Intercellular lipid flow coordinates tissue-scale lipid gradients in plants

Intercellular lipid flow coordinates tissue-scale lipid gradients in plants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Intercellular lipid flow coordinates tissue-scale lipid gradients in plants Yvon Jaillais, Chloe Beziat, Vincent Bayle, Frederique Rozier, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8163315/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The lipid composition of biological membranes dictates cellular states and functions, yet how cells coordinate their lipid profiles across tissues remains unclear. In plant roots, the lipid phosphatidylserine forms a tissue-scale localization gradient that regulates Rho GTPase signaling in response to auxin and osmotic stress1–6. How this gradient emerges is unknown. Here, we show that direct phospholipid exchange between cells coordinates these lipid patterns. Combining in vivo phospholipid phototracing and localized induction of phosphatidylserine synthesis, we found that lipids move between cells through the numerous plasmodesmata-membrane bridges connecting them. The phosphatidylserine gradient arises from local synthesis by PHOSPHATIDYLSERINE SYNTHASE1 within a restricted epidermal zone, combined with intercellular phosphatidylserine diffusion. Producing and receiving cells exhibit distinct subcellular phosphatidylserine localization patterns, together generating the tissue-scale lipid gradient. These results reveal that direct cell-cell membrane connections act as conduits for lipid flow, coordinating membrane composition and signaling capacity across tissues. Biological sciences/Plant sciences/Plant cell biology Biological sciences/Plant sciences/Plant cell biology/Protein trafficking in plants Biological sciences/Cell biology/Organelles/Endoplasmic reticulum Figures Figure 1 Figure 2 Figure 3 Figure 4 Main text Membrane lipids are organized as gradients within cells 6–10 . These gradients arise as lipids are transported from their sites of synthesis to different membrane compartments via vesicular and non-vesicular pathways. The resulting non-uniform lipid distribution among membrane compartments governs protein localization and, ultimately, cellular functions 9,11,12 . A classic example of cellular lipid gradient is the anionic phospholipid phosphatidylserine (PS), which is present as a gradient in post-Golgi membranes 8 . Genetically-encoded biosensors for PS preferentially localize at the plasma membrane in macrophage cells, then to a lesser degree to early endosomes and then to late endosomes and lysosomes 13 . This intracellular PS gradient is largely conserved in plants 1 , and remarkably extends beyond single-cell level, to establish a differential localization pattern across the root epidermis 2,6 . Specifically, PS localizes to both the plasma membrane and endosomes in meristematic cells 1 , but becomes increasingly restricted to endosomes in the transition and elongation zone 1,2,5 . This spatial redistribution links intracellular lipid gradients to tissue-level organization 5,6 . The tissue-scale PS gradient is functionally important for Rho GTPase signaling 14 . Upon activation by the plant hormone auxin or osmotic stress, RHO-RELATED PROTEIN OF PLANTS6 (ROP6) clusters into nanodomains at the plasma membrane, a step required for signaling 2,3,15,16 . ROP6 signaling output scales with the numbers of ROP6 nanoclusters, which depends on the amount of PS at the plasma membrane 2–4,14 . As such, at the same concentration of auxin, ROP6 nanoclustering is high in the meristematic zone of the root, where PS accumulates at the plasma membrane, and reduced in the elongation zone, where PS is prevalently localized in intracellular compartments 2 . The downstream ROP6 outputs, such as the regulation of intracellular trafficking and microtubule dynamics, similarly follow the tissue-wide PS gradient, being stronger in the meristematic zone and lower in the elongation zone 2,5 . While intracellular lipid gradients are well characterized in eukaryotic cells, the mechanisms by which lipid patterns are coordinated across cells within tissue remain largely unknown. This question is particularly compelling in plants, where nearly every single cell within the plant body is connected to its neighbors by hundreds to thousands of plasmodesmata intercellular bridges 17,18 . These bridges not only create cytosolic but also membrane continuity, of the plasma membrane and the endoplasmic reticulum, between cells 17–19 . While the cytosolic route is well documented and provides a major pathway for cell-to-cell molecular exchanges, the membrane route remains largely unexplored, especially when comes to lipid transport. Lipids diffuse laterally within the membrane planes, with diffusion coefficients 10 to 100 times greater than most membrane proteins 11,19 . This typically allows them to explore about 0.1 to 1 µm 2 /s. Given that meristematic cells are 10–20 µm in each dimension, membrane continuity provided by plasmodesmata could in principle, support direct and rapid lipid exchange between cells. With this in mind, we investigated the question of cell-to-cell lipid trafficking and the establishment of lipid gradients across tissues using the Arabidopsis thaliana root tip as a model. Phospholipids can traffic cell-to-cell within the root meristem To evaluate whether lipids can diffuse cell-to-cell within plant organs, we screened for a fluorescent phospholipid that accumulated within the endomembrane system in Arabidopsis roots upon exogenous delivery. While most fluorescently labeled lipids that we tested were not readily taken up by roots, we found that 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phosphocholine (TopFluor-PC) accumulated within the endomembrane system in meristematic cells ( Fig. 1a ). TopFluor, a hydrophobic derivative of BODIPY, is attached to the end of a saturated 11-carbon acyl chain. BODIPY- and to a greater degree TopFluor-labeled lipid analogs were shown to best mirror the dynamics of natural lipids 20 . Using confocal microscopy, we found that TopFluor-PC labeled the perinuclear and cortical ER in root meristematic cells ( Fig. 1a , arrow and arrowhead, respectively), as well as intracellular dots ( Fig. 1a , asterisk). Colocalization with a red ER membrane marker, DERLIN1-mScarlet, and line-scan analysis confirmed the overlap between TopFluor-PC-labeled membranes and DERLIN1 in the cortical and perinuclear ER ( Fig. 1a ). The intracellular dots labeled by TopFluor-PC did not colocalize with DERLIN1, excluding the possibility that they could be ER bodies induced by the exogenous TopFluor-PC treatment. To confirm that TopFluor-PC was not heavily metabolized into different lipid species, we extracted all phospholipids from seedlings after TopFluor-PC treatment and performed High Performance Thin Layer Chromatography (HPTLC) analyses of the main phospholipid species. We found that the main lipid specie labeled with TopFluor in these plants was indeed PC, confirming that we can use TopFluor-PC to analyze the dynamic behavior of PC in the ER ( Fig. 1a and Extended Data Fig. 1a ). We also found traces of TopFluor in low-migrating phospholipids that likely correspond to PS ( Extended Fig. 1b ). This result is consistent with the fact that PC is an abundant lipid used to synthesize the minor anionic lipid PS 21,22 . This observation confirms that the cellular machinery can use TopFluor-PC and reinforces the idea that TopFluor-PC is incorporated into the endogenous membrane phospholipid pool. To determine whether TopFluor-PC can move between plant cells, we next performed fluorescent recovery after photobleaching (FRAP) ( Extended Data Fig. 1c ). As a positive control, we used carboxyfluorescein diacetate (CFDA). CFDA is a membrane-permeable compound cleaved by intracellular esterases after entering the cytosol. This creates a membrane-impermeable cytosolic fluorescent probe that moves through plasmodesmata cytoplasmic sleeve, using the cytosolic path 23,24 . Consistent with its cytosolic location, CFDA labeled the cytoplasm and nucleoplasm of Arabidopsis root meristematic cells ( Fig. 1c and Supplementary Video 1 ). As reported before 23,24 , we found a rapid fluorescence recovery of CFDA fluorescence in the bleached cell, showing that it can diffuse between neighboring cells in the root meristem ( Fig. 1c-e and Supplementary Video 1 ). We also found recorded fluorescence recovery when using TopFluor-PC ( Fig. 1c-e and Supplementary Video 2 ). By contrast, a membrane-bound ER protein, CINNAMATE 4-HYDROXYLASE-GFP (C4H-GFP), did not show any fluorescence recovery in the bleached cells ( Fig. 1c-e and Supplementary Video 3 ). These results suggest that the TopFluor-PC lipid diffused from cell-to-cell while the C4H-GFP transmembrane protein did not. The observable recovery of TopFluor-PC was in the perinuclear ER ( Fig. 1c arrowhead ), suggesting that TopFluor-PC uses the ER membrane as a conduit for diffusion to neighboring cells. TopFluor-PC fluorescence recovery was slower than that of CFDA ( Fig. 1d and e ). Although a direct comparison is difficult given that TopFluor-PC molecular mass is twice that of CFDA, a slower diffusion of TopFluor-PC is consistent with the notion that it is embedded in the ER membrane and, as such, moves more slowly along the 2D membrane plane, than a free-diffusing cytosolic component such as CFDA. Altogether, FRAP analyses demonstrate that TopFluor-PC can move between cells within intact root tissues, which is in line with the diffusion of lipophilic molecules recorded in vitro , in cultured soybean cells 25 . Phospholipids diffuses between cells along plasmodesmata membranes Next, we asked whether TopFluor-PC uses plasmodesmata as a conduit for intercellular diffusion. To address this question, we reasoned that if TopFluor-PC diffuses through plasmodesmata, its level of connectivity to neighboring cells should reflect the number of plasmodesmata within a given wall. Using serial-block face electron microscopy, we quantified the number of plasmodesmata on the lateral and apico-basal sides of epidermal meristematic cells. This analysis showed that the number of plasmodesmata at the apico-basal interfaces is 4.46 ± 0.31 times higher than at the lateral interfaces ( Fig. 2a-d ), confirming previous analyses 26 . More membrane conduits at the apico-basal interfaces should translate into more connectivity to apical and basal neighbors compared to lateral neighbors. To analyze TopFluor-PC connectivity, we used a variant of FRAP called Fluorescence Loss In Photobleaching (FLIP) ( Extended Data Fig. 1d ). In FLIP, we repeatedly bleach a small region of interest within a target cell, which gradually induces the loss of fluorescence in that cell. If this cell is connected, its neighbors will also lose fluorescence, quantitatively depending on their connectivity with the bleached cell. To account for the natural bleaching occurring during imaging, we also measured the fluorescence in a distant cell in the same root (control cell). We found that all cells surrounding the bleached cell lost fluorescence to a much greater degree than the control cells ( Fig. 2e-g and Supplementary Video 4 ), confirming the diffusion of TopFluor-PC between adjacent cells. Furthermore, neighbor cells connected through apico-basal walls presented a faster loss of fluorescence than neighboring cells connected through lateral walls (which presented less plasmodesmata) ( Fig. 2e-g ). Thus, lipid movement is correlated with plasmodesmata number. To confirm that TopFluor-PC diffuses through plasmodesmata, we wondered whether callose, a known regulator of cell-cell molecular trafficking might regulate its diffusion 17,18 . We genetically manipulated callose levels by expressing the gain-of-function callose synthase 3 gene ( cals3m ) under the control of an inducible system 23,27 . After 12 hours of induction, we quantified an increase in callose deposits in the root epidermis ( Extended Data Fig. 2a ). Under this condition, FRAP experiments revealed an absence of fluorescence recovery of TopFluor-PC, showing that callose deposition drastically reduced TopFluor-PC intercellular diffusion ( Fig. 2h-i, Extended Data Fig. 2b and Supplementary Video 5 ). Thus, altering plasmodesmata transport capacity impact lipid movement, confirming that this lipid diffuses through plasmodesmata. At this stage, two scenarios were still possible for the diffusion of TopFluor-PC through plasmodesmata. In the first scenario, TopFluor-PC diffuses laterally in the plane of the ER membrane from one cell to its neighbor. In the second scenario, TopFluor-PC in the membrane is not able to diffuse from cell-to-cell, but is taken up by cytoplasmic shuttle proteins or vesicles, which themselves can diffuse through the cytoplasmic sleeve of plasmodesmata. Both scenarios are compatible with the connectivity observed by FRAP, FLIP and the effect of callose induction on their diffusion. We consider the second scenario less likely, given that i) fluorescence recovery happens at the ER in our FRAP experiments and ii) intracellular dots labeled by TopFluor-PC do not move from cell to cell in this assay ( Fig. 1c and Supplementary Video 2 ). To unambiguously distinguished between these two scenarios, we took advantage of the recently described mctp3 mctp4 double mutant 23,28,29 . This mutant target ER/plasma membrane tethering elements from the MULTIPLE C2 DOMAINS TRANSMEMBRANE PROTEINS (MCTP) family. The mctp3 mctp4 double mutant presents fewer plasmodesmata than the wild-type 28 . However, the lack of tethering elements in this mutant induces a constitutively open cytoplasmic sleeve and enhances cytoplasmic connectivity despite the low number of plasmodesmata 23 . We thus reasoned that if TopFluor-PC uses the ER as a membrane conduit for diffusion in plasmodesmata (scenario 1), it should diffuse more slowly in the mctp3 mctp4 double mutant, this mutant having fewer membrane connections between cells. By contrast, if TopFluor-PC uses the cytoplasmic connection for diffusion in plasmodesmata (scenario 2), its diffusion should be faster in the mctp3 mctp4 double mutant, as demonstrated for the cytoplasmic components DRONPA-s and CFDA 23 . FRAP analysis showed that TopFluor-PC diffused more slowly in mctp3 mctp4 double mutant compared to the wild type ( Fig. 2j-k ), confirming that it uses the ER membrane as a conduit for intercellular movement. To conclude, our FRAP and FLIP analyses together with genetic manipulation of plasmodesmata show that TopFluor-PC diffuses from cell-to-cell in the root meristem using membrane-continuity established by plasmodesmata intercellular bridges. Endogenous phosphatidylserine is produced locally but is distributed broadly across the root tip Cell-to-cell diffusion of lipids opens up the possibility of forming tissue-level gradient, provided that the lipid source is locally confined. To address this, we next turn our attention to PS, an endogenously synthesized phospholipid important for signaling 1,2,4,6,14 . In Arabidopsis thaliana , PS is produced from the abundant PC by a single enzyme name PHOSPHATIDYLSERINE SYNTHASE1 (PSS1) 1,21 . PS is synthesized by PSS1 in the ER (see colocalization of PSS1 with the ER membrane marker DDRGK1, Extended Data Fig. 3a ) before being transported to the plasma membrane and endosomes 1 . PS is detected throughout the root, from elongation to the meristem zone ( Fig. 3a ). This is particularly evident when comparing the localization of the PS biosensor mCITRINE-2xPH EVECTIN2 (2xPH EVCT2 ) in the wild type and pss1 mutant. Indeed, in the wild type, 2xPH EVCT2 labels both the plasma membrane and intracellular compartments ( Fig. 3a ), while in the pss1 mutant, the sensor remains cytosolic and nucleoplasmic ( Fig. 3b ). Although PS is broadly distributed, its synthesis is spatially restricted. Analysis of the pPSS1::2xmCITRINE-NLS transcriptional reporter line revealed promoter activity confined to the epidermis and absent from the root tip ( Fig. 3c ). Thus, despite its broad distribution in the root, PS synthesis occurs only in a subset of epidermal cells, consistent with a non-cell-autonomous mechanism. Phosphatidylserine exhibits non-cell autonomous trafficking To investigate whether PS can move from its site of synthesis to neighboring cells, we established a tractable genetic system to study PS diffusion within root tissue.Specifically, we designed a system to restore PS synthesis in specific cell types within the pss1 mutant, which completely lacks PS 1,4 ( Fig. 3d ). The presence or absence of PS in adjacent cells was monitored using the 2xPH EVCT2 genetically encoded fluorescent PS sensor, which localizes to the plasma membrane and endosomes in PS-containing cells, but remains cytosolic in cells devoid of PS, ( Fig. 3a-b ) 1,30 . This system allowed us to control the sites of PS synthesis while monitoring its distribution at subcellular resolution. To restrict PS biosynthesis to specific cells, we expressed the PSS1 protein under the control of several tissue-specific promoters active in the root meristem: the atrichoblast epidermis (non-hair cells, GLABRA2 promoter, pGL2 ), cortex ( PEP promoter, pPEP ), endodermis ( SCARECROW promoter, pSCR ), and stele ( SHORT ROOT promoter, pSHR ) in the pss1 mutant expressing the mCITRINE-2xPH EVCT2 reporter. PSS1 was fused to mCHERRY to visualize its expression and localization pattern. With this set up, we have a unique opportunity to trace both the mobility of the enzyme PSS1 and its lipid product PS across tissues ( Fig. 3d ). In this collection of Arabidopsis transgenic lines, PSS1-mCHERRY was always expressed in the expected tissue, ruling out that the PSS1 mRNA or the PSS1 protein were able to move within the root meristem ( Fig. 3e-f ). We therefore next looked whether the lipid product, PS, could move from the site of synthesis to neighboring cells. For that, we analyzed the localization of the mCITRINE-2xPH EVCT2 sensor in non-PS producing epidermal cells. We found that mCITRINE-2xPH EVCT2 was localized at the PM and intracellular compartments in all epidermal cells at the root tips, despite the lack of PS synthesis in these cells ( Fig. 3e and g ). For example, when PSS1-mCHERRY was expressed in internal tissues of the root meristem, such as cortex ( pPEP ), endodermis ( pSCR ), or stele ( pSHR ), mCITRINE-2xPH EVCT2 localized at the PM and intracellular compartments in 100% of epidermal cell analyzed ( Fig. 3e-g ), suggesting that PS diffusion was possible between the concentric tissue layers of the root. Similarly, when PSS1-mCHERRY was expressed in atrichoblast cells ( pGL2 ), mCITRINE-2xPH EVCT2 localization was rescued in all cells of the epidermis, including non-PS synthetizing trichoblasts cells ( Fig. 3f-g ), suggesting that PS can also diffuse laterally within the same tissue layer. By contrast to meristematic expression of PSS1 , the expression of PSS1-mCHERRY in differentiated root epidermal cells (under the expression of the EXP7 promoter, pEXP7 ) or phloem companion cells (phloem CC, under the expression of the SUC2 promoter, pSUC2 ) did not complement 2xPH EVCT2 localization, which remained soluble in the root meristem of the pss1 mutant ( Fig. 3f-g ). Taken together, these results indicate that PS can move at relatively short distance within the root tip of Arabidopsis but does not efficiently diffuse out of differentiated tissues or on longer distances. To make sure that the complementation of 2xPH EVCT2 localization in non-PS synthetizing cells was not caused by the movement of PSS1 enzyme to neighboring cells, we fused PSS1 with three mCHERRY (PSS1-3xmCHERRY). This larger fusion exceeds the size exclusion limit of plasmodesmata, effectively preventing intercellular movement of the protein 31–33 . We found similar results with pSHR::PSS1-3xmCHERRY than with pSHR::PSS1-mCHERRY ( Extended Data Fig. 3b ), demonstrating that the rescue of 2xPH EVCT2 localization in the pss1 mutant was indeed not triggered by PSS1 protein movement. Together, these results confirm that PS can move away from its site of synthesis to other cells in the Arabidopsis root meristem. A combination of tissue-specific biosynthesis and diffusion creates the PS localization gradient at the tissue level Our data show that both TopFluor-PC and PS are able to move between cells in the root tip. Given that molecular gradients are frequently established via diffusion from a source, we asked whether intercellular diffusion of PS contributes to the formation of the tissue-scale PS gradient in the root epidermis, a gradient previously reported to be critical for signaling 2,5,6 . Using both transcriptional ( pPSS1::2xmCITRINE-NLS ) and translational ( pPSS1::PSS1-mCITRINE ) reporters, we precisely mapped PSS1 expression along the root epidermis. ( Fig. 4a-c ). Both reporters revealed a progressive increase starting at the end of the meristem and increasing in the transition and elongation zones of the root ( Fig. 4b-c ). This expression pattern closely aligns with the previously reported PS localization gradient in the epidermis 2 , with the PS sensor 2xPH EVCT2 evenly distributed between the plasma membrane and intracellular compartments in the meristematic zone and progressively shifting its localization predominantly in intracellular compartments in the elongation zone. These results suggest that PS distribution across the epidermis might be determined by the spatial interplay of PSS1-mediated PS synthesis and PS cell-cell diffusion. According to this model, the expression of PSS1 would favor the intracellular localization of PS. By contrast, in meristematic cells that do not synthesize PS due to the lack of PSS1 expression, the main source of PS is diffusion, which would favor plasma membrane accumulation of the lipid. To test this model, we took advantage of our PSS1 tissue-specific expression lines. We compared the plasma membrane and endosomal accumulation of the PS sensor 2xPH EVCT2 in the epidermis of the root when PSS1 was expressed in the same cells (i.e., the epidermis, pPSS1 ) or in neighboring cells (i.e. in the cortex, pPEP ). As expected, expression of PSS1-mCHERRY under its endogenous promoter recapitulated the previously reported PS gradient 2 ( Fig.4d ), with 2xPH EVCT2 predominantly at the plasma membrane in the meristem, where PSS1 is absent, and accumulation in intracellular compartments in the transition zone, where PSS1 is expressed ( Fig. 4d-e ). When PSS1-mCHERRY was expressed in the cortex (pPEP::PSS1-mCHERRY/ pss1 ), so that all epidermal cells received PS exclusively via diffusion, 2xPH EVCT2 showed enhanced plasma membrane localization in both the meristem and transition zones ( Fig. 4d-e ).Thus, by expressing PSS1 in the cortex, we perturb the establishment of the PS subcellular pattern in another tissue, the epidermis. Next, we did the reverse experiment, and we addressed the impact of ectopically expressing PSS1 in all epidermal cells including the meristematic ones that normally do not express it (using a pUBQ10::PSS1-mCHERRY/pss1 line). By doing so, we observed a reverse PS localization pattern with 2xPH EVCT2 reporter accumulating in intracellular compartments rather than the plasma membrane in the meristematic zone, in contrast to the wild-type situation ( Extended Data Fig. 4a ). Hence, producing PS directly in epidermal cells of the meristematic zone, which normally receives the lipid by diffusion, shifted its localization to endosomes (similar to the transition zone, where PS is produced in wild-type conditions). Of note, driving the expression of PSS1 under the UBQ10 promotor not only abolished the PS concentration