1 Title: Symplastic communication in organ formation and tissue patterning. Authors: Sofia Oterob Yrjo Helariuttab,c Yoselin Benitez-Alfonsoa a Centre for Plant Sciences, School of Biology, University of Leeds, Leeds, LS2 9JT. UK. b Sainsbury Laboratory, University of Cambridge, Bateman Street, Cambridge, CB2 1LR. UK. c Institute of Biotechnology, University of Helsinki, PO Box 65, Helsinki, FIN-00014. Finland. Corresponding author: Yoselin Benitez-Alfonso Tel: 44-113-343 2811 email: y.benitez-alfonso@leeds.ac.uk Short title: Role of plasmodesmata in organ development 2 Abstract Communication between cells is a crucial step to coordinate organ formation and tissue patterning. In plants, the intercellular transport of metabolites and signalling molecules occur symplastically through membranous structures (named plasmodesmata) that traverse the cell wall to connect the cytoplasm and endoplasmic reticulum of neighbouring cells. This review aims to highlight the importance of symplastic communication in plant development. We revisit current literature reporting the effects of changing plasmodesmata in cell morphogenesis, organ initiation and meristem maintenance and comment on recent work involving the identification of novel plasmodesmata regulators and of mobile developmental proteins and RNA molecules. New opportunities for unravelling the dynamic regulation and function of plasmodesmata are also discussed. Introduction At the end of the XIX century, plants were thought to be a mere aggregation of isolated cells; however, Eduard Tangl completely shifted the paradigm when he observed cytoplasmic intercellular connections in cotyledons of the tree Strychnos nux-vomica [1]. His discovery demonstrated that, despite the cell wall, plant cells communicate to each other and form higher order structures that characterize organisms. Some years later, in 1901, Strasburger named these connections plasmodesmata (PD), etymologically fluid bonds [1,2]. In simple terms, PD are channels made of plasma membrane (PM) that provide cytoplasmic and membranous continuity between neighbouring cells forming the symplasm (Figure 1A). Microscopically these channels appear as concentric cylinders, due to the presence of the desmotubule (DT), a structure derived from endoplasmic reticulum (ER) that becomes trapped in the middle of the channel during cytokinesis [3,4]. Symplastic molecular transport mainly occurs through the cytoplasmic sleeve: the space left between the PM and the DT. Alternative methods for transport can be proposed including diffusion in the ER/DT lumen and lateral segregation of proteins in the PM and ER membrane but their contribution to symplastic intercellular communication is not well defined [5-7] (Figure 1A). The capacity of molecules to move through PD cytoplasmic sleeve depends on their size and shape and on the cell type and/or developmental stage where they appear. This is because PD number and size exclusion limit (SEL, the maximum molecular 3 size allowed through any specific pore) are developmentally (and environmentally) regulated [8]. Symplastic molecular transport is extremely important in the phloem, where PD connect companion cells (CC), sieve elements (SE) and the surrounding tissues to regulate communication of metabolites and signals between distant organs [9]. It is also essential in the meristems where transcription factors (and other signalling molecules) move to determine cell fate and tissue development [10,11]. Here we summarize information from recent papers supporting the role of PD in organ formation, meristem development and tissue patterning. Three current topics will be discussed: the regulation of PD during organ development, the role of PD- located receptor proteins in this process and the identification of novel mobile developmental regulators (non-cell autonomous proteins and RNA molecules). Plasmodesmata regulation during organ formation and vascular patterning As a key factor in cell-to-cell communication, PD-cytoplasmic aperture is tightly regulated (Figure 1). This is, at least partly, achieved by modifications of the surrounding cell walls, which have different composition (and properties) in the microdomains that are in contact with PD, such as enrichment in certain pectic polysaccharides and callose [4]. Callose (a β-1,3-glucan polymer) is found delimiting PD sites and its accumulation greatly influences transport through the channel by imposing physical constrictions on PD-cytoplasmic aperture (Figure 1B). Callose synthases (CALS) and β-1,3 glucanases (PdBG) that localize at PD sites were identified, providing the machinery for dynamic regulation of callose turnover in situ. Altering the expression of these enzymes affects PD transport capacity, thus cell-to- cell symplastic connectivity (Figure 1B). Indeed, gain-of-function mutations in CALS3 trigger an excessive accumulation of callose at PD impairing root organ development by blocking the transport of transcription factors and miRNAs (such as SHORT- ROOT and microRNA165) that determine the correct formation of the vascular tissue [12]. Further studies using transgenic lines expressing a strongly