RESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/metabolismo , Meristema/metabolismo , Nitrilas , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Nicho de Células-Tronco/genéticaRESUMO
Brassinosteroids (BRs) are steroid hormones that are essential for plant growth and development. These hormones control the division, elongation and differentiation of various cell types throughout the entire plant life cycle. Our current understanding of the BR signaling pathway has mostly been obtained from studies using Arabidopsis thaliana as a model. In this context, the membrane steroid receptor BRI1 (BRASSINOSTEROID INSENSITIVE 1) binds directly to the BR ligand, triggering a signal cascade in the cytoplasm that leads to the transcription of BR-responsive genes that drive cellular growth. However, recent studies of the primary root have revealed distinct BR signaling pathways in different cell types and have highlighted cell-specific roles for BR signaling in controlling adaptation to stress. In this Review, we summarize our current knowledge of the spatiotemporal control of BR action in plant growth and development, focusing on BR functions in primary root development and growth, in stem cell self-renewal and death, and in plant adaption to environmental stress.
Assuntos
Aclimatação , Arabidopsis/fisiologia , Brassinosteroides/metabolismo , Transdução de Sinais , Estresse Fisiológico , Proteínas de Arabidopsis/fisiologia , Diferenciação Celular , Regulação da Expressão Gênica de Plantas , Ligantes , Desenvolvimento Vegetal , Reguladores de Crescimento de Plantas/fisiologia , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas/fisiologia , Proteínas Quinases/fisiologiaRESUMO
Understanding stem cell regulatory circuits is the next challenge in plant biology, as these cells are essential for tissue growth and organ regeneration in response to stress. In the Arabidopsis primary root apex, stem cell-specific transcription factors BRAVO and WOX5 co-localize in the quiescent centre (QC) cells, where they commonly repress cell division so that these cells can act as a reservoir to replenish surrounding stem cells, yet their molecular connection remains unknown. Genetic and biochemical analysis indicates that BRAVO and WOX5 form a transcription factor complex that modulates gene expression in the QC cells to preserve overall root growth and architecture. Furthermore, by using mathematical modelling we establish that BRAVO uses the WOX5/BRAVO complex to promote WOX5 activity in the stem cells. Our results unveil the importance of transcriptional regulatory circuits in plant stem cell development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Divisão Celular , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Meristema/genética , Meristema/metabolismo , Nitrilas , Raízes de Plantas/genética , Raízes de Plantas/metabolismoRESUMO
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.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Software , Algoritmos , Hipocótilo/crescimento & desenvolvimento , Fenótipo , Raízes de Plantas/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Arabidopsis/citologia , Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Comunicação Parácrina , Regeneração , Transdução de Sinais , Nicho de Células-Tronco , Proteínas de Arabidopsis/metabolismo , Microambiente Celular , Dano ao DNA , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana/metabolismo , Meristema/citologia , Meristema/metabolismo , Modelos Biológicos , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Plântula/citologia , Plântula/metabolismo , Transcrição GênicaRESUMO
The transcription factor BRI1-EMS-SUPRESSOR 1 (BES1) is a master regulator of brassinosteroid (BR)-regulated gene expression. BES1 together with BRASSINAZOLE-RESISTANT 1 (BZR1) drive activated or repressed expression of several genes, and have a prominent role in negative regulation of BR synthesis. Here, we report that BES1 interaction with TOPLESS (TPL), via its ERF-associated amphiphilic repression (EAR) motif, is essential for BES1-mediated control of organ boundary formation in the shoot apical meristem and the regulation of quiescent center (QC) cell division in roots. We show that TPL binds via BES1 to the promoters of the CUC3 and BRAVO targets and suppresses their expression. Ectopic expression of TPL leads to similar organ boundary defects and alterations in QC cell division rate to the bes1-d mutation, while bes1-d defects are suppressed by the dominant interfering protein encoded by tpl-1, with these effects respectively correlating with changes in CUC3 and BRAVO expression. Together, our data unveil a pivotal role of the co-repressor TPL in the shoot and root meristems, which relies on its interaction with BES1 and regulation of BES1 target gene expression.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Meristema/embriologia , Meristema/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Divisão Celular , Flores/fisiologia , Dosagem de Genes , Regulação da Expressão Gênica de Plantas , Organogênese , Fenótipo , Folhas de Planta/embriologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transcrição GênicaRESUMO
