BACKGROUND
plants use environmental signals to modify and adapt their growth and development according to local climatic/ecological conditions.
a major step in plant life cycle is the transition from the vegetative to the reproductive stage that controls flowering, a critical trait for plant fitness.
seasonal cues, such as temperature and day-length, ensure that flowering coincides with favorable conditions to escape stress and maximize photosynthesis and seed production
<cit> .
in temperate climate, certain species, including the temperate cereals  and dicot species  need to be exposed to a period of prolonged low winter temperatures to accelerate the progression from vegetative to reproductive growth, a process known as vernalization
<cit> .
vernalization requirement is an adaptive trait that prevents flowering initiation prior to winter, which would otherwise result in severe frost damages on fragile flower meristems.
the prolonged cold period is remembered over time, maintaining flowering stimulation when temperatures rise back during spring
<cit> .
the vernalization signal is transmissible through mitosis but is reset in the next sexual generation therefore ensuring that descendants will be themselves able to respond to vernalization.
in arabidopsis, cold induces a transcriptional repression of the repressor of floral initiation flowering locus c .
this repression is mediated by an epigenetic-based regulation in which the flc chromatin state is switched from an actively transcribed state  to a repressed state
<cit> .
this transcriptional repression is then maintained over cell divisions by mitotic inheritance of the repressive histone modifications
<cit> , but is reset during reproduction, thus allowing progeny to be competent to respond to vernalization
<cit> .
molecular basis of such transient memory of a cold period during plant development has been recently described in other species.
in temperate cereals such as the triticeae, response to vernalization is mediated by the stable induction of a floral activator, vernalization <dig>
<cit> .
vrn <dig> encodes a fruitfull-like mads-box transcription factor that is required for the initiation of reproductive development at the shoot apex
<cit> .
vrn <dig> is central in the vernalization pathway
<cit>  as it down regulates the floral repressor vernalization <dig>  but also interacts with other flowering pathways, allowing long-day induction of the floral activator flowering locus t-like  <dig>  to accelerate subsequent stages of floral development
<cit> .
the expression of vrn <dig> is induced by cold treatment, is maintained when cold treatment is released, and is reset in the next generation
<cit> , some characteristics that point out to a possible epigenetic regulation.
the epigenetic regulation of hvvrn <dig>  the vrn <dig> gene of barley , has been assessed through the analysis of histone post-translational modifications
<cit> .
vernalization increases active histone marks for transcription  in two regions located in exon one and the beginning of intron  <dig>  while decreasing silent marks  in  <dig> regions located from the promoter to the end of intron  <dig>  in a more recent study
<cit> , histone acetylation of intron  <dig> was also shown to be involved in the regulation of the gene: acetylation levels of histones  <dig> and  <dig> increase during cold treatment in intron  <dig>  and sodium butyrate, a histone deacetylation inhibitor, induces an increase in hvvrn <dig> expression.
altogether, this suggests that in barley vernalization induces histone modifications associated with an active chromatin state, which correlates with an increase in vrn <dig> transcripts.
these changes are retained posterior to vernalization, providing a molecular hypothesis for the epigenetic-based memory of vernalization in barley.
in hexaploid wheat , analysis of h3k4me <dig> and h3k27me <dig> histone modifications of the tavrn <dig> promoter region  in winter and spring wheat revealed no significant changes for h3k27me <dig> following vernalization in both genotypes
<cit> .
however, vernalization caused an enrichment of h3k4me <dig> in winter wheat while a decrease of this histone modification was observed in spring wheat.
altogether, these results suggest that vernalization promotes an active state of the tavrn <dig> chromatin in winter wheat and a reduction of this active state in spring wheat.
these results are consistent with the relative abundance of tavrn <dig> mrna in winter and spring wheat and suggest that the wheat vernalization responsive gene vrn <dig> is regulated, at least in part, by histone methylation at the promoter.
genetic studies have shown that, besides the promoter region, internal regions of vrn-a <dig> such as intron  <dig> are also involved in the diffe rential regulation observed between spring and winter wheat
<cit> .
this, together with the results obtained for barley, suggests that an epigenetic regulation of the internal regions of the gene could be involved in the regulation of vrn-a <dig>
however, these studies of the vrn <dig> regulation were limited to the analysis of histone marks, and did not allow for testing the potential role of dna methylation in the cereal vernalization process.
while dna methylation could not explain the expression changes observed for flc in arabidopsis <cit> , studies on sugar beet  revealed that the flowering repressor bvflc is hypermethylated and decreases during vernalization in genotypes sensitive to vernalization, this effect being more pronounced for longer duration of cold treatment
<cit> .
the differences observed between arabidopsis and sugar beet suggest that the ability to respond to vernalization may have been acquired through different molecular mechanisms within dicotyledonous plants and highlight the need to better characterize the role of dna methylation in the molecular mechanisms involved in the response to vernalization for other species.
in particular, the role played by dna methylation in the vernalization response of monocotyledonous plants such as cereals needs to be investigated.
dna methylation on cytosines is a well studied epigenetic mark in plants.
it is involved in the regulation and transcriptional silencing of genes and transposable elements , respectively, and plays major roles in cellular differentiation and genome evolution
<cit> .
inherited methylation changes also likely play a role in plant phenotypic evolution
<cit> .
while dna methylation mainly occurs at cg sites in animal genomes, it can affect cytosines in cg, chg and chh contexts  in plants
<cit> .
repeated sequences such as tes are heavily methylated at the three cytosine contexts, whereas dna methylation in the body of the genes  is less dense and mainly limited to cg sites
<cit> .
whereas the mechanisms underlying the establishment and maintenance of dna methylation are now relatively well understood, little is known about their dynamics during the plant and animal life time.
several studies have focused on the biological relevance of dna methylation dynamics in plant response to biotic stresses
<cit>  but its relevance in the response to abiotic cues remains largely unknown.
in this article, we investigated the effect of vernalization on the dna methylation profile of the vrn-a <dig> gene in winter wheat.
we focused on studying the cellular memory of cold-induced dna methylation after the environmental stimulus was removed.
for this, we analyzed the dna methylation pattern of vrn-a <dig> on plants submitted or not to 45 days-long cold treatment inducing the expression of vrn-a <dig> gene and placed back in greenhouse conditions for  <dig> additional days.
our study indicates that cold induces an increase in dna methylation within the body of the vrn-a <dig> gene, which is reset in the next generation, thus highlighting a first case of environmentally-induced epigenetic change in wheat.
whether the observed increase in dna methylation explains the increase in expression of the vrn-a <dig> gene or is simply correlative remains to be elucidated.
nevertheless, the finding that cold-induced hypermethylation specifically affects non cg sites located in te fragments from the  <dig>  kb long intron  <dig> suggests that te fragments may facilitate the dna hypermethylation process observed.
