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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.

In many eukaryotes, cytokinesis proceeds in two successive steps: first, ingression of the cleavage furrow and second, abscission of the intercellular bridge. In animal cells, the actomyosin contractile ring is involved in the first step, while the endosomal sorting complex required for transport (ESCRT), which participates in various membrane fusion/fission events, mediates the second step. Intriguingly, in archaea, ESCRT is involved in cytokinesis, raising the hypothesis that the function of ESCRT in eukaryotic cytokinesis descended from the archaeal ancestor. In eukaryotes other than animals, the roles of ESCRT in cytokinesis have been poorly understood. To explore the primordial core mechanisms for eukaryotic cytokinesis, we investigated ESCRT functions in the unicellular red alga Cyanidioschyzon merolae that diverged early in eukaryotic evolution. C. merolae provides an excellent experimental system. The cell has a simple organelle composition. The genome (16.5 Mb, 5335 genes) has been completely sequenced, transformation methods are established, and the cell cycle is synchronized by a light and dark cycle. Similar to animal and fungal cells, C. merolae cells divide through furrowing at the division site followed by abscission of the intercellular bridge. However, they lack an actomyosin contractile ring. The proteins that comprise ESCRT-I–IV, the four subcomplexes of ESCRT, are partially conserved in C. merolae. Immunofluorescence of native or tagged proteins localized the homologs of the five ESCRT-III components [charged multivesicular body protein (CHMP) 1, 2, and 4–6], apoptosis-linked gene-2-interacting protein X (ALIX), the ESCRT-III adapter, and the main ESCRT-IV player vacuolar protein sorting (VPS) 4, to the intercellular bridge. In addition, ALIX was enriched around the cleavage furrow early in cytokinesis. When the ESCRT function was perturbed by expressing dominant-negative VPS4, cells with an elongated intercellular bridge accumulated, a phenotype resulting from abscission failure. Our results showed that ESCRT mediates cytokinetic abscission in C. merolae. The fact that ESCRT plays a role in cytokinesis in archaea, animals, and early diverged alga C. merolae supports the hypothesis that the function of ESCRT in cytokinesis descended from archaea to a common ancestor of eukaryotes.

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.

Triacylglycerols (TAGs) produced by microalgae are a promising biofuel resource, but low productivity is still a problem for practical production. Understanding the underlying molecular mechanisms of TAG production is essential to improve productivity, which remains mostly in a primitive stage. Recently, a nuclear gene encoding a chloroplast inner membrane protein, AtFAX1, which is involved in the export of fatty acids (FAs) from the chloroplast, was identified and analyzed in Arabidopsis thaliana. In this study, we identified and analyzed a nuclear-encoded homolog of AtFAX1, named CmFAX1, from the unicellular red alga Cyanidioschyzon merolae. CmFAX1 was localized around the chloroplast envelope membrane in C. merolae, and heterologous expression in budding yeast suggested a role in FA traffic through membranes. Overexpression of CmFAX1 in C. merolae cells resulted in a 0.4-fold decrease in intracellular free FAs and a 2.4-fold increase in intracellular TAGs under normal growth conditions, suggesting that export of the free FAs produced in the chloroplast was enhanced and they were consumed for TAG synthesis in the cytosol. The accumulated TAGs in the CmFAX1 overexpression strain included short chain FAs, 14:0 and 14:1, which were not observed in the TAGs of the control strain and are suitable for biojet/biodiesel production because of their low cloud points. Overproduction of CmFAX1 resulted in higher cell yields after exponential growth phase culture. This was probably because of decreased photosynthetic pigments (chlorophyll a) and increased light permeability into the highly dense cell culture, which are beneficial characteristics for increased biomass production. These results indicate that CmFAX1 could be a useful target protein for improvement of algal biofuel production.

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.