Studies On The Mechanism Of Regulations Of Ethylene, NO And H₂O₂ In The Adaptation Of Arabidopsis Callus To Salt Stress

In the present study,we used the calli from wild-type Arabidopsis(Arabidopsis thaliana,WT)and ethylene-insensitive mutant etr1-3 to study their physiological responses to salt stress,and further investigated the regulative roles of ethylene and NO under salt stress.In addition,the present study was aimed to investigate the relationship between H₂O₂ and ethylene in the induction of alternative pathway(AP) in Arabidopsis calli from WT and etr1-3 under salt stress.And an effort was also made to demonstrate the possible regulation and physiological function of AP under salt stress.The main results were summarized as follows:1.Results showed that ethylene-insensitive mutant etr1-3 was more sensitive to salt stress than WT.Under 100 mM NaCl,etr1-3 callus displayed a greater electrolyte leakage and Na⁺/K⁺ ratio but a lower plasma membrane(PM)H⁺-ATPase activity than those of WT callus.Application of exogenous 1-aminocyclopropane-1 -carboxylic acid(ACC,an ethylene precursor)or sodium nitroprusside(SNP,a NO donor)alleviated NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and an increased PM H⁺-ATPase activity in WT callus but not in etr1-3 callus.The SNP actions in NaCl stress were attenuated by a specific NO scavenger PTIO(2-phenyl-4, 4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde)or an ethylene biosynthesis inhibitor AOA(aminooxyacetic acid)in WT callus.Under 100 mM NaCl,the NO accumulation and ethylene emission appeared at early time,and NO production greatly stimulated ethylene emission in WT callus.In addition,ethylene induced the expression of PM H⁺-ATPase genes under salt stress.Furthermore,the recovery experiment showed mat NaCl-induced injury was reversible,as signaled by the similar recovery of Na⁺/K⁺ ratio and PM H⁺-ATPase activity in WT callus.Taken together,the results indicate that ethylene and NO cooperate in stimulating PM H⁺-ATPase activity to modulate ion homeostasis for salt tolerance,and ethylene may be a part of the downstream signal molecular in NO action.2.The capacity of AP and the contribution of AP to the total respiration were significantly induced in the presence of 100 mM NaCl for 48 h in WT Arabidopsis callus but only slightly induced in an ethylene-insensitive mutant,etr1-3.Endogenous ethylene emission was enhanced in WT Arabidopsis callus under salt stress.In addition,application of 1-aminocyclopropane-1-carboxylic acid(ACC,an ethylene precursor)in the presence of NaCl further increased AP capacity in WT callus but had little effects on that of etr1-3 callus,suggesting ethylene is required for the induction of AP.On the other hand,limiting endogenous ethylene production by aminooxyacetic acid(AOA,an ethylene biosynthesis inhibitor)in WT callus eliminated the NaCl-induced increase of ethylene emission and inhibited the induction of AP under salt stress.H₂O₂ enhanced ethylene production while ethylene reduced H₂O₂ generation in WT callus under salt stress.Inhibition of the AP pathway by salicylhydroxamic acid under salt stress resulted in more severe membrane damage as indicated by higher levels of H₂O₂,TBARS and electrolyte leakage in WT callus. Taken together,these results suggest AP can play a role in antioxidant protection under salt stress,and ethylene may be the direct inducer of AP.