Dimitra Papaefthimiou

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.

Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes.

Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a copalyl diphosphate synthase (RoCPS1) and two kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts geranylgeranyl diphosphate (GGDP) to copalyl diphosphate (CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal CDP to produce an abietane diterpene. Comparison to the already characterized diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the sclareol synthase from Salvia sclarea, RoKSL1/2 are diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split.

Morphology, phylogeny and toxin analysis of Pseudo-nitzschia pseudodelicatissima (Bacillariophyceae) isolated from the Thermaikos Gulf, Greece

Moschandreou K.K., Papaefthimiou D., Katikou P., Kalopesa E., Panou A. and Nikolaidis G. 2010. Morphology, phylogeny and toxin analysis of Pseudo-nitschia pseudodelicatissima (Bacillariophyceae) isolated from the Thermaikos Gulf, Greece. Phycologia 49: 260–273. DOI: 10.2216/09-42.1 Pseudo-nitzschia pseudodelicatissima is a diatom species that has been associated with the production of the neurotoxin domoic acid (DA), causative agent of amnesic shellfish poisoning. However, the taxonomic revision of this species complex resulted in uncertain identity and, subsequently, toxicity of the strains tentatively identified under light microscope as P. pseudodelicatissima. Six strains isolated from the Thermaikos Gulf during the period from August to October 2008 were cultured and tested for production of DA. All strains were studied in morphological detail using electron microscopy and were identified as P. pseudodelicatissima. The identification was supported by phylogenetic analyses of the large subunit (LSU) and internal transcribed spacer (ITS) ribosomal rDNA regions, along with the secondary structure of ITS-2 rDNA region. DA was detected in all cultures and in three of the strains in quantifiable amounts up to 3.08 ng ml−1. Two of the strains were studied over the course of a batch culture growth experiment and DA was detected from the mid-exponential until late-stationary phases; the maximum whole-culture DA levels were measured in late-exponential to early-stationary phases. This study confirms the production of DA by P. pseudodelicatissima in culture.

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.

Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae : Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis

Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.

Genus Cistus: a model for exploring labdane-type diterpenes' biosynthesis and a natural source of high value products with biological, aromatic, and pharmacological properties.

The family Cistaceae (Angiosperm, Malvales) consists of 8 genera and 180 species, with 5 genera native to the Mediterranean area (Cistus, Fumara, Halimium, Helianthemum, and Tuberaria). Traditionally, a number of Cistus species have been used in Mediterranean folk medicine as herbal tea infusions for healing digestive problems and colds, as extracts for the treatment of diseases, and as fragrances. The resin, ladano, secreted by the glandular trichomes of certain Cistus species contains a number of phytochemicals with antioxidant, antibacterial, antifungal, and anticancer properties. Furthermore, total leaf aqueous extracts possess anti-influenza virus activity. All these properties have been attributed to phytochemicals such as terpenoids, including diterpenes, labdane-type diterpenes and clerodanes, phenylpropanoids, including flavonoids and ellagitannins, several groups of alkaloids and other types of secondary metabolites. In the past 20 years, research on Cistus involved chemical, biological and phylogenetic analyses but recent investigations have involved genomic and molecular approaches. Our lab is exploring the biosynthetic machinery that generates terpenoids and phenylpropanoids, with a goal to harness their numerous properties that have applications in the pharmaceutical, chemical and aromatic industries. This review focuses on the systematics, botanical characteristics, geographic distribution, chemical analyses, biological function and biosynthesis of major compounds, as well as genomic analyses and biotechnological approaches of the main Cistus species found in the Mediterranean basin, namely C. albidus, C. creticus, C. crispus, C. parviflorus, C. monspeliensis, C. populifolius, C. salviifolius, C. ladanifer, C. laurifolius, and C. clusii.

Inter- and intra-specific diversity of Pseudo-nitzschia (Bacillariophyceae) in the northeastern Mediterranean

A total of 92 cultured Pseudo-nitzschia strains, established between March 2007 and April 2010 from Greek coastal waters, were identified by using morphological and molecular (ITS2 region) characters. Twelve species were identified, among which P. brasiliana, P. subpacifica and P. hasleana were detected for the first time near Greek and East Mediterranean coasts. One P. delicatissima-like morphotype and another strain that closely resembled P. dolorosa were also found. Morphology and ITS2 phylogeny indicated that the level of diversity within the genus is higher than previously estimated (e.g. among P. delicatissima-like diatoms). Additionally, the ITS2 phylogeny revealed genetic variation within species, indicative of geographical differentiation (in P. brasiliana, P. fraudulenta, P. arenysensis, P. delicatissima and P. galaxiae). The majority of the cultured strains were also tested for domoic acid (DA) production. Strains of only three species, P. pseudodelicatissima, P. galaxiae and P. pungens var. pungens, were found capable of producing DA.

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.

Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast

Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities.

The study of a SPATULA-like bHLH transcription factor expressed during peach (Prunus persica) fruit development

Extensive studies on the dry fruits of the model plant arabidopsis (Arabidopsis thaliana) have revealed various gene regulators of the development and dehiscence of the siliques. Peach pericarp is analogous to the valve tissues of the arabidopsis siliques. The stone (otherwise called pit) in drupes is formed through lignification of the fruit endocarp. The lignified endocarp in peach can be susceptible to split-pit formation under certain genetic as well as environmental factors. This phenomenon delays processing of the clingstone varieties of peach and causes economical losses for the peach fruit canning industry. The fruitfull (FUL) and shatterproof (SHP) genes are key MADS-box transcription protein coding factors that control fruit development and dehiscence in arabidopsis by promoting the expression of basic helix-loop-helix (bHLH) transcription factors like Spatula (SPT) and Alcatraz (ALC). Results from our previous studies on peach suggested that temporal regulation of PPERFUL and PPERSHP gene expression may be involved in the regulation of endocarp margin development. In the present study a PPERSPATULA-like (PPERSPT) gene was cloned and characterized. Comparative analysis of temporal regulation of PPERSPT gene expression during pit hardening in a resistant and a susceptible to split-pit variety, suggests that this gene adds one more component to the genes network that controls endocarp margins development in peach. Taking into consideration that no ALC-like genes have been identified in any dicot plant species outside the Brassicaceae family, where arabidopsis belongs, PPERSPT may have additional role(s) in peach that are fulfilled in arabidopsis by ALC.