Adoración Zafra

ReprOlive: a database with linked data for the olive tree (Olea europaea L.) reproductive transcriptome.

Plant reproductive transcriptomes have been analyzed in different species due to the agronomical and biotechnological importance of plant reproduction. Here we presented an olive tree reproductive transcriptome database with samples from pollen and pistil at different developmental stages, and leaf and root as control vegetative tissues http://reprolive.eez.csic.es). It was developed from 2,077,309 raw reads to 1,549 Sanger sequences. Using a pre-defined workflow based on open-source tools, sequences were pre-processed, assembled, mapped, and annotated with expression data, descriptions, GO terms, InterPro signatures, EC numbers, KEGG pathways, ORFs, and SSRs. Tentative transcripts (TTs) were also annotated with the corresponding orthologs in Arabidopsis thaliana from TAIR and RefSeq databases to enable Linked Data integration. It results in a reproductive transcriptome comprising 72,846 contigs with average length of 686 bp, of which 63,965 (87.8%) included at least one functional annotation, and 55,356 (75.9%) had an ortholog. A minimum of 23,568 different TTs was identified and 5,835 of them contain a complete ORF. The representative reproductive transcriptome can be reduced to 28,972 TTs for further gene expression studies. Partial transcriptomes from pollen, pistil, and vegetative tissues as control were also constructed. ReprOlive provides free access and download capability to these results. Retrieval mechanisms for sequences and transcript annotations are provided. Graphical localization of annotated enzymes into KEGG pathways is also possible. Finally, ReprOlive has included a semantic conceptualisation by means of a Resource Description Framework (RDF) allowing a Linked Data search for extracting the most updated information related to enzymes, interactions, allergens, structures, and reactive oxygen species.

Peroxisomal Localization of CuZn Superoxide Dismutase in the Male Reproductive Tissues of the Olive Tree

Superoxide dismutases (SODs) are a class of antioxidant enzymes which catalyze the dismutation of superoxide into oxygen and hydrogen peroxide, therefore controlling cellular levels of Reactive Oxygen Species (ROS). In the mature pollen grains of the olive tree, the presence of several forms of CuZn-SOD and the cytosolic localization of the enzyme have been described. The present study was aimed to elucidate the adaptation of the oxidative metabolism to the changing conditions occurring during the course of olive pollen formation, hydration and pollen tube emergence and growth. We used a polyclonal antibody (raised against a KLH-linked synthetic peptide including a consensus sequence for CuZn-SODs in olive pollen) in immunocytochemical experiments carried out by Fluorescence (FM) and Transmission Electron Microcopy (TEM).

Patterns of ROS Accumulation in the Stigmas of Angiosperms and Visions into Their Multi-Functionality in Plant Reproduction

Accumulation of reactive oxygen species (ROS) in the stigma of several plant species has been investigated. Four developmental stages (unopened flower buds, recently opened flowers, dehiscent anthers, and flowers after fertilization) were analyzed by confocal laser scanning microscopy using the ROS-specific probe DCFH2-DA. In all plants scrutinized, the presence of ROS in the stigmas was detected at higher levels during those developmental phases considered “receptive” to pollen interaction. In addition, these molecules were also present at early (unopened flower) or later (post-fertilization) stages, by following differential patterns depending on the different species. The biological significance of the presence ROS may differ between these stages, including defense functions, signaling and senescence. Pollen-stigma signaling is likely involved in the different mechanisms of self-incompatibility in these plants. The study also register a general decrease in the presence of ROS in the stigmas upon pollination, when NO is supposedly produced in an active manner by pollen grains. Finally, the distribution of ROS in primitive Angiosperms of the genus Magnolia was determined. The production of such chemical species in these plants was several orders of magnitude higher than in the remaining species evoking a massive displacement toward the defense function. This might indicate that signaling functions of ROS/NO in the stigma evolved later, as fine tune likely involved in specialized interactions like self-incompatibility.

Automated identification of reference genes based on RNA-seq data

BackgroundGene expression analyses demand appropriate reference genes (RGs) for normalization, in order to obtain reliable assessments. Ideally, RG expression levels should remain constant in all cells, tissues or experimental conditions under study. Housekeeping genes traditionally fulfilled this requirement, but they have been reported to be less invariant than expected; therefore, RGs should be tested and validated for every particular situation. Microarray data have been used to propose new RGs, but only a limited set of model species and conditions are available; on the contrary, RNA-seq experiments are more and more frequent and constitute a new source of candidate RGs.ResultsAn automated workflow based on mapped NGS reads has been constructed to obtain highly and invariantly expressed RGs based on a normalized expression in reads per mapped million and the coefficient of variation. This workflow has been tested with Roche/454 reads from reproductive tissues of olive tree (Olea europaea L.), as well as with Illumina paired-end reads from two different accessions of Arabidopsis thaliana and three different human cancers (prostate, small-cell cancer lung and lung adenocarcinoma). Candidate RGs have been proposed for each species and many of them have been previously reported as RGs in literature. Experimental validation of significant RGs in olive tree is provided to support the algorithm.ConclusionRegardless sequencing technology, number of replicates, and library sizes, when RNA-seq experiments are designed and performed, the same datasets can be analyzed with our workflow to extract suitable RGs for subsequent PCR validation. Moreover, different subset of experimental conditions can provide different suitable RGs.

Pollen Allergenicity is Highly Dependent on the Plant Genetic Background: The “Variety”/“Cultivar” Issues

© 2012 de Dios Alche et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pollen Allergenicity is Highly Dependent on the Plant Genetic Background: The “Variety”/“Cultivar” Issues

Automatic Workflow for the Identification of Constitutively-Expressed Genes Based on Mapped NGS Reads

Expression analyses such as quantitative and/or real-time PCR require the use of reference genes for normalization in order to obtain reliable assessments. The expression levels of these reference genes must remain constant in all different experimental conditions and/or tissues under study. Traditionally, housekeeping genes have been used for this purpose, but most of them have been reported to vary their expression levels under some experimental conditions. Consequently, the election of the best reference genes should be tested and validated in every experimental scenario. Microarray data are not always available for the search of appropriate reference genes, but NGS experiments are increasingly common. For this reason, an automatic workflow based on mapped NGS reads is presented with the aim of obtaining putative reference genes for a giving species in the experimental conditions of interest. The calculation of the coefficient of variation (CV) and a simple, normalized expression value such as RPKM per transcript allows for filtering and selecting those transcripts expressed homogeneously and consistently in all analyzed conditions. This workflow has been tested with Roche/454 reads obtained from olive (Olea europaea L.) pollen and pistil at different developmental stages, as well as with Illumina paired-end reads from two different accessions of Arabidopsis thaliana. Some of the putative candidate reference genes have been experimentally validated.

Identification of Distinctive Variants of the Olive Pollen Allergen Ole e 5 (Cu,Zn Superoxide Dismutase) throughout the Analysis of the Olive Pollen Transcriptome

Ole e 5 is an olive pollen allergen displaying high identity with Cu,Zn superoxide dismutases. Previous studies characterized biochemical variability in this allergen, which may be of relevance for allergy diagnosis and therapy. The generation of an olive pollen transcriptome allowed us to identify eight Ole e 5 sequences, one of them including a 24 nt deletion. Further in silico analysis permitted designing primers for PCR amplification and cloning from both cDNA and gDNA. A large number of sequences were retrieved, which experimentally validated the predictive NGS sequences, including the deleted enzyme. The PCR-obtained sequences were used for further scrutiny, including sequence aligment and phylogenetic analysis. Two model sequences (a complete sequence and a deleted one) were used to perform 3-D modeling and a prediction of the T- and B-cell epitopes. These predictions interestingly foreseed relevant differences in the antigenicity/allergenicity of both molecules. Clinical relevance of differences is discussed.

