Nitric oxide mediates positive regulation of Nostoc flagelliforme polysaccharide yield via potential S-nitrosylation of G6PDH and UGDH.

Based on our previous findings that salicylic acid and jasmonic acid increased Nostoc flagelliforme polysaccharide yield by regulating intracellular nitric oxide (NO) levels, the mechanism through which NO affects polysaccharide biosynthesis in Nostoc flagelliforme was explored from the perspective of S-nitrosylation (SNO). The addition of NO donor and scavenger showed that intracellular NO had a significant positive effect on the polysaccharide yield of N. flagelliforme. To explore the mechanism, we investigated the relationship between NO levels and the activity of several key enzymes involved in polysaccharide biosynthesis, including fructose 1,6-bisphosphate aldolase (FBA), glucokinase (GK), glucose 6-phosphate dehydrogenase (G6PDH), mitochondrial isocitrate dehydrogenase (ICDH), and UDP-glucose dehydrogenase (UGDH). The enzymatic activities of G6PDH, ICDH, and UGDH were shown to be significantly correlated with the shifts in intracellular NO levels. For further validation, G6PDH, ICDH, and UGDH were heterologously expressed in Escherichia coli and purified via Ni+-NAT affinity chromatography, and subjected to a biotin switch assay and western blot analysis, which revealed that UGDH and G6PDH were susceptible to SNO. Furthermore, mass spectrometry analysis of proteins treated with S-nitrosoglutathione (GSNO) identified the SNO modification sites for UGDH and G6PDH as cysteine 423 and cysteine 249, respectively. These findings suggest that NO modulates polysaccharide biosynthesis in N. flagelliforme through SNO of UGDH and G6PDH. This reveals a potential mechanism through which NO promotes polysaccharide synthesis in N. flagelliforme, while also providing a new strategy for improving the industrial production of polysaccharides.

Carbon Metabolism and the ROS Scavenging System Participate in Nostoc flagelliforme's Adaptive Response to Dehydration Conditions through Protein Acetylation.

Acetylation represents an extensively occurring protein post-translational modification (PTM) that plays a key role in many cellular physiological and biochemical processes. However, studies on PTMs such as acetylation of lysine (LysAc) in cyanobacteria are still rare. In this study, a quantitative LysAc approach (acetylome) on the strains of Nostoc flagelliforme subjected to different dehydration treatments was conducted. We observed that starch contents were significantly accumulated due to dehydration treatments, and we identified 2474 acetylpeptides and 1060 acetylproteins based on acetylome analysis. Furthermore, an integrative analysis was performed on acetylome and nontargeted metabolism, and the results showed that many KEGG terms were overlapped for both omics analyses, including starch and sucrose metabolism, transporter activity, and carbon metabolism. In addition, time series clustering was analyzed, and some proteins related to carbon metabolism and the ROS scavenging system were significantly enriched in the list of differentially abundant acetylproteins (DAAPs). These protein expression levels were further tested by qPCR. A working model was finally proposed to show the biological roles of protein acetylation from carbon metabolism and the ROS scavenging system in response to dehydration in N. flagelliforme. We highlighted that LysAc was essential for the regulation of key metabolic enzymes in the dehydration stress response.

Proteome profiling reveals changes in energy metabolism, transport and antioxidation during drought stress in Nostoc flagelliforme.

BACKGROUND: Drought is an important abiotic stress that constrains the growth of many species. Despite extensive study in model organisms, the underlying mechanisms of drought tolerance in Nostoc flagelliforme remain elusive. RESULTS: We characterized the drought adaptation of N. flagelliforme by a combination of proteomics and qRT-PCR. A total of 351 differentially expressed proteins involved in drought stress adaptation were identified. It was found that the expression of several nutrient influx transporters was increased, including molybdate ABC transporter substrate binding protein (modA), sulfate ABC transporter substrate-binding protein (sbp) and nitrate ABC transporter (ntrB), while that of efflux transporters for toxic substances was also increased, including arsenic transporting ATPase (ArsA), potassium transporter (TrkA) and iron ABC transporter substrate-binding protein (VacB). Additionally, photosynthetic components were reduced while sugars built up during drought stress. Non-enzymatic antioxidants, orange carotenoid protein (OCP) homologs, cytochrome P450 (CYP450), proline (Pro) and ascorbic acid (AsA) were all altered during drought stress and may play important roles in scavenging reactive oxygen species (ROS). CONCLUSION: In this study, N. flagelliforme may regulates its adaptation to drought stress through the changes of protein expression in photosynthesis, energy metabolism, transport, protein synthesis and degradation and antioxidation. HIGHLIGHTS: • A total of 351 DEPs involved in adaptation to drought stress were identified. • Changes in the expression of six OCP homologs were found in response to drought stress. • Differential expression of transporters played an important role in drought stress adaptation. • Most PSII proteins were downregulated, while PSI proteins were unchanged in response to drought stress. • Sugar metabolism was upregulated in response to drought stress.

