Plasmodesmata mediate cell-to-cell transport of brassinosteroid hormones.

Brassinosteroids (BRs) are steroidal phytohormones that are essential for plant growth, development and adaptation to environmental stresses. BRs act in a dose-dependent manner and do not travel over long distances; hence, BR homeostasis maintenance is critical for their function. Biosynthesis of bioactive BRs relies on the cell-to-cell movement of hormone precursors. However, the mechanism of the short-distance BR transport is unknown, and its contribution to the control of endogenous BR levels remains unexplored. Here we demonstrate that plasmodesmata (PD) mediate the passage of BRs between neighboring cells. Intracellular BR content, in turn, is capable of modulating PD permeability to optimize its own mobility, thereby manipulating BR biosynthesis and signaling. Our work uncovers a thus far unknown mode of steroid transport in eukaryotes and exposes an additional layer of BR homeostasis regulation in plants.

Geometry and cellular function of organelle membrane interfaces.

A vast majority of cellular processes take root at the surface of biological membranes. By providing a two-dimensional platform with limited diffusion, membranes are, by nature, perfect devices to concentrate signaling and metabolic components. As such, membranes often act as "key processors" of cellular information. Biological membranes are highly dynamic and deformable and can be shaped into curved, tubular, or flat conformations, resulting in differentiated biophysical properties. At membrane contact sites, membranes from adjacent organelles come together into a unique 3D configuration, forming functionally distinct microdomains, which facilitate spatially regulated functions, such as organelle communication. Here, we describe the diversity of geometries of contact site-forming membranes in different eukaryotic organisms and explore the emerging notion that their shape, 3D architecture, and remodeling jointly define their cellular activity. The review also provides selected examples highlighting changes in membrane contact site architecture acting as rapid and local responses to cellular perturbations, and summarizes our current understanding of how those structural changes confer functional specificity to those cellular territories.

Intercellular trafficking via plasmodesmata: molecular layers of complexity.

Plasmodesmata are intercellular pores connecting together most plant cells. These structures consist of a central constricted form of the endoplasmic reticulum, encircled by some cytoplasmic space, in turn delimited by the plasma membrane, itself ultimately surrounded by the cell wall. The presence and structure of plasmodesmata create multiple routes for intercellular trafficking of a large spectrum of molecules (encompassing RNAs, proteins, hormones and metabolites) and also enable local signalling events. Movement across plasmodesmata is finely controlled in order to balance processes requiring communication with those necessitating symplastic isolation. Here, we describe the identities and roles of the molecular components (specific sets of lipids, proteins and wall polysaccharides) that shape and define plasmodesmata structural and functional domains. We highlight the extensive and dynamic interactions that exist between the plasma/endoplasmic reticulum membranes, cytoplasm and cell wall domains, binding them together to effectively define plasmodesmata shapes and purposes.

Multiple C2 domains and transmembrane region proteins (MCTPs) tether membranes at plasmodesmata.

In eukaryotes, membrane contact sites (MCS) allow direct communication between organelles. Plants have evolved a unique type of MCS, inside intercellular pores, the plasmodesmata, where endoplasmic reticulum (ER)-plasma membrane (PM) contacts coincide with regulation of cell-to-cell signalling. The molecular mechanism and function of membrane tethering within plasmodesmata remain unknown. Here, we show that the multiple C2 domains and transmembrane region protein (MCTP) family, key regulators of cell-to-cell signalling in plants, act as ER-PM tethers specifically at plasmodesmata. We report that MCTPs are plasmodesmata proteins that insert into the ER via their transmembrane region while their C2 domains dock to the PM through interaction with anionic phospholipids. A Atmctp3/Atmctp4 loss of function mutant induces plant developmental defects, impaired plasmodesmata function and composition, while MCTP4 expression in a yeast Δtether mutant partially restores ER-PM tethering. Our data suggest that MCTPs are unique membrane tethers controlling both ER-PM contacts and cell-to-cell signalling.

Plasma Membrane-Associated Receptor-like Kinases Relocalize to Plasmodesmata in Response to Osmotic Stress.

