High-throughput characterization of cortical microtubule arrays response to anisotropic tensile stress.

BACKGROUND: Plants can perceive and respond to mechanical signals. For instance, cortical microtubule (CMT) arrays usually reorganize following the predicted maximal tensile stress orientation at the cell and tissue level. While research in the last few years has started to uncover some of the mechanisms mediating these responses, much remains to be discovered, including in most cases the actual nature of the mechanosensors. Such discovery is hampered by the absence of adequate quantification tools that allow the accurate and sensitive detection of phenotypes, along with high throughput and automated handling of large datasets that can be generated with recent imaging devices. RESULTS: Here we describe an image processing workflow specifically designed to quantify CMT arrays response to tensile stress in time-lapse datasets following an ablation in the epidermis - a simple and robust method to change mechanical stress pattern. Our Fiji-based workflow puts together several plugins and algorithms under the form of user-friendly macros that automate the analysis process and remove user bias in the quantification. One of the key aspects is also the implementation of a simple geometry-based proxy to estimate stress patterns around the ablation site and compare it with the actual CMT arrays orientation. Testing our workflow on well-established reporter lines and mutants revealed subtle differences in the response over time, as well as the possibility to uncouple the anisotropic and orientational response. CONCLUSION: This new workflow opens the way to dissect with unprecedented detail the mechanisms controlling microtubule arrays re-organization, and potentially uncover the still largely elusive plant mechanosensors.

Characterising the mechanics of cell-cell adhesion in plants.

Cell-cell adhesion is a fundamental feature of multicellular organisms. To ensure multicellular integrity, adhesion needs to be tightly controlled and maintained. In plants, cell-cell adhesion remains poorly understood. Here, we argue that to be able to understand how cell-cell adhesion works in plants, we need to understand and quantitatively measure the mechanics behind it. We first introduce cell-cell adhesion in the context of multicellularity, briefly explain the notions of adhesion strength, work and energy and present the current knowledge concerning the mechanisms of cell-cell adhesion in plants. Because still relatively little is known in plants, we then turn to animals, but also algae, bacteria, yeast and fungi, and examine how adhesion works and how it can be quantitatively measured in these systems. From this, we explore how the mechanics of cell adhesion could be quantitatively characterised in plants, opening future perspectives for understanding plant multicellularity.

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.

Effects of Arabidopsis wall associated kinase mutations on ESMERALDA1 and elicitor induced ROS.

Angiosperm cell adhesion is dependent on interactions between pectin polysaccharides which make up a significant portion of the plant cell wall. Cell adhesion in Arabidopsis may also be regulated through a pectin-related signaling cascade mediated by a putative O-fucosyltransferase ESMERALDA1 (ESMD1), and the Epidermal Growth Factor (EGF) domains of the pectin binding Wall associated Kinases (WAKs) are a primary candidate substrate for ESMD1 activity. Genetic interactions between WAKs and ESMD1 were examined using a dominant hyperactive allele of WAK2, WAK2cTAP, and a mutant of the putative O-fucosyltransferase ESMD1. WAK2cTAP expression results in a dwarf phenotype and activation of the stress response and reactive oxygen species (ROS) production, while esmd1 is a suppressor of a pectin deficiency induced loss of adhesion. Here we find that esmd1 suppresses the WAK2cTAP dwarf and stress response phenotype, including ROS accumulation and gene expression. Additional analysis suggests that mutations of the potential WAK EGF O-fucosylation site also abate the WAK2cTAP phenotype, yet only evidence for an N-linked but not O-linked sugar addition can be found. Moreover, a WAK locus deletion allele has no effect on the ability of esmd1 to suppress an adhesion deficiency, indicating WAKs and their modification are not a required component of the potential ESMD1 signaling mechanism involved in the control of cell adhesion. The WAK locus deletion does however affect the induction of ROS but not the transcriptional response induced by the elicitors Flagellin, Chitin and oligogalacturonides (OGs).

External Mechanical Cues Reveal a Katanin-Independent Mechanism behind Auxin-Mediated Tissue Bending in Plants.

Tissue folding is a central building block of plant and animal morphogenesis. In dicotyledonous plants, hypocotyl folds to form hooks after seedling germination that protects their aerial stem cell niche during emergence from soil. Auxin response factors and auxin transport are reported 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 the presence or absence of external mechanical cues.

