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Regulation of KNOLLE Syntaxin

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Regulation of KNOLLE Syntaxin RESEARCH ARTICLE 3001 Cell cycle-independent expression of the Arabidopsis cytokinesis-specific syntaxin KNOLLE results in mistargeting to the plasma membrane and is not sufficient for cytokinesis Axel Völker1, York-Dieter Stierhof2 and Gerd Jürgens1,* 1Zentrum für Molekularbiologie der Pflanzen, Entwicklungsgenetik, Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany 2Zentrum für Molekularbiologie der Pflanzen, Mikroskopie, Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany *Author for correspondence (e-mail: [email protected]) Accepted 21 May 2001 Journal of Cell Science 114, 3001-3012 (2001) © The Company of Biologists Ltd SUMMARY The Arabidopsis KNOLLE gene encodes a cytokinesis- cells, whereas no mislocalisation was observed in specific syntaxin that localises to the plane of division and proliferating cells. By comparative in situ hybridisation to mediates cell-plate formation. KNOLLE mRNA and embryo sections, the 35S promoter yielded, relative to the protein expression is tightly regulated during the cell cycle. endogenous KNOLLE promoter, low levels of KNOLLE To explore the significance of this regulation, we expressed mRNA accumulation in proliferating cells that were KNOLLE protein under the control of two constitutive insufficient to rescue cytokinesis-defective knolle mutant promoters, the flower-specific AP3 and the cauliflower embryos. Our results suggest that in wild type, strong mosaic virus 35S promoter. The transgenic plants expression of KNOLLE protein during M phase is developed normally, although KNOLLE mRNA and protein necessary to ensure efficient vesicle fusion during accumulated to high levels in non-proliferating cells cytokinesis. and protein was incorporated into membranes. Immunolocalisation studies in transgenic seedling roots revealed mistargeting of KNOLLE protein to the plasma Key words: Arabidopsis, Cytokinesis, Syntaxin, KNOLLE, membrane in tip-growing root hairs and in expanding root Expression INTRODUCTION KNOLLE and KEULE, result in cytokinesis defects, such as enlarged cells with incomplete cell walls and more than one Cytokinesis partitions the cytoplasm of the dividing cell, which nucleus (Lukowitz et al., 1996; Assaad et al., 1996; Nacry et requires targeting of membrane vesicles to the plane of al., 2000). KNOLLE encodes a cytokinesis-specific syntaxin division. In yeast and animal cells, a contractile actomyosin (Lukowitz et al., 1996; Lauber et al., 1997). KEULE is a ring supports ingrowth of the existing plasma membrane, and member of the Sec1 family of syntaxin-binding proteins that this cleavage furrow is expanded by the fusion of membrane interacts with KNOLLE in vitro and in vivo, and mutations in vesicles that are delivered along furrow microtubule arrays both genes result in the accumulation of unfused cytokinetic (reviewed by Robinson and Spudich, 2000; Straight and Field, vesicles (Assaad et al., 2000; Lauber et al., 1997; Waizenegger 2000). By contrast, plant cells form the partitioning plasma et al., 2000). Whereas the KEULE gene appears to be expressed membrane de novo from the centre to the periphery of the cell in both proliferating and non-proliferating cells (Assaad et al., (reviewed by Staehelin and Hepler, 1996; Heese et al., 1998). 2000), the expression of KNOLLE is tightly regulated during Golgi-derived vesicles are transported along the microtubules the cell cycle. KNOLLE mRNA accumulates transiently in of a plant-specific cytoskeletal array, the phragmoplast, to the proliferating cells, giving a patchy pattern that reflects plane of cell division where they fuse with one another to form asynchrony of cell division in the embryo (Lukowitz et al., a transient membrane-bounded compartment, the cell plate, 1996). KNOLLE protein accumulates only during M phase, which matures into a cell wall with flanking plasma initially in patches presumed to represent Golgi stacks, then membranes. The lateral expansion of the cell plate is mediated localises to the forming cell plate during telophase and by the transformation of the phragmoplast from a compact disappears at the end of cytokinesis (Lauber et al., 1997). The array into a widening hollow cylindrical structure that delivers tight regulation of KNOLLE expression is reminiscent of the additional vesicles to the growing edge of the cell plate until synthesis and degradation of mitotic cyclins (Ito, 2000). the latter fuses with the parental plasma membrane (Samuels KNOLLE syntaxin appears to be involved in all sporophytic et al., 1995). Thus, plant cytokinesis is a special case of vesicle cell divisions as well as in endosperm cellularisation (Lauber trafficking and fusion. et al., 1997). Mutations in several genes of Arabidopsis, including