**4. Histone methylation changes associated with abiotic stress conditions**

Abiotic stresses such as heat, cold, drought, salinity and nutrient deficiency are inherent to every ecosystem and essentially unavoidable. Abiotic stresses are considered the most harmful factors in terms of growth and productivity of crops worldwide [54, 55], especially when they occur in combinations [56]. Here, we summarize and discuss various studies in order to clarify the functional involvement of different histone methylation marks in setting up plant respons‐ es to adverse environmental growth conditions.

### **4.1. Histone methylation/demethylation and the plant stress hormone ABA**

plants, SDG8 may establish a 'permissive' chromatin structure at some *R* genes by methylating H3K36, thus ensuring their basal expression and their transcriptional inducibility upon need. Similarly as *atx1* and *sdg8* mutants, loss-of-function mutants for the putative HKMT *SDG7* were also found to be more sensitive to *Pst* infection than wild-type plants [44]. The expression of other *R* genes seems to be under the control of histone methylation. Indeed, enhanced downy mildew 2 (EDM2) impacts disease resistance by controlling levels of H3K9me2 at an alternative polyadenylation site in the immune receptor gene *RPP7*, thus regulating the balance between full-length *RPP7* transcripts and prematurely polyadenylated transcripts, which do not encode the *RPP7* immune receptor [45, 46]. EDM2, as an epigenetic 'reader', contains two stretches of atypical PHD-finger motifs known to dock specifically several forms of methylated or unmethylated lysine residues on histones [47]. Besides this, EDM2 was also proposed to cooperate within a large protein complex with EMSY-like (AtEML) members, harbouring an

Agenet domain related to the Tudor domain family of epigenetic 'readers' [48].

pathogen, while its mutation reduces the plant resistance [49].

36 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

pathogen infections.

Apart from *Arabidopsis*, little is known about the regulatory role of histone methylation in the defence against pathogen attack in other plant species. In rice, the JmjC protein gene *JMJ705* encoding a histone lysine demethylase that specifically reverses H3K27me2/3 was found induced during infection with the bacterial pathogen *Xanthomonas oryzae*. JMJ705 was further involved in the dynamic removal of the basal H3K27me3 over defence-related genes, thereby increasing their basal expression and/or potentiating their higher expression upon biotic stress. Interestingly, the *JMJ705* overexpression resulted in an enhanced resistance to the bacterial

**3.2. Histone methylation/demethylation in the defence against necrotrophic pathogens**

While to combat biotrophic pathogens the plant activates mainly the SA signalling pathway, the activation of the JA/ET signalling pathway is prominent to mediate defences against necrotrophic pathogens and herbivorous insect attacks [50]. The involvement of histone methylation in the defence against necrotrophic pathogens is far less documented as compared with the defence against biotrophic pathogens. Besides being more susceptible to *Pst* [43], *sdg8* mutants were also reported to be more sensitive to necrotrophic fungal pathogens such as *Alternaria brassicicola* (*Alt*) and *Botrytis cinerea* [51]. This increased susceptibility was the consequence of the inefficient transcriptional induction of different genes along the JA/ET signalling pathway that was correlated with a stably weak level of H3K36me3 at these genes. Inversely, in wild-type plants, H3K36me3 together with gene expression were increased upon *Alt* infection or stimulation with exogenous MeJA. Under resting conditions, a similarly weak level of H3K36me3 was correlated with a reduced basal expression in *sdg8*. On that account, H3K36 methylation was proposed to act as a 'permissive' mark correlated with gene activity and readily in place at a subset of JA/ET signalling-related genes to raise their rapid and efficient transcriptional induction when required [52]. Interestingly, a stable and very low level of H3K27me3 was detected in defence effector genes. Because H3K27me3 is often associated with epigenetic silencing [53], this low H3K27me3 level may provide these genes with a reduced probability for undesired silencing, thus participating in the reactivity of plants to The phytohormone abscisic acid (ABA) is a crucial signalling molecule playing versatile functions in regulating many developmental processes, including seed dormancy and germination [57, 58]. ABA also plays a pivotal role in adaptive stress processes, integrating both biotic and abiotic environmental constraints in a complex network of interacting path‐ ways with crosstalks at different levels [59–61]. Currently, ABA is considered as a global regulator of stress responses that can dominantly control the switch in priority between the responses to biotic or abiotic stress, allowing plants to respond to the most severe threat [62].

The transition from heterotrophic to autotrophic development at the post-germinative stage (i.e. embryonic state) is highly vulnerable to osmotic stress [63]. During a period of osmotic stress, ABA promotes the expression of transcription factors such as ABI3 and ABI5, which in turn delay germination and lead to osmotolerance and survival [64]. In *Arabidopsis*, mutation in *PICKLE* (*PKL*), encoding a putative chromatin modifier, results in increased and abnormally sustained expression of *ABI3* and *ABI5* in response to exogenous ABA treatment. This sustained expression was correlated with reduced levels of H3K9me2 and H3K27me2, two methylation marks found in the chromatin of silent genes [65]. Based on these results, it was suggested that PKL might act on *ABI3* and *ABI5* to promote directly or indirectly the formation of a repressed chromatin state through a so-far-unknown mechanism.

In adult plants, the establishment of a response and tolerance to drought stress by ABA has been extensively studied and is well discussed in several outstanding reviews [58, 66]. Briefly, under drought conditions, water stress perception triggers ABA biosynthesis and increased tissue ABA accumulation, resulting in stomatal closure and reduced transpiration. Among major enzymes involved in the ABA biosynthesis pathway, NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) is thought to be the rate-limiting enzyme [67]. In *Arabidopsis*, the increased transcription of *NCED3* upon dehydration was correlated with the binding of the HKMT ATX1 and the increased level of H3K4me3 at *NCED3* [68]. Therefore, the loss-offunction *atx1* mutant showed less tolerance to dehydration, notably because of the lower enrichment of RNA Polymerase II (RNAPII) and H3K4me3 at *NCED3* under stress. ATX1 modified H3K4me3 may thus have an important function in the transcriptional regulation of *NCED3*. However, it is still unclear whether this function is directly linked with the transcrip‐ tional induction or is more related to the reinforcement of the increased transcription upon stress perception.

#### **4.2. Histone methylation/demethylation in response to water stresses**

Water stresses including drought or submergence are major environmental factors limiting plant growth and crop productivity worldwide [69, 70]. Consequently, plants have evolved a variety of biochemical and physiological mechanisms to respond/adapt to these stresses [71, 72]. In the following section, we distinctly address the involvement of histone methylation in responses to drought and submergence.

