Gibberellins and ovule number: a molecular mechanism

Tesis por compendio[ES] Como precursores de las semillas, los óvulos representan un órgano fundamental durante el ciclo de vida de las plantas. Debido a su importancia, el desarrollo del óvulo ha sido estudiado durante décadas desde un punto de vista morfológico y molecular, lo que ha permitido dilucidar la compleja e intrincada red de regulación genética que lo rige. En concreto, la iniciación del óvulo está controlada por las hormonas vegetales auxinas, citoquininas y brasinoesteroides (BRs), siendo todas ellas reguladoras positivas del número de óvulos. Recientemente demostramos que las giberelinas (GAs) modulan negativamente el número de óvulos mediante la desestabilización de las proteínas DELLA. Sin embargo, aún debe aclararse cómo encajan las GAs y las proteínas DELLA en el modelo regulador de la iniciación de los óvulos. El trabajo presentado en esta tesis doctoral tiene como objetivo aclarar el mecanismo molecular por el cual las GAs actúan en la iniciación del óvulo. Después de una introducción general, en el Capítulo 1 mostramos que tanto las GAs como los BRs regulan el número de óvulos en Arabidopsis independientemente de los niveles de actividad de la otra hormona, lo que sugiere que las GAs y los BRs actúan de forma independiente para controlar la iniciación del óvulo. En el Capítulo 2 proporcionamos evidencias genéticas y moleculares que apuntan a que las proteínas DELLA participan en la iniciación de los óvulos mediante su interacción con el factor de transcripción CUC2 en las células placentarias. En conjunto, los hallazgos presentados aquí nos han permitido integrar a las GAs y proteínas DELLA en la red genética que guía el inicio de los primordios de óvulos. Una discusión final destaca las preguntas abiertas que aún deben abordarse para comprender completamente el control hormonal de la iniciación de los óvulos en las plantas.[CAT] Com a precursors de les llavors, els òvuls representen un òrgan fonamental durant el cicle de vida de les plantes. A causa de la seva importància, el desenvolupament de l'òvul ha estat estudiat durant dècades des d'un punt de vista morfològic i molecular, el que ha permès dilucidar la complexa i intricada xarxa de regulació genètica que el regeix. En concret, la iniciació del òvul està controlada per les hormones vegetals auxines, citoquinines i brasinoesteroides (BRs), sent totes elles reguladores positives del nombre d'òvuls. Recentment demostrem que les gibberel·lines (GAs) modulen negativament el nombre d'òvuls mitjançant la desestabilització de les proteïnes DELLA. No obstant, encara s'ha d'aclarir com encaixen les GAs i les proteïnes DELLA al model regulador de la iniciació dels òvuls. El treball presentat en aquesta tesi doctoral té com a objectiu aclarir el mecanisme molecular pel qual les GAs actuen a la iniciació de l'òvul. Després d'una introducció general, al Capítol 1 mostrem que tant les GAs com els BRs regulen el nombre d'òvuls a Arabidopsis independentment dels nivells d'activitat de l'altra hormona, cosa que suggereix que les GAs i els BRs actuen de forma independent per controlar la iniciació de l'òvul. Al Capítol 2 proporcionem evidències genètiques i moleculars que apunten que les proteïnes DELLA participen en la iniciació dels òvuls mitjançant la seva interacció amb el factor de transcripció CUC2 a les cèl·lules placentàries. En conjunt, els descobriments presentats ací ens han permès integrar les GAs i proteïnes DELLA a la xarxa genètica que guia l'inici dels primordis d'òvuls. Una discussió final destaca les preguntes obertes que encara cal abordar per comprendre completament el control hormonal de la iniciació dels òvuls a les plantes.[EN] As precursors of seeds, ovules represent a fundamental organ during the plant life cycle. Due to their importance, ovule development has been studied for decades from a morphological and molecular point of view, allowing the elucidation of a complex and intricate gene regulatory network governing it. Specifically, ovule initiation is controlled by the plant hormones auxins, cytokinins and brassinosteroids (BRs), all of them being positive regulators of ovule number. Recently, we demonstrated that gibberellins (GAs) negatively module ovule number by the destabilization of DELLA proteins. However, how GAs and DELLA proteins fit in the regulatory model for ovule initiation still needs to be clarified. The work presented in this PhD thesis aims to clarify the molecular mechanism by which GAs act in ovule initiation. After a comprehensive introduction, we show in Chapter 1 that both GAs and BRs regulate ovule number in Arabidopsis regardless of the activity levels of the other hormone, suggesting that GAs and BRs act independently to control ovule initiation. In Chapter 2 we provide genetic and molecular evidence pointing to DELLA proteins participating in ovule initiation by the interaction with the CUC2 transcription factor in placental cells. Collectively, the findings presented here allowed us to integrate GAs and DELLA proteins in the gene regulatory network guiding ovule primordia initiation. A final discussion highlights open questions that still need to be addressed to fully understand the hormonal control of ovule initiation in plants.La realización de esta Tesis Doctoral ha sido posible gracias a un contrato para la Formación de Personal Investigador de la Universidad Politécnica de Valencia (durante un año y medio) y a un contrato para la Formación de Profesorado Universitario (FPU18/00331) del Ministerio de Universidades (durante dos años y medio). Las estancias breves en Chile y Francia fueron posible gracias a la financiación H2020-MSCA-RISE-2014 y a una ayuda EMBO Short-Term (STF 8961), respectivamente. El trabajo experimental ha sido financiado por los proyectos BIO2017-83138-R y PID2020-113920RB-100 del Ministerio de Ciencia e Innovación y AICO/2020/256 de la Generalitat Valenciana.Barro Trastoy, D. (2022). Gibberellins and ovule number: a molecular mechanism [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/187756Compendi

On the way to ovules: The hormonal regulation of ovule development

[EN] This review focuses on the hormonal regulation of ovule development, especially on ovule initiation, patterning, and morphogenesis. Understanding of the genetic and molecular basis of ovule development is essential from both the scientific and economic perspective. The ovule represents an attractive system to study lateral organ development in plants, and, since ovules are the precursors of seeds, full comprehension of this process can be the key to the improvement of crops, especially those depending on high production of seeds and grains. Ovule initiation, patterning, and morphogenesis are governed by complex genetic and hormonal networks involving auxins, cytokinins, brassinosteroids, and gibberellins. These coordinate the determination of the ovule number, size, and shape through the regulation of the number of ovule primordia that arise from the placenta and/or ensuring their correct development into mature functional ovules. Here we summarize the current knowledge of how ovules are formed, paying special attention to the roles of these four plant hormones.This work was supported by the Spanish Ministry for Science and Innovation-FEDER under [grant BIO2017-83138R].Barro-Trastoy, D.; Gómez, MD.; Tornero Feliciano, P.; Perez Amador, MA. (2020). On the way to ovules: The hormonal regulation of ovule development. Critical Reviews in Plant Sciences. 