Blocking ER export of the Golgi SNARE SYP31 affects plant growth

We recently identified a novel and transplantable di-acidic motif (EXXD) that facilitates ER export of the Golgi syntaxin SYP31 (type IV protein) and which may function also for type I and type II proteins in plants. By mutagenesis of Arabidopsis thaliana SYP31 and live cell imaging experiments in tobacco leaf epidermal cells, we determined that replacing the MELAD sequence of SYP31 with gagag retained SYP31 in the ER, which demonstrates that the di-acidic motif ELAD is critical for SYP31 ER export. To investigate whether blockage of a Golgi SNARE in the ER have consequences for plant growth, we produced tobacco plants stably overexpressing either the wild type MELAD or the mutant gagag form of SYP31. Whereas tobacco plants overexpressing the wild-type SYP31 developed to set seed, tobacco plants overexpressing the mutant form gagag rapidly became chlorotic, ceased their growth and invariably died after several weeks. This indicated that retention of overexpressed SYP31 in the ER is likely toxic for the secretory pathway and, therefore, plant development. Putative explanations for this observation are discussed taking into account SNARE properties and possible interactions.Key words: plant growth, ER-golgi interface, ER export, Golgi SNAREs, SYP31, SNARE interactions, di-acidic motifSNAREs (soluble N-ethyl-maleimide sensitive factor attachment receptor proteins) are components of the molecular machinery that facilitates vesicular transport in the secretory pathway of eukaryotic cells,^1,2 and are critical for numerous plant physiological functions.^1,3 SYP31 is a type IV syntaxin localized at the Golgi and it is required for anterograde traffic from the ER to the Golgi.^4,5 In the search for putative ER export signals in the sequence of SYP31, we identified a di-acidic motif (M) ELAD(G) of the type EXXD.⁶ This di-acidic motif was essential for ER export and Golgi targeting of SYP31, and we suggested an interaction of this motif with the COPII machinery (reviewed in ref. ⁶). To investigate whether blocking a Golgi SNARE in the ER may affect plant growth, we have produced transgenic tobacco plants overexpressing either the wild type MELAD or the mutant gagag form of A. thaliana SYP31.     

Evolutionarily diverse SYP1 Qa‐SNAREs jointly sustain pollen tube growth in Arabidopsis

Intracellular membrane fusion is effected by SNARE proteins that reside on adjacent membranes and form bridging trans‐SNARE complexes. Qa‐SNARE members of the Arabidopsis SYP1 family are involved in membrane fusion at the plasma membrane or during cell plate formation. Three SYP1 family members have been classified as pollen‐specific as inferred from gene expression profiling studies, and two of them, SYP124 and SYP125, are confined to angiosperms. The SYP124 gene appears genetically unstable, whereas its sister gene SYP125 shows essentially no variation among Arabidopsis accessions. The third pollen‐specific member SYP131 is sister to SYP132, which appears evolutionarily conserved in the plant lineage. Although evolutionarily diverse, the three SYP1 proteins are functionally overlapping in that only the triple mutant syp124 syp125 syp131 shows a specific and severe male gametophytic defect. While pollen development and germination appear normal, pollen tube growth is arrested during passage through the style. Our results suggest that angiosperm pollen tubes employ a combination of ancient and modern Qa‐SNARE proteins to sustain their growth‐promoting membrane dynamics during the reproductive process.     

The syntaxins SYP31 and SYP81 control ER-Golgi trafficking in the plant secretory pathway

Overexpression of the Golgi and endoplasmic reticulum (ER) syntaxins SYP31 and SYP81 strongly inhibits constitutive secretion. By comparing the secreted reporter alpha-amylase with the ER-retained reporter alpha-amylase-HDEL, it was concluded that SYP81 overexpression inhibits both retrograde and anterograde transport, while SYP31 overexpression mainly affected anterograde transport. Of the other interacting SNAREs investigated, only the overexpression of MEMB11 led to an inhibition of protein secretion. Although the position of a fluorescent tag does not influence the correct localization of the fusion protein, only N-terminal-tagged SYP31 retained the ability of the untagged SNARE to inhibit transport. C-terminal-tagged SYP31 failed to exhibit this effect. Overexpression of both wild-type and N-terminal-tagged syntaxins caused standard Golgi marker proteins to redistribute into the ER. Nevertheless, green fluorescent protein (GFP)-SYP31 was still visible as fluorescent punctae, which, unlike SYP31-GFP, were resistant to brefeldin A treatment. Immunogold electron microscopy showed that endogenous SYP81 is not only present at the ER but also in the cis Golgi, indicating that this syntaxin cycles between these two organelles. However, when expressed at non-inhibitory levels, YFP-SYP81 was seen to locate principally to subdomains of the ER. These punctate structures were physically separated from the Golgi, suggesting that they might possibly reflect the position of ER import sites.     