The Overlooked Genetic Diversity in the Dryland Soil Surface-Dwelling Cyanobacterium Nostoc flagelliforme as Revealed by the Marker Gene wspA.

Biodiversity is recognized to be relatively low in the dryland ecosystem. However, we might overlook the accumulating genetic variation in those dryland micro-populations, which should eventually increase the dryland biodiversity. In the xeric steppes of western and northwestern China, there are two soil surface-dwelling and genetically close cyanobacterial species, Nostoc commune and Nostoc flagelliforme. They respectively exhibit lamellate and filamentous colony shapes. Their individual colony is consisted of hundreds of trichomes and the common exopolysaccharide matrix. N. flagelliforme is exclusively distributed in the dryland and supposed to be evolved from N. commune. We previously reported that the morphological diversity of N. flagelliforme colonies was very limited, being either cylindrical or strip-like. In this communication, we performed single-nucleotide polymorphism (SNP) analysis of the marker gene wspA as well as phylogenetic analysis of the WspA protein in N. flagelliforme colonies to gain insights into its genetic diversity. SNP analysis suggested that there existed plentiful nucleotide variations in the individual colonies and meanwhile these variations shared certain evolutionary regularity. Phylogenetic analysis of the deduced proteins from the cloned wspA sequences suggested that the relatively regular variations were possibly dispersed in the N. flagelliforme populations of different regions. Thus, these results presented a scenario of the underestimated genetic diversity hidden behind the limited morphotype of dryland cyanobacteria. Maybe, we can consider the individual cyanobacterial colony as a potential biodiversity pool in the drylands.

Orange and red carotenoid proteins are involved in the adaptation of the terrestrial cyanobacterium Nostoc flagelliforme to desiccation.

The remarkable drought-resistance of the terrestrial cyanobacterium Nostoc flagelliforme (N. flagelliforme) has attracted attention for many years. In this study, we purified a group of red proteins that accumulate in dried field samples of N. flagelliforme. These red proteins contain canthaxanthin as the bound chromophore. Native-PAGE analysis revealed that the purified red proteins resolved into six visible red bands and were composed of four helical carotenoid proteins (HCPs), HCP1, HCP2, HCP3, and HCP6 (homologs to the N-terminal domain of the orange carotenoid protein (OCP)). Seven genes encode homologs of the OCP in the genome of N. flagelliforme: two full-length ocp genes (ocpx1 and ocpx2), four N-terminal domain hcp genes (hcp1, hcp2, hcp3, and hcp6), and one C-terminal domain ccp gene. The expression levels of hcp1, hcp2, and hcp6 were highly dependent on the water status of field N. flagelliforme samples, being downregulated during rehydration and upregulated during subsequent dehydration. Transcripts of ocpx2 were dominant in the dried field samples, which we confirmed by detecting the presence of OCPx2-derived peptides in the purified red proteins. The results shed light on the relationship between carotenoid-binding proteins and the desiccation resistance of terrestrial cyanobacteria, and the physiological functions of carotenoid-binding protein complexes in relation to desiccation are discussed.

Profiling of Small Molecular Metabolites in Nostoc flagelliforme during Periodic Desiccation.

