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UBC Theses and Dissertations

Discovery of nitrogen starvation detecting and signaling mutants in Chlamydomonas reinhardtii Munz, Jacob Alexander


Nitrogen (N) is a fundamental macronutrient that constitutes nucleic/amino acids. Therefore, organisms require signaling mechanisms to sense external and internal N and regulate essential processes like cell growth, development, and energy metabolism. The responses to changes in cellular N status in bacteria/fungi are mediated by monitoring metabolites of the N assimilation pathway ending with glutamine synthetase (GS) and glutamate synthase (GOGAT) enzymes, forming the GS/GOGAT cycle. In photosynthetic eukaryotes, N assimilation relies on the GS/GOGAT cycle, however, whether similar signaling mechanisms are operant remains unknown. Breakthroughs in the N status signaling of bacteria/fungi were made by utilizing N sources that activated N starvation responses during growth, defined as 'derepressive'. While it is known that NH₄⁺ and NO₃- repress N starvation responses in photosynthetic eukaryotes, no derepressive N sources have been established. The unicellular green alga, Chlamydomonas reinhardtii, can survive on a wider array of N sources than land plants, of which arginine-feeding was known to derepress gametogenesis, a N starvation-specific response. This work investigated the use of arginine as a derepressive N source in photosynthetic eukaryotes and documented that N starvation responses were activated (Chapter 3 and 4). Using this derepressive condition, screens for defective N starvation responses collected mutants displaying nitrogen insensitivity (nsi) or constitutive nitrogen starvation responses (cns). Five nsi mutants disrupted the same region, NSI1, encoding a divergent glutamine synthetase (GLN4) (Chapter 5). NSI1 homologs were found in chlorophytes, and sequence features common to this family suggested it is unlikely to catalyze the reduction of NH₄⁺ but may detect N status via its potential binding to glutamate (Chapter 5). The cns mutants suggested a negative regulation of N starvation responses, whose further study may elucidate how NH₄⁺ rapidly represses N starvation responses (Chapter 6). Collectively, the work done in this thesis has led to the discovery of a hypothetical signaling network that responds to cellular N status in photosynthetic eukaryotes involving a detection mechanism of N flux through the GS/GOGAT cycle, comparable to the N status signaling counterparts in bacteria/fungi. Whether an analogous system for N status signaling exists in land plants remains to be investigated.

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