The nutrient-responsive regulation of cell division and quiescence in Chlamydomonas reinhardtii

Faced with adverse conditions, nearly all cells are able to cease energy-costly processes associated with the cell division cycle and enter a reversible alternative state known as quiescence (G0). In the green microalga Chlamydomonas reinhardtii, the entry into and exit from such a non-dividing state is mediated in response to the availability of nutrients, such as nitrogen (N). Following N starvation, a whole-sale reprogramming of intracellular processes takes place in order to maximize the chances of survival, including the repression of cell cycle-related genes, the multi-level downregulation of photosynthesis, the induction of autophagy, and the diversion of carbon (C) towards triacylglycerols (TAG).One protein implicated in the regulation of nutrient-responsive quiescence-associated transcriptomic programs in Chlamydomonas is Compromised Hydrolysis of TAG 7 (CHT7). The CHT7 protein contains cysteine-rich motifs that are often collectively referred to as the CXC domain, which is widely known for its ability to bind DNA in a sequence-specific manner in animals. A number of CXC domain-containing proteins in both animals and plants have been identified as core constituents of transcriptional regulatory complexes termed DREAM (DP, RB, E2F and Myb-MuvB) complexes, which act to coordinate cell cycle-related gene expression in response to various signals present during quiescence, cell proliferation and differentiation, and development. Through the generation of newly introgressed cell-walled lines and the detailed examination of the cht7 mutant at cellular levels, my dissertation work identified novel quiescence-related transcriptional defects in cht7 during N starvation, including derepression of DNAmetabolism and cell cycle-related genes and downregulation of oxidoreductase- and nutrient transporter-encoding genes. Largely due to their inability to repress S/M (DNA synthesis/mitosis) phase-specific gene expression, the cht7 cells were unable to properly arrest nuclear replication and cell division following N removal. The loss of viability during N starvation and the abortive divisions during N refeeding were identified as causal for the observed delay in resuming growth and the reduction in colony formation in cht7 following N resupply.The homolog of the retinoblastoma tumor suppressor (RB) protein in Chlamydomonas, MAT3, and CHT7 were shown to coimmunoprecipitated together during N-replete synchronized growth and following N deprivation, and several phosphorylated isoforms of CHT7 were identified under these conditions. Despite the presence of the presumed DNA-binding function of the CXC domain in CHT7, the largely disordered C-terminal half of CHT7 with predicted protein-binding domains, and not the proposed CXC DNA-binding domain, was essential for the ability of CHT7 to form stable complexes, and to restore the cellular phenotypes and transcript levels back to wild-type levels in the cht7 mutant. Taken together, while the mechanism of CHT7 function likely differs from that of canonical CXC domain proteins, CHT7 is necessary for the establishment of an effective quiescent state and thus, the nutrient-mediated life-cycle transitions in Chlamydomonas. A better understanding of how the cell division cycle is regulated in response to nutrient scarcity may improve the prospects of biofuels and biomass production in microalgae.