GSE51602
Systems-level analysis of N-starvation induced modifications of carbon metabolism in a Chlamydomonas starchless mutant
To understand the molecular basis underlying increased triacylglycerol accumulation in starchless Chlamydomonas mutants, we undertook comparative time course transcriptomics of sta6 (CC-4348), a cw strain purported to represent the parental STA6 strain (CC-4349), and 3 independent STA6 strains (STA6-C2, STA6-C4 and STA6-C6, generated by complementation of sta6, CC-4565, –4566 and –4567) in the context of nitrogen deprivation. Despite the N starvation induced dramatic remodeling of the transcriptome, there are relatively few differences (5 x 102) between sta6 and STA6, the most dramatic of which are in the abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically ICL1, MAS1, TAL1, FBP1 and PCK1, suggestive of increased carbon movement towards hexosephosphate in sta6 by up-regulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities. Targeted metabolite analysis indicates increased succinate and malate, increased Glc-6-P and decreased fructose-1,6-bisphosphate, which documents the impact of these changes. Comparisons of the independent datasets in multiple strains allowed the delineation of a sequence of events in a global N-starvation response in Chlamydomonas, starting within minutes with up-regulation of alternative N assimilation routes and carbohydrate synthesis and subsequently a more gradual up-regulation of genes encoding enzymes of TAG synthesis. Finally, genome re-sequencing analysis indicated that i) the deletion in sta6 extends into the neighboring gene, RBO1, and ii) a commonly-used STA6 strain (CC-4349) as well as the sequenced reference (CC-503) are not congenic with respect to sta6 (CC-4348), underscoring the importance of using complemented strains for more rigorous assignment of phenotype to genotype. Overall design: The transcriptomes of five different Chlamydomonas strains (wild type CC-4349, sta6 mutant (CC-4348),and three indepdent STA6 strains (STA6-C2, STA6-C4 and STA6-C6) are profiled by RNA-Seq in timecourse experiments after the transition to minus nitrogen