Abscisic acid catabolism generates phaseic acid, a molecule able to activate a subset of ABA receptors

Rodríguez Egea, PL. (2016). Abscisic acid catabolism generates phaseic acid, a molecule able to activate a subset of ABA receptors. Molecular Plant. 9(11):1448-1450. doi:10.1016/j.molp.2016.09.009S1448145091

Biotecnología agrícola para mejorar la tolerancia a sequía y salinidad

Rodríguez Egea, PL.; Pardo Muñoz, JM. (2016). Biotecnología agrícola para mejorar la tolerancia a sequía y salinidad. SEBBM. Revista de la Sociedad Española de Bioquímica y Biología Molecular. 188:21-24. http://hdl.handle.net/10251/98795S212418

News on ABA transport, protein degradation and ABFs/WRKYs in ABA signalling

[EN] 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 (TFs) 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 Educacion y Ciencia and Fondo Europeo de Desarrollo Regional grant BIO2008-00221 to P.L.R. Critical reading by Dr. M. Holdsworth is acknowledged.Antoni, R.; Rodríguez, L.; González Guzmán, M.; Pizzio Bianchi, GA.; Rodríguez Egea, PL. (2011). News on ABA transport, protein degradation and ABFs/WRKYs in ABA signalling. Current Opinion in Plant Biology. 14(5):547-553. https://doi.org/10.1016/j.pbi.2011.06.004S54755314

Selective Inhibition of Clade A Phosphatases Type 2C by PYR/PYL/RCAR Abscisic Acid Receptors

[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.).Antoni-Alandes, R.; González Guzmán, M.; Rodriguez, L.; Rodrigues, A.; Pizzio Bianchi, GA.; Rodríguez Egea, PL. (2012). Selective Inhibition of Clade A Phosphatases Type 2C by PYR/PYL/RCAR Abscisic Acid Receptors. Plant Physiology. 158(2):970-980. https://doi.org/10.1104/pp.111.188623S970980158

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

[EN] 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.This work was supported by National Science Center (grant 2011/03/B/NZ3/00297 to GD). Funding in the laboratory of Pedro L. Rodriguez was provided by grant BIO2014-52537-R.Krzywinska, E.; Kulik, A.; Bucholc, M.; Antolín Fernández, M.; Rodríguez Egea, PL.; Dobrowolska, G. (2016). Protein phosphatase type 2C PP2CA together with ABI1 inhibits SnRK2.4 activity and regulates plant responses to salinity. Plant Signaling and Behavior. 11(12). https://doi.org/10.1080/15592324.2016.1253647S111

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

[EN] 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 thismechanismhas been extensively studied, how PP2Cs are regulated at the protein level is only starting to be explored. Arabidopsis thaliana 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 RGLG1 and RGLG5 stabilizes endogenous PP2CA and diminishes ABA-mediated responses. Moreover, the reduced response to ABA in germination assays is suppressed in the rglg1 amiR (artificial microRNA)-rglg5 pp2ca-1 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 amechanismfor activation of the ABA pathway by controlling PP2C half-life.We thank Andreas Bachmair for the rglg1 mutant, Sean R. Cutler for the pyr1 pyl1 pyl2 pyl4 seeds, Dapeng Zhang for the transgenic material harboring ABI2, Hongwei Guo and Jianmin Zhou for the pCAMBIA1300-Nluc and pCAMBIA1300-Cluc vectors, and John Olson for assistance in English editing. Work in C.A.'s laboratory was supported by grants from the National Key Basic Science "973" Program (Grant 2012CB114006), the National Natural Science Foundation (Grants 31272023, 31170231, and 90817001) of the Chinese government, and by the State Key Laboratory of Protein and Plant Gene Research, Peking University. Work in P.L.R.'s laboratory was supported by Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Cientificas (Grant BIO2014-52537-R).Wu, Q.; Zhang, X.; Peirats-Llobet, M.; Belda Palazón, B.; Wang, X.; Cui, S.; Yu, X.... (2016). Ubiquitin Ligases RGLG1 and RGLG5 Regulate Abscisic Acid Signaling by Controlling the Turnover of Phosphatase PP2CA. Plant Cell. 28(9):2178-2196. https://doi.org/10.1105/tpc.16.003642178219628

