A putative TRAPPII tethering factor is required for cell plate assembly during cytokinesis in Arabidopsis

Authors


Author for correspondence:
Farhah F. Assaad
Tel: +49 8161 715437
Email: farhah.assaad@wzw.tum.de

Summary

  • At the end of the cell cycle, the plant cell wall is deposited within a membrane compartment referred to as the cell plate. Little is known about the biogenesis of this transient membrane compartment.
  • We have positionally cloned and characterized a novel Arabidopsis gene, CLUB, identified by mutation.
  • CLUB/AtTRS130 encodes a putative TRAPPII tethering factor. club mutants are seedling-lethal and have a canonical cytokinesis-defective phenotype, characterized by the appearance of bi- or multinucleate cells with cell wall stubs, gaps and floating walls. Confocal microscopy showed that in club mutants, KNOLLE-positive vesicles formed and accumulated at the cell equator throughout cytokinesis, but failed to assemble into a cell plate. Similarly, electron micrographs showed large vesicles loosely connected as patchy, incomplete cell plates in club root tips. Neither the formation of KNOLLE-positive vesicles nor the delivery of these vesicles to the cell equator appeared to be perturbed in club mutants. Thus, the primary defect in club mutants appears to be an impairment in cell plate assembly.
  • As a putative tethering factor required for cell plate biogenesis, CLUB/AtTRS130 helps to define the identity of this membrane compartment and comprises an important handle on the regulation of cell plate assembly.

Introduction

The plant cell wall is laid down in two successive stages in a narrow window of time during cytokinesis. In the first stage, vesicles fuse to produce a membrane network, the cell plate. In the second stage, the cell wall is assembled within this membrane network. According to traditional models, Golgi-derived vesicles carrying cell wall materials are transported to the equator of a dividing cell during anaphase. This targeted vesicle transport is facilitated by a plant-specific cytoskeletal array referred to as the phragmoplast. Vesicle fusion gives rise to a novel membrane-bound compartment, the cell plate, in which the new cell wall is assembled (Samuels et al., 1995; Seguí-Simarro et al., 2004). A genetic dissection of plant cytokinesis in somatic cells has broken the process down into four steps, each of which is characterized by a specific class of mutants. In the first step, the plane of division is determined. This step is perturbed in mutants such as fass, tonneau, tangled and discordia (Torres-Ruiz & Jürgens, 1994; Walker et al., 2007; Azimzadeh et al., 2008; Wright et al., 2009). In the second step, the phragmoplast and cell plate are assembled and guided to the division site established during preprophase. Mutants affecting these steps in the execution of cytokinesis are characterized by multinucleate cells with gapped or incomplete cross walls (Söllner et al., 2002). The cyd mutant of pea, and the CYD, KNOLLE, KEULE, HINKEL, PLEIADE, RSH and RUNKEL genes of Arabidopsis fall into this class (Liu et al., 1995; Lukowitz et al., 1996; Assaad et al., 2001; Strompen et al., 2002; Müller et al., 2004; Cannon et al., 2008; Krupnova et al., 2009). In the third stage of cytokinesis, the cross wall is deposited and subsequently remodeled to form a primary cell wall. Genes in the third class include cell wall mutants such as korrigan, procuste, rsw1/rms, cyt and massue (Nickle & Meinke, 1998; Nicol et al., 1998; Fagard et al., 2000; Williamson et al., 2001; Thiele et al., 2009). The fourth and final stage of cytokinesis involves cell plate maturation and the loss of juvenile traits. Mutants in the ESCRT (endosomal sorting complexes required for transport; Schellmann & Pimpl, 2009) machinery, required for sorting proteins targeted for degradation into luminal vesicles of multivesicular bodies (MVBs), have been reported to have cytokinesis defects (Spitzer et al., 2006, 2009). Localization studies and the advent of reverse genetics have recently added additional genes to the meager list of genes identified in forward genetic screens (reviewed by Jürgens, 2005; Chow et al., 2008; Fujimoto et al., 2008).

KNOLLE and KEULE, among the first trafficking genes identified, are required for the fusion of vesicles to form a cell plate (Lukowitz et al., 1996; Waizenegger et al., 2000; Assaad et al., 2001). As a considerable number of trafficking proteins have been shown to play a role in cytokinesis, or to localize to the cell plate, we give a very brief synopsis of the literature on trafficking below (recently reviewed by Assaad, 2009). Vesicle trafficking is a highly regulated process involving vesicle formation, transport, tethering, docking and fusion (Mellman & Warren, 2000). Tethering is a process that brings and holds vesicles in close proximity to their target membranes. There are two classes of tethering factors. The first class consists of long coiled-coil proteins, and the second of large multisubunit complexes (Grosshans et al., 2006). Tethering factors reside on a specific cellular compartment and mediate a specific series of membrane fusion events in the secretory pathway. In order to function as tethers, tethering factors need to be attached to donor and acceptor membranes. Rab GTPases play a critical role in the tethering reaction in that they capture tethering factors when they are in their Guanidine Triphosphate (GTP) (but not Guanidine Diphosphate (GDP)) bound forms. Rab proteins belong to a family of small GTP-binding proteins, which function as molecular switches that cycle between ‘active’ and ‘inactive’ states. This cycle is linked to the binding and hydrolysis of GTP (Cai et al., 2007). Two post-Golgi, trans-Golgi network (TGN) Rab GTPases, RabA2 and RabA3, have recently been shown to be required for cytokinesis (Chow et al., 2008).

