Mikio Tsuzuki

Utilization of a chloroplast membrane sulfolipid as a major internal sulfur source for protein synthesis in the early phase of sulfur starvation in Chlamydomonas reinhardtii.

Information is limited on sulfur (S)‐sources inside plant cells for synthesis of the proteins for acclimation to S‐starvation. We found that a green alga, Chlamydomonas reinhardtii, when transferred to S‐starved conditions, degrades 85% of a chloroplast membrane lipid, sulfoquinovosyl diacylglycerol (SQDG), to redistribute its S to a large part of protein fraction as early as by 6 h. Furthermore, the degradation of SQDG preceded that of proteins such as ribulose bisphosphate carboxylase/oxygenase, the candidates of internal S‐sources. SQDG was thus demonstrated to yield a major internal S‐source for protein synthesis during the early phase of acclimation process to S‐starvation.

Sequential accumulation of starch and lipid induced by sulfur deficiency in Chlorella and Parachlorella species

The influence of sulfur deficiency on biomass production was analyzed in the four Chlorellaceae species, Chlorella vulgaris, Chlorella sorokiniana, Chlorella lobophora, and Parachlorella kessleri. Culturing under sulfur-deficient conditions promoted transient accumulation of starch followed by a steady increase in lipid storage. Transmission electron microscopy indicated an increase and decrease in starch granules and subsequent enlargement of lipid droplets under sulfur-deficient conditions. Chlorellaceae spp. accumulated 1.5-2.7-fold higher amounts of starch and 1.5-2.4-fold higher amounts of lipid under sulfur-deficient conditions than under sulfur-sufficient conditions. More than 75% of the fatty acids that accumulated in Chlorellaceae spp. under the sulfur-sufficient condition were unsaturated and culturing under sulfur-deficient conditions increased the saturated fatty acid content from 24.3% to 59.7% only in P. kessleri. These results indicate that the sequential accumulation of starch and lipid is a response to the sulfur depletion that commonly occurs in Chlorellaceae spp.

Rapid Biotransformation of Arsenate into Oxo-Arsenosugars by a Freshwater Unicellular Green Alga, Chlamydomonas reinhardtii

We examined the short-term metabolic processes of arsenate for 24 h in a freshwater unicellular green alga, Chlamydomonas reinhardtii wild-type strain CC-125. The arsenic species in the algal extracts were identified by high-performance liquid chromatography/inductively coupled plasma mass spectrometry after water extraction using a sonicator. Speciation analyses of arsenic showed that the levels of arsenite, arsenate, and methylarsonic acid in the cells rapidly increased for 30 min to 1 h, and those of dimethylarsinic acid and oxo-arsenosugar-glycerol also tended to increase continuously for 24 h, while that of oxo-arsenosugar-phosphate was quite low and fluctuated throughout the experiment. These results indicate that this alga can rapidly biotransform arsenate into oxo-arsenosugar-glycerol for at least 10 min and then oxo-arsenosugar-phosphate through both reduction of incorporated arsenate to arsenite and methylation of arsenite and/or arsenate retained in the cells to dimethylarsinic acid via methylarsonic acid as an possible intermediate.

Upregulation of PG synthesis on sulfur-starvation for PS I in Chlamydomonas.

Sulfur(S)-starvation was previously shown to induce the degradation of an acidic lipid in chloroplasts, sulfoquinovosyl diacylglycerol (SQDG), to yield a major internal S-source in a green alga, Chlamydomonas reinhardtii. We here found that the synthesis of phosphatidylglycerol (PG), the other acidic lipid in chloroplasts, is activated to elevate its content up to a level that just compensates for the loss of SQDG. Similar activation of PG synthesis was also observed in an SQDG-deficient mutant under S-replete conditions, which led us to propose that upregulation of PG synthesis under S-starved conditions occurs through direct sensing of SQDG-loss, but not of S-starvation. Moreover, thylakoid membranes isolated from S-starved cells were reduced in photosystem I activity on treatment with phospholipase A(2) that specifically broke down PG, which suggested a critical role of PG that is increased under S-starved conditions in the maintenance of the photosystem I activity.

