RESUMO
Growth regulation tailors development in plants to their environment. A prominent example of this is the response to gravity, in which shoots bend up and roots bend down1. This paradox is based on opposite effects of the phytohormone auxin, which promotes cell expansion in shoots while inhibiting it in roots via a yet unknown cellular mechanism2. Here, by combining microfluidics, live imaging, genetic engineering and phosphoproteomics in Arabidopsis thaliana, we advance understanding of how auxin inhibits root growth. We show that auxin activates two distinct, antagonistically acting signalling pathways that converge on rapid regulation of apoplastic pH, a causative determinant of growth. Cell surface-based TRANSMEMBRANE KINASE1 (TMK1) interacts with and mediates phosphorylation and activation of plasma membrane H+-ATPases for apoplast acidification, while intracellular canonical auxin signalling promotes net cellular H+ influx, causing apoplast alkalinization. Simultaneous activation of these two counteracting mechanisms poises roots for rapid, fine-tuned growth modulation in navigating complex soil environments.
Assuntos
Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Prótons , Transdução de Sinais , Álcalis , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Ativação Enzimática , Proteínas F-Box/metabolismo , Concentração de Íons de Hidrogênio , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Apical growth in plants initiates upon seed germination, whereas radial growth is primed only during early ontogenesis in procambium cells and activated later by the vascular cambium1. Although it is not known how radial growth is organized and regulated in plants, this system resembles the developmental competence observed in some animal systems, in which pre-existing patterns of developmental potential are established early on2,3. Here we show that in Arabidopsis the initiation of radial growth occurs around early protophloem-sieve-element cell files of the root procambial tissue. In this domain, cytokinin signalling promotes the expression of a pair of mobile transcription factors-PHLOEM EARLY DOF 1 (PEAR1) and PHLOEM EARLY DOF 2 (PEAR2)-and their four homologues (DOF6, TMO6, OBP2 and HCA2), which we collectively name PEAR proteins. The PEAR proteins form a short-range concentration gradient that peaks at protophloem sieve elements, and activates gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by the HD-ZIP III proteins, well-known polarity transcription factors4-the expression of which is concentrated in the more-internal domain of radially non-dividing procambial cells by the function of auxin, and mobile miR165 and miR166 microRNAs. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, and thereby establish a negative-feedback loop that forms a robust boundary that demarks the zone of cell division. Taken together, our data establish that during root procambial development there exists a network in which a module that links PEAR and HD-ZIP III transcription factors integrates spatial information of the hormonal domains and miRNA gradients to provide adjacent zones of dividing and more-quiescent cells, which forms a foundation for further radial growth.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Câmbio/crescimento & desenvolvimento , Câmbio/genética , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Câmbio/citologia , Câmbio/metabolismo , Divisão Celular/genética , Sinais (Psicologia) , Citocininas/metabolismo , Ácidos Indolacéticos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Floema/citologia , Floema/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Transdução de Sinais , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
In vascular plants, the root endodermis surrounds the central vasculature as a protective sheath that is analogous to the polarized epithelium in animals, and contains ring-shaped Casparian strips that restrict diffusion. After an initial lag phase, individual endodermal cells suberize in an apparently random fashion to produce 'patchy' suberization that eventually generates a zone of continuous suberin deposition. Casparian strips and suberin lamellae affect paracellular and transcellular transport, respectively. Most angiosperms maintain some isolated cells in an unsuberized state as so-called 'passage cells', which have previously been suggested to enable uptake across an otherwise-impermeable endodermal barrier. Here we demonstrate that these passage cells are late emanations of a meristematic patterning process that reads out the underlying non-radial symmetry of the vasculature. This process is mediated by the non-cell-autonomous repression of cytokinin signalling in the root meristem, and leads to distinct phloem- and xylem-pole-associated endodermal cells. The latter cells can resist abscisic acid-dependent suberization to produce passage cells. Our data further demonstrate that, during meristematic patterning, xylem-pole-associated endodermal cells can dynamically alter passage-cell numbers in response to nutrient status, and that passage cells express transporters and locally affect the expression of transporters in adjacent cortical cells.
Assuntos
Arabidopsis/anatomia & histologia , Arabidopsis/citologia , Padronização Corporal , Citocininas/metabolismo , Difusão , Endoderma/citologia , Endoderma/metabolismo , Transdução de Sinais , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Diferenciação Celular , Endoderma/anatomia & histologia , Ácidos Indolacéticos/metabolismo , Meristema/anatomia & histologia , Meristema/citologia , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Células Vegetais/metabolismoRESUMO
In this Letter, owing to a copying error in Illustrator, the two centre panels in Extended Data Fig. 7a were identical. This error has been corrected online. The old, incorrect Extended Data Fig. 7 is shown in the Supplementary Information to this Amendment for transparency. Some typos ('occurence') in Figs. 1, 2 and 3 have also been corrected and the publication details for ref. 32 have been added.