The mass spectrometry-based metabolomics approach has become a powerful tool for the quantitative analysis of small-molecule metabolites in biological samples. Nostoc flagelliforme, an edible cyanobacterium with herbal value, serves as an unexploited bioresource for small molecules. In natural environments, N. flagelliforme undergoes repeated cycles of rehydration and dehydration, which are interrupted by either long- or short-term dormancy. In this study, we performed an untargeted metabolite profiling of N. flagelliforme samples at three physiological states: Dormant (S1), physiologically fully recovered after rehydration (S2), and physiologically partially inhibited following dehydration (S3). Significant metabolome differences were identified based on the OPLS-DA (orthogonal projections to latent structures discriminant analysis) model. In total, 183 differential metabolites (95 up-regulated; 88 down-regulated) were found during the rehydration process (S2 vs. S1), and 130 (seven up-regulated; 123 down-regulated) during the dehydration process (S3 vs. S2). Thus, it seemed that the metabolites' biosynthesis mainly took place in the rehydration process while the degradation or possible conversion occurred in the dehydration process. In addition, lipid profile differences were particularly prominent, implying profound membrane phase changes during the rehydration-dehydration cycle. In general, this study expands our understanding of the metabolite dynamics in N. flagelliforme and provides biotechnological clues for achieving the efficient production of those metabolites with medical potential.

Comparative proteomic analysis of Nostoc flagelliforme reveals the difference in adaptive mechanism in response to different ultraviolet-B radiation treatments.

Nostoc flagelliforme is a pioneer organism in the desert and highly resistant to ultraviolet B (UV-B) radiation, while the involved adaptive mechanism has not been fully explored yet. To elucidate the responsive mechanism, two doses of UV-B radiation (low: 1 W/m2 and high: 5 W/m2) were irradiated for 6 h and 48 h, respectively, and their effects on global metabolism in N. flagelliforme were comprehensively investigated. In this study, we used iTRAQ-based proteomic approach to explore the proteomes of N. flagelliforme, and 151, 172, 124 and 148 differentially expressed proteins were identified under low and high UV-B doses for 6 h and 48 h, respectively. Functional classification analysis showed these proteins were mainly involved in photosynthesis, amino acid metabolism, antioxidant activity and carbohydrate metabolism. Further analysis revealed that UV-B imposed restrictions on primary metabolism including photosynthesis, Calvin cycle, and amino acid metabolism, and cells started defense mechanism through repair of DNA and protein damage, increasing antioxidant activity, and accumulating extracellular polysaccharides to minimize the damage. Moreover, high UV-B dose imposed more severe restrictions and activated stronger defense mechanism compared with low dose. The results would improve the understanding of molecular mechanisms of UV-B-stress adaption in N. flagelliforme.

Scytonemin Plays a Potential Role in Stabilizing the Exopolysaccharidic Matrix in Terrestrial Cyanobacteria.

Cyanobacteria are photosynthetic oxygen-evolving prokaryotes that are distributed in diverse habitats. They synthesize the ultraviolet (UV)-screening pigments, scytonemin (SCY) and mycosporine-like amino acids (MAAs), located in the exopolysaccharide (EPS) matrix. Multiple roles for both pigments have gradually been recognized, such as sunscreen ability, antioxidant activity, and heat dissipation from absorbed UV radiation. In this study, a filamentous terrestrial cyanobacterium Nostoc flagelliforme was used to evaluate the potential stabilizing role of SCY on the EPS matrix. SCY (∼3.7 %) was partially removed from N. flagelliforme filaments by rinsing with 100 % acetone for 5 s. The physiological damage to cells resulting from this treatment, in terms of photosystem II activity parameter Fv/Fm, was repaired after culturing the sample for 40 h. The physiologically recovered sample was further desiccated by natural or rapid drying and then allowed to recovery for 24 h. Compared with the normal sample, a relatively slower Fv/Fm recovery was observed in the SCY-partially removed sample, suggesting that the decreased SCY concentration in the EPS matrix caused cells to suffer further damage upon desiccation. In addition, the SCY-partially removed sample could allow the release of MAAs (∼25 %) from the EPS matrix, while the normal sample did not. Therefore, damage caused by drying of the former resulted from at least the reduction of structural stability of the EPS matrix as well as the loss of partial antioxidant compounds. Considering that an approximately 4 % loss of SCY led to this significant effect, the structurally stabilizing potential of SCY on the EPS matrix is crucial for terrestrial cyanobacteria survival in complex environments.

Ecophysiology of gelatinous Nostoc colonies: unprecedented slow growth and survival in resource-poor and harsh environments.