#### *4.2.1. Drought stress*

Using *Arabidopsis*, the molecular response to water deficit was found to rely notably on the transcriptional regulation of stress-inducible genes with products thought to function in drought tolerance and response [73]. Using ChIP analyses in 15-day-old *Arabidopsis* seedlings, the level of the active mark H3K4me3 was found gradually enriched in response to dehydra‐ tion stress, preferentially on the coding region of four drought-inducible genes (*RD29A*, *RD29B*, *RD20* and *RAP2.4*), and was correlated with their upregulation [74]. Consistent with this result, ATX1 was reported to be required for the efficient transcriptional induction of *RD29A* and *RD29B* during a dehydration stress response in an ABA-independent manner [68]. Also, for unknown reasons, it is worth noting that upon exogenous ABA treatment the transcriptional induction of *RD29A* and *RD29B* was stronger in *atx1* mutants than in wild-type plants (their basal transcript levels were lower in *atx1* than in wild-type, while their transcript levels upon ABA treatment were identical [68]. Next, for the ABA-dependent *RD29A* and ABAindependent *RAP2.4* genes, a time lag was observed between their transcriptional induction and the increase in H3K4me3 [74]. Based on these findings and the rapid saturation in RNAPII enrichment compared with H3K4me3 (i.e. already saturated 1 h after stress exposure for RNAPII, while H3K4me3 was still increasing up to 5 h), Kim et al. [74] concluded that the H3K4me3 enrichment may be established, gradually, in response to drought stress after full transcriptional activation of *RD29A* and *RAP2.4*. Because the timing of H3K4me3 enrichment followed subsequent to the RNAPII enrichment, H3K4me3 might be dispensable for the initiation of transcription. Finally, the gradual increase of H3K4me3 further indicates that the longer the stress lasts, the more H3K4me3 will be enriched, suggesting that the epigenetic responsiveness must depend on the intensity of a stress [75].

In a similar approach but using ChIP-Seq, van Dijk et al. [76] established the whole-genome distribution patterns of H3K4me1, H3K4me2 and H3K4me3 in 4-week-old rosette *Arabidop‐ sis* leaves under dehydration stress conditions. They also observed a strong correlation between H3K4me3 abundance and transcripts levels from responding genes. Indeed, among the most strongly downregulated genes, an increase in H3K4me1 and a decrease in H3K4me3 were detected, suggesting the involvement of a histone demethylase in modulating the expression of a subset of stress-responsive genes. Supporting this finding, a putative PKDM7 subfamily-like H3K4 demethylase homologue and two putative demethylase enzymes containing a JmjC domain were found to be drought*-*inducible in two barley cultivars and in young peanut plants, respectively [77, 78]. Surprisingly, in contrast to the classical genomewide H3K4me3 enrichment around the transcriptional start site observed in all other eukar‐ yotes [79], H3K4me3 displayed a broader distribution on dehydration and ABA-inducible genes. Such an unusual profile may reflect a function not strictly related to transcription initiation, as will be discussed later in the stress memory section.

Through a genome-wide approach in rice seedling, Zong et al. [80] also uncovered a weak but positive correlation between H3K4me3 enrichment and the transcript level of some droughtresponsive genes under drought stress. This correlation was extended to many genes involved in stress-related metabolite and hormone signalling pathways, further supporting the role played by H3K4me3 in the stress response [80]. However, because H3K4me3 is not the only histone mark for gene activation, this weak correlation may reflect that other active histone marks may also play important roles in regulating gene expression in response to stress in rice. Although these large data sets have provided much information on drought responses in rice, more detailed analyses will be required to elucidate whether the observed variations in H3K4 methylation are a cause or a consequence of the transcriptional changes triggered by water stress. Moreover, identifying key histone modification enzymes is indispensable to better understand the transcriptional regulatory network of the abiotic stress response.

#### *4.2.2. Submergence*

**4.2. Histone methylation/demethylation in response to water stresses**

38 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

responsiveness must depend on the intensity of a stress [75].

responses to drought and submergence.

*4.2.1. Drought stress*

Water stresses including drought or submergence are major environmental factors limiting plant growth and crop productivity worldwide [69, 70]. Consequently, plants have evolved a variety of biochemical and physiological mechanisms to respond/adapt to these stresses [71, 72]. In the following section, we distinctly address the involvement of histone methylation in

Using *Arabidopsis*, the molecular response to water deficit was found to rely notably on the transcriptional regulation of stress-inducible genes with products thought to function in drought tolerance and response [73]. Using ChIP analyses in 15-day-old *Arabidopsis* seedlings, the level of the active mark H3K4me3 was found gradually enriched in response to dehydra‐ tion stress, preferentially on the coding region of four drought-inducible genes (*RD29A*, *RD29B*, *RD20* and *RAP2.4*), and was correlated with their upregulation [74]. Consistent with this result, ATX1 was reported to be required for the efficient transcriptional induction of *RD29A* and *RD29B* during a dehydration stress response in an ABA-independent manner [68]. Also, for unknown reasons, it is worth noting that upon exogenous ABA treatment the transcriptional induction of *RD29A* and *RD29B* was stronger in *atx1* mutants than in wild-type plants (their basal transcript levels were lower in *atx1* than in wild-type, while their transcript levels upon ABA treatment were identical [68]. Next, for the ABA-dependent *RD29A* and ABAindependent *RAP2.4* genes, a time lag was observed between their transcriptional induction and the increase in H3K4me3 [74]. Based on these findings and the rapid saturation in RNAPII enrichment compared with H3K4me3 (i.e. already saturated 1 h after stress exposure for RNAPII, while H3K4me3 was still increasing up to 5 h), Kim et al. [74] concluded that the H3K4me3 enrichment may be established, gradually, in response to drought stress after full transcriptional activation of *RD29A* and *RAP2.4*. Because the timing of H3K4me3 enrichment followed subsequent to the RNAPII enrichment, H3K4me3 might be dispensable for the initiation of transcription. Finally, the gradual increase of H3K4me3 further indicates that the longer the stress lasts, the more H3K4me3 will be enriched, suggesting that the epigenetic

In a similar approach but using ChIP-Seq, van Dijk et al. [76] established the whole-genome distribution patterns of H3K4me1, H3K4me2 and H3K4me3 in 4-week-old rosette *Arabidop‐ sis* leaves under dehydration stress conditions. They also observed a strong correlation between H3K4me3 abundance and transcripts levels from responding genes. Indeed, among the most strongly downregulated genes, an increase in H3K4me1 and a decrease in H3K4me3 were detected, suggesting the involvement of a histone demethylase in modulating the expression of a subset of stress-responsive genes. Supporting this finding, a putative PKDM7 subfamily-like H3K4 demethylase homologue and two putative demethylase enzymes containing a JmjC domain were found to be drought*-*inducible in two barley cultivars and in young peanut plants, respectively [77, 78]. Surprisingly, in contrast to the classical genomewide H3K4me3 enrichment around the transcriptional start site observed in all other eukar‐ yotes [79], H3K4me3 displayed a broader distribution on dehydration and ABA-inducible

