Morphogenesis at the shoot meristem.

Shoot apical meristems are populations of stem cells which initiate the aerial parts of higher plants. Work during the last decades has revealed a complex network of molecular regulators, which control both meristem maintenance and the production of different types of organs. The behavior of this network in time and space is defined by the local interactions between regulators and also involves hormonal regulation. In particular, auxin and cytokinin are intimately implicated in the coordination of gene expression patterns. To control growth patterns at the shoot meristem the individual components of the network influence directions and rates of cell growth. This requires interference with the mechanical properties of the cells. How this complex multiscale process, characterized by multiple feedbacks, is controlled remains largely an open question. Fortunately, genetics, live imaging, computational modelling and a number of other recently developed tools offer interesting albeit challenging perspectives.

Les méristèmes apicaux caulinaires sont des populations de cellules souches qui initient les parties aériennes des plantes supérieures. Les travaux des dernières décennies ont révélé un réseau complexe de régulateurs moléculaires, qui contrôlent à la fois la maintenance des méristèmes et la production de différents types d'organes. Le comportement de ce réseau dans le temps et dans l'espace est défini par les interactions locales entre régulateurs et implique également une régulation hormonale. En particulier, l'auxine et la cytokinine sont intimement impliquées dans la coordination de l'expression génique. Pour contrôler la morphogenèse au niveau du méristème caulinaire, les éléments individuels du réseau influencent les directions et les taux de croissance cellulaire. Cela nécessite une interférence avec les propriétés mécaniques des cellules. Comment ce processus multi-échelle complexe, caractérisé par de multiples rétroactions, est contrôlé reste largement une question ouverte et sa compréhension représente un défi majeur. Heureusement, la génétique, l'imagerie in vivo, la modélisation informatique et un certain nombre d'autres outils récemment développés offrent des perspectives intéressantes.

Jan Traas.

Interview with Jan Traas, who studies shoot meristem function at the École normale supérieure de Lyon.

A 3D gene expression atlas of the floral meristem based on spatial reconstruction of single nucleus RNA sequencing data.

Cellular heterogeneity in growth and differentiation results in organ patterning. Single-cell transcriptomics allows characterization of gene expression heterogeneity in developing organs at unprecedented resolution. However, the original physical location of the cell is lost during this methodology. To recover the original location of cells in the developing organ is essential to link gene activity with cellular identity and function in plants. Here, we propose a method to reconstruct genome-wide gene expression patterns of individual cells in a 3D flower meristem by combining single-nuclei RNA-seq with microcopy-based 3D spatial reconstruction. By this, gene expression differences among meristematic domains giving rise to different tissue and organ types can be determined. As a proof of principle, the method is used to trace the initiation of vascular identity within the floral meristem. Our work demonstrates the power of spatially reconstructed single cell transcriptome atlases to understand plant morphogenesis. The floral meristem 3D gene expression atlas can be accessed at http://threed-flower-meristem.herokuapp.com .

Benchmarking of deep learning algorithms for 3D instance segmentation of confocal image datasets.

Segmenting three-dimensional (3D) microscopy images is essential for understanding phenomena like morphogenesis, cell division, cellular growth, and genetic expression patterns. Recently, deep learning (DL) pipelines have been developed, which claim to provide high accuracy segmentation of cellular images and are increasingly considered as the state of the art for image segmentation problems. However, it remains difficult to define their relative performances as the concurrent diversity and lack of uniform evaluation strategies makes it difficult to know how their results compare. In this paper, we first made an inventory of the available DL methods for 3D cell segmentation. We next implemented and quantitatively compared a number of representative DL pipelines, alongside a highly efficient non-DL method named MARS. The DL methods were trained on a common dataset of 3D cellular confocal microscopy images. Their segmentation accuracies were also tested in the presence of different image artifacts. A specific method for segmentation quality evaluation was adopted, which isolates segmentation errors due to under- or oversegmentation. This is complemented with a 3D visualization strategy for interactive exploration of segmentation quality. Our analysis shows that the DL pipelines have different levels of accuracy. Two of them, which are end-to-end 3D and were originally designed for cell boundary detection, show high performance and offer clear advantages in terms of adaptability to new data.

Stable establishment of organ polarity occurs several plastochrons before primordium outgrowth in Arabidopsis.

