Understanding The Molecular Mechanism Of The Natural Cell Death Of Clc2clc3 Double Mutant In Arabidopsis

In plants,clathrin-dependent membrane trafficking is critical for a numerous biological processes.Clathrin in Arabidopsis consists of three light chain subunits(CLC1,CLC2 and CLC3)and two heavy chain subunits(CHC1 and CLC2).Natural cell death was observed on the leaves of clc2clc3 mutant Accompanying the cell death,the Pathogenesis-related(PR)gene induction,MAPK activation and enhanced resistance to Pseudomonas syringae DC3000 were also observed in clc2clc3 mutant.However,the molecular mechanism of the natural cell death and enhanced resistance is not understood.As both salicylic acid(SA)and hydrogen peroxide(H2O2)have been implicated in cell death and defense responses,we determine to investigate the roles of these two molecules in the cell death and activated defense responses in clc2clc3 mutant using genetic approach.Isochorismate Synthase 1(ICS1)plays an important role in SA biosynthesis of salicylic acid and it accounts for over 90%pathogen-induced SA production.Pathogen-induced production of H2O2 is catalyzed by plasma-membrane(PM)-anchored respiratory burst oxidase homologue(Rboh)protein complex,which is also called NADPH oxidase.NADPH oxidase consists of ten members,among which RbohD plays a key role in production of H2O2.Therefore,we generated sid2clc2clc3 and rbohDclc2clc3 triple mutants by crossing to examine the effects of SA or H2O2 production on the the natural cell death and activated defense responses observed in clc2clc3 mutant.Interestingly,we found that the natural cell death and activated defense responses of clc2clc3 was almost fully reversed by either ICS1 or RbohD mutation.In addition,DAB staining indicated that the significantly higher H2O2 level in clc2clc3 double mutant was restored to WT level in rbohDclc2clc3 triple mutant Together,our results indicated that clathin plays negative roles in production of both SA and H2O2 and the misregulated levels of SA and H2O2 are responsible for the cell death and activated defense resposnse observed in clc2clc3 double mutant.