RESULTS
overall dna methylation pattern of vrn-a <dig> in non-vernalized plants
dna methylation pattern of the vrn-a <dig> gene was investigated using the bisulfite conversion method, which allows for the quantitative detection of methylation level for each cytosine of a pcr-amplified fragment.
to analyze the overall methylation pattern of the vrn-a <dig> gene, we analyzed  <dig> fragments located along the vrn-a <dig> gene, covering 21% of this 12 kb gene .
dna methylation of these  <dig> fragments was investigated on two genotypes, three biological repetitions and two treatments .
because these experiments would be facilitated by the use of a cost- and labor-effective method to quantify dna methylation, we used direct reading of pcr sanger sequence chromatograms to quantify dna methylation at each cytosine site after bisulfite conversion and pcr amplification .
design of primers fitting the bisulfite treatment requirements for a hexaploid genome was not trivial, and about 50% of the pcr designed were either not amplifying or leading to poor sequencing traces.
in particular, the middle of intron  <dig> and the region located between exons  <dig> and  <dig> could not be covered by bisulfite-pcr assays.
we therefore used methylsensitive pcr assays  to investigate the dna methylation profile of these regions .
we also used this technique to validate results for  <dig> bisulfite-pcr fragments with contrasted dna methylation patterns .
vrn-a1
bold letters in the sequence represent primer positions matching cytosines, which were considered as non methylated and therefore converted into thymines after bisulfite treatment .
primer pairs that were not genome a specific; fragment for which several primer pairs with different c/t content were designed ; while corresponding pcr amplicon contains a fragment of the jorge transposon, the reverse primer does not overlap the annotation of this element.
the closest jorge element found shares only 76% nucleotide identity with this amplicon; while the two primers are included within the sumaya annotation, the closest sumaya element found shares only 81% nucleotide identity with this amplicon.
arrowheads indicate enzymes cutting sites and bold cs show methylation sensitive sites.
vrn-a1
msp
bst
*pcr primers from
<cit> .
abbreviations: n.a.
: not analyzed.
analysis of these  <dig> fragments  on one plant from the g <dig> genotype grown in non vernalizing conditions revealed that most fragments  were not methylated.
for the remaining fragments, dna methylation was either restricted to cg sites , or was observed in all cg, chg and chh contexts  and  <dig>  k).
these results indicate that the dna methylation of the vrn-a <dig> gene is heterogeneous along its sequence, with an absence of methylation at the start and end, while the region extending from the middle of intron  <dig> to the start of exon  <dig> is highly methylated .
dna methylation rates at the different cytosine contexts  were similar to what has been observed in arabidopsis and maize .
the black box indicated as “prom” highlights position of the promoter region as described in
<cit> .
grey boxes represent exons.
dashed frame indicates position of the deletion in the langdon allele .
purple lines highlight positions of transposable element sequences, with corresponding class and name indicated below.
brown and pink lines represents the fragments studied using bisulfite-based and restriction enzyme-based techniques, respectively.
corresponding fragment names are given above each line.
vernalization induces both hypermethylation at the end of vrn-a <dig> intron  <dig> and gene transcription increase
comparison of dna methylation profiles of one vernalized and one non-vernalized plant of the g <dig> genotype for all bisulfite pcr fragments revealed a difference of methylation level only for fragments  <dig>  k and  <dig>  k. further investigation of the dna methylation pattern of these two fragments on two additional biological replicates of genotype g <dig> and three biological replicates of genotype g <dig> showed that patterns were homogeneous among non-vernalized plants, and among vernalized plants, and confirmed the difference observed between vernalized and non vernalized plants .
cg sites show an average methylation rate higher than 90% in both conditions, with no significant variation between treatments.
in contrast, chg and chh sites showed a significant increase in dna methylation in vernalized plants .
analysis of cytosines taken individually  revealed that all cg sites are methylated above 90% in both conditions, while all chg sites are methylated in non vernalized plants but show a significant increase in dna methylation following vernalization  for which the increase is visible but not significant).
interestingly, while only a small fraction of chh sites is methylated  and  <dig>  k, respectively), no additional methylated site was found.
rather, a significant increase of dna methylation was observed for almost all sites that were methylated in non-vernalized conditions .
comparison of the two genotypes revealed very similar methylation patterns: only sites  <dig> and  <dig> of fragment  <dig>  k showed significant differences between the two genotypes .
taken together, these results reveal that, at the end of intron  <dig>  vernalized plant show an increase in methylation at chg and chh sites that is restricted to the sites that are also partially methylated in non-vernalized plants.
on the contrary, almost all cg sites are highly methylated in both conditions, and are therefore not impacted by the vernalization treatment.
these observations were confirmed on a second set of experiments , indicating that the shift in dna methylation observed is repeatable.
because the expression of the vrn-a <dig> gene is known to increase following vernalization
<cit> , our data suggested a positive correlation between dna methylation and gene transcription.
to identify whether the dna methylation shift observed following vernalization was correlated to the expression of the vnr-a <dig> gene, we performed qrt-pcr for both vernalized and non-vernalized conditions, using the same leaf powder used for dna methylation analyses.
qpcr results indicate a  <dig> ct change between vernalized and non vernalized plants, pointing to a  <dig> fold increase of vrn-a <dig> leaf mrna levels following vernalization .
this expression increase is associated with the dna methylation shift observed, with vrn-a <dig> being expressed when the gene shows higher gene body methylation.
the high level of dna methylation observed in vernalized plants is maintained through mitosis but is reset in the next generation
to verify the results obtained on 4th leaf and to study the mitotic and transgenerational transmission of the modifications in dna methylation observed following vernalization, a second set of plants was grown up to flowering maturity.
dna methylation pattern of fragments  <dig>  k and  <dig>  k was analyzed on fourth leaf and flag leaf samples .
no significant difference was observed between the 4th leaf and the flag leaf samples , thus revealing that the dna methylation profile observed is maintained through mitosis.
vernalized plants of this second set were selfed, and the progeny was analyzed in non-vernalizing conditions at the fourth leaf stage .
dna methylation profile analysis of fragments  <dig>  k and  <dig>  k revealed that all cytosines show a methylation profile typical of non vernalized plants , therefore providing evidence that the increase in dna methylation induced by vernalization is reset in the next generation.
the sequence of winter allele of vrn-a <dig> harbor te fragments not found in vrn-b <dig> and vrn-d <dig> copies
te annotation within the vrn-a <dig> winter genotype sequence using the most recently updated wheat transposable element database
<cit>  revealed the presence of several te fragments, most of which are located within the second half of intron  <dig> .
interestingly, the region covered by these tes is the region showing the highest methylation level, harboring methylated cs from cg context, but also from chg and chh contexts.
in particular, fragments  <dig>  k and  <dig>  k that show a difference in dna methylation level between vernalized and non-vernalized plants also harbor pieces of tes: amplicon  <dig>  contains a fragment of the cacta-like tir transposon jorge  while amplicon  <dig>  k is fully covered by a piece of the gypsy-like ltr retrotransposon sumaya .
annotation of vrn-b <dig> and vrn-d <dig> genes and comparative analysis with vrn-a <dig> showed that the sumaya element is specific from the a genome vrn <dig> copy .
comparison of the jorge and sumaya fragments located in vrn-a <dig> with sequences from other jorge and sumaya elements extracted from chromosome 3b revealed that they share 76% and 81% identity with the closest copy, respectively.
this, together with the fact that they are truncated tes, suggests that these sequences have evolved from ancient insertions.
