Mechanisms Of Submerged Plant Hydrilla Verticillata(L.f.)Royle Response To Nh₄⁺-N Stress

The phenomenon of eutrophication gains people's attention around the world,not only for its fast and seriously development,but also for its jeopardizing effect,such as hazard to health,destruction of environments and obstruction to social progress.Currently,the fresh water eutrophication is becoming serious,animals and aquatic plants,especially submerged plants,were severely damaged in China.Submerged plants play a key role in aquatic environment,including providing food and shelter for the fish and participating in the carbon nitrogen and phosphorus cycle in water.Hence the recovery of submerged vegetation is an effective measure to control water eutrophication.This paper studied the mechanism of Hydrilla verticillata(L.f.)Royle in response to NH4+-N stress,which help to enhance the understand of the mechanism of eutrophic water submerged plants' die,the prevention and control of eutrophic water,ecological restoration and management,and provide a theoretical basis.H.verticillata were treated with different concentrations of NH4+-N,NO3--N and PO43--P,different light quality,light intensity and dissolved oxygen(DO)(hypoxic or anoxic)conditions for 10 days.The plant height,photosynthetic pigment content,antioxidase activity and other physiological indexes changes in H.verticillata were determined.The results showed that high level of NH4+-N(>2.00 mg L-1),yellow light,the 5%light intensity below normal condition and low DO(<0.2 mg L-1)inhibited the growth of H.verticillata.H.verticillata were treated with different concentrations of NH4+-N to investigate the physiological response.Results showed that NH4+-N in high concentration(>2 mg L-1)significantly reduced photosynthetic pigment content,the oxygen evolving rate of photosynthesis,the accumulation of intracellular soluble sugar and soluble protein content;increased the accumulation of proline,the rate of oxygen consumption in respiration and key enzymes activity invoved in nitrogen assimilation;triggered the lost of the carbon and nitrogen metabolism balance and the accumulation of the in vivo ROS and MDA,the lipid peroxidation product,leading to the oxidative stress.The antioxidant enzyme activity were determined,the active electrophoretic patterns were identified,the non enzymatic antioxidant levels were measured to study the antioxidant response of H.verticillata treated with different concentrations of NH4+-N;And the addition of the treatments of BSO,GSH synthesis inhibitor,were to investigate the effects of GSH in H.verticillata against oxidative stress caused by NH4+-N toxicity.Results showed that the activity of antioxidant enzymes in H.verticillata tended to increase with the increase of NH4+-N concentration,and indicated that ROS accumulation in H.verticillata was not caused by anti-oxidase activity inhibition after the treatment of NH4+-N.The improvement of the antioxidant defense ability was a response to the oxidation stress caused by NH4+-N stress on H.verticillata,Under NH4+-N stress,AsA and GSH in H.verticillata functioned together in resistance to oxidative stress,but AsA content decreased with the increase of NH4+-N concentration when GSH content increased.While GSH content increased significantly with the concentration increase,after BSO treatment in H.verticillata,carbon and nitrogen metabolism imbalance increased and oxidation stress enhanced caused by NH4+-N stress,which indicated that GSH played an important role in H.verticillata against oxidative stress caused by NH4+-N toxicity.The inhibitor of NADPH oxidase and H2O2 scavenger were used in the treatment of H.verticillata under nitrogen stress,preliminary proved that ROS production were related to NADPH oxidase.H2O2 and O2·-content related to NADPH oxidase accounted for about 40%of H2O2 and O2·-content in H.verticillata under ammonia nitrogen stress;through dark treatment on the H.verticillata under ammonia nitrogen stress,H2O2 and O2·-derived by chloroplast accounted for more than 50%of the total content of H.verticillata,indicating that the chloroplast is the main source of ROS in H.verticillata under NH4+-N stress.Observed by transmission electron microscope,H2O2 and O2·-were accumulated in cell membrane and the inner wall of the extracellular space,O2·-were also accumulated in the chloroplast of H.verticillata.Percoll cell separating liquid were used in separation of the chloroplasts in H.verticillata under NH4+-N stress,O2·-generation rate,H2O2 accumulation and antioxidant system response of H.verticillata chloroplast was studied,the results also proved that NH4+-N stress can significantly enhance the H.verticillata chloroplast O2·-and H2O2 accumulation,and chloroplast antioxidase activity were induced significantly enhancement.The high accumulation of H2O2 in the chloroplast may be related to the high SOD activity and the restricted AsA/GSH cycle.In the treatment of NH4+-N,AsA accumulation were significantly reduced,which may lead to APX mediated diminished capacity of the clearance ROS.GSH can be directly involved in the removal and detoxification of ROS,GSH content in chloroplast can enhance the resistance to high NH4+-N stress on H.verticillata.NH4+-N stress induced the upregulation of cytosolic and chloroplastic NAD biosynthesis key enzymes,which explained the reason of the high accumulation of NAD(H)and NADP(H)in H.verticillata.To understand the mechanism involved in response to NH4+-N stress,the proteomic approach was used to investigate the protein expression of H.verticillata induced by different concentration of NH4+-N stress-H.verticillara.91 spots were found differently expressed in the NH4+-N-stressed shoots.The 91 differentially-expressed protein spots were analyzed by MALDI-TOF MS and a total of 52 proteins(53 spots)were identified,in which 44 proteins were up-regulated.The up-regulated prpteins related to photosynthesis,electron transport,biosynthesis,proteolysis,energy metabolism and other unknown proteins;6 proteins were down-regulated,which related to the Calvin cycle and chlorophyll synthesis;3 proteins involved in the antioxidant effect emerged.The differentially expressed of these key enzymes may resulted in pigment content decreased,protein degradation and the inhibition of photosynthesis in H.verticillata.