Vesicle targeting and membrane fusion require a specific ‘lock and key’ interaction between cognate t-SNAREs on target membranes and v-SNAREs on vesicle membranes. SNARE interactions, however, are not sufficiently selective to fully account for the specificity and fidelity of membrane fusion, which are ensured by key regulators such as tethering factors, Rab GTPases and Sec1/Munc18 SM proteins (Cai et al., 2007). KNOLLE encodes a t-SNARE and KEULE encodes a Sec1 (or SM) protein (Lukowitz et al., 1996; Assaad et al., 2001). Sec1 proteins gate membrane fusion by specifically promoting interactions between cognate SNAREs, while reducing promiscuous interactions between noncognate SNAREs (Shen et al., 2007).

Although a number of trafficking genes implicated in cytokinesis have been identified, a large number of questions about the membrane dynamics of plant cytokinesis remain unanswered. Other than two t-SNAREs, KNOLLE and NPSN11 (Lauber et al., 1997; Zheng et al., 2002), it is unclear which v- and t-SNAREs define the vesicle and target membranes in fusion events at the cell plate. Is cytokinesis the result of homotypic fusion among like vesicles, or are there different types of vesicles, derived from the Golgi and the cell surface? The relative contribution of Golgi-derived vesicles vs endosomes to the cell plate is a subject of debate and remains to be established (Dhonukshe et al., 2006; Reichardt et al., 2007; Chow et al., 2008). The identity of the cell plate as a target membrane that is initially distinct from, yet after cell wall maturation identical to, the plasma membrane remains to be clearly defined. Furthermore, tethering factors required for cell plate formation have not been identified, yet such factors would shed light on the identity of the cell plate and on the cell cycle regulation of its assembly. In this study we identify a tethering factor that is required for cell plate assembly.

Materials and Methods

Lines

Owing to the lethality of club mutants, all lines were propagated as heterozygotes. club-1 (U57) was identified in a screen of ethyl methanesulfonate (EMS)-mutagenized Landsberg erecta (Mayer et al., 1991; Söllner et al., 2002). Insertion mutants, including club-2, in the recombination interval defined by mapping were found on the websites http://www.arabidopsis.org (Swarbreck et al., 2008) or http://signal.salk.edu (Alonso et al., 2003), and were obtained from the NASC stock center (http://arabidopsis.info; Scholl et al., 2000). The club-2 insertion line is in Columbia. The wild-type ecotypes used as controls were Columbia and Landsberg erecta. club mutant seed will be made available on request.

Positional cloning of CLUB

For mapping, the club-1 allele in Landsberg erecta was crossed to wild-type Columbia. Bulk segregation analysis (as described by Lukowitz et al., 2000) mapped CLUB to the lower arm of chromosome V and this was confirmed by genotyping 100 F2 plants with simple sequence length polymorphism (SSLP) markers on chromosome V. For fine mapping, recombinants were selected by genotyping flanking SSLP markers in an additional 1900 F2 segregants. Recombinants were defined as individuals in which the genotype of the two SSLP markers flanking CLUB on either side differed. DNA preparation for the preselection of recombinants was carried out on 1900 F2 plants by alkaline lysis as described by Lukowitz et al. (2000). For the analysis of breakpoints in the relevant recombinants, DNA CTAB minipreps were prepared as described by Assaad et al. (2001). The Monsanto Arabidopsis polymorphism and Ler sequence database (http://www.arabidopsis.org) was used for identifying polymorphisms (Jander et al., 2002). The mapping and sequencing primers we designed as well as the protocols for PCR analysis are described in Supporting Information Table S1 and S2, and in Methods S1.

Antibody stains and confocal microscopy

Antibody stains were carried out as described by Völker et al. (2001) on the root tips of 5-d-old seedlings grown on MS (Sigma, http://www.sigmaaldrich.com) agar medium supplemented with 1% sucrose. The KNOLLE rabbit polyclonal antiserum was generated as described previously (Lauber et al., 1997). Monoclonal anti-tubulin antibody (Sigma) was used at a dilution of 1 : 2500. Goat anti-rabbit monoclonal antibody was coupled to Alexa-m488 (Molecular Probes, Eugene OR, USA, http://www.invitrogen.com) and goat anti-mouse secondary antibody was coupled to Cy3 (Dianova, Hamburg, Germany, http://www.dianova.de). Nuclei were stained with 1 mg ml−1 DAPI (4′,6-diamidino-2-phenylindole, Sigma). Slides were analyzed with a Fluoview 1000 confocal laser scanning microscope (Olympus, Hamburg, Germany, http://www.olympus.de). A 40× water immersion 0.9 numerical aperture objective (Olympus) was used, and scanning was carried out with four- to sixfold electronic magnification. Images were acquired and processed with the Fluoview 1000 acquisition software (Olympus) and subsequently processed using Adobe Photoshop. Images were assembled using Adobe Illustrator. (http://www.abode.com).

