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Regular Paper Differential Expression Control and Polarized Distribution of Plasma Membrane-Resident SYP1 SNAREs in Arabidopsis thaliana Kazuhiko Enami1, Mie Ichikawa1, Tomohiro Uemura2, Natsumaro Kutsuna3,4, Seiichiro Hasezawa3,4, Tsuyoshi Nakagawa5, Akihiko Nakano2,6 and Masa H. Sato1,* 1Laboratory of Cellular Dynamics, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5, Shimogamonakaragi-cho, Sakyo-ku, Kyoto, 606-8522 Japan 2Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan 3Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, 277-8562 Japan 4Institute for Bioinformatics Research and Development (BIRD), Japan Science and Technology Agency (JST), Japan 5Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, Matsue, 690-8504 Japan 6Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, 351-0198 Japan Membrane trafficking to the plasma membrane (PM) is a highly organized process which enables plant cells to build up their bodies. SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) genes, which encode the proteins involved in membrane trafficking, are much more abundant in the Arabidopsis genome than in that of any other eukaryote. We have previously shown that a large number of SNARE molecules in the Arabidopsis cell are localized predominantly on the PM. In the present study, in order to elucidate the physiological function of each PM-localized SNARE, we analyzed the spatiotemporal expression profiling of nine SYP1s that are resident in the PM of Arabidopsis, and used the information thus acquired to generate transgenic Arabidopsis plants expressing green fluorescent protein-fused Qa-SNAREs under control of their authentic promoters. Among the nine SYP1s, only SYP132 is expressed ubiquitously in all tissues throughout plant development. The expression patterns of the other SYP1s, in contrast, are tissue specific, and all different from one another. A particularly noteworthy example is SYP123, which is predominantly expressed in root hair cells during root development, and shows a focal accumulation pattern at the tip region of root hairs. These results suggest that SYP132 is involved in constitutive membrane trafficking to the PM throughout plant development, while the other SYP1s are involved in membrane trafficking events such as root formation or tip growth of root hair, with some redundancy. Keywords: Arabidopsis • Membrane traffic • Plasma membrane • SNARE • Tip growth Abbreviations: DIC, differential interference contrast; FTFLP, fluorescent tagging of full-length proteins; GFP, green fluorescent protein; mRFP, monomeric red fluorescent protein; ORF, open reading frame; PM, plasma membrane; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TT-PCR, triple-template PCR. Introduction SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) molecules play an essential role in the membrane fusion event that occurs at the final step of membrane trafficking. SNAREs are one of the most conservative molecules in eukaryotes, and constitute a large superfamily found in all eukaryotes. They are classified as either Q-SNARE (Qa, Qb and Qc) or R-SNARE according to their conserved residues within the SNARE motif (Fasshauer et al. 1998, Bock et al. 2001). During the membrane fusion process, a specific set of three distinct Q-SNARE molecules and one R-SNARE molecule form a transSNARE complex on the membrane of each organelle. In order to complete the process of *Corresponding author: E-mail, [email protected]; Fax, +81-75-703-5448. Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197, available online at www.pcp.oxfordjournals.org © The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] 280 Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008. Spatiotemporal expression control of PM SNAREs membrane fusion on a particular organelle, the appropriate set of SNARE molecules is required in the transport pathway to that organelle. The family of SNAREs found in Arabidopsis is encoded by >60 genes (Yoshizawa et al. 2006, Sanderfoot 2007; reviewed in Lipka et al. 2007); this is a greater number of SNARE genes than that found in any yeast or animal. We have previously determined the subcellular localization of almost all SNARE molecules in Arabidopsis by transient expression analysis of green fluoresent protein (GFP)-fused SNARE molecules, and found that many SNARE molecules are localized on the plasma membrane (PM). Among the 17 Qa-SNARE group proteins, a total of nine (SYP111, SYP112, SYP121, SYP122, SYP123, SYP124, SYP125, SYP131 and SYP132) are localized on the PM (Uemura et al. 2004). Functional analyses have already been performed for some of these PM-Qa SNAREs. The KNOLLE/SYP111 protein, for example, is specifically expressed during mitosis and relocated to the forming cell plate, thereby functioning in cytokinesis (Lukowitz et al. 1996, Lauber et al. 1997). As for SYP121, an ortholog of it was first isolated from tobacco as an ABA response-related molecule, NtSyr1 (Leyman et al. 1999). The expression of the cytosolic domain of NtSyr1 or SYP121 causes inhibition of accurate secretion of secGFP and