gradient, but this condition was also showed to perturb the ROP6 response within the root and its downstream signaling 2 . Altogether, these experiments confirm that PS diffusion favors its plasma membrane accumulation in cells lacking PSS1 expression, while local PS production drives its endosomal accumulation in the producing cells ( Fig. 4f ). From these experiments, we concluded that PS diffusion, in conjunction with the tissue and zone-specific expression of the PS biosynthetic enzyme, creates supracellular lipid patterns within plant roots ( Fig. 4f ) relevant for Rho GTPases regulation at the tissue level 2,4 . Conclusions Already 20 years ago, Joost Holthuis and Tim Levine hypothesized that the ER functioned “as a passive, long-range, highly efficient, pan-cellular lipid-distribution system in some way analogous to the information superhighway” 11 . Here, we extend this concept to the tissue and organ scales by showing that in plants, the ER is not only a pan-cellular but also a supracellular lipid distribution system. In the case of PS, it remains to be determined whether it uses the ER, the plasma membrane or both as a conduit within plasmodesmata for diffusion. Indeed, PS is made in the lumen of the ER, where it cannot be detected by cytosolic PS sensors. PS could thus move from cell to cell by diffusing within the luminal leaflet of the ER. However, since PS eventually accumulates at the cell surface, PS could also use the plasma membrane as a route to neighboring cells. It is a common theme in cell biology and for the regulation of lipid homeostasis, in particular, to observe a physical separation between the site of synthesis in the cell and the site of action. This separation offers tremendous opportunity for regulation, both during the production of molecules and also later at their sites of action. Here, we propose that plants take advantage of this concept, but that, thanks to plasmodesmata, they not only use it inside the cells but also between cells. In addition to phospholipids, we predict that plasmodesmata might be used as an intercellular conduit for other hydrophobic molecules such as the brassinosteroid hormones, known to diffuse through plasmodesmata 34 . Furthermore, our finding opens the possibility of direct exchanges of lipids between cells, via other types of membrane-lined cellular junctions, such as tunneling nanotubes in animal cells. Declarations Acknowledgments: We thank Jürgen Kleine-Vehn for providing the DERLIN1-mScarlet ER membrane marker, Yasin Dagdas the DDRGK1-mCherry ER membrane marker, Abel Rosado for providing the C4H-GFP ER membrane marker, Marie Barberon for providing the PDR9:iCals3m lines, Lothar Kalmbach for the pLOK180 plasmid gift, Jean Christophe Mulatier for help in preparing lipids, Claire Lionnet for help in microscopy, the SiCE group, Marie-Cécile Caillaud, Ziqiang P. Li and Benoit Landrein for discussion, comments, and technical help, the LBI-PLATIM-MICROSCOPY for assistance with imaging. Funding : This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (projects 101001097-LIPIDEV to Y.J. and 772103-BRIDGING to E.M.B.), the National Agency for Research (grants ANR-18-CE13-0016 STAYING-TIGHT to E.M.B. and Y.J.), the French Government in the framework of the IdEX Bordeaux University ‘‘Investments for the Future’’ program/GPR Bordeaux Plant Sciences (E.M.B.), and by a EMBO Long-Term Fellowship to C.B. (ALTF 1018-2019). TLC analyses were performed at the Bordeaux Metabolome platform supported by MetaboHub (grant no ANR-11-INBS-0010 to L.F.) Author contributions: Conceptualization: C.B, E.M.B, Y.J. Methodology: C.B, H.M, V.B, M.P.P, E.M.B, Y.J., L.F. Investigation: C.B, H.M, V.B, M.P.P Visualization: C.B, H.M Funding acquisition: C.B, E.M.B, Y.J. Project administration: E.M.B, Y.J. Supervision: E.M.B, Y.J. Writing – original draft: C.B, E.M.B, Y.J. Writing – review & editing: C.B, H.M, V.B, M.P.P, E.M.B, Y.J. Competing interests: Authors declare that they have no competing interests. Data and materials availability & correspondence: All data are available in the main text or the supplementary materials and all material are available upon request to E.M.B. and Y.J. References Platre, M. P. et al. A Combinatorial Lipid Code Shapes the Electrostatic Landscape of Plant Endomembranes. Developmental Cell 1–28 (2018). Platre, M. P. et al. Developmental control of plant Rho GTPase nano-organization by the lipid phosphatidylserine. Science (New York, NY) 364 , 57–62 (2019). Smokvarska, M. et al. A Plasma Membrane Nanodomain Ensures Signal Specificity during Osmotic Signaling in Plants. Current Biology 1–26 (2020). Smokvarska, M. et al. The receptor kinase FERONIA regulates phosphatidylserine localization at the cell surface to modulate ROP signaling. Sci Adv 9 , eadd4791 (2023). Boutté, Y. & Jaillais, Y. Metabolic Cellular Communications: Feedback Mechanisms between Membrane Lipid Homeostasis and Plant Development. Developmental Cell 1–12 (2020). Dubois, G. A. & Jaillais, Y. Anionic phospholipid gradients: an uncharacterized frontier of the plant endomembrane network. Plant Physiology 185 , 577–592 (2021). van Meer, G., Voelker, D. R. & Feigenson, G. W. Membrane lipids: where they are and how they behave. Nature Reviews Molecular Cell Biology 9 , 112–124 (2008). Bigay, J. & Antonny, B. Curvature, Lipid Packing, and Electrostatics of Membrane Organelles: Defining Cellular Territories in Determining Specificity. Developmental Cell 23 , 886–895 (2012). Holthuis, J. C. M. & Menon, A. K. Lipid landscapes and pipelines in membrane homeostasis. Nature 510 , 48–57 (2014). Iglesias-Artola, J. M. et al. Quantitative imaging of lipid transport in mammalian cells. Nature 646 , 474–482 (2025). Holthuis, J. C. M. & Levine, T. P. Lipid traffic: floppy drives and a superhighway. Nature Reviews Molecular Cell Biology 6 , 209–220 (2005). Wong, L. H., Gatta, A. T. & Levine, T. P. Lipid transfer proteins: the lipid commute via shuttles, bridges and tubes. Nature Reviews Molecular Cell Biology 1–17 (2018). Yeung, T. et al. Membrane phosphatidylserine regulates surface charge and protein localization. Science (New York, NY) 319 , 210–213 (2008). Smokvarska, M., Jaillais, Y. & Martinière, A. Function of membrane domains in Rho-Of-Plant signaling. PLANT PHYSIOLOGY (2021). Gorgues, L. et al. GEF14 acts as a specific activator of the plant osmotic signaling pathway by controlling ROP6 nanodomain formation. EMBO Rep https://doi.org/10.1038/s44319-025-00412-w (2025) doi:10.1038/s44319-025-00412-w. Pan, X. et al. Auxin-induced signaling protein nanoclustering contributes to cell polarity formation. Nature Communications 1–14 (2020). Bayer, E. M. & Benitez-Alfonso, Y. Plasmodesmata: Channels Under Pressure. Annual Review of Plant Biology 75 , 291–317 (2024). Tee, E. E. & Faulkner, C. Plasmodesmata and intercellular molecular traffic control. New Phytologist 243 , 32–47 (2024). Béziat, C. & Jaillais, Y. Should I stay or should I go: the functional importance and regulation of lipid diffusion in biological membranes. Journal of Experimental Botany erad032 (2023) doi:10.1093/jxb/erad032. Kay, J. G., Koivusalo, M., Ma, X., Wohland, T. & Grinstein, S. Phosphatidylserine dynamics in cellular membranes. Molecular biology of the cell 23 , 2198–2212 (2012). Yamaoka, Y. et al. PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana. The Plant Journal 67 , 648–661 (2011). Colin, L. A. & Jaillais, Y. Phospholipids across scales: lipid patterns and plant development. Curr Opin Plant Biol 53 , 1–9 (2019). Pérez-Sancho, J. et al. Plasmodesmata act as unconventional membrane contact sites regulating intercellular molecular exchange in plants. Cell 188 , 958-977.e23 (2025). Nicolas, W. J. et al. Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves. Nature Plants 1–11 (2017). Grabski, S., De Feijter, A. W. & Schindler, M. Endoplasmic Reticulum Forms a Dynamic Continuum for Lipid Diffusion between Contiguous Soybean Root Cells. THE PLANT CELL ONLINE 5 , 25–38 (1993). Zhu, T., Lucas, W. J. & Rost, T. L. Directional cell-to-cell communication in the Arabidopsis root apical meristem I. An ultrastructural and functional analysis. Protoplasma 203 , 35–47 (1998). Vatén, A. et al. Callose Biosynthesis Regulates Symplastic Trafficking during Root Development. Developmental Cell 21 , 1144–1155 (2011). Li, Z. P. et al. Plant plasmodesmata bridges form through ER-dependent incomplete cytokinesis. Science 386 , 538–545 (2024). Brault, M. L. et al. Multiple C2 domains and transmembrane region proteins ( MCTPs) tether membranes at plasmodesmata. EMBO reports 172 , 1061 (2019). Platre, M. P. & Jaillais, Y. Exogenous treatment of Arabidopsis seedlings with lyso-phospholipids for the inducible complementation of lipid mutants. STAR Protocols 2 , 100626 (2021). Wu, X. et al. Modes of intercellular transcription factor movement in the Arabidopsis apex. Development 130 , 3735–3745 (2003). Kim, I., Cho, E., Crawford, K., Hempel, F. D. & Zambryski, P. C. Cell-to-cell movement of GFP during embryogenesis and early seedling development in Arabidopsis. Proceedings of the National Academy of Sciences 102 , 2227–2231 (2005). Kim, J. Y., Yuan, Z., Cilia, M., Khalfan-Jagani, Z. & Jackson, D. Intercellular trafficking of a KNOTTED1 green fluorescent protein fusion in the leaf and shoot meristem of Arabidopsis. Proc Natl Acad Sci U S A 99 , 4103–4108 (2002). Wang, Y. et al. Plasmodesmata mediate cell-to-cell transport of brassinosteroid hormones. Nat Chem Biol 1–11 (2023) doi:10.1038/s41589-023-01346-x. Additional Declarations There is NO Competing Interest. Supplementary Files 19112025BeziatLipidDiffusionExtendeddata.pdf Supplementary Materials Materials and Methods Extended Data Figure 1 to 4 MOVIES1.avi Supplementary Video 1: FRAP analysis of CFDA dynamics. MOVIES2.avi Supplementary Video 2: FRAP analysis of TopFluor-PC dynamics. MOVIES3.avi Supplementary Video 3: FRAP analysis of C4H-GFP dynamics. MOVIES4.avi Supplementary Video 4: FLIP analysis of TopFluor-PC dynamics. MOVIES5.avi Supplementary Video 5: FRAP analysis of TopFluor-PC dynamics upon callose induction by estradiol in PDR9::cals3m line. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8163315","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":551111326,"identity":"17abfa5f-7252-4885-9570-932bc87a1713","order_by":0,"name":"Yvon Jaillais","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIie3QMUvEMBQH8Bci7ZLerSkW6kfIpAjH+VUMhbqJ4CIoGhDi0i9wIPoVDoTDMSXQLv0AHb1vcN3uJs3T6YZQR8H8hwcvvB95CUBIyJ8NAzaJAQzcYEcV1ukoiSiSDjvyTVI1dhESIPoXRPRF8wHZPIsoXde7l9llrojmwzvwE48RfXkhgBVusUjYZFVeC0N0uuiAZ8ZDuu6YA6P4FmHJysplvtaHiYZ77lvshzw4Em/q3fOnfHWLIeFe0lZIrCNMmEQZqcwISVtd8nPWIrmyrCnk0pDH04XmXjKxtOFDdXs2jZ/ehu3d3C1G637QMy85wm+R1f7hgZv2AYBcYd3uH9KNdz4kJCTkP+YLkqFQBLLDy/sAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4923-883X","institution":"Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, INRAE","correspondingAuthor":true,"prefix":"","firstName":"Yvon","middleName":"","lastName":"Jaillais","suffix":""},{"id":551111327,"identity":"aef70c2a-273a-434a-a4aa-bfe10d3ceb2e","order_by":1,"name":"Chloe Beziat","email":"","orcid":"","institution":"University of Natural Resources and Life Sciences, Vienna","correspondingAuthor":false,"prefix":"","firstName":"Chloe","middleName":"","lastName":"Beziat","suffix":""},{"id":551111328,"identity":"adf5405e-5943-4e73-a8b8-4644c4a3d4fd","order_by":2,"name":"Vincent Bayle","email":"","orcid":"https://orcid.org/0000-0002-3747-9604","institution":"ENS Lyon - CNRS","correspondingAuthor":false,"prefix":"","firstName":"Vincent","middleName":"","lastName":"Bayle","suffix":""},{"id":551111329,"identity":"c52db724-a2b8-42c6-b988-69771f2dbea5","order_by":3,"name":"Frederique Rozier","email":"","orcid":"","institution":"CNRS/Ecole Normale Supérieure de Lyon","correspondingAuthor":false,"prefix":"","firstName":"Frederique","middleName":"","lastName":"Rozier","suffix":""},{"id":551111330,"identity":"e19b5369-ad69-456c-865d-bdc829525a48","order_by":4,"name":"Matthieu Platre","email":"","orcid":"https://orcid.org/0000-0002-4934-3050","institution":"INRAE","correspondingAuthor":false,"prefix":"","firstName":"Matthieu","middleName":"","lastName":"Platre","suffix":""},{"id":551111331,"identity":"0f3b42b9-c874-49b2-ae80-ef535d99e527","order_by":5,"name":"Hortense Moreau","email":"","orcid":"","institution":"CNRS, Université de Bordeaux","correspondingAuthor":false,"prefix":"","firstName":"Hortense","middleName":"","lastName":"Moreau","suffix":""},{"id":551111332,"identity":"a188249d-f1ca-47f5-82d0-fc0d0b3ed8ff","order_by":6,"name":"Laetitia Fouillen","email":"","orcid":"https://orcid.org/0000-0002-1204-9296","institution":"University of Bordeaux","correspondingAuthor":false,"prefix":"","firstName":"Laetitia","middleName":"","lastName":"Fouillen","suffix":""},{"id":551111333,"identity":"2af365f4-ed36-4f00-8b86-944baf50baca","order_by":7,"name":"Emmanuelle Bayer","email":"","orcid":"https://orcid.org/0000-0001-8642-5293","institution":"CNRS, Université de Bordeaux","correspondingAuthor":false,"prefix":"","firstName":"Emmanuelle","middleName":"","lastName":"Bayer","suffix":""}],"badges":[],"createdAt":"2025-11-20 10:18:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8163315/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8163315/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96903138,"identity":"d3276105-8027-40ba-80a2-61030e219421","added_by":"auto","created_at":"2025-11-27 11:49:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":373168,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA fluorescently labeled PC accumulates in the ER membrane and diffuses from cell to cell. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Airyscan confocal pictures of root meristematic cells treated with TopFluor-PC (green) and expressing the ER membrane marker DERLIN1-mScarlet(magenta) and merge of the two markers. Gray values of the two markers were plotted along the white line: TopFluor-PC (green), DERLIN1-mScarlet\u003cem\u003e \u003c/em\u003e(magenta). Scale bar: 5 µm. White arrows highlight the perinuclear ER, white arrowheads the cortical ER and white asterisks the intracellular compartments labeled by TopFluor-PC (\u003cstrong\u003eb\u003c/strong\u003e) HPTLC of lipid extracts visualized either at 302 nm (left, all lipid species being labeled by primuline), or at 525 nm (right, showing only TopFluor-labeled lipids). Std: standards (PE: phosphatidylethanolamine, PG: phosphatidylglycerol, PA: phosphatidic acid, PI: phosphatidylinositol, PC: Phosphatidylcholine). The lane labeled “TopFluor-PC” was loaded with pure TopFluor-PC, while the lane labeled “total lipids” was loaded with a total lipid extract of roots pretreated with TopFluor-PC. The black arrowhead indicates TopFluor-PC. (\u003cstrong\u003ec\u003c/strong\u003e) Representative pictures of root epidermal cells of wild-type treated with CFDA, TopFluor-PC, or expressing C4H-GFP, each labeled with Propidium Iodide during FRAP experiments. The dotted square represents the photobleached region. The white arrowhead depicts the reappearance of the perinuclear ER membrane. Scale bar: 10 µm. (\u003cstrong\u003ed\u003c/strong\u003e) Fluorescence quantification of wild-type treated with CFDA (N=29 roots, n=29 cells), TopFluor-PC (N=36 roots, n=36 cells) or expressing C4H-GFP (N=40 roots, n=40 cells) during FRAP timelapse and (\u003cstrong\u003ee\u003c/strong\u003e) their respective mobile fraction (% of fluorescence recovery at 100 seconds). Bars indicate the mean and SD (Kruskal Wallis test followed by multiple post hoc test, *** p\u0026lt;0,001 for all).\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-8163315/v1/770652808b69e9749691a750.png"},{"id":96920901,"identity":"6a9c02f5-ffa7-4a0d-8564-6b1a313e7746","added_by":"auto","created_at":"2025-11-27 14:15:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":387670,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTopFluor-PC diffuses through plasmodesmata. (a)\u003c/strong\u003e Serial Block Face Scanning Electron Microscopy pictures of the cell/cell interface in wild-type root epidermal cell and projection of the apico-basal cell-cell interface (\u003cstrong\u003eb\u003c/strong\u003e) and lateral cell-cell interface (\u003cstrong\u003ec\u003c/strong\u003e) each showing plasmodesmata as black dots. Scale bars: 2 µm. (\u003cstrong\u003ed\u003c/strong\u003e) Quantification of plasmodesmata numbers at each cell-cell interface. Bars indicate the mean and SD (Kruskal Wallis test followed by multiple post hoc test, ***p\u0026lt;0,001). \u003cstrong\u003e(e)\u003c/strong\u003e Representative pictures of wild-type root epidermal cells labeled with propidium iodide and treated with TopFluor-PC during repetitive bleaching in fluorescence loss in photobleaching (FLIP) timelapse. The bleached ROI is indicated by dashed line, \u003cem\u003ea\u003c/em\u003e: apical cell, \u003cem\u003eb\u003c/em\u003e: basal cell, \u003cem\u003el\u003c/em\u003e: lateral cells, \u003cem\u003ebl\u003c/em\u003e: bleached cell. Scale bar: 10 µm \u003cstrong\u003e(f)\u003c/strong\u003e Quantification of fluorescence intensity in each indicated cells in wild type treated with TopFluor-PC (N=30 roots, n=30 cells) during FLIP timelapse and (\u003cstrong\u003eg\u003c/strong\u003e) % of fluorescence loss at 200 seconds. Bars indicate the mean and SD (Kruskal Wallis test followed by multiple post hoc test, p values in Extended Data Fig. 2c). \u003cstrong\u003e(h)\u003c/strong\u003e Fluorescence quantification of \u003cem\u003epPDR9:XVE-Cals3m\u003c/em\u003e root cells treated with TopFluor-PC and DMSO (N=28 roots, n=28 cells) or estradiol (N=36 roots, n=36 cells) during FRAP timelapse and (\u003cstrong\u003ei\u003c/strong\u003e) their respective mobile fraction (% of fluorescence recovery at 100 seconds). Bars indicate the mean and SD (Kruskal Wallis test followed by multiple post hoc tests, p value *** p\u0026lt;0,001). \u003cstrong\u003e(j)\u003c/strong\u003e Fluorescence quantification of WT\u003cem\u003e \u003c/em\u003e(N=28 roots, n=28 cells) and \u003cem\u003emctp3 mctp4 \u003c/em\u003e(N=26 roots, n=26 cells) root epidermal cells treated with TopFluor-PC during FRAP timelapse and (\u003cstrong\u003ek\u003c/strong\u003e) their respective mobile fraction (% of fluorescence recovery at 100 seconds). Bars indicate the mean and SD (Kruskal Wallis test followed by multiple post hoc test, p value *p=0,028).\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-8163315/v1/9c730cbf1c949036cdc8dfb1.png"},{"id":96903141,"identity":"0d862bba-fdfb-4005-9690-edbe3d1d3f0b","added_by":"auto","created_at":"2025-11-27 11:49:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":376991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePS is produced in a subset of cells at the root tip and can diffuses throughout the meristem.\u003c/strong\u003e \u003cstrong\u003e(a-b) \u003c/strong\u003eSchematic representation (left) and confocal pictures (right) of the localization of the PS sensor mCitrine-2PH(EVCT2) in the Arabidopsis root in the wild type (\u003cstrong\u003ea\u003c/strong\u003e) and the \u003cem\u003epss1\u003c/em\u003e mutant (\u003cstrong\u003eb\u003c/strong\u003e). (\u003cstrong\u003ec\u003c/strong\u003e) Representative confocal pictures of the transcriptional reporter line \u003cem\u003epPSS1::2xmCitrine-NLS \u003c/em\u003elabeled with propidium iodide to highlight cell contour. Image on the left show a section through the root meristem, image on the right shows a section through the epidermis and cortex of the transition zone. ep = epidermis, c = cortex, e = endodermis, s = stele. (\u003cstrong\u003ed\u003c/strong\u003e) Schematic representation if PS is able to move from cell-to-cell (left) or not (right) of the localization of the PS sensor mCitrine-2PH(EVCT2) in the Arabidopsis root of \u003cem\u003epss1\u003c/em\u003e expressing \u003cem\u003epSHR::PSS1-mCHERRY\u003c/em\u003e(stele). (\u003cstrong\u003ee\u003c/strong\u003e) Merged confocal picture (left) showing the localization of the PS sensor mCitrine-2PH(EVCT2) (green) and \u003cem\u003epSHR::PSS1-mCHERRY \u003c/em\u003eexpression pattern\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e(magenta) and close-up view of the PS sensor mCitrine-2PH(EVCT2) in the epidermis. (\u003cstrong\u003ef\u003c/strong\u003e) Representative pictures of the expression pattern of \u003cem\u003epGL2::PSS1-mCHERRY\u003c/em\u003e, \u003cem\u003epPEP::PSS1-mCHERRY\u003c/em\u003e, \u003cem\u003epSCR::PSS1-mCHERRY, pSUC2:PSS1-mCHERRY, pEXP7A::PSS1-mCHERRY\u003c/em\u003e and (\u003cstrong\u003eg\u003c/strong\u003e) localization of the PS sensor mCitrine-2PH(EVCT2) in the meristem epidermis in the \u003cem\u003epss1\u003c/em\u003emutant background expressing the constructs indicated at the top. From a to f, scale bars: 20 µm.\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-8163315/v1/c9f50c1bb3c65813bc55df5d.png"},{"id":96903140,"identity":"05df69ce-ce73-4a80-8817-72030f564b3f","added_by":"auto","created_at":"2025-11-27 11:49:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":328572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTissue-specific expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePSS1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in conjunction with phosphatidylserine diffusion regulates the PS developmental gradient in roots. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Schematic representation of \u003cem\u003ePSS1\u003c/em\u003e expression pattern in \u003cem\u003eArabidopsis thaliana \u003c/em\u003eroot. The green arrowhead highlights the epidermis-specific expression of \u003cem\u003ePSS1\u003c/em\u003e and the different shades of green represent the \u003cem\u003ePSS1\u003c/em\u003e expression gradient being high in the Elongation Zone (EZ), intermediate in the Transition Zone (TZ) and low the Meristematic Zone (MZ). (\u003cstrong\u003eb-c\u003c/strong\u003e) Close up of \u003cem\u003ePSS1\u003c/em\u003e promoter activity (\u003cem\u003epPSS1::2xmCitrine-NLS, \u003c/em\u003ePI counterstaining in magenta, \u003cstrong\u003eb\u003c/strong\u003e) or PSS1 protein accumulation (\u003cem\u003epPSS1::PSS1-mCitrine, \u003c/em\u003e\u003cstrong\u003ec\u003c/strong\u003e)\u003cem\u003e \u003c/em\u003ein the epidermis along the different zones of the root (left) and related quantification of fluorescence intensity. n=100 cells from 10 roots (Kruskal Wallis test followed by multiple post hoc test, p value \u003cstrong\u003eb\u003c/strong\u003e: for all *** p\u0026lt;0,001 excepted for MZ compared to TZ: *p value=0,025, \u003cstrong\u003ec\u003c/strong\u003e: for all *** p\u0026lt;0,001). (\u003cstrong\u003ed\u003c/strong\u003e) Representative pictures of the PS sensor mCitrine-2PH(EVCT2) localization\u003cem\u003e \u003c/em\u003ein the root epidermal tissue of \u003cem\u003epPSS1::PSS1-mCHERRY/pss1\u003c/em\u003e and \u003cem\u003epPEP::PSS1-mCHERRY\u003c/em\u003e/\u003cem\u003epss1\u003c/em\u003ein both the meristematic (MZ) and transition zones (TZ), scale bar: 5 µm, and (\u003cstrong\u003ee\u003c/strong\u003e) ratios of the PS sensor mCitrine-2PH(EVCT2)plasma membrane/cytoplasm in \u003cem\u003epPSS1::PSS1-mCHERRY/pss1\u003c/em\u003emeristematic zone (MZ, n=98 cells) and in transition zone (TZ, n=100), and in \u003cem\u003epPEP::PSS1-mCHERRY\u003c/em\u003e/\u003cem\u003epss1\u003c/em\u003e meristematic zone (MZ, n=99 cells) and in transition zone (TZ, n=101). Bars indicate the mean and SD. Kruskal Wallis test was performed *** p value\u0026lt;0,001). (\u003cstrong\u003ef\u003c/strong\u003e) Cartoon representing a model for the creation of PS localization gradient in Arabidopsis root. Intra: intracellular compartments.