Functional characterization of anion channels of the SLAC/SLAH family in Arabidopsis Thaliana.

Memoria presenta para optar al títuto de Doctor por la Escuela Internacional de Posgrado - Universidad de Sevilla (US)El crecimiento óptimo de las plantas requiere del aporte sincronizado de cloruro (Cl¿) y nitrato (NO3¿), pero la acumulación excesiva de Cl¿ en órganos aéreos puede producir toxicidad iónica. La toma neta de ambos aniones en el simplasto de la raíz resulta del equilibrio entre: i) la entrada activa mediante cotransporte de protones de alta y baja afinidad no identificados aún; ii) la salida pasiva mediada por canales aniónicos desconocidos. Tras su adquisición, los nutrientes se retienen en la raíz o se transportan a la parte aérea a través del xilema. La translocación de Cl¿ al xilema es un mecanismo clave en la regulación de la acumulación de este anión en la parte aérea. En este trabajo hemos caracterizado los genes AtSLAH1 y AtSLAH4 que se expresan en la raíz de Arabidopsis thaliana y que codifican canales aniónicos de tipo lento de la familia SLAC/SLAH, y hemos descrito su función biológica. Las líneas mutantes interrumpidas en dichos genes muestran alteraciones en el desarrollo, cuya manifestación depende de la dosis de Cl¿ aplicada en el medio de cultivo, proporcionando así evidencia de una interacción entre la nutrición por Cl¿ y la función de SLAH1 y SLAH4. En las muestras de savia del xilema obtenidas de las plantas mutantes slah1-2, el contenido de Cl¿, pero no el de NO3¿, se reduce un 50%. En la raíz, la expresión de AtSLAH1 se localiza específicamente en las células del periciclo adyacentes a los vasos del xilema, donde este gen se coexpresa con AtSLAH3, otro miembro de la familia SLAC/SLAH. Los estudios con las líneas de mutantes slah3 y con la doble mutante slah1-slah3 indican que la regulación correcta de la translocación de Cl¿ al xilema de la raíz requiere la actividad conjunta de ambos canales. SLAH1 no transporta aniones per se, pero coexpresado en ovocitos de Xenopus con el canal de NO3¿/Cl¿ SLAH3 verificamos que interaccionan físicamente y permite la activación de SLAH3 en ausencia de quiasas y NO3¿ extracelular.N

Chloride regulates leaf cell size and water relations in tobacco plants

19 páginas.-- 9 figuras.-- 5 tablas.-- 77 referencias.-- Supplementary Data: Supplementary_figures_S1_S7___Tables_S1_S7.pdfChloride (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 role as an unspecific cell osmoticum, no clear biological roles have been explicitly associated with Cl– when accumulated to macronutrient concentrations. To address this question, the glycophyte tobacco (Nicotiana tabacum L. var. Habana) has been treated with a basal nutrient solution supplemented with one of three salt combinations containing the same cationic balance: Cl–-based (CL), nitrate-based (N), and sulphate+phosphate-based (SP) treatments. Under non-saline conditions (up to 5mM Cl–) and no water limitation, 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 1–5mM 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. In contrast to Cl–, these effects were not observed for essential anionic macronutrients such as nitrate, sulphate, and phosphate. We propose that the abundant uptake and accumulation of Cl– responds to adaptive functions improving water homeostasis in higher plants.This work was supported by the Spanish Ministry of Science and Innovation-FEDER grant AGL2009-08339/AGR. The help, expertise, and technical assistance of C. Rivero, A. Vázquez, S. Luque, B.J. Sañudo, F.J. Durán, Y. Pinto, and J. Espartero are gratefully acknowledged. We would like to extend our gratitude to the valuable reviews and contributions by the anonymous referees and the editor, Timothy Colmer, which helped us to improve the manuscript substantially.Peer reviewe

Vacuolar control of stomatal opening revealed by 3D imaging of the guard cells

Abstract Land plants regulate their photosynthesis and water transpiration by exchanging gases (CO2 and H2Ovapour) with the atmosphere. These exchanges take place through microscopic valves, called stomata, on the leaf surface. The opening of the stomata is regulated by two guard cells that actively and reversibly modify their turgor pressure to modulate the opening of the stomatal pores. Stomatal function depends on the regulation of the ion transport capacities of cell membranes as well as on the modification of the subcellular organisation of guard cells. Here we report how the vacuolar and cytosolic compartments of guard cells quantitatively participate in stomatal opening. We used a genetically encoded biosensor to visualise changes in ionic concentration during stomatal opening. The 3D reconstruction of living guard cells shows that the vacuole is the responsible for the change in guard cell volume required for stomatal opening

Dynamic measurement of cytosolic pH and [NO 3 − ] uncovers the role of the vacuolar transporter AtCLCa in cytosolic pH homeostasis

International audienceIon transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile, the understanding of their exact functions in cellular homeostasis is limited by the difficulty of monitoring their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides invaluable tools to tackle this challenging issue. AtCLCa (Arabidopsis thaliana Chloride Channel a) is a vacuolar NO3-/H+ exchanger regulating stomata aperture in Athaliana Here, we used a genetically encoded biosensor, ClopHensor, reporting the dynamics of cytosolic anion concentration and pH to monitor the activity of AtCLCa in vivo in Arabidopsis guard cells. We first found that ClopHensor is not only a Cl- but also, an NO3- sensor. We were then able to quantify the variations of NO3- and pH in the cytosol. Our data showed that AtCLCa activity modifies cytosolic pH and NO3- In an AtCLCa loss of function mutant, the cytosolic acidification triggered by extracellular NO3- and the recovery of pH upon treatment with fusicoccin (a fungal toxin that activates the plasma membrane proton pump) are impaired, demonstrating that the transport activity of this vacuolar exchanger has a profound impact on cytosolic homeostasis. This opens a perspective on the function of intracellular transporters of the Chloride Channel (CLC) family in eukaryotes: not only controlling the intraorganelle lumen but also, actively modifying cytosolic conditions

Chloride nutrition at macronutrient levels regulates plant development, water balance and drought resistance of tobacco plants

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 2013Peer Reviewe

Chloride as a Beneficial Macronutrient in Higher Plants: New Roles and Regulation.

Chloride (Cl-) has traditionally been considered a micronutrient largely excluded by plants due to its ubiquity and abundance in nature, its antagonism with nitrate (NO3-), and its toxicity when accumulated at high concentrations. In recent years, there has been a paradigm shift in this regard since Cl- has gone from being considered a harmful ion, accidentally absorbed through NO3- transporters, to being considered a beneficial macronutrient whose transport is finely regulated by plants. As a beneficial macronutrient, Cl- determines increased fresh and dry biomass, greater leaf expansion, increased elongation of leaf and root cells, improved water relations, higher mesophyll diffusion to CO2, and better water- and nitrogen-use e ciency. While optimal growth of plants requires the synchronic supply of both Cl- and NO3- molecules, the NO3-/Cl- plant selectivity varies between species and varieties, and in the same plant it can be modified by environmental cues such as water deficit or salinity. Recently, new genes encoding transporters mediating Cl- influx (ZmNPF6.4 and ZmNPF6.6), Cl- efflux (AtSLAH3 and AtSLAH1), and Cl- compartmentalization (AtDTX33, AtDTX35, AtALMT4, and GsCLC2) have been identified and characterized. These transporters have proven to be highly relevant for nutrition, long-distance transport and compartmentalization of Cl-, as well as for cell turgor regulation and stress tolerance in plants

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

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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

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