In many species, leaves are initiated at the flanks of shoot meristems. Subsequent growth usually occurs mainly in the plane of the leaf blade, which leads to the formation of a bifacial leaf with dorsoventral identities. In a classical set of surgical experiments in potato meristems, Sussex provided evidence that dorsoventrality depends on a signal emanating from the meristem center. Although these results could be reproduced in tomato, this concept has been debated. We revisited these experiments in Arabidopsis, in which a range of markers are available to target the precise site of ablation. Using specific markers for organ founder cells and dorsoventral identity, we were unable to perturb the polarity of leaves and sepals long before organ outgrowth. Although results in Solanaceae suggested that dorsoventral patterning was unstable during early development, we found that, in Arabidopsis, the local information contained within and around the primordium is able to withstand major invasive perturbations, long before polarity is fully established.

Visualization of cortical microtubule networks in plant cells by live imaging and immunostaining.

Cortical microtubules (CMTs) play pivotal roles during plant cell growth and division. The organization of CMTs undergoes important changes during different cellular and developmental processes. Here, we describe two methods for the visualization of CMT organization in plant cells using confocal laser scanning microscopy. CMT networks in the outer tissue layers can be directly visualized by live imaging of a fluorescent reporter line, and a protocol combining sectioning and immunostaining is applied for visualization of CMTs throughout tissues. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).

A multiscale analysis of early flower development in Arabidopsis provides an integrated view of molecular regulation and growth control.

We have analyzed the link between the gene regulation and growth during the early stages of flower development in Arabidopsis. Starting from time-lapse images, we generated a 4D atlas of early flower development, including cell lineage, cellular growth rates, and the expression patterns of regulatory genes. This information was introduced in MorphoNet, a web-based platform. Using computational models, we found that the literature-based molecular network only explained a minority of the gene expression patterns. This was substantially improved by adding regulatory hypotheses for individual genes. Correlating growth with the combinatorial expression of multiple regulators led to a set of hypotheses for the action of individual genes in morphogenesis. This identified the central factor LEAFY as a potential regulator of heterogeneous growth, which was supported by quantifying growth patterns in a leafy mutant. By providing an integrated view, this atlas should represent a fundamental step toward mechanistic models of flower development.

How Mechanical Forces Shape Plant Organs.

Plants produce organs of various shapes and sizes. While much has been learned about genetic regulation of organogenesis, the integration of mechanics in the process is also gaining attention. Here, we consider the role of forces as instructive signals in organ morphogenesis. Turgor pressure is the primary cause of mechanical signals in developing organs. Because plant cells are glued to each other, mechanical signals act, in essence, at multiple scales, through cell wall contiguity and water flux. In turn, cells use such signals to resist mechanical stress, for instance, by reinforcing their cell walls. We show that the three elemental shapes behind plant organs - spheres, cylinders and lamina - can be actively maintained by such a mechanical feedback. Combinations of this 3-letter alphabet can generate more complex shapes. Furthermore, mechanical conflicts emerge at the boundary between domains exhibiting different growth rates or directions. These secondary mechanical signals contribute to three other organ shape features - folds, shape reproducibility and growth arrest. The further integration of mechanical signals with the molecular network offers many fruitful prospects for the scientific community, including the role of proprioception in organ shape robustness or the definition of cell and organ identities as a result of an interplay between biochemical and mechanical signals.

The Mechanical Feedback Theory of Leaf Lamina Formation.

The appearance of leaves with flattened laminae about 400 million years (Myr) ago had broad impacts on the Earth's ecosystem. The influential telome theory presents a model for this evolutionary transition, although it lacks plausible molecular evidence. Recently, microtubule-mediated mechanical feedback was proposed as a parsimonious alternative mechanism to explain leaf blade evolution.

Microtubule-Mediated Wall Anisotropy Contributes to Leaf Blade Flattening.

Plant organs can adopt a wide range of shapes, resulting from highly directional cell growth and divisions. We focus here on leaves and leaf-like organs in Arabidopsis and tomato, characterized by the formation of thin, flat laminae. Combining experimental approaches with 3D mechanical modeling, we provide evidence that leaf shape depends on cortical microtubule mediated cellulose deposition along the main predicted stress orientations, in particular, along the adaxial-abaxial axis in internal cell walls. This behavior can be explained by a mechanical feedback and has the potential to sustain and even amplify a preexisting degree of flatness, which in turn depends on genes involved in the control of organ polarity and leaf margin formation.

Cellular Heterogeneity in Pressure and Growth Emerges from Tissue Topology and Geometry.

