Publications by authors named "Pedro L Rodriguez"

87 Publications

Low ABA concentration promotes root growth and hydrotropism through relief of ABA INSENSITIVE 1-mediated inhibition of plasma membrane H-ATPase 2.

Sci Adv 2021 Mar 17;7(12). Epub 2021 Mar 17.

Center for Plant Water-Use and Nutrition Regulation and College of Life Sciences, Joint International Research Laboratory of Water and Nutrient in Crops, Fujian Agriculture and Forestry University, Jinshan Fuzhou 350002, China.

The quadruple mutant () seedlings lacking key negative regulators of ABA signaling, namely, clade A protein phosphatases type 2C (PP2Cs), show more apoplastic H efflux in roots and display an enhanced root growth under normal medium or water stress medium compared to the wild type. The presence of low ABA concentration (0.1 micromolar), inhibiting PP2C activity via monomeric ABA receptors, enhances root apoplastic H efflux and growth of the wild type, resembling the phenotype in normal medium. seedlings also demonstrate increased hydrotropism compared to the wild type in obliquely-oriented hydrotropic experimental system, and asymmetric H efflux in root elongation zone is crucial for root hydrotropism. Moreover, we reveal that ABA-insensitive 1, a key PP2C in ABA signaling, interacts directly with the C terminus of plasma membrane H-dependent adenosine triphosphatase 2 (AHA2) and dephosphorylates its penultimate threonine residue (Thr), whose dephosphorylation negatively regulates AHA2.
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http://dx.doi.org/10.1126/sciadv.abd4113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968848PMC
March 2021

PYL8 ABA receptors of Phoenix dactylifera play a crucial role in response to abiotic stress and are stabilized by ABA.

J Exp Bot 2021 Feb;72(2):757-774

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-Universidad Politécnica de Valencia, Valencia, Spain.

The identification of those prevalent abscisic acid (ABA) receptors and molecular mechanisms that trigger drought adaptation in crops well adapted to harsh conditions such as date palm (Phoenix dactylifera, Pd) sheds light on plant-environment interactions. We reveal that PdPYL8-like receptors are predominantly expressed under abiotic stress, with Pd27 being the most expressed receptor in date palm. Therefore, subfamily I PdPYL8-like receptors have been selected for ABA signaling during abiotic stress response in this crop. Biochemical characterization of PdPYL8-like and PdPYL1-like receptors revealed receptor- and ABA-dependent inhibition of PP2Cs, which triggers activation of the pRD29B-LUC reporter in response to ABA. PdPYLs efficiently abolish PP2C-mediated repression of ABA signaling, but loss of the Trp lock in the seed-specific AHG1-like phosphatase PdPP2C79 markedly impairs its inhibition by ABA receptors. Characterization of Arabidopsis transgenic plants that express PdPYLs shows enhanced ABA signaling in seed, root, and guard cells. Specifically, Pd27-overexpressing plants showed lower ABA content and were more efficient than the wild type in lowering transpiration at negative soil water potential, leading to enhanced drought tolerance. Finally, PdPYL8-like receptors accumulate after ABA treatment, which suggests that ABA-induced stabilization of these receptors operates in date palm for efficient boosting of ABA signaling in response to abiotic stress.
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http://dx.doi.org/10.1093/jxb/eraa476DOI Listing
February 2021

A Luciferase Reporter Assay to Identify Chemical Activators of ABA Signaling.

Methods Mol Biol 2021 ;2213:113-121

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas, Universidad Politécnica de Valencia (IBMCP-CSIC-UPV), Valencia, Spain.

Plant stress tolerance relies on intricate signaling networks that are not fully understood. Several plant hormones are involved in the adaptation to different environmental conditions. Abscisic acid (ABA) has an essential role in stress tolerance, especially in the adaptation to drought. During the last years, chemical genomics has gained attention as an alternative approach to decipher complex traits. Additionally, chemical-based strategies have been very useful to untangle genetic redundancy, which is hard to address by other approaches such as classical genetics. Here, we describe the use of an ABA-inducible luciferase (LUC) reporter line for the high-throughput identification of chemical activators of the ABA signaling pathway. In this assay, seven-day-old pMAPKKK18-LUC seedlings are grown on 96-well plates and treated with test compounds. Next, the activity of the LUC reporter is quantified semiautomatically by image analysis. Candidate compounds able to activate the reporter are thus identified and subjected to a secondary screen by analyzing their effect on ABA-related phenotypes (e.g., inhibition of seed germination). This assay is fast, high-throughput, nondestructive, semiquantitative and can be applied to any other luciferase reporter lines, making it ideal for forward chemical genetic screenings.
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http://dx.doi.org/10.1007/978-1-0716-0954-5_10DOI Listing
March 2021

Identification of ABA Receptor Agonists Using a Multiplexed High-Throughput Chemical Screening.

Methods Mol Biol 2021 ;2213:99-111

Innopharma Screening Platform, BioFarma Research group, Centro de Investigación en Medicina Molecular y Enfermedades Crónicas (CiMUS), Universidad de Santiago de Compostela, Santiago de Compostela, Spain.