<cit> .
purple lines highlight positions of transposable element sequences as defined by our annotation, with corresponding class and name shown below.
blue lines represent the annotation of two sine elements that could not be found in our analysis but were described in genebank accession ay <dig>
<cit> .
light blue parallelograms represent conserved regions as defined by our gevo analysis.
discussion
the vrn-a <dig> gene is body methylated and harbors additional non-cg methylation in intron 1
the  <dig> assays  applied over the whole vrn-a <dig> gene sequence revealed that this gene shows a non homogeneous and contrasted methylation pattern: while the 5′ and 3′ extremities are not methylated, the inside of the gene is highly methylated .
in particular, methylation at cg sites shows a bell shape pattern with high methylation inside the gene and a low or absent methylation level at the extremities, a pattern commonly observed in plants and animals, and that is referred to as “gene body methylation”
<cit> .
in a. thaliana, body-methylated genes tend to be longer and contain more exons than unmethylated genes
<cit> .
accordingly, vrn-a <dig> is a long gene, with a total size of  <dig>  bp and  <dig> exons  and  <dig>  k) which involves almost complete methylation at cg sites and intermediate levels of methylation at chg and chh sites, a pattern that is commonly associated with te sequences.
while genes are mainly methylated at cg sites through the action of met1
<cit> , dna methylation at tes is reinforced by the action of the rna-directed dna methylation  machinery
<cit> , which involves the production of  <dig> nt heterochromatic small rnas that direct the de novo methylation machinery to te sequences at cg, chg and chh sites.
this epigenetic mechanism limits te transposition and therefore protects the genome from insertional mutations
<cit> .
indeed the annotation of the vrn-a <dig> sequence revealed the presence of several te fragments in the second half of intron  <dig>  i.e.
in the region showing heavy cg, chg and chh methylation.
hence, the vrn-a <dig> gene is globally devoid of rddm marks except in regions intimately associated with te-related sequences.
while this type of pattern has been observed at the genome wide level in arabidopsis <cit>  the detailed gene level characterization of both vrn-a <dig> methylation pattern and te annotation makes it a good model to investigate the possible role of such intronic methylation in complex genomes.
vernalization hypermethylates vrn-a <dig> at specific chg and chh sites
comparison of the dna methylation profile of vrn-a <dig> in vernalized and non-vernalized plants revealed a globally invariable pattern over most of the studied areas.
a noticeable exception is the highly methylated area of intron  <dig>  which exhibits a high level of dna methylation in non-vernalized plants and an even higher methylation level in vernalized plants.
this increase does not affect cg sites, which are almost fully methylated in non-vernalized plants and therefore likely saturated.
rather, it occurs at non-cg sites, especially chg sites and some chh sites .
the observation of a cold-induced hypermethylation at the vrn-a <dig> gene raises the question of its specificity, that is, whether the hypermethylation observed is a specific response of the vrn-a <dig> gene, or a consequence of a genome-wide methylation increase at non cg sites following vernalization.
environmental stress is known to induce changes in dna methylation at the genome-wide level.
in particular, a decrease in dna methylation has been observed at the genome-wide level following vernalization in wheat
<cit> .
cold therefore induces a hypermethylation of vrn-a <dig> which does not follow the hypomethylation trend observed at the whole genome level as reported in the study of sherman and talbert
<cit> .
in this study, v <dig> vernalization conditions are very close to the conditions used in our study , the only difference being the tissue used  and lighting .
such a cold-induced genome-wide hypomethylation has also been reported in maize
<cit> , suggesting it is a common response in cereals.
hence, genome-wide hypomethylation also likely applies to our experiment, and points out to a specific regulation of the vrn-a <dig> gene, or of a limited set of wheat genes/genomic regions.
similarly, induction of the asr <dig> gene following drought stress in tomato has recently been associated with a change in methylation occurring within its body
<cit> .
although the changes observed are different , these observations suggest that dna methylation could participate in the regulation of genes involved in specific stress response.
interestingly, while in the asr <dig> case site specificity was difficult to assess due to high methylation levels in control conditions, the methylation increase observed for vrn-a <dig> is clearly site-specific and restricted to the sites that are already methylated in non-vernalizing conditions.
the absence of additional methylated sites suggests a reinforcement of existing methylation signal rather than the establishment of a new methylation signal.
dna methylation and histone modifications at the promoter
the absence of dna methylation in the promoter of vrn-a <dig> in both vernalizing and control conditions suggests that the expression change of vrn-a <dig> does not occur through dna methylation of its promoter.
both in wheat and barley, previous studies have shown that histone modification at the promoter region is involved in the transcriptional regulation of the vrn <dig> gene: together with an increase in expression, vernalization treatment induces an increase in the active chromatin mark h3k4me <dig> and a decrease in the silent mark h3k27me <dig> in the barley vernalization-responsive variety sonja
<cit>  and increases h3k4me <dig> in the norstar winter wheat cultivar
<cit> .
combination of these sets of information suggests that the vrn-a <dig> transcriptional regulation at the promoter would be mediated by histone post-translational modifications rather than dna methylation.
hence, even though histone post-translational modification and dna methylation are to some extent interlinked
<cit> , they could be disconnected in the case of the cold-response regulation of vrn-a <dig>  however, distinction between the three vrn <dig> homeologous copies is not clear in the oury-diallo study
<cit> , thus making it difficult to assess whether the histone mark change observed is occurring at the vrn-a <dig> gene promoter, at another of the vrn <dig> gene promoters, or at all vrn <dig> genes promoters simultaneously.
for these reasons, additional studies on both histone marks and dna methylation need to be performed for this gene on the same plant material, to test for a disconnection between dna methylation and histone marks.
methylation of te fragments in intron  <dig> does not follow the same trend as other tes but likely involves the rddm pathway
vrn-a <dig> is body methylated at cg sites, but this methylation profile is not modified by cold treatment.
therefore, although vrn-a <dig> is body methylated at cg, this body methylation is not associated with the expression change observed between vernalized and non vernalized plants.
on the other hand, significant vernalization-induced changes in dna methylation were found in intron  <dig> within a region harboring te fragments, and occurred at chg and chh sites.
interestingly, te sequences usually show a decrease in methylation and an increase in expression  following biotic and abiotic stresses
<cit> .
moreover, tes were shown to be transcriptionally activated by vernalization treatment in wheat
<cit> .
as transcriptional activation of tes is usually associated with a release of their silencing, our observation that the two te fragments are hypermethylated following cold treatment suggests they are regulated by a different process.
the observation that hypermethylation occurs at cytosines from chg and chh contexts points out that the rna-directed dna methylation  pathway is likely involved in the hypermethylation process.
interestingly, hypermethylation is restricted to regions bearing te fragments and targets specific chg and chh sites that show some methylation in non vernalizing conditions.