Submergence is a complex stress that encompasses many changes in environmental factors, including light intensity, pH and dissolved oxygen concentration. Alcoholic fermentation is important for the survival of plants especially under anaerobic environments [81]. In rice, *alcohol dehydrogenase 1* (*ADH1*) and *pyruvate decarboxylase 1* (*PDC1*) genes are involved in this anaerobic metabolism and their expression is reversibly induced (i.e. activated upon submer‐ gence and repressed upon re-aeration) [82]. Using these two genes as a model, Tsuji et al. [83] observed that the level of H3K4 methylation, specifically at both the 5′- and 3′-coding regions of *ADH1* and *PDC1*, was changed from a dimethylated state to a trimethylated state upon their transcriptional upregulation in response to submergence. This change was reverted back to its initial level following re-aeration, indicating that in this particular case, H3K4me3 does not serve as a memory mark of a prior transcriptional activity. Similarly to drought, these results highlight the dynamic and reversible change of histone H3K4 methylation at stress-related genes in response to the occurrence and disappearance of a stress.

#### **4.3. Histone methylation/demethylation in response to salt stress**

Salinity is also a serious factor affecting plants in several ways (i.e. water stress, ion toxicity, nutritional disorders, oxidative stress, alteration of metabolic processes, membrane disorga‐ nization, genotoxicity, reduction of cell division and expansion), thus limiting plant growth, development and survival [84]. In *Arabidopsis*, a NaCl or ABA treatment has been shown to induce the transcription of a range of abiotic stress-responsive genes (*ABI1*, *ABI2*, *KAT1*, *KAT2*, *DREB2A*, *RD29A* and *RD29B*). Using ChIP, this induction was found significantly correlated with an increase in the active mark H3K4me3 and a decrease in the repressive mark H3K9me2 [85]. Also, suggesting a link between different histone PTMs, mutations in the histone deacetylase HDA6 partially suppressed the H3K4me3 increase observed in response to stress, while the H3K9me2 decrease was not affected. Since histone acetylation and H3K4 trimethy‐ lation are often associated with gene activation [86], the repressive function of HDA6 may suggest that acetylation on histone H3 is required for K4 methylation to occur [87]. Unfortu‐ nately, because ChIP experiments were not normalised against the total H3 density, it is difficult to attribute the decrease in H3K9me2 to either the active removal of methyl groups by a histone demethylases or the active removal of nucleosome in response to a stress. Supporting the second possibility, the nucleosome density of two other drought stressinducible genes (*RD20* and *RAP2.4*) was found to gradually decrease in response to drought stress [74].

In soya bean and in response to a high NaCl concentration, these histone methylation marks were also found altered at some salinity-induced transcription factors (i.e. MYB, b-ZIP and AP2/DREB family members) that were primarily identified by microarray analysis [88]. For some genes, their transcriptional induction was correlated with an increased level of histone acetylation and H3K4me3, accompanied or not with a reduced level of DNA methylation and H3K9me2 in various parts of the promoter or coding regions. For other genes, DNA methyl‐ ation had no influence on histone methylation. This work perfectly reflects the heterogeneity of the effect of salinity on histone methylation and DNA methylation, and supports the role(s) of histone methylation changes in the expression of some transcription factors important for salinity tolerance.

As mentioned above, H3K4me3 was found to be involved in the transcriptional induction of stress-responsive gene upon salt stress exposure. In plants, the JmjC-domain-containing histone demethylases JMJ14, JMJ15 and JMJ18 have been reported to display an H3K4me2/3 demethylase activity as well as to regulate diverse aspects of chromatin function and devel‐ opment [89–95]. Recently, the overexpression of *JMJ15* was reported to preferentially down‐ regulate many stress-related genes preferentially marked by H3K4me2/3 and to enhance salt stress tolerance [96]. In contrast, the loss-of-function mutant was more sensitive to salt. Despite the fact that an increased JMJ15 level may regulate stress-responsive gene transcription programmes in *Arabidopsis*, the role of H3K4me3 resetting in these processes is still very elusive.

Besides histone lysine methylation, arginine methylation was also involved in establishing the transcriptional response to salt stress. The protein arginine methyltransferase 5 (PRMT5), also named Shk1 kinase-binding protein1 (SKB1), is a type II methyltransferase that catalyses symmetric H4R3 dimethylation, a repressive mark known to promote flowering through the repression of the floral repressor *FLOWERING LOCUS C* (*FLC*) in *Arabidopsis* [20, 97]. Inter‐ estingly, gain-of-function *SKB1* mutants showed an enhanced salt stress tolerance and sensitivity to ABA [98]. As a consequence of PRMT5 disassociating from chromatin, the H4R3sme2 level at stress-responsive genes was reduced during salt stress, resulting in their induced expression. Suggesting an additional function of PRMT5 on non-histone proteins, the methylation level of the U6 small nuclear ribonucleoprotein Sm-like4 (LSM4, a core protein of the spliceosome) was increased in response to salt stress and ABA. Since splicing defects were observed in the *prmt5*/*skb1* and *lsm4* mutants, with both of them being hypersensitive to salt stress, authors proposed that PRMT5 might mediate plant development and salt response by altering the methylation status of H4R3me2 and LSM4, linking transcriptional regulation to pre-mRNA splicing [98].

#### **4.4. Histone methylation/demethylation in response to temperature**

In plants, temperature stresses are classically classified into different types according to temperature exposure, which may be warm, high, chilling or freezing temperature. Due to global warming and because temperature stress greatly affects plant growth and development, immunity and circadian rhythm, and poses a serious threat to the global food supply, the genetic mechanisms of plant responses to heat have been well studied. Plants exposed to temperature stresses modulate the transcription of a large number of genes involved in distinct biochemical and physiological response pathways and networks of phytohormones or secondary metabolites, ultimately leading to increased tolerance to hazardous temperature stresses [99–102]. The role played by histone methylation during the plant response to a heat or a cold stress is discussed separately hereafter.