Small molecules that can activate abscisic acid (ABA) receptors represent valuable probes to study ABA perception and signaling. Additionally, these compounds have the potential to be used in the field to counteract the negative effect of drought stress on plant productivity. The PYR/PYL ABA receptors, in their ligand-bound conformation, inactivate protein phosphatases 2C (PP2Cs), triggering physiological responses that are essential for plant adaptation to environmental stresses, including drought. Based on this ligand-induced PP2C inactivation mechanism, we have developed an in vitro assay for the identification of ABA-receptor agonists by high-throughput screening of chemical libraries. The assay allows simultaneous use of different ABA receptors, increasing the chances to find new agonists and eliminates the need for parallel screening. In this chapter, we describe detailed procedures for the identification of ABA agonists using this multiplexed assay in a medium- (96-well plates) or a high-throughput (384-well plates) setup.
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http://dx.doi.org/10.1007/978-1-0716-0954-5_9DOI Listing
March 2021

A dual function of SnRK2 kinases in the regulation of SnRK1 and plant growth.

Nat Plants 2020 11 19;6(11):1345-1353. Epub 2020 Oct 19.

Instituto Gulbenkian de Ciência, GREEN-IT Bioresources for Sustainability, Oeiras, Portugal.

Adverse environmental conditions trigger responses in plants that promote stress tolerance and survival at the expense of growth. However, little is known of how stress signalling pathways interact with each other and with growth regulatory components to balance growth and stress responses. Here, we show that plant growth is largely regulated by the interplay between the evolutionarily conserved energy-sensing SNF1-related protein kinase 1 (SnRK1) protein kinase and the abscisic acid (ABA) phytohormone pathway. While SnRK2 kinases are main drivers of ABA-triggered stress responses, we uncover an unexpected growth-promoting function of these kinases in the absence of ABA as repressors of SnRK1. Sequestration of SnRK1 by SnRK2-containing complexes inhibits SnRK1 signalling, thereby allowing target of rapamycin (TOR) activity and growth under optimal conditions. On the other hand, these complexes are essential for releasing and activating SnRK1 in response to ABA, leading to the inhibition of TOR and growth under stress. This dual regulation of SnRK1 by SnRK2 kinases couples growth control with environmental factors typical for the terrestrial habitat and is likely to have been critical for the water-to-land transition of plants.
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http://dx.doi.org/10.1038/s41477-020-00778-wDOI Listing
November 2020

PYL8 ABA receptors of Phoenix dactylifera play a crucial role in response to abiotic stress and are stabilized by ABA (118).

J Exp Bot 2020 Oct 16. Epub 2020 Oct 16.

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-Universidad Politécnica de Valencia, Valencia, Spain.

The identification of those prevailing ABA receptors and molecular mechanisms that trigger drought adaptation in crops well adapted to harsh conditions such as date palm (Phoenix dactylifera, Pd) sheds light on plant-environment interactions. We reveal that PdPYL8-like receptors are predominantly expressed under abiotic stress, being Pd27 the most expressed receptor in date palm. Therefore, subfamily I PdPYL8-like receptors have been selected for ABA signaling during abiotic stress response in this crop. Biochemical characterization of PdPYL8-like and PdPYL1-like receptors revealed receptor- and ABA-dependent inhibition of PP2Cs, which triggers activation of the pRD29B-LUC reporter in response to ABA. PdPYLs efficiently abolish PP2C-mediated repression of ABA signaling, but loss of the Trp lock in the seed-specific AHG1-like phosphatase PdPP2C79 markedly impairs its inhibition by ABA receptors. Characterization of Arabidopsis transgenic plants that express PdPYLs shows enhanced ABA signaling in seed, root and guard cells. Specifically, Pd27 overexpressing (OE) plants showed lower ABA content and were more efficient than wild type to lower transpiration at negative soil water potential, leading to enhanced drought tolerance. Finally, PdPYL8-like receptors accumulate after ABA treatment, which suggests that ABA-induced stabilization of these receptors operates in date palm for efficient boosting of ABA signaling in response to abiotic stress.
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http://dx.doi.org/10.1093/jxb/eraa476DOI Listing
October 2020

Plant Osmotic Stress Signaling: MAPKKKs Meet SnRK2s.

Trends Plant Sci 2020 12 21;25(12):1179-1182. Epub 2020 Sep 21.

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas - Universidad Politécnica de Valencia, 46022, Valencia, Spain. Electronic address:

Osmotic stress signaling in higher plants is crucial to cope with abiotic stress. RAF-like MAPKKKs are activated by hyperosmotic stress and activate downstream ABA-unresponsive and ABA-activated SnRK2s, integrating early osmotic stress and ABA signaling cascades. The connection of B2/B3/B4 RAF-like MAPKKKs with SnRK2s is a new paradigm in signal transduction.
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http://dx.doi.org/10.1016/j.tplants.2020.09.003DOI Listing
December 2020

The Role of ABA in Plant Immunity is Mediated through the PYR1 Receptor.

Int J Mol Sci 2020 Aug 14;21(16). Epub 2020 Aug 14.

Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-C.S.I.C, Ciudad Politécnica de la Innovación, Edificio 8E, 46000 Valencia, Spain.

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, -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.
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http://dx.doi.org/10.3390/ijms21165852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461614PMC
August 2020

Drug Discovery for Thirsty Crops.

Trends Plant Sci 2020 09 18;25(9):844-846. Epub 2020 Jul 18.

