Corrigendum: Low-Temperature Adaptation of the Snow Alga Chlamydomonas nivalis Is Associated With the Photosynthetic System Regulatory Process.

[This corrects the article DOI: 10.3389/fmicb.2020.01233.].

Low-Temperature Adaptation of the Snow Alga Chlamydomonas nivalis Is Associated With the Photosynthetic System Regulatory Process.

The alga Chlamydomonas nivalis thrives in polar snow fields and on high-altitude mountain tops, and contributes significantly on primary production in the polar regions, however, the mechanisms underlying this adaptation to low temperatures are unknown. Here, we compared the growth, photosynthetic activity, membrane lipid peroxidation, and antioxidant activity of C. nivalis with those of the model alga C. reinhardtii, under grow temperature and low temperatures. C. nivalis maintained its photosynthetic activity in these conditions by reducing the light-harvesting ability of photosystem II and enhancing the cyclic electron transfer around photosystem I, both of which limited damage to the photosystem from excess light energy and resulted in ATP production, supporting cellular growth and other physiological processes. Furthermore, the increased cyclic electron transfer rate, carotenoid content, and antioxidant enzyme activities jointly regulated the reactive oxygen species levels in C. nivalis, enabling recovery from excess excitation energy and reduced photooxidative damage to the cell. Therefore, we propose a model in which adaptive mechanisms related to photosynthetic regulation promote the survival and even blooming of C. nivalis under polar environment, suggesting that C. nivalis can provide organic carbon sources as an important primary producer for other surrounding life in the polar regions for maintaining ecosystem.

The Small Regulatory Antisense RNA PilR Affects Pilus Formation and Cell Motility by Negatively Regulating pilA11 in Synechocystis sp. PCC 6803.

Pili are found on the surface of many bacteria and play important roles in cell motility, pathogenesis, biofilm formation, and sensing and reacting to environmental changes. Cell motility in the model cyanobacterium Synechocystis sp. PCC 6803 relies on expression of the putative pilA9-pilA10-pilA11-slr2018 operon. In this study, we identified the antisense RNA PilR encoded in the noncoding strand of the prepilin-encoding gene pilA11. Analysis of overexpressor [PilR(+)] and suppressor [PilR(-)] mutant strains revealed that PilR is a direct negative regulator of PilA11 protein. Although overexpression of PilR did not affect cell growth, it greatly reduced levels of pilA11 mRNA and protein and decreased both the thickness and number of pili, resulting in limited cell motility and small, distinct colonies. Suppression of PilR had the opposite effect. A hypothetical model on the regulation of pilA9-pilA10-pilA11-slr2018 operon expression by PilR was proposed. These results add a layer of complexity to the mechanisms controlling pilA11 gene expression and cell motility, and provide novel insights into how sRNA and the intergenic region secondary structures can work together to discoordinatly regulate target gene in an operon in cyanobacterium.

Ammonium Nitrogen Tolerant Chlorella Strain Screening and Its Damaging Effects on Photosynthesis.

Nitrogen is an essential nutrient element. Ammonium nitrogen, one of the most common nitrogen sources, is found in various habitats, especially wastewater. However, excessive amounts of ammonium nitrogen can be toxic to phytoplankton, higher plants, fish, and other animals, and microorganisms. In this study, we explored the tolerance of green algae to ammonium nitrogen using 10 Chlorella strains. High concentrations of ammonium nitrogen directly inhibited the growth of Chlorella, but the degree of inhibition varied by strain. With the EC50 of 1.6 and 0.4 g L-1, FACHB-1563 and FACHB-1216, respectively had the highest and lowest tolerance to ammonium nitrogen among all strains tested, suggesting that FACHB-1563 could potentially be used to remove excess ammonium nitrogen from wastewater in bioremediation efforts. Two strains with the highest and lowest tolerance to ammonium nitrogen were selected to further explore the inhibitory effect of ammonium nitrogen on Chlorella. Analysis of chlorophyll fluorescence, oxygen evolution, and photosynthesis proteins via immunoblot showed that photosystem II (PSII) had been damaged when exposed to high levels of ammonium nitrogen, with the oxygen-evolving complex as the primary site, and electron transport from Q A - to QB was subsequently inhibited by this treatment. A working model of ammonium nitrogen competition between N assimilation and PSII damage is proposed to elucidate that the assimilation rate of ammonium nitrogen by algae strains determines the tolerance of cells to ammonium nitrogen toxicity.

