BRAVO self-confined expression through WOX5 in the Arabidopsis root stem-cell niche

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

Spatiotemporal analysis of brassinosteroid signaling in the vascular stem cells

Aquesta tesi doctoral descriu diversos avenços conceptuals per a la comprensió molecular de la senyalització de brassinosteroides en el nínxol de cèl·lules mare de l'arrel d'Arabidopsis thaliana. Els brassinoesteroides són les hormones esteroides de les plantes que juguen un paper important en el creixement i desenvolupament vegetal. En l'arrel primària d'Arabidopsis, els brassinoesteroides estan involucrats en el desenvolupament del meristem i manteniment de les cèl·lules mare. Les cèl·lules mare són les cèl·lules més indiferenciades, aquestes es van dividint i diferenciant per generar els diferents tipus cel·lulars de l'arrel. Aquests processos estan estretament controlats per factors interns i externs. El baix nombre de cada població de cèl·lules mare fa complicat el seu estudi de manera individual, per tant, el desenvolupament de mètodes amb resolució per estudiar tipus cel·lulars i fins i tot cèl·lules individuals representa una oportunitat única per investigar aquesta població cel·lular tan escassa. En aquesta tesi doctoral, utilitzem una estratègia multidisciplinària, que inclou genètica, anàlisi transcriptòmica i models matemàtics, per identificar les característiques moleculars de les cèl·lules mare de l'arrel enfocant-nos en el paper dels brassinoesteroides en aquestes cèl·lules. Defectes en els processos de creixement i desenvolupament vegetal generalment són deguts a defectes en el creixement de l'arrel principal. Com que la quantificació precisa de la longitud de l'arrel requereix molt de temps, en aquesta tesi doctoral es descriu el desenvolupament de l'eina MyROOT per a la mesura d'arrels d'Arabidopsis d'una manera semiautomàtica (Capítol 2). A més a més, els resultats presentats en aquesta tesi revelen quin és el paper dels brassinoesteroides en el nínxol de cèl·lules mare. Mitjançant una estratègia de biologia de sistemes s'ha estudiat el paper del factor de transcripció BRAVO, regulat per brassinoesteroides, juntament amb WOX5 en el creixement i desenvolupament de l'arrel (Capítol 3). Igualment, una aproximació específica per tipus cel·lulars ha revelat la resposta transcripcional mediada per BRAVO al centre quiescent i en les cèl·lules mare vasculars adjacents (Capítol 4). Finalment, l'ús de seqüenciació massiva de l'RNA (RNAseq) amb resolució cel·lular ha estat implementat per generar el primer atles transcriptòmic del nínxol de cèl·lules mare de l'arrel. Aquesta aproximació ha permès identificar les característiques moleculars de les cèl·lules mare i la presència de diferents poblacions d'aquestes en el domini d'expressió de BRAVO (Capítol 5). Aquesta tesi doctoral avança en el coneixement de les cèl·lules mare de les plantes i posa de manifest la necessitat d'estratègies multidisciplinàries per descobrir processos fonamentals de el desenvolupament vegetal.Esta tesis doctoral reporta avances conceptuales en la respuesta molecular mediada por la ruta de señalización de los brasinoesteroides en el nicho de células madre de Arabidopsis thaliana. Los brasinoesteroides son las hormonas esteroideas de las plantas y juegan un papel importante en el crecimiento y desarrollo vegetal. En la raíz primaria de Arabidopsis, los brasinoesteroides están involucrados en el desarrollo del meristemo y mantenimiento de las células madre. En el nicho de células madre, las células madre son las células más indiferenciadas que se van dividiendo y diferenciando para generar los distintos tipos celulares de la raíz. Estos procesos están estrechamente controlados por factores internos y externos. El bajo número de cada población de células madre hace complicado su estudio individualmente, por lo tanto, el desarrollo de métodos con resolución para estudiar tipos celulares e incluso células individualmente representa una oportunidad única para investigar esta población celular tan escasa. En esta tesis doctoral, utilizamos una estrategia multidisciplinar, que incluye genética, análisis