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Plant growth is driven by apical meristems at the shoot and root growth points, which comprise continuously active stem cell populations. While many of the key factors involved in homeostasis of the shoot apical meristem (SAM) have been extensively studied under artificial constant growth conditions, only little is known how variations in the environment affect the underlying regulatory network. To shed light on the responses of the SAM to ambient temperature, we combined 3D live imaging of fluorescent reporter lines that allowed us to monitor the activity of two key regulators of stem cell homeostasis in the SAM namelyCLAVATA3(CLV3)andWUSCHEL (WUS),with computational image analysis to derive morphological and cellular parameters of the SAM. WhereasCLV3expression marks the stem cell population,WUSpromoter activity is confined to the organizing center (OC), the niche cells adjacent to the stem cells, hence allowing us to record on the two central cell populations of the SAM. Applying an integrated computational analysis of our data we found that variations in ambient temperature not only led to specific changes in spatial expression patterns of key regulators of SAM homeostasis, but also correlated with modifications in overall cellular organization and shoot meristem morphology.

Building a safe space for stem cells The meristem, the collection of stem cells that builds plants, is resistant to viral infection. Wu et al. now show that WUSCHEL, a transcription factor that helps to sustain stem cell production in the shoot apical meristem of Arabidopsis , also protects that stem cell domain from viruses. WUSCHEL inhibited viral protein synthesis by repressing methyltransferases that regulate ribosomal RNA processing and ribosome stability. Science , this issue p. 227

Fluorescent imaging, especially in living tissue, has become a key method in modern life sciences, with the development of new tools for sample preparation, imaging, and data analysis continuously advancing our understanding of biological principles. Here, we present our strategy for in vivo imaging of the Arabidopsis shoot apical meristem (SAM), a central structure in plant development. We implement simplifications to previously published workflows and present a novel approach to subsequentially image the meristem from multiple angles at high resolution. This tool may represent a valuable resource for shoot meristem-centered research in general, but also for studies on plasmodesmata or intercellular connectivity within the SAM: via the analysis of fluorescently labeled plasmodesmata-localized proteins, via the tracing of fluorescent dyes, via analyzing the cell-to-cell mobility of fluorescently labeled proteins, but also via the analysis of morphological features of meristematic cells in mutants or upon perturbation of symplastic connectivity.

Plants continuously adjust organ initiation and growth to endogenous and environmental signals. At the heart of this postembryonic mode of development are stem cells, whose activity is influenced by a large diversity of signaling pathways. We have shown previously that the TARGET OF RAPAMYCIN (TOR) kinase network controls expression of WUSCHEL (WUS), a transcriptional master regulator of stem cells in the shoot apical meristem (SAM), by integrating metabolic- and light signals. While this provided a framework how environmental parameters determine shoot development, the mechanisms linking TOR and WUS activity remained unresolved. Here we show that TOR controls the accumulation of trans-zeatin, the cytokinin species mainly responsible for shoot development, by translational repression of RNAs encoding cytokinin degrading CYTOKININ OXIDASES/DEHYDROGENASE (CKX) enzymes. Thus, our work not only sheds light on how plants are able to quickly adjust stem cell activity in response to their environment, but also opens new avenues for studying the mechanisms translating TOR kinase activity into relevant biological output.

Embryogenesis of flowering plants is initiated by polarization of the zygote, a prerequisite for correct axis formation in the embryo. The daughter cells of the asymmetric zygote division form the pro-embryo and the mostly extra-embryonic suspensor.1 The suspensor plays a pivotal role in nutrient and hormone transport and rapid growth of the embryo.2,3 Zygote polarization is controlled by a MITOGEN-ACTIVATING PROTEIN (MAP) kinase signaling pathway including the MAPKK kinase (MAP3K) YODA (YDA)4 and the upstream membrane-associated proteins BRASINOSTEROID SIGNALING KINASE 1 (BSK1) and BSK2.5,6 Furthermore, suspensor development is controlled by cysteine-rich peptides of the EMBRYO SURROUNDING FACTOR 1 (ESF1) family.7 While they act genetically upstream of YDA, the corresponding receptor to perceive these potential ligands is unknown. In other developmental processes, such as stomata development, YDA activity is controlled by receptor kinases of the ERECTA family (ERf).8-12 While the receptor kinases upstream of BSK1/2 in the embryo have so far not been identified,1 YDA is in part activated by the sperm cell-derived BSK family member SHORT SUSPENSOR (SSP) that represents a naturally occurring, constitutively active variant of BSK1.5,13 It has been speculated that SSP might be a paternal component of a parental tug-of-war controlling resource allocation toward the embryo.2,13 Here, we show that in addition to SSP, the receptor kinase ERECTA plays a crucial role in zygote polarization as a maternally contributed part of the embryonic YDA pathway. We conclude that two independent parental contributions initiate zygote polarization and control embryo development.

