Analysis of metabolic dynamics during drought stress in Arabidopsis plants

Altres ajuts: we acknowledge support from the CERCA Programme/Generalitat de CatalunyaDrought 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 perspective

Paracrine brassinosteroid signaling at the stem cell niche controls cellular regeneration

Stem cell regeneration is crucial for both cell turnover and tissue healing in multicellular organisms. In Arabidopsis roots, a reduced group of cells known as the quiescent center (QC) act as a cell reservoir for surrounding stem cells during both normal growth and in response to external damage. Although cells of the QC have a very low mitotic activity, plant hormones such as brassinosteroids (BRs) can promote QC divisions. Here, we used a tissue-specific strategy to investigate the spatial signaling requirements of BR-mediated QC divisions. We generated stem cell niche-specific receptor knockout lines by placing an artificial microRNA against BRI1 (BRASSINOSTEROID INSENSITIVE 1) under the control of the QC-specific promoter WOX5. Additionally, QC-specific knock-in lines for BRI1 and its downstream transcription factor BES1 (BRI1-EMS-SUPPRESOR1) were also created using the WOX5 promoter. By analyzing the roots of these lines, we show that BES1-mediated signaling cell-autonomously promotes QC divisions, that BRI1 is essential for sensing nearby inputs and triggering QC divisions and that DNA damage promotes BR-dependent paracrine signaling in the stem cell niche as a prerequisite to stem cell replenishment

Control of abiotic stress responses by brassinosteroids receptors in Arabidopsis thaliana

La presente tesis doctoral reporta nuevas funciones de los receptores de brassinosteroides (BRs) en el control de respuestas a estrés abiótico en la planta modelo Arabidopsis thaliana. Los BRs son hormonas esteroides que desempeñan roles esenciales en el crecimiento y desarrollo de la planta, así como en su adaptación al estrés ambiental. Se sabe que la aplicación exógena de BRs dota a los cultivos de protección frente a estreses abióticos, tales como estrés salino, frío o sequía, pero aún se desconocen los mecanismos que gobiernan estas respuestas. La activación de los componentes de la señalización mediada por BRs no han conseguido dotar de la resistencia observada con aplicaciones exógenas. Los roles putativos de los receptores de BRs bajo estrés arrojarian información clave para desentrañar los mecanismos de adaptación a estrés mediados por BRs, pero estos han permanecido inexplorados. Aquí, usamos un enfoque multidisciplinar, incluyendo genética, análisis multiómico y bioinformática, para descifrar los roles de los receptores de BR frente a estreses abióticos, tales como daño al ADN, estrés osmótico y sequía. Los resultados presentados en esta tesis desvelan el papel del control espacio-temporal de la señalización de BRs en respuesta a estrés abiótico. El análisis fisiológico de raíces de Arabidopsis reveló que los receptores de BRs son necesarios para la regeneración celular de las células madre de la raíz tras daño al ADN. Además el análisis multiómico de plantas expuestas a sequía mostró que la sobre expresión del receptor BRI1-like 3 (BRL3), específico de tejidos vasculares, promueve una firma transcriptomica y metabolómica alterada que alivia los efectos negativos de la sequía y los desacopla de la parada del crecimiento. La mayor parte de los distintivos ómicos encontrados en estas plantas son específicos de floema. El enfoque bioinformático usado para desgranar el control transcripcional específico de tejido, fue además implementado en una herramienta web extensible a cualquier otro organismo. Finalmente, mediante un enfoque de biología estructural, encontramos una pequeña Receptor-Like Kinase (RLK), cuya interacción con BRL3 es más favorable que con el correceptor canónico BAK1. De hecho, este candidato se ha implicado recientemente en la respuesta a estrés osmótico, lo que sugiere vías alternativas activadas por BRs que controlan la respuestas a estrés abiótico. En resumen, la presente tesis doctoral avanza sobre las funciones de los receptores de BRs que promueven el crecimiento y supervivencia de la planta bajo estrés abiótico. La señalización paracrina de los BRs en el nicho de células madre de la raíz y la adaptación metabólica dirigida desde los tejidos vasculares ilustran la importancia de analizar las respuestas específicas de tejidos. El presente estudio también arroja nuevas ideas para futuras investigaciones en los mecanismos mediados por los BRs que contribuyen a la adaptación de las plantas.The present PhD thesis dissertation reports new functions for Brasssinosteroids receptors controlling abiotic stress responses in Arabidopsis thaliana. Brassinosteroids (BRs) are the steroid hormones of plants. BRs play essential roles in plant growth and development and plant adaptation to stress. In this direction, exogenous application of BRs provide crop protection against abiotic stresses, such as salt, cold or drought stress, yet the mechanisms governing these responses have remained unknown. Activation of signaling downstream components failed to provide the resistance observed with exogenous applications. The putative roles of BR receptors under stress stand out as key information for dissecting the BR-driven mechanism of stress adaptation but they have remained very unexplored. Here, we use an interdisciplinary approach, including genetics, multiomics analyses and bioinformatics, to decipher the roles of BR receptors in front of abiotic stresses such as DNA damage, osmotic stress and drought. The results presented in this thesis uncover a role for the spatiatiotemporal control of BR signaling in response to abiotic stress. Physiological analysis of Arabidopsis roots revealed that BR receptors are required for cellular regeneration of the root stem cells after DNA damage. Moreover, the multiomic analysis of plants exposed to drought showed that the overexpression of the vascular-specific BRI1-like 3 (BRL3) receptor lead to an altered transcriptional and metabolic signature that alleviate the detrimental effects of drought and decouple drought tolerance from growth arrest. A major part of omics hallmarks found in these plants are phloem-specific. The bioinformatic approach used to disentangle tissue-specific transcriptional control was further implemented in a web tool, expandable to any plant specie. Finally, through a structural biology approach we found a small Receptor-Like Kinase (RLK) whose interaction with BRL3 is more favorable than the canonical co-receptor BAK1. Indeed, this candidate has been recently involved in response to osmotic stress, which suggest alternative BR-activated pathways that control abiotic stress responses. Overall, the present PhD thesis advances the roles of BR receptors to support plant growth and survival under abiotic stress. BRs paracrine signaling at the root stem cell niche and the metabolic adaptation driven from vascular tissues illustrate the importance of dissecting plant tissue-specific responses. The study presented here also opens new windows for further investigation on mechanisms triggered by BR-receptor that contribute to plant adaptation