BACKGROUND: The cyanobacterial genus Nostoc includes several species forming centimetre-large gelatinous colonies in nutrient-poor freshwaters and harsh semi-terrestrial environments with extended drought or freezing. These Nostoc species have filaments with normal photosynthetic cells and N2-fixing heterocysts embedded in an extensive gelatinous matrix of polysaccharides and many other organic substances providing biological and environmental protection. Large colony size imposes constraints on the use of external resources and the gelatinous matrix represents extra costs and reduced growth rates. SCOPE: The objective of this review is to evaluate the mechanisms behind the low rates of growth and mortality, protection against environmental hazards and the persistence and longevity of gelatinous Nostoc colonies, and their ability to economize with highly limiting resources. CONCLUSIONS: Simple models predict the decline in uptake of dissolved inorganic carbon (DIC) and a decline in the growth rate of spherical freshwater colonies of N. pruniforme and N. zetterstedtii and sheet-like colonies of N. commune in response to a thicker diffusion boundary layer, lower external DIC concentration and higher organic carbon mass per surface area (CMA) of the colony. Measured growth rates of N. commune and N. pruniforme at high DIC availability comply with general empirical predictions of maximum growth rate (i.e. doubling time 10-14 d) as functions of CMA for marine macroalgae and as functions of tissue thickness for aquatic and terrestrial plants, while extremely low growth rates of N. zetterstedtii (i.e. doubling time 2-3 years) are 10-fold lower than model predictions, either because of very low ambient DIC and/or an extremely costly colony matrix. DIC uptake is limited by diffusion at low concentrations for all species, although they exhibit efficient HCO3(-) uptake, accumulation of respiratory DIC within the colonies and very low CO2 compensation points. Long light paths and light attenuation by structural substances in large Nostoc colonies cause lower quantum efficiency and assimilation number and higher light compensation points than in unicells and other aquatic macrophytes. Extremely low growth and mortality rates of N. zetterstedtii reflect stress-selected adaptation to nutrient- and DIC-poor temperate lakes, while N. pruniforme exhibits a mixed ruderal- and stress-selected strategy with slow growth and year-long survival prevailing in sub-Arctic lakes and faster growth and shorter longevity in temperate lakes. Nostoc commune and its close relative N. flagelliforme have a mixed stress-disturbance strategy not found among higher plants, with stress selection to limiting water and nutrients and disturbance selection in quiescent dry or frozen stages. Despite profound ecological differences between species, active growth of temperate specimens is mostly restricted to the same temperature range (0-35 °C; maximum at 25 °C). Future studies should aim to unravel the processes behind the extreme persistence and low metabolism of Nostoc species under ambient resource supply on sediment and soil surfaces.

Salicylic Acid and Jasmonic Acid Increase the Polysaccharide Production of Nostoc flagelliforme via the Regulation of the Intracellular NO Level.

To significantly improve the polysaccharide production of Nostoc flagelliforme, a total of 12 chemicals were evaluated for their effects on polysaccharide accumulation. The results showed that salicylic acid and jasmonic acid increased the accumulation of the polysaccharides in N. flagelliforme significantly, by more than 20%. Three polysaccharides, namely control-capsule polysaccharide, salicylic acid-capsule polysaccharide, and jasmonic acid-capsule polysaccharide, were extracted and purified from N. flagelliforme under normal, salicylic acid, and jasmonic acid culture conditions, respectively. Their chemical compositions slightly differed regarding the total sugar and uronic acid contents, with average molecular weights of 2.06 × 103, 2.16 × 103 and 2.04 × 103 kDa, respectively. They presented similar Fourier transform infrared spectra and no significant difference in antioxidant activity. It was revealed that the salicylic acid and jasmonic acid significantly increased the level of nitric oxide. By investigating the effects of the exogenous nitric oxide scavenger and nitric oxide donor on the nitric oxide levels and polysaccharide yield of N. flagelliforme, the results showed that the increase in intracellular nitric oxide levels might be an important factor promoting the accumulation of polysaccharides. These findings provide a theoretical foundation for enhancing the yield of secondary metabolites by regulating the intracellular nitric oxide levels.

Effect of Drought Stress on Degradation and Remodeling of Membrane Lipids in Nostoc flagelliforme.