transcriptómicos y modelos matemáticos, para identificar las características moleculares de las células madre de la raíz y enfocándonos al papel de los brasinoesteroides en esas células. Defectos en procesos de crecimiento y desarrollo vegetal se reflejan generalmente en defectos en el crecimiento de la raíz principal. Como la cuantificación precisa de la longitud de la raíz requiere mucho tiempo, en esta tesis doctoral se describe el desarrollo de la herramienta MyROOT para la medida de raíces de Arabidopsis de una forma semiautomática (Capítulo 2). Además, los resultados presentados en esta tesis revelan el papel de los brasinoesteroides en el nicho de células madre. Una estrategia de biología de sistemas revela el papel del factor de transcripción BRAVO, regulado por brasinoesteroides, junto con WOX5 en el crecimiento y desarrollo de la raíz (Capítulo 3). Igualmente, una aproximación específica para tipos celulares revela la respuesta transcripcional mediada por BRAVO en el centro quiescente y en las células madre vasculares adyacentes (Capítulo 4). Por último, el uso de RNAseqs con resolución celular ha sido implementado para generar el que creemos es el primer atlas transcriptómico del nicho de células madre de la raíz. Esta aproximación ha permitido identificar las características moleculares de las células madre y la presencia de diferentes poblaciones de estas células en el dominio de expresión de BRAVO (Capítulo 5). Esta tesis doctoral avanza en el conocimiento de las células madre de las plantas y pone de manifiesto la necesidad de estrategias multidisciplinares para descubrir procesos fundamentales del desarrollo vegetal.This PhD thesis dissertation reports a number conceptual advances for the molecular understanding of brassinosteroid signaling in the root stem cell niche of Arabidopsis thaliana. Brassisnosteroids are the plant steroid hormones that play important roles in plant growth and development. In the Arabidopsis primary root, brassinosteroids are involved in meristem development and stem cell maintenance. At the root stem cell niche, stem cells are the more undifferentiated cells that divide and differentiate to give rise to the distinct cell types of the root. These processes are tightly controlled by internal and external factors. The low number each stem cell population makes it difficult to study them individually, therefore, the advent of cell-type and single-cell specific approaches represents a unique opportunity to investigate this rare cell population. In this PhD thesis, we used an interdisciplinary approach, including genetics, transcriptomics analysis and mathematical modelling, to identify the molecular signatures of the root stem cells with a focus on the role of brassinosteroid hormones in those cells. Defects in growth and development processes is often reflected in abnormal primary root growth. As the accurate quantification of plant primary root length is time consuming, in this PhD dissertation, we describe the development of MyROOT software for the semi-automatic measurement of Arabidopsis primary roots (Chapter 2). In addition, the results presented in this thesis uncover the role of brassinosteroids in the stem cell niche. A systems biology approach revealed a role of the brassinosteroid-mediated BRAVO transcription factor together with WOX5 in overall root growth and development (Chapter 3). Moreover, cell-type specific transcriptomic analysis uncover the transcriptional response mediated by BRAVO in the QC and adjacent vascular stem cells (Chapter 4). Finally, the use of single-cell RNAseq has been implemented to generate to our knowledge the first transcriptomic atlas of the root stem cell niche. This approach allowed to characterize the molecular signatures of the stem cells and to find novel stem cell populations within the BRAVO expression domain (Chapter 5). Overall, the present PhD thesis advances in the understanding of stem cells in plants and expose the necessity of multidisciplinary approaches to uncover fundamental biological questions in plant development.Universitat Autònoma de Barcelona. Programa de Doctorat en Biologia i Biotecnologia Vegeta