\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-8163315/v1/c991ce3da207e2a988b53882.png"},{"id":96923329,"identity":"65f1d0c4-33d7-4b1f-9b05-6c95fd66760d","added_by":"auto","created_at":"2025-11-27 14:21:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2519403,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8163315/v1/7d88c6e0-f553-4cf2-9dd0-a6aad1a1e311.pdf"},{"id":96903142,"identity":"6f725bf9-9ef6-4626-8f8a-a0e9c9eff8b4","added_by":"auto","created_at":"2025-11-27 11:49:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3395612,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Materials\u003c/p\u003e\n\u003cp\u003eMaterials and Methods\u003c/p\u003e\n\u003cp\u003eExtended Data Figure 1 to 4\u003c/p\u003e","description":"","filename":"19112025BeziatLipidDiffusionExtendeddata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8163315/v1/d093567e6a0d25be28769b1f.pdf"},{"id":96903148,"identity":"3658d1df-6f29-4e8a-aefa-5ec1d6cde307","added_by":"auto","created_at":"2025-11-27 11:49:01","extension":"avi","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":889410,"visible":true,"origin":"","legend":"Supplementary Video 1: FRAP analysis of CFDA dynamics.","description":"","filename":"MOVIES1.avi","url":"https://assets-eu.researchsquare.com/files/rs-8163315/v1/573bbd7359ad0567c4c25636.avi"},{"id":96903143,"identity":"44dc88c4-d4ef-4b2d-9bab-71a0182a62c6","added_by":"auto","created_at":"2025-11-27 11:49:00","extension":"avi","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6667292,"visible":true,"origin":"","legend":"Supplementary Video 2: FRAP analysis of TopFluor-PC dynamics.","description":"","filename":"MOVIES2.avi","url":"https://assets-eu.researchsquare.com/files/rs-8163315/v1/f2365a56c64c14389de29aed.avi"},{"id":96920210,"identity":"474019f5-8847-4c95-ac51-182a1f13a2c8","added_by":"auto","created_at":"2025-11-27 14:14:56","extension":"avi","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":7685002,"visible":true,"origin":"","legend":"Supplementary Video 3: FRAP analysis of C4H-GFP dynamics.","description":"","filename":"MOVIES3.avi","url":"https://assets-eu.researchsquare.com/files/rs-8163315/v1/49ccac00dcaab9ae346ff5c5.avi"},{"id":96903145,"identity":"9da19ce4-836a-4eb9-9895-8ffede3db4bb","added_by":"auto","created_at":"2025-11-27 11:49:00","extension":"avi","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1855804,"visible":true,"origin":"","legend":"Supplementary Video 4: FLIP analysis of TopFluor-PC dynamics.","description":"","filename":"MOVIES4.avi","url":"https://assets-eu.researchsquare.com/files/rs-8163315/v1/381199ce88317e3454dfaf27.avi"},{"id":96919834,"identity":"5383d494-68cf-4f26-bd8f-df4dba4460dd","added_by":"auto","created_at":"2025-11-27 14:14:32","extension":"avi","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2778194,"visible":true,"origin":"","legend":"Supplementary Video 5: FRAP analysis of TopFluor-PC dynamics upon callose induction by estradiol in PDR9::cals3m line.","description":"","filename":"MOVIES5.avi","url":"https://assets-eu.researchsquare.com/files/rs-8163315/v1/255458c429abcd3fe951f485.avi"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Intercellular lipid flow coordinates tissue-scale lipid gradients in plants","fulltext":[{"header":"Main text","content":"\u003cp\u003eMembrane lipids are organized as gradients within cells\u003csup\u003e6–10\u003c/sup\u003e. These gradients arise as lipids are transported from their sites of synthesis to different membrane compartments via vesicular and non-vesicular pathways. The resulting non-uniform lipid distribution among membrane compartments governs protein localization and, ultimately, cellular functions\u003csup\u003e9,11,12\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eA classic example of cellular lipid gradient is the anionic phospholipid phosphatidylserine (PS), which is present as a gradient in post-Golgi membranes\u003csup\u003e8\u003c/sup\u003e. Genetically-encoded biosensors for PS preferentially localize at the plasma membrane in macrophage cells, then to a lesser degree to early endosomes and then to late endosomes and lysosomes\u003csup\u003e13\u003c/sup\u003e. This intracellular PS gradient is largely conserved in plants\u003csup\u003e1\u003c/sup\u003e, and\u0026nbsp;remarkably extends beyond single-cell level, to establish a differential localization pattern across the \u0026nbsp;root epidermis\u003csup\u003e2,6\u003c/sup\u003e.\u0026nbsp;Specifically, PS localizes to both the plasma membrane and endosomes in meristematic cells\u003csup\u003e1\u003c/sup\u003e, but becomes increasingly restricted to endosomes in the transition and elongation zone\u003csup\u003e1,2,5\u003c/sup\u003e. This spatial redistribution links intracellular lipid gradients to tissue-level organization\u003csup\u003e5,6\u003c/sup\u003e.\u0026nbsp;The tissue-scale PS gradient is functionally important for Rho GTPase signaling\u003csup\u003e14\u003c/sup\u003e. Upon activation by the plant hormone auxin or osmotic stress, RHO-RELATED PROTEIN OF PLANTS6 (ROP6) clusters into nanodomains at the plasma membrane, a step required for signaling\u003csup\u003e2,3,15,16\u003c/sup\u003e. ROP6 signaling output scales with the numbers of ROP6 nanoclusters, which depends on the amount of PS at the plasma membrane\u003csup\u003e2–4,14\u003c/sup\u003e. As such, at the same concentration of auxin, ROP6 nanoclustering is high in the meristematic zone of the root, where PS accumulates at the plasma membrane, and reduced in the elongation zone, where PS is prevalently localized in intracellular compartments\u003csup\u003e2\u003c/sup\u003e. The downstream ROP6 outputs, such as the regulation of intracellular trafficking and microtubule dynamics,\u0026nbsp;similarly follow the tissue-wide PS gradient, being stronger in the meristematic zone and lower in the elongation zone\u003csup\u003e2,5\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile intracellular lipid gradients are well characterized in eukaryotic cells, the mechanisms by which lipid patterns are coordinated\u0026nbsp;across cells\u0026nbsp;within tissue remain largely unknown. This question is particularly compelling in plants, where nearly every single cell within the plant body is connected to its neighbors by hundreds to thousands of\u0026nbsp;plasmodesmata intercellular bridges\u003csup\u003e17,18\u003c/sup\u003e. These bridges not only create cytosolic but also membrane continuity, of the plasma membrane and the endoplasmic reticulum, between cells\u003csup\u003e17–19\u003c/sup\u003e. While the cytosolic route is well documented and provides a major pathway for cell-to-cell molecular exchanges, the membrane route remains largely unexplored, especially when comes to lipid transport.\u0026nbsp;Lipids diffuse laterally within the membrane planes, with diffusion coefficients 10 to 100 times greater than most membrane proteins\u003csup\u003e11,19\u003c/sup\u003e. This typically allows them to explore about 0.1 to 1 µm\u003csup\u003e2\u003c/sup\u003e/s. Given that meristematic cells are 10–20 µm in each dimension, membrane continuity provided by plasmodesmata could in principle, support direct and rapid lipid exchange between cells.\u0026nbsp;With this in mind, we investigated the question of cell-to-cell lipid trafficking and the establishment of lipid gradients across tissues using the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e root tip as a model.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhospholipids can traffic cell-to-cell within the root meristem\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate whether lipids can diffuse cell-to-cell within plant organs, we screened for a fluorescent phospholipid that accumulated within the endomembrane system in Arabidopsis roots upon exogenous delivery. While most fluorescently labeled lipids that we tested were not readily taken up by roots, we found that 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phosphocholine (TopFluor-PC) accumulated within the endomembrane system in meristematic cells (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). TopFluor, a hydrophobic derivative of BODIPY, is attached to the end of a saturated 11-carbon acyl chain. BODIPY- and to a greater degree TopFluor-labeled lipid analogs were shown to best mirror the dynamics of natural lipids\u003csup\u003e20\u003c/sup\u003e. Using confocal microscopy, we found that TopFluor-PC labeled the perinuclear and cortical ER in root meristematic cells (\u003cstrong\u003eFig. 1a\u003c/strong\u003e, arrow and arrowhead, respectively), as well as intracellular dots (\u003cstrong\u003eFig. 1a\u003c/strong\u003e, asterisk). Colocalization with a red ER membrane marker, DERLIN1-mScarlet, and line-scan analysis confirmed the overlap between TopFluor-PC-labeled membranes and DERLIN1 in the cortical and perinuclear ER (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). The intracellular dots labeled by TopFluor-PC did not colocalize with DERLIN1, excluding the possibility that they could be ER bodies induced by the exogenous TopFluor-PC treatment. To confirm that TopFluor-PC was not heavily metabolized into different lipid species, we extracted all phospholipids from seedlings after TopFluor-PC treatment and performed High Performance Thin Layer Chromatography (HPTLC) analyses of the main phospholipid species. We found that the main lipid specie labeled with TopFluor in these plants was indeed PC, confirming that we can use TopFluor-PC to analyze the dynamic behavior of PC in the ER (\u003cstrong\u003eFig. 1a and Extended Data Fig. 1a\u003c/strong\u003e). We also found traces of TopFluor in low-migrating phospholipids that likely correspond to PS (\u003cstrong\u003eExtended\u003c/strong\u003e \u003cstrong\u003eFig. 1b\u003c/strong\u003e). This result is consistent with the fact that PC is an abundant lipid used to synthesize the minor anionic lipid PS\u003csup\u003e21,22\u003c/sup\u003e. This observation confirms that the cellular machinery can use TopFluor-PC and reinforces the idea that TopFluor-PC is incorporated into the endogenous membrane phospholipid pool.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine whether TopFluor-PC can move between plant cells, we next performed fluorescent recovery after photobleaching (FRAP) (\u003cstrong\u003eExtended\u003c/strong\u003e \u003cstrong\u003eData Fig. 1c\u003c/strong\u003e). As a positive control, we used carboxyfluorescein diacetate (CFDA). CFDA is a membrane-permeable compound cleaved by intracellular esterases after entering the cytosol. This creates a membrane-impermeable cytosolic fluorescent probe that moves through plasmodesmata cytoplasmic sleeve, using the cytosolic path\u003csup\u003e23,24\u003c/sup\u003e. Consistent with its cytosolic location, CFDA labeled the cytoplasm and nucleoplasm of Arabidopsis root meristematic cells (\u003cstrong\u003eFig. 1c and Supplementary Video 1\u003c/strong\u003e). As reported before\u003csup\u003e23,24\u003c/sup\u003e, we found a rapid fluorescence recovery of CFDA fluorescence in the bleached cell, showing that it can diffuse between neighboring cells in the root meristem (\u003cstrong\u003eFig. 1c-e and Supplementary Video 1\u003c/strong\u003e). We also found recorded fluorescence recovery when using TopFluor-PC (\u003cstrong\u003eFig. 1c-e and Supplementary Video 2\u003c/strong\u003e). By contrast, a membrane-bound ER protein, CINNAMATE 4-HYDROXYLASE-GFP (C4H-GFP), did not show any fluorescence recovery in the bleached cells (\u003cstrong\u003eFig. 1c-e and Supplementary Video 3\u003c/strong\u003e). These results suggest that the TopFluor-PC lipid diffused from cell-to-cell while the C4H-GFP transmembrane protein did not. The observable recovery of TopFluor-PC was in the perinuclear ER (\u003cstrong\u003eFig. 1c arrowhead\u003c/strong\u003e), suggesting that TopFluor-PC uses the ER membrane as a conduit for diffusion to neighboring