Molecular mechanism for the interaction between gibberellin and brassinosteroid signaling pathways in Arabidopsis

[EN] Plant development is modulated by the convergence of multiple environmental and endogenous signals, and the mechanisms that allow the integration of different signaling pathways is currently being unveiled. A paradigmatic case is the concurrence of brassinosteroid (BR) and gibberellin (GA) signaling in the control of cell expansion during photomorphogenesis, which is supported by physiological observations in several plants but for which no molecular mechanism has been proposed. In this work, we show that the integration of these two signaling pathways occurs through the physical interaction between the DELLA protein GAI, which is a major negative regulator of the GA pathway, and BRASSINAZOLE RESISTANT1 (BZR1), a transcription factor that broadly regulates gene expression in response to BRs. We provide biochemical evidence, both in vitro and in vivo, indicating that GAI inactivates the transcriptional regulatory activity of BZR1 upon their interaction by inhibiting the ability of BZR1 to bind to target promoters. The physiological relevance of this interaction was confirmed by the observation that the dominant gai-1 allele interferes with BR-regulated gene expression, whereas the bzr1-1D allele displays enhanced resistance to DELLA accumulation during hypocotyl elongation. Because DELLA proteins mediate the response to multiple environmental signals, our results provide an initial molecular framework for the integration with BRs of additional pathways that control plant development.We thank the Nottingham Arabidopsis Stock Centre, Tai-ping Sun, Zhi-Yong Wang, Yanhai Yin, Ana Cano-Delgado, Luis Lopez-Molina, and Francois Parcy for providing seeds or reagents; Laura Garcia-Carcel and Gaston Pizzio for help in the early stages of this work; and Salome Prat for fruitful discussions, sharing unpublished results, and careful reading of the manuscript. J.G.-B. holds a Consejo Superior de Investigaciones Cientificas Fellowship of the Joint Admissions Exercise Predoctoral Program. E. G. M. is recipient of a postdoctoral "Juan de la Cierva" contract from the Spanish Ministry of Science and Innovation. A. L. was supported in part by a fellowship of the Fondo per gli Investimenti della Ricerca di Base Progetto Giovani of the Italian Ministry of Education, University, and Research. Work in the authors' laboratory was funded by Spanish Ministry of Science and Innovation Grants BIO2007-60923, BIO2010-15071, and CSD2007-00057 and by Generalitat Valenciana Grants ACOMP/2010/190 and PROMETEO/2010/020. Rothamsted Research is funded by the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom.Gallego Bartolomé, J.; Minguet, EG.; Grau Enguix, F.; Abbas, M.; Locascio, AAM.; Thomas, SG.; Alabadí Diego, D.... (2012). Molecular mechanism for the interaction between gibberellin and brassinosteroid signaling pathways in Arabidopsis. Proceedings of the National Academy of Sciences. 109(33):13446-13451. https://doi.org/10.1073/pnas.1119992109S134461345110933Depuydt, S., & Hardtke, C. S. (2011). Hormone Signalling Crosstalk in Plant Growth Regulation. Current Biology, 21(9), R365-R373. doi:10.1016/j.cub.2011.03.013Alabadi, D., Blazquez, M. A., Carbonell, J., Ferrandiz, C., & Perez-Amador, M. A. (2009). Instructive roles for hormones in plant development. The International Journal of Developmental Biology, 53(8-9-10), 1597-1608. doi:10.1387/ijdb.072423daHartwell, L. H., Hopfield, J. J., Leibler, S., & Murray, A. W. (1999). From molecular to modular cell biology. Nature, 402(S6761), C47-C52. doi:10.1038/35011540Kuppusamy, K. T., Walcher, C. L., & Nemhauser, J. L. (2008). Cross-regulatory mechanisms in hormone signaling. Plant Molecular Biology, 69(4), 375-381. doi:10.1007/s11103-008-9389-2Jaillais, Y., & Chory, J. (2010). Unraveling the paradoxes of plant hormone signaling integration. Nature Structural & Molecular Biology, 17(6), 642-645. doi:10.1038/nsmb0610-642Sun, T. (2011). The Molecular Mechanism and Evolution of the