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High-temperature effect on genes engaged in DNA methylation Arabidopsis

Research article

High-temperature effect on genes engaged in DNA methylation and affected by DNA methylation in Arabidopsis

Mladen Naydenov a ,Vesselin Baev a ,Elena Apostolova a ,Nadezhda Gospodinova a ,Gaurav Sablok b ,Mariyana Gozmanova a ,Galina Yahubyan a ,*

a Department of Plant Physiology and Molecular Biology,University of Plovdiv,24Tsar Assen St,4000Plovdiv,Bulgaria b

Department of Biodiversity and Molecular Ecology,Fondazione Edmund Mach,IASMA,San Michele 38010,Italy

a r t i c l e i n f o

Article history:

Received 2October 2014Accepted 31December 2014Available online 1January 2015Keywords:Heat stress

DNA methylation Gene expression PolIV PolV

a b s t r a c t

Along with its essential role in the maintenance of genome integrity,DNA methylation takes part in regulation of genes which are important for plant development and stress response.In plants,DNA methylation process can be directed by small RNAs in process known as RNA-directed DNA methylation (RdDM)involving two plant-speci ?c RNA polymerases e PolIV and PolV.The aim of the present study was to investigate the effect of heat stress on the expression of genes encoding key players in DNA methylation e DNA methyltransferase (MET1,CMT3,and DRM2),the largest subunits of PoIIV and PolV (NRPD1and NRPE1respectively)and the DNA demethylase ROS1.We also examined the high-temperature effect on two protein-coding genes e At3g50770and At5g43260whose promoters contain transposon insertions and are affected by DNA-methylation,as well as on the AtSN1,a SINE-like retrotransposon.To assess the involvement of PolIV and PolV in heat stress response,the promoter methylation status and transcript levels of these genes were compared between wild type and double mutant lacking NRPD1and NRPE1.The results demonstrate coordinated up-regulation of the DRM2,NRPD1and NRPE1in response to high temperature and suggest that PolIV and/or PolV might be required for the induction of DRM2expression under heat stress.The ROS1expression was con ?rmed to be suppressed in the mutant lacking active PolIV and PolV that might be a consequence of abolished DNA methylation.The increased expression of At3g50770in response to elevated temperature correlated with reduced promoter DNA methylation,while the stress response of At5g43260did not show inverse correlation between promoter methylation and gene expression.Our results also imply that PolIV and/or PolV could regulate gene expression under stress conditions not only through RdDM but also by acting in other regulatory processes.

1.Introduction

Methylation of cytosine to methylcytosine is a common DNA modi ?cation for plants,animals and fungi.DNA methylation can generally be found in transposable elements (TEs)and repetitive elements as well as in gene bodies in Arabidopsis (Cokus et al.,2008;Lister et al.,2008).DNA methylation of TEs and repeats in-activates their transcription and is an evolutionary mechanism of defense against sel ?sh DNA.Gene-body methylation was found to correlate with high expression levels (Tran et al.,2005;Zemach et al.,2010;Zhang et al.,2006)or,alternatively,DNA methylation can de ?ne exons boundaries or regulate alternative splicing,

because it has been observed that exons are more highly methyl-ated than introns (Feng et al.,2010;Laurent et al.,2010).

DNA methylation occurs in three different sequence contexts:CG,CNG (where N is any base)and asymmetric CHH (where H ?A,T,or C),and is catalyzed by DNA methyltransferase enzymes (DNMT).They can either methylate DNA,establishing a new methylation state (de novo methylation),or maintain preexisting methylation (Feng et al.,2010).The maintenance of CG methylation relies on site symmetry and requires the highly conservative mammalian DNMT1homologue e Methylase 1(MET1)in Arabi-dopsis (Bestor and Verdine,1994;Cao and Jacobsen,2002b;Finnegan et al.,1996;Kishimoto et al.,2001).De novo methylation occurs in CNG and CHH context.Chromomethylase 3(CMT3)is a chromodomain containing plant speci ?c enzyme (Henikoff and Comai,1998)that is responsible for the CNG methylation and

*Corresponding author.

E-mail address:galina.yahubyan@https://www.wendangku.net/doc/3510060100.html, (G.