cells. TopFluor-PC fluorescence recovery was slower than that of CFDA (\u003cstrong\u003eFig. 1d and e\u003c/strong\u003e). Although a direct comparison is difficult given that TopFluor-PC molecular mass is twice that of CFDA, a slower diffusion of TopFluor-PC is consistent with the notion that it is embedded in the ER membrane and, as such, moves more slowly along the 2D membrane plane, than a free-diffusing cytosolic component such as CFDA. Altogether, FRAP analyses demonstrate that TopFluor-PC can move between cells within intact root tissues, which is in line with the diffusion of lipophilic molecules recorded \u003cem\u003ein vitro\u003c/em\u003e, in cultured soybean cells\u003csup\u003e25\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhospholipids diffuses between cells along plasmodesmata membranes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we asked whether TopFluor-PC uses plasmodesmata as a conduit for intercellular diffusion. To address this question, we reasoned that if TopFluor-PC diffuses through plasmodesmata, its level of connectivity to neighboring cells should reflect the number of plasmodesmata within a given wall. Using serial-block face electron microscopy, we quantified the number of plasmodesmata on the lateral and apico-basal sides of epidermal meristematic cells. This analysis showed that the number of plasmodesmata at the apico-basal interfaces is 4.46 ± 0.31 times higher than at the lateral interfaces (\u003cstrong\u003eFig. 2a-d\u003c/strong\u003e), confirming previous analyses\u003csup\u003e26\u003c/sup\u003e. More membrane conduits at the apico-basal interfaces should translate into more connectivity to apical and basal neighbors compared to lateral neighbors. To analyze TopFluor-PC connectivity, we used a variant of FRAP called Fluorescence Loss In Photobleaching (FLIP) (\u003cstrong\u003eExtended Data Fig. 1d\u003c/strong\u003e). In FLIP, we repeatedly bleach a small region of interest within a target cell, which gradually induces the loss of fluorescence in that cell. If this cell is connected, its neighbors will also lose fluorescence, quantitatively depending on their connectivity with the bleached cell. To account for the natural bleaching occurring during imaging, we also measured the fluorescence in a distant cell in the same root (control cell). We found that all cells surrounding the bleached cell lost fluorescence to a much greater degree than the control cells (\u003cstrong\u003eFig. 2e-g and Supplementary Video 4\u003c/strong\u003e), confirming the diffusion of TopFluor-PC between adjacent cells. Furthermore, neighbor cells connected through apico-basal walls presented a faster loss of fluorescence than neighboring cells connected through lateral walls (which presented less plasmodesmata) (\u003cstrong\u003eFig. 2e-g\u003c/strong\u003e). Thus, lipid movement is correlated with plasmodesmata number.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo confirm that TopFluor-PC diffuses through plasmodesmata, we wondered whether callose, a known regulator of cell-cell molecular trafficking might regulate its diffusion\u003csup\u003e17,18\u003c/sup\u003e. We genetically manipulated callose levels by expressing the gain-of-function \u003cem\u003ecallose synthase 3\u003c/em\u003e gene (\u003cem\u003ecals3m\u003c/em\u003e) under the control of an inducible system\u003csup\u003e23,27\u003c/sup\u003e. After 12 hours of induction, we quantified an increase in callose deposits in the root epidermis (\u003cstrong\u003eExtended Data Fig. 2a\u003c/strong\u003e). Under this condition, FRAP experiments revealed an absence of fluorescence recovery of TopFluor-PC, showing that callose deposition drastically reduced TopFluor-PC intercellular diffusion (\u003cstrong\u003eFig. 2h-i, Extended Data Fig. 2b and Supplementary Video 5\u003c/strong\u003e). Thus, altering plasmodesmata transport capacity impact lipid movement, confirming that this lipid diffuses through plasmodesmata.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt this stage, two scenarios were still possible for the diffusion of TopFluor-PC through plasmodesmata. In the first scenario, TopFluor-PC diffuses laterally in the plane of the ER membrane from one cell to its neighbor. In the second scenario, TopFluor-PC in the membrane is not able to diffuse from cell-to-cell, but is taken up by cytoplasmic shuttle proteins or vesicles, which themselves can diffuse through the cytoplasmic sleeve of plasmodesmata. Both scenarios are compatible with the connectivity observed by FRAP, FLIP and the effect of callose induction on their diffusion. We consider the second scenario less likely, given that i) fluorescence recovery happens at the ER in our FRAP experiments and ii) intracellular dots labeled by TopFluor-PC do not move from cell to cell in this assay (\u003cstrong\u003eFig. 1c and Supplementary Video 2\u003c/strong\u003e). To unambiguously distinguished between these two scenarios, we took advantage of the recently described \u003cem\u003emctp3 mctp4\u003c/em\u003e double mutant\u003csup\u003e23,28,29\u003c/sup\u003e. This mutant target ER/plasma membrane tethering elements from the MULTIPLE C2 DOMAINS TRANSMEMBRANE PROTEINS (MCTP) family. The \u003cem\u003emctp3 mctp4\u003c/em\u003e double mutant presents fewer plasmodesmata than the wild-type\u003csup\u003e28\u003c/sup\u003e. However, the lack of tethering elements in this mutant induces a constitutively open cytoplasmic sleeve and enhances cytoplasmic connectivity despite the low number of plasmodesmata\u003csup\u003e23\u003c/sup\u003e. We thus reasoned that if TopFluor-PC uses the ER as a membrane conduit for diffusion in plasmodesmata (scenario 1), it should diffuse more slowly in the \u003cem\u003emctp3 mctp4\u0026nbsp;\u003c/em\u003edouble mutant, this mutant having fewer membrane connections between cells. By contrast, if TopFluor-PC uses the cytoplasmic connection for diffusion in plasmodesmata (scenario 2), its diffusion should be faster in the \u003cem\u003emctp3 mctp4\u003c/em\u003e double mutant, as demonstrated for the cytoplasmic components DRONPA-s and CFDA\u003csup\u003e23\u003c/sup\u003e. FRAP analysis showed that TopFluor-PC diffused more slowly in \u003cem\u003emctp3 mctp4\u003c/em\u003e double mutant compared to the wild type (\u003cstrong\u003eFig. 2j-k\u003c/strong\u003e), confirming that it uses the ER membrane as a conduit for intercellular movement. To conclude, our FRAP and FLIP analyses together with genetic manipulation of plasmodesmata show that TopFluor-PC diffuses from cell-to-cell in the root meristem using membrane-continuity established by plasmodesmata intercellular bridges.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndogenous phosphatidylserine is produced locally but is distributed broadly across the root tip\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell-to-cell diffusion of lipids opens up the possibility of forming tissue-level gradient, provided that the lipid source is locally confined. To address this, we next turn our attention to PS,\u0026nbsp;an endogenously synthesized phospholipid important for signaling\u003csup\u003e1,2,4,6,14\u003c/sup\u003e.\u0026nbsp;In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, PS is produced from the abundant PC by a single enzyme name PHOSPHATIDYLSERINE SYNTHASE1 (PSS1)\u003csup\u003e1,21\u003c/sup\u003e.\u0026nbsp;PS is synthesized by PSS1 in the ER (see colocalization of PSS1 with the ER membrane marker DDRGK1, \u003cstrong\u003eExtended\u003c/strong\u003e \u003cstrong\u003eData Fig. 3a\u003c/strong\u003e) before being transported to the plasma membrane and endosomes\u003csup\u003e1\u003c/sup\u003e. PS is detected throughout the root, from elongation to the meristem zone (\u003cstrong\u003eFig. 3a\u003c/strong\u003e). This is particularly evident when comparing the localization of the PS biosensor mCITRINE-2xPH\u003csup\u003eEVECTIN2\u003c/sup\u003e (2xPH\u003csup\u003eEVCT2\u003c/sup\u003e) in the wild type and \u003cem\u003epss1\u003c/em\u003e mutant. Indeed, in the wild type, 2xPH\u003csup\u003eEVCT2\u0026nbsp;\u003c/sup\u003elabels both the plasma membrane and intracellular compartments (\u003cstrong\u003eFig. 3a\u003c/strong\u003e), while in the \u003cem\u003epss1\u003c/em\u003e mutant, the sensor remains cytosolic and nucleoplasmic (\u003cstrong\u003eFig. 3b\u003c/strong\u003e).\u0026nbsp;Although PS is broadly distributed, its synthesis is spatially restricted. Analysis of the \u003cem\u003epPSS1::2xmCITRINE-NLS\u0026nbsp;\u003c/em\u003etranscriptional reporter line revealed promoter activity confined to the epidermis and absent from the root tip\u0026nbsp;(\u003cstrong\u003eFig. 3c\u003c/strong\u003e). Thus, despite its broad distribution in the root, PS synthesis occurs only in a subset of epidermal cells, consistent with a non-cell-autonomous mechanism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhosphatidylserine exhibits non-cell autonomous trafficking\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether PS can move from its site of synthesis to neighboring cells, we established a tractable genetic system to study PS diffusion within root tissue.Specifically, we designed a system to restore PS synthesis in specific cell types within the \u003cem\u003epss1\u003c/em\u003e mutant, which completely lacks PS\u003csup\u003e1,4\u003c/sup\u003e (\u003cstrong\u003eFig. 3d\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe presence or absence of PS in adjacent cells was monitored using the 2xPH\u003csup\u003eEVCT2\u0026nbsp;\u003c/sup\u003egenetically encoded fluorescent PS sensor, which localizes to the plasma membrane and endosomes in PS-containing cells, but remains cytosolic in cells devoid of PS, (\u003cstrong\u003eFig. 3a-b\u003c/strong\u003e)\u003csup\u003e1,30\u003c/sup\u003e. This system allowed us to control the sites of PS synthesis while monitoring its distribution at subcellular resolution. To restrict PS biosynthesis to specific cells, we expressed the PSS1 protein under the control of several tissue-specific promoters active in the root meristem: the atrichoblast epidermis (non-hair cells, \u003cem\u003eGLABRA2\u003c/em\u003e promoter, \u003cem\u003epGL2\u003c/em\u003e), cortex (\u003cem\u003ePEP\u003c/em\u003e promoter, \u003cem\u003epPEP\u003c/em\u003e), endodermis (\u003cem\u003eSCARECROW\u003c/em\u003e promoter, \u003cem\u003epSCR\u003c/em\u003e), and stele (\u003cem\u003eSHORT ROOT\u003c/em\u003e promoter, \u003cem\u003epSHR\u003c/em\u003e) in the \u003cem\u003epss1\u003c/em\u003e mutant expressing the mCITRINE-2xPH\u003csup\u003eEVCT2\u003c/sup\u003e reporter. PSS1 was fused to mCHERRY to visualize its expression and localization pattern. With this set up, we have a unique opportunity to trace both the mobility of the enzyme PSS1 and its lipid product PS across tissues (\u003cstrong\u003eFig. 3d\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn this collection of Arabidopsis transgenic lines, PSS1-mCHERRY was always expressed in the expected tissue, ruling out that the \u003cem\u003ePSS1\u003c/em\u003e mRNA or the PSS1 protein were able to move within the root meristem (\u003cstrong\u003eFig. 3e-f\u003c/strong\u003e). We therefore next looked whether the lipid product, PS, could move from the site of synthesis to neighboring cells. For that, we analyzed the localization of the mCITRINE-2xPH\u003csup\u003eEVCT2\u003c/sup\u003e sensor in non-PS producing epidermal cells. We found that mCITRINE-2xPH\u003csup\u003eEVCT2\u003c/sup\u003e was localized at the PM and intracellular compartments in all epidermal cells at the root tips, despite the lack of PS synthesis in these cells (\u003cstrong\u003eFig. 3e and g\u003c/strong\u003e). For example, when PSS1-mCHERRY was expressed in internal tissues of the root meristem, such as cortex (\u003cem\u003epPEP\u003c/em\u003e), endodermis (\u003cem\u003epSCR\u003c/em\u003e), or stele (\u003cem\u003epSHR\u003c/em\u003e), mCITRINE-2xPH\u003csup\u003eEVCT2\u003c/sup\u003e localized at the PM and intracellular compartments in 100% of epidermal cell analyzed (\u003cstrong\u003eFig. 3e-g\u003c/strong\u003e), suggesting that PS diffusion was possible between the concentric tissue layers of the root. Similarly, when PSS1-mCHERRY was expressed in atrichoblast cells (\u003cem\u003epGL2\u003c/em\u003e), mCITRINE-2xPH\u003csup\u003eEVCT2\u003c/sup\u003e localization was rescued in all cells of the epidermis, including non-PS synthetizing trichoblasts cells (\u003cstrong\u003eFig. 3f-g\u003c/strong\u003e), suggesting that PS can also diffuse laterally within the same tissue layer. By contrast to meristematic expression of \u003cem\u003ePSS1\u003c/em\u003e, the expression of PSS1-mCHERRY in differentiated root epidermal cells (under the expression of the \u003cem\u003eEXP7\u003c/em\u003e promoter, \u003cem\u003epEXP7\u003c/em\u003e) or phloem companion cells (phloem CC, under the expression of the \u003cem\u003eSUC2\u003c/em\u003e promoter, \u003cem\u003epSUC2\u003c/em\u003e) did not complement 2xPH\u003csup\u003eEVCT2\u003c/sup\u003e localization, which remained soluble in the root meristem of the \u003cem\u003epss1\u003c/em\u003e mutant (\u003cstrong\u003eFig. 3f-g\u003c/strong\u003e). Taken together, these results indicate that PS can move at relatively short distance within the root tip of Arabidopsis but does not efficiently diffuse out of differentiated tissues or on longer distances.