Search

Póster presentado en el SEB’s Annual Meeting 2-5 July (2019) Seville, Spain, Chloride (Cl-) has been recently described as a beneficial macronutrient in well-irrigated plants, playing specific roles in promoting plant growth, photosynthetic performance and water-use efficiency (WUE). Therefore, a new research framework is opened in the search for adaptive mechanisms that regulate water homeostasis and, in particular, the plant ability to withstand water deficit. Drought is now recognized as the abiotic stress that most importantly affects global crop productivity. Understanding how plants use water for optimal biomass production has become a fundamental issue worldwide. In this work, we aim to elucidate whether Cl- nutrition at macronutrient levels stimulates drought resistance and which physiological mechanisms are involved. To that end, tobacco plants were treated for 30 days with three nutritional treatments added to the basal solution: CL (5 mM Cl-), N (5 mM nitrate), and SP (1.25 mM sulphate and 1.875 mM phosphate), containing all of them the same cationic balance. Then, plants were subjected to two irrigation regimes for 20 days: control (100% field capacity) and moderate drought (60% field capacity). Results showed that, in comparison to SP and N treatments, Cl- application (CL treatment) improved plant growth, leaf development, water balance parameters and WUE under drought conditions. Interestingly, N-treated plants exhibited the strongest growth reduction during drought, showing higher stress symptoms caused by an inefficient water use. Therefore, we propose that the abundant uptake and accumulation of Cl- responds to an adaptive function that improves water relations and drought resistance in higher plants.

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: Chloride (Cl-) is a micronutrient that accumulates to macronutrient levels since it is normally available in nature and actively taken up by higher plants. Besides a supposedly unspecific role as cell osmoticum, no clear biological roles had been explicitly associated with Cl- when accumulated to macronutrient concentrations until our group published a novel perspective on the matter, in which we proposed Cl- to be both a micronutrient and a beneficial macronutrient for higher plants (Franco-Navarro et al. 2016). We showed that Cl- specifically stimulated higher leaf cell size and led to a moderate increase of plant fresh and dry biomass mainly due to higher shoot expansion. When applied in the 1-5 mM range, Cl- played specific roles in regulating leaf osmotic potential and turgor, allowing plants to improve leaf water balance at both cellular and whole-plant level. Regarding the ion content Cl¿, which is not assimilated, was preferentially accumulated over other anionic macronutrients like NO3-, SO42- and PO43- that are assimilated in anabolic processes. Our data supported the NO3¿ diversion hypothesis by which macronutrient accumulation of Cl¿ prevents vacuolar compartmentalization of NO3¿ as an osmolyte or charge-balancing molecule, facilitating its assimilation and increasing plant biomass. This would represent a radical change in the perception of Cl¿ as an NO3¿ antagonist. Our working hypothesis and the objective of this work was to verify whether or not Cl- is a nutrient that promotes a more efficient use of nitrogen in two different species (tobacco and cherry tomato plants) of the Solanaceae family. 2. Material and Methods: Tobacco plants (Nicotiana tabacum L. var. Habana) and Cherry tomato plants (Solanum lycopersicum L. cv Zarina) were alternatively treated with a basal nutrient solution supplemented with three salt combinations containing the same cationic balance: 5 mM Cl--based (CL), 5 mM nitrate-based (N) and sulphate + phosphate-based (SP) treatments. Experimental design, greenhouse conditions, nutrient list of the irrigation treatments, harvesting procedures, anion determination and statistical analysis are fully described at Franco-Navarro et al. (2016). The equations that determine NUE parameters are described in Ríos et al. (2010). 3. Results: Plant biomass, anion content and NUE parameters will be presented. 4. Conclusions: The NO3¿ diversion hypothesis is clearly supported by a better NUE in Cl-treated plants coinciding a boost in biomass production. All parameters measured were quite similar in two different species from the same family. This gives light to a brand-new interpretation of Cl- properties as a beneficial macronutrient in higher plants, and therefore a potential resource in agricultural production of both crops. Franco-Navarro et al. (2016). J. Exp. Bot., 67. Pages 873-891 Ríos et al. (2010). J. plant growth regul., 29(2). Pages 164-170 This work was supported by the Spanish Ministry of Science and Innovation-FEDER grants AGL2009-08339/AGR and AGL2015-71386-R., This work was supported by the Spanish Ministry of Science and Innovation-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.