39(5):431-456. https://doi.org/10.1080/07352689.2020.1820203S431456395Aida, M., & Tasaka, M. (2006). Genetic control of shoot organ boundaries. Current Opinion in Plant Biology, 9(1), 72-77. doi:10.1016/j.pbi.2005.11.011Aida, M., Ishida, T., Fukaki, H., Fujisawa, H., & Tasaka, M. (1997). Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. The Plant Cell, 9(6), 841-857. doi:10.1105/tpc.9.6.841Armenta-Medina, A., & Gillmor, C. S. (2019). Genetic, molecular and parent-of-origin regulation of early embryogenesis in flowering plants. Plant Development and Evolution, 497-543. doi:10.1016/bs.ctdb.2018.11.008Azhakanandam, S., Nole-Wilson, S., Bao, F., & Franks, R. G. (2008). SEUSSandAINTEGUMENTAMediate Patterning and Ovule Initiation during Gynoecium Medial Domain Development    . Plant Physiology, 146(3), 1165-1181. doi:10.1104/pp.107.114751Baker, C. C., Sieber, P., Wellmer, F., & Meyerowitz, E. M. (2005). The early extra petals1 Mutant Uncovers a Role for MicroRNA miR164c in Regulating Petal Number in Arabidopsis. Current Biology, 15(4), 303-315. doi:10.1016/j.cub.2005.02.017Balasubramanian, S., & Schneitz, K. (2000). NOZZLE regulates proximal-distal pattern formation, cell proliferation and early sporogenesis during ovule development in Arabidopsis thaliana. Development, 127(19), 4227-4238. doi:10.1242/dev.127.19.4227Balasubramanian, S., & Schneitz, K. (2002). NOZZLE links proximal-distal and adaxial-abaxial pattern formation during ovule development in Arabidopsis thaliana. Development, 129(18), 4291-4300. doi:10.1242/dev.129.18.4291Bao, F., Azhakanandam, S., & Franks, R. G. (2009). SEUSSandSEUSS-LIKETranscriptional Adaptors Regulate Floral and Embryonic Development in Arabidopsis. Plant Physiology, 152(2), 821-836. doi:10.1104/pp.109.146183Barro‐Trastoy, D., Carrera, E., Baños, J., Palau‐Rodríguez, J., Ruiz‐Rivero, O., Tornero, P., … Pérez‐Amador, M. A. (2020). Regulation of ovule initiation by gibberellins and brassinosteroids in tomato and Arabidopsis: two plant species, two molecular mechanisms. The Plant Journal, 102(5), 1026-1041. doi:10.1111/tpj.14684Bartrina, I., Otto, E., Strnad, M., Werner, T., & Schmülling, T. (2011). Cytokinin Regulates the Activity of Reproductive Meristems, Flower Organ Size, Ovule Formation, and Thus Seed Yield in Arabidopsis thaliana      . The Plant Cell, 23(1), 69-80. doi:10.1105/tpc.110.079079Becker, A. (2020). A molecular update on the origin of the carpel. Current Opinion in Plant Biology, 53, 15-22. doi:10.1016/j.pbi.2019.08.009Bencivenga, S., Simonini, S., Benková, E., & Colombo, L. (2012). The Transcription Factors BEL1 and SPL Are Required for Cytokinin and Auxin Signaling During Ovule Development in Arabidopsis. The Plant Cell, 24(7), 2886-2897. doi:10.1105/tpc.112.100164Benková, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertová, D., Jürgens, G., & Friml, J. (2003). Local, Efflux-Dependent Auxin Gradients as a Common Module for Plant Organ Formation. Cell, 115(5), 591-602. doi:10.1016/s0092-8674(03)00924-3BERRY, P. M., & SPINK, J. H. (2009). Understanding the effect of a triazole with anti-gibberellin activity on the growth and yield of oilseed rape (Brassica napus). The Journal of Agricultural Science, 147(3), 273-285. doi:10.1017/s0021859609008491BOUTTIER, C., & MORGAN, D. G. (1992). Ovule Development and Determination of Seed Number Per Pod in Oilseed Rape (Brassica napusL.). Journal of Experimental Botany, 43(5), 709-714. doi:10.1093/jxb/43.5.709Bowman, J. L., Smyth, D. R., & Meyerowitz, E. M. (1991). Genetic interactions among floral homeotic genes of Arabidopsis. Development, 112(1), 1-20. doi:10.1242/dev.112.1.1Brambilla, V., Battaglia, R., Colombo, M., Masiero, S., Bencivenga, S., Kater, M. M., & Colombo, L. (2007). Genetic and Molecular Interactions between BELL1 and MADS Box Factors Support Ovule Development inArabidopsis. The Plant Cell, 19(8), 2544-2556. doi:10.1105/tpc.107.051797Broadhvest, J., Baker, S. C., & Gasser, C. S. (2000). SHORT INTEGUMENTS 2 Promotes Growth During Arabidopsis Reproductive Development. Genetics, 155(2), 899-907. doi:10.1093/genetics/155.2.899Brumos, J., Robles, L. M., Yun, J., Vu, T. C., Jackson, S., Alonso, J. M., & Stepanova, A. N. (2018). Local Auxin Biosynthesis Is a Key Regulator of Plant Development. Developmental Cell, 47(3), 306-318.e5. doi:10.1016/j.devcel.2018.09.022Carter, B., Henderson, J. T., Svedin, E., Fiers, M., McCarthy, K., Smith, A., … Ogas, J. (2016). Cross-Talk Between Sporophyte and Gametophyte Generations Is Promoted by CHD3 Chromatin Remodelers in Arabidopsis thaliana. Genetics, 203(2), 817-829. doi:10.1534/genetics.115.180141Ceccato, L., Masiero, S., Sinha Roy, D., Bencivenga, S., Roig-Villanova, I., Ditengou, F. A., … Colombo, L. (2013). Maternal Control of PIN1 Is Required for Female Gametophyte Development in Arabidopsis. PLoS ONE, 8(6), e66148. doi:10.1371/journal.pone.0066148Chevalier, D., Batoux, M., Fulton, L., Pfister, K., Yadav, R. K., Schellenberg, M., & Schneitz, K. (2005). STRUBBELIG defines a receptor kinase-mediated signaling pathway regulating organ development in Arabidopsis. Proceedings of the National Academy of Sciences, 102(25), 9074-9079. doi:10.1073/pnas.0503526102Christensen, C. A., King, E. J., Jordan, J. R., & Drews, G. N. (1997). Megagametogenesis in Arabidopsis wild type and the Gf mutant. Sexual Plant Reproduction, 10(1), 49-64. doi:10.1007/s004970050067Conner, J., & Liu, Z. (2000). LEUNIG, a putative transcriptional corepressor that regulates AGAMOUS expression during flower development. Proceedings of the National Academy of Sciences, 97(23), 12902-12907. doi:10.1073/pnas.230352397Cucinotta, M., Colombo, L., & Roig-Villanova, I. (2014). Ovule development, a new model for lateral organ formation. Frontiers in Plant Science, 5. doi:10.3389/fpls.2014.00117Cucinotta, M., Di Marzo, M., Guazzotti, A., de Folter, S., Kater, M. M., & Colombo, L. (2020). Gynoecium size and ovule number are interconnected traits that impact seed yield. Journal of Experimental Botany, 71(9), 2479-2489. doi:10.1093/jxb/eraa050Cucinotta, M., Manrique, S., Cuesta, C., Benkova, E., Novak, O., & Colombo, L. (2018). CUP-SHAPED COTYLEDON1 (CUC1) and CUC2 regulate cytokinin homeostasis to determine ovule number in Arabidopsis. Journal of Experimental Botany, 69(21), 5169-5176. doi:10.1093/jxb/ery281Cucinotta, M., Manrique, S., Guazzotti, A., Quadrelli, N. E., Mendes, M. A., Benkova, E., & Colombo, L. (2016). Cytokinin response factors integrate auxin and cytokinin pathways for female reproductive organ development. Development. doi:10.1242/dev.143545Davière, J.