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

BACKGROUND: Epigenetic 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. RESULTS: Four 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. CONCLUSION: This 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.

Barley polyamine oxidase isoforms 1 and 2, a peculiar case of gene duplication.

Polyamine oxidases (PAOs, EC 1.5.3.11) are key enzymes responsible for the terminal catabolism of polyamines in plants, bacteria and protozoa. In barley, two PAO isoforms (HvPAO1 and HvPAO2) have been previously analyzed as regards their tissue expression and subcellular localization. Only the major isoform HvPAO2 has been biochemically characterized up to now. In order to study the ear-specific expression of the HvPAO1 isoform in detail, RT-PCR analysis was performed in barley on the whole ear and on various ear tissues. Moreover, HvPAO1promoter::GUS transient expression was examined in barley developing caryopses at 30-day postfertilization. Results from these analyses have demonstrated that the HvPAO1 gene is specifically expressed in all the ear organs analyzed (i.e. basal lemma, rachis, awn, embryo-deprived caryopsis, embryo and sterile spikelets), at variance with the HvPAO2 gene, which is expressed at high levels in sterile spikelets and at very low levels in embryos. We purified HvPAO1 from barley immature caryopses and characterized its catalytic properties. Furthermore, we carried out in vitro synthesis of HvPAO1 protein in a cell-free translation system. The HvPAO1 enzymes purified from immature caryopses and in vitro synthesized showed the same catalytic properties, in particular, an optimum at pH 7.0 for Spd and Spm oxidation and comparable Km values for both substrates, i.e. 0.89x10(-5) M and 0.5x10(-5) M for Spd and Spm, respectively. It has been found that HvPAO1 enzyme activity significantly differs in substrate specificity and pH optimum when compared with the major isoform HvPAO2. As a whole, these data strongly suggest that, in barley, the two PAO genes evolved separately, after a duplication event, to code for two distinct tissue-specific enzymes, and they are likely to play different physiological roles.

Genetics of barley hooded suppression.

The molecular basis of the barley dominant Hooded (K) mutant is a duplication of 305 bp in intron IV of the homeobox gene Bkn3. A chemical mutagenesis screen was carried out to identify genetical factors that participate in Bkn3 intron-mediated gene regulation. Plants from recurrently mutagenized KK seeds were examined for the suppression of the hooded awn phenotype induced by the K allele and, in total, 41 suK (suppressor of K) recessive mutants were identified. Complementation tests established the existence of five suK loci, and alleles suKB-4, suKC-33, suKD-25, suKE-74, and suKF-76 were studied in detail. All K-suppressed mutants showed a short-awn phenotype. The suK loci have been mapped by bulked segregant analysis nested in a standard mapping procedure based on AFLP markers. K suppressor loci suKB, B, E, and F all map in a short interval of chromosome 7H, while the locus suKD is assigned to chromosome 5H. A complementation test between the four suK mutants mapping on chromosome 7H and the short-awn mutant lks2, located nearby, excluded the allelism between suK loci and lks2. The last experiment made clear that the short-awn phenotype of suK mutants is due to a specific dominant function of the K allele, a function that is independent from the control on hood formation. The suK loci are discussed as candidate participants in the regulation of Bkn3 expression.