Light and electron microscopy

Embryos and seedlings for light and electron microscopy were fixed as described by Schumann et al. (2007). Infiltration was as described by Assaad et al. (1996). Five-day-old seedlings were used for light and electron micrographs. Transmission electron micrographs were taken with an EM 912 electron microscope (Zeiss, Oberkochen, Germany, http://www.zeiss.com) equipped with an integrated OMEGA energy filter operated at 80 kV in the zero loss mode. For scanning electron microscopy of fresh material, samples were placed on to stubs, and examined immediately in low vacuum with a Zeiss (LEO) VP 438 scanning electron microscope operated at 15 kV. Electron micrographs were digitally recorded from the BSE-signal.

Results

The embryo and seedling phenotypes of club mutants

A large collection of cytokinesis-defective mutants was identified in a screen for mutations affecting pattern formation and morphogenesis in the Arabidopsis seedling (Mayer et al., 1991). In addition to keule there were a number of mutants that had very similar seedling phenotypes but that were not in the same complementation group (Söllner et al., 2002). Among these was a mutant we named club-1, which is the English for ‘keule’ and which refers to the amorphous, club-like appearance of the mutant seedlings. Fig. 1 depicts the seedling phenotype of the club-1 mutant. Note the reduced growth of the root and shoot apices (Fig. 1b).

Figure 1.

club-1 seedling phenotype. (a) Wild-type, (b) club-1.

CLUB encodes a conserved subunit of the TRAPPII tethering complex

A mapping population of 2000 F2 plants localized CLUB to a 38 kb region at the bottom of chromosome V (Fig. 2a). The 38 kb interval contains 12 genes (Fig. 2b). We sequenced all 12 candidates in club-1 and found a single EMS-induced mutation in At5g54440, which inserts an opal terminator in the fourth exon of the predicted coding sequences (Fig. 2c, Table 1). In parallel, 32 T-DNA insertion lines were ordered from the SALK, GABI and INRA collections. One insertion line in At5g54440 (club-2, Fig. 2c, Table 1) shows a clear club phenotype (Figs 3–5). None of the other lines examined to date show the club phenotype. In an analysis of 93 club-2 plants, the T-DNA insertion showed an absolute segregation with the mutant phenotype (see Fig. S1). Complementation analysis showed that club-1, an EMS-induced allele in the Landsberg erecta background, and club-2, a T-DNA insertion in a Columbia background, are allelic, as evidenced by the presence of mutants in the F1 of a cross. We have characterized club-1 and club-2 by light, electron and confocal microscopy and found that both alleles have identical cytokinesis defects (see Figs 3–5). We have concluded that At5g54440 encodes CLUB, as evidenced by fine mapping to 38 kb, the sequencing and characterization of two null alleles and the segregation of the club-2 T-DNA with the mutant phenotype.

Figure 2.

CLUB encodes a putative TRAPPII tethering factor. (a) PCR-based markers (Supporting Information, Table S1) are shown in the top row. Mb, megabases 22.07–22.20 on chromosome V; Rec, number of recombinants in a population of 2000 plants. For further details see the Materials and Methods section, as well as Table S1. Fine mapping defined a 38 kb interval spanning CLUB. (b) Position of At5g54440 in the 38 kb interval defined by mapping. (c) The CLUB gene. A single open reading frame (ORF) is depicted by a line, and the exons are drawn as boxes. Two alleles, depicted by a dot for an ethyl methanesulfonate (EMS)-induced stop codon and by a triangle for a T-DNA insertion, show a club phenotype (Table 1). The scale is for nucleotides in bp. (d) The CLUB protein. Top: the conserved Interpro PTHR13251 domain that comprises the Trs130p signature is depicted by a white rectangle that spans the entire protein. The small black rectangle depicts a tetratricopeptide repeat (TRP)-like domain. The scale is for amino acid (a.a.) residues. Bottom: regions of homology with yeast and mammalian Trs130 orthologs are depicted by bars. For further details see the Materials and Methods section.

Table 1.   Two club alleles
AllelePolymorphismStock, accessionPosition in genePosition on ORFaPlate phenotype
  1. aNucleotide position on the full-length genomic sequence, including introns, starting from the start (ATG) codon. The polymorphisms are depicted graphically in Fig. 2(c).

