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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.

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.

Rice (Oryza sativa) stands among the world's most important crop species. Rice is salt sensitive, and 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 Salt Overly Sensitive 1 (SOS1) is the sole Na+ efflux transporter that has been genetically characterized to date. Here, the importance of SOS1-facilitated Na+ flux in the salt tolerance of rice was analyzed in a reversegenetics approach. A sos1 loss-of-function mutant displayed exceptional salt sensitivity that was correlated with excessive Na+ intake and impaired Na+ loading into the xylem, thus indicating that SOS1 controls net root Na+ uptake and long-distance Na+ transport to shoots. The acute Na+ sensitivity of sos1 plants at low NaCl concentrations allowed analysis of the transcriptional response to sodicity stress without effects of the osmotic stress intrinsic to high-salinity treatments. In contrast with that in the wild type, sos1 mutant roots displayed preferential down-regulation of stress-related genes in response to salt treatment, despite the greater intensity of stress experienced by the mutant. These results suggest there is impaired stress detection or an inability to mount a comprehensive response to salinity in sos1. In summary, the plasma membrane Na+/H+ exchanger SOS1 plays a major role in the salt tolerance of rice by controlling Na+ homeostasis and possibly contributing to the sensing of sodicity stress.

Rice (Oryza sativa) stands among the world's most important crop species. Rice is salt-sensitive and 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 is the sole Na+ efflux transporter that has been genetically characterized to date. Here, the importance of SOS1-facilitated Na+ flux in the salt tolerance of rice was analyzed in a reverse-genetics approach. A sos1 loss-of-function mutant displayed exceptional salt sensitivity that was correlated with excessive Na+ intake and impaired Na+ loading into the xylem, thus indicating that SOS1 controls net root Na+ uptake and long-distance Na+ transport to shoots. The acute Na+ sensitivity of sos1 plants at low NaCl concentrations allowed analysis of the transcriptional response to sodicity stress without effects of the osmotic stress intrinsic to high-salinity treatments. In contrast to that in the wild type, sos1 mutant roots displayed preferential down-regulation of stress-related genes in response to salt treatment, despite the greater intensity of stress experienced by the mutant. These results suggest there is impaired stress detection or an inability to mount a comprehensive response to salinity in sos1. In summary, the plasma membrane Na+/H+ exchanger SOS1 plays a major role in the salt tolerance of rice by controlling Na+ homeostasis and possibly contributing to the sensing of sodicity stress.

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

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.

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 oo, Brumós et al. (2010). Plant Cell Env, 33. Pages 2012-2027. - Franco-Navarro et al. (2016) J Exp Bot, 67. Pages 873-891.

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 homo

Higher plants take up nutrients via the roots and load them into xylem vessels for translocation to the shoot. After uptake, anions have to be channeled toward the root xylem vessels. Thereby, xylem parenchyma and pericycle cells control the anion composition of the root-shoot xylem sap [1, 2, 3, 4, 5 and 6]. The fact that salt-tolerant genotypes possess lower xylem-sap Cl− contents compared to salt-sensitive genotypes [7, 8, 9 and 10] indicates that membrane transport proteins at the sites of xylem loading contribute to plant salinity tolerance via selective chloride exclusion. However, the molecular mechanism of xylem loading that lies behind the balance between NO3− and Cl− loading remains largely unknown. Here we identify two root anion channels in Arabidopsis, SLAH1 and SLAH3, that control the shoot NO3−/Cl− ratio. The AtSLAH1 gene is expressed in the root xylem-pole pericycle, where it co-localizes with AtSLAH3. Under high soil salinity, AtSLAH1 expression markedly declined and the chloride content of the xylem sap in AtSLAH1 loss-of-function mutants was half of the wild-type level only. SLAH3 anion channels are not active per se but require extracellular nitrate and phosphorylation by calcium-dependent kinases (CPKs) [ 11, 12 and 13]. When co-expressed in Xenopus oocytes, however, the electrically silent SLAH1 subunit gates SLAH3 open even in the absence of nitrate- and calcium-dependent kinases. Apparently, SLAH1/SLAH3 heteromerization facilitates SLAH3-mediated chloride efflux from pericycle cells into the root xylem vessels. Our results indicate that under salt stress, plants adjust the distribution of NO3− and Cl− between root and shoot via differential expression and assembly of SLAH1/SLAH3 anion channel subunits.