this suggests that te fragments could act as facilitators of dna methylation recruitment
<cit> : presence of te fragments would trigger recruitment of the rddm machinery in control conditions, and this pathway would be even more active following cold conditions.
however, because rddm requires multiple forms of rna polymerase activity , polymerase v , but also polymerase ii  itself
<cit> ), additional investigation is required to establish whether the cold-induced methylation occurs through a higher level of poliv/polv transcription at te fragments, or due to a higher polii transcription of the gene.
while jorge is a very abundant family that is estimated to harbor hundred thousands of copies in the wheat genome sharing over 90% identity with their consensus sequence , the fragment located on vrn-a <dig> shares at most 76% identity with other jorge copies  and is likely derived from an old insertion event.
the sumaya family is a middle repetitive element with less than  <dig> copies in the wheat genome , and these copies share only 75% with their consensus, suggesting that this family did not amplify recently.
the fragment of sumaya ltr found in vrn-a <dig> shares at most 81% identity with another sumaya copy, and is therefore also an ancient insertion.
because both insertions are old and degenerated, occurrence of the rddm pathway on these elements could be disconnected from this of the other members of these te families.
comparative analysis of the two te fragments where hypermethylation occurs revealed that the jorge-like transposon is conserved among the three vrn <dig> copies, while the sumaya-like ltr retrotransposon fragment is specific to vrn-a <dig> .
both elements are conserved between t. aestivum a genome and t. monococcum which diverged less than  <dig>  my ago
<cit> .
this suggests they could be selectively maintained, and opens the possibility that this te-related hypermethylation might play a role in the regulation of the gene, or in another unknown process.
methylation of intron  <dig> and gene expression
we describe the first case of a mitotically stable increase in dna methylation in a gene intron, which is reset in the following generation.
however, it remains unclear whether the changes in dna methylation observed are a consequence of the high vrn-a <dig> expression level induced by cold or if dna methylation might play an active role in setting vrn-a <dig> expression levels following cold treatment.
several studies in wheat and barley support a functional role of promoter histone modification in setting vrn <dig> expression in the triticea
<cit> .
but deletion of vrn- <dig> intron  <dig> is sufficient to induce a spring phenotype
<cit> , thus suggesting a role of this intron in the regulation of vrn- <dig>  although the case of vrn-a <dig> is not clear in hexaploid wheat , the observation that deletion of part of vrn-a <dig> intron  <dig> induces a spring phenotype in tetraploid wheat triticum turgidum  supports the importance of this intron for the regulation of the vrn-a <dig> response to cold
<cit> .
the recent expression comparison of vrn-a <dig> expression in near-isogenic lines of wheat carrying different alleles of the gene clarified that the promoter insertion allele has a larger effect on gene expression than the intron deletion
<cit> .
altogether, this suggests that histone-based regulation of gene transcription occurring at the promoter is likely explaining most of the expression phenotype observed for vrn-a <dig>  but this does not rule out the possibility of redundant switches in which intron  <dig> could participate in the regulation of the gene, with a lesser extent.
oliver et al.
<cit>  also report that the langdon allele , while still inducible by cold, has a  <dig> fold higher basal expression level than the wild type triple dirk allele .
interestingly, alignment comparison of these two allele sequences reveals that deletion in the langdon allele extends from nucleotide  <dig> to nucleotide  <dig> on the wild type triple dirk allele, thus including fragments  <dig>  k and  <dig>  k .
hence, while not critical for the low-temperature response, the hypermethylated region observed could play a role in the regulation of the vrn-a <dig> gene.
but what role?
recently, methylation at chhs in the close vicinity of genes was shown to positively correlate with gene expression in maize, suggesting that this methylation mark may be interlinked with gene expression
<cit> .
in arabidopsis, dna methylation at cg and chg sites in the large ibm <dig> intron was shown to positively correlate with accumulation of the long transcriptional form of the gene, thus suggesting that intronic dna methylation at cg and chg sites may be required for proper ibm1-l transcript elongation
<cit> .
dna methylation was proposed to prevent spurious expression from cryptic intragenic promoters
<cit>  or enhance accurate splicing of primary transcripts
<cit> .
interestingly, analysis of genes with long introns in arabidopsis revealed that those harboring chg methylation also tend to have te insertions
<cit> .
the ibm <dig> protein, which is enriched at intronic tes and which mutation affects transcription of genes with long, te-rich introns, was proposed to avoid premature transcription termination around heterochromatic domains by suppressing antisense transcription from cryptic promoters
<cit> .
presence of te fragments may indeed increase the potential for cryptic transcripts, and the hypermethylation observed in vrn-a <dig> intron could be involved in the taming of such transcripts.
because intron  <dig> is  <dig>  kb long, its splicing may also be more challenging to accurately complete than the one of smaller introns.
could the inducible hypermethylation observed in te fragments of intron  <dig> be associated with its splicing?
in honeybee, dna methylation in exon was shown to be positively associated with retention
<cit> .
in plants, a recent study demonstrated that the novel pre-mrna splicing factor zop <dig> is involved in both pre-mrna splicing and the rddm pathway in arabidopsis and proposed that the splicing machinery may be involved in promoting rddm and transcriptional silencing.
a recent study indicated that inclusion of introns in transgenes can increase the transcripts levels of the transgenes in arabidopsis <cit> , suggesting a possible role of the splicing machinery in the regulation of rna transcript levels.
altogether, these studies suggest that the interconnection between dna methylation, pre-mrna splicing and transcripts levels may be more complex than previously anticipated.
further characterization of how the dna methylation pattern observed impacts the splicing of vrn-a <dig> intron  <dig> will likely help better understand this interconnection.
in the langdon allele, the  <dig>  kb deletion reduces intron size and deletes te fragments, thus reducing both intron size  and probability for cryptic promoters.
does the cold-induced hypermethylation observed reduce the impact of splicing and cryptic transcription effects?
te fragments are remnants that are not able to move, therefore their silencing is not critical to avoid transposition.
however, presence of such fragments within introns both enlarge their size  and enhance chances for the emergence of cryptic promoters .
while additional studies are needed to further investigate the role of non cg methylation in introns, our study raises the following question: is de novo dna methylation at intronic te fragments a remaining trace of original te silencing, or is it involved in decreasing te-derived transcriptional effects?
CONCLUSIONS
our study reveals that vernalization induces hypermethylation of vrn-a <dig> at specific non cg sites located in te fragments of large intron  <dig>  which is associated with gene expression.
while additional analyses are needed to investigate whether this hypermethylation is a by-product of gene expression or participates to its regulation, even to a low extent, our study provides the first detailed dna methylation characterization of vrn-a <dig>  observation of a cold-induced dna methylation shift in two winter genotypes following mild vernalization treatment opens the way to examining this dna methylation pattern in a broader set of vernalization conditions and using more time points , as well as in multiple genotypes , thus providing a starting point to investigate the biological role of dna methylation in the wheat vernalization response.
our findings on vrn-a <dig> also open interesting questions on the role of intronic chh methylation, in particular those which are large and contain te fragments.
in particular, whether the hypermethylation observed in vrn-a <dig> is also found in vrn-1b and vrn-1d copies remains to be elucidated.
if a similar hypermethylation is found in all three copies, investigation of the connection of this hypermethylation to presence of other tes in the b and d copies will help get insights into the underlying mechanism.