MyROOT : a method and software for the semiautomatic measurement of primary root length in Arabidopsis seedlings

Root analysis is essential for both academic and agricultural research. Despite the great advances in root phenotyping and imaging, calculating root length is still performed manually and involves considerable amounts of labor and time. To overcome these limitations, we developed MyROOT, a software for the semiautomatic quantification of root growth of seedlings growing directly on agar plates. Our method automatically determines the scale from the image of the plate, and subsequently measures the root length of the individual plants. To this aim, MyROOT combines a bottom-up root tracking approach with a hypocotyl detection algorithm. At the same time as providing accurate root measurements, MyROOT also significantly minimizes the user intervention required during the process. Using Arabidopsis, we tested MyROOT with seedlings from different growth stages and experimental conditions. When comparing the data obtained from this software with that of manual root measurements, we found a high correlation between both methods (R2 = 0.997). When compared with previous developed software with similar features (BRAT and EZ-Rhizo), MyROOT offered an improved accuracy for root length measurements. Therefore, MyROOT will be of great use to the plant science community by permitting high-throughput root length measurements while saving both labor and time

BES1 regulates the localization of the brassinosteroid receptor BRL3 within the provascular tissue of the Arabidopsis primary root

Brassinosteroid (BR) hormones are important regulators of plant growth and development. Recent studies revealed the cell-specific role of BRs in vascular and stem cell development by the action of cell-specific BR receptor complexes and downstream signaling components in Arabidopsis thaliana. Despite the importance of spatiotemporal regulation of hormone signaling in the control of plant vascular development, the mechanisms that confer cellular specificity to BR receptors within the vascular cells are not yet understood. The present work shows that BRI1-like receptor genes 1 and 3 (BRL1 and BRL3) are differently regulated by BRs. By using promoter deletion constructs of BRL1 and BRL3 fused to GFP/GUS (green fluorescent protein/β-glucuronidase) reporters in Arabidopsis, analysis of their cell-specific expression and regulation by BRs in the root apex has been carried out. We found that BRL3 expression is finely modulated by BRs in different root cell types, whereas the location of BRL1 appears to be independent of this hormone. Physiological and genetic analysis show a BR-dependent expression of BRL3 in the root meristem. In particular, BRL3 expression requires active BES1, a central transcriptional effector within the BRI1 pathway. ChIP analysis showed that BES1 directly binds to the BRRE present in the BRL3 promoter region, modulating its transcription in different subsets of cells of the root apex. Overall our study reveals the existence of a cell-specific negative feedback loop from BRI1-mediated BES1 transcription factor to BRL3 in phloem cells, while contributing to a general understanding of the spatial control of steroid signaling in plant development

MyROOT 2.0 : An automatic tool for high throughput and accurate primary root length measurement

The automatic measurement of external physical traits (i.e. phenotyping) of plant organs, such as root length -which is highly correlated with plant viability- is one of the current bottlenecks in academic and agricultural research. Although many root length measurement software tools are available to the community, plant scientists often find their usability is limited, the measurements they provide are not accurate enough or they are too limited to specific image characteristics. In response to that, this work describes MyROOT 2.0, an automatic software tool jointly developed by plant scientists and computer vision engineers to create a high throughput root length measurement tool that reduces user intervention to a minimum. Using Arabidopsis thaliana seedlings grown on agar plates as a case study, MyROOT 2.0 is capable of detecting the root regions of interest in a fully automatic manner with an accuracy of 98%. Furthermore, this work also presents previously unreported experiments to evaluate several constituting modules of MyROOT 2.0, such as the ability to determine image scale automatically with subpixel accuracy, or the influence of training the hypocotyl detector using wildtype or mutant samples. Finally, when compared to state-of-the-art root length measurement software tools, MyROOT 2.0 achieves the highest root detection rate, obtaining measurements which are four times more accurate than its competitors. This makes MyROOT 2.0 an attractive tool for high throughput root phenotyping

MyROOT 2.0 : An automatic tool for high throughput and accurate primary root length measurement

