Auxin-mediated stress relaxation in pericycle and endoderm remodeling drives lateral root initiation.

Plant development relies on the precise coordination of cell growth, which is influenced by the mechanical constraints imposed by rigid cell walls. The hormone auxin plays a crucial role in regulating this growth by altering the mechanical properties of cell walls. During the postembryonic formation of lateral roots, pericycle cells deep within the main root are triggered by auxin to resume growth and divide to form a new root. This growth involves a complex interplay between auxin, growth, and the resolution of mechanical conflicts with the overlying endodermis. However, the exact mechanisms by which this coordination is achieved are still unknown. Here, we propose a model that integrates tissue mechanics and auxin transport, revealing a connection between the auxin-induced relaxation of mechanical stress in the pericycle and auxin signaling in the endodermis. We show that the endodermis initially limits the growth of pericycle cells, resulting in a modest initial expansion. However, the associated stress relaxation is sufficient to redirect auxin to the overlying endodermis, which then actively accommodates the growth, allowing for the subsequent development of the lateral root. Our model uncovers that increased pericycle turgor and decreased endodermal resistance license expansion of the pericycle and how the topology of the endodermis influences the formation of the new root. These findings highlight the interconnected relationship between mechanics and auxin flow during lateral root initiation, emphasizing the vital role of the endodermis in shaping root development through mechanotransduction and auxin signaling.

Tunable recurrent priming of lateral roots in Arabidopsis: More than just a clock?

Lateral root (LR) formation in Arabidopsis is a continuous, repetitive, post-embryonic process regulated by a series of coordinated events and tuned by the environment. It shapes the root system, enabling plants to efficiently explore soil resources and adapt to changing environmental conditions. Although the auxin-regulated modules responsible for LR morphogenesis and emergence are well documented, less is known about the initial priming. Priming is characterised by recurring peaks of auxin signalling, which, once memorised, earmark cells to form the new LR. We review the recent experimental and modelling approaches to understand the molecular processes underlying the recurring LR formation. We argue that the intermittent priming of LR results from interweaving the pattern of auxin flow and root growth together with an oscillatory auxin-modulated transcriptional mechanism and illustrate its long-range sugar-mediated tuning by light.

GreenGate 2.0: Backwards compatible addons for assembly of complex transcriptional units and their stacking with GreenGate.

Molecular cloning is a crucial technique in genetic engineering that enables the precise design of synthetic transcriptional units (TUs) and the manipulation of genomes. GreenGate and several other modular molecular cloning systems were developed about ten years ago and are widely used in plant research. All these systems define grammars for assembling transcriptional units from building blocks, cloned as Level 0 modules flanked by four-base pair overhangs and recognition sites for a particular Type IIs endonuclease. Modules are efficiently assembled into Level 1 TUs in a hierarchical assembly process, and Level 2 multigene constructs are assembled by stacking Level 1 TUs. GreenGate is highly popular but has three main limitations. First, using ad-hoc overhangs added by PCR and classical restriction/ligation prevents the efficient use of a one-pot, one-step reaction to generate entry clones and domesticate internal sites; second, a Level 1 TU is assembled from a maximum of six modules, which may be limiting for applications such as multiplex genome editing; third, the generation of Level 2 assemblies is sequential and inefficient. GreenGate 2.0 (GG2.0) expands GreenGate features. It introduces additional overhangs, allowing for the combination of up to 12 Level 0 modules in a Level 1 TU. It includes a Universal Entry Generator plasmid (pUEG) to streamline the generation of Level 0 modules. GG2.0 introduces GreenBraid, a convenient method for stacking transcriptional units iteratively for multigene assemblies. Importantly, GG2.0 is backwards compatible with most existing GreenGate modules. Additionally, GG2.0 includes Level 0 modules for multiplex expression of guide RNAs for CRISPR/Cas9 genome editing and pre-assembled Level 1 vectors for dexamethasone-inducible gene expression and ubiquitous expression of plasma membrane and nuclear fluorescent markers. GG2.0 streamlines and increases the versatility of assembling complex transcriptional units and their combination.

Increased gene expression variability hinders the formation of regional mechanical conflicts leading to reduced organ shape robustness.

