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.

The cell biology of primary cell walls during salt stress.

Salt stress simultaneously causes ionic toxicity, osmotic stress, and oxidative stress, which directly impact plant growth and development. Plants have developed numerous strategies to adapt to saline environments. Whereas some of these strategies have been investigated and exploited for crop improvement, much remains to be understood, including how salt stress is perceived by plants and how plants coordinate effective responses to the stress. It is, however, clear that the plant cell wall is the first contact point between external salt and the plant. In this context, significant advances in our understanding of halotropism, cell wall synthesis, and integrity surveillance, as well as salt-related cytoskeletal rearrangements, have been achieved. Indeed, molecular mechanisms underpinning some of these processes have recently been elucidated. In this review, we aim to provide insights into how plants respond and adapt to salt stress, with a special focus on primary cell wall biology in the model plant Arabidopsis thaliana.

Imaging the living plant cell: From probes to quantification.

At the center of cell biology is our ability to image the cell and its various components, either in isolation or within an organism. Given its importance, biological imaging has emerged as a field of its own, which is inherently highly interdisciplinary. Indeed, biologists rely on physicists and engineers to build new microscopes and imaging techniques, chemists to develop better imaging probes, and mathematicians and computer scientists for image analysis and quantification. Live imaging collectively involves all the techniques aimed at imaging live samples. It is a rapidly evolving field, with countless new techniques, probes, and dyes being continuously developed. Some of these new methods or reagents are readily amenable to image plant samples, while others are not and require specific modifications for the plant field. Here, we review some recent advances in live imaging of plant cells. In particular, we discuss the solutions that plant biologists use to live image membrane-bound organelles, cytoskeleton components, hormones, and the mechanical properties of cells or tissues. We not only consider the imaging techniques per se, but also how the construction of new fluorescent probes and analysis pipelines are driving the field of plant cell biology.

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.

How Mechanical Forces Shape Plant Organs.

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

The plasma membrane as a mechanotransducer in plants.

The plasma membrane is a physical boundary made of amphiphilic lipid molecules, proteins and carbohydrates extensions. Its role in mechanotransduction generates increasing attention in animal systems, where membrane tension is mainly induced by cortical actomyosin. In plant cells, cortical tension is of osmotic origin. Yet, because the plasma membrane in plant cells has comparable physical properties, findings from animal systems likely apply to plant cells too. Recent results suggest that this is indeed the case, with a role of membrane tension in vesicle trafficking, mechanosensitive channel opening or cytoskeleton organization in plant cells. Prospects for the plant science community are at least three fold: (i) to develop and use probes to monitor membrane tension in tissues, in parallel with other biochemical probes, with implications for protein activity and nanodomain clustering, (ii) to develop single cell approaches to decipher the mechanisms operating at the plant cell cortex at high spatio-temporal resolution, and (iii) to revisit the role of membrane composition at cell and tissue scale, by considering the physical implications of phospholipid properties and interactions in mechanotransduction.

La membrane plasmique est une barrière physique constituée de molécules lipidiques amphiphiles, de protéines et de prolongements glucidiques. Son rôle dans la mécanotransduction suscite une attention croissante dans les systèmes animaux, où la tension membranaire est principalement induite par l'actomyosine corticale. Dans les cellules végétales, la tension corticale est d'origine osmotique. Cependant, comme la membrane plasmique des cellules végétales a des propriétés physiques comparables, les résultats obtenus dans les systèmes animaux s'appliquent probablement aussi aux cellules végétales. Des résultats récents suggèrent que c'est effectivement le cas, avec un rôle de la tension membranaire dans le trafic des vésicules, l'ouverture des canaux mécanosensibles ou l'organisation du cytosquelette dans les cellules végétales. Les perspectives pour la communauté scientifique végétale sont au moins de trois ordres : (i) développer et utiliser des sondes pour évaluer la tension membranaire dans les tissus, en parallèle avec d'autres sondes biochimiques, avec des implications pour l'activité des protéines et le regroupement en nanodomaines, (ii) développer des approches unicellulaires pour déchiffrer les mécanismes opérant au niveau du cortex cellulaire des plantes à haute résolution spatio-temporelle, et (iii) réexaminer le rôle de la composition de la membrane à l'échelle de la cellule et du tissu, en considérant les implications physiques des propriétés et des interactions des phospholipides dans la mécanotransduction.

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.

Phospholipids across scales: lipid patterns and plant development.

Phospholipids are major building blocks of cell membranes and as such they have a key structural role in maintaining their integrity as a hydrophobic barrier. However, phospholipids not only have structural but also regulatory functions that are involved in a myriad of signaling pathways. Integrative approaches in plants recently revealed that certain phospholipids have distinct patterns of accumulation at the tissue or organ scales, which turned out to be important cues in a developmental context. Using examples on different phospholipid classes, including phosphatidylinositol-4,5-bisphosphate, phosphatidylserine, phosphatidylcholine, and phosphatidic acid, we review how spatio-temporal lipid patterns arise at the organismal level and what are their downstream consequences on plant development.