BUSCADOR RECOLECTA

Incluye 7 ficheros de datos, Background: The increased selection pressure of the herbicide glyphosate has played a role in the evolution of glyphosate-resistance in weedy species, an issue that is becoming a threat to global agriculture. The molecular components involved in the cellular toxicity response to this herbicide at the expression level are still unidentified. Results: In this study, we identify the protein kinase GCN2 as a cellular component that fosters the action of glyphosate in the model plant Arabidopsis thaliana. Comparative studies using wild-type and gcn2 knock-out mutant seedlings show that the molecular programme that the plant deploys after the treatment with the herbicide, is compromised in gcn2. Moreover, gcn2 adult plants show a lower inhibition of photosynthesis, and both seedlings and adult gcn2 plants accumulate less shikimic acid than wild-type after treatment with glyphosate. Conclusions: These results points to an unknown GCN2-dependent factor involved in the cascade of events triggered by glyphosate in plants. Data suggest either that the herbicide does not equally reach the target-enzyme in a gcn2 background, or that a decreased flux in the shikimate pathway in a gcn2 plants minimize the impact of enzyme inhibition., This work was mainly supported by the Universidad Politecnica de Valencia (PAID2011-16) and the Ministerio Español de Ciencia y Tecnología (BFU2011-22526). The work was partially supported through a grant from the Ministerio Español de Ciencia y Tecnología (AGL-2010-18621).

[EN] Membrane-delimited events play a crucial role for ABA signaling and PYR/PYL/RCAR ABA receptors, clade
A PP2Cs and SnRK2/CPK kinases modulate the activity of different plasma membrane components involved
in ABA action. Therefore, the turnover of PYR/PYL/RCARs in the proximity of plasma membrane might be a
step that affects receptor function and downstream signaling. In this study we describe a single-subunit
RING-type E3 ubiquitin ligase RSL1 that interacts with the PYL4 and PYR1 ABA receptors at the plasma
membrane. Overexpression of RSL1 reduces ABA sensitivity and rsl1 RNAi lines that impair expression of
several members of the RSL1/RFA gene family show enhanced sensitivity to ABA. RSL1 bears a C-terminal
transmembrane domain that targets the E3 ligase to plasma membrane. Accordingly, bimolecular fluorescent
complementation (BiFC) studies showed the RSL1–PYL4 and RSL1–PYR1 interaction is localized to
plasma membrane. RSL1 promoted PYL4 and PYR1 degradation in vivo and mediated in vitro ubiquitylation
of the receptors. Taken together, these results suggest ubiquitylation of ABA receptors at plasma
membrane is a process that might affect their function via effect on their half-life, protein interactions or
trafficking., This work was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Cientificas (grants BIO2011-23446 to P.L.R.; BFU2011-22526 to R.S.; fellowship to L.R.; fellowship UPV to L.L-O; JAE-DOC contract to M.G.G.). We acknowledge Professor Joerg Kudla (University of Munster, Germany) for kindly providing plasma membrane marker OFP-TM23. Technical assistance of Maria A. Fernandez is greatly acknowledged.

[EN] We have identified QDR2 in a screening for genes able to confer tolerance to sodium and/or lithium stress upon overexpression. Qdr2 is a multidrug transporter of the major facilitator superfamily, originally described for its ability to transport the antimalarial drug quinidine and the herbicide barban. In order to identify its physiological substrate, we have screened for phenotypes dependent on QDR2 and found that Qdr2 is able to transport monovalent and divalent cations with poor selectivity, as shown by growth tests and the determination of internal cation content. Moreover, strains overexpressing or lacking QDR2 also exhibit phenotypes when reactive oxygen species producing agents, such as hydrogen peroxide or menadione, were added to the growth medium. We have also found that the presence of copper and hydrogen peroxide repress the expression of QDR2. In addition, the copper uptake of a qdr2 mutant strain is similar to a wild type, but the extrusion is clearly impaired. Based on our results, we propose that free divalent copper is the main physiological substrate of Qdr2. As copper is a substrate for several redox reactions that occur within the cytoplasm, this function in copper homeostasis explains its role in the oxidative stress response, We are indebted to Prof. A. Maquieira for technical assistance in copper measurements. This work was supported by grants PAID-06-10-1496 of the Universitat Politecnica de Valencia (Valencia, Spain), PROMETEO/2010/038 of the 'Conselleria de Educacion' (Valencia, Spain), by grant BFU2011-30197-C03-03 and by grant BFU2011-22526 from the Ministerio de Ciencia e Innovacion (Madrid, Spain).

