymatic activities of PP1c and PP2Ac are known to be regulated by their association with different regulatory subunits. I also isolated and characterized cDNA and genomic DNA sequences for the 55-kDa B regulatory subunit of PP2A (OsRPP2AB). The cDNA contains an open reading frame that encodes a predicted protein of 531 amino acid residues with a molecular weight of 59.7 kDa. Sequence analysis of the rice OsRPP2AB cDNA showed three unique regions of amino acid insertions that also exist in Arabidopsis but not in the mammalian B regulatory subunits. Southern blot analysis indicated that there are at least two homologous genes in the rice genome. Northern blot analysis showed two different sized transcripts (2.1 and 1.5 Kb) in seed, shoot and root tissues. Sequencing of OsRPP2AB genomic (c. a. 9.4 kb) indicated there are fourteen exons in the coding region.;Finally, I optimized the particle bombardment conditions and carried out the cotransformation experiments to test the in vivo function of OsPP1c and OsPP2Ac gene products in the GA-dependent gene expression of alpha-amylase. The preliminary result showed that overexpression of anti-sense PP2Ac or PP1c cDNAs increases the GA-induction of GUS activity of a barley alpha-amylase-GUS reporter gene. On the other hand, overexpression of sense PP1c or PP2Ac cDNA results in the reduction of the GUS activity.