Molecular genetic analysis of phosphorus metabolism in the green alga, Chlamydomonas reinhardtii

Phosphate (P_i) is an important fertilizer utilized in agriculture to increase crop yields, but significant amounts of P_i leach from agricultural fields and accumulate in aquatic ecosystems, triggering rapid algal proliferation and subsequent eutrophication. Efficient use of P _i will have significant economic and ecological consequences, yet little is known about how photosynthetic eukaryotes regulate P_i metabolism. The unicellular green alga, Chlamydomonas reinhardtii, was used as a model species: (1) to define responses of photosynthetic eukaryotes to P_i deprivation and (2) to isolate regulators of P _i metabolism. C. reinhardtii acclimates to P_i starvation by inducing ‘nutrient-specific’ responses such as secretion of alkaline phosphatase activity and accumulation of a high-affinity P_i uptake system. It also induces ‘general’ responses, which are observed during a variety of starvation conditions. General responses include the cessation of cell division and the down-regulation of photosynthesis.; The down-regulation of photosynthesis was studied in detail. The rate of photosynthetic oxygen evolution declined approximately 75% after four days of P_i starvation. Quantification of the partial reactions of photosynthetic electron transport during P_i starvation demonstrated that PSI activity was unaffected whereas PSII activity was significantly reduced. The decline in PSII activity correlated with a decline in both the maximal quantum efficiency of PSII and the accumulation of PSII Q_B-nonreducing centers (PSII centers capable of performing a charge separation but unable to reduce the plastoquinone pool). During sulfur starvation these two responses were correlated with cell survival.; To elucidate the control of both the specific and general responses, I developed screens to isolate mutants abnormal in their responses to environmental levels of P_i. One of the mutants, psr1, was defective in the specific responses and exhibited no increase in extracellular phosphatase activity or P_i uptake upon starvation but was able to down-regulate photosynthesis and survive extended periods of P_i starvation. Other mutants were defective in the general responses and died more rapidly in response to P_i starvation.; Using direct complementation, the psr1 mutant phenotype was complemented, and the Psr1 gene was characterized. Sequence analysis suggested that Psr1 was a transcription factor, and immunocytochemical studies indicated that Psr1 was nuclear-localized, consistent with it being a transcriptional regulator of P_i metabolism. Furthermore, the primary sequence of Psr1 is similar to a number of plant proteins of unknown function; it is possible that these Psr1-like proteins could control P metabolism in angiosperms. In conclusion, this dissertation contains a characterization of the responses of C. reinhardtii to P_i starvation, isolation of mutants aberrant in their responses to P_i starvation, and characterization of the first cloned regulator of P_i metabolism in a photosynthetic eukaryote.