Plant roots grow due to cell division in the meristem and subsequent cell elongation and differentiation, a tightly coordinated process that ensures growth and adaptation to the changing environment. How the newly formed cells decide to stop elongating becoming fully differentiated is not yet understood. To address this question, we established a novel approach that combines the quantitative phenotypic variability of wild-type Arabidopsis roots with computational data from mathematical models. Our analyses reveal that primary root growth is consistent with a Sizer mechanism, in which cells sense their length and stop elongating when reaching a threshold value. The local expression of brassinosteroid receptors only in the meristem is sufficient to set this value. Analysis of roots insensitive to BR signaling and of roots with gibberellin biosynthesis inhibited suggests distinct roles of these hormones on cell expansion termination. Overall, our study underscores the value of using computational modeling together with quantitative data to understand root growth.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Modelos Teóricos , Arabidopsis/citologia , Arabidopsis/metabolismo , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Giberelinas/farmacologia , Meristema/citologia , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Fenótipo , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismoRESUMO
Auxin is an essential hormone for plant growth and development. Auxin influx carriers AUX1/LAX transport auxin into the cell, while auxin efflux carriers PIN pump it out of the cell. It is well established that efflux carriers play an important role in the shoot vascular patterning, yet the contribution of influx carriers to the shoot vasculature remains unknown. Here, we combined theoretical and experimental approaches to decipher the role of auxin influx carriers in the patterning and differentiation of vascular tissues in the Arabidopsis inflorescence stem. Our theoretical analysis predicts that influx carriers facilitate periodic patterning and modulate the periodicity of auxin maxima. In agreement, we observed fewer and more spaced vascular bundles in quadruple mutants plants of the auxin influx carriers aux1lax1lax2lax3. Furthermore, we show AUX1/LAX carriers promote xylem differentiation in both the shoot and the root tissues. Influx carriers increase cytoplasmic auxin signaling, and thereby differentiation. In addition to this cytoplasmic role of auxin, our computational simulations propose a role for extracellular auxin as an inhibitor of xylem differentiation. Altogether, our study shows that auxin influx carriers AUX1/LAX regulate vascular patterning and differentiation in plants.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Membrana Transportadoras/genética , Xilema/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Diferenciação Celular/genética , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos , Proteínas de Membrana Transportadoras/metabolismo , Desenvolvimento Vegetal/genética , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Xilema/crescimento & desenvolvimentoRESUMO
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/ß-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.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Proteínas Nucleares/fisiologia , Raízes de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/fisiologia , Brassinosteroides/metabolismo , Proteínas de Ligação a DNA , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/fisiologia , Receptores de Superfície Celular/fisiologiaRESUMO
Brassinosteroid (BR) hormones are primarily perceived at the cell surface by the leucine-rich repeat receptor-like kinase brassinosteroid insensitive1 (BRI1). In Arabidopsis thaliana, BRI1 has two close homologs, BRI1-LIKE1 (BRL1) and BRL3, respectively, which are expressed in the vascular tissues and regulate shoot vascular development. Here, we identify novel components of the BRL3 receptor complex in planta by immunoprecipitation and mass spectrometry analysis. Whereas BRI1 associated kinase1 (BAK1) and several other known BRI1 interactors coimmunoprecipitated with BRL3, no evidence was found of a direct interaction between BRI1 and BRL3. In addition, we confirmed that BAK1 interacts with the BRL1 receptor by coimmunoprecipitation and fluorescence microscopy analysis. Importantly, genetic analysis of brl1 brl3 bak1-3 triple mutants revealed that BAK1, BRL1, and BRL3 signaling modulate root growth and development by contributing to the cellular activities of provascular and quiescent center cells. This provides functional relevance to the observed protein-protein interactions of the BRL3 signalosome. Overall, our study demonstrates that cell-specific BR receptor complexes can be assembled to perform different cellular activities during plant root growth, while highlighting that immunoprecipitation of leucine-rich repeat receptor kinases in plants is a powerful approach for unveiling signaling mechanisms with cellular resolution in plant development.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Transdução de Sinais , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Ciclo Celular , Cromatografia Líquida , Genes Reporter , Complexos Multiproteicos , Mutação , Fenótipo , Floema/citologia , Floema/genética , Floema/crescimento & desenvolvimento , Floema/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão , Espectrometria de Massas em TandemRESUMO
