Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis.

The presence of diffuse morphogen gradients in tissues supports a view in which growth is locally homogenous. Here we challenge this view: we used a high-resolution quantitative approach to reveal significant growth variability among neighboring cells in the shoot apical meristem, the plant stem cell niche. This variability was strongly decreased in a mutant impaired in the microtubule-severing protein katanin. Major shape defects in the mutant could be related to a local decrease in growth heterogeneity. We show that katanin is required for the cell's competence to respond to the mechanical forces generated by growth. This provides the basis for a model in which microtubule dynamics allow the cell to respond efficiently to mechanical forces. This in turn can amplify local growth-rate gradients, yielding more heterogeneous growth and supporting morphogenesis.

A transient radial cortical microtubule array primes cell division in Arabidopsis.

Although the formation of new walls during plant cell division tends to follow maximal tensile stress direction, analyses of individual cells over time reveal a much more variable behavior. The origin of such variability as well as the exact role of interphasic microtubule behavior before cell division have remained mysterious so far. To approach this question, we took advantage of the Arabidopsis stem, where the tensile stress pattern is both highly anisotropic and stable. Although cortical microtubules (CMTs) generally align with maximal tensile stress, we detected a specific time window, ca. 3 h before cell division, where cells form a radial pattern of CMTs. This microtubule array organization preceded preprophase band (PPB) formation, a transient CMT array predicting the position of the future division plane. It was observed under different growth conditions and was not related to cell geometry or polar auxin transport. Interestingly, this cortical radial pattern correlated with the well-documented increase of cytoplasmic microtubule accumulation before cell division. This radial organization was prolonged in cells of the trm678 mutant, where CMTs are unable to form a PPB. Whereas division plane orientation in trm678 is noisier, we found that cell division symmetry was in contrast less variable between daughter cells. We propose that this "radial step" reflects a trade-off in robustness for two essential cell division attributes: symmetry and orientation. This involves a "reset" stage in G2, where an increased cytoplasmic microtubule accumulation transiently disrupts CMT alignment with tissue stress.

Contribution of vasculature to stem integrity in Arabidopsis thaliana.

In plants, coordinated growth is important for organ mechanical integrity because cells remain contiguous through their walls. So far, defects in inflorescence stem integrity in Arabidopsis thaliana have mainly been related to epidermal defects. Although these observations suggest a growth-limiting function at the stem cortex, deeper layers of the stem could also contribute to stem integrity. The nac secondary cell wall thickening promoting factor1 (nst1) nst3 double-mutant background is characterized by weaker vascular bundles without cracks. By screening for the cracking phenotype in this background, we identified a regulator of stem cracking, the transcription factor INDETERMINATE DOMAIN9 (IDD9). Stem cracking was not caused by vascular bundle breakage in plants that expressed a dominant repressor version of IDD9. Instead, cracking emerged from increased cell expansion in non-lignified interfascicular fiber cells that stretched the epidermis. This phenotype could be enhanced through CLAVATA3-dependent cell proliferation. Collectively, our results demonstrate that stem integrity relies on three additive mechanical components: the epidermis, which resists inner cell growth; cell proliferation in inner tissues; and growth heterogeneity associated with vascular bundle distribution in deep tissues.

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.

Fifteen compelling open questions in plant cell biology.

As scientists, we are at least as excited about the open questions-the things we do not know-as the discoveries. Here, we asked 15 experts to describe the most compelling open questions in plant cell biology. These are their questions: How are organelle identity, domains, and boundaries maintained under the continuous flux of vesicle trafficking and membrane remodeling? Is the plant cortical microtubule cytoskeleton a mechanosensory apparatus? How are the cellular pathways of cell wall synthesis, assembly, modification, and integrity sensing linked in plants? Why do plasmodesmata open and close? Is there retrograde signaling from vacuoles to the nucleus? How do root cells accommodate fungal endosymbionts? What is the role of cell edges in plant morphogenesis? How is the cell division site determined? What are the emergent effects of polyploidy on the biology of the cell, and how are any such "rules" conditioned by cell type? Can mechanical forces trigger new cell fates in plants? How does a single differentiated somatic cell reprogram and gain pluripotency? How does polarity develop de-novo in isolated plant cells? What is the spectrum of cellular functions for membraneless organelles and intrinsically disordered proteins? How do plants deal with internal noise? How does order emerge in cells and propagate to organs and organisms from complex dynamical processes? We hope you find the discussions of these questions thought provoking and inspiring.

