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During the last years, a great effort has been dedicated at the development and employment of diverse approaches for achieving more stress-tolerant and climate-flexible crops and sustainable yield increases to meet the food and energy demands of the future. The ongoing climate change is in fact leading to more frequent extreme events with a negative impact on food production, such as increased temperatures, drought, soil salinization as well as invasive arthropod pests and diseases. In this review, diverse "green strategies" (e.g., chemical priming, root-associated microorganisms), and advanced technologies (e.g., genome editing, high-throughput phenotyping) are described on the basis of the most recent research evidence. Particularly, attention has been focused on the potential use in a context of sustainable and climate-smart agriculture (the so called "next agriculture generation") to improve plant tolerance and resilience to abiotic and biotic stresses. In addition, the gap between the results obtained in controlled experiments and those from application of these technologies in real field conditions (lab to field step) is also discussed. This article is protected by copyright. All rights reserved.

Soil flooding is a compound abiotic stress that alters soil properties and limits atmospheric gas diffusion (O2 and CO2) to the roots. The involvement of abscisic acid (ABA) in the regulation of soil flooding-specific genetic and metabolic responses has been scarcely studied despite its key importance as regulator in other abiotic stress conditions. To attain this objective, wild type and ABA-deficient tomatoes were subjected to short-term (24 h) soil waterlogging. After this period, gas exchange parameters were reduced in the wild type but not in ABA-deficient plants that always had higher E and gs. Transcript and metabolite alterations were more intense in waterlogged tissues, with genotype-specific variations. Waterlogging reduced the ABA levels in the roots while inducing PYR/PYL/RCAR ABA receptors and ABA-dependent transcription factor transcripts, of which induction was less pronounced in the ABA-deficient genotype. Ethylene/O2-dependent genetic responses (ERFVIIs, plant anoxia survival responses, and genes involved in the N-degron pathway) were induced in hypoxic tissues independently of the genotype. Interestingly, genes encoding a nitrate reductase and a phytoglobin involved in NO biosynthesis and scavenging and ERFVII stability were induced in waterlogged tissues, but to a lower extent in ABA-deficient tomato. At the metabolic level, flooding-induced accumulation of Ala was enhanced in ABA-deficient lines following a differential accumulation of Glu and Asp in both hypoxic and aerated tissues, supporting their involvement as sources of oxalacetate to feed the tricarboxylic acid cycle in waterlogged tissues and constituting a potential advantage upon long periods of soil waterlogging. The promoter analysis of upregulated genes indicated that the production of oxalacetate from Asp via Asp oxidase, energy processes such as acetyl-CoA, ATP, and starch biosynthesis, and the lignification process were likely subjected to ABA regulation. Taken together, these data indicate that ABA depletion in waterlogged tissues acts as a positive signal, inducing several specific genetic and metabolic responses to soil flooding., This work was supported by the Spanish Ministerio de Economía y Competitividad, Universitat Jaume I and Generalitat Valenciana/Fondo Europeo de Desarrollo Regional (FEDER), co-funded through grant nos. AGL2016-76574-R, UJI-B2016-23, UJI-B2016-24, IDIFEDER/2018/010, and UJI-B2019-24 to AG-C, VA, and MG-G, respectively. CD was supported by UJI PICD program. MG-G and JM were supported by Ramon y Cajal contracts from Spanish Ministerio de Economía y Competitividad (RYC-2016-19325 and RYC-2017-23645, respectively). JR was supported by a Juan de la Cierva-Formación contract from the Spanish Ministerio de Economía y Competitividad (FJCI-2016-28601).

Interaction between plants and their environment is changing as a consequence of the climate change and global warming, increasing the performance and dispersal of some pest species which become invasive species. Tetranychus evansi also known as the tomato red spider mite, is an invasive species which has been reported to increase its performance when feeding in the tomato cultivar Moneymaker (MM) under water deficit conditions. In order to clarify the underlying molecular events involved, we examined early plant molecular changes occurring on MM during T. evansi infestation alone or in combination with moderate drought stress. Hormonal profiling of MM plants showed an increase in abscisic acid (ABA) levels in drought-stressed plants while salicylic acid (SA) levels were higher in drought-stressed plants infested with T. evansi, indicating that SA is involved in the regulation of plant responses to this stress combination. Changes in the expression of ABA-dependent DREB2, NCED1, and RAB18 genes confirmed the presence of drought-dependent molecular responses in tomato plants and indicated that these responses could be modulated by the tomato red spider mite. Tomato metabolic profiling identified 42 differentially altered compounds produced by T. evansi attack, moderate drought stress, and/or their combination, reinforcing the idea of putative manipulation of tomato plant responses by tomato red spider mite. Altogether, these results indicate that the tomato red spider mite acts modulating plant responses to moderate drought stress by interfering with the ABA and SA hormonal responses, providing new insights into the early events occurring on plant biotic and abiotic stress interaction., This work was supported by a Young Investigator project (AGL2015-73235-JIN) and a Ramon y Cajal contract (RYC-2016-19325) from the Ministerio de Economía y Competitividad (MINECO) and Fondo Europeo de Desarrollo Regional (FEDER), and Universitat Jaume grant (UJI-B2019-24) to MG-G; by a Grant from INIA (GENOMITE, Proposal No. 618105 FACCE Era Net Plus-Food security, Agriculture, Climate Change) to FO; by MINECO, FEDER, Generalitat Valenciana and Universitat Jaume I through grants nos. AGL201676574-R, IDIFEDER 2018-10 and UJI-B2016- 23/UJI-B2016-24 to AG-C and VA. We are grateful to Maria Navajas for kindly providing the T. evansi population used in this study. The authors greatly acknowledge the central facilities (Servei Central d’Instrumentació Científica, SCIC) of the Universitat Jaume I where the phytohormone and metabolic profiling measurements were carried out.

The management of the medfly, Ceratitis capitata, in Spanish citrus crops relies mainly on the use of insecticides and the release of sterile males. However, the development of resistance to different insecticides in field populations, including lambda-cyhalothrin, implies a threat for the sustainable control of this pest. The inheritance, fitness cost, and management of lambda-cyhalothrin resistance were examined in the laboratory-selected W-1K&lambda, strain. We have demonstrated that lambda-cyhalothrin resistance in W-1K&lambda, is autosomic, completely dominant, and polygenic. In addition, individuals from W-1K&lambda, showed a lower embryo to pupal viability, a slower developmental time from egg to pupae, and an increase in adults&rsquo, weight and longevity. We did not find significant trade-offs in the activity of digestive hydrolytic enzymes, with the exception of higher &alpha, amylase activity in W-1K&lambda, females. A comparative study with different insecticide treatment strategies showed that lambda-cyhalothrin resistance increased when several consecutive treatments with this insecticide were applied. However, the alternation of this insecticide with spinosad was enough to delay the development of resistance. Our results indicate that the rotation of lambda-cyhalothrin with spinosad&mdash, a practice already used in some fields&mdash, may contribute to prevent the development of resistance.