  2. ORF, open reading frame.

club-1EMS (U57)4th Exon1658Seedling lethal
club-2SALK_039353N53935314th Intron5302Seedling lethal
Figure 3.

 Cell plate assembly during cytokinesis in wild-type and club-2 root tips. DAPI (4′,6-diamidino-2-phenylindole)/nucleus (blue, first column); KNOLLE protein (green, second column); microtubules (MT, red, third column) and merge (fourth column). The cell cycle stage is indicated on the left and the genotype in the first column. In all cases, representative cells are shown (Table 2). Note that cell plate formation, as judged with the KNOLLE antibody, is complete by anaphase in the wild-type (j) but not in club, even at telophase (n, v). Both club alleles were indistinguishable in this respect, such that, for consistency, all club panels (e–h, m–p, u–x) are club-2. See the Results section for a detailed description. Bars, 5 μm.

Figure 4.

 Cytokinesis defects in club root tips. Electron micrographs of 5-d-old Arabidopsis root tips. (a) Wild-type. Two largely nonvacuolate cells in which two nuclei (N) are fully separated by a cross wall (CW). Note the regular cell shapes and complete walls. (b–e) club-1. White arrows point to gaps in the cross wall. N, nucleus; S, cell wall stub. (b, c) Overviews of club-1 root tips showing frequent cell wall gaps (arrows) and stubs. (b) In the top right cell, two nuclei are found in a single cell; no cross-wall cell can be seen, other than a cell wall stub to the right. Below, an arrow points to the large gap between two juxtaposed wall stubs (S). (d) A ‘floating wall’, flanked by arrows on either side, is depicted, which, in the plane of the section, does not appear to be connected to the parental cell wall on either side; (d) is an enlargement of (c), in a different section. In (e), the left-hand arrow points to a gap in the cross wall that incompletely separates two daughter nuclei.

Figure 5.

 Incomplete cell plate assembly in club mutants of Arabidopsis. (a) club-2, (b) wild-type, (c–e) club-1. D, dictyosome (Golgi); M, mitochondria; N, nucleus; P, proplastid; V, vacuole. White arrows point to gaps in the cell plate or cross wall (CW). (a) An incomplete, patchy cell plate or cross wall. The membrane compartments constituting the patchy cell plate are larger and somewhat darker in appearance than the Golgi-derived vesicles that surround an adjacent dictyosome (D). Panel (d) is an enlarged version of the cell on the right in (c), and (e) is a magnification of the boxed area in (c). (c) Two adjacent, largely avacuolate cells, in which one cell (left) has two nuclei that are not separated by a cross wall, and the other (right) has two nuclei incompletely separated by a string of loose compartments or vesicles (arrows in c, d). These untethered vesicles or compartments have the appearance of beads on a string. The nuclei (N) in this cell, however, have decondensed chromosomes and nuclear envelopes. Thus, and in contrast to the wild-type (b), the nuclear cycle is complete but the cell plate has failed to assemble.

CLUB encodes a large 1280 aa protein with a conserved PTHR13251 domain that comprises the signature of the transport protein particle TRAPPII C10 subunit, which corresponds to Trs130p in yeast, and TMEM1 in mammals (Cox et al., 2007). This domain is depicted by a white rectangle that spans the entire length of the protein in Fig. 2(d). In both club-1 and club-2, the PTHR13251 domain would be disrupted. Thus, both alleles are presumably null and, consistently, both had the same phenotype. CLUB also has a TPR/PPR (tetratricopeptide repeat) motif (depicted by a black rectangle in Fig. 2d) that could be involved in the binding of proteins and nucleic acids. It has homologs in all other plants whose genomes have been sequenced. Multiple sequence alignment analysis of Trs130-related proteins across kingdoms revealed three domains, spanning 33–314 amino acids (Cox et al., 2007). These are consistent with our own phylogenetic analyses, and in Fig. 2(d) we depict three highly conserved domains shared by CLUB and its yeast and mammalian homologs.

There are two TRAPP complexes in yeast, TRAPP I and TRAPP II. These complexes have seven common subunits, while three additional subunits, including Trs130p, are only found in the TRAPP II complex (Cox et al., 2007; Sacher et al., 2008). Eight of these subunits have as yet uncharacterized Arabidopsis homologues. A thorough phylogenetic analysis of the TRAPPII complex as well as a list of known subunits and their gene identities for 41 species, including Arabidopsis, have been published (Cox et al., 2007). The mammalian and yeast TRAPP I complexes have recently been characterized, and shown to reside on Golgi membranes where they tether (or capture) endoplasmic reticulum (ER)-derived vesicles (Kim et al., 2006; Cai et al., 2008; Sacher et al., 2008). The role of the TRAPP II complex remains somewhat more elusive than that of TRAPP I. In yeast, this may be to regulate exit from the Golgi, and/or to tether vesicles that recycle from endosomes to a late Golgi compartment (Cai et al., 2005; Morozova et al., 2006). Thus, while TRAPP I tethers ER vesicles at the cis face of the Golgi, TRAPP II may tether Golgi or recycling vesicles at the TGN (Cai et al., 2005). TRAPP II mutants of yeast affect endocytosis and secretion, consistent with the localization of the yeast TRAPPII complex to the TGN (Cai et al., 2005).