SummaryA key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the root vasculature, a miRNA gradient controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. Here, we show that a functional miRNA gradient requires an opposing gradient of ARGONAUTE10, which sequesters miRNAs to protect HD-ZIP III transcripts from degradation. In the absence of ARGONAUTE10, xylem precursor cells undergo periclinal divisions that lead to continuous strands of differentiated xylem elements at ectopic positions. Notably, periclinal daughter cells maintain xylem identity even when they are located outside of the xylem axis, resulting in disrupted tissue boundaries. We further demonstrate that ARGONAUTE10 and HD-ZIP IIIs buffer cytokinin signalling to control formative cell divisions, providing a framework for integration of phytohormone and miRNA-mediated patterning.

Cell wall recalcitrance is a major constraint for the exploitation of lignocellulosic biomass as a renewable resource for energy and bio-based products. Transcriptional regulators of the lignin biosynthetic pathway represent promising targets for tailoring lignin content and composition in plant secondary cell walls. However, knowledge about the transcriptional regulation of lignin biosynthesis in lignocellulosic feedstocks, such as Miscanthus, is limited. In Miscanthus leaves, MsSCM1 and MsMYB103 are expressed at growth stages associated with lignification. The ectopic expression of MsSCM1 and MsMYB103 in N. benthamiana leaves was sufficient to trigger secondary cell wall deposition with distinct sugar and lignin compositions. Moreover, RNA-seq analysis revealed that the transcriptional responses to MsSCM1 and MsMYB103 overexpression showed an extensive overlap with the response to the NAC master transcription factor MsSND1, but were distinct from each other, underscoring the inherent complexity of secondary cell wall formation. Furthermore, conserved and previously described promoter elements as well as novel and specific motifs could be identified from the target genes of the three transcription factors. Together, MsSCM1 and MsMYB103 represent interesting targets for manipulations of lignin content and composition in Miscanthus towards a tailored biomass.

Embryogenesis of flowering plants is initiated by polarization of the zygote, a prerequisite for correct axis formation in the embryo. Zygote polarity and the decision between embryonic and non-embryonic development in the daughter cells is controlled by a MITOGEN-ACTIVATING PROTEIN (MAP) kinase signaling pathway including the MAPKK kinase YODA (YDA). Upstream of YDA act two members of the BRASINOSTEROID SIGNALING KINASE (BSK) family, BSK1 and BSK2. These membrane-associated proteins serve as signaling relay linking receptor kinase complexes with intracellular signaling cascades. The receptor kinases acting upstream of BSK1 and BSK2 in the zygote, however, have so far not been identified. Instead, YDA can in part be activated by the non-canonical BSK family member SHORT SUSPENSOR (SSP) that is contributed by the sperm cell during fertilization. Here, we show that the receptor kinase ERECTA plays a crucial role in zygote polarization as maternally contributed part of the embryonic YDA pathway. SSP on the other hand provides an independent, paternal input to YDA activation, outlining a Y-shaped pathway. We conclude that two independent parental contributions initiate zygote polarization and control suspensor formation and embryonic development.

Quantitative analysis of plant and animal morphogenesis requires accurate segmentation of individual cells in volumetric images of growing organs. In the last years, deep learning has provided robust automated algorithms that approach human performance, with applications to bio-image analysis now starting to emerge. Here, we present PlantSeg, a pipeline for volumetric segmentation of plant tissues into cells. PlantSeg employs a convolutional neural network to predict cell boundaries and graph partitioning to segment cells based on the neural network predictions. PlantSeg was trained on fixed and live plant organs imaged with confocal and light sheet microscopes. PlantSeg delivers accurate results and generalizes well across different tissues, scales, acquisition settings even on non plant samples. We present results of PlantSeg applications in diverse developmental contexts. PlantSeg is free and open-source, with both a command line and a user-friendly graphical interface.

