Study On Nitric Oxide Signal Production And Its Action Mechanism Of Cyanobacteria Stressed By Enhanced UV-B

In recent years, continuous depletion of the stratospheric ozone layer by atmospheric pollutants, in particular chlorofluorocarbons (CFCs), has resulted in an increasing incidence of solar UV-B irradiation (280-320 nm) at the Earth's surface. Besides exerting a variety of adverse effects on terrestrial vegetation, enhanced UV-B radiation is also known to penetrate fairly deep in freshwater and marine environments and resulted in the same effect to aquatic creature. Enhanced UV-B radiation resulted in great effects to cyanobacteria, especially which shallow water species and exist in rice field.Plants can produce nitric oxide (NO) induced by enhanced UV-B. Act as a new signal molecule, NO involved in response of plants to stress and there are a lot of relative studies in high plants. Although NO can act as a signal molecule taking part in the response of cells to UV-B stress in plants, animals and eukaryote of single cell, its role under enhanced UV-B irradiation in prokaryote of single cell remains unclear. It is essential to study whether or not there are NO production and the relationship between NO signal molecule and metabolism, between NO signal and oxidative injury induced by UV-B radiation in cyanobacteria. In this paper, we researched NO production and it's producing way, measured the effects of NO on the activity of anti-oxidanse and oxidant, the effects of NO on other metabolism and preliminarily clarified the mechanism by treated Spirulina platensis 794 cells with enhanced UV-B and different chemicals. The study results can act as a science basis for further research in this field. The results as following:1. NO production and its production way in Spirulina platensis 794 cells had been studied by treated with different UV-B intensity stress and different chemicals. Results shown that Spirulina platensis 794 cells produced and released NO signal molecular and quantity of NO production was related to UV-B intensity and time under enhanced UV-B radiation. NO production increased along with increase of UV-B intensity and time. The study results first revealed that as high eukaryote, prokaryote ofsingle cell-cyanobacteria could respond to enhanced UV-B stress and produce NO signal molecular. NO probably mediate a series of physiological and biochemical processes to make cyanobacteria responding to enhanced UV-B stress. These results provide scientifical basis and theoretical guide for further studing in this field.2.By measured and analyzed NOS activity, NR activity, NiR activity, PAL activity and quantity of NO2"#1 NO3",it was found that NO was produced through NOS way, NR way, NiR way and PAL way in Spirulina platensis 794 cells under enhanced UV-B radiation. NO synthesized and released was not by one way, but many ways, which related and restricted each other and accomplished moderate NO synthesis and release all together, so to response to UV-B stress further. Contribution of each enzyme to total NO production will be dependent on cells growth stage and utilizabiiity of substances and factors in cells and it can be changed by action of factors for NO to adjust cellular physiological activity. This research explored out NO production way in enhanced UV-B stressed cyanobacteria and illustrated the correlation and complexity of NO production processes in cyanobacteria. It not only has important theoretical value but established scientific base for further researching signal conduction of NO and its regulation mechanism to metabolic processes. At the same time, it provide scientific basis for establishing the mechanism of signal production net in cells.3.To study the role of NO on enhanced UV-B radiation (280-320nm)-induced damage of cyanobacterium, the growth, the content of pigment and the anti-oxidative activity of Spirulina platensis 794 cells were investigated under enhanced UV-B radiation and different chemical treatments with or without UV-B radiation for 6h. The changes of chlorosphyll-a, malondialdehyde (MDA) contents and biomass confirmed that 0.5 mM sodium nitroprusside (SNP), a donor of nitric oxide (NO), could markedly alleviate the damage of chlorosphyll-a, the loss of biomass and the accumulation of malondialdehyde (MDA) caused by enhanced ultraviolet-B. Specifically, the biomass and the chlorophyll-a content in S.platensis-794 cells decreased 40 per cent and 42 per cent respectively under enhanced ultraviolet-B stress alone, while they decreased only 10 per cent and 18.4 per cent in the cells treated with ultraviolet-B irradiation and 0.5mM SNP together.4.Further studies proved that NO treatment significantly increased the activities of superoxide dismutase (SOD) and catalase (CAT), and decreased the accumulation of O2" in enhanced ultraviolet-B-irritated cells. The increased folds of SOD and CAT activity are 0.95 and 6.73 respectively. Meanwhile, the accumulation of reduced glutathione (GSH) increased while treated with 0.5mM SNP in normal S. platensis cells, but SNP treatment could decrease the increasing of reduced glutathione (GSH) in enhanced UV-B stressed S. platensis cells. These results thus suggest that NO can strongly alleviate oxidative damage caused by UV-B stress by increasing the activities of SOD, POD, CAT and the accumulation of GSH, by eliminating O2" in S.platensis-794 cells. In addition, the difference of NO origin between plants and cyanobacteria was discussed. The results abundantly confirmed that cyanobacteria could clean free radicals produced by substance oxidation processes without delay through increase anti-oxidase activity and anti-oxide substance content to make cells and active moleculars, especially key moleculars in cells avoid injury. Also, it further confirmed that NO could act as a second messenger exist below the UV-B signal to function in signal production and regulate enzyme metabolic level and substance transformation processes in cells and had a protect effect to cells. The results illustrated that if endogenous NO level rise by adjusting metabolic processes, the adapting ability of cells to stress will increase.5.Exposure of N2-fixing cyanobacterium Spirulina platensis 794 to enhanced ultraviolet-B radiation (0.6 