Biology subjects

Hamant, O.

Publications and source records attributed to Hamant, O..

At least 19 recordsLinked to original sources

bioRxiv2026

Live confocal imagining of cellular touch responses upon local quantifiable mechanical stimulation in plant cells

Plant cells grow and differentiate in an ever-changing environment characterized by transient signals and stimuli. The ability of plant cells to perceive, integrate, and dynamically respond to these stimuli underpins a plant adaptation and survival. Among the plethora of complex stimuli plant cells are exposed to, several stimuli have a mechanical component, for example, wind, touch, contact with insects, penetration of pathogens, and even intrinsic mechanical stresses arising during tissue growth. Despite the presence of a load-bearing cell wall that separates cells from the environment and fixes their location within a tissue, plant cells are responsive to mechanical stimuli. However, several questions remain unanswered around how mechanical stimuli are perceived and translated into cellular responses. Here we establish a system enabling application of quantifiable localized mechanical stress while simultaneously capturing cellular responses with high spatio-temporal resolution using confocal imaging. We show that this system enables estimation of locally applied pressure and provides access to the temporal and spatial details of subcellular events in intact living tissues upon touch. We propose that observing these subcellular events at high spatiotemporal resolution and linking their dynamics to the intensity of mechanical stimuli may uncover the molecular mechanisms underlying plant cell responses to touch.

plant biology↗

bioRxiv2026

Interlayer Communication Integrates Genetic and Mechanical Signals for Robust Leaf Morphogenesis

Leaf development requires coordinated growth across distinct tissue layers, integrating genetic regulation with mechanical coupling to achieve proper organ shaping. However, the mechanisms coordinating differentiation and growth across layers to ensure coherent leaf morphogenesis remain poorly understood. Therefore, we investigated the role of TCP4 as a potential central regulator of interlayer communication coupling genetic and mechanical signals during Arabidopsis leaf development. Three-dimensional analyses revealed pronounced asymmetry in the expression of TCP4 and its negative regulator, MIR319C, across developing primordia, establishing spatially patterned differentiation cues. Tissue-specific perturbations demonstrated that TCP4 activity in either epidermis or subepidermis modulates cell proliferation and expansion across layers through both protein mobility and mobility-independent signalling mechanisms. Strong epidermal TCP4 induction arrested leaf development by prematurely suppressing proliferation and preventing symmetry breaking, thereby locking primordia in cylindrical states. Live imaging revealed fundamental reprogramming of growth anisotropy, with TCP4 driving cortical microtubule alignment, increasing cell wall stiffness, and establishing layer-specific pectin patterns. Together, these findings establish TCP4 as an integrator of genetic and mechanical signals across leaf tissue layers, coupling transcriptional programmes to cytoskeletal organisation, cell wall remodelling, and cross-layer mechanical feedback.

plant biology↗

bioRxiv2025

MAP65-1/PRC1 reinforces microtubules through nucleation

Microtubules are dynamic cytoskeletal filaments that help shape cells and guide their response to external cues, including mechanical stress. How cells reorganize and reinforce microtubule arrays under stress remains poorly understood. Here, we show that MAP65-1/PRC1 promotes microtubule nucleation on pre-existing lattices in vitro, particularly on microtubules with lattice defects and damage sites associated with mechanical stress. MAP65-1 preferentially binds bent microtubules both in vitro and in cells, likely because bending induces lattice damage, enhancing nucleation on and bundling of these microtubules in vitro. This creates a potential feedback loop where mechanical stress promotes MAP65-1 binding, which in turn stabilizes microtubules by nucleation and bundling and reinforces the alignment of the array with mechanical stress. Thus, we reveal a previously unknown role for MAP65/PRC1 proteins in lattice-based nucleation and suggest a mechanism by which cells record and respond to mechanical stress through microtubule reorganization.

