ABI4 Mediates Ascorbic Acid Biosynthesis To Regulate Salt Tolerance In Arabidopsis

Salt stress triggers osmotic stress and ion toxicity in plants,resulting in oxidative stress,accumulation of reactive oxygen species(ROS),and inhibition of plant growth.As an important antioxidant,the ascorbic acid(As A)plays core roles in the regulation of plant normal growth and stress tolerance.As the key transcription factor of abscisic acid(ABA)signaling pathway,ABA INSENSITIVE4(ABI4)plays roles in the regulation of plant growth and response to drought,salinity and other stresses.Our previous studies have proved that ABA regulates As A biosynthesis through ABI4-affected VITAMIN C DEFECTIVE 2(VTC2)expression,a key gene of As A biosynthetic.Compared with wild type Col-0,the salt tolerance in abi4 mutant was enhanced but decreased in the transgenic plants that overexpressing ABI4.It is still unclear about the underlying molecular mechanism of ABI4-regulated salt tolerance.Thus,this study will uncover the function of ABI4-mediated As A biosynthesis in Arabidopsis salt tolerance,In this study,we found that exogenous As A treatment could recover the salt tolerance of ABI4-OEM1/5 overexpressing transgenic lines.Meanwhile,the transgenic plants accumulated less As A and more ROS,indicating that the removal of ROS by As A is related to ABI4-mediated growth inhibition under salinity stress.Quantitative PCR(q PCR)analysis showed that the expression of ABI4 increased in the early stage of salt stress,and the expression of VTC2 decreased accordingly.Meanwhile,Western blot analysis showed that the signal of GFP driven by VTC2 promoter on Col-0 background is decreased by salt stress,but the induction was obviously changed when the GFP was driven by VTC2 promoter in abi4 mutant background.In order to prove the regulation of ABI4 in VTC2 transcription,we further verified the inhibition of VTC2 transcription by ABI4 through transient expression in tobacco leaves and Arabidopsis protoplasts.Also,we evidenced that ABI4 was directly combined with VTC2 gene promoter-154 bp site CCAC element to inhibit its expression,using electrophoretic mobility shift assay(EMSA).This observation was confirmed by the results of GFP fluorescence in roots of transgenic materials driven by VTC2 promoter in the background of wild type and abi4 mutant,showing that the fluorescence signal of VTC2-GFP in abi4 mutant was stronger than that in wild type,indicating that ABI4 inhibits VTC2 transcription in vivo under salt stress.Furthermore,the salt tolerance of abi4vtc2 double mutant is similar to that of vtc2,indicating that VTC2 is genetically located in the downstream of ABI4.In conclusion,we found that abi4 mutant displays enhanced salt tolerance with increasedAs A content and decreased ROS accumulated induced by high level of VTC2 expression.However,the VTC2 expression in ABI4-overexpressing transgenic materials was inhibited,resulting in decreased As A level and increased ROS accumulation,which made the transgenic materials slightly smaller under normal condition and enhanced sensibility to salinity stress.Further research shown that ABI4 was induced whereas VTC2 expression was suppressed under salinity stress,with the inhibition of VTC2 expression by ABI4,caused reduced As A production and increased ROS accumulation.Therefore,this study elucidates the molecular mechanism that ABI4 regulates As A biosynthesis and ROS accumulation in response to salt stress in plants,and thus affecting the growth and development of seedlings.In different biological processes,ethylene and ABA show antagonistic or synergistic regulatory relationship.Ethylene can regulate As A biosynthesis and ROS elimination under salt stress.It was preliminarily proved that ABI4 was regulated by ethylene and ABA,providing a theoretical basis for revealing ABI4 regulation of plant salt tolerance.