Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform.

BACKGROUND: Testing an ever-increasing number of CRISPR components is challenging when developing new genome engineering tools. Plant biotechnology has few high-throughput options to perform iterative design-build-test-learn cycles of gene-editing reagents. To bridge this gap, we develop ITER (Iterative Testing of Editing Reagents) based on 96-well arrayed protoplast transfections and high-content imaging. RESULTS: We validate ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors (ABEs), allowing one optimization cycle - from design to results - within 3 weeks. Given that previous LbCas12a-ABEs have low or no activity in plants, we use ITER to develop an optimized LbCas12a-ABE. We show that sequential improvement of five components - NLS, crRNA, LbCas12a, adenine deaminase, and linker - leads to a remarkable increase in activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirm the activity of LbCas12a-ABE at endogenous targets in protoplasts and obtain base-edited plants in up to 55% of stable wheat transformants and the edits are transmitted to T1 progeny. We leverage these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE demonstrating that the optimizations can be broadly applied to the Cas12a toolbox. CONCLUSION: Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants. We use ITER to create an efficient Cas12a-ABE by iteratively testing a large panel of vector components. ITER will likely be useful to create and optimize genome editing reagents in a wide range of plant species.

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.

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.

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

Clustered regularly interspaced short palindromic repeat (CRISPR)-associated systems have revolutionized genome engineering by facilitating a wide range of targeted DNA perturbations. These systems have resulted in the development of powerful new screens to test gene functions at the genomic scale. While there is tremendous potential to map and interrogate gene regulatory networks at unprecedented speed and scale using CRISPR screens, 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 describing the use of this strategy to generate mutant knockout collections or to diversify DNA sequences. In addition, we provide insight into 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 functions 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 numerous genomic profiles that have been generated over the past two decades, the implementation of CRISPR screens offers new opportunities to analyze plant genomes at deeper resolution and will lead to great advances in functional and synthetic biology.

HY5 and phytochrome activity modulate shoot-to-root coordination during thermomorphogenesis in Arabidopsis.

Temperature is one of the most impactful environmental factors to which plants adjust their growth and development. Although the regulation of temperature signaling has been extensively investigated for the aerial part of plants, much less is known and understood about how roots sense and modulate their growth in response to fluctuating temperatures. Here, we found that shoot and root growth responses to high ambient temperature are coordinated during early seedling development in Arabidopsis A shoot signaling module that includes HY5, the phytochromes and the PIFs exerts a central function in coupling these growth responses and maintaining auxin levels in the root. In addition to the HY5/PIF-dependent shoot module, a regulatory axis composed of auxin biosynthesis and auxin perception factors controls root responses to high ambient temperature. Taken together, our findings show that shoot and root developmental responses to temperature are tightly coupled during thermomorphogenesis and suggest that roots integrate energy signals with local hormonal inputs.

Local HY5 Activity Mediates Hypocotyl Growth and Shoot-to-Root Communication.

Plants optimize their growth in fluctuating environments using information acquired by different organs. This information is then transmitted through the rest of the plant using both short- and long-distance signals, including hormones and mobile proteins. Although a few of these signals have been characterized, long-distance signaling is not well understood in plants. Recently, the light-regulated transcription factor HY5 was reported to move from the shoot to the root to regulate root growth. We generated a cell-type specifically expressed HY5 fusion protein that could not be detected outside the tissue in which it was targeted. By expressing this DOF-HY5 protein in specific cell types of the hypocotyl, we showed that its local activity was sufficient to regulate hypocotyl growth. We also found that, although DOF-HY5 was expressed specifically in the shoot and not detected in the roots, it could rescue hy5 growth defects in primary roots but not in lateral roots. We therefore conclude that HY5 protein mobility is not required in the hypocotyl or for shoot-to-root communication. Our results indicate that a signal downstream of, or in parallel with, HY5 in the shoot is mobile and links shoot and root growth.

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.

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.

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

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.

An apical hypoxic niche sets the pace of shoot meristem activity.

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.

A Comprehensive Toolkit for Inducible, Cell Type-Specific Gene Expression in Arabidopsis.

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.

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.

Control of plant cell fate transitions by transcriptional and hormonal signals.

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.

Arabidopsis HECATE genes function in phytohormone control during gynoecium development.

The fruit, which develops from the fertilised gynoecium formed in the innermost whorl of the flower, is the reproductive organ and one of the most complex structures of an angiosperm plant. Phytohormones play important roles during flower and fruit patterning, morphogenesis and growth, and there is emerging evidence for a cross-talk between different classes of plant hormones throughout these processes. Here, we show that the bHLH transcription factors HECATE 1 (HEC1), HEC2 and HEC3, which have previously been identified as essential components of transmitting tract formation, affect both auxin and cytokinin responses during reproductive tissue development. We find that HEC1 interacts with SPATULA (SPT) to control carpel fusion and that both transcription factors restrict sensitivity to cytokinin in the gynoecium. In addition, HEC1 is tightly integrated into the auxin-signalling network at the levels of biosynthesis, transport and transcriptional response. Based on this data, we propose that HEC1 acts as a local modulator of auxin and cytokinin responses to control gynoecium development in Arabidopsis.

The never-ending story: from pluripotency to plant developmental plasticity.

Plants are sessile organisms, some of which can live for over a thousand years. Unlike most animals, plants employ a post-embryonic mode of development driven by the continuous activity of pluripotent stem cells. Consequently, plants are able to initiate new organs over extended periods of time, and many species can readily replace lost body structures by de novo organogenesis. Classical studies have also shown that plant tissues have a remarkable capacity to undergo de-differentiation and proliferation in vitro, highlighting the fact that plant cell fate is highly plastic. This suggests that the mechanisms regulating fate transitions must be continuously active in most plant cells and that the control of cellular pluripotency lies at the core of diverse developmental programs. Here, we review how pluripotency is established in plant stem cell systems, how it is maintained during development and growth and re-initiated during regeneration, and how these mechanisms eventually contribute to the amazing developmental plasticity of plants.

O cell, where art thou? The mechanisms of shoot meristem patterning.

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.

A regulatory framework for shoot stem cell control integrating metabolic, transcriptional, and phytohormone signals.

Plants 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.