Microtubule Response to Tensile Stress Is Curbed by NEK6 to Buffer Growth Variation in the Arabidopsis Hypocotyl.

Growth variability generates mechanical conflicts in tissues. In plants, cortical microtubules usually align with maximal tensile stress direction, thereby mechanically reinforcing cell walls, and channeling growth rate and direction. How this is achieved remains largely unknown and likely involves microtubule regulators. The NIMA-related microtubule-associated kinase NEK6 phosphorylates tubulin, leading to the depolymerization of a subset of microtubules. We found that cortical microtubules exhibit a hyper-response to mechanical stress in the nek6 mutant. This response contributes to local cell protrusions in slow-growing regions of the nek6 mutant hypocotyl. When growth amplitude is higher, the hyper-alignment of microtubules leads to variable, stop-and-go, phenotypes, resulting in wavy hypocotyl shapes. After gravistimulation or touch, the nek6 mutant also exhibits a hyperbent hypocotyl phenotype, consistent with an enhanced perception of its own deformation. Strikingly, we find that NEK6 exhibits a novel form of polarity, being recruited at the ends of a subset of microtubules at cell edges. This pattern can be modified after local ablation, matching the new maximal tensile stress directions. We propose that NEK6 depolymerizes cortical microtubules that best align with maximal tensile stress to generate a noisier network of microtubules. This prevents an overreaction of microtubules to growth fluctuations and, instead, promotes the buffering of growth variations.

Polar expedition: mechanisms for protein polar localization.

Most cells show asymmetry in their shape or in the organization of their components that results in poles with different properties. This is a fundamental feature that participates in modulating the development of an organism and its responses to external stimuli. In plants, a number of proteins that are important for developmental and physiological processes have been shown to display polar localization. However, how these polarities are established, maintained, or dynamically modulated is still largely unclear for most of these proteins. In this review we report recent updates on the mechanisms of polar protein localization, focusing on a subset of these proteins that are the focus of current research efforts.

Feeling Stressed or Strained? A Biophysical Model for Cell Wall Mechanosensing in Plants.

Mechanical signals have recently emerged as a major cue in plant morphogenesis, notably influencing cytoskeleton organization, gene expression, protein polarity, or cell division. Although many putative mechanosensing proteins have been identified, it is unclear what mechanical cue they might sense and how this would occur. Here we briefly explain the notions of mechanical stress and strain. We present the challenges to understand their sensing by plants, focusing on the cell wall and the plasma membrane, and we review putative mechanosensing structures. We propose minimal biophysical models of mechanosensing, revealing the modes of mechanosensing according to mechanosensor lifetime, threshold force for mechanosensor dissociation, and type of association between the mechanosensor and the cell wall, as the sensor may be associated to a major load-bearing structure such as cellulose or to a minor load-bearing structure such as pectins or the plasma membrane. Permanent strain, permanent expansion, and relatively slow variations thereof are sensed in all cases; variations of stress are sensed in all cases; permanent stress is sensed only in the following specific cases: sensors associated to minor load-bearing structures slowly relaxing in a growing wall, long-lived sensors with high dissociation force and associated to major-load-bearing structures, and sensors with low dissociation force associated to major-load-baring structures behaving elastically. We also find that all sensors respond to variations in the composition or the mechanical properties of the cell wall. The level of sensing is modulated by the properties of all of mechanosensor, cell wall components, and plasma membrane. Although our models are minimal and not fully realistic, our results yield a framework to start investigating the possible functions of putative mechanosensors.

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.

Mechanical Conflicts in Twisting Growth Revealed by Cell-Cell Adhesion Defects.

