RESUMO
BACKGROUND: Summer squash (Cucurbita pepo: Cucurbitaceae) are a popular horticultural crop for which there is insufficient genomic and transcriptomic information. Gene expression atlases are crucial for the identification of genes expressed in different tissues at various plant developmental stages. Here, we present the first comprehensive gene expression atlas for a summer squash cultivar, including transcripts obtained from seeds, shoots, leaf stem, young and developed leaves, male and female flowers, fruits of seven developmental stages, as well as primary and lateral roots. RESULTS: In total, 27,868 genes and 2352 novel transcripts were annotated from these 16 tissues, with over 18,000 genes common to all tissue groups. Of these, 3812 were identified as housekeeping genes, half of which assigned to known gene ontologies. Flowers, seeds, and young fruits had the largest number of specific genes, whilst intermediate-age fruits the fewest. There also were genes that were differentially expressed in the various tissues, the male flower being the tissue with the most differentially expressed genes in pair-wise comparisons with the remaining tissues, and the leaf stem the least. The largest expression change during fruit development was early on, from female flower to fruit two days after pollination. A weighted correlation network analysis performed on the global gene expression dataset assigned 25,413 genes to 24 coexpression groups, and some of these groups exhibited strong tissue specificity. CONCLUSIONS: These findings enrich our understanding about the transcriptomic events associated with summer squash development and ripening. This comprehensive gene expression atlas is expected not only to provide a global view of gene expression patterns in all major tissues in C. pepo but to also serve as a valuable resource for functional genomics and gene discovery in Cucurbitaceae.
Assuntos
Cucurbita , Cucurbita/genética , Flores/genética , Frutas/genética , Regulação da Expressão Gênica de Plantas , Polinização , RNA-SeqRESUMO
Cytosine methylation is an epigenetic modification with essential roles in diverse plant biological processes including vegetative and reproductive development and responsiveness to environmental stimuli. A dynamic process involving DNA methyltransferases and DNA demethylases establishes cytosine DNA methylation levels and distribution along the genome. A DNA demethylase gene from barley (Hordeum vulgare), DEMETER (HvDME), the homologue of the Arabidopsis thaliana DME (AtDME), has been characterized previously and found to respond to drought conditions. Here, the promoter of the HvDME gene was analysed further by in silico and DNA methylation analysis. The effect of drought conditions on the DNA methylation status of HvDME was investigated at single-cytosine resolution using bisulfite sequencing. It was demonstrated that the HvDME promoter can be divided into two discrete regions, in terms of DNA methylation level and density; a relatively unmethylated region proximal to the translational start site that is depleted of non-CG (CHG, CHH) methylation and another distal region, approximately 1500 bp upstream of the translational start site, enriched in CG, as well as non-CG methylation. Drought stress provoked alterations in the methylation status of the HvDME promoter distal region, whereas the DNA methylation of the proximal region remained unaffected. Computational analysis of the HvDME promoter revealed the presence of several putative regulatory elements related to drought responsiveness, as well as transposable elements (TEs) that may affect DNA methylation. Overall, our results expand our investigations of the epigenetic regulation of the HvDME gene in response to drought stress in barley and may contribute to further understanding of the epigenetic mechanisms underlying abiotic stress responses in barley and other cereals.
Assuntos
Hordeum , Metilação de DNA , Secas , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Hordeum/genéticaRESUMO
An amplicon metagenomic approach based on the ITS1 region of fungal rDNA was employed to identify the composition of fungal communities associated with diseases of pear fruits during postharvest storage. The sampled fruits were harvested at an orchard using routine management practices involving treatments with various chemical fungicides and were transferred to a storage packinghouse. Effective tags of reading sequences clustered into 53 OTUs whereas Ascomycota was the dominant phylum (83.4%) followed by Basidiomycota (15.8%). Our results revealed that four genera, Penicillium, Rhodotorula, Alternaria and Cladosporium were the most abundant representing 59-95% of the relative abundance per sample. The interruption of chemical treatments during the last month before harvest altered the structure of the fungal community of fruits among untreated and treated samples, mainly in cases of relative abundance of Penicillium and Rhodotorula genera. We hypothesize that various antagonistic interactions might occur on fruit surfaces among the detected fungal genera whose relative abundances were affected by fungicide treatments. Interestingly, some common pre- and postharvest pear fungal pathogens were either less present (such as Moniliana), or undetected (such as Aspergillus, Venturia and Septoria) in untreated and treated samples.
