[Show abstract][Hide abstract] ABSTRACT: Σε καρπούς από 15 ποικιλίες Ευρωπαϊκής και 28 ποικιλίες Ιαπωνικής δαμασκηνιάς που διατηρούνται στην Ελληνική Τράπεζα Γενετικού Υλικού, στη Νάουσα έγιναν αναλύσεις ποιοτικών χαρακτηριστικών όπως το νωπό βάρος καρπού, παράμετροι χρώματος CIELAB L, a* και b* σε σάρκα και φλοιό, συνολικά διαλυτά στερεά (ΣΔΣ), ογκομετρούμενη οξύτητα (ΟΟ), συγκέντρωση συνολικών φαινολών και συνολική αντιοξειδωτική ικανότητα χρησιμοποιώντας τις μεθόδους DPPH+ (2,2-diphenyl-1- picrylhydrazyl) και FRAP (Ferric Reducing Antioxidant Power) σε σάρκα και φλοιό. Ανάμεσα στις μελετούμενες ποικιλίες ήταν και οι Ελληνικής καταγωγής ποικιλίες δαμασκηνιάς Ασβεστοχωρίου, Πραούστι, Μπαρδάκι σφαιρικό, Μπαρδάκι μακρόστενο, Μαύρα Σκοπέλου, Ξινά Σκοπέλου και Αυγάτη. Η συγκέντρωση συνολικών φαινολών ήταν από 2,2 μέχρι 14,6 φορές μεγαλύτερη στο φλοιό (152.1-1797.3 mg ισοδύναμα γαλλικού οξέος 100-1 g νωπού βάρους), σε σύγκριση με τη σάρκα (55.9-352.3 mg 100-1 g). Βρέθηκε μεγάλη παραλλακτικότητα σε όλα τα μελετώμενα ποιοτικά χαρακτηριστικά. Οι ποικιλίες Ιαπωνικής δαμασκηνιάς είχαν μεγαλύτερο βάρος καρπού (64%), συνολικές φαινόλες στο φλοιό (57%) και DPPH+ αντιοξειδωτική ικανότητα (43%), σε σύγκριση με τις Ευρωπαϊκές ποικιλίες. Η ευρέως καλλιεργούμενη ποικιλία Black Amber ξεχώρισε για τις ιδιαίτερα υψηλές τιμές αντιοξειδωτικών ουσιών. Επίσης, πραγματοποιήθηκε γενοτυπική ανάλυση των ποικιλιών συνδυάζοντας την εφαρμογή επτά μικροδορυφορικών μοριακών δεικτών (Μικροδορυφόροι- Απλές Επαναλαμβανόμενες Αλληλουχίες- Simple Sequence Repeats- SSRs) με τη μέθοδο τήξης σε υψηλή ανάλυση (HRM). Η σύγκριση των καμπυλών τήξης σε υψηλή ανάλυση που προέκυψαν κατέληξε στην κατασκευή δεντρογράμματος όπου οι γενότυποι διακρίνονται σε τέσσερις κλάδους ένας εκ των οποίων περιλαμβάνει όλες τις ελληνικές ποικιλίες εκτός της «Αυγάτης Σκοπέλου». Σημειώνεται ότι τέσσερις μικροδορυφορικοί δείκτες φάνηκε να είναι αρκετοί για τη διάκριση των γενοτύπων ενώ η χρήση των επτά οδήγησε σε περισσότερο εμπεριστατωμένη διάκριση. Τα αποτελέσματα επιβεβαιώθηκαν τόσο με ανάλυση κυρίων συντεταγμάνων (PCoA) όσο επίσης και με ανάλυση κατά Bays.