To relate gene networks and organ shape, one needs to address two wicked problems: i) Gene expression is often variable locally, and shape is reproducible globally; ii) gene expression can have cascading effects on tissue mechanics, with possibly counterintuitive consequences for the final organ shape. Here, we address such wicked problems, taking advantage of simpler plant organ development where shape only emerges from cell division and elongation. We confirm that mutation in VERNALIZATION INDEPENDENCE 3 (VIP3), a subunit of the conserved polymerase-associated factor 1 complex (Paf1C), increases gene expression variability in Arabidopsis. Then, we focused on the Arabidopsis sepal, which exhibits a reproducible shape and stereotypical regional growth patterns. In vip3 sepals, we measured higher growth heterogeneity between adjacent cells. This even culminated in the presence of negatively growing cells in specific growth conditions. Interestingly, such increased local noise interfered with the stereotypical regional pattern of growth. We previously showed that regional differential growth at the wild-type sepal tip triggers a mechanical conflict, to which cells resist by reinforcing their walls, leading to growth arrest. In vip3, the disturbed regional growth pattern delayed organ growth arrest and increased final organ shape variability. Altogether, we propose that gene expression variability is managed by Paf1C to ensure organ robustness by building up mechanical conflicts at the regional scale, instead of the local scale.

Alexis Maizel.

Interview with Alexis Maizel, who studies plant morphogenesis at Heidelberg University.

TOR acts as a metabolic gatekeeper for auxin-dependent lateral root initiation in Arabidopsis thaliana.

Plant organogenesis requires matching the available metabolic resources to developmental programs. In Arabidopsis, the root system is determined by primary root-derived lateral roots (LRs), and adventitious roots (ARs) formed from non-root organs. Lateral root formation entails the auxin-dependent activation of transcription factors ARF7, ARF19, and LBD16. Adventitious root formation relies on LBD16 activation by auxin and WOX11. The allocation of shoot-derived sugar to the roots influences branching, but how its availability is sensed for LRs formation remains unknown. We combine metabolic profiling with cell-specific interference to show that LRs switch to glycolysis and consume carbohydrates. The target-of-rapamycin (TOR) kinase is activated in the lateral root domain. Interfering with TOR kinase blocks LR initiation while promoting AR formation. The target-of-rapamycin inhibition marginally affects the auxin-induced transcriptional response of the pericycle but attenuates the translation of ARF19, ARF7, and LBD16. TOR inhibition induces WOX11 transcription in these cells, yet no root branching occurs as TOR controls LBD16 translation. TOR is a central gatekeeper for root branching that integrates local auxin-dependent pathways with systemic metabolic signals, modulating the translation of auxin-induced genes.

PLETHORA-WOX5 interaction and subnuclear localization control Arabidopsis root stem cell maintenance.

Maintenance and homeostasis of the stem cell niche (SCN) in the Arabidopsis root is essential for growth and development of all root cell types. The SCN is organized around a quiescent center (QC) maintaining the stemness of cells in direct contact. The key transcription factors (TFs) WUSCHEL-RELATED HOMEOBOX 5 (WOX5) and PLETHORAs (PLTs) are expressed in the SCN where they maintain the QC and regulate distal columella stem cell (CSC) fate. Here, we describe the concerted mutual regulation of the key TFs WOX5 and PLTs on a transcriptional and protein interaction level. Additionally, by applying a novel SCN staining method, we demonstrate that both WOX5 and PLTs regulate root SCN homeostasis as they control QC quiescence and CSC fate interdependently. Moreover, we uncover that some PLTs, especially PLT3, contain intrinsically disordered prion-like domains (PrDs) that are necessary for complex formation with WOX5 and its recruitment to subnuclear microdomains/nuclear bodies (NBs) in the CSCs. We propose that this partitioning of PLT-WOX5 complexes to NBs, possibly by phase separation, is important for CSC fate determination.

Microtubule-based perception of mechanical conflicts controls plant organ morphogenesis.

