Revealing the effects of two DNA methyltransferase inhibitors on DNA methylation and carotenoid metabolism in Chlamydomonas reinhardtii by comparative transcriptome and physiological analysis

DNA methylation plays an important role in cell growth and development. However, little is known about the possible functions of DNA methylation in microalgae. Here, two DNA methyltransferase (DNMT) inhibitors (5-azacytidine and zebularine) were used to treat Chlamydomonas reinhardtii to investigate the effects of DNA methylation on microalgae. 5-Azacytidine (5-Azac) showed the promoting effects on cell growth and was benefit to the accumulation of pigments, while zebularine (Zeb) has adverse effects on the physiology of algal cells. However, the genomes of C. reinhardtii treated with two DNMT inhibitors unexpectedly showed slightly increased 5-mC levels. According to the results of transcriptome sequencing to algal cells treated with Zeb and 5-Azac, two DNMT inhibitors were found to up-regulate the transcription levels of genes involved in the cytosine methylation pathway and down-regulate genes involved in the cytosine demethylation pathway, which may result in higher 5-mC levels in genome. The increased content of carotenoids by 5-Azac may be due to the up-regulated transcription levels of carotenogenic genes. In addition, the up-regulated transcription levels of carotenoid degradation-related genes may be responsible for the reduced carotenoid content by Zeb. Finally, a carotenoid 9,10(9′,10′)-cleavage dioxygenase 1 (CrCCD1) gene was isolated from C. reinhardtii, and its important role in carotenoid degradation was demonstrated. In summary, this study revealed the physiological changes and molecular mechanisms of C. reinhardtii under the effects of two DNMT inhibitors, providing valuable information for subsequent studies on DNA methylation in microalgae.