Arabidopsis Qa-SNARE SYP2 proteins localized to different subcellular regions function redundantly in vacuolar protein sorting and plant development

SYP2 proteins are a sub-family of Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) that may be responsible for protein trafficking between pre-vacuolar compartments (PVC) and vacuoles. Arabidopsis thaliana SYP22/VAM3/SGR3 and SYP21/PEP12 proteins function independently, but are both reported to be essential for male gametophytic viability. Here, we systematically examined the redundancy of three SYP2 paralogs (i.e. SYP21, 22 and 23) using a Col-0 ecotype harboring a SYP2 paralog (SYP23/PLP) that lacked a transmembrane domain. Surprisingly, no visible phenotypes were observed, even in the double knockout syp21/pep12 syp23/plp. Deficiency of either SYP21/PEP12 or SYP23/PLP in the syp22 background resulted in a defect in vacuolar protein sorting, characterized by abnormal accumulation of protein precursors in seeds. SYP21/PEP12 knockdown enhanced the syp22 phenotype (i.e. semi-dwarfism, poor leaf vein development and abnormal development of myrosin cells), and additional knockout of SYP23/PLP further aggravated the phenotype. A GFP-SYP23/PLP fusion localized to the cytosol, but not to the PVC or vacuolar membrane, where SYP21/PEP12 or SYP22/VAM3, respectively, were localized. Immunoprecipitation analysis showed that SYP23/PLP interacted with the vacuolar Qb- and Qc-SNAREs, VTI11 and SYP5, respectively, suggesting that SYP23/PLP is able to form a SNARE complex anchoring the membrane. Unexpectedly, we found that expression of multiple copies of a genomic fragment of SYP23/PLP suppressed the abnormal syp22-3 phenotype. Thus, SYP2 proteins, including cytosolic SYP23/PLP, appear to function redundantly in vacuolar trafficking and plant development.     

The physiological role of SYP4 in the salinity and osmotic stress tolerances

The trans-Golgi network (TGN) contains multiple sorting domains and acts as the compartment for cargo sorting. Recent evidence indicates that the TGN also functions as an early endosome, the first compartment in the endocytic pathway in plants. The SYP4 group, plant Qa-SNAREs localized on the TGN, regulates both secretory and vacuolar transport pathways. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance to fungal pathogens. However, the physiological role of SYP4 in abiotic stress remains unknown. Here, we report the phenotypes of a syp4-mutant in regard to salinity and osmotic response, and describe the physiological roles of the SYP4 group in the abiotic stress response.     

Myrosin Cell Development Is Regulated by Endocytosis Machinery and PIN1 Polarity in Leaf Primordia of Arabidopsis thaliana

Myrosin cells, which accumulate myrosinase to produce toxic compounds when they are ruptured by herbivores, form specifically along leaf veins in Arabidopsis thaliana. However, the mechanism underlying this pattern formation is unknown. Here, we show that myrosin cell development requires the endocytosis-mediated polar localization of the auxin-efflux carrier PIN1 in leaf primordia. Defects in the endocytic/vacuolar SNAREs (syp22 and syp22 vti11) enhanced myrosin cell development. The syp22 phenotype was rescued by expressing SYP22 under the control of the PIN1 promoter. Additionally, myrosin cell development was enhanced either by lacking the activator of endocytic/vacuolar RAB5 GTPase (VPS9A) or by PIN1 promoter-driven expression of a dominant-negative form of RAB5 GTPase (ARA7). By contrast, myrosin cell development was not affected by deficiencies of vacuolar trafficking factors, including the vacuolar sorting receptor VSR1 and the retromer components VPS29 and VPS35, suggesting that endocytic pathway rather than vacuolar trafficking pathway is important for myrosin cell development. The phosphomimic PIN1 variant (PIN1-Asp), which is unable to be polarized, caused myrosin cells to form not only along leaf vein but also in the intervein leaf area. We propose that Brassicales plants might arrange myrosin cells near vascular cells in order to protect the flux of nutrients and water via polar PIN1 localization.     