[EN] Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified and named salt resistant (sre). The sre mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1 and sre1-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of the new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a Km value for xanthoxin of 19 µM and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC-mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin-->abscisic aldehyde-->ABA transition as the last steps of the major ABA biosynthetic pathway. The abscisic aldehyde oxidase 3 (AAO3) gene product of A. thaliana catalyzes the final step in abscisic acid (ABA) biosynthesis. An aao3-1 mutant in a Landsberg erecta genetic background exhibited a wilty phenotype in rosette leaves, whereas seed dormancy was not affected (Seo et al., 2000a). Therefore, it was speculated that a different aldehyde oxidase would be the major contributor to ABA biosynthesis in seeds (Seo et al., 2000a). Through a screening based on germination under high-salt concentration, we isolated two mutants in a Columbia genetic background, initially named sre2-1 and sre2-2. Complementation tests with different ABA-deficient mutants indicated that sre2-1 and sre2- 2 mutants were allelic to aao3-1, and therefore they were renamed as aao3-2 and aao3-3, respectively. Indeed, molecular characterization of the aao3-2 mutant revealed a T-DNA insertional mutation that abolished the transcription of AAO3 gene, while sequence analysis of AAO3 in aao3-3 mutant revealed a deletion of three nucleotides and several missense mutations. Physiological characterization of aao3-2 and aao3-3 mutants revealed a wilty phenotype and osmotolerance in germination assays. In contrast to aao3-1, both aao3-2 and aao3-3 mutants showed a reduced dormancy. Accordingly, ABA levels were reduced in dry seeds and rosette leaves of both aao3-2 and aao3-3. Taken together, these results indicate that AAO3 gene product plays a major role in seed ABA biosynthesis., [ES] El ácido abscísico (ABA) es una fitohormona implicada en el control de procesos vegetales esenciales, principalmente el desarrollo de la semilla así como la respuesta de las plantas a diferentes estreses ambientales, particularmente frío, sequía y salinidad. En el presente trabajo se ha realizado la búsqueda en colecciones de Arabidopsis thaliana mutagenizadas con T-DNA de mutantes capaces de germinar en condiciones de alta sal (NaCl), identificándose tres loci mutantes, inicialmente denominados sre1, sre2 y sre3 (“salt resistant”). Los mutantes sre, además de su capacidad para germinar y establecer plántula en condiciones de estrés osmótico, mostraban una germinación resistente a paclobutrazol, latencia muy reducida, mayor transpiración y niveles de ABA en hojas de roseta significativamente menores que plantas silvestres. El análisis de complementación reveló que los mutantes sre1 eran alélicos al mutante aba2-1, los mutante sre2 eran alélicos al mutante aao3-1 y el mutante sre3 era alélico al mutante aba1-5. Así, el ABA bloquea la germinación y el establecimiento de la plántula en condiciones de bajo potencial hídrico, por lo que mutantes capaces de germinar en condiciones de estrés osmótico representan frecuentemente mutantes defectivos en la biosíntesis de ABA. Mediante una combinación de las estrategias de mapeo posicional y gen candidato se llevó a cabo la identificación del gen ABA2 y de las mutaciones sre1-1/aba2-11 y sre1- 2/aba2-12. El gen ABA2 (At1g52340) codifica una alcohol deshidrogenasa de cadena corta dependiente de NAD+ (SDR). La mutación sre1-1/aba2-11 es debida a una deleción de 53 pb que también supone un cambio en la pauta de lectura, generando un alelo nulo de ABA2. La mutación sre1-2/aba2-12 conduce a la sustitución Gly28Arg, afectando al sitio de unión del coenzima. Mediante un análisis HPLC-MS se ha demostrado que la proteína recombinante ABA2 cataliza la conversión de xantoxina en aldehído abscísico en una reacción dependiente de NAD+ . Los resultados obtenidos en el presente trabajo establecen inequívocamente que la conversión de xantoxina en aldehído abscísico representa el penúltimo paso de la ruta de biosíntesis de ABA y que este es catalizado por el producto génico del gen ABA2 (At1g52340). El último paso de la ruta de biosíntesis de ABA es la conversión de aldehído abscísico en ácido abscísico. Este paso está catalizado en Arabidopsis por el producto génico del gen AAO3. Los mutantes sre2-1/aao3-2 y sre2-2/aao3-3, igual que el mutante aao3-1, muestran una reducción en los niveles de ABA en hojas de roseta y una mayor transpiración que las plantas silvestres. Además, y contrariamente el mutante aao3-1, muestran un fenotipo de osmotolerancia en germinación y establecimiento de plántula, germinación resistente a paclobutrazol, latencia reducida y poseen una reducción del 65% en los niveles de ABA en semillas. La caracterización molecular de la mutación sre2-1/aao3-2 revela una inserción de T-DNA que elimina la transcripción del gen AAO3, constituyendo por tanto un alelo nulo. La secuenciación del gen AAO3 en el mutante sre2-2/aao3-3 revela una deleción de tres nucleótidos y varias mutaciones de cambio de sentido. La evidencia genética y bioquímica resultante del análisis de los mutantes sre2-1/aao3-2 y sre2-2/aaao3-3 indica que lesiones en el gen AAO3 conducen a niveles reducidos de ABA y fenotipos característicos en semillas. Por consiguiente, queda demostrado que el gen AAO3 desempeña un papel crucial en la biosíntesis de ABA en semillas., [CA] L´àcid abscísic (ABA) és una fitohormona que ha estat implicada en el control de processos vegetals essencials, principalment en el desenvolupament de la llavor així com en la resposta de les plantes a diferents estressos ambientals, particularment fred, sequera i salinitat. En el present treball de tesi doctoral s´ha fet la recerca en coleccions d´Arabidopsis thaliana mutagenizades amb T-DNA de mutants capaços de germinar en condicions d´elevada sal (NaCl), identificant-se tres loci mutants, inicialment denominats sre1,sre2 i sre3 (“salt resistant”). Els mutants sre, a més de la seva capacitat per a germinar i establir plàntula en condicions d´estrès osmòtic, mostraven una germinació resistent a paclobutrazol, latència molt reduïda, major transpiració i nivells d´ABA en fulles de roseta significativament menors que les plantes silvestres. L´anàlisi de complementació va mostrar que els mutants sre1 eren al·lèlics al mutant aba2-1, els mutants sre2 eren al·lèlics al mutant aao3-1 i el mutant sre3 era al·lèlic al mutant aba1-5. Així, l´ABA bloqueja la germinació i l´establiment de la plàntula en condicions de baix potencial hídric, i aleshores mutants capaços de germinar en condicions d´estrès osmòtic representen normalment mutants amb defectes en la biosíntesi d´ABA. Mitjançant una combinació de les estratègies de mapeig posicional i gen candidat s´ha realitzat la identificació del gen ABA2 y de les mutacions sre1-1/aba2- 11 y sre1-2/aba2-12.El gen ABA2 (At1g52340) codifica una alcohol deshidrogenasa de cadena curta dependent de NAD+ (SDR). La mutació sre1-1/aba2-11 es deguda a una deleció de 53 pb que també suposa un camvi en la pauta de lectura, generant un al·lel nul de ABA2. La mutació sre1-2/aba2-12 produeix a la substitució Gly28Arg, afectant al lloc d´unió del coenzima. Mitjançant un anàlisi HPLC-MS s´ha demostrat que la proteïna recombinant ABA2 catalitza la conversió de xantoxina en aldehid abscísic en una reacció dependent de NAD+ . Els resultats obtinguts en el present treball estableixen inequívocament que la conversió de xantoxina en aldehíd abscísic representa el penúltim pas de la ruta de biosíntesi d´ABA i que està catalitzat pel producte gènic del gen ABA2 (At1g52340). L´últim pas de la ruta de biosíntesi d´ABA és la conversió de l´aldehid abscísic en àcid abscísic. Aquest pas està catalitzat en A. thaliana pel producte gènic del gen AAO3. Els mutants sre2-1/aao3-2 y sre2-2/aao3-3, igual que el mutant aao3-1, mostraven una reducció en els nivells d´ABA en fulles de roseta i una major transpiració que las plantes silvestres. A més, i contràriament al mutant aao3-1, mostraven un fenotip de osmotolerancia en germinació i establiment de plàntula, germinació resistent a paclobutrazol, latència reduïda i posseeixen una reducció del 65% en els nivells d´ABA en llavors. La caracterització molecular de la mutació sre2-1/aao3-2 revela una inserció de T-DNA que elimina la transcripció del gen AAO3, constituint por tant un al·lel nul. La secuenciació del gen AAO3 en el mutant sre2-2/aao3-3 va revelar una deleció de tres nucleòtids i vàries mutacions de canvi de sentit. L´evidencia genètica i bioquímica resultant de l´anàlisi dels mutants sre2- 1/aao3-2 i sre2-2/aaao3-3 indica que lesions en el gen AAO3 condueixen a nivells reduïts d´ABA i fenotips característics en llavors. Aleshores, queda demostrat que el gen AAO3 poseeix un paper crucial en la biosíntesi d´ABA en llavors.