￼￼Η εφαρμογή του συνδυασμού SSR/HRM αποτελεί μία εύκολη και
￼γρήγορη εργαστηριακή πρακτική σχετικά μικρού κόστους την οποία προτείνουμε για
[Show abstract][Hide abstract] ABSTRACT: The epigenetic structure in wild plant populations remains largely unknown despite the substantial interest in evaluating epigenetic diversity in non-model organisms living in nature. The forces that shape natural epigenetic variation continue to be elusive and the potential challenge of neo-Darwinian evolutionary theory is still an open question. In this work, we estimated epigenetic diversity and differentiation within and between five natural wild cherry (Prunus avium L.) populations in northern Greece. By using a combination of either EcoRI/HpaII, or EcoRI/MspI restriction enzymes, we found low epigenetic polymorphism and weak epigenetic structure. In total, 324 methylation-sensitive amplified polymorphic (MSAP) marker bands were detected across 93 wild cherry individuals examined. They represented 272 methylation-susceptible and 52 non-methylated epiloci. The total 5′-CCGG-methylation level, ranged from 37.05% to 59.39%, presenting an overall mean of 49.67% and the difference among populations was significant. The relative levels of full-methylation (m-subepiloci), hemi-methylation (h-subepiloci) and non-methylation (n-subepiloci), presented an overall mean of 47.07%, 30.99% and 21.92% respectively. Most of the epigenetic variation (97%) resided within populations (ΦST = 0.017; p < 0.001). Epigenetic and genetic diversity did not differ significantly and were not significantly correlated. Epigenetic variation was not congruent to genetic variation that was assessed in the same populations and individuals by ISSR markers and by the S-locus. This study supports the thesis that epigenetic variation is uncoupled from genetic variation. It presents a first insight into the partitioning of epigenetic diversity within and among natural wild cherry populations.
Full-text · Article · Aug 2015 · Tree Genetics & Genomes
[Show abstract][Hide abstract] ABSTRACT: The involvement of glutathione transferases (GSTs) in plant’s tolerance to abiotic stresses has been extensively studied; however, the metabolic changes occurring in the plants with altered GSTs expression have not been studied in detail. We have previously demonstrated that GmGSTU4 overexpression in tobacco plants conferred increased tolerance to herbicides, partly through its peroxidase activity. Here, we investigated GmGSTU4 transcriptional response to abiotic and chemical stimuli in soybean. Transgenic tobacco plants overexpressing GmGSTU4 were also evaluated regarding their phenotypic and metabolomics responses under salt stress. GmGSTU4 expression was highly induced after salt stress and atrazine treatment. Tobacco plants overexpressing GmGSTU4 were highly tolerant to 150 mM NaCl in vitro. Metabolomics comparison of plants growing under optimal conditions, indicating a shift of the transgenic plants metabolism towards the metabolic profiles observed under stress, increased concentration of precursors of glutathione biosynthesis and hexose concentration reduction. Under salt stress, transgenic plants maintained their cellular homeostasis in contrast to wild-type plants which exhibited deregulated energy metabolism. The metabolic response of the transgenic plants was characterized by higher concentration of protective metabolites such as proline and trehalose and greater induction of the oxidative pentose phosphate pathway. These results confirm GmGSTU4 contribution to salt stress tolerance, and outline a regulatory role that primes plants towards the up-regulation of protective and detoxification mechanisms under abiotic stress.
Full-text · Article · May 2015 · Acta Physiologiae Plantarum
[Show abstract][Hide abstract] ABSTRACT: Plant cytosolic glutathione transferases (GSTs) belong to an ancient enzyme superfamily with multiple and diverse functions which are important in counteracting biotic and abiotic stress. GSTs catalyze the conjugation of xenobiotics and endogenous electrophilic compounds with glutathione (GSH), leading to their detoxification. GSTs not only catalyze detoxification reactions but they are also involved in GSH-dependent isomerization reactions, in GSH-dependent reduction of organic hydroperoxides, biosynthesis of secondary metabolites, and exhibit thioltransferase and dehydroascorbate reductase activity. The applications of ‘omics’ technologies have allowed the classification of GSTs and the study of their evolution and sequence diversity, while enzymology has provided powerful insights into their catalytic role. This review focuses on plant GSTs, and attempts to give an overview of the new insights into their catalytic function and biological role in biotic and abiotic stress tolerance mechanisms in plants.
