Hablando de sexo en plantas: ¡Vamos al grano!

In plants, development and sexual reproduction are key steps for the generation of the next generation and for the genetic recombination resulting from the meiotic process to take place. In addition, it should not be forgotten that in many cases the production of many species of agricultural interest exploits the partial or final result of the reproductive process, as flowers, the complete fruit or the seed are often collected. Higher plants have been evolving towards the generation of increasingly reduced gametophytic (haploid) phases, which include the embryonic sac and the pollen grain as the expression of the female and male gametophytes respectively. Understanding the physiology of the pollen grain is one of the most exciting issues in reproductive biologyEn las plantas, el desarrollo y la reproducción sexual son pasos clave para la obtención de la generación siguiente y para que tenga lugar la recombinación genética resultante del proceso meiótico. Además, no hay que olvidar que en muchos casos la producción de muchas especies de interés agrícola explota el resultado parcial o final del proceso reproductivo, ya que con frecuencia se recolectan las flores, el fruto completo o la semilla. Las plantas supe- riores han ido evolucionando hacia la generación de fases gametofíticas (haploides) cada vez más reducidas, que incluyen el saco embrionario y el grano de polen como expresión de los gametofitos femenino y masculino respectivamente. La comprensión de la fisiología del grano de polen es una de las cuestiones más apasionantes de la biología reproductiv

Application of X-Ray microanalysis, diffraction and cytochemical techniques in the study of the structure and chemical composition of inclusions in Olea europaea leaves

4 páginas, 11 figuras.-- Trabajo presentado al EMAG-MICRO 89 celebrado en Londres (Inglaterra) en Septiembre de 1989.Two types of inclusions have been found in mesophyll cells oÍ leaves of Olea eurooaea. The first type is located in the vacuole, and the application of X-Ray microanalysis, X-Ray diffraction and cytochemical techniques shown that these inclusions are composed of calcium oxalate. The second type of inclusion is intranuclear and its proteic nature is demonstrated by means of light microscopy stains. These crystal structures are probably well ordered in three dimensions.Peer reviewe

Cellular localization of ROS and NO in olive reproductive tissues during flower development

14 pages, 9 figures, 14 additional files.[Background] Recent studies have shown that reactive oxygen species (ROS) and nitric oxide (NO) are involved in the signalling processes taking place during the interactions pollen-pistil in several plants. The olive tree (Olea europaea L.) is an important crop in Mediterranean countries. It is a dicotyledonous species, with a certain level of self-incompatibility, fertilisation preferentially allogamous, and with an incompatibility system of the gametophytic type not well determined yet. The purpose of the present study was to determine whether relevant ROS and NO are present in the stigmatic surface and other reproductive tissues in the olive over different key developmental stages of the reproductive process. This is a first approach to find out the putative function of these signalling molecules in the regulation of the interaction pollen-stigma.[Results] The presence of ROS and NO was analyzed in the olive floral organs throughout five developmental stages by using histochemical analysis at light microscopy, as well as different fluorochromes, ROS and NO scavengers and a NO donor by confocal laser scanning microscopy. The "green bud" stage and the period including the end of the "recently opened flower" and the "dehiscent anther" stages displayed higher concentrations of the mentioned chemical species. The stigmatic surface (particularly the papillae and the stigma exudate), the anther tissues and the pollen grains and pollen tubes were the tissues accumulating most ROS and NO. The mature pollen grains emitted NO through the apertural regions and the pollen tubes. In contrast, none of these species were detected in the style or the ovary.[Conclusion] The results obtained clearly demonstrate that both ROS and NO are produced in the olive reproductive organs in a stage- and tissue- specific manner. The biological significance of the presence of these products may differ between early flowering stages (defence functions) and stages where there is an intense interaction between pollen and pistil which may determine the presence of a receptive phase in the stigma. The study confirms the enhanced production of NO by pollen grains and tubes during the receptive phase, and the decrease in the presence of ROS when NO is actively produced.This work was supported by research projects P06-AGR-01719 (Junta de Andalucía) and BFU2008-00629 (MCI). AZ thanks the CSIC for providing a JAE grant.Peer reviewe