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

[EN] The hab1-1abi1-2abi2-2pp2ca-1 quadruple mutant (Qabi2-2) 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 Qabi2-2 phenotype in normal medium. Qabi2-2 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 Arabidopsis ABA-insensitive 1, a key PP2C in ABA signaling, interacts directly with the C terminus of Arabidopsis plasma membrane H+-dependent adenosine triphosphatase 2 (AHA2) and dephosphorylates its penultimate threonine residue (Thr(947)), whose dephosphorylation negatively regulates AHA2.We are grateful for grant support from the National Key R&D Program of China (2017YFE0118100 and 2018YFD02003025), National Natural Science Foundation of China (nos. 31600209, 31761130073, and 31872169), a Newton Advanced Fellowship (NSFC-RS: NA160430), Fujian Province Education Department Funding (JK2017015), and Research Grant of FAFU (KXGH17005). Work in the laboratory of P.L.R. was supported by the Ministerio de Ciencia, Innovacion y Universidades (MICIU), grant BIO2017-82503-R.Miao, R.; Yuan, W.; Wang, Y.; Garcia-Maquilon, I.; Dang, X.; Li, Y.; Zhang, J.... (2021). Low ABA concentration promotes root growth and hydrotropism through relief of ABA INSENSITIVE 1-mediated inhibition of plasma membrane H+-ATPase 2. Science Advances. 7(12):1-14. https://doi.org/10.1126/sciadv.abd411311471

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

[EN] 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 half-life of key negative regulators of hormone signaling. For ABA signaling, 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 they are predominantly found in plasma membrane and 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 plasma membrane. ABA inhibits myristoylation of RGLG1 through downregulation of Nmyristoyltransferase1 (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 RGLG1Gly2Ala -mutated in the Nterminal myristoylation site- shows constitutive nuclear localization and causes enhanced response to ABA and salt/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 re-localize 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.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 through grants BIO2014-52537-R and BIO2017-82503-R. This work was also funded by grants from the Deutsche Forschungsgemeinschaft (DFG) Ku931/4-1 to J. K., and BA4742/1-2 to O.B. B.B. was funded by Programa VALi+ d GVA APOSTD/2017/039. J.J. was supported by an FPI contract from MINECOBelda Palazón, B.; Julian, J.; Coego, A.; Wu, Q.; Zhang, X.; Batistic, O.; Alquraishi, SA.... (2019). ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA. 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The PYL4 A194T mutant uncovers a key role of PYL4-PP2CA interaction for ABA signaling and plant drought resistance

[EN] 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 PYL4(A194T) 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: PYL4(A194T) 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:PYL4(A194T) 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.Pizzio Bianchi, GA.; Rodriguez, L.; Antoni-Alandes, R.; Gonzalez Guzman, M.; Yunta, C.; Merilo, E.; Kollist, H.... (2013). The PYL4 A194T mutant uncovers a key role of PYL4-PP2CA interaction for ABA signaling and plant drought resistance. Plant Physiology. 163(1):441-455. doi:10.​1104/​pp.​113.​224162S441455163

PYRABACTIN RESISTANCE1-LIKE8 plays an important role for the regulation of abscisic acid signaling in root

[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.).Antoni-Alandes, R.; Gonzalez Guzman, M.; Rodriguez, L.; Peirats-Llobet, M.; Pizzio Bianchi, GA.; Fernández, MA.; De Winne, N.... (2013). PYRABACTIN RESISTANCE1-LIKE8 plays an important role for the regulation of abscisic acid signaling in root. Plant Physiology. 161(2):931-941. doi:10.1104/pp.112.208678S931941161