The automatic measurement of external physical traits (i.e. phenotyping) of plant organs, such as root length -which is highly correlated with plant viability- is one of the current bottlenecks in academic and agricultural research. Although many root length measurement software tools are available to the community, plant scientists often find their usability is limited, the measurements they provide are not accurate enough or they are too limited to specific image characteristics. In response to that, this work describes MyROOT 2.0, an automatic software tool jointly developed by plant scientists and computer vision engineers to create a high throughput root length measurement tool that reduces user intervention to a minimum. Using Arabidopsis thaliana seedlings grown on agar plates as a case study, MyROOT 2.0 is capable of detecting the root regions of interest in a fully automatic manner with an accuracy of 98%. Furthermore, this work also presents previously unreported experiments to evaluate several constituting modules of MyROOT 2.0, such as the ability to determine image scale automatically with subpixel accuracy, or the influence of training the hypocotyl detector using wildtype or mutant samples. Finally, when compared to state-of-the-art root length measurement software tools, MyROOT 2.0 achieves the highest root detection rate, obtaining measurements which are four times more accurate than its competitors. This makes MyROOT 2.0 an attractive tool for high throughput root phenotyping

BES1 regulates the localization of the brassinosteroid receptor BRL3 within the provascular tissue of the Arabidopsis primary root

Brassinosteroid (BR) hormones are important regulators of plant growth and development. Recent studies revealed the cell-specific role of BRs in vascular and stem cell development by the action of cell-specific BR receptor complexes and downstream signaling components in Arabidopsis thaliana. Despite the importance of spatiotemporal regulation of hormone signaling in the control of plant vascular development, the mechanisms that confer cellular specificity to BR receptors within the vascular cells are not yet understood. The present work shows that BRI1-like receptor genes 1 and 3 (BRL1 and BRL3) are differently regulated by BRs. By using promoter deletion constructs of BRL1 and BRL3 fused to GFP/GUS (green fluorescent protein/β-glucuronidase) reporters in Arabidopsis, analysis of their cell-specific expression and regulation by BRs in the root apex has been carried out. We found that BRL3 expression is finely modulated by BRs in different root cell types, whereas the location of BRL1 appears to be independent of this hormone. Physiological and genetic analysis show a BR-dependent expression of BRL3 in the root meristem. In particular, BRL3 expression requires active BES1, a central transcriptional effector within the BRI1 pathway. ChIP analysis showed that BES1 directly binds to the BRRE present in the BRL3 promoter region, modulating its transcription in different subsets of cells of the root apex. Overall our study reveals the existence of a cell-specific negative feedback loop from BRI1-mediated BES1 transcription factor to BRL3 in phloem cells, while contributing to a general understanding of the spatial control of steroid signaling in plant development

BES1 regulates the localization of the brassinosteroid receptor BRL3 within the provascular tissue of the Arabidopsis primary root

Brassinosteroid (BR) hormones are important regulators of plant growth and development. Recent studies revealed the cell-specific role of BRs in vascular and stem cell development by the action of cell-specific BR receptor complexes and downstream signaling components in Arabidopsis thaliana. Despite the importance of spatiotemporal regulation of hormone signaling in the control of plant vascular development, the mechanisms that confer cellular specificity to BR receptors within the vascular cells are not yet understood. The present work shows that BRI1-like receptor genes 1 and 3 (BRL1 and BRL3) are differently regulated by BRs. By using promoter deletion constructs of BRL1 and BRL3 fused to GFP/GUS (green fluorescent protein/β-glucuronidase) reporters in Arabidopsis, analysis of their cell-specific expression and regulation by BRs in the root apex has been carried out. We found that BRL3 expression is finely modulated by BRs in different root cell types, whereas the location of BRL1 appears to be independent of this hormone. Physiological and genetic analysis show a BR-dependent expression of BRL3 in the root meristem. In particular, BRL3 expression requires active BES1, a central transcriptional effector within the BRI1 pathway. ChIP analysis showed that BES1 directly binds to the BRRE present in the BRL3 promoter region, modulating its transcription in different subsets of cells of the root apex. Overall our study reveals the existence of a cell-specific negative feedback loop from BRI1-mediated BES1 transcription factor to BRL3 in phloem cells, while contributing to a general understanding of the spatial control of steroid signaling in plant development