\u003c/p\u003e\n\u003cp\u003eTo make sure that the complementation of 2xPH\u003csup\u003eEVCT2\u003c/sup\u003e localization in non-PS synthetizing cells was not caused by the movement of PSS1 enzyme to neighboring cells, we fused PSS1 with three mCHERRY (PSS1-3xmCHERRY). This larger fusion exceeds the size exclusion limit of plasmodesmata, effectively preventing intercellular movement of the protein\u003csup\u003e31–33\u003c/sup\u003e. We found similar results with \u003cem\u003epSHR::PSS1-3xmCHERRY\u003c/em\u003e than with\u003cem\u003e\u0026nbsp;pSHR::PSS1-mCHERRY\u003c/em\u003e (\u003cstrong\u003eExtended\u003c/strong\u003e \u003cstrong\u003eData Fig. 3b\u003c/strong\u003e), demonstrating that the rescue of 2xPH\u003csup\u003eEVCT2\u003c/sup\u003e localization in the \u003cem\u003epss1\u003c/em\u003e mutant was indeed not triggered by PSS1 protein movement. Together, these results confirm that PS can move away from its site of synthesis to other cells in the Arabidopsis root meristem.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA combination of tissue-specific biosynthesis and diffusion creates the PS localization gradient at the tissue level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur data show that both TopFluor-PC and PS are able to move between cells in the root tip. Given that molecular gradients are frequently established via diffusion from a source, we asked whether intercellular diffusion of PS contributes to the formation of the tissue-scale PS gradient in the root epidermis, a gradient previously reported to be critical for signaling\u003csup\u003e2,5,6\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUsing both transcriptional (\u003cem\u003epPSS1::2xmCITRINE-NLS\u003c/em\u003e) and translational (\u003cem\u003epPSS1::PSS1-mCITRINE\u003c/em\u003e) reporters, we precisely mapped PSS1 expression along the root epidermis. (\u003cstrong\u003eFig. 4a-c\u003c/strong\u003e). Both reporters revealed a progressive increase\u0026nbsp;starting at the end of the meristem and increasing in the transition and elongation zones of the root (\u003cstrong\u003eFig. 4b-c\u003c/strong\u003e). This expression pattern closely aligns with the previously reported PS localization gradient in the epidermis\u003csup\u003e2\u003c/sup\u003e, with the PS sensor \u0026nbsp;2xPH\u003csup\u003eEVCT2\u0026nbsp;\u003c/sup\u003eevenly distributed between the plasma membrane and intracellular compartments in the meristematic zone and progressively shifting its localization predominantly in intracellular compartments in the elongation zone. These results suggest that PS distribution across the epidermis might be determined by the spatial interplay of PSS1-mediated PS synthesis and PS cell-cell diffusion. According to this model, the expression of \u003cem\u003ePSS1\u003c/em\u003e would favor the intracellular localization of PS. By contrast, in meristematic cells that do not synthesize PS due to the lack of \u003cem\u003ePSS1\u003c/em\u003e expression, the main source of PS is diffusion, which would favor plasma membrane accumulation of the lipid.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo test this model, we took advantage of our \u003cem\u003ePSS1\u003c/em\u003e tissue-specific expression lines. We compared the plasma membrane and endosomal accumulation of the PS sensor 2xPH\u003csup\u003eEVCT2\u003c/sup\u003e in the epidermis of the root when \u003cem\u003ePSS1\u003c/em\u003e was expressed in the same cells (i.e., the epidermis, \u003cem\u003epPSS1\u003c/em\u003e) or in neighboring cells (i.e. in the cortex, \u003cem\u003epPEP\u003c/em\u003e). As expected, expression of PSS1-mCHERRY under its endogenous promoter recapitulated the previously reported PS gradient\u003csup\u003e2\u003c/sup\u003e (\u003cstrong\u003eFig.4d\u003c/strong\u003e), with 2xPH\u003csup\u003eEVCT2\u003c/sup\u003e predominantly at the plasma membrane in the meristem, where \u003cem\u003ePSS1\u003c/em\u003e is absent, and accumulation in intracellular compartments in the transition zone, where \u003cem\u003ePSS1\u003c/em\u003e is expressed (\u003cstrong\u003eFig. 4d-e\u003c/strong\u003e). When PSS1-mCHERRY was expressed in the cortex (pPEP::PSS1-mCHERRY/\u003cem\u003epss1\u003c/em\u003e), so that all epidermal cells received PS exclusively via diffusion, 2xPH\u003csup\u003eEVCT2\u003c/sup\u003e showed enhanced plasma membrane localization in both the meristem and transition zones (\u003cstrong\u003eFig. 4d-e\u003c/strong\u003e).Thus, by expressing \u003cem\u003ePSS1\u003c/em\u003e in the cortex, we perturb the establishment of the PS subcellular pattern in another tissue, the epidermis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we did the reverse experiment, and we addressed the impact of ectopically expressing \u003cem\u003ePSS1\u003c/em\u003e in all epidermal cells including the meristematic ones that normally do not express it (using a \u003cem\u003epUBQ10::PSS1-mCHERRY/pss1\u0026nbsp;\u003c/em\u003eline). By doing so, we observed a reverse PS localization pattern with 2xPH\u003csup\u003eEVCT2\u003c/sup\u003e reporter accumulating in intracellular compartments rather than the plasma membrane in the meristematic zone, in contrast to the wild-type situation (\u003cstrong\u003eExtended Data Fig. 4a\u003c/strong\u003e). Hence, producing PS directly in epidermal cells of the meristematic zone, which normally receives the lipid by diffusion, shifted its localization to endosomes (similar to the transition zone, where PS is produced in wild-type conditions). Of note, driving the expression of PSS1 under the \u003cem\u003eUBQ10\u003c/em\u003e promotor not only abolished the PS concentration gradient, but this condition was also showed to perturb the ROP6 response within the root and its downstream signaling\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAltogether, these experiments confirm that PS diffusion favors its plasma membrane accumulation in cells lacking \u003cem\u003ePSS1\u0026nbsp;\u003c/em\u003eexpression, while local PS production drives its endosomal accumulation in the producing cells (\u003cstrong\u003eFig. 4f\u003c/strong\u003e). From these experiments, we concluded that PS diffusion, in conjunction with the tissue and zone-specific expression of the PS biosynthetic enzyme, creates supracellular lipid patterns within plant roots (\u003cstrong\u003eFig. 4f\u003c/strong\u003e) relevant for Rho GTPases regulation at the tissue level\u003csup\u003e2,4\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAlready 20 years ago, Joost Holthuis and Tim Levine hypothesized that the ER functioned “as a passive, long-range, highly efficient, pan-cellular lipid-distribution system in some way analogous to the information superhighway”\u003csup\u003e11\u003c/sup\u003e. Here, we extend this concept to the tissue and organ scales by showing that in plants, the ER is not only a pan-cellular but also a supracellular lipid distribution system. In the case of PS, it remains to be determined whether it uses the ER, the plasma membrane or both as a conduit within plasmodesmata for diffusion. Indeed, PS is made in the lumen of the ER, where it cannot be detected by cytosolic PS sensors. PS could thus move from cell to cell by diffusing within the luminal leaflet of the ER. However, since PS eventually accumulates at the cell surface, PS could also use the plasma membrane as a route to neighboring cells.\u003c/p\u003e\n\u003cp\u003eIt is a common theme in cell biology and for the regulation of lipid homeostasis, in particular, to observe a physical separation between the site of synthesis in the cell and the site of action. This separation offers tremendous opportunity for regulation, both during the production of molecules and also later at their sites of action. Here, we propose that plants take advantage of this concept, but that, thanks to plasmodesmata, they not only use it inside the cells but also between cells. In addition to phospholipids, we predict that plasmodesmata might be used as an intercellular conduit for other hydrophobic molecules such as the brassinosteroid hormones, known to diffuse through plasmodesmata\u003csup\u003e34\u003c/sup\u003e. Furthermore, our finding opens the possibility of direct exchanges of lipids between cells, via other types of membrane-lined cellular junctions, such as tunneling nanotubes in animal cells. \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Jürgen Kleine-Vehn for providing the DERLIN1-mScarlet ER membrane marker, Yasin Dagdas the DDRGK1-mCherry ER membrane marker, Abel Rosado for providing the C4H-GFP ER membrane marker, Marie Barberon for providing the \u003cem\u003ePDR9:iCals3m\u003c/em\u003e lines, Lothar Kalmbach for the \u003cem\u003epLOK180\u003c/em\u003e plasmid gift, Jean Christophe Mulatier for help in preparing lipids, Claire Lionnet for help in microscopy, the SiCE group, Marie-Cécile Caillaud, Ziqiang P. Li and Benoit Landrein for discussion, comments, and technical help, the LBI-PLATIM-MICROSCOPY for assistance with imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (projects 101001097-LIPIDEV to Y.J. and 772103-BRIDGING to E.M.B.), the National Agency for Research (grants ANR-18-CE13-0016 STAYING-TIGHT to E.M.B. and Y.J.), the French Government in the framework of the IdEX Bordeaux University ‘‘Investments for the Future’’ program/GPR Bordeaux Plant Sciences (E.M.B.), and by a EMBO Long-Term Fellowship to C.B. (ALTF 1018-2019). TLC analyses were performed at the Bordeaux Metabolome platform supported by MetaboHub (grant no ANR-11-INBS-0010 to L.F.)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: C.B, E.M.B, Y.J.\u003c/p\u003e\n\u003cp\u003eMethodology: C.B, H.M, V.B, M.P.P, E.M.B, Y.J., L.F.\u003c/p\u003e\n\u003cp\u003eInvestigation: C.B, H.M, V.B, M.P.P\u003c/p\u003e\n\u003cp\u003eVisualization: C.B, H.M\u003c/p\u003e\n\u003cp\u003eFunding acquisition: C.B, E.M.B, Y.J.\u003c/p\u003e\n\u003cp\u003eProject administration: E.M.B, Y.J.\u003c/p\u003e\n\u003cp\u003eSupervision: E.M.B, Y.J.\u003c/p\u003e\n\u003cp\u003eWriting – original draft: C.B, E.M.B, Y.J.\u003c/p\u003e\n\u003cp\u003eWriting – review \u0026amp; editing: C.B, H.M, V.B, M.P.P, E.M.B, Y.J.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e Authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability \u0026amp; correspondence:\u003c/strong\u003e All data are available in the main text or the supplementary materials and all material are available upon request to E.M.B. and Y.J.