Nostoc flagelliforme is a kind of terrestrial edible cyanobacteria with important ecological and economic value which has developed special mechanisms to adapt to drought conditions. However, the specific mechanism of lipidome changes in drought tolerance of N. flagelliforme has not been well understood. In this study, the ultra-high-performance liquid chromatography and mass spectrometry were employed to analyze the lipidome changes of N. flagelliforme under dehydration. A total of 853 lipid molecules were identified, of which 171 were significantly different from that of the control group. The digalactosyldiacylglycerol/monogalactosyldiacylglycerol (DGDG/MGDG) ratio was increased. The amount of wax ester (WE) was sharply decreased during drought stress, while Co (Q10) was accumulated. The levels of odd chain fatty acids (OCFAs) were increased under dehydration, positively responding to drought stress according to the energy metabolism state. In conclusion, the lipidomic data corroborated that oxidation, degradation, and biosynthesis of membrane lipids took place during lipid metabolism, which can respond to drought stress through the transformation of energy and substances. Besides, we constructed a lipid metabolic model demonstrating the regulatory mechanism of drought stress in N. flagelliforme. The present study provides insight into the defense strategies of cyanobacteria in lipid metabolic pathways.

Exogenous melatonin improves salt tolerance mainly by regulating the antioxidant system in cyanobacterium Nostoc flagelliforme.

Melatonin is a multifunctional nontoxic bio-stimulant or signaling molecule, generally distributing in different animal and plant organs for invigorating numerous physiological processes against abiotic stresses. In this study, we investigated the potential impact of melatonin on the cyanobacterium Nostoc flagelliforme when exposed to salt stress according to some biochemical and physiological parameters, such as relative electrolyte leakage, PSII activity, and photosynthetic pigments including chlorophyll a, phycocyanobilin, and phycoerythrobilin. We found that melatonin could also maintain K+ homeostasis in salt-stressed N. flagelliforme. These above results confirmed melatonin had multiple functions in hyperosmotic stress and ion stress caused by salinity. Notably, we observed melatonin could regulate the reactive oxygen species (ROS) signal and distinctly decrease the content of hydrogen peroxide and superoxide anion in salt-stressed cells, which were largely attributed to the increased antioxidant enzymes activities including catalase, superoxide dismutase, ascorbate peroxidase, and glutathione reductase. Finally, qRT-PCR analysis showed that melatonin stimulated the expression of antioxidant genes (NfCAT, NfSOD, and NfGR). In general, our findings demonstrate melatonin has beneficial effects on N. flagelliforme under salt stress by intensively regulating antioxidant system.

Nostoc flagelliforme capsular polysaccharides from different culture conditions improve hyperlipidemia and regulate intestinal flora in C57BL/6J mice to varying degrees.

Two capsular polysaccharides (WL-CPS-1 and GLU-CPS-1) purified from Nostoc flagelliforme under normal and mixotrophic culture conditions were used to investigate the hypolipidemic activity and effect on intestinal flora in C57BL/6J mice respectively. Their molecular weight and monosaccharide composition have been determined in previous studies. They both improved the lipid level by affecting the expression of lipid metabolism genes. They down-regulated the TNF-α and IL-1ß levels in serum and up-regulated the activity of antioxidant enzymes in liver thus decreased the atherosclerosis index and MDA content. They up-regulated the short chain fatty acids (SCFAs) synthesis. They decreased the abundance of pathogenic bacteria and increased the abundance of probiotics positively correlated with SCFAs. Compared with WL-CPS-1, GLU-CPS-1 exhibited higher in vivo activity and enriched Odoribacter and Alloprevotella correlating with the gene expression of lipid metabolism, suggesting that the bioactivity of polysaccharides could be regulated by culture conditions. These findings contributed to application of N. flagelliforme polysaccharides with higher activity in hypolipidemia by adjusting culture conditions.

POTASSIUM ALLEVIATES THE INHIBITORY ACTION OF AMMONIUM UPON THE PHOTOSYNTHETIC RECOVERY OF NOSTOC FLAGELLIFORME (CYANOPHYCEAE) DURING REHYDRATION1.

