Summary Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] is a low abundant membrane lipids essential for plasma membrane function. In plants, mutations in PI4P 5-kinases (PIP5K) suggest that PI(4,5)P 2 production is involved in development, immunity and reproduction. However, phospholipid synthesis is highly intricate. It is thus likely that steady-state depletion of PI(4,5)P 2 triggers confounding indirect effects. Furthermore, inducible tools available in plants, allow to increase but not decrease PI(4,5)P2, and no PIP5K inhibitors are available. Here, we introduce iDePP ( I nducible Dep letion of PI (4,5)P 2 in P lants), a system for the inducible and tunable depletion of PI(4,5)P 2 in plants in less than three hours. Using this strategy, we confirm that PI(4,5)P 2 is critical for various aspects of plant development, including root growth, root hair elongation and organ initiation. We show that PI(4,5)P 2 is required to recruit various endocytic proteins, including AP2, to the plasma membrane, and thus to regulate clathrin-mediated endocytosis. Finally, we uncover that inducible PI(4,5)P 2 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)P 2 in given cellular or developmental responses, but also to evaluate the importance of this lipid in protein localization.

Membrane lipids, and especially phosphoinositides, are differentially enriched within the eukaryotic endomembrane system. This generates a landmark code by modulating the properties of each membrane. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] specifically accumulates at the plasma membrane in yeast, animal, and plant cells, where it regulates a wide range of cellular processes including endocytic trafficking. However, the functional consequences of mispatterning PI(4,5)P2 in plants are unknown. Here, we functionally characterized the putative phosphoinositide phosphatase SUPPRESSOR OF ACTIN9 (SAC9) in Arabidopsis thaliana (Arabidopsis). We found that SAC9 depletion led to the ectopic localization of PI(4,5)P2 on cortical intracellular compartments, which depends on PI4P and PI(4,5)P2 production at the plasma membrane. SAC9 localizes to a subpopulation of trans-Golgi Network/early endosomes that are enriched in a region close to the cell cortex and that are coated with clathrin. Furthermore, it interacts and colocalizes with Src Homology 3 Domain Protein 2 (SH3P2), a protein involved in endocytic trafficking. In the absence of SAC9, SH3P2 localization is altered and the clathrin-mediated endocytosis rate is reduced. Together, our results highlight the importance of restricting PI(4,5)P2 at the plasma membrane and illustrate that one of the consequences of PI(4,5)P2 misspatterning in plants is to impact the endocytic trafficking.

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 Organism: A. thaliana surface charge (Hammond et al., 2012), a basic property of the plasma membrane, which recruits many 36 proteins through electrostatic interaction (Platre and Jaillais, 2017). 37 38In plant cells, less is known about PI(4,5)P2. Intriguingly, it seems that some functions of PI(4,5)P2 39 in animals are not conserved in plants. For example, plasma membrane surface charges relies on PI4P, 40 phosphatidylserine (PS) and phosphatidic acid (PA), but not PI(4,5)P2 (Simon et al., 2016a; Platre and 41 Jaillais, 2017). Yet, mutants in PI4P 5-kinases (PIP5K) suggest that PI(4,5)P2 production is essential 42 and has critical roles in development, immunity and reproduction (Heilmann, 2016; Noack and Jaillais, 43 2017a). However, i) the synthesis of each phosphoinositide species is tied to each other and to other 44 lipids, ii) PI(4,5)P2 has pleiotropic cellular functions and iii) PI(4,5)P2 interacts with many different 45 proteins. Thus, it is likely that steady-state depletion of PI(4,5)P2 triggers a range of indirect effects 46 involving several types cellular and developmental compensatory mechanisms. It is therefore essential 47

Cell division is a tightly regulated mechanism, notably in tissues where malfunctions can lead to tumour formation or developmental defects. This is particularly true in land plants, where cells cannot relocate and therefore cytokinesis determines tissue topology. In plants, cell division is executed in radically different manners than in animals, with the appearance of new structures and the disappearance of ancestral mechanisms. Whilst F-actin and microtubules closely co-exist, recent studies mainly focused on the involvement of microtubules in this key process. Here, we used a root tracking system to image the spatio-temporal dynamics of both F-actin reporters and cell division markers in dividing cells embedded in their tissues. In addition to the F-actin accumulation at the phragmoplast, we observed and quantified a dynamic apico-basal enrichment of F-actin from the prophase/metaphase transition until the end of the cytokinesis.