Precise coordination between cells and tissues is essential for differential growth in plants. During lateral root formation in Arabidopsis thaliana, the endodermis is actively remodeled to allow outgrowth of the new organ. Here, we show that microtubule arrays facing lateral root founder cells display a higher order compared to arrays on the opposite side of the same cell, and this asymmetry is required for endodermal remodeling and lateral root initiation. We identify that MICROTUBULE ASSOCIATED PROTEIN 70-5 (MAP70-5) is necessary for the establishment of this spatially defined microtubule organization and endodermis remodeling and thus contributes to lateral root morphogenesis. We propose that MAP70-5 and cortical microtubule arrays in the endodermis integrate the mechanical signals generated by lateral root outgrowth, facilitating the channeling of organogenesis.

Integration of Cell Growth and Asymmetric Division during Lateral Root Initiation in Arabidopsis thaliana.

Lateral root formation determines to a large extent the ability of plants to forage their environment and thus their growth. In Arabidopsis thaliana and other angiosperms, lateral root initiation requires radial cell expansion and several rounds of anticlinal cell divisions that give rise to a central core of small cells, which express different markers than the larger surrounding cells. These small central cells then switch their plane of divisions to periclinal and give rise to seemingly morphologically similar daughter cells that have different identities and establish the different cell types of the new root. Although the execution of these anticlinal and periclinal divisions is tightly regulated and essential for the correct development of the lateral root, we know little about their geometrical features. Here, we generate a four-dimensional reconstruction of the first stages of lateral root formation and analyze the geometric features of the anticlinal and periclinal divisions. We identify that the periclinal divisions of the small central cells are morphologically dissimilar and asymmetric. We show that mother cell volume is different when looking at anticlinal vs. periclinal divisions and the repeated anticlinal divisions do not lead to reduction in cell volume, although cells are shorter. Finally, we show that cells undergoing a periclinal division are characterized by a strong cell expansion. Our results indicate that cells integrate growth and division to precisely partition their volume upon division during the first two stages of lateral root formation.

Accurate and versatile 3D segmentation of plant tissues at cellular resolution.

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.

To move or not to move: roles and specificity of plant RNA mobility.

Intercellular communication in plants coordinates cellular functions during growth and development, and in response to environmental cues. RNAs figure prominently among the mobile signaling molecules used. Many hundreds of RNA species move over short and long distances, and can be mutually exchanged in biotic interactions. Understanding the specificity determinants of RNA mobility and the physiological relevance of this phenomenon are areas of active research. Here, we highlight the recent progress in our knowledge of small RNA and messenger RNA movement. Particular emphasis is given to novel insight into the specificity determinants of messenger RNA mobility, the role of small RNA movement in development, and the specificity of RNA exchange in plant-plant and plant-microbe interactions.

Novel Imaging Modalities Shedding Light on Plant Biology: Start Small and Grow Big.

The acquisition of quantitative information on plant development across a range of temporal and spatial scales is essential to understand the mechanisms of plant growth. Recent years have shown the emergence of imaging methodologies that enable the capture and analysis of plant growth, from the dynamics of molecules within cells to the measurement of morphometricand physiological traits in field-grown plants. In some instances, these imaging methods can be parallelized across multiple samples to increase throughput. When high throughput is combined with high temporal and spatial resolution, the resulting image-derived data sets could be combined with molecular large-scale data sets to enable unprecedented systems-level computational modeling. Such image-driven functional genomics studies may be expected to appear at an accelerating rate in the near future given the early success of the foundational efforts reviewed here. We present new imaging modalities and review how they have enabled a better understanding of plant growth from the microscopic to the macroscopic scale.

Early developmental plasticity of lateral roots in response to asymmetric water availability.

Root branching is influenced by the soil environment and exhibits a high level of plasticity. We report that the radial positioning of emerging lateral roots is influenced by their hydrological environment during early developmental stages. New lateral root primordia have both a high degree of flexibility in terms of initiation and development angle towards the available water. Our observations reveal how the external hydrological environment regulates lateral root morphogenesis.

Cell Death in Cells Overlying Lateral Root Primordia Facilitates Organ Growth in Arabidopsis.

