The long-term objectives of this proposal ar to understand global control of gene expression in plants. In particular we are investigating the mechanisms which coordinate the expression of genes belonging to integrated amino acid biosynthetic pathways. Insights into this process are beginning to emerge from recent work in our laboratory on the gene families for glutamine synthetase (GS) and asparagine synthetase (AS). The integrated expression of these key enzymes serves to regulate nitrogen assimilation and transport in plants. Our studies have revealed that individual members of the GS and AS gene families encode distinct products which are differentially expressed in particular cell-types, at particular times in development, and in response to environmental signals. Light induces the expression of the nuclear gene for chloroplast GS2 and concomitantly represses the expression of the AS1 gene. The switch from GS2 expression in the light to AS1 expression in the dark is dramatic and has biological significance to nitrogen economy in plants. We propose to elucidate the novel mechanism of light- repressed expression of AS1 with particular attention to its potential as an inducible promoter system. Several models will be tested to determine whether light-activation of GS2 and light- repression of AS1 are mediated by a """"""""repressor/activator"""""""" protein or by distinct factors. GS and AS gene expression are also coordinated when nitrogen must be mobilized from nitrogen-rich sources to metabolic sinks. Accordingly, genes for cytosolic GS3A and AS1 are coordinately induced during germination and in nitrogen-fixing root nodules. We propose to identify factors involved in the phloem- specific and developmentally-regulated expression of these genes. Preliminary results suggest that a number of factors interact with the promoters of the GS and AS genes and suggest that the mechanisms underlying the integrated regulation of these genes are complex and multifaceted. The proposed studies on factors which coordinate the regulation of GS and AS genes during development will provide some of the first insights into global control of gene expression in plants.
The specific aims of this proposal are to: 1) Define Light Regulatory Elements (LREs) involved in activation of GS2 expression and/or repression (nLRE) of AS1 expression by light using a reporter gene system in transgenic plants. We will elucidate the novel mechanism of light-repressed gene expression and develop AS1 for use as a """"""""dark"""""""" inducible promoter system in plants. 2) Determine the nature of unique binding activities in """"""""light"""""""" and """"""""dark"""""""" extracts detected with cis-elements of the GS2 and AS1 promoters. 3) Identify DNA sequences involved in phloem-specific and developmentally regulated expression of GS3A and AS1 in cotyledons and nitrogen- fixing nodules. 4) Detect phloem-specific DNA binding activities utilizing a novel purification procedure. 5) Characterize cDNA clones encoding DNA binding protein factors and determine the nature of factor:factor interactions. 6) Test ability of DNA-binding factor to activate or repress transcription in vitro. 7) Determine cell- specific and in vivo regulation of cloned factor. 8) Test models for integrated GS and AS regulation by ectopic expression of factor(s) in transgenic plants harboring GS-GUS and AS-GUS transgenes. 9) Determine whether ectopic expression of factor modulates endogenous GS and AS gene expression and/or affects nitrogen assimilation.
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