Current overview of S-nitrosoglutathione (GSNO) in higher plants

S-nitrosoglutathione is a nitric oxide-derived molecule, generated by the interaction of nitric oxide (NO) with reduced glutathione (GSH) in a process called S-nitrosylation (Figure 1). The reaction appears to take place either through the formation of N2O3 or the addition of NO to a glutathionyl radical formed during this reaction (Broniowska et al., 2013). GSNO is regarded as an intracellular NO reservoir as well as a vehicle of NO throughout the cell, which enables NO biological activity to expand. GSNO is also considered to be the most abundant low-molecular-mass (LMM) S-nitrosothiol (SNO). This family includes other molecules such as S-nitrosocysteine (CySNO) and S-nitrosocysteinylglycine (GlyCySNO), which have been the subject of less study in the field of plant research. There is another group of SNOs called high-molecular mass (HMM) SNOs which are produced by NO binding to sulfhydryl (-SH) groups present in specific cysteine residues of proteins. Figure 1 shows a simple model of GSNO metabolism and its interactions with other molecules in cells where different reactions including S-nitrosylation, S-transnitrosation, and S-glutathionylation are involved (Hogg, 2002; Martínez-Ruiz and Lamas, 2007). In plants, research has focused on the importance of total SNOs in specific stress situations (Feechan et al., 2005; Chaki et al., 2011a) and on the identification of the potential protein targets of S-nitrosylation as this kind of post-translational modification can alter the function of the affected proteins (Astier et al., 2012). Initial studies in this area exogenously applied GSNO in order to identify the pool of potential protein candidates (Lindermayr et al., 2005). However, less attention has been paid to the abundance, distribution, and modulation of endogenous GSNO under natural and stress conditions. In this article, we will provide a current overview of GSNO in higher plants. [EN]Work in our laboratories is supported by ERDF-cofinanced grants from the Ministry of Science and Innovation (BIO2012-33904 and BFU2011-22779)Peer reviewe

The use of in situ hybridisation and immunocytochemistry to characterise coiled bodies in plant meiocytes

2 páginas, 2 figuras.-- Trabajo presentado a la XI European Conference on Electron Microscopy- EUREM11 celebrada en Dublín (Irlanda) del 26 al 30 de Agosto de 1996.This study was supported by project DGICYT PB92-0079-CO3-O3.Peer reviewe

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.This study was supported by the following European Regional Development Fund co-financed grants: MCINN BFU2011-22779, RTC-2015-4181-2, CSIC-201540E065, CICE (Junta de Andalucía) P2010-CVI15767, P2010-AGR6274, P2011-CVI-7487, and the MINECO/CSIC agreement RECUPERA 2020.Peer reviewedPeer Reviewe

Preliminary characterization of ssps (Seed Storage Proteins) in Argania spinosa L.

4 páginas, 2 figuras, 1 tabla.-- Comunicación presentada al 3rd International SMBBM Congress, IUBMB Special Meeting & 6th FASBMB Congress, celebrado en Marrakech (Marruecos) del 20 al 24 de Abril de 2009.SSPs (Seed Storage Proteins) of the 11S type have been preliminary characterized in the seeds of the argan tree, an endemic species from Morocco. Protein extracts from mature seeds were prepared by using different solutions in order to assess the solubility of the major protein forms. SSPs of the 11S type were classified as albumins according to the further SDS-PAGE analysis of these extracts. The combination of both reducing- and non-reducing conditions for the SDS-PAGE analysis, together with immunoblot experiments allowed us to determine the presence of three precursor forms of these proteins (pro1, pro2 and pro3), which are composed of six individual peptides (p1 to p6) in different combinations.This work was funded by Spanish BFU2004-00601/BFI and BFU2008-00629 projects. M. Allach thanks the research bursary granted by UNESCO/L´ORÉAL and L´ORÉAL Morocco.Peer reviewe

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. [EN]European Regional Development Fund (ERDF) co-financed grants: BFU2008-006292, BFU2011-22779, CSIC-201540E065, and RECUPERA2020-3.1.4.Peer reviewe

Involvement of Climatic Factors in the Allergen Expression in Olive Pollen

This work was supported by the Spanish Ministry of Science and Innovation (MICINN) (ERDF-cofinanced projects AGL2008-00517, BFU2011-22779 and PIE-200840I186) and the Junta de Andalucía (ERDF-cofinanced projects P2010-CVI5767 and P2010-AGR6274).Peer reviewe

A Protocol for Minimal Single Protein Labeling with CyDye Fluors for Live Cell Internalization Assays

Individual proteins chemically labeled with fluorescent dyes can be localized and tracked in real-time experiments in order to get insights about the site and molecular mechanism of action. Here, we have adapted a protocol that was originally developed for two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) applications, to label proteins with CyDye fluors for single-molecule internalization assays in living cells. This “minimal labeling” method offers a number of advantages including specificity and known stoichiometry, simplicity, high reproducibility, and sensitivity and allows multiplexing while minimizing perturbations of the biological system. Moreover, since only a single lysine (Lys) residue per protein molecule is labeled, this method is also quantitative. To validate experimentally our protocol, we carried out the fluorescent labeling of IBB1, a major soybean protease isoinhibitor of the Bowman-Birk family that is currently being investigated as colorectal chemopreventive agent. Then, we analyzed the in vivo internalization dynamics of the labeled IBB1 protein in human colorectal adenocarcinoma HT29 cells