activated version of CALS3 in specific tissue types and under inducible promoters reveal the importance of callose regulation in the formation of lateral roots and in the transport of hormones involved in vascular patterning and meristem maintenance [12-15]. Characterization of loss-of-function mutations and RNAi lines in CALS10 and CALS7 support the role of callose, and PD, in organ formation and patterning. Mutants in CALS10 (also known as Glucan-Synthase-Like 8 or GSL8) display stomata clustering, presumably 4 due to the unrestricted mobility of the bHLH transcription factor SPEECHLESS (SPCH) which controls asymmetric cell division in leaves to establish stomata cell fate [16,17]. Separate research, using an inducible RNAi line, identified the role of GSL8 in hypocotyl bending in response to phototropism, a phenotype associated with auxin gradient formation [18]. On the other hand, decreasing CALS7 expression affects the formation of sieve pores: a special type of enlarged PD found at the cell plate of adjacent SE, leading to defective phloem transport, reduced seedling height and aborted embryos among other defects [19,20]. Similarly, a CALS mutant in maize, named tie-dyed2 (tdy-2), is affected in vascular development, specifically in the connections between CC and SE [21]. Correspondingly, phloem export is blocked leading to an increase in the accumulation of starch and sucrose in leaves. The importance of PdBG in callose regulation during organ formation and vascular patterning was also revealed through the analysis of mutant phenotypes [14,22,23]. Mutants in PdBG1 and PdBG2 (pdbg1,2) are affected in root development showing abnormal clustering of lateral root primordia in Arabidopsis [14]. Orthologous genes in Populus are regulated by photoperiod, chilling and gibberellins and are involved in the opening of PD for the transport of the FLOWERING LOCUS T (FT), a protein that regulates flowering but also dormancy release at the shoot apex in poplar [24]. PdBG are attached to the PD- PM subdomains by a glycosylphosphatidylinositol (GPI) anchor. These PD- PM subdomains are enriched in sterol and highly glycosylated sphingolipids [25]. Altering this composition, using inhibitors of sterol biosynthesis, was shown to affect PdBG localization, increase callose, decrease symplastic transport and to impair root meristem development. In addition to callose, other factors/activities can strongly influence PD transport during organ development. Some of these factors emerged from the analysis of the PD proteome [26] while others were identified in independent studies. For example, the GERMIN-LIKE PROTEIN 1 (GLP1), identified by immunoprecipitation with the non-cell autonomous Phloem Protein 16 from Cucurbita maxima (CmPP16), was found to regulate PD permeability in Arabidopsis [27]. Expressing tagged versions of these proteins in Arabidopsis (named PDGLP1 and PDGLP2) results in short meristems, reduced primary root growth and increased lateral root length, suggesting defects in the relative distribution of photosynthates between primary and lateral roots [27]. 5 In a separate approach, Vilaine and collaborators reported the identification of NHL26, a phloem-specific protein that localizes to PD [28]. Overexpression (OE) of NHL26 reduces root and seed biomass, increases fresh weight of rosette leaves and delays growth, senescence and flowering [28]. These plants also accumulate sugars in mature source leaves which correlate with a reduction of these metabolites in phloem sap. The evidences suggest a function for NHL26 in controlling sugar export between CC and the SE through regulating PD permeability [27]. Also influencing phloem export, overexpression of the PD-located rice gene Grain Setting Defect1 (GSD1) causes sugar accumulation in leaves and reduces panicle size and grain setting [29]. GSD1 encodes a putative remorin, which are plant- specific proteins of unknown function that appear attached to PM lipid-raft domains and enriched around PD in Solanaceae [30]. Remorins encode conserved coiled-coil domains presumably involved in protein-protein interactions. The research indicates that GSD1 interacts with other PD proteins, including Actin 1 (ACT1), to regulate PD permeability and phloem transport. Another protein influencing phloem transport is the choline transporter-like protein, CHER1 [31]. CHER1 localizes to the trans-Golgi network, the cell plate and polarly at the incipient sieve plates during early SE differentiation. The mutant cher1 displays a short root phenotype characterized by a short meristem size. This is accompanied by blocked connectivity between the protophloem and the root meristem, discontinuous phloem differentiation, longer retention of the desmotubule in the pores, reduced sieve plate area and decreased pore density [31]. The mechanism underlying these effects is unknown but the results point to CHER1 as one of the major regulators in sieve pore formation, a process that involves PD modifications. Taken together the research demonstrates the importance of PD regulation in organ formation, vascular patterning and in the phloem transport of resources that determine