The long term goal of this research is to define the regulation of a coordinately regulated set of genes, those which code for proteins inducible by the hormone abscisic acid (ABA) in cotton embryogenesis. These genes are likely to be induced in many developmental and evironmental contexts and are probably under the control of more than one inducing agent. In this respect it is an excellent model for studying the control of genes expressed in normal human development. Fifteen mRNA sequences have been cloned and characterized. At least 10 encode ABA inducible polypeptides and eight of these are from multigene families. During normal embryogenesis these mRNAs fall into three regulation classes indicative of coordinate regulation. The studies described here are designed to further evaluate the extent and relationship of the different regulation patterns.
Our aims i nclude: 1) Complete the analysis, by RNA dot hybridization and electrophoresis of in vivo synthesized protein, of the kinetics of ABA-induced mRNAs and proteins and their dose response relationships in the excised embryo system and in the in ovulo culture system. These studies should further define the extent of coordination of their expression. 2) Use recently developed techniques to allow reliable deatection of expression at the RNA or protein level of these sequences in nonembryogenic tissues, especially in plants treated with exogenous ABA, heat shock, cold adapted, and water stressed. Induction in cotton cell culture will also be attempted. If positive results are obtained in any situation, quantitative analysis of mRNA expression will be performed as in 1) above. 3) Development of polyclonal antibodies which should be sequence group-specific. We will use as antigens protein synthesized in E. coli which contain expression vector-cloned cDNA recombinants. Clones will be group-specific or gene-specific, depending on the cross-reactivity of the resulting antibodies. 4) Isolation of gene-specific probes. 5) Measure the mRNA abundance and protein synthesis in vivo of the whole family or of individual family members in order to investigate in detail the magnitude of translational level control, especially where it is considered very likely: during the ABA induction of two particular mRNA families. 6) Determine if any multimeric proteins or complexes may exist involving ABA-inducible proteins. If so this could provide a rational basis for regulative and structural homologies already observed among them.