Looking beyond the gene network - metabolic and mechanical cell drivers of leaf morphogenesis.

The above-ground organs in plants display a rich diversity, yet they grow to characteristic sizes and shapes. Organ morphogenesis progresses through a sequence of key events, which are robustly executed spatiotemporally as an emerging property of intrinsic molecular networks while adapting to various environmental cues. This Review focuses on the multiscale control of leaf morphogenesis. Beyond the list of known genetic determinants underlying leaf growth and shape, we focus instead on the emerging novel mechanisms of metabolic and biomechanical regulations that coordinate plant cell growth non-cell-autonomously. This reveals how metabolism and mechanics are not solely passive outcomes of genetic regulation but play instructive roles in leaf morphogenesis. Such an integrative view also extends to fluctuating environmental cues and evolutionary adaptation. This synthesis calls for a more balanced view on morphogenesis, where shapes are considered from the standpoints of geometry, genetics, energy and mechanics, and as emerging properties of the cellular expression of these different properties.

Stem integrity in Arabidopsis thaliana requires a load-bearing epidermis.

Because plant cells are glued to each other via their cell walls, failure to coordinate growth among adjacent cells can create cracks in tissues. Here, we find that the unbalanced growth of inner and outer tissues in the clavata3 de-etiolated3 (clv3 det3) mutant of Arabidopsis thaliana stretched epidermal cells, ultimately generating cracks in stems. Stem growth slowed before cracks appeared along clv3 det3 stems, whereas inner pith cells became drastically distorted and accelerated their growth, yielding to stress, after the appearance of cracks. This is consistent with a key role of the epidermis in restricting growth. Mechanical property measurements recorded using an atomic force microscope revealed that epidermal cell wall stiffness decreased in det3 and clv3 det3 epidermises. Thus, we hypothesized that stem integrity depends on the epidermal resistance to mechanical stress. To formally test this hypothesis, we used the DET3 gene as part of a tissue-specific strategy to complement cell expansion defects. Epidermis-driven DET3 expression restored growth and restored the frequency of stem cracking to 20% of the clv3 det3 mutant, demonstrating the DET3-dependent load-bearing role of the epidermis.

FERONIA and microtubules independently contribute to mechanical integrity in the Arabidopsis shoot.

To survive, cells must constantly resist mechanical stress. In plants, this involves the reinforcement of cell walls, notably through microtubule-dependent cellulose deposition. How wall sensing might contribute to this response is unknown. Here, we tested whether the microtubule response to stress acts downstream of known wall sensors. Using a multistep screen with 11 mutant lines, we identify FERONIA (FER) as the primary candidate for the cell's response to stress in the shoot. However, this does not imply that FER acts upstream of the microtubule response to stress. In fact, when performing mechanical perturbations, we instead show that the expected microtubule response to stress does not require FER. We reveal that the feronia phenotype can be partially rescued by reducing tensile stress levels. Conversely, in the absence of both microtubules and FER, cells appear to swell and burst. Altogether, this shows that the microtubule response to stress acts as an independent pathway to resist stress, in parallel to FER. We propose that both pathways are required to maintain the mechanical integrity of plant cells.

Sepal shape variability is robust to cell size heterogeneity in Arabidopsis.

How organisms produce organs with robust shapes and sizes is still an open question. In recent years, the Arabidopsis sepal has been used as a model system to study this question because of its highly reproducible shape and size. One interesting aspect of the sepal is that its epidermis contains cells of very different sizes. Previous reports have qualitatively shown that sepals with more or less giant cells exhibit comparable final size and shape. Here, we investigate this question using quantitative approaches. We find that a mixed population of cell size modestly contribute to the normal width of the sepal but is not essential for its shape robustness. Furthermore, in a mutant with increased cell and organ growth variability, the change in final sepal shape caused by giant cells is exaggerated but the shape robustness is not affected. This formally demonstrates that sepal shape variability is robust to cell size heterogeneity.