-M., & Achard, P. (2016). A Pivotal Role of DELLAs in Regulating Multiple Hormone Signals. Molecular Plant, 9(1), 10-20. doi:10.1016/j.molp.2015.09.011Denay, G., Chahtane, H., Tichtinsky, G., & Parcy, F. (2017). A flower is born: an update on Arabidopsis floral meristem formation. Current Opinion in Plant Biology, 35, 15-22. doi:10.1016/j.pbi.2016.09.003Elliott, R. C., Betzner, A. S., Huttner, E., Oakes, M. P., Tucker, W. Q., Gerentes, D., … Smyth, D. R. (1996). AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth. The Plant Cell, 8(2), 155-168. doi:10.1105/tpc.8.2.155Endress, P. K. (2011). Angiosperm ovules: diversity, development, evolution. Annals of Botany, 107(9), 1465-1489. doi:10.1093/aob/mcr120Enugutti, B., & Schneitz, K. (2013). Genetic analysis of ectopic growth suppression during planar growth of integuments mediated by the Arabidopsis AGC protein kinase UNICORN. BMC Plant Biology, 13(1), 2. doi:10.1186/1471-2229-13-2Enugutti, B., Kirchhelle, C., Oelschner, M., Torres Ruiz, R. A., Schliebner, I., Leister, D., & Schneitz, K. (2012). Regulation of planar growth by the Arabidopsis AGC protein kinase UNICORN. Proceedings of the National Academy of Sciences, 109(37), 15060-15065. doi:10.1073/pnas.1205089109Erbasol Serbes, I., Palovaara, J., & Groß-Hardt, R. (2019). Development and function of the flowering plant female gametophyte. Plant Development and Evolution, 401-434. doi:10.1016/bs.ctdb.2018.11.016Eshed, Y., Baum, S. F., Perea, J. V., & Bowman, J. L. (2001). Establishment of polarity in lateral organs of plants. Current Biology, 11(16), 1251-1260. doi:10.1016/s0960-9822(01)00392-xFavaro, R., Pinyopich, A., Battaglia, R., Kooiker, M., Borghi, L., Ditta, G., … Colombo, L. (2003). MADS-Box Protein Complexes Control Carpel and Ovule Development in Arabidopsis. The Plant Cell, 15(11), 2603-2611. doi:10.1105/tpc.015123Ferreira, L. G., de Alencar Dusi, D. M., Irsigler, A. S. T., Gomes, A. C. M. M., Mendes, M. A., Colombo, L., & de Campos Carneiro, V. T. (2017). GID1 expression is associated with ovule development of sexual and apomictic plants. Plant Cell Reports, 37(2), 293-306. doi:10.1007/s00299-017-2230-0Franks, R. G., Wang, C., Levin, J. Z., & Liu, Z. (2002). SEUSS, a member of a novel family of plant regulatory proteins, represses floral homeotic gene expression withLEUNIG. Development, 129(1), 253-263. doi:10.1242/dev.129.1.253Fridman, Y., & Savaldi-Goldstein, S. (2013). Brassinosteroids in growth control: How, when and where. Plant Science, 209, 24-31. doi:10.1016/j.plantsci.2013.04.002Friedt, W., Tu, J., & Fu, T. (2018). Academic and Economic Importance of Brassica napus Rapeseed. The Brassica napus Genome, 1-20. doi:10.1007/978-3-319-43694-4_1Gaiser, J. C., Robinson-Beers, K., & Gasser, C. S. (1995). The Arabidopsis SUPERMAN Gene Mediates Asymmetric Growth of the Outer Integument of Ovules. The Plant Cell, 7(3), 333. doi:10.2307/3869855Galbiati, F., Sinha Roy, D., Simonini, S., Cucinotta, M., Ceccato, L., Cuesta, C., … Colombo, L. (2013). An integrative model of the control of ovule primordia formation. The Plant Journal, 76(3), 446-455. doi:10.1111/tpj.12309Gallego-Giraldo, C., Hu, J., Urbez, C., Gomez, M. D., Sun, T., & Perez-Amador, M. A. (2014). Role of the gibberellin receptors GID1 during fruit-set in Arabidopsis. The Plant Journal, 79(6), 1020-1032. doi:10.1111/tpj.12603Gasser, C. S., & Skinner, D. J. (2019). Development and evolution of the unique ovules of flowering plants. Plant Development and Evolution, 373-399. doi:10.1016/bs.ctdb.2018.10.007Gifford, M. L., Dean, S., & Ingram, G. C. (2003). TheArabidopsis ACR4gene plays a role in cell layer organisation during ovule integument and sepal margin development. Development, 130(18), 4249-4258. doi:10.1242/dev.00634Goldental-Cohen, S., Israeli, A., Ori, N., & Yasuor, H. (2017). Auxin Response Dynamics During Wild-Type and entire Flower Development in Tomato. Plant and Cell Physiology, 58(10), 1661-1672. doi:10.1093/pcp/pcx102Gomez, M. D., Barro-Trastoy, D., Escoms, E., Saura-Sánchez, M., Sánchez, I., Briones-Moreno, A., … Perez-Amador, M. A. (2018). Gibberellins negatively modulate ovule number in plants. Development. doi:10.1242/dev.163865Gomez, M. D., Barro-Trastoy, D., Fuster-Almunia, C., Tornero, P., Alonso, J. M., & Perez-Amador, M. A. (2020). Gibberellin-mediated RGA-LIKE1 degradation regulates embryo sac development in Arabidopsis. Journal of Experimental Botany, 71(22), 7059-7072. doi:10.1093/jxb/eraa395Gómez, M. D., Fuster-Almunia, C., Ocaña-Cuesta, J., Alonso, J. M., & Pérez-Amador, M. A. (2019). RGL2 controls flower development, ovule number and fertility in Arabidopsis. Plant Science, 281, 82-92. doi:10.1016/j.plantsci.2019.01.014Gomez, M. D., Urbez, C., Perez-Amador, M. A., & Carbonell, J. (2011). Characterization of constricted fruit (ctf) Mutant Uncovers a Role for AtMYB117/LOF1 in Ovule and Fruit Development in Arabidopsis thaliana. PLoS ONE, 6(4), e18760. doi:10.1371/journal.pone.0018760Gomez, M. D., Ventimilla, D., Sacristan, R., & Perez-Amador, M. A. (2016). Gibberellins Regulate Ovule Integument Development by Interfering with the Transcription Factor ATS. Plant Physiology, 172(4), 2403-2415. doi:10.1104/pp.16.01231Gonçalves, B., Hasson, A., Belcram, K., Cortizo, M., Morin, H., Nikovics, K., … Arnaud, N. (2015). A conserved role forCUP-SHAPED COTYLEDONgenes during ovule development. The Plant Journal, 83(4), 732-742. doi:10.1111/tpj.12923Grobeta-Hardt, R. (2002). WUSCHEL signaling functions in interregional communication during Arabidopsis ovule development. Genes & Development, 16(9), 1129-1138. doi:10.1101/gad.225202Hashimoto, K., Miyashima, S., Sato-Nara, K., Yamada, T., & Nakajima, K. (2018). Functionally Diversified Members of the MIR165/6 Gene Family Regulate Ovule Morphogenesis in Arabidopsis thaliana. Plant and Cell Physiology, 59(5), 1017-1026. doi:10.1093/pcp/pcy042Hauser, B. A., He, J. Q., Park, S. O., & Gasser, C. S. (2000). TSO1 is a novel protein that modulates cytokinesis and cell expansion in Arabidopsis. Development, 127(10), 2219-2226. doi:10.1242/dev.127.10.2219Hedden, P., & Sponsel, V. (2015). A Century of Gibberellin Research. Journal of Plant Growth Regulation, 34(4), 740-760. doi:10.1007/s00344-015-9546-1Heisler, M. G., & Byrne, M. E. (2020). Progress in understanding the role of auxin in lateral organ development in plants. Current Opinion in Plant Biology, 53, 73-79. doi:10.1016/j.pbi.2019.10.007Heisler, M. G., Ohno, C., Das, P., Sieber, P., Reddy, G. V., Long, J. A., & Meyerowitz, E. M. (2005). Patterns of Auxin Transport and Gene Expression during Primordium Development Revealed by Live Imaging of the Arabidopsis Inflorescence Meristem. Current Biology, 15(21), 1899-1911. doi:10.1016/j.cub.2005.09.052Hibara, K., Takada, S., & Tasaka, M. (2003). CUC1 gene activates the expression of SAM-related genes to induce adventitious shoot formation. The Plant Journal, 36(5), 687-696. doi:10.1046/j.1365-313x.2003.01911.xHill, T. A., Broadhvest, J., Kuzoff, R. K., & Gasser, C. S. (2006). Arabidopsis SHORT INTEGUMENTS 2 Is a Mitochondrial DAR GTPase. Genetics, 174(2), 707-718. doi:10.1534/genetics.106.060657Huang, H.-Y., Jiang, W.-B., Hu, Y.-W., Wu, P., Zhu, J.-Y., Liang, W.-Q., … Lin, W.-H. (2013). BR Signal Influences Arabidopsis Ovule and Seed Number through Regulating Related Genes Expression by BZR1. Molecular Plant, 6(2), 456-469. doi:10.1093/mp/sss070Hwang, I., Sheen, J., & Müller, B. (2012). Cytokinin Signaling Networks. Annual Review of Plant Biology, 63(1), 353-380. doi:10.1146/annurev-arplant-042811-105503Ishida, T., Aida, M., Takada, S., & Tasaka, M. (2000). Involvement of CUP-SHAPED COTYLEDON Genes in Gynoecium and Ovule Development in Arabidopsis thaliana. Plant and Cell Physiology, 41(1), 60-67. doi:10.1093/pcp/41.1.60Jia, D., Chen, L., Yin, G., Yang, X., Gao, Z., Guo, Y., … Tang, W. (2020). Brassinosteroids regulate outer ovule integument growth in part via the control ofINNER NO OUTERby BRASSINOZOLE‐RESISTANT family transcription factors. Journal of Integrative Plant Biology, 62(8), 1093-1111. doi:10.1111/jipb.12915Jung, J.-H., & Park, C.