cell biology↗

bioRxiv2025

A molecular glue: LecRKI.9 creates stiff plasma membrane cell wall connections

Water stress challenges plasma membrane - wall attachment. When plants are exposed to strong hyperosmotic stress, water exits the cell leading to plasmolysis, where the plasma membrane detaches partially from the cell wall and reveals discrete and persistent attachment sites in the form of Hechtian strands. Although these structures have been observed in several species for more than a century, the molecular bases behind plasma membrane - wall attachment remains elusive. Through a screen of candidate proteins, we reveal that the overexpression of two lectin receptor-like proteins (LecRK-I.9 and LecTM) increases the density of plasma membrane - wall connections in N. Benthamiana. The extracellular lectin domain of LecRK-I.9 was able to remain in the wall during plasmolysis. Conversely, deletion of the lectin domain in the LecRK-I.9 overexpressor restored the density of Hechtian strands to WT levels. In Arabidopsis, upon hyperosmotic stress, LecRK-I.9 formed largely immobile clusters, whereas lectin-deleted versions of LecRK-I.9 clusters were mobile. Cluster density correlated with predicted tensile stress levels and mechanical reinforcement in the wall before plasmolysis, consistent with a scenario in which attachment of the lectin domain also reflects the wall properties. Last, while overexpressing LecRK-I.9 conferred resistance to low water potential conditions, deletion of the lectin domain in the LecRK-I.9 overexpressor restored a WT response to water stress. Altogether, this demonstrates that the lectin domain of LecRK-I.9 creates persistent plasma membrane - wall attachment sites, with physiological relevance for plant resistance to water stress. SIGNIFICANCE STATEMENTAs observed since the mid-19th century, when placed in hyperosmotic conditions, plant cells undergo plasmolysis and form thin membraneous threads called Hechtian strands. These structures reflect the presence of persistent plasma membrane - cell wall attachment sites. Yet, the molecular components behind these sites are unknown. Through a candidate screen approach, we identify and demonstrate that the lectin domain of a receptor-like kinase provides such molecular glue. We also show that the clustering of the receptor is patterned and correlate with mechanically reinforced cell walls. Conversely, we find that the promotion of attachment increases resistance to water stress. This work revisits an old plant cell biology classic, and opens new avenues of research for plant adaptation to their environment.

plant biology↗

bioRxiv2025

Root hair growth phases are coordinated by cytoskeleton, nucleus dynamics and cell mechanics in Arabidopsis

Polar cell growth is a fundamental process across organisms, yet its coordination with nuclear movement and cytoskeleton dynamics remains underexplored. Focusing on Arabidopsis thaliana root hairs, we investigate these processes using high-resolution live imaging within microfluidics-based experiments. By incorporating data on cytoskeletal dynamics, nuclear positioning, and tip growth into a mathematical model, we analyse how their interactions shape the different growth phases that we reveal for the first time in this study. Chemical treatments and mutant analyses further support our model, revealing that timely cytoskeletal changes drive transitions between these growth phases, and correlate with shifts in nuclear movement and morphology. This regulation suggests a microtubule-actin crosstalk in the root hair subapical region. Additionally, we present novel findings on vacuole movement and cell stiffness, further refining our understanding of tip growth dynamics. Collectively, our work provides a comprehensive framework for understanding how transitions between growth phases are orchestrated in plant tip-growing cells.

cell biology↗

bioRxiv2024

Dynamic cortical behavior of plant protoplasts reveals unexpected similarities between plant and animal cells

In contrast to animal cells with their contractile cortical cytoskeleton, plant cells are encased in walls and are usually thought to behave like stiff, pressurized vessels. When digesting the cell wall, plant protoplasts form spherical shapes, further consolidating this model. Here we challenge this apparent opposition between animal and plant cells. We show that plant protoplasts can form protrusions. More precisely, using plasma membrane markers, we reveal that 27 {+/-} 1% of the protoplasts show dot-like protrusions and 16 {+/-} 7% of protoplasts show long (typically 1-20 m, up to 200 m) protrusions that we name "filopods". We demonstrate that this behavior is independent of the plant species, the membrane marker or the osmolyte. Protrusions can host large and long structures, such as microtubule bundles, which in turn can impact the mechanical behavior of the protrusions. We find that filopods can form de novo when increasing osmolarity, that they most likely move passively, and that they can attach to artificial surfaces. Altogether, these observations show that forming and retaining protrusions is not exclusive to animal cells. This calls for revisiting the dynamics and probing ability of the plant cell cortex in different osmotic and mechanical environments.

plant biology↗

bioRxiv2024

CUP-SHAPED COTYLEDON2 activates MIR319C transcription and promotes cell proliferation in Arabidopsis leaf primordia