RESUMO
The roots of lycophytes branch through dichotomy or bifurcation, during which the root apex splits into two daughter roots. This is morphologically distinct from lateral root (LR) branching in the extant euphyllophytes, with LRs developing along the root axis at different distances from the apex. Although the process of root bifurcation is poorly understood, such knowledge can be important, because it may represent an evolutionarily ancient strategy that roots recruited to form new stem cells or meristems. In this study, we examined root bifurcation in the lycophyte Selaginella moellendorffii. We characterized an in vitro developmental time frame based on repetitive apex bifurcations, allowing us to sample different stages of dichotomous root branching and analyze the root meristem and root branching in S. moellendorffii at the microscopic and transcriptomic level. Our results showed that, in contrast to previous assumptions, initial cells (ICs) in the root meristem are mostly not tetrahedral but rather show an irregular shape. Tracking down the early stages of root branching argues for the occurrence of a symmetric division of the single IC, resulting in two apical stem cells that initiate root meristem bifurcation. Moreover, we generated a S. moellendorffii root branching transcriptome that resulted in the delineation of a subset of core meristem genes. The occurrence of multiple putative orthologs of meristem genes in this dataset suggests the presence of conserved pathways in the control of meristem and root stem cell establishment or maintenance.
Assuntos
Selaginellaceae , Selaginellaceae/genética , Meristema/metabolismo , Transcriptoma/genética , Raízes de Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Transcriptional networks are crucial to integrate various internal and external signals into optimal responses during plant growth and development. In Arabidopsis thaliana, primary root vasculature patterning and proliferation are controlled by a network centred around the basic Helix-Loop-Helix transcription factor complex, formed by TARGET OF MONOPTEROS 5 (TMO5) and LONESOME HIGHWAY (LHW), which control cell proliferation and division orientation by modulating the cytokinin response and other downstream factors. Despite recent progress, many aspects of the TMO5/LHW pathway are not fully understood. In particular, the upstream regulators of TMO5/LHW activity remain unknown. Here, using a forward genetics approach to identify new factors of the TMO5/LHW pathway, we discovered a novel function of the MYB-type transcription factor, MYB12. MYB12 physically interacts with TMO5 and dampens the TMO5/LHW-mediated induction of direct target gene expression, as well as the periclinal/radial cell divisions. The expression of MYB12 is activated by the cytokinin response, downstream of TMO5/LHW, resulting in a novel MYB12-mediated negative feedback loop that restricts TMO5/LHW activity, to ensure optimal cell proliferation rates during root vascular development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Meristema , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raízes de Plantas/metabolismo , Retroalimentação , Transativadores/genética , Regulação da Expressão Gênica de Plantas , Arabidopsis/metabolismo , Divisão Celular , Citocininas/metabolismoRESUMO
The visualization of hormonal signaling input and output is key to understanding how multicellular development is regulated. The plant signaling molecule auxin triggers many growth and developmental responses, but current tools lack the sensitivity or precision to visualize these. We developed a set of fluorescent reporters that allow sensitive and semiquantitative readout of auxin responses at cellular resolution in Arabidopsis thaliana. These generic tools are suitable for any transformable plant species.
Assuntos
Arabidopsis/genética , Genes Reporter , Ácidos Indolacéticos/metabolismo , Elementos de Resposta/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ácidos Indolacéticos/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Imagem Molecular/métodos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Transdução de Sinais/genéticaRESUMO
Plant cells cannot rearrange their positions; therefore, sharp tissue boundaries must be accurately programmed. Movement of the cell fate regulator SHORT-ROOT from the stele to the ground tissue has been associated with transferring positional information across tissue boundaries. The zinc finger BIRD protein JACKDAW has been shown to constrain SHORT-ROOT movement to a single layer, and other BIRD family proteins were postulated to counteract JACKDAW's role in restricting SHORT-ROOT action range. Here, we report that regulation of SHORT-ROOT movement requires additional BIRD proteins whose action is critical for the establishment and maintenance of the boundary between stele and ground tissue. We show that BIRD proteins act in concert and not in opposition. The exploitation of asymmetric redundancies allows the separation of two BIRD functions: constraining SHORT-ROOT spread through nuclear retention and transcriptional regulation of key downstream SHORT-ROOT targets, including SCARECROW and CYCLIND6. Our data indicate that BIRD proteins promote formative divisions and tissue specification in the Arabidopsis thaliana root meristem ground tissue by tethering and regulating transcriptional competence of SHORT-ROOT complexes. As a result, a tissue boundary is not "locked in" after initial patterning like in many animal systems, but possesses considerable developmental plasticity due to continuous reliance on mobile transcription factors.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Meristema/citologia , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Meristema/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismoRESUMO
In Arabidopsis, more than 1000 putative small signalling peptides have been predicted, but very few have been functionally characterized. One class of small post-translationally modified signalling peptides is the C-TERMINALLY ENCODED PEPTIDE (CEP) family, of which one member has been shown to be involved in regulating root architecture. This work applied a bioinformatics approach to identify more members of the CEP family. It identified 10 additional members and revealed that this family only emerged in flowering plants and was absent from extant members of more primitive plants. The data suggest that the CEP proteins form two subgroups according to the CEP domain. This study further provides an overview of specific CEP expression patterns that offers a comprehensive framework to study the role of the CEP signalling peptides in plant development. For example, expression patterns point to a role in aboveground tissues which was corroborated by the analysis of transgenic lines with perturbed CEP levels. These results form the basis for further exploration of the mechanisms underlying this family of peptides and suggest their putative roles in distinct developmental events of higher plants.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Peptídeos/genética , Transdução de Sinais , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Evolução Biológica , Embriófitas/citologia , Embriófitas/efeitos dos fármacos , Embriófitas/genética , Embriófitas/crescimento & desenvolvimento , Magnoliopsida/citologia , Magnoliopsida/efeitos dos fármacos , Magnoliopsida/genética , Magnoliopsida/crescimento & desenvolvimento , Dados de Sequência Molecular , Família Multigênica , Peptídeos/metabolismo , Fenótipo , Filogenia , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/citologia , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Estrutura Terciária de ProteínaRESUMO
In the Arabidopsis root, growth is sustained by the meristem. Signalling from organiser cells, also termed the quiescent centre (QC), is essential for the maintenance and replenishment of the stem cells. Here, we highlight three publications from the founder of the concept of the stem cell niche in Arabidopsis and a pioneer in unravelling regulatory modules governing stem cell specification and maintenance, as well as tissue patterning in the root meristem: Ben Scheres. His research has tremendously impacted the plant field. We have selected three publications from the Scheres legacy, which can be considered a breakthrough in the field of plant developmental biology. van den Berg et al. (1995) and van den Berg et al. (1997) uncovered that positional information-directed patterning. Sabatini et al. (1999), discovered that auxin maxima determine tissue patterning and polarity. We describe how simple but elegant experimental designs have provided the foundation of our current understanding of the functioning of the root meristem.
RESUMO
During plant development, a precise balance of cytokinin is crucial for correct growth and patterning, but it remains unclear how this is achieved across different cell types and in the context of a growing organ. Here we show that in the root apical meristem, the TMO5/LHW complex increases active cytokinin levels via two cooperatively acting enzymes. By profiling the transcriptomic changes of increased cytokinin at single-cell level, we further show that this effect is counteracted by a tissue-specific increase in CYTOKININ OXIDASE 3 expression via direct activation of the mobile transcription factor SHORTROOT. In summary, we show that within the root meristem, xylem cells act as a local organizer of vascular development by non-autonomously regulating cytokinin levels in neighbouring procambium cells via sequential induction and repression modules.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Citocininas , Raízes de Plantas/crescimento & desenvolvimento , Proteínas de Arabidopsis , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Oxirredutases , Transdução de Sinais , TransativadoresRESUMO
Spontaneously arising channels that transport the phytohormone auxin provide positional cues for self-organizing aspects of plant development such as flexible vasculature regeneration or its patterning during leaf venation. The auxin canalization hypothesis proposes a feedback between auxin signaling and transport as the underlying mechanism, but molecular players await discovery. We identified part of the machinery that routes auxin transport. The auxin-regulated receptor CAMEL (Canalization-related Auxin-regulated Malectin-type RLK) together with CANAR (Canalization-related Receptor-like kinase) interact with and phosphorylate PIN auxin transporters. camel and canar mutants are impaired in PIN1 subcellular trafficking and auxin-mediated PIN polarization, which macroscopically manifests as defects in leaf venation and vasculature regeneration after wounding. The CAMEL-CANAR receptor complex is part of the auxin feedback that coordinates polarization of individual cells during auxin canalization.