the development of sink (developing) and source (photosynthetic) tissues. The mechanisms controlling PD aperture during organ formation and vascular patterning are mostly unknown but require the participation of multiple proteins, that either alone or assembled, modify PD structure, transport capacity and/or their differentiation into sieve pores. Receptor proteins act at plasmodesmata to regulate organ development 6 Research on receptor proteins that target and/or interact at PD to function in developmental signalling is gaining momentum [32]. Analysis of the PD proteome in Arabidopsis identified three receptor-like kinases (RLKs) and a number of receptor- like proteins including the PD-Located Proteins (PDLPs) and the chitin receptor-like protein LYM2 [26 ,32]. In rice, a candidate gene approach was undertaken to investigate the cell wall proteome leading to the identification of 15 putative RLKs, six of which were confirmed to target PD using fluorescent tagging assays [33]. Although most of the research in PD-located receptor proteins focuses on their function in plant-pathogen interactions, new cumulative data support their role in development. For example, PDLP overexpression impairs plant growth and recent work links this receptor to the regulation of callose at PD [34-36]. The Arabidopsis RLK protein STRUBBELIG (SUB), involved in organ formation and tissue morphogenesis, also localizes at PD, where it physically interacts with the protein QUIRKY (QKY) to act non-cell autonomously in the regulation of flower shape, leaf symmetry and integument development [37]. QKY homolog FT- INTERACTING PROTEIN 1 (FTIP1) also accumulates at PD in phloem cells and regulates the transport of the flowering signal FT [38]. Interaction of receptor proteins is also proposed as a mechanism to regulate the PD transport of factors maintaining stem cell fate in the apical meristems (Figure 2). In the shoot apical meristem (SAM), the receptor protein CLAVATA 1 (CLV1) is activated by the small peptide CLV3 which act as a signalling ligand to regulate the expression domain of the mobile stem cell transcription factor WUSCHEL (WUS) [39,40]. In the root meristem, the PD-located receptor kinase CRINKLY4 (ACR4 in Arabidopsis) interacts with CLV1 upon perception of the CLV3-like peptide CLE40p to trigger differentiation of the columella [41]. Although the mechanism is unknown, the results suggest that the formation of ACR4/CLV1 complexes restrict the movement of developmental regulators (WUS-like factors) necessary to maintain stem cell fate [41]. This elegant research highlights the similarities in the mechanisms that regulate intercellular signalling, stem cell maintenance and differentiation in roots and shoots and the importance of PD-located receptor proteins in this process. The model proposed in Figure 2 aims to illustrate this idea: signals that move in the apoplast bind specific receptors that interact at PD to modulate the symplastic transport of proteins that determine cell fate during organ development. Work undertaken by different research groups aims to identify the 7 signalling ligands, the receptors proteins and the mobile factors involved in this mechanism. Identification and developmental function of novel mobile proteins and RNAs Defects in PD structure and connectivity restrict the cell-to-cell transport of transcription factors and a range of RNA molecules (messenger RNAs (mRNAs), short interference RNAs (siRNA), microRNAs (miRNAs) and trans-acting siRNAs (TasiRNAs)) that coordinate development (for a recent review consult [42-44]). The list of transcriptional and signalling factors likely transported through PD is continuously growing (Table 1). Reports demonstrating the requirement of proper PD regulation for the transport of well characterized transcription factors, such as SHORTROOT (SHR) [12,45-47], are added to new studies suggesting the intercellular mobility of other important developmental regulators, such as ANGUSTIFOLIA3 (AN3) which moves from the mesophyll to control epidermal cell proliferation in leaves [48]. A genomic screen of transcription factors in Arabidopsis identified 22 mobile factors distributed within the homeobox, GRAS, and MYB families [49]. Further characterization of the Dof transcription factor AtDof4.1, isolated in this screen, supports intercellular transport through PD and identified a small motif sufficient to confer mobility to otherwise cell-autonomous proteins [50]. New evidence also suggests the intercellular mobility of PLETHORA2 (PLT2), an auxin-induced transcription factor necessary to establish the different developmental domains in Arabidopsis root [51]. Using a combination of modelling and experimental approaches, the authors propose that intercellular movement of PLT2, and dilution of its concentration due to root growth, are both necessary to generate a longitudinal gradient that defines root zonation [51]. Related to vascular development, Zhou et al. (2013) identified two new transcription factors (AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 3 and 4, AHL3/AHL4) that move and interact in the stele to regulate non-cell autonomously the development of the xylem [52]. More knowledge has been gathered on the structural requirements for protein trafficking through PD. In the SAM, the transport of WUS was found to be an intrinsic property (independent of the cellular context) that is coded in multiple domains of the protein [40]. Conversely, the intercellular transport of the maize protein KNOTTED1 (KN1), and its ortholog in Arabidopsis STM, involved in SAM maintenance, is determined by specific signatures present in the homeodomain sequence [53,54]. 