There are two main types of root systems in flowering plants, namely taproot systems of dicots and fibrous root systems found in monocots. Despite this fundamental split, our current knowledge of cellular and molecular mechanism driving root development is mainly based on studies of the dicot model Arabidopsis. However, the world major crops are monocots and little is known about the transcriptional programs underlying cell-type specification in this clade. Here, we report the transcriptomes of more than 20 000 single cells derived from root tips of two agronomically important rice cultivars. Using combined computational and experimental analyses we were able to robustly identify most of the major cell types and define novel cell-type-specific marker genes for both cultivars. Importantly, we found divergent cell types associated with specific regulatory programs, including phytohormone biosynthesis, signaling, and response, which were well conserved between the two rice cultivars. In addition, we detected substantial differences between the cell-type transcript profiles of Arabidopsis and rice. These species-specific features emphasize the importance of analyzing tissues across diverse model species, including rice. Taken together, our study provides insight into the transcriptomic landscape of major cell types of rice root tip at single-cell resolution and opens new avenues to study cell-type specification, function, and evolution in plants.

Stem cells are one of the foundational evolutionary novelties that allowed the independent emergence of multicellularity in the plant and animal lineages. In plants, the homeodomain (HD) transcription factor WUSCHEL (WUS) is essential for the maintenance of stem cells in the shoot apical meristem. WUS has been reported to bind to diverse DNA motifs and to act as transcriptional activator and repressor. However, the mechanisms underlying this remarkable behavior have remained unclear. Here, we quantitatively delineate WUS binding to three divergent DNA motifs and resolve the relevant structural underpinnings. We show that WUS exhibits a strong binding preference for TGAA repeat sequences, while retaining the ability to weakly bind to TAAT elements. This behavior is attributable to the formation of dimers through interactions of specific residues in the HD that stabilize WUS DNA interaction. Our results provide a mechanistic basis for dissecting WUS dependent regulatory networks in plant stem cell control., WUSCHEL is a homeodomain transcription factor that is essential for stem cell maintenance in the plant shoot apical meristem. Here, via structural and biochemical approaches, Sloan et al. show that strong WUSCHEL binding to preferential target motifs can be attributed to dimer formation that stabilizes DNA binding.

Quantitative analysis of plant and animal morphogenesis requires accurate segmentation of individual cells in volumetric images of growing organs. In the last years, deep learning has provided robust automated algorithms that approach human performance, with applications to bio-image analysis now starting to emerge. Here, we present PlantSeg, a pipeline for volumetric segmentation of plant tissues into cells. PlantSeg employs a convolutional neural network to predict cell boundaries and graph partitioning to segment cells based on the neural network predictions. PlantSeg was trained on fixed and live plant organs imaged with confocal and light sheet microscopes. PlantSeg delivers accurate results and generalizes well across different tissues, scales, and acquisition settings. We present results of PlantSeg applications in diverse developmental contexts. PlantSeg is free and open-source, with both a command line and a user-friendly graphical interface.

The phloem tissue mediates long-distance transport of energy metabolites along plant bodies and, therefore, is central for plant performance. However, mechanisms initiating the transition of undifferentiated stem cells to cells specialized in metabolite transport are unknown. Here we identify the ubiquitously expressed PHD-finger protein OBERON3 (OBE3) to be essential for phloem formation. We show that OBE3 directly interacts with the SUPPRESSOR OF MAX2 1-LIKE 5 (SMXL5) protein specifically expressed during early phloem development. Both proteins co-localize in nuclei of phloem stem cells and, together with the SMXL5 homologs SMXL3 and SMXL4 , promote the establishment of phloem-specific cellular signatures in a cell-autonomous manner. These signatures include expression of OCTOPUS ( OPS ), BREVIS RADIX ( BRX ), BARELY ANY MERISTEM3 ( BAM3 ), and COTYLEDON VASCULAR PATTERN2 ( CVP2 ) genes acting as mediators of phloem differentiation. Consistently, genetic analyses show that SMXL5 acts upstream and independently of OPS and BRX functions. Based on our findings, we conclude that the formation of an OBE3/SMXL5 protein complex specifically in nuclei of early phloem cells is essential for establishing a phloem-specific developmental program.