Regulation of synthesis and degradation of a sulfolipid under sulfur‐starved conditions and its physiological significance in Chlamydomonas reinhardtii

Regulation of synthesis and degradation of sulfoquinovosyl diacylglycerol (SQDG), one of the membrane lipids that construct thylakoids, under sulfur (S)-starved conditions and its physiological significance were explored in a green alga, Chlamydomonas reinhardtii. Here, we used sac1 and sac3 mutants defective in response to ambient S-status to characterize the system of known induction of SQDG degradation by S starvation that ensures a major S source for protein synthesis. The SQDG synthesis system was monitored in the wild type during S starvation. An SQDG-deficient mutant, hf-2, was utilized to discover functions where SQDG metabolism participates during S starvation. The induction of SQDG degradation was largely repressed in both sac1 and sac3 mutants. The SQDG synthesis capacity was increased by 40% after S starvation, with a sixfold elevation in the mRNA level of the SQD1 gene for SQDG synthesis. Compared with the wild type, hf-2 had decreased protein accumulation, photosystem (PS) I stability and growth rate. A role of SQDG as an S storage lipid is fulfilled under the control of both SAC1 and SAC3 genes, and it is essential for proper protein synthesis in acclimatization of cells to S starvation. The enhancement in SQDG synthesis may reflect the importance of SQDG as the membrane lipid that stabilizes the PSI complex.

Diversity of reaction characteristics of glucan branching enzymes and the fine structure of α-glucan from various sources.

To investigate the functional properties of 10 α-glucan branching enzymes (BEs) from various sources, we determined the chain-length distribution of BE enzymatic products and their phosphorylase-limit dextrins (Φ-LD). All BEs could be classified into either of the three rice BE isozymes: OsBEI, OsBEIIa, or OsBEIIb. Escherichia coli BE (EcoBE) had the same enzymatic properties as OsBEI, while Synechococcus elongatus BE (ScoBE) and Chlorella kessleri BE (ChlBE) had BEIIb-type properties. Human BE (HosBE), yeast BE (SacBE), and two Porphyridium purpureum BEs (PopBE1 and PopBE2) exhibited the OsBEIIa-type properties. Analysis of chain-length profile of Φ-LD of the BE reaction products revealed that EcoBE, ScoBE, PopBE1, and PopBE2 preferred A-chains as acceptors, while OsBEIIb used B-chains more frequently than A-chains. Both EcoBE and ScoBE specifically formed the branch linkages at the third glucose residue from the reducing end of the acceptor chain. The present results provide evidence for the first time that great variation exists as to the preference of BEs for their acceptor chain, either A-chain or B-chain. In addition, EcoBE and ScoBE recognize the location of branching points in their acceptor chain during their branching reaction. Nevertheless, no correlation exists between the primary structure of BE proteins and their enzymatic characteristics.

The primitive rhodophyte Cyanidioschyzon merolae contains a semiamylopectin-type, but not an amylose-type, α-glucan

The storage glucans of Cyanidioschyzon merolae [clade L-1 (cyanidian algae), order Porphyridiales, subclass Bangiophycidae], which is considered to be one of the most primitive rhodophytes, were analyzed to understand the early evolution of the glucan structure in the Rhodophyta. Chain-length distribution analysis of the glucans of cyanidian algae demonstrated that while the glucans of Cyanidium caldarium and Galdieria sulphuraria are of the glycogen type, those of C. merolae are of the semiamylopectin type, as in other lineages of the Rhodophyta. Gel permeation chromatography, however, showed that the glucans of C. merolae do not include amylose, being different from those of other Bangiophycidae species. Identification by MALDI-TOF-MS and enzyme assaying of glucan granule-bound proteins indicated that phosphorylase, but not starch synthase, is included. Thus, C. merolae has an unusual glucan and bound-protein composition for the Bangiophycidae, appearing to be a member of the Florideophycidae. The finding that the alga does not contain amylose or the related enzyme, granule-bound starch synthase, is, however, consistent with previously reported results of molecular phylogenetic analysis of starch synthases. Our results support an evolutionary scenario defined by the loss of starch and reversion to glycogen synthesis during the evolution of cyanidian algae, and suggest the possibility that a C. merolae-like primitive rhodophyte might have evolved into the Florideophycidae.