The use of dwarfing rootstocks represents an optimum mechanism to control the vigour of fruit tree species. Exceptionally, olive trees are grown on their own roots and due to excessive vigour, only a very limited number of cultivars have been successfully adapted to high-density orchards. Through prospection and phenotyping of different wild subspecies, a wide range of genetic variability of the species Olea europaea is being explored to identify genotypes that can be used as rootstocks to optimize intensive olive cultivation (e.g. low vigour, low susceptibility to Verticillium dahliae and high tolerance to abiotic stress). Here, early vigour parameters were studied in genotypes belonging to the subsp. europaea (var. sylvestris), guanchica, cuspidata and crosses of cuspidata x sylvestris. Homogeneous plants were obtained from in-vitro germinated seedlings and further micropropagation. After ex-vitro acclimatization and soil planting, plant height, diameter of the main axis at different heights and length of the longest shoot were measured eight months later. Significant correlations were obtained among the different vigour parameters and high values of broad sense heritability were obtained for all of them. A wide variability in vigour parameters was observed with some clear differences regarding the origin of the plant materials. Thus, the lowest vigour was obtained in sylvestris genotypes from Jaen (Spain) and Marrakech (Morocco) provenances, while the highest vigour was obtained in sylvestris genotypes from Cadiz (Spain) and genotypes from crosses cuspidata x sylvestris. However, a wide variability was obtained in all cases, particularly in genotypes from guanchica origin. Future works will be carried out in order to determine the relationship among vigour parameters measured at different plant age and to define the most efficient strategies for selection at early stages of breeding programs. Funding source: RECUPERA-2020 and contract agreement between IFAPA and Viveros Sevilla (CAICEM15-02).

In the agronomic context, chloride (Cl) has been generally considered a toxic anion rather than a plant nutrient (Colmenero-Flores et al, 2007; Brumós et al, 2009; Brumós et al, 2010). However, we have recently shown that in addition to an essential micronutrient, chloride is a beneficial macronutrient (Franco-Navarro et al, 2016). Under non-saline conditions Clspecifically stimulates higher leaf cell size and leads to a moderate increase of plant fresh and dry biomass mainly due to higher shoot expansion. Clplays specific roles in regulating leaf osmotic potential and turgor, allowing plants to improve leaf water balance parameters. In addition, Clregulates 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). The following open questions about chloride accumulation at macronutrient levels will be addressed: i) does Climprove resistance to water deficit?; ii) why Cl- mediated reduction of stomatal conductance does not result in reduction of net photosynthetic rate?; iii) why nitrate assimilation is not adversely affected?; iv) How is Claccumulation and Cl/NO3 - interaction regulated at the molecular level? We propose that the abundant uptake and accumulation of Clresponds to adaptive functions improving water homeostasis, drought tolerance, and nitrate- and Carbon-use efficiency in plants. In addition, molecular mechanisms regulating the distribution of Clbetween root and shoot according to changing environmental conditions will be proposed. Brumos, J., J.M. Colmenero-Flores, A. Conesa, P. Izquierdo, G. Sanchez, D.J. Iglesias, M.F. Lopez-Climent, A. Gomez-Cadenas, and M. Talon. 2009. Membrane transporters and carbon metabolism implicated in chloride homeostasis differentiate salt stress responses in tolerant and sensitive Citrus rootstocks. Functional & Integrative Genomics 9:293-309. Brumós, J., M. Talón, Bouhlal, R. and J.M. Colmenero-Flores. 2010. Cl- homeostasis in includer and excluder citrus rootstocks: transport mechanisms and identification of candidate genes. Plant, Cell & Environment 33:2012-2027. Colmenero-Flores, J.M., G. Martinez, G. Gamba, N. Vazquez, D.J. Iglesias, J. Brumos, and M. Talon. 2007. Identification and functional characterization of cation-chloride cotransporters in plants. The Plant Journal 50:278-292. Franco-Navarro, J.D., J. Brumós, M.A. Rosales, P. Cubero, M. Talón, and J.M. Colmenero-Flores. 2016. Chloride regulates leaf cell size and water relations in tobacco plants. Journal of Experimental Botany. 67:873-891.