8 Concretely, two evolutionary conserved surface residues, an arginine and a leucine, were found essential for intercellular transport [54]. In addition, a chaperonin complex was identified as crucial to refold homeodomain proteins after translocation and this mechanism also seems involved in regulating the transport of viral movement proteins [55,56]. The importance of mobile RNA molecules in tissue patterning and organ development emerged from recent publications. Thousands of transcripts moving in the phloem of Arabidopsis and other plant species were identified using different strategies (such as grafting, translocation of RNA between host and parasitic plants and phloem sap analysis) [57-61]. These phloem RNA molecules move long- distances and between CC and SE, by diffusion or in complex with RNA-binding proteins, to regulate development in target tissues. However questions remain regarding the selectivity for phloem translocation or the final destination of these transcripts [57]. The transport of miRNA and siRNA molecules can also be phloem independent [62]. Research on miR394 suggests that it moves from the L1 layer of the SAM to the inner stem cell layers to repress LEAF CURLING RESPONSIVENESS (LCR, a gene involved in leaf and shoot meristem development) acting as a positional cue to maintain shoot stem cell activity [63,64]. Also important for patterning, miR165/166 move between cell layers in embryos and root meristem to regulate CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIP III) proteins [47,65]. In turn, tasiR-ARF, a trans-acting siRNA that targets AUXIN RESPONSE FACTOR 3 (ARF3) and ARF4, diffuses from the adaxial to the abaxial side to establish leaf polarity [62,66]. siRNA can also move long-distances in a phloem-independent pathway from root to shoot [67]. When produced in roots, siRNA moves to the shoot by a combination of short-range cell-to-cell communication events and amplification of the signal in all cells en route. Interestingly, the spreading of the silencing signal is affected in mutants in a hydrogen peroxide (H2O2)-producing type III peroxidase (named RCI3) concordant with previous research indicating the role of H2O2 on regulating PD permeability [68]. Together, the findings support the involvement of PD in the transport of siRNA and their role in developmental signalling [67]. Conclusions and future perspectives 9 The role of PD in organ development and tissue patterning is now well-established. However, knowledge on the mechanisms regulating PD structure and transport capacity and on the specific signatures/modifications that determine the mobility of developmental factors is still sparse. Protein-protein interactions at PD sites and crosstalk between the symplastic and the apoplastic pathway for molecular transport are proposed to occur in the apical meristems to regulate stem cell fate and organogenesis. Similar mechanisms might play a role in other developmental and morphogenetic processes such as the post-embryonic initiation of secondary meristems and meristemoids. An effective pathway to regulate development is through modifications in hormonal transport. PD mediates the phloem- transport of cytokinins [14] but their contribution to the establishment of auxin gradients is still under discussion. Research using moss and a combination of computational and experimental approaches, has shed some light on this conundrum [69]. In Physcomitrella, bi-directional diffusion of auxin through PD is required to generate realistic branching patterns in silico and altering PD connectivity (by inhibition of callose) is sufficient to inhibit branching in vivo. How conserved is this mechanism in higher plants is still unknown but mosses emerged from these studies as a suitable system to understand PD roles in development [70]. Independent lines of research demonstrate the importance of PD in regulating auxin response in planta. Grafting experiments between Arabidopsis and Nicotiana benthamiana, showed that Aux/IAA transcripts (key regulators of auxin-responsive genes) move from mature leaves to roots, to regulate the initiation of lateral roots [71]. In addition, the phloem transport of Cyclophilin 1 (Cyp1) from a wildtype scion to a mutant rootstock restores auxin signalling and lateral root development in the tomato diageotropica (dgt) mutant which is normally auxin insensitive. This effect is dependent on light intensity suggesting that movement of Cyp1 can be involved in coordinating shoot-root relations in response to the plant photosynthetic status [72]. Experimental approaches combined with predictions from mathematical simulations