Contributions of lignification, tissue arrangement patterns, and cross-sectional area to whole-stem mechanical properties in Arabidopsis thaliana.

Plant cells withstand mechanical stress originating from turgor pressure by robustly maintaining the mechanical properties of the cell wall. This applies at the organ scale as well; many plant stems act as pressurized cylinders, where the epidermis is under tension and inner tissues are under compression. The clavata3 de-etiolated3 (clv3-8 det3-1) double mutant of Arabidopsis thaliana displays cracks in its stems because of a conflict between the mechanical properties of the weak epidermis and over-proliferation of inner stem tissues. In this work, we conducted three-point bending tests on various Arabidopsis thaliana mutants, including those displaying the stem cracking phenotype, to examine the differences in their mechanical properties. The clv3-8 det3-1 double mutant exhibited reduced stem stiffness, consistent with reduced differentiation due to the clv3-8 mutation. Yet, in clv3-8, stem cross-sectional area was increased associating with the increase in vascular bundle number, and stem cross-sections displayed various shapes. To uncouple the contribution of geometry and cell-wall differentiation to the mechanical properties of the whole stems, we tested the contribution of lignified fibers to stem stiffness. In order to suppress lignin deposition in stems genetically, we generated multiple higher-order mutants by crossing clv3-8 and/or det3-1 with nst1-1 nst3-1, in which lignin deposition is suppressed. Stem stiffness was reduced markedly in all nst1-1 nst3-1 mutant backgrounds. Overall, our results suggest that stem stiffness relies on the presence of differentiated, lignified, fiber tissue as well as on the alignment or spatial distribution of vascular bundles within the stem organ.

Tissue folding at the organ-meristem boundary results in nuclear compression and chromatin compaction.

Artificial mechanical perturbations affect chromatin in animal cells in culture. Whether this is also relevant to growing tissues in living organisms remains debated. In plants, aerial organ emergence occurs through localized outgrowth at the periphery of the shoot apical meristem, which also contains a stem cell niche. Interestingly, organ outgrowth has been proposed to generate compression in the saddle-shaped organ-meristem boundary domain. Yet whether such growth-induced mechanical stress affects chromatin in plant tissues is unknown. Here, by imaging the nuclear envelope in vivo over time and quantifying nucleus deformation, we demonstrate the presence of active nuclear compression in that domain. We developed a quantitative pipeline amenable to identifying a subset of very deformed nuclei deep in the boundary and in which nuclei become gradually narrower and more elongated as the cell contracts transversely. In this domain, we find that the number of chromocenters is reduced, as shown by chromatin staining and labeling, and that the expression of linker histone H1.3 is induced. As further evidence of the role of forces on chromatin changes, artificial compression with a MicroVice could induce the ectopic expression of H1.3 in the rest of the meristem. Furthermore, while the methylation status of chromatin was correlated with nucleus deformation at the meristem boundary, such correlation was lost in the h1.3 mutant. Altogether, we reveal that organogenesis in plants generates compression that is able to have global effects on chromatin in individual cells.

Cortical tension overrides geometrical cues to orient microtubules in confined protoplasts.

In plant cells, cortical microtubules (CMTs) generally control morphogenesis by guiding cellulose synthesis. CMT alignment has been proposed to depend on geometrical cues, with microtubules aligning with the cell long axis in silico and in vitro. Yet, CMTs are usually transverse in vivo, i.e., along predicted maximal tension, which is transverse for cylindrical pressurized vessels. Here, we adapted a microwell setup to test these predictions in a single-cell system. We confined protoplasts laterally to impose a curvature ratio and modulated pressurization through osmotic changes. We find that CMTs can be longitudinal or transverse in wallless protoplasts and that the switch in CMT orientation depends on pressurization. In particular, longitudinal CMTs become transverse when cortical tension increases. This explains the dual behavior of CMTs in planta: CMTs become longitudinal when stress levels become low, while stable transverse CMT alignments in tissues result from their autonomous response to tensile stress fluctuations.