Cell-to-cell heterogeneity prevails in many systems, as exemplified by cell growth, although the origin and function of such heterogeneity are often unclear. In plants, growth is physically controlled by cell wall mechanics and cell hydrostatic pressure, alias turgor pressure. Whereas cell wall heterogeneity has received extensive attention, the spatial variation of turgor pressure is often overlooked. Here, combining atomic force microscopy and a physical model of pressurized cells, we show that turgor pressure is heterogeneous in the Arabidopsis shoot apical meristem, a population of stem cells that generates all plant aerial organs. In contrast with cell wall mechanical properties that appear to vary stochastically between neighboring cells, turgor pressure anticorrelates with cell size and cell neighbor number (local topology), in agreement with the prediction by our model of tissue expansion, which couples cell wall mechanics and tissue hydraulics. Additionally, our model predicts two types of correlations between pressure and cellular growth rate, where high pressure may lead to faster- or slower-than-average growth, depending on cell wall extensibility, yield threshold, osmotic pressure, and hydraulic conductivity. The meristem exhibits one of these two regimes, depending on conditions, suggesting that, in this tissue, water conductivity may contribute to growth control. Our results unravel cell pressure as a source of patterned heterogeneity and illustrate links between local topology, cell mechanical state, and cell growth, with potential roles in tissue homeostasis.

Xyloglucans and Microtubules Synergistically Maintain Meristem Geometry and Phyllotaxis.

The shoot apical meristem (SAM) gives rise to all aerial plant organs. Cell walls are thought to play a central role in this process, translating molecular regulation into dynamic changes in growth rate and direction, although their precise role in morphogenesis during organ formation is poorly understood. Here, we investigated the role of xyloglucans (XyGs), a major, yet functionally poorly characterized, wall component in the SAM of Arabidopsis (Arabidopsis thaliana). Using immunolabeling, biochemical analysis, genetic approaches, microindentation, laser ablation, and live imaging, we showed that XyGs are important for meristem shape and phyllotaxis. No difference in the Young's modulus (i.e. an indicator of wall stiffness) of the cell walls was observed when XyGs were perturbed. Mutations in enzymes required for XyG synthesis also affect other cell wall components such as cellulose content and pectin methylation status. Interestingly, control of cortical microtubule dynamics by the severing enzyme KATANIN became vital when XyGs were perturbed or absent. This suggests that the cytoskeleton plays an active role in compensating for altered cell wall composition.

Simulating Turgor-Induced Stress Patterns in Multilayered Plant Tissues.

The intertwining between mechanics and developmental biology is extensively studied at the shoot apical meristem of land plants. Indeed, plant morphogenesis heavily relies on mechanics; tissue deformations are fueled by turgor-induced forces, and cell mechanosensitivity plays a major regulatory role in this dynamics. Since measurements of forces in growing meristems are still out of reach, our current knowledge relies mainly on theoretical and numerical models. So far, these modeling efforts have been mostly focusing on the epidermis, where aerial organs are initiated. In many models, the epidermis is assimilated to its outermost cell walls and described as a thin continuous shell under pressure, thereby neglecting the inner walls. There is, however, growing experimental evidence suggesting a significant mechanical role of these inner walls. The aim of this work is to investigate the influence of inner walls on the mechanical homeostasis of meristematic tissues. To this end, we simulated numerically the effect of turgor-induced loading, both in realistic flower buds and in more abstract structures. These simulations were performed using finite element meshes with subcellular resolution. Our analysis sheds light on the mechanics of growing plants by revealing the strong influence of inner walls on the epidermis mechanical stress pattern especially in negatively curved regions. Our simulations also display some strong and unsuspected features, such as a correlation between stress intensity and cell size, as well as differential response to loading between epidermal and inner cells. Finally, we monitored the time evolution of the mechanical stresses felt by each cell and its descendants during the early steps of flower morphogenesis.

Regulation of plant cell wall stiffness by mechanical stress: a mesoscale physical model.