 plants use environmental signals to modify and adapt their growth and development according to local climatic/ecological conditions. a major step in plant life cycle is the transition from the vegetative to the reproductive stage that controls flowering, a critical trait for plant fitness. seasonal cues, such as temperature and day-length, ensure that flowering coincides with favorable conditions to escape stress and maximize photosynthesis and seed production. in temperate climate, certain species, including the temperate cereals and dicot species need to be exposed to a period of prolonged low winter temperatures to accelerate the progression from vegetative to reproductive growth, a process known as vernalization. vernalization requirement is an adaptive trait that prevents flowering initiation prior to winter, which would otherwise result in severe frost damages on fragile flower meristems. the prolonged cold period is remembered over time, maintaining flowering stimulation when temperatures rise back during spring. the vernalization signal is transmissible through mitosis but is reset in the next sexual generation therefore ensuring that descendants will be themselves able to respond to vernalization. in arabidopsis, cold induces a transcriptional repression of the repressor of floral initiation flowering locus c. this repression is mediated by an epigenetic-based regulation in which the flc chromatin state is switched from an actively transcribed state to a repressed state. this transcriptional repression is then maintained over cell divisions by mitotic inheritance of the repressive histone modifications, but is reset during reproduction, thus allowing progeny to be competent to respond to vernalization. molecular basis of such transient memory of a cold period during plant development has been recently described in other species. in temperate cereals such as the triticeae, response to vernalization is mediated by the stable induction of a floral activator, vernalization. vrn encodes a fruitfull-like mads-box transcription factor that is required for the initiation of reproductive development at the shoot apex. vrn is central in the vernalization pathway as it down regulates the floral repressor vernalization but also interacts with other flowering pathways, allowing long-day induction of the floral activator flowering locus t-like to accelerate subsequent stages of floral development. the expression of vrn is induced by cold treatment, is maintained when cold treatment is released, and is reset in the next generation, some characteristics that point out to a possible epigenetic regulation. the epigenetic regulation of hvvrn the vrn gene of barley, has been assessed through the analysis of histone post-translational modifications. vernalization increases active histone marks for transcription in two regions located in exon one and the beginning of intron while decreasing silent marks in regions located from the promoter to the end of intron in a more recent study, histone acetylation of intron was also shown to be involved in the regulation of the gene: acetylation levels of histones and increase during cold treatment in intron and sodium butyrate, a histone deacetylation inhibitor, induces an increase in hvvrn expression. altogether, this suggests that in barley vernalization induces histone modifications associated with an active chromatin state, which correlates with an increase in vrn transcripts. these changes are retained posterior to vernalization, providing a molecular hypothesis for the epigenetic-based memory of vernalization in barley. in hexaploid wheat, analysis of h3k4me and h3k27me histone modifications of the tavrn promoter region in winter and spring wheat revealed no significant changes for h3k27me following vernalization in both genotypes. however, vernalization caused an enrichment of h3k4me in winter wheat while a decrease of this histone modification was observed in spring wheat. altogether, these results suggest that vernalization promotes an active state of the tavrn chromatin in winter wheat and a reduction of this active state in spring wheat. these results are consistent with the relative abundance of tavrn mrna in winter and spring wheat and suggest that the wheat vernalization responsive gene vrn is regulated, at least in part, by histone methylation at the promoter. genetic studies have shown that, besides the promoter region, internal regions of vrn-a such as intron are also involved in the diffe rential regulation observed between spring and winter wheat. this, together with the results obtained for barley, suggests that an epigenetic regulation of the internal regions of the gene could be involved in the regulation of vrn-a however, these studies of the vrn regulation were limited to the analysis of histone marks, and did not allow for testing the potential role of dna methylation in the cereal vernalization process. while dna methylation could not explain the expression changes observed for flc in arabidopsis, studies on sugar beet revealed that the flowering repressor bvflc is hypermethylated and decreases during vernalization in genotypes sensitive to vernalization, this effect being more pronounced for longer duration of cold treatment. the differences observed between arabidopsis and sugar beet suggest that the ability to respond to vernalization may have been acquired through different molecular mechanisms within dicotyledonous plants and highlight the need to better characterize the role of dna methylation in the molecular mechanisms involved in the response to vernalization for other species. in particular, the role played by dna methylation in the vernalization response of monocotyledonous plants such as cereals needs to be investigated. dna methylation on cytosines is a well studied epigenetic mark in plants. it is involved in the regulation and transcriptional silencing of genes and transposable elements, respectively, and plays major roles in cellular differentiation and genome evolution. inherited methylation changes also likely play a role in plant phenotypic evolution. while dna methylation mainly occurs at cg sites in animal genomes, it can affect cytosines in cg, chg and chh contexts in plants. repeated sequences such as tes are heavily methylated at the three cytosine contexts, whereas dna methylation in the body of the genes is less dense and mainly limited to cg sites. whereas the mechanisms underlying the establishment and maintenance of dna methylation are now relatively well understood, little is known about their dynamics during the plant and animal life time. several studies have focused on the biological relevance of dna methylation dynamics in plant response to biotic stresses but its relevance in the response to abiotic cues remains largely unknown. in this article, we investigated the effect of vernalization on the dna methylation profile of the vrn-a gene in winter wheat. we focused on studying the cellular memory of cold-induced dna methylation after the environmental stimulus was removed. for this, we analyzed the dna methylation pattern of vrn-a on plants submitted or not to 45 days-long cold treatment inducing the expression of vrn-a gene and placed back in greenhouse conditions for additional days. our study indicates that cold induces an increase in dna methylation within the body of the vrn-a gene, which is reset in the next generation, thus highlighting a first case of environmentally-induced epigenetic change in wheat. whether the observed increase in dna methylation explains the increase in expression of the vrn-a gene or is simply correlative remains to be elucidated. nevertheless, the finding that cold-induced hypermethylation specifically affects non cg sites located in te fragments from the  kb long intron suggests that te fragments may facilitate the dna hypermethylation process observed.  