A chloroplast retrograde signal, 3’phosphoadenosine 5’-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination

Organelle-nuclear retrograde signaling regulates gene expression, but its roles in specialized cells and integration with hormonal signaling remain enigmatic. Here we show that the SAL1-PAP (3'-phosphoadenosine 5'- phosphate) retrograde pathway interacts with abscisic acid (ABA) signaling to regulate stomatal closure and seed germination in Arabidopsis. Genetically or exogenously manipulating PAP bypasses the canonical signaling components ABA Insensitive 1 (ABI1) and Open Stomata 1 (OST1); priming an alternative pathway that restores ABA-responsive gene expression, ROS bursts, ion channel function, stomatal closure and drought tolerance in ost1-2. PAP also inhibits wild type and abi1-1 seed germination by enhancing ABA sensitivity. PAP-XRN signaling interacts with ABA, ROS and Ca2+; up-regulating multiple ABA signaling components, including lowly-expressed Calcium Dependent Protein Kinases (CDPKs) capable of activating the anion channel SLAC1. Thus, PAP exhibits many secondary messenger attributes and exemplifies how retrograde signals can have broader roles in hormone signaling, allowing chloroplasts to fine-tune physiological responses.Wannarat Pornsiriwong, Gonzalo M Estavillo, Kai Xun Chan, Estee E Tee, Diep Ganguly, Peter A Crisp, Su Yin Phua, Chenchen Zhao, Jiaen Qiu, Jiyoung Park, Miing Tiem Yong, Nazia Nisar, Arun Kumar Yadav, Benjamin Schwessinger, John Rathjen, Christopher I Cazzonelli, Philippa B Wilson, Matthew Gilliham, Zhong-Hua Chen, Barry J Pogso

The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling

[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.Bueso Ródenas, E.; Rodriguez, L.; Lorenzo Orts, L.; González Guzmán, M.; Sayas Montañana, EM.; Muñoz Bertomeu, J.; Ibañez, C.... (2014). The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling. Plant Journal. 80(6):1057-1071. https://doi.org/10.1111/tpj.12708S10571071806Antoni, R., Gonzalez-Guzman, M., Rodriguez, L., Peirats-Llobet, M., Pizzio, G. A., Fernandez, M. A., … Rodriguez, P. L. (2012). 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.112.208678Barberon, M., Zelazny, E., Robert, S., Conéjéro, G., Curie, C., Friml, J., & Vert, G. (2011). Monoubiquitin-dependent endocytosis of the IRON-REGULATED TRANSPORTER 1 (IRT1) transporter controls iron uptake in plants. Proceedings of the National Academy of Sciences, 108(32), E450-E458. doi:10.1073/pnas.1100659108Batistič, O., Sorek, N., Schültke, S., Yalovsky, S., & Kudla, J. (2008). Dual Fatty Acyl Modification Determines the Localization and Plasma Membrane Targeting of CBL/CIPK Ca2+ Signaling Complexes in Arabidopsis. The Plant Cell, 20(5), 1346-1362. doi:10.1105/tpc.108.058123Batistič, O., Rehers, M., Akerman, A., Schlücking, K., Steinhorst, L., Yalovsky, S., & Kudla, J. (2012). S-acylation-dependent association of the calcium sensor CBL2 with the vacuolar membrane is essential for proper abscisic acid responses. Cell Research, 22(7), 1155-1168. doi:10.1038/cr.2012.71Belda-Palazón, B., Ruiz, L., Martí, E., Tárraga, S., Tiburcio, A. F., Culiáñez, F., … Ferrando, A. (2012). Aminopropyltransferases Involved in Polyamine Biosynthesis Localize Preferentially in the Nucleus of Plant Cells. PLoS ONE, 7(10), e46907. doi:10.1371/journal.pone.0046907Brandt, B., Brodsky, D. E., Xue, S., Negi, J., Iba, K., Kangasjarvi, J., … Schroeder, J. I. (2012). Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. Proceedings of the National Academy of Sciences, 109(26), 10593-10598. doi:10.1073/pnas.1116590109Bueso, E., Ibañez, C., Sayas, E., Muñoz-Bertomeu, J., Gonzalez-Guzmán, M., Rodriguez, P. L., & Serrano, R. (2014). A forward genetic approach in Arabidopsis thaliana identifies a RING-type ubiquitin ligase as a novel determinant of seed longevity. Plant Science, 215-216, 110-116. doi:10.1016/j.plantsci.2013.11.004Capili, A. D., Edghill, E. ., Wu, K., & Borden, K. L. . (2004). Structure of the C-terminal RING Finger from a RING-IBR-RING/TRIAD Motif Reveals a Novel Zinc-binding Domain Distinct from a RING. Journal of Molecular Biology, 340(5), 1117-1129. doi:10.1016/j.jmb.2004.05.035Curtis, M. D., & Grossniklaus, U. (2003). A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta. Plant Physiology, 133(2), 462-469. doi:10.1104/pp.103.027979Cutler, S. R., Rodriguez, P. L., Finkelstein, R. R., & Abrams, S. R. (2010). Abscisic Acid: Emergence of a Core Signaling Network. Annual Review of Plant Biology, 61(1), 651-679. doi:10.1146/annurev-arplant-042809-112122Czechowski, T., Stitt, M., Altmann, T., Udvardi, M. K., & Scheible, W.-R. (2005). Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis. Plant Physiology, 139(1), 5-17. doi:10.1104/pp.105.063743Demir, F., Horntrich, C., Blachutzik, J. O., Scherzer, S., Reinders, Y., Kierszniowska, S., … Kreuzer, I. (2013). Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3. Proceedings of the National Academy of Sciences, 110(20), 8296-8301. doi:10.1073/pnas.1211667110French, A. P., Mills, S., Swarup, R., Bennett, M. J., & Pridmore, T. P. (2008). Colocalization of fluorescent markers in confocal microscope images of plant cells. Nature Protocols, 3(4), 619-628. doi:10.1038/nprot.2008.31Friml, J. (2010). Subcellular trafficking of PIN auxin efflux carriers in auxin transport. European Journal of Cell Biology, 89(2-3), 231-235. doi:10.1016/j.ejcb.2009.11.003Fujii, H., & Zhu, J.-K. (2009). Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress. Proceedings of the National Academy of Sciences, 106(20), 8380-8385. doi:10.1073/pnas.0903144106Fujita, Y., Nakashima, K., Yoshida, T., Katagiri, T., Kidokoro, S., Kanamori, N., … Yamaguchi-Shinozaki, K. (2009). Three SnRK2 Protein Kinases are the Main Positive Regulators of Abscisic Acid Signaling in Response to Water Stress in Arabidopsis. Plant and Cell Physiology, 50(12), 2123-2132. doi:10.1093/pcp/pcp147Geiger, D., Scherzer, S., Mumm, P., Stange, A., Marten, I., Bauer, H., … Hedrich, R. (2009). Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair. Proceedings of the National Academy of Sciences, 106(50), 21425-21430. doi:10.1073/pnas.0912021106Geiger, D., Scherzer, S., Mumm, P., Marten, I., Ache, P., Matschi, S., … Hedrich, R. (2010). Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+affinities. Proceedings of the National Academy of Sciences, 107(17), 8023-8028. doi:10.1073/pnas.0912030107Geiger, D., Maierhofer, T., AL-Rasheid, K. A. S., Scherzer, S., Mumm, P., Liese, A., … Hedrich, R. (2011). Stomatal Closure by Fast Abscisic Acid Signaling Is Mediated by the Guard Cell Anion Channel SLAH3 and the Receptor RCAR1. Science Signaling, 4(173), ra32-ra32. doi:10.1126/scisignal.2001346Geldner, N., & Jürgens, G. (2006). Endocytosis in signalling and development. Current Opinion in Plant Biology, 9(6), 589-594. doi:10.1016/j.pbi.2006.09.011Geldner, N., Dénervaud-Tendon, V., Hyman, D. L., Mayer, U., Stierhof, Y.-D., & Chory, J. (2009). Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. The Plant Journal, 59(1), 169-178. doi:10.1111/j.1365-313x.2009.03851.xGonzalez-Guzman, M., Pizzio, G. A., Antoni, R., Vera-Sirera, F., Merilo, E., Bassel, G. W., … Rodriguez, P. L. (2012). Arabidopsis PYR/PYL/RCAR Receptors Play a Major Role in Quantitative Regulation of Stomatal Aperture and Transcriptional Response to Abscisic Acid. The Plant Cell, 24(6), 2483-2496. doi:10.1105/tpc.112.098574Hirsch, C., Gauss, R., Horn, S. C., Neuber, O., & Sommer, T. (2009). The ubiquitylation machinery of the endoplasmic reticulum. Nature, 458(7237), 453-460. doi:10.1038/nature07962Hua, Z., & Vierstra, R. D. (2011). The Cullin-RING Ubiquitin-Protein Ligases. Annual Review of Plant Biology, 62(1), 299-334. doi:10.1146/annurev-arplant-042809-112256Irigoyen, M. L., Iniesto, E., Rodriguez, L., Puga, M. I., Yanagawa, Y., Pick, E., … Rubio, V. (2014). Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis. The Plant Cell, 26(2), 712-728. doi:10.1105/tpc.113.122234Jones, A. M., Xuan, Y., Xu, M., Wang, R.-S., Ho, C.-H., Lalonde, S., … Frommer, W. B. (2014). Border Control--A Membrane-Linked Interactome of Arabidopsis. Science, 344(6185), 711-716. doi:10.1126/science.1251358Kasai, K., Takano, J., Miwa, K., Toyoda, A., & Fujiwara, T. (2010). High Boron-induced Ubiquitination Regulates Vacuolar Sorting of the BOR1 Borate Transporter inArabidopsis thaliana. Journal of Biological Chemistry, 286(8), 6175-6183. doi:10.1074/jbc.m110.184929Kim, D.-Y., Scalf, M., Smith, L. M., & Vierstra, R. D. (2013). Advanced Proteomic Analyses Yield a Deep Catalog of Ubiquitylation Targets in Arabidopsis. The Plant Cell, 25(5), 1523-1540. doi:10.1105/tpc.112.108613Kollist, H., Nuhkat, M., & Roelfsema, M. R. G. (2014). Closing gaps: linking elements that control stomatal movement. New Phytologist, 203(1), 44-62. doi:10.1111/nph.12832Kosarev, P., Mayer, K. F., & Hardtke, C. S. (2002). Genome Biology, 3(4), research0016.1. doi:10.1186/gb-2002-3-4-research0016Lee, S. C., Lan, W., Buchanan, B. B., & Luan, S. (2009). A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells. Proceedings of the National Academy of Sciences, 106(50), 21419-21424. doi:10.1073/pnas.0910601106Liu, L., Zhang, Y., Tang, S., Zhao, Q., Zhang, Z., Zhang, H., … Xie, Q. (2010). An efficient system to detect protein ubiquitination by agroinfiltration inNicotiana benthamiana. The Plant Journal, 61(5), 893-903. doi:10.1111/j.1365-313x.2009.04109.xLyzenga, W. J., & Stone, S. L. (2011). Abiotic stress tolerance mediated by protein ubiquitination. Journal of Experimental Botany, 63(2), 599-616. doi:10.1093/jxb/err310MacGurn, J. A., Hsu, P.