1. Background and Objectives: Chloride (Cl-) is a micronutrient that accumulates to macronutrient levels since it is normally available in nature and actively taken up by higher plants. Besides a supposedly unspecific role as cell osmoticum, no clear biological roles had been explicitly associated with Cl- when accumulated to macronutrient concentrations until our group published a novel perspective on the matter, in which we proposed Cl- to be both a micronutrient and a beneficial macronutrient in higher plants (Franco-Navarro et al. 2016). We showed that Cl- specifically stimulated higher leaf cell size and led to a moderate increase of plant fresh and dry biomass mainly due to higher shoot expansion. When applied in the 1-5 mM range, Cl- played specific roles in regulating leaf osmotic potential and turgor, allowing plants to improve leaf water balance parameters. In addition, Cl- also altered water relations at the whole plant level through reduction of plant transpiration. This was a consequence of a lower stomatal conductance, which resulted in lower water loss and greater photosynthetic and integrated water-use efficiency. It was unclear however how the Cl-dependent reduction of stomatal conductance did not result in reduction of the plant photosynthetic efficiency. Our working hypothesis and the objective of this work was to verify whether the reduction in stomatal conductance is compensated by an increase in mesophyll diffusion conductance to CO2. 2. Material and Methods: Tobacco (Nicotiana tabacum L. var. Habana) plants were alternatively treated with a basal nutrient solution supplemented with three salt combinations containing the same cationic balance: 5 mM Cl-based (CL), 5 mM nitrate-based (N) and sulphate + phosphate-based (SP) treatments. Experimental design, greenhouse conditions, nutrient list of the irrigation treatments, harvesting procedures, leaf gas-exchange measurements and statistical analyses are described in Franco-Navarro et al. (2016). Mesophyll conductance (gm) was estimated from simultaneous measurements of A/Ci response curves and chlorophyll fluorescence measurements by the variable j method (Ethier & Livingston, 2004). 3. Results: Stomatal aperture size and density as well as mesophyll diffusion conductance parameters have been quantified. 4. Conclusions: The more efficient water-use efficiency and mesophyll diffusion conductance resulting from leaf anatomical ad subcellular modifications give light to a brand-new interpretation of Cl- properties as a beneficial macronutrient in higher plants. Franco-Navarro et al. (2016). J. Exp. Bot., 67. Pages 873-891 Ethier & Livingston (2004) Plant Cell Environ., 27. Pages 137¿153, This work was supported by the Spanish Ministry of Science and Innovation-FEDER grants AGL2009-08339/AGR and AGL2015-71386-R.

Elucidation of genomic sequences is an essential and necessary first step to identify genetic variants associated with agronomic traits of interest. The Citruseq Consortium, a joint effort developed by public institutions and private companies has sequenced the genomes of 150 citrus varieties with the Illumina platform at high average coverage. Paired end genomic DNA libraries of 100 (2x) bp reads and with an average insert size of 500 bp were constructed. The haploid genome of Citrus clementine (www.phytozome.com) was used as reference genome. More than 150 Citrus genomes including rootstocks and species of mandarins, oranges, lemons, grapefruits and limes were sequenced. The consortium also generated a web portal that allows searching and sequence comparisons among approximately 8 million of gene variants and 45 billion of base pairs. Initial analyses suggest that the stored information can help unravel fundamental aspects of biological and evolutionary interest such as the origin of citrus. From the commercial standpoint this information may help to authenticate varieties, to improve marker-assisted breeding and to identify genes of major agronomic interest.

6 páginas.-- 5 figuras.-- 9 referencias.-- Poster presentado en el XII Luso-Spanish Symposium on Plant Water Relations – Water to Feed the World. 30th of September – 3rd of October (Evora) Portugal, Cl- is a strange micronutrient since actual Cl- concentration in plants is about two orders of magnitude higher than the content required as essential micronutrient. This accumulation requires a high cost of energy, and since Cl- is a major osmotically active solute in the vacuole, we propose that Cl- plays a role in the regulation of water balance in plants. We show here that, when accumulated to macronutrient levels, Cl- specifically regulates leaf cell elongation and water balance parameters, improving water relations at both the leaf tissue and the whole plant levels, increasing drought resistance in higher plants., This work was supported by the Spanish Ministry of Science and Innovation-FEDER grant AGL2009-08339/AGR.

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.

