Ecological Effect And Molecular Mechanism Of Ammonium Toxicity On Cyanobacteria

With the human intensification of agricultural and industrial activities, the amount of nitrogen input by human has been increasing, which consequently results in some significant negative ecological effects, such as eutrophication, cyanobacterial blooms, decline of biodiversity and so on. Ammonium accounts for a large proportion to the total nitrogen created by human. It is one of the major and preferred nutrients for plants, however, it is known to be toxic to many organisms at high concentration. Prevoius studies of ammonium toxicity were mainly focused on higher plants, relatively less attention has been paid in cyanobacteria, and the cyanobacterial ecological effects caused by ammonium toxicity had not been attracted human’s special concern. Based on above background, present study explored the cause of reduction of Nostoc sp.(Ge-Xian-Mi) resource—one kind of edible cyanbacteria and its ammonium toxicity mechanism, analyzed the role of ammonium in the succession of cyanobacterial blooms and the distribution of common algal species in shallow freshwater lakes, investigated the mechanism of ammonium toxicity on PSII of Synechocystis sp. PCC6803, and the function of psbAl gene on the resisting to ammonium toxicity damage, the detailed content and results of this study is as follows,1. Effects of two fertilizers, NH4Cl and KCl, on the growth of the edible cyanobacterium Ge-Xian-Mi (Nostoc) and other four cyanobacterial strains were compared. Their growth was decreased by at least65%at10mmol·L-1NH4Cl, but no inhibitory effect was observed at the same level of KCl. Meanwhile, they exhibited a great variation of sensitivity to NH4+toxicity in the order: Ge-Xian-Mi> Anabaena azotica FACHB118> Microcystis aeruginosa FACHB905> Microcystis aeruginosa FACHB315> Synechococcus FACHB805. The relative growth rate96-h EC50value of NH4+for Ge-Xian-Mi was1.105mmol L-1, which was much less than NH4+concentration in many agricultural soils (2to20mmol L’1). These indicated that the use of ammonium as nitrogen fertilizer was responsible for the reduced resource of Ge-Xian-Mi in the paddy field. After96h exposure to1mmol L-1NH4Cl, the photosynthetic rate, Fv/Fm value, saturating irradiance for photosynthesis and PSII activity of Ge-Xian-Mi colonies were remarkably decreased. The chlorophyll synthesis of Ge-Xian-Mi was more sensitive to NH4+toxicity than phycobiliproteins. Thus, its functional absorption cross section of PSII was increased markedly at NH4Cl levels≥1mmol·L-1and the electron transport on the acceptor side of PSII was significantly accelerated by NH4Cl addition≥3mmol·L-1. Dark respiration of Ge-Xian-Mi was significantly increased by246%and384%at5and 10mmol L-1NH4Cl respectively. The rapid fluorescence rise kinetic suggested that the oxygen-evolving complex of PSII was the inhibitory site of NH4-2. The sensitivity of Ge-Xian-Mi (Nostoc) to ammonium toxicity under two limiting light intensities (40and100μmol photons m-2s-) were compared. After96h exposure to2mmol L-1NH4Cl, their growth were decreased to44.3%and-16.0%of the control under low light and high one respectively. The rapid fluorescence rise kinetic showed that the oxygen-evolving complex of PSII was much greatly damaged upon5mmol L-1NH4+addition under high light. The relative growth rate96-h EC50value showed that Ge-Xian-Mi was more sensitive to NH4+toxicity under high light compared to low one during the limiting light climate. With the treatment of5mmol L-1NH4+during60min, the connectivity factor, Fv/Fm value and electron transport rate between PSII and PSI decreased much faster, and the functional absorption cross section of PSII increased much faster, under high light than that of low light. Therefore, much more degree of damage in donor side with the treatment of ammonium upon high light may cause the generation of highly reaction P680, which will oxidate the β-carotene, damage the reaction centre, reduce the nonphotochemical fluorescence quenching, and aggravate the damage of reaction centre. Meanwhile, the lincomycin addition experiment indicated NH4+triggered photodamage response for accelerating the damage and less inhibiton of repair the of PSII of Nostoc sp.(Ge-Xian-Mi).3. With the human intensification of agricultural and industrial activities, large amount of reduced nitrogen enter into the biosphere, which consequently results in the development of global eutrophication and cyanobacterial blooms. However, no research had reported the direct effect of ammonia toxicity on the algal succession in freshwater lakes. In this study, we investigated the ammonia toxicity to nineteen algal species or strains to test the hypothesis that ammonia may regulate the succession of cyanobacterial blooms and the distribution of common algal species in freshwater lakes. The bloom-forming cyanobacterium Microcystis aeruginosa PCC7806suffered from ammonia toxicity at high pH value and light intensity conditions. Low NH4CI concentration (0.06mmol L-1) resulted in the decrease of operational PSII quantum yield by50%compared to the control exposed to1000μmol photons m-2s-1for one hour at pH9.0±0.2, which can be reached in freshwater lakes. Furthermore, the tolerant abilities to NH3toxicity of eighteen freshwater algal species or strains were as follows:hypertrophication species> eutrophication species> mesotrophication species> oligotrophication species. The different sensitivities of NH3toxicity in the present study could well explain the distributing rule of common algal species in the freshwater lakes of different trophic states. Meanwhile, the cyanobacterial bloom (e.g. M. aeruginosa) always happened at the low concentration of ammonia in summer, and disappeared with the decrease of ammonia. This may be attributed to the toxic effect of ammonia to M. aeruginosa in spring (the average and maximum ammonia concentration were0.08mmol L-1, and0.72mmol L-1in33Chinese lakes), and the low level of NH3-N in summer and fall in the lakes might be used as preferred nitrogen nutrition by M. aeruginosa, rather than with toxicity. Therefore, ammonia could be a key factor to determine the distribution of common algal species and cyanobacterial bloom in the freshwater systems.4. The effects of ammonia on the photodamage of PSII and repair of damaged PSII in cyanobacterium Synechocystis sp. strain PCC6803were investigated in this study. Ammonia was shown to accelerate the decrease of PSII activity through a mechanism that directly caused photodamage of PSII, rather than inhibition of the repair of photodamaged PSII. Isolated thylakoid membranes lost PSII activity upon ammonia treatment, with the oxygen-evolving complex being the primary target for ammonia toxicity. The D1protein of photosynthetic organisms is known to be rapidly turned over in the light. Previous studies indicated that the psbAl gene encoded one form of Dl protein that was not expressed in wild-type Synechocystis6803. The present investigation shows that its expression is transiently induced by high light. Synechocystis wild-type cells are more resistant to high light and ammonia than a psbAl-deficient mutant, indicating that the psbAl gene is not redundant, but could play a role in protecting PSII against high light and ammonia toxicity.