AtNHX5 and AtNHX6 Are Required for the Subcellular Localization of the SNARE Complex That Mediates the Trafficking of Seed Storage Proteins in Arabidopsis

The SNARE complex composed of VAMP727, SYP22, VTI11 and SYP51 is critical for protein trafficking and PSV biogenesis in Arabidopsis. This SNARE complex directs the fusion between the prevacuolar compartment (PVC) and the vacuole, and thus mediates protein trafficking to the vacuole. In this study, we examined the role of AtNHX5 and AtNHX6 in regulating this SNARE complex and its function in protein trafficking. We found that AtNHX5 and AtNHX6 were required for seed production, protein trafficking and PSV biogenesis. We further found that the nhx5 nhx6 syp22 triple mutant showed severe defects in seedling growth and seed development. The triple mutant had short siliques and reduced seed sets, but larger seeds. In addition, the triple mutant had numerous smaller protein storage vacuoles (PSVs) and accumulated precursors of the seed storage proteins in seeds. The PVC localization of SYP22 and VAMP727 was repressed in nhx5 nhx6, while a significant amount of SYP22 and VAMP727 was trapped in the Golgi or TGN in nhx5 nhx6. AtNHX5 and AtNHX6 were co-localized with SYP22 and VAMP727. Three conserved acidic residues, D164, E188, and D193 in AtNHX5 and D165, E189, and D194 in AtNHX6, were essential for the transport of the storage proteins, indicating the importance of exchange activity in protein transport. AtNHX5 or AtNHX6 did not interact physically with the SNARE complex. Taken together, AtNHX5 and AtNHX6 are required for the PVC localization of the SNARE complex and hence its function in protein transport. AtNHX5 and AtNHX6 may regulate the subcellular localization of the SNARE complex by their transport activity.     

AtVPS45 Is a Positive Regulator of the SYP41/SYP61/VTI12 SNARE Complex Involved in Trafficking of Vacuolar Cargo

We report a functional characterization of AtVPS45 (for vacuolar protein sorting 45), a protein from the Sec1/Munc18 family in Arabidopsis (Arabidopsis thaliana) that interacts at the trans-Golgi network (TGN) with the SYP41/SYP61/VTI12 SNARE complex. A null allele of AtVPS45 was male gametophytic lethal, whereas stable RNA interference lines with reduced AtVPS45 protein levels had stunted growth but were viable and fertile. In the silenced lines, we observed defects in vacuole formation that correlated with a reduction in cell expansion and with autophagy-related defects in nutrient turnover. Moreover, transport of vacuolar cargo with carboxy-terminal vacuolar sorting determinants was blocked in the silenced lines, suggesting that AtVPS45 functions in vesicle trafficking to the vacuole. These trafficking defects are similar to those observed in vti12 mutants, supporting a functional relationship between AtVPS45 and VTI12. Consistent with this, we found a decrease in SYP41 protein levels coupled to the silencing of AtVPS45, pointing to instability and malfunction of the SYP41/SYP61/VTI12 SNARE complex in the absence of its cognate Sec1/Munc18 regulator. Based on its localization on the TGN, we hypothesized that AtVPS45 could be involved in membrane fusion of retrograde vesicles recycling vacuolar trafficking machinery. Indeed, in the AtVPS45-silenced plants, we found a striking alteration in the subcellular fractionation pattern of vacuolar sorting receptors, which are required for sorting of carboxy-terminal vacuolar sorting determinant-containing cargo. We propose that AtVPS45 is essential for recycling of the vacuolar sorting receptors back to the TGN and that blocking this step underlies the defects in vacuolar cargo trafficking observed in the silenced lines.     