© 2020 by the authors., ABA is involved in plant responses to a broad range of pathogens and exhibits complex antagonistic and synergistic relationships with salicylic acid (SA) and ethylene (ET) signaling pathways, respectively. However, the specific receptor of ABA that triggers the positive and negative responses of ABA during immune responses remains unknown. Through a reverse genetic analysis, we identified that PYR1, a member of the family of PYR/PYL/RCAR ABA receptors, is transcriptionally upregulated and specifically perceives ABA during biotic stress, initiating downstream signaling mediated by ABA-activated SnRK2 protein kinases. This exerts a damping effect on SA-mediated signaling, required for resistance to biotrophic pathogens, and simultaneously a positive control over the resistance to necrotrophic pathogens controlled by ET. We demonstrated that PYR1-mediated signaling exerted control on a priori established hormonal cross-talk between SA and ET, thereby redirecting defense outputs. Defects in ABA/PYR1 signaling activated SA biosynthesis and sensitized plants for immune priming by poising SA-responsive genes for enhanced expression. As a trade-off effect, pyr1-mediated activation of the SA pathway blunted ET perception, which is pivotal for the activation of resistance towards fungal necrotrophs. The specific perception of ABA by PYR1 represented a regulatory node, modulating different outcomes in disease resistance., This research was founded by the Spanish AEI agency by grant BIO2017-82503-R to P.L.R. and by grant RTI2018-098501-B-I00 to P.V.

Background: Characterization of the dynamic response of plant transpiration to decreasing soil water content in a reproducible way is required for the correct phenotyping of traits related to water saving strategies. Nowadays, an increasing number of automated high throughput platforms are available, but their development requires a great economic investment and it is not always desirable/feasible to outsource these analyses. We propose a mediumthroughput protocol to characterize transpiration responses to decreasing soil moisture in a quantitative and highly reproducible way with a minimum investment of resources. Results: The quantitative characterization of plant responses to a decreasing soil water content using our pheno‑ typing platform has showed high reproducibility between diferent experiments. The proposed irrigation strategy allowed us to harvest plants ranging from a well-watered condition to the loss-of-turgor point in a predictable and controlled way. Coupling this protocol with hormone profling allows investigation of hormonal responses (metabo‑ lite accumulation as well as plant sensitivity) to water stress. As a proof-of-concept, we have characterized the dynamic responses of leaf transpiration to decreasing soil water contents in an abscisic acid (ABA) defcient genotype (aba1-1) as well as in genotypes with altered sensitivity to ABA (abi1-1 and hab1-1abi1-1), which are insensitive and hypersensitive to ABA, respectively. Conclusions: This protocol allows for assessment of quantitative diferences in rosette transpiration responses to water depletion in both ABA biosynthesis mutants and genotypes with altered sensitivity to the hormone. Data indicate a correlation between ABA levels and/or hormone perception and growth rate and/or water content. The protocol guarantees the correct application of water stress to adult plants, which is essential to understand responses of mutants and/or natural accessions.

[EN] Recently, we described the ubiquitylation of PYL4 and PYR1 by the RING E3 ubiquitin ligase RSL1 at the plasma membrane of Arabidopsis thaliana. This suggested that ubiquitylated abscisic acid (ABA) receptors might be targeted to the vacuolar degradation pathway because such ubiquitylation is usually an internalization signal for the endocytic route. Here, we show that FYVE1 (previously termed FREE1), a recently described component of the endosomal sorting complex required for transport (ESCRT) machinery, interacted with RSL1-receptor complexes and recruited PYL4 to endosomal compartments. Although the ESCRT pathway has been assumed to be reserved for integral membrane proteins, we show the involvement of this pathway in the degradation of ABA receptors, which can be associated with membranes but are not integral membrane proteins. Knockdown fyve1 alleles are hypersensitive to ABA, illustrating the biological relevance of the ESCRT pathway for the modulation of ABA signaling. In addition, fyve1 mutants are impaired in the targeting of ABA receptors for vacuolar degradation, leading to increased accumulation of PYL4 and an enhanced response to ABA. Pharmacological and genetic approaches revealed a dynamic turnover of ABA receptors from the plasma membrane to the endosomal/vacuolar degradation pathway, which was mediated by FYVE1 and was dependent on RSL1. This process involves clathrin-mediated endocytosis and trafficking of PYL4 through the ESCRT pathway, which helps to regulate the turnover of ABA receptors and attenuate ABA signaling, Work in P.L.R.'s laboratory was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Cientificas (Grant BIO2014-52537-R). Work in R.T.M.'s laboratory was supported by a grant from the Natural Sciences and Engineering Research Council of Canada and a University of Guelph Research Chair. Work in J.L.'s laboratory was supported by the Ministerio de Ciencia e Innovacion and Fondo Europeo de Desarrollo Regional Grants BIO2011-27526 and BIO2014-56067-P L.R. was supported by a FPI fellowship and M.G.-G. by a JAE-DOC research contract. Work in L.J.'s laboratory was supported by grants from the Research Grants Council of Hong Kong (C4011-14R and AoE/M-05/12). We thank S.Y. Bednarek for the ProCLC2: CLC2-mOrange construct.

Climate change is expected to increase drought periods and the performance and dispersal of some invasive species such as Tetranychus evansi, which has been reported to take advantage of the nutritional changes induced by water-shortage on the tomato cultivar Moneymaker (MM). We have examined the implications for mite’s biology of four accessions of the drought-adapted tomatoes, “Tomàtiga de Ramellet” (TR), under moderate drought stress. Mite performance was enhanced by drought in two accessions (TR61 and TR154), but not in the other two accessions (TR58 and TR126). We selected one accession of each outcome (i.e., TR154 and TR126) to further analyze plant nutritional parameters. We found that free sugars and most essential amino acids for mites were induced by drought and/or mite infestation on MM and TR154 plants, whereas sugars were not altered and a reduced number of essential amino acids were induced by drought in TR126. Remarkably, mite performance was enhanced by leaf infiltration of free sugars, essential amino acids mixture, and L-proline on well-watered MM and by free sugars on drought-stressed TR126 plants. These results indicate a positive link between the induction of soluble carbohydrates and amino acids used by the plant for osmotic adjustment and mite performance. The effects of drought and/or mite infestation on the defense response of plants was analyzed at three levels: phytohormone accumulation, the transcript levels of marker genes linked to jasmonates (JAs), salicylic acid (SA), and abscisic acid (ABA) pathways, and the activity of defense proteins. The ability of T. evansi to downregulate the accumulation of defense-related phytohormones was noted on MM and the two TR accessions analyzed (TR126 and TR154), though differences in the induction of protein defense genes and activities by drought and/or mite infestation were observed among them. These results emphasize the importance of studying plant biotic and abiotic stress factors in combination and provides an experimental framework for screening drought-tolerant tomato accessions that will be also resistant to herbivore mites.

