| With the expansion of producing and living activities, the proportion of artificial total nitrogen in the nature nitrogen deposition rises rapidly, of which ammonia occupies a large proportion. Ammonium is often used as main nitrogen nutrition for plants preferentially, whereas a high concentration of ammonia is toxic to organisms, decreasing the biodiversity, and even pushing ecosystem to the verge of collapse. Taking an important participant in algal bloom, cyanobacteria share similar photosystems with higher plants as more primitive photoautotroph. Study on the relationship between ammonia toxicity and cyanobacteria can not only reflect the the mechanism of plant response to high ammonia concentration directly, but also provide a basis for the study of effects on the whole ecological system, which lay a theoretical foundation to take measures. PsbO, PsbU, PsbV, PsbP, PsbQ are extrinsic proteins of OEC, which located on the thylakoid membrane, that are essential part of OEC, maintaining the OEC activities and stabilizing PSII function in tolerance of high urea or low Cl-/Ca2+ medium. Research suggests OEC is the initial damage point of ammonia toxicity on PSII, but no research reported the responses of extrinsic proteins and the mechanisms of ammonia toxicity.In present research, the genes coding those proteins were knocking out individually, and those proteins'functions and molecular mechanisms during tolerance of ammonia toxicity were focused in Synechocystis sp. PCC 6803, which is a kind of photoautotrophic model organism. By comparation of the variation of Fv/Fm value and growth phenotype in different mutants and wild-type strains on various ammonia concentrations for 1 h and 48 h, it confirmed that psbO-, psbU, psbV mutants were more sensitive than wild-type strain. The significant roles that PsbO?PsbU?PsbV playing in ammonia tolecance were proved by the complementation experiments. Chlorophyll a transient fluorescence showed an increase of fluorescence at 300 ?s in psbU and psbV mutants treated with ammonia, suggesting that the damage of the OEC, and the psbO-mutant showed a high 300 ?s fluorescence value at normal growth condition, indicating its OEC was damaged seriously.77 K fluorescence showed a blue shift of PSII in these mutants, indicating instability of PSII in the absence of extrinsic proteins. Blue shifts appearing in the fluorescence of all strains with ammonia treatments on wild-type for 1 h showed the PSII disassembled. With ammonia treatment for 1 h, the expressions of psbO?psbV genes tended to decreased, whereas psbU gene was up-regulated expressing. With exposure to 20 mmol L-1 ammonia for 8?24?48?72 h, PsbO protein content decreased in cells, reflected on the thylakoid membranes, whereas PsbU protein showed an increase both in total protein and soluble partial but a decrease on membranes, indicating that ammonia treatment weakened the combination of PsbU to the membrane and dissociated it to the lumen side. Ammonia treatment showed little impacts on PsbV content and the combination to the membrane. Analysis on transcriptome of wild-type cells after 1 h ammonia treatment showed that expressions of psbO gene, genes relative to pigments, ATPase and most genes coding PSI down-regulated, whereas genes coding proteins related with PSII turnover up-regulated. In order to resist ammonia toxicity, cells decreased the expression of PSI and reduced light harvest to accelerate the PSII recovery and to balance of redox state of photosystems.Comprehensively, PsbO, PsbU, PsbV showed important function in maintaining the stability of PSII during the tolerance of ammonia toxicity. Ammonia treatment inhibited PsbO expression in cells, weakened the combination of PsbU to the thylakoid membranes, and reduce the protection on PSII. |
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