GA–GID1–DELLA Signaling Module in Plants. Current Biology, 21(9), R338-R345. doi:10.1016/j.cub.2011.02.036Hou, X., Lee, L. Y. C., Xia, K., Yan, Y., & Yu, H. (2010). DELLAs Modulate Jasmonate Signaling via Competitive Binding to JAZs. Developmental Cell, 19(6), 884-894. doi:10.1016/j.devcel.2010.10.024Fonseca, S., Chico, J. M., & Solano, R. (2009). The jasmonate pathway: the ligand, the receptor and the core signalling module. Current Opinion in Plant Biology, 12(5), 539-547. doi:10.1016/j.pbi.2009.07.013Frigerio, M., Alabadí, D., Pérez-Gómez, J., García-Cárcel, L., Phillips, A. L., Hedden, P., & Blázquez, M. A. (2006). Transcriptional Regulation of Gibberellin Metabolism Genes by Auxin Signaling in Arabidopsis. Plant Physiology, 142(2), 553-563. doi:10.1104/pp.106.084871Vert, G., Walcher, C. L., Chory, J., & Nemhauser, J. L. (2008). Integration of auxin and brassinosteroid pathways by Auxin Response Factor 2. Proceedings of the National Academy of Sciences, 105(28), 9829-9834. doi:10.1073/pnas.0803996105Nemhauser, J. L., Mockler, T. C., & Chory, J. (2004). Interdependency of Brassinosteroid and Auxin Signaling in Arabidopsis. PLoS Biology, 2(9), e258. doi:10.1371/journal.pbio.0020258Clouse, S. D. (2011). Brassinosteroid Signal Transduction: From Receptor Kinase Activation to Transcriptional Networks Regulating Plant Development. The Plant Cell, 23(4), 1219-1230. doi:10.1105/tpc.111.084475Tanaka, K., Nakamura, Y., Asami, T., Yoshida, S., Matsuo, T., & Okamoto, S. (2003). Physiological Roles of Brassinosteroids in Early Growth of Arabidopsis: Brassinosteroids Have a Synergistic Relationship with Gibberellin as well as Auxin in Light-Grown Hypocotyl Elongation. Journal of Plant Growth Regulation, 22(3), 259-271. doi:10.1007/s00344-003-0119-3Alabadí, D., Gil, J., Blázquez, M. A., & García-Martínez, J. L. (2004). Gibberellins Repress Photomorphogenesis in Darkness. Plant Physiology, 134(3), 1050-1057. doi:10.1104/pp.103.035451Li, J., Nagpal, P., Vitart, V., McMorris, T. C., & Chory, J. (1996). A Role for Brassinosteroids in Light-Dependent Development of Arabidopsis. Science, 272(5260), 398-401. doi:10.1126/science.272.5260.398Szekeres, M., Németh, K., Koncz-Kálmán, Z., Mathur, J., Kauschmann, A., Altmann, T., … Koncz, C. (1996). Brassinosteroids Rescue the Deficiency of CYP90, a Cytochrome P450, Controlling Cell Elongation and De-etiolation in Arabidopsis. Cell, 85(2), 171-182. doi:10.1016/s0092-8674(00)81094-6Chory, J., Nagpal, P., & Peto, C. A. (1991). Phenotypic and Genetic Analysis of det2, a New Mutant That Affects Light-Regulated Seedling Development in Arabidopsis. The Plant Cell, 445-459. doi:10.1105/tpc.3.5.445Cheadle, C., Vawter, M. P., Freed, W. J., & Becker, K. G. (2003). Analysis of Microarray Data Using Z Score Transformation. The Journal of Molecular Diagnostics, 5(2), 73-81. doi:10.1016/s1525-1578(10)60455-2Koornneef, M., & van der Veen, J. H. (1980). Induction and analysis of gibberellin sensitive mutants in Arabidopsis thaliana (L.) heynh. Theoretical and Applied Genetics, 58(6), 257-263. doi:10.1007/bf00265176Davière, J.-M., de Lucas, M., & Prat, S. (2008). Transcriptional factor interaction: a central step in DELLA function. Current Opinion in Genetics & Development, 18(4), 295-303. doi:10.1016/j.gde.2008.05.004Leivar, P., Tepperman, J. M., Monte, E., Calderon, R. H., Liu, T. L., & Quail, P. H. (2009). Definition of Early Transcriptional Circuitry Involved in Light-Induced Reversal of PIF-Imposed Repression of Photomorphogenesis in Young Arabidopsis Seedlings. The Plant Cell, 21(11), 3535-3553. doi:10.1105/tpc.109.070672Shin, J., Kim, K., Kang, H., Zulfugarov, I. S., Bae, G., Lee, C.-H., … Choi, G. (2009). Phytochromes promote seedling light responses by inhibiting four negatively-acting phytochrome-interacting factors. Proceedings of the National Academy of Sciences, 106(18), 7660-7665. doi:10.1073/pnas.0812219106Silverstone, A. L. (2001). Repressing a Repressor: Gibberellin-Induced Rapid Reduction of the RGA Protein in Arabidopsis. THE PLANT CELL ONLINE, 13(7), 1555-1566. doi:10.1105/tpc.13.7.1555Dill, A., Jung, H.