Positional information is essential for coordinating the development of multicellular organisms. In plants, positional information provided by the hormone auxin regulates rhythmic organ production at the shoot apex, but the spatio-temporal dynamics of auxin gradients is unknown. We used quantitative imaging to demonstrate that auxin carries high-definition graded information not only in space but also in time. We show that, during organogenesis, temporal patterns of auxin arise from rhythmic centrifugal waves of high auxin travelling through the tissue faster than growth. We further demonstrate that temporal integration of auxin concentration is required to trigger the auxin-dependent transcription associated with organogenesis. This provides a mechanism to temporally differentiate sites of organ initiation and exemplifies how spatio-temporal positional information can be used to create rhythmicity., eLife digest Plants, like animals and many other multicellular organisms, control their body architecture by creating organized patterns of cells. These patterns are generally defined by signal molecules whose levels differ across the tissue and change over time. This tells the cells where they are located in the tissue and therefore helps them know what tasks to perform. A plant hormone called auxin is one such signal molecule and it controls when and where plants produce new leaves and flowers. Over time, this process gives rise to the dashing arrangements of spiraling organs exhibited by many plant species. The leaves and flowers form from a relatively small group of cells at the tip of a growing stem known as the shoot apical meristem. Auxin accumulates at precise locations within the shoot apical meristem before cells activate the genes required to make a new leaf or flower. However, the precise role of auxin in forming these new organs remained unclear because the tools to observe the process in enough detail were lacking. Galvan-Ampudia, Cerutti et al. have now developed new microscopy and computational approaches to observe auxin in a small plant known as Arabidopsis thaliana. This showed that dozens of shoot apical meristems exhibited very similar patterns of auxin. Images taken over a period of several hours showed that the locations where auxin accumulated were not fixed on a group of cells but instead shifted away from the center of the shoot apical meristems faster than the tissue grew. This suggested the cells experience rapidly changing levels of auxin. Further experiments revealed that the cells needed to be exposed to a high level of auxin over time to activate genes required to form an organ. This mechanism sheds a new light on how auxin regulates when and where plants make new leaves and flowers. The tools developed by Galvan-Ampudia, Cerutti et al. could be used to study the role of auxin in other plant tissues, and to investigate how plants regulate the response to other plant hormones.

Coordination of fate transition and cell division is crucial to maintain the plant architecture and to achieve efficient production of plant organs. In this paper, we analysed the stem cell dynamics at the shoot apical meristem (SAM) that is one of the plant stem cells locations. We designed a mathematical model to elucidate the impact of hormonal signaling on the fate transition rates between different zones corresponding to slowly dividing stem cells and fast dividing transit amplifying cells. The model is based on a simplified two-dimensional disc geometry of the SAM and accounts for a continuous displacement towards the periphery of cells produced in the central zone. Coupling growth and hormonal signaling results in a nonlinear system of reaction-diffusion equations on a growing domain with the growth rate depending on the model components. The model is tested by simulating perturbations in the level of key transcription factors that maintain SAM homeostasis. The model provides new insights on how the transcription factor HECATE is integrated in the regulatory network that governs stem cell differentiation., Author summary Plants continuously generate new organs such as leaves, roots and flowers. This process is driven by stem cells which are located in specialized regions, so-called meristems. Dividing stem cells give rise to offspring that, during a process referred to as cell fate transition, become more specialized and give rise to organs. Plant architecture and crop yield crucially depend on the regulation of meristem dynamics. To better understand this regulation, we develop a computational model of the shoot meristem. The model describes the meristem as a two-dimensional disk that can grow and shrink over time, depending on the concentrations of the signalling factors in its interior. This allows studying how the non-linear interaction of multiple transcription factors is linked to cell division and fate-transition. We test the model by simulating perturbations of meristem signals and comparing them to experimental data. The model allows simulating different hypotheses about signal effects. Based on the model we study the specific role of the transcription factor HECATE and provide new insights in its action on cell dynamics and in its interrelation with other known transcription factors in the meristem.

Highlights • The shoot apical meristem is a highly dynamic stem cell system. • Environmental as well as endogenous parameters are actively sensed. • Complex signaling pathways continuously modulate stem cell behaviour. • Mechanisms of signal integration and decision making are still poorly understood. • Cell type specific genetic approaches will be required to decode the regulatory logic., During plant development, organ morphology and body architecture are dynamically adjusted in response to a changing environment. This developmental plasticity is based on precisely controlled maintenance of primary, as well as programmed initiation of pluripotent stem cell populations during secondary- and de novo meristem formation (reviewed in [1, 2, 3]). Plant stem cells are found exclusively in specific locations that are defined by relative position within the growing tissue. It follows that stem cell fate is primarily instructed by endogenous signals that dynamically define the stem cell niche in response to tissue topography [4]. Furthermore, plant stem cell activity is strongly dependent on developmental stage, suggesting that they are sensitive to long range signaling from distant organs, including the root [5,6••]. And finally, environmental signals exert a major influence allowing plants to cope with the plethora of highly variable environmental parameters during their life-cycle [7]. Integrating tissue level positional information with long range developmental cues, as well as environmental signals requires intricate molecular mechanisms that allow to filter, classify, and balance diverse inputs and translate them into appropriate local cell behavior. In this short review, we aim to highlight advances in identifying the relevant signals, their mode of action, as well as the mechanisms of information processing in stem cells of the shoot apical meristem (SAM).