changes in the mobility of GFP–KAT1 on the PM, suggesting that SYP121 is involved in secretion and is required for cellular growth and ion homeostasis (Geelen et al. 2002, Sutter et al. 2006, Tyrrell et al. 2007). SYP121 was also identified as the molecule that is involved in non-host resistance against powdery mildew; a mutation in AtPEN1/SYP121 and its barley ortholog HvROR2 allowed enhanced penetration of the powdery mildew fungus Blumeria graminis f. sp. hordei (Collins et al. 2003). SYP122, a paralog of SYP121, is phosphorylated in response to elicitor flagellin (Nuhse et al. 2003). SYP121 and SYP122 are induced by the stimulation of fungal, bacterial and/or viral pathogens; at the time of non-host fungal infection, these molecules accumulate at the infection site to form papilla (Assaad et al. 2004). A syp121 syp122 double mutant exhibited a dwarfed and necrotic phenotype, suggesting that these two PM SNARE molecules have redundant functions not only in plant immunity but also in general secretion events (Assaad et al. 2004, Zhang et al. 2007). Recent progress in PM Qa-SNARE research has revealed that SYP132 orthologs also play roles in bacterial defense and symbiosome definition in Nicotiana benthamiana and Medicago truncatula, respectively (Catalano et al. 2007, Kalde et al. 2007). These data suggest that PM-resident Qa-SNARE molecules function in different membrane trafficking pathways leading to the particular domains of the PM surface, with some redundancy so that they can transport various functional molecules in a polarized or non-polarized manner. Although some PM Qa-SNARE molecules have been well characterized, no PM Qa-SNARE involved in polarized membrane trafficking to the PM, such as that involved in tip growth, has been identified so far. In order to identify some of the Qa-SNARE molecules involved in these processes, we generated transgenic plants expressing GFP-fused PM Qa-SYP1s under control of their own promoters, using the fluorescent tagging of full-length proteins (FTFLP) technique (Tian et al. 2004). This technique offers us a strong advantage in analyzing the expression patterns of the target genes and the subcellular distributions of their protein products simultaneously. Here we report that one SYP12 molecule, SYP123, shows strong focal accumulation on the tip region of the root hair, suggesting that it might be involved in the polarized elongation process of root hairs. In contrast, SYP132 is ubiquitously expressed in all tissues so far examined, and is localized uniformly on the PM. We also observed complicated tissue-specific expression patterns for each GFP–SYP1 SNARE, indicating that the physiological roles of PM Qa-SNAREs are probably independent of each other, although with partial redundancy. Results Expression profiles of SYP1 genes in various Arabidopsis organs Based on a phylogenic analysis of the SNAREs found in green plants, Sanderfoot (2007) categorized the PM-resident SYP1 clade into two large groups, SYP12 and SYP13. The SYP12 group is further divided into four distinct subgroups. Up to five subgroups of Qa-SNAREs may exist on the PM, namely SYP13 (SYP131 and SYP132), PEN1/ROR2 (SYP121 and SYP122), SYP124 (SYP123, SYP124 and SYP125), SYP11/ KNOLLE (SYP111/KNOLLE) and SYP112. Using the Genevestigator V3 microarray database (Zimmermann et al. 2004, Hruz et al. 2008), we analyzed the tissue-specific gene expression patterns of all SYP1 group SNAREs. As shown in Supplementary Fig. S1, SYP111 is highly expressed in organs containing dividing cells, such as root tip, silique and shoot apex, whereas its functional paralog, SYP112, is expressed to a lesser degree throughout all tissues so far examined. The PEN1/ROR2 subgroup Qa-SNAREs, SYP121 and SYP122, show similar expression patterns, but the expression of SYP121 is slightly higher than that of SYP122 in the lateral root cap. SYP123, one of the SYP124 subgroup SNAREs, is predominantly expressed in particular root tissues including the elongation zone, root hair zone and the lateral root. In contrast, the other SYP124 subgroup SNAREs, SYP124 and SYP125, are expressed only in pollen. One SYP13 subgroup SNARE, SYP131, is also specifically expressed in pollen, whereas SYP132, another member of this subgroup, shows a ubiquitous expression throughout all tissues. Thus, these Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008. 