The microRNA miR319 regulates leaf size in diverse plant species by reducing the level of the target transcripts that encode JAW-TCPs, the transcription factors (TF) that restrict leaf size by committing the proliferating pavement cells to differentiation. MIR319C, one of the three miR319-producing genes in Arabidopsis, is expressed throughout the incipient leaf primordia, and its expression domain gets restricted to the base at later stages, partly due to its transcriptional repression by JAW-TCPs. However, the factors that activate and maintain MIR319C expression in leaf primordia are yet unknown. Here, we identify the CUP-SHAPED COTYLEDON2 (CUC2) TF as a direct activator of MIR319C transcription. Using a yeast one-hybrid (Y1H) screen, we identified several NAC domain TFs as potential regulators of MIR319C. Subsequent ex vivo binding and transactivation assays suggested that CUC2 binds to a distal promoter region of the MIR319C locus and activates its transcription. Mutants with compromised CUC2 and MIR319C activities resulted in smaller leaves with fewer cells. Detailed morphometric analysis of higher order CUC2 and MIR319 loss-of-function mutants highlighted the crucial role of the CUC2-MIR319 module in maintaining the duration of cell proliferation in leaf primordia. Additionally, the phenotype of mutants with altered CUC2 and MIR319/JAW-TCP activities demonstrated that CUC2 enhances leaf size through the MIR319C-JAW-TCP pathway. Overall, our findings uncovered a novel role for CUC2 in sustaining cell division by activating MIR319C transcription in the leaf primordia.

plant biology↗

bioRxiv2024

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 with very different sizes. Previous reports had 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. Main conclusionA mixed population of cells with varied sizes plays a limited role in ensuring the symmetrical shape of the sepal, and is not essential for sepal shape robustness in Arabidopsis.

plant biology↗

bioRxiv2023

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.

plant biology↗

bioRxiv2023

Puzzle cell shape emerges from the interaction of growth with mechanical constraints

Puzzle-shaped epidermal cells not only reduce mechanical stress during organ growth but also record the expansion history of the tissue in their outlines. By combining mechanical simulations with time-lapse imaging, we show that transitions from directional to isotropic expansion induce new lobes to form along the previous growth axis, and that switching the order of anisotropic and isotropic phases yields hybrid shapes that reliably preserve those transitions. In maize, model predictions and live imaging coincide precisely, and in Ara-bidopsis, final lobe patterns correlate more with growth history than with cell size alone. Ge-netic or pharmacological disruption of lobe formation constrains leaf expansion or drives compensatory elongation, which underscores a mechanical function. A broad survey of living and fossil vascular plants reveals that the mechanism to make puzzle-shaped cells is both widespread and developmentally plastic, suggesting that single snapshots of leaves can give insight into their growth history. Together, these findings demonstrate that puzzle cells trans-form cell geometry into a living record of how tissues grow.

plant biology↗

bioRxiv2023

Water fluxes contribute to growth patterning in shoot meristems

In multicellular organisms, localized tissue outgrowth creates a new water sink thereby modifying hydraulic patterns at the organ level. These fluxes are often considered passive by-products of development and their patterning and potential contribution to morphogenesis remains largely unexplored. Here, we generated a complete map of cell volumetric growth and deformation across the shoot apex in Arabidopsis thaliana. Within the organ-meristem boundary, we found that a subpopulation of cells next to fast-growing cells experiences volumetric shrinkage. To understand this process, we used a vertex-based model integrating mechanics and hydraulics, informed by the measured growth rates. Organ outgrowth simulations revealed the emerging water fluxes and predicted water deficit with volume loss for a few cells at the boundary. Consistently, in planta, a water-soluble dye is preferentially allocated to fast-growing tissues and fails to enter the boundary domain. Analysis of intact meristems further validated our model by revealing cell shrinkage next to fast-growing cells in different contexts of tissue surface curvature and cell deformation. A molecular signature of water deficit at the boundary further confirmed our conclusion. Taken together, we propose a model where the differential sink strength of emerging organs prescribes the hydraulic patterns that define the boundary domain at the shoot apex.

plant biology↗

bioRxiv2022

Paf1C denoises transcription and growth patterns to achieve organ shape reproducibility

