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The firefly luciferase (Luc) reporter assay is a powerful tool used to analyze promoter activities in living cells. In this report, we established a firefly Luc reporter assay system in the unicellular model red alga Cyanidioschyzon merolae. A nitrite reductase (NIR) promoter-Luc fusion gene was integrated into the URA5.3 genomic region to construct the C. merolae NIR-Luc strain. Luc activities in the NIR-Luc strain were increased, correlating with the accumulation of endogenous NIR transcripts in response to nitrogen depletion. Luc activity was also significantly increased by the overexpression of the MYB1 gene, which encodes a transcription factor responsible for NIR promoter activation. Thus, our results demonstrate the utility of the Luc reporter system in C. merolae.

Microalgae accumulate triacylglycerols (TAGs), a promising feedstock for biodiesel production, under unfavorable environmental or stress conditions for their growth. Our previous analyses revealed that only transcripts of CmGPAT1 and CmGPAT2, both encoding glycerol-3-phosphate acyltransferase, were increased among fatty acid and TAG synthesis genes under TAG accumulation conditions in the red alga Cyanidioschyzon merolae. In this study, to investigate the role of these proteins in TAG accumulation in C. merolae, we constructed FLAG-fused CmGPAT1 and CmGPAT2 overexpression strains. We found that CmGPAT1 overexpression resulted in marked accumulation of TAG even under normal growth conditions, with the maximum TAG productivity increased 56.1-fold compared with the control strain, without a negative impact on algal growth. The relative fatty acid composition of 18:2 in the TAGs and the sn−1/sn−3 positions were significantly increased compared with the control strain, suggesting that CmGPAT1 had a substrate preference for 18:2. Immunoblot analysis after cell fractionation and immunostaining analysis demonstrated that CmGPAT1 localizes in the endoplasmic reticulum (ER). These results indicate that the reaction catalyzed by the ER-localized CmGPAT1 is a rate-limiting step for TAG synthesis in C. merolae, and would be a potential target for improvement of TAG productivity in microalgae.

The nuclear genome of the unicellular red alga Cyanidioschyzon merolae can be modified by homologous recombination with exogenously introduced DNA. However, it is presently difficult to modify multiple chromosome loci because of the limited number of available positive selectable markers. In this study, we constructed a modified URA5.3 gene (URA5.3T), which can be repeatedly used for nuclear genome transformation, as well as two plasmid vectors for 3× FLAG- or 3× Myc-epitope tagging of nuclear-encoded proteins using URA5.3T. In the URA5.3T marker, the promoter region and open reading frame were located between directly repeated URA5.3 terminator sequences, and the URA5.3 gene can be eliminated by 5-fluoroorotic acid selection through homologous recombination. To demonstrate the utility of the constructed system, a 3× FLAG-tag and 3× Myc-tag were introduced at the C-termini of two of the six Rab proteins in C. merolae, CmRab18 and CmRab7, respectively, and the differential expression levels were successfully monitored by immunoblot analysis using these epitope tags. The URA5.3T marker's introduction and elimination cycle can be repeated. Thus, we have constructed a marker recycling system for C. merolae nuclear transformation. A novel procedure to obtain a high plating efficiency of C. merolae cells on solid gellan gum plates is also presented.

Chloroplasts are plant organelles that carry out oxygenic photosynthesis. Chloroplast biogenesis depends upon chloroplast ribosomes and their translational activity. However, regulation of chloroplast ribosome biogenesis remains an important unanswered question. In this study, we found that inhibition of target of rapamycin (TOR), a general eukaryotic checkpoint kinase, results in a decline in chloroplast ribosomal RNA (rRNA) transcription in the unicellular red alga, Cyanidioschyzon merolae. Upon TOR inhibition, transcriptomics and other analyses revealed increased expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) gene (CmRSH4b), which encodes a homolog of the guanosine 3'-diphosphate 5'-diphosphate (ppGpp) synthetases that modulate rRNA synthesis in bacteria. Using an Escherichia coli mutant lacking ppGpp, CmRSH4b was demonstrated to have ppGpp synthetase activity. Expression analysis of a green fluorescent protein-fused protein indicated that CmRSH4b localizes to the chloroplast, and overexpression of the CmRSH4b gene resulted in a decrease of chloroplast rRNA synthesis concomitant with growth inhibition and reduction of chloroplast size. Biochemical analyses using C. merolae cell lysates or purified recombinant proteins revealed that ppGpp inhibits bacteria-type RNA polymerase-dependent chloroplast rRNA synthesis as well as a chloroplast guanylate kinase. These results suggest that CmRSH4b-dependent ppGpp synthesis in chloroplasts is an important regulator of chloroplast rRNA transcription. Nuclear and mitochondrial rRNA transcription were both reduced by TOR inhibition, suggesting that the biogeneses of the three independent ribosome systems are interconnected by TOR in plant cells.