Yahubyan).Contents lists available at ScienceDirect

Plant Physiology and Biochemistry

journal h omepage:www.elsevier.co m/lo

cate/plaphy

https://www.wendangku.net/doc/3510060100.html,/10.1016/j.plaphy.2014.12.022

Plant Physiology and Biochemistry 87(2015)102e 108

reveals some locus-speci?c effects on asymmetric methylation (Bartee and Bender,2001;Cao and Jacobsen,2002a;Lindroth et al., 2001;McCallum et al.,2000).The de novo methytransferases e Domains Rearranged Methyltransferase DRM1and DRM2,are ho-mologues of the mammalians DNMT3and are responsible for asymmetric methylation In Arabidopsis(Cao et al.,2000).Similarly to CMT3,DRM methyltransferases have locus-speci?c effects on the CNG methylation(Cao and Jacobsen,2002a).In nondividing cells, CG methylation can be removed through active demethylation resulting from the action of the DNA glycosylase/lyases,Repressor Of Silencing1(ROS1)and Demeter(DME)(Choi et al.,2002; Kinoshita et al.,2004).

RNA-directed DNA methylation(RdDM)is a plant-speci?c DNA methylation pathway,in which short interfering RNA(siRNA) molecules guide cytosine de novo methylation(Law and Jacobsen, 2010).RNA-dependent RNA polymerase2(RDR2),Dicer-like3 (DCL3),Argonaute4(AGO4)and two types of DNA-dependent RNA polymerases e Polymerase IV(PolIV)and Polymerase V(PolV)are main components in this pathway.RDR2makes PolIV transcripts double-stranded and the endonuclease DCL3cleaves double-stranded RNAs to24nt siRNAs(Xie et al.,2004).In turn,AGO4,as a component of the RNA-induced silencing complex,binds siRNAs and targets cognate DNA sequence for methylation(Chan et al., 2005;Huettel et al.,2007).The plant speci?c PolIV and PolV are distinguished by their unique largest subunits,NRPD1and NRPE1, and act at different steps and loci of the RdDM pathway.While PolIV is needed to produce or amplify the siRNA trigger(Smith et al.,2007),PolV generally acts downstream of this step to facili-tate de novo methylation at the siRNA-targeted site(Kanno et al., 2005;Pontier et al.,2005).RdDM may be passively lost in dividing cells if siRNA signal is withdrawn(Chan et al.,2005).

Along with its essential role in the maintenance of genome integrity through silencing transposons and harmful DNA,DNA methylation takes also part in regulation of genes that are impor-tant for plant physiology and development(Bartee and Bender, 2001;McCallum et al.,2000).Environmental stress can induce chromatin structure changes like histone modi?cations and DNA methylation/demethylation.In maize roots,cold stress leads to a reduction of methylation in the DNA of the nucleosome's cores which correlates with increased expression of ZmMI1which con-tains part of the coding region of a putative protein and part of a retrotransposon-like sequence(Steward et al.,2000).Changes in the ambient conditions may also activate transposons which are normally inactivated by epigenetic silencing through methylation of their promoters(Grandbastien,2004).Heat stress mobilizes ONSEN retrotransposons,making them transcriptionally active,so they synthesize extrachromosomal DNA copies in Arabidopsis seedlings(Ito et al.,2011).Cold stress results in hypomethylation and transposition of Tam-3transposon in Antirrhinum majus (Hashida et al.,2006).Drought and salt stress induced CNG hypermethylation of the satellite DNA in the facultative halophyte Mesembryanthemum crystallinum that in turn leads to a metabolic switch from C3to CAM photosynthesis mode(Dyachenko et al., 2006).