Assuntos
Arabidopsis/enzimologia , Ácidos Indolacéticos/metabolismo , Proteínas Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Proteínas de Membrana Transportadoras/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Quinases/genética , Fatores de Transcrição/metabolismoRESUMO
Optimal plant growth is hampered by deficiency of the essential macronutrient phosphate in most soils. Plant roots can, however, increase their root hair density to efficiently forage the soil for this immobile nutrient. By generating and exploiting a high-resolution single-cell gene expression atlas of Arabidopsis roots, we show an enrichment of TARGET OF MONOPTEROS 5/LONESOME HIGHWAY (TMO5/LHW) target gene responses in root hair cells. The TMO5/LHW heterodimer triggers biosynthesis of mobile cytokinin in vascular cells and increases root hair density during low-phosphate conditions by modifying both the length and cell fate of epidermal cells. Moreover, root hair responses in phosphate-deprived conditions are TMO5- and cytokinin-dependent. Cytokinin signaling links root hair responses in the epidermis to perception of phosphate depletion in vascular cells.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Meristema/crescimento & desenvolvimento , Floema/crescimento & desenvolvimento , Fosfatos/deficiência , Epiderme Vegetal/crescimento & desenvolvimento , Transativadores/fisiologia , Xilema/crescimento & desenvolvimento , Arabidopsis/citologia , Arabidopsis/genética , Citocininas/biossíntese , Citocininas/genética , Meristema/citologia , Meristema/metabolismo , Floema/citologia , Floema/metabolismo , Epiderme Vegetal/citologia , Epiderme Vegetal/genética , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Xilema/citologia , Xilema/metabolismoRESUMO
To create a three-dimensional structure, plants rely on oriented cell divisions and cell elongation. Oriented cell divisions are specifically important in procambium cells of the root to establish the different vascular cell types [1, 2]. These divisions are in part controlled by the auxin-controlled TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) transcription factor complex [3-7]. Loss-of-function of tmo5 or lhw clade members results in strongly reduced vascular cell file numbers, whereas ectopic expression of both TMO5 and LHW can ubiquitously induce periclinal and radial cell divisions in all cell types of the root meristem. TMO5 and LHW interact only in young xylem cells, where they promote expression of two direct target genes involved in the final step of cytokinin (CK) biosynthesis, LONELY GUY3 (LOG3) and LOG4 [8, 9] Therefore, CK was hypothesized to act as a mobile signal from the xylem to trigger divisions in the neighboring procambium cells [3, 6]. To unravel how TMO5/LHW-dependent cytokinin regulates cell proliferation, we analyzed the transcriptional responses upon simultaneous induction of both transcription factors. Using inferred network analysis, we identified AT2G28510/DOF2.1 as a cytokinin-dependent downstream target gene. We further showed that DOF2.1 controls specific procambium cell divisions without inducing other cytokinin-dependent effects such as the inhibition of vascular differentiation. In summary, our results suggest that DOF2.1 and its closest homologs control vascular cell proliferation, thus leading to radial expansion of the root.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Proliferação de Células/genética , Citocininas/metabolismo , Fatores Genéricos de Transcrição/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Câmbio/fisiologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Transativadores/genética , Transativadores/metabolismo , Fatores Genéricos de Transcrição/metabolismo , Xilema/fisiologiaRESUMO
Electrochemistry constitutes a mild, green and versatile activation method of organic molecules. Despite these innate advantages, its widespread use in organic chemistry has been hampered due to technical limitations, such as mass and heat transfer limitations which restraints the scalability of electrochemical methods. Herein, we describe an undivided-cell electrochemical flow reactor with a flexible reactor volume. This enables its use in two different modes, which are highly relevant for flow chemistry applications, including a serial (volume ranging from 88 µL/channel up to 704 µL) or a parallel mode (numbering-up). The electrochemical flow reactor was subsequently assessed in two synthetic transformations, which confirms its versatility and scale-up potential.
RESUMO
Protein complex formation has been extensively studied using Förster resonance energy transfer (FRET) measured by Fluorescence Lifetime Imaging Microscopy (FLIM). However, implementing this technology to detect protein interactions in living multicellular organism at single-cell resolution and under native condition is still difficult to achieve. Here we describe the optimization of the labeling conditions to detect FRET-FLIM in living plants. This study exemplifies optimization procedure involving the identification of the optimal position for the labels either at the N or C terminal region and the selection of the bright and suitable, fluorescent proteins as donor and acceptor labels for the FRET study. With an effective optimization strategy, we were able to detect the interaction between the stem cell regulators SHORT-ROOT and SCARECROW at endogenous expression levels in the root pole of living Arabidopsis embryos and developing lateral roots by FRET-FLIM. Using this approach we show that the spatial profile of interaction between two transcription factors can be highly modulated in reoccurring and structurally resembling organs, thus providing new information on the dynamic redistribution of nuclear protein complex configurations in different developmental stages. In principle, our optimization procedure for transcription factor complexes is applicable to any biological system.
RESUMO
The plant vascular system develops from a handful of provascular initial cells in the early embryo into a whole range of different cell types in the mature plant. In order to account for such proliferation and to generate this kind of diversity, vascular tissue development relies on a large number of highly oriented cell divisions. Different hormonal and genetic pathways have been implicated in this process and several of these have been recently interconnected. Nevertheless, how such networks control the actual division plane orientation and how they interact with the generic cell cycle machinery to coordinate these divisions remains a major unanswered question.