CLUB is required for cell plate assembly

The orthology between CLUB and conserved TRAPPII subunits in yeast, combined with the highly informative cytokinesis defect of club mutants (see Figs 4 and 5 below), have prompted us to postulate that CLUB acts to tether vesicles to the cell plate. We therefore predicted that cell plate formation might be perturbed in club mutants. As described later, we addressed cell plate assembly with both confocal and electron microscopy.

We first studied cell plate formation in club root tips by immunofluorescence. The assembly of the membrane networks that support cell wall formation was followed with a rabbit polyclonal antibody to the syntaxin KNOLLE, required for vesicle fusion at the cell plate (Lauber et al., 1997). In parallel, nuclei were stained with DAPI and microtubules with a monoclonal antibody to monitor cell cycle stages. In an analysis of 202 mutant and 162 wild-type cytokinetic cells, we found a clear difference between club and the wild-type (Fig. 3). This is tabulated in Table 2 and described in detail for each cell cycle stage as follows:

Table 2.   Confocal analysis of wild-type and club cytokinetic Arabidopsis root tip cells
 Cell plate assembly (%)Vesicle abundance (%)Total cells (n)
Cell plate absentPatchily assembledIntermediate or thinCompleteNoneFewIntermediateAbundant
  1. The cell cycle stage is given at the top and numbers that differ considerably between the mutant and wild-type are in bold. The stages of cell plate assembly were designated ‘absent’ when there was no cell plate, patchily assembled when only patches of a plate could be seen, intermediate if there were numerous vesicles and/or a thin plate, and complete if the cell plate was contiguous and extended. Vesicle abundance refers to the number of vesicles (none, few, intermediate, abundant) that could be seen at the equator and throughout the cell. The right column gives the total number of cells of a given genotype at a given cell cycle stage.

Late anaphase
Wild-type4.51.513.780.334.827.331.86.166
club-122.227.833.316.75.65.661.127.718
club-223.225.330.321.27.05.034.453.699
Telophase
Wild-type02.111.486.555.420.522.91.296
club-130.020.030.020.00040.060.010
club-222.737.332.08.02.71.332.064.075

Metaphase: KNOLLE-positive vesicles have accumulated by this stage, which is characterized by the alignment of chromosomes at the cell equator and the presence of the spindle apparatus. In a quantitative analysis of 28 wild-type and 20 club metaphase cells, we recorded KNOLLE-positive vesicles as being frequent or abundant in 75% of wild-type and 85% of mutant cells. Thus, we found no difference in the abundance of KNOLLE-positive vesicles at the beginning of the cell cycle (Fig. 3a–h).

Anaphase: As soon as the two daughter nuclei have reached the opposite poles of the cell, the spindle disintegrates and phragmoplast microtubules can be seen to form two bands at the equator of the cell. Throughout anaphase the chromosomes still appear condensed (Fig. 3i,m). In 80.3% of wild-type cell plates we observed at this stage, a complete cell plate (Fig. 3j; Table 2) had formed between the phragmoplast microtubules (Fig. 3k). In the majority of club anaphase mutants, however, only loose or patchily assembled vesicles were found at this stage (Fig. 3n; Table 2).

Telophase: We identified telophase cells based on a DAPI stain showing an ovoid nucleus in which the DNA is decondensed and the nucleoli are visible (Fig. 3u), together with microtubules that had reorganized to the leading edges of the phragmoplast (Fig. 3s,w). By telophase, the α-KNOLLE labeling of the plate stretched across wild-type cells (Fig. 3r), but club mutants still predominantly accumulated unassembled vesicles (Fig. 3v; Table 2). KNOLLE-positive, completely formed cell plates were scored in only 8% of club-2 mutant telophase cells, as opposed to 86.5% in the wild-type (Table 2). In the remaining wild-type plates, the vesicles we saw were likely to be ones already recycling away from the cell plate, as can be seen at the end of cytokinesis (Thiele et al., 2009). Loose KNOLLE-positive vesicles were abundant in 60–64% of club telophase cells, but in only 1.2% of the wild-type cells at this stage (Table 2).

Conclusion: Whereas in the wild-type, the cell plate was fully assembled by anaphase, club mutants failed to assemble a cell plate throughout cytokinesis. Rather, loose KNOLLE-positive vesicles accumulated in club mutant cells throughout mitosis, and were at best only patchily assembled into incomplete plate fragments at the cell equator. This differs from the wild-type, in which KNOLLE-labeled vesicles were initially only visible during metaphase and early anaphase and were then seen to assemble into a contiguous cell plate.