#### *4.4.1. Heat stress*

suggest that acetylation on histone H3 is required for K4 methylation to occur [87]. Unfortu‐ nately, because ChIP experiments were not normalised against the total H3 density, it is difficult to attribute the decrease in H3K9me2 to either the active removal of methyl groups by a histone demethylases or the active removal of nucleosome in response to a stress. Supporting the second possibility, the nucleosome density of two other drought stressinducible genes (*RD20* and *RAP2.4*) was found to gradually decrease in response to drought

40 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

In soya bean and in response to a high NaCl concentration, these histone methylation marks were also found altered at some salinity-induced transcription factors (i.e. MYB, b-ZIP and AP2/DREB family members) that were primarily identified by microarray analysis [88]. For some genes, their transcriptional induction was correlated with an increased level of histone acetylation and H3K4me3, accompanied or not with a reduced level of DNA methylation and H3K9me2 in various parts of the promoter or coding regions. For other genes, DNA methyl‐ ation had no influence on histone methylation. This work perfectly reflects the heterogeneity of the effect of salinity on histone methylation and DNA methylation, and supports the role(s) of histone methylation changes in the expression of some transcription factors important for

As mentioned above, H3K4me3 was found to be involved in the transcriptional induction of stress-responsive gene upon salt stress exposure. In plants, the JmjC-domain-containing histone demethylases JMJ14, JMJ15 and JMJ18 have been reported to display an H3K4me2/3 demethylase activity as well as to regulate diverse aspects of chromatin function and devel‐ opment [89–95]. Recently, the overexpression of *JMJ15* was reported to preferentially down‐ regulate many stress-related genes preferentially marked by H3K4me2/3 and to enhance salt stress tolerance [96]. In contrast, the loss-of-function mutant was more sensitive to salt. Despite the fact that an increased JMJ15 level may regulate stress-responsive gene transcription programmes in *Arabidopsis*, the role of H3K4me3 resetting in these processes is still very

Besides histone lysine methylation, arginine methylation was also involved in establishing the transcriptional response to salt stress. The protein arginine methyltransferase 5 (PRMT5), also named Shk1 kinase-binding protein1 (SKB1), is a type II methyltransferase that catalyses symmetric H4R3 dimethylation, a repressive mark known to promote flowering through the repression of the floral repressor *FLOWERING LOCUS C* (*FLC*) in *Arabidopsis* [20, 97]. Inter‐ estingly, gain-of-function *SKB1* mutants showed an enhanced salt stress tolerance and sensitivity to ABA [98]. As a consequence of PRMT5 disassociating from chromatin, the H4R3sme2 level at stress-responsive genes was reduced during salt stress, resulting in their induced expression. Suggesting an additional function of PRMT5 on non-histone proteins, the methylation level of the U6 small nuclear ribonucleoprotein Sm-like4 (LSM4, a core protein of the spliceosome) was increased in response to salt stress and ABA. Since splicing defects were observed in the *prmt5*/*skb1* and *lsm4* mutants, with both of them being hypersensitive to salt stress, authors proposed that PRMT5 might mediate plant development and salt response by altering the methylation status of H4R3me2 and LSM4, linking transcriptional regulation to

stress [74].

salinity tolerance.

elusive.

pre-mRNA splicing [98].

Heat stress during seed development decreases the seed size in many cereals, resulting in severe yield losses [103, 104]. In rice, a molecular mechanism involving the putative rice polycomb repressive complex 2 (PRC2) gene *Fertilization-Independent Endosperm1* (*OsFIE1*) was suggested as being a potential key component involved in regulating the thermal sensitivity of seed enlargement during endosperm development [105]. When developing seeds were exposed to a heat stress, both DNA methylation and H3K9 methylation were reduced on *OsFIE1* resulting in its derepression. Under heat stress, syncytial stage-specific MADS-box genes involved in seed size regulation were precociously repressed, due to the increased deposition of H3K27me3 silencing marks by the PRC2 complex [105]. In the unicellular green alga *Chlamydomonas reinhardtii*, histone modification was also affected by heat stress [106]. Indeed, after heat stress, the level of H3K4me1 was found decreased and the level of histone acetylation increased at promoter regions of active genes compared with inactive regions. As a hypothesis, authors proposed that upon heat stress, the heat shock transcription factor HSF1 might promote chromatin remodelling and RNAPII recruitment for transcription initiation/ elongation [106]. Finally, while addressing molecular mechanisms of the response of cotton anthers to high temperature, two jmjC domain-containing genes, putatively involved in histone demethylation, were found significantly repressed during anther development under heat stress [107].

Because euchromatin is gene rich and usually transcriptionally active, investigation about the role of histone methylation in temperature stress acclimation was largely centred on euchro‐ matin-associated coding regions. Focusing on the transcriptionally silent heterochromatin, mainly constituted of repetitive DNA sequences, some works demonstrated the transcriptional activation of normally silent transposable element embedded within heterochromatic regions under stress conditions [108]. Intriguingly, such activation under heat stress can occur without alteration of DNA methylation and with only minor changes in both H3K9me2 and H3K4me3 [109, 110]. In summary, these works suggest that temperature stress-mediated transcription of tandem-repeat elements might play a vital role in the adaptation of plants to temperature stimuli, offering an efficient mechanism by which heat or cold could promote the expression of some stress-responsive genes. Upon activation and when inserted into or very close to a gene, such transposable elements could interfere with the expression of this gene, giving rise to deleterious mutations, genetic instability or positive contribution to gene regulation and adaptation [111].

#### *4.4.2. Cold stress*

The increased tolerance of plants to cold is referred as 'cold acclimation'. Cold acclimation differs from vernalization, as the last one requires a long-term exposure to cold temperatures, while cold acclimation can be achieved in a couple of days under non-freezing low tempera‐ tures [112]. Locally, histone methylation changes in cold-responsive genes were addressed in *Arabidopsis*. Upon cold stress, the repressive mark H3K27me3 decreased, in both a histone occupancy-dependent (i.e. arising from the lowering nucleosome density) and -independent (i.e. as the result of the activity of a not yet identified histone demethylase) manner, on the cold-responsive genes, cold-regulated 15A (*COR15A*) and galactinol synthase 3 (*ATGOLS3*; [113]). Interestingly, the decrease in H3K27me3 upon stimulation occurred more gradually than their rapid transcriptional induction, so that their activation may not be inhibited by H3K27me3 itself but rather lead to the removal of H3K27me3. Also, while the transcription of *COR15A* and *ATGOLS3* was completely repressed to the initial level upon returning plants to normal growth conditions, the H3K27me3 decrease was maintained. Given that this decrease does not affect the transcriptional induction of *COR15A* and *ATGOLS3* upon re-exposure to cold temperatures, such chromatin change can so far only be view as a 'reminiscence' of a recent transcriptional activity and not as a stress memory implicated in a gene priming process.

In maize during cold stress, changes in histone modifications, including the heterochromatic marks H3K9me2 and DNA methylation, were assessed through a genome-wide approach [114]. The more detailed analysis of the two knob-associated tandem-repetitive sequences, the 180-bp repeat and the 350-bp repeat termed TR-1, demonstrated that their selectively and transiently cold-activated transcription was correlated with a decreased H3K9me2 and DNA methylation, together with an increased H3K9 acetylation. Such cold-induced transcriptional activation of tandem repeats is selective and transient, and the silencing state is recovered as the treatment continues.

