Aliki Kapazoglou

Epigenetic chromatin modifiers in barley: IV. The study of barley Polycomb group (PcG) genes during seed development and in response to external ABA

BackgroundEpigenetic phenomena have been associated with the regulation of active and silent chromatin states achieved by modifications of chromatin structure through DNA methylation, and histone post-translational modifications. The latter is accomplished, in part, through the action of PcG (Polycomb group) protein complexes which methylate nucleosomal histone tails at specific sites, ultimately leading to chromatin compaction and gene silencing. Different PcG complex variants operating during different developmental stages have been described in plants. In particular, the so-called FIE/MEA/FIS2 complex governs the expression of genes important in embryo and endosperm development in Arabidopsis. In our effort to understand the epigenetic mechanisms regulating seed development in barley (Hordeum vulgare), an agronomically important monocot plant cultivated for its endosperm, we set out to characterize the genes encoding barley PcG proteins.ResultsFour barley PcG gene homologues, named HvFIE, HvE(Z), HvSu(z)12a, and HvSu(z)12b were identified and structurally and phylogenetically characterized. The corresponding genes HvFIE, HvE(Z), HvSu(z)12a, and HvSu(z)12b were mapped onto barley chromosomes 7H, 4H, 2H and 5H, respectively. Expression analysis of the PcG genes revealed significant differences in gene expression among tissues and seed developmental stages and between barley cultivars with varying seed size. Furthermore, HvFIE and HvE(Z) gene expression was responsive to the abiotic stress-related hormone abscisic acid (ABA) known to be involved in seed maturation, dormancy and germination.ConclusionThis study reports the first characterization of the PcG homologues, HvFIE, HvE(Z), HvSu(z)12a and HvSu(z)12b in barley. All genes co-localized with known chromosomal regions responsible for malting quality related traits, suggesting that they might be used for developing molecular markers to be applied in marker assisted selection. The PcG differential expression pattern in different tissues and seed developmental stages as well as in two barley cultivars with different seed size is suggestive of a role for these genes in barley seed development. HvFIE and HvE(Z) were also found to be induced by the plant hormone ABA implying an association with ABA-mediated processes during seed development, germination and stress response.

Epigenetic chromatin modifiers in barley: I. Cloning, mapping and expression analysis of the plant specific HD2 family of histone deacetylases from barley, during seed development and after hormonal treatment.

Epigenetic phenomena have been associated with modifications of chromatin structure. These are achieved, in part, by histone post-translational modifications including acetylations and deacetylations, the later being catalyzed by histone deacetylaces (HDACs). Eukaryotic HDACs are grouped into three major families, RPD3/HDA1, SIR2 and the plant-specific HD2. HDAC genes have been analyzed from model plants such as Arabidopsis, rice and maize and have been shown to be involved in various cellular processes including seed development, vegetative and reproductive growth and responses to abiotic and biotic stress, but reports on HDACs from other crops are limited. In this work two full-length cDNAs (HvHDAC2-1 and HvHDAC2-2) encoding two members of the plant-specific HD2 family, respectively, were isolated and characterized from barley (Hordeum vulgare), an agronomically important cereal crop. HvHDAC2-1 and HvHDAC2-2 were mapped on barley chromosomes 1H and 3H, respectively, which could prove useful in developing markers for marker-assisted selection in breeding programs. Expression analysis of the barley HD2 genes demonstrated that they are expressed in all tissues and seed developmental stages examined. Significant differences were observed among tissues and seed stages, and between cultivars with varying seed size, suggesting an association of these genes with seed development. Furthermore, the HD2 genes from barley were found to respond to treatments with plant stress-related hormones such as jasmonic acid (JA), abscisic acid (ABA) and salicylic acid (SA) implying an association of these genes with plant resistance to biotic and abiotic stress. The expression pattern of HD2 genes suggests a possible role for these genes in the epigenetic regulation of seed development and stress response.

