Abstract
Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. DGAT1 and DGTT1 also show increased mRNA abundance in other TAG-accumulating conditions (minus sulfur, minus phosphorus, minus zinc, and minus iron). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared with the parent strain, CC-4425, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analyses suggest than a SQUAMOSA promoter-binding protein domain transcription factor, whose mRNA increases precede that of lipid biosynthesis genes like DGAT1, is a candidate regulator of the nitrogen deficiency responses. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared with the parental strain in nitrogen-starvation conditions and is unaffected by other nutrient stresses, suggesting the specificity of this regulator for nitrogen-deprivation conditions.
Highlights
Nitrogen-starvation and other stresses induce triacylglycerol (TAG) accumulation in algae, but the relevant enzymes and corresponding signal transduction pathways are unknown
When we monitored transcript abundance for the three acyltransferases, we noted that all three respond to low nitrogen content and that the abundances of PDAT1 and DGAT1 mRNAs were very similar, again suggesting that these three enzymes are responding to nitrogen deficiency (Fig. 3)
Work with other organisms, including S. cerevisiae, Arabidopsis, and mammals, has identified key enzymes, which enable the identification of homologs and orthologs in algae that genome sequences are available or are in the pipeline [1]
Summary
Nitrogen-starvation and other stresses induce triacylglycerol (TAG) accumulation in algae, but the relevant enzymes and corresponding signal transduction pathways are unknown. DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. Genes representing a third class (type-3) of DGAT, which is a soluble cytosolic enzyme, have been identified in Arachis hypogaea (peanut) [37] and Arabidopsis thaliana [38] Another major contributor to TAG synthesis is phospholipid:diacylglycerol acyltransferase (PDAT), which is an acyl-CoA independent enzyme that transfers the acyl group from the sn-2 position of a phospholipid to the sn-3 position of a diacylglycerol. To assess the role of these proteins in TAG accumulation in Chlamydomonas, we used RNA-Seq to describe the transcriptome during a time course of nitrogen starvation. The functions of DGTT1 and PDAT1 were validated by heterologous complementation in yeast, and the respective contributions of the enzymes encoded by PDAT1 and of the candidate regulator were assessed in loss of function mutants
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