#### **5. Histone methylation as a memory mark of stress**

In animals, the formation of memory immune cells after primary antigen recognition confers long-lasting resistance, resulting in an accelerated and a more effective immune response in case of second exposure. Despite the absence of such memory immune cells, plants often acquire a systemic immunity to further infections after a primary localized infection [115]. This requires the accumulation of the plant hormone SA in systemic tissues and is called systemic acquired resistance (reviewed in [116]. The SAR is also associated with gene priming in systemic tissues, in which defence genes will be expressed more rapidly and robustly in case of a second attack [117]. At the transcriptional level, gene expression is primarily influenced by the chromatin structure, which in turn is controlled partly by processes, often referred to as 'epigenetic' processes, which can be transmitted through mitosis and/or inherited through meiosis [118]. Therefore, chromatin remodelling through histone methylation offers a potential mechanism for short-/long-term stress memory within the lifespan of an individual, referred to as somatic memory, and/or across generations, referred to as transgenerational memory.

#### **5.1. Somatic stress memory**

gene, such transposable elements could interfere with the expression of this gene, giving rise to deleterious mutations, genetic instability or positive contribution to gene regulation and

42 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

The increased tolerance of plants to cold is referred as 'cold acclimation'. Cold acclimation differs from vernalization, as the last one requires a long-term exposure to cold temperatures, while cold acclimation can be achieved in a couple of days under non-freezing low tempera‐ tures [112]. Locally, histone methylation changes in cold-responsive genes were addressed in *Arabidopsis*. Upon cold stress, the repressive mark H3K27me3 decreased, in both a histone occupancy-dependent (i.e. arising from the lowering nucleosome density) and -independent (i.e. as the result of the activity of a not yet identified histone demethylase) manner, on the cold-responsive genes, cold-regulated 15A (*COR15A*) and galactinol synthase 3 (*ATGOLS3*; [113]). Interestingly, the decrease in H3K27me3 upon stimulation occurred more gradually than their rapid transcriptional induction, so that their activation may not be inhibited by H3K27me3 itself but rather lead to the removal of H3K27me3. Also, while the transcription of *COR15A* and *ATGOLS3* was completely repressed to the initial level upon returning plants to normal growth conditions, the H3K27me3 decrease was maintained. Given that this decrease does not affect the transcriptional induction of *COR15A* and *ATGOLS3* upon re-exposure to cold temperatures, such chromatin change can so far only be view as a 'reminiscence' of a recent transcriptional activity and not as a stress memory implicated in a gene priming process.

In maize during cold stress, changes in histone modifications, including the heterochromatic marks H3K9me2 and DNA methylation, were assessed through a genome-wide approach [114]. The more detailed analysis of the two knob-associated tandem-repetitive sequences, the 180-bp repeat and the 350-bp repeat termed TR-1, demonstrated that their selectively and transiently cold-activated transcription was correlated with a decreased H3K9me2 and DNA methylation, together with an increased H3K9 acetylation. Such cold-induced transcriptional activation of tandem repeats is selective and transient, and the silencing state is recovered as

In animals, the formation of memory immune cells after primary antigen recognition confers long-lasting resistance, resulting in an accelerated and a more effective immune response in case of second exposure. Despite the absence of such memory immune cells, plants often acquire a systemic immunity to further infections after a primary localized infection [115]. This requires the accumulation of the plant hormone SA in systemic tissues and is called systemic acquired resistance (reviewed in [116]. The SAR is also associated with gene priming in systemic tissues, in which defence genes will be expressed more rapidly and robustly in case of a second attack [117]. At the transcriptional level, gene expression is primarily influenced by the chromatin structure, which in turn is controlled partly by processes, often referred to

**5. Histone methylation as a memory mark of stress**

adaptation [111].

*4.4.2. Cold stress*

the treatment continues.

In *Arabidopsis*, a priming event, either treatment with BTH or infection with *Pseudomonas syringae pv. maculicola*, systematically resulted in an increase in the level of H3K4me2/me3 at defence gene promoters (namely the WRKY transcription factors *WRKY6*, *WRKY29* and *WRKY52*) that are normally found on active genes, while the genes remain inactive [119]. More interestingly, this increase also occurs in leaves distal to localized foliar infection. Hence, even if the histone-modifying enzyme involved in this process remains unidentified, results from Jaskiewicz et al. [119] clearly suggest that histone methylation might create a 'memory' of the primary infection that is associated with an amplified reaction to a second stress stimulus. Further, Luna et al. [120] observed that promoters of SA-inducible *PR1*, *WRKY6* and *WRKY53* in the progeny of *Pst*-inoculated *Arabidopsis* plants were enriched with acetylated histone, while the promoter of the JA-inducible gene *PLANT DEFENSIN1.2* (*PDF1.2*) showed an increased level of H3K27me3. For decades, the signalling protein *NON EXPRESSOR OF PR1* (NPR1) has been implicated in mediating SAR induction [115] and also the crosstalk between SA- and JA/ET-dependent defence pathways, enabling plants to mount an appropriate defence reaction, depending on the nature of the attacker and the stage of infection [121, 122]. More recently, NPR1 has been proposed to play a critical role in the expression of the transgenera‐ tional SAR as progeny from *npr1* failed to develop transgenerational defence phenotypes and failed to present enrichment for H3K27me3 at the *PDF1.2* promoter [120]. Together, these findings suggested that one or more systemic signals are stored as an immune memory on defence-related gene promoters in the form of histone modifications, thus providing the plant with a life-long protection, which can be transmitted to subsequent generations.

