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Characterization of Chlamydomonas reinhardtii ammonium insensitive mutants

University of British Columbia

Nitrogen is an essential macronutrient for biosynthesis, as it is a building block for amino acids and nucleic acids in living organisms. Photoautotrophic plants acquire nitrogen from environmental ammonium, nitrate, and urea. Plants surveil multiple nitrogen sources and prefer the most economical source, ammonium, that consumes the least energy for assimilation. The ammonium preference is observed as it represses the expression of genes for assimilating non-preferred nitrogen sources, contributing to nitrogen catabolite repression (NCR). NCR-target genes are also up-regulated under nitrogen-starvation. My thesis investigates the mechanistic link between ammonium repression and nitrogen-starvation responses by characterizing Chlamydomonas reinhardtii mutants defective in nitrogen-starvation responses. Previously nine mutants were collected for their constitutive activation of nitrogen-starvation response marker NIT1 gene as nitrogen insensitive (nin) 1 ~ 9, of which nin1 shows the strongest nitrogen insensitive phenotype. nin1 harbors an insertional mutation at the HLH10 gene encoding a bHLH family transcriptional factor. Transformation of the wild-type copy HLH10 reverses the nin1 phenotype, confirming the insertion at HLH10 as the causative mutation for the nitrogen-insensitive nin1 phenotype. To test whether Nin1 is essential for repressing other nitrogen-starvation phenotypes, ammonium-grown nin1 cells were examined for gametogenesis whose activation relies solely on nitrogen-starvation. 26% of ammonium-grown nin1 cells show gamete activity and form the zygote-pellicle. To substantiate the role of Nin1, the genome-wide transcriptome of nin1 was analyzed, collecting 409 genes that are normally up-regulated in nitrogen-starved cells but constitutively expressed in ammonium-grown nin1 cells. The inclusion of NCR genes in the up-regulated genes in nin1 suggests that Nin1 negatively regulates NCR genes in nitrogen repletion. To investigate whether Nin1 is involved in ammonium repression to nitrate-triggered responses mediated by an RWP-RK family transcription factor, Nit2, nin1 in the NIT2 genetic background shows typical nitrate-dependent activation but fails to show ammonium repression of Nit2 target genes. This result indicates that Nin1 is necessary for the suppression of Nit2 target genes by ammonium. In conclusion, my thesis provides a mechanistic link between nitrogen-starvation responses and ammonium repression. It suggests that plants may use ammonium or its direct derivatives for regulating NCR genes and nitrogen-starvation responses.

Text

2024-01-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/77075

Botany

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/