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas - Universidad Politécnica de Valencia, 46022, Valencia, Spain.

Following virtual screening and structure-based ligand optimization, researchers have developed opabactin (OP), an abscisic acid (ABA)-receptor agonist with tenfold greater in vivo activity than ABA. This new ligand surpasses previous agonists for its potency and bioactivity on staple crops. OP leads a new class of agrochemicals designed to protect crops from drought.
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http://dx.doi.org/10.1016/j.tplants.2020.07.001DOI Listing
September 2020

Degradation of Abscisic Acid Receptors Through the Endosomal Pathway.

Methods Mol Biol 2020 ;2177:35-48

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-Universidad Politécnica de Valencia, Valencia, Spain.

Turnover of membrane proteins or soluble proteins associated to plasma membrane involves clathrin-mediated endocytosis (CME), endosomal trafficking, and vacuolar degradation. Thus, endocytic and endosomal trafficking regulate numerous physiological processes, including mineral transport, hormone signaling, and pathogen response. Abscisic acid (ABA) signaling is triggered upon ABA perception by PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR), which are soluble proteins that can associate to membrane by interaction with members of the C2-domain ABA-related (CAR) protein family and the RING finger of seed longevity (RSL1) E3 ubiquitin ligase. Half-life of PYR/PYL/RCAR ABA receptors is regulated by ubiquitination and degradation in different subcellular compartments. In particular, pharmacological, genetic, and cell biology approaches have been used to study the different steps that encompass from CME to receptor degradation in the vacuole. In this chapter, we will focus on (1) coimmunoprecipitation (co-IP) assays of clathrin heavy chain (CHC) subunits together with HA-tagged PYL4 ABA receptor and (2) analysis of PYL4 delivery to the vacuole using the TMD23-Ub marker.
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http://dx.doi.org/10.1007/978-1-0716-0767-1_4DOI Listing
March 2021

Evaluation of Contamination Risk by the cobas e 602 Serology Module Before Viral Load Testing on the cobas 6800 System.

Sex Transm Dis 2020 05;47(5S Suppl 1):S32-S34

Medical and Scientific Affairs, Roche Molecular Systems, Pleasanton, CA.

Background: Diagnosis of HCV, HBV, and HIV involves antibody screening followed by confirmation and/or treatment decision using nucleic acid tests. However, minimal data exist evaluating the risk of nucleic acid cross-contamination on serology devices upstream of molecular testing despite the potential clinical and laboratory workflow advantages of single specimen vial testing for both procedures.

Methods: We conducted a checkerboard study investigating the potential risk of HCV, HBV, and HIV nucleic acid cross-contamination on 480 negative specimens by a serology screening instrument that uses disposable tips for sample transfer, rather than a fixed needle, before molecular testing.

Results: Nucleic acid contamination was observed in 0 of 480 negative specimens when processed with alternating high-titer HCV, HBV, or HIV specimens on the serology platform.

Conclusions: This study suggests that specimens analyzed by a serology instrument using disposable tips for sample transfer may be suitable for direct primary specimen reflex testing by a sensitive nucleic acid confirmatory test.
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http://dx.doi.org/10.1097/OLQ.0000000000001125DOI Listing
May 2020

RBR-Type E3 Ligases and the Ubiquitin-Conjugating Enzyme UBC26 Regulate Abscisic Acid Receptor Levels and Signaling.

Plant Physiol 2020 04 7;182(4):1723-1742. Epub 2019 Nov 7.

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-Uiversidad Politécnica de Valencia, 46022 Valencia, Spain

The turnover of abscisic acid (ABA) signaling core components modulates the plant's response to ABA and is regulated by ubiquitination. We show that Arabidopsis () RING Finger ABA-Related1 (RFA1) and RFA4 E3 ubiquitin ligases, members of the RING between RING fingers (RBR)-type RSL1/RFA family, are key regulators of ABA receptor stability in root and leaf tissues, targeting ABA receptors for degradation in different subcellular locations. RFA1 is localized both in the nucleus and cytosol, whereas RFA4 shows specific nuclear localization and promotes nuclear degradation of ABA receptors. Therefore, members of the RSL1/RFA family interact with ABA receptors at plasma membrane, cytosol, and nucleus, targeting them for degradation via the endosomal/vacuolar RSL1-dependent pathway or 26S proteasome. Additionally, we provide insight into the physiological function of the relatively unexplored plant RBR-type E3 ligases, and through mutagenesis and biochemical assays we identified cysteine-361 in RFA4 as the putative active site cysteine, which is a distinctive feature of RBR-type E3 ligases. Endogenous levels of PYR1 and PYL4 ABA receptors were higher in the double mutant than in wild-type plants. UBC26 was identified as the cognate nuclear E2 enzyme that interacts with the RFA4 E3 ligase and forms UBC26-RFA4-receptor complexes in nuclear speckles. Loss-of-function alleles and the double mutant showed enhanced sensitivity to ABA and accumulation of ABA receptors compared with the wild type. Together, our results reveal a sophisticated mechanism by which ABA receptors are targeted by ubiquitin at different subcellular locations, in which the complexity of the ABA receptor family is mirrored in the partner RBR-type E3 ligases.
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http://dx.doi.org/10.1104/pp.19.00898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140949PMC
April 2020

The role of Arabidopsis ABA receptors from the PYR/PYL/RCAR family in stomatal acclimation and closure signal integration.