Control of abiotic stress responses by brassinosteroids receptors in Arabidopsis thaliana

La presente tesis doctoral reporta nuevas funciones de los receptores de brassinosteroides (BRs) en el control de respuestas a estrés abiótico en la planta modelo Arabidopsis thaliana. Los BRs son hormonas esteroides que desempeñan roles esenciales en el crecimiento y desarrollo de la planta, así como en su adaptación al estrés ambiental. Se sabe que la aplicación exógena de BRs dota a los cultivos de protección frente a estreses abióticos, tales como estrés salino, frío o sequía, pero aún se desconocen los mecanismos que gobiernan estas respuestas. La activación de los componentes de la señalización mediada por BRs no han conseguido dotar de la resistencia observada con aplicaciones exógenas. Los roles putativos de los receptores de BRs bajo estrés arrojarian información clave para desentrañar los mecanismos de adaptación a estrés mediados por BRs, pero estos han permanecido inexplorados. Aquí, usamos un enfoque multidisciplinar, incluyendo genética, análisis multiómico y bioinformática, para descifrar los roles de los receptores de BR frente a estreses abióticos, tales como daño al ADN, estrés osmótico y sequía. Los resultados presentados en esta tesis desvelan el papel del control espacio-temporal de la señalización de BRs en respuesta a estrés abiótico. El análisis fisiológico de raíces de Arabidopsis reveló que los receptores de BRs son necesarios para la regeneración celular de las células madre de la raíz tras daño al ADN. Además el análisis multiómico de plantas expuestas a sequía mostró que la sobre expresión del receptor BRI1-like 3 (BRL3), específico de tejidos vasculares, promueve una firma transcriptomica y metabolómica alterada que alivia los efectos negativos de la sequía y los desacopla de la parada del crecimiento. La mayor parte de los distintivos ómicos encontrados en estas plantas son específicos de floema. El enfoque bioinformático usado para desgranar el control transcripcional específico de tejido, fue además implementado en una herramienta web extensible a cualquier otro organismo. Finalmente, mediante un enfoque de biología estructural, encontramos una pequeña Receptor-Like Kinase (RLK), cuya interacción con BRL3 es más favorable que con el correceptor canónico BAK1. De hecho, este candidato se ha implicado recientemente en la respuesta a estrés osmótico, lo que sugiere vías alternativas activadas por BRs que controlan la respuestas a estrés abiótico. En resumen, la presente tesis doctoral avanza sobre las funciones de los receptores de BRs que promueven el crecimiento y supervivencia de la planta bajo estrés abiótico. La señalización paracrina de los BRs en el nicho de células madre de la raíz y la adaptación metabólica dirigida desde los tejidos vasculares ilustran la importancia de analizar las respuestas específicas de tejidos. El presente estudio también arroja nuevas ideas para futuras investigaciones en los mecanismos mediados por los BRs que contribuyen a la adaptación de las plantas.The present PhD thesis dissertation reports new functions for Brasssinosteroids receptors controlling abiotic stress responses in Arabidopsis thaliana. Brassinosteroids (BRs) are the steroid hormones of plants. BRs play essential roles in plant growth and development and plant adaptation to stress. In this direction, exogenous application of BRs provide crop protection against abiotic stresses, such as salt, cold or drought stress, yet the mechanisms governing these responses have remained unknown. Activation of signaling downstream components failed to provide the resistance observed with exogenous applications. The putative roles of BR receptors under stress stand out as key information for dissecting the BR-driven mechanism of stress adaptation but they have remained very unexplored. Here, we use an interdisciplinary approach, including genetics, multiomics analyses and bioinformatics, to decipher the roles of BR receptors in front of abiotic stresses such as DNA damage, osmotic stress and drought. The results presented in this thesis uncover a role for the spatiatiotemporal control of BR signaling in response to abiotic stress. Physiological analysis of Arabidopsis roots revealed that BR receptors are required for cellular regeneration of the root stem cells after DNA damage. Moreover, the multiomic analysis of plants exposed to drought showed that the overexpression of the vascular-specific BRI1-like 3 (BRL3) receptor lead to an altered transcriptional and metabolic signature that alleviate the detrimental effects of drought and decouple drought tolerance from growth arrest. A major part of omics hallmarks found in these plants are phloem-specific. The bioinformatic approach used to disentangle tissue-specific transcriptional control was further implemented in a web tool, expandable to any plant specie. Finally, through a structural biology approach we found a small Receptor-Like Kinase (RLK) whose interaction with BRL3 is more favorable than the canonical co-receptor BAK1. Indeed, this candidate has been recently involved in response to osmotic stress, which suggest alternative BR-activated pathways that control abiotic stress responses. Overall, the present PhD thesis advances the roles of BR receptors to support plant growth and survival under abiotic stress. BRs paracrine signaling at the root stem cell niche and the metabolic adaptation driven from vascular tissues illustrate the importance of dissecting plant tissue-specific responses. The study presented here also opens new windows for further investigation on mechanisms triggered by BR-receptor that contribute to plant adaptation