Effects of ammonium on the photosynthetic recovery of Nostoc flagelliforme Berk. et M. A. Curtis were assayed when being rehydrated in low-K+ or high-K+ medium. Its photosynthetic recovery was K+ limited after 3 years of dry storage. The potassium absorption of N. flagelliforme reached the maximum after 3 h rehydration in low-K+ medium but at 5 min in high-K+ medium. The K+ content of N. flagelliforme rehydrated in high-K+ medium was much higher than that in low-K+ medium. The maximal PSII quantum yield (Fv /Fm ) value of N. flagelliforme decreased significantly when samples were rehydrated in low-K+ medium treated with 5 mM NH4 Cl. However, the treatment of 20 mM NH4 Cl had little effect on its Fv /Fm value in high-K+ medium. The relative Fv /Fm 24 h EC50 (concentration at which 50% inhibition occurred) value of NH4+ in high-K+ medium (64.35 mM) was much higher than that in low-K+ medium (22.17 mM). This finding indicated that high K+ could alleviate the inhibitory action of NH4+ upon the photosynthetic recovery of N. flagelliforme during rehydration. In the presence of 10 mM tetraethylammonium chloride (TEACl), the relative Fv /Fm 24 h EC50 value of NH4+ was increased to 46.34 and 70.78 mM, respectively, in low-K+ and high-K+ media. This observation suggested that NH4+ entered into N. flagelliforme cells via the K+ channel. Furthermore, NH4+ could decrease K+ absorption in high-K+ medium.

Enhancement of exopolysaccharides production and reactive oxygen species level of Nostoc flagelliforme in response to dehydration.

Nostoc flagelliforme is a remarkable drought-resistant terrestrial cyanobacterium whose exopolysaccharides (EPS) have been found to exert important physiological and ecological functions, and the EPS are known to improve soil physicochemical properties. In this study, we used physiological and molecular methods to investigate the influences of three moisture loss levels on EPS production and the antioxidant system in N. flagelliforme. The aim was to reveal the EPS production mechanism involved in the gene differential expression and antioxidant system of N. flagelliforme in response to drought. Our results showed that EPS contents increased by 13% and 22% after 6-h and 48-h dehydration (6HAD and 48HAD) compared with 4-h rehydration (4HAR), respectively. The same trends were also detected for most EPS synthesis genes, especially glycosyltransferases. Furthermore, the intracellular reactive oxygen species (ROS) levels in N. flagelliforme were generally higher at 6HAD and 48HAD than at 4HAR. Superoxide dismutase (SOD) and peroxidase (POD) activities were restricted in N. flagelliforme under 6HAD and 48HAD compared with 4HAR, but the opposite result was found in catalase (CAT) activity. These results provide a new foundation for understanding the mechanism of EPS accumulation in N. flagelliforme in response to drought.

Global analysis of protein succinylation modification of Nostoc flagelliforme in response to dehydration.

Nostoc flagelliforme is a type of terrestrial cyanobacteria that is distributed in arid or semi-arid steppes in China. To research the molecular mechanisms underlying the adaptation of N. flagelliforme to drought stress, the succinylated expression profile and changes in N. flagelliforme that resulted as a response to dehydration were analyzed by label-free proteomics. A total of 1149 succinylated sites, 1128 succinylated peptides, and 396 succinylated proteins were identified. Succinylated proteins were differentially involved in photosynthesis and energy metabolism, as well as in reactive oxygen species (ROS) scavenging. Motif-X analysis of succinylated sites determined a succinylation motif [KxxG]. N. flagelliforme adapts to dehydration by increasing glucose metabolism and pentose phosphate pathway flux, and decreasing photosynthetic rate, which some of the key proteins were succinylated. ROS scavenging was mainly involved in the regulation of the enzyme antioxidant defense system and non-enzymatic antioxidant defense system through succinylation modification, thus eliminating excessive ROS. Protein succinylation of N. flagelliforme may play an important regulatory role in response to dehydration. The results are foundational, as they can inform future research into the mechanisms involved in the succinylation regulation mechanism of N. flagelliforme in response to dehydration. SIGNIFICANCE: The global succinylation network involved in response to dehydration in N. flagelliforme has been established. We found that many succinylated proteins were involved in photosynthesis, glucose metabolism and antioxidation. The global survey of succinylated proteins and the changes of succinylated levels in response to dehydration provided effective information for the drought tolerance mechanism in N. flagelliforme.

Complementary UV-absorption of mycosporine-like amino acids and scytonemin is responsible for the UV-insensitivity of photosynthesis in Nostoc flagelliforme.