Nat Plants 2019 09 26;5(9):1002-1011. Epub 2019 Aug 26.

Institute for Molecular Plant Physiology and Biophysics, Biocenter, University Würzburg, Würzburg, Germany.

Stomata are microscopic pores found on the surfaces of leaves that act to control CO uptake and water loss. By integrating information derived from endogenous signals with cues from the surrounding environment, the guard cells, which surround the pore, 'set' the stomatal aperture to suit the prevailing conditions. Much research has concentrated on understanding the rapid intracellular changes that result in immediate changes to the stomatal aperture. In this study, we look instead at how stomata acclimate to longer timescale variations in their environment. We show that the closure-inducing signals abscisic acid (ABA), increased CO, decreased relative air humidity and darkness each access a unique gene network made up of clusters (or modules) of common cellular processes. However, within these networks some gene clusters are shared amongst all four stimuli. All stimuli modulate the expression of members of the PYR/PYL/RCAR family of ABA receptors. However, they are modulated differentially in a stimulus-specific manner. Of the six members of the PYR/PYL/RCAR family expressed in guard cells, PYL2 is sufficient for guard cell ABA-induced responses, whereas in the responses to CO, PYL4 and PYL5 are essential. Overall, our work shows the importance of ABA as a central regulator and integrator of long-term changes in stomatal behaviour, including sensitivity, elicited by external signals. Understanding this architecture may aid in breeding crops with improved water and nutrient efficiency.
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http://dx.doi.org/10.1038/s41477-019-0490-0DOI Listing
September 2019

Arabidopsis ALIX Regulates Stomatal Aperture and Turnover of Abscisic Acid Receptors.

Plant Cell 2019 10 30;31(10):2411-2429. Epub 2019 Jul 30.

Centro Nacional de Biotecnología, 28049 Madrid, Spain

The plant endosomal trafficking pathway controls the abundance of membrane-associated soluble proteins, as shown for abscisic acid (ABA) receptors of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) family. ABA receptor targeting for vacuolar degradation occurs through the late endosome route and depends on FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FYVE1) and VACUOLAR PROTEIN SORTING23A (VPS23A), components of the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT-I (ESCRT-I) complexes. FYVE1 and VPS23A interact with ALG-2 INTERACTING PROTEIN-X (ALIX), an ESCRT-III-associated protein, although the functional relevance of such interactions and their consequences in cargo sorting are unknown. In this study we show that Arabidopsis () ALIX directly binds to ABA receptors in late endosomes, promoting their degradation. Impaired ALIX function leads to altered endosomal localization and increased accumulation of ABA receptors. In line with this activity, partial loss-of-function mutants display ABA hypersensitivity during growth and stomatal closure, unveiling a role for the ESCRT machinery in the control of water loss through stomata. ABA-hypersensitive responses are suppressed in plants impaired in PYR/PYL/RCAR activity, in accordance with ALIX affecting ABA responses primarily by controlling ABA receptor stability. ALIX-1 mutant protein displays reduced interaction with VPS23A and ABA receptors, providing a molecular basis for ABA hypersensitivity in mutants. Our findings unveil a negative feedback mechanism triggered by ABA that acts via ALIX to control the accumulation of specific PYR/PYL/RCAR receptors.
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http://dx.doi.org/10.1105/tpc.19.00399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790096PMC
October 2019

The MATH-BTB BPM3 and BPM5 subunits of Cullin3-RING E3 ubiquitin ligases target PP2CA and other clade A PP2Cs for degradation.

Proc Natl Acad Sci U S A 2019 07 15;116(31):15725-15734. Epub 2019 Jul 15.

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientificas-Universidad Politecnica de Valencia, ES-46022 Valencia, Spain;

Early abscisic acid signaling involves degradation of clade A protein phosphatases type 2C (PP2Cs) as a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of PP2C activity. At later steps, ABA induces up-regulation of transcripts and protein levels as a negative feedback mechanism. Therefore, resetting of ABA signaling also requires PP2C degradation to avoid excessive ABA-induced accumulation of PP2Cs. It has been demonstrated that ABA induces the degradation of existing ABI1 and PP2CA through the PUB12/13 and RGLG1/5 E3 ligases, respectively. However, other unidentified E3 ligases are predicted to regulate protein stability of clade A PP2Cs as well. In this work, we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the multimeric cullin3 (CUL3)-RING-based E3 ligases (CRL3s), as PP2CA-interacting proteins. BPM3 and BPM5 interact in the nucleus with PP2CA as well as with ABI1, ABI2, and HAB1. BPM3 and BPM5 accelerate the turnover of PP2Cs in an ABA-dependent manner and their overexpression leads to enhanced ABA sensitivity, whereas plants show increased accumulation of PP2CA, ABI1 and HAB1, which leads to global diminished ABA sensitivity. Using biochemical and genetic assays, we demonstrated that ubiquitination of PP2CA depends on BPM function. Given the formation of receptor-ABA-phosphatase ternary complexes is markedly affected by the abundance of protein components and ABA concentration, we reveal that BPMs and multimeric CRL3 E3 ligases are important modulators of PP2C coreceptor levels to regulate early ABA signaling as well as the later desensitizing-resetting steps.
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http://dx.doi.org/10.1073/pnas.1908677116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681733PMC
July 2019

The fungal sesquiterpenoid pyrenophoric acid B uses the plant ABA biosynthetic pathway to inhibit seed germination.

