Plants utilize light as an important signal to regulate both developmental and metabolic processes. The long term goal of this project is to understand the mechanisms by which this signal is transmitted to the nucleus to affect changes in the transcription of specific genes. These studies should also provide general information about the mechanisms of plant gene expression. The experiments described in this proposal are designed to identify and characterize components of the light signal transduction pathway that link photoperception with a transcriptional response. We have previously developed pea rbcS-3A as a model system for the study of light-responsive transcription. This gene encodes the small subunit of ribulose-1,5-bisphosphate carboxylase, a key enzyme in photosynthetic carbon assimilation; its transcription is induced thirty- five fold by light. The proposed studies will integrate biochemical, molecular and genetic approaches and will be based on the following strategies: (1) We have previously demonstrated a pivotal role for the nuclear factor GT-1 in the modulation of light-responsive transcription. We have recently isolated cDNA clones from both tobacco and Arabidopsis that encode GT-1. We will use these sequences to characterize the encoded proteins. These experiments will provide information on the mechanisms by which GT-1 mediates its effects. Additionally, we will use GT-1 as a biochemical substrate to identify further components of the light signal transduction chain. (2) A recently defined nuclear factor, GAF-1, that binds upstream of rbcS-3A will be further characterized. GAF-1 is present in greater abundance in nuclear extracts prepared from light-grown as opposed to dark-adapted plants. We propose to isolate cDNA clones encoding this factor in order to determine the mechanism by which its light- dependent DNA binding activity is modulated. (3) Arabidopsis thaliana plants carrying a det-1 mutation bypass the normal requirement for light to initiate developmental processes. We will use these plants to characterize specific components of the light responsive transcriptional machinery. Defined DNA sequence elements will be assayed in a det-1 mutant background; these experiments should provide information about the contribution of specific sequence elements to light-responsive and developmentally- regulated gene expression. Additionally, we will assess the status of GT-1 and GAF-1 DNA binding activities in det-1 plants to determine whether the genetic lesion affects these specific components of the transcriptional machinery. (4) Intermediates of the light-signal transduction pathway will be identified by screening mutagenized Arabidopsis plants for those which show aberrant expression of light-regulated genes. These studies will be done using transgenic plants carrying constructs in which both complex promoters and short defined DNA sequences are fused to luciferase reporter gene. Use of these gene fusions should lead to the identification of novel mutants, including some that effect DNA binding proteins as well as other that affect additional components of the light-signal transduction pathways. These mutants will be subjected to genetic analyses to define the mutant phenotype. (5) The mutant genes will be isolated by a combination of genetic and molecular approaches for analysis of their encoded proteins. They will also be used to study the expression of the wildtype genes as well as the effects of their expression in transgenic plants.
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