SNARE SYP132 mediates divergent traffic of plasma membrane H+-ATPase AHA1 and antimicrobial PR1 during bacterial pathogenesis

The vesicle trafficking SYNTAXIN OF PLANTS132 (SYP132) drives hormone-regulated endocytic traffic to suppress the density and function of plasma membrane (PM) H⁺-ATPases. In response to bacterial pathogens, it also promotes secretory traffic of antimicrobial pathogenesis-related (PR) proteins. These seemingly opposite actions of SYP132 raise questions about the mechanistic connections between the two, likely independent, membrane trafficking pathways intersecting plant growth and immunity. To study SYP132 and associated trafficking of PM H⁺-ATPase 1 (AHA1) and PATHOGENESIS-RELATED PROTEIN1 (PR1) during pathogenesis, we used the virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) bacteria for infection of Arabidopsis (Arabidopsis thaliana) plants. SYP132 overexpression suppressed bacterial infection in plants through the stomatal route. However, bacterial infection was enhanced when bacteria were infiltrated into leaf tissue to bypass stomatal defenses. Tracking time-dependent changes in native AHA1 and SYP132 abundance, cellular distribution, and function, we discovered that bacterial pathogen infection triggers AHA1 and SYP132 internalization from the plasma membrane. AHA1 bound to SYP132 through its regulatory SNARE Habc domain, and these interactions affected PM H⁺-ATPase traffic. Remarkably, using the Arabidopsis aha1 mutant, we discovered that AHA1 is essential for moderating SYP132 abundance and associated secretion of PR1 at the plasma membrane for pathogen defense. Thus, we show that during pathogenesis SYP132 coordinates AHA1 with opposing effects on the traffic of AHA1 and PR1.

Coordination between SNARE SYP132 and plasma membrane H⁺-ATPase AHA1 moderates SNARE abundance during pathogenesis with opposing effects on trafficking of AHA1 and antimicrobial pathogenesis-related protein 1.     

COPII genes SEC31A/B are essential for gametogenesis and interchangeable in pollen development in Arabidopsis

In eukaryotes, coat protein complex II (COPII) vesicles mediate anterograde traffic from the endoplasmic reticulum to the Golgi apparatus. Compared to yeasts, plants have multiple COPII coat proteins; however, the functional diversity among them is less well understood. SEC31A and SEC31B are outer coat proteins found in COPII vesicles in Arabidopsis. In this study, we explored the function of SEC31A and compared it with that of SEC31B from various perspectives. SEC31A was widely expressed, but at a significantly lower level than SEC31B. SEC31A-mCherry and SEC31B-GFP exhibited a high co-localization rate in pollen, but a lower rate in growing pollen tubes. The sec31a single mutant exhibited normal growth. SEC31A expression driven by the SEC31B promoter rescued the pollen abortion and infertility observed in sec31b. A sec31asec31b double mutant was unavailable due to lethality of the sec31asec31b gametophyte. Transmission electron microscopy revealed that one quarter of male gametogenesis was arrested at the uninuclear microspore stage, while confocal laser scanning microscopy showed that 1/4 female gametophyte development was suspended at the functional megaspore stage in sec31a-1/+sec31b-3/+ plants. Our study highlights the essential role of SEC31A/B in gametogenesis and their interchangeable functions in pollen development.     

The trafficking protein SYP121 of Arabidopsis connects programmed stomatal closure and K⁺ channel activity with vegetative growth

The vesicle‐trafficking protein SYP121 (SYR1/PEN1) was originally identified in association with ion channel control at the plasma membrane of stomatal guard cells, although stomata of the Arabidopsis syp121 loss‐of‐function mutant close normally in ABA and high Ca²⁺. We have now uncovered a set of stomatal phenotypes in the syp121 mutant that reduce CO₂ assimilation, slow vegetative growth and increase water use efficiency in the whole plant, conditional upon high light intensities and low relative humidity. Stomatal opening and the rise in stomatal transpiration of the mutant was delayed in the light and following Ca²⁺‐evoked closure, consistent with a constitutive form of so‐called programmed stomatal closure. Delayed reopening was observed in the syp121, but not in the syp122 mutant lacking the homologous gene product; the delay was rescued by complementation with wild‐type SYP121 and was phenocopied in wild‐type plants in the presence of the vesicle‐trafficking inhibitor Brefeldin A. K⁺ channel current that normally mediates K⁺ uptake for stomatal opening was suppressed in the syp121 mutant and, following closure, its recovery was slowed compared to guard cells of wild‐type plants. Evoked stomatal closure was accompanied by internalisation of GFP‐tagged KAT1 K⁺ channels in both wild‐type and syp121 mutant guard cells, but their subsequently recycling was slowed in the mutant. Our findings indicate that SYP121 facilitates stomatal reopening and they suggest that K⁺ channel traffic and recycling to the plasma membrane underpins the stress memory phenomenon of programmed closure in stomata. Additionally, they underline the significance of vesicle traffic for whole‐plant water use and biomass production, tying SYP121 function to guard cell membrane transport and stomatal control.     