Mycosporine-like amino acids (MAAs) and scytonemin are UV-screening compounds that have presumably appeared early in the history of life and are widespread in cyanobacteria. Natural colonies of the UV-insensitive Nostoc flagelliforme were found to be especially rich in MAAs (32.1 mg g DW(-1)), concentrated in the glycan sheath together with scytonemin. MAAs are present in the form of oligosaccharide-linked molecules. Photosystem II activity, measured using PAM fluorescence and oxygen evolution, was used as a most sensitive physiological parameter to analyse the effectiveness of UV-protection. Laboratory experiments were performed under controlled conditions with a simulated solar radiation specifically deprived of UV-wavebands with cut-off filters (295, 305, 320, 345 and 395 nm). The UV-insensitivity of N. flagelliforme was found to cover the whole UV-A (315-400 nm) and UV-B (280-320 nm) range and is almost certainly due to the complementary UV-absorption of MAAs and scytonemin. The experimental approach used is proposed to be suitable for the comparison of the UV-protection ability in organisms that differ in their complement of UV-sunscreen compounds. Furthermore, this study performed with a genuinely terrestrial organism points to the relevance of marine photoprotective compounds for life on Earth, especially for the colonization of terrestrial environments.

Improved production of echinenone and canthaxanthin in transgenic Nostoc sp. PCC 7120 overexpressing a heterologous crtO gene from Nostoc flagelliforme.

Echinenone and canthaxanthin are important carotenoid pigments with food and industrial applications. Biosynthesis of echinenone and/or canthaxanthin is catalyzed by ß-carotene ketolase (CrtO), with ß-carotene as the substrate. In this study, we generated transgenic Nostoc sp. PCC 7120 overexpressing a heterologous crtO gene from Nostoc flagelliforme and evaluated the productivity of both pigments. Normal (BG11 medium, 30 °C) and osmotic stress (BG11 medium supplemented with 0.4 M mannitol, 30 °C) conditions were used for cultivation. As compared to control strain, production of echinenone and canthaxanthin in transgenic strain were respectively increased by more than 16 % and 80 %, under either normal or osmotic stress conditions. Especially upon the stress condition, higher proportion of echinenone and canthaxanthin in total pigments was achieved, which should be beneficial for downstream separation and purification. In addition, transgenic strain showed drought tolerance and could revive from desiccation treatment after rewetting. Thus, this study provided technical clues for production of both pigments in engineered cyanobacteria as well as for cyanobacterial anhydrobiotic engineering.

Dryland cyanobacterial exopolysaccharides show protection against acid deposition damage.

Soil surface-dwelling cyanobacteria constitute an important part of the dryland ecosystem. The exopolysaccharide (EPS) matrix they establish plays multiple roles in helping cells cope with harsh environments and also improves soil physicochemical properties. Anthropogenic atmospheric nitrogen or sulfur depositions have arisen as an important environmental change in drylands. The acid moisture derived from the depositions will be absorbed by cyanobacterial EPS matrix and thus may pose a threat to cells. In this communication, we evaluated this potential impact in a dryland cyanobacterium, Nostoc flagelliforme, which is a representative polysaccharide-rich species and shows remarkable resistance to desiccation stress. A strong and resilient pH buffering property was found for the EPS matrix, mainly of the polysaccharide's role, and this could protect the cells from acid damage of pH 4-6, a general acidity range of rainwater in the world. Unlike in acid aquatic environments, terrestrial xeric environments ensure N. flagelliforme unlikely to undertake lasting severe acidification. Thus, protection of the EPS matrix for dryland cyanobacteria would be conducive to sustain their growth and ecological roles in face of atmospheric acid pollution.

ROS Is a Factor Regulating the Increased Polysaccharide Production by Light Quality in the Edible Cyanobacterium Nostoc flagelliforme.

To explore the regulatory factor of light quality affecting exopolysaccharide (EPS) production, transcriptome analysis of Nostoc flagelliforme cells exposed to red light (R), blue light (B), and mixed light (B/R = 15:7) (BR) with white fluorescent light as control was performed. The differentially expressed genes mainly enriched in carbohydrate metabolism and energy metabolism. Significant enrichment in the oxidation-reduction process and energy metabolism indicated that intracellular redox homeostasis was disrupted. An assay of reactive oxygen species (ROS) and malondialdehyde contents demonstrated light quality induced oxidative stress. To illustrate the relationship between ROS level and EPS accumulation, the effects of the exogenous addition of ROS scavenger N-acetyl cysteine and inducer H2O2 on the oxidation-reduction level and EPS production were compared. The results revealed that light quality regulated EPS biosynthesis via the intracellular ROS level directly other than oxidative stress. Understanding such relationships might provide guidance for efficient EPS production to regulate the intracellular redox level.