In multicellular systems, all cells exhibit transcriptional noise. However, its exact contribution to morphogenesis often remains unclear, especially in animals where cells can also move. Here we take advantage of walled plant cells, where transcriptional noise happens in tissues with a fixed topology. Using synchronously growing guard cells in stomata, we demonstrate that mutation in VIP3, a subunit of the conserved polymerase-associated factor 1 complex (Paf1C), increases transcriptional noise in Arabidopsis. This conclusion could be generalized to other group of cells at the shoot apex. Such noise translates into growth and shape defects. Indeed, in vip3 sepals, we measured higher growth heterogeneity between adjacent cells, with molecular evidence of increased local mechanical conflicts. This even culminated with the presence of negatively growing cells in specific growth conditions. Interestingly, such increased local noise makes the regional pattern of growth less clear-cut. Reduced regional conflicts lead to delay in organ growth arrest, ultimately making final organ shapes and sizes more variable. We propose that transcriptional noise is managed by Paf1C to ensure organ robustness by building up mechanical conflicts at the regional scale, instead of the local scale. HIGHLIGHTSO_LIPaf1C controls transcriptional noise in Arabidopsis C_LIO_LIPaf1C reduces growth heterogeneity and dampens local mechanical conflicts C_LIO_LIImpairing both Paf1C and microtubules can trigger negative growth in Arabidopsis C_LIO_LIScaling up local conflicts to regional ones with sharp boundaries contributes to organ shape robustness C_LI

developmental biology↗

bioRxiv2021

Turning plants from passive to active material: FERONIA and microtubules independently contribute to mechanical feedback

To survive, cells must constantly resist mechanical stress. In plants, this involves the reinforcement of cell walls, notably through microtubule-dependent cellulose deposition, and thus wall sensing. Several receptor-like kinases have been proposed to act as mechanosensors. Here we tested whether the microtubule response to stress acts downstream of known wall sensors. Using a multi-step screen with eleven mutant lines, we identify FERONIA as the primary candidate for controlling the microtubule response to stress. However, when performing mechanical perturbations, we show that the microtubule response to stress can be independent from 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 swell and burst like soap bubbles. 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 key components to turn plant cells from passive to active material.

plant biology↗

bioRxiv2020

Developmental constraints modulate reproductive fate and plasticity within the Arabidopsis ovule primordium

In multicellular organisms, sexual reproduction requires the separation of the germline from the soma. In flowering plants, the first cells of the germline, so-called spore mother cells (SMCs), differentiate as the reproductive organs form. Here, we explored how organ growth influences and contributes to SMC differentiation. We generated a collection of 92 annotated 3D images capturing ovule primordium ontogeny at cellular resolution in Arabidopsis. We identified a spatio-temporal pattern of cell divisions that acts in a domain-specific manner as the primordium forms, which is coupled with the emergence of a single SMC. Using tissue growth models, we uncovered plausible morphogenetic principles involving a spatially confined growth signal, differential mechanical properties, and cell growth anisotropy. Our analysis also reveals 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 this fate restriction process in katanin mutants. Altogether, our study suggests that tissue geometry canalizes and modulates reproductive cell fate in the Arabidopsis ovule primordium.

plant biology↗

bioRxiv2020

iDePP: a genetically encoded system for the inducible depletion of PI(4,5)P2 in Arabidopsis thaliana.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a low abundant lipid present at the plasma membrane of eukaryotic cells. Extensive studies in animal cells revealed the pleiotropic functions of PI(4,5)P2. In plant cells, PI(4,5)P2 is involved in various cellular processes including the regulation of cell polarity and tip growth, clathrin-mediated endocytosis, polar auxin transport, actin dynamics or membrane-contact sites. To date, most studies investigating the role of PI(4,5)P2 in plants have relied on mutants lacking enzymes responsible for PI(4,5)P2 synthesis and degradation. However, such genetic perturbations only allow steady-state analysis of plants undergoing their life cycle in PI(4,5)P2 deficient conditions and the corresponding mutants are likely to induce a range of non-causal (untargeted) effects driven by compensatory mechanisms. In addition, there are no small molecule inhibitors that are available in plants to specifically block the production of this lipid. Thus, there is currently no system to fine tune PI(4,5)P2 content in plant cells. Here we report a genetically encoded and inducible synthetic system, iDePP (Inducible Depletion of PI(4,5)P2 in Plants), that efficiently removes PI(4,5)P2 from the plasma membrane in different organs of Arabidopsis thaliana, including root meristem, root hair and shoot apical meristem. We show that iDePP allows the inducible depletion of PI(4,5)P2 in less than three hours. Using this strategy, we reveal that PI(4,5)P2 is critical for cortical microtubule organization. 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 but also to evaluate the importance of this lipid in protein localization. Research OrganismA. thaliana

