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

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

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

This work was funded by ERDF (co)-financed projects P2010-CVI5767, P2010-AGR6274, BFU2011-22779, P2011-CVI-7487 and PEOPLE-IOF/1526.Peer reviewe

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

Differential characteristics of olive pollen from different cultivars: Biological and clinical implications

The olive tree is grown in many parts of the world. Its germplasm is very broad, with 250 varieties in Spain alone. Variations in the ability of pollen to germinate have been studied in detail and show conspicuous differences between varieties. However, commercial olive pollen from cultivars whose origin is unknown is the material that is commonly used for clinical and biological studies. We aim to assess the putative heterogeneity of olive cultivars with regard to the presence of several pollen allergens and to determine whether these differences have biological and clinical relevance. Previous studies show that most allergens isolated and characterized to date are highly polymorphic. Olive cultivars display wide differences in the expression levels of many allergens and in the number and molecular characteristics of the allergen isoforms expressed. These differences are maintained over the years, and are intrinsic to the genetics of each cultivar. Such broad polymorphism seems to be involved in the physiology of the olive reproductive system, which might include the adaptation of the plant to different environmental conditions, the establishment of the compatibility system, and pollen performance. The differences in allergen composition in cultivars, particularly in the Ole e 1 allergen, are responsible for the important differences in the allergenic potency of the extracts. These fi ndings could have a number of implications for the diagnosis and therapy of olive pollen allergy. We discuss how cultivar differences affect extract quality, diagnostic and therapeutic effi cacy and safety, and the development of new vaccines based on the use of recombinant allergens.El olivo es un cultivo ampliamente representado en el mundo. Su germoplasma es muy amplio, con 250 variedades sólo en España. La capacidad del polen para germinar, que presenta notables diferencias entre variedades, ha sido estudiada en detalle. El material usado comúnmente para estudios clínicos y biológicos es, sin embargo, polen comercial de cultivares de origen desconocido. Nuestro objetivo es evaluar la posible heterogeneidad de los cultivares de olivo en relación a la presencia de varios alérgenos del polen, y determinar si esas diferencias tienen relevancia biológica y clínica. Estudios previos muestran que la mayor parte de los alérgenos aislados y caracterizados hasta la fecha son altamente polimórfi cos. Los cultivares de olivo muestran amplias diferencias en los niveles de expresión de muchos alérgenos, así como en el número y características moleculares de las isoformas alergénicas expresadas. Estas diferencias se mantienen a lo largo de años, y son intrínsecas a la genética de cada cultivar. Este amplio polimorfi smo parece estar implicado en la fi siología del sistema reproductivo del olivo, en relación con la adaptación de la planta a diferentes condiciones ambientales, el establecimiento de un sistema de compatibilidad, y el dinamismo del polen. Las diferencias en la composición alergénica de los cultivares, particularmente en cuanto al alérgeno Ole e 1, son responsables de las importantes diferencias en la potencia alergénica de los extractos. Estos hallazgos pueden tener numerosas implicaciones en la diagnosis y terapia de la alergia al polen del olivo. Discutimos cómo las diferencias entre cultivares afectan a la calidad del polen, a la efi cacia y seguridad del diagnóstico y la terapia, así como al desarrollo de nuevas vacunas basadas en el uso de alérgenos recombinantes

Estudios sobre la reproducción sexual en el olivo

Trabajo presentado a las VI Jornadas Internacionales de Olivar Ecológico. Ecoliva, celebradas en Puente de Génave (Jaén) en 2007.El olivo es una planta de gran importancia agronómica en los países mediterráneos, y muy especialmente en nuestra región. Nuestro grupo viene tradicionalmente estudiando numerosos aspectos de la reproducción sexual de dicha especie, tales como el desarrollo del polen, sus características (viabilidad y capacidad de germinación), el desarrollo del gineceo, los procesos de fertilización, la formación y maduración del fruto, etc. Para estos estudios utilizamos distintos métodos que incluyen desde la observación morfológica mediante técnicas de microscopía óptica, microscopía electrónica de transmisión, barrido y microscopía confocal, hasta técnicas de aislamiento y caracterización de proteínas y genes implicados en dichos procesos. Todos estos estudios nos están ayudando a comprender el sistema de reproducción sexual del olivo, una planta en la que la presencia de mecanismos de autoincompatibilidad polen-pistilo con bases aparentemente genéticas y ambientales, está empezando a ser estudiada. Otra línea de investigación en curso en nuestro grupo es aquella que se refiere al estudio de determinadas proteínas del grano de polen con funciones clave en su desarrollo y en la germinación y crecimiento del tubo polínico, y que además exhiben carácter alergénico para algunos individuos. Las características moleculares y el contenido de estos alérgenos varían significativamente entre cultivares de olivo, lo cual hace especialmente interesante su estudio en la fisiología del grano de polen, además de las implicaciones clínicas que de ello se derivan.Los resultados obtenidos en este campo se engloban en dos proyectos de investigación (AGL2003-00719 y BFU 2004-00601/BFI). Los resultados descritos han sido obtenidos gracias a la financiación de diversas instituciones públicas, esencialmente, los proyectos del Plan Nacional de I+D+i mencionados anteriormente y varios planes de formación de personal investigador de dicho programa, así como de la AECI y del programa I3P-CSIC.Peer reviewe