-M. (2006). MIR166/165 genes exhibit dynamic expression patterns in regulating shoot apical meristem and floral development in Arabidopsis. Planta, 225(6), 1327-1338. doi:10.1007/s00425-006-0439-1Kelley, D. R., Arreola, A., Gallagher, T. L., & Gasser, C. S. (2012). ETTIN (ARF3) physically interacts with KANADI proteins to form a functional complex essential for integument development and polarity determination in Arabidopsis. Development, 139(6), 1105-1109. doi:10.1242/dev.067918Kelley, D. R., Skinner, D. J., & Gasser, C. S. (2009). Roles of polarity determinants in ovule development. The Plant Journal, 57(6), 1054-1064. doi:10.1111/j.1365-313x.2008.03752.xKhan, S. U., Yangmiao, J., Liu, S., Zhang, K., Khan, M. H. U., Zhai, Y., … Zhou, Y. (2019). Genome-wide association studies in the genetic dissection of ovule number, seed number, and seed weight in Brassica napus L. Industrial Crops and Products, 142, 111877. doi:10.1016/j.indcrop.2019.111877Klucher, K. M., Chow, H., Reiser, L., & Fischer, R. L. (1996). The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floral homeotic gene APETALA2. The Plant Cell, 8(2), 137-153. doi:10.1105/tpc.8.2.137Krizek, B. A. (1999). Ectopic expression ofAINTEGUMENTA inArabidopsis plants results in increased growth of floral organs. Developmental Genetics, 25(3), 224-236. doi:10.1002/(sici)1520-6408(1999)25:33.0.co;2-yKrizek, B. A., Blakley, I. C., Ho, Y., Freese, N., & Loraine, A. E. (2020). The Arabidopsis transcription factor AINTEGUMENTA orchestrates patterning genes and auxin signaling in the establishment of floral growth and form. The Plant Journal, 103(2), 752-768. doi:10.1111/tpj.14769Larsson, E., Roberts, C. J., Claes, A. R., Franks, R. G., & Sundberg, E. (2014). Polar Auxin Transport Is Essential for Medial versus Lateral Tissue Specification and Vascular-Mediated Valve Outgrowth in Arabidopsis Gynoecia. Plant Physiology, 166(4), 1998-2012. doi:10.1104/pp.114.245951Larsson, E., Vivian-Smith, A., Offringa, R., & Sundberg, E. (2017). Auxin Homeostasis in Arabidopsis Ovules Is Anther-Dependent at Maturation and Changes Dynamically upon Fertilization. Frontiers in Plant Science, 8. doi:10.3389/fpls.2017.01735Laufs, P., Peaucelle, A., Morin, H., & Traas, J. (2004). MicroRNA regulation of the CUC genes is required for boundary size control in Arabidopsis meristems. Development, 131(17), 4311-4322. doi:10.1242/dev.01320Lee, D.-K., Geisler, M., & Springer, P. S. (2009). LATERAL ORGAN FUSION1 and LATERAL ORGAN FUSION2function in lateral organ separation and axillary meristem formation in Arabidopsis. Development, 136(14), 2423-2432. doi:10.1242/dev.031971Leyser, O. (2017). Auxin Signaling. Plant Physiology, 176(1), 465-479. doi:10.1104/pp.17.00765Li, L., Qin, G., Tsuge, T., Hou, X., Ding, M., Aoyama, T., … Qu, L. (2008). SPOROCYTELESSmodulatesYUCCAexpression to regulate the development of lateral organs in Arabidopsis. New Phytologist, 179(3), 751-764. doi:10.1111/j.1469-8137.2008.02514.xLiao, S., Wang, L., Li, J., & Ruan, Y.-L. (2020). Cell Wall Invertase Is Essential for Ovule Development through Sugar Signaling Rather Than Provision of Carbon Nutrients. Plant Physiology, 183(3), 1126-1144. doi:10.1104/pp.20.00400Lieber, D., Lora, J., Schrempp, S., Lenhard, M., & Laux, T. (2011). Arabidopsis WIH1 and WIH2 Genes Act in the Transition from Somatic to Reproductive Cell Fate. Current Biology, 21(12), 1009-1017. doi:10.1016/j.cub.2011.05.015Lituiev, D. S., Krohn, N. G., Müller, B., Jackson, D., Hellriegel, B., Dresselhaus, T., & Grossniklaus, U. (2013). Theoretical and experimental evidence indicates that there is no detectable auxin gradient in the angiosperm female gametophyte. Development, 140(22), 4544-4553. doi:10.1242/dev.098301Liu, H.-H., Xiong, F., Duan, C.-Y., Wu, Y.-N., Zhang, Y., & Li, S. (2019). Importin β4 Mediates Nuclear Import of GRF-Interacting Factors to Control Ovule Development in Arabidopsis. Plant Physiology, 179(3), 1080-1092. doi:10.1104/pp.18.01135Liu, Z., Franks, R. G., & Klink, V. P. (2000). Regulation of Gynoecium Marginal Tissue Formation by LEUNIG and AINTEGUMENTA. The Plant Cell, 12(10), 1879-1891. doi:10.1105/tpc.12.10.1879Lora, J., Yang, X., & Tucker, M. R. (2019). Establishing a framework for female germline initiation in the plant ovule. Journal of Experimental Botany, 70(11), 2937-2949. doi:10.1093/jxb/erz212Mallory, A. C., Dugas, D. V., Bartel, D. P., & Bartel, B. (2004). MicroRNA Regulation of NAC-Domain Targets Is Required for Proper Formation and Separation of Adjacent Embryonic, Vegetative, and Floral Organs. Current Biology, 14(12), 1035-1046. doi:10.1016/j.cub.2004.06.022La Rosa, N. M. -d., Sotillo, B., Miskolczi, P., Gibbs, D. J., Vicente, J., Carbonero, P., … Blazquez, M. A. (2014). Large-Scale Identification of Gibberellin-Related Transcription Factors Defines Group VII ETHYLENE RESPONSE FACTORS as Functional DELLA Partners. PLANT PHYSIOLOGY, 166(2), 1022-1032. doi:10.1104/pp.114.244723Marsch-Martínez, N., & de Folter, S. (2016). Hormonal control of the development of the gynoecium. Current Opinion in Plant Biology, 29, 104-114. doi:10.1016/j.pbi.2015.12.006Matilla, A. J. (2019). Seed coat formation: its evolution and regulation. Seed Science Research, 29(4), 215-226. doi:10.1017/s0960258519000254McAbee, J. M., Hill, T. A., Skinner, D. J., Izhaki, A., Hauser, B. A., Meister, R. J., … Gasser, C. S. (2006). ABERRANT TESTA SHAPEencodes a KANADI family member, linking polarity determination to separation and growth of Arabidopsis ovule integuments. The Plant Journal, 46(3), 522-531. doi:10.1111/j.1365-313x

Regulation of ovule initiation by gibberellins and brassinosteroids in tomato and Arabidopsis: two plant species, two molecular mechanisms

This is the peer reviewed version of the following article: Barro¿Trastoy, D., Carrera, E., Baños, J., Palau-Rodríguez, J., Ruiz-Rivero, O., Tornero, P., Alonso, J.M., López-Díaz, I., Gómez, M.D. and Pérez-Amador, M.A. (2020), Regulation of ovule initiation by gibberellins and brassinosteroids in tomato and Arabidopsis: two plant species, two molecular mechanisms. Plant J, 102: 1026-1041, which has been published in final form at https://doi.org/10.1111/tpj.14684. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] Ovule primordia formation is a complex developmental process with a strong impact on the production of seeds. In Arabidopsis this process is controlled by a gene network, including components of the signalling pathways of auxin, brassinosteroids (BRs) and cytokinins. Recently, we have shown that gibberellins (GAs) also play an important role in ovule primordia initiation, inhibiting ovule formation in both Arabidopsis and tomato. Here we reveal that BRs also participate in the control of ovule initiation in tomato, by promoting an increase on ovule primordia formation. Moreover, molecular and genetic analyses of the co-regulation by GAs and BRs of the control of ovule initiation indicate that two different mechanisms occur in tomato and Arabidopsis. In tomato, GAs act downstream of BRs. BRs regulate ovule number through the downregulation of GA biosynthesis, which provokes stabilization of DELLA proteins that will finally promote ovule primordia initiation. In contrast, in Arabidopsis both GAs and BRs regulate ovule number independently of the activity levels of the other hormone. Taken together, our data strongly suggest that different molecular mechanisms could operate in different plant species to regulate identical developmental processes even, as for ovule primordia initiation, if the same set of hormones trigger similar responses, adding a new level of complexity.We wish to thank B. Janssen (Horticulture and Food Research Institute, New Zealand) for the pBJ60 shuttle vector, C. Ferrandiz and M. Colombo (IBMCP, CSIC-UPV, Valencia, Spain) for their help in the generation of 35S:ANT lines and L.E.P. Peres (Universidade de Sao Paulo, Brazil) for the tomato mutant lines. Our thanks also go to C. Fuster for technical assistance. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness-FEDER (BIO2017-83138R) to MAPA and from NSF (DBI-0820755, MCB-1158181, and IOS-1444561) to JMA.Barro-Trastoy, D.; Carrera, E.; Baños, J.; Palau-Rodríguez, J.; Ruiz-Rivero, O.; Tornero Feliciano, P.; Alonso, JM.... (2020). Regulation of ovule initiation by gibberellins and brassinosteroids in tomato and Arabidopsis: two plant species, two molecular mechanisms. The Plant Journal. 102(5):1026-1041. https://doi.org/10.1111/tpj.14684S102610411025Azhakanandam, S., Nole-Wilson, S., Bao, F., & Franks, R. G. (2008). SEUSSandAINTEGUMENTAMediate Patterning and Ovule Initiation during Gynoecium Medial Domain Development    . Plant Physiology, 146(3), 1165-1181. doi:10.1104/pp.107.114751Bai, M.-Y., Shang, J.-X., Oh, E., Fan, M., Bai, Y., Zentella, R., … Wang, Z.-Y. (2012). Brassinosteroid, gibberellin and phytochrome impinge on a common transcription module in Arabidopsis. Nature Cell Biology, 14(8), 810-817. doi:10.1038/ncb2546Baker, S. C., Robinson-Beers, K., Villanueva, J. M., Gaiser, J. C., & Gasser, C. S. (1997). Interactions Among Genes Regulating Ovule Development in Arabidopsis thaliana. Genetics, 145(4), 1109-1124. doi:10.1093/genetics/145.4.1109Bartrina, I., Otto, E., Strnad, M., Werner, T., & Schmülling, T. (2011). Cytokinin Regulates the Activity of Reproductive Meristems, Flower Organ Size, Ovule Formation, and Thus Seed Yield in Arabidopsis thaliana      . The Plant Cell, 23(1), 69-80. doi:10.1105/tpc.110.079079Belkhadir, Y., & Jaillais, Y. (2015). The molecular circuitry of brassinosteroid signaling. New Phytologist, 206(2), 522-540. doi:10.1111/nph.13269Bencivenga, S., Simonini, S., Benková, E., & Colombo, L. (2012). The Transcription Factors BEL1 and SPL Are Required for Cytokinin and Auxin Signaling During Ovule Development in Arabidopsis. The Plant Cell, 24(7), 2886-2897. doi:10.1105/tpc.112.100164Brumos, J., Zhao, C., Gong, Y., Soriano, D., Patel, A. P., Perez-Amador, M. A., … Alonso, J. M. (2019). An Improved Recombineering Toolset for Plants. The Plant Cell, 32(1), 100-122. doi:10.1105/tpc.19.00431Carrera, E., Ruiz-Rivero, O., Peres, L. E. P., Atares, A., & Garcia-Martinez, J. L. (2012). Characterization of the procera Tomato Mutant Shows Novel Functions of the SlDELLA Protein in the Control of Flower Morphology, Cell Division and Expansion, and the Auxin-Signaling Pathway during Fruit-Set and Development    . Plant Physiology, 160(3), 1581-1596. doi:10.1104/pp.112.204552Carvalho, R. F., Campos, M. L., Pino, L. E., Crestana, S. L., Zsögön, A., Lima, J. E., … Peres, L. E. (2011). Convergence of developmental mutants into a single tomato model system: «Micro-Tom» as an effective toolkit for plant development research. Plant Methods, 7(1). doi:10.1186/1746-4811-7-18Chory, 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, 3(5), 445. doi:10.2307/3869351Clough, S. J., & Bent, A. F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana. The Plant Journal, 16(6), 735-743. doi:10.1046/j.1365-313x.1998.00343.xClouse, 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.111.084475Cucinotta, M., Colombo, L., & Roig-Villanova, I. (2014). Ovule development, a new model for lateral organ formation. Frontiers in Plant Science, 5. doi:10.3389/fpls.2014.00117Cucinotta, M., Manrique, S., Guazzotti, A., Quadrelli, N. E., Mendes, M. A., Benkova, E., & Colombo, L. (2016). Cytokinin response factors integrate auxin and cytokinin pathways for female reproductive organ development. Development. doi:10.1242/dev.143545Davière, J.-M., & Achard, P. (2016). A Pivotal Role of DELLAs in Regulating Multiple Hormone Signals. Molecular Plant, 9(1), 10-20. doi:10.1016/j.molp.2015.09.011De Vleesschauwer, D., Van Buyten, E., Satoh, K., Balidion, J., Mauleon, R., Choi, I.-R., … Höfte, M. (2012). Brassinosteroids Antagonize Gibberellin- and Salicylate-Mediated Root Immunity in Rice      . Plant Physiology, 158(4), 1833-1846. doi:10.1104/pp.112.193672Dorcey, E., Urbez, C., Blázquez, M. A., Carbonell, J., & Perez-Amador, M. A. (2009). Fertilization-dependent auxin response in ovules triggers fruit development through the modulation of gibberellin metabolism in Arabidopsis. The Plant Journal, 58(2), 318-332. doi:10.1111/j.1365-313x.2008.03781.xFujioka, S., Li, J., Choi, Y. H., Seto, H., Takatsuto, S., Noguchi, T., … Sakurai, A. (1997). The Arabidopsis deetiolated2 mutant is blocked early in brassinosteroid biosynthesis. The Plant Cell, 9(11), 1951-1962. doi:10.1105/tpc.9.11.1951Galbiati, F., Sinha Roy, D., Simonini, S., Cucinotta, M., Ceccato, L., Cuesta, C., … Colombo, L. (2013). An integrative model of the control of ovule primordia formation. The Plant Journal, 76(3), 446-455. doi:10.1111/tpj.12309Gallego-Bartolome, J., Minguet, E. G., Grau-Enguix, F., Abbas, M., Locascio, A., Thomas, S. G., … Blazquez, M. A. (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. doi:10.1073/pnas.1119992109García-Hurtado, N., Carrera, E., Ruiz-Rivero, O., López-Gresa, M. P., Hedden, P., Gong, F., & García-Martínez, J. L. (2012). The characterization of transgenic tomato overexpressing gibberellin 20-oxidase reveals induction of parthenocarpic fruit growth, higher yield, and alteration of the gibberellin biosynthetic pathway. Journal of Experimental Botany, 63(16), 5803-5813. doi:10.1093/jxb/ers229Gleave, A. P. (1992). A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Molecular Biology, 20(6), 1203-1207. doi:10.1007/bf00028910Gomez, M. D., Ventimilla, D., Sacristan, R., & Perez-Amador, M. A. (2016). Gibberellins Regulate Ovule Integument Development by Interfering with the Transcription Factor ATS. Plant Physiology, 172(4), 2403-2415. doi:10.1104/pp.16.01231He, J.-X., Gendron, J. M., Sun, Y., Gampala, S. S. L., Gendron, N., Sun, C. Q., & Wang, Z.-Y. (2005). BZR1 Is a Transcriptional Repressor with Dual Roles in Brassinosteroid Homeostasis and Growth Responses. Science, 307(5715), 1634-1638. doi:10.1126/science.1107580Huang, H.-Y., Jiang, W.-B., Hu, Y.-W., Wu, P., Zhu, J.-Y., Liang, W.-Q., … Lin, W.-H. (2013). BR Signal Influences Arabidopsis Ovule and Seed Number through Regulating Related Genes Expression by BZR1. Molecular Plant, 6(2), 456-469. doi:10.1093/mp/sss070Kurepin, L. V., Joo, S.-H., Kim, S.-K., Pharis, R. P., & Back, T. G. (2011). Interaction of Brassinosteroids with Light Quality and Plant Hormones in Regulating Shoot Growth of Young Sunflower and Arabidopsis Seedlings. Journal of Plant Growth Regulation, 31(2), 156-164. doi:10.1007/s00344-011-9227-7Li, Q.-F., Wang, C., Jiang, L., Li, S., Sun, S. S. M., & He, J.-X. (2012). An Interaction Between BZR1 and DELLAs Mediates Direct Signaling Crosstalk Between Brassinosteroids and Gibberellins in Arabidopsis. Science Signaling, 5(244). doi:10.1126/scisignal.2002908Li, X.-J., Chen, X.-J., Guo, X., Yin, L.-L., Ahammed, G. J., Xu, C.-J., … Yu, J.