The Mechanisms Of Hydrogen Sulfide In Mediating The Protective Effects Of Heme Oxygenase On Rice(Oryza Sativa)Tolerance Against Ammonium Stress

In animals,heme oxygenase(HO,EC 1.14.99.3)has been considered as a crucial antioxidant enzyme,which catalyzes the oxidative cleavage of heme to carbon monoxide(CO),biliverdin(BV)and free iron.Ample studies have confirmed that HO plays an important role in the antioxidant defense and growth in plants.In rice,PHOTOPERIOD SENSITIVITY 5(OsSE5)was assumed to encode OsHO1 with high similarity to Arabidopsis HO1 Hydrogen sulfide(H2S)was the third gasotransmitter after nitric oxide(NO)and CO.Studies in plants have shown that H2S can alleviates injury and toxicity caused by the stress conditions,and participates in specific physiological and metabolic processes.In this work,the rice wild-type(Oryza sativa L.),OsSE5 overexpression lines(35S:OsSE5-1)and OsSE5-RNAi transgenic lines(OsSE5-RNAi-1)were used to investigate the effects of H2S on rice physiological and biochemistry charcteristics,as well as the relationship between H2S and OsSE5 under ammonium stress.The main results were as follows:1.Levels of ammonium content and total L-DES activity were increased in a dose-dependent manner after NH4CI treatment ranging from 2.5 to 20 mM.When supplied with sodium hydrosulfide(NaHS,a well-known H2S donor;100 μM),rice plants displayed enhanced tolerance to ammonium.This was supported by alleviation of root growth inhibition,reduced lipid peroxidation and decreased ammonium content,while NH4Cl-triggerd toxic symptoms were further aggravated by addition of hypotaurine(HT,a H2S scavenger)or DL-propargylglycine(PAG,a L-DES biosynthesis inhibitor).We further observed that the activities of antioxidant and ammonium assimilation enzymes were significantly enhanced by the pretreatment of NaHS,which therefore increased the contents of total nitrogen and free amino acids.2.The sensitivity of 35S:OsSE5-1,wild-type and OsSE5-RNAi-1 plants to high ammonium stress was increased.The root growth inhibition,lipid peroxidation and ammonium content in 35S:OsSE5-1 plants were less than that of wild-type and OsSE5-RNAi-1 plants.The activities of L-DES were induced by high concentration of ammonium in rice roots.Compared with wild-type plants,the L-DES activity of 35S:OsSE5-1 plants was significantly higher than that of OsSE5-RNAi-1.Further results showed that compared with wild-type plants,35S:OsSE5-1 plants had higher ammonium assimilation enzymatic activities than OsSE5-RNAi-1,as well as higher contents of nitrogen and free amino acids,indicating that 35S:OsSE5-1 lines could reduce the content of ammonium by enhancing the ammonium assimilation capacity in order to adapt to the ammonium stress.3.Pharmacological experiments showed that NH4+-tolerant phenotypes of 35S:OsSE5-1 plants was blocked by the application of HT/PAG,and pretreat with NaHS can rescue NH4+-sensitive phenotypes of OsSE5-RNAi-1 plants.Our results further showed that pretreatment with HT or PAG decreased the antioxidant and ammonium assimilation enzymatic activities as well as nitrogen content,while these parameters were strengthened in OsSE5-RNAi-1 plants by the pretreatment with NaHS.4.Taken together,our findings indicate that H2S plays a crucial role in rice response to ammonium stress.H2S could alleviated the ammonium-triggered lipid peroxidation by upregulating the antioxidant system,and assimilated excessive ammonia into amino acids by regulating the ammonia assimilation system,thus improve the efficiency of ammonia assimilation.Moreover,OsSE5 could enhance ammonium tolerance by upregulating ammonia assimilation system in rice,which was mediated by L-DES-related H2S production.