In roots, stem cells in the cambium region form vascular tissues needed for the long-distance transport of water and nutrients. How these stem cells are specified and regulated has now been illuminated. Stem cells identified that form vascular tissue in plants.

Auxin represents one of the most potent and most versatile hormonal signals in the plant kingdom. Built on a simple core of only a few dedicated components, the auxin signaling system plays important roles for diverse aspects of plant development, physiology, and defense. Key to the diversity of context-dependent functional outputs generated by cells in response to this small molecule are gene duplication events and sub-functionalization of signaling components on the one hand, and a deep embedding of the auxin signaling system into complex regulatory networks on the other hand. Together, these evolutionary innovations provide the mechanisms to allow each cell to display a highly specific auxin response that suits its individual requirements. In this review, we discuss the regulatory networks connecting auxin with a large number of diverse pathways at all relevant levels of the signaling system ranging from biosynthesis to transcriptional response.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness., Spatial control of auxin signaling maintains a balance between stem-cell self-renewal and differentiation at the plant shoot apex. Here Ma et al. show that rheostatic control of auxin response by the WUSCHEL transcription factor maintains stem cells by conferring resistance to auxin mediated differentiation.

A key question in developmental biology is how morphogenetic regulators control patterning. Recent findings have raised an important question: do morphogenetic signals carry information not only in space, as originally proposed in the morphogen concept, but also in time? The hormone auxin is an essential plant morphogenetic regulator that drives rhythmic organogenesis at the shoot apical meristem. Here, we used a quantitative imaging approach to map auxin distribution and response. We demonstrate the existence of high-definition spatio-temporal auxin distribution in the meristem. We provide evidence that developing organs are auxin-emitting centers that could allow self-sustained distribution of auxin through a structured auxin transport network converging on the meristem center. We finally demonstrate that regulation of histone acetylation allows cells to measure the duration of the exposition to auxin preceding organ initiation, providing a remarkable example of how both spatial and temporal morphogenetic information generates rhythmic patterning.

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.

Among the trending topics in the life sciences, stem cells have received a fair share of attention in the public debate - mostly in connection with their potential for biomedical application and therapies. While the promise of organ regeneration and the end of cancer have captured our imagination, it has gone almost unnoticed that plant stem cells represent the ultimate origin of much of the food we eat, the oxygen we breathe, as well the fuels we burn. Thus, plant stem cells may be ranked among the most important cells for human well-being. Research by many labs in the last decades has uncovered a set of independent stem cell systems that fulfill the specialized needs of plant development and growth in four dimensions. Surprisingly, the cellular and molecular design of these systems is remarkably similar, even across diverse species. In some long-lived plants, such as trees, plant stem cells remain active over hundreds or even thousands of years, revealing the exquisite precision in the underlying control of proliferation, self-renewal and differentiation. In this minireview, we introduce the basic features of the three major plant stem cell systems building on these facts, highlight their modular design at the level of cellular layout and regulatory underpinnings and briefly compare them with their animal counterparts.

The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH.

Shoot regeneration can be achieved in vitro through a two‐step process involving the acquisition of pluripotency on callus‐induction media (CIM) and the formation of shoots on shoot‐induction media. Although the induction of root‐meristem genes in callus has been noted recently, the mechanisms underlying their induction and their roles in de novo shoot regeneration remain unanswered. Here, we show that the histone acetyltransferase HAG1/AtGCN5 is essential for de novo shoot regeneration. In developing callus, it catalyzes histone acetylation at several root‐meristem gene loci including WOX5 , WOX14 , SCR , PLT1 , and PLT2 , providing an epigenetic platform for their transcriptional activation. In turn, we demonstrate that the transcription factors encoded by these loci act as key potency factors conferring regeneration potential to callus and establishing competence for de novo shoot regeneration. Thus, our study uncovers key epigenetic and potency factors regulating plant‐cell pluripotency. These factors might be useful in reprogramming lineage‐specified plant cells to pluripotency.