Membranes lipids, and especially phosphoinositides, are differentially enriched within the eukaryotic endomembrane system. This generates a landmark code by modulating the properties of each membrane. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] specifically accumulates at the plasma membrane in yeast, animal and plant cells, where it regulates a wide range of cellular processes including endocytosis. However, the functional consequences of mispatterning PI(4,5)P2 in plants are unknown. Here, we functionally characterized the phosphoinositide phosphatase SUPPRESSOR OF ACTIN9 (SAC9) in Arabidopsis thaliana (Arabidopsis). We found that SAC9 depletion led to the ectopic localization of PI(4,5)P2 on cortical intracellular compartments, which depends on PI4P and PI(4,5)P2 production at the plasma membrane. SAC9 localizes to a subpopulation of trans-Golgi Network/early endosomes that are spatially restricted to a region close to the cell cortex and that are coated with clathrin. Furthermore, it interacts and colocalizes with the endocytic component Src Homology 3 Domain Protein 2 (SH3P2). In the absence of SAC9, SH3P2 localization is altered and the clathrin mediated endocytosis rate is significantly reduced. Thus, SAC9 is required to maintain efficient endocytic uptake, highlighting the importance of restricting the PI(4,5)P2 pool at the plasma membrane for the proper regulation of endocytosis in plants.

Plant cytokinesis, which fundamentally differs from that in animals, requires the outward expansion of a plasma membrane precursor named the cell plate. How the transition from a cell plate to a plasma membrane occurs remains poorly understood. Here, we report that the acquisition of plasma membrane identity occurs through lateral patterning of the phosphatidylinositol 4,5-bisphosphate PI(4,5)P 2 at the newly formed cell plate membrane. There, the phosphoinositide phosphatase SAC9 emerges as a key regulator, colocalizing with and regulating the function of the microtubule-associated protein MAP65-3 at the cell plate leading zone. In sac9-3 mutant, the polar distribution of PI(4,5)P 2 at the cell plate is altered, leading to ectopic recruitment of the cytokinesis apparatus and formation of an additional cell plate insertion site. We propose that at the cell plate, SAC9 drives the depletion of PI(4,5)P 2 , which acts as a polar cue to spatially separate cell plate expansion from the acquisition of plasma membrane identity during final step of cytokinesis.

Plant cytokinesis, which fundamentally differs from that in animals, involves de novo assembly of a plasma membrane precursor named the cell plate. How the transition from the cell plate to a plasma membrane occurs at the end of the plant cytokinesis remains poorly understood. Here, we describe with unprecedented spatiotemporal precision, the acquisition of plasma membrane identity upon cytokinesis through the lateral patterning of phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 at the newly formed cell plate membrane. We show that during late cytokinesis, opposing polarity domains are formed along the cell plate. The exclusion of PI(4,5)P2 from the leading edge of the cell plate is controlled by SAC9, a putative phosphoinositide phosphatase. SAC9 colocalizes with MAP65-3, a key regulator of cytokinesis, at the cell plate leading zone and regulates its function. In the sac9-3 mutant, the polar distribution of PI(4,5)P2 at the cell plate is altered, leading to de-novo recruitment of the cytokinesis apparatus and to the formation of an additional, ectopic cell plate insertion site. We proposed that PI(4,5)P2 acts as a polar cue to spatially separate the expansion and maturation domains of the forming cell plate during the final steps of cytokinesis.

During the life cycle of any multicellular organism, cell division contributes to the proliferation of the cell in the tissues as well as the generation of specialized cells, both necessary to form a functional organism. Therefore, the mechanisms of cell division need to be tightly regulated, as malfunctions in their control can lead to tumor formation or developmental defects. This is particularly true in land plants, where cells cannot relocate and therefore cytokinesis is key for morphogenesis. In the green lineage, cell division is executed in radically different manners than animals, with the appearance of new structures (the preprophase band (PPB), cytokinetic the cell plate and phragmoplast), and the disappearance of ancestral mechanisms (cleavage, centrosomes). While F-actin and microtubules closely co-exist to allow the orientation and the progression of the plant cell division, recent studies mainly focused on the involvement of microtubules in this key process. Here, we used our recently developed root tracking system to follow actin dynamics in dividing Arabidopsis meristematic root cells. In this study, we imaged in time and space the fluorescent-tagged F-actin reporter Lifeact together with cell division markers in dividing cells embedded in their tissues. In addition to the F-actin accumulation in the phragmoplasts, we observed and quantified a dynamic apical-basal enrichment of the F-actin during cytokinesis. The role and the possible actors responsible for F-actin dynamics during cytokinesis are discussed.