It has been shown that differential expression of endogenes and transgenes in response to stress can be regulated by RdDM of TEs which reside in promoter regions(Grandbastien,2004;Kashkush et al.,2003;Steward et al.,2002).Recently,it was reported that read-through transcription from transposon remnants result both in up-and down-regulation of adjacent protein-coding genes e the pentatricopeptide repeat(PPR)-containing protein gene and several auxin-responsive genes(Popova et al.,2013).In our previ-ous study,we determined that the promoter regions of81abiotic stress-responsive genes contain TE insertions,which are potential targets for24nt siRNAs and DNA methylation in Arabidopsis(Baev et al.,2010).In the present study,we examined the high-temperature effect on expression of key DNA-methylation related genes,and on promoter methylation status and transcript levels of two protein-coding genes,At3g50770and At5g43260,which are under control of promoter regions containing TE insertions(Baev et al.,2010).The AtSN1was chosen in order to test more general effect of elevated temperature on DNA methylation and expression of repeated endogenous DNA.To assess the involvement of the plant-speci?c RNA polymerases in stress response,the transcript levels of these genes and the methylation status of their promoter regions were compared between the wild type(Col-0)and the nrpd1a-1nrpd1b-1double mutant that lacks the largest subunits of PolIV(NRPD1)and Pol V(NRPE1).

2.Materials and methods

2.1.Plant material and stress treatment

Seeds of Arabidopsis thaliana(Col-0),wild type and the nrpd1a-1 nrpd1b-1double mutant provided by https://www.wendangku.net/doc/3510060100.html,grange(Pontier et al., 2005)were planted on soil and strati?ed at4 C for4days.Seed-lings were grown in a Percival chamber at21 C,16h light(100 umol mà2sà1)/8h dark for5weeks.For high-temperature treat-ment,plants were transferred to36 C.Plant material(rosette leaves)of four plants was taken at different time points e0,6,24 and48h after the beginning of stress experiment.The experiment was performed three times with nearly identical results.

2.2.Total RNA and DNA extraction

Total RNA was extracted using RNeasy Plant Mini Kit(Qiagen) and treated with DNase(Qiagen)according to the manufacturer's instructions.RNA samples from all time points were used for cDNA synthesis and qRT-PCR expression analysis.Total DNA was extrac-ted using DNeasy Plant Mini Kit(Qiagen)according to the manu-facturer's instructions.DNA samples from all time points were digested with McrBC and used for methylation-sensitive qPCR. 2.3.qRT-PCR

cDNA synthesis:1m g of RNA was reverse transcribed with RevertAid reverse Transcriptase(Thermo Scienti?c)under following conditions:65 C for5min,4 C for5min;42 C for 60min;?nal inactivation at70 C for10min.

Ampli?cation:PCR ampli?cation was performed using a stan-dard SYBR Green protocol(Fermentas)on7500Real-time PCR machine(Applied Biosystems).All reactions were carried out in a total volume of25m l and contained5m l of diluted cDNA,1.5m l of primer mix to?nal concentration0.6m M,4.5m l of Nuclease-free water and12.5m l of SYBR Green mix with ROX.PCR conditions were as follows:50 C for2min,95 C for10min,followed by40 ampli?cation cycles of95 C for15s and60 C for1min.All re-actions were performed in triplicates.The housekeeping gene-EF1a was used as endogenous control for normalization (Czechowski et al.,2005),and the untreated sample(0h of treat-ment)was accepted as reference(RQ j1).Ct values were calculated using7500software v.2.0.1(ABI).Relative quantitation of gene expression(RQ)was determined with the equation:E to the power of e D Ct of gene of interest/E to the power ofàD Ct of housekeeping gene,where E is the primer ef?ciency measured by standard curve experiment with serial dilutions and D Ct is the difference between the Ct values of target gene for each sample and for the reference sample.All primer sequences are given in Supplemental?le1.

M.Naydenov et al./Plant Physiology and Biochemistry87(2015)102e108103

2.4.DNA digestion and methylation-sensitive qPCR

DNA digestion :40ng of genomic DNA were digested with McrBC (NEB),which recognizes and cleaves DNA containing at least two 5-methylcytosines preceded by a purine and located in one of CG,CNG,and CNN sequence contexts.McrBC digestion was performed at 37 C for 16h,according to the manufacturer's conditions.In mock sample,the incubation was performed with 1m l of 50%Glycerol instead of enzyme.

