Caracterización molecular de la S-adenosilmetionina sintetasa y de la glioxalasa

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.Peer reviewe

Caracterización molecular de las S-Adenosilmetionina sintetasa y de la Glioxalasa I de Lycopersicon esculentum análisis de su respuesta a estrés salino

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 país

The SOS system is essential for the salt tolerance of rice

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.Peer Reviewe

Molecular characterization of glyoxalase-I from a higher plant; upregulation by stress

A cDNA, GLX1, encoding glyoxalase-I was isolated by differential screening of salt-induced genes in tomato. Glyoxalases-I and-II are ubiquitous enzymes whose functions are not clearly understood. They may serve to detoxify methylglyoxal produced from triosephosphates in all cells. The protein encoded by GLX1 shared 49.4% and 58.5% identity with glyoxalase-I isolated from bacteria and human, respectively. Furthermore, yeast cells expressing GLX1 showed a glyoxalase-I specific activity 20-fold higher than non-transformed cells. Both GLX1 mRNA and glyoxalase-I polypeptide levels increased 2- to 3-fold in roots, stems and leaves of plants treated with either NaCl, mannitol, or abscisic acid. Immunohistochemical localization indicated that glyoxalase-I was expressed in all cell types, with preferential accumulation in phloem sieve elements. This expression pattern was not appreciably altered by salt-stress. We suggest that the increased expression of glyoxalase-I may be linked to a higher demand for ATP generation and to enhanced glycolysis in salt-stressed plants.Peer reviewe

Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress

NaCl stress causes the accumulation of several mRNAs in tomato seedlings. An upregulated cDNA clone, SAM1, was found to encode a S-adenosyl-L-methionine synthetase enzyme (AdoMet synthetase). Expression of the cDNA SAM1 in a yeast mutant lacking functional SAM genes resulted in high AdoMet synthetase activity and AdoMet accumulation. We show that tomato plants contain at least four SAM isogenes. Clones corresponding to isogenes SAM2 and SAM3 have also been isolated and sequenced. they encode predicted polypeptides 95% and 92% identical, respectively, to the SAM1-encoded AdoMet Synthetase. RNA hybridization analysis showed a differential response of SAM genes to salt and other stress treatments. SAM1 and SAM3 mRNAs accumulated in the root in response to NaCl, mannitol or ABA treatments. SAM1 mRNA accumulated also in leaf tissue. These increases of mRNA level were apparent as soon as 8 h after the initiation of the salt treatment and were maintained for at least 3 days. A possible role for AdoMet synthetases in the adaptation to salt stress is discussed.Peer reviewe

Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation

8 páginas.-- 4 figuras.-- 40 referencias.-- Supplemental Information includes Supplemental Experimental Procedures, four figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.cub.2016.06.045 .-- Article in press as: Cubero-Font et al., Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation, Current Biology (2016), http://dx.doi.org/10.1016/j.cub.2016.06.045We appreciate J.D. Franco-Navarro, Inmaculada Flores, and F.J. Durán for the technical assistance provided to this work. Investigations of plant ion channels in Xenopus oocytes adhere to the regulations and provisions of the Animal Protection Act and the Experimental Animals Ordinance. Permission for keeping African clawfrog Xenopus laevis and using Xenopus oocytes exists at the Julius-von-Sachs Institute, University Würzburg and is registered and oversight at/from the district government of Unterfranken, Germany.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.Supported by the Spanish Ministry of Science and Innovation FEDER grants AGL2009-08339/AGR and AGL2015-71386-R. R.H. and D.G. were supported by the German Research Foundation (DFG) within the SFB/TR166 “ReceptorLight” project B8. P.C.-F. had fellowship support from the Spanish National Research Council (CSIC) and the German Academic Exchange Service (DAAD). R.H. and K.A.S.A.-R. were further supported by the International Research Group Program (project IRG14-08) of the Deanship of Scientific Research, King Saud University.Peer reviewe

Differential regulation of Chloride vs. Nitrate transport in plants: biological relevance and molecular mechanisms

Peer Reviewe

Functional characterization of the root anion channels SLAH1 and SLAH4: involvement in Cl- and NO3- nutrition and interaction with abiotic stress

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, MurciaWe 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.Peer Reviewe

Iidentification of low-vigour rootstocks in a collection of wild olive genotypes

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 cultivarsPeer reviewe

Choride as a beneficial macronutrient in plants: biological functions and regulation

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.Peer Reviewe