A crucial question in developmental biology is how cell growth is coordinated in living tissue to generate complex and reproducible shapes. We address this issue here in plants, where stiff extracellular walls prevent cell migration and morphogenesis mostly results from growth driven by turgor pressure. How cells grow in response to pressure partly depends on the mechanical properties of their walls, which are generally heterogeneous, anisotropic and dynamic. The active control of these properties is therefore a cornerstone of plant morphogenesis. Here, we focus on wall stiffness, which is under the control of both molecular and mechanical signaling. Indeed, in plant tissues, the balance between turgor and cell wall elasticity generates a tissue-wide stress field. Within cells, mechano-sensitive structures, such as cortical microtubules, adapt their behavior accordingly and locally influence cell wall remodeling dynamics. To fully apprehend the properties of this feedback loop, modeling approaches are indispensable. To that end, several modeling tools in the form of virtual tissues have been developed. However, these models often relate mechanical stress and cell wall stiffness in relatively abstract manners, where the molecular specificities of the various actors are not fully captured. In this paper, we propose to refine this approach by including parsimonious biochemical and biomechanical properties of the main molecular actors involved. Through a coarse-grained approach and through finite element simulations, we study the role of stress-sensing microtubules on organ-scale mechanics.

Evidence for the Regulation of Gynoecium Morphogenesis by ETTIN via Cell Wall Dynamics.

ETTIN (ETT) is an atypical member of the AUXIN RESPONSE FACTOR family of transcription factors that plays a crucial role in tissue patterning in the Arabidopsis (Arabidopsis thaliana) gynoecium. Though recent insights have provided valuable information on ETT's interactions with other components of auxin signaling, the biophysical mechanisms linking ETT to its ultimate effects on gynoecium morphology were until now unknown. Here, using techniques to assess cell-wall dynamics during gynoecium growth and development, we provide a coherent body of evidence to support a model in which ETT controls the elongation of the valve tissues of the gynoecium through the positive regulation of pectin methylesterase (PME) activity in the cell wall. This increase in PME activity results in an increase in the level of demethylesterified pectins and a consequent reduction in cell wall stiffness, leading to elongation of the valves. Though similar biophysical mechanisms have been shown to act in the stem apical meristem, leading to the expansion of organ primordia, our findings demonstrate that regulation of cell wall stiffness through the covalent modification of pectin also contributes to tissue patterning within a developing plant organ.

Transcriptional induction of cell wall remodelling genes is coupled to microtubule-driven growth isotropy at the shoot apex in Arabidopsis.

The shoot apical meristem of higher plants continuously generates new tissues and organs through complex changes in growth rates and directions of its individual cells. Cell growth, which is driven by turgor pressure, largely depends on the cell walls, which allow cell expansion through synthesis and structural changes. A previous study revealed a major contribution of wall isotropy in organ emergence, through the disorganization of cortical microtubules. We show here that this disorganization is coupled with the transcriptional control of genes involved in wall remodelling. Some of these genes are induced when microtubules are disorganized and cells shift to isotropic growth. Mechanical modelling shows that this coupling has the potential to compensate for reduced cell expansion rates induced by the shift to isotropic growth. Reciprocally, cell wall loosening induced by different treatments or altered cell wall composition promotes a disruption of microtubule alignment. Our data thus indicate the existence of a regulatory module activated during organ outgrowth, linking microtubule arrangements to cell wall remodelling.

Mechanical signaling in plant morphogenesis.

To control changes in shape during development, the molecular regulatory networks have to interact with the mechanical, structural components of the individual cells, in particular the cytoskeleton and the cell wall. A widely accepted hypothesis proposes that molecular regulation interferes with wall synthesis and stiffness, causing the wall polymers to yield to the internal turgor pressure. However, growth is not only the result of a rigid molecular program instructing the cells precisely what to do. Local differences in growth rates between neighboring cells generate mechanical constraints that can feed back on the regulatory networks and the cytoskeleton. A number of components involved in the perception of these constraints have been identified, although their precise function remains to be determined.

Organogenesis at the Shoot Apical Meristem.

Lateral organ initiation at the shoot apical meristem involves complex changes in growth rates and directions, ultimately leading to the formation of leaves, stems and flowers. Extensive molecular analysis identifies auxin and downstream transcriptional regulation as major elements in this process. This molecular regulatory network must somehow interfere with the structural elements of the cell, in particular the cell wall, to induce specific morphogenetic events. The cell wall is composed of a network of rigid cellulose microfibrils embedded in a matrix composed of water, polysaccharides such as pectins and hemicelluloses, proteins, and ions. I will discuss here current views on how auxin dependent pathways modulate wall structure to set particular growth rates and growth directions. This involves complex feedbacks with both the cytoskeleton and the cell wall.

Plant Development: From Dynamics to Mechanics.

A new study analyses the complex changes in shape occurring during petal development in snapdragon. Combining simulations with quantitative analysis leads to a new model, where molecular regulators control overall organ shape through mechanical conflicts operating at the level of entire tissues.