Jm'V1) for 2h and 4h resulted in decrease of nitrogenase activity by 86.03 per cent and 86.62 per cent respectively and a dose dependent inhibition was observed in UV-B irradiated S.platensis 794 cells. Culture cells were treated with enhanced UV-B and 0.5 mM SNP for 6h increased nitrogenase activity by 47.3 per cent as compared with UV-B treated control cells. SNP apparently counteracted UV-B stress-caused decrease of nitrogenase activity. Similarly, NAC (a free radical scavenger) significantly increased nitrogenase activity but PTIO (a NO scavenger) decreased nitrogenase activity in UV-B treated S. platensis 794 cells, whichindicate that NO and free radical scavenger NAC may counteract loss of nitrogenase activity caused by enhanced UV-B radiation. The results indicated that enhanced UV-B stress decreased the key enzyme- nitrogenase activity and first proved that NO signal molecular had counteractive action on enhanced ultraviolet-B induced decrease of cyanobacterial nitrogenase activity. This further indicated that NO signal molecular has protect effect on key molecular in cells by signal production processes and this will provide scientific guide for research in metablism of nitrogen fixation biology in stressed cyanobacteria.6.The activity of UV-B-inhibited nitrogenase did not recover upon transfer of exposed cells to fluorescent light, suggesting that the inhibition may be due to specific inactivation of the enzyme. By employment of inhibitors of protein synthesis and PS-II activity, it was demonstrated that reductant and ATP limit nitrogenase activity in cyanobacterium S.platensis cells, which illustrated that nitrogenase requires abundant supply of reductant and ATP for maintain its activity and further confirmed the mechanism of nitrogenase requires a continued and abundant supply of suitable reductant and ATP for conversion of N2 to NH3. These results illustrated that the decrease of nitrogenase activity was majorely due to specifilic inactivity of enzyme and inactivity of nitrogenase correlated to the lack of reductant and ATP in cells, this suggested that the decrease of nitrogenase activity induced by enhanced UV-B could correlated to the effect of enhanced UV-B on photobiothesis in cells and also suggested that the protective effect of NO on nitrogenase could correlated to the protective effect of NO on photobiothesis membrance of cells.7.Further results indicated that enhanced UV-B treatment increased nitrate reductase activity, SNP and NAC could further significantly increase the activity of nitrate reductase induced by enhanced UV-B radiation. In summary, NO had a counteractive action to changes of N2-fixing involved enzymes induced by enhanced ultraviolet-B radiation in S.platensis. The results indicated that UV-B stress and exogenous NO had effect on key enzyme activity in cells and revealed the important role of nitrate reductase in response of cells to stress. It further suggested that nitratereductase activity way is maybe a principal way of NO production in S.platensis cells and also proved the feedback regulation metablism of NO signal production processes. 8.We fist found that cyanobacteria cells could product and accumulate PAs under enhanced UV-B stress and researched the effect of NO on the production and accumulation of PAs in cells. Production and accumulation of PAs were researched in Spirulina platensis cells by treated with different UV-B radiation and different chemicals, results shown that under enhanced UV-B, Spirulina platensis cells could product and accumulate PAs and the quantities of PAs were related to UV-B intensity and time. Production of PAs was very sensitive to UV-B radiation and Cad and Put were the most ones among four PAs. Quantity of Cad and Spd were highest among four PAs, which up to 7.29 times and 2.07 times respectively compared with control cells. Spd+Spm and Spd+Spm+Cad content increased and the rates of (Spd+Spm )/Put and (Spd+Spm+Cad)/Put increased 6.06 and 29.48 respectively in enhanced UV-B stressed Spirulina platensis cells.We first found that cyanobacteria could product and accumulate Pas and proved that cyanobacteria could increase signal conduction and anti-oxide ability to avoid injury to cells by increase production and accumulatation of anti-oxide substance/signal substance Pas under enhanced UV-B stress. Further results revealed that NO signal resulted in the production of Pas signal substance, suggesting that NO is important signal molecular of Pas synthesis and it exist up the Pas signal conduction chain, indicating that the increase of rate of (Spd+Spm) /Putand (Spd+Spm+Cad) /Put correlated to the increase of anti-oxide ability of cells.9.Production of MAA was researched in Spirulina platensis cells by treated with different UV-B radiation and different chemicals, results shown that under enhanced UV-B, Spirulina platensis cells could product and accumulate MAA and the quantities of MAA was related to UV-B intensity and time. Also, under enhanced UV-B, Spirulina platensis cells produced two MAAs, shinorine and sporphyra-334 and quantity of sporphyra-334 was obviously over shinorine. The results showed that Spirulina platensis could filter away excessive UV-B radiation by synthesis and accumulating screen/protect substance MAA to protect cells, this abundantly illustratedthat cyanobacteria has protect metablism by itself and high adaption ability to stress.10. Further study indicated that NO signal molecular and free radicals scavenger NAC could adjust the synthesis and accumulation of MAA, NO signal and PAs could act each other to adjust production and accumulation of MAA all together. Further results revealed that NO could acted as a second messenger acting key role in signal conduction of cells and illustrated the correlation of signals and the mechanism of net regulation of metabolic adjusting in cells.Sum up, this paper first researched and found that prokaryote of single cell-cyanobacteria could produce and release NO signal molecular and explored out NO production way. It aboudantly proved the effect of NO signal molecular on anti-oxide system and nitrogen metabolism of enhanced UV-B stressed cyanobacteria. These results provide scientifical basis and theoretical guide for further studing in this field.