To confirm the cell plate phenotype, we then carried out electron micrscopy on root tips. Electron micrographs showed a dramatic perturbation of cross-wall formation in club root tips. Canonical cytokinesis defects such as bi- or multinucleate cells, cell wall stubs and gaps are shown in Fig. 4 and the incompletely assembled cell plates that characterize club mutants are shown in Fig. 5. Figs 4(b) and (c) show two different club-1 mutants with multiple defects, including stubs and gaps (arrows). In Fig. 4(b), a binucleate meristematic cell (towards the top of the panel) with a cell wall stub (S) can be seen. In an adjacent cell (at the lower right), one cross wall is incomplete, leaving two stubs (S) on either side of the cell with a large gap (arrow) in between. To the left, an adjoining cell has an incomplete cross wall leaving a gap (arrow) on one side of the cell, as also shown enlarged in a different sample in Fig. 4(e). In Fig. 4(d), a ‘floating wall’ can be seen, which in the plane of division does not appear to be attached to the parental wall on either side (arrows). A similarly aberrant cell wall fragment can be seen in the boxed area in Fig. 5(c), which is enlarged in Fig. 5(e). These canonical cytokinesis defects are in stark contrast to the wild-type shown in panel (a), in which the daughter nuclei are completely separated by a cross wall.

In Figs 5(a), (c) and (d), a patchy alignment of large vesicles and/or membrane compartments presumably correspond to incomplete cell plates. The numerous gaps in a club-2 patchily assembled cell plate are designated by white arrows in Fig. 5(a). While the cell plate has failed to assemble fully, the formation of Golgi-derived vesicles (surrounding a dictyosome) did not appear to be impaired in club mutants (Fig. 5a). Fig. 5(c) depicts two adjacent cells, of which one (left) has two nuclei that are not separated by a cross wall, and the other (right) has two nuclei incompletely separated by a string of loose compartments or vesicles (arrows in panels c and d). These untethered vesicles or compartments have the appearance of beads on a string. The nuclei in this cell, however, have decondensed chromosomes and nuclear envelopes, such that cell plate or cross-wall formation should be complete, as in the wild-type (Fig. 5b).

Collectively, these binucleate cells, cell wall stubs, gaps and patchy cross walls in meristematic (nonvacuolate) cells define club mutants as being cytokinesis-defective. In both the confocal images and the electron micrographs, the observed defects in cell plate assembly as well as the high number of untethered vesicles present during cytokinesis in club mutants show that CLUB is required for cell plate biogenesis.

CLUB is required for transmission via the pollen

To assess T-DNA transmission in the club-2 insertion line, DNA was isolated from viable plants grown on soil, and the presence or absence of the T-DNA was monitored via both PCR (Fig. S1) and phenotyping. For a seedling lethal mutation, one would expect 67% hemizygous and 33% wild-type individuals in the progeny of a selfed hemizygous line. For club-2, we observed only 51% hemizygous segregants. Thus, this insertion line shows reduced transmission of the mutant allele. In reciprocal crosses between Columbia and club-2, transmission via the female gametophyte was not reduced, as evidenced by 50% transmission rates when the insertion mutant was the recipient in the cross (Table 3). By contrast, the transmission rate was reduced to 33% (Table 3) when the insertion mutant was used as a pollen donor. This indicates that the reduction in transmission occurs specifically via the pollen. Expression profiling of CLUB (At5g54440) in silico (http://www.genevestigator.ethz.ch, Hruz et al., 2008) shows a significant up-regulation in embryos, shoot apices, inflorescences, root hairs, and root tips (Fig. S2). We have, consistently, documented phenotypes in root hairs (Söllner et al., 2002) and root tips and in the male gametophyte. CLUB is also required during embryogenesis, as evidenced by the presence of mutant embryos in dissected siliques (F Assaad, unpublished). Thus, both expression profiling and phenotypic analysis suggest that CLUB is required not only for cytokinesis but also for a broader range of processes pertaining to plant growth.

Table 3.   Reduced transmission of the club-2 insertion
 WT (%)Hemizygous (%)Total
  1. aHemizygous segregants in the progeny of a hemizygous line grown on soil (%). In the absence of defects in transmission of the T-DNA insertion, and given that the mutation is lethal, 67% would be expected.

  2. bIn this cross, a club-2 hemizygous line is the recipient.

  3. cA club-2 hemizygous line is the pollen donor in this backcross to the wild-type (WT).