are useful for determining the significance of sRNA and protein movement in developmental signalling. Using these approaches, it was shown that a gradient in the distribution of miR165/166 can be translated into sharp boundaries in the expression of its target PHABULOSA (PHB) to determine vascular pattern [73]. Computational models to predict the importance of PD in the establishment of small molecular gradients have also been formulated but quantitative data to test these 10 models are still missing [44,68]. Advances in microscopy techniques to determine PD transport parameters, the phenotypic analysis of mutants in PD form and function and the discovery of tools to modify PD permeability will be essential to make progress in this field. Acknowledgements Research work on Y.B-A laboratory is funded by grants EP/MO27740/1 from the EPSRC. Y. H laboratory is funded by the Academy of Finland Centre of Excellence programme, the Gatsby Foundation, University of Helsinki, the European Research Council Advanced Investigator Grant Symdev (No. 323052) and Tekes (the Finnish Funding Agency for Technology and Innovation). 11 Table 1. List of mobile proteins (blue shaded cells) and sRNAs (in green) studied in the last five years with a function in organ development and patterning. Mobile factor1 Direction of movement Function References2 AHL3/AHL4 From procambium cells to the xylem Xylem specification [52] PLT2 Longitudinally from the root meristem forming a gradient Longitudinal root zonation [51],[74] SHR From the stele into the endodermis Ground tissue formation [12],[45],[46] WUS From the organizing centre to L1, L2 layers in the shoot Meristem maintenance [40] FT From leaf and cotyledons to the SAM Transition to flowering [38] SPCH Cell-to-cell diffusion in the leaf epidermis of chorus Stomata cell fate [16] AtDof4.1 From pericycle to endodermis/cortex in roots Unknown [50] KN1/STM Broadly in the SAM Meristem maintenance [54] AN3 From the mesophyll to the epidermis in leaves Leaf development [48] Cyp1 From leaves to root in tomato Regulation of root growth [70] miR165/6 From endodermis into the stele Xylem specification [47],[65] miR394 From L1 into inner layers in the shoot meristem Meristem maintenance [63,64] tasiR-ARF From the adaxial to the abaxial side of the leaf Establishment of leaf polarity [66] IAA18 and IAA28 mRNA From mature leaves to roots Lateral root formation [69] Artificial siRNA From root to shoot Gene silencing [75] 1Full name of mobile factors is provided in the text. 2Citations are included in the reference list. 12 Figure Caption Figure 1. Transport pathways and PD regulation by callose. (A) Intercellular transport occur through PD cytoplasmic sleeve (black arrows), by diffusion in the lumen of the endoplasmic reticulum (ER) and the desmotubule (DT) (orange arrows) and, potentially, by lateral segregation in the membranes (green discontinuous arrows). Plasma membrane (PM), cell wall (CW) and PD-cytoplasmic aperture (in discontinuous blue) are indicated. (B) PD transport is regulated by the deposition of callose in the surrounding cell wall. Callose is produced at PD sites from UDP- glucose by callose synthases (CALS) and degraded to glucose subunits by PD- located beta-1,3 glucanases (PdBG). Callose turnover depends on the activity of these enzymes. High levels of callose restricts PD-cytoplasmic aperture blocking molecular transport thus cell-to-cell symplastic connectivity. Figure 2. Hypothetical model that illustrates the role of receptor proteins in regulating PD connectivity and stem cell differentiation. Open PD allows the transport of developmental proteins such as transcription factors involved in stem cell fate specification. Upon perception of signalling molecules specific PD-located receptors (such as ACR4) and membrane RLKs (such as CLV1) relocate at PD proximity and form complexes that trigger a cascade of unknown events (discontinuous arrows) that either modify protein movement capacity or PD aperture (potentially through changing callose as in Figure 1B). Restricted intercellular communication (in red) of stem cell factors leads to the activation of the stem cell differentiation program. Reference and recommended reading Papers of special interest (*) or outstanding interest (**) are highlighted. 1. Kokowski M: The Global and the local: the history of science and the cultural integration of Europe. TProceedings of the 2nd ICESHS 2006. 2. Oparka KJ, Roberts AG: Plasmodesmata. A not so open-and-shut case. Plant Physiol 2001, 125:123-126. 13 3. Knox K, Wang P, Kriechbaumer V, Tilsner J, Frigerio L, Sparkes I, Hawes C, Oparka K: Putting the Squeeze on Plasmodesmata: A Role for Reticulons in Primary Plasmodesmata Formation. Plant Physiol 2015, 168:1563-1572. ** This elegant piece of work characterizes, for the first time, the role of reticulons in the formation of the desmotubule during PD biogenesis in primary cell walls. Two members of this family of proteins were found to move in developing cell plate to determine the position where new PD is formed. 4. Knox JP, Benitez-Alfonso Y: Roles and regulation of plant cell walls surrounding plasmodesmata. Curr Opin Plant Biol 2014, 22:93-100. 