Plasmodesmata act as key elements in intercellular communication, coordinating processes related to plant growth, development, and responses to environmental stresses. While many of the developmental, biotic, and abiotic signals are primarily perceived at the plasma membrane (PM) by receptor proteins, plasmodesmata also cluster receptor-like activities; whether these two pathways interact is currently unknown. Here, we show that specific PM-located Leu-rich-repeat receptor-like-kinases, Qian Shou kinase (QSK1) and inflorescence meristem kinase2, which under optimal growth conditions are absent from plasmodesmata, rapidly relocate and cluster to the pores in response to osmotic stress. This process is remarkably fast, is not a general feature of PM-associated proteins, and is independent of sterol and sphingolipid membrane composition. Focusing on QSK1, previously reported to be involved in stress responses, we show that relocalization in response to mannitol depends on QSK1 phosphorylation. Loss-of-function mutation in QSK1 results in delayed lateral root (LR) development, and the mutant is affected in the root response to mannitol stress. Callose-mediated plasmodesmata regulation is known to regulate LR development. We found that callose levels are reduced in the qsk1 mutant background with a root phenotype resembling ectopic expression of PdBG1, an enzyme that degrades callose at the pores. Both the LR and callose phenotypes can be complemented by expression of wild-type and phosphomimic QSK1 variants, but not by phosphodead QSK1 mutant, which fails to relocalize at plasmodesmata. Together, the data indicate that reorganization of receptor-like-kinases to plasmodesmata is important for the regulation of callose and LR development as part of the plant response to osmotic stress.

Specific membrane lipid composition is important for plasmodesmata function in Arabidopsis.

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of "native" PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the ß-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes.

Shaping intercellular channels of plasmodesmata: the structure-to-function missing link.

Plasmodesmata (PD) are a hallmark of the plant kingdom and a cornerstone of plant biology and physiology, forming the conduits for the cell-to-cell transfer of proteins, RNA and various metabolites, including hormones. They connect the cytosols and endomembranes of cells, which allows enhanced cell-to-cell communication and synchronization. Because of their unique position as intercellular gateways, they are at the frontline of plant defence and signalling and constitute the battleground for virus replication and spreading. The membranous organization of PD is remarkable, where a tightly furled strand of endoplasmic reticulum comes into close apposition with the plasma membrane, the two connected by spoke-like elements. The role of these structural features is, to date, still not completely understood. Recent data on PD seem to point in an unexpected direction, establishing a close parallel between PD and membrane contact sites and defining plasmodesmal membranes as microdomains. However, the implications of this new viewpoint are not fully understood. Aided by available phylogenetic data, this review attempts to reassess the function of the different elements comprising the PD and the relevance of membrane lipid composition and biophysics in defining specialized microdomains of PD, critical for their function.

Integration of transport-based models for phyllotaxis and midvein formation.

The plant hormone auxin mediates developmental patterning by a mechanism that is based on active transport. In the shoot apical meristem, auxin gradients are thought to be set up through a feedback loop between auxin and the activity and polar localization of its transporter, the PIN1 protein. Two distinct molecular mechanisms for the subcellular polarization of PIN1 have been proposed. For leaf positioning (phyllotaxis), an "up-the-gradient" PIN1 polarization mechanism has been proposed, whereas the formation of vascular strands is thought to proceed by "with-the-flux" PIN1 polarization. These patterning mechanisms intersect during the initiation of the midvein, which raises the question of how two different PIN1 polarization mechanisms may work together. Our detailed analysis of PIN1 polarization during midvein initiation suggests that both mechanisms for PIN1 polarization operate simultaneously. Computer simulations of the resulting dual polarization model are able to reproduce the dynamics of observed PIN1 localization. In addition, the appearance of high auxin concentration in our simulations throughout the initiation of the midvein is consistent with experimental observation and offers an explanation for a long-standing criticism of the canalization hypothesis; namely, how both high flux and high concentration can occur simultaneously in emerging veins.

Plasmodesmata: Channels Under Pressure.

Multicellularity has emerged multiple times in evolution, enabling groups of cells to share a living space and reducing the burden of solitary tasks. While unicellular organisms exhibit individuality and independence, cooperation among cells in multicellular organisms brings specialization and flexibility. However, multicellularity also necessitates intercellular dependence and relies on intercellular communication. In plants, this communication is facilitated by plasmodesmata: intercellular bridges that allow the direct (cytoplasm-to-cytoplasm) transfer of information between cells. Plasmodesmata transport essential molecules that regulate plant growth, development, and stress responses. They are embedded in the extracellular matrix but exhibit flexibility, adapting intercellular flux to meet the plant's needs. In this review, we delve into the formation and functionality of plasmodesmata and examine the capacity of the plant communication network to respond to developmental and environmental cues. We illustrate how environmental pressure shapes cellular interactions and aids the plant in adapting its growth. Expected final online publication date for the Annual Review of Plant Biology, Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Toward uncovering an operating system in plant organs.

Molecular motifs can explain information processing within single cells, while how assemblies of cells collectively achieve this remains less well understood. Plant fitness and survival depend upon robust and accurate decision-making in their decentralised multicellular organ systems. Mobile agents, including hormones, metabolites, and RNAs, have a central role in coordinating multicellular collective decision-making, yet mechanisms describing how cell-cell communication scales to organ-level transitions is poorly understood. Here, we explore how unified outputs may emerge in plant organs by distributed information processing across different scales and using different modalities. Mathematical and computational representations of these events are also explored toward understanding how these events take place and are leveraged to manipulate plant development in response to the environment.