Assuntos
Microbiologia de Alimentos , Frutas/microbiologia , Fungos , Metagenômica , Micobioma , Pyrus/microbiologia , Fungos/classificação , Fungos/efeitos dos fármacos , Fungos/genética , Fungicidas Industriais/farmacologia , Micobioma/efeitos dos fármacos , Micobioma/genéticaRESUMO
Adverse environmental conditions such as UV radiation induce oxidative and aging events leading to severe damage to human skin cells. Natural products such as plant extracts have been implicated in the skin anti-oxidant and anti-aging cellular protection against environmental stress. Moreover, environmental factors have been shown to impact chromatin structure leading to altered gene expression programs with profound changes in cellular functions. In this study, we assessed the in vitro effect of a leaf extract from Vitis vinifera L. on UV-stressed primary human dermal fibroblasts, focusing on gene expression and DNA methylation as an epigenetic factor. Expression analysis of two genes known to be implicated in skin anti-aging, SIRT1and HSP4, demonstrated significant induction in the presence of the extract under normal or UVA conditions. In addition, DNA methylation profiling of SIRT1 and HSP47 promoters showed that the V. vinifera L. extract induced changes in the DNA methylation pattern of both genes that may be associated with SIRT1 and HSP47 gene expression. Our study shows for the first time transcriptional and DNA methylation alterations on human skin fibroblasts exposed to UV stress and suggest a protective effect of a V. vinifera extract possibly through transcriptional regulation of critical skin anti-aging genes.
Assuntos
Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Extratos Vegetais/farmacologia , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Vitis/química , Antioxidantes/farmacologia , Células Cultivadas , Metilação de DNA , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Pele/citologia , Pele/metabolismo , Raios UltravioletaRESUMO
Beans are one of the most important staple crops in the world. Runner bean (Phaseolus coccineus L.) is a small-scale agriculture crop compared to common bean (Phaseolusvulgaris). Beans have been introduced to Europe from the Central America to Europe and since then they have been scattered to different geographical regions. This has resulted in the generation of numerous local cultivars and landraces with distinguished characters and adaptive potential. To identify and characterize the underlying genomic variation of two very closely related runner bean cultivars, we performed RNA-Seq with de novo transcriptome assembly in two landraces of P. coccineus, 'Gigantes' and 'Elephantes' phenotypically distinct, differing in seed size and shape. The cleaned reads generated 37,379 and 37,774 transcripts for 'Gigantes' and 'Elephantes,' respectively. A total of 1896 DEGs were identified between the two cultivars, 1248 upregulated in 'Elephantes' and 648 upregulated in 'Gigantes.' A significant upregulation of defense-related genes was observed in 'Elephantes,' among those, numerous members of the AP2-EREBP, WRKY, NAC, and bHLH transcription factor families. In total, 3956 and 4322 SSRs were identified in 'Gigantes' and 'Elephantes,' respectively. Trinucleotide repeats were the most dominant repeat motif, accounting for 41.9% in 'Gigantes' and 40.1% in 'Elephantes' of the SSRs identified, followed by dinucleotide repeats (29.1% in both cultivars). Additionally, 19,281 putative SNPs were identified, among those 3161 were non-synonymous, thus having potential functional implications. High-confidence non-synonymous SNPs were successfully validated with an HRM assay, which can be directly adopted for P. coccineus molecular breeding. These results significantly expand the number of polymorphic markers within P. coccineus genus, enabling the robust identification of runner bean cultivars, the construction of high-resolution genetic maps, potentiating genome-wide association studies. They finally contribute to the genetic reservoir for the improvement of the closely related and intercrossable Phaseolus vulgaris.
