Functional innovations of PIN auxin transporters mark crucial evolutionary transitions during rise of flowering plants

Flowering plants display the highest diversity among plant species and have notably shaped terrestrial landscapes. Nonetheless, the evolutionary origin of their unprecedented morphological complexity remains largely an enigma. Here, we show that the coevolution of cis-regulatory and coding regions of PIN-FORMED (PIN) auxin transporters confined their expression to certain cell types and directed their subcellular localization to particular cell sides, which together enabled dynamic auxin gradients across tissues critical to the complex architecture of flowering plants. Extensive intraspecies and interspecies genetic complementation experiments with PINs from green alga up to flowering plant lineages showed that PIN genes underwent three subsequent, critical evolutionary innovations and thus acquired a triple function to regulate the development of three essential components of the flowering plant Arabidopsis: shoot/root, inflorescence, and floral organ. Our work highlights the critical role of functional innovations within the PIN gene family as essential prerequisites for the origin of flowering plants

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

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

Auxin-regulated reversible inhibition of TMK1 signaling by MAKR2 modulates the dynamics of root gravitropism

Plants are able to orient their growth according to gravity, which ultimately controls both shoot and root architecture.1 Gravitropism is a dynamic process whereby gravistimulation induces the asymmetric distribution of the plant hormone auxin, leading to asymmetric growth, organ bending, and subsequent reset of auxin distribution back to the original pre-gravistimulation situation.1, 2, 3 Differential auxin accumulation during the gravitropic response depends on the activity of polarly localized PIN-FORMED (PIN) auxin-efflux carriers.1, 2, 3, 4 In particular, the timing of this dynamic response is regulated by PIN2,5,6 but the underlying molecular mechanisms are poorly understood. Here, we show that MEMBRANE ASSOCIATED KINASE REGULATOR2 (MAKR2) controls the pace of the root gravitropic response. We found that MAKR2 is required for the PIN2 asymmetry during gravitropism by acting as a negative regulator of the cell-surface signaling mediated by the receptor-like kinase TRANSMEMBRANE KINASE1 (TMK1).2,7, 8, 9, 10 Furthermore, we show that the MAKR2 inhibitory effect on TMK1 signaling is antagonized by auxin itself, which triggers rapid MAKR2 membrane dissociation in a TMK1-dependent manner. Our findings suggest that the timing of the root gravitropic response is orchestrated by the reversible inhibition of the TMK1 signaling pathway at the cell surface

Structural insights into PYR/PYL/RCAR ABA receptors and PP2Cs

[EN] 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 ABA-dependent 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/RCAR-ABA-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. (C) 2010 Elsevier Ireland Ltd. All rights reserved.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 InfrastructureAction PCUBE under the FP7 “Capacities” specific program.Santiago Cuéllar, J.; Dupeux, F.; Betz, K.; Antoni-Alandes, R.; González Guzmán, M.; Rodriguez, L.; Márquez, JA.... (2012). Structural insights into PYR/PYL/RCAR ABA receptors and PP2Cs. Plant Science. 182:3-11. https://doi.org/10.1016/j.plantsci.2010.11.014S31118

C2-Domain Abscisic Acid-Related Proteins Mediate the Interaction of PYR/PYL/RCAR Abscisic Acid Receptors with the Plasma Membrane and Regulate Abscisic Acid Sensitivity in Arabidopsis

Supplemental Data: http://www.plantcell.org/content/26/12/4802/suppl/DC1© 2014 American Society of Plant BiologistsMembrane-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.).Rodriguez, L.; Gonzalez Guzman, M.; Díaz, M.; Rodrigues, A.; Izquierdo Garcia, AC.; Peirats-Llobet, M.; Fernández López, MA.... (2014). C2-Domain Abscisic Acid-Related Proteins Mediate the Interaction of PYR/PYL/RCAR Abscisic Acid Receptors with the Plasma Membrane and Regulate Abscisic Acid Sensitivity in Arabidopsis. Plant Cell. 26(12):4802-4820. doi:10.1105/tpc.114.129973S48024820261