Many plants grow organs and tissues with twisted shapes. Arabidopsis mutants with impaired microtubule dynamics exhibit such a phenotype constitutively. Although the activity of the corresponding microtubule regulators is better understood at the molecular level, how large-scale twisting can emerge in the mutants remains largely unknown. Classically, oblique cortical microtubules would constrain the deposition of cellulose microfibrils in cells, and such conflicts at the cell level would be relaxed at the tissue scale by supracellular torsion. This model implicitly assumes that cell-cell adhesion is a key step to transpose local mechanical conflicts into a macroscopic twisting phenotype. Here we tested this prediction using the quasimodo1 mutant, which displays cell-cell adhesion defects. Using the spriral2/tortifolia1 mutant with hypocotyl helical growth, we found that qua1-induced cell-cell adhesion defects restore straight growth in qua1-1 spr2-2. Detached cells in qua1-1 spr2-2 displayed helical growth, confirming that straight growth results from the lack of mechanical coupling between cells rather than a restoration of SPR2 activity in the qua1 mutant. Because adhesion defects in qua1 depend on tension in the outer wall, we also showed that hypocotyl twisting in qua1-1 spr2-2 could be restored when decreasing the matrix potential of the growth medium, i.e., by reducing the magnitude of the pulling force between adjacent cells, in the double mutant. Interestingly, the induction of straight growth in qua1-1 spr2-2 could be achieved beyond hypocotyls, as leaves also displayed a flat phenotype in the double mutant. Altogether, these results provide formal experimental support for a scenario in which twisted growth in spr2 mutant would result from the relaxation of local mechanical conflicts between adjacent cells via global organ torsion.

An Image Analysis Pipeline to Quantify Emerging Cracks in Materials or Adhesion Defects in Living Tissues.

Microcracks in materials reflect their mechanical properties. The quantification of the number or orientation of such cracks is thus essential in many fields, including engineering and geology. In biology, cracks in soft tissues can reflect adhesion defects, and the analysis of their pattern can help to deduce the magnitude and orientation of tensions in organs and tissues. Here, we describe a semi-automatic method amenable to analyze cell separations occurring in the epidermis of Arabidopsis thaliana seedlings. Our protocol is applicable to any image exhibiting small cracks, and thus also adapted to the analysis of emerging cracks in animal tissues and materials.

A tension-adhesion feedback loop in plant epidermis.

Mechanical forces have emerged as coordinating signals for most cell functions. Yet, because forces are invisible, mapping tensile stress patterns in tissues remains a major challenge in all kingdoms. Here we take advantage of the adhesion defects in the Arabidopsis mutant quasimodo1 (qua1) to deduce stress patterns in tissues. By reducing the water potential and epidermal tension in planta, we rescued the adhesion defects in qua1, formally associating gaping and tensile stress patterns in the mutant. Using suboptimal water potential conditions, we revealed the relative contributions of shape- and growth-derived stress in prescribing maximal tension directions in aerial tissues. Consistently, the tension patterns deduced from the gaping patterns in qua1 matched the pattern of cortical microtubules, which are thought to align with maximal tension, in wild-type organs. Conversely, loss of epidermis continuity in the qua1 mutant hampered supracellular microtubule alignments, revealing that coordination through tensile stress requires cell-cell adhesion.

The parts of a plant that protrude from the ground are constantly shaken by the wind, applying forces to the plant that it must be able to resist. Indeed, mechanical forces are crucial for the development, growth and life of all organisms and can trigger certain behaviours or the production of particular molecules: for example, forces that bend a plant trigger gene activity that ultimately makes the stem more rigid. Mechanical forces can also originate from inside the organism. For example, the epidermal cells that cover the surface of a plant are placed under tension by the cells in the underlying layers of the plant as they grow and expand. The exact pattern of forces in the plant epidermis was not known because they cannot be directly seen, although scientists have tried to map them using theoretical and computational modeling. A mutant form of the Arabidopsis plant is unable to produce some of the molecules that allow epidermal cells to adhere to each other. Verger et al. placed the mutants in different growth conditions that lowered the pressure inside the plant, and consequently reduced the tension on the epidermal cells. This partly restored the ability of epidermal cells to adhere to each other, although gaps remained between cells in regions of the plant that have been predicted to be under high levels of tension. Verger et al. could therefore use the patterns of the gaps to map the forces across the epidermis, opening the path for the study of the role of these forces in plant development. Further experiments showed that cell adhesion defects prevent the epidermal cells from coordinating how they respond to mechanical forces. There is therefore a feedback loop in the plant epidermis: cell-cell connections transmit tension across the epidermis, and, in turn, tension is perceived by the cells to alter the strength of those connections. The results presented by Verger et al. suggest that plants use tension to monitor the adhesion in the cell layer that forms an interface with the environment. Other organisms may use similar processes; this theory is supported by the fact that sheets of animal cells use proteins that are involved in both cell-cell adhesion and the detection of tension. The next challenge is to analyse how tension in the epidermis affects developmental processes and how a plant responds to its environment.