This work was funded by grants from MINECO (BFU2014-59796-R to A. A. and BIO2014-52537-R to P.L.R.). J.L.-J was supported by a Juan de la Cierva contract from MINECO and by the Marie Sklodowska-Curie Action H2020-MSCA-IF-2015-707477.

15 pags, 8 figs, Because abscisic acid (ABA) is recognized as the critical hormonal regulator of plant stress physiology, elucidating its signaling pathway has raised promise for application in agriculture, for instance through genetic engineering of ABA receptors. PYRABACTIN RESISTANCE1/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS ABA receptors interact with high affinity and inhibit clade A phosphatases type-2C (PP2Cs) in an ABA-dependent manner. We generated an allele library composed of 10,000 mutant clones of Arabidopsis (Arabidopsis thaliana) PYL4 and selected mutations that promoted ABA-independent interaction with PP2CA/ABA-HYPERSENSITIVE3. In vitro protein-protein interaction assays and size exclusion chromatography confirmed that PYL4A194T was able to form stable complexes with PP2CA in the absence of ABA, in contrast to PYL4. This interaction did not lead to significant inhibition of PP2CA in the absence of ABA; however, it improved ABA-dependent inhibition of PP2CA. As a result, 35S: PYL4A194T plants showed enhanced sensitivity to ABA-mediated inhibition of germination and seedling establishment compared with 35S:PYL4 plants. Additionally, at basal endogenous ABA levels, whole-rosette gas exchange measurements revealed reduced stomatal conductance and enhanced water use efficiency compared with nontransformed or 35S:PYL4 plants and partial up-regulation of two ABA-responsive genes. Finally, 35S:PYL4A194T plants showed enhanced drought and dehydration resistance compared with nontransformed or 35S:PYL4 plants. Thus, we describe a novel approach to enhance plant drought resistance through allele library generation and engineering of a PYL4 mutation that enhances interaction with PP2CA. 2013 American Society of Plant Biologists. All Rights Reserved., This work was supported by the Ministerio de Ciencia e Innovación, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Científicas (grant nos. BIO2011–23446 to P.L.R and BFU2011–25384 to A.A., fellowships to R.A. and L.R., and a Juan de la Cierva contract to M.G.-G.) and the Estonian Ministry of Science and Education and European Regional Fund (IUT2–21 and Center of Excellence Environ to E.M. and H.K.).

31 p.-11 fig.-2 tab.+ Erratum (2 p.) Papanikolaou, Alexie et al., Background: The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control., Results: The 479 Mb medfly genome is sequenced from adult flies from lines inbred for 20 generations. A highquality assembly is achieved having a contig N50 of 45.7 kb and scaffold N50 of 4.06 Mb. In-depth curation of more than 1800 messenger RNAs shows specific gene expansions that can be related to invasiveness and host adaptation, including gene families for chemoreception, toxin and insecticide metabolism, cuticle proteins, opsins, and aquaporins. We identify genes relevant to IPM control, including those required to improve SIT., Conclusions: The medfly genome sequence provides critical insights into the biology of one of the most serious and widespread agricultural pests. This knowledge should significantly advance the means of controlling the size and invasive potential of medfly populations. Its close relationship to Drosophila, and other insect species important to agriculture and human health, will further comparative functional and structural studies of insect genomes that should broaden our understanding of gene family evolution, Support of this project was provided by the U.S. Department of Agriculture(USDA), Agricultural Research Service (ARS), Animal and Plant Health Inspection Service (APHIS), and National Institute of Food and Agriculture(NIFA)-Biotechnology Risk Assessment Grants Program (grant #2011-39211-30769 to AMH) for funding the initial phase of this project, and to the National Institutes of Health (NIH)-National Human Genome Research Institute (NHGRI) for funding the medfly genome sequencing, assembly and Maker 2.0 automated annotation as part of the i5K 30 genome pilot project (grant #U54 HG003273 to RAG). The NIH Intramural Research Program, National Library of Medicine funded the NCBI Gnomon annotation and the USDA-National Agricultural Library (NAL) provided support for the WebApollo curation website, with support for manual curation training (to MM-T) provided by NIGMS (grant #5R01GM080203),NHGRI (grant #5R01HG004483), and the U.S. Department of Energy(contract #DE-AC02-05CH11231). Support was provided for: toxin metabolism and insecticide resistance gene studies from MINECO,Spain (AGL2013-42632-R to FO and PH-C); microRNAs, horizontal gene transfer and bacterial contaminant studies from the European Social Fund and National Strategic Reference Framework-THALES (MIS375869 to KB, GT, AGH, and KM) and the U.S. National Science Foundation(DEB 1257053 to JHW); cuticle protein gene studies from USDA-NIFA(grant #2016-67012-24652 to AJR); sex-determination studies from L.R. Campania (grant 5/02, 2008 to GS); male reproduction and sexual differentiation studies from the FAO/IAEA (Technical Contract No: 16966 to GGa) and Cariplo IMPROVE (to FS); and programmed cell death gene studies and genomic data analysis (to MFS) from the Emmy Noether program, DFG(SCHE 1833/1-1) and the LOEWE Center for Insect Biotechnology & Bioresources grant of the Hessen State Ministry of Higher Education, Research and the Arts(HMWK), Germany and from the USDA-NIFA-Biotechnology Risk Assessment Grants Program (grant #2015-33522-24094 to AMH).

[EN] Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca2+ are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca2+ signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca2+-dependent recruitment of the pyrabactin resistance 1/PYR1like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca2+ sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca2+-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress., A.A. and J.A.M. thank the European Syncrotron Radiation Facility and EMBL for access to the synchrotron radiation source. This work was funded by Ministerio de Economia y Competitividad (MINECO) Grants BFU2014-59796-R (to A.A.), BFU2011-28184-C02 (to M.J.S.-B.), and BIO2014-52537-R (to P.L.R.) and Comunidad de Madrid Grant S2010/BMD-2457 (to A.A and M.M.). M.J.S.-B. is supported by Ramon y Cajal Contract RYC-2008-03449 from MINECO and M.D. by a fellowship from Senacyt-Ifarhu. Access to the High Throughput Crystallization facility at European Molecular Biology Laboratory (EMBL) Grenoble was supported by the European Community's Seventh Framework Programme through the Protein Production Platform project (P-CUBE) Grant 227764.

[EN] Optimal response to drought is critical for plant survival and will affect biodiversity and crop performance during climate change. Mitotically heritable epigenetic or dynamic chromatin state changes have been implicated in the plant response to the drought stress hormone abscisic acid (ABA). The Arabidopsis SWI/SNF chromatin-remodeling ATPase BRAHMA (BRM) modulates response to ABA by preventing premature activation of stress response pathways during germination. We show that core ABA signaling pathway components physically interact with BRM and post-translationally modify BRM by phosphorylation/dephosphorylation. Genetic evidence suggests that BRM acts downstream of SnRK2.2/2.3 kinases, and biochemical studies identified phosphorylation sites in the C-terminal region of BRM at SnRK2 target sites that are evolutionarily conserved. Finally, the phosphomimetic BRMS1760D (S1762D) mutant displays ABA hypersensitivity. Prior studies showed that BRM resides at target loci in the ABA pathway in the presence and absence of the stimulus, but is only active in the absence of ABA. Our data suggest that SnRK2-dependent phosphorylation of BRM leads to its inhibition, and PP2CA-mediated dephosphorylation of BRM restores the ability of BRM to repress ABA response. These findings point to the presence of a rapid phosphorylation-based switch to control BRM activity; this property could be potentially harnessed to improve drought tolerance in plants., Work in Dr. Rodriguez's laboratory was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Cientificas (grant BIO2014-52537-R). M.P.-L. and L.R. were supported by FPI fellowships, and M.G.-G. by a JAE-DOC research contract. Funding of chromatin research in the Wagner laboratory is supported by National Science Foundation grant MCB-0925071.