Understanding the context-specific role of gene function is a key objective of modern biology. To this end, we generated a resource for inducible cell type-specific transactivation in Arabidopsis ( Arabidopsis thaliana ) based on the well-established combination of the chimeric GR-LhG4 transcription factor and the synthetic pOp promoter. Harnessing the flexibility of the GreenGate cloning system, we produced a comprehensive set of transgenic lines termed GR-LhG4 driver lines targeting most tissues in the Arabidopsis shoot and root with a strong focus on the indeterminate meristems. When we combined these transgenic lines with effectors under the control of the pOp promoter, we observed tight temporal and spatial control of gene expression. In particular, inducible expression in F1 plants obtained from crosses of driver and effector lines allows for rapid assessment of the cell type-specific impact of an effector with high temporal resolution. Thus, our comprehensive and flexible method is suitable for overcoming the limitations of ubiquitous genetic approaches, the outputs of which often are difficult to interpret due to the widespread existence of compensatory mechanisms and the integration of diverging effects in different cell types.

Complex multicellular organisms evolved on Earth in an oxygen-rich atmosphere1; their tissues, including stem-cell niches, require continuous oxygen provision for efficient energy metabolism2. Notably, the maintenance of the pluripotent state of animal stem cells requires hypoxic conditions, whereas higher oxygen tension promotes cell differentiation3. Here we demonstrate, using a combination of genetic reporters and in vivo oxygen measurements, that plant shoot meristems develop embedded in a low-oxygen niche, and that hypoxic conditions are required to regulate the production of new leaves. We show that hypoxia localized to the shoot meristem inhibits the proteolysis of an N-degron-pathway4,5 substrate known as LITTLE ZIPPER 2 (ZPR2)—which evolved to control the activity of the class-III homeodomain-leucine zipper transcription factors6–8—and thereby regulates the activity of shoot meristems. Our results reveal oxygen as a diffusible signal that is involved in the control of stem-cell activity in plants grown under aerobic conditions, which suggests that the spatially distinct distribution of oxygen affects plant development. In molecular terms, this signal is translated into transcriptional regulation by the N-degron pathway, thereby linking the control of metabolic activity to the regulation of development in plants. Hypoxia in the shoot meristem of Arabidopsis links the regulation of metabolic activity to development by inhibiting proteolysis of a substrate of the N-degron pathway, which controls class-III homeodomain-leucine zipper transcription factors.

SummaryDuring the plant life cycle, diverse signalling inputs are continuously integrated and engage specific genetic programs depending on the cellular or developmental context. Consistent with an important role in this process, HECATE (HEC) bHLH transcription factors display diverse functions, from photomorphogenesis to the control of shoot meristem dynamics and gynoecium patterning. However, the molecular mechanisms underlying their functional versatility and the deployment of specific HEC sub-programs still remain elusive.To address this issue, we systematically identified proteins with the capacity to interact with HEC1, the best characterized member of the family, and integrated this information with our data set of direct HEC1 target genes. The resulting core genetic modules were consistent with specific developmental functions of HEC1, including its described activities in light signalling, gynoecium development and auxin homeostasis. Importantly, we found that in addition,HECgenes play a role in the modulation of flowering time and uncovered that their role in gynoecium development may involve the direct transcriptional regulation ofNGATHA1 (NGA1)andNGA2genes. NGA factors were previously shown to contribute to fruit development, but our data now show that they also modulate stem cell homeostasis in the SAM.Taken together, our results suggest a molecular network underlying the functional versatility of HEC transcription factors. Our analyses have not only allowed us to identify relevant target genes controlling shoot stem cell activity and a so far undescribed biological function of HEC1, but also provide a rich resource for the mechanistic elucidation of further context dependent HEC activities.Significance statementAlthough many transcription factors display diverse regulatory functions during plant development, our understanding of the underlying mechanisms remains poor. Here, by reconstructing the regulatory modules orchestrated by the bHLH transcription factor HECATE1 (HEC1), we defined its regulatory signatures and delineated a regulatory network that provides a molecular basis for its functional versatility. In addition, we uncovered a function forHECgenes in modulating flowering time and further identified downstream signalling components balancing shoot stem cell activity.

Understanding the context-specific role of gene function is a key objective of modern biology. To this end, we generated a resource for inducible cell-type specific trans-activation based on the well-established combination of the chimeric GR-LhG4 transcription factor and the syntheticpOppromoter. Harnessing the flexibility of the GreenGate cloning system, we produced a comprehensive set of GR-LhG4 driver lines targeting most tissues in theArabidopsisshoot and root with a strong focus on the indeterminate meristems. We show that, when combined with effectors under control of thepOppromoter, tight temporal and spatial control of gene expression is achieved. In particular, inducible expression in F1 plants obtained from crosses of driver and effector lines allows rapid assessment of the cell type-specific impact of an effector with high temporal resolution. Thus, our comprehensive and flexible toolbox is suited to overcome the limitations of ubiquitous genetic approaches, the outputs of which are often difficult to interpret due to widespread existence of compensatory mechanisms and the integration of diverging effects in different cell types.One sentence summary: A set of lines enabling spatio-temporal control of gene expression in Arabidopsis.