[EN] A fungal gene encoding a transcription factor is expressed from its own promoter in Arabidopsis phloem and improves drought tolerance by reducing transpiration and increasing osmotic potential.

Horizontal gene transfer from unrelated organisms has occurred in the course of plant evolution, suggesting that some foreign genes may be useful to plants. The CtHSR1 gene, previously isolated from the halophytic yeast Candida tropicalis, encodes a heat-shock transcription factor-related protein. CtHSR1, with expression driven by its own promoter or by the Arabidopsis UBQ10 promoter, was introduced into the model plant Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation and the resulting transgenic plants were more tolerant to drought than controls. Fusions of the CtHSR1 promoter with beta-glucuronidase reporter gene indicated that this fungal promoter drives expression to phloem tissues. A chimera of CtHSR1 and green fluorescence protein is localized at the cell nucleus. The physiological mechanism of drought tolerance in transgenic plants is based on reduced transpiration (which correlates with decreased opening of stomata and increased levels of jasmonic acid) and increased osmotic potential (which correlates with increased proline accumulation). Transcriptomic analysis indicates that the CtHSR1 transgenic plants overexpressed a hundred of genes, including many relevant to stress defense such as LOX4 (involved in jasmonic acid synthesis) and P5CS1 (involved in proline biosynthesis). The promoters of the induced genes were enriched in upstream activating sequences for water stress induction. These results demonstrate that genes from unrelated organisms can have functional expression in plants from its own promoter and expand the possibilities of useful transgenes for plant biotechnology., We acknowledge support by Grants BFU2011-22526 of the Spanish MICINN (Madrid, Spain) and PROMETEO II 2014-041 of Generalitat Valenciana (Valencia, Spain). J. M.-B. was supported by a Juan de la Cierva contract of the Spanish MICINN. A. A. was supported by a short-term EMBO fellowship to visit the laboratory of R. Serrano. We thank Dr. Jose Maria Belles (IBMCP, Valencia, Spain) for assistance in the determination of sugars, Dr. Isabel Lopez-Diaz and Dr. Esther Carrera for the hormone analysis carried out at the Plant Hormone Quantification Service of IBMCP and Prof. Jorg Kudla (Westfalische Wilhelms-Universitat, Munster, Germany) for the pGPTVII.Hyg.P<INF>UBQ10</INF>::MCS plasmid.

[EN] One of the main mechanisms regulating translation is the one based on the phosphorylation of the alpha subunit of the translation initiation factor 2 (eIF2 alpha) by the general control non-repressive 2 (GCN2) protein kinase. In yeast, this kinase binds to two scaffold proteins (GCN1 and GCN20), facilitating its activation on translating ribosomes. The homology of the three proteins exists in Arabidopsis. In this species, whereas the kinase is activated under several stress situations, the involvement of the scaffold proteins in those processes is controversial, and a new role for GCN1 in translation, independent of the phosphorylation of eIF2 alpha, has been proposed. Arabidopsis presents five genes with homology to GCN20 (ABCF1 to 5) in its genome. We show here that any of these five genes is needed for eIF2 alpha phosphorylation. Furthermore, plant phenotypes under abiotic stresses and chloroplast development suggest that ABCF3 is functionally linked with GCN1, but not with GCN2. Finally, gcn1 and abcf3 mutants share similar transcriptional reprogramming, affecting photosynthesis and stress responses. The common downregulation of regulators of the flagellin receptor FLS2 in both mutants suggest that the observed defect in pathogen-associated molecular pattern (PAMP)-induced stomatal closure of these two mutants could be mediated by these proteins., This work was funded by the Spanish Ministerio de Ciencia e Innovacion (MICINN), reference BFU2011-22526. Vigya Kesari thanks the EC for an Erasmus Mundus postdoctoral fellowship.

Background: The increased selection pressure of the herbicide glyphosate has played a role in the evolution of glyphosate-resistance in weedy species, an issue that is becoming a threat to global agriculture. The molecular components involved in the cellular toxicity response to this herbicide at the expression level are still unidentified.