Besides being involved in defence priming related to biotic stress, histone methylation was also proposed as a priming strategy against drought. To further explore the functional impact of histone methylation on biotic stress responses in *Arabidopsis*, Kim et al. [123] followed chromatin dynamics of several drought genes (*RD20*, *RD29A* and *AtGOLS2*) and a rehydra‐ tion-inducible gene (*ProDH*) during drought and rehydration. As previously discussed, a strong correlation was observed between H3K4me3 enrichment (i.e. especially in gene bodies) and transcription for drought genes upon drought [74, 123]. Such a correlation was also detected for the rehydration gene upon rehydration [123]. Suggesting a memory role for H3K4me3, RNAPII rapidly disappeared after rehydration at drought genes, while H3K4me3 was gradually decreased. Concomitantly, by training plants with up to four successive drought treatments, Ding et al. [124] uncovered the existence of two distinct subsets of genes within the dehydration stress–response gene fraction. The 'non-trainable' genes (e.g. the ABAindependent *RD29A* and *COR15A*) have repetitively similar transcription rates during each stress treatment, while 'trainable' ones (e.g. the ABA-dependent *RD29B* and *RAB18*) increased the magnitude of their subsequent transcriptional response, relative to their initial stress response. Using ChIP, Ding et al. [124] observed that the H3K4me3 enrichment at 'trainable' genes, especially in gene bodies, was atypically retained from the preceding transcription after rehydration. Even more interestingly, the RNAPII phosphorylated at C-terminal domain (CTD) repeat serine 5 (Ser5P; associated with transcription initiation) was found stalled on these genes as a memory mark from a previously transcribed state. In contrast to 'trainable' genes, the stress-induced H3K4me3 and Ser5P enrichment at 'non-trainable' genes was decreased to its basal level during recovery [124]. Moreover, this transcriptional memory can persist in the absence of inducing signals at least for 5 days, but is lost after 7 days. Supporting a specific role for H3K4me3 in stress memory, other active chromatin marks such as acetylation of histones H3 and H4 were found rapidly increased at drought genes upon stress and decreased at comparable levels as before induction quickly after recovery [123, 124]. Consistent with an activating role of ATX1 at dehydration stress–response genes [68, 125, 126], dehydra‐ tion-induced transcript levels were diminished in *atx1* plants [127]. However, ATX1 does not seem to have a critical impact on drought stress memory in *Arabidopsis*. Indeed, while being less increased than in wild-type plants, trainable genes still produced increased transcripts in trained, relative to untrained, *atx1* plants and retained high H3K4me3 levels during the watered recovery states. ATX1, ATX2, SDG25 and SET DOMAIN GROUP2/ATX-RELATED3 (SDG2/ATXR3) belong to the same class III of H3K4me3 methyltransferases and are thought to act, partially redundantly, as H3K4 methyltransferase [128–134]. More recently, SDG2 has been found to be essential for the full transcriptional activation of various hormone-responsive genes upon hormone treatment (i.e. including the ABA-dependant *RD29A*) via its H3K4 trimethyltransferase activity [135]. It is, therefore, likely that the other class III HKMT might also contribute to the drought stress memory. In summary, these results suggest that in addition to be a good marker of gene activation when found around promoter and 5′ regions of genes, H3K4me3 might also play a role in establishing a transcriptional short-term somatic memory of drought stress when found in gene bodies.

Using a large-scale approach, the distribution of H3K4me2, H3K4me3, H3K9me2 and H3K27me3 was analysed in *Arabidopsis* seedlings, which have been treated with mild salt stress in the seedling stage, resulting in an increased tolerance upon an additional salt stress application [136]. At low resolution in primed seedlings, H3K4me2 and H3K4me3 most commonly consisted of higher peaks of pre-existing enriched histone modification domains, named islands, whereas H3K9me2 produced the least differences. By contrast, majority of differences in H3K27me3 resulted from a higher number of islands with lower genome coverage. At high resolution, changes in H3K27me3 were already detectable a few hours after salt addition, suggesting that demethylation of H3K27me3 operates at a speed that is compa‐ rable to that of transcriptional regulation. Interestingly, this effect fades over time; however, it is still clearly visible after a 10-day-growth period in control conditions. In response to a second stress treatment, genes with high responsiveness, such as *HKT1* (i.e. encoding a rootspecific Na transporter) and *PIP2E* (i.e. encoding a plasma membrane aquaporin), experienced a decrease of H3K27me3, whereas genes with lower responsiveness, such as *GH3.1* and *GH3.3* (i.e. encoding auxin and JA-amino acid-conjugating enzymes, respectively), experienced an increase of H3K27me3. Conversely, another group reported that in plants that have experi‐ enced several exposures to dehydration stress no significant change in the level of H3K27me3 could be detected on trainable and non-trainable genes, or during transcriptionally active/ inactive gene states [137]. However, the high H3K27me3 level present at inactive dehydration stress memory genes did not interfere with the transition to an active transcription and with the accumulation of H3K4me3 [138]. Together, the function of H3K27me3 in genes that dynamically change transcription seems to depend on the type of environmental stimuli.

In contrast to H3K27me3, the higher level of H3K4me3 retained at the trainable gene *RD29B*, when its transcription is low, further supports the idea that H3K4me3 works as a 'memory' histone mark of a previously active state [138]. Generally, H3K4me3 and H3K27me3 play antagonistic roles in gene transcription and are therefore mutually repulsive at developmental genes [37, 139]. Seemingly, the presence of both marks, referred as 'bivalent domains', was first described in mammalian stem cells and was proposed to represent a pluripotent chro‐ matin state that poises genes for activation upon appropriate developmental cues [140, 141]. Further work is required to determine whether H3K4me3 and H3K27me3 co-exist at certain genes whose expression is rapidly altered in response to environmental stimuli.

Because plant stress research has traditionally focused on single stresses, we separately described priming in response to abiotic or biotic environmental cues. However, in nature, plants are constantly exposed to mild environmental stresses during their lifetime. While testing how different environmental histories can affect the response of the plant to a subse‐ quent biotic stress, Singh et al. [142] reported that *Arabidopsis* plants exposed to a recurrent abiotic stress (i.e. heat, cold or salt) were more resistant to *Pst* than plants grown in a more stable environment. This enhanced resistance was due to the priming of commonly used marker genes of pattern-triggered immunity (PTI; *WRKY53*, *FLG22-INDUCED RECEPTOR KINASE1* (*FRK1*) and *NDR1*/*HIN1-LIKE10* (*NHL10*)). Indeed, enrichment for epigenetic marks associated with transcriptional activation, such as H3K4me2 and H3K4me3, at PTI-responsive genes was observed after the exposure to recurrent stress, resulting in an enrichment of RNA polymerase II and a primed transcription in response to a subsequent bacterial infection. Collectively, these works on somatic stress memory promote the idea that, in plant, the environmental history can shape/modulate the response to stress, providing a mechanistic link between histone methylation and gene priming.