J Exp Bot 2019 10;70(19):5487-5494

Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte Sant'Angelo, Napoli, Italy.

Pyrenophoric acid (P-Acid), P-Acid B, and P-Acid C are three phytotoxic sesquiterpenoids produced by the ascomycete seed pathogen Pyrenophora semeniperda, a fungus proposed as a mycoherbicide for biocontrol of cheatgrass, an extremely invasive weed. When tested in cheatgrass bioassays, these metabolites were able to delay seed germination, with P-Acid B being the most active compound. Here, we have investigated the cross-kingdom activity of P-Acid B and its mode of action, and found that it activates the abscisic acid (ABA) signaling pathway in order to inhibit seedling establishment. P-Acid B inhibits seedling establishment in wild-type Arabidopsis thaliana, while several mutants affected in the early perception as well as in downstream ABA signaling components were insensitive to the fungal compound. However, in spite of structural similarities between ABA and P-Acid B, the latter is not able to activate the PYR/PYL family of ABA receptors. Instead, we have found that P-Acid B uses the ABA biosynthesis pathway at the level of alcohol dehydrogenase ABA2 to reduce seedling establishment. We propose that the fungus P. semeniperda manipulates plant ABA biosynthesis as a strategy to reduce seed germination, increasing its ability to cause seed mortality and thereby increase its fitness through higher reproductive success.
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http://dx.doi.org/10.1093/jxb/erz306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6793445PMC
October 2019

The plant ESCRT component FREE1 shuttles to the nucleus to attenuate abscisic acid signalling.

Nat Plants 2019 05 8;5(5):512-524. Epub 2019 Apr 8.

Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University (SCNU), Guangzhou, China.

The endosomal sorting complex required for transport (ESCRT) machinery has been well documented for its function in endosomal sorting in eukaryotes. Here, we demonstrate an up-to-now unknown and non-endosomal function of the ESCRT component in plants. We show that FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body biogenesis, plays additional functions in the nucleus in transcriptional inhibition of abscisic acid (ABA) signalling. Following ABA treatment, SNF1-related protein kinase 2 (SnRK2) kinases phosphorylate FREE1, a step requisite for ABA-induced FREE1 nuclear import. In the nucleus, FREE1 interacts with the basic leucine zipper transcription factors ABA-RESPONSIVE ELEMENTS BINDING FACTOR4 and ABA-INSENSITIVE5 to reduce their binding to the cis-regulatory sequences of downstream genes. Collectively, our study demonstrates the crosstalk between endomembrane trafficking and ABA signalling at the transcriptional level and highlights the moonlighting properties of the plant ESCRT subunit FREE1, which has evolved unique non-endosomal functions in the nucleus besides its roles in membrane trafficking in the cytoplasm.
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http://dx.doi.org/10.1038/s41477-019-0400-5DOI Listing
May 2019

ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA.

Plant J 2019 06 12;98(5):813-825. Epub 2019 Mar 12.

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientificas-Universidad Politecnica de Valencia, ES-46022, Valencia, Spain.

Hormone- and stress-induced shuttling of signaling or regulatory proteins is an important cellular mechanism to modulate hormone signaling and cope with abiotic stress. Hormone-induced ubiquitination plays a crucial role to determine the half-life of key negative regulators of hormone signaling. For ABA signaling, the degradation of clade-A PP2Cs, such as PP2CA or ABI1, is a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of PP2C activity. ABA promotes the degradation of PP2CA through the RGLG1 E3 ligase, although it is not known how ABA enhances the interaction of RGLG1 with PP2CA given that they are predominantly found in the plasma membrane and the nucleus, respectively. We demonstrate that ABA modifies the subcellular localization of RGLG1 and promotes nuclear interaction with PP2CA. We found RGLG1 is myristoylated in vivo, which facilitates its attachment to the plasma membrane. ABA inhibits the myristoylation of RGLG1 through the downregulation of N-myristoyltransferase 1 (NMT1) and promotes nuclear translocation of RGLG1 in a cycloheximide-insensitive manner. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP2CA protein levels and the formation of RGLG1-receptor-phosphatase complexes. We show that RGLG1 mutated at the N-terminal myristoylation site shows constitutive nuclear localization and causes an enhanced response to ABA and salt or osmotic stress. RGLG1/5 can interact with certain monomeric ABA receptors, which facilitates the formation of nuclear complexes such as RGLG1-PP2CA-PYL8. In summary, we provide evidence that an E3 ligase can dynamically relocalize in response to both ABA and increased levels of its target, which reveals a mechanism to explain how ABA enhances RGLG1-PP2CA interaction and hence PP2CA degradation.
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http://dx.doi.org/10.1111/tpj.14274DOI Listing
June 2019

Wounding-Induced Stomatal Closure Requires Jasmonate-Mediated Activation of GORK K Channels by a Ca Sensor-Kinase CBL1-CIPK5 Complex.

Dev Cell 2019 01 6;48(1):87-99.e6. Epub 2018 Dec 6.