-S., & Sun, T. -p. (2001). The DELLA motif is essential for gibberellin-induced degradation of RGA. Proceedings of the National Academy of Sciences, 98(24), 14162-14167. doi:10.1073/pnas.251534098Alabadí, D., Gallego-Bartolomé, J., Orlando, L., García-Cárcel, L., Rubio, V., Martínez, C., … Blázquez, M. A. (2007). Gibberellins modulate light signaling pathways to prevent Arabidopsis seedling de-etiolation in darkness. The Plant Journal, 53(2), 324-335. doi:10.1111/j.1365-313x.2007.03346.xYin, Y., Wang, Z.-Y., Mora-Garcia, S., Li, J., Yoshida, S., Asami, T., & Chory, J. (2002). BES1 Accumulates in the Nucleus in Response to Brassinosteroids to Regulate Gene Expression and Promote Stem Elongation. Cell, 109(2), 181-191. doi:10.1016/s0092-8674(02)00721-3He, J.-X., Gendron, J. M., Yang, Y., Li, J., & Wang, Z.-Y. (2002). The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis. Proceedings of the National Academy of Sciences, 99(15), 10185-10190. doi:10.1073/pnas.152342599Wang, Z.-Y., Nakano, T., Gendron, J., He, J., Chen, M., Vafeados, D., … Chory, J. (2002). Nuclear-Localized BZR1 Mediates Brassinosteroid-Induced Growth and Feedback Suppression of Brassinosteroid Biosynthesis. Developmental Cell, 2(4), 505-513. doi:10.1016/s1534-5807(02)00153-3Ryu, H., Kim, K., Cho, H., Park, J., Choe, S., & Hwang, I. (2007). Nucleocytoplasmic Shuttling of BZR1 Mediated by Phosphorylation Is Essential in Arabidopsis Brassinosteroid Signaling. The Plant Cell, 19(9), 2749-2762. doi:10.1105/tpc.107.053728Gampala, S. S., Kim, T.-W., He, J.-X., Tang, W., Deng, Z., Bai, M.-Y., … Wang, Z.-Y. (2007). An Essential Role for 14-3-3 Proteins in Brassinosteroid Signal Transduction in Arabidopsis. Developmental Cell, 13(2), 177-189. doi:10.1016/j.devcel.2007.06.009De Lucas, M., Davière, J.-M., Rodríguez-Falcón, M., Pontin, M., Iglesias-Pedraz, J. M., Lorrain, S., … Prat, S. (2008). A molecular framework for light and gibberellin control of cell elongation. Nature, 451(7177), 480-484. doi:10.1038/nature06520Sun, T., & Gubler, F. (2004). MOLECULAR MECHANISM OF GIBBERELLIN SIGNALING IN PLANTS. Annual Review of Plant Biology, 55(1), 197-223. doi:10.1146/annurev.arplant.55.031903.141753He, J.-X. (2005). BZR1 Is a Transcriptional Repressor with Dual Roles in Brassinosteroid Homeostasis and Growth Responses. Science, 307(5715), 1634-1638. doi:10.1126/science.1107580Sun, Y., Fan, X.-Y., Cao, D.-M., Tang, W., He, K., Zhu, J.-Y., … Wang, Z.-Y. (2010). Integration of Brassinosteroid Signal Transduction with the Transcription Network for Plant Growth Regulation in Arabidopsis. Developmental Cell, 19(5), 765-777. doi:10.1016/j.devcel.2010.10.010Triezenberg, S. J., Kingsbury, R. C., & McKnight, S. L. (1988). Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes & Development, 2(6), 718-729. doi:10.1101/gad.2.6.718Feng, S., Martinez, C., Gusmaroli, G., Wang, Y., Zhou, J., Wang, F., … Deng, X. W. (2008). Coordinated regulation of Arabidopsis thaliana development by light and gibberellins. Nature, 451(7177), 475-479. doi:10.1038/nature06448Gallego-Bartolomé, J., Arana, M. V., Vandenbussche, F., Žádníková, P., Minguet, E. G., Guardiola, V., … Blázquez, M. A. (2011). Hierarchy of hormone action controlling apical hook development in Arabidopsis. The Plant Journal, 67(4), 622-634. doi:10.1111/j.1365-313x.2011.04621.xGallego-Bartolomé, J., Alabadí, D., & Blázquez, M. A. (2011). DELLA-Induced Early Transcriptional Changes during Etiolated Development in Arabidopsis thaliana. PLoS ONE, 6(8), e23918. doi:10.1371/journal.pone.0023918Steber, C. M., & McCourt, P. (2001). A Role for Brassinosteroids in Germination in Arabidopsis. Plant Physiology, 125(2), 763-769. doi:10.1104/pp.125.2.763Yin, Y., Vafeados, D., Tao, Y., Yoshida, S., Asami, T., & Chory, J. (2005). A New Class of Transcription Factors Mediates Brassinosteroid-Regulated Gene Expression in Arabidopsis. Cell, 120(2), 249-259. doi:10.1016/j.cell.2004.11.044Müller, B., & Sheen, J. (2008). Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis. Nature, 453(7198), 1094-1097. doi:10.1038/nature0694