[EN] Abscisic acid (ABA) is a key hormone for plant growth, development, and stress adaptation. Perception of ABA through four types of receptors has been reported. We show here that impairment of ABA perception through the PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) branch reduces vegetative growth and seed production and leads to a severe open stomata and ABA-insensitive phenotype, even though other branches for ABA perception remain functional. An Arabidopsis thaliana sextuple mutant impaired in six PYR/PYL receptors, namely PYR1, PYL1, PYL2, PYL4, PYL5, and PYL8, was able to germinate and grow even on 100 mu M ABA. Whole-rosette stomatal conductance (Gst) measurements revealed that leaf transpiration in the sextuple pyr/pyl mutant was higher than in the ABA-deficient aba3-1 or ABA-insensitive snrk2.6 mutants. The gradually increasing Gst values of plants lacking three, four, five, and six PYR/PYLs indicate quantitative regulation of stomatal aperture by this family of receptors. The sextuple mutant lacked ABA-mediated activation of SnRK2s, and ABA-responsive gene expression was dramatically impaired as was reported in snrk2.2/2.3/2.6. In summary, these results show that ABA perception by PYR/PYLs plays a major role in regulation of seed germination and establishment, basal ABA signaling required for vegetative and reproductive growth, stomatal aperture, and transcriptional response to the hormone., We thank Joseph Ecker and the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants, the Arabidopsis Biological Resource Center/Nottingham Arabidopsis Stock Centre for distributing these seeds, and Sean Cutler for the pyr1-1, 114 and 1124 mutants. This study was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Cientificas (grant BIO2011-23446 to P. L. R; fellowship to R. A.; Juan de la Cierva contract to M. G.-G.), by the Estonian Ministry of Science and Education (ETF7763 and ETF9208, SF0180071s07), by the European Regional Fund (Center of Excellence in Environmental Adaptation), and by a Marie Curie Incoming International Fellowship to G. W. B.

[EN] Clade A protein phosphatases type 2C (PP2Cs) are negative regulators of abscisic acid (ABA) signaling that are inhibited in an ABA-dependent manner by PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) intracellular receptors. We provide genetic evidence that a previously uncharacterized member of this PP2C family in Arabidopsis (Arabidopsis thaliana), At5g59220, is a negative regulator of osmotic stress and ABA signaling and that this function was only apparent when double loss-of-function mutants with pp2ca-1/ahg3 were generated. At5g59220-green fluorescent protein and its close relative PP2CA-green fluorescent protein showed a predominant nuclear localization; however, hemagglutinin-tagged versions were also localized to cytosol and microsomal pellets. At5g59220 was selectively inhibited by some PYR/PYL ABA receptors, and close relatives of this PP2C, such as PP2CA/ABA-HYPERSENSITIVE GERMINATION3 (AHG3) and AHG1, showed a contrasting sensitivity to PYR/PYL inhibition. Interestingly, AHG1 was resistant to inhibition by the PYR/PYL receptors tested, which suggests that this seed-specific phosphatase is still able to regulate ABA signaling in the presence of ABA and PYR/PYL receptors and therefore to control the highly active ABA signaling pathway that operates during seed development. Moreover, the differential sensitivity of the phosphatases At5g59220 and PP2CA to inhibition by ABA receptors reveals a functional specialization of PYR/PYL ABA receptors to preferentially inhibit certain PP2Cs., This work was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Cientificas (grant nos. BIO2008-00221 and BIO2011-23446 to P. L. R; fellowships to R.A. and L.R.; Juan de la Cierva contract to M.G.-G.).

The recent identification of abscisic acid (ABA) transporters provides an important insight into the delivery of ABA from the vascular system and its uptake by target cells. A putative connection with PYR/PYL receptors is envisaged, linking ABA uptake and intracellular perception by a fast and efficient mechanism. Downstream signaling of the core pathway involves regulation of ABA-responsive element binding factors (ABFs/AREBs) through phosphorylation, ubiquitination and sumoylation in the case of ABI5. Several E3 ligases appear to regulate ABA signaling either positively or negatively, although relatively few targets are known yet. ABFs/AREBs are themselves subjected to transcriptional regulation, and some transcription factors harboring the WRKY domain (WRKYs) appear to regulate their expression through W-box sequences present in the promoters of ABFs/AREBs., This work was supported by Ministerio de Educación y Ciencia and Fondo Europeo de Desarrollo Regional grant BIO2008-00221 to P.L.R.

The plant hormone abscisic acid (ABA) plays a crucial role in the control of the stress response and the regulation of plant growth and development. ABA binding to PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS intracellular receptors leads to inhibition of key negative regulators of ABA signaling, i.e. clade A protein phosphatases type 2C (PP2Cs) such as ABA-INSENSITIVE1 and HYPERSENSITIVE TO ABA1 (HAB1), causing the activation of the ABA signaling pathway. To gain further understanding on the mechanism of hormone perception, PP2C inhibition, and its implications for ABA signaling, we have performed a structural and functional analysis of the PYR1-ABA-HAB1 complex. Based on structural data, we generated a gain-of-function mutation in a critical residue of the phosphatase, hab1W385A, which abolished ABA-dependent receptor-mediated PP2C inhibition without impairing basal PP2C activity. As a result, hab1W385A caused constitutive inactivation of the protein kinase OST1 even in the presence of ABA and PYR/PYL proteins, in contrast to the receptor-sensitive HAB1, and therefore hab1W385A qualifies as a hypermorphic mutation. Expression of hab1W385A in Arabidopsis (Arabidopsis thaliana) plants leads to a strong, dominant ABA insensitivity, which demonstrates that this conserved tryptophan residue can be targeted for the generation of dominant clade A PP2C alleles. Moreover, our data highlight the critical role of molecular interactions mediated by tryptophan-385 equivalent residues for clade A PP2C function in vivo and the mechanism of ABA perception and signaling.

[EN] Abscisic acid (ABA) is a phytohormone that inhibits growth and enhances adaptation to stress in plants. ABA perception and signaling rely on its binding to receptors of the pyrabactin resistance1/PYR1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) family, the subsequent inhibition of clade A type 2C protein phosphatases (PP2Cs), and the phosphorylation of ion channels and transcription factors by protein kinases of the SnRK2 family. Nitric oxide (NO) may inhibit ABA signaling because NO-deficient plants are hypersensitive to ABA. Regulation by NO often involves posttranslational modification of proteins. Mass spectrometry analysis of ABA receptors expressed in plants and recombinant receptors modified in vitro revealed that the receptors were nitrated at tyrosine residues and S-nitrosylated at cysteine residues. In an in vitro ABA-induced, PP2C inhibition assay, tyrosine nitration reduced receptor activity, whereas S-nitrosylated receptors were fully capable of ABA-induced inhibition of the phosphatase. PYR/PYL/RCAR proteins with nitrated tyrosine, which is an irreversible covalent modification, were poly-ubiquitylated and underwent proteasome-mediated degradation. We propose that tyrosine nitration, which requires NO and superoxide anions, is a rapid mechanism by which NO limits ABA signaling under conditions in which NO and reactive oxygen species are both produced., This work was supported by the Ministry of Science and Innovation (MICINN) (Spain) grants BIO2008-00839 and BIO2011-27526 (to J.L.) and BIO2011-23446 (to P.L.R.).

