**2. Chromatin structure and histone methylation/demethylation**

In eukaryotes, genomic DNA in the cell nucleus is packaged in a complex and evolutionarily conserved structure named chromatin, with nucleosome as the basic unit. The nucleosome complex contains about 160–241 base pairs (bp) of DNA, a nucleosome core particle and the H1 linker histone. The nucleosome core particle is composed of an octamer of core histones, consisting of two H3–H4 dimers associated with two H2A–H2B dimers. About 146 bp of DNA is wrapped in ~1.65 negatively supercoiled circles around the histone octamer, while the linker DNA associated with H1 varies in length from 8 to 114 bp [7]. At first sight, the chromatin as it is described appears as a barrier, restricting the access of all kinds of enzymes that process the DNA. However, nucleosomes are not merely static but highly dynamic entities. Indeed, nucleosomes can be moved, stabilized/destabilized, disassembled/reassembled at particular genome locations in response to specific environmental signals or developmental cues [8]. This dynamic leads to a wide range of chromatin condensation states modulating the DNA accessibility, with euchromatin, being relaxed, and heterochromatin, being compacted. Therefore, in eukaryotic cells, an intimate connection exists between the structural organiza‐ tion of the genome and its functioning. For this reason, the level of chromatin condensation is directly related to all aspects of DNA metabolism, thus playing a major role in regulating transcription, DNA replication, DNA repair, recombination, transposition and chromosome segregation. In plants, changes in the chromatin structure were reported to affect various biological processes such as root growth, flowering, organogenesis, gametophyte or embryo formation [9–11].

**1. Introduction**

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

demethylation.

Stress, as we currently think of it, is a highly subjective phenomenon defined as a state of threatened homeostasis. Depending on their nature, external stresses are usually divided into biotic (i.e. herbivorous insects and pathogens such as fungi, bacteria and viruses) or abiotic (i.e. including, among others, high or low temperature, submergence or drought and salinity). During their lifetime, all living organisms inevitably and constantly face all sorts of environ‐ mental stresses that often occur suddenly and/or simultaneously. Classically, different strategies can be applied to minimize deleterious effects of stresses, such as resistance, tolerance, avoidance or escape. Being sessile, plants cannot escape and are therefore more prone to the deleterious effect of unfavourable environmental growth conditions. Because responses are critical to ensure their survival, plants have developed specific and efficient strategies that allow them to precisely perceive different environmental stresses and respond and/or adapt to them [1, 2]. In addition to preformed defence traits, plants have evolved inducible defence strategies. Indeed, upon perception, each stress will raise a complex and more or less specific repertoire of cellular and molecular responses implemented by the plant to minimize or prevent damage. Particularly, the stimulation of a given stress-signalling pathway after pathogen detection will be integrated into the plant cell nucleus through a set of regulatory transcription factor cascades, which prioritizes defence over growth-related cellular functions, while conserving enough valuable resources for survival and reproduction [3, 4]. Supporting the idea that the capacity of a plant to rapidly reprogramme its gene expression at the transcriptional level is an essential and common component of all plant response strategies to stress and disturbance; more than 1,000 transcription factors were found to be involved in stress responses [5, 6]. Because eukaryotic genes function in the context of chromatin, modifications and remodelling of the chromatin configuration from permissive for transcription to restrictive, and vice versa, may be an integral part of mechanisms involved in this vital transcriptional reprogramming. In this chapter, we review and discuss the current knowledge about the functional impact of chromatin changes on the transcriptional regulation of genes under different stress conditions, with particular emphasis on histone methylation/