8 páginas, 1 figura, 15 referencias.-- cmartin@irnase.csic.es, In the field, crops are routinely subjected to a combination of different abiotic stresses. Particularly, in arid and semi-arid areas many crops encounter combinations of drought, heat, and salinity that lead to the subsequent overproduction of reactive oxygen species (ROS). Agronomic and physiological studies have revealed that the response of plants to a combination of different abiotic stresses cannot be directly extrapolated from the response of plants to each of the different stresses applied individually (Mittler 2006 and references therein). The same is true at the molecular level: large-scale microarray experiments, in which several abiotic and biotic stresses were studied either in combination or as a single stress, concluded that about 60% of the total transcripts expressed cannot be predicted from the corresponding single stress experiments (Rasmussen et al. 2013). These complex interactions may account for: i) the difficulty of extrapolating the results of laboratory research to the field (e.g. tolerance phenotypes of many transgenic plant lines); ii) the function of many still uncharacterized plant genes, ~13% in Arabidopsis thaliana (Rasmussen et al. 2013); and iii) the unexpectedly high proportion of A. thaliana mutant lines exhibiting tolerant or sensitive phenotypes to specific combinations of different abiotic stress treatments (Luhua et al. 2012). Therefore, a combination of stresses should be regarded as a new state of abiotic stress in plants that requires new acclimation responses. These findings highlight the importance of studying genetic, biochemical, metabolic and physiological aspects of combined stresses in model and agronomic plant species to facilitate the development of crops with enhanced tolerance to field stress conditions.

Chloride (Cl-) is considered to be a strange micronutrient since actual Cl- concentrations in plants is 10-100 times higher than the content required as essential micronutrient, (Marschner, 1995; Brumós et al, 2010), whereas all the other mineral micronutrients (B, Cu, Fe, Mn, Mo, Ni, Zn) are present at much lower concentrations in plant tissues (1-5 orders of magnitude below). Since Cl- uptake and transport is an energetically expensive process (White and Broadley 2001; Brumós et al, 2010), we propose that Cl-, when accumulated to concentrations typical of the content of a macronutrient, plays a poorly understood biological role, not critical under normal growth conditions. Since Cl- appears to be particularly well suited to accomplish osmoregulatory functions, the proposed biological role could be related to the regulation of water balance at both the cell and the whole plant level. There is little experimental evidence in this regard since: i) it is unclear in which extent Cl- is specifically required to fulfil osmoregulatory roles or whether other anions, like nitrate, phosphate, sulphate, and organic acids can replace chloride in such functions; ii) usually the role of Cl- is not adequately differentiated from that of their accompanying cations; iii) the concepts linking Cl- homeostasis with osmotic/turgor regulation have been frequently discussed in the context of halophyte species and in glycophytes under salt stress conditions (Flowers et al, 1988), what have led to some confusion in the context of Cl- nutrition. We intend to establish the role of Cl- in glycophyte plants when accumulated to macronutrient levels, and we will present results showing that under non-saline conditions (1-5 mM external Cl- concentrations) and no water limitation, Cl- specifically promotes the growth of tobacco plants through mechanisms regulating leaf cell elongation and water relations. Furthermore, under water deficit conditions, Cl--treated plants exhibit drought resistance due to the sum of stress avoidance (reduced estomatal water loss) and tolerance (probably due to higher solute accumulation) mechanisms. - Brumós J., Talón M., Bouhlal R.Y.M. & Colmenero-Flores J.M. (2010) Cl- homeostasis in includer and excluder citrus rootstocks: transport mechanisms and identification of candidate genes. Plant Cell Env, 33, 2012-2027. - Marschner H. (1995) Mineral Nutrition of Higher Plants, 2nd ed. (Second Edition ed.). Academic Press, London. - Flowers T.J. (1988) Chloride as a nutrient and as an osmoticum. In: Advances in plant nutrition (ed L.A. Tinker B), pp. 55-78. Praeger, New York., ENVIRONMENT WORKSHOPS 2013 “GENOMIC, PHYSIOLOGICAL AND BREEDING APPROAHES FOR ENHANCING DROUGHT RESISTANCE IN CROPS ” Baeza, Spain, 23–25 September 2013

CITRUSEQ/CITRUSGENN is a public-private consortium comprised of nine mayor institutions ANECOOP, ICCSA, Centro Principe Felipe, IRNASE, Fundacion RuralCaja, IVIA, GCM, SNFLSource Citrus Genesis, and Eurosemillas, aimed to provide scientific knowledge to develop genomics and biotechnological tools to facilitate the generation and selection of new lines and varieties of citrus. Activities in this initiative are centered in the sequencing of citrus varieties and rootstocks and the identification of genes, markers and alleles related to citrus fruit quality, production and tolerance to environmental adverse conditions. This goal is supported by complementary strategies including the translational identification of genes of potential agricultural interest. Further developments will involve the identification of variants of genes with agronomic interest in citrus and subsequently the technological application of this information through the use of genotyping ¿chips¿ and genotyping by sequencing platforms on segregating populations. Ongoing and new breeding programs have been included in the project to provide source material for these analyses. In this communication the efforts we are developing in several areas of citrus genomics will be revised and an overview of current progress and results will be presented.