[Show abstract][Hide abstract] ABSTRACT: The glutathione transferases (GSTs) are members of a superfamily of enzymes with pivotal role in the detoxification of both xenobiotic and endogenous compounds. In this work, the generation and characterization of transgenic tobacco plants over-expressing tau glutathione transferases from Citrus sinensis (CsGSTU1 and CsGSTU2) and several cross-mutate forms of these genes are reported. Putative transformed plants were verified for the presence of the transgenes and the relative quantification of transgene copy number was evaluated by Taqman real time PCR. The analysis of gene expression revealed that transformed plants exhibit high levels of CsGSTU transcription suggesting that the insertion of the transgenes occurred in transcriptional active regions of the tobacco genome. In planta studies demonstrate that transformed tobacco plants gain tolerance against fluorodifen. Simultaneously, the wild type CsGSTU genes were in vitro expressed and their kinetic properties were determined using fluorodifen as substrate. The results show that CsGSTU2 follows a Michaelis–Menten hyperbolic kinetic, whereas CsGSTU1 generates a sigmoid plot typical of the regulatory enzymes, thus suggesting that when working at sub-lethal fluorodifen concentrations CsGSTU2 can counteract the herbicide injury more efficiently than the CsGSTU1. Moreover, the transgenic tobacco plant over-expressing CsGSTs exhibited both drought and salinity stress tolerance. However, as we show that CsGSTUs do not function as glutathione peroxidase in vitro, the protective effect against salt and drought stress is not due to a direct scavenging activity of the oxidative stress byproducts. The transgenic tobacco plants, which are described in the present study, can be helpful for phytoremediation of residual xenobiotics in the environment and overall the over-expression of CsGSTUs can be helpful to develop genetically modified crops with high resistance to abiotic stresses.
[Show abstract][Hide abstract] ABSTRACT: Identification of genotypes in Sideritis is complicated owing to the morphological similarity and common occurrence of natural hybridisation within Sideritis species. Species- and genotype-specific DNA markers are very useful for plant identification, breeding and preservation programs. Herein, a real-time polymerase chain reaction (PCR) of ITS2 barcode region coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on seven Sideritis species growing in Greece. The HRM assay developed in this study is a rapid and straightforward method for the identification and discrimination of the investigated Sideritis species. This assay is simple compared to other genotyping methods as it does not require DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of Sideritis species.
Full-text · Article · May 2014 · Molecular Biology Reports
[Show abstract][Hide abstract] ABSTRACT: Transcription factor function is crucial for eukaryotic systems. The presence of transcription factor families in genomes represents a significant technical challenge for functional studies. To understand their function, we must understand how they evolved and maintained by organisms. Based on genome scale searches for homologs of LEAFY COTYLEDON-LIKE (L1L; AtNF-YB6), NF-YB transcription factor, we report the discovery and annotation of a complete repertoire of thirteen novel genes that belong to the L1L paralogous gene family of Solanum lycopersicum. Gene duplication events within the species resulted in the expansion of the L1L family. Sequence and structure-based phylogenetic analyses revealed two distinct groups of L1Ls in tomato. Natural selection appears to have contributed to the asymmetric evolution of paralogs. Our results point to key differences among SlL1L paralogs in the presence of motifs, structural features, cysteine composition and expression patterns during plant and fruit development. Furthermore, differences in the binding domains of L1L members suggest that some of them evolved new binding specificities. These results reveal dramatic functional diversification of L1L paralogs for their maintenance in tomato genome. Our comprehensive insights on tomato L1L family should provide the basis for further functional and genetic experimentation.
[Show abstract][Hide abstract] ABSTRACT: The universal stress proteins (USPs) comprise a large family of proteins that has been found and characterized in a variety of organisms including plants. Intrinsic domains of these proteins are considered as one of the most ancient domains in all life forms. The first member of the USP family was isolated and extensively characterized in bacteria during the 1990s, demonstrating that the USP-containing proteins are phosphoproteins responsive to a variety of abiotic stress stimuli. Since then, many other members of the family have been characterized. Here, we present the isolation and characterization of two USP genes from Gossypium hirsutum, the most widely cultivated cotton species that has a serious impact in world’s economy. The two cotton USP genes, each in the form of two alleles, possess the same structural organization with their Arabidopsis homologues, possessing one intron sequence separating the coding sequence into two unequal parts, a large 5′-end part and a small 3′-end tail. In silico amino acid sequence analysis and three-dimensional modeling show the high levels of conservation in the USP domain, an indication that the cotton GhUSPs may function in a variety of developmental and stress-induced pathways. Moreover, expression analysis showed that while both are expressed in stems and meristems, although at different levels, only one of them is expressed in flowers. Upon salt treatment, both GhUSPs are transcriptionally activated, with the GhUSP1 reaching a peak within 30 min after the application of the stress stimuli, whereas activation of the GhUSP2 lasts longer.