Molekulare Pflanzenphysiologie & Biophysik, Julius-Maximilians-Universität Würzburg, Julius-von-Sachs-Platz 2, Würzburg 97082, Germany. Electronic address:

Guard cells integrate various hormone signals and environmental cues to balance plant gas exchange and transpiration. The wounding-associated hormone jasmonic acid (JA) and the drought hormone abscisic acid (ABA) both trigger stomatal closure. In contrast to ABA however, the molecular mechanisms of JA-induced stomatal closure have remained largely elusive. Here, we identify a fast signaling pathway for JA targeting the K efflux channel GORK. Wounding triggers both local and systemic stomatal closure by activation of the JA signaling cascade followed by GORK phosphorylation and activation through CBL1-CIPK5 Ca sensor-kinase complexes. GORK activation strictly depends on plasma membrane targeting and Ca binding of CBL1-CIPK5 complexes. Accordingly, in gork, cbl1, and cipk5 mutants, JA-induced stomatal closure is specifically abolished. The ABA-coreceptor ABI2 counteracts CBL1-CIPK5-dependent GORK activation. Hence, JA-induced Ca signaling in response to biotic stress converges with the ABA-mediated drought stress pathway to facilitate GORK-mediated stomatal closure upon wounding.
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http://dx.doi.org/10.1016/j.devcel.2018.11.014DOI Listing
January 2019

PYL8 mediates ABA perception in the root through non-cell-autonomous and ligand-stabilization-based mechanisms.

Proc Natl Acad Sci U S A 2018 12 27;115(50):E11857-E11863. Epub 2018 Nov 27.

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-Universidad Politécnica de Valencia, ES-46022 Valencia, Spain;

The phytohormone abscisic acid (ABA) plays a key role regulating root growth, root system architecture, and root adaptive responses, such as hydrotropism. The molecular and cellular mechanisms that regulate the action of core ABA signaling components in roots are not fully understood. ABA is perceived through receptors from the PYR/PYL/RCAR family and PP2C coreceptors. PYL8/RCAR3 plays a nonredundant role in regulating primary and lateral root growth. Here we demonstrate that ABA specifically stabilizes PYL8 compared with other ABA receptors and induces accumulation of PYL8 in root nuclei. This requires ABA perception by PYL8 and leads to diminished ubiquitination of PYL8 in roots. The ABA agonist quinabactin, which promotes root ABA signaling through dimeric receptors, fails to stabilize the monomeric receptor PYL8. Moreover, a PYL8 mutant unable to bind ABA and inhibit PP2C is not stabilized by the ligand, whereas a PYL8 mutant is more stable than PYL8 at endogenous ABA concentrations. The PYL8 transcript was detected in the epidermis and stele of the root meristem; however, the PYL8 protein was also detected in adjacent tissues. Expression of PYL8 driven by tissue-specific promoters revealed movement to adjacent tissues. Hence both inter- and intracellular trafficking of PYL8 appears to occur in the root apical meristem. Our findings reveal a non-cell-autonomous mechanism for hormone receptors and help explain the nonredundant role of PYL8-mediated root ABA signaling.
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http://dx.doi.org/10.1073/pnas.1815410115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294950PMC
December 2018

The Xerobranching Response Represses Lateral Root Formation When Roots Are Not in Contact with Water.

Curr Biol 2018 10 27;28(19):3165-3173.e5. Epub 2018 Sep 27.

Earth and Life Institute, Université catholique de Louvain, 1348 Louvain-la-Neuve, Belgium. Electronic address:

Efficient soil exploration by roots represents an important target for crop improvement and food security [1, 2]. Lateral root (LR) formation is a key trait for optimizing soil foraging for crucial resources such as water and nutrients. Here, we report an adaptive response termed xerobranching, exhibited by cereal roots, that represses branching when root tips are not in contact with wet soil. Non-invasive X-ray microCT imaging revealed that cereal roots rapidly repress LR formation as they enter an air space within a soil profile and are no longer in contact with water. Transcript profiling of cereal root tips revealed that transient water deficit triggers the abscisic acid (ABA) response pathway. In agreement with this, exogenous ABA treatment can mimic repression of LR formation under transient water deficit. Genetic analysis in Arabidopsis revealed that ABA repression of LR formation requires the PYR/PYL/RCAR-dependent signaling pathway. Our findings suggest that ABA acts as the key signal regulating xerobranching. We conclude that this new ABA-dependent adaptive mechanism allows roots to rapidly respond to changes in water availability in their local micro-environment and to use internal resources efficiently.
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http://dx.doi.org/10.1016/j.cub.2018.07.074DOI Listing
October 2018

The Cys-Arg/N-End Rule Pathway Is a General Sensor of Abiotic Stress in Flowering Plants.

Curr Biol 2017 Oct 12;27(20):3183-3190.e4. Epub 2017 Oct 12.