Cyanobacteria produce arsenosugars

Environmental context Although arsenic is known to accumulate in both marine and freshwater ecosystems, the pathways by which arsenic is accumulated and transferred in freshwater systems are reasonably unknown. This study revealed that freshwater cyanobacteria have the ability to produce arsenosugars from inorganic arsenic compounds. The findings suggest that not only algae, but cyanobacteria, play an important role in the arsenic cycle of aquatic ecosystems. Abstract Metabolic processes of incorporated arsenate in axenic cultures of the freshwater cyanobacteria Synechocystis sp. PCC 6803 and Nostoc (Anabaena) sp. PCC 7120 were examined. Analyses of arsenic compounds in cyanobacterial extracts using a high-performance liquid chromatography–inductively coupled plasma mass spectrometry system showed that both strains have an ability to biotransform arsenate into oxo-arsenosugar-glycerol within 20 min through (1) reduction of incorporated arsenate to arsenite and (2) methylation of produced arsenite to dimethylarsinic acid by methylarsonic acid as a possible intermediate product. In addition, Synechocystis sp. PCC 6803 cells are able to biosynthesise oxo-arsenosugar-phosphate from incorporated arsenate. These findings suggest that arsenosugar formation as well as arsenic methylation in cyanobacteria possibly play a significant role in the global arsenic cycle.

Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii.

Triacylglycerol (TG) synthesis is induced for energy and carbon storage in algal cells under nitrogen(N)-starved conditions, and helps prevent reactive oxygen species (ROS) production through fatty acid synthesis that consumes excessive reducing power. Here, the regulatory mechanism for the TG content in sulfur(S)-starved cells of Chlamydomonas reinhardtii was examined, in comparison to that in N- or phosphorus(P)-starved cells. S- and N- starved cells exhibited markedly increased TG contents with up-regulation of mRNA levels of diacylglycerol acyltransferase (DGAT) genes. S-Starvation also induced expression of the genes for phosphatidate synthesis. In contrast, P-starved cells exhibited little alteration of the TG content with almost no induction of these genes. The results implied deficient nutrient-specific regulation of the TG content. An arg9 disruptant defective in arginine synthesis, even without nutritional deficiencies, exhibited an increased TG content upon removal of supplemented arginine, which repressed protein synthesis. Repression of protein synthesis thus seemed crucial for TG accumulation in S- or N- starved cells. Meanwhile, the results of inhibitor experiments involving cells inferred that TG accumulation during S-starvation is supported by photosynthesis and de novo fatty acid synthesis. During S-starvation, sac1 and snrk2.2 disruptants, which are defective in the response to the ambient S-status, accumulated TG at lower and higher levels, respectively, than the wild type. The sac1 and snrk2.2 disruptants showed no or much greater up-regulation of DGAT genes, respectively. In conclusion, TG synthesis would be activated in S-starved cells, through the diversion of metabolic carbon-flow from protein to TG synthesis, and simultaneously through up-regulation of the expression of a particular set of genes for TG synthesis at proper levels through the actions of SAC1 and SNRK2.2.

Light-induced gene expression of fructose 1,6-bisphosphate aldolase during heterotrophic growth in a cyanobacterium, Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 exhibits light‐activated heterotrophic growth (LAHG) under dark conditions with glucose as a carbon source. The light activation is remarkable at a late period of photoautotrophic preculture, such as the late‐linear and stationary growth phases. To understand the physiological effects of light irradiation and glucose under LAHG conditions, their effects on the expression of soluble proteins were analyzed by means of 2D‐PAGE. Various soluble proteins, which were minimal under photoautotrophic preculture conditions, were observed clearly under LAHG conditions, suggesting that proteins were synthesized actively under these conditions. Fructose 1,6‐bisphosphate aldolase, one of the glycolytic enzymes, was found to be induced under LAHG conditions on 2D‐PAGE. The activity of fructose 1,6‐bisphosphate aldolase, which had decreased during photoautotrophic preculture, also increased under LAHG conditions, similar to the mRNA level of the encoding gene, fbaA. In addition, we found that a deletion mutant of sll1330, a putative gene containing a helix‐turn‐helix DNA‐binding motif, could not grow under LAHG conditions, whereas it could grow photoautotrophically. The increases in the protein level of FbaA and fbaA gene expression observed in wild‐type cells under LAHG conditions were greatly inhibited in the deletion mutant. These results suggest that the regulation of fbaA gene expression by way of sll1330 is one of the important processes in Synechocystis sp. PCC 6803 under light pulse LAHG conditions.