281 K. Enami et al. PM Qa-SNAREs are expressed differently among Arabidopsis organs, suggesting that these SNAREs might have distinct functions in plant development. B A SYP1 genes are expressed differently during different periods of root development We generated transgenic Arabidopsis plants expressing GFPfused PM-resident SYP1 SNAREs under control of their native promoters, and simultaneously observed the expression patterns and subcellular localization with confocal laser microscopy. Among the PM-resident Qa-SNARE molecules, five (SYP111, SYP121, SYP122, SYP123 and SYP132) were expressed in certain root tissues of 5-day-old seedlings. The GFP fluorescence of SYP111/KNOLLE was detected exclusively in dividing cells in the root tip region (Fig. 1A). The majority of expressed SYP111 proteins were predominantly localized to the forming cell plates during cytokinesis (arrows in Fig. 1C), although faint fluorescent signals were also observed on the intracellular punctate structures around the cell plate in the same cells (arrowheads in Fig. 1C). These punctate GFP signals partially overlapped with the fluorescence of an endocytosis marker, FM4-64 (Fig. 1E), indicating that GFP–SYP111 was localized to various punctate organelles including the Golgi and trans-Golgi network, as well as to the endosomes (Reichardt et al. 2007). No intracellular signals were detected in non-dividing cells anywhere in the root tissues including the root tip. The localization pattern of GFP–SYP111 observed here was completely consistent with the localization pattern of authentic SYP111 that was shown in an indirect immunofluorescent experiment (Volker et al. 2001). These results imply that our own promoter system revealed the precise subcellular localizations and tissue-specific expression of SNARE molecules throughout the plant body. In contrast to SYP111 with its characteristic restricted expression pattern, SYP132 was expressed ubiquitously in almost all cells in the root tissues, including the root apical meristem, epidermis, cortex, endodermis, stele and root hair cell (Fig. 1F, H, I). Although in non-dividing cells the fluorescence of GFP–SYP132 was only detected on the PM, in dividing cells its fluorescence was also observed in cytosolic punctate structures in addition to the cell plates (asterisks in Fig. 1F). It has been reported elsewhere that PM-localized Qa-SNARE molecules can be detected in FM4-64-positive endosomes (Uemura et al. 2004); accordingly, we believe that the GFP– SYP132-containing punctate structures we observed were the endosomes. SYP121 was expressed predominantly in the epidermal cells and lateral root cap cells in the root tip region (Fig. 2A). The expression in lateral root cap cells observed with GFP fluorescence is consistent with the microarray data 282 C D F E G * * * * * H * ep co en st I Fig. 1 Expression patterns of GFP–SYP111 and GFP–SYP132 in root. (A and B) A projection image of confocal sections of the fluorescence of GFP–SYP111 (A), and the corresponding DIC image (B) in a 5-dayold seedling of a transgenic plant expressing GFP–SYP111 under control of the SYP111 promoter. (C–E) Co-localization of GFP–SYP111 with FM4-64 in the dividing cell. Higher magnification images of the confocal sections of dividing cells expressing GFP–SYP111 were taken 1 h after the FM4-64 treatment. A merged image of GFP (C) and FM464 (D) is depicted in (E). Two fluorescence signals were overlapped at the cell plates (arrows) as well as at the subcellular punctate structures (arrowheads). (F–I) Confocal section images of GFP– SYP132 fluorescence in the root (F, H, I) and the corresponding DIC images (G). The root tip region (F, G) and mature root region (H, I) of the 5-day-old seedlings were observed. Asterisks in F indicate dividing cells. ep, epidermis; co, cortex; en, endodermis; st, stele. Bars = 20 µm (A, F, H, I) and 10 µm (C). Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008. Spatiotemporal expression control of PM SNAREs ep ep A B lrc lrc E ep C en en ep F D G * H * * * * * * I ep co en * * * st J ep co en * K st * en L ep en ep Fig. 2 SYP121 and SYP122 are differentially expressed in root tissues. The confocal images of GFP fluorescence (A, C, E, G, I, K) and the DIC (B, D, F, H, J, L) images of root sections of 5-day-old transgenic plants expressing either GFP–SYP121 (A, B, E, F, I, J) or GFP–SYP122 (C, D, G, H, K, L); root tip (A–D), root hair elongation zone (E–H), mature root (I–L). Projection images are shown in E and G. Asterisks indicate the point where root hairs start to bulge. lrc, lateral root cap; ep, epidermis; co, cortex; en, endodermis; st, stele. Bars = 20 µm. (Supplementary Fig. S1). The epidermal expression of SYP121 was observed in the whole root, but the intensity of the fluorescence decreased during root development. Particularly in root hair cells, GFP fluorescence was weaker in the differentiation zone, where root hairs begin to develop (Fig. 2E and Supplementary Fig. S2A–C). Finally, weak GFP signals were constantly observed in all types of cells in various tissues, including the cortex, endodermis and stele, in addition to the epidermal cells in the mature root region (Fig. 2I). Notably, GFP–SYP121 formed patch-like structures which were scattered on the PM in addition to its normal PM localization (Fig. 2E). Longitudinal imaging of GFP– SYP121 in the root showed that these patch-like structures protruded slightly into the cytosolic region from the PM (Supplementary Fig. S2A–C). Time-lapse observation revealed that several of these dot structures of GFP–SYP121 moved dynamically along the surface of PM (Supplementary Fig. S3, arrows), whereas predominant fluorescent dots were less mobile on the PM (Supplementary Fig S3, arrowheads). The expression profile of GFP-tagged SYP122 during root development was completely different from that of SYP121. In the root tip region, no