Divide and connect: divorce by mutual consent, keeping in touch by desideratum.

Cell division is fundamental for living organisms, sustaining their growth and development. During cell division a single mother cell will duplicate its genome and organelles, and give rise to two independent entities that will eventually split apart in a tightly regulated process called abscission or the final-cut. In multicellular organisms, newly born daughter cells split apart while they simultaneously need to maintain contact for intercellular communication. In this mini-review, I discuss this fascinating paradox of how cells across kingdoms combine the need to divide with the need to connect.

La division cellulaire est fondamentale pour les organismes vivants, soutenant leur croissance et leur développement. Au cours de la division cellulaire, une seule cellule mère va dupliquer son génome et ses organites, et donner naissance à deux entités indépendantes qui vont finalement se séparer dans un processus étroitement régulé appelé abscission ou la coupe finale. Chez les organismes multicellulaires, les cellules filles nouvellement nées se séparent alors qu'elles doivent simultanément maintenir le contact pour établir une communication intercellulaire. Dans cette mini-revue, je discute de ce paradoxe fascinant qui montre comment les cellules de tous les règnes combinent le besoin de se diviser avec le besoin de se connecter.

Plasmodesmata Ultrastructure Determination Using Electron Tomography.

Plant plasmodesmata (PD) are complex intercellular channels consisting of a thin endoplasmic reticulum (ER) tubule enveloped by the plasma membrane (PM). PD were first observed by electron microscopy about 50 years ago and, since, numerous studies in transmission and scanning electron microscopy have provided important information regarding their overall organization, revealing at the same time their diversity in terms of structure and morphology. However, and despite the fact that PD cell-cell communication is of critical importance for plant growth, development, cellular patterning, and response to biotic and abiotic stresses, linking their structural organization to their functional state has been proven difficult. This is in part due to their small size (20-50 nm in diameter) and the difficulty to resolve these structures in three dimensions at nanometer resolution to provide details of their internal organization.In this protocol, we provide in detail a complete process to produce high-resolution transmission electron tomograms of PD. We describe the preparation of the plant sample using high-pressure cryofixation and cryo-substitution. We also describe how to prepare filmed grids and how to cut and collect the sections using an ultramicrotome. We explain how to acquire a tilt series and how to reconstruct a tomogram from it using the IMOD software. We also give a few guidelines on segmentation of the reconstructed tomogram.

Isolation of Plasmodesmata Membranes for Lipidomic and Proteomic Analysis.

Plasmodesmata (PD) are membranous intercellular nanochannels crossing the plant cell wall to connect adjacent cells in plants. Our understanding of PD function heavily relies on the identification of their molecular components, these being proteins or lipids. In that regard, proteomic and lipidomic analyses of purified PD represent a crucial strategy in the field. Here we describe a simple two-step purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells suitable for "omic" approaches. The first step of this procedure consists on isolating pure cell walls containing intact PD, followed by a second step which involves an enzymatic degradation of the wall matrix to release PD membranes. The PD-enriched fraction can then serve to identify the lipid and protein composition of PD using lipidomic and proteomic approaches, which we also describe in this method article.

Specific targeting of a plasmodesmal protein affecting cell-to-cell communication.

Plasmodesmata provide the cytoplasmic conduits for cell-to-cell communication throughout plant tissues and participate in a diverse set of non-cell-autonomous functions. Despite their central role in growth and development and defence, resolving their modus operandi remains a major challenge in plant biology. Features of protein sequences and/or structure that determine protein targeting to plasmodesmata were previously unknown. We identify here a novel family of plasmodesmata-located proteins (called PDLP1) whose members have the features of type I membrane receptor-like proteins. We focus our studies on the first identified type member (namely At5g43980, or PDLP1a) and show that, following its altered expression, it is effective in modulating cell-to-cell trafficking. PDLP1a is targeted to plasmodesmata via the secretory pathway in a Brefeldin A-sensitive and COPII-dependent manner, and resides at plasmodesmata with its C-terminus in the cytoplasmic domain and its N-terminus in the apoplast. Using a deletion analysis, we show that the single transmembrane domain (TMD) of PDLP1a contains all the information necessary for intracellular targeting of this type I membrane protein to plasmodesmata, such that the TMD can be used to target heterologous proteins to this location. These studies identify a new family of plasmodesmal proteins that affect cell-to-cell communication. They exhibit a mode of intracellular trafficking and targeting novel for plant biology and provide technological opportunities for targeting different proteins to plasmodesmata to aid in plasmodesmal characterisation.

Arabidopsis plant homeodomain finger proteins operate downstream of auxin accumulation in specifying the vasculature and primary root meristem.

