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Wild subspecies of Olea europaea constitute a source of genetic variability with huge potential for olive breeding to face global changes in Mediterranean-climate regions. We intend to identify wild olive genotypes with optimal adaptability to different environmental conditions to serve as a source of rootstocks and resistance genes for olive breeding. The SILVOLIVE collection includes 146 wild genotypes representative of the six O. europaea subspecies and early-generations hybrids. These genotypes came either from olive germplasm collections or from direct prospection in Spain, continental Africa and the Macaronesian archipelago. The collection was genotyped with plastid and nuclear markers, confirming the origin of the genotypes and their high genetic variability. Morphological and architectural parameters were quantified in 103 genotypes allowing the identification of three major groups of correlative traits including vigor, branching habits and the belowground-to-aboveground ratio. The occurrence of strong phenotypic variability in these traits within the germplasm collection has been shown. Furthermore, wild olive relatives are of great significance to be used as rootstocks for olive cultivation. Thus, as a proof of concept, different wild genotypes used as rootstocks were shown to regulate vigor parameters of the grafted cultivar “Picual” scion, which could improve the productivity of high-density hedgerow orchards. Fondos FEDER de la Unión Europea y Consejo Superior de Investigaciones Científicas de España (CSIC)-RECUPERA-2020-20134R089 Grupos Operativos Intra-autonómicos fondos europeos FEDER y Ministerio de Agricultura y Pesca, Alimentación y Medio Ambiente de España-FEDER-MAPAMA 201600200066 29 Consejo Superior de Investigaciones Científicas Proyectos Intramurales CSIC-201640E069, CSIC-201740E041 y CSIC201940E077

Póster presentado en Olivebioteq 2018, the 6th International Conference on Olive Management and Olive Products, held in Seville, Spain, on 15th-19th October 2018., In recent years a change in the traditional cropping model under rain-fed regime is being transformed into intensive and superintensive olive groves undergoing fertilization and irrigation. This means the use of low vigor cultivars of early entry into production and higher branching habit like Arbequina, currently the most used cultivar in super high-density plantations. This cultivation system excludes the possibility of using varieties of greater vigour, but of high socioeconomic importance. The growing use of very few cultivars of reduced vigor is leading to a progressive genetic impoverishment of the crop. We intend to take advantage of the genetic variability present in the wild subspecies of Olea europaea to identify and characterize wild olive genotypes of reduced vigor with optimal adaptability to diverse soils and adverse environmental conditions that can be used as rootstocks for intensive and super-intensive olive tree cultivation. The SILVOLIVE collection consists of 149 genotypes from all known subspecies of Olea europaea described so far, including the subspecies: europaea, laperrinei, cuspidata, cerasiformis, guanchica and maroccana. These genotypes were prospected from world olive germplasm collections (Córdoba and Marrakech) and different regions of Spain, continental Africa and the Macaronesian archipelago. The genetic variability of the collection was addressed with nuclear and plastidial molecular markers. Early vigour parameters were studied in 103 genotypes of 13-14 months old age belonging to all the subspecies. Different anatomical traits regarding root, shoot and stem growth, branching patterns and internodal elongation were quantified. A wide variability in vigour parameters was observed, including genotypes from very low to very high vigour. Future works will be carried out to determine the capacity of selected genotypes used as rootstocks to reduce the vigour of different olive cultivars

Ponencia presentada en el Workshop on Agricultural Biotechnology, Corea 20-21 feb. (2017) organizado por el Systems & Synthetic Agrobiotech Center, Rice (Oryza sativa L.) stands among the world's most important and salt-sensitive crop species. The undue accumulation of sodium ions (Na+) in shoots has the strongest negative correlation with rice productivity under long-term salinity. The plasma membrane Na/H exchanger protein SOS1, the only Na+ efflux transporter that has to date been genetically characterized in plants, controls the net Na+ uptake by roots and the long-distance transport to shoots in several species. Hence, we have analyzed the importance of Na+ fluxes governed by the SOS system in the salt tolerance of rice by a reverse-genetics approach. Loss-of-function mutant in the SOS1 gene displayed exceptional salt sensitivity in hydroponic culture and in experimental paddies. Salt sensitivity correlated with excessive Na+ intake and the interruption of Na+ and K+ loading into the xylem, in agreement with the expression pattern of the SOS1 gene and the function of the SOS1 protein in active Na+ efflux. Contrary to most analyses of the transcriptional response of plants to high salinity in which it is not feasible to separate the response to the osmotic and the ionic components of this multifaceted stress, the extreme Na+-sensitivity of the sos1 mutant plant offered the unique advantage of inspecting the transcriptional response specific to sodicity stress at low external salt concentrations. Whereas the sos1 mutation induced minor transcriptional changes in control conditions, its transcriptional impact was largely specific to the salt-induced response and affected to almost 20% of the expressed genes. Notably, the majority (64%) of these salt-responsive genes were down-regulated in the sos1 root relative to the control. This biased transcriptional response to salinity supports a regulatory role for the SOS1 protein.