fluorescence was observed in any cells examined (Fig. 2C). Instead, GFP fluorescence appeared in the endodermal cells at the differentiation zone (Fig. 2G). In the mature root region, a weak expression of GFP–SYP122 was observed in the cortex and epidermis, in addition to the strong expression in the endodermal cells (Fig. 2K and Supplementary Fig. S2D–F). Thus the expression patterns and intensities of these PM Qa-SNAREs varied according to cell types in the root section, although SNAREs were uniformly localized to the PM of all types of cells in root so far examined. Intriguingly, the expression pattern of GFP–SYP123 is notably different from those of the other PM-resident QaSNARE molecules described above. No expression of GFP– SYP123 was observed at the root tip region (data not shown), but, after the root hair bulge rose at the differentiate region, GFP fluorescence was specifically observed in the root hair cells (Fig. 3D). The high-resolution expression analysis of Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008. 283 K. Enami et al. A B C E D 10 80 150 220 Fig. 3 GFP–SYP123 is predominantly expressed in root hair cells and accumulates in the root hair tip region. Five-day-old seedlings expressing GFP–SYP123 were observed and analyzed. Section images of a single root hair cell (A–C) and projection image of the root hair elongation region (D). As root hairs elongate, a strong GFP signal is observed at the PM of the root hair region. Note that GFP fluorescence can be observed after the root hair bulges emerged. The region surrounded by a dotted square in F was analyzed and constructed into a 3D image with ReantLight software (E). Color represents normalized fluorescence intensity (10–220 arbitrary units) which correlates with GFP–SYP123 density, estimated by 3D deconvolution to reduce the bias from point-spread function anisotropy (Sano et al. 2008). The intensity seen in the root hair (from sky-blue to red) was higher than that in the base cell (blue) and peaked at the tip of the root hair (from green to red). Bars = 20 µm. root tissues also revealed that SYP123 is enriched in root hair cells (Brady et al. 2007). Although the fluorescence signals of GFP–SYP123 continued to be visible in mature root hair cells, their intensity was significantly reduced (data not shown). No expression of GFP–SYP123 was observed in any other tissues, not even in non-root hair cells. It should be noted that once the root hair elongation began at the root hair cells, GFP fluorescence accumulated predominantly in the PM of the developing root hair region, especially at the tip of the root hair (Fig. 3A–C). 3D image construction using ReantLight software provided us with a clear view of the strong accumulation of GFP–SYP123 at the tip region of each root hair (Fig. 3D, E). This distribution pattern of SYP123 in root hair cells is obviously different from that of SYP132 (compare Fig. 3E with Fig. 1I or Supplementary Fig. S4). These data suggest that the expression of SYP123 is specific to root hair cells, and that it might be involved in root hair elongation. 284 Expression profiles of SYP1 genes in the aerial part of Arabidopsis plants Next, we observed the aerial part of each transgenic plant expressing GFP-fused PM Qa-SNAREs. GFP–SYP111 was specifically expressed and localized to the developing cell plates in the dividing cells during guard cell development, which suggests that SYP111 might also be involved in the formation of guard cells (Supplementary Fig. S5A). Both SYP121 and SYP122 were uniformly localized to the PM of epidermal cells in mature rosette leaves, even in the absence of any fungal infection (Supplementary Fig. S5D, E). SYP132 showed a strong steady-state expression in all rosette leaf cells throughout all stages of leaf development (Supplementary Fig. S5G). In the GFP–SYP123-expressing plant, on the other hand, clear GFP fluorescence was only observed in the epidermal cells of young rosette leaves (Supplementary Fig. S5F). The intensity of GFP–SYP123 fluorescence attenuated gradually during leaf development, and only faint Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008. Spatiotemporal expression control of PM SNAREs signals of this SNARE remained in the mature epidermal cells of rosette leaves (data not shown). SYP121, SYP123 and SYP132 were also expressed in trichome cells of rosette leaves and the inflorescence stem. The GFP fluorescence of these GFP–SNAREs was observed uniformly on the PM of the cells (Supplementary Fig. S5H–J). In the inflorescence stem, expression of the SYP12 subgroup SNAREs (SYP121 and SYP123) was restricted to the epidermal cell layer, whereas the fluorescence of GFP– SYP132 was observed not only in epidermal cells but also in mesophyll cells (Supplementary Fig. S6A–C). No GFP fluorescence on the PM was ever observed in guard cells or trichome cells of wild-type Arabidopsis (Supplementary Fig. S7). In flower tissues, the expression of GFP–SYP121, GFP– SYP123 and GFP–SYP132 was detected in epidermal cells. Strong expression of GFP–SYP123 was observed in epidermal cells around the top of the stigma, while none was detected in