In the past decade, the importance of the niche to provide regulatory inputs to balance stem cell self-renewal and differentiation has become clear. However, the regulatory interplay between stem cells and their niche at the whole genome level is still poorly understood. To elucidate the mechanisms controlling stem cells and their progenies as they progress through their developmental program at the transcriptional level, we recorded the regulatory program of two independent cell lineages in the Drosophila testis stem cell model. To this end, we identified genes active in the soma or germline as well as genome-wide binding profiles of two essential transcription factors, Zfh-1 and Abd-A, expressed in somatic support cells and crucial for fate acquisition of both cell lineages. Our data identified key roles for TOR signalling, signal processing V-ATPase proton pumps and the nuclear transport engaged nucleoporins and we demonstrate their importance in controlling germline maintenance, proliferation and differentiation from the support side. To make our dataset publicly available and support quick and intuitive data mining, we generated an interactive online analysis tool. Applying our tool for comparative analysis, we uncovered conserved core gene sets of adult stem cells across species boundaries. We have tested the functional relevance of these genes in the Drosophila testis and intestine and find a striking over-representation of stem cell defects when the corresponding genes were depleted. In summary, our dataset and interactive platform represents a versatile tool for identifying novel gene networks active in diverse stem cell types and provides a valuable resource for elucidating the multifaceted regulatory inputs required to guide proper stem cell behaviour.

Spatial organization of signalling events of the phytohormone auxin is fundamental for maintaining a dynamic transition from plant stem cells to differentiated descendants. The cambium, the stem cell niche mediating wood formation, fundamentally depends on auxin signalling but its exact role and spatial organization is obscure. Here we show that, while auxin signalling levels increase in differentiating cambium descendants, a moderate level of signalling in cambial stem cells is essential for cambium activity. We identify the auxin-dependent transcription factor ARF5/MONOPTEROS to cell-autonomously restrict the number of stem cells by directly attenuating the activity of the stem cell-promoting WOX4 gene. In contrast, ARF3 and ARF4 function as cambium activators in a redundant fashion from outside of WOX4-expressing cells. Our results reveal an influence of auxin signalling on distinct cambium features by specific signalling components and allow the conceptual integration of plant stem cell systems with distinct anatomies., Auxin activity controls plant stem cell function. Here the authors show that in the cambium, moderate auxin activity restricts cambial stem cell number via ARF5-dependent repression of the stem‐cell‐promoting factor WOX4, while ARF3 and ARF4 promote cambial activity outside of the WOX4‐expression domain.

In the past decade, the importance of the niche to provide regulatory inputs to balance stem cell self-renewal and differentiation has become clear. However, the regulatory interplay between stem cells and their niche at the whole genome level is still poorly understood. To elucidate the mechanisms controlling stem cells and their progenies as they progress through their developmental program at the transcriptional level, we recorded the regulatory program of two independent cell lineages in theDrosophilatestis stem cell model. To this end, we identified genes active in the soma or germline as well as genome-wide binding profiles of two essential transcription factors, Zfh-1 and Abd-A, expressed in somatic support cells and crucial for fate acquisition of both cell lineages. Our data identified key roles for TOR signalling, signal processing V-ATPase proton pumps and the nuclear transport engaged nucleoporins and we demonstrate their importance in controlling germline maintenance, proliferation and differentiation from the support side. To make our dataset publicly available and support quick and intuitive data mining, we generated an interactive online analysis tool. Applying our tool for comparative analysis, we uncovered conserved core gene sets of adult stem cells across species boundaries. We have tested the functional relevance of these genes in theDrosophilatestis and intestine and find a striking overrepresentation of stem cell defects when the corresponding genes were depleted. In summary, our dataset and interactive platform represents a versatile tool for identifying novel gene networks active in diverse stem cell types and provides a valuable resource for elucidating the multifaceted regulatory inputs required to guide proper stem cell behaviour.

