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

Legumbres: el pan del pobre

Domesticated at the same time as the first cereals and other carbohydrate-rich crops, legumes have maintained close ties with these in all aspects of human life: in the land, traditions, and as food for man or animal feed. However, in modern farming they are only considered to be second class crops, in spite of continuous calls for their use in balanced diets and in crop rotations to increase soil fertility by fixing atmospheric nitrogen, avoiding excess use of synthetic fertilizers. Because of their high protein content they were known as “the poorman’s meat”. Moreover, their ability to fertilize soil was greatly valued by ancient agronomists since Greek and Roman times. Rather than focusing on the technical aspects of legumes, the present article considers the relationship between man and legumes from different perspectives, including their role in the History of Science, being protagonists of fundamental studies such as those carried out by Mendel, Galton and Johannsen, as well as the first description of a QTL.<br><br>Domesticadas a la par que los primeros cereales y otros cultivos ricos en carbohidratos, compañeras inseparables de ellos en la tierra, en las costumbres, en la mesa y en el pesebre, en la agricultura moderna las leguminosas no son, sin embargo, más que secundarias o terciarias, a pesar de las llamadas a su consumo en dietas equilibradas y a la necesidad de su inclusión en la “alimentación” del suelo, es decir, en las rotaciones que incrementen la fertilidad del mismo de forma natural. Su riqueza en proteínas hizo que se llamaran “el pan del pobre”; su capacidad de fertilizar la tierra fijando nitrógeno atmosférico fue recomendación constante de los autores agrícolas desde los tiempos de Grecia y Roma. En el presente artículo se presentan las leguminosas no en sus aspectos técnicos sino en su relación con el Hombre desde diversos puntos de vista, incluyendo el papel que han representado en la Historia de la Ciencia al haber sido protagonista de estudios trascendentales como los de Mendel, Galton y Johannsen y la descripción del primer QTL entre otros

Anion channel SLAH3 is a regulatory target of chitin receptor-associated kinase PBL27 in microbial stomatal closure

In plants, antimicrobial immune responses involve the cellular release of anions and are responsible for the closure of stomatal pores. Detection of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) induces currents mediated via slow-type (S-type) anion channels by a yet not understood mechanism. Here, we show that stomatal closure to fungal chitin is conferred by the major PRRs for chitin recognition, LYK5 and CERK1, the receptor-like cytoplasmic kinase PBL27, and the SLAH3 anion channel. PBL27 has the capacity to phosphorylate SLAH3, of which S127 and S189 are required to activate SLAH3. Full activation of the channel entails CERK1, depending on PBL27. Importantly, both S127 and S189 residues of SLAH3 are required for chitin-induced stomatal closure and anti-fungal immunity at the whole leaf level. Our results demonstrate a short signal transduction module from MAMP recognition to anion channel activation, and independent of ABA-induced SLAH3 activation

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

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

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: Novel Functions in Water Relations

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)Peer Reviewe