the stigmatic papilla at flowering stage 11 (Supplementary Fig. S5K). In mature pollen grains, SYP132 was expressed with clear PM localization (Fig. 4G), although other SYP1s which were expressed in vegetative tissues were not expressed in pollen grains (data not shown). In contrast, SYP124, SYP125 and SYP131 were specifically expressed in the pollen grains (Fig. 4A, C, E). Intriguingly, these SNAREs did not show clear subcellular localization patterns on the PM. Instead, faint GFP signals were observed throughout the cytoplasm of pollen grains. We compared these GFP signals with the fluorescence pattern of a vacuolar membrane marker, monomeric red fluorescent protein (mRFP)– AtVam3 in pollen grains, and found that the fluorescence seems not to be localized to particular organelle membranes such as the vacuolar membrane (Fig. 4I, J). Intriguingly, the fluorescence of GFP–SYP124 and GFP–SYP125 localized to the tip region of pollen tubes during pollen tube elongation, not to the cytoplasm of pollen grains (Fig. 4B, D). The localizations of GFP–SYP124 and GFP–SYP125 in growing pollen tubes are a little bit different. Namely, GFP–SYP125 localized to the apical region of the pollen tube, while GFP–SYP124 localized to the subapical region of the tip. In contrast, the fluorescence of GFP–SYP131 and GFP–SYP132 localized uniformly to the PMs not only of pollen tubes but also of pollen grains (Fig. 4F, H). A B C D E F * * * * * * * G H I J * * Expression profiles of SYP1 genes in embryonic cells We examined the expression patterns of GFP–SYP1s during embryonic development. Only SYP111 and SYP132 were expressed in embryos at any stage of development. GFP– SYP111 was highly expressed in leaf primordia cells (Fig. 5A), probably due to the high dividing frequency of the cells in this region. Strong GFP fluorescence was observed in the forming cell plates, while relatively weak fluorescence was observed in the mature PM (Fig. 5C). Meanwhile, GFP–SYP132 showed constitutive expression throughout the development of Fig. 4 Localizations of fluorescent-tagged SYP1s in pollen grains and elongating pollen tubes. Mature pollen grains were scattered onto coverslips from anthers of the transgenic plants at flowering stage 14. These pollen grains were isolated from the transgenic plants expressing GFP–SYP124 (A), GFP–SYP125 (C), GFP–SYP131 (E), GFP–SYP132 (G) and mRFP–AtVam3 (I and J). The localization of each GFP-tagged SYP1 during pollen tube elongation is shown for GFP–SYP124 (B), GFP–SYP125, (D) GFP–SYP131 (F) and GFP–SYP132 (H). Asterisks indicate the pollen grains that are lacking fluorescence, because the transgenic plants expressing GFP–SYP131 and mRFP– AtVam3 were hemizygous. Bars = 20 µm. Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008. 285 K. Enami et al. A B C D E F Fig. 5 SYP111 and SYP132 were expressed in developing embryos. Immature embryos were isolated from their seed coats, and GFP fluorescence of embryos was observed with confocal microscopy. GFP fluorescence of confocal sections (A, C, E) and corresponding DIC (B, D, F) images represent mid-torpedo embryos expressing GFP– SYP111 (A–D) and late heart embryos expressing GFP–SYP132 (E–F). Bars = 40 µm (A) and 20 µm (C, E). embryos, and was localized uniformly on the PM of all embryonic cells (Fig. 5E). No other SYP1s were expressed during the development of embryos (data not shown). Discussion Our gene expression analysis of PM Qa-SNAREs reveals that these SNAREs have different expression profiles during different developmental stages. Analysis using microarray data restricts the resolution of expression pattern analysis to the organ level; the FTFLP procedure, in contrast, allows us to analyze tissue specificities at a resolution of the level of one cell and to observe the polarized distributions of these SNAREs on the PM simultaneously. In our analysis, no expression of GFP–SYP112 was observed in any tissues examined, probably because SYP112 286 has a low expression level under normal conditions (Supplementary Fig. S1). Although SYP112 is the closest paralog of SYP111 and can completely complement the knolle phenotype when this transgene is expressed under the control of a SYP111 promoter (Muller et al. 2003), it is likely to be latent under normal growth conditions. SYP121 and SYP122 are the closest paralogs among the PM Qa-SNAREs, and both belong to the PEN1/ROR2 subgroup (Sanderfoot 2007). SYP121/PEN1 was first reported as Nt-SYR1, a syntaxin involved in ABA-related ion channel response in tobacco (Leyman et al. 1999), and was later proposed to mediate pre-invasive immunity against the non-host barley powdery mildew B. graminis in Arabidopsis (Collins et al. 2003). SYP122 has also been reported to be responsive to pathogen infection. syp121 syp122 double mutants exhibit a dwarfed and necrotic growth phenotype (Assaad et al. 2004). Recently, these two Qa-SNAREs were reported to function as negative regulators of defense signaling pathways, including the salicylic acid, jasmonic acid and ethylene signaling pathways (Zhang et al. 2007). In addition, each has a different relationship with the tomato Rab11 