Comparative metabolic profiling of the lipid-producing green microalga Chlorella reveals that nitrogen and carbon metabolic pathways contribute to lipid metabolism.

Microalgae are a promising feedstock for biofuel production. Microalgal metabolic pathways are heavily influenced by environmental factors. For instance, lipid metabolism can be induced by nitrogen-limiting conditions. However, the underlying mechanisms of lipid biosynthesis are unclear. In this study, we analyzed the global metabolic profiles of three genetically closely related Chlorella strains (C1, C2, and C3) with significant differences in lipid productivity to identify the contributions of key metabolic pathways to lipid metabolism. We found that nitrogen obtained from amino acid catabolism was assimilated via the glutamate-glutamine pathway and then stored as amino acids and intermediate molecules (particularly proline, alanine, arginine, succinate, and gamma-aminobutyrate) via the corresponding metabolic pathways, which led to carbon-nitrogen disequilibrium. Excess carbon obtained from photosynthesis or glycolysis was re-distributed into carbon-containing compounds, such as glucose-6-phosphate, fructose-6-phosphate, phosphoenolpyruvate, lactate, citrate, 3-hydroxybutyrate, and leucine, and then diverted into lipid metabolism for the production of storage lipids via the gamma-aminobutyrate pathway, glycolysis, and the tricarboxylic acid cycle. These results were substantiated in the model green alga Chlamydomonas reinhardtii by analyzing various mutants deficient in glutamate synthase/NADH-dependent, glutamate synthase/Fd-dependent, glutamine synthetase, aspartate aminotransferase, alanine aminotransferase, pyruvate kinase, and citrate synthase. Our study suggests that not only carbon but also nitrogen assimilation and distribution pathways contribute to lipid biosynthesis. Furthermore, these findings may facilitate genetic engineering efforts to enhance microalgal biofuel production.

Small Antisense RNA RblR Positively Regulates RuBisCo in Synechocystis sp. PCC 6803.

Small regulatory RNAs (sRNAs) function as transcriptional and post-transcriptional regulators of gene expression in organisms from all domains of life. Cyanobacteria are thought to have developed a complex RNA-based regulatory mechanism. In the current study, by genome-wide analysis of differentially expressed small RNAs in Synechocystis sp. PCC 6803 under high light conditions, we discovered an asRNA (RblR) that is 113nt in length and completely complementary to its target gene rbcL, which encodes the large chain of RuBisCO, the enzyme that catalyzes carbon fixation. Further analysis of the RblR(+)/(-) mutants revealed that RblR acts as a positive regulator of rbcL under various stress conditions; Suppressing RblR adversely affects carbon assimilation and thus the yield, and those phenotypes of both the wild type and the overexpressor could be downgraded to the suppressor level by carbonate depletion, indicated a regulatory role of RblR in CO2 assimilation. In addition, a real-time expression platform in Escherichia coli was setup and which confirmed that RblR promoted the translation of the rbcL mRNA into the RbcL protein. The present study is the first report of a regulatory RNA that targets RbcL in Synechocystis sp. PCC 6803, and provides strong evidence that RblR regulates photosynthesis by positively modulating rbcL expression in Synechocystis.

Dynamic Changes of IsiA-Containing Complexes during Long-Term Iron Deficiency in Synechocystis sp. PCC 6803.