Emerging roles of vascular brassinosteroid receptors of the BRI1-like family

Brassinosteroids (BRs) are essential hormones for plant growth and development that are perceived at the plasma membrane by a group of Leucine-Rich Repeat Receptor-Like Kinases (LRR–RLKs) of the BRASSINOSTEROID INSENSITIVE 1 (BRI1) family. The BRI1 receptor was first discovered by genetic screenings based on the dwarfism of BR-deficient plants. There are three BRI1 homologs, named BRI1-like 1, 2 and 3 (BRLs), yet only BRL1 and BRL3 behave as functional BR receptors. Whereas the BRI1 pathway operates in the majority of cells to promote growth, BRL receptor signaling operates under specific spatiotemporal constraints. Despite a wealth of information on the BRI1 pathway, data on specific BRL pathways and their biological relevance is just starting to emerge. Here, we systematically compare BRLs with BRI1 to identify any differences that could account for specific receptor functions. Understanding how vascular and cell-specific BRL receptors orchestrate plant development and adaptation to the environment will help shed light on membrane signaling and cell communication in plants, while opening up novel possibilities to improve stress adaptation without penalizing growth.A.I.C-D. is a recipient of a Spanish Ministry of Economy and Competitiveness and a European Research Council (FEDER-BIO2016-78150-), ERC Consolidator Grant (ERC-2015-CoG – 683163). F.L.E. PhD thesis is funded by the FEDER-BIO2016-78150-P grant in A.I.C-D laboratory.Peer reviewe

Control of abiotic stress responses by brassinosteroids receptors in Arabidopsis thaliana /

