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Drought represents a major threat to food security. Mechanistic data describing plantresponses to drought have been studied extensively and genes conferring drought resistancehave been introduced into crop plants. However, plants with enhanced drought resistanceusually display lower growth, highlighting the need for strategies to uncouple droughtresistance from growth. Here, we show that overexpression of BRL3, a vascular-enrichedmember of the brassinosteroid receptor family, can confer drought stress tolerance inArabidopsis. Whereas loss-of-function mutations in the ubiquitously expressed BRI1 receptorleads to drought resistance at the expense of growth, overexpression of BRL3 receptorconfers drought tolerance without penalizing overall growth. Systematic analyses reveal thatupon drought stress, increased BRL3 triggers the accumulation of osmoprotectant metabo-lites including proline and sugars. Transcriptomic analysis suggests that this results fromdifferential expression of genes in the vascular tissues. Altogether, this data suggests thatmanipulating BRL3 expression could be used to engineer drought tolerant crops.

Sorghum (Sorghum bicolor) is an emerging cereal crop in temperate climates due to its high drought tolerance and other valuable traits. Genetic transformation is an important tool for the improvement of cereals. However, sorghum is recalcitrant to genetic transformation which is almost only successful in warmer climates. Here, we test the application of two new techniques for sorghum transformation in temperate climates, namely transient transformation by Agrobacterium tumefaciens-mediated agroinfiltration and stable transformation using gold particle bombardment and leaf whorls as explants. We optimized the transient transformation method, including post-infiltration incubation of plants in the dark and using Agrobacterium grown on plates with a high cell density (OD600 = 2.0). Expression of the green fluorescence protein (GFP)-tagged endogenous sorghum gene SbDHR2 was achieved with low transformation efficiency, and our results point out a potential weakness in using this approach for localization studies. Furthermore, we succeeded in the production of callus and somatic embryos from leaf whorls, although no genetic transformation was accomplished with this method. Both methods show potential, even if they seem to be influenced by climatic conditions and therefore need further optimization to be applied routinely in temperate climates.

Sorghum (Sorghum bicolor) is an emerging cereal crop in temperate climates due to its high drought tolerance and other valuable traits. Genetic transformation is an important tool for the improvement of cereals. However, sorghum is recalcitrant to genetic transformation which is almost only successful in warmer climates. Here, we test the application of two new techniques for sorghum transformation in temperate climates, namely transient transformation by Agrobacterium tumefaciens–mediated agroinfiltration and stable transformation using gold particle bombardment and leaf whorls as explants. We optimized the transient transformation method, including post-infiltration incubation of plants in the dark and using Agrobacterium grown on plates with a high cell density (OD600 = 2.0). Expression of the green fluorescence protein (GFP)-tagged endogenous sorghum gene SbDHR2 was achieved with low transformation efficiency, and our results point out a potential weakness in using this approach for localization studies. Furthermore, we succeeded in the production of callus and somatic embryos from leaf whorls, although no genetic transformation was accomplished with this method. Both methods show potential, even if they seem to be influenced by climatic conditions and therefore need further optimization to be applied routinely in temperate climates.

