Molecular Cloning of Phycobilisome Polypeptides

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A major question in plant biology concerns the role of light in regulation of chloroplast gene expression and the mechanisms for positive and negative control of chloroplast genes are incompletely understood. The unicellular rhodophyte, Cyanidium caldarium, has a number of properties which make it a good system for investigating photoregulated chloroplast genes. The light harvesting antenna in this organism is comprised of phycobilisomes, which are macromolecular assemblies containing allophycocyanin, phycocyanin and several linker polypeptides. Cells grown in the dark contain a small etioplast which develops into a large chloroplast in the light. This is accompanied by 50 fold increases in the levels of mRNAs for apc, cpc, and other chloroplast genes, such as psaA, psbA and rbcL. Turnover rates of chloroplast photogenes will be determined in dark and light to determine whether accumulation of transcripts in light results from increased transcription activity, changes in mRNA stability, or both. In addition, heme promotes accumulation of transcripts for certain chloroplast photogenes in the dark and mechanisms underlying this process will be investigated (Aim 1). Experiments will be performed to determine whether genes for linker polypeptides are photoregulated and whether levels of transcripts of these genes are modulated by heme (Aim 2). Genes for allophycocyanin and phycocyanin will be cloned and sequenced in order to identify possible light- and heme-responsive regulatory elements (Aim 3). A homologous in vitro transcription system will be developed to investigate factors involved in regulation of chloroplast photogenes.