Paf1c defects challenge the robustness of flower meristem termination in Arabidopsis thaliana.

Although accumulating evidence suggests that gene regulation is highly stochastic, genetic screens have successfully uncovered master developmental regulators, questioning the relationship between transcriptional noise and intrinsic robustness of development. To identify developmental modules that are more or less resilient to large-scale genetic perturbations, we used the Arabidopsis polymerase II-associated factor 1 complex (Paf1c) mutant vip3, which is impaired in several RNA polymerase II-dependent transcriptional processes. We found that the control of flower termination was not as robust as classically pictured. In angiosperms, the floral female organs, called carpels, display determinate growth: their development requires the arrest of stem cell maintenance. In vip3 mutant flowers, carpels displayed a highly variable morphology, with different degrees of indeterminacy defects up to wild-type size inflorescence emerging from carpels. This phenotype was associated with variable expression of two key regulators of flower termination and stem cell maintenance in flowers, WUSCHEL and AGAMOUS The phenotype was also dependent on growth conditions. Together, these results highlight the surprisingly plastic nature of stem cell maintenance in plants and its dependence on Paf1c.

Microtubule self-organisation during seed germination in Arabidopsis.

BACKGROUND: Upon water uptake and release of seed dormancy, embryonic plant cells expand, while being mechanically constrained by the seed coat. Cortical microtubules (CMTs) are key players of cell elongation in plants: their anisotropic orientation channels the axis of cell elongation through the guidance of oriented deposition of load-bearing cellulose microfibrils in the cell wall. Interestingly, CMTs align with tensile stress, and consistently, they reorient upon compressive stress in growing hypocotyls. How CMTs first organise in germinating embryos is unknown, and their relation with mechanical stress has not been investigated at such an early developing stage. RESULTS: Here, we analysed CMT dynamics in dormant and non-dormant Arabidopsis seeds by microscopy of fluorescently tagged microtubule markers at different developmental time points and in response to abscisic acid and gibberellins. We found that CMTs first appear as very few thick bundles in dormant seeds. Consistently, analysis of available transcriptome and translatome datasets show that limiting amounts of tubulin and microtubule regulators initially hinder microtubule self-organisation. Seeds imbibed in the presence of gibberellic acid or abscisic acid displayed altered microtubule organisation and transcriptional regulation. Upon the release of dormancy, CMTs then self-organise into multiple parallel transverse arrays. Such behaviour matches the tensile stress patterns in such mechanically constrained embryos. This suggests that, as CMTs first self-organise, they also align with shape-derived tensile stress patterns. CONCLUSIONS: Our results provide a scenario in which dormancy release in the embryo triggers microtubule self-organisation and alignment with tensile stress prior to germination and anisotropic growth.

Phyllotactic regularity requires the Paf1 complex in Arabidopsis.

In plants, aerial organs are initiated at stereotyped intervals, both spatially (every 137° in a pattern called phyllotaxis) and temporally (at prescribed time intervals called plastochrons). To investigate the molecular basis of such regularity, mutants with altered architecture have been isolated. However, most of them only exhibit plastochron defects and/or produce a new, albeit equally reproducible, phyllotactic pattern. This leaves open the question of a molecular control of phyllotaxis regularity. Here, we show that phyllotaxis regularity depends on the function of VIP proteins, components of the RNA polymerase II-associated factor 1 complex (Paf1c). Divergence angles between successive organs along the stem exhibited increased variance in vip3-1 and vip3-2 compared with the wild type, in two different growth conditions. Similar results were obtained with the weak vip3-6 allele and in vip6, a mutant for another Paf1c subunit. Mathematical analysis confirmed that these defects could not be explained solely by plastochron defects. Instead, increased variance in phyllotaxis in vip3 was observed at the meristem and related to defects in spatial patterns of auxin activity. Thus, the regularity of spatial, auxin-dependent, patterning at the meristem requires Paf1c.