Chloride (Cl-) has been generally considered a toxic anion rather than a plant nutrient. However we have recently shown that, in addition to an essential micronutrient, Cl- is a beneficial macronutrient (Franco-Navarro et al, 2016). Under non-saline conditions (1-5 mM), Cl- specifically stimulates higher leaf cell size and leads to a moderate increase of plant fresh and dry biomass mainly due to higher shoot expansion. Chloride plays specific roles in regulating leaf osmotic potential and turgor, allowing plants to improve leaf water balance parameters. In addition, Cl- regulates water relations at the whole plant level through reduction of plant transpiration. This is a consequence of a lower stomatal conductance, which results in lower water loss and greater photosynthetic and integrated water-use efficiency (Franco-Navarro et al, 2016). We are currently studying the interaction of Cl- homeostasis with: i) plant development; ii) water deficit tolerance; iii) carbon and nitrate (NO3-) assimilation. These points will be addressed at the physiological level, and data concerning the molecular regulation of Cl- nutrition and Cl-/NO3- interaction (Cubero-Font et al., 2016) will be presented. - Franco-Navarro et al. (2016) Chloride regulates leaf cell size and water relations in tobacco plants. Journal of Experimental Botany 67 (3): 873-891. - Cubero-Font et al. (2016) Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation. Current Biology 26 (16): 2213-2220., MINECO-FEDER grants AGL2009-08339/AGR and AGL2015-71386-R.

1. Background and Objectives: Although classified as a micronutrient, plants accumulate chloride (Cl-) to levels that are typical of the content of a macronutrient. This enables plants to improve cell osmoregulation, water relations and growth under optimal growing conditions (Franco-Navarro et al., 2016). Under salt stress conditions Cl- impairs nitrate (NO3-) nutrition due, apparently, to competition for membrane transport mechanisms. Chloride loading into root xylem vessels is a key mechanism regulating shoot Cl- accumulation (Brumós et al., 2010). To maintain an appropriate balance between both anions, xylem loading of NO3- and Cl- has to be tightly regulated. Given the thermodynamics of anion transport, release of Cl- and NO3- into the root xylem is highly likely to be electrochemically passive and, therefore, facilitated by plasma membrane anion channels that are not yet identified. The NO3- and Cl- conductance of xylem parenchyma cells registered by early patch clamp studies are reminiscent to electrical characteristics of the recently identified anion channels from the slow-type (SLAC/SLAH) family, consisting of five members in Arabidopsis thaliana. To determine the involvement of the slow-type channels in plant Cl- homeostasis, a member of the gene family predominantly expressed in the root has been functionally characterized 2. Material and Methods: Tissue and cell-specific expression pattern of the gene was determined in different transgenic lines of Arabidopsis thaliana Col-0 expressing the chimeric GUS::GFP marker gene under the control of the native gene promoter. Gene expression in response to different abiotic stress and nutritional treatments was quantified by Quantitative Real Time-PCR (qPCR). Homozygous knock-out mutant plants were phenotyped according to plant growth, shoot Cl- content and xylem sap Cl- concentration. Electrophysiological activity of the gene product was characterized through functional expression of the gene in Xenopus laevis oocytes and Double-Electrode Voltage-Clamp (DEVC) measurements. 3. Results: The following results regarding the functional characterization of a gene encoding a slow-type root anion channel will be presented: cell-type expression, gene regulation, knockout mutant phenotypes, as well as electrophysiological and regulatory mechanisms involved in protein activity regulation. 4. Conclusions: A novel anion channel with a pivotal role in Cl- homeostasis in higher plants has been characterized., Brumós et al. (2010). Plant Cell Env, 33. Pages 2012-2027. - Franco-Navarro et al. (2016) J Exp Bot, 67. Pages 873-891., Acknowledgments: JMC-F was supported by the Spanish Ministry of Science and Innovation-FEDER grants AGL2009-08339/AGR and AGL2015-71386-R and PC-F had fellowships support from the Spanish National Research Council (CSIC) and the German Academic Exchange Service (DAAD)

