The NAD Kinase Slr0400 Functions as a Growth Repressor in Synechocystis sp. PCC 6803.

NADP+, the phosphorylated form of nicotinamide adenine dinucleotide (NAD), plays an essential role in many cellular processes. NAD kinase (NADK), which is conserved in all living organisms, catalyzes the phosphorylation of NAD+ to NADP+. However, the physiological role of phosphorylation of NAD+ to NADP+ in the cyanobacterium Synechocystis remains unclear. In this study, we report that slr0400, an NADK-encoding gene in Synechocystis, functions as a growth repressor under light-activated heterotrophic growth conditions and light and dark cycle conditions in the presence of glucose. We show, via characterization of NAD(P)(H) content and enzyme activity, that NAD+ accumulation in slr0400-deficient mutant results in the unsuppressed activity of glycolysis and tricarboxylic acid (TCA) cycle enzymes. In determining whether Slr0400 functions as a typical NADK, we found that constitutive expression of slr0400 in an Arabidopsis nadk2-mutant background complements the pale-green phenotype. Moreover, to determine the physiological background behind the growth advantage of mutants lacking slr04000, we investigated the photobleaching phenotype of slr0400-deficient mutant under high-light conditions. Photosynthetic analysis found in the slr0400-deficient mutant resulted from malfunctions in the Photosystem II (PSII) photosynthetic machinery. Overall, our results suggest that NADP(H)/NAD(H) maintenance by slr0400 plays a significant role in modulating glycolysis and the TCA cycle to repress the growth rate and maintain the photosynthetic capacity.

Specific Incorporation of Polyunsaturated Fatty Acids into the sn-2 Position of Phosphatidylglycerol Accelerates Photodamage to Photosystem II under Strong Light.

Free fatty acids (FFAs) are generated by the reaction of lipases with membrane lipids. Generated polyunsaturated fatty acids (PUFAs) containing more than two double bonds have toxic effects in photosynthetic organisms. In the present study, we examined the effect of exogenous FFAs in the growth medium on the activity of photosystem II (PSII) under strong light in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). PUFAs but not monounsaturated fatty acids accelerated the rate of photodamage to PSII by inactivating electron transfer at the oxygen-evolving complex. Moreover, supplemented PUFAs were specifically incorporated into the sn-2 position of phosphatidylglycerol (PG), which usually contains C16 fatty acids at the sn-2 position in Synechocystis cells. The disruption of the gene for an acyl-ACP synthetase reduced the effect of PUFAs on the photoinhibition of PSII. Thus, the specific incorporation of PUFAs into PG molecules requires acyl-ACP synthetase and leads to an unstable PSII, thereby accelerating photodamage to PSII. Our results are a breakthrough into elucidating the molecular mechanism of the toxicity of PUFAs to photosynthetic organisms.

Strain, cell density, and nutrient condition affect patterns of diurnal vertical migration and superoxide production in a red-tide alga.

A red tide occurs when cell densities of autotrophic microalgae and some heterotrophic protists increase dramatically and thereby change the color of the sea. Red tides sometimes have negative impacts on human activities, such as fisheries and tourism. Most red-tide flagellates display diurnal vertical migration (DVM) in which cells normally migrate upward during the day and downward at night. This behavior promotes active growth, due to the effective acquisition of nutrients and light, as well as population density increase and cell aggregation. However, the factors and their interactions influencing DVM remain to be clarified, such that no algorithm exists that can precisely simulate the DVM pattern and the development of a red tide in the field. Chattonella marina complex (hereafter Chattonella) is a representative microalga of harmful red tides and some previous studies has suggested that Chattonella's DVM plays important roles in development of a red tide. Chattonella can produce a large amount of superoxide (•O2 -), which is responsible for the regulation of various physiological processes as well as its toxicity against microorganisms and animals. In the present study, we examined the effects of strain, growth phase, cell density, and nutrient deficiency on the pattern of DVM. In addition, we also measured the •O2 - level in most experiments to assess the relationship between DVM and •O2 - production. Some strains displayed clear DVM, whereas others aggregated at the surface all day in a fixed condition. Strains' DVM patterns did not show a relationship with •O2 - production. Moreover, the DVM became less clear at high cell density and in nitrogen- or phosphorus-depleted conditions. Although a previous study reported that the •O2 - production rate increased during the light period and decreased during the dark period, regardless of cell density, the diurnal pattern of •O2 - became less clear at a higher cell density in a Chattonella strain used in the present study. Our findings indicate that DVM and •O2 - production by a Chattonella population composed of various strains can change across developmental phases and environmental conditions. This characteristic may produce adaptability in species and increase the chances of a massive population increase.

Recovery of photosynthesis after long-term storage in the terrestrial cyanobacterium Nostoc commune.