#### **5.2. Transgenerational stress memory**

the magnitude of their subsequent transcriptional response, relative to their initial stress response. Using ChIP, Ding et al. [124] observed that the H3K4me3 enrichment at 'trainable' genes, especially in gene bodies, was atypically retained from the preceding transcription after rehydration. Even more interestingly, the RNAPII phosphorylated at C-terminal domain (CTD) repeat serine 5 (Ser5P; associated with transcription initiation) was found stalled on these genes as a memory mark from a previously transcribed state. In contrast to 'trainable' genes, the stress-induced H3K4me3 and Ser5P enrichment at 'non-trainable' genes was decreased to its basal level during recovery [124]. Moreover, this transcriptional memory can persist in the absence of inducing signals at least for 5 days, but is lost after 7 days. Supporting a specific role for H3K4me3 in stress memory, other active chromatin marks such as acetylation of histones H3 and H4 were found rapidly increased at drought genes upon stress and decreased at comparable levels as before induction quickly after recovery [123, 124]. Consistent with an activating role of ATX1 at dehydration stress–response genes [68, 125, 126], dehydra‐ tion-induced transcript levels were diminished in *atx1* plants [127]. However, ATX1 does not seem to have a critical impact on drought stress memory in *Arabidopsis*. Indeed, while being less increased than in wild-type plants, trainable genes still produced increased transcripts in trained, relative to untrained, *atx1* plants and retained high H3K4me3 levels during the watered recovery states. ATX1, ATX2, SDG25 and SET DOMAIN GROUP2/ATX-RELATED3 (SDG2/ATXR3) belong to the same class III of H3K4me3 methyltransferases and are thought to act, partially redundantly, as H3K4 methyltransferase [128–134]. More recently, SDG2 has been found to be essential for the full transcriptional activation of various hormone-responsive genes upon hormone treatment (i.e. including the ABA-dependant *RD29A*) via its H3K4 trimethyltransferase activity [135]. It is, therefore, likely that the other class III HKMT might also contribute to the drought stress memory. In summary, these results suggest that in addition to be a good marker of gene activation when found around promoter and 5′ regions of genes, H3K4me3 might also play a role in establishing a transcriptional short-term somatic

Using a large-scale approach, the distribution of H3K4me2, H3K4me3, H3K9me2 and H3K27me3 was analysed in *Arabidopsis* seedlings, which have been treated with mild salt stress in the seedling stage, resulting in an increased tolerance upon an additional salt stress application [136]. At low resolution in primed seedlings, H3K4me2 and H3K4me3 most commonly consisted of higher peaks of pre-existing enriched histone modification domains, named islands, whereas H3K9me2 produced the least differences. By contrast, majority of differences in H3K27me3 resulted from a higher number of islands with lower genome coverage. At high resolution, changes in H3K27me3 were already detectable a few hours after salt addition, suggesting that demethylation of H3K27me3 operates at a speed that is compa‐ rable to that of transcriptional regulation. Interestingly, this effect fades over time; however, it is still clearly visible after a 10-day-growth period in control conditions. In response to a second stress treatment, genes with high responsiveness, such as *HKT1* (i.e. encoding a rootspecific Na transporter) and *PIP2E* (i.e. encoding a plasma membrane aquaporin), experienced a decrease of H3K27me3, whereas genes with lower responsiveness, such as *GH3.1* and *GH3.3* (i.e. encoding auxin and JA-amino acid-conjugating enzymes, respectively), experienced an

memory of drought stress when found in gene bodies.

44 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

The transgenerational stress memory refers to the transmittance of certain environmental responses from one generation to the next, thus providing the offspring of environmentally challenged plants with an adaptive advantage for better fitness (i.e. improve plant stress tolerance and impart developmental flexibility; [143]). Compared with DNA methylation and RNA interference (RNAi), very few studies suggest the involvement of histone methyltrans‐ ferases and histone methylation changes in this process [144]. In *Arabidopsis*, changes in DNA methylation, histone modifications and gene expression were followed in the progeny of plants exposed to salt stress over one generation [145]. Although the DNA from the progeny of plants exposed to salt stress was globally hypomethylated, the majority of genes and promoters causing methylation changes were hypermethylated and lowly expressed. In addition, DNA hypermethylation was correlated with an increased level of the repressive mark H3K9me2. Among these hypermethylated genes, a large number was encoding different histone meth‐ yltransferases, such as the *Arabidopsis SU(VAR)3-9* homologues *SUVH2*, *SUVH5*, *SUVH8*, involved in H3K9 methylation, or the PRC2 subunit *CURLY LEAF* (*CLF*), involved in H3K27 methylation. Following this work, the progeny of heat-stressed plants was used to explore epigenetic variations under both normal and stressed conditions, in comparison to the progeny of control plants [146]. Similarly to salt stress, the progeny of plants exposed to heat stress had a global decrease of genomic DNA methylation and a reduced expression of several *SUVH* genes, which correlated with their enrichment in H3K9me2. Together, the hypermethylation of SUVH genes in the progeny of stressed plants may represent a protective mechanism against hypermethylation of the entire genome. Interestingly, in both works [145, 146], the transposon expression was elevated in the progeny of stressed plants. Because main targets of the SUVH pathway are transposable elements [24], the authors proposed that a decrease in the expression of *SUVH* genes might contribute to transposon activation, which at opportune times can create intragenomic potential upon transposition to facilitate adaptation in response to environmen‐ tal changes [147]. In summary, these works suggested a role for histone methylation in the inheritance of stress memory; however, whether histone methylation changes are heritable through multiple generations and whether they sustain the acquisition of adaptive traits is still a matter of debate [148]. However, the evidence to date favours the view that stress-induced transgenerational changes in chromatin might increase the survival chances of the plant species, rather than each individual, by broadening the phenotypic plasticity and the genetic variation within the population [149, 150].

#### **6. Discussion and perspectives**

Recent advances, especially in *Arabidopsis*, have uncovered that chromatin remodelling through histone methylation changes are not only restricted to developmental needs but also an integral part of the very complex cascade of events that lead to abiotic/biotic stress tolerance, resistance and short-/long-term memory. Currently, a preliminary view is emerging, indicat‐ ing that histone methylation changes, providing specific chromatin configurations, can be classified into several interrelated categories when involved in stress responses (Fig. 1): (*i*) histone methylation changes that are basally present on stress-related genes to establish a 'permissive' chromatin state that may either limit the spreading of repressive chromatin marks and/or potentiate a rapid transcriptional induction upon need; (*ii*) histone methylation changes that are transiently induced from an inactive or a permissive chromatin state by stress, to either facilitate the transcriptional initiation and/or reinforce transcription of stress-responding genes, and finally, histone methylation changes that are established in response to a stress; (*iii*) maintained for a certain time during the lifespan of an individual (i.e. somatic memory) or (*iv*) transmitted to one or more subsequent generations (i.e. transgenerational memory).