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

[EN] 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 PYL85KR 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.Work in the P.L.R. and F.M. laboratories was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Cientificas Grants BIO2014-52537-R and BIO2017-82503-R (to P.L.R.) and BIO2015-64307-R (to F.M.). J.L.-J. was supported by a Juan de la Cierva contract from Ministerio de Economia y Competitividad (MINECO) and by the Marie Sklodowska-Curie Action H2020-MSCA-IF-2015-707477. B.B.-P. was funded by Programa VALi+d GVA APOSTD/2017/039. J.J. was supported by a FPI contract from MINECO and M.A.F. by a Formacion de Profesorado Universitario contract from MINECO. D.D. and M.J.B. were supported by Biotechnology and Biological Sciences Research Council Grant BB/M002136/1 and Leverhulme Trust Grant RPG-2016-409.Belda-Palazón, B.; Gonzalez-Garcia, M.; Lozano Juste, J.; Coego Gonzalez, A.; Antoni-Alandes, R.; Julian-Valenzuela, J.; Peirats-Llobet, M.... (2018). PYL8 mediates ABA perception in the root through non-cell-autonomous and ligand-stabilization-based mechanisms. Proceedings of the National Academy of Sciences of the United States of America (Online). 115(50):E11857-E11863. https://doi.org/10.1073/pnas.1815410115SE11857E1186311550Ubeda-Tomás, S., Beemster, G. T. S., & Bennett, M. J. (2012). Hormonal regulation of root growth: integrating local activities into global behaviour. Trends in Plant Science, 17(6), 326-331. doi:10.1016/j.tplants.2012.02.002Bao, Y., Aggarwal, P., Robbins, N. E., Sturrock, C. J., Thompson, M. C., Tan, H. Q., … Dinneny, J. R. (2014). Plant roots use a patterning mechanism to position lateral root branches toward available water. Proceedings of the National Academy of Sciences, 111(25), 9319-9324. doi:10.1073/pnas.1400966111Dietrich, D., Pang, L., Kobayashi, A., Fozard, J. A., Boudolf, V., Bhosale, R., … Bennett, M. J. (2017). Root hydrotropism is controlled via a cortex-specific growth mechanism. Nature Plants, 3(6). doi:10.1038/nplants.2017.57Harris, J. (2015). Abscisic Acid: Hidden Architect of Root System Structure. Plants, 4(3), 548-572. doi:10.3390/plants4030548Spollen, W. G., LeNoble, M. E., Samuels, T. D., Bernstein, N., & Sharp, R. E. (2000). Abscisic Acid Accumulation Maintains Maize Primary Root Elongation at Low Water Potentials by Restricting Ethylene Production. Plant Physiology, 122(3), 967-976. doi:10.1104/pp.122.3.967Sharp, R. E. (2004). Root growth maintenance during water deficits: physiology to functional genomics. Journal of Experimental Botany, 55(407), 2343-2351. doi:10.1093/jxb/erh276Deak, K. I., & Malamy, J. (2005). Osmotic regulation of root system architecture. The Plant Journal, 43(1), 17-28. doi:10.1111/j.1365-313x.2005.02425.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.112.098574Duan, L., Dietrich, D., Ng, C. H., Chan, P. M. Y., Bhalerao, R., Bennett, M. J., & Dinneny, J. R. (2013). Endodermal ABA Signaling Promotes Lateral Root Quiescence during Salt Stress in Arabidopsis Seedlings. The Plant Cell, 25(1), 324-341. doi:10.1105/tpc.112.107227Feng, W., Lindner, H., Robbins, N. E., & Dinneny, J. R. (2016). Growing Out of Stress: The Role of Cell- and Organ-Scale Growth Control in Plant Water-Stress Responses. The Plant Cell, 28(8), 1769-1782. doi:10.1105/tpc.16.00182Geng, Y., Wu, R., Wee, C. W., Xie, F., Wei, X., Chan, P. M. Y., … Dinneny, J. R. (2013). A Spatio-Temporal Understanding of Growth Regulation during the Salt Stress Response in Arabidopsis. The Plant Cell, 25(6), 2132-2154. doi:10.1105/tpc.113.112896Takahashi, N., Goto, N., Okada, K., & Takahashi, H. (2002). Hydrotropism in abscisic acid, wavy, and gravitropic mutants of Arabidopsis thaliana. Planta, 216(2), 203-211. doi:10.1007/s00425-002-0840-3Antoni, 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., Vermeer, J. E. M., De Bellis, D., Wang, P., Naseer, S., Andersen, T. G., … Geldner, N. (2016). Adaptation of Root Function by Nutrient-Induced Plasticity of Endodermal Differentiation. Cell, 164(3), 447-459. doi:10.1016/j.cell.2015.12.021Ondzighi-Assoume, C. A., Chakraborty, S., & Harris, J. M. (2016). Environmental Nitrate Stimulates Abscisic Acid Accumulation in Arabidopsis Root Tips by Releasing It from Inactive Stores. The Plant Cell, 28(3), 729-745. doi:10.1105/tpc.15.00946Irigoyen, 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.113.122234Bueso, E., Rodriguez, L., Lorenzo-Orts, L., Gonzalez-Guzman, M., Sayas, E., Muñoz-Bertomeu, J., … Rodriguez, P. L. (2014). The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling. The Plant Journal, 80(6), 1057-1071. doi:10.1111/tpj.12708Knoblich, J. A. (2005). Pins for spines. Nature Cell Biology, 7(12), 1057-1058. doi:10.1038/ncb1205-1057Zhang, H., Han, W., De Smet, I., Talboys, P., Loya, R., Hassan, A., … Wang, M.