plant biology↗

bioRxiv2020

Timing of organ initiation is crucial for robust organ size

AbstractOrgans precisely regulate their size and shape to ensure proper function1-6. The contribution of organ initiation timing to final organ size and shape is often masked by compensatory adjustments to growth later in development7-9. Here we show that DEVELOPMENT RELATED MYB-LIKE1 (DRMY1) is required for both proper organ initiation timing and growth leading to robust sepal size in Arabidopsis. Within each drmy1 flower, the initiation of some sepals is variably delayed. Late-initiating sepals in drmy1 mutants remain smaller throughout development resulting in variability in sepal size. DRMY1 focuses the spatiotemporal signaling patterns of the plant hormones auxin and cytokinin, which jointly control the timing of sepal initiation. Contrary to expectation, our findings demonstrate that timing of organ initiation contributes to robust organ size throughout development.

plant biology↗

bioRxiv2020

External mechanical cues reveal core molecular pathway behind tissue bending in plants

Tissue folding is a central building block of plant and animal morphogenesis. In dicotyledonous plants, hypocotyl folds to form hook after seedling germination that protects their aerial stem cell niche during emergence from soil. Auxin response factors and auxin transport are classically thought to play a key role in this process. Here we show that the microtubule-severing enzyme katanin contributes to hook formation. However, by exposing hypocotyls to external mechanical cues mimicking the natural soil environment, we reveal that auxin response factors ARF7/ARF19, auxin influx carriers, and katanin are dispensable for apical hook formation, indicating that these factors primarily play the role of catalyzers of tissue bending in the absence of external mechanical cues. Instead, our results reveal the key roles of the non-canonical TMK-mediated auxin pathway, PIN efflux carriers and cellulose microfibrils as components of the core pathway behind hook formation in presence or absence of external mechanical cues.

plant biology↗

bioRxiv2017

The self-organization of plant microtubules in three dimensions enables stable cortical localization and sensitivity to external cues

Many cell functions rely on the ability of microtubules to self-organize as complex networks. In plants, cortical microtubules are essential to determine cell shape as they guide the deposition of cellulose microfibrils, and thus control mechanical anisotropy in the cell wall. Here we analyze how, in turn, cell shape may influence microtubule behavior. Using a computational model of microtubules enclosed in a three-dimensional space, We show that the microtubule network has spontaneous configurations that could explain many experimental observations without resorting to specific regulation. In particular, we find that the preferred localization of microtubules at the cortex emerges from directional persistence of the microtubules, combined with their growth mode. We identified microtubule parameters that seem relatively insensitive to cell shape, such as length or number. In contrast, microtubule array anisotropy depends strongly on local curvature of the cell surface and global orientation follows robustly the longest axis of the cell. Lastly, we found that the network is capable of reorienting toward weak external directional cues. Altogether our simulations show that the microtubule network is a good transducer of weak external polarity, while at the same time, it easily reaches stable global configurations.\n\nAuthor summaryPlants exhibit an astonishing diversity in architecture and shape. A key to such diversity is the ability of their cells to coordinate and grow to reach a broad spectrum of sizes and shapes. Cell growth in plants is guided by the microtubule cytoskeleton. Here, we seek to understand how microtubules self-organize close to the cell surface. We build upon previous two-dimensional models and we consider microtubules as lines growing in three dimensions, accounting for interactions between microtubules or between microtubules and the cell surface. We show that microtubule arrays are able to adapt to various cell shapes and to reorient in response to factors such as signals or environment. Altogether, our results help to understand how the microtubule cytoskeleton contributes to the diversity of plant shapes and to how these shapes adapt to environment.

cell biology↗