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.

Debunking the idea of biological optimisation: quantitative biology to the rescue.

The idea that plants would be efficient, frugal or optimised echoes the recurrent semantics of 'blueprint' and 'program' in molecular genetics. However, when analysing plants with quantitative approaches and systems thinking, we instead find that plants are the results of stochastic processes with many inefficiencies, incoherence or delays fuelling their robustness. If one had to highlight the main value of quantitative biology, this could be it: plants are robust systems because they are not efficient. Such systemic insights extend to the way we conduct plant research and opens plant science publication to a much broader framework.

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.

A 3-component module maintains sepal flatness in Arabidopsis.

As in origami, morphogenesis in living systems heavily relies on tissue curving and folding, through the interplay between biochemical and biomechanical cues. In contrast, certain organs maintain their flat posture over several days. Here we identified a pathway, which is required for the maintenance of organ flatness, taking the sepal, the outermost floral organ, in Arabidopsis as a model system. Through genetic, cellular and mechanical approaches, our results demonstrate that global gene expression regulator VERNALIZATION INDEPENDENCE 4 (VIP4) fine-tunes the mechanical properties of sepal cell walls and maintains balanced growth on both sides of the sepals, mainly by orchestrating the distribution pattern of AUXIN RESPONSE FACTOR 3 (ARF3). vip4 mutation results in softer cell walls and faster cell growth on the adaxial sepal side, which eventually cause sepals to bend outward. Downstream of VIP4, ARF3 works through modulating auxin signaling to down-regulate pectin methylesterase VANGUARD1, resulting in decreased cell wall stiffness. Our work unravels a 3-component module, which relates hormonal patterns to organ curvature, and actively maintains sepal flatness during its growth.

The plasma membrane - cell wall nexus in plant cells: focus on the Hechtian structure.

Across all kingdoms of life, cells secrete an extracellular polymer mesh that in turn feeds back onto them. This entails physical connections between the plasma membrane and the polymer mesh. In plant cells, one connection stands out: the Hechtian strand which, during plasmolysis, reflects the existence of a physical link between the plasma membrane of the retracting protoplast and the cell wall. The Hechtian strand is part of a larger structure, which we call the Hechtian structure, that comprises the Hechtian strand, the Hechtian reticulum and the Hechtian attachment sites. Although it has been observed for more than 100 years, its molecular composition and biological functions remain ill-described. A comprehensive characterization of the Hechtian structure is a critical step towards understanding this plasma membrane-cell wall connection and its relevance in cell signaling. This short review intends to highlight the main features of the Hechtian structure, in order to provide a clear framework for future research in this under-explored and promising field.

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.

Why is FERONIA pleiotropic?

The plant cell wall has many roles: structure, hydraulics, signalling and immunity. Monitoring its status is therefore essential for plant life. Among many candidate cell wall sensors, FERONIA, a member of the Catharanthus roseus receptor-like kinase-1-like kinase (CrRLK1L) family, has received considerable attention, notably because of its numerous interactors and its implication in many biological pathways. Conversely, such an analytical dissection may blur its core function. Here we revisit the array of feronia phenotypes as an attempt to identify a unifying feature behind the plethora of biological and biochemical functions. We propose that the contribution of FERONIA in monitoring turgor-dependent cell wall tension may explain its pleiotropy.

The 1972 Meadows report: A wake-up call for plant science.

The 1972 Meadows report, 'the limits to growth', predicted a global socio-economic tipping point during the twenty-first century. Now supported by 50 years of empirical evidence, this work is a tribute to systems thinking and an invitation to take the current environmental crisis for what it is: neither a transition nor a bifurcation, but an inversion. For instance, we used matter (e.g., fossil fuel) to save time; we will use time to preserve matter (e.g., bioeconomy). We were exploiting ecosystems to fuel production; production will feed ecosystems. We centralised to optimise; we will decentralise to support resilience. In plant science, this new context calls for new research on plant complexity (e.g., multiscale robustness and benefits of variability), also extending to new scientific approaches (e.g., participatory research, art and science). Taking this turn reverses many paradigms and becomes a new responsibility for plant scientists as the world becomes increasingly turbulent.

Discretizing the cellular bases of plant morphogenesis: Emerging properties from subcellular and noisy patterning.

A fundamental question in biology is how multicellular organisms robustly shape their organs. In the past decade, much progress has been made not just in identifying biochemical and biophysical factors underpinning morphogenesis, but also in analyzing their spatio-temporal dynamics. A remarkable outcome of such analyses is that morphogenesis involves high levels of heterogeneity and fluctuations at local scales. Although this could be considered as white noise to be averaged over time, there is increasing evidence that these heterogeneities and fluctuations are instructive cues for development. In this review, we highlight some of the new questions that such heterogeneities raise for plant morphogenesis. We also investigate their effects across scales, focusing on how subcellular heterogeneities contribute to organ shape robustness and evolvability.

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.

Single-Cell Confinement Methods to Study Plant Cytoskeleton.

Progress in cytoskeletal research in animal systems has been accompanied by the development of single-cell systems (e.g., fibroblasts in culture). Single-cell systems exist for plant research, but the presence of a cell wall hinders the possibility to relate cytoskeleton dynamics to changes in cell shape or in mechanical stress pattern. Here we present two protocols to confine wall-less plant protoplasts in microwells with defined geometries. Either protocol might be more or less adapted to the question at hand. For instance, when using microwells made of agarose, the composition of the well can be easily modified to analyze the impact of biochemical cues. When using microwells in a stiff polymer (NOA73), protoplasts can be pressurized, and the wall of the well can be coated with cell wall components. Using both protocols, we could analyze microtubule and actin dynamics in vivo while also revealing the relative contribution of geometry and stress in their self-organization.

