Photosynthesis provides energy for nearly all forms of life on earth. To harvest light energy, photosynthesis requires chlorophyll. The biosynthetic pathway for chlorophyll has been studied for decades, but relatively little is known about the regulation of each enzymatic step, or the regulation of the production of the enzymes for each step. The penultimate step in chlorophyll synthesis is the reduction of protochlorophyllide to chlorophyllide. Two separate enzymes can carry out this reduction step. They are differentiated based on the fact that one enzyme NADPH: protochlorophyllide oxidoreductase (POR) requires red light (RL) for its activity. The other enzyme is the dark operative protochlorophyllide oxidoreductase (DPOR), which does not require light for its activity. The organism to be studied in this project, Fremyella diplosiphon, is capable of maintaining photosynthetic activity in green light due to the presence of specific proteins in the light harvesting antennae that absorb green light. However, in the absence of red light, chlorophyll synthesis cannot rely on the activity of POR, and likely must rely exclusively on the activity of DPOR. Recent microarray analysis of gene expression in F. diplosiphon in red light versus green light has shown that the gene encoding one subunit of DPOR is expressed 2.2 fold higher in green light than in red light. This suggests that F. diplosiphon is transcriptionally regulating DPOR so that it is more abundant when POR is non-functional and may increase the fitness of this organism by allowing it to maintain photosynthetic efficiency in the absence of red light. Of the cyanobacterial species currently used to study photosynthesis, only F. diplosiphon has identified photoreceptors that regulate gene expression in response to red light and green light, has been shown to transcriptionally regulate a DPOR subunit in response to light quality, and for which there are advanced molecular genetic tools that make it amenable to molecular analysis. The goals of this project are to identify the genes encoding POR and DPOR in F. diplosiphon, determine the light qualities under which the genes encoding POR and DPOR are expressed and investigate the role known photoreceptors and regulatory proteins play in their expression. The findings will provide the first data from cyanobacteria on the patterns of expression and regulation of DPOR and POR, two central enzymes in the chlorophyll biosynthetic pathway. The results will provide important new perspectives on the mechanisms and regulation of an important step in the chlorophyll biosynthetic pathway in a large, ecologically and evolutionarily important group of photosynthetic organisms. The broader impact of this work is that identification of these genes and analysis of their expression patterns will allow the testing of the selective advantage of maintaining these two enzymes in a fluctuating light environment. This may ultimately add to the understanding of the evolution of chlorophyll synthesis in higher plants. These activities will also provide an excellent platform for education of undergraduates in molecular biology, photobiology and microbiology.