The stress hormone abscisic acid (ABA) induces expression of defense 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, over-expression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than 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 inhibits the root plasma membrane H+-ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (H+ efflux from seedling roots). This inhibition involves the core ABA signaling 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 indicate that ABA, acting through the PYR/PYL/RCAR receptors, induces membrane hyperpolarization (due to K+ efflux through 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 are different from those in guard cells. ABA does not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H+-ATPase in roots and SnRK2.2 phosphorylates in vitro the C-terminal regulatory domain of H+-ATPase while the guard cell kinase SnRK2.6/OST1 does not., This work was funded by grants BFU2011-22526 (to RS) and BIO2011-23446 (to PLR) of the Spanish ‘Ministerio de Economía 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 Científicas’, Madrid, Spain.

Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calcium-dependent interactions of PYR/PYL ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL subcellular localization and positively regulates ABA signaling., We thank Joerg Kudla (University of Munster) for kindly providing plasma membrane markers. 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 and BFU2011-25384 to A. A.; fellowships to L.R., R.A., and A.C.I.-G.; BES-2009- 016569; JAE-DOC contract to M.G.-G.) as well as the Senacyt-Ifarhu (Panama) (fellowship to M.D.).

HAB1 was originally cloned on the basis of sequence homology to ABI1 and ABI2, and indeed, a multiple sequence alignment of 32 Arabidopsis protein phosphatases type-2C (PP2Cs) reveals a cluster composed by the four closely related proteins, ABI1, ABI2, HAB1 and At1g17550 (here named HAB2). Characterisation of transgenic plants harbouring a transcriptional fusion ProHAB1: green fluorescent protein (GFP) indicates that HAB1 is broadly expressed within the plant, including key target sites of abscisic acid (ABA) action as guard cells or seeds. The expression of the HAB1 mRNA in vegetative tissues is strongly upregulated in response to exogenous ABA. In this work, we show that constitutive expression of HAB1 in Arabidopsis under a cauliflower mosaic virus (CaMV) 35S promoter led to reduced ABA sensitivity both in seeds and vegetative tissues, compared to wild-type plants. Thus, in the field of ABA signalling, this work represents an example of a stable phenotype in planta after sustained overexpression of a PP2C genes. Additionally, a recessive T-DNA insertion mutant of HAB1 was analysed in this work, whereas previous studies of recessive alleles of PP2C genes were carried out with intragenic revertants of the abi1-1 and abi2-1 mutants that carry missense mutations in conserved regions of the PP2C domain. In the presence of exogenous ABA, hab1-1 mutant shows ABA-hypersensitive inhibition of seed germination; however, its transpiration rate was similar to that of wild-type plants. The ABA-hypersensitive phenotype of hab1-1 seeds together with the reduced ABA sensitivity of 35S:HAB1 plants are consistent with a role of HAB1 as a negative regulator of ABA signalling. Finally, these results provide new genetic evidence on the function of a PP2C in ABA signalling.

[EN] Natural genetic variation is crucial for adaptability of plants to different environments. Seed dormancy prevents precocious germination in unsuitable conditions and is an adaptation to a major macro-environmental parameter, the seasonal variation in temperature and day length. Here we report the isolation of IBO, a quantitative trait locus (QTL) that governs c. 30% of germination rate variance in an Arabidopsis recombinant inbred line (RIL) population derived from the parental accessions Eilenburg-0 (Eil-0) and Loch Ness-0 (Lc-0).IBO encodes an uncharacterized phosphatase 2C-related protein, but neither the Eil-0 nor the Lc-0 variant, which differ in a single amino acid, have any appreciable phosphatase activity in in vitro assays. However, we found that the amino acid change in the Lc-0 variant of the IBO protein confers reduced germination rate. Moreover, unlike the Eil-0 variant of the protein, the Lc-0 variant can interfere with the activity of the phosphatase 2C ABSCISIC ACID INSENSITIVE 1 in vitro. This suggests that the Lc-0 variant possibly interferes with abscisic acid signaling, a notion that is supported by physiological assays. Thus, we isolated an example of a QTL allele with a nonsynonymous amino acid change that might mediate local adaptation of seed germination timing.

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondary metabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Here we show that, in the legume Medicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membrane-anchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues., This research has received funding from the Agency for Innovation by Science and Technology in Flanders (‘Strategisch Basisonderzoek’ Combiplan project SBO040093), the European Union Seventh Framework Programme FP7/2007-2013 under grant agreement number 222716 –SMARTCELL and the Spanish Ministerio de Economía y Competitividad under grant BFU2011-24208. T.M. and N.D.G. are indebted to the VIB International PhD Fellowship Program and the Agency for Innovation by Science and Technology for predoctoral fellowships, respectively. J.P. and S.L. are postdoctoral fellows of the Research Foundation Flanders (FWO).

[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] Seed longevity is important to preserve crops and wild plants and it is limited by progressive cellular damage (aging) during storage. The induction of cellular stress defenses and the formation of the seed coat are crucial protecting events during seed development, a process mediated in Arabidopsis thaliana by the transcription factors LEC1, LEC2, FUS3 and the abscisic acid-activated ABI3. In order to identify novel determinants of seed longevity we have screened an activation-tagging mutant collection of Arabidopsis and isolated a dominant mutant with increased seed longevity under both natural and accelerated aging conditions. Molecular characterization indicates that the mutant phenotype is caused by over-expression of the At2g26130 gene encoding a RING-type zinc finger putative ubiquitin ligase. Loss of function of this gene in a T-DNA insertion mutant resulted in decreased seed longevity. We named this important gene for seed longevity RSL1 (from Ring finger of Seed Longevity1) and we could demonstrate ubiquitin ligase activity with the recombinant protein. Morphological alterations in shoot tissues of the RSL1 over-expressing plants and analysis of gibberellins levels suggest that RSL1 may increase gibberellins responses by some unknown mechanism. These results validate the forward genetic approach to seed longevity and anticipate the identification of many novel determinants of this important trait. (C) 2013 Elsevier Ireland Ltd. All rights reserved., This work was funded by grant EUI2009-03985 of the Spanish "Ministerio de Economia y Competividad" (MINECO, Madrid). J.M.-B. was supported by a contract (Juan de la Cierva) of the Spanish MINECO. M.G.-G. was supported by a contract (JAE-DOC) of the Spanish Consejo Superior de Investigaciones Cientificas (CSIC, Madrid). We thank Dr Judy Callis for kindly lending us the 6xHis-AtUBC8 plas mid.

In the sequences released by the Arabidopsis Genome Initiative (AGI), we have discovered a new large gene family (48 genes as of July 2000). A detailed computational and biochemical analysis of the predicted gene products reveals a novel family of plant F-box proteins, where the amino (N)-terminal F-box motif is followed by four kelch repeats and a characteristic carboxy-terminal domain. F-box proteins are an expanding family of eukaryotic proteins, which have been shown in some cases to be critical for the controlled degradation of cellular regulatory proteins via the ubiquitin pathway. The F-box motif of the At5g48990 gene product, a member of the family, was shown to be functionally active by its ability to mediate the in vitro interaction between At5g48990 and ASK1 proteins. F-box proteins specifically recruit the targets to be ubiquitinated, mainly through protein-protein interaction modules such as WD-40 domains or leucine-rich repeats (LRRs). The kelch repeats of the family described here form a potential protein-protein interaction domain, as molecular modelling of the kelch repeats according to the galactose oxidase crystal structure (the only solved structure containing kelch repeats) predicts a β-propeller. The identification of this family of F-box proteins greatly expands the field of plant F-box proteins and suggests that controlled degradation of cellular proteins via the ubiquitin pathway could play a critical role in multiple plant cellular processes.