[EN] Brassinosteroids have been proven to be of great importance for the development of plants but also in the adaptation of plants to different biotic and abiotic stresses. Our previous work in Arabidopsis showed that BRL3, a discretely expressed Brassinosteroid receptor, intervenes in the adaptative response of plants to drought, and BRL3 overexpressing plants were tolerant to drought. Therefore, we wanted to test whether this adaptative mechanism is conserved in one of the most important crops in arid and semiarid zones of the planet, Sorghum bicolor. In the first chapter, we identified the two Brassinosteroid receptor genes of Sorghum bicolor. Their genetic sequence and the protein 3D structure of BRI1 was analysed showing a high degree of conservation. Furthermore, we tested their functionality through heterologous complementation in Arabidopsis. In the second chapter, a mutagenized collection was screened in an effort to isolate six sorghum brassinosteroid receptor mutants. The plants were backcrossed to eliminate background mutations and to obtain segregant populations for plant phenotyping. Furthermore, different lines were subjected to drought and elevated temperature stresses, showing that both receptors control abiotic stress responses. The third chapter constitutes an effort to establish sorghum transformation protocol in our laboratory. Sorghum is known to be a recalcitrant specie for transformation. Thus, we established a collaboration with the laboratory of Prof. Ian Godwin, in Australia. The results obtained at CRAG during two years were compared with the results obtained during an international stay in QAAFI (Australia). We obtained transgenic sorghum plants, which will be a useful tool for further research. Overall, the aim of the present PhD thesis is to contribute to the translational research from molecular biology to agriculture. Towards this direction, we managed to set Sorghum research at CRAG, adapt protocols from Arabidopsis to Sorghum, and, [ES] Los brasinosteroides son fitothormonas esenciales para el desarrollo de las plantas y su adaptación a diferentes tipos de estrés ambiental. Nuestro trabajo previo en Arabidopsis demostró que BRL3, uno de los receptores de brasinosteroides, interviene en la respuesta adaptativa de estas plantas a la sequía. Las plantas sobreexpresoras de BRL3 toleran mejor la sequía sin penalizar el rendimiento. Por lo tanto, queríamos comprobar si este mecanismo de adaptación está conservado en uno de los cultivos más importantes en las zonas áridas y semiáridas del planeta, el Sorghum bicolor. En el primer capítulo, identificamos los dos genes receptores de brasinosteroides en el genoma de Sorghum bicolor. Su secuencia genética y la estructura proteica 3D de BRI1 fue analizada mostrando un alto grado de conservación. Además, comprobamos que son funcionales mediante complementación heteróloga en Arabidopsis. En el segundo capítulo, se identificaron varios alelos mutantes en los receptores de brasinosteroides a partir de una colección existente de mutantes. Las plantas fueron retrocruzadas dos veces, para eliminar mutaciones en el fondo genético y así obtener poblaciones segregantes para el fenotipado de las plantas. Asimismo, diferentes de estas líneas fueron sometidas a caracterización fisiológica, en ensayos de estrés por sequía y a temperaturas elevadas. En el tercer capítulo se reportan los avances hacia el establecimiento de un protocolo de transformación de sorgo en nuestro laboratorio. El sorgo es conocido como una especie recalcitrante a transformación. Así pues, establecimos una colaboración con el laboratorio del Prof. Ian Godwin, en Australia. Los resultados obtenidos en el CRAG durante dos años diferentes fueron comparados con los resultados obtenidos durante mi estancia internacional en QAAFI (Australia). Obtuvimos plantas transgénicas de sorgo que serán útiles para investigaciones futuras. En general, el objetivo de esta tesis doctoral es contribuir a la biología, [CA] La importància dels brasinosteroides per al desenvolupament de les plantes i la seva adaptació a diferents tipus d'estrès, biòtic i abiòtic, ha estat demostrada. El nostre treball previ amb Arabidopsis va demostrar que BRL3, un dels receptors de brasinosteroides discretament expressat, intervé en la resposta adaptativa d'aquestes plantes a sequera, i plantes sobreexpressores de BRL3 toleren millor la sequera. Per tant, volíem comprovar si aquest mecanisme d'adaptació està conservat en un dels cultius més importants a les zones àrides i semiàrides del planeta, Sorghum bicolor. Al primer capítol, identifiquem els dos gens receptors de brasinosteroides a Sorghum bicolor. La seqüència genètica i l'estructura proteica 3D de BRI1 va ser analitzada mostrant un alt grau de conservació. A més, vam comprobar que són funcionals mitjançant complementació heteròloga a Arabidopsis. Al segon capítol, una col·lecció mutagenitzada va ser cribada amb l'objectiu d'aïllar sis mutants de receptors de brasinosteroides. Les plantes van ser retrocreuades per eliminar mutacions en el fons genètic i així obtenir poblacions segregants per al fenotipat de les plantes. Així mateix, diferents d'aquestes línies van ser sotmeses a estrès per sequera ia temperatures elevades. El tercer capítol es reporten els esforços per establir el protocol de transformació de sorgo al nostre laboratori. El sorgo és conegut com una espècie recalcitrant a transformació. Així doncs, vam establir una col·laboració amb el laboratori del Prof. Ian Godwin, a Austràlia. Els resultats obtinguts al CRAG durant dos anys diferents van ser comparats amb els resultats obtinguts durant una estada internacional a QAAFI (Austràlia). Vam obtenir plantes transgèniques de sorgo que seran útils per a altres investigacions. En general, l'objectiu d'aquesta tesi doctoral és contribuir a la recerca translativa de biologia molecular cap a l'agricultura. En aquesta direcció, aconseguim establir la investigació sobre sorgo al CRAG, a

