Cadmium-Induced DNA Demethylation Mediates Rice Cadmium Tolerance By OsHMT And OsCTF

With the rapid development of modern industry and urbanization,as well as overuse of fertilizers and pesticides,soil pollution has become seriously environmental problems.One of the major contaminants in soil is the accumulation of heavy metals such cadmium(Cd)in crops and through food chains to strenten human health.Thus,it is imperative to develop strategies and estabolish effective methodology to lower accumulation of toxic metals in crops.The first step is to investigate the the mechanism for heavy metal uptake,translocation and accumulation in crops.In this study,we investigated Cd accumulation,detoxification and regulatory mechanisms in rice plants using multiple approches including biochemistry,molecular biology,physiology,genetics and high-throughput sequencing.The goal of the study is to uncover a mechanism underlying plant resistance or tolerance to the toxic metal Cd.We first constructed DNA methylated and transcriptome libraries in Cd-exposed rice plants.Using the bisulfate-sequencing(Bis-seq),we developed and presented the first genome-wide single-base-resolution maps of DNA methylation marks in Cd-exposed rice.Widespread differences were identified in CG and non-CG methylation marks between the Cd-exposed and Cd-free(control)rice.The major result we found is that Cd exposure is able to induce the genome-wide demethylation at CG Transcription of most of DNA methyltransferases,histone methyltransferases and DNA demethylase was differentially affected by Cd.In some special regions such as upstream,genebody and downstream of genes,we found more genes hypermethylated than those hypomethylated under Cd exposure.By cross analysis of these loci that were differentially methylated and differentially expressed under Cd stress,a set of genes such as for metal transporters and antioxidance were identified.A stringent group of 2092 genes(p<0.05,1.5-fold change)with a strong preference of differential expression in Cd-exposed rice was identified.Some of the genes were validated in a subset of loss of function mutants defective in DNA methylation/demethylation.Supply of 5-azacytidine(Aza,inhibitor of DNA methylation)promoted rice growth and reduced Cd content.These results indicate that Cd-induced DNA demethylation could be responsible for tolerance of rice to Cd toxicity.To specify the regulatory mechanism,we functionally characterized two representitive genes encoding HMT(heavy metal transport/detoxification protein)and OsCTF(Cadmium tolerance factor).OsHMT is constitutively expressed in many tissues of rice,but the higher expression level was detected in roots and sheaths of rice.Subcellular localization analysis reveals that OsHMT is mainly located in cytoplasm.Expression of OsHMT in yeast cells enhanced the tolerance of Cd and Nn,leading to increased accumulation of Cd and decreased concentration of Mn in the yeast cells.OsHMT overexpression in rice resulted in accumulating more Cd or Mn but promoting tolerance to Cd and Mn,while knockout of OsHMT led to a significant reduction in growth under Cd stress and toxic Mn concentrations in rice.A in vitro assay using metal-chelate affinity chromatography demonstrated that OsHMT proteins can be directly bound to Cd(?)and Mn(?),and the binding activity is independent of glutathione(GSH).Finally,we identified the correlation between DNA demethylation and transcript levels of OsHMT under Cd stress.We detected a lower level of methylation marks at the promoter region of OsHMT.We also found a lower level of H3K9me2(for histome methylation)at the region compared to control.These DNA demethylation marks were validated by a group of epigenic mutants of OsMETl,SDG714 and OsROSl in which expression of OsHMT was enhanced while the corresponding methylation levels were reduced.Overall,these results indicate that Cd-induced demethylation at the upstream of OsHMT should be responsible for rice tolerance to Cd.To validate the mechanism by which OsHMT was transcriptionally regulated,we further characterized another metal transporter gene OsCTF.Like OsHMT,OsCTF is constitutively expressed in many tissues of rice,but localized at the plasma membrane.Expression of OsCTF in yeast shows Cd(?)efflux,leading to a decrease in the content of Cd in yeast.Knockout and RNA interference of OsCTF resulted in a Cd sensitive phenotype.Complementation of OsCTF into the Osctf mutant can restore the phenotype.Inductively coupled plasma-atomic emission spectrometry(ICP-AES)(for metal determination)analysis shows that Osctf mutant accumulated more Cd in shoots and roots.We also identified the DNA methylation and transcript levels of OsCTF in rice plants when exposed to Cd,and found both DNA and histome demethylation(H3K9me2)at promoter and down-stream regions.Several mutants defective in OsMET1,SDG714 and OsDRM2 were used and found to be increased transcripts of OsHMT in these mutants.