Plant survival under environmental stress requires the integration of multiple signaling pathways into a coordinated response, but the molecular mechanisms underlying this integration are poorly understood. Stress-derived energy deprivation activates the Snf1-related protein kinases1 (SnRK1s), triggering a vast transcriptional and metabolic reprogramming that restores homeostasis and promotes tolerance to adverse conditions. Here, we show that two clade A type 2C protein phosphatases (PP2Cs), established repressors of the abscisic acid (ABA) hormonal pathway, interact with the SnRK1 catalytic subunit causing its dephosphorylation and inactivation. Accordingly, SnRK1 repression is abrogated in double and quadruple pp2c knockout mutants, provoking, similarly to SnRK1 overexpression, sugar hypersensitivity during early seedling development. Reporter gene assays and SnRK1 target gene expression analyses further demonstrate that PP2C inhibition by ABA results in SnRK1 activation, promoting SnRK1 signaling during stress and once the energy deficit subsides. Consistent with this, SnRK1 and ABA induce largely overlapping transcriptional responses. Hence, the PP2C hub allows the coordinated activation of ABA and energy signaling, strengthening the stress response through the cooperation of two key and complementary pathways., E.B.-G. was supported by grants from Marie Curie IRG, the EMBO Installation program, Marie Curie Actions FP7-People-2010-ITN, the Fundação para a Ciência e a Tecnologia (FCT-PTDC/AGR-AAM/104939/2008), and the Portugal-Spain Bilateral Collaboration program Ações integradas (Ação E-26/10). A.C. was supported by SFRH/BPD/47280/2008, C.M. was supported by SFRH/BD/33563/2008, L.M. was supported by SFRH/BD/51627/2011, and P.C. was supported by SFRH/BPD/79255/2011. A. Rabissi was supported by a Generalitat de Catalunya PhD grant (FI-AR067443). P.L.R. was supported by the Ministerio de Ciencia e Innovación (grants BIO2011-23446 and PT2009-0155), R.A. was supported by the Junta para Ampliación de Estudios e Investigaciones Científicas-Consejo Superior de Investigaciones Cientificas fellowship, and M.G.-G. was supported by a Juan de la Cierva contract.

[EN] Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABA-INSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches. We also discovered that PYR/PYL receptors and clade A PP2Cs are crucial for the hydrotropic response that takes place to guide root growth far from regions with low water potential. Thus, an ABA-hypersensitive pp2c quadruple mutant showed enhanced hydrotropism, whereas an ABA-insensitive sextuple pyr/pyl mutant showed reduced hydrotropic response, indicating that ABA-dependent inhibition of PP2Cs by PYR/PYLs is required for the proper perception of a moisture gradient., This work was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Cientificas (grant no. BIO2011-23446 to P. L. R.; fellowships to R. A., L. R., and M. P.-L.; Juan de la Cierva contract to M.G.-G.).

[EN] Rapid stomatal closure induced by changes in the environment, such as elevation of CO2, reduction of air humidity, darkness, and pulses of the air pollutant ozone (O-3), involves the SLOW ANION CHANNEL1 (SLAC1). SLAC1 is activated by OPEN STOMATA1 (OST1) and Ca2+-dependent protein kinases. OST1 activation is controlled through abscisic acid (ABA)-induced inhibition of type 2 protein phosphatases (PP2C) by PYRABACTIN RESISTANCE/REGULATORY COMPONENTS OF ABA RECEPTOR (PYR/RCAR) receptor proteins. To address the role of signaling through PYR/RCARs for whole-plant steady-state stomatal conductance and stomatal closure induced by environmental factors, we used a set of Arabidopsis (Arabidopsis thaliana) mutants defective in ABA metabolism/signaling. The stomatal conductance values varied severalfold among the studied mutants, indicating that basal ABA signaling through PYR/RCAR receptors plays a fundamental role in controlling whole-plant water loss through stomata. PYR/RCAR-dependent inhibition of PP2Cs was clearly required for rapid stomatal regulation in response to darkness, reduced air humidity, and O-3. Furthermore, PYR/RCAR proteins seem to function in a dose-dependent manner, and there is a functional diversity among them. Although a rapid stomatal response to elevated CO2 was evident in all but slac1 and ost1 mutants, the bicarbonate-induced activation of S-type anion channels was reduced in the dominant active PP2C mutants abi1-1 and abi2-1. Further experiments with a wider range of CO2 concentrations and analyses of stomatal response kinetics suggested that the ABA signalosome partially affects the CO2-induced stomatal response. Thus, we show that PYR/RCAR receptors play an important role for the whole-plant stomatal adjustments and responses to low humidity, darkness, and O-3 and are involved in responses to elevated CO2., This work was supported by the Estonian Ministry of Science and Education (grant no. IUT2-21), by the European Regional (Center of Excellence in Environmental Adaptation) and Social (Mobilitas Top Researchers grant no. MTT9) Fund, by the National Science Foundation (grant no. MCB0918220 to J.I.S.) and the National Institutes of Health (grant no. R01GM060396 to J.I.S.), by Shanxi Scholarship Council of China (grant no. 2011-012 to S.X.), by Shanxi Technology Foundation and Natural Science Foundation of Shanxi (grant no. 2012011006-4 to S.X.), and by Ministerio de Economia y Competitividad (grant no. BIO2011-23446 to P.L.R.).

Abscisic acid (ABA) plays an essential function in plant physiology since it is required for biotic and abiotic stress responses as well as control of plant growth and development. A new family of soluble ABA receptors, named PYR/PYL/RCAR, has emerged as ABA sensors able to inhibit the activity of specific protein phosphatases type-2C (PP2Cs) in an ABAdependent manner. The structural and functional mechanism by which ABA is perceived by these receptors and consequently leads to inhibition of the PP2Cs has been recently elucidated. The module PYR/PYL/RCARABA- PP2C offers an elegant and unprecedented mechanism to control phosphorylation signaling cascades in a ligand-dependent manner. The knowledge of their three-dimensional structures paves the way to the design of ABA agonists able to modulate the plant stress response., We are grateful to the European Synchrotron Radiation Facility (ESRF) and the EMBL for access to macromolecular crystallography beam lines. Work in the laboratory of Dr Rodriguez is supported by grant BIO2008- 00221 from Ministerio de Educación y Ciencia and Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Científicas (fellowships to JS, RA and LR; Juan de la Cierva contract to MGG). Access to the high Throughput Crystallization facility of the Partnership for Structural Biology in Grenoble (PSB) (https://htxlab.embl.fr ) was supported by the European Community- Research Infrastructure Action PCUBE under the FP7 "Capacities" specific program.

4 pags, 3 figs, 1 tab, An uncharacterized protein from Arabidopsis thaliana consisting of a single C2 domain (At3g17980) was cloned into the pETM11 vector and expressed in Escherichia coli, allowing purification to homogeneity in a single chromatographic step. Good-quality diffracting crystals were obtained using vapour-diffusion techniques. The crystals diffracted to 2.2 Å resolution and belonged to space group P212121, with unit-cell parameters a = 35.3, b = 88.9, c = 110.6 Å. A promising molecular-replacement solution has been found using the structure of the C2 domain of Munc13-C2b (PDB entry 3kwt) as the search model. © 2011 International Union of Crystallography. All rights reserved., his work was funded by grants BFU2008-00368/BMC, BFU2011-25384 and CSD2006-00015 (Factoría de Cristalización) of the Spanish ‘Plan Nacional’ (MICINN) to AA. MD was supported by fellowship SENACYT-IFARHU of Panama–‘Programa de Investigadores’.