Biología reproductiva del olivo (BRO-EEZ-CSIC)

Trabajo presentado a las III Jornadas Nacionales del grupo de Horticultura de la SECH, celebradas en Sevilla el 6 y 7 de octubre de 2011.Peer reviewe

Microscopy to study plant sexual reproduction

This work has been supported by numerous research projects. Currently active projects include EFDF confunded grants BFU2011-22779 (Ministerio de Economía y Competitividad), P2010-CVI5767, P2010-AGR6274 and P2011-CVI7487 (Junta de Andalucía), and PEOPLE-PIOF-GA-2011-301550 (European Research Council)Peer reviewe

An overview of ROS and RNS metabolism in the olive reproductive process

The olive tree (Olea europaea L.) has a high economical and social importance in the Mediterranean area. Spain as the main world producer, and specially Andalusia, has very significant positions regarding these aspects. The peculiarities of the olive tree in relation with the reproductive process make its study an important tool to achieve satisfactory yield production. We are beginning to understand that Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), far from their traditional roles related to toxicity and stress, have much to say in Plant Reproductive Biology as in the many processes that have been described to by regulated by these two groups of molecules. As a first approach, and aimed to study the overall implications of ROS and RNS in the Reproductive Biology of Plants, we used flowers from a variety of Higher Plant families with different types of stigmas and self-incompatibility systems (as well as an ancient representative of Angiosperms) at different developmental stages. We analysed the presence of ROS (H2O2)/NO in these stigmas by fluorescence stereomicroscopy. Next, we performed a detailed and individualised detection of H2O2, O2 - and NO in the olive stigmas, anthers and pollen grains over several developmental stages under stereomicroscopy and high-resolution confocal laser scanning microscopy by using highly selective probes.El olivo (Olea europaea L.) posee una gran importancia económica y social en el área mediterránea. España como el mayor productor mundial, y especialmente Andalucía, tiene una posición muy significativa en relación con estos aspectos. Las peculiaridades del olivo en relación a los procesos reproductivos hacen de su estudio una importante herramienta para alcanzar una producción satisfactoria. Estamos empezando a conocer que las especies de oxígeno reactivo (ROS) y las especies de nitrógeno reactivo (RNS), lejos de sus tradicionales funciones implicadas en toxicidad y estrés, tienen mucho que decir en la Biología Reproductiva de las Plantas, tanto como en los numerosos procesos regulados por estos dos grupos de moléculas. Como una primera aproximación, y con objeto de estudiar las implicaciones generales de las ROS y RNS in la Biología Reproductiva de las Plantas, usamos flores de varias familias de Plantas Superiores con diferentes tipos de estigmas y de sistemas de autoincompatibilidad (así como un representante primigenio de las Angiospermas) en diferentes estadios de desarrollo. Analizamos la presencia de ROS (H2O2)/NO en dichos estigmas mediante estereomicroscopía de fluorescencia. A continuación, realizamos una detección detallada e individualizada de H2O2, O2 - y NO en los estigmas, anteras y granos de polen de olivo en diferentes estadios de desarrollo mediante técnicas de estereomicroscopía y microscopía láser confocal de alta definición, usando sondas altamente selectivas.Tesis Univ. Granada. Programa Oficial de Doctorado en Biología Fundamental y de SistemasEl trabajo ha sido financiado parcialmente con fondos FEDER: proyectos BFU 2004-00601/BFI y BFU2008-00629 (Plan Nacional I+D), proyectos P2010-AGR6274 y P2011-CVI7487 (Junta de Andalucía) y un Convenio de Cooperación Científica y Técnica entre el CSIC y la Academia de Ciencias de la República Checa (Ref. 2010CZ0001)