The primary goal of this project is to understand how genes involved in two important metabolic pathways, glycolysis and the photosynthetic carbon fixation cycle, are regulated in higher plants. A second and more long range goal is to understand how nuclear and chloroplast genomes interact in controlling chloroplast function. Nuclear genes encoding Arabidopsis cytosolic (GapC) and chloroplast (GapA and GapB) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), will be used as markers to address the following questions: (i). How are these genes regulated under different physiological conditions? (ii). What are the mechanisms of light stimulated and tissue specific expression of GapA and GapB genes? (iii). What is the molecular basis of the light signal transduction? To achieve these goals, the following experiments are proposed. Expression of GAPDH genes in wild-type and mutant Arabidopsis plants defective in light response will be compared under different light conditions. In addition, a genetic scheme has been devised to select regulatory mutations affecting GAPDH gene expression. These experiments should provide information on functional roles of light receptors in regulating gene expression and on intermediate steps between signal reception and actual gene activation. To identify cis- and trans-regulatory elements, binary vectors linking the promoter regions of Gap genes with a marker have been constructed. Agrobacterium-mediated transformation will be used to make transgenic plants containing these constructs. Deletional and mutational analyses of these transgenic plants should allow identification of the cis-acting regulatory elements of these genes. Biochemical methods will then be used to identify and purify trans-acting regulatory proteins. Aside from the practical purpose of crop improvement, the proposed research should improve our understanding of: (i) modes of regulation of gene expression, (ii) mechanisms of signal transduction, (iii) the molecular basis of development in higher plants. Although these questions are common and can be studied in many organisms, the ease of regeneration of the whole plant from a single cell and the high efficiency of introducing foreign DNA into plant cells through Ti- plasmid DNA mediated transformation make higher plants unique systems in which to study these problems in vivo.
