UC San Diego

Diatoms have evolved complex processes called carbon concentrating mechanisms (CCMs) to maintain high photosynthesis rates by accumulating CO2 at the site of Rubisco, the key enzyme in carbon fixation. Many CCM proteins are known to be regulated in response to environmental pH, CO2 and HCO3- levels via the cyclic adenosine monophosphate (cAMP) signaling pathway. However, the regulatory pathways are poorly understood. The goal of my thesis was to explore a potential role soluble adenylyl cyclase (sAC) has in regulating photosynthesis by the marine diatom Thalassiosira pseudonana. This enzyme is stimulated by HCO3- to produce cAMP, and deemed an evolutionary conserved CO2/pH/HCO3- sensor. Two sAC-like genes, Thaps3-24827 and Thaps3-22442, were cloned into fcp-sAC-eGFP expression vectors that add enhanced green fluorescent protein (eGFP) tag with the purpose of studying their subcellular localizations. However, this approached failed, likely due to the large size of the genes; ~6.8 for Thaps3-24827 and~5.8 kilobase pairs for Thaps3-22442. The functional role of sAC in modulating diatom photosynthesis was studied by measuring oxygen (O2) production rates in the absence and presence of KH7, a small molecule that specifically inhibits sAC. Gross O2 production (GOP) was inhibited by KH7 in a dose-dependent fashion, and effects were partially reversible following a washout. The degree of KH7 inhibition was variable between cultures, and 10µM inhibited GOP between ~50-90%. Additionally, 20µM KH7 affected O2 consumption rate. These results suggest that sAC may act as a CO2/pH/HCO3- sensor that regulates photosynthesis in diatoms, perhaps by modulating CCM-related proteins.