  4. In both reciprocal crosses, 50% hemizygous progeny would be expected. Note that transmission rates are reduced in crosses in the first and third rows, but not the second. This discrepancy between expected and observed transmission rates is indicated by bold text in the third column.

club-2 selfeda495187
club-2b × Col505096
Col × club-2c673394

Discussion

We have characterized an Arabidopsis gene, CLUB, that encodes a putative TRAPPII tethering factor required for cytokinesis. The CLUB protein shares homology with characterized, conserved subunits of the TRAPPII tethering complex of yeast and mammalian cells. Canonical cytokinesis defects, including bi- or multinucleate cells, cell wall stubs and gaps as well as floating walls, were observed in club seedlings. Confocal microscopy revealed that, in contrast to the wild-type, a large number of untethered or unfused KNOLLE-positive vesicles accumulated at the equator of dividing cells throughout cytokinesis in club mutants. Similarly, electron microscopy showed that membrane compartments or vesicles were sometimes aligned along the cell equator, but failed to assemble into a contiguous plate. The resulting mutant plates resemble beads on a string. This is a unique feature of club mutants, as we have not observed a ‘beads on a string’ plate phenotype at this frequency in any other cytokinesis-defective mutant we have analyzed, including keule mutants defective in vesicle fusion at the cell plate (Assaad et al., 1996; Waizenegger et al., 2000; Assaad et al., 2001; Söllner et al., 2002; Thiele et al., 2009). Our interpretation of these findings is that the primary defect in club mutants is a failure to assemble the cell plate.

In addition to their cytokinesis defects, club mutants have been reported to have root hair tip growth defects (Söllner et al., 2002). In this study, we show a reduced transmission of the club-2 insertion via the pollen. Expression profiling showed an up-regulation of CLUB in rapidly cycling tissues as well as in root hairs. It appears that CLUB is required for a broad range of processes requiring rapid, differential growth.

CLUB encodes a putative tethering factor. Tethering is a term that has been used to describe the initial interaction of a transport vesicle with its target membrane. Our confocal analysis and electron micrographs are consistent with a block in vesicle tethering on two counts. First, the presence of loose vesicles throughout cytokinesis in club mutants suggests that it is the initial interaction between such vesicles that is impaired. Second, as the large vesicles or membrane compartments (the ‘beads’, as it were) building the incomplete cell plates characteristic of club mutants differ in their appearance from the vesicles surrounding the adjacent dictyomsomes and from the vesicles in the vicinity of the plate, it appears that the post-Golgi vesicles undergo a limited degree of homotypic fusion at the cell plate. The SNARE (KNOLLE) and SM (KEULE) machinery should still be intact in club mutants, and these should allow for a basal degree of fusion in the absence of the tethering apparatus. The role of the tethering apparatus would, in fact, be to increase the efficiency and speed of such fusion events, without being absolutely required. While we cannot formally distinguish between vesicle tethering and fusion in our experiments, our findings suggest that it is the initial connection between post-Golgi or KNOLLE-positive vesicles that is impaired and provide functional evidence that CLUB acts as a tethering factor.

The yeast and mammalian TRAPPII tethering complexes and the club phenotype

CLUB bears significant sequence homology to a conserved subunit, Trs130, of the TRAPPII tethering complexes of yeast and mammals. trs130 mutants of yeast display general defects in secretion, as well as defects in membrane recycling along an endocytotic route from the plasma membrane via the early endosome to the late Golgi and back to the plasma membrane (Cai et al., 2005). Defects in secretion have been visualized in yeast via the analysis of defects in the glycosylation and sorting of proteins of the secretory pathway (Cai et al., 2005). Recycling along an endocytotic route was assayed with green fluorescent protein fusions to marker proteins targeted to regions of polarized growth or to the plasma membrane (Cai et al., 2005). Trs130p colocalizes with late Golgi and early endosomal markers, but not with PI(3)P-containing endosomal membranes (Cai et al., 2005). These findings are consistent with a role of yeast Trs130p in post-Golgi traffic, as well as traffic between the Golgi and early endosomes. Thus, TRAPP II may tether Golgi or recycling vesicles at the TGN, thereby potentially regulating exit from the Golgi as well as endocytosis (Cai et al., 2005).

While the yeast TRAPPII complex is thought to regulate exit from the Golgi, the mammalian TRAPPII complex has been proposed to mediate tethering of vesicles to early Golgi membranes (Yamasaki et al., 2009). The function of the TRAPPII complex in yeast and mammals is controversial, with different results obtained by different groups working on the same yeast proteins and discrepancies between yeast and mammalian homologs. Discrepancies within the yeast literature may be the result of differences in complex purification and assay conditions (Sacher et al., 2008), but the different roles of the yeast and mammalian TRAPPII complexes may be real and related to fundamental differences in the architecture of the ER–Golgi pathway between different kingdoms (Yamasaki et al., 2009).