5. Barton DA, Cole L, Collings DA, Liu DY, Smith PM, Day DA, Overall RL: Cell- to-cell transport via the lumen of the endoplasmic reticulum. Plant J 2011, 66:806-817. 6. Brunkard JO, Runkel AM, Zambryski PC: The cytosol must flow: intercellular transport through plasmodesmata. Curr Opin Cell Biol 2015, 35:13-20. 7. Boutte Y, Moreau P: Plasma membrane partitioning: from macro-domains to new views on plasmodesmata. Front Plant Sci 2014, 5:128. 8. Sevilem I, Yadav SR, Helariutta Y: Plasmodesmata: channels for intercellular signaling during plant growth and development. Methods Mol Biol 2015, 1217:3-24. 9. Heo JO, Roszak P, Furuta KM, Helariutta Y: Phloem development: current knowledge and future perspectives. Am J Bot 2014, 101:1393-1402. 10. Benitez-Alfonso Y: Symplastic intercellular transport from a developmental perspective. J Exp Bot 2014, 65:1857-1863. 11. Jackson D: Plasmodesmata spread their influence. F1000Prime Rep 2015, 7:25. 12. Vaten A, Dettmer J, Wu S, Stierhof YD, Miyashima S, Yadav SR, Roberts CJ, Campilho A, Bulone V, Lichtenberger R, et al.: Callose biosynthesis regulates symplastic trafficking during root development. Dev Cell 2011, 21:1144-1155. 13. Sevilem I, Miyashima S, Helariutta Y: Cell-to-cell communication via plasmodesmata in vascular plants. Cell Adh Migr 2013, 7:27-32. 14. Benitez-Alfonso Y, Faulkner C, Pendle A, Miyashima S, Helariutta Y, Maule A: Symplastic intercellular connectivity regulates lateral root patterning. Dev Cell 2013, 26:136-147. 14 15. Bishopp A, Lehesranta S, Vaten A, Help H, El-Showk S, Scheres B, Helariutta K, Mahonen AP, Sakakibara H, Helariutta Y: Phloem-transported cytokinin regulates polar auxin transport and maintains vascular pattern in the root meristem. Curr Biol 2011, 21:927-932. 16. Guseman JM, Lee JS, Bogenschutz NL, Peterson KM, Virata RE, Xie B, Kanaoka MM, Hong Z, Torii KU: Dysregulation of cell-to-cell connectivity and stomatal patterning by loss-of-function mutation in Arabidopsis chorus (glucan synthase-like 8). Development 2010, 137:1731-1741. 17. De Storme N, De Schrijver J, Van Criekinge W, Wewer V, Dormann P, Geelen D: GLUCAN SYNTHASE-LIKE8 and STEROL METHYLTRANSFERASE2 are required for ploidy consistency of the sexual reproduction system in Arabidopsis. Plant Cell 2013, 25:387-403. 18. Han X, Hyun TK, Zhang M, Kumar R, Koh EJ, Kang BH, Lucas WJ, Kim JY: Auxin-callose-mediated plasmodesmal gating is essential for tropic auxin gradient formation and signaling. Dev Cell 2014, 28:132-146. 19. Xie B, Wang X, Zhu M, Zhang Z, Hong Z: CalS7 encodes a callose synthase responsible for callose deposition in the phloem. Plant J 2011, 65:1-14. 20. Barratt DH, Kolling K, Graf A, Pike M, Calder G, Findlay K, Zeeman SC, Smith AM: Callose synthase GSL7 is necessary for normal phloem transport and inflorescence growth in Arabidopsis. Plant Physiol 2011, 155:328-341. 21. Slewinski TL, Baker RF, Stubert A, Braun DM: Tie-dyed2 encodes a callose synthase that functions in vein development and affects symplastic trafficking within the phloem of maize leaves. Plant Physiol 2012, 160:1540-1550. 22. Levy A, Erlanger M, Rosenthal M, Epel BL: A plasmodesmata-associated beta-1,3-glucanase in Arabidopsis. Plant J 2007, 49:669-682. 23. Zavaliev R, Levy A, Gera A, Epel BL: Subcellular dynamics and role of Arabidopsis beta-1,3-glucanases in cell-to-cell movement of tobamoviruses. Mol Plant Microbe Interact 2013, 26:1016-1030. 24. Rinne PL, Welling A, Vahala J, Ripel L, Ruonala R, Kangasjarvi J, van der Schoot C: Chilling of dormant buds hyperinduces FLOWERING LOCUS T and recruits GA-inducible 1,3-beta-glucanases to reopen signal conduits and release dormancy in Populus. Plant Cell 2011, 23:130-146. 25. Grison MS, Brocard L, Fouillen L, Nicolas W, Wewer V, Dormann P, Nacir H, Benitez-Alfonso Y, Claverol S, Germain V, et al.: Specific membrane lipid 15 composition is important for plasmodesmata function in Arabidopsis. Plant Cell 2015, 27:1228-1250. *This manuscript reports, for the first time, the composition of PD membranous microdomains. A high content in sterol and sphingolipids, characteristic of lipid raft domains, distinguishes PD from other PM regions. Inhibition of sterol biosynthesis disturbs the localization and function of PD proteins leading to defective intercellular transport. 26. Fernandez-Calvino L, Faulkner C, Walshaw J, Saalbach G, Bayer E, Benitez-Alfonso Y, Maule A: Arabidopsis plasmodesmal proteome. PLoS One 2011, 6:e18880. 27. Ham BK, Li G, Kang BH, Zeng F, Lucas WJ: Overexpression of Arabidopsis plasmodesmata germin-like proteins disrupts root growth and development. Plant Cell 2012, 24:3630-3648. 28. Vilaine F, Kerchev P, Clement G, Batailler B, Cayla T, Bill L, Gissot L, Dinant S: Increased expression of a phloem membrane protein encoded by NHL26 alters phloem export and sugar partitioning in Arabidopsis. Plant Cell 2013, 25:1689-1708. 29. Gui J, Liu C, Shen J, Li L: Grain setting defect1, encoding a remorin protein, affects the grain setting in rice through regulating plasmodesmatal conductance. Plant Physiol 2014, 166:1463-1478. *A remorin protein, encoded by GSD1, was localized at the PM and the PD of phloem CC in rice. GSD1 regulates PD aperture and the phloem- translocation of photoassimilates which affects grain setting. 