By probing plant cDNA libraries sequentially with two partially degenerate oligonucleotides corresponding to highly conserved domains of mammalian serine protein kinases, a clone (PVPK-1) that encodes a putative plant protein kinase has recently been identifed. This clone has striking homology to mammalian cAMP- dependent protein kinases. This development now provides the basis for examination of the structure, organization and regulation of plant protein kinase gene families and dissection of the function of individual protein kinases in specific signal transduction pathways that regulate development, mediate environmental responses and integrate metabolism. Specific objectives are: (a) use of these and existing cloned sequences to determine the organization of families and sub- families of protein kinase genes; (b) isolation, characterization and nucleotide sequence analysis of genomic sequences corresponding to cloned PVPK-1 cDNA; (c) generation of specific antibodies against PVPK-1 protein kinase; (d) immunoaffinity purification of the biochemical properties of PVPK-1 protein kinase; and (e) analysis of function by expression of wild-type and mutant PVPK-1 genes in protoplasts and in stable transgenic plants. Relatively little is known at the molecular level about signal transduction and response-coupling mechanisms that control plant growth and development. Protein kinase-mediated phosphorylation of specific proteins is a major strategy for regulation of protein and enzyme activity in the transduction of environmental, developmental and metabolic signals in animals and simple eukaryotes. In higher plants, while protein phosphorylation has been observed in vivo and protein kinase activity in vitro, little information is available on the molecular properties of plant protein kinases, their number and diversity with respect to regulatory inputs and protein substrate outputs, or the function of protein kinases in specific signal transduction pathways. This research begins to fill this information gap.