School of Biosciences, University of Nottingham, Loughborough LE12 5RD, UK. Electronic address:

Abiotic stresses impact negatively on plant growth, profoundly affecting yield and quality of crops. Although much is known about plant responses, very little is understood at the molecular level about the initial sensing of environmental stress. In plants, hypoxia (low oxygen, which occurs during flooding) is directly sensed by the Cys-Arg/N-end rule pathway of ubiquitin-mediated proteolysis, through oxygen-dependent degradation of group VII Ethylene Response Factor transcription factors (ERFVIIs) via amino-terminal (Nt-) cysteine [1, 2]. Using Arabidopsis (Arabidopsis thaliana) and barley (Hordeum vulgare), we show that the pathway regulates plant responses to multiple abiotic stresses. In Arabidopsis, genetic analyses revealed that response to these stresses is controlled by N-end rule regulation of ERFVII function. Oxygen sensing via the Cys-Arg/N-end rule in higher eukaryotes is linked through a single mechanism to nitric oxide (NO) sensing [3, 4]. In plants, the major mechanism of NO synthesis is via NITRATE REDUCTASE (NR), an enzyme of nitrogen assimilation [5]. Here, we identify a negative relationship between NR activity and NO levels and stabilization of an artificial Nt-Cys substrate and ERFVII function in response to environmental changes. Furthermore, we show that ERFVIIs enhance abiotic stress responses via physical and genetic interactions with the chromatin-remodeling ATPase BRAHMA. We propose that plants sense multiple abiotic stresses through the Cys-Arg/N-end rule pathway either directly (via oxygen sensing) or indirectly (via NO sensing downstream of NR activity). This single mechanism can therefore integrate environment and response to enhance plant survival.
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http://dx.doi.org/10.1016/j.cub.2017.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668231PMC
October 2017

Structure of Ligand-Bound Intermediates of Crop ABA Receptors Highlights PP2C as Necessary ABA Co-receptor.

Mol Plant 2017 09 20;10(9):1250-1253. Epub 2017 Jul 20.

Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas, 28006 Madrid, Spain. Electronic address:

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http://dx.doi.org/10.1016/j.molp.2017.07.004DOI Listing
September 2017

Root hydrotropism is controlled via a cortex-specific growth mechanism.

Nat Plants 2017 May 8;3:17057. Epub 2017 May 8.

Department of Plant Biotechnology and Bioinformatics, Ghent University, (Technologiepark 927), 9052 Ghent, Belgium.

Plants can acclimate by using tropisms to link the direction of growth to environmental conditions. Hydrotropism allows roots to forage for water, a process known to depend on abscisic acid (ABA) but whose molecular and cellular basis remains unclear. Here we show that hydrotropism still occurs in roots after laser ablation removed the meristem and root cap. Additionally, targeted expression studies reveal that hydrotropism depends on the ABA signalling kinase SnRK2.2 and the hydrotropism-specific MIZ1, both acting specifically in elongation zone cortical cells. Conversely, hydrotropism, but not gravitropism, is inhibited by preventing differential cell-length increases in the cortex, but not in other cell types. We conclude that root tropic responses to gravity and water are driven by distinct tissue-based mechanisms. In addition, unlike its role in root gravitropism, the elongation zone performs a dual function during a hydrotropic response, both sensing a water potential gradient and subsequently undergoing differential growth.
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http://dx.doi.org/10.1038/nplants.2017.57DOI Listing
May 2017

Depletion of abscisic acid levels in roots of flooded Carrizo citrange (Poncirus trifoliata L. Raf. × Citrus sinensis L. Osb.) plants is a stress-specific response associated to the differential expression of PYR/PYL/RCAR receptors.

Plant Mol Biol 2017 Apr 3;93(6):623-640. Epub 2017 Feb 3.

Ecofisiologia i Biotecnologia Department Ciències Agràries i del Medi Natural, Universitat Jaume I, 12071, Castelló de la Plana, Spain.

Soil flooding reduces root abscisic acid (ABA) levels in citrus, conversely to what happens under drought. Despite this reduction, microarray analyses suggested the existence of a residual ABA signaling in roots of flooded Carrizo citrange seedlings. The comparison of ABA metabolism and signaling in roots of flooded and water stressed plants of Carrizo citrange revealed that the hormone depletion was linked to the upregulation of CsAOG, involved in ABA glycosyl ester (ABAGE) synthesis, and to a moderate induction of catabolism (CsCYP707A, an ABA 8'-hydroxylase) and buildup of dehydrophaseic acid (DPA). Drought strongly induced both ABA biosynthesis and catabolism (CsNCED1, 9-cis-neoxanthin epoxycarotenoid dioxygenase 1, and CsCYP707A) rendering a significant hormone accumulation. In roots of flooded plants, restoration of control ABA levels after stress release was associated to the upregulation of CsBGLU18 (an ABA β-glycosidase) that cleaves ABAGE. Transcriptional profile of ABA receptor genes revealed a different induction in response to soil flooding (CsPYL5) or drought (CsPYL8). These two receptor genes along with CsPYL1 were cloned and expressed in a heterologous system. Recombinant CsPYL5 inhibited ΔNHAB1 activity in vitro at lower ABA concentrations than CsPYL8 or CsPYL1, suggesting its better performance under soil flooding conditions. Both stress conditions induced ABA-responsive genes CsABI5 and CsDREB2A similarly, suggesting the occurrence of ABA signaling in roots of flooded citrus seedlings. The impact of reduced ABA levels in flooded roots on CsPYL5 expression along with its higher hormone affinity reinforce the role of this ABA receptor under soil-flooding conditions and explain the expression of certain ABA-responsive genes.
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http://dx.doi.org/10.1007/s11103-017-0587-7DOI Listing
April 2017

Protein phosphatase type 2C PP2CA together with ABI1 inhibits SnRK2.4 activity and regulates plant responses to salinity.