Plant meristems carry pools of continuously active stem cells, whose activity is controlled by developmental and environmental signals. After stem cell division, daughter cells that exit the stem cell domain acquire transit amplifying cell identity before they are incorporated into organs and differentiate. In this study, we used an integrated approach to elucidate the role of HECATE (HEC) genes in regulating developmental trajectories of shoot stem cells in Arabidopsis thaliana. Our work reveals that HEC function stabilizes cell fate in distinct zones of the shoot meristem thereby controlling the spatio-temporal dynamics of stem cell differentiation. Importantly, this activity is concomitant with the local modulation of cellular responses to cytokinin and auxin, two key phytohormones regulating cell behaviour. Mechanistically, we show that HEC factors transcriptionally control and physically interact with MONOPTEROS (MP), a key regulator of auxin signalling, and modulate the autocatalytic stabilization of auxin signalling output., eLife digest Unlike animals, plants continuously generate new organs that make up their body. At the core of this amazing capacity lie tissues called meristems, which are found at the growing tips of all plants. Meristems contain dividing stem cells. The daughters of these stem cells pass through nearby regions called transition domains. Over time, they change – or differentiate – to go on to become part of tissues like leaves, roots, stems, shoots, flowers or fruits. Stem cell differentiation has a direct impact on a plant’s architecture and eventually its reproductive success. For crops, these factors determine yield. This means that understanding this aspect of plant development is central to basic and applied plant biology. Many factors required for shoot meristem activity have been identified, with a focus so far on the processes that control the identity of the cells produced. Now, Gaillochet et al. have asked which genes are responsible for controlling when stem cells in meristems differentiate. The analysis focused on the meristem that makes all the above ground parts of model plant Arabidopsis thaliana – the shoot apical meristem. Gaillochet et al. found that HECATE genes (or HEC for short) control the timing of stem cell differentiation by regulating the balance between the activities of two plant hormones: cytokinin and auxin. These genes promote cytokinin signals at the centre of the meristem, and dampen auxin response at the edges. This acts to slow down cell differentiation in two key transition domains of the shoot meristem. These new findings provide a molecular framework that now can be further investigated in crop plants to try to improve their yield. The findings also lay the foundation for studies of animals that may define common principles shared among stem cell systems in organisms that diverged over a billion years ago.

Plants develop postembryonically from pools of continuously active stem cells embedded in specialized tissues called meristems, which are located at the growing points of shoot and root. How these stem cells are established, maintained and guided towards differentiation within the highly dynamic shoot apical meristem is only beginning to emerge. At the core of the complex regulatory system are spatially distinct subdomains within the shoot apex, in which cells carry out defined functions, despite highly similar phenotypes. Spatial and temporal control of these domains appears to rely on an elaborate network of phytohormone signaling, transcriptional loops and intercellular trafficking of key regulators. In this review, we aim at summarizing and connecting the mechanisms underlying the spatial organization of the shoot apical meristem and the sequence of molecular events occurring during the life of a shoot cell, from its birth towards its differentiation.

This project aims at analyzing the causal structure of genetic regulatory networks of stem cells of plants using novel causal inference techniques to be developed here. Known methods for causal inference from statistical data usually require a large number of samples. Our preliminary work shows that it is in principle possible to infer causal relations from sample size one if the variables are high-dimensional, since algorithmic information provides additional hints on causal directions. Recent advances in genomic methods have allowed the simultaneous quantification of all genes in an organism. To identify the causal relation between individual transcripts, we will use inducible expression to analyze the effect of the homeodomain transcription factor WUSCHEL on the regulatory network of plant stem cell control. After appropriate clustering of the genes, we obtain a causal network between extremely high-dimensional variables, to which algorithmic information theory based methods can be applied. The inferred causal relation will then be tested by advanced experiments.

Identifying the transcription factors (TFs) and associated networks involved in stem cell regulation is key for understanding the initiation and growth of plant tissues and organs. Although many TFs have been shown to have a role in the Arabidopsis root stem cells, a comprehensive view of the transcriptional signature of the stem cells is lacking. In this work, we used spatial and temporal transcriptomic data to predict interactions among the genes involved in stem cell regulation. For this, we transcriptionally profiled several stem cell populations and developed a gene regulatory network (GRN) inference algorithm that combines clustering with Dynamic Bayesian Network (DBN) inference. We leveraged the topology of our networks to infer potential key regulators. The results presented in this work show that our combination of molecular biology approaches, computational biology and mathematical modeling was key to identify candidate factors that function in the stem cells. Specifically, through experimental validation and mathematical modeling, we identified PERIANTHIA (PAN) as an important molecular regulator of quiescent center (QC) function.