-Q. (2015). DWARFoverexpression induces alteration in phytohormone homeostasis, development, architecture and carotenoid accumulation in tomato. Plant Biotechnology Journal, 14(3), 1021-1033. doi:10.1111/pbi.12474Liu, Z., Franks, R. G., & Klink, V. P. (2000). Regulation of Gynoecium Marginal Tissue Formation by LEUNIG and AINTEGUMENTA. The Plant Cell, 12(10), 1879-1891. doi:10.1105/tpc.12.10.1879Marti, E. (2006). Genetic and physiological characterization of tomato cv. Micro-Tom. Journal of Experimental Botany, 57(9), 2037-2047. doi:10.1093/jxb/erj154Mizukami, Y., & Fischer, R. L. (2000). Plant organ size control: AINTEGUMENTA regulates growth and cell numbers during organogenesis. Proceedings of the National Academy of Sciences, 97(2), 942-947. doi:10.1073/pnas.97.2.942Montoya, T., Nomura, T., Yokota, T., Farrar, K., Harrison, K., Jones, J. G. D., … Bishop, G. J. (2005). Patterns of Dwarf expression and brassinosteroid accumulation in tomato reveal the importance of brassinosteroid synthesis during fruit development. The Plant Journal, 42(2), 262-269. doi:10.1111/j.1365-313x.2005.02376.xMüller, C. J., Larsson, E., Spíchal, L., & Sundberg, E. (2017). Cytokinin-Auxin Crosstalk in the Gynoecial Primordium Ensures Correct Domain Patterning. Plant Physiology, 175(3), 1144-1157. doi:10.1104/pp.17.00805Murashige, T., & Skoog, F. (1962). A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum, 15(3), 473-497. doi:10.1111/j.1399-3054.1962.tb08052.xOlimpieri, I., Siligato, F., Caccia, R., Soressi, G. P., Mazzucato, A., Mariotti, L., & Ceccarelli, N. (2007). Tomato fruit set driven by pollination or by the parthenocarpic fruit allele are mediated by transcriptionally regulated gibberellin biosynthesis. Planta, 226(4), 877-888. doi:10.1007/s00425-007-0533-zPaz-Ares, J., & The REGIA Consortium. (2002). REGIA, An EU Project on Functional Genomics of Transcription Factors fromArabidopsis thaliana. Comparative and Functional Genomics, 3(2), 102-108. doi:10.1002/cfg.146Peng, J., Carol, P., Richards, D. E., King, K. E., Cowling, R. J., Murphy, G. P., & Harberd, N. P. (1997). The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses . Genes & Development, 11(23), 3194-3205. doi:10.1101/gad.11.23.3194Reyes-Olalde, J. I., Zuñiga-Mayo, V. M., Chávez Montes, R. A., Marsch-Martínez, N., & de Folter, S. (2013). Inside the gynoecium: at the carpel margin. Trends in Plant Science, 18(11), 644-655. doi:10.1016/j.tplants.2013.08.002Sabelli, P. A., & Larkins, B. A. (2009). The Development of Endosperm in Grasses. Plant Physiology, 149(1), 14-26. doi:10.1104/pp.108.129437Schneitz, K., Baker, S. C., Gasser, C. S., & Redweik, A. (1998). Pattern formation and growth during floral organogenesis: HUELLENLOS and AINTEGUMENTA are required for the formation of the proximal region of the ovule primordium in Arabidopsis thaliana. Development, 125(14), 2555-2563. doi:10.1242/dev.125.14.2555Schneitz, K., Hulskamp, M., & Pruitt, R. E. (1995). Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue. The Plant Journal, 7(5), 731-749. doi:10.1046/j.1365-313x.1995.07050731.xSeo, M., Jikumaru, Y., & Kamiya, Y. (2011). Profiling of Hormones and Related Metabolites in Seed Dormancy and Germination Studies. Methods in Molecular Biology, 99-111. doi:10.1007/978-1-61779-231-1_7Serrani, J. C., Sanjuán, R., Ruiz-Rivero, O., Fos, M., & García-Martínez, J. L. (2007). Gibberellin Regulation of Fruit Set and Growth in Tomato. Plant Physiology, 145(1), 246-257. doi:10.1104/pp.107.098335Serrani, J. C., Carrera, E., Ruiz-Rivero, O., Gallego-Giraldo, L., Peres, Lá. E. P., & García-Martínez, J. L. (2010). Inhibition of Auxin Transport from the Ovary or from the Apical Shoot Induces Parthenocarpic Fruit-Set in Tomato Mediated by Gibberellins    . Plant Physiology, 153(2), 851-862. doi:10.1104/pp.110.155424Sun, T. (2010). Gibberellin-GID1-DELLA: A Pivotal Regulatory Module for Plant Growth and Development. Plant Physiology, 154(2), 567-570. doi:10.1104/pp.110.161554Sun, 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.2011.02.036Tanaka, 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/s00344-003-0119-3Tang, Y., Liu, H., Guo, S., Wang, B., Li, Z., Chong, K., & Xu, Y. (2017). OsmiR396d Affects Gibberellin and Brassinosteroid Signaling to Regulate Plant Architecture in Rice. Plant Physiology, 176(1), 946-959. doi:10.1104/pp.17.00964Tong, H., Xiao, Y., Liu, D., Gao, S., Liu, L., Yin, Y., … Chu, C. (2014). Brassinosteroid Regulates Cell Elongation by Modulating Gibberellin Metabolism in Rice    . The Plant Cell, 26(11), 4376-4393. doi:10.1105/tpc.114.132092Truernit, E., Bauby, H., Dubreucq, B., Grandjean, O., Runions, J., Barthélémy, J., & Palauqui, J.-C. (2008). High-Resolution Whole-Mount Imaging of Three-Dimensional Tissue Organization and Gene Expression Enables the Study of Phloem Development and Structure inArabidopsis . The Plant Cell, 20(6), 1494-1503. doi:10.1105/tpc.107.056069Tursun, B., Cochella, L., Carrera, I., & Hobert, O. (2009). A Toolkit and Robust Pipeline for the Generation of Fosmid-Based Reporter Genes in C. elegans. PLoS ONE, 4(3), e4625. doi:10.1371/journal.pone.0004625Unterholzner, S. J., Rozhon, W., Papacek, M., Ciomas, J., Lange, T., Kugler, K. G., … Poppenberger, B. (2015). Brassinosteroids Are Master Regulators of Gibberellin Biosynthesis in Arabidopsis. The Plant Cell, 27(8), 2261-2272. doi:10.1105/tpc.15.00433Wang, 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)00153-3Xiao, H., Radovich, C., Welty, N., Hsu, J., Li, D., Meulia, T., & van der Knaap, E. (2009). Integration of tomato reproductive developmental landmarks and expression profiles, and the effect of SUN on fruit shape. BMC Plant Biology, 9(1). doi:10.1186/1471-2229-9-49Xiao, Y., Liu, D., Zhang, G., Tong, H., & Chu, C. (2017). Brassinosteroids Regulate OFP1, a DLT Interacting Protein, to Modulate Plant Architecture and Grain Morphology in Rice. Frontiers in Plant Science, 8. doi:10.3389/fpls.2017.0169

Gibberellin-mediated RGA-LIKE1 degradation regulates embryo sac development in Arabidopsis

[EN] Ovule development is essential for plant survival, as it allows correct embryo and seed development upon fertilization. The female gametophyte is formed in the central area of the nucellus during ovule development, in a complex developmental programme that involves key regulatory genes and the plant hormones auxins and brassinosteroids. Here we provide novel evidence of the role of gibberellins (GAs) in the control of megagametogenesis and embryo sac development, via the GA-dependent degradation of RGA-LIKE1 (RGL1) in the ovule primordia. YPet-rgl1.17 plants, which express a dominant version of RGL1, showed reduced fertility, mainly due to altered embryo sac formation that varied from partial to total ablation. YPet-rgl1.17 ovules followed normal development of the megaspore mother cell, meiosis, and formation of the functional megaspore, but YPet-rgl1.17 plants had impaired mitotic divisions of the functional megaspore. This phenotype is RGL1-specific, as it is not observed in any other dominant mutants of the DELLA proteins. Expression analysis of YPet-rgl1.17 coupled to in situ localization of bioactive GAs in ovule primordia led us to propose a mechanism of GA-mediated RGL1 degradation that allows proper embryo sac development. Taken together, our data unravel a novel specific role of GAs in the control of female gametophyte development.We wish to thank the IBMCP microscopy facility, and Ms J. Yun for technical assistance. We also thank Jennifer Nemhauser (University of Washington, USA) for the HACR sensor. Cambridge proofreading (https://proofreading.org/order/) provided proofreading and editing of this manuscript. This work was supported by grants from the Spanish Ministry for Science and Innovation-FEDER [BIO2017-83138R] to MAP-A and National Science Foundation [MCB-0923727] to JMA. MAP-A received a fellowship of the `Salvador de Madariaga' program from Spanish Ministry of Science and Innovation. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Gomez, MD.; Barro-Trastoy, D.; Fuster Almunia, C.; Tornero Feliciano, P.; Alonso, JM.; Perez Amador, MA. (2020). Gibberellin-mediated RGA-LIKE1 degradation regulates embryo sac development in Arabidopsis. Journal of Experimental Botany. 