Histone Methylation - A Cornerstone for Plant Responses to Environmental Stresses? http://dx.doi.org/10.5772/61733 47

causing methylation changes were hypermethylated and lowly expressed. In addition, DNA hypermethylation was correlated with an increased level of the repressive mark H3K9me2. Among these hypermethylated genes, a large number was encoding different histone meth‐ yltransferases, such as the *Arabidopsis SU(VAR)3-9* homologues *SUVH2*, *SUVH5*, *SUVH8*, involved in H3K9 methylation, or the PRC2 subunit *CURLY LEAF* (*CLF*), involved in H3K27 methylation. Following this work, the progeny of heat-stressed plants was used to explore epigenetic variations under both normal and stressed conditions, in comparison to the progeny of control plants [146]. Similarly to salt stress, the progeny of plants exposed to heat stress had a global decrease of genomic DNA methylation and a reduced expression of several *SUVH* genes, which correlated with their enrichment in H3K9me2. Together, the hypermethylation of SUVH genes in the progeny of stressed plants may represent a protective mechanism against hypermethylation of the entire genome. Interestingly, in both works [145, 146], the transposon expression was elevated in the progeny of stressed plants. Because main targets of the SUVH pathway are transposable elements [24], the authors proposed that a decrease in the expression of *SUVH* genes might contribute to transposon activation, which at opportune times can create intragenomic potential upon transposition to facilitate adaptation in response to environmen‐ tal changes [147]. In summary, these works suggested a role for histone methylation in the inheritance of stress memory; however, whether histone methylation changes are heritable through multiple generations and whether they sustain the acquisition of adaptive traits is still a matter of debate [148]. However, the evidence to date favours the view that stress-induced transgenerational changes in chromatin might increase the survival chances of the plant species, rather than each individual, by broadening the phenotypic plasticity and the genetic

46 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

Recent advances, especially in *Arabidopsis*, have uncovered that chromatin remodelling through histone methylation changes are not only restricted to developmental needs but also an integral part of the very complex cascade of events that lead to abiotic/biotic stress tolerance, resistance and short-/long-term memory. Currently, a preliminary view is emerging, indicat‐ ing that histone methylation changes, providing specific chromatin configurations, can be classified into several interrelated categories when involved in stress responses (Fig. 1): (*i*) histone methylation changes that are basally present on stress-related genes to establish a 'permissive' chromatin state that may either limit the spreading of repressive chromatin marks and/or potentiate a rapid transcriptional induction upon need; (*ii*) histone methylation changes that are transiently induced from an inactive or a permissive chromatin state by stress, to either facilitate the transcriptional initiation and/or reinforce transcription of stress-responding genes, and finally, histone methylation changes that are established in response to a stress; (*iii*) maintained for a certain time during the lifespan of an individual (i.e. somatic memory) or (*iv*)

transmitted to one or more subsequent generations (i.e. transgenerational memory).

variation within the population [149, 150].

**6. Discussion and perspectives**

**Figure 1. Hypothetical model depicting the role of histone methylation/demethylation in regulating plant stress re‐ sponses. (A)** Plants exposed either to biotic or abiotic stresses integrate the signal stress into the cell nuclei, where it affects the chromatin structure through histone methylation changes. According to our knowledge, these histone meth‐ ylation changes can be classified into several interrelated categories. **(B)** The permissive state represents a more loos‐ ened chromatin state that will either offer a protection against repressive marks (represented with nucleosomes in red) and/or potentiates a rapid transcriptional induction upon stress induction. **(C)** The induced state represents histone methylation changes that are transiently induced by a stress signal. If methylation changes occur early during the stress-response process, they might participate to the transcriptional induction of stress-responding genes, while if they occur later, they might reinforce the transcription of stress-responding genes. Both permissive and the induced states can be maintained allowing a faster and/or stronger transcriptional induction of stress-responding genes upon a subsequent challenge. The memorized chromatin state can be maintained **(D)** for a certain time during the lifespan of an individual and referred as the somatic memory, or **(E)** transmitted to one or more subsequent generations and refer‐ red as the transgenerational memory.

Nonetheless, this emerging view is facing many gaps, inaccuracies and divergences, mainly related to numerous difficulties inherent to the study of such a dynamic and acute process. In this respect, plants in nature are usually challenged simultaneously by different kinds of stresses. Responses to these stress combinations are largely controlled by different signalling pathways that can interact in a non-additive manner, producing effects that could not have been predicted from the study of either stress individually [151, 152]. The occurrence of simultaneous biotic and abiotic stresses introduces an added degree of complexity that requires stresses to be imposed simultaneously and to treat each set of environmental condi‐ tions as an entirely new stress. For this reason and to clarify the mechanism behind the regulation of stress responses by histone methylation changes, there is a strong necessity to intensify our investigations. For instance, the correlation between histone methylation/ demethylation and stress responses remains elusive and clarifications will require in-depth dynamic approaches based on comparative analyses of both epigenomes and transcriptomes during stress responses. In parallel, current knowledge about the corresponding histonemodifying enzymes is still largely missing. This lack of knowledge is pending on the identi‐ fication of different stress-responsive histone modifiers and will require large-scale screens and genetic analyses for the sensitivity of different histone methyltransferases/demethylases mutants to various stresses, combined or not. Among other factors governing stress-induced chromatin changes, almost nothing is known about the specific reader/effector that will recognize particular histone methylation sites in order to determine their functional and structural outcome. An effort in this direction will most likely benefit the comprehensive understanding of the fundamental mechanisms connecting histone methylation changes with the modulation of transcription of stress-responsive genes, subsequently enabling plant to withstand stress. Higher-resolution chromatin studies are undoubtedly required to reveal the targeted stress-responsive genes and the specific sites of histone methylation/demethylation. Nevertheless, investigation of the direct effects of histone methylation/demethylation in plants is difficult. One reason is that plant genomes harbour high-copy number of histone genes (e.g. the *Arabidopsis* genome comprises 47 genes that encode 33 different core histone proteins; www.chromdb.org) and the incorporation/modification of such variants can result in the formation of chromatins with particular properties and functions [153–155]. Although ChIP assays have proven valuable in helping to identify histone methylation changes, many antibodies used to detect these changes have been so far unable to distinguish between different variants. New technologies (e.g. generation of mutants with point mutations targeting amino acid in the N-terminal tail of histone using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPER-associated (Cas) system) [156] will need to be explored to unravel histone methylation changes of specific histone variants and their functions. Another challenge is that plants consist of many functionally specialized tissues and cell types, each with its own unique epigenome, transcriptome and proteome. Until now, histone methylation changes induced by stresses were exclusively addressed in entire plant or organs, meaning that the obtained profiles most likely reflect the consensus of multiple tissue- or cell-specific profiles that may differ. New methods allowing the mapping of chromatin features in specific tissue/cell types such as the one described by Wang and Deal [157] will be decisive for determining the cell-/tissue-specific chromatin alterations involved in a particular stress response. Finally, as plants have finite resources that must be balanced between growth and defence against stresses, often resulting in a growth or yield penalty, histone methylation changes in response to stress should be integrated in a more global developmental view, taking into account the involvement of several histone methyltransfer‐ ases/demethylases in various processes such as root growth, flowering time, floral organo‐ genesis, gametophyte or embryo formation [33]. Finally, understanding such regulatory network is an essential step to provide both novel paradigms and potential tools for further exploitation towards sustainable agriculture.