Syntaxins are components of SNARE complexes that play 3002 JOURNAL OF CELL SCIENCE 114 (16) an important role in membrane fusion events (reviewed by MATERIALS AND METHODS Jahn and Südhof, 1999). The SNARE core complex consists of three or four proteins that form a four-helix bundle: a Plant material, growth conditions and in vitro culture bipartite t-SNARE on the target membrane, which consists of Arabidopsis thaliana ecotypes Wassilewskija (WS), Landsberg/ a syntaxin and a SNAP25 protein or two t-SNARE light-chain Niederzenz (Ler/Nd) heterozygous for the knolle mutation X37-2 proteins, interacts with the v-SNARE synaptobrevin on the (Lukowitz et al., 1996) and the EMS-induced knolle allele UU1319 vesicle membrane (Clague and Herrmann, 2000). There are (kindly provided by U. Mayer) were grown on soil at 18°C, as numerous members of each SNARE protein family in yeast, described previously (Mayer et al., 1991). The Arabidopsis cell suspension culture (Fuerst et al., 1996) was a gift from the John Innes animals and plants that have been implicated in diverse vesicle Centre (Norwich, UK). The in vitro culture was done in petri dishes trafficking pathways between membrane compartments (for containing 1% Select Agar (Gibco BRL, Karlsruhe, Germany) and reviews on plant SNAREs, see Blatt et al., 1999; Sanderfoot 0.5× or 1× Murashige and Skoog (MS) salts (Ducheva, Haarlem, The et al., 2000). In general, syntaxins and synaptobrevins Netherlands) at 18°C under constant illumination. To induce root hair involved in a particular pathway appear more closely related formation, 1% sucrose was added. To determine the organisation of to functional counterparts in different organisms than to vacuoles in root hairs, seedlings were grown on 0.5× MS medium family members involved in a different pathway within the containing no, 1% or 3% sucrose. Callus induction of knolle-X37-2 same organism. The original SNARE hypothesis postulated mutant seedlings carrying the 35S::KN transgene was carried out that specific pairs of cognate syntaxins and synaptobrevins with modified callus-inducing (1 mg/l 2.4D, 0.25 mg/l kinetin) or provide specificity to vesicle trafficking (Söllner et al., 1993a; shoot-inducing (0.5 mg/l NAA, 0.25 mg/l kinetin) media (Soni et al., 1995). Söllner et al., 1993b). This idea was challenged in recent in vitro interaction studies that provided evidence for Plant transformation and selection of transgenic plants promiscuity among interacting SNARE partners (Fasshauer et WS and knolle-X37-2 (Ler/Nd) heterozygous plants were transformed al., 1999). However, thorough analyses of yeast SNARE by a modified transformation protocol (Bechtold and Pelletier, 1998; interactions in liposome assays have indicated a high degree Clough and Bent, 1998), using a combination of vacuum infiltration of specificity of interaction between syntaxins and and additional dip transformation one week later. One hundred to 150 synaptobrevins (Fukuda et al., 2000; McNew et al., 2000; plants were transformed with an Agrobacterium GV3101 culture bearing the desired transgene. Infiltration medium consisted of 0.5× Parlati et al., 2000). × µ KNOLLE is a distant member of the plasma membrane MS salts, 1 Gamborg B5 vitamins (Ducheva), 5% sucrose, 0.044 M benzyl aminopurine (Sigma) pH 5.7 with KOH, and 0.005% SILWET subgroup of the syntaxin family but has no close counterpart L77 (Osi Specialities). T1 seeds were bulk-harvested (each seed among yeast or animal syntaxins (Lukowitz et al., 1996; representing a single transformation event; Bechtold et al., 2000; Sanderfoot et al., 2000). However, syntaxins with analogous Desfeux et al., 2000; Ye et al., 1999), sown on soil and selected for roles in membrane fusion during cellularisation or cytokinesis transformants by spraying BASTA® (183 g/l Glufosinate, AgrEvo™; have been described in animals. The Drosophila syntaxin 1 Düsseldorf, Germany; 1:1000) twice. BASTA®-resistant plants were gene is required for cellularisation of the blastoderm embryo, genotyped for KNOLLE by PCR with the primers X37-2C and X37- as well as for neural development (Burgess et al., 1997). 2D (Lukowitz et al., 1996), which amplify a 0.7 kb fragment from Likewise, the Caenorhabditis syn-4 gene is involved in X37-2 and a 1.7 kb fragment from wild type. Seeds containing knolle mutant embryos are shrunken and darker than wild-type seeds. For embryo cleavage divisions but also plays a role in nuclear × membrane reformation (Jantsch-Plunger and Glotzer, 1999). confirmation of the genotype, mutant seeds were germinated on 0.5 MS salts, 1% Select Agar plates, and seedlings were examined for the In contrast to the other two syntaxins, KNOLLE is required knolle mutant phenotype. only for de novo formation of the partitioning plasma Plants heterozygous for kn-X37-2 were transformed
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