specific aims): The investigator proposes to continue her research effort in understanding how phytochrome regulates gene expression. In the past, this investigator has successfully identified several promoter elements involved in phytochrome control of transription activity in both positively and negatively light regulated genes in Lemna and Arabidopsis. Further, a gene encodes a defined DNA element-binding protein CA1 have been cloned and one of its putative interactive partner, a CKII beta subunit, have been isolated using yeast two hybrid screen. The proposed work continues and expands those studies in three major areas. First, investigation of the regulation and function of CA1, a transcription factor that binds to a region of the Arabidopsis Lhcb1*3 that is necessary for phytochrome regulation. This will be achieved by investigating: (1) the expression, activity, cellular localization and their possible light regulation mediated by phytochrome; (2) possible covalent modification involved in light control of CA1 activity mediated by phytochrome; (3) significance of CA1 interaction with beta subunit of CKII, and (4) characterization of the phenotypic effects of CA1 overexpression. The second area (Objectives 2, 3, and 4) will be further analysis of promoter characterization and transcription factors identification. Specifically, previous experiment suggested that there are other regions in Arabidopsis Lhcb1*3 promoter beside CA1-binding site is also important for its phytochrome modulation. Two regions with sequence similarity to other phytochrome responsive elements will be focused on in future analysis. Also, transcription factors with capability to interact with the defined lemna Lhcb2*1 promoter's phytochrome regulatory regions will be identified, in addition to identify additional phytochrome regulatory regions in the same promoter. Further, homologous and heterologous transient assay systems will be developed for investigating the in vivo interactions of the transcription factors and the promoters they bind. The third major aspect of the investigation is the isolation and characterization of mutants in the phytochrome signaling pathway of Arabidopsis that specifically affect the phytochrome regulation of transcription of Lhcb genes. For this purpose, a Lhcb promoter-Nitrate reductase fusion has been used as a counter-selection and some promising putative mutant lines have been isolated. The investigator proposes to not only characterize those available mutants, but further isolate and characterize new mutants. The eventual goal here is to clone and characterize the genes identified.
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