-C., & Emr, S. D. (2012). Ubiquitin and Membrane Protein Turnover: From Cradle to Grave. Annual Review of Biochemistry, 81(1), 231-259. doi:10.1146/annurev-biochem-060210-093619Manzano, C., Abraham, Z., López-Torrejón, G., & Del Pozo, J. C. (2008). Identification of ubiquitinated proteins in Arabidopsis. Plant Molecular Biology, 68(1-2), 145-158. doi:10.1007/s11103-008-9358-9Oñate-Sánchez, L., & Vicente-Carbajosa, J. (2008). DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques. BMC Research Notes, 1(1), 93. doi:10.1186/1756-0500-1-93Osakabe, Y., Yamaguchi-Shinozaki, K., Shinozaki, K., & Tran, L.-S. P. (2013). ABA control of plant macroelement membrane transport systems in response to water deficit and high salinity. New Phytologist, 202(1), 35-49. doi:10.1111/nph.12613Peyroche, A., Antonny, B., Robineau, S., Acker, J., Cherfils, J., & Jackson, C. L. (1999). Brefeldin A Acts to Stabilize an Abortive ARF–GDP–Sec7 Domain Protein Complex. Molecular Cell, 3(3), 275-285. doi:10.1016/s1097-2765(00)80455-4Pizzio, G. A., Rodriguez, L., Antoni, R., Gonzalez-Guzman, M., Yunta, C., Merilo, E., … Rodriguez, P. L. (2013). The PYL4 A194T Mutant Uncovers a Key Role of PYR1-LIKE4/PROTEIN PHOSPHATASE 2CA Interaction for Abscisic Acid Signaling and Plant Drought Resistance. Plant Physiology, 163(1), 441-455. doi:10.1104/pp.113.224162Pollier, J., Moses, T., González-Guzmán, M., De Geyter, N., Lippens, S., Bossche, R. V., … Goossens, A. (2013). The protein quality control system manages plant defence compound synthesis. Nature, 504(7478), 148-152. doi:10.1038/nature12685Popper, Z. A., Michel, G., Hervé, C., Domozych, D. S., Willats, W. G. T., Tuohy, M. G., … Stengel, D. B. (2011). Evolution and Diversity of Plant Cell Walls: From Algae to Flowering Plants. Annual Review of Plant Biology, 62(1), 567-590. doi:10.1146/annurev-arplant-042110-103809Van Der Reijden, B. A., Erpelinck-Verschueren, C. A. J., BOB LÖWENBERG, & Jansen, J. H. (1999). TRIADs: A new class of proteins with a novel cysteine-rich signature. Protein Science, 8(7), 1557-1561. doi:10.1110/ps.8.7.1557Richardson, L. G. L., Howard, A. S. M., Khuu, N., Gidda, S. K., McCartney, A., Morphy, B. J., & Mullen, R. T. (2011). Protein–Protein Interaction Network and Subcellular Localization of the Arabidopsis Thaliana ESCRT Machinery. Frontiers in Plant Science, 2. doi:10.3389/fpls.2011.00020Robinson, D. G., Langhans, M., Saint-Jore-Dupas, C., & Hawes, C. (2008). BFA effects are tissue and not just plant specific. Trends in Plant Science, 13(8), 405-408. doi:10.1016/j.tplants.2008.05.010Saez, A., Robert, N., Maktabi, M. H., Schroeder, J. I., Serrano, R., & Rodriguez, P. L. (2006). Enhancement of Abscisic Acid Sensitivity and Reduction of Water Consumption in Arabidopsis by Combined Inactivation of the Protein Phosphatases Type 2C ABI1 and HAB1. Plant Physiology, 141(4), 1389-1399. doi:10.1104/pp.106.081018Santiago, J., Rodrigues, A., Saez, A., Rubio, S., Antoni, R., Dupeux, F., … Rodriguez, P. L. (2009). Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs. The Plant Journal, 60(4), 575-588. doi:10.1111/j.1365-313x.2009.03981.xSantner, A., & Estelle, M. (2009). Recent advances and emerging trends in plant hormone signalling. Nature, 459(7250), 1071-1078. doi:10.1038/nature08122Saracco, S. A., Hansson, M., Scalf, M., Walker, J. M., Smith, L. M., & Vierstra, R. D. (2009). Tandem affinity purification and mass spectrometric analysis of ubiquitylated proteins in Arabidopsis. The Plant Journal, 59(2), 344-358. doi:10.1111/j.1365-313x.2009.03862.xSato, A., Sato, Y., Fukao, Y., Fujiwara, M., Umezawa, T., Shinozaki, K., … Uozumi, N. (2009). Threonine at position 306 of the KAT1 potassium channel is essential for channel activity and is a target site for ABA-activated SnRK2/OST1/SnRK2.6 protein kinase. Biochemical Journal, 424(3), 439-448. doi:10.1042/bj20091221Scheuring, D., Künzl, F., Viotti, C., Yan, M. S. W., Jiang, L., Schellmann, S., … Pimpl, P. (2012). Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway. BMC Plant Biology, 12(1), 164. doi:10.1186/1471-2229-12-164Sirichandra, C., Gu, D., Hu, H.-C., Davanture, M., Lee, S., Djaoui, M., … Kwak, J. M. (2009). Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase by OST1 protein kinase. FEBS Letters, 583(18), 2982-2986. doi:10.1016/j.febslet.2009.08.033Smalle, J., & Vierstra, R. D. (2004). THE UBIQUITIN 26S PROTEASOME PROTEOLYTIC PATHWAY. Annual Review of Plant Biology, 55(1), 555-590. doi:10.1146/annurev.arplant.55.031903.141801Spitzer, C., Reyes, F. C., Buono, R., Sliwinski, M. K., Haas, T. J., & Otegui, M. S. (2009). The ESCRT-Related CHMP1A and B Proteins Mediate Multivesicular Body Sorting of Auxin Carriers in Arabidopsis and Are Required for Plant Development. The Plant Cell, 21(3), 749-766. doi:10.1105/tpc.108.064865Teis, D., Saksena, S., & Emr, S. D. (2009). SnapShot: The ESCRT Machinery. Cell, 137(1), 182-182.e1. doi:10.1016/j.cell.2009.03.027Umezawa, T., Sugiyama, N., Mizoguchi, M., Hayashi, S., Myouga, F., Yamaguchi-Shinozaki, K., … Shinozaki, K. (2009). Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis. Proceedings of the National Academy of Sciences, 106(41), 17588-17593. doi:10.1073/pnas.0907095106Vierstra, R. D. (2009). The ubiquitin–26S proteasome system at the nexus of plant biology. Nature Reviews Molecular Cell Biology, 10(6), 385-397. doi:10.1038/nrm2688Viotti, C., Bubeck, J., Stierhof, Y.-D., Krebs, M., Langhans, M., van den Berg, W., … Schumacher, K. (2010). Endocytic and Secretory Traffic in Arabidopsis Merge in the Trans-Golgi Network/Early Endosome, an Independent and Highly Dynamic Organelle. The Plant Cell, 22(4), 1344-1357. doi:10.1105/tpc.109.072637Vlad, F., Rubio, S., Rodrigues, A., Sirichandra, C., Belin, C., Robert, N., … Merlot, S. (2009). Protein Phosphatases 2C Regulate the Activation of the Snf1-Related Kinase OST1 by Abscisic Acid in Arabidopsis. The Plant Cell, 21(10), 3170-3184. doi:10.1105/tpc.109.069179Voinnet, O., Rivas, S., Mestre, P., & Baulcombe, D. (2003). Retracted: An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. The Plant Journal, 33(5), 949-956. doi:10.1046/j.1365-313x.2003.01676.xZhao, Q., Tian, M., Li, Q., Cui, F., Liu, L., Yin, B., & Xie, Q. (2013). A plant-specificin vitroubiquitination analysis system. The Plant Journal, 74(3), 524-533. doi:10.1111/tpj.1212