overall dna methylation pattern of vrn-a in non-vernalized plants dna methylation pattern of the vrn-a gene was investigated using the bisulfite conversion method, which allows for the quantitative detection of methylation level for each cytosine of a pcr-amplified fragment. to analyze the overall methylation pattern of the vrn-a gene, we analyzed fragments located along the vrn-a gene, covering 21% of this 12 kb gene. dna methylation of these fragments was investigated on two genotypes, three biological repetitions and two treatments. because these experiments would be facilitated by the use of a cost- and labor-effective method to quantify dna methylation, we used direct reading of pcr sanger sequence chromatograms to quantify dna methylation at each cytosine site after bisulfite conversion and pcr amplification. design of primers fitting the bisulfite treatment requirements for a hexaploid genome was not trivial, and about 50% of the pcr designed were either not amplifying or leading to poor sequencing traces. in particular, the middle of intron and the region located between exons and could not be covered by bisulfite-pcr assays. we therefore used methylsensitive pcr assays to investigate the dna methylation profile of these regions. we also used this technique to validate results for bisulfite-pcr fragments with contrasted dna methylation patterns. vrn-a1 bold letters in the sequence represent primer positions matching cytosines, which were considered as non methylated and therefore converted into thymines after bisulfite treatment. primer pairs that were not genome a specific; fragment for which several primer pairs with different c/t content were designed; while corresponding pcr amplicon contains a fragment of the jorge transposon, the reverse primer does not overlap the annotation of this element. the closest jorge element found shares only 76% nucleotide identity with this amplicon; while the two primers are included within the sumaya annotation, the closest sumaya element found shares only 81% nucleotide identity with this amplicon. arrowheads indicate enzymes cutting sites and bold cs show methylation sensitive sites. vrn-a1 msp bst *pcr primers from. abbreviations: n.a.: not analyzed. analysis of these fragments on one plant from the g genotype grown in non vernalizing conditions revealed that most fragments were not methylated. for the remaining fragments, dna methylation was either restricted to cg sites, or was observed in all cg, chg and chh contexts and  k). these results indicate that the dna methylation of the vrn-a gene is heterogeneous along its sequence, with an absence of methylation at the start and end, while the region extending from the middle of intron to the start of exon is highly methylated. dna methylation rates at the different cytosine contexts were similar to what has been observed in arabidopsis and maize. the black box indicated as “prom” highlights position of the promoter region as described in. grey boxes represent exons. dashed frame indicates position of the deletion in the langdon allele. purple lines highlight positions of transposable element sequences, with corresponding class and name indicated below. brown and pink lines represents the fragments studied using bisulfite-based and restriction enzyme-based techniques, respectively. corresponding fragment names are given above each line. vernalization induces both hypermethylation at the end of vrn-a intron and gene transcription increase comparison of dna methylation profiles of one vernalized and one non-vernalized plant of the g genotype for all bisulfite pcr fragments revealed a difference of methylation level only for fragments  k and  k. further investigation of the dna methylation pattern of these two fragments on two additional biological replicates of genotype g and three biological replicates of genotype g showed that patterns were homogeneous among non-vernalized plants, and among vernalized plants, and confirmed the difference observed between vernalized and non vernalized plants. cg sites show an average methylation rate higher than 90% in both conditions, with no significant variation between treatments. in contrast, chg and chh sites showed a significant increase in dna methylation in vernalized plants. analysis of cytosines taken individually revealed that all cg sites are methylated above 90% in both conditions, while all chg sites are methylated in non vernalized plants but show a significant increase in dna methylation following vernalization for which the increase is visible but not significant). interestingly, while only a small fraction of chh sites is methylated and  k, respectively), no additional methylated site was found. rather, a significant increase of dna methylation was observed for almost all sites that were methylated in non-vernalized conditions. comparison of the two genotypes revealed very similar methylation patterns: only sites and of fragment  k showed significant differences between the two genotypes. taken together, these results reveal that, at the end of intron vernalized plant show an increase in methylation at chg and chh sites that is restricted to the sites that are also partially methylated in non-vernalized plants. on the contrary, almost all cg sites are highly methylated in both conditions, and are therefore not impacted by the vernalization treatment. these observations were confirmed on a second set of experiments, indicating that the shift in dna methylation observed is repeatable. because the expression of the vrn-a gene is known to increase following vernalization, our data suggested a positive correlation between dna methylation and gene transcription. to identify whether the dna methylation shift observed following vernalization was correlated to the expression of the vnr-a gene, we performed qrt-pcr for both vernalized and non-vernalized conditions, using the same leaf powder used for dna methylation analyses. qpcr results indicate a ct change between vernalized and non vernalized plants, pointing to a fold increase of vrn-a leaf mrna levels following vernalization. this expression increase is associated with the dna methylation shift observed, with vrn-a being expressed when the gene shows higher gene body methylation. the high level of dna methylation observed in vernalized plants is maintained through mitosis but is reset in the next generation to verify the results obtained on 4th leaf and to study the mitotic and transgenerational transmission of the modifications in dna methylation observed following vernalization, a second set of plants was grown up to flowering maturity. dna methylation pattern of fragments  k and  k was analyzed on fourth leaf and flag leaf samples. no significant difference was observed between the 4th leaf and the flag leaf samples, thus revealing that the dna methylation profile observed is maintained through mitosis. vernalized plants of this second set were selfed, and the progeny was analyzed in non-vernalizing conditions at the fourth leaf stage. dna methylation profile analysis of fragments  k and  k revealed that all cytosines show a methylation profile typical of non vernalized plants, therefore providing evidence that the increase in dna methylation induced by vernalization is reset in the next generation. the sequence of winter allele of vrn-a harbor te fragments not found in vrn-b and vrn-d copies te annotation within the vrn-a winter genotype sequence using the most recently updated wheat transposable element database revealed the presence of several te fragments, most of which are located within the second half of intron. interestingly, the region covered by these tes is the region showing the highest methylation level, harboring methylated cs from cg context, but also from chg and chh contexts. in particular, fragments  k and  k that show a difference in dna methylation level between vernalized and non-vernalized plants also harbor pieces of tes: amplicon contains a fragment of the cacta-like tir transposon jorge while amplicon  k is fully covered by a piece of the gypsy-like ltr retrotransposon sumaya. annotation of vrn-b and vrn-d genes and comparative analysis with vrn-a showed that the sumaya element is specific from the a genome vrn copy. comparison of the jorge and sumaya fragments located in vrn-a with sequences from other jorge and sumaya elements extracted from chromosome 3b revealed that they share 76% and 81% identity with the closest copy, respectively. this, together with the fact that they are truncated tes, suggests that these sequences have evolved from ancient insertions.. purple lines highlight positions of transposable element sequences as defined by our annotation, with corresponding class and name shown below. blue lines represent the annotation of two sine elements that could not be found in our analysis but were described in genebank accession ay. light blue parallelograms represent conserved regions as defined by our gevo analysis. discussion the vrn-a gene is body methylated and harbors additional non-cg methylation in intron 1 the assays applied over the whole vrn-a gene sequence revealed that this gene shows a non homogeneous and contrasted methylation pattern: while the 5′ and 3′ extremities are not methylated, the inside of the gene is highly methylated. in particular, methylation at cg sites shows a bell shape pattern with high methylation inside the gene and a low or absent methylation level at the extremities, a pattern commonly observed in plants and animals, and that is referred to as “gene body methylation”. in a. thaliana, body-methylated genes tend to be longer and contain more exons than unmethylated genes. accordingly, vrn-a is a long gene, with a total size of  bp and exons and  k) which involves almost complete methylation at cg sites and intermediate levels of methylation at chg and chh sites, a pattern that is commonly associated with te sequences. while genes are mainly methylated at cg sites through the action of met1, dna methylation at tes is reinforced by the action of the rna-directed dna methylation machinery, which involves the production of nt heterochromatic small rnas that direct the de novo methylation machinery to te sequences at cg, chg and chh sites. this epigenetic mechanism limits te transposition and therefore protects the genome from insertional