HECATE factors control cell fate transitions and organ patterning in Arabidopsis thaliana

Throughout their life span, plants keep the ability to generate new tissues and organs. This remarkable developmental property relies on the continuous activity of pluripotent stem cells localized in meristems, which generate cell progenies acquiring specific cellular identities. Thus, the regulatory processes controlling the progression of stem cell lineages and their final differentiation are essential to establish the whole body plan and to ultimately define plant reproductive success. The integration of phytohormonal signals like auxin or cytokinin with key transcriptional regulators is central for balancing stem cell activity and differentiation (reviewed in Gaillochet and Lohmann, 2015), however our current understanding of the regulatory interactions mediating this molecular communication remains elusive. In this study, we used an integrated approach–including live-cell imaging, computational modeling, genome-wide profiling and genetic functional characterization–to investigate the function of the bHLH transcription factors HECATE (HEC) in controlling stem cell homeostasis and organ patterning. We found that HEC regulatory function is highly versatile and tightly interacts with cytokinin and auxin signalling pathways under multiple developmental contexts. We show in the shoot apical meristem that HEC function regulates the timing of stem cell differentiation by locally promoting cytokinin at the centre of the meristem and repressing auxin signals at the periphery. In contrast, we found that HEC genes pattern style differentiation at the gynoecium by regulating auxin flow and by buffering cytokinin responses. Using a gene network reconstruction approach, we started to unravel the regulatory interactions mediating HEC functional versatility and identified NGATHA transcription factors as relevant direct targets controlling shoot meristem activity. Together, our findings refine the molecular and developmental framework for shoot meristem activity and gynoecium differentiation

The receptor kinase SRF3 coordinates iron- level and flagellin dependent defense and growth responses in plants

Iron is critical for host–pathogen interactions. While pathogens seek to scavenge iron to spread, the host aims at decreasing iron availability to reduce pathogen virulence. Thus, iron sensing and homeostasis are of particular importance to prevent host infection and part of nutritional immunity. While the link between iron homeostasis and immunity pathways is well established in plants, how iron levels are sensed and integrated with immune response pathways remains unknown. Here we report a receptor kinase SRF3, with a role in coordinating root growth, iron homeostasis and immunity pathways via regulation of callose synthases. These processes are modulated by iron levels and rely on SRF3 extracellular and kinase domains which tune its accumulation and partitioning at the cell surface. Mimicking bacterial elicitation with the flagellin peptide flg22 phenocopies SRF3 regulation upon low iron levels and subsequent SRF3-dependent responses. We propose that SRF3 is part of nutritional immunity responses involved in sensing external iron levels

CRISPR screens in plants : approaches, guidelines, and future prospects

CRISPR-Cas systems have revolutionized genome engineering by facilitating a wide range of targeted DNA perturbations. These systems have resulted in new powerful screens to test gene functions at the genomic scale. While there is tremendous potential for CRISPR screens to map and interrogate gene regulatory networks at unprecedented speed and scale, their implementation in plants remains in its infancy. Here we discuss the general concepts, tools and workflows for establishing CRISPR screens in plants and analyze the handful of recent reports using this strategy to generate mutant knockout collections or diversify DNA sequences. In addition, we provide insight on how to design CRISPR knockout screens in plants given the current challenges and limitations and examine multiple design options. Finally, we discuss the unique multiplexing capabilities of CRISPR screens to investigate redundant gene function in highly duplicated plant genomes. Combinatorial mutant screens have the potential to routinely generate higher-order mutant collections and facilitate the characterization of gene networks. By integrating this approach with the large resource of genomic profiles that were generated in the last two decades, the implementation of CRISPR screens offers new opportunities to analyze plant genomes at deeper resolution and will greatly advance plant functional and synthetic biology

Simple and efficient modification of Golden Gate design standards and parts using oligo stitching

The assembly of DNA parts is a critical aspect of contemporary biological research. Gibson assembly and Golden Gate cloning are two popular options. Here, we explore the use of single stranded DNA oligos with Gibson assembly to augment Golden Gate cloning workflows in a process called "oligo stitching". Our results show that oligo stitching can efficiently convert Golden Gate parts between different assembly standards and directly assemble incompatible Golden Gate parts without PCR amplification. Building on previous reports, we show that it can also be used to assemble de novo sequences. As a final application, we show that restriction enzyme recognition sites can be removed from plasmids and utilize the same concept to perform saturation mutagenesis. Given oligo stitching's versatility and high efficiency, we expect that it will be a useful addition to the molecular biologist's toolbox

Mathematical modeling of plant cell fate transitions controlled by hormonal signals.

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

A molecular network for functional versatility of HECATE transcription factors

During the plant life cycle, diverse signaling 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) basic helix–loop–helix 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 subprograms 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 signaling, gynoecium development and auxin homeostasis. Importantly, we found that HEC genes also play a role in the modulation of flowering time, and uncovered that their role in gynoecium development may involve the direct transcriptional regulation of NGATHA1 (NGA1) and NGA2 genes. NGA factors were previously shown to contribute to fruit development, but our data now show that they also modulate stem cell homeostasis in the shoot apical meristem. Taken together, our results delineate 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.</p

A molecular network for functional versatility of HECATE transcription factors

During the plant life cycle, diverse signaling 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) basic helix–loop–helix 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 subprograms 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 signaling, gynoecium development and auxin homeostasis. Importantly, we found that HEC genes also play a role in the modulation of flowering time, and uncovered that their role in gynoecium development may involve the direct transcriptional regulation of NGATHA1 (NGA1) and NGA2 genes. NGA factors were previously shown to contribute to fruit development, but our data now show that they also modulate stem cell homeostasis in the shoot apical meristem. Taken together, our results delineate 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

A Regulatory Framework for Shoot Stem Cell Control Integrating Metabolic, Transcriptional, and Phytohormone Signals

SummaryPlants continuously maintain pluripotent stem cells embedded in specialized tissues called meristems, which drive long-term growth and organogenesis. Stem cell fate in the shoot apical meristem (SAM) is controlled by the homeodomain transcription factor WUSCHEL (WUS) expressed in the niche adjacent to the stem cells. Here, we demonstrate that the bHLH transcription factor HECATE1 (HEC1) is a target of WUS and that it contributes to SAM function by promoting stem cell proliferation, while antagonizing niche cell activity. HEC1 represses the stem cell regulators WUS and CLAVATA3 (CLV3) and, like WUS, controls genes with functions in metabolism and hormone signaling. Among the targets shared by HEC1 and WUS are phytohormone response regulators, which we show to act as mobile signals in a universal feedback system. Thus, our work sheds light on the mechanisms guiding meristem function and suggests that the underlying regulatory system is far more complex than previously anticipated

WUSCHEL acts as an auxin response rheostat to maintain apical stem cells in Arabidopsis

International audienceTo 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