Results: In this study, we identify the protein kinase GCN2 as a cellular component that fosters the action of glyphosate in the model plant Arabidopsis thaliana. Comparative studies using wild-type and gcn2 knock-out mutant seedlings show that the molecular programme that the plant deploys after the treatment with the herbicide, is compromised in gcn2. Moreover, gcn2 adult plants show a lower inhibition of photosynthesis, and both seedlings and adult gcn2 plants accumulate less shikimic acid than wild-type after treatment with glyphosate.

Conclusions: These results points to an unknown GCN2-dependent factor involved in the cascade of events triggered by glyphosate in plants. Data suggest either that the herbicide does not equally reach the target-enzyme in a gcn2 background, or that a decreased flux in the shikimate pathway in a gcn2 plants minimize the impact of enzyme inhibition., p This work was mainly supported by the Universidad Politecnica de Valencia (PAID2011-16) and the Ministerio Espanol de Ciencia y Tecnologia (BFU2011-22526). The work was partially supported through a grant from the Ministerio Espanol de Ciencia y Tecnologia (AGL-2010-18621).

[EN] The stress hormone abscisic acid (ABA) induces expression of defence genes in many organs, modulates ion
homeostasis and metabolism in guard cells, and inhibits germination and seedling growth. Concerning the latter
effect, several mutants of Arabidopsis thaliana with improved capability for H+
efflux (wat1-1D, overexpression of
AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type. This suggested that ABA could inhibit
H+
efflux (H+
-ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to
test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system.
ABA inhibited the root plasma-membrane H+
-ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in
vivo (H+
efflux from seedling roots). This inhibition involved the core ABA signalling elements: PYR/PYL/RCAR ABA
receptors, ABA-inhibited protein phosphatases (HAB1), and ABA-activated protein kinases (SnRK2.2 and SnRK2.3).
Electrophysiological measurements in root epidermal cells indicated that ABA, acting through the PYR/PYL/RCAR
receptors, induced membrane hyperpolarization (due to K+
efflux through the GORK channel) and cytosolic acidification.
This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the H+
-ATPase by
ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells. ABA
did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H+
-ATPase
in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of H+
-ATPase while the guard-cell
kinase SnRK2.6/OST1 did not., This work was funded by grants BFU2011-22526 (to RS) and BIO2011-23446 (to PLR) of the Spanish 'Ministerio de Economia y Competitividad', Madrid, Spain, and grant PROMETEO/2010/038 (to RS) of the 'Generalitat Valenciana', Valencia, Spain. MGG was funded by a JAE-DOC contract of the Spanish 'Consejo Superior de Investigaciones Cientificas', Madrid, Spain. We thank Dr Toshinori Kinoshita (Nagoya University, Nagoya, Japan) for the rabbit antibody against the last 9 aa of AHA2 H<SUP>+</SUP>-ATPase with the penultimate Thr947 phosphorylated. We also thank the Proteomics Facility of the 'Centro Nacional de Biotecnologia', Madrid, Spain, for the attempts to identify the phosphorylation site of the H<SUP>+</SUP>-ATPase.

[EN] We have compared the toxicity, mutagenicity and transport in Saccharomyces cerevisiae of three DNA-intercalating fluorescent dyes widely used to stain DNA in gels. Safety data about ethidium bromide (EtBr) are contradictory, and two compounds of undisclosed structure (Redsafe and Gelred) have been proposed as safe alternatives. Our results indicate that all three compounds inhibit yeast growth, with Gelred being the most inhibitory and also the only one causing cell death. EtBr and Gelred, but not Redsafe, induce massive formation of petite (non-respiratory) mutants, but only EtBr induces massive loss of mitochondrial DNA. All three compounds increase reversion of a chromosomal point mutation (lys2-801(amber)), with Gelred being the most mutagenic and Redsafe the least. These dyes are all cationic and are probably taken by cells through non-selective cation channels. We could measure the glucose-energized transport of EtBr and Gelred inside the cells, while uptake of Redsafe was below our detection limit. We conclude that although all three compounds are toxic and mutagenic in the yeast system, Redsafe is the safest for yeast, probably because of very limited uptake by these cells. Copyright (c) 2015 John Wiley & Sons, Ltd., This work was funded by the Ministerio de Ciencia e Innovacion, Madrid, Spain (Grant No. BFU2011-22526).