4 páginas.-- 3 figuras.-- 11 referencias.-- Póster presentado en el XI Simposio Hispano-Portugués de Relaciones Hídricas en las Plantas, Sevilla 17-20 Sep. (2012), Although Cl- has been characterized as a micronutrient, we have observed that when available in the millimolar range (e.g. 1-5 mM), higher plants accumulate Cl- to levels that are typical of the content of a macronutrient (Plant Cell Env. 2010, 33: 2012-27). Since this requires a considerable cost of energy, we speculate whether Cl- might play a poorly understood function in plants when accumulated to macronutrient levels. Given that Cl- is a major osmotically active solute in the plant vacuole, we propose that this element alter plant water relation mechanisms. Besides promoting plant growth and dry weight, we observed that chloride nutrition in the millimolar range improved water parameters like the relative water content, leaf succulence and water use efficiency. Under conditions of water deficit chloride-treated plants exhibited an improved regulation of the water balance and drought-tolerance. According to the data obtained, we propose that critical factors behind these phenomena are an improved osmotic regulation, a reduced transpiration and developmental alterations., This work was financed by ‘MICINN’ (AGL2009-08339; Spain)

En los últimos años, el uso de marcadores moleculares que identifican cambios polimórficos a nivel del ADN, está jugando un papel cada vez mayor en las aplicaciones biotecnológicas de todo tipo así como en los estudios básicos de genética. Sin embargo, todos los esfuerzos realizados hasta la fecha para discriminar variedades de cítricos próximas y, en particular, variedades derivadas por mutaciones espontáneas e inducidas, han resultado infructuosos. En este contexto, el consorcio CITRUSEQ formado por 3 instituciones públicas, el Instituto Valenciano de Investigaciones Agrarias, el IRNASE del Consejo Superior de Investigaciones Científicas y la Fundación Centro de Investigación Príncipe Felipe y 6 entidades privadas: Eurosemillas, S.L., Investigación Citrícola Castellón S.A., Anecoop, Special Newfruit Licensing Mediterraneo S.L., GMC Variedades Vegetales A.I.E y Fundación Ruralcaja está desarrollando un número considerable de aplicaciones biotecnológicas basadas en el conocimiento del genoma de los cítricos entre las que se incluyen protocolos y procedimientos de identificación inequívoca de especies y variedades de agrios. En este artículo describimos la estrategia de detección y validación de marcadores de ADN derivados de la comparación genómica que permiten mediante análisis de PCR la discriminación rápida, eficaz y asequible de variedades comerciales generadas por mutaciones espontáneas e inducidas.

Poster presentado en the XII International Citrus Congress. Valencia, Spain 18th–23rd November 2012, While the molecular response of salinized plants in the short-medium term (hours-days) has been broadly studied, the knowledge about the nature of the genes involved in maintaining homeostatic conditions the long term (months-years) has remained elusive. With this aim, we have analyzed the transcriptome of leaves from citrus plants acclimated to moderate salinity (2 years with NaCl 30 mM). Through functional genomics, using the 7K cDNA chip from ¿Consorcio Valenciano de Genómica Funcional de Cítricos¿, the transcriptome of citrus plants acclimatized to salinity was analyzed in mature leaves (8 months) and immature leaves (2 months). Although immature leaves accumulated low levels of chloride (0.51%±0.06), they exhibited high responsiveness to salinity (1,211 differential-responsive genes) compared with mature leaves, which cumulated higher chloride levels (1.05% ± 0.01), and showed a lower number of differentially-responsive genes (100 genes). Immature leaves induced functional categories that were not induced in mature leaves, like ¿cell wall biosynthesis¿, ¿metabolism¿, ¿defense¿, ¿secretion and membrane traffic¿, ¿water transport¿ and ¿antioxidant activity¿. On the other hand, the degree of coincidence was higher in the group of genes that were repressed by salinity. Both mature and immature leaves repressed genes mostly involved in ¿stress response¿. We highlight how the same plant organ in different developmental stages show huge differences in the amount and nature of genes which are responding to an abiotic stimulus.