71(22):7059-7072. https://doi.org/10.1093/jxb/eraa395S705970727122Bai, M.-Y., Shang, J.-X., Oh, E., Fan, M., Bai, Y., Zentella, R., … Wang, Z.-Y. (2012). Brassinosteroid, gibberellin and phytochrome impinge on a common transcription module in Arabidopsis. Nature Cell Biology, 14(8), 810-817. doi:10.1038/ncb2546Battaglia, R., Brambilla, V., & Colombo, L. (2008). Morphological analysis of female gametophyte development in thebel1 stk shp1 shp2mutant. Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, 142(3), 643-649. doi:10.1080/11263500802411098Beeckman, T., De Rycke, R., Viane, R., & Inzé, D. (2000). Histological Study of Seed Coat Development in Arabidopsis thaliana. Journal of Plant Research, 113(2), 139-148. doi:10.1007/pl00013924Bencivenga, S., Simonini, S., Benková, E., & Colombo, L. (2012). The Transcription Factors BEL1 and SPL Are Required for Cytokinin and Auxin Signaling During Ovule Development in Arabidopsis. The Plant Cell, 24(7), 2886-2897. doi:10.1105/tpc.112.100164Brumos, J., Zhao, C., Gong, Y., Soriano, D., Patel, A. P., Perez-Amador, M. A., … Alonso, J. M. (2019). An Improved Recombineering Toolset for Plants. The Plant Cell, 32(1), 100-122. doi:10.1105/tpc.19.00431Clough, S. J., & Bent, A. F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana. The Plant Journal, 16(6), 735-743. doi:10.1046/j.1365-313x.1998.00343.xCoen, O., Lu, J., Xu, W., De Vos, D., Péchoux, C., Domergue, F., … Magnani, E. (2019). Deposition of a cutin apoplastic barrier separating seed maternal and zygotic tissues. BMC Plant Biology, 19(1). doi:10.1186/s12870-019-1877-9Cucinotta, M., Di Marzo, M., Guazzotti, A., de Folter, S., Kater, M. M., & Colombo, L. (2020). Gynoecium size and ovule number are interconnected traits that impact seed yield. Journal of Experimental Botany, 71(9), 2479-2489. doi:10.1093/jxb/eraa050Davière, J.-M., & Achard, P. (2013). Gibberellin signaling in plants. Development, 140(6), 1147-1151. doi:10.1242/dev.087650Davière, J.-M., & Achard, P. (2016). A Pivotal Role of DELLAs in Regulating Multiple Hormone Signals. Molecular Plant, 9(1), 10-20. doi:10.1016/j.molp.2015.09.011Dill, 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.251534098Ferreira, L. G., de Alencar Dusi, D. M., Irsigler, A. S. T., Gomes, A. C. M. M., Mendes, M. A., Colombo, L., & de Campos Carneiro, V. T. (2017). GID1 expression is associated with ovule development of sexual and apomictic plants. Plant Cell Reports, 37(2), 293-306. doi:10.1007/s00299-017-2230-0Fleck, B., & Harberd, N. P. (2002). Evidence that theArabidopsisnuclear gibberellin signalling protein GAI is not destabilised by gibberellin. The Plant Journal, 32(6), 935-947. doi:10.1046/j.1365-313x.2002.01478.xGallego-Bartolome, J., Minguet, E. G., Grau-Enguix, F., Abbas, M., Locascio, A., Thomas, S. G., … Blazquez, M. A. (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. doi:10.1073/pnas.1119992109Gallego-Bartolome, J., Minguet, E. G., Marin, J. A., Prat, S., Blazquez, M. A., & Alabadi, D. (2010). Transcriptional Diversification and Functional Conservation between DELLA Proteins in Arabidopsis. Molecular Biology and Evolution, 27(6), 1247-1256. doi:10.1093/molbev/msq012Gomez, M. D., Barro-Trastoy, D., Escoms, E., Saura-Sánchez, M., Sánchez, I., Briones-Moreno, A., … Perez-Amador, M. A. (2018). Gibberellins negatively modulate ovule number in plants. Development. doi:10.1242/dev.163865G�mez, M. D., Beltr�n, J.-P., & Ca�as, L. A. (2004). The pea END1 promoter drives anther-specific gene expression in different plant species. Planta, 219(6), 967-981. doi:10.1007/s00425-004-1300-zGómez, M. D., Fuster-Almunia, C., Ocaña-Cuesta, J., Alonso, J. M., & Pérez-Amador, M. A. (2019). RGL2 controls flower development, ovule number and fertility in Arabidopsis. Plant Science, 281, 82-92. doi:10.1016/j.plantsci.2019.01.014Gomez, M. D., Ventimilla, D., Sacristan, R., & Perez-Amador, M. A. (2016). Gibberellins Regulate Ovule Integument Development by Interfering with the Transcription Factor ATS. Plant Physiology, 172(4), 2403-2415. doi:10.1104/pp.16.01231Hedden, P., & Sponsel, V. (2015). A Century of Gibberellin Research. Journal of Plant Growth Regulation, 34(4), 740-760. doi:10.1007/s00344-015-9546-1Khakhar, A., Leydon, A. R., Lemmex, A. C., Klavins, E., & Nemhauser, J. L. (2018). Synthetic hormone-responsive transcription factors can monitor and re-program plant development. eLife, 7. doi:10.7554/elife.34702Koorneef, M., Elgersma, A., Hanhart, C. J., Loenen-Martinet, E. P., Rijn, L., & Zeevaart, J. A. D. (1985). A gibberellin insensitive mutant of Arabidopsis thaliana. Physiologia Plantarum, 65(1), 33-39. doi:10.1111/j.1399-3054.1985.tb02355.xKurihara, D., Mizuta, Y., Sato, Y., & Higashiyama, T. (2015). ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging. Development. doi:10.1242/dev.127613Lee, S. (2002). Gibberellin regulates Arabidopsis seed germination via RGL2, a GAI/RGA-like gene whose expression is up-regulated following imbibition. Genes & Development, 16(5), 646-658. doi:10.1101/gad.969002Li, Q.-F., Wang, C., Jiang, L., Li, S., Sun, S. S. M., & He, J.-X. (2012). An Interaction Between BZR1 and DELLAs Mediates Direct Signaling Crosstalk Between Brassinosteroids and Gibberellins in Arabidopsis. Science Signaling, 5(244). doi:10.1126/scisignal.2002908Lieber, D., Lora, J., Schrempp, S., Lenhard, M., & Laux, T. (2011). Arabidopsis WIH1 and WIH2 Genes Act in the Transition from Somatic to Reproductive Cell Fate. Current Biology, 21(12), 1009-1017. doi:10.1016/j.cub.2011.05.015Lora, J., Herrero, M., Tucker, M. R., & Hormaza, J. I. (2016). The transition from somatic to germline identity shows conserved and specialized features during angiosperm evolution. New Phytologist, 216(2), 495-509. doi:10.1111/nph.14330Murashige, T., & Skoog, F. (1962). A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum, 15(3), 473-497. doi:10.1111/j.1399-3054.1962.tb08052.xPeng, J., Carol, P., Richards, D. E., King, K. E., Cowling, R. J., Murphy, G. P., & Harberd, N. P. (1997). The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses . Genes & Development, 11(23), 3194-3205. doi:10.1101/gad.11.23.3194Pinto, S. C., Mendes, M. A., Coimbra, S., & Tucker, M. R. (2019). Revisiting the Female Germline and Its Expanding Toolbox. Trends in Plant Science, 24(5), 455-467. doi:10.1016/j.tplants.2019.02.003Schneitz, K., Hulskamp, M., & Pruitt, R. E. (1995). Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue. The Plant Journal, 7(5), 731-749. doi:10.1046/j.1365-313x.1995.07050731.xErbasol Serbes, I., Palovaara, J., & Groß-Hardt, R. (2019). Development and function of the flowering plant female gametophyte. Plant Development and Evolution, 401-434. doi:10.1016/bs.ctdb.2018.11.016Sun, 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.2011.02.036Tucker, M. R., Okada, T., Hu, Y., Scholefield, A., Taylor, J. M., & Koltunow, A. M. G. (2012). Somatic small RNA pathways promote the mitotic events of megagametogenesis during female reproductive development in Arabidopsis. Development, 139(8), 1399-1404. doi:10.1242/dev.075390Ursache, R., Andersen, T. G., Marhavý, P., & Geldner, N. (2018). A protocol for combining fluorescent proteins with histological stains for diverse cell wall components. The Plant Journal, 93(2), 399-412. doi:10.1111/tpj.13784Villanueva, J. M., Broadhvest, J., Hauser, B. A., Meister, R. J., Schneitz, K., & Gasser, C. S. (1999). INNER NO OUTER regulates abaxial- adaxial patterning in Arabidopsis ovules. Genes & Development, 13(23), 3160-3169. doi:10.1101/gad.13.23.3160Wen, C.