Iron stress-induced protein A (IsiA), a major chlorophyll-binding protein in the thylakoid membrane, is significantly induced under iron deficiency conditions. Using immunoblot analysis and 77 K fluorescence spectroscopy combined with sucrose gradient fractionation, we monitored dynamic changes of IsiA-containing complexes in Synechocystis sp. PCC 6803 during exposure to long-term iron deficiency. Within 3 days of exposure to iron deficiency conditions, the initially induced free IsiA proteins preferentially conjugated to PS I trimer to form IsiA18-PS I trimers, which serve as light energy collectors for efficiently transmitting energy to PS I. With prolonged iron deficiency, IsiA proteins assembled either into IsiA aggregates or into two other types of IsiA-PS I supercomplexes, namely IsiA-PS I high fluorescence supercomplex (IHFS) and IsiA-PS I low fluorescence supercomplex (ILFS). Further analysis revealed a role for IsiA as an energy dissipater in the IHFS and as an energy collector in the ILFS. The trimeric structure of PS I mediated by PsaL was found to be indispensable for the formation of IHFS/ILFS. Dynamic changes in IsiA-containing complexes in cyanobacteria during long-term iron deficiency may represent an adaptation to iron limitation stress for flexible light energy distribution, which balances electron transfer between PS I and PS II, thus minimizing photooxidative damage.

The Acceptor Side of Photosystem II Is the Initial Target of Nitrite Stress in Synechocystis sp. Strain PCC 6803.

Nitrite, a common form of inorganic nitrogen (N), can be used as a nitrogen source through N assimilation. However, high levels of nitrite depress photosynthesis in various organisms. In this study, we investigated which components of the photosynthetic electron transfer chain are targeted by nitrite stress in Synechocystis sp. strain PCC 6803 cells. Measurements of whole-chain and photosystem II (PSII)-mediated electron transport activities revealed that high levels of nitrite primarily impair electron flow in PSII. Changes in PSII activity in response to nitrite stress occurred in two distinct phases. During the first phase, which occurred in the first 3 h of nitrite treatment, electron transfer from the primary quinone acceptor (QA) to the secondary quinone acceptor (QB) was retarded, as indicated by chlorophyll (Chl) a fluorescence induction, S-state distribution, and QA- reoxidation tests. In the second phase, which occurred after 6 h of nitrite exposure, the reaction center was inactivated and the donor side of photosystem II was inhibited, as revealed by changes in Chl fluorescence parameters and thermoluminescence and by immunoblot analysis. Our data suggest that nitrite stress is highly damaging to PSII and disrupts PSII activity by a stepwise mechanism in which the acceptor side is the initial target. IMPORTANCE In our previous studies, an alga-based technology was proposed to fix the large amounts of nitrite that are released from NOX-rich flue gases and proved to be a promising industrial strategy for flue gas NOX bioremediation (W. Chen et al., Environ Sci Technol 50:1620-1627, 2016, https://doi.org/10.1021/acs.est.5b04696; X. Zhang et al., Environ Sci Technol 48:10497-10504, 2014, https://doi.org/10.1021/es5013824). However, the toxic effects of high concentrations of nitrite on algal cells remain obscure. The analysis of growth rates, photochemistry, and protein profiles in our study provides important evidence that the inhibition by nitrite occurs in two phases: in the first phase, electron transfer between QA- and QB is retarded, whereas in the second, the donor side of PSII is affected. This is an excellent example of investigating the "early" inhibitory effects (i.e., within the first 6 h) on the PSII electron transfer chain in vivo This paper provides novel insights into the mechanisms of nitrite inhibition of photosynthesis in an oxygenic phototrophic cyanobacterium.

Thf1 interacts with PS I and stabilizes the PS I complex in Synechococcus sp. PCC7942.