Endoreplication mediates cell size control via mechanochemical signaling from cell wall.

Endoreplication is an evolutionarily conserved mechanism for increasing nuclear DNA content (ploidy). Ploidy frequently scales with final cell and organ size, suggesting a key role for endoreplication in these processes. However, exceptions exist, and, consequently, the endoreplication-size nexus remains enigmatic. Here, we show that prolonged tissue folding at the apical hook in Arabidopsis requires endoreplication asymmetry under the control of an auxin gradient. We identify a molecular pathway linking endoreplication levels to cell size through cell wall remodeling and stiffness modulation. We find that endoreplication is not only permissive for growth: Endoreplication reduction enhances wall stiffening, actively reducing cell size. The cell wall integrity kinase THESEUS plays a key role in this feedback loop. Our data thus explain the nonlinearity between ploidy levels and size while also providing a molecular mechanism linking mechanochemical signaling with endoreplication-mediated dynamic control of cell growth.

Shoot meristem progenitors emerge from mechanical heterogeneities.

In this issue of Developmental Cell, Varapparambath et al. identify a new module that is instrumental for the selection of meristem progenitor cells in undifferentiated tissues. This module is triggered by mechanical feedback, and it integrates extracellular matrix modification, polar hormone transport, and transcription factors.

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.

Plant cell walls as mechanical signaling hubs for morphogenesis.

The instructive role of mechanical cues during morphogenesis is increasingly being recognized in all kingdoms. Patterns of mechanical stress depend on shape, growth and external factors. In plants, the cell wall integrates these three parameters to function as a hub for mechanical feedback. Plant cells are interconnected by cell walls that provide structural integrity and yet are flexible enough to act as both targets and transducers of mechanical cues. Such cues may act locally at the subcellular level or across entire tissues, requiring tight control of both cell-wall composition and cell-cell adhesion. Here we focus on how changes in cell-wall chemistry and mechanics act in communicating diverse cues to direct growth asymmetries required for plant morphogenesis. We explore the role of cellulose microfibrils, microtubule arrays and pectin methylesterification in the transduction of mechanical cues during morphogenesis. Plant hormones can affect the mechanochemical composition of the cell wall and, in turn, the cell wall can modulate hormone signaling pathways, as well as the tissue-level distribution of these hormones. This also leads us to revisit the position of biochemical growth factors, such as plant hormones, acting both upstream and downstream of mechanical signaling. Finally, while the structure of the cell wall is being elucidated with increasing precision, existing data clearly show that the integration of genetic, biochemical and theoretical studies will be essential for a better understanding of the role of the cell wall as a hub for the mechanical control of plant morphogenesis.

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.

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.

Organ geometry channels reproductive cell fate in the Arabidopsis ovule primordium.

In multicellular organisms, sexual reproduction requires the separation of the germline from the soma. In flowering plants, the female germline precursor differentiates as a single spore mother cell (SMC) as the ovule primordium forms. Here, we explored how organ growth contributes to SMC differentiation. We generated 92 annotated 3D images at cellular resolution in Arabidopsis. We identified the spatio-temporal pattern of cell division that acts in a domain-specific manner as the primordium forms. Tissue growth models uncovered plausible morphogenetic principles involving a spatially confined growth signal, differential mechanical properties, and cell growth anisotropy. Our analysis revealed that SMC characteristics first arise in more than one cell but SMC fate becomes progressively restricted to a single cell during organ growth. Altered primordium geometry coincided with a delay in the fate restriction process in katanin mutants. Altogether, our study suggests that tissue geometry channels reproductive cell fate in the Arabidopsis ovule primordium.

Inducible depletion of PI(4,5)P2 by the synthetic iDePP system in Arabidopsis.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a low-abundance membrane lipid essential for plasma membrane function1,2. In plants, mutations in phosphatidylinositol 4-phosphate (PI4P) 5-kinases (PIP5K) suggest that PI(4,5)P2 production is involved in development, immunity and reproduction3-5. However, phospholipid synthesis is highly intricate6. It is thus likely that steady-state depletion of PI(4,5)P2 triggers confounding indirect effects. Furthermore, inducible tools available in plants allow PI(4,5)P2 to increase7-9 but not decrease, and no PIP5K inhibitors are available. Here, we introduce iDePP (inducible depletion of PI(4,5)P2 in plants), a system for the inducible and tunable depletion of PI(4,5)P2 in plants in less than three hours. Using this strategy, we confirm that PI(4,5)P2 is critical for various aspects of plant development, including root growth, root-hair elongation and organ initiation. We show that PI(4,5)P2 is required to recruit various endocytic proteins, including AP2-µ, to the plasma membrane, and thus to regulate clathrin-mediated endocytosis. Finally, we find that inducible PI(4,5)P2 perturbation impacts the dynamics of the actin cytoskeleton as well as microtubule anisotropy. Together, we propose that iDePP is a simple and efficient genetic tool to test the importance of PI(4,5)P2 in given cellular or developmental responses, and also to evaluate the importance of this lipid in protein localization.

Fluctuations shape plants through proprioception.

Plants constantly experience fluctuating internal and external mechanical cues, ranging from nanoscale deformation of wall components, cell growth variability, nutating stems, and fluttering leaves to stem flexion under tree weight and wind drag. Developing plants use such fluctuations to monitor and channel their own shape and growth through a form of proprioception. Fluctuations in mechanical cues may also be actively enhanced, producing oscillating behaviors in tissues. For example, proprioception through leaf nastic movements may promote organ flattening. We propose that fluctuation-enhanced proprioception allows plant organs to sense their own shapes and behave like active materials with adaptable outputs to face variable environments, whether internal or external. Because certain shapes are more amenable to fluctuations, proprioception may also help plant shapes to reach self-organized criticality to support such adaptability.