Plant Signal Behav 2016 12;11(12):e1253647

a Institute of Biochemistry and Biophysics, Polish Academy of Sciences , Warsaw , Poland.

Protein phosphatases 2C (PP2Cs) are important regulators of plant responses to abiotic stress. It is established that clade A PP2Cs inhibit ABA-activated SNF1-related protein kinases 2 (SnRK2s). Our recently published results show that ABI1, a member of clade A of PP2C is also a negative regulator of SnRK2.4, a kinase not activated in response to ABA. Here, we show that another member of this clade - PP2CA, interacts with and inhibits SnRK2.4. The salt-induced SnRK2.4/SnRK2.10 activity is higher in abi1-2 pp2ca-1 mutant than in wild type or single abi1 or pp2ca mutants, indicating that both phosphatases are inhibitors of SnRK2.4 and are at least partially redundant. Moreover, PP2CA together with ABI1 and SnRK2.4 regulates root growth in response to salinity.
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http://dx.doi.org/10.1080/15592324.2016.1253647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225939PMC
December 2016

ESCRT-I Component VPS23A Affects ABA Signaling by Recognizing ABA Receptors for Endosomal Degradation.

Mol Plant 2016 12 14;9(12):1570-1582. Epub 2016 Nov 14.

State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, P. R. China. Electronic address:

Recent discovery of PYR/PYL/RCAR-type abscisic acid (ABA) receptors has become one of most significant advances in plant science in the past decade. In mammals, endosomal sorting acts as an important pathway to downregulate different types of receptors, but its role in plant hormone signaling is poorly understood. Here, we report that an ubiquitin E2-like protein, VPS23A, which is a key component of ESCRT-I, negatively regulates ABA signaling. VPS23A has epistatic relationship with PYR/PYL/RCAR-type ABA receptors and disruption of VPS23A enhanced the activity of key kinase OST1 in the ABA signaling pathway under ABA treatment. Moreover, VPS23A interacts with PYR1/PYLs and K63-linked diubiquitin, and PYL4 possesses K63-linked ubiquitinated modification in vivo. Further analysis revealed that VPS23A affects the subcellular localization of PYR1 and the stability of PYL4. Taken together, our results suggest that VPS23A affects PYR1/PYL4 via vacuole-mediated degradation, providing an advanced understanding of both the turnover of ABA receptors and ESCRTs in plant hormone signaling.
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http://dx.doi.org/10.1016/j.molp.2016.11.002DOI Listing
December 2016

Abscisic Acid Catabolism Generates Phaseic Acid, a Molecule Able to Activate a Subset of ABA Receptors.

Mol Plant 2016 11 29;9(11):1448-1450. Epub 2016 Sep 29.

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientificas-Universidad Politecnica de Valencia, 46022 Valencia, Spain. Electronic address:

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http://dx.doi.org/10.1016/j.molp.2016.09.009DOI Listing
November 2016

Pre-mRNA splicing repression triggers abiotic stress signaling in plants.

Plant J 2017 01 17;89(2):291-309. Epub 2017 Jan 17.

Laboratory for Genome Engineering, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Saudi Arabia.

Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress- and ABA-responsive reporters RD29A::LUC and MAPKKK18::uidA in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2C phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling and post-transcriptional regulation in plants.
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http://dx.doi.org/10.1111/tpj.13383DOI Listing
January 2017

Ubiquitin Ligases RGLG1 and RGLG5 Regulate Abscisic Acid Signaling by Controlling the Turnover of Phosphatase PP2CA.

Plant Cell 2016 Sep 30;28(9):2178-2196. Epub 2016 Aug 30.

The State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, School of Agriculture Science, Peking University, Beijing 100871, P.R. China

Abscisic acid (ABA) is an essential hormone for plant development and stress responses. ABA signaling is suppressed by clade A PP2C phosphatases, which function as key repressors of this pathway through inhibiting ABA-activated SnRK2s (SNF1-related protein kinases). Upon ABA perception, the PYR/PYL/RCAR ABA receptors bind to PP2Cs with high affinity and biochemically inhibit their activity. While this mechanism has been extensively studied, how PP2Cs are regulated at the protein level is only starting to be explored. RING DOMAIN LIGASE5 (RGLG5) belongs to a five-member E3 ubiquitin ligase family whose target proteins remain unknown. We report that RGLG5, together with RGLG1, releases the PP2C blockade of ABA signaling by mediating PP2CA protein degradation. ABA promotes the interaction of PP2CA with both E3 ligases, which mediate ubiquitination of PP2CA and are required for ABA-dependent PP2CA turnover. Downregulation of and stabilizes endogenous PP2CA and diminishes ABA-mediated responses. Moreover, the reduced response to ABA in germination assays is suppressed in the (artificial microRNA) triple mutant, supporting a functional link among these loci. Overall, our data indicate that RGLG1 and RGLG5 are important modulators of ABA signaling, and they unveil a mechanism for activation of the ABA pathway by controlling PP2C half-life.
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http://dx.doi.org/10.1105/tpc.16.00364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059804PMC
September 2016