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners.     

Arabidopsis SNAREs SYP61 and SYP121 Coordinate the Trafficking of Plasma Membrane Aquaporin PIP2;7 to Modulate the Cell Membrane Water Permeability

Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive movement of water and small neutral solutes through biological membranes. Here, we report that post-Golgi trafficking of PIP2;7 in Arabidopsis thaliana involves specific interactions with two syntaxin proteins, namely, the Qc-SNARE SYP61 and the Qa-SNARE SYP121, that the proper delivery of PIP2;7 to the plasma membrane depends on the activity of the two SNAREs, and that the SNAREs colocalize and physically interact. These findings are indicative of an important role for SYP61 and SYP121, possibly forming a SNARE complex. Our data support a model in which direct interactions between specific SNARE proteins and PIP aquaporins modulate their post-Golgi trafficking and thus contribute to the fine-tuning of the water permeability of the plasma membrane.     

Synaptotagmin 5 Controls SYP132-VAMP721/722 Interaction for Arabidopsis Immunity to Pseudomonas syringae pv tomato DC3000

Vesicle-associated membrane proteins 721 and 722 (VAMP721/722) are secretory vesicle-localized arginine-conserved soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) to drive exocytosis in plants. They are involved in diverse physiological processes in plants by interacting with distinct plasma membrane (PM) syntaxins. Here, we show that synaptotagmin 5 (SYT5) is involved in plant defense against Pseudomonas syringae pv tomato (Pst) DC3000 by regulating SYP132-VAMP721/722 interactions. Calcium-dependent stimulation of in vitro SYP132-VAMP722 interaction by SYT5 and reduced in vivo SYP132-VAMP721/722 interaction in syt5 plants suggest that SYT5 regulates the interaction between SYP132 and VAMP721/722. We interestingly found that disease resistance to Pst DC3000 bacterium but not to Erysiphe pisi fungus is compromised in syt5 plants. Since SYP132 plays an immune function to bacteria, elevated growth of surface-inoculated Pst DC3000 in VAMP721/722-deficient plants suggests that SYT5 contributes to plant immunity to Pst DC3000 by promoting the SYP132-VAMP721/722 immune secretory pathway.     

Localization of arabidopsis SYP125 syntaxin in the plasma membrane sub-apical and distal zones of growing pollen tubes

Tip growth in pollen tubes occurs by continuous vesicle secretion and delivery of new wall material, but the exact sub-cellular location of endocytic and exocytic domains remains unclear. Here we studied the localization of the Arabidopsis thaliana pollen specific syntaxin SYP125 using GFP-fusion constructs expressed in Nicotiana tobaccum pollen tubes. In agreement with the predicted role for syntaxins, SYP125 was found to be associated with the plasma membrane and apical vesicles in growing cells. At the plasma membrane, SYP125 was asymmetrically localized with a higher labeling 20–35 µm behind the apex, a distribution which is distinct from SYP124, another pollen-specific syntaxin. Competition with a related dominant negative mutant affected the specific distribution of SYP125 but not tip growth. Co-expression of the phosphatidylinositol-4-monophosphate-5-kinase 4 (PIP5K4) or of the small GTPase Rab11 perturbed polarity and the normal distribution of GFP-SYP but did not inhibit the accumulation in vesicles or at the plasma membrane.Taken together, our results corroborates previous observations that in normal growing pollen tubes, the asymmetric distribution of syntaxins helps to define exocytic sub-domains but requires the involvement of additional signaling and functional mechanisms, namely phosphoinositides and small GTPases. The localization of syntaxins at different membrane domains likely depends on the interaction with specific partners not yet identified.Key words: [Ca²⁺]_c, endocytosis, exocytosis, secretion, syntaxins, tip growth     

Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the SNARE SYP121

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K⁺ channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K⁺ channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.     