**2. Chromatin structure and histone methylation/demethylation**

In eukaryotes, genomic DNA in the cell nucleus is packaged in a complex and evolutionarily conserved structure named chromatin, with nucleosome as the basic unit. The nucleosome complex contains about 160–241 base pairs (bp) of DNA, a nucleosome core particle and the H1 linker histone. The nucleosome core particle is composed of an octamer of core histones, consisting of two H3–H4 dimers associated with two H2A–H2B dimers. About 146 bp of DNA is wrapped in ~1.65 negatively supercoiled circles around the histone octamer, while the linker DNA associated with H1 varies in length from 8 to 114 bp [7]. At first sight, the chromatin as it is described appears as a barrier, restricting the access of all kinds of enzymes that process the DNA. However, nucleosomes are not merely static but highly dynamic entities. Indeed, In the nucleosome core particle, histones H2A, H2B, H3 and H4 possess two common regions, a histone-fold domain and a histone tail. The histone-fold domain is the most conserved region and the main element of histone dimerization [12]. The tail protrudes from the nucleosome core particle and is more variable and unstructured than the fold [13]. All four core histones have an N-terminal tail domain, but only histone H2A has an additional long C-terminal tail. Histone tails are extremely basic due to their particularly high content in basic amino acid residues, such as lysine and arginine [14]. Resulting positive charges allow them to closely associate with the negatively charged nucleosomal DNA through electrostatic interactions [15]. In addition, histone tails, especially N-terminal ones, may undergo diverse types of posttranslational modifications such as acetylation or methylation. The great diversity of these modifications as well as the high number of amino acid residues that can be modified within histone tails, and the correlation between these modifications and various nuclear processes, lead to the hypothesis that the specific combination of histone modifications constitute a histone 'code' [16].

Technically, these histone marks can be localized by chromatin immunoprecipitation (ChIP) using specific antibodies against the modification [17]. Briefly, protein–DNA interactions are stabilized by cross-linking with formaldehyde; chromatin is sheared into small pieces to facilitate analysis and then immunoprecipitated using an antibody raised against a specific histone modification. Following enrichment, cross links are reversed to release DNA, which is then quantified by polymerase chain reaction (PCR) to measure the relative amount of the specific histone mark on selected plant genes. ChIP can also be combined with microarray hybridization (ChIP-chip) or high-throughput sequencing (ChIP-Seq), allowing the genomewide discovery of DNA–histone modification interactions.

Methylation is the most abundant one compared with other histone PTMs. It can occur at both lysine (K) and arginine (R) residues of core histone tails. Further extending the indexing potential of this modification, mono-, di- and trimethylation of lysine and mono- and dime‐ thylation (symmetric or asymmetric) of arginine are common at N-terminal tails of H2A, H2B, H3 and H4. Although histone acetylation is generally associated with active gene transcription, histone methylation can be associated with either active or silent gene expression, depending upon the histone, the methylated residue or the level (mono-, di- or tri-) of methylation. In *Arabidopsis*, genome-wide analyses revealed that trimethylations of H3K4 and H3K36 (H3K4me3 and H3K36me3) are generally enriched at actively transcribed genes, whereas H3K27me3 is associated with repressed genes and H3K9me2 and H4K20me1 are enriched at constitutive heterochromatin and silenced transposons [18]. For histone arginine methylation, a definitive role has not yet been clearly established. However, because the level of symmetric H3R2me2 and H4R3me2 was negatively correlated with the level of H3K4me3, a well-known mark reflecting active transcription, high levels of H3R2me2 and H4R3me2 are thought to cause transcriptional repression [19–21]. In contrast, asymmetric H4R3me2 was associated with gene activation [22, 23].

Histone methylation is relatively stable and can be established on lysine and arginine by two distinct families of enzymes, the histone lysine methyltransferases (HKMTs), all containing the evolutionary conserved catalytic SET domain in plants [24], and the protein arginine methyltransferases (PRMTs) [25], respectively. As a counterpart, methyl groups on histone can also be removed by at least two evolutionarily conserved classes of histone demethylases, the lysine-specific demethylase1 (LSD1) type and the Jumonji C (JmjC) domain-containing demethylases [26]. Histone methyltransferases and demethylases are well conserved in angiosperms and have been identified and classified on the basis of phylogenetic analyses and domain organization in several plants, including *Arabidopsis*, maize, tomato, rice, grapevine and *Brassica rapa*, [27–32]. However, cellular and molecular functions of many of these modifiers have not yet been addressed.

Although histone acetylation can directly modulate the chromatin structure, arginine and lysine methylation of histone tails can promote or prevent the docking of key transcriptional effector molecules, named readers, needed to 'translate' the code in order to determine the functional and structural outcome of the corresponding PTMs. Just as there are a large number of PTMs on histone tails, there are also numerous protein domains that recognize and bind to particular PTMs on these tails. For example, PTM-recognition domains such as plant homeo‐ domain (PHD) fingers, chromodomains and Tudor domains all recognize methylated lysine residues [33].