Protein phosphatases with Kelch-like domains (PPKL) are members of the phosphoprotein phosphatases family present only in plants and alveolates. PPKL have been described as positive effectors of brassinosteroid (BR) signaling in plants. Most of the evidence supporting this role has been gathered using one of the four homologs in Arabidopsis (Arabidopsis thaliana), brassinosteroid-insensitive1 suppressor (BSU1). We reappraised the roles of the other three members of the family, BSL1, BSL2, and BSL3, through phylogenetic, functional, and genetic analyses. We show that BSL1 and BSL2/BSL3 belong to two ancient evolutionary clades that have been highly conserved in land plants. In contrast, BSU1-type genes are exclusively found in the Brassicaceae and display a remarkable sequence divergence, even among closely related species. Simultaneous loss of function of the close paralogs BSL2 and BSL3 brings about a peculiar array of phenotypic alterations, but with marginal effects on BR signaling; loss of function of BSL1 is, in turn, phenotypically silent. Still, the products of these three genes account for the bulk of PPKL-related activity in Arabidopsis and together have an essential role in the early stages of development that BSU1 is unable to supplement. Our results underline the functional relevance of BSL phosphatases in plants and suggest that BSL2/BSL3 and BSU1 may have contrasting effects on BR signaling. Given that BSU1-type genes have likely undergone a functional shift and are phylogenetically restricted, we caution that inferences based on these genes to the whole family or to other species may be misleading.
Assuntos
Evolução Molecular , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Plantas/enzimologia , Plantas/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Arabidopsis/genética , Brassinosteroides/farmacologia , Flores/anatomia & histologia , Flores/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Mutação/genética , Fenótipo , Filogenia , Proteínas de Plantas/metabolismo , Plantas/efeitos dos fármacos , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologiaRESUMO
Global climate change and a growing population require tackling the reduction in arable land and improving biomass production and seed yield per area under varying conditions. One of these conditions is suboptimal water availability. Here, we review some of the classical approaches to dealing with plant response to drought stress and we evaluate how research on RECEPTOR-LIKE KINASES (RLKs) can contribute to improving plant performance under drought stress. RLKs are considered as key regulators of plant architecture and growth behavior, but they also function in defense and stress responses. The available literature and analyses of available transcript profiling data indeed suggest that RLKs can play an important role in optimizing plant responses to drought stress. In addition, RLK pathways are ideal targets for nontransgenic approaches, such as synthetic molecules, providing a novel strategy to manipulate their activity and supporting translational studies from model species, such as Arabidopsis thaliana, to economically useful crops.
Assuntos
Secas , Fenômenos Fisiológicos Vegetais , Proteínas Quinases/fisiologia , Projetos de Pesquisa , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/metabolismo , Biossíntese de Proteínas , Estresse FisiológicoRESUMO
Brassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cell elongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Our analyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size, indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, the expression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activity accounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activity of the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiation phenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BR signalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which also negatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal and differentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Divisão Celular , Colestanóis/metabolismo , Meristema/crescimento & desenvolvimento , Reguladores de Crescimento de Plantas/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Esteroides Heterocíclicos/metabolismo , Arabidopsis/citologia , Brassinosteroides , Diferenciação Celular , Meristema/citologia , Mitose , Proteínas Mutantes , Fitosteróis , Células-TroncoRESUMO
Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Carbocianinas/química , Membrana Celular/metabolismo , Colestanóis/metabolismo , Endocitose , Corantes Fluorescentes/química , Proteínas Quinases/metabolismo , Transdução de Sinais , Arabidopsis/enzimologia , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Brassinosteroides/química , Brassinosteroides/metabolismo , Membrana Celular/ultraestrutura , Colestanóis/química , Relação Dose-Resposta a Droga , Endossomos/enzimologia , Endossomos/metabolismo , Endossomos/ultraestrutura , Proteínas de Fluorescência Verde/genética , Cinética , Meristema/enzimologia , Meristema/metabolismo , Meristema/ultraestrutura , Microscopia Confocal , Estrutura Molecular , Reguladores de Crescimento de Plantas , Proteínas Quinases/genética , Transporte Proteico , Plântula/enzimologia , Plântula/metabolismo , Plântula/ultraestrutura , Vacúolos/enzimologia , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