Póster presentado en el XIV Simposio Hispano-Luso de Nutrición Mineral de las Plantas. (nutriPLANTA 2012, Madrid 23-26 julio de 2012

Although Cl- has been characterized as a micronutrient, we have observed that when available in the millimolar range (e.g. 1-5 mM), higher plants accumulate Cl- to levels that are typical of the content of a macronutrient (Plant Cell Env. 2010, 33: 2012-27). Since this requires a considerable cost of energy, we speculate whether Cl- might play a poorly understood function in plants when accumulated to macronutrient levels. Given that Cl- is a major osmotically active solute in the plant vacuole, we propose that this element improves osmoregulatory and plant water relation mechanisms. To elucidate this hypothesis, tobacco plants were grown for 9 weeks with a basal nutrient solution (BS) and subjected to different treatments: 5 mM Clsolution (Cl); 5 mM NO3- (N); and 5 mM SO4 2- and PO33- (SP). Compared to BS and SP plants, chloride nutrition in the millimolar range promoted plant growth in terms of dry weight and total leaf area. This, together with the observation that Cl- was preferentially included in growing and reproductive organs, suggested a role in plant development. In addition, compared to BS, SP and N plants, Cl plants exhibited the best water parameters., Financed by ‘MICINN’ (AGL2009-08339; Spain).

La mejora de los cultivos cítricos en general está muy mermada debido entre otros obstáculos a la combinación tan inusual de características reproductivas y biológicas que tienen lugar en los cítricos. Las aproximaciones genómicas, por otro lado, permiten vislumbrar desarrollos biotecnológicos que podrían facilitar o al menos acortar los periodos de mejora en este cultivo. En este contexto, el Centro de Genómica del Instituto Valenciano de Investigaciones Agrarias (IVIA), está desarrollando la propuesta Citruseq, un proyecto de colaboración público-privado, liderado por la Fundación Ruralcaja Grupo CRM,, en el que también intervienen otras instituciones públicas como el Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS, CSIC) y la Fundación de la Comunidad Valenciana Centro de Investigación Príncipe Felipe (CPF), y cinco empresas del sector cítricola español como Eurosemillas, S.L., Investigación Citrícola Castellón S.A., Anecoop S. Coop., Special Newfruit Licensing Mediterraneo S.L. / Citrus Genesis S.L., y GCM Variedades Vegetales. La propuesta Citruseq, que lleva por título ¿Secuenciación, genotipado, y desarrollo de herramientas genómicas para la mejora de cítricos ¿está financiada por el Ministerio de Ciencia e Innovación a través de los Proyectos Singulares y Estratégicos y por las entidades privadas del propio consorcio. Uno de los objetivos principales del proyecto es la dilucidación de la secuencia del genoma de las variedades de cítricos más importantes de la citricultura española, información que en definitiva permitirá la identificación de genes responsables de la calidad y de la producción. Otro de los objetivos fundamentales de la propuesta es la generación directa de nuevas variedades mediante metodologías de irradiación e hibridación. El proyecto dedica además un gran esfuerzo al desarrollo de herramientas biotecnológicas que aceleren la obtención y autentificación de variedades., Esta investigación está financiada por el Ministerio de Ciencia e Innovación a través del proyecto CITRUSEQ, PSE-060000-2009-8.

The survival of the citrus industry is critically dependent on genetically superior cultivars. Improvements in these traits through traditional techniques are unfortunately extremely difficult due to the unusual combination of biological characteristics of citrus. Genomic science holds promise of improvements in breeding and the main goal of our group is to develop genomics tools for the generation of new genotypes. We pursue the identification of candidate genes, alleles and genotypes improving citrus fruit quality and performance, correlating phenotypic analyses, metabolic profiling and gene expression. At completion, genes and alleles with major functions in nutritional quality and stress tolerance could be selected and genotypes with improved fruit composition searched among existing or generated collections. This goal is supported by two complementary strategies the identification of genes of agricultural interest by the use of mutants and microarrays and the elucidation of the citrus genome sequence. In this communication the efforts we are developing in this first approach are revised while the other strategy will be resumed elsewhere.

The survival of the citrus industry is critically dependent on genetically superior cultivars. Improvements in these traits through traditional techniques are unfortunately extremely difficult due to the unusual combination of biological characteristics of citrus. Genomic science holds promise of improvements in breeding and the main goal of our group is to develop genomics tools for the generation of new genotypes. We pursue the identification of candidate genes, alleles and genotypes improving citrus fruit quality and performance, correlating phenotypic analyses, metabolic profiling and gene expression. At completion, genes and alleles with major functions in nutritional quality and stress tolerance could be selected and genotypes with improved fruit composition searched among existing or generated collections. This goal is supported by two complementary strategies the identification of genes of agricultural interest by the use of mutants and microarrays and the elucidation of the citrus genome sequence. In this communication the efforts we are developing in this first approach are revised while the other strategy will be resumed elsewhere