No preview · Article · Dec 2013 · Plant Molecular Biology Reporter
[Show abstract][Hide abstract] ABSTRACT: Dactylis glomerata L. is an important forage species in the Mediterranean region and in regions having a similar climate. Genetic material from three locations covering the north, central and south Greece was studied, using morphological traits, SSRs and ISSRs molecular markers. Morphological analysis revealed differences among the studied geographic locations for all the studied morphological traits except the number of reproductive tillers. Moreover, the highest phenotypic variation was observed on the accessions from south and the lowest on the accessions from north. Although the results of the molecular markers are indicative, a high level of genetic diversity at the species level was revealed by ISSRs, GST=0.291 and SSRs, FST=0.186. Analysis of molecular variance showed that high level of genetic diversity existed within populations 62% and 83% rather than among populations 38% and 17% for ISSRs and SSRs respectively. Cluster analysis divided the three populations in two groups, with the population originating from the island of Crete clustered in one group and the populations from north Greece (Taxiarchis) and central Greece (Pertouli) in a second. Generally, the results suggest that SSRs are more informative than ISSRs regarding the genetic variation within a population, whereas the ISSRs were more informative regarding the genetic diversity among populations However, regarding the diversity (genotypic and phenotypic) of the studied locations, similar trend was observed by morphological traits and microsatellite-based (SSR/ISSR) markers.
Preview · Article · Dec 2013 · Genetics and molecular research: GMR
[Show abstract][Hide abstract] ABSTRACT: The transition to flowering is one of the most important developmental decisions made by plants. At the molecular level, many genes coordinate this transition. Among these, genes encoding for phosphatidylethanolamine-binding proteins (PEBPs) play important roles in regulating flower time and the fate of inflorescence meristem. To investigate the role of PEBPs in an industrially important crop cultivated for its nutritional and medicinal properties, the monocotyledonous species Crocus sativus L., we have isolated three FLOWERING LOCUS T (FT)-like genes designated as CsatFT1-like, CsatFT2-like, and CsatFT3-like. The isolated genes maintain the exon/intron organization of FT-like genes and encode proteins similar to the members of the PEBP family. Phylogenetic and amino acid analysis at critical positions confirmed that the isolated sequence belongs to the FT clade of the PEBP family phylogeny distinctly from the TERMINAL FLOWER 1 (TFL1) and MOTHER OF FT AND TFL1 clades. Expression analysis indicated differences in the expression of the three FT-like genes in different organs and different expressions during the day–night diurnal clock. Additionally, analysis of isolated promoter sequences using computational methods reveals the preservation of common binding motifs in FT-like promoters from other species, thus suggesting their importance among plant species.
No preview · Article · Dec 2013 · Plant Molecular Biology Reporter
[Show abstract][Hide abstract] ABSTRACT: Epigenetic 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.
An 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[prime] and 3[prime] Long Terminal Repeats (LTR) of a Copia retrotransposon element within the 3[prime]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.
A 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.
Full-text · Article · Oct 2013 · BMC Plant Biology
[Show abstract][Hide abstract] ABSTRACT: Glutathione transferases (GSTs, EC 18.104.22.168) are ubiquitous proteins in plants that play important roles in stress tolerance and in the detoxification of toxic chemicals and metabolites. In this study, we systematically examined the catalytic diversification of a GST isoenzyme from Phaseolus vulgaris (PvGST) which is induced under biotic stress treatment (Uromyces appendiculatus infection). The full-length cDNA of this GST isoenzyme (termed PvGSTU3-3) with complete open reading frame, was isolated using RACE-RT and showed that the deduced amino acid sequence shares high homology with the tau class plant GSTs. PvGSTU3-3 catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance is the finding that the enzyme shows high antioxidant catalytic function and acts as hydroperoxidase, thioltransferase, and dehydroascorbate reductase. In addition, its K m for GSH is about five to ten times lower compared to other plant GSTs, suggesting that PvGSTU3-3 is able to perform efficient catalysis under conditions where the concentration of reduced glutathione is low (e.g., oxidative stress). Its ability to conjugate GSH with isothiocyanates may provide an additional role for this enzyme to act as a regulator of the released isothiocyanates from glucosinolates as a response of biotic stress. Molecular modeling showed that PvGSTU3-3 shares the same overall fold and structural organization with other plant cytosolic GSTs, with major differences at their hydrophobic binding sites (H-sites) and some differences at the level of C-terminal domain and the linker between the C- and N-terminal domains. PvGSTU3-3, in general, exhibits restricted ability to bind xenobiotics in a nonsubstrate manner, suggesting that the biological role of PvGSTU3-3, is restricted mainly to the catalytic function. Our findings highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in Phaseolus vulgaris.