1. Background and Objectives: In the agronomic context, chloride (Cl-) has been generally considered a toxic anion rather than a plant nutrient (Brumós et al, 2010). However we have recently shown that, in addition to an essential micronutrient, Cl- is a beneficial macronutrient (Franco-Navarro et al, 2016). Under non-saline conditions (1-5 mM), Cl- specifically stimulates higher leaf cell size and leads to a moderate increase of plant fresh and dry biomass mainly due to higher shoot expansion. Chloride plays specific roles in regulating leaf osmotic potential and turgor, allowing plants to improve leaf water balance parameters. In addition, Cl- regulates water relations at the whole plant level through reduction of plant transpiration. This is a consequence of a lower stomatal conductance, which results in lower water loss and greater photosynthetic and integrated water-use efficiency (Franco-Navarro et al, 2016). 2. Material and Methods: Tobacco plants were predominantly used for physiological measurements; Arabidopsis thaliana plants were used for the molecular characterization of genes involved in Cl- transport. Physiological methods used in this work include quantification of: nutrients content; plant biomass; anatomic parameters (leaf area, leaf cell size and density, chloroplast size and density and stomatal opening); water parameters (water content, relative water content, succulence, water potential, osmotic potential, turgor and water use efficiency); photosynthetic parameters (photosynthetic rate, stomatal conductance, intrinsic WUE and mesophyll conductance to CO2). Molecular methods used in this work include: tissue and cell-specific expression pattern of the candidate gene determined in transgenic lines of Arabidopsis thaliana expressing the chimeric GUS::GFP gene marker under the control of the gene promoter; gene expression response to abiotic stress and nutritional treatments quantified by Quantitative Real Time-PCR (qPCR); and phenotype of the homozygous mutant line (growth, shoot Cl- content and xylem sap Cl- concentration). 3. Results: The following open questions about chloride accumulation at macronutrient levels will be addressed: i) are Cl--treated plants more resistant to water deficit?; ii) why photosynthetic rate is not reduced as a result of lower stomatal conductance?; iii) why nitrate assimilation is not adversely affected?; iv) How is Cl- accumulation and Cl-/NO3- interaction regulated at the molecular level? 4. Conclusions: We propose that the abundant uptake and accumulation of Cl- responds to adaptive functions improving water homeostasis, drought tolerance, and nitrate- and carbon-use efficiency in plants. Brumós et al. (2010). Plant Cell Env, 33. Pages 2012-2027. Franco-Navarro et al. (2016) J Exp Bot, 67. Pages 873-891., We acknowledge the projects funded by the Spanish Ministry of Science and Innovation-FEDER grants AGL2009-08339/AGR and AGL2015-71386-R.

9 figuras.-- 7 referencias.-- Poster presentado en el Área temática de Estrés Abiótico de la XII Reunión de Biología Molecular de Plantas 11-13 de junio de 2014 Cartagena, Murcia, We aim to characterize genes and plasma membrane (PM) transporters involved in the uptake and long-distance transport of chloride (Cl-; Colmenero-Flores et al, 2007; Brumós et al, 2009; Brumós et al, 2010). Under most circumstances, at PM potentials more negative than -50 mV, chloride channels mediate passive Cl- efflux. In peripherall root cell layers, Cl- channles have been proposed to make a considerable contribution to net Cl- uptake, which results from combined activities of influx (active) and efflux transport mechanisms. In the root vasculature, different Cl- conductance activities measured in xylem parenchyma cells participate in root-to-shoot translocation of Cl- and nitrate (NO3-). PM anion channels can be broadly classified on the basis of their voltage dependence into depolarization- and hyperpolarization-activated channels. Depolarization-activated anion channels can be subdivided further based on their kinetics and gating properties into R(rapid)-type, S(slow)-type, and outwardly rectifying anion channels. The physiological role of R- and S-type channels was elucidated in guard cells using electrophysiological analyses, where R- and S-type channels were respectively named QUAC, for QUick Anion Channel, and SLAC, for SLow Anion Channel. These channels, activated by the drought hormone abscisic acid (ABA), are involved in the early steps leading to stomata closure (Geiger et al, 2009; Dreyer et al, 2012). The molecular nature of the guard cell slow anion channel has been uncovered and the gene encoding for this transporter was named SLAC1 (Negi et al., 2008). SLAH3 (SLAC1 Homolog 3) represents a second S-type channel present in guard cells which exhibits a higher preference for NO3- (Geiger et al., 2011). Other members of the SLAC family, SLAH1 and SLAH4 (SLAC1 Homolog 1 and 4) may encode for root isoforms of the S-type channels (Brumos et al, 2010). Under this hypothesis, we have initiated the molecular characterization of AtSLAH1 and AtSLAH4 genes. Data concerning gene expression, abiotic stress response and knock-out phenotypes will be presented.