Departament responsable de la tesi: Departament de Biologia Animal, de Biologia Vegetal i d'Ecologia.La presente tesis doctoral reporta nuevas funciones de los receptores de brassinosteroides (BRs) en el control de respuestas a estrés abiótico en la planta modelo Arabidopsis thaliana.Los BRs son hormonas esteroides que desempeñan roles esenciales en el crecimiento y desarrollo de la planta, así como en su adaptación al estrés ambiental. Se sabe que la aplicación exógena de BRs dota a los cultivos de protección frente a estreses abióticos, tales como estrés salino, frío o sequía, pero aún se desconocen los mecanismos que gobiernan estas respuestas. La activación de los componentes de la señalización mediada por BRs no han conseguido dotar de la resistencia observada con aplicaciones exógenas. Los roles putativos de los receptores de BRs bajo estrés arrojarian información clave para desentrañar los mecanismos de adaptación a estrés mediados por BRs, pero estos han permanecido inexplorados. Aquí, usamos un enfoque multidisciplinar, incluyendo genética, análisis multiómico y bioinformática, para descifrar los roles de los receptores de BR frente a estreses abióticos, tales como daño al ADN, estrés osmótico y sequía. Los resultados presentados en esta tesis desvelan el papel del control espacio-temporal de la señalización de BRs en respuesta a estrés abiótico. El análisis fisiológico de raíces de Arabidopsis reveló que los receptores de BRs son necesarios para la regeneración celular de las células madre de la raíz tras daño al ADN. Además el análisis multiómico de plantas expuestas a sequía mostró que la sobre expresión del receptor BRI1-like 3 (BRL3), específico de tejidos vasculares, promueve una firma transcriptomica y metabolómica alterada que alivia los efectos negativos de la sequía y los desacopla de la parada del crecimiento. La mayor parte de los distintivos ómicos encontrados en estas plantas son específicos de floema. El enfoque bioinformático usado para desgranar el control transcripcional específico de tejido, fue además implementado en una herramienta web extensible a cualquier otro organismo. Finalmente, mediante un enfoque de biología estructural, encontramos una pequeña Receptor-Like Kinase (RLK), cuya interacción con BRL3 es más favorable que con el correceptor canónico BAK1. De hecho, este candidato se ha implicado recientemente en la respuesta a estrés osmótico, lo que sugiere vías alternativas activadas por BRs que controlan la respuestas a estrés abiótico.En resumen, la presente tesis doctoral avanza sobre las funciones de los receptores de BRs que promueven el crecimiento y supervivencia de la planta bajo estrés abiótico. La señalización paracrina de los BRs en el nicho de células madre de la raíz y la adaptación metabólica dirigida desde los tejidos vasculares ilustran la importancia de analizar las respuestas específicas de tejidos. El presente estudio también arroja nuevas ideas para futuras investigaciones en los mecanismos mediados por los BRs que contribuyen a la adaptación de las plantas.The present PhD thesis dissertation reports new functions for Brasssinosteroids receptors controlling abiotic stress responses in Arabidopsis thaliana. Brassinosteroids (BRs) are the steroid hormones of plants. BRs play essential roles in plant growth and development and plant adaptation to stress. In this direction, exogenous application of BRs provide crop protection against abiotic stresses, such as salt, cold or drought stress, yet the mechanisms governing these responses have remained unknown. Activation of signaling downstream components failed to provide the resistance observed with exogenous applications. The putative roles of BR receptors under stress stand out as key information for dissecting the BR-driven mechanism of stress adaptation but they have remained very unexplored. Here, we use an interdisciplinary approach, including genetics, multiomics analyses and bioinformatics, to decipher the roles of BR receptors in front of abiotic stresses such as DNA damage, osmotic stress and drought. The results presented in this thesis uncover a role for the spatiatiotemporal control of BR signaling in response to abiotic stress. Physiological analysis of Arabidopsis roots revealed that BR receptors are required for cellular regeneration of the root stem cells after DNA damage. Moreover, the multiomic analysis of plants exposed to drought showed that the overexpression of the vascular-specific BRI1-like 3 (BRL3) receptor lead to an altered transcriptional and metabolic signature that alleviate the detrimental effects of drought and decouple drought tolerance from growth arrest. A major part of omics hallmarks found in these plants are phloem-specific. The bioinformatic approach used to disentangle tissue-specific transcriptional control was further implemented in a web tool, expandable to any plant specie. Finally, through a structural biology approach we found a small Receptor-Like Kinase (RLK) whose interaction with BRL3 is more favorable than the canonical co-receptor BAK1. Indeed, this candidate has been recently involved in response to osmotic stress, which suggest alternative BR-activated pathways that control abiotic stress responses. Overall, the present PhD thesis advances the roles of BR receptors to support plant growth and survival under abiotic stress. BRs paracrine signaling at the root stem cell niche and the metabolic adaptation driven from vascular tissues illustrate the importance of dissecting plant tissue-specific responses. The study presented here also opens new windows for further investigation on mechanisms triggered by BR-receptor that contribute to plant adaptation

Transcriptomic study of Arabidopsis roots overexpressing the brassinosteroid receptor BRL3, in control conditions and under severe drought [Dataset]