Plant Physiology: FERONIA Defends the Cell Walls against Corrosion.

A new study uncovers the role of wall sensing and remodeling in the plant response to salt stress, identifying the FERONIA receptor kinase as a key player in that process, likely through direct sensing of cell wall pectins.

Why plants make puzzle cells, and how their shape emerges.

The shape and function of plant cells are often highly interdependent. The puzzle-shaped cells that appear in the epidermis of many plants are a striking example of a complex cell shape, however their functional benefit has remained elusive. We propose that these intricate forms provide an effective strategy to reduce mechanical stress in the cell wall of the epidermis. When tissue-level growth is isotropic, we hypothesize that lobes emerge at the cellular level to prevent formation of large isodiametric cells that would bulge under the stress produced by turgor pressure. Data from various plant organs and species support the relationship between lobes and growth isotropy, which we test with mutants where growth direction is perturbed. Using simulation models we show that a mechanism actively regulating cellular stress plausibly reproduces the development of epidermal cell shape. Together, our results suggest that mechanical stress is a key driver of cell-shape morphogenesis.

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.

Cell adhesion in plants is under the control of putative O-fucosyltransferases.

Cell-to-cell adhesion in plants is mediated by the cell wall and the presence of a pectin-rich middle lamella. However, we know very little about how the plant actually controls and maintains cell adhesion during growth and development and how it deals with the dynamic cell wall remodeling that takes place. Here we investigate the molecular mechanisms that control cell adhesion in plants. We carried out a genetic suppressor screen and a genetic analysis of cell adhesion-defective Arabidopsis thaliana mutants. We identified a genetic suppressor of a cell adhesion defect affecting a putative O-fucosyltransferase. Furthermore, we show that the state of cell adhesion is not directly linked with pectin content in the cell wall but instead is associated with altered pectin-related signaling. Our results suggest that cell adhesion is under the control of a feedback signal from the state of the pectin in the cell wall. Such a mechanism could be necessary for the control and maintenance of cell adhesion during growth and development.

A galactosyltransferase acting on arabinogalactan protein glycans is essential for embryo development in Arabidopsis.

Arabinogalactan proteins (AGPs) are a complex family of cell-wall proteoglycans that are thought to play major roles in plant growth and development. Genetic approaches to studying AGP function have met limited success so far, presumably due to redundancy within the large gene families encoding AGP backbones. Here we used an alternative approach for genetic dissection of the role of AGPs in development by modifying their glycan side chains. We have identified an Arabidopsis glycosyltransferase of CAZY family GT31 (AtGALT31A) that galactosylates AGP side chains. A mutation in the AtGALT31A gene caused the arrest of embryo development at the globular stage. The presence of the transcript in the suspensor of globular-stage embryos is consistent with a role for AtGALT31A in progression of embryo development beyond the globular stage. The first observable defect in the mutant is perturbation of the formative asymmetric division of the hypophysis, indicating an essential role for AGP proteoglycans in either specification of the hypophysis or orientation of the asymmetric division plane.

[The necklace from the 660 grave in Megara Iblea]. / Sotto la protezione delle eliadi la collana della tomba 660 di Megara Iblea.

The article analyzes the 660 grave in Megara Iblea, a Greek colony in Sicily, in which a woman has been buried. On her breast a magnificent neckless was found, made of amulets recalling the travel of the sun during the summer solstice. Some objects allude to solar cults (a cock; round pendants), others seem to came from Gallia and Macedonia (summer far West and East), others recall archeological contexts such as tombs in Marvinci, in the Vardar Valley, and allude to relations with female practices of medicine and magic and to female roles characterized by extraordinary powers, due to being descendants of the Sun god. These solar symbols, joint with the discovery of many little objects, typical of children burials, allow to hypotize a relation with the cult of Mater Matuta and seem to point out a difficult or anomalous pregnancy or birth.