homolog (Rehman et al. 2008). These data suggest that the two syntaxins have overlapping functions not only in pathogen immunity but also in the general secretion pathway in plants. The present study supports this hypothesis by showing that, while the localization and expression profiles of SYP121 and SYP122 were almost identical in mature tissues, their expression patterns in developing root tissues were slightly different: SYP121 was expressed in the epidermal cells and lateral root cap cells in the root tip, while SYP122 was expressed in the endodermal cells in the differentiation zone; these observations were almost consistent with available microarray data (see Supplementary Fig. S1). This difference in expression may suggest that these two Qa-SNARE molecules have different functions in early root development at distinct cell layers. SYP123 was specifically expressed in the root hair cells and showed focal accumulation to the tip region of the growing root hairs. Root hairs and pollen tubes typically exhibit a polarized cell expansion known as tip growth. Polarized vesicle trafficking to the tip region of growing root hairs or pollen tubes is essential for this process (Xu and Scheres 2005, Šamaj et al. 2006). Although small GTPases of the Rab, Arf and Rho/Rac families (as well as their regulatory proteins) have been shown to participate in tip growth (Campanoni and Blatt 2007, Cheung and Wu 2008), so far no SNARE molecules have been reported to be involved in this growth. This is the first report that SYP123 may be involved in the tip growth of root hairs. Another significant observation we made was that SYP124 and SYP125 are expressed in pollen grains only. We found that these SNAREs did not show clear localization on the PM; rather they were dispersed thoughout the cytoplasm. Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008. Spatiotemporal expression control of PM SNAREs This localization pattern might indicate that these SNAREs exist on transport vesicles, rather than on any particular organelle. Yet Qa-SNAREs are generally described as being preferentially localized on the target membrane compartments, not on transport vesicles. This discrepancy must be resolved in further examinations. In summary, SYP132 is the only SYP1 family molecule that is expressed constitutively in any of the tissues we examined. In pollen, however, its GFP signal was relatively low compared with that of SYP131, a pollen-specific SYP13 subgroup component; this observation is supported by the published microarray data (Supplementary Fig. S1). These findings may indicate the function of the SYP13 subgroup, namely its general role in constitutive membrane trafficking to the PM, specifically in the transport of housekeeping molecules, in all plant cell types, with SYP132 predominating in vegetative tissues. Other PM-resident Qa-SNAREs, on the other hand, may have diverse functions depending on the type of membrane trafficking required in each cell type, with partial redundancy. In particular, we found in this study that SYP123 is specifically expressed in root hair cells and accumulates at the tip region of root hairs (Fig. 6). Our present study strongly implies that multiple membrane trafficking routes to the PM exist in the same and/or different cell types in an arrangement which confers complexity to the process of plant development. Recently, it has been reported that the R-SNARE VAMP721/722 associates with SYP121/PEN1 and SNAP33 to form a SNARE complex during plant immune responses (Kwon et al. 2008). In future studies, it will be necessary to determine which SNARE complexes are involved in each of the membrane trafficking steps to the PM. Materials and Methods Plant materials and growth conditions Arabidopsis thaliana ecotype Columbia (Col-0) seeds were surface sterilized and grown on Jiffy-7 peat pellets for appropriate periods under continuous light at 23°C. For the observation of roots, seeds were placed on MS–agar plates (2.1 g l–1 Murashige–Skoog salt, 1% sucrose, 1.5 mg l–1 thiamine, 2.5 mg l–1 nicotinic acid, 0.25 mg l–1 pyridoxine and 1.5% agar, pH 5.8). After vernalization for 4 d, seeds were germinated under the conditions described above, on vertically oriented plates. Plasmid construction and plant transformation The translational fusions between GFP and the SYP1s were generated using the FTFLP method described by Tian et al. (2004) with some modifications. Briefly, for each SYP1, about 2,300 bp of an upstream region sequence with 5′-CACC, an sGFP(S65T) sequence with a GGSG linker, and an SYP1 open reading frame (ORF) followed by 1,000 bp of a downstream Pollen-grain SYP124 SYP132 SYP125 SYP131 Flowering stem (epidermis) SYP121 SYP123 Rosette leaf (pavement cells) SYP121 SYP122 SYP123 Ubiquitous expression (endodermis) mature root SYP123 (root hair) root hair elongation SYP111 (phragmoplast) root tip (epidermis) SYP122 SYP121 Fig. 6 Schematic illustration of the expression profiles of Arabidopsis PM-Qa SYP1s. SYP132 was constitutively expressed in all tissues examined in this study. sequence were each amplified separately by PCR. Then the three DNA fragments