The terrestrial cyanobacterium Nostoc commune is an anhydrobiotic organism with extreme longevity. Recovery of photosynthesis by rehydration was examined using our laboratory stocks of dry N. commune thalli after long-term storage in a desiccated state. In the samples stored at room temperature for over 8 years, photosynthetic oxygen evolution was barely detectable, whereas oxygen consumption was recovered. There was an exceptional case in which photosynthetic oxygen evolution recovered after 8 years of storage at room temperature. Both photosynthetic oxygen evolution and respiratory oxygen consumption were recovered in dry thalli stored at -20°C for over 15 years. Consistent with the recovery of photosynthetic oxygen evolution, Fv/Fm was detected in the samples stored at -20°C at levels similar to those of freshly collected N. commune colonies. Carotenoids, scytonemin and chlorophyll a appeared to be intact in the dry thalli stored at -20°C, but ß-carotene was not detected in the samples stored at room temperature. α-Tocopherol was intact in the samples stored at -20°C but was degraded in the samples stored at room temperature. These results suggest that dry thalli of N. commune are capable of sustaining biological activities for a long time, although they are gradually damaged when stored at room temperature.

Superoxide Production by the Red Tide-Producing Chattonella marina Complex (Raphidophyceae) Correlates with Toxicity to Aquacultured Fishes.

The marine raphidophyte Chattonella marina complex forms red tides, causing heavy mortalities of aquacultured fishes in temperate coastal waters worldwide. The mechanism for Chattonella fish mortality remains unresolved. Although several toxic chemicals have been proposed as responsible for fish mortality, the cause is still unclear. In this study, we performed toxicity bioassays with red sea bream and yellowtail. We also measured biological parameters potentially related to ichthyotoxicity, such as cell size, superoxide (O2•-) production, and compositions of fatty acids and sugars, in up to eight Chattonella strains to investigate possible correlations with toxicity. There were significant differences in moribundity rates of fish and in all biological parameters among strains. One strain displayed no ichthyotoxicity even at high cell densities. Strains were categorized into three groups based on cell length, but this classification did not significantly correlate with ichthyotoxicity. O2•- production differed by a factor of more than 13 between strains at the late exponential growth phase. O2•- production was significantly correlated with ichthyotoxicity. Differences in fatty acid and sugar contents were not related to ichthyotoxicity. Our study supports the hypothesis that superoxide can directly or indirectly play an important role in the Chattonella-related mortality of aquacultured fishes.

Nutrient deficiency stimulates the production of superoxide in the noxious red-tide-forming raphidophyte Chattonella antiqua.

The raphidophyte Chattonella antiqua is a single-celled alga that forms 'red tides' in coastal areas. C. antiqua produces superoxide anions (O2-), the excessive production of which has been associated with fish mortality. It is suggested that putative NADPH oxidase in the outer membrane oxidizes intracellular NADPH to produce O2- and secrete it externally. Earlier studies revealed that photosynthetic electron transport, a major producer of NADPH in photosynthetic organisms, is involved in the production of O2- in C. antiqua but the details of the O2- production mechanism have yet to be elucidated. Since nutrient deficiency adversely affects the formation of blooms of C. antiqua, in this study, we examined the effects of nutrient deficiency on O2- production in C. antiqua. When cells were grown under nitrogen (N)- or phosphorus (P)-deficient conditions, the production of O2- was stimulated. In particular, the extracellular levels of O2- under N- or P-deficient conditions were high during the dark period when photosynthetic activities in terms of actual quantum efficiency and photochemical quenching were low. The extracellular levels of O2- under the nutrient-deficient conditions were unaffected by the presence of 3-(3,4-dichlorophenyl)-1,1­dimethylurea (DCMU), an inhibitor of photosynthetic electron transport, but decreased when the nutrients were present. Furthermore, the intracellular ratio of NADPH to NADP+ under N- or P-deficient conditions was higher than that under nutrient-replete conditions. These observations suggest that another metabolic pathway, independent of photosynthesis, provides NADPH for the production of O2- under nutrient deficiency.

Extracellular secretion of superoxide is regulated by photosynthetic electron transport in the noxious red-tide-forming raphidophyte Chattonella antiqua.

The raphidophyte Chattonella antiqua is a noxious red-tide-forming alga that harms fish culture and the aquatic environment. Chattonella antiqua produces and secretes superoxide anions (O2-), and excessive secretion of O2- into the water has been associated with fish mortality. It is known that strong light stimulates the production of O2- in Chattonella spp. but the mechanism of the light-induced production of O2- remains to be clarified. In the present study, we examined the effects of light on extracellular levels of O2- and photosynthesis in C. antiqua. Extracellular levels of O2- rose during growth under high-intensity light, and the level of O2- was correlated with the photosynthetic parameter qP, which reflects the rate of transport of electrons downstream of photosystem II. The production of O2- was inhibited in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthetic electron transport, suggesting that reducing power derived from electron transport might be required for the production of O2-. By contrast, the production of O2- was enhanced in the presence of glycolaldehyde, an inhibitor of the Calvin-Benson cycle, suggesting that the accumulation of NADPH might stimulate the production of O2-. Thus, it is likely that the production of O2- is regulated by photosynthesis in C. antiqua.