BACKGROUND: cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is a commonly used technique for genome-wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequence information are obtained for a large number of differentially expressed gene tags. However, most of the gene tags do not correspond to full-length (FL) coding sequences, which is a prerequisite for subsequent functional analysis. METHODOLOGY: A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incomplete cDNAs. The method was applied to screen for the FL open reading frames of a selection of 163 cDNA-AFLP tags from three different medicinal plants, leading to the identification of 109 (67%) FL clones. Furthermore, the protocol allows for the use of multiple probes in a single hybridization event, thus significantly increasing the throughput when screening for rare transcripts. CONCLUSIONS: The presented strategy offers an efficient method for the conversion of incomplete expressed sequence tags (ESTs), such as cDNA-AFLP tags, to FL-coding sequences.

Triterpene saponins are a class of plant natural products with a wide range of bioactivities, which makes them an interesting research subject. The small tree Maesa lanceolata, growing in African countries, is used in traditional medicine against various diseases. In previous work a triterpenoid saponin mixture was isolated from the leaves of M. lanceolata and the compounds were identified as closely related oleanane type triterpenes [Apers, S., Foriers, A., Sindambiwe, J.B., Vlietinck, A., Pieters, L., 1998. Separation of a triterpenoid saponin mixture from Maesa lanceolata: semi preparative reversed-phase wide pore high performance liquid chromatography with temperature control. J. Pharm. Biomed. Anal. 18, 737; Apers, S., De Bruyne, T.E., Claeys, M., Vlietinck, A.J., Pieters, L.A.C., 1999. New acylated triterpenoid saponins from Maesa lanceolata. Phytochemistry 52, 1121]. The compounds showed virucidal, haemolytic, molluscicidal and antiangiogenic activity [Apers, S., Baronikova, S., Sindambiwe, J.B., Witvrouw, M., De Clercq, E., Vanden Berghe, D., Van Marck, E., Vlietinck, A., Pieters, L., 2001. Antiviral, haemolytic and molluscicidal activities of triterpenoid saponins from Maesa lanceolata: establishment of structure-activity relationships. Planta Med. 67, 528; Apers, S., Burgermeister, J., Baronikova, S., Vermeulen, P., Paper, D., Van Marck, E., Vlietinck, A.J., Pieters, L.A.C., 2002. Antiangiogenic activity of natural products: in vivo and in vitro test models. J. Pharm. Belg. 57 (Hors-serie 1), 47]. Here we report the development of an extraction and quantification method to analyse saponin compounds in roots and leaves of M. lanceolata. After a purification step using C(18) solid phase extraction (SPE) cartridges, the samples were analysed on a LC-UV/MS system. The identification of the peaks from the different saponins was confirmed based on the retention time and mass spectrum. The quantification was performed using the UV signals. The standard oleanolic acid curve was linear over a concentration range of 2.8-140.0mug/mL. The recovery from the leaves was 94.5%. The precision of the method with respect to time and concentration was acceptable, with relative standard deviation (RSD%) values of 4.9 and 4.3, respectively.

Once the plant coenzyme A (CoA) biosynthetic pathway has been elucidated by comparative genomics, it is feasible to analyze the physiological relevance of CoA biosynthesis in plant life. To this end, we have identified and characterized Arabidopsis (Arabidopsis thaliana) T-DNA knockout mutants of two CoA biosynthetic genes, HAL3A and HAL3B. The HAL3A gene encodes a 4′-phosphopantothenoyl-cysteine decarboxilase that generates 4′-phosphopantetheine. A second gene, HAL3B, whose gene product is 86% identical to that of HAL3A, is present in the Arabidopsis genome. HAL3A appears to have a predominant role over HAL3B according to their respective mRNA expression levels. The hal3a-1, hal3a-2, and hal3b mutants were viable and showed a similar growth rate as that in wild-type plants; in contrast, a hal3a-1 hal3b double mutant was embryo lethal. Unexpectedly, seedlings that were null for HAL3A and heterozygous for HAL3B (aaBb genotype) displayed a sucrose (Suc)-dependent phenotype for seedling establishment, which is in common with mutants defective in β-oxidation. This phenotype was genetically complemented in aaBB siblings of the progeny and chemically complemented by pantethine. In contrast, seedling establishment of Aabb plants was not Suc dependent, proving a predominant role of HAL3A over HAL3B at this stage. Total fatty acid and acyl-CoA measurements of 5-d-old aaBb seedlings in medium lacking Suc revealed stalled storage lipid catabolism and impaired CoA biosynthesis; in particular, acetyl-CoA levels were reduced by approximately 80%. Taken together, these results provide in vivo evidence for the function of HAL3A and HAL3B, and they point out the critical role of CoA biosynthesis during early postgerminative growth.

Much of the literature on the phytohormone abscisic acid (ABA) describes it as a mediator in triggering plant responses to environmental stimuli, as well as a growth inhibitor. ABA-deficient mutants, however, display a stunted phenotype even under well-watered conditions and high relative humidity, which suggests that growth promotion may also be one of the roles of endogenous ABA. Zeaxanthin epoxidase, the product of the ABA1 gene of Arabidopsis thaliana, catalyses the epoxidation of zeaxanthin to antheraxanthin and violaxanthin, generating the epoxycarotenoid precursor of the ABA biosynthetic pathway. This paper gives a description of the molecular and phenotypic characterization of a large series of mutant alleles of the ABA1 gene, which cause different degrees of ABA deficiency, four of them previously isolated (aba1-1, aba1-3, aba1-4, and aba1-6) and the remaining five novel (san ˜ 1-1, san ˜ 1-2, san ˜ 1-3, san ˜ 1-4 ,a ndsre3). Molecular analysis of these alleles provides insights into the domains in which they compromise zeaxanthin epoxidase function. The size of the leaves, inflorescences, and flowers of these mutants is reduced, and their rosettes have lower fresh and dry weights than their wild types, as a result of a diminished cell size. Low concentrations of exogenous ABA increase the fresh weight of mutant and wild-type plants, as well as the dry weight of the mutants. The leaves of aba1 mutants are abnormally shaped and fail to develop clearly distinct spongy and palisade mesophyll layers. Taken together, these phenotypic traits indicate, as suggested by previous authors, that ABA acts as a growth promoter during vegetative development. The abnormal shape and internal structure of the leaves of aba1 mutants suggests, in addition, a role for ABA in organogenesis.

9 páginas, 3 figuras, 3 tablas -- PAGS nros. 325-333, The abscisic aldehyde oxidase 3 (AAO3) gene product of Arabidopsis catalyzes the final step in abscisic acid (ABA) biosynthesis. An aao3-1 mutant in a Landsberg erecta genetic background exhibited a wilty phenotype in rosette leaves, whereas seed dormancy was not affected (Seo et al., 2000a). Therefore, it was speculated that a different aldehyde oxidase would be the major contributor to ABA biosynthesis in seeds (Seo et al., 2000a). Through a screening based on germination under high-salt concentration, we isolated two mutants in a Columbia genetic background, initially named sre2-1 and sre2-2 (for salt resistant). Complementation tests with different ABA-deficient mutants indicated that sre2-1 and sre2-2 mutants were allelic to aao3-1, and therefore they were renamed as aao3-2 and aao3-3, respectively. Indeed, molecular characterization of the aao3-2 mutant revealed a T-DNA insertional mutation that abolished the transcription of AAO3 gene, while sequence analysis of AAO3 in aao3-3 mutant revealed a deletion of three nucleotides and several missense mutations. Physiological characterization of aao3-2 and aao3-3 mutants revealed a wilty phenotype and osmotolerance in germination assays. In contrast to aao3-1, both aao3-2 and aao3-3 mutants showed a reduced dormancy. Accordingly, ABA levels were reduced in dry seeds and rosette leaves of both aao3-2 and aao3-3. Taken together, these results indicate that AAO3 gene product plays a major role in seed ABA biosynthesis, This work was supported by the Ministerio de Ciencia y Tecnologia (grant no. BIO2002–03090) and FEDER