María José Jiménez-Quesada

NADPH oxidase activity in pollen tubes is affected by calcium ions, signaling phospholipids and Rac/Rop GTPases

Reactive oxygen species (ROS) generated by NADPH oxidase (NOX) are crucial for tip growth of pollen tubes. However, the regulation of NOX activity in pollen tubes remains unknown. Using purified plasma membrane fractions from tobacco and olive pollen and tobacco BY-2 cells, we demonstrate that pollen NOX is activated by calcium ions and low abundant signaling phospholipids, such as phosphatidic acid and phosphatidylinositol 4,5-bisphosphate in vitro and in vivo. Our data also suggest possible synergism between Ca(2+) and phospholipid-mediated NOX activation in pollen. Rac/Rop small GTPases are also necessary for normal pollen tube growth and have been proposed to regulate ROS production in root hairs. We show here elevated ROS formation in pollen tubes overexpressing wild-type NtRac5 and constitutively active NtRac5, while overexpression of dominant-negative NtRac5 led to a decrease of ROS in pollen tubes. We also show that PA formed by distinct phospholipases D (PLD) is involved in pathways both upstream and downstream of NOX-mediated ROS generation and identify NtPLDδ as a PLD isoform acting in the ROS response pathway.

NADPH Oxidase-Dependent Superoxide Production in Plant Reproductive Tissues.

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.

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).

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.

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.

S-nitroso- and nitro- proteomes in the olive (Olea europaea L.) pollen. Predictive versus experimental data by nano-LC-MS

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.

Identification and in silico Analysis of NADPH Oxidase Homologues Involved in Allergy from an Olive Pollen Transcriptome

Reactive oxygen species generated by pollen NADPH oxidases are present in numerous allergenic pollen species. The superoxide generated by this enzyme has been suggested as a key actor in the induction of allergic inflammation. However, this enzyme has been characterized in Arabidopsis thaliana pollen only, where two pollen-specific genes (RbohH and RbohJ) have been described. The olive (Olea europaea L.) pollen is an important source of allergy in Mediterranean countries. We have assembled and annotated an olive pollen transcriptome, which allowed us to determine the presence of at least two pollen-specific NADPH oxidase homologues. Primers were designed to distinguish between the two homologues, and full-length sequences were obtained through a PCR strategy. Complete in silico analysis of such sequences, including phylogeny, 3-D modeling of the N-terminus, and prediction of cellular localization and post-translational modifications was carried out with the purpose of shed light into the involvement of olive pollen-intrinsic NADPH oxidases in triggering allergy symptoms.