30. Raffaele S, Bayer E, Lafarge D, Cluzet S, German Retana S, Boubekeur T, Leborgne-Castel N, Carde JP, Lherminier J, Noirot E, et al.: Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement. Plant Cell 2009, 21:1541-1555. 31. Dettmer J, Ursache R, Campilho A, Miyashima S, Belevich I, O'Regan S, Mullendore DL, Yadav SR, Lanz C, Beverina L, et al.: CHOLINE TRANSPORTER-LIKE1 is required for sieve plate development to mediate long-distance cell-to-cell communication. Nat Commun 2014, 5:4276. 16 *In this work, a member of the choline transporter-like family was identified to be required for proper sieve pore formation during the differentiation of SE. The mutant phenotype suggests that PD structural modifications and phloem connectivity are affected. 32. Faulkner C, Petutschnig E, Benitez-Alfonso Y, Beck M, Robatzek S, Lipka V, Maule AJ: LYM2-dependent chitin perception limits molecular flux via plasmodesmata. Proc Natl Acad Sci U S A 2013, 110:9166-9170. 33. Jo Y, Cho WK, Rim Y, Moon J, Chen XY, Chu H, Kim CY, Park ZY, Lucas WJ, Kim JY: Plasmodesmal receptor-like kinases identified through analysis of rice cell wall extracted proteins. Protoplasma 2011, 248:191-203. 34. Thomas CL, Bayer EM, Ritzenthaler C, Fernandez-Calvino L, Maule AJ: Specific targeting of a plasmodesmal protein affecting cell-to-cell communication. PLoS Biol 2008, 6:e7. 35. Lee JY, Wang X, Cui W, Sager R, Modla S, Czymmek K, Zybaliov B, van Wijk K, Zhang C, Lu H, et al.: A plasmodesmata-localized protein mediates crosstalk between cell-to-cell communication and innate immunity in Arabidopsis. Plant Cell 2011, 23:3353-3373. 36. Caillaud MC, Wirthmueller L, Sklenar J, Findlay K, Piquerez SJ, Jones AM, Robatzek S, Jones JD, Faulkner C: The plasmodesmal protein PDLP1 localises to haustoria-associated membranes during downy mildew infection and regulates callose deposition. PLoS Pathog 2014, 10:e1004496. 37. Vaddepalli P, Herrmann A, Fulton L, Oelschner M, Hillmer S, Stratil TF, Fastner A, Hammes UZ, Ott T, Robinson DG, et al.: The C2-domain protein QUIRKY and the receptor-like kinase STRUBBELIG localize to plasmodesmata and mediate tissue morphogenesis in Arabidopsis thaliana. Development 2014, 141:4139-4148. **The manuscript describes interactions between QKY and the receptor-like kinase SUB both acting non-cell autonomously to regulate flower development. Interestingly both QKY and SUB are found in close contact at PD, suggesting a link between signal transduction and the regulation of cell-to-cell communication through PD. 17 38. Liu L, Liu C, Hou X, Xi W, Shen L, Tao Z, Wang Y, Yu H: FTIP1 is an essential regulator required for florigen transport. PLoS Biol 2012, 10:e1001313. 39. Yadav RK, Perales M, Gruel J, Girke T, Jonsson H, Reddy GV: WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex. Genes Dev 2011, 25:2025-2030. 40. Daum G, Medzihradszky A, Suzaki T, Lohmann JU: A mechanistic framework for noncell autonomous stem cell induction in Arabidopsis. Proc Natl Acad Sci U S A 2014, 111:14619-14624. **The authors show that WUS, a key transcription factor involved in stem cell maintenance in the SAM, moves through PD. The mobility of the protein is encoded in multiple domains of its sequence. 41. Stahl Y, Grabowski S, Bleckmann A, Kuhnemuth R, Weidtkamp-Peters S, Pinto KG, Kirschner GK, Schmid JB, Wink RH, Hulsewede A, et al.: Moderation of Arabidopsis root stemness by CLAVATA1 and ARABIDOPSIS CRINKLY4 receptor kinase complexes. Curr Biol 2013, 23:362-371. 42. Gallagher KL, Sozzani R, Lee CM: Intercellular protein movement: deciphering the language of development. Annu Rev Cell Dev Biol 2014, 30:207-233. 43. Han X, Kumar D, Chen H, Wu S, Kim JY: Transcription factor-mediated cell- to-cell signalling in plants. J Exp Bot 2014, 65:1737-1749. 44. Kawade K, Tanimoto H: Mobility of signaling molecules: the key to deciphering plant organogenesis. J Plant Res 2015, 128:17-25. 45. Helariutta Y, Fukaki H, Wysocka-Diller J, Nakajima K, Jung J, Sena G, Hauser MT, Benfey PN: The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell 2000, 101:555-567. 46. Nakajima K, Sena G, Nawy T, Benfey PN: Intercellular movement of the putative transcription factor SHR in root patterning. Nature 2001, 413:307-311. 47. Carlsbecker A, Lee JY, Roberts CJ, Dettmer J, Lehesranta S, Zhou J, Lindgren O, Moreno-Risueno MA, Vaten A, Thitamadee S, et al.: Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate. Nature 2010, 465:316-321. 18 48. Kawade K, Horiguchi G, Usami T, Hirai MY, Tsukaya H: ANGUSTIFOLIA3 signaling coordinates proliferation between clonally distinct cells in leaves. Curr Biol 2013, 23:788-792. 49. Rim Y, Huang L, Chu H, Han X, Cho WK, Jeon CO, Kim HJ, Hong JC, Lucas WJ, Kim JY: Analysis of Arabidopsis transcription factor families revealed extensive capacity for cell-to-cell movement as well as discrete trafficking patterns. Mol Cells 2011, 32:519-526. 50. Chen H, Ahmad M, Rim Y, Lucas WJ, Kim JY: Evolutionary and molecular analysis of Dof transcription factors identified a conserved motif for intercellular protein trafficking. New Phytol 2013, 198:1250-1260. 