Role Of DNA Demethylation In Response To Cadmium Stress In Arabidopsis Plants

Prevention of cadmium accumulation in crops by proper measures is considered as a promising strategy for safe crop production in the soils with slight or moderate Cd contamination.The mechanism of Cd uptake and accumulation in plants is an important theoretical basis for ensuring a successful application of above strategy.Recent studies showed that epigenetic modifications,particularly DNA methylation,may play an important role in plant response to Cd.The homeostasis and pattern of DNA methylation in plants are mainly regulated by DNA methylation and DNA demethylation.However,the change of genomic DNA methylation pattern in response to Cd stress is largely unknown.Furthermore,whether the Cd-altered DNA methylation acts a role in regulating Cd tolerance and Cd accumulation in plants also remains unclear.Therefore,in this study,the Arabidopsis was used as a test plant to investigate the above two questions.The main findings are summarized as follows:The whole-genome bisulfate sequencing?WGBS?was used to investigate the dynamic changes in pattern of global DNA methylation upon Cd stress in Columbia ecotype?Col-0?Arabidopsis plants.After Cd treatment,the average global DNA methylation was increased,while the methylation at CHG and CHH is more sensitive in comparison with the methylation at CG.By using a sliding window approach,5,806 differentially methylated regions?DMRs?in the genome was identified in response to Cd stress.Moreover,all the DMRs were CHG and CHH DMRs.After classifying all of the DMRs into different genomic categories,most of them were found to be overlapped with the transposable elements?TEs?,similar to that of TEs distribution in the genome,indicating that Cd exposure increased DNA methylation,especially in CHG and CHH and TEs.Furthermore,the effect of Cd stress on the expression level of genes involved in epigenetic process was investigated using RNA-sequencing and RT-q PCR.We found that three demethylase genes?ROS1,DML2,and DML3?were strongly repressed by Cd.Therefore,the Cd-induced DMRs in Col-0 and rdd triple mutants,which loss the functions of ROS1,DML2,and DML3,were compared by a complete linkage clustering analysis,principal component analysis,and heatmaps of the correlation coefficient.The results showed that the DNA methylation profiles of roots were very similar between the Cd-exposed Col-0 plants and rdd mutants from either Cd-free or Cd-exposed treatment.These results suggested that the Cd-induced DMRs are likely associated with the inhibition of ROS1/DML2/DML3-mediated DNA demethylation.Col-0 and rdd mutants were then used to investigate a role of DNA demethylation in Cd tolerance.Upon the treatment with Cd concentration at 20?M or 40?M,the rdd plants had better growth than those of Col-0,whereas the application of DNA methylation inhibitor 5-aza minimized the difference in the growth between Col-0 and rdd plants.This result indicated that Cd-induced DNA hypermethylation may favor Cd tolerance.We also analyzed the functional redundancies of ROS1,DML2,and DML3 in the plant response to Cd stress.Upon 40??M Cd exposure,the root length of ros1 single mutant,ros1/dml2,dml2/dml3,and ros1/dml3 double mutants was longer than that of Col-0,but was still shorter than that of rdd triple mutant,while the root length of dml2 and dml3 single mutants was similar to that of Col-0 plant.These results suggested that ROS1,DML2,and DML3 play partially redundant role in negatively regulating the plant tolerance to Cd stress,with the highest contribution from ROS1.By analyzing the Cd in plant tissues,we found that the rdd mutant had a significantly lower Cd concentration in the roots than that in Col-0 plant,although no difference in the shoot Cd concentration was observed between those two lines.In contrast,the Col-0 plants treated with DNA methylation inhibitor 5-aza had higher Cd concentration in their roots than that in plants not treated with the inhibitor,although the inhibitor had little effect on the shoot Cd concentration.These results suggested that an elevation in DNA methylation facilitate to prevent higher Cd accumulation in root tissues.The net flux of Cd²⁺was measured using a non-invasive microelectrode ion flux measurement system.We found that there were no statistically significant differences in the net Cd²⁺influx rate between rdd mutant and Col-0 plant.5-aza treatment also had little effect on the net Cd²⁺influx rate.These results indicated that changes in DNA methylation had little impact on Cd uptake in the roots.Therefore,the negative effect of DNA methylation on root Cd accumulation is not associated with an alteration in root Cd uptake,but is probably attributed to a dilution effect resulted from more biomass production due to greater tolerance against Cd stress.Based on above results,we next investigated the mechanism of the negative effect of ROS1,DML2,and DML3 on plant Cd tolerance.The transcriptome sequencing showed that there was a significant difference of the expression pattern of the genes involved in the process of cellular response to Fe starvation after Cd treatment between Col-0 and rdd.Among the process,eight genes,b HLH38,b HLH100,b HLH101,PYE,MTP3,IREG1,IREG2,and IRT1,have been characterized to be involved in Fe uptake,trafficking,and homeostasis.The expression levels of the above genes were higher in the roots of rdd than those of Col-0 plants.In addition,a higher Fe concentration was observed in the roots of rdd in comparison to Col-0,suggesting that an elevation in DNA methylation might be associated with an improved Fe status in the roots.Furthermore,limited Fe supply?1?M Fe?which minimized the difference in Fe status between Col-0 and rdd plants,also minimized the difference in the Cd tolerance between these two plant lines.Above results provide evidence for the notion that the improved Fe status in roots might be responsible for the enhanced Cd tolerance due to an elevation in DNA methylation.In conclusion,Cd stress resulted in an increase in global genome-wide DNA methylation in the roots by inhibiting the expression of DNA demethylase genes ROS1/DML2/DML3.This improved plant Fe nutrition by upregulating the genes related to Fe uptake,trafficking,and homeostasis,resulting in an improvement in Cd tolerance in the plants.