Investigating the Role of MicroRNAs in the Response to Nitrogen Deprivation in the Green Alga Chlamydomonas reinhardtii

Microalgae are gaining attention as a potential feedstock for the production of biodiesel, mainly derived from triacylglycerols (TAG). In many algae, TAG synthesis increases dramatically upon certain stresses but this is often accompanied by growth retardation. Rational improvements to strain productivity are limited by the scant knowledge on algal lipid metabolism and gene regulatory mechanisms. In this context, systems-level approaches aimed at understanding and modeling metabolic and regulatory networks may enable hypothesis-driven genetic engineering strategies. The green microalga Chlamydomonas reinhardtii accumulates significant amounts of TAGs under nutrient starvation and provides a genetically tractable model for manipulating biosynthetic pathways. In order to gain insight into Chlamydomonas TAG metabolism and regulation, we have examined both the transcriptome of strain CC-125 grown photoautotrophically in nutrient-replete or nitrogen-depleted media and the corresponding changes in microRNA population. While the production of microRNAs (miRNAs) by Chlamydomonas reinhardtii has been established for several years, little is known about how they target transcripts for regulation or what roles they play in cellular processes, in particular whether they play a role in regulating the accumulation of TAG in nitrogen-depleted media. To characterize functional miRNAs in Chlamydomonas, we identified small RNAs associated with Flag-tagged-AGO3 by affinity purification and deep sequencing in cells grown heterotrophically and cells grown photoautotrophically in nitrogen-replete and nitrogen-deplete media and used these small RNAs to determine changes in the miRNA populations across these three conditions. We determined the role that these miRNAs play in regulating the response to nitrogen-deplete media by searching the genes that are differentially expressed in that condition for potential targets of these miRNA