Brassinosteroid (BR) hormones are essential for plant growth and development. In Arabidopsis, the general understanding of BR signaling has been greatly attained by genetic and biochemical approaches that led to the identification of central BR signaling components, from the BRI1 receptor at the plasma membrane to downstream acting BR-regulated BRZ1 and BES1 transcription factors in the nuclei. Recently, an emerging trend is being established to further advance our understanding of the BR signaling pathway in plant development. Scientists have turned on the microscope lens turret to revisit the pleiotropic phenotypes of the BR mutants at a higher magnification, uncovering novel and specific cellular defects in the plant. In-depth phenotypic analysis in combination with the search for cell-specific signaling components that are responsible for those particular defects in the mutants are leading to: (1) definition of novel roles for BRs in vascular development, (2) unraveling BR function in cell division through quantitative analysis of Arabidopsis root growth, (3) establishment of a molecular connection between known patterning and BR-signaling components in organ boundary and stomata development and (4) development of novel strategies toward the identification of BR signaling components with spatiotemporal resolution. In this review, we highlight the importance of these emerging studies to investigate the spatiotemporal control of BR pathways in plant development.
Assuntos
Arabidopsis/fisiologia , Brassinosteroides/metabolismo , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/metabolismo , Transdução de Sinais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mutação , Especificidade de Órgãos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/fisiologia , Feixe Vascular de Plantas/genética , Feixe Vascular de Plantas/crescimento & desenvolvimento , Feixe Vascular de Plantas/fisiologiaRESUMO
Brassinosteroid (BR) hormones control plant growth through acting on both cell expansion and division. Here, we examined the role of BRs in leaf growth using the Arabidopsis BR-deficient mutant constitutive photomorphogenesis and dwarfism (cpd). We show that the reduced size of cpd leaf blades is a result of a decrease in cell size and number, as well as in venation length and complexity. Kinematic growth analysis and tissue-specific marker gene expression revealed that the leaf phenotype of cpd is associated with a prolonged cell division phase and delayed differentiation. cpd-leaf-rescue experiments and leaf growth analysis of BR biosynthesis and signaling gain-of-function mutants showed that BR production and BR receptor-dependent signaling differentially control the balance between cell division and expansion in the leaf. Investigation of cell cycle markers in leaves of cpd revealed the accumulation of mitotic proteins independent of transcription. This correlated with an increase in cyclin-dependent kinase activity, suggesting a role for BRs in control of mitosis.
Assuntos
Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Brassinosteroides/biossíntese , Divisão Celular , Folhas de Planta/citologia , Folhas de Planta/crescimento & desenvolvimento , Transdução de Sinais , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Brassinosteroides/farmacologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Mitose/efeitos dos fármacos , Mutação/genética , Fenótipo , Folhas de Planta/efeitos dos fármacos , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
RESUMO
The plant vascular system provides transport and support capabilities that are essential for plant growth and development, yet the mechanisms directing the arrangement of vascular bundles within the shoot inflorescence stem remain unknown. We used computational and experimental biology to evaluate the role of auxin and brassinosteroid hormones in vascular patterning in Arabidopsis. We show that periodic auxin maxima controlled by polar transport and not overall auxin levels underlie vascular bundle spacing, whereas brassinosteroids modulate bundle number by promoting early procambial divisions. Overall, this study demonstrates that auxin polar transport coupled to brassinosteroid signaling is required to determine the radial pattern of vascular bundles in shoots.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Colestanóis/metabolismo , Ácidos Indolacéticos/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Transdução de Sinais , Esteroides Heterocíclicos/metabolismo , Arabidopsis/citologia , Transporte Biológico , Brassinosteroides , Contagem de Células , Modelos Biológicos , Mutação/genética , Brotos de Planta/citologia , Brotos de Planta/metabolismoRESUMO
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.