[EN] The present PhD thesis dissertation reports novel mechanisms for stem cell divisions mediated by brassinosteroids (BRs) upon DNA damage conditions in plants. BRs are steroid hormones involved in multiple process of plant growth and development, and the adaptation to the environment. One of the processes that are known to be regulated by BRs is the stem cell divisions. Plants stem cells are in niches, which are mainly found in the root and shoot apexes. At the core of the root stem cell niche it is placed the quiescent center (QC), a group of cells with very low mitotic activity that maintain the undifferentiated status of surrounding stem cells and act as a cell reservoir. QC cells only trigger their divisions when need to replenish the stem cells, for example, after a DNA damage. BR signaling is in charge of triggering these QC divisions, but the exact mechanisms of how this process is regulated is still unknown. Here, we use an interdisciplinary approach, using Arabidopsis thaliana as a model system, including molecular genetics, physiology and bioinformatics to decipher the role of BR receptors upon DNA damage regulating the QC divisions. The results presented in this thesis dissertation uncover novel roles for the BR-receptor kinase BRL3 (BRI1-like 3) protein in DNA damage and DNA repair machinery in plants. This is important in order to control DNA repair mechanisms and cell cycle progression in the root meristem, which requires a balance of these processes to ensure plant adaptation to adverse conditions. We found that, at the root apex, BRL3 downstream signaling events modulate an enzyme that is specifically expressed in the QC cells, the RNR2A (RIBONUCLEOTIDE REDUCTASE 2A), in charge of maintaining dNTPs (deoxynucleotide triphosphates) supply during DNA synthesis. Overall, we found that RNR2A is crucial for a proper QC division in response to DNA damage conditions. Moreover, we also discovered that BRL3 pathway is also involved in the triggering of ROS, [ES] La presente tesis doctoral describe nuevos mecanismos mediados por brasinoesteroides (BRs) para controlar las divisiones de las células madre en condiciones de daño en el ADN en las plantas. Los BRs son hormonas esteroideas involucradas en múltiples procesos de crecimiento y de desarrollo en las plantas, y en la adaptación al medio ambiente. Se sabe que la división de las células madre está regulada por BRs. Las células madre de las plantas se encuentran en nichos, que se localizan principalmente en los ápices de las raíces y de los tallos. En el centro del nicho de células madre de la raíz se encuentra el centro quiescente (QC), un grupo de células con muy baja actividad mitótica, que mantiene el estado indiferenciado de las células madre circundantes y actúa como un reservorio celular. Las células del QC solo se dividen cuando necesitan reponer las células madre, por ejemplo, después de un daño en el ADN. Las señales mediadas por BR son las encargadas de activar estas divisiones del QC pero aún se desconocen los mecanismos exactos de cómo se regula este proceso. En este trabajo, utilizamos un enfoque interdisciplinario, utilizando Arabidopsis thaliana como sistema modelo, que incluye genética molecular, fisiología y bioinformática, para descifrar el papel de los receptores de los BRs en respuesta al daño al ADN y en la regulación de las divisiones del QC. Los resultados presentados en esta tesis descubren nuevos roles para la proteína quinasa BRL3 (BRI1-like 3) en el daño al ADN y la maquinaria de reparación del ADN en las plantas. Esto es importante para controlar los mecanismos de reparación del ADN y la progresión en el ciclo celular del meristemo de la raíz, lo que requiere un equilibrio de estos procesos para asegurar la adaptación de la planta a condiciones adversas. Encontramos que, en el ápice de la raíz, los eventos de señalización aguas abajo de BRL3 modulan una enzima que se expresa específicamente en las células del QC, la RNR2A (RIBONUCLEOTIDE RE, [CA] La present tesi doctoral descriu nous mecanismes regulats per brassinosteroides (BRs) per controlar les divisions de les cèl·lules mare en condicions de dany al DNA a les plantes. Les BRs són hormones esteroides implicades en múltiples processos de creixement i de desenvolupament de les plantes, i en l'adaptació al medi ambient. Se sap que les divisions de les cèl·lules mare estan regulades per BRs. Les cèl·lules mare de les plantes es troben en nínxols, que es troben principalment als àpexs de les arrels i de les tiges. Al centre del nínxol de les cèl·lules mare de l'arrel està el centre quiescent (QC), un grup de cèl·lules amb una activitat mitòtica molt baixa que manté l'estat indiferenciat de les cèl·lules mare circumdants i actua com a reservori cel·lular. Les cèl·lules del QC només activen les seves divisions quan necessiten reemplenar les cèl·lules mare, per exemple, després d'un dany al DNA. La senyalització per BRs s'encarrega d'activar aquestes divisions del QC, però encara es desconeixen els mecanismes exactes de com es regula aquest procés. En aquest treball, utilitzem un enfocament interdisciplinari, utilitzant Arabidopsis thaliana com a sistema model, que inclou genètica molecular, fisiologia i bioinformàtica, per desxifrar el paper dels receptors de BRs en resposta al dany al DNA i en la regulació de les divisions del QC. Els resultats presentats en aquesta tesi descobreixen noves funcions per a la proteïna quinasa BRL3 (BRI1-like 3) en el dany al DNA i en la maquinària de reparació de l'ADN a les plantes. Això és important per controlar els mecanismes de reparació del DNA i la progressió del cicle cel·lular al meristema de l'arrel, que requereix un equilibri d'aquests processos per garantir l'adaptació de la planta a condicions adverses. Vam trobar que, a l'àpex de l'arrel, els esdeveniments de senyalització aigües avall de BRL3 modulen un enzim que s'expressa específicament a les cèl·lules del QC, la RNR2A (RIBONUCLEOTIDE REDUCTASE 2A), encarre