Overexpression of trans‐Golgi network t‐SNAREs rescues vacuolar trafficking and TGN morphology defects in a putative tethering factor mutant

The trans‐Golgi network (TGN) is a major site for sorting of cargo to either the vacuole or apoplast. The TGN‐localized coiled‐coil protein TNO1 is a putative tethering factor that interacts with the TGN t‐SNARE SYP41 and is required for correct localization of the SYP61 t‐SNARE. An Arabidopsis thaliana tno1 mutant is hypersensitive to salt stress and partially mislocalizes vacuolar proteins to the apoplast, indicating a role in vacuolar trafficking. Here, we show that overexpression of SYP41 or SYP61 significantly increases SYP41–SYP61 complex formation in a tno1 mutant, and rescues the salt sensitivity and defective vacuolar trafficking of the tno1 mutant. The TGN is disrupted and vesicle budding from Golgi cisternae is reduced in the tno1 mutant, and these defects are also rescued by overexpression of SYP41 or SYP61. Our results suggest that the trafficking and Golgi morphology defects caused by loss of TNO1 can be rescued by increasing SYP41–SYP61 t‐SNARE complex formation, implicating TNO1 as a tethering factor mediating efficient vesicle fusion at the TGN.     

Differential Expression Control and Polarized Distribution of Plasma Membrane-Resident SYP1 SNAREs in Arabidopsis thaliana

Membrane trafficking to the plasma membrane (PM) is a highlyorganized process which enables plant cells to build up theirbodies. SNARE (soluble N-ethylmaleimide-sensitive factor attachmentprotein receptor) genes, which encode the proteins involvedin membrane trafficking, are much more abundant in the Arabidopsisgenome than in that of any other eukaryote. We have previouslyshown that a large number of SNARE molecules in the Arabidopsiscell are localized predominantly on the PM. In the present study,in order to elucidate the physiological function of each PM-localizedSNARE, we analyzed the spatiotemporal expression profiling ofnine SYP1s that are resident in the PM of Arabidopsis, and usedthe information thus acquired to generate transgenic Arabidopsisplants expressing green fluorescent protein-fused Qa-SNAREsunder control of their authentic promoters. Among the nine SYP1s,only SYP132 is expressed ubiquitously in all tissues throughoutplant development. The expression patterns of the other SYP1s,in contrast, are tissue specific, and all different from oneanother. A particularly noteworthy example is SYP123, whichis predominantly expressed in root hair cells during root development,and shows a focal accumulation pattern at the tip region ofroot hairs. These results suggest that SYP132 is involved inconstitutive membrane trafficking to the PM throughout plantdevelopment, while the other SYP1s are involved in membranetrafficking events such as root formation or tip growth of roothair, with some redundancy.     

A SNARE complex unique to seed plants is required for protein storage vacuole biogenesis and seed development of Arabidopsis thaliana

The SNARE complex is a key regulator of vesicular traffic, executing membrane fusion between transport vesicles or organelles and target membranes. A functional SNARE complex consists of four coiled-coil helical bundles, three of which are supplied by Q-SNAREs and another from an R-SNARE. Arabidopsis thaliana VAMP727 is an R-SNARE, with homologs only in seed plants. We have found that VAMP727 colocalizes with SYP22/ VAM3, a Q-SNARE, on a subpopulation of prevacuolar compartments/endosomes closely associated with the vacuolar membrane. Genetic and biochemical analyses, including examination of a synergistic interaction of vamp727 and syp22 mutations, histological examination of protein localization, and coimmunoprecipitation from Arabidopsis lysates indicate that VAMP727 forms a complex with SYP22, VTI11, and SYP51 and that this complex plays a crucial role in vacuolar transport, seed maturation, and vacuole biogenesis. We suggest that the VAMP727 complex mediates the membrane fusion between the prevacuolar compartment and the vacuole and that this process has evolved as an essential step for seed development.