Assuntos
Produtos Agrícolas/genética , Variação Genética , Genoma de Planta , Phaseolus/genética , Transcriptoma , Marcadores Genéticos , Anotação de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genéticaRESUMO
Plant glutathione transferases (EC 2.5.1.18, GSTs) are an ancient, multimember and diverse enzyme class. Plant GSTs have diverse roles in plant development, endogenous metabolism, stress tolerance, and xenobiotic detoxification. Their study embodies both fundamental aspects and agricultural interest, because of their ability to confer tolerance against biotic and abiotic stresses and to detoxify herbicides. Here we review the biotechnological applications of GSTs towards developing plants that are resistant to biotic and abiotic stresses. We integrate recent discoveries, highlight, and critically discuss the underlying biochemical and molecular pathways involved. We elaborate that the functions of GSTs in abiotic and biotic stress adaptation are potentially a result of both catalytic and non-catalytic functions. These include conjugation of reactive electrophile species with glutathione and the modulation of cellular redox status, biosynthesis, binding, and transport of secondary metabolites and hormones. Their major universal functions under stress underline the potential in developing climate-resilient cultivars through a combination of molecular and conventional breeding programs. We propose that future GST engineering efforts through rational and combinatorial approaches, would lead to the design of improved isoenzymes with purpose-designed catalytic activities and novel functional properties. Concurrent GST-GSH metabolic engineering can incrementally increase the effectiveness of GST biotechnological deployment.
Assuntos
Biotecnologia/métodos , Glutationa Transferase/metabolismo , Biodegradação Ambiental , Reguladores de Crescimento de Plantas/metabolismoRESUMO
KEY MESSAGE: A selection-free, highly efficient targeted mutagenesis approach based on a novel ZFN monomer arrangement for genome engineering in tomato reveals plant trait modifications. How to achieve precise gene targeting in plants and especially in crops remains a long-sought goal for elucidating gene function and advancing molecular breeding. To address this issue, zinc finger nuclease (ZFN)-based technology was developed for the Solanum lycopersicum seed system. A ZFN architecture design with an intronic sequence between the two DNA recognition sites was evaluated for its efficiency in targeted gene mutagenesis. Custom engineered ZFNs for the developmental regulator LEAFY-COTYLEDON1-LIKE4 (L1L4) coding for the ß subunit of nuclear factor Y, when transiently expressed in tomato seeds, cleaved the target site and stimulated imperfect repair driven by nonhomologous end-joining, thus, introducing mutations into the endogenous target site. The successful in planta application of the ZFN platform resulted in L1L4 mutations which conferred heterochronic phenotypes during development. Our results revealed that sequence changes upstream of the DNA binding domain of L1L4 can lead to phenotypic diversity including fruit organ. These results underscore the utility of engineered ZFN approach in targeted mutagenesis of tomato plant which may accelerate translational research and tomato breeding.
Assuntos
Endonucleases/metabolismo , Marcação de Genes/métodos , Genes de Plantas , Engenharia Genética/métodos , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Dedos de Zinco/genética , Sequência de Bases , Frutas/anatomia & histologia , Regulação da Expressão Gênica de Plantas , Testes Genéticos , Solanum lycopersicum/crescimento & desenvolvimento , Mutação/genética , Fenótipo , Plantas Geneticamente ModificadasRESUMO
Upland cotton (Gossypium hirsutum L.) is an important fiber crop species, which is intensively plagued by a plethora of phytopathogenic fungi such as Fusarium oxysporum f. sp. vasinfectum (Fov) causing severe wilt disease. Resistance gene analogs (RGAs) are the largest class of potential resistance (R) genes depicting highly conserved domains and structures in plants. Additionally, RGAs are pivotal components of breeding projects towards host disease resistance, serving as useful functional markers linked to R genes. In this study, a cloning approach based on conserved RGAs motifs was used in order to amplify 38 RGAs from two upland cotton cultivars differing in their Fov susceptibility. Besides, we assessed the phylogenetic expansion and the evolutionary pressures acting upon 127 RGA homologues, which were previously deposited in GenBank along with the 38 RGAs from this study. A total of 165 RGAs sequences were clustered according to their BLAST(P) similarities in ten paralogous genes groups (PGGs). These RGAs exhibited intensive signs of positive selection as it was revealed by inferring various maximum likelihood analyses. The results showed robust signs of positive selection, acting in almost all PGGs across the phylogeny. The evolutionary analysis revealed the existence of 42 positively selected residue sites across the PGG lineages, putatively affecting their ligand-binding specificities. As RGAs derived markers are in close linkage to R genes, these results could be used in ongoing breeding programs of upland cotton.
RESUMO
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.
Assuntos
Leucemia Linfocítica Crônica de Células B/classificação , Leucemia Linfocítica Crônica de Células B/genética , Técnicas de Diagnóstico Molecular/métodos , Terapia de Alvo Molecular , Receptores de Antígenos de Linfócitos B/genética , Sequência de Aminoácidos , Rearranjo Gênico do Linfócito B/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Receptores de Antígenos de Linfócitos B/metabolismo , Hipermutação Somática de Imunoglobulina/genéticaRESUMO
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.