51. Mahonen AP, ten Tusscher K, Siligato R, Smetana O, Diaz-Trivino S, Salojarvi J, Wachsman G, Prasad K, Heidstra R, Scheres B: PLETHORA gradient formation mechanism separates auxin responses. Nature 2014, 515:125-129. **In this manuscript the authors show that there is a gradient of PLT2 that determine the root longitudinal zonation pattern and that intercellular diffusion of the protein is required to create this gradient. The authors suggest that coordination between auxin and PLT gradients is important to regulate root response to environmental cues while maintaining a robust developmental program. 52. Zhou J, Wang X, Lee JY, Lee JY: Cell-to-cell movement of two interacting AT-hook factors in Arabidopsis root vascular tissue patterning. Plant Cell 2013, 25:187-201. 53. Kim JY, Rim Y, Wang J, Jackson D: A novel cell-to-cell trafficking assay indicates that the KNOX homeodomain is necessary and sufficient for intercellular protein and mRNA trafficking. Genes Dev 2005, 19:788-793. 54. Chen H, Jackson D, Kim JY: Identification of evolutionarily conserved amino acid residues in homeodomain of KNOX proteins for intercellular trafficking. Plant Signal Behav 2014, 9:e28355. 55. Xu XM, Wang J, Xuan Z, Goldshmidt A, Borrill PG, Hariharan N, Kim JY, Jackson D: Chaperonins facilitate KNOTTED1 cell-to-cell trafficking and stem cell function. Science 2011, 333:1141-1144. 56. Fichtenbauer D, Xu XM, Jackson D, Kragler F: The chaperonin CCT8 facilitates spread of tobamovirus infection. Plant Signal Behav 2012, 7:318-321. 19 57. Notaguchi M: Identification of phloem-mobile mRNA. J Plant Res 2015, 128:27-35. 58. Zhang C, Han L, Slewinski TL, Sun J, Zhang J, Wang ZY, Turgeon R: Symplastic phloem loading in poplar. Plant Physiol 2014, 166:306-313. 59. Spiegelman Z, Golan G, Wolf S: Don't kill the messenger: Long-distance trafficking of mRNA molecules. Plant Sci 2013, 213:1-8. 60. Hannapel DJ, Sharma P, Lin T: Phloem-mobile messenger RNAs and root development. Front Plant Sci 2013, 4:257. 61. Ham BK, Li G, Jia W, Leary JA, Lucas WJ: Systemic delivery of siRNA in pumpkin by a plant PHLOEM SMALL RNA-BINDING PROTEIN 1- ribonucleoprotein complex. Plant J 2014, 80:683-694. 62. Hisanaga T, Miyashima S, Nakajima K: Small RNAs as positional signal for pattern formation. Curr Opin Plant Biol 2014, 21:37-42. 63. Knauer S, Holt AL, Rubio-Somoza I, Tucker EJ, Hinze A, Pisch M, Javelle M, Timmermans MC, Tucker MR, Laux T: A protodermal miR394 signal defines a region of stem cell competence in the Arabidopsis shoot meristem. Dev Cell 2013, 24:125-132. 64. Song XF, Yu DL, Xu TT, Ren SC, Guo P, Liu CM: Contributions of individual amino acid residues to the endogenous CLV3 function in shoot apical meristem maintenance in Arabidopsis. Mol Plant 2012, 5:515-523. 65. Miyashima S, Koi S, Hashimoto T, Nakajima K: Non-cell-autonomous microRNA165 acts in a dose-dependent manner to regulate multiple differentiation status in the Arabidopsis root. Development 2011, 138:2303- 2313. 66. Chitwood DH, Timmermans MC: Small RNAs are on the move. Nature 2010, 467:415-419. 67. Liang D, White RG, Waterhouse PM: Mobile gene silencing in Arabidopsis is regulated by hydrogen peroxide. PeerJ 2014, 2:e701. 68. Rutschow HL, Baskin TI, Kramer EM: Regulation of solute flux through plasmodesmata in the root meristem. Plant Physiol 2011, 155:1817-1826. 69. Coudert Y, Palubicki W, Ljung K, Novak O, Leyser O, Harrison CJ: Three ancient hormonal cues co-ordinate shoot branching in a moss. Elife 2015, 4. 20 70. Kitagawa M, Fujita T: A model system for analyzing intercellular communication through plasmodesmata using moss protonemata and leaves. J Plant Res 2015, 128:63-72. 71. Notaguchi M, Wolf S, Lucas WJ: Phloem-mobile Aux/IAA transcripts target to the root tip and modify root architecture. J Integr Plant Biol 2012, 54:760- 772. 72. Spiegelman Z, Ham BK, Zhang Z, Toal TW, Brady SM, Zheng Y, Fei Z, Lucas WJ, Wolf S: A tomato phloem-mobile protein regulates the shoot-to-root ratio by mediating the auxin response in distant organs. Plant J 2015. *In this paper the authors show that a cyclophilin protein, Cyp1, moves in the phloem from leaves to roots and that transport is modulated in response to light. Cyp1 movement is sufficient to reactivate auxin signalling and lateral root development in the dgt mutant background. A role for CYP1 is proposed in coordinating shoot and root growth in response to light. 73. Muraro D, Mellor N, Pound MP, Help H, Lucas M, Chopard J, Byrne HM, Godin C, Hodgman TC, King JR, et al.: Integration of hormonal signaling networks and mobile microRNAs is required for vascular patterning in Arabidopsis roots. Proc Natl Acad Sci U S A 2014, 111:857-862. 74. Galinha C, Hofhuis H, Luijten M, Willemsen V, Blilou I, Heidstra R, Scheres B: PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development. Nature 2007, 449:1053-1057. 75. Liang D, White RG, Waterhouse PM: Gene silencing in Arabidopsis spreads from the root to the shoot, through a gating barrier, by template-dependent, nonvascular, cell-to-cell movement. Plant Physiol 2012, 159:984-1000.