In Arabidopsis thaliana, auxin is a key regulator of tissue patterning in the developing embryo. We have identified a group of proteins that act downstream of auxin accumulation in auxin-mediated root and vascular development in the embryo. Combined mutations in OBERON1 (OBE1) and OBERON2 (OBE2) give rise to obe1 obe2 double mutant seedlings that closely phenocopy the monopteros (mp) mutant phenotype, with an absence of roots and defective development of the vasculature. We show that, in contrast to the situation in mp mutants, obe1 obe2 double mutant embryos show auxin maxima at the root pole and in the provascular region, and that the SCF(TIR1) pathway, which translates auxin accumulation into transcriptional activation of auxin-responsive genes, remains intact. Although we focus on the impact of obe mutations on aspects of embryo development, the effect of such mutations on a broad range of auxin-related gene expression and the tissue expression patterns of OBE genes in seedlings suggest that OBE proteins have a wider role to play in growth and development. We suggest that OBE1 and OBE2 most likely control the transcription of genes required for auxin responses through the action of their PHD finger domains.

Quantification of Protein Enrichment at Plasmodesmata.

Intercellular communication plays a crucial role in the establishment of multicellular organisms by organizing and coordinating growth, development and defence responses. In plants, cell-to-cell communication takes place through nanometric membrane channels called plasmodesmata (PD). Understanding how PD dictate cellular connectivity greatly depends on a comprehensive knowledge of the molecular composition and the functional characterization of PD components. While proteomic and genetic approaches have been crucial to identify PD-associated proteins, in vivo fluorescence microscopy combined with fluorescent protein tagging is equally crucial to visualise the subcellular localisation of a protein of interest and gain knowledge about their dynamic behaviour. In this protocol we describe in detail a robust method for quantifying the degree of association of a given protein with PD, through ratiometric fluorescent intensity using confocal microscopy. Although developed for N. benthamiana and Arabidopsis, this protocol can be adapted to other plant species.

Dare to change, the dynamics behind plasmodesmata-mediated cell-to-cell communication.

Plasmodesmata pores control the entry and exit of molecules at cell-to-cell boundaries. Hundreds of pores perforate the plant cell wall, connecting cells together and establishing direct cytosolic and membrane continuity. This ability to connect cells in such a way is a hallmark of plant physiology and is thought to have allowed sessile multicellularity in Plantae kingdom. Indeed, plasmodesmata-mediated cell-to-cell signalling is fundamental to many plant-related processes. In fact, there are so many facets of plant biology under the control of plasmodesmata that it is hard to conceive how such tiny structures can do so much. While they provide 'open doors' between cells, they also need to guarantee cellular identities and territories by selectively transporting molecules. Although plasmodesmata operating mode remains difficult to grasp, little by little plant scientists are divulging their secrets. In this review, we highlight novel functions of cell-to-cell signalling and share recent insights into how plasmodesmata structural and molecular signatures confer functional specificity and plasticity to these unique cellular machines.

Auxin transport-feedback models of patterning in plants.

Many patterning events in plants are regulated by the phytohormone auxin. In fact, so many things are under the influence of auxin that it seems difficult to understand how a single hormone can do so much. Auxin moves throughout the plant via a network of specialized membrane-bound import and export proteins, which are often differentially expressed and polarized depending on tissue type. Here, we review simulation models of pattern formation that are based on the control of these transporters by auxin itself. In these transport-feedback models, diversity in patterning comes not from the addition of more morphogens, but rather by varying the mechanism that regulates the transporters.

Lipids or Proteins: Who Is Leading the Dance at Membrane Contact Sites?

Understanding the mode of action of membrane contact sites (MCSs) across eukaryotic organisms at the near-atomic level to infer function at the cellular and tissue levels is a challenge scientists are currently facing. These peculiar systems dedicated to inter-organellar communication are perfect examples of cellular processes where the interplay between lipids and proteins is critical. In this mini review, we underline the link between membrane lipid environment, the recruitment of proteins at specialized membrane domains and the function of MCSs. More precisely, we want to give insights on the crucial role of lipids in defining the specificity of plant endoplasmic reticulum (ER)-plasma membrane (PM) MCSs and we further propose approaches to study them at multiple scales. Our goal is not so much to go into detailed description of MCSs, as there are numerous focused reviews on the subject, but rather try to pinpoint the critical elements defining those structures and give an original point of view by considering the subject from a near-atomic angle with a focus on lipids. We review current knowledge as to how lipids can define MCS territories, play a role in the recruitment and function of the MCS-associated proteins and in turn, how the lipid environment can be modified by proteins.

Publisher Correction: Sphingolipid biosynthesis modulates plasmodesmal ultrastructure and phloem unloading.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.