Ampli ?cation :PCR ampli ?cation was performed using a stan-dard SYBR Green protocol (Fermentas)on StepOnePlus Real-time PCR machine (Applied Biosystems).All reactions were performed in a total volume of 15m l and contained 50pg of DNA (digested or undigested),0.9m l of primer mix (to ?nal concentration 0.6m M),4.5of Nuclease-free water and 7.5m l of SYBR Green with ROX.PCR conditions were as follows:50 C for 2min,95 C for 10min,fol-lowed by 40ampli ?cation cycles of 95 C for 15s and 60 C for 1min.All reactions were performed in triplicates.The undigested (mock)sample for each time point was used as reference sample.Relative quantitation of methylation (RQ)was calculated using the StepOnePlus (ABI)Ct Values and the equation:E to the power of e D Ct of gene of interest/E to the power of àD Ct of endogenous control,where E is the primer ef ?ciency measured by standard curve experiment with serial dilutions and D Ct is the difference between the Ct values for mock sample and for digested sample.

Primer pairs were designed to ?ank the TE of At3g50770pro-moter and each of the two TEs of the At5g43260promoter (Figs.3A and 4A,Supplemental File 1),and the primers for AtSN1methyl-ation were according (Hamilton et al.,2002).

2.5.Software and web based analysis tools

Masking of TEs in analyzed promoter regions was performed using the software starPRO DB v1.0http://bioinfo1.uni-plovdiv.bg/cgi-bin/starpro/(Baev et al.,2010)and Arabidopsis epigenome map.Sequence data were retrieved from The Arabidopsis Infor-mation Resource (TAIR,https://www.wendangku.net/doc/3510060100.html, ).Primers were designed using the Geneious.Pro.v4.8.5software and https://www.wendangku.net/doc/3510060100.html, based tool Primer-BLAST.

3.Results

3.1.High-temperature response of key genes engaged in DNA methylation

To explore the effect of high temperature on key genes engaged in DNA methylation,we analyzed the expression levels of several genes closely related to this process:the DNA methyltransferase genes e MET1,CMT3,and DRM2,the genes encoding the largest subunits of Polymerase IV and V (NRPD1and NRPE1)and the gene encoding ROS1DNA demethylase.mRNA steady-state abundance was measured during high-temperature treatment (0h,6h,24h and 48h)by RT-qPCR (Fig.1).Expression analysis revealed that the DNA methylation-related genes responded to elevated temperature speci ?cally in the wild type:(1)the expression of DRM2,NRPD1and NRPE1,and to a lower extent that of ROS1,was stably up-regulated in the course of high-temperature treatment;(2)the transcript levels of MET1and CMT3were increased during the ?rst 6h of treatment but the longer treatment resulted in decreased transcript levels compared to untreated sample.

In the nrpd1a-1nrpd1b-1double mutant,

high-temperature

Fig.1.High-temperature response of DNA-methylation related genes.Expression pattern of Methylase1(MET1),(Chromomethylase 3)CMT3,Domains Rearranged Methyl-transferase 2(DRM2),the largest subunit of Polymerase IV (NRPD1)and of Polymerase V (NRPE1)and Repressor Of Silencing 1(ROS1)was analyzed by qRT-PCR.Expression is represented as fold change of expression relative to non-stressed controls.Representative experiment from 3independent replicates is shown.Error bars indicate SD of

replicates.

Fig.2.Response of the retrotransposon AtSN1to high-temperature stress in wild-type and nrpd1a-1nrpd1b-1mutant.(A)Promoter analysis:relative DNA methylation was assayed by qPCR of undigested and McrBC-digested DNA.(B)Transcript analysis:relative expression levels were assayed by qRT-PCR.Expression levels were normalized to non-stressed controls.Representative experiment from 3independent replicates is shown.Error bars indicate SD of replicates.

M.Naydenov et al./Plant Physiology and Biochemistry 87(2015)102e 108

104

treatment resulted in expression pro ?les,which differed from those of wild type (Fig.1):(1)the strong stimulating effect of high temperature on DRM2and MET1expression that was observed in wild-type was strongly suppressed in the mutant;(2)the

ROS1

Fig.3.Response of the Calmodulin-like 41(CML41)encoding gene (At3g50770)to high-temperature stress in wild-type and nrpd1a-1nrpd1b-1mutant.(A)Gene structure e data were obtained from the Arabidopsis epigenome map and RepeatMasker Pre-masked Genomes.Arrows indicate the position of the primers used for the following qPCR and qRT-PCR.(B)Promoter analysis:relative DNA methylation was assayed by qPCR of undigested and McrBC-digested DNA.(C)Transcript analysis:relative expression levels were assayed by qRT-PCR.Expression levels were normalized to non-stressed controls.Representative experiment from 3independent replicates is shown.Error bars indicate SD of

replicates.