Plant organ growth is widely accepted to be determined by cell division and cell expansion, but, unlike that in animals, the contribution of cell elimination has rarely been recognized. We investigated this paradigm during Arabidopsis lateral root formation, when the lateral root primordia (LRP) must traverse three overlying cell layers within the parent root. A subset of LRP-overlying cells displayed the induction of marker genes for cell types undergoing developmental cell death, and their cell death was detected by electron, confocal, and light sheet microscopy techniques. LRP growth was delayed in cell-death-deficient mutants lacking the positive cell death regulator ORESARA1/ANAC092 (ORE1). LRP growth was restored in ore1-2 knockout plants by genetically inducing cell elimination in cells overlying the LRP or by physically killing LRP-overlying cells by ablation with optical tweezers. Our results support that, in addition to previously discovered mechanisms, cell elimination contributes to regulating lateral root emergence.

Cytoskeleton Dynamics Are Necessary for Early Events of Lateral Root Initiation in Arabidopsis.

How plant cells re-establish differential growth to initiate organs is poorly understood. Morphogenesis of lateral roots relies on the asymmetric cell division of initially symmetric founder cells. This division is preceded by the tightly controlled asymmetric radial expansion of these cells. The cellular mechanisms that license and ensure the coordination of these events are unknown. Here, we quantitatively analyze microtubule and F-actin dynamics during lateral root initiation. Using mutants and pharmacological and tissue-specific genetic perturbations, we show that dynamic reorganization of both microtubule and F-actin networks is necessary for the asymmetric expansion of the founder cells. This cytoskeleton remodeling intertwines with auxin signaling in the pericycle and endodermis in order for founder cells to acquire a basic polarity required for initiating lateral root development. Our results reveal the conservation of cell remodeling and polarization strategies between the Arabidopsis zygote and lateral root founder cells. We propose that coordinated, auxin-driven reorganization of the cytoskeleton licenses asymmetric cell growth and divisions during embryonic and post-embryonic organogenesis.

ARF5/MONOPTEROS directly regulates miR390 expression in the Arabidopsis thaliana primary root meristem.

The root meristem is organized around a quiescent center (QC) surrounded by stem cells that generate all cell types of the root. In the transit-amplifying compartment, progeny of stem cells further divides prior to differentiation. Auxin controls the size of this transit-amplifying compartment via auxin response factors (ARFs) that interact with auxin response elements (AuxREs) in the promoter of their targets. The microRNA miR390 regulates abundance of ARF2, ARF3, and ARF4 by triggering the production of trans-acting (ta)-siRNA from the TAS3 precursor. This miR390/TAS3/ARF regulatory module confers sensitivity and robustness to auxin responses in diverse developmental contexts and organisms. Here, we show that miR390 is expressed in the transit-amplifying compartment of the root meristem where it modulates response to exogenous auxin. We show that a single AuxRE located in miR390 promoter is necessary for miR390 expression in this compartment and identify that ARF5/MONOPTEROS (MP) binds miR390 promoter via the AuxRE. We show that interfering with ARF5/MP-dependent auxin signaling attenuates miR390 expression in the transit-amplifying compartment of the root meristem. Our results show that ARF5/MP regulates directly the expression of miR390 in the root meristem. We propose that ARF5, miR390, and the ta-siRNAs-regulated ARFs modulate the response of the transit-amplifying region of the meristem to exogenous auxin.

EXPANSIN A1-mediated radial swelling of pericycle cells positions anticlinal cell divisions during lateral root initiation.

In plants, postembryonic formation of new organs helps shape the adult organism. This requires the tight regulation of when and where a new organ is formed and a coordination of the underlying cell divisions. To build a root system, new lateral roots are continuously developing, and this process requires the tight coordination of asymmetric cell division in adjacent pericycle cells. We identified EXPANSIN A1 (EXPA1) as a cell wall modifying enzyme controlling the divisions marking lateral root initiation. Loss of EXPA1 leads to defects in the first asymmetric pericycle cell divisions and the radial swelling of the pericycle during auxin-driven lateral root formation. We conclude that a localized radial expansion of adjacent pericycle cells is required to position the asymmetric cell divisions and generate a core of small daughter cells, which is a prerequisite for lateral root organogenesis.