Thylakoid formation1 protein (Thf1) is a multifunctional protein that is conserved in all photosynthetic organisms. In this study, we used the model cyanobacterium Synechococcus sp. PCC7942 (hereafter Synechococcus) to show that the level of Thf1 is altered in response to various stress conditions. Although this protein has been reported to be involved in thylakoid formation, the thylakoid membrane in the thf1 deletion strain (ΔThf1) was not affected. Compared with the WT, ΔThf1 showed reduced PS II activity, with increased levels of D1 under high light (HL) conditions, which was resulted from blocked D1 degradation by the FtsH protease and thus inhibits PS II repair. PS I was found to be more seriously affected than PS II in ΔThf1, even under low light conditions, suggesting that PS I damage could be the primary effect of thf1 deletion in Synechococcus. Further analysis revealed that the ΔThf1 mutant had a lower PS I subunit content and lower PS I stability under HL conditions. Further sucrose gradient fractionation of the membrane protein complexes and crosslinking and immunoblot analysis indicated that Thf1 interacts with PS I. Together, our results reveal that Thf1 interacts with PS I and thereby stabilizes PS I in Synechococcus.

Improved Productivity of Neutral Lipids in Chlorella sp. A2 by Minimal Nitrogen Supply.

Nitrogen starvation is an efficient environmental pressure for increasing lipid accumulation in microalgae, but it could also significantly lower the biomass productivity, resulting in lower lipid productivity. In this study, green alga Chlorella sp. A2 was cultivated by using a minimal nitrogen supply strategy under both laboratory and outdoor cultivation conditions to evaluate biomass accumulation and lipid production. Results showed that minimal nitrogen supply could promote neutral lipid accumulation of Chlorella sp. A2 without a significant negative effect on cell growth. In laboratory cultivation mode, alga cells cultured with 18 mg L(-1) d(-1) urea addition could generate 74 and 416% (w/w) more neutral lipid productivity than cells cultured with regular BG11 and nitrogen starvation media, respectively. In outdoor cultivation mode, lipid productivity of cells cultured with 18 mg L(-1) d(-1) urea addition is approximately 10 and 88% higher than the one with regular BG11 and nitrogen starvation media, respectively. Notably, the results of photosynthetic analysis clarified that minimal nitrogen supply reduced the loss of photosynthetic capacity to keep CO2 fixation during photosynthesis for biomass production. The minimal nitrogen supply strategy for microalgae cultivation could promote neutral lipid accumulation without a significant negative effect on cell growth, resulting in a significant improvement in the lipid productivity.

The acclimation of Chlorella to high-level nitrite for potential application in biological NOx removal from industrial flue gases.

Nitrogen oxides (NOx) are the components of fossil flue gas that give rise to the greatest environmental concerns. This study evaluated the ability of the green algae Chlorella to acclimate to high level of NOx and the potential utilization of Chlorella strains in biological NOx removal (DeNOx) from industrial flue gases. Fifteen Chlorella strains were subject to high-level of nitrite (HN, 176.5 mmolL(-1) nitrite) to simulate exposure to high NOx. These strains were subsequently divided into four groups with respect to their ability to tolerate nitrite (excellent, good, fair, and poor). One strain from each group was selected to evaluate their photosynthetic response to HN condition, and the nitrite adaptability of the four Chlorella strains were further identified by using chlorophyll fluorescence. The outcome of our experiments shows that, although high concentrations of nitrite overall negatively affect growth and photosynthesis of Chlorella strains, the degree of nitrite tolerance is a strain-specific feature. Some Chlorella strains have an appreciably higher ability to acclimate to high-level of nitrite. Acclimation is achieved through a three-step process of restrict, acclimate, and thriving. Notably, Chlorella sp. C2 was found to have a high tolerance and to rapidly acclimate to high concentrations of nitrite; it is therefore a promising candidate for microalgae-based biological NOx removal.

An informatics-based analysis of developments to date and prospects for the application of microalgae in the biological sequestration of industrial flue gas.