Assuntos
Código de Barras de DNA Taxonômico/métodos , Técnicas de Genotipagem/métodos , Sideritis/classificação , Sideritis/genética , Sequência de Bases , Primers do DNA/genética , DNA de Plantas/genética , DNA Espaçador Ribossômico/genética , Marcadores Genéticos , Grécia , Dados de Sequência Molecular , Filogenia , Filogeografia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de SequênciaRESUMO
An outbreak situation of human listeriosis requires a fast and accurate protocol for typing Listeria monocytogenes. Existing techniques are either characterized by low discriminatory power or are laborious and require several days to give a final result. Polymerase chain reaction (PCR) coupled with high resolution melting (HRM) analysis was investigated in this study as an alternative tool for a rapid and precise genotyping of L. monocytogenes isolates. Fifty-five isolates of L. monocytogenes isolated from poultry carcasses and the environment of four slaughterhouses were typed by HRM analysis using two specific markers, internalin B and ssrA genes. The analysis of genotype confidence percentage of L. monocytogenes isolates produced by HRM analysis generated dendrograms with two major groups and several subgroups. Furthermore, the analysis of the HRM curves revealed that all L. monocytogenes isolates could easily be distinguished. In conclusion, HRM was proven to be a fast and powerful tool for genotyping isolates of L. monocytogenes.
RESUMO
Sireviruses are one of the three genera of Copia long terminal repeat (LTR) retrotransposons, exclusive to and highly abundant in plants, and with a unique, among retrotransposons, genome structure. Yet, perhaps due to the few references to the Sirevirus origin of some families, compounded by the difficulty in correctly assigning retrotransposon families into genera, Sireviruses have hardly featured in recent research. As a result, analysis at this key level of classification and details of their colonization and impact on plant genomes are currently lacking. Recently, however, it became possible to accurately assign elements from diverse families to this genus in one step, based on highly conserved sequence motifs. Hence, Sirevirus dynamics in the relatively obese maize genome can now be comprehensively studied. Overall, we identified >10 600 intact and approximately 28 000 degenerate Sirevirus elements from a plethora of families, some brought into the genus for the first time. Sireviruses make up approximately 90% of the Copia population and it is the only genus that has successfully infiltrated the genome, possibly by experiencing intense amplification during the last 600 000 years, while being constantly recycled by host mechanisms. They accumulate in chromosome-distal gene-rich areas, where they insert in between gene islands, mainly in preferred zones within their own genomes. Sirevirus LTRs are heavily methylated, while there is evidence for a palindromic consensus target sequence. This work brings Sireviruses in the spotlight, elucidating their lifestyle and history, and suggesting their crucial role in the current genomic make-up of maize, and possibly other plant hosts.
Assuntos
Evolução Molecular , Genoma de Planta , Retroelementos , Zea mays/genética , Algoritmos , Metilação de DNA , DNA de Plantas/genética , Filogenia , Análise de Sequência de DNARESUMO
Chronic lymphocytic leukemia (CLL) patients assigned to stereotyped subset 4 possess distinctive patterns of intraclonal diversification (ID) within their immunoglobulin (IG) genes. Although 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 eight 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 not been selected. 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.
Assuntos
Evolução Clonal , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos B/genética , Humanos , Fenômenos Imunogenéticos , Leucemia Linfocítica Crônica de Células B/imunologiaRESUMO
BACKGROUND: 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. RESULTS: 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' 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. CONCLUSION: 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.