28 days old root system were collected from soil, quickly washed in water and flash-frozen. Experiment with a bifactorial design. Factor one is the genotype, which include WT (Col-0) and 35S:BRL3. Factor two is the condition, which include control (Properly watered) and 5 days of drought (water-hold) conditions. 3 Biological replicates were collected per each genotype and condition. -- Organism: Arabidopsis thaliana. -- Experiment type: Expression profiling by high throughput sequencing.Resources available on the publisher's site: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119382Drought is the primary cause of global agricultural losses and represents a major threat to worldwide food security. Currently, plant biotechnology stands out as the most promising strategy to increase crop growth in rain-fed conditions. The main mechanisms underlying drought resistance have been uncovered by studies of plant physiology and by engineering crops with drought-resistant genes. However, plants with enhanced drought resistance usually display lower levels of growth, highlighting the need to search for novel strategies capable of uncoupling drought resistance from growth. Here, we show that the brassinosteroid family of receptors, in addition to promoting growth, guides phenotypic adaptation to a great variety of drought stress traits analyzed herein. Whilst mutations in the ubiquitously localized BRI1 receptor pathway show an enhanced drought resistance at the expense of plant growth, we found that vascular-enriched BRL3 receptors confer drought tolerance without penalizing overall growth. Systematic analyses reveal that upon drought stress the BRL3 receptor pathway triggers the synthesis and mobilization of osmoprotectant metabolites, mainly proline and sugars. This preferentially occurs in the vascular tissues of the roots and favors overall plant growth. Altogether, our results uncover a new role for the spatial control of BR signaling in drought tolerance, and offer a novel strategy to address food security issues in an increasingly water-limited climate.Peer reviewe

The primary root of Sorghumbicolor (L. Moench) as a model system to study brassinosteroid signaling in crops

Roots anchor plants to the soil and are essential for a successful plant growth and adaptation to the environment. Research on the primary root in the plant model system Arabidopsis thaliana has yielded important advances in the molecular and cellular understanding of root growth and development. Several studies have uncovered how the hormones brassinosteroids (BRs) control cell cycle and differentiation programs through different cell-specific signaling pathways that are key for root growth and development. Currently, an important challenge resides in the translation of the current knowledge on Arabidopsis roots into agronomically valuable species to improve the agricultural production and to meet the food security goals of the millennium. In this chapter, we characterize the primary root apex of the cereal Sorghum bicolor (L. Moench) (sorghum), analyze the physiological response of sorghum roots to BRs, and examine the phylogeny of the BRASSINOSTEROID INSENSITIVE1-like receptor family in Arabidopsis and its orthologous genes in sorghum. Overall, we support the use of sorghum as a suitable crop model species for the study of BR signaling in root growth and development. The methods presented enable any laboratory worldwide to use sorghum primary roots as a favorite organ for the study of growth and development in crops.We acknowledge the financial support from the Spanish Ministry of Economy and Competitiveness, through the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016–2019 (SEV-2015-0533).” N.F. is indebted to the “Fundación Renta Corporation” charity and F.L-E and D.B-E. are funded by a BIO2013-43873 grant and a “Retos Colaboración” project (RTC-2014-1916-2) from the Spanish Ministry of Economy and Competitiveness, respectively. A.I.C.-D. is the recipient of a European Research Council, ERC Consolidator Grant (ERC-2015-CoG – 683163).Peer reviewe

Paracrine brassinosteroid signaling at the stem cell niche controls cellular regeneration

Stem cell regeneration is crucial for both cell turnover and tissue healing in multicellular organisms. In Arabidopsis roots, a reduced group of cells known as the quiescent center (QC) act as a cell reservoir for surrounding stem cells during both normal growth and in response to external damage. Although cells of the QC have a very low mitotic activity, plant hormones such as brassinosteroids (BRs) can promote QC divisions. Here, we used a tissue-specific strategy to investigate the spatial signaling requirements of BR-mediated QC divisions. We generated stem cell niche-specific receptor knockout lines by placing an artificial microRNA against BRI1 (BRASSINOSTEROID INSENSITIVE 1) under the control of the QC-specific promoter WOX5. Additionally, QC-specific knock-in lines for BRI1 and its downstream transcription factor BES1 (BRI1-EMS-SUPPRESOR1) were also created using the WOX5 promoter. By analyzing the roots of these lines, we show that BES1-mediated signaling cell-autonomously promotes QC divisions, that BRI1 is essential for sensing nearby inputs and triggering QC divisions and that DNA damage promotes BR-dependent paracrine signaling in the stem cell niche as a prerequisite to stem cell replenishment