Fig.4.Response of At5g43260to high-temperature stress in wild-type and nrpd1a-1nrpd1b-1mutant.(A)Gene structure e data were obtained from the Arabidopsis epigenome map and RepeatMasker Pre-masked Genomes.Arrows indicate the position of the primers used for the following qPCR and qRT-PCR.(B)Promoter analysis:relative DNA methylation was assayed by qPCR of undigested and McrBC-digested DNA.(C)Transcript analysis:relative expression levels were assayed by qRT-PCR.Expression levels were normalized to non-stressed controls.Representative experiment from 3independent replicates is shown.Error bars indicate SD of replicates.

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expression was almost completely abolished in the mutant.

3.2.High-temperature response of genes affected by DNA-methylation

3.2.1.Response of the retrotransposon AtSN1

Previous?ndings have demonstrated that temperature stress changes the activity of LINE and copia-like retrotransposons in A.thaliana(Cavrak et al.,2014;Lang-Mladek et al.,2010;Matsunaga et al.,2012;Tittel-Elmer et al.,2010).Here,we tested the effect of high temperature on methylation and transcript levels of the SINE-like retrotransposon,AtSN1.Methylation sensitive-qPCR showed that temperature elevation resulted in enhanced DNA methylation of AtSN1with a peak at24h of treatment(Fig.2A).The AtSN1 transcript levels were down-regulated throughout the entire period of heat treatment(48h).The AtSN1methylation was strongly reduced in the double mutant compared to wild type due to the lack of active PolIV and PolV in the mutant.The AtSN1 transcript levels were down-regulated in the mutant compared to wild type before and up to24h of heat application,but high accumulation of AtSN1transcripts(over4times higher than that of untreated sample)was observed in the double mutant at48h of treatment(Fig.2B).

3.2.2.Response of the protein-coding genes At3g50770and

At5g43260

At3g50770encodes Calmodulin-like41(CML41)protein that was identi?ed as being induced by oligogalacturonides,oligosac-charides derived from the plant cell wall,and by the bacterial ?agellin peptide Flg22,general elicitors of the basal defense response in plants.(Denoux et al.,2008).The At3g50770promoter contains a DNA type TE insertion(At3TE76530),which lies very closely to the transcriptional start site(Fig.3A).We have previously reported that the DNA methylation of the TE region was RdDM dependent(Baev et al.,2010).Here,the exposure of Arabidopsis to high temperature resulted in a strong increase of At3g50770tran-script levels with a peak at the24h of treatment(over5.5times higher than those of untreated sample)(Fig.3C).The high-temperature pro?les of gene expression and promoter DNA methylation revealed that the enhanced expression was accom-panied by reduced promoter methylation levels(Fig.3B).The lack of PolIV and PolV resulted in highly reduced DNA methylation of the At3g50770promoter region in the double mutant compared to wild type(Fig.3B).

At5g43260encodes chaperon-like protein,there are two TE insertions in its promoter region which are located approximately at1200bp and1900bp upstream of the Transcriptional Start Site (TSS)and?ank a region massively targeted by siRNAs.The two TEs belong to the DNA TE families ATREP6and ATREP8and are RdDM dependent(Fig.4A).High temperature up-regulated the At5g43260 transcript levels,which reached their maximum at24h of treat-ment(over 2.5times higher than those of untreated sample) (Fig.4C).The methylation levels of the two TE-derived regions were higher compared to untreated sample during high-temperature treatment(Fig.4B,C).These?ndings did not show the expected inverse correlation between the levels of promoter methylation and transcript levels of At5g43260.The comparison of the wild type and double mutant showed that the methylation of the two TE insertions was highly reduced in the double mutant,while the transcript levels were unchanged when plants were grown at normal temperature(Fig.4C).So,it is dif?cult to see any direct relationship between methylation status of the two TE regions of the promoter and gene expression,and that could be due to the remote position of TE insertions from the TSS.4.Discussion

A number of studies have been devoted to the elucidation of impact of temperature stress on protein-coding gene expression,TE activity and heterochromatic silencing(Ito et al.,2011;Tittel-Elmer et al.,2010;Zeller et al.,2009),yet there is a limited documentation on the involvement of the genes encoding important components of DNA-methylation machinery in heat stress response.