The excessive emission of flue gas contributes to air pollution, abnormal climate change, global warming, and sea level rises associated with glacial melting. With the ability to utilize NOx as a nitrogen source and to convert solar energy into chemical energy via CO2 fixation, microalgae can potentially reduce air pollution and relax global warming, while also enhancing biomass and biofuel production as well as the production of high-value-added products. This informatics-based review analyzes the trends in the related literature and in patent activity to draw conclusions and to offer a prospective view on the developments of microalgae for industrial flue gas biosequestration. It is revealed that in recent years, microalgal research for industrial flue gas biosequestration has started to attract increasing attention and has now developed into a hot research topic, although it is still at a relatively early stage, and needs more financial and policy support in order to better understand microalgae and to develop an economically viable process. In comparison with onsite microalgal CO2 capture, microalgae-based biological DeNOx appears to be a more realistic and attractive alternative that could be applied to NOx treatment.

Effective Biological DeNOx of Industrial Flue Gas by the Mixotrophic Cultivation of an Oil-Producing Green Alga Chlorella sp. C2.

Nitrogen oxides (NOx) are the components of fossil flue gas that result in the most serious environmental concerns. We previously showed that the biological removal of NOx by microalgae appears superior to traditional treatments. This study optimizes the strategy for the microalgal-based DeNOx of flue gas by fed-batch mixotrophic cultivation. By using actual flue gas fixed salts (FGFS) as the nitrogen supply, the mixotrophical cultivation of the green alga Chlorella sp. C2 with high NOx absorption efficiency was optimized in a stepwise manner in a 5 L bioreactor and resulted in a maximum biomass productivity of 9.87 g L(-1) d(-1). The optimized strategy was further scaled up to 50 L, and a biomass productivity of 7.93 g L(-1) d(-1) was achieved, with an overall DeNOx efficiency of 96%, along with an average nitrogen CR of 0.45 g L(-1) d(-1) and lipid productivity of 1.83 g L(-1) d(-1). With an optimized mixotrophical cultivation, this study further proved the feasibility of using Chlorella for the combination of efficient biological DeNOx of flue gas and microalgae-based products production. Thus, this study shows a promising industrial strategy for flue gas biotreatment in plants with limited land area.

Ca(2+)-regulated cyclic electron flow supplies ATP for nitrogen starvation-induced lipid biosynthesis in green alga.

We previously showed that both the linear photosynthetic electron transportation rate and the respiration rate dropped significantly during N starvation-induced neutral lipid accumulation in an oil-producing microalga, Chlorella sorokiniana, and proposed a possible role for cyclic electron flow (CEF) in ATP supply. In this study, we further exploited this hypothesis in both Chlorella sorokiniana C3 and the model green alga Chlamydomonas. We found that both the rate of CEF around photosystem I and the activity of thylakoid membrane-located ATP synthetase increased significantly during N starvation to drive ATP production. Furthermore, we demonstrated that the Chlamydomonas mutant pgrl1, which is deficient in PGRL1-mediated CEF, accumulated less neutral lipids and had reduced rates of CEF under N starvation. Further analysis revealed that Ca(2+) signaling regulates N starvation-induced neutral lipid biosynthesis in Chlamydomonas by increasing calmodulin activity and boosting the expression of the calcium sensor protein that regulates Pgrl1-mediated CEF. Thus, Ca(2+)-regulated CEF supplies ATP for N starvation-induced lipid biosynthesis in green alga. The increased CEF may re-equilibrate the ATP/NADPH balance and recycle excess light energy in photosystems to prevent photooxidative damage, suggesting Ca(2+)-regulated CEF also played a key role in protecting and sustaining photosystems.

