Mitochondria And Apoplastic H₂O₂ Impacts Disease Resistance In Arabidopsis

Riboflavin is the precursor of flavin mononucleotide?FMN?and flavin adenine dinucleotide?FAD?,essential cofactors for many metabolic enzymes that catalyze a variety of biochemical reactions.Previously we showed that free flavin?riboflavin,FMN,and FAD?concentrations were decreased in leaves of transgenic Arabidopsis plants expressingRfBP.Here we report that RfBP-induced H₂O₂ presumably results from electron leakage at the METC and this source of H₂O₂ contributes to the enhanced diseasesresistance.We examine whether the cellular localization of Hpal,a harpin protein produced by the ricebacterial leaf blight pathogen,impacts H₂O₂ production and pathogen resistance in Arabidopsis thaliana.In addition,Hpal localization accompanied H₂O₂ accumulation in both the apoplast and cytoplasm of SHETAt plants but only in the cytoplasm of HETAt plants.Apoplastic H₂O₂ production via nicotinamide adenine dinucleotide phosphate?NADPH?oxidase?NOX?located in the plasma membrane is a commonfeature of plant defenses.After being applied to the wild-type plant,Hpal localized to apoplasts and stimulated H₂O₂ production as in SHETAt plants.In both plants,inhibiting apoplastic H₂O₂ generation abrogated both cytoplasmic H₂O₂ accumulation and plant resistance to bacterial pathogens.1.Down-regulation of free riboflavin content induces hydrogenperoxide and a pathogen defense in ArabidopsisIn the RfBP⁺Arabidopsis plants,RfBP localizes to chloroplasts and binds with riboflavin,resulting in significant decreases of free riboflavin concentrations.This change accompanies an elevation in the cytosolic level of H₂O₂.All these RfBP-conferred responses can be eliminated by nullifying RfBP production under RtBP+ background,and the RfBP gene silencing?RfBP-?Arabidopsis lines resemble the wild-type?WT?plant in riboflavin and H₂O₂ concentrations.Thus,the alteration of flavin content is an initial force for H₂O₂ generation in the plant cytosol.Here,we show that changing riboflavin content affects H₂O₂ accumulation and a pathogen defense in Arabidopsis thaliana.RfBP-induced H₂O₂ presumably results from electron leakage at METC and this source of H₂O₂ contributes to the enhanced pathogen defense phenotype.In this study,we elucidate that leaf riboflavin contentdownregulation by RfBP induces H₂O₂ generation presumably through electron leakage from METC and this source of H₂O₂ causes a promoting effect on pathogen defense in Arabidopsis.2.Apoplastic harpin protein Hpalplays an important role in H₂O₂ generation and pathogen resistance in ArabidopsisHarpin proteins secreted by phytopathogenic bacteria have been shown to activate the plant defensepathway,which involves transduction of a H₂O₂ signal generated in the apoplast.However,the way in which harpins are recognized in the pathway and what role the apoplastic H₂O₂ plays in plant defenses are unclear.Here,we examine whether the extracellular localization of Hpal impacts H₂O₂productionand pathogen resistance in Arabidopsis thaliana.Transformation with the hpal gene and hpal fused to an apoplastic localization signal?shpal?generated hpaland shpal-expressing transgenic A.thaliana?HETAt and SHETAt?plants,respectively.Hpal was associated with the apoplast in SHETAt plants but localized inside the cell in HETAt plants.In addition,Hpal localization accompanied H₂O₂ accumulation in both the apoplast and cytoplasm of SHETAt plants but only in the cytoplasm of HETAt plants.After being applied to the wild-type plant,Hpal localized to apoplasts and stimulated H₂O₂ production as in SHETAt plants.In both plants,inhibiting apoplastic H₂O₂ generation abrogated both cytoplasmic H₂O₂ accumulation and plant resistance to bacterial pathogens.These results suggest the possibility that the apoplastic H₂O₂ is subject to a cytoplasmic translocation for participation in the pathogen defense.We proved that Hpal can interact with AtPIP1;4 and OsPIP1;3?unpublished?.The relationship among Hpa1,AtPIP1;4/OsPIP1;3 and H₂O₂is meaningful.We also have carried out the preliminary study on Hpal-OsPIPl;3 interaction domain.