Functional Analysis Of ERF5 Gene Responding To Cadmium Stress In Arabidopsis

Heavy mental cadmium is toxic to organisms,cadmium not only slowing the growth of plants,but also can accumulate with the delivery of food chains,bringing irreversible damage to animals.Our experiment found that Arabidopsis AtERF5 is participate in regulating plant response to cadmium stress,the potential molecular mechanism of AtERF5 response to cadmium stress was also discussed.The main results are as follows:1.Loss-of-function mutant of AtERF5 gene,erf5-1,was tolerant to heavy metal cadmium;using AtERF5 gene to complement the mutant erf5-1,the tolerant phenotype disappeared;and the AtERF5 overexpression plants are sensitive to cadmium stress.Based on phenotypic results,we conclude that AtERF5 gene negatively regulates plant responds to cadmium stress.2.AtERF5 was found located in nucleus by tobacco subcellular localization;the expression level in root is the highest in Arabidopsis by Pro:ERF5-GUS plants GUS staining.3.Under cadmium treatment,cadmium content in roots and shoots of wild type(WT),erf5-1,ERF5OE2-2 and ERF5OE3-4 plants was measured.It was found that the cadmium content in erf5-1 mutants is the highest,and the cadmium content in ERF5OE2-2 and ERF5OE3-4 overexpression plants is the lowest,indicating that AtERF5 may mainly regulate plant response to cadmium stress through cadmium chelation pathway.4.Treating WT and erf5-1 mutant with buthionine sulfoximine(BSO)and cadmium at the same time,the cadmium tolerance phenotype of erf5-1 mutant disappeared.The glutathione(GSH)/ phytochelatin(PC)content in roots of WT?erf5-1?ERF5OE2-2 and ERF5OE3-4 plants was also measured,it was found that after cadmium treatment,the content of GSH decreased while the content of PC increased,among which,the erf5-1 mutant has the highest content of PC,while ERF5OE2-2 and ERF5OE3-4 overexpression has the lowest content of PC.The results indicating that AtERF5 responds to cadmium stress though the GSH dependent PC synthesis pathway.5.Under cadmium treatment,the expression level of GSH1?GSH2?PCS1 and PCS2 in roots of WT?erf5-1?ERF5OE2-2 and ERF5OE3-4 plants was measured.It was found that the expression level in erf5-1 mutants is the highest,and the expression level in ERF5OE2-2 and ERF5OE3-4 overexpression plants is the lowest.It was concluded that AtERF5 inhibited the expression of GSH/PC synthesis genes,thereby negatively regulating the GSH-dependent PC synthesis pathway.6.AtERF5 could inhibit the transcription activity of GSH1,GSH2,PCS1 and PCS2 by tobacco transient expression system analysis;AtERF5 was found can directly bind to the promoter of GSH1,GSH2 and PCS2 by ChIP-qPCR,thereby inhibiting the transcriptional activity of GSH1,GSH2 and PCS2.In summary,the transcription factor AtERF5 could directly bind to the promoter of GSH/PC biosynthesis pathway related genes GSH1,GSH2 and PCS2,inhibit the expression of GSH/PC biosynthesis pathway related genes,and thus negatively regulate the accumulation and tolerance to cadmium in Arabidopsis.