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePlatre, M. P. \u003cem\u003eet al.\u003c/em\u003e A Combinatorial Lipid Code Shapes the Electrostatic Landscape of Plant Endomembranes. \u003cem\u003eDevelopmental Cell\u003c/em\u003e 1\u0026ndash;28 (2018).\u003c/li\u003e\n\u003cli\u003ePlatre, M. P. \u003cem\u003eet al.\u003c/em\u003e Developmental control of plant Rho GTPase nano-organization by the lipid phosphatidylserine. \u003cem\u003eScience (New York, NY)\u003c/em\u003e \u003cstrong\u003e364\u003c/strong\u003e, 57\u0026ndash;62 (2019).\u003c/li\u003e\n\u003cli\u003eSmokvarska, M. \u003cem\u003eet al.\u003c/em\u003e A Plasma Membrane Nanodomain Ensures Signal Specificity during Osmotic Signaling in Plants. \u003cem\u003eCurrent Biology\u003c/em\u003e 1\u0026ndash;26 (2020).\u003c/li\u003e\n\u003cli\u003eSmokvarska, M. \u003cem\u003eet al.\u003c/em\u003e The receptor kinase FERONIA regulates phosphatidylserine localization at the cell surface to modulate ROP signaling. \u003cem\u003eSci Adv\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, eadd4791 (2023).\u003c/li\u003e\n\u003cli\u003eBoutt\u0026eacute;, Y. \u0026amp; Jaillais, Y. Metabolic Cellular Communications: Feedback Mechanisms between Membrane Lipid Homeostasis and Plant Development. \u003cem\u003eDevelopmental Cell\u003c/em\u003e 1\u0026ndash;12 (2020).\u003c/li\u003e\n\u003cli\u003eDubois, G. A. \u0026amp; Jaillais, Y. Anionic phospholipid gradients: an uncharacterized frontier of the plant endomembrane network. \u003cem\u003ePlant Physiology\u003c/em\u003e \u003cstrong\u003e185\u003c/strong\u003e, 577\u0026ndash;592 (2021).\u003c/li\u003e\n\u003cli\u003evan Meer, G., Voelker, D. R. \u0026amp; Feigenson, G. W. Membrane lipids: where they are and how they behave. \u003cem\u003eNature Reviews Molecular Cell Biology\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 112\u0026ndash;124 (2008).\u003c/li\u003e\n\u003cli\u003eBigay, J. \u0026amp; Antonny, B. Curvature, Lipid Packing, and Electrostatics of Membrane Organelles: Defining Cellular Territories in Determining Specificity. \u003cem\u003eDevelopmental Cell\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 886\u0026ndash;895 (2012).\u003c/li\u003e\n\u003cli\u003eHolthuis, J. C. M. \u0026amp; Menon, A. K. Lipid landscapes and pipelines in membrane homeostasis. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e510\u003c/strong\u003e, 48\u0026ndash;57 (2014).\u003c/li\u003e\n\u003cli\u003eIglesias-Artola, J. M. \u003cem\u003eet al.\u003c/em\u003e Quantitative imaging of lipid transport in mammalian cells. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e646\u003c/strong\u003e, 474\u0026ndash;482 (2025).\u003c/li\u003e\n\u003cli\u003eHolthuis, J. C. M. \u0026amp; Levine, T. P. Lipid traffic: floppy drives and a superhighway. \u003cem\u003eNature Reviews Molecular Cell Biology\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 209\u0026ndash;220 (2005).\u003c/li\u003e\n\u003cli\u003eWong, L. H., Gatta, A. T. \u0026amp; Levine, T. P. Lipid transfer proteins: the lipid commute via shuttles, bridges and tubes. \u003cem\u003eNature Reviews Molecular Cell Biology\u003c/em\u003e 1\u0026ndash;17 (2018).\u003c/li\u003e\n\u003cli\u003eYeung, T. \u003cem\u003eet al.\u003c/em\u003e Membrane phosphatidylserine regulates surface charge and protein localization. \u003cem\u003eScience (New York, NY)\u003c/em\u003e \u003cstrong\u003e319\u003c/strong\u003e, 210\u0026ndash;213 (2008).\u003c/li\u003e\n\u003cli\u003eSmokvarska, M., Jaillais, Y. \u0026amp; Martini\u0026egrave;re, A. Function of membrane domains in Rho-Of-Plant signaling. \u003cem\u003ePLANT PHYSIOLOGY\u003c/em\u003e (2021).\u003c/li\u003e\n\u003cli\u003eGorgues, L. \u003cem\u003eet al.\u003c/em\u003e GEF14 acts as a specific activator of the plant osmotic signaling pathway by controlling ROP6 nanodomain formation. \u003cem\u003eEMBO Rep\u003c/em\u003e https://doi.org/10.1038/s44319-025-00412-w (2025) doi:10.1038/s44319-025-00412-w.\u003c/li\u003e\n\u003cli\u003ePan, X. \u003cem\u003eet al.\u003c/em\u003e Auxin-induced signaling protein nanoclustering contributes to cell polarity formation. \u003cem\u003eNature Communications\u003c/em\u003e 1\u0026ndash;14 (2020).\u003c/li\u003e\n\u003cli\u003eBayer, E. M. \u0026amp; Benitez-Alfonso, Y. Plasmodesmata: Channels Under Pressure. \u003cem\u003eAnnual Review of Plant Biology\u003c/em\u003e \u003cstrong\u003e75\u003c/strong\u003e, 291\u0026ndash;317 (2024).\u003c/li\u003e\n\u003cli\u003eTee, E. E. \u0026amp; Faulkner, C. Plasmodesmata and intercellular molecular traffic control. \u003cem\u003eNew Phytologist\u003c/em\u003e \u003cstrong\u003e243\u003c/strong\u003e, 32\u0026ndash;47 (2024).\u003c/li\u003e\n\u003cli\u003eB\u0026eacute;ziat, C. \u0026amp; Jaillais, Y. Should I stay or should I go: the functional importance and regulation of lipid diffusion in biological membranes. \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e erad032 (2023) doi:10.1093/jxb/erad032.\u003c/li\u003e\n\u003cli\u003eKay, J. G., Koivusalo, M., Ma, X., Wohland, T. \u0026amp; Grinstein, S. Phosphatidylserine dynamics in cellular membranes. \u003cem\u003eMolecular biology of the cell\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 2198\u0026ndash;2212 (2012).\u003c/li\u003e\n\u003cli\u003eYamaoka, Y. \u003cem\u003eet al.\u003c/em\u003e PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana. \u003cem\u003eThe Plant Journal\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 648\u0026ndash;661 (2011).\u003c/li\u003e\n\u003cli\u003eColin, L. A. \u0026amp; Jaillais, Y. Phospholipids across scales: lipid patterns and plant development. \u003cem\u003eCurr Opin Plant Biol\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 1\u0026ndash;9 (2019).\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;rez-Sancho, J. \u003cem\u003eet al.\u003c/em\u003e Plasmodesmata act as unconventional membrane contact sites regulating intercellular molecular exchange in plants. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e188\u003c/strong\u003e, 958-977.e23 (2025).\u003c/li\u003e\n\u003cli\u003eNicolas, W. J. \u003cem\u003eet al.\u003c/em\u003e Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves. \u003cem\u003eNature Plants\u003c/em\u003e 1\u0026ndash;11 (2017).\u003c/li\u003e\n\u003cli\u003eGrabski, S., De Feijter, A. W. \u0026amp; Schindler, M. Endoplasmic Reticulum Forms a Dynamic Continuum for Lipid Diffusion between Contiguous Soybean Root Cells. \u003cem\u003eTHE PLANT CELL ONLINE\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 25\u0026ndash;38 (1993).\u003c/li\u003e\n\u003cli\u003eZhu, T., Lucas, W. J. \u0026amp; Rost, T. L. Directional cell-to-cell communication in the Arabidopsis root apical meristem I. An ultrastructural and functional analysis. \u003cem\u003eProtoplasma\u003c/em\u003e \u003cstrong\u003e203\u003c/strong\u003e, 35\u0026ndash;47 (1998).\u003c/li\u003e\n\u003cli\u003eVat\u0026eacute;n, A. \u003cem\u003eet al.\u003c/em\u003e Callose Biosynthesis Regulates Symplastic Trafficking during Root Development. \u003cem\u003eDevelopmental Cell\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 1144\u0026ndash;1155 (2011).\u003c/li\u003e\n\u003cli\u003eLi, Z. P. \u003cem\u003eet al.\u003c/em\u003e Plant plasmodesmata bridges form through ER-dependent incomplete cytokinesis. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e386\u003c/strong\u003e, 538\u0026ndash;545 (2024).\u003c/li\u003e\n\u003cli\u003eBrault, M. L. \u003cem\u003eet al.\u003c/em\u003e Multiple C2 domains and transmembrane region proteins ( MCTPs) tether membranes at plasmodesmata. \u003cem\u003eEMBO reports\u003c/em\u003e \u003cstrong\u003e172\u003c/strong\u003e, 1061 (2019).\u003c/li\u003e\n\u003cli\u003ePlatre, M. P. \u0026amp; Jaillais, Y. Exogenous treatment of Arabidopsis seedlings with lyso-phospholipids for the inducible complementation of lipid mutants. \u003cem\u003eSTAR Protocols\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 100626 (2021).\u003c/li\u003e\n\u003cli\u003eWu, X. \u003cem\u003eet al.\u003c/em\u003e Modes of intercellular transcription factor movement in the \u003cem\u003eArabidopsis\u003c/em\u003e apex. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e130\u003c/strong\u003e, 3735\u0026ndash;3745 (2003).\u003c/li\u003e\n\u003cli\u003eKim, I., Cho, E., Crawford, K., Hempel, F. D. \u0026amp; Zambryski, P. C. Cell-to-cell movement of GFP during embryogenesis and early seedling development in Arabidopsis. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e102\u003c/strong\u003e, 2227\u0026ndash;2231 (2005).\u003c/li\u003e\n\u003cli\u003eKim, J. Y., Yuan, Z., Cilia, M., Khalfan-Jagani, Z. \u0026amp; Jackson, D. Intercellular trafficking of a KNOTTED1 green fluorescent protein fusion in the leaf and shoot meristem of Arabidopsis. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 4103\u0026ndash;4108 (2002).\u003c/li\u003e\n\u003cli\u003eWang, Y. \u003cem\u003eet al.\u003c/em\u003e Plasmodesmata mediate cell-to-cell transport of brassinosteroid hormones. \u003cem\u003eNat Chem Biol\u003c/em\u003e 1\u0026ndash;11 (2023) doi:10.1038/s41589-023-01346-x.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8163315/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8163315/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The lipid composition of biological membranes dictates cellular states and functions, yet how cells coordinate their lipid profiles across tissues remains unclear. In plant roots, the lipid phosphatidylserine forms a tissue-scale localization gradient that regulates Rho GTPase signaling in response to auxin and osmotic stress1–6. How this gradient emerges is unknown. Here, we show that direct phospholipid exchange between cells coordinates these lipid patterns. Combining in vivo phospholipid phototracing and localized induction of phosphatidylserine synthesis, we found that lipids move between cells through the numerous plasmodesmata-membrane bridges connecting them. The phosphatidylserine gradient arises from local synthesis by PHOSPHATIDYLSERINE SYNTHASE1 within a restricted epidermal zone, combined with intercellular phosphatidylserine diffusion. Producing and receiving cells exhibit distinct subcellular phosphatidylserine localization patterns, together generating the tissue-scale lipid gradient. These results reveal that direct cell-cell membrane connections act as conduits for lipid flow, coordinating membrane composition and signaling capacity across tissues.","manuscriptTitle":"Intercellular lipid flow coordinates tissue-scale lipid gradients in plants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-27 11:48:56","doi":"10.21203/rs.3.rs-8163315/v1","editorialEvents":[],"status":"published","journal":{"display":false,"email":"[email protected]","identity":"nature","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nature","sideBox":"Learn more about [Nature](http://www.nature.com/nature/)","snPcode":"","submissionUrl":"","title":"Nature","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"08b0a9bb-8d32-4b79-8c7f-e3167d4f166b","owner":[],"postedDate":"November 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58643805,"name":"Biological sciences/Plant sciences/Plant cell biology"},{"id":58643806,"name":"Biological sciences/Plant sciences/Plant cell biology/Protein trafficking in plants"},{"id":58643807,"name":"Biological sciences/Cell biology/Organelles/Endoplasmic reticulum"}],"tags":[],"updatedAt":"2026-01-08T15:26:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-27 11:48:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8163315","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8163315","identity":"rs-8163315","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}