Epigenetic chromatin modifiers in barley: III. Isolation and characterization of the barley GNAT-MYST family of histone acetyltransferases and responses to exogenous ABA.

Histone acetylation is a vital mechanism for the activation of chromatin and the corresponding expression of genes competing the action of histone deacetylation and leading to chromatin inactivation. Histone acetyltransferases (HATs) comprise a superfamily including the GNAT/MYST, CBP and TF(II)250 families. Histone acetyltransferases have been well studied in Arabidopsis but information from agronomically important crops is limited. In the present work three full-length sequences encoding members of the GNAT/MYST family, namely HvMYST, HvELP3 and HvGCN5, respectively, were isolated and characterized from barley (Hordeum vulgare L.), a crop of high economic value. Expression analysis of the barley GNAT/MYST genes revealed significant quantitative differences in different seed developmental stages and between cultivars with varying seed size and weight, suggesting an association of these genes with barley seed development. Furthermore, all three HvGNAT/MYST genes were inducible by the stress-related phytohormone abscisic acid (ABA) involved in seed maturation, dormancy and germination, implying a possible regulation of these genes by ABA, during barley seed development, germination and stress response.

Epigenetic Chromatin Modifiers in Barley: II. Characterization and Expression Analysis of the HDA1 Family of Barley Histone Deacetylases During Development and in Response to Jasmonic Acid

Epigenetic regulation of gene expression plays an important role in various aspects of eukaryotic development and is associated with modifications of chromatin structure. These are accomplished, in part, through the reversible process of histone acetylation/deacetylation, catalyzed by histone acetyltransferases (HATs), and histone deacetylases (HDACs), respectively. Eukaryotic HDACs are grouped in three major families, RPD3/HDA1 (thereafter cited as HDA1), SIR2 and the plant-specific HD2. Histone deacetylase genes have been studied in Arabidopsis and rice, but little is known about these genes from important crop plants. In this work, cDNAs encoding members of the HDA1 family and representing all four classes, Class I, Class II, Class III, and Class IV, were isolated and characterized from barley (Hordeum vulgare), a cereal crop of high agronomic importance. Expression analysis of the barley HDA1 family genes, HvHDAC1-I-1, HvHDAC1-I-3, HvHDAC1-II-1, HvHDAC1-III-1, and HvHDAC1-IV-1 demonstrated that they are expressed in all tissues and seed developmental stages examined. Differences in transcript abundance both in vegetative and reproductive tissues were observed among the different genes suggesting functional diversification of the HDA1 members. Differential expression was also evidenced for some of the HDA1 genes in two barley cultivars differing in various characteristics, such as seed size and resistance to stress, implying a possible association of these genes with different traits. Furthermore, the HDA1 genes were found to respond to the stress-related hormone jasmonic acid (JA), suggesting an association of these genes with barley responsiveness to biotic and abiotic stress. The expression pattern of the HDA1 genes suggests possible roles in the epigenetic regulation of barley development and stress response.

The study of a barley epigenetic regulator, HvDME, in seed development and under drought