When the levee of sympathetic outflow breaks

In this issue of Immunity, Bi et al. identify a microglia-neuron signaling axis that is critical for maintaining central control of the sympathetic nervous system. They find that platelet growth factor B released by microglia acts on neurons via PDGFRα to regulate sympathetic outflow. Disrupting this pathway leads to neuronal excitability, highlighting a promising therapeutic target to modulate sympathetic outflow and reduce hypertension

‘God’s Truth’: Kant, Mill and moral epistemology in Oliver Twist

This essay aims to demonstrate how Dickens’s search for ‘truth’ (and his understanding of what that abstraction consists of) entered into and emerged from one of the key philosophical discussions of the early nineteenth century: namely whether moral knowledge is the sum of one’s experiences or whether there are such things as a priori or ‘natural’ principles of ethics that transcend human practice

Single-cell transcriptomics of the developing lateral geniculate nucleus reveals insights into circuit assembly and refinement

Coordinated changes in gene expression underlie the early patterning and cell-type specification of the central nervous system. However, much less is known about how such changes contribute to later stages of circuit assembly and refinement. In this study, we employ single-cell RNA sequencing to develop a detailed, whole-transcriptome resource of gene expression across four time points in the developing dorsal lateral geniculate nucleus (LGN), a visual structure in the brain that undergoes a well-characterized program of postnatal circuit development. This approach identifies markers defining the major LGN cell types, including excitatory relay neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells. Most cell types exhibit significant transcriptional changes across development, dynamically expressing genes involved in distinct processes including retinotopic mapping, synaptogenesis, myelination, and synaptic refinement. Our data suggest that genes associated with synapse and circuit development are expressed in a larger proportion of nonneuronal cell types than previously appreciated. Furthermore, we used this single-cell expression atlas to identify the Prkcd-Cre mouse line as a tool for selective manipulation of relay neurons during a late stage of sensory-driven synaptic refinement. This transcriptomic resource provides a cellular map of gene expression across several cell types of the LGN, and offers insight into the molecular mechanisms of circuit development in the postnatal brain

Sensory Experience Engages Microglia to Shape Neural Connectivity through a Non-Phagocytic Mechanism.

Sensory experience remodels neural circuits in the early postnatal brain through mechanisms that remain to be elucidated. Applying a new method of ultrastructural analysis to the retinogeniculate circuit, we find that visual experience alters the number and structure of synapses between the retina and the thalamus. These changes require vision-dependent transcription of the receptor Fn14 in thalamic relay neurons and the induction of its ligand TWEAK in microglia. Fn14 functions to increase the number of bulbous spine-associated synapses at retinogeniculate connections, likely contributing to the strengthening of the circuit that occurs in response to visual experience. However, at retinogeniculate connections near TWEAK-expressing microglia, TWEAK signals via Fn14 to restrict the number of bulbous spines on relay neurons, leading to the elimination of a subset of connections. Thus, TWEAK and Fn14 represent an intercellular signaling axis through which microglia shape retinogeniculate connectivity in response to sensory experience

Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q.

Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gα(q) activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gβγ and Gα(q) contribute to activation of PLCβ3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gβγ and Gα(q) on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent