Search

Reactive oxygen species (ROS) are produced in the olive reproductive organs as the result of intense metabolism. ROS production and pattern of distribution depend on the developmental stage, supposedly playing a broad panel of functions, which include defense and signaling between pollen and pistil. Among ROS-producing mechanisms, plasma membrane NADPH-oxidase activity is being highlighted in plant tissues, and two enzymes of this type have been characterized in Arabidopsis thaliana pollen (RbohH and RbohJ), playing important roles in pollen physiology. Besides, pollen from different species has shown distinct ROS production mechanism and patterns of distribution. In the olive reproductive tissues, a significant production of superoxide has been described. However, the enzymes responsible for such generation are unknown. Here, we have identified an Rboh-type gene (OeRbohH), mainly expressed in olive pollen. OeRbohH possesses a high degree of identity with RbohH and RbohJ from Arabidopsis, sharing most structural features and motifs. Immunohistochemistry experiments allowed us to localize OeRbohH throughout pollen ontogeny as well as during pollen tube elongation. Furthermore, the balanced activity of tip-localized OeRbohH during pollen tube growth has been shown to be important for normal pollen physiology. This was evidenced by the fact that overexpression caused abnormal phenotypes, whereas incubation with specific NADPH oxidase inhibitor or gene knockdown lead to impaired ROS production and subsequent inhibition of pollen germination and pollen tube growth.

[Background] Gene 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., [Results] An 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., [Conclusion] Regardless 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.

The data presented here are related to the research article entitled “Generation of nitric oxide by olive (Olea europaea L.) pollen during in vitro germination and assessment of the S-nitroso- and nitro-proteomes by computational predictive methods” doi:10.1016/j.niox.2017.06.005 (Jimenez-Quesada et al., 2017) [1]. Predicted cysteine S-nitrosylation and Tyr-nitration sites in proteins derived from a de novo assembled and annotated pollen transcriptome from olive tree (Olea europaea L.) were obtained after using well-established predictive tools in silico. Predictions were performed using both default and highly restrictive thresholds. Numerous gene products identified with these characteristics are listed here. An experimental validation of the data, consisting in nano-LC-MS (Liquid Chromatography-Mass Spectrometry) determination of olive pollen proteins after immunoprecipitation with antibodies to anti-S-nitrosoCys and anti-3-NT (NitroTyrosine) allowed identification of numerous proteins subjected to these two post-translational modifications, which are listed here together with information regarding their cross-presence among the predictions.

Nitric oxide is recognized as a signaling molecule involved in a broad range of physiological processes in plants including sexual reproduction. NO has been detected in the pollen grain at high levels and regulates pollen tube growth. Previous studies demonstrated that NO as well as ROS are produced in the olive reproductive tissues in a stage- and tissue-specific manner. The aim of this study was to assess the production of NO throughout the germination of olive (Olea europaea L.) pollen in vitro. The NO fluorescent probe DAF-2DA was used to image NO production in situ, which was correlated to pollen viability. Moreover, by means of a fluorimetric assay we showed that growing pollen tubes release NO. GSNO -a mobile reservoir of NO, formed by the S-nitrosylation of NO with reduced glutathione (GSH) - was for the first time detected and quantified at different stages of pollen tube growth using a LC-ES/MS analysis. Exogenous NO donors inhibited both pollen germination and pollen tube growth and these effects were partially reverted by the specific NO-scavenger c-PTIO. However, little is known about how NO affects the germination process. With the aim of elucidating the putative relevance of protein S-nitrosylation and Tyr-nitration as important post-translational modifications in the development and physiology of the olive pollen, a de novo assembled and annotated reproductive transcriptome from olive was challenged in silico for the putative capability of transcripts to become potentially modified by S-nitrosylation/Tyr-nitration according to well-established criteria. Numerous gene products with these characteristics were identified, and a broad discussion as regards to their potential role in plant reproduction was built after their functional classification. Moreover, the importance of both S-nitrosylation/Tyr-nitrations was experimentally assessed and validated by using Western blotting, immunoprecipitation and proteomic approaches.

In the life cycle of a flowering plant, the male gametophyte (pollen grain) produced in the anther reaches the stigmatic surface and initiates the pollen–pistil interaction, an important step in plant reproduction, which ultimately leads to the delivery of two sperm cells to the female gametophyte (embryo sac) inside the ovule. The pollen tube undergoes a strictly apical expansion characterized by a high growth rate, whose targeting should be tightly regulated. A continuous exchange of signals therefore takes place between the haploid pollen and diploid tissue of the pistil until fertilization. In compatible interactions, theses processes result in double fertilization to form a zygote (2n) and the triploid endosperm. Among the large number of signaling mechanisms involved, the redox network appears to be particularly important. Respiratory burst oxidase homologs (Rbohs) are superoxide-producing enzymes involved in a broad range of processes in plant physiology. In this study, we review the latest findings on understanding Rboh activity in sexual plant reproduction, with a particular focus on the male gametophyte from the anther development stages to the crowning point of fertilization. Rboh isoforms have been identified in both the male and female gametophyte and have proven to be tightly regulated. Their role at crucial points such as proper growth of pollen tube, self-incompatibility response and eventual fertilization is discussed. [EN]

Enzyme activities regulating ROS metabolism in the olive seeds are relatively unknown, in spite of the economical importance of this tree and the increasing interest of these seeds for multiple purposes. The aim of this work was to provide a comprehensive analysis of the genes encoding major antioxidant enzymes in mature seed. For this purpose, we have used transcriptomic sequence information available through our own experiments and in specific databases (Olea EST). Numerous sequences have been retrieved and analysed by using basic descriptive tools. Expression of transcripts encoding several of these enzymes has been carried out. These include catalases, superoxide dismutase, dehydroascorbate reductase, GSNO reductase, NADPH oxidase, lipoxygenase and thioredoxins. Cellular localization of several of these enzymes and histochemical detection of enzyme activities have been also carried out. We discuss how these enzyme activities may be involved in the olive seed metabolism through germination, and how they may contribute to the antioxidant capacity, stability and organoleptic properties of olive oils.

Olive transcriptome has been recently built as the result of library screening through the use of different NGS strategies. Such transcriptome mainly relies on vegetative reads. However, the peculiarity of the reproductive tissues, and the widely reported presence of numerous tissue-specific transcripts in both stigma and pollen grains made us to attempt a similar approach in the later tissues. mRNA was isolated from olive stigmas and pollen grains at different developmental stages and during in vitro pollen germination, respectively. cDNA libraries were built from the isolated mRNA, and subjected to 454/Roche Titanium+ sequencing. Moreover, NGS readings were combined with additional sequences obtained from subtractive libraries, in order to produce a de novo assembly of the olive reproductive transcriptome. Several public software and other tools developed by authors were used for this purpose. Sequences were assembled together into 58 098 (stigma) and 26 422 (pollen) contigs, respectively. Functional annotation is currently underway, however numerous gene products have been previously analysed, particularly those corresponding to major enzymes regulating ROS metabolism. Some of them, including catalases, superoxide dismutase, dehydroascorbate reductase, GSNO reductase, NADPH oxidase, lipoxygenase and thioredoxins, have been further analysed using basic descriptive tools.

Transcriptome analysis of the olive stigma and pollen grain was carried out at different developmental stages and during in vitro pollen germination, respectively. Isolated mRNA was used to build cDNA libraries, which were subjected to 454+ sequencing. Readings were combined with sequences obtained from subtractive libraries, in order to produce a de novo assembly of the olive reproductive transcriptome. The presence of transcripts corresponding to major enzymes regulating ROS metabolism was assessed. Sequences corresponding to catalases, superoxide dismutase, dehydroascorbate reductase, GSNO reductase, NADPH oxidase, lipoxygenase and thioredoxins were retrieved and analysed using basic descriptive tools. Expression of transcripts in pollen and pistil was analysed by semiquantitative RT-PCR. Cellular localization of several enzymes was also carried out by using immunocytochemistry. We discuss how these enzyme activities may be involved in the olive pollen metabolism through germination, in the olive stigma over the periods before, after and during receptivity, and how they may contribute to the interaction pollen-stigma.

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).

The presence of high levels of NO has been described in several pollen grains, where their presence has been shown to regulate pollen tube growth and orientation. Here we have assessed the production of NO during in vitro germination in the olive pollen by using exogenous NO donors like sodium nitroprusside and S-nitrosoglutathione. Both chemical induced a remarkable inhibition of pollen germination and tube growth, which were partially reverted by the addition of the specific NO-scavenger carboxy-PTIO (cPTIO). The presence and localization of NO was determined either by using a fluorimetric assay and by confocal microscopy using the fluorescent probe diaminoflurescein-2 diacetate (DAF-2DA). As olive pollen is also a highly allergenic source, the putative role of NO in eliciting the hypersensitive response in humans is discussed.

ROS and NO are low molecular weight molecules implicated in plant signalling. Their relevant implication in pollen tube growth and reorientation has been shown particularly in plant model species. In olive reproductive tissues, their presence has been described in a stage- and tissue- specific manner. Accurate detection and quantification of minor changes in ROS/NO and the chemical species resulting or interacting with their metabolism in reproductive tissues is a challenging task at present. The goal of the present study was to elucidate the implication of the NO metabolism in the process of olive pollen germination in vítro. For this purpose we used a recently published method based in the use of liquid chromatography-electrospray/mass spectrometry to detect different antioxidant metabolites in volved in NO metabolism. We have analysed the levels of key molecules like GSH (reduced glutathione), GSSH (oxidized glutathione), GSNO {Snitrosoglutathione) and ascorbic acid (vitamin e) in the mature pollen, and the pollen grain and pollen tube at different times of in vitro germination. These data correspond to both dormant and activated metabolism, respectively. The results revealed remarkalble changes in the levels of GSNO, which is a cellular NO reservoir, throughout the germination process. These data confirm the involvement of NO and antioxidant molecules in the pollen tube growth, as observed previously by using CLSM, and point to potential new specific roles for these molecules as signalling intermediates.

Olive pollen is a major cause of allergy in Mediterranean countries. Numerous studies are devoted to the characterization of allergy-eliciting proteins from this allergenic source. However, recent research is focusing on the study of other non-allergenic protein and non-protein components modulating the allergic immune response. These molecules include pollen-associated lipid mediators, adenosine, proteases and oxidases, particularly NAD(P)H oxidase (NOX) among others. The production of superoxide by NOX activity in various allergenic pollens has been recently described. NOX activity has been shown to induce oxidative stress in cultured airway epithelium, driving to allergic inflammation. We report here the presence, molecular characteristics and expression of a NOX enzyme in olive pollen. Moreover, we describe the presence, activity and cellular localization of a well characterized allergenic protein from olive pollen (Ole e 5) displaying relevant homology with Cu/Zn SODs from different species, and a Mn-SOD also from olive pollen. The incidence of Ole e 5 in type I hypersensitivity reactions to olive pollen has been reported to be 35%, whereas several Mn-SODs have also been identified as allergens in different species. In addition to their intrinsic allergenic properties, both enzymes may regulate the levels of NOX-generated superoxide, therefore modulating the inflammation response to this pollen. A model of the interactions occurring among these molecules themselves, their substrates/products and the immune system is proposed.