Drought is a major environmental stress that limits growth and productivity in agricultural ecosystems limiting crop yield worldwide. Breeding crops for enhanced drought tolerance is a priority to preserve food security on the increasing world population. Recent work in Arabidopsis has shown that vascular brassinosteroid receptor BRL3 (Brassinosteroid insensitive like-3) transcriptionally controls the production of osmoprotectant metabolites that confer drought resistance without penalizing growth, offering new and exciting possibilities for biotechnological improvement of drought-resistant crops. In cereals, understanding transcriptional responses to drought is an essential step for the production of gene-edited drought-resistant cereals. In this chapter, we present a method to analyze the transcriptional responses to drought in Sorghum bicolor (L.) Moench, our cereal of choice. Among the genes we tested, we found that drought marker gene SbDHN1 has a 1000-fold increase only after 1 day of drought, bringing possibilities for the development of molecular sensors for testing drought. Overall, this analysis is useful to set up conditions of high-throughput transcriptomic analysis of drought stressed plants before drought phenotype is observed.

Drought is a major cause of agricultural losses worldwide. Climate change will intensify drought episodes threatening agricultural sustainability. Gaining insights into drought response mechanisms is vital for crop adaptation to climate emergency. To date, only few studies report comprehensive analyses of plant metabolic adaptation to drought. Here, we present a multifactorial metabolomic study of early-mid drought stages in the model plant Arabidopsis thaliana. We sampled root and shoot tissues of plants subjected to water withholding over a six-day time course, including brassinosteroids receptor mutants previously reported to show drought tolerance phenotypes. Furthermore, we sequenced the root transcriptome at basal and after 5 days drought, allowing direct correlation between metabolic and transcriptomic changes and the multi-omics integration. Significant abiotic stress signatures were already activated at basal conditions in a vascular-specific receptor overexpression (BRL3ox). These were also rapidly mobilized under drought, revealing a systemic adaptation strategy driven from inner tissues of the plant. Overall, this dataset provides a significant asset to study drought metabolic adaptation and allows its analysis from multiple perspectives.

In animals and plants, stem-cell niches are local microenvironments that are tightly regulated to preserve their unique identity while communicating with adjacent cells that will give rise to specialized cell types. In the primary root of Arabidopsis thaliana, two transcription factors, BRAVO and WOX5, among others, are expressed in the stem-cell niche. Intriguingly, BRAVO, a repressor of quiescent center divisions, confines its own gene expression to the stem-cell niche, as evidenced in a bravo mutant background. Here, we propose through mathematical modeling that BRAVO confines its own expression domain to the stem-cell niche by attenuating a WOX5-dependent diffusible activator of BRAVO. This negative feedback drives WOX5 activity to be spatially restricted as well. The results show that WOX5 diffusion and sequestration by binding to BRAVO are sufficient to drive the experimentally observed confined BRAVO expression at the stem-cell niche. We propose that the attenuation of a diffusible activator can be a general mechanism acting at other stem-cell niches to spatially confine genetic activity to a small region while maintaining signaling within them and with the surrounding cells.