Clones of cells switch from reduction to enhancement of size variability in Arabidopsis sepals.

Organs form with remarkably consistent sizes and shapes during development, whereas a high variability in growth is observed at the cell level. Given this contrast, it is unclear how such consistency in organ scale can emerge from cellular behavior. Here, we examine an intermediate scale, the growth of clones of cells in Arabidopsis sepals. Each clone consists of the progeny of a single progenitor cell. At early stages, we find that clones derived from a small progenitor cell grow faster than those derived from a large progenitor cell. This results in a reduction in clone size variability, a phenomenon we refer to as size uniformization. By contrast, at later stages of clone growth, clones change their growth pattern to enhance size variability, when clones derived from larger progenitor cells grow faster than those derived from smaller progenitor cells. Finally, we find that, at early stages, fast growing clones exhibit greater cell growth heterogeneity. Thus, cellular variability in growth might contribute to a decrease in the variability of clones throughout the sepal.

ImageJ SurfCut: a user-friendly pipeline for high-throughput extraction of cell contours from 3D image stacks.

BACKGROUND: Many methods have been developed to quantify cell shape in 2D in tissues. For instance, the analysis of epithelial cells in Drosophila embryogenesis or jigsaw puzzle-shaped pavement cells in plant epidermis has led to the development of numerous quantification methods that are applied to 2D images. However, proper extraction of 2D cell contours from 3D confocal stacks for such analysis can be problematic. RESULTS: We developed a macro in ImageJ, SurfCut, with the goal to provide a user-friendly pipeline specifically designed to extract epidermal cell contour signals, segment cells in 2D and analyze cell shape. As a reference point, we compared our output to that obtained with MorphoGraphX (MGX). While both methods differ in the approach used to extract the layer of signal, they output comparable results for tissues with shallow curvature, such as pavement cell shape in cotyledon epidermis (as quantified with PaCeQuant). SurfCut was however not appropriate for cell or tissue samples with high curvature, as evidenced by a significant bias in shape and area quantification. CONCLUSION: We provide a new ImageJ pipeline, SurfCut, that allows the extraction of cell contours from 3D confocal stacks. SurfCut and MGX have complementary advantages: MGX is well suited for curvy samples and more complex analyses, up to computational cell-based modeling on real templates; SurfCut is well suited for rather flat samples, is simple to use, and has the advantage to be easily automated for batch analysis of images in ImageJ. The combination of these two methods thus provides an ideal suite of tools for cell contour extraction in most biological samples, whether 3D precision or high-throughput analysis is the main priority.

Does resource availability help determine the evolutionary route to multicellularity?

Genetic heterogeneity and homogeneity are associated with distinct sets of adaptive advantages and bottlenecks, both in developmental biology and population genetics. Whereas populations of individuals are usually genetically heterogeneous, most multicellular metazoans are genetically homogeneous. Observing that resource scarcity fuels genetic heterogeneity in populations, we propose that monoclonal development is compatible with the resource-rich and stable internal environments that complex multicellular bodies offer. In turn, polyclonal development persists in tumors and in certain metazoans, both exhibiting a closer dependence on external resources. This eco-evo-devo approach also suggests that multicellularity may originally have emerged through polyclonal development in early metazoans, because of their reduced shielding from environmental fluctuations.

Developing a 'thick skin': a paradoxical role for mechanical tension in maintaining epidermal integrity?

Plant aerial epidermal tissues, like animal epithelia, act as load-bearing layers and hence play pivotal roles in development. The presence of tension in the epidermis has morphogenetic implications for organ shapes but it also constantly threatens the integrity of this tissue. Here, we explore the multi-scale relationship between tension and cell adhesion in the plant epidermis, and we examine how tensile stress perception may act as a regulatory input to preserve epidermal tissue integrity and thus normal morphogenesis. From this, we identify parallels between plant epidermal and animal epithelial tissues and highlight a list of unexplored questions for future research.