Studies in electron transport during photosynthesis
Cyanobacteria play a huge role in global ecology, accounting for ~50% of photosynthesis in the oceans and ~25% globally. In photosynthesis, the plastoquinone (PQ) pool and cytochrome (Cyt) bf complex transfer electrons between two photochemical reaction centers (PS II and I). The Cyt bf complex serves in both photosynthetic linear and cyclic as well as respiratory electron transfer chains of cyanobacteria and in redox sensing that signals adjustments in photosynthesis. The ATP and NAD(P)H generated by photosynthesis drive conversion of atmospheric CO2 into carbohydrates. These carbohydrates provide energy for dark respiratory and fermentative metabolism. Cyanobacteria have persisted on earth for billions of years and are well adapted to many extreme and fluctuating environments such as those under high solar irradiance, temperature extremes, CO2 or other nutrient limitation, and dark aerobic or anaerobic conditions. Electron transfer and metabolic pathways that allow cyanobacteria to adjust to such conditions are poorly understood. A central hypothesis of the proposed research is that the redox status of the PQ pool and the Cyt bf high- and low-potential electron transfer chains serves important roles in regulating gene expression and thereby the activities of metabolic pathways in response to environmental perturbations. Components downstream of PS I, or oxygen radicals from the bf complex or PS I, might also participate in redox signaling. The research seeks to test these hypotheses and elucidate the mechanism and targets of redox signaling of gene expression in cyanobacterial adaptation.
The research will employ the marine cyanobacterium Synechococcus PCC 7002, which tolerates extreme light intensities and is amenable to genetics and time-resolved spectroscopy. Specific objectives are: 1) Investigation of global gene expression profiles in native and mutant Synechococcus under several environmental conditions, redox states of the PQ pool and Cyt bf complex high- and low-potential chains, and NAD(P)H levels. Conditions to be tested include phototrophy with high and low CO2, dark aerobic and anaerobic incubations, and deprivation of selected nutrients. Preliminary microarray data show that the redox states of the PQ pool and/or Cyt bf electron transfer chains control the expression of numerous genes. Tiny “millichip” microarrays (with Mike Sussman, UW Madison) will be used for inexpensive, detailed tracking of genome-wide gene-expression in several environments and mutants including Cyt bf low- and high-potential chain and NAD(P)H dehydrogenase mutants. 2) Investigation of photosynthesis and redox parameters, electron transfer kinetics, and key metabolites in wild type and mutant Synechococcus to establish links between cellular physiology and gene expression profiles. A unique, BioLogic JTS-10 kinetics spectrophotometer will be used to investigate photosynthesis reactions in living cells. Mass spectrometry will be used to assess NAD(P)H and key metabolite pools. Oxygen, CO2, and H2 will be measured by gas chromatography. 3) Construction of new mutants to begin testing the roles of possible components or targets of redox regulation identified from the microarray data. 4) Mass spectrometry experiments to investigate protein responses in selected mutants and environmental conditions. Ultimately, knowledge of protein and metabolite levels, as well as gene expression, will be essential for understanding redox regulation and adaptive strategies of cyanobacteria.
Broader impacts. The research is significant for understanding mechanisms by which globally important cyanobacteria adapt to changing environments and for development of biofuels applications. Current global energy consumption is ~14 terawatts (TW = 1012 Watts = 1012 Joule s-1) with anticipated growth to at least 40 TW by 2050. Because of their high solar energy conversion efficiency and rapid growth rates, microalgae hold enormous potential for generating renewable, carbon-neutral biofuels. Such applications will require detailed, fundamental knowledge of electron transport and metabolic pathways and their regulation. The proposed research will contribute to this knowledge. Research and teaching are closely linked at UW Oshkosh and our NSF-funded Proteomics and Functional-Genomics Core Facility and other resources provide excellent opportunities for student research. The PI has mentored numerous undergraduate and Master's students, including members of underrepresented groups. Most have entered further graduate or medical programs or found employment in biotechnology fields. The research on cyanobacterial electron transfer pathways is multifaceted and will provide continued, excellent and exciting research opportunities for students.