Effects of Phosphorylation of ß Subunits of Phycocyanins on State Transition in the Model Cyanobacterium Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 (hereafter Synechocystis) is a model cyanobacterium and has been used extensively for studies concerned with photosynthesis and environmental adaptation. Although dozens of protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues have been predicted, only a few substrate proteins are known in Synechocystis. In this study, we report 194 in vivo phosphorylation sites from 149 proteins in Synechocystis, which were identified using a combination of peptide pre-fractionation, TiO(2) enrichment and liquid chromatograpy-tandem mass spectrometry (LC-MS/MS) analysis. These phosphorylated proteins are implicated in diverse biological processes, such as photosynthesis. Among all identified phosphoproteins involved in photosynthesis, the ß subunits of phycocyanins (CpcBs) were found to be phosphorylated on Ser22, Ser49, Thr94 and Ser154. Four non-phosphorylated mutants were constructed by using site-directed mutagenesis. The in vivo characterization of the cpcB mutants showed a slower growth under high light irradiance and displayed fluorescence quenching to a lower level and less efficient energy transfer inside the phycobilisome (PBS). Notably, the non-phosphorylated mutants exhibited a slower state transition than the wild type. The current results demonstrated that the phosphorylation status of CpcBs affects the energy transfer and state transition of photosynthesis in Synechocystis. This study provides novel insights into the molecular mechanisms of protein phosphorylation in the regulation of photosynthesis in cyanobacteria and may facilitate the elucidation of the entire regulatory network by linking kinases to their physiological substrates.

A novel high light-inducible carotenoid-binding protein complex in the thylakoid membranes of Synechocystis PCC 6803.

Synechocystis sp. PCC 6803 is a model cyanobacterium extensively used to study photosynthesis. Here we reveal a novel high light-inducible carotenoid-binding protein complex (HLCC) in the thylakoid membranes of Synechocystis PCC 6803 cells exposed to high intensity light. Zeaxanthin and myxoxanthophyll accounted for 29.8% and 54.8%, respectively, of the carotenoids bound to the complex. Using Blue-Native PAGE followed by 2D SDS-PAGE and mass spectrometry, we showed that the HLCC consisted of Slr1128, IsiA, PsaD, and HliA/B. We confirmed these findings by SEAD fluorescence cross-linking and anti-PsaD immuno-coprecipitation analyses. The expression of genes encoding the protein components of the HLCC was enhanced by high light illumination and artificial oxidative stress. Deletion of these proteins resulted in impaired state transition and increased sensitivity to oxidative and/or high light stress, as indicated by increased membrane peroxidation. Therefore, the HLCC protects thylakoid membranes from extensive photooxidative damage, likely via a mechanism involving state transition.

Acetylome analysis reveals the involvement of lysine acetylation in photosynthesis and carbon metabolism in the model cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria are the oldest known life form inhabiting Earth and the only prokaryotes capable of performing oxygenic photosynthesis. Synechocystis sp. PCC 6803 (Synechocystis) is a model cyanobacterium used extensively in research on photosynthesis and environmental adaptation. Posttranslational protein modification by lysine acetylation plays a critical regulatory role in both eukaryotes and prokaryotes; however, its extent and function in cyanobacteria remain unexplored. Herein, we performed a global acetylome analysis on Synechocystis through peptide prefractionation, antibody enrichment, and high accuracy LC-MS/MS analysis; identified 776 acetylation sites on 513 acetylated proteins; and functionally categorized them into an interaction map showing their involvement in various biological processes. Consistent with previous reports, a large fraction of the acetylation sites are present on proteins involved in cellular metabolism. Interestingly, for the first time, many proteins involved in photosynthesis, including the subunits of phycocyanin (CpcA, CpcB, CpcC, and CpcG) and allophycocyanin (ApcA, ApcB, ApcD, ApcE, and ApcF), were found to be lysine acetylated, suggesting that lysine acetylation may play regulatory roles in the photosynthesis process. Six identified acetylated proteins associated with photosynthesis and carbon metabolism were further validated by immunoprecipitation and Western blotting. Our data provide the first global survey of lysine acetylation in cyanobacteria and reveal previously unappreciated roles of lysine acetylation in the regulation of photosynthesis. The provided data set may serve as an important resource for the functional analysis of lysine acetylation in cyanobacteria and facilitate the elucidation of the entire metabolic networks and photosynthesis process in this model cyanobacterium.

Evaluation of an oil-producing green alga Chlorella sp. C2 for biological DeNOx of industrial flue gases.