-H. (2010). ABA promotes quiescence of the quiescent centre and suppresses stem cell differentiation in the Arabidopsis primary root meristem. The Plant Journal, 64(5), 764-774. doi:10.1111/j.1365-313x.2010.04367.xBelda-Palazon, B., Rodriguez, L., Fernandez, M. A., Castillo, M.-C., Anderson, E. M., Gao, C., … Rodriguez, P. L. (2016). FYVE1/FREE1 Interacts with the PYL4 ABA Receptor and Mediates Its Delivery to the Vacuolar Degradation Pathway. The Plant Cell, 28(9), 2291-2311. doi:10.1105/tpc.16.00178Yu, F., Lou, L., Tian, M., Li, Q., Ding, Y., Cao, X., … Xie, Q. (2016). ESCRT-I Component VPS23A Affects ABA Signaling by Recognizing ABA Receptors for Endosomal Degradation. Molecular Plant, 9(12), 1570-1582. doi:10.1016/j.molp.2016.11.002Santiago, 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.xSzostkiewicz, I., Richter, K., Kepka, M., Demmel, S., Ma, Y., Korte, A., … Grill, E. (2010). Closely related receptor complexes differ in their ABA selectivity and sensitivity. The Plant Journal, 61(1), 25-35. doi:10.1111/j.1365-313x.2009.04025.xOkamoto, M., Peterson, F. C., Defries, A., Park, S.-Y., Endo, A., Nambara, E., … Cutler, S. R. (2013). Activation of dimeric ABA receptors elicits guard cell closure, ABA-regulated gene expression, and drought tolerance. Proceedings of the National Academy of Sciences, 110(29), 12132-12137. doi:10.1073/pnas.1305919110Cao, M., Liu, X., Zhang, Y., Xue, X., Zhou, X. E., Melcher, K., … Xu, Y. (2013). An ABA-mimicking ligand that reduces water loss and promotes drought resistance in plants. Cell Research, 23(8), 1043-1054. doi:10.1038/cr.2013.95Castillo, M.-C., Lozano-Juste, J., González-Guzmán, M., Rodriguez, L., Rodriguez, P. L., & León, J. (2015). Inactivation of PYR/PYL/RCAR ABA receptors by tyrosine nitration may enable rapid inhibition of ABA signaling by nitric oxide in plants. Science Signaling, 8(392), ra89-ra89. doi:10.1126/scisignal.aaa7981Wu, S., & Gallagher, K. L. (2014). The movement of the non-cell-autonomous transcription factor, SHORT-ROOT relies on the endomembrane system. The Plant Journal, 80(3), 396-409. doi:10.1111/tpj.12640Nakajima, K., Sena, G., Nawy, T., & Benfey, P. N. (2001). Intercellular movement of the putative transcription factor SHR in root patterning. Nature, 413(6853), 307-311. doi:10.1038/35095061Gallagher, K. L., Paquette, A. J., Nakajima, K., & Benfey, P. N. (2004). Mechanisms Regulating SHORT-ROOT Intercellular Movement. Current Biology, 14(20), 1847-1851. doi:10.1016/j.cub.2004.09.081Pálfy, M., Reményi, A., & Korcsmáros, T. (2012). Endosomal crosstalk: meeting points for signaling pathways. Trends in Cell Biology, 22(9), 447-456. doi:10.1016/j.tcb.2012.06.004Christmann, A., Hoffmann, T., Teplova, I., Grill, E., & Müller, A. (2004). Generation of Active Pools of Abscisic Acid Revealed by In Vivo Imaging of Water-Stressed Arabidopsis. Plant Physiology, 137(1), 209-219. doi:10.1104/pp.104.053082Kim, T.-H., Hauser, F., Ha, T., Xue, S., Böhmer, M., Nishimura, N., … Schroeder, J. I. (2011). Chemical Genetics Reveals Negative Regulation of Abscisic Acid Signaling by a Plant Immune Response Pathway. Current Biology, 21(11), 990-997. doi:10.1016/j.cub.2011.04.045Waadt, R., Hitomi, K., Nishimura, N., Hitomi, C., Adams, S. R., Getzoff, E. D., & Schroeder, J. I. (2014). FRET-based reporters for the direct visualization of abscisic acid concentration changes and distribution in Arabidopsis. eLife, 3. doi:10.7554/elife.01739Jones, A. M., Danielson, J. Å., ManojKumar, S. N., Lanquar, V., Grossmann, G., & Frommer, W. B. (2014). Abscisic acid dynamics in roots detected with genetically encoded FRET sensors. eLife, 3. doi:10.7554/elife.01741Zhao, Y., Xing, L., Wang, X., Hou, Y.-J., Gao, J., Wang, P., … Zhu, J.-K. (2014). The ABA Receptor PYL8 Promotes Lateral Root Growth by Enhancing MYB77-Dependent Transcription of Auxin-Responsive Genes. Science Signaling, 7(328), ra53-ra53. doi:10.1126/scisignal.2005051Peirats-Llobet, M., Han, S.-K., Gonzalez-Guzman, M., Jeong, C. W., Rodriguez, L., Belda-Palazon, B., … Rodriguez, P. L. (2016). A Direct Link between Abscisic Acid Sensing and the Chromatin-Remodeling ATPase BRAHMA via Core ABA Signaling Pathway Components. Molecular Plant, 9(1), 136-147. doi:10.1016/j.molp.2015.10.003Moes, D., Himmelbach, A., Korte, A., Haberer, G., & Grill, E. (2008). Nuclear localization of the mutant protein phosphatase abi1 is required for insensitivity towards ABA responses in Arabidopsis. The Plant Journal, 54(5), 806-819. doi:10.1111/j.1365-313x.2008.03454.xLynch, T., Erickson, B. J., & Finkelstein, R. R. (2012). Direct interactions of ABA-insensitive(ABI)-clade protein phosphatase(PP)2Cs with calcium-dependent protein kinases and ABA response element-binding bZIPs may contribute to turning off ABA response. Plant Molecular Biology, 80(6), 647-658. doi:10.1007/s11103-012-9973-

Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling

[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.Diaz, M.; Sanchez-Barrena, MJ.; Gonzalez Rubio, JM.; Rodríguez Solovey, LN.; Fernández, D.; Antoni-Alandes, R.; Yunta, C.... (2016). Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling. Proceedings of the National Academy of Sciences. 113(3):E396-E405. https://doi.org/10.1073/pnas.1512779113SE396E4051133Serrano, R., & Rodriguez-Navarro, A. (2001). 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The global retinoblastoma outcome study : a prospective, cluster-based analysis of 4064 patients from 149 countries

DATA SHARING : The study data will become available online once all analyses are complete.BACKGROUND : Retinoblastoma is the most common intraocular cancer worldwide. There is some evidence to suggest that major differences exist in treatment outcomes for children with retinoblastoma from different regions, but these differences have not been assessed on a global scale. We aimed to report 3-year outcomes for children with retinoblastoma globally and to investigate factors associated with survival. METHODS : We did a prospective cluster-based analysis of treatment-naive patients with retinoblastoma who were diagnosed between Jan 1, 2017, and Dec 31, 2017, then treated and followed up for 3 years. Patients were recruited from 260 specialised treatment centres worldwide. Data were obtained from participating centres on primary and additional treatments, duration of follow-up, metastasis, eye globe salvage, and survival outcome. We analysed time to death and time to enucleation with Cox regression models. FINDINGS : The cohort included 4064 children from 149 countries. The median age at diagnosis was 23·2 months (IQR 11·0–36·5). Extraocular tumour spread (cT4 of the cTNMH classification) at diagnosis was reported in five (0·8%) of 636 children from high-income countries, 55 (5·4%) of 1027 children from upper-middle-income countries, 342 (19·7%) of 1738 children from lower-middle-income countries, and 196 (42·9%) of 457 children from low-income countries. Enucleation surgery was available for all children and intravenous chemotherapy was available for 4014 (98·8%) of 4064 children. The 3-year survival rate was 99·5% (95% CI 98·8–100·0) for children from high-income countries, 91·2% (89·5–93·0) for children from upper-middle-income countries, 80·3% (78·3–82·3) for children from lower-middle-income countries, and 57·3% (52·1-63·0) for children from low-income countries. On analysis, independent factors for worse survival were residence in low-income countries compared to high-income countries (hazard ratio 16·67; 95% CI 4·76–50·00), cT4 advanced tumour compared to cT1 (8·98; 4·44–18·18), and older age at diagnosis in children up to 3 years (1·38 per year; 1·23–1·56). For children aged 3–7 years, the mortality risk decreased slightly (p=0·0104 for the change in slope). INTERPRETATION : This study, estimated to include approximately half of all new retinoblastoma cases worldwide in 2017, shows profound inequity in survival of children depending on the national income level of their country of residence. In high-income countries, death from retinoblastoma is rare, whereas in low-income countries estimated 3-year survival is just over 50%. Although essential treatments are available in nearly all countries, early diagnosis and treatment in low-income countries are key to improving survival outcomes.The Queen Elizabeth Diamond Jubilee Trust and the Wellcome Trust.https://www.thelancet.com/journals/langlo/homeam2023Paediatrics and Child Healt