[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.
[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 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.
[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.
[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.
Genetics and molecular research: GMR 12/2013; · 0.85 Impact Factor
[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.
[Show abstract][Hide abstract] ABSTRACT: Glutathione transferases (GSTs, EC 220.127.116.11) 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.
Applied biochemistry and biotechnology 10/2013; · 1.94 Impact Factor
[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.
[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.
PLoS ONE 04/2013; 8(4):e60945. · 3.53 Impact Factor
[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.
[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.
[Show abstract][Hide abstract] ABSTRACT: Histone H3 lysine 4 methylations catalyzed by histone lysine methyltransferases (HKMTs), like the Arabidopsis thaliana ATX1 and ATX2, are important epigenetic modifications related to chromatin decondensation and gene activation. In order to study this epigenetic mechanism in monocot cereal plants, we performed homology searches of ATX1 and ATX2 against the Brachypodium distachyon L. Beauv and rice (Oryza sativa L. spp. japonica) genomes, discovering single homologues for each cereal crop representing both Arabidopsis sequences. Using this information, we employed the rolling circle amplification — rapid amplification of cDNA ends (RCA-RACE) method to isolate, clone and characterize HvTX1 from RNA extracted from barley (Hordeum vulgare L.) tissues and studied its expression during seed development and under drought stress. The cloned cDNA sequence contained a 3 093 bp ORF homologous to ATX1 and ATX2. Characterization of the translated HvTX1 transcript sequence revealed the multi-domain nature of the putative protein, including all conserved regions characteristic for ATX1 and ATX2. By comparative genomic analysis and homology searches in EST databases we located, with high probability, the gene coding for HvTX1 on the barley chromosome 5H. Constant elevation of HvTX1 expression was observed during seed development. Expression of HvTX1 after drought stress was analyzed by quantitative real-time polymerase chain reaction (qPCR) in two different barley cultivars with varying drought stress tolerance, revealing HvTX1 drought-induction in a tolerance-specific manner.
Biologia Plantarum 12/2012; 56(4). · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The entire publicly available set of 37 genome sequences from the bacterial order Chlamydiales has been subjected to comparative analysis in order to reveal the salient features of this pangenome and its evolutionary history. Over 2,000 protein families are detected across multiple species, with a distribution consistent to other studied pangenomes. Of these, there are 180 protein families with multiple members, 312 families with exactly 37 members corresponding to core genes, 428 families with peripheral genes with varying taxonomic distribution and finally 1,125 smaller families. The fact that, even for smaller genomes of Chlamydiales, core genes represent over a quarter of the average protein complement, signifies a certain degree of structural stability, given the wide range of phylogenetic relationships within the group. In addition, the propagation of a corpus of manually curated annotations within the discovered core families reveals key functional properties, reflecting a coherent repertoire of cellular capabilities for Chlamydiales. We further investigate over 2,000 genes without homologs in the pangenome and discover two new protein sequence domains. Our results, supported by the genome-based phylogeny for this group, are fully consistent with previous analyses and current knowledge, and point to future research directions towards a better understanding of the structural and functional properties of Chlamydiales.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: MADS-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. RESULTS: Here 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. CONCLUSIONS: Two 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.
[Show abstract][Hide abstract] ABSTRACT: Genes in the phosphatidyl-ethanolamine-binding protein (PEBP) family are instrumental in regulating the fate of meristems and flowering time. To investigate the role of these genes in the monocotyledonous plant Crocus (Crocus sativus L), an industrially important crop cultivated for its nutritional and medicinal properties, we have cloned and characterized a CENTRORADIALIS/TERMINAL FLOWER1 (CEN/TFL1) like gene, named CsatCEN/TFL1-like, the first reported CEN/TFL1 gene characterized from such a perennial geophyte. Sequence analysis revealed that CsatCEN/TFL1 shows high similarity to its homologous PEBP family genes CEN/TFL1, FT and MFT from a variety of plant species and maintains the same exon/intron organization. Phylogenetic analysis of the CsatCEN/TFL1 amino acid sequence confirmed that the isolated sequences belong to the CEN/TFL1 clade of the PEBP family. CsatCEN/TFL1 transcripts could be detected in corms, flower and flower organs but not in leaves. An alternative spliced transcript was also detected in the flower. Comparison of expression levels of CsatCEN/TFL1 and its alternative spliced transcript in wild type flower and a double flower mutant showed no significant differences. Overexpression of CsatCEN/TFL1 transcript in Arabidopsis tfl1 plants reversed the phenotype of early flowering and terminal flowering of the tfl1 plants to a normal one. Computational analysis of the obtained promoter sequences revealed, next to common binding motifs in CEN/TFL1-like genes as well as other flowering gene promoters, the presence of two CArG binding sites indicative of control of CEN/TFL1 by MADS-box transcription factors involved in crocus flowering and flower organ formation.
[Show abstract][Hide abstract] ABSTRACT: Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of "CLL-biased" features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.