-K., & Chang, C. (2002). Arabidopsis RGL1 Encodes a Negative Regulator of Gibberellin Responses. The Plant Cell, 14(1), 87-100. doi:10.1105/tpc.010325Wu, J., Mohamed, D., Dowhanik, S., Petrella, R., Gregis, V., Li, J., … Gazzarrini, S. (2020). Spatiotemporal Restriction of FUSCA3 Expression by Class I BPCs Promotes Ovule Development and Coordinates Embryo and Endosperm Growth. The Plant Cell, 32(6), 1886-1904. doi:10.1105/tpc.19.00764Yang, W.-C., Shi, D.-Q., & Chen, Y.-H. (2010). Female Gametophyte Development in Flowering Plants. Annual Review of Plant Biology, 61(1), 89-108. doi:10.1146/annurev-arplant-042809-112203Yang, W.-C., Ye, D., Xu, J., & Sundaresan, V. (1999). The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein. Genes & Development, 13(16), 2108-2117. doi:10.1101/gad.13.16.2108Zhao, L., He, J., Cai, H., Lin, H., Li, Y., Liu, R., … Qin, Y. (2014). Comparative expression profiling reveals gene functions in female meiosis and gametophyte development in Arabidopsis. The Plant Journal, 80(4), 615-628. doi:10.1111/tpj.12657Zhou, R., Benavente, L. M., Stepanova, A. N., & Alonso, J. M. (2011). A recombineering-based gene tagging system for Arabidopsis. The Plant Journal, 66(4), 712-723. doi:10.1111/j.1365-313x.2011.04524.

Estudio de la Función de las Giberelinas en la Determinación del Número de Óvulos en Plantas

La formación de óvulos y semillas es un proceso esencial para las plantas, ya que asegura su correcta reproducción, y para el ser humano, pues tiene una gran importancia económica al repercutir directamente en el rendimiento de muchos cultivos. La formación de óvulos se trata, además, de un proceso de desarrollo complejo en el que participan varias redes génicas y hormonales. Actualmente se conocen algunos de los genes clave implicados en el control de la iniciación y desarrollo de los óvulos en Arabidopsis thaliana. Entre los componentes genéticos más relevantes se encuentran AINTEGUMENTA (ANT) y CUP-SHAPE COTYLEDON1 y 2 (CUC1 y 2), así como algunos de los componentes de las vías de señalización de auxinas, brasinoesteroides y citoquininas. Curiosamente, a pesar de estar involucradas en multitud de procesos del desarrollo de las plantas, hasta ahora no ha habido evidencias que involucrasen a las giberelinas (GAs) y sus reguladores negativos de señalización, las proteínas DELLA, en el desarrollo de los óvulos. No obstante, recientemente se han aportado evidencias que apuntan a que estas hormonas no solo podrían estar involucradas en el desarrollo de los óvulos, sino también en la determinación de su número en Arabidopsis, posiblemente a través de CUC2. Este Trabajo Final de Máster tiene como objetivo general profundizar en el mecanismo molecular por el cual las GAs y las proteínas DELLA determinan la formación de los óvulos. Para ello se caracterizó la interacción genética entre las proteínas DELLA y CUC en la determinación del número de óvulos. Asimismo, también se realizaron estudios para identificar el posible cross-talk de las GAs con las auxinas y los brasinoesteroides en la iniciación de los óvulos. Finalmente, se proporcionan evidencias que apuntan a que las GAs, a través de las DELLA, podrían actuar como reguladores del número de óvulos no sólo en Arabidopsis, sino también en colza (Brassica napus), una planta de interés agronómico.The ovule and seed formation are essential processes for plants since they ensure their correct reproduction; and for the human being, since they have a great economic importance as they directly affect many crops production. Ovule formation is also a complex development process that involves several gene and hormonal networks. Currently, some of the key genes involved in the control of the ovule initiation and development in Arabidopsis thaliana are known. The most relevant genetic components are AINTEGUMENTA (ANT) and CUP-SHAPE COTYLEDON1 and 2 (CUC1 and 2), as well as some of the auxins, brassinosteroids, and cytokinins signaling pathways components. Interestingly, despite being involved in many processes of plant development, until now there has been no evidence that involved the gibberellins (GAs) and their negative signaling regulators, the DELLA proteins, in the development of the ovules. However, it has been recently provided evidence that suggests that these hormones could not only be involved in the development of the ovules but also in the determination of their number in Arabidopsis, possibly through CUC2. This Master's Final Project aims to deepen the molecular mechanism by which GAs and DELLA proteins determine the ovule formation. To this end, the genetic interaction between the DELLA and CUC proteins in the determination of the ovule number was characterized. Likewise, studies were also carried out to identify the possible cross-talk of the GAs with auxins and brassinosteroids in the ovule initiation. Finally, it is provided evidence that the GAs, through the DELLA proteins, could act as regulators of ovule number not only in Arabidopsis but also in rapeseed (Brassica napus), a plant of agronomic interest.Barro Trastoy, D. (2018). Estudio de la Función de las Giberelinas en la Determinación del Número de Óvulos en Plantas. http://hdl.handle.net/10251/97984TFG

Gibberellins regulate ovule number through a DELLA¿CUC2 complex in Arabidopsis

[EN] Ovule development is a key process for plant reproduction, helping to ensure correct seed production. Several molecular factors and plant hormones such as gibberellins are involved in ovule initiation and development. Gibberellins control ovule development by the destabilization of DELLA proteins, whereas DELLA activity has been shown to act as a positive factor for ovule primordia emergence. But the molecular mechanism by which DELLA acts in ovule primordia initiation remained unknown. In this study we report that DELLA proteins participate in ovule initiation by the formation of a protein complex with the CUC2 transcription factor. The DELLA protein GAI requires CUC2 to promote ovule primordia formation, through the direct GAI-CUC2 interaction in placental cells that would determine the boundary regions between ovules during pistil development. Analysis of GAI-CUC2 interaction and co-localization in the placenta supports this hypothesis. Moreover, molecular analysis identified a subset of the loci for which the GAI protein may act as a transcriptional co-regulator in a CUC2-dependent manner. The DELLA-CUC2 complex is a component of the gene regulatory network controlling ovule primordia initiation in Arabidopsis.We wish to thank Dr. N. Arnaud (INRAE-Versailles, France) for the pCUC1:CUC1-GFP and pCUC2:CUC2-VENUS lines, Dr. D. Alabadi (IBMCP, Valencia, Spain) for the 35S:YFP-M5GAI line and GAI deletions in pDEST32, and Dr. S. Prat (CNB-Madrid, Spain) for the M5-DELLA clones in pGBKT7. We also thank Ms. C. Fuster for her excellent technical assistance and the IBMCP Bioinformatics Core Service for helping in the data processing. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness-FEDER (BIO2017-83138R) and Spanish Ministry of Science and Innovation-AEI (PID2020-113920RB-I00) to MAP-A; DB-T was the recipient of a pre-doctoral fellowship from the Spanish Ministry of Universities (FPU18/00331).Barro-Trastoy, D.; Gómez, MD.; Blanco-Touriñán, N.; Tornero Feliciano, P.; Perez Amador, MA. (2022). Gibberellins regulate ovule number through a DELLA-CUC2 complex in Arabidopsis. The Plant Journal. 110(1):43-57. https://doi.org/10.1111/tpj.156074357110