Functional Research Of Ethylene Response Factor ERF4/ERF72 Involved In Iron Deficiency Response Of Apple Rootstocks

China’s apple cultivation area and production rank first in the world.In the north of our country,the influence of calcareous soil is often encountered,which leads to the decrease of the quality and yield of apple,and it seriously restricted the development of apple industry.Therefore,it is more and more important to explore the regulation of iron in plants,especially in the fruit trees.Over the years,many researches have carried on the mechanism of plant resistance to iron deficiency,including physiological and biochemical,functional gene cloning,and the research of signal transduction has been carried out.It is also known that ethylene signaling is involved in the regulation of iron deficiency response in plants.However,there is no report on how the ERF family is involved in the response to iron deficiency.In this study,we used the apple rootstocks with different iron tolerance under iron deficiency and exogenous reagent treatment,to investigate the function of transcription factor ERF4 and ERF72 in the ethylene signal transduction induced by iron deficiency and in comparing the differences between different rootstocks.The main results are as follow:Ethylene was produced in roots of Malus xiaojinensis Cheng et Jiang and Malus baccata Borkh under early iron deficiency,but with the increase of iron deficiency time,the ethylene production in M.xiaojinensis root decreased,and the ethylene accumulation of M.baccata root cells increasedThe response patterns in different iron deficiency tolerant apple rootstocks have been showed differently under different exogenous ethylene treatments.The additional ethylene inhibitor AVG(0 μM Fe + 10 μM AVG)can promote M.xiaojinensis chlorosis symptoms to be increased,and the chlorophyll content to be decreased significantly.Additional ethephon(40 μM Fe + 100 mg/L ACP)did not make the phenotype and chlorophyll content of plants to be significantly changed.At the same time,exogenous ethylene made other iron absorption parameters such as root ferric reductase(FCR)activity,rhizosphere pH value,root hair zone H+ velocity,the iron transporter gene IRT1 and hydrogen excretion gene HA2 expression and active Fe content in roots and leaves of M.xiaojinensis to be significantly influenced.But the iron deficiency response of M.baccata was not obvious by ethylene action.Transcription factor ERF4 and ERF72 were selected as the key research object through the iron deficiency test of M.xiaojinensis and M.baccata.The expression of MxERF4 and MxERF72 decreased during the first 6 d under iron deficiency in M.xiaojinensis,and MxERF4 and MxERF72 expression significantly increased in response to Fe deficiency,compared with Fe sufficiency on the ninth day.The expression patterns of genes(MxEIN3,MxEIL1,and MxFIT)which play an important role in positive regulation of iron deficiency and iron uptake related genes(MxIRTl and MxHA2)were opposite to that of MxERF4.The inhibition of transcriptional activation of MxERF4 was realized by the amino acid C terminal EAR-motif(LDLNL)inhibitory domain by yeast one-hybrid.The promoters of MxIRTl and MxHA2 was inhibited in the tobacco leaves by coinfiltrated with agrobacterium containing MxERF4 and MxFIT.Yeast two-hybrid and bimolecular fluorescence complementation(BiFC)assays showed that MxERF4 could interact with MxFIT and MxIR02.Furthermore,the positive regulation effect of these two transcription factors on MxIRT1 and MxHA2 were inhibited.This study displayed that MxIRTl and MxHA2 expression,active iron content and H+ efflux in M.xiaojinensis root were increased significantly after silencing MxERF4 by VIGS.This study also found that MxERF72 could interact with MxERF4,MxFIT and MxIR02.MxERF72 could not only directly bind to MxHA2 promoter,but alsoit inhibited the expression of MxHA2 by MxFIT.ERF4 and ERF72 genes in M.xiaojinensis and M.baccata were silenced by VIGS,rhizosphere pH value in M.baccata was significantly lower than in M.xiaojinensis.The effect of iron absorption in interference ERF4 treatment group was more significant than that of ERF72 treatment group.This study also found that iron deficiency produces ethylene and it can alleviate the disintegrate of M.xiaojinensis chloroplast,while MxERF4 and MxERF72 could effect chlorophyll content.The phenotypic,physiological and molecular studies of Arabidopsis mutants revealed that ERF4 and ERF72 play an important role in the response to iron deficiency.We proposed that ethylene induced by iron deficiency lead to the increase of the expression of AtERF4 and AtERF72.AtERF4 negatively regulated iron deficiency response by combining AtIRT1 and AtCLHl promoter,and AtERF72 negatively regulated AtIRTl,AtHA2 and AtCLH1 expression.