mutations. indeed the annotation of the vrn-a sequence revealed the presence of several te fragments in the second half of intron i.e. in the region showing heavy cg, chg and chh methylation. hence, the vrn-a gene is globally devoid of rddm marks except in regions intimately associated with te-related sequences. while this type of pattern has been observed at the genome wide level in arabidopsis the detailed gene level characterization of both vrn-a methylation pattern and te annotation makes it a good model to investigate the possible role of such intronic methylation in complex genomes. vernalization hypermethylates vrn-a at specific chg and chh sites comparison of the dna methylation profile of vrn-a in vernalized and non-vernalized plants revealed a globally invariable pattern over most of the studied areas. a noticeable exception is the highly methylated area of intron which exhibits a high level of dna methylation in non-vernalized plants and an even higher methylation level in vernalized plants. this increase does not affect cg sites, which are almost fully methylated in non-vernalized plants and therefore likely saturated. rather, it occurs at non-cg sites, especially chg sites and some chh sites. the observation of a cold-induced hypermethylation at the vrn-a gene raises the question of its specificity, that is, whether the hypermethylation observed is a specific response of the vrn-a gene, or a consequence of a genome-wide methylation increase at non cg sites following vernalization. environmental stress is known to induce changes in dna methylation at the genome-wide level. in particular, a decrease in dna methylation has been observed at the genome-wide level following vernalization in wheat. cold therefore induces a hypermethylation of vrn-a which does not follow the hypomethylation trend observed at the whole genome level as reported in the study of sherman and talbert. in this study, v vernalization conditions are very close to the conditions used in our study, the only difference being the tissue used and lighting. such a cold-induced genome-wide hypomethylation has also been reported in maize, suggesting it is a common response in cereals. hence, genome-wide hypomethylation also likely applies to our experiment, and points out to a specific regulation of the vrn-a gene, or of a limited set of wheat genes/genomic regions. similarly, induction of the asr gene following drought stress in tomato has recently been associated with a change in methylation occurring within its body. although the changes observed are different, these observations suggest that dna methylation could participate in the regulation of genes involved in specific stress response. interestingly, while in the asr case site specificity was difficult to assess due to high methylation levels in control conditions, the methylation increase observed for vrn-a is clearly site-specific and restricted to the sites that are already methylated in non-vernalizing conditions. the absence of additional methylated sites suggests a reinforcement of existing methylation signal rather than the establishment of a new methylation signal. dna methylation and histone modifications at the promoter the absence of dna methylation in the promoter of vrn-a in both vernalizing and control conditions suggests that the expression change of vrn-a does not occur through dna methylation of its promoter. both in wheat and barley, previous studies have shown that histone modification at the promoter region is involved in the transcriptional regulation of the vrn gene: together with an increase in expression, vernalization treatment induces an increase in the active chromatin mark h3k4me and a decrease in the silent mark h3k27me in the barley vernalization-responsive variety sonja and increases h3k4me in the norstar winter wheat cultivar. combination of these sets of information suggests that the vrn-a transcriptional regulation at the promoter would be mediated by histone post-translational modifications rather than dna methylation. hence, even though histone post-translational modification and dna methylation are to some extent interlinked, they could be disconnected in the case of the cold-response regulation of vrn-a however, distinction between the three vrn homeologous copies is not clear in the oury-diallo study, thus making it difficult to assess whether the histone mark change observed is occurring at the vrn-a gene promoter, at another of the vrn gene promoters, or at all vrn genes promoters simultaneously. for these reasons, additional studies on both histone marks and dna methylation need to be performed for this gene on the same plant material, to test for a disconnection between dna methylation and histone marks. methylation of te fragments in intron does not follow the same trend as other tes but likely involves the rddm pathway vrn-a is body methylated at cg sites, but this methylation profile is not modified by cold treatment. therefore, although vrn-a is body methylated at cg, this body methylation is not associated with the expression change observed between vernalized and non vernalized plants. on the other hand, significant vernalization-induced changes in dna methylation were found in intron within a region harboring te fragments, and occurred at chg and chh sites. interestingly, te sequences usually show a decrease in methylation and an increase in expression following biotic and abiotic stresses. moreover, tes were shown to be transcriptionally activated by vernalization treatment in wheat. as transcriptional activation of tes is usually associated with a release of their silencing, our observation that the two te fragments are hypermethylated following cold treatment suggests they are regulated by a different process. the observation that hypermethylation occurs at cytosines from chg and chh contexts points out that the rna-directed dna methylation pathway is likely involved in the hypermethylation process. interestingly, hypermethylation is restricted to regions bearing te fragments and targets specific chg and chh sites that show some methylation in non vernalizing conditions. this suggests that te fragments could act as facilitators of dna methylation recruitment: presence of te fragments would trigger recruitment of the rddm machinery in control conditions, and this pathway would be even more active following cold conditions. however, because rddm requires multiple forms of rna polymerase activity, polymerase v, but also polymerase ii itself ), additional investigation is required to establish whether the cold-induced methylation occurs through a higher level of poliv/polv transcription at te fragments, or due to a higher polii transcription of the gene. while jorge is a very abundant family that is estimated to harbor hundred thousands of copies in the wheat genome sharing over 90% identity with their consensus sequence, the fragment located on vrn-a shares at most 76% identity with other jorge copies and is likely derived from an old insertion event. the sumaya family is a middle repetitive element with less than copies in the wheat genome, and these copies share only 75% with their consensus, suggesting that this family did not amplify recently. the fragment of sumaya ltr found in vrn-a shares at most 81% identity with another sumaya copy, and is therefore also an ancient insertion. because both insertions are old and degenerated, occurrence of the rddm pathway on these elements could be disconnected from this of the other members of these te families. comparative analysis of the two te fragments where hypermethylation occurs revealed that the jorge-like transposon is conserved among the three vrn copies, while the sumaya-like ltr retrotransposon fragment is specific to vrn-a. both elements are conserved between t. aestivum a genome and t. monococcum which diverged less than my ago. this suggests they could be selectively maintained, and opens the possibility that this te-related hypermethylation might play a role in the regulation of the gene, or in another unknown process. methylation of intron and gene expression we describe the first case of a mitotically stable increase in dna methylation in a gene intron, which is reset in the following generation. however, it remains unclear whether the changes in dna methylation observed are a consequence of the high vrn-a expression level induced by cold or if dna methylation might play an active role in setting vrn-a expression levels following cold treatment. several studies in wheat and barley support a functional role of promoter histone modification in setting vrn expression in the triticea. but deletion of vrn- intron is sufficient to induce a spring phenotype, thus suggesting a role of this intron in the regulation of vrn- although the case of vrn-a is not clear in hexaploid wheat, the observation that deletion of part of vrn-a intron induces a spring phenotype in tetraploid wheat triticum turgidum supports the importance of this intron for the regulation of the vrn-a response to cold. the recent expression comparison of vrn-a expression in near-isogenic lines