BackgroundEpigenetic factors such as DNA methylation and histone modifications regulate a wide range of processes in plant development. Cytosine methylation and demethylation exist in a dynamic balance and have been associated with gene silencing or activation, respectively. In Arabidopsis, cytosine demethylation is achieved by specific DNA glycosylases, including AtDME (DEMETER) and AtROS1 (REPRESSOR OF SILENCING1), which have been shown to play important roles in seed development. Nevertheless, studies on monocot DNA glycosylases are limited. Here we present the study of a DME homologue from barley (HvDME), an agronomically important cereal crop, during seed development and in response to conditions of drought.ResultsAn HvDME gene, identified in GenBank, was found to encode a protein with all the characteristic modules of DME-family DNA glycosylase proteins. Phylogenetic analysis revealed a high degree of homology to other monocot DME glycosylases, and sequence divergence from the ROS1, DML2 and DML3 orthologues. The HvDME gene contains the 5′ and 3′ Long Terminal Repeats (LTR) of a Copia retrotransposon element within the 3′ downstream region. HvDME transcripts were shown to be present both in vegetative and reproductive tissues and accumulated differentially in different seed developmental stages and in two different cultivars with varying seed size. Additionally, remarkable induction of HvDME was evidenced in response to drought treatment in a drought-tolerant barley cultivar. Moreover, variable degrees of DNA methylation in specific regions of the HvDME promoter and gene body were detected in two different cultivars.ConclusionA gene encoding a DNA glycosylase closely related to cereal DME glycosylases was characterized in barley. Expression analysis during seed development and under dehydration conditions suggested a role for HvDME in endosperm development, seed maturation, and in response to drought. Furthermore, differential DNA methylation patterns within the gene in two different cultivars suggested epigenetic regulation of HvDME. The study of a barley DME gene will contribute to our understanding of epigenetic mechanisms operating during seed development and stress response in agronomically important cereal crops.

Cloning and characterization of SOC1 homologs in barley (Hordeum vulgare) and their expression during seed development and in response to vernalization

A number of genes are involved in the vernalization pathway, such as VRN1, VRN2 and VRN3/FT1, whose function has been studied in barley and wheat. However, the function of the flowering and vernalization integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) has not been well studied in Triticeae, and particularly in barley. Herein, we cloned and characterized two barley SOC1-like homologs, HvSOC1-like1 and HvSOC1-like2. Primary sequence analysis of the predicted HvSOC1-like1 and HvSOC1-like2 proteins showed that they are members of the type II MADS-box protein family. Phylogenetic analysis placed the predicted proteins with other SOC1 and SOC1-like proteins from different species neighboring those from other cereal plant species. Primary and secondary structures of the predicted proteins are conserved to each other and more distant to the recently identified barley ODDSOC1 proteins. Genomic organization of HvSOC1-like1 is very similar to the Arabidopsis and Brachypodium SOC1 genes and localized in highly syntenic chromosomal regions. Regulatory cis-acting elements detected in the HvSOC1-like1 promoter include the CArG-box, implicated in the regulation of SOC1 expression in Arabidopsis. Both HvSOC1-like1 and HvSOCI-like2 are expressed in vegetative and reproductive tissues and at different stages of seed development. Both are upregulated in a particular seed developmental stage suggesting their possible implication in seed development. Furthermore, HvSOC1-like1 was induced in two winter barley cultivars after vernalization treatment pointing to its probable involvement in the vernalization process. The study of the SOC1 genes reported here opens the way for a better understanding of both the vernalization process and seed development and germination in this important cereal crop.

The study of two barley Type I-like MADS-box genes as potential targets of epigenetic regulation during seed development

BackgroundMADS-box genes constitute a large family of transcription factors functioning as key regulators of many processes during plant vegetative and reproductive development. Type II MADS-box genes have been intensively investigated and are mostly involved in vegetative and flowering development. A growing number of studies of Type I MADS-box genes in Arabidopsis, have assigned crucial roles for these genes in gamete and seed development and have demonstrated that a number of Type I MADS-box genes are epigenetically regulated by DNA methylation and histone modifications. However, reports on agronomically important cereals such as barley and wheat are scarce.ResultsHere we report the identification and characterization of two Type I-like MADS-box genes, from barley (Hordeum vulgare), a monocot cereal crop of high agronomic importance. Protein sequence and phylogenetic analysis showed that the putative proteins are related to Type I MADS-box proteins, and classified them in a distinct cereal clade. Significant differences in gene expression among seed developmental stages and between barley cultivars with varying seed size were revealed for both genes. One of these genes was shown to be induced by the seed development- and stress-related hormones ABA and JA whereas in situ hybridizations localized the other gene to specific endosperm sub-compartments. The genomic organization of the latter has high conservation with the cereal Type I-like MADS-box homologues and the chromosomal position of both genes is close to markers associated with seed quality traits. DNA methylation differences are present in the upstream and downstream regulatory regions of the barley Type I-like MADS-box genes in two different developmental stages and in response to ABA treatment which may be associated with gene expression differences.ConclusionsTwo barley MADS-box genes were studied that are related to Type I MADS-box genes. Differential expression in different seed developmental stages as well as in barley cultivars with different seed size was evidenced for both genes. The two barley Type I MADS-box genes were found to be induced by ABA and JA. DNA methylation differences in different seed developmental stages and after exogenous application of ABA is suggestive of epigenetic regulation of gene expression. The study of barley Type I-like MADS-box genes extends our investigations of gene regulation during endosperm and seed development in a monocot crop like barley.

