With their high photosynthetic efficiency and the ability to synthesize triacylglycerol (TAG), unicellular microalgae have an important ecological role, as well as value for production of triacylglycerol lipids that can be converted to biodiesel. The recent state of knowledge about microalgal lipid metabolism had been deduced from that of a model seed plant, Arabidopsis. However, recent studies have revealed that aspects of lipid metabolism differ between microalgae and Arabidopsis. Investigating these differences was a cornerstone of this study, using Chlamydomonas, a representative of microalgae, and Arabidopsis. Two major approaches were undertaken: forward and reverse genetics. The forward genetic screening used insertional mutagenesis of Chlamydomonas and focused on a knockout mutation of a gene, which proved to be an orthologue of the Arabidopsis TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2). The tgd2 mutant exhibits increases in cellular concentrations of phosphatidic acid (PtdOH) and triacylglycerol (TAG); the latter contains signature fatty acids of monogalactosyldiacylglycerol (MGDG), pointing to its likely origin of synthesis. The mutant also experiences low viability in extended culture. Similar to AtTGD2, CrTGD2 is located in the chloroplast inner envelope membrane and binds PtdOH in vitro. Radioactive labeling experiments suggest that CrTGD2 functions in transferring a lipid precursor, presumably PtdOH, from the outer chloroplast envelope into the chloroplast. This study shows that, in contrast to prevailing assumptions, Chlamydomonas is able to import lipids from the endoplasmic reticulum (ER) to the chloroplast, and utilizes the eukaryotic pathway to synthesize galactoglycerolipids. The reverse genetics investigation focused on CrLIP4, a putative TAG lipase. CrLIP4 is an orthologue of a major Arabidopsis TAG lipase. Reverse transcription PCR revealed that the CrLIP4 transcript is reduced in abundance during N deprivation when TAG accumulates. Down-regulation of this gene through an artificial microRNA construct resulted in delayed TAG degradation. Expression of CrLIP4 in Escherichia coli alters the pattern of neutral lipids. Recombinant CrLIP4 exhibited TAG lipase activity. These results show that CrLIP4 has TAG lipase activity both in in vitro and in vivo. In summary, two Arabidopsis orthologues in Chlamydomonas were characterized through forward and reverse genetic approaches. The results elaborate and refine our understanding of Chlamydomonas lipid metabolism, and are likely relevant for other unicellular microalgae.
Read