Histological features of the olive seed and presence of 7S-type seed storage proteins as hallmarks of the olive fruit development

The production of olive oil is an important economic engine in the Mediterranean area. Nowadays, olive oil is obtained mainly by mechanical processes, by using the whole fruit as the primary raw material. Although the mesocarp is the main source of lipids contributing to olive oil formation, the seed also contributes to the olive oil composition and attributes. The olive seed is also becoming an interesting emerging material itself when obtained after alternative processing of the olive fruit. Such seed is used for the production of differential oil and a unique flour among other bioactive products, with increasing uses and applications in cosmetics, nutrition, and health. However, olive seed histology has been poorly studied to date. A complete description of its anatomy is described for the first time in the present study by using the ‘Picual’ cultivar as a model to study the development of the different tissues of the olive seed from 60 to 210 days after anthesis. A deep analysis of the seed coats, endosperm storage tissue and the embryo during their development has been performed. Moreover, a panel of other olive cultivars has been used to compare the weight contribution of the different tissues to the seed, seed weight variability and the number of seeds per fruit. In addition to the histological features, accumulation of seed storage proteins of the 7S-type (β-conglutins) in the seed tissues has been assessed by both biochemical and immunocytochemical methods. These hallmarks will help to settle the basis for future studies related to the location of different metabolites along the olive seed and mesocarp development, and therefore helping to assess the appropriate ripening stage for different commercial and industrial purposes.

Identification of novel superoxide dismutase isoenzymes in the olive ( Olea europaea L.) pollen

BackgroundAmong antioxidant enzymes, the superoxide dismutase (SOD) family is a major actor in catalysing the disproportionation of superoxide. Apart from its role as antioxidant, these enzymes have a role in cell signalling, and Cu,Zn-SOD proteins are also major pollen allergens. In order to deepen our understanding of the SOD isoenzymes present in olive pollen and to analyse the molecular variability of the pollen Cu,Zn-SOD family, we carried out biochemical, transcriptomic and localization studies of pollen grains from different olive cultivars and other allergenic species.ResultsOlive pollen showed a high rate of total SOD activity in all cultivars assayed, which did not correlate with pollen viability. Mass spectrometry analysis together with activity assays and Western blotting experiments enabled us to identify new forms of Cu,Zn-SOD enzyme (including chloroplastidic and peroxisomal forms) as well as differentially expressed Mn-, Fe- and Cu,Zn-SOD isoenzymes among the pollen of different olive cultivars and allergenic species. Ultrastructural localization of Cu,Zn-SOD revealed its plastidial localization in the pollen grain. We also identified the occurrence of a shorter form of one of the cytosolic Cu,Zn-SOD enzymes, likely as the result of alternative splicing. This shorter enzyme showed lower SOD activity as compared to the full length form.ConclusionsThe presence of multiple SOD isoenzymes in the olive pollen could be related to the need of finely tuning the ROS metabolism during the transition from its quiescent condition at maturity to a highly metabolically active state at germination.

Identification and in silico Analysis of Glutathione Reductase Transcripts Expressed in Olive (Olea europaea L.) Pollen and Pistil

Glutathione (GSH) protects proteins against oxidation of their thiol-containing groups, by alternatively becoming the subject of oxidation, forming glutathione disulfide (GSSG). Appropriate GSH:GSSG levels are maintained by glutathione reductase (GR), a homodimeric flavoprotein which uses NADPH to reduce one GSSG molecule to two of GSH. This enzyme has been characterized in several species, and described as highly conserved, with two isoforms only. Heterogeneity and discticntiveness of plant reproductive tissues led us to investigate the presence of GR sequences. A de novo assembled and annotated olive reproductive transcriptome was subjected to screening, which allowed us to identify at least 11 GR homologues (1 pollen-specific and 10 from pistils). Primers were designed, and full-length sequences were obtained through PCR. In silico analysis, including phylogeny, 3-D modeling of N-terminus, and prediction of cellular localization and post-translational modifications was carried out to shed light into the involvement of olive pollen-intrinsic GR in reproductive development.