In this study, we present a novel assay for TRAPPII function, namely the biogenesis of a specialized, transient cell compartment. Although the cell plate is a plant-specific compartment, it has the advantage that its assembly can be readily visualized in space and in time and that it is clearly distinct from the Golgi stacks. It is becoming widely accepted that the cell plate is a TGN-derived compartment (Dettmer et al., 2006; Reichardt et al., 2007; Chow et al., 2008). The club cell plate phenotype therefore argues for a function at the TGN, consistent with the work of Cai et al. (2005) and Morozova et al. (2006) on the CLUB homologue Trs130p in yeast. To our knowledge, there has not to date been any evidence in yeast or mammalian cells that the TRAPPII complex is required for the biogenesis of a cell compartment. There is, however, one such report for the conserved TRAPPI subunit mBet3p, which is thought to be required for the biogenesis of pre-Golgi structures referred to as intermediate compartments or vesicular tubular clusters that lie adjacent to the transitional ER (Yu et al., 2006). In plants, the homotypic fusion and vacuole protein sorting (HOPS)/vacuole protein sorting (VPS) tethering complex, which resides on the tonoplast and prevacuolar compartment, has been shown to be required for the biogenesis of the vacuole (Rojo et al., 2001, 2003).

The yeast literature at times describes the TRAPPII complex as residing on late Golgi membranes, and as being required for exit from the Golgi and/or for post-Golgi traffic (Cai et al., 2005). In our confocal analysis of club mutants, the timing of the appearance of KNOLLE-positive vesicles, and the abundance of such vesicles, did not differ from the wild-type. Similarly, in electron micrographs of club mutants, the frequent appearance of Golgi-derived vesicles (surrounding dictyosomes) suggests that the formation of Golgi-derived vesicles is not impaired in club mutants. We conclude that the primary defect in our plant TRAPPII mutant lies not in exit from the Golgi, but in the tethering of vesicles to what is most likely a TGN compartment.

CLUB comprises a novel handle on the cell plate

The TGN is emerging as a major hub for the flow and sorting of information from and to the cell surface (Dettmer et al., 2006; Lam et al., 2007; Otegui & Spitzer, 2008; Staehelin & Kang, 2008). As an early endosome, the TGN is the first point of entry into the secretory pathway for information from the cell surface. Genome analysis has highlighted the sophisticated and differentiated nature of membrane trafficking in plants (Sanderfoot et al., 2000; Assaad, 2001a). Interestingly, the expansion of plant-trafficking proteins occurs in specific gene families involved in TGN function or post-Golgi traffic (Sanderfoot et al., 2000; Assaad, 2001a). The most striking example of this is the Rab family of small GTPases. Indeed, there are 26 Rab GTPases required for TGN function and post-Golgi traffic in Arabidopsis, compared with only one in fission yeast, two in budding yeast and three in the human genome (Vernoud et al., 2003; Chow et al., 2008). Rab GTPases are important in vesicle tethering and docking, and TRAPP I and II subunits have been shown to act as nucleotide exchangers for Rab GTPases (Kim et al., 2006; Morozova et al., 2006; Cai et al., 2008). In budding yeast (Jones et al., 2000; Morozova et al., 2006), the TRAPPII complex has been shown to act as a GEF for the two late Golgi Rab GTPases YPT31 and YPT32, which regulate exit from the Golgi. The plant homologs of these two Rabs are the expanded Rab A family, of which two family members have recently been shown to reside on the cell plate (Chow et al., 2008). Thus, the analysis of club mutants is entirely compatible with a role for the TRAPPII complex at the TGN.

There are two tethering complexes known to play a role at the TGN in yeast, the TRAPPII and the GARP complex (Koumandou et al., 2007). While the TRAPPII complex has been implicated in both exo- and endocytosis (Cai et al., 2005), the GARP complex has only been shown to be required for the tethering of endocytotic vesicles to the TGN (Conibear et al., 2003). The Arabidopsis GARP complex has been shown to play a role in pollen tip growth (Lobstein et al., 2004; Guermonprez et al., 2008). As the relative contribution of Golgi-derived and endocytosed molecules to the cell plate remains to be determined, it will be interesting to explore the different roles of the TRAPPII and GARP complexes in cytokinesis.

One of the most important questions in plant cytokinesis is the cell cycle regulation of cell plate assembly. Tethers mediate the first contact between donor and acceptor membranes, and cell cycle regulation is more likely to occur at the tethering step than at the level of membrane fusion. CLUB, required for cell plate biogenesis, provides a potentially important and novel handle on the cell cycle regulation of cross-wall deposition.

Acknowledgements

Farhah Assaad is especially grateful to Erwin Grill for his encouragement and useful suggestions. For TEM analyses, Silvia Dobler and Connie Niemann are gratefully acknowl-edged for excellent technical assistance. Caroline Klaus tended to our plants. Thanks to Theis Stüven, Peter Zehetmayer, and to numerous members of the botany and Genetics departments, and to the Schneitz and Schwechheimer laboratories for their input. We are grateful to TAIR (http://arabidopsis.org), to the NASC stock center (http://arabidopsis.org) and to the SSP consortium (http://signal.salk.edu) for bioinformatic resources and T-DNA lines. This work was funded by a Deutsche Forschungsgemeinschaft grant, AS 110/4-4, to FA.

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