The Methylation Patterns Of Acid Invertase Gene Promoters From Cu-tolerant And Non-tolerant Populations Of Elsholtzia Haichowensis Under Copper Stress

Although metal toxicity restricts the growth of sensitive species,some plant species called metallophytes have developed tolerance capabilities which enable them to survive and thrive in metal-enriched soils.Elsholtzia haichowensis Sun is a well-known indicator of Cu mines,and its metallicolous population(MP)and non-metallicolous population(NMP)were discovered in the areas along the middle and lower streams of Yangtze River in China.In our previous studies,acid invertases(including vacuolar invertase and cell wall invertase)were found to be crucial for metallophyte plants to govern root growth and root/shoot biomass allocation,and at least partly associated with Cu tolerance by supplying carbon and energy for tolerance mechanisms.These results suggested that the varied enzyme activities and gene transcription between the contrasting populations of the metallophytes under Cu stress were not relevant to the amino acid divergences in the proteins,but some other mechanisms,such as cis-regulatory divergences(genetic and epigenetic)in promoters might be involved.DNA methylation is one of the important epigenetic regulations in all organisms,which play a central role in gene expression.DNAmethylation in promoter region is mainly associated with transcriptional repression and is highly tissue-specific in plants.Environmental stimuli,such as heavy metals and salt stress,are known to affect the extent of DNA methylation throughout the genome and at specific loci by their interaction with the plant.As investigations into DNA methylation under heavy metals stress have relied on the use of methylation-sensitive restriction enzymes and detailed information on the methylation status of each cytosine is lacking,bisulphite sequencing is a preferable method to provide a single-base resolution methylation map of selected promoters.In this study,we attempted to clarify whether Cu stress contribute to the distinction of DNA methylation levels and patterns in the promoters of acid invertase genes between Cu-tolerant and non-tolerant populations of E.haichowensis,thus to detect if any epigenetic mechanisms could underlie the gene expression.The main results in this study are as follows:NMP and MP plants of E.haichowensis both displayed high methylation levels at CG sites within the vINV promoter in the controls and the Cu treatments.However,methylation levels at CHG and CHH sties were low in both populations.No significant difference of methylation levels was detected between the populations for all contexts.The vINV promoter contained 18 CGs,21 CHGs and 217 CHHs.15 out of 18 CG sites were all hypermethylated and located from 1484 to 1000 bp upstream of the ATG start codon,including a CG-rich region.A special CHG site located at 1061bp upstream of the ATG start codon displayed significant difference in methylation state between two populations under Cu stress compared with control.No significant difference of overall methylation levels occurred in cwINV promoter between two populations without Cu treatment.Three types of cytosine methylation levels all decreased slightly under Cu treatment for both populations,except CHH type of NMP which did not change much.The cwINV promoter contained 25 CGs,45 CHGs and 222 CHHs.All CG sites as well as most CHG and CHH sites within cwINVpromoter were hypomethylated in NMP and MP plants.Remarkably,several CHH sites located at-1478,-1447,-1256,-1181,-1068,-842 bp of promoter region displayed discrepancy of methylation state between two populations.Four of these differential methylated CHH sites were within or in the vicinity of several predicted cis-regulatory elements in the cwINV promoter,including UTR Py-rich stretch,Skn-1_motif,CARE-motif and TATC-box.CG methylation was relatively conserved in the vINV and cwINV promoter under Cu stress,while methylation at CHG and CHH sites was more prone to perturbation by Cu stress.Interestingly,under Cu stress,methylation of these individual sites seemed to maintain stable in MP,while it declined or increased drastically in NMP.This implied that MP might have evolved modulating mechanisms due to long-term Cu stress including the change of methylation pattern in specific DNA sequences and cytosine sites.