NOx, a significant portion of fossil fuel flue gases, are among the most serious environmental issues in the world and must be removed in an additional costly gas treatment step. This study evaluated the growth of the green alga Chlorella sp. C2 under a nitrite-simulated NOx environment and the removal rates of actual flue gas fixed salts (FGFSs) from Sinopec's Shijiazhuang refinery along with lipid production. The results showed that nitrite levels lower than 176.5 mM had no significant adverse effects on the cell growth and photosynthesis of Chlorella sp. C2, demonstrating that this green alga could utilize nitrite and NOx as a nitrogen source. High concentrations of nitrite (88.25-176.5 mM) also resulted in the accumulation of neutral lipids. A 60% nitrite removal efficiency was obtained together with the production of 33% algae lipids when cultured with FGFS. Notably, the presence of nitrate in the FGFS medium significantly enhanced the nitrite removal capability, biomass and lipid production. Thus, this study may provide a new insight into the economically viable application of microalgae in the synergistic combination of biological DeNOx of industrial flue gases and biodiesel production.

Deep sequencing-based identification of small regulatory RNAs in Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 is a genetically tractable model organism for photosynthesis research. The genome of Synechocystis sp. PCC 6803 consists of a circular chromosome and seven plasmids. The importance of small regulatory RNAs (sRNAs) as mediators of a number of cellular processes in bacteria has begun to be recognized. However, little is known regarding sRNAs in Synechocystis sp. PCC 6803. To provide a comprehensive overview of sRNAs in this model organism, the sRNAs of Synechocystis sp. PCC 6803 were analyzed using deep sequencing, and 7,951,189 reads were obtained. High quality mapping reads (6,127,890) were mapped onto the genome and assembled into 16,192 transcribed regions (clusters) based on read overlap. A total number of 5211 putative sRNAs were revealed from the genome and the 4 megaplasmids, and 27 of these molecules, including four from plasmids, were confirmed by RT-PCR. In addition, possible target genes regulated by all of the putative sRNAs identified in this study were predicted by IntaRNA and analyzed for functional categorization and biological pathways, which provided evidence that sRNAs are indeed involved in many different metabolic pathways, including basic metabolic pathways, such as glycolysis/gluconeogenesis, the citrate cycle, fatty acid metabolism and adaptations to environmentally stress-induced changes. The information from this study provides a valuable reservoir for understanding the sRNA-mediated regulation of the complex physiology and metabolic processes of cyanobacteria.

Ca2+ signal transduction related to neutral lipid synthesis in an oil-producing green alga Chlorella sp. C2.

Changes in the cytosolic Ca(2+) levels and the role of Ca(2+) signal transduction in neutral lipid synthesis in Chlorella sp. C2 under nitrogen starvation conditions were investigated. The results detected by using the scanning ion-selective electrode technique demonstrate that nitrogen starvation induced significant Ca(2+) influx across the plasma membrane into cells. Ca(2+) fluorescence imaging and flow cytometry were used to estimate the effect of this Ca(2+) influx on the generation of the Ca(2+) signal, and the results showed that the cytosolic Ca(2+) concentration increased transiently and then remained at a stable, high level when the cells were exposed to nitrogen starvation. However, the increase could be inhibited by pre-treatment with the Ca(2+) channel blockers ruthenium red, verapamil and GdCl3, indicating that both the influx of Ca(2+) from the extracellular space via Ca(2+) channels that are localized in the plasma membrane and the release of Ca(2+) from intracellular calcium storage via the internal calcium store were required for the generation and transduction of the Ca(2+) signal. During nitrogen starvation, neutral lipid synthesis in Chlorella sp. C2 in response to stress conditions was also inhibited to differing degrees by pre-treatment with the three Ca(2+) channel blockers, demonstrating the regulation of Ca(2+) via these Ca(2+) channels in neutral lipid synthesis. The results suggested that by transduction of extracellular stress signals into the cell and the regulation of the Ca(2+) signal in neutral lipid synthesis, Ca(2+) signal transduction played important roles in the response mechanism of Chlorella sp. C2 to nitrogen starvation.