Sodium homeostasis in plants is controlled by Na/H antiporters in the plasma membrane and tonoplast. In Arabidopsis thaliana, the Na/H antiporter SOS1 regulates sodium efflux in roots and the long-distance transport of sodium from roots to shoots. SOS1 activity is regulated through phosphorylation by the protein kinase SOS2 together with the calcium-sensing regulatory subunit SOS3. Functional homologues of the SOS genes have been identified in rice. OsSOS1, OsCIPK24 and OsCBL4 are the rice orthologues of Arabidopsis SOS1, SOS2 and SOS3 genes, respectively. They suppressed the salt sensitivity of the corresponding mutants of Arabidopsis. We have used reverse genetics to demostrate the importance of the SOS system in the salt tolerance of rice plants. Mutant lines in public rice collections bearing gene disruptions in OsSOS1, OsCBL24 and OsCBL4 that had been created by insertion of T-DNA or retrotransposons, have been analyzed for salt tolerance. Our data indicates that mutants with reduced activity of OsSOS1 or OsCIPK24 are indeed salt sensitive. The gene expression pattern of OsSOS1 will be analyzed using promoter::GUS transcriptional fusions to corroborate a role in sodium efflux and long-distance transport in rice plants. SOS1 proteins contain self-inhibitory domains located at their carboxy termini. The truncation of this inhibitory domain in OsSOS1 resulted in a much greater transport activity and enhanced salt tolerance in yeast cells, that were both independent of CIPK24/CBL4.

La productividad de las plantas está limitada por diferentes tipos de factores ambientales, entendiendo por “estrés” cualquier factor que inhibe el crecimiento de la planta. Así, el estrés debido a sequía, salinidad, carencia de nutrientes minerales, variaciones extremas de temperatura y pH, metales pesados, etc., es común en todo el mundo. Estos tipos de estrés abiótico han causado muchos problemas económicos y sociales, especialmente en países en vías de desarrollo. Según Dudal (1976), tan sólo el 10% de los terrenos de cultivo en todo el mundo está libre de estos tipos de estrés abiótico. El estrés salino y la sequía son, de todos ellos, los factores más extendidos que limitan la productividad de las tierras de cultivo. El problema de la salinización es especialmente grave en regiones áridas y semiáridas donde las escasas precipitaciones condicionan una alta dependencia del regadío, de modo que las sales (principalmente sodio) contenidas en el agua de riego no tardan en acumularse en los suelos bajo esas condiciones climáticas. Las elevadas concentraciones de sales en los suelos producen elevadas presiones osmóticas, reduciendo la disponibilidad del agua para las plantas: al mismo tiempo, iones como el sodio o el cloruro pueden ser tóxicos a elevadas concentraciones (Epstein et al., 1980).Estamos tratando con uno de los problemas más antiguos para el desarrollo de las civilizaciones. Así, por ejemplo, los registros históricos muestran cambios en los recursos agrícolas en la antigua Mesopotamia, que pasó del cultivo del trigo al de la cebada (más resistente a la salinidad) conforme el suelo fértil pero sin el drenaje se salinizó progresivamente. Pero el proceso de salinización ha seguido extendiéndose implacablemente a lo largo de la historia, y en la actualidad existen pocos países que practiquen la agricultura de regadío sin estar afectados por este problema. Se ha estimado (Christiansen, 1982) que de toda la tierra disponible para la agricultura en el mundo (unas 14*109 ha), 6*109 ha son áridas o semiáridas y aproximadamente 1*109 ha están afectadas por exceso de sales. Los terrenos basados en agricultura de regadío ocupan aproximadamente 0.23*109 ha, de los cuales un tercio parece estar afectado por salinidad (Epstein et al., 1980). En España la extensión de los suelos afectados por la salinidad se estimó en 840.000 ha hace unos años (Alberto et al., 1983), siendo las zonas más afectadas las marismas del Sur, Alicante, Murcia y Almería, así como el área delimitada por las provincias de Zaragoza, Navarra, Huesca y Lérida.