Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and β-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase.

RNA interferences in the unicellular green alga, Chlamydomonas reinhardtii, can be silenced. We have used the silencing of a transgene (aadA) that confers resistance to spectinomycin to investigate the mechanisms responsible for silencing by an artificial inverted repeat (IR) of the aadA gene. The IR construct provided strong silencing, but the RNAi efficiency varied among subclones of a single RNAi-transformed strain with successive cell divisions. Northern blot analyses revealed an inverse correlation between the copy number of the hairpin RNA and the spectinomycin resistance of the subclones. There is an inverse correlation between the efficiency of RNAi and the frequency of methylated CpG (*CpG) in the silenced region. No significant methylated cytosine was observed in the target aadA gene, which suggests the absence of RNA-directed DNA methylation in trans. Several experiments suggest the existence of an intrinsic IR sequence-dependent but a transcription-independent DNA methylation system in C. reinhardtii. The correlation between the *CpG levels and the IR transcript implies the existence of IR DNA-dependent DNA methylation. Treatment of RNAi-induced cells with a histone deacetylase inhibitor, Trichostatin A, rapidly increased the amount of the hairpin RNA and suggests that transcription of the silencer construct was repressed by *CpG-related silencing mechanisms.