No preview · Article · Oct 2013 · Applied biochemistry and biotechnology
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) patients assigned to stereotyped subset #4 possess distinctive patterns of intraclonal diversification (ID) within their immunoglobulin (IG) genes. Though highly indicative of an ongoing response to antigen(s), the critical question concerning the precise timing of antigen involvement is unresolved. Hence, we conducted a large-scale longitudinal study of 8 subset #4 cases totaling 511 and 398 subcloned IG heavy and kappa sequences. Importantly, we could establish a hierarchical pattern of subclonal evolution, thus revealing which somatic hypermutations were negatively or positively selected. In addition, distinct clusters of subcloned sequences with cluster-specific mutational profiles were observed initially, however at later time-points the minor cluster had often disappeared and hence been selected against. Despite the high intensity of ID, it was remarkable that certain residues remained essentially unaltered. These novel findings strongly support a role for persistent antigen stimulation in the clonal evolution of CLL subset #4.
Full-text · Article · Aug 2013 · Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220bp and 77bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products.
[Show abstract][Hide abstract] ABSTRACT: Fast and accurate detection of plant species and their hybrids using molecular tools will facilitate the assessment and monitoring of local biodiversity in an era of climate and environmental change. Herein, we evaluate the utility of the plastid trnL marker for species identification applied to Mediterranean pines (Pinus spp.). Our results indicate that trnL is a very sensitive marker for delimiting species biodiversity. Furthermore, High Resolution Melting (HRM) analysis was exploited as a molecular fingerprint for fast and accurate discrimination of Pinus spp. DNA sequence variants. The trnL approach and the HRM analyses were extended to wood samples of two species (Pinus nigra and Pinus sylvestris) with excellent results, congruent to those obtained using leaf tissue. Both analyses demonstrate that hybrids from the P. brutia (maternal parent) × P. halepensis (paternal parent) cross, exhibit the P. halepensis profile, confirming paternal plastid inheritance in Group Halepensis pines. Our study indicates that a single one-step reaction method and DNA marker are sufficient for the identification of Mediterranean pines, their hybrids and the origin of pine wood. Furthermore, our results underline the potential for certain DNA regions to be used as novel biological information markers combined with existing morphological characters and suggest a relatively reliable and open taxonomic system that can link DNA variation to phenotype-based species or hybrid assignment status and direct taxa identification from recalcitrant tissues such as wood samples.
[Show abstract][Hide abstract] ABSTRACT: Microsatellites are codominant molecular genetic markers, which are universally dispersed within genomes. These markers are highly popular because of their high level of polymorphism, relatively small size, and rapid detection protocols. They are widely used in a variety of fundamental and applied fields of biological sciences for plants and animal studies. Microsatellites are also extensively used in the field of agriculture, where they are used in characterizing genetic materials, plant selection, constructing dense linkage maps, mapping economically important quantitative traits, identifying genes responsible for these traits. In addition microsatellites are used for marker-assisted selection in breeding programs, thus speeding up the process. In this chapter, genomic distribution, evolution, and practical applications of microsatellites are considered, with special emphasis on plant breeding and agriculture. Moreover, novel advances in microsatellite technologies are also discussed.
No preview · Article · Apr 2013 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Glutathione transferases (GSTs) belong to a super-family of multifunctional proteins. GSTs play a key role in cellular detoxification from xenobiotic substances like herbicides, secondary metabolites and toxic degradation products resulting from oxidative stress and cellular metabolism. Furthermore, environmental conditions generate oxidative stress, the products of which have to be detoxified by plants.
It is anticipated that environmental stresses will worsen over the following years due to climate change. Hence, plants must adapt rapidly to the new environmental conditions in order to both survive and satisfy the constantly increasing human demand for agricultural products. Genetic engineering has been successfully used to develop plants resistant to stresses and, having taken all the necessary precautions, could offer a solution as it can help to develop plants with desirable traits in a short period of time.
We present here the use of GST isoenzymes in the development of transgenic plants. Although transgenic plants over-expressing various GSTs have been used for “in planta” evaluation of the enzymes used in response to different stresses, the results show that GSTs could be of great value for generating stress tolerant plants. However, the literature is limited and more studies should be performed in order to exploit their full potential.