From epigenetics to epigenomics and their implications in plant breeding

Publisher Summary Epigenetics involves the mitotically and meiotically heritable variation that does not entail a change in DNA. This chapter describes the transition from epigenetics to epigenomics, together with the genome-wide methods used and the data, resources, and tools available in exploring, integrating, and extracting useful information. It begins with a brief description of the different epigenetic mechanisms. Finally, the role of epigenomics in plant development, phenotypic variation, response to environmental cues and stresses, and the implication of different epigenetic phenomena in plant breeding are discussed. Work in epigenomics is reinforcing the view for the whole genome, providing significant outputs. The very recent sequence of the maize genome opens new avenues to this research. It is the largest plant genome to be fully sequenced and characterized. It contains a high percentage of transposable elements (TEs) of different types, providing an excellent opportunity to study and understand the role of different TEs in eukaryotic large genome organization and function, but essentially it provides an understanding of the function of epigenomic mechanisms as TE regulators at a genomic level.

Epigenetic Chromatin Regulators as Mediators of Abiotic Stress Responses in Cereals

Plants are constantly exposed to environmental changes and have to adapt to a multitude of abiotic and biotic stresses. Due to their sessile nature plants had to develop sophisticated ways to respond and adapt to a variety of external stress factors that would otherwise compromise proper development, reproductive success and ultimately survival. Years of rigorous research have demonstrated that abiotic stress such as drought, high salinity, temperature extremes, UV irradiation and oxidative stress, affect various cellular processes in plants and induce alterations in gene expression programmes in order to activate the plants defense mechanisms to survival. Extensive studies based on forward genetic, reverse genetics, and biochemical investigations of individual loci as well as genome-wide approaches, especially in the model-plant Arabidopsis, have revealed a plethora of genes that are involved in abiotic stress response pathways and acquisition of stress tolerance. These include a wide range of stress-responsive genes encoding transcription factors and functional proteins whose transcription is altered during abiotic stress [1]. Growing evidence from recent studies has indicated that regulation of expression of stressresponsive genes is often accomplished by epigenetic mechanisms which modulate chromatin structure or regulate the level of mRNA accumulation at the postranscriptional level [2; 3; 4]. In eukaryotes nuclear DNA is organized in chromatin, a tightly packed higher order structure which permits genomic DNA to fit within the nucleus. The fundamental unit of chromatin is the nucleosome which is composed of 147 base pairs of DNA that is wrapped almost twice around an octamer of histone proteins. The octamer consists of two copies of each of histone H2A, H2B, H3 and H4. Chromatin higher-order structure switches between condensed and relaxed states and plays a crucial role in the epigenetic regulation of gene expression [5Kouzarides 2007]. Alterations in chromatin structure affect the accessibility of the transcriptional machinery (transcription factors, RNA polymerase) to nucleosomal DNA and determine the levels of gene expression in response to developmental and environmental stimuli. Chromatin modulation is achieved by a variety of mechanisms including: DNA methylation catalyzed by DNA cytosine methyltransferases, histone post-translational modifications catalyzed by a wide range of enzymes specific for each modification, alterations in histoneDNA interactions which facilitate nucleosome sliding and are catalyzed by chromatin