4.1.Heat stress stimulates DRM2,NRPD1and NRPE1expression

One of the main?ndings of the present study was the heat-induced expression of DRM2,NRPD1and NRPE1,suggesting that the RdDM pathway was mobilized by high temperature to reinforce stabilization of DNA methylation.The simultaneous increase of ROS1and DRM2expression,which was observed in response to elevated temperature,could result in target-speci?c deposition and removal of DNA methylation.There are increasing evidences for DNA-methylation changes and altered expression of DNA-methylation related enzymes in response to abiotic and biotic stress(Bilichak et al.,2012;Boyko et al.,2010;Dowen et al.,2012; Ou et al.,2012).For example the rice orthologous genes of DRM2, DRM2-1and DRM2-2,were reported to be up-regulated by heavy metal stress(Ou et al.,2012).Stress-induced DNA methylation was shown to be impaired in dcl3de?ciency mutant(Boyko et al.,2010). Thus,the RdDM pathway,which can operate in all sequence context methylation and accounts for approximately30%of all DNA methylation in Arabidopsis(Cokus et al.,2008;Lister et al.,2008), seems to play an indispensable role in plant response to stress factors.

4.2.PolIV and/or PolV might be required for the induction of DRM2 expression under heat stress,and for ROS1expression under normal temperature

The coordinated expression of DRM2,NRPD1and NRPE1in response to high-temperature stress was clearly seen not only in wild type but also in the double mutant,in which the de?ciency of the largest subunits of PolIV and PolV repressed the stimulating effect of high temperature on DRM2expression.Previous study showed that some proteins of the RdDM pathway were mis-localized in PolIV-null mutants(Pontes et al.,2006)that indicated the importance of PolIV for the pathway stability(Haag and Pikaard,2011).In addition to that,our observation suggests that PolIV and/or PolV might be required for the induction of DRM2 expression under heat stress.The observed almost completely abolished ROS1expression in the double mutant con?rms the previous report for down-regulated ROS1transcript levels in the single mutants nrpd1,nrpe1and nrpd2(Huettel et al.,2006).Taken together,our mutant analysis comes in line with the assumption of Huettel and coworkers that plant speci?c RNA polymerases can cooperate with either DNA methyltransferases to catalyze cytosine methylation,or DNA glycosylases to catalyze cytosine demethylation.

4.3.RdDM machinery could interact with other factors to regulate AtSN1expression at elevated temperature

High temperature caused increased DNA methylation of AtSN1 that resulted in its transcriptional repression throughout the entire period of treatment(48h).The increased AtSN1methylation correlated with the up-regulated transcript levels of DRM2,NRPD1 and NRPE1(Figs.1and2).Taken together,these results reveal the importance of RdDM for A tSN1silencing in plants exposed to heat stress.Notably,expression analysis revealed increased AtSN1

M.Naydenov et al./Plant Physiology and Biochemistry87(2015)102e108 106

transcript levels in the double mutant after longer heat exposure that cannot be linked to DNA methylation(Fig.2).AtSN1dere-pression has been previously observed in several?owering mu-tants grown under normal temperature and,together with other TEs,was inked to RNA-mediated chromatin silencing(Baurle and Dean,2008).The AtSN1response reminds of the heat response of another TE e ONSEN,whose activation was linked to general loss of epigenetic control and required heat shock transcriptional factors (Cavrak et al.,2014).Likely,the RdDM machinery interacts with other factors(most likely such as chromatin remodelers or/and transcriptional factors)to regulate A tSN1expression at elevated temperature.