of wheat carrying different alleles of the gene clarified that the promoter insertion allele has a larger effect on gene expression than the intron deletion. altogether, this suggests that histone-based regulation of gene transcription occurring at the promoter is likely explaining most of the expression phenotype observed for vrn-a but this does not rule out the possibility of redundant switches in which intron could participate in the regulation of the gene, with a lesser extent. oliver et al. also report that the langdon allele, while still inducible by cold, has a fold higher basal expression level than the wild type triple dirk allele. interestingly, alignment comparison of these two allele sequences reveals that deletion in the langdon allele extends from nucleotide to nucleotide on the wild type triple dirk allele, thus including fragments  k and  k. hence, while not critical for the low-temperature response, the hypermethylated region observed could play a role in the regulation of the vrn-a gene. but what role? recently, methylation at chhs in the close vicinity of genes was shown to positively correlate with gene expression in maize, suggesting that this methylation mark may be interlinked with gene expression. in arabidopsis, dna methylation at cg and chg sites in the large ibm intron was shown to positively correlate with accumulation of the long transcriptional form of the gene, thus suggesting that intronic dna methylation at cg and chg sites may be required for proper ibm1-l transcript elongation. dna methylation was proposed to prevent spurious expression from cryptic intragenic promoters or enhance accurate splicing of primary transcripts. interestingly, analysis of genes with long introns in arabidopsis revealed that those harboring chg methylation also tend to have te insertions. the ibm protein, which is enriched at intronic tes and which mutation affects transcription of genes with long, te-rich introns, was proposed to avoid premature transcription termination around heterochromatic domains by suppressing antisense transcription from cryptic promoters. presence of te fragments may indeed increase the potential for cryptic transcripts, and the hypermethylation observed in vrn-a intron could be involved in the taming of such transcripts. because intron is kb long, its splicing may also be more challenging to accurately complete than the one of smaller introns. could the inducible hypermethylation observed in te fragments of intron be associated with its splicing? in honeybee, dna methylation in exon was shown to be positively associated with retention. in plants, a recent study demonstrated that the novel pre-mrna splicing factor zop is involved in both pre-mrna splicing and the rddm pathway in arabidopsis and proposed that the splicing machinery may be involved in promoting rddm and transcriptional silencing. a recent study indicated that inclusion of introns in transgenes can increase the transcripts levels of the transgenes in arabidopsis, suggesting a possible role of the splicing machinery in the regulation of rna transcript levels. altogether, these studies suggest that the interconnection between dna methylation, pre-mrna splicing and transcripts levels may be more complex than previously anticipated. further characterization of how the dna methylation pattern observed impacts the splicing of vrn-a intron will likely help better understand this interconnection. in the langdon allele, the kb deletion reduces intron size and deletes te fragments, thus reducing both intron size and probability for cryptic promoters. does the cold-induced hypermethylation observed reduce the impact of splicing and cryptic transcription effects? te fragments are remnants that are not able to move, therefore their silencing is not critical to avoid transposition. however, presence of such fragments within introns both enlarge their size and enhance chances for the emergence of cryptic promoters. while additional studies are needed to further investigate the role of non cg methylation in introns, our study raises the following question: is de novo dna methylation at intronic te fragments a remaining trace of original te silencing, or is it involved in decreasing te-derived transcriptional effects?  our study reveals that vernalization induces hypermethylation of vrn-a at specific non cg sites located in te fragments of large intron which is associated with gene expression. while additional analyses are needed to investigate whether this hypermethylation is a by-product of gene expression or participates to its regulation, even to a low extent, our study provides the first detailed dna methylation characterization of vrn-a observation of a cold-induced dna methylation shift in two winter genotypes following mild vernalization treatment opens the way to examining this dna methylation pattern in a broader set of vernalization conditions and using more time points, as well as in multiple genotypes, thus providing a starting point to investigate the biological role of dna methylation in the wheat vernalization response. our findings on vrn-a also open interesting questions on the role of intronic chh methylation, in particular those which are large and contain te fragments. in particular, whether the hypermethylation observed in vrn-a is also found in vrn-1b and vrn-1d copies remains to be elucidated. if a similar hypermethylation is found in all three copies, investigation of the connection of this hypermethylation to presence of other tes in the b and d copies will help get insights into the underlying mechanism. 
while previous studies in wheat and barley have revealed the functional role of histone modification in setting vrn expression, other mechanisms might also be involved. we provide the first dna methylation analysis of the vrn-a gene, and describe the existence of methylation at cg but also at non cg sites. interestingly, cold induces a site-specific hypermethylation at these non cg sites. the finding that this shift occurs at non cg sites in a te-rich region opens interesting questions onto the possible consequences of this type of methylation in gene expression. while cg sites show a bell-shape profile typical of gene-body methylation, non cg methylation is restricted to the large intron in a region harboring fragments of transposable elements. the genetic bases of vernalization response have been largely studied in wheat, leading to the characterization of a regulation pathway that involves the key gene vernalization. in wheat, winter cultivars require vernalization to initiate flowering, making vernalization requirement a trait of key importance in wheat agronomy. these results demonstrate that vrn-a has a particular dna methylation pattern, exhibiting rapid shift within the life cycle of a winter wheat plant following exposure to particular environmental conditions. here, we were interested in determining whether the cold-induced expression of the wheat vrn-a gene is associated with a change in dna methylation. this increase in dna methylation is transmitted through mitosis, and is reset to its original level after sexual reproduction. vrn1vernalizationdna methylationnon cg methylationwinter wheattransposable elementtriticum aestivumcoldintronjorgesumaya
 certain temperate species require prolonged exposure to low temperature to initiate transition from vegetative growth to flowering, a process known as vernalization. in wheat, winter cultivars require vernalization to initiate flowering, making vernalization requirement a trait of key importance in wheat agronomy. the genetic bases of vernalization response have been largely studied in wheat, leading to the characterization of a regulation pathway that involves the key gene vernalization. while previous studies in wheat and barley have revealed the functional role of histone modification in setting vrn expression, other mechanisms might also be involved. here, we were interested in determining whether the cold-induced expression of the wheat vrn-a gene is associated with a change in dna methylation.  we provide the first dna methylation analysis of the vrn-a gene, and describe the existence of methylation at cg but also at non cg sites. while cg sites show a bell-shape profile typical of gene-body methylation, non cg methylation is restricted to the large intron in a region harboring fragments of transposable elements. interestingly, cold induces a site-specific hypermethylation at these non cg sites. this increase in dna methylation is transmitted through mitosis, and is reset to its original level after sexual reproduction.  these results demonstrate that vrn-a has a particular dna methylation pattern, exhibiting rapid shift within the life cycle of a winter wheat plant following exposure to particular environmental conditions. the finding that this shift occurs at non cg sites in a te-rich region opens interesting questions onto the possible consequences of this type of methylation in gene expression. vrn1vernalizationdna methylationnon cg methylationwinter wheattransposable elementtriticum aestivumcoldintronjorgesumaya 