Assuntos
Secas , Epigênese Genética , Genes de Plantas/genética , Hordeum/crescimento & desenvolvimento , Hordeum/genética , Sementes/crescimento & desenvolvimento , Sementes/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Simulação por Computador , Metilação de DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genômica , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , Estresse Fisiológico/genéticaRESUMO
We examined 807 productive IGHV-IGHD-IGHJ gene rearrangements from mantle cell lymphoma (MCL) cases, by far the largest series to date. The IGHV gene repertoire was remarkably biased, with IGHV3-21, IGHV4-34, IGHV1-8, and IGHV3-23 accounting for 46.3% of the cohort. Eighty-four of 807 (10.4%) cases, mainly using the IGHV3-21 and IGHV4-34 genes, were found to bear stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences and were placed in 38 clusters. Notably, the MCL stereotypes were distinct from those reported for chronic lymphocytic leukemia. Based on somatic hypermutation (SHM) status, 238/807 sequences (29.5%) carried IGHV genes with 100% germ line identity; the remainder (569/807; 70.5%) exhibited different SHM impact, ranging from minimal (in most cases) to pronounced. Shared replacement mutations across the IGHV gene were identified for certain subgroups, especially those using IGHV3-21, IGHV1-8, and IGHV3-23. Comparison with other entities, in particular CLL, revealed that several of these mutations were "MCL-biased." In conclusion, MCL is characterized by a highly restricted immunoglobulin gene repertoire with stereotyped VH CDR3s and very precise SHM targeting, strongly implying a role for antigen-driven selection of the clonogenic progenitors. Hence, an antigen-driven origin of MCL could be envisaged, at least for subsets of cases.
Assuntos
Epitopos/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Imunogenética , Linfoma de Célula do Manto/etiologia , Linfoma de Célula do Manto/imunologia , Sequência de Aminoácidos , Análise por Conglomerados , Estudos de Coortes , Epitopos/fisiologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B/fisiologia , Genes de Imunoglobulinas/genética , Genes de Imunoglobulinas/fisiologia , Humanos , Imunogenética/métodos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Linfoma de Célula do Manto/genética , Dados de Sequência MolecularRESUMO
BACKGROUND: The adulteration of high-priced olive oil with low-cost oils and the fraudulent labelling of oil products make the identification and traceability of vegetable oil species in the food chain very important. This paper describes a high-resolution melting analysis-based method using chloroplast barcoding regions as target (Bar-HRM) to obtain barcoding information for the major vegetable oil species and to quantitatively identify the botanical origin of plant oils. The detection of adulteration of olive oil with canola oil was used as a case study. RESULTS: The proposed method was capable of distinguishing among different vegetable oil species and detecting a level of 1% (w/w) of canola oil in olive oil. CONCLUSION: Bar-HRM analysis is a more accurate, faster and less costly alternative method to authenticate vegetable oils, including olive oil, and to detect mixtures of oils.
Assuntos
Código de Barras de DNA Taxonômico , DNA de Plantas/análise , Inspeção de Alimentos/métodos , Rotulagem de Alimentos , Frutas/química , Olea/química , Óleos de Plantas/química , Brassica napus/química , Brassica napus/metabolismo , DNA de Plantas/metabolismo , Ácidos Graxos Monoinsaturados/análise , Ácidos Graxos Monoinsaturados/química , Contaminação de Alimentos , Toxicologia Forense/métodos , Frutas/metabolismo , Grécia , Limite de Detecção , Desnaturação de Ácido Nucleico , Olea/metabolismo , Azeite de Oliva , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Óleo de Brassica napus , Reação em Cadeia da Polimerase em Tempo Real , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
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.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica de Plantas , Hordeum/crescimento & desenvolvimento , Hordeum/genética , Proteínas de Domínio MADS/genética , Sementes/crescimento & desenvolvimento , Sementes/genética , Ácido Abscísico/farmacologia , Sequência de Aminoácidos , Ciclopentanos/farmacologia , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Epigênese Genética/efeitos dos fármacos , Éxons/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Hordeum/efeitos dos fármacos , Íntrons/genética , Proteínas de Domínio MADS/química , Proteínas de Domínio MADS/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Oxilipinas/farmacologia , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sequências Reguladoras de Ácido Nucleico/genética , Sementes/efeitos dos fármacos , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris.
Assuntos
Biocatálise/efeitos dos fármacos , Glutationa Transferase/biossíntese , Glutationa Transferase/química , Phaseolus/efeitos dos fármacos , Phaseolus/enzimologia , Piridinas/farmacologia , Sequência de Aminoácidos , Aminoácidos/metabolismo , Clonagem Molecular , Dinitroclorobenzeno/química , Dinitroclorobenzeno/metabolismo , Eletroforese em Gel de Poliacrilamida , Indução Enzimática/efeitos dos fármacos , Glutationa/metabolismo , Glutationa Transferase/isolamento & purificação , Isotiocianatos/química , Isotiocianatos/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Piridinas/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Especificidade por Substrato/efeitos dos fármacosRESUMO
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.