4.4.The increased expression of At3g50770in response to elevated temperature correlates with reduced promoter DNA methylation, and suggests that PolIV and/or PolV may regulate gene expression not only through RdDM but also by acting in other regulatory processes

The observed results indicate a negative correlation between the promoter methylation and transcript levels of At3g50770,and reveal a role of DNA methylation for transcriptional regulation of CML41in response to elevated temperature.Recently,using mu-tants lacking CG or non-CG methylation,it was demonstrated that DNA methylation modulated expression of some pathogen-responsive genes(Dowen et al.,2012).The lack of PolIV and PolV resulted in highly reduced DNA methylation of the At3g50770 promoter region in the double mutant compared to wild type (Fig.3B).We expected for the double mutant that the highly reduced promoter methylation would result in At3g50770reac-tivation under normal conditions and that effect would persist at elevated temperature.In fact,the At3g50770transcript levels did not differ signi?cantly between the wild type and double mutant when plants were grown under normal temperature conditions (Fig.3C).Interestingly,unlike the observed strong stimulating ef-fect of high temperature on At3g50770expression in wild type,the transcript levels were slightly altered by high temperature in the double mutant(Fig.3C)suggesting a role for PolIV and/or PolV in regulatory mechanism different than DNA-methylation.The At3g50770promoter reminds that of the FWA gene,which has tandem repeats that are targeted by siRNAs and RdDM,but in contrast to At3g50770,the FWA transgene silencing was released of nrpd1and nrpe1mutants(Pontier et al.,2005).Our?ndings indi-cate that the high-temperature dependent At3g50770up-regulation requires PolIV and/or PolV.It seems that these pro-teins take part not only in RdDM of the gene promoter but also in a regulatory process that could not directly be linked to promoter DNA methylation.

4.5.The At5g43260response to elevated temperature does not show inverse correlation between promoter methylation and gene expression but yet requires PolIV and/or PolV

Notably,the At5g43260expression pro?les differed signi?cantly between the wild type and double mutant treated with high tem-perature(Fig.4C)e the stimulating effect of high temperature on At5g43260expression in the wild type was completely abolished in the double mutant.This observation is similar to that we described above for At3g50770expression and supports the involvement of PolIV and/or PolV in high-temperature stress response of At5g43260through a regulatory mechanism distinct from pro-moter methylation.Since it has been shown that body methylation has only minor but positive effects on levels of gene expression (Zilberman et al.,2007),one can speculate that high temperature could alter methylation within coding regions(or body methylation)of these genes via PolIV or PolV-mediated mecha-nism.Besides,several studies of nrpe1mutants show that loss of PolV might have roles in heterochromatin organization and chro-matin remodeling pathways that are independent of RdDM pathway(Douet et al.,2009;Pontes et al.,2009).We found a number of cis-regulatory elements in the TE-related insertions of the promoters of the two genes.It could be possible that high-temperature induced changes of RdDM(and of related siRNAs) and chromatin structure alter the interactions between those ele-ments and speci?c regulatory factors.

5.Conclusions

The current study demonstrates that key genes of plant DNA methylation/demethylation pathway respond speci?cally to elevated temperature that may result in target-speci?c deposition and removal of DNA methylation.It provides supportive evidences for the involvement of plant speci?c RNA polymerases in plant stress response.The comparison of methylation and transcript levels of analyzed genes in the wild type and double mutant in-dicates that PolIV and/or PolV could regulate stress-responsive gene expression through RdDM and thus enlarges the list of genes affected by RdDM upon heat stress(Popova et al.,2013).In addition,our analysis suggests that the plant speci?c RNA poly-merases might act under heat stress in regulatory mechanisms different from RdDM.It will be of great importance to analyze nrpd1and nrpe1single mutants in order to distinguish the partic-ular role of each of the two RNA polymerases in stress response and to go further in elucidating other possible mechanisms of their action.

Contributions

M.N.,G.Y.designed research,M.N.,E.A.,M.G,N.G.carried out experiments,V.B performed bioinformatics analyses,G.Y.wrote the paper.

Acknowledgment

We thank Thierry Lagrange and lab colleagues from the LGDP, University of Perpignan,France,for providing mutant seed stocks. This work was partially supported by Bulgarian Ministry of Edu-cation and Sciences Grant DO02-279,DKOF7RP02/20and EC FP7 REGPOT Project BioSupport.

Appendix A.Supplementary data

Supplementary data related to this article can be found at http:// https://www.wendangku.net/doc/3510060100.html,/10.1016/j.plaphy.2014.12.022.

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