were conjugated in order, using the triple-template PCR (TT-PCR) method to form a single DNA fragment which carries the SYP1 ORF with the GFP sequence at the 5′ end in tandem. In the case of mRFP–AtVam3, about 3,200 bp of an upstream region sequence followed by an mRFP sequence was used for its construction. Whole constructs were designed to express the SNARE molecule fused with fluorescent protein at the N-terminus. The fragment was subcloned into pENTR/D-TOPO (Invitrogen, Carlsbad, CA, USA), sequence verified, and then transferred into a binary vector pGWB1 (Nakagawa et al. 2007) according to the Gateway method. The sequences of primers used to generate constructs are listed in Supplementary Table S1. These constructs were introduced into Agrobacterium tumefaciens strain C58 Rifr/pGV2260; subsequently, Arabidopsis wild-type plants (Col-0) were transformed by the floral dipping method (Clough and Bent 1998). Screening of transgenic plants was performed on MS plates containing 50 µg ml–1 hygromycin. T2 lines which showed a segregation Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008. 287 K. Enami et al. ratio of 3 : 1 for antibiotic resistance were used for further experiments. Confocal microscopy GFP fluorescence signals and differential interference contrast (DIC) images were obtained using the Nikon ECLIPSE E600 laser scanning microscope equipped with the C1siready confocal system (Nikon, Tokyo, Japan) and an argon laser. The endocytosis marker FM 4–64 (Molecular Probes, Eugene, OR, USA) was treated for 1 h before observation at a final concentration of 30 µM, and 1 g l–1 of propidium iodide solution was used to stain the cell wall. Pollen germination was performed as described previously (Bovid and McCormick 2007). The collected images were processed using Nikon EZ-C1 software. 3D analysis of GFP–SYP1 distribution To examine the distribution of GFP–SYP1 in 3D, a 3D surface model of a root hair was reconstructed from a series of confocal images and the intensities of GFP signals were mapped on the reconstructed model. As a preprocessing step to reduce the noise, a Gaussian filter was applied using ImageJ 1.39J (http://rsb.info.nih.gov/ij/). The 3D reconstruction and intensity mapping was performed according to the technique employed by Kutsuna and Hasezawa (2005) using ReantLight software (http://hasezawa.ib.k.u-tokyo.ac.jp/zp/ Kbi/ReantLight). Once the 3D models were reconstructed, 3D images were rendered interactively using ParaView 3.2.1 (http://www.paraview.org). Analysis of microarray data Microarray expression analysis data for nine PM Qa-SNAREs were obtained using the Genevestigator V3 microarray database and analysis toolbox (Zimmermann et al. 2004, Hruz et al. 2008). The Genevestigator program is available on the web site at (https://www.genevestigator.ethz.ch/gv/index.jsp). Supplementary data Supplementary data are available at PCP online. Funding Monbukagakusho (the Japanese Ministry of Education, Culture, Sport, Science and Technology) grant-in-aid for Basic Science Research (C) and a grant-in-aid for Scientific Research on Priority Areas (to M.H.S.); the Yamada Science Foundation (to M.H.S.); Japan Society for the Promotion of Science (JSPS) Research Fellowships for Young Scientists grant-in-aid (to T.U.); Nara Institute of Science and Technology (supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan) grant-in-aid for Scientific Research for Plant Graduate Students (to K.E.). 288 Acknowledgments We thank Dr. G. Jürgens for critical reading of our manuscript. We also thank Dr. Y. Niwa for generously providing the sGFP vector. References Assaad, F.F., Qiu, J.L., Youngs, H., Ehrhardt, D., Zimmerli, L., Kalde, M., et al. (2004) The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae. Mol. Biol. Cell 15: 5118–5129. Bock, J.B., Matern, H.T., Peden, A.A. and Scheller, R.H. (2001) A genomic perspective on membrane compartment organization. Nature 409: 839–841. Bovid, L.C. and McCormick, S. (2007) Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana. Plant J. 52: 570–582. Brady, S.M., Orlando, D.A., Lee, J., Wang, J.Y., Koch, J., Dinneny, J.R., et al. (2007) A high-resolution root spatiotemporal map reveals dominant expression patterns. Science 318: 801–806. Campanoni, P. and Blatt, M.R. (2007) Membrane trafficking and polar growth in root hairs and pollen tubes. J. Exp. Bot. 58: 65–74. Catalano, C.M., Czymmek, K.J., Gann, J.G. and Sherrier, D.J. (2007) Medicago truncatula syntaxin SYP132 defines the symbiosome membrane and infection droplet membrane in root nodules. Planta 225: 541–550. Cheung, A.Y. and Wu, H.M. (2008) Structural and signaling networks for the polar cell growth machinery in pollen tubes. Annu. Rev. Plant Biol. 59: 541–572. Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–743. Collins, N.C., Thordal-Christensen, H., Lipka, V., Bau, S., Kombrink, E., Qiu, J.L., et al. (2003) SNARE-protein-mediated disease resistance at the plant cell wall. Nature 425: 973–977. Fasshauer, D., Sutton, R.B., Brunger, A.T. and Jahn, R. (1998) Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc. Natl Acad. Sci. USA 95: 15781–15786. Geelen, D., Leyman, B., Batoko, H., Di Sansebastiano, G.P., Moore, I. and Blatt, M.R. (2002) The abscisic acid-related SNARE homolog NtSyr1 contributes to secretion and growth: evidence from competition with its cytosolic domain. Plant Cell 14: 387–406. Hruz, T., Laule, O., Szabo, G., Wessendorp, F., Bleuler, S., Oertle, L., et al. (2008) Genevestigator V3: a reference expression database for the meta-analysis of transcriptome. Adv. Bioinform. 420–747. Kalde, M., Nuhse, T.S., Findlay, K. and Peck, S.C. (2007) The syntaxin SYP132 contributes to plant resistance against bacteria and secretion of pathogenesis-related protein 1. Proc. Natl Acad. Sci. USA 104: 11850–11855. Kutsuna, N. and Hasezawa, S. (2005) Morphometrical study of plant vacuolar dynamics in single cells using three-dimensional reconstruction from optical sections. Microsc. Res. Tech. 68: 296–306. Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008. Spatiotemporal expression control of PM SNAREs Kwon, C., Neu, C., Pajonk, S., Yun, H.S., Lipka, U., Humphry, M., et al. (2008) Co-option of a default secretory pathway for plant immune responses. Nature 451: 835–840. Lauber, M.H., Waizenegger, I., Steinmann, T., Schwarz, H., Mayer, U., Hwang, I., et al. (1997) The Arabidopsis KNOLLE protein is a cytokinesis-specific syntaxin. J. Cell Biol. 139: 1485–1493. Leyman, B., Geelen, D., Quintero, F.J. and Blatt, M.R. (1999) A tobacco syntaxin with a role in hormonal control of guard cell ion channels. Science 283: 537–540. Lipka, V., Kwon, C. and Panstruga, R. (2007) SNARE-Ware: the role of SNARE-domain proteins in plant biology. Annu. Rev. Cell Dev. Biol. 23: 147–174. Lukowitz, W., Mayer, U. and Jürgens, G. (1996) Cytokinesis in the Arabidopsis embryo involves the syntaxin-related KNOLLE gene product. Cell 84: 61–71. Muller, I., Wagner, W., Volker, A., Schellmann, S., Nacry, P., Kuttner, F., et al. (2003) Syntaxin specificity of cytokinesis in Arabidopsis. Nat. Cell Biol. 5: 531–534. Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., et al. (2007) Development of series of Gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J. Biosci. Bioeng. 104P 34–41. Nuhse, T.S., Boller, T. and Peck, S.C. (2003) A plasma membrane syntaxin is phosphorylated in response to the bacterial elicitor flagellin. J. Biol. Chem. 278: 45248–45254. Rehman, R.U., Stigliano, E., Lycett, G.W., Sticher, L., Sbano, F., Faraco, M., et al. (2008) Tomato Rab11a characterization evidenced a difference between SYP121-dependent and SYP122-dependent exocytosis. Plant Cell Physiol. 49: 751–766. Reichardt, I., Stierhof, Y.D., Mayer, U., Richter, S., Schwarz, H., Schumacher, K., et al. (2007) Plant cytokinesis requires de novo secretory trafficking but not endocytosis. Curr. Biol. 17: 2047–2053. Šamaj, J., Műller, J., Beck, M., Böhm, N. and Menzel, D. (2006) Vesicular trafficking, cytoskeleton and signaling in root hairs and pollen tubes. Trends Plant Sci. 11: 549–600. Sanderfoot, A. (2007) Increases in the number of SNARE genes parallels the rise of multicellularity among the green plants. Plant Physiol. 144: 6–17. Sano, T., Kutsuna, N., Hasezawa, S. and Tanaka, Y. (2008) Membrane trafficking in guard cells during stomatal movement. Plant Signal. Behav. 3: 233–235. Sutter, J.U., Campanoni, P., Tyrrell, M. and Blatt, M.R. (2006) Selective mobility and sensitivity to SNAREs is exhibited by the Arabidopsis KAT1 K+ channel at the plasma membrane. Plant Cell 18: 935–954. Tian, G.W., Mohanty, A., Chary, S.N., Li, S., Paap, B., Drakakaki, G., et al. (2004) High-throughput fluorescent tagging of full-length Arabidopsis gene products in planta. Plant Physiol. 135: 25–38. Tyrrell, M., Campanoni, P., Sutter, J.U., Pratelli, R., Paneque, M., Sokolovski, S., et al. (2007) Selective targeting of plasma membrane and tonoplast traffic by inhibitory (dominant-negative) SNARE fragments. Plant J. 51: 1099–1115. Uemura, T., Ueda, T., Ohniwa, R.L., Nakano, A., Takeyasu, K. and Sato, M.H. (2004) Systematic analysis of SNARE molecules in Arabidopsis: dissection of the post-Golgi network in plant cells. Cell Struct. Funct. 29: 49–65. Volker, A., Stierhof, Y.D. and Jürgens, G. (2001) Cell cycle-independent expression of the Arabidopsis cytokinesis-specific syntaxin KNOLLE results in mistargeting to the plasma membrane and is not sufficient for cytokinesis. J. Cell Sci. 114: 3001–3012. Xu, J. and Scheres, B. (2005) Curr. Opin. Plant Biol. 8(6): 613–618. Yoshizawa, A.C., Kawashima, S., Okuda, S., Fujita, M., Itoh, M., Moriya, Y., et al. (2006) Extracting sequence motifs and the phylogenetic features of SNARE-dependent membrane traffic. Traffic 7: 1104–1118. Zhang, Z., Feechan, A., Pedersen, C., Newman, M.A., Qiu, J.L., Olesen, K.L., et al. (2007) A SNARE-protein has opposing functions in penetration resistance and defence signaling pathways. Plant J. 49: 302–312. Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L. and Gruissem, W. (2004) GENEVESTIGATOR. Arabidopsis database and analysis toolbox. Plant Physiol. 136: 2621–2632. (Received September 28, 2008; Accepted December 15, 2008) Plant Cell Physiol. 50(2): 280–289 (2009) doi:10.1093/pcp/pcn197 © The Author 2008. 289