Cytokinesis-Defective Mutants of Arabidopsis1 Rosi So¨llner, Gerti Gla¨sser, Gehard Wanner, Chris R. Somerville, Gerd Ju¨rgens, and Farhah F. Assaad* Genetics and Microbiology Institute, Ludwig Maximillian University, Maria Ward Strasse 1a, 80638 Munich, Germany (R.S., G.G., F.F.A.); Botany Institute, Ludwig Maximillian University, Menzingerstrasse 67, 80638 Munich, Germany (G.W.); Carnegie Institution, Department of Plant Biology, 260 Panama Street, Stanford, California 94305 (C.R.S.); and Lehrstuhl fu¨r Entwicklungsgenetik, Universita¨t Tu¨bingen, D–72076 Tu¨bingen, Germany (G.J.)
We have identified mutations in six previously uncharacterized genes of Arabidopsis, named club, bublina, massue, rod, bloated, and bims, that are required for cytokinesis. The mutants are seedling lethal, have morphological abnormalities, and are characterized by cell wall stubs, gapped walls, and multinucleate cells. In these and other respects, the new mutants are phenotypically similar to knolle, keule, hinkel, and pleiade mutants. The mutants display a gradient of stomatal phenotypes, correlating roughly with the severity of their cytokinesis defect. Similarly, the extent to which the different mutant lines were capable of growing in tissue culture correlated well with the severity of the cytokinesis defect. Phenotypic analysis of the novel and previously characterized loci indicated that the secondary consequences of a primary defect in cytokinesis include anomalies in body organization, organ number, and cellular differentiation, as well as organ fusions and perturbations of the nuclear cycle. Two of the 10 loci are required for both cytokinesis and root hair morphogenesis. The results have implications for the identification of novel cytokinesis genes and highlight the mechanistic similarity between cytokinesis and root hair morphogenesis, two processes that result in a rapid deposition of new cell walls via polarized secretion.
During the process of cell division, cells undergo four key transitions: entry into S phase (DNA replication), entry into mitosis, exit from mitosis, and the onset and execution of cytokinesis, the partitioning of the cytoplasm after nuclear division. The key regulators of entry into S phase and mitosis appear largely conserved among plant, yeast, and animal cells (Assaad, 2001b). By contrast, the last phase of the cell cycle seems different in plants as compared with fungi and animals (Assaad, 2001b). Polo kinases and septins and the CDC15 protein, which is required for mitotic exit and/or cytokinesis, are present in all sequenced eukaryotes with the notable exception of Arabidopsis (Song and Lee, 2001; Assaad, 2001b). Localization and phosphorylation studies, and the study of dominant negative mutants have highlighted the importance of a mitogenactivated protein kinase cascade in the regulation of plant cytokinesis (Bo¨gre et al., 1999; Nishihama et al., 2001). Plant cytokinesis has many unique features and can be considered as a form of polarized secretion 1 This work was supported by a European Union Biotechnology Program Framework IV grant (to G.J.), by the U.S. Department of Energy (grant no. DOE–FG02– 00ER20133 to C.R.S.), and by the Deutsche Forschungsgemeinschaft (grant no. AS110/2–1 to F.F.A.). F.F.A. was supported by a long-term European Molecular Biology Organization fellowship and a Hochschulsonderprogram stipend from the University of Munich. * Corresponding author; e-mail
[email protected]; fax 650 –325– 6857. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.004184.
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(Assaad, 2001a). At the end of anaphase, Golgiderived secretory vesicles carrying cell wall materials are transported to the equator of a dividing cell. Fusion of these vesicles gives rise to a membranebound compartment, the cell plate. The cell plate expands until it reaches the division site on the mother cell wall (Ehrhardt and Cutler, 2002). Once this attachment has taken place, the cell plate undergoes a complex process of maturation during which callose is replaced by cellulose and pectin (Samuels et al., 1995, and refs. therein). Two plant-specific cytoskeletal arrays of microtubules and actin filaments, the preprophase band and the phragmoplast, play central roles in the orientation and expansion of the cell plate and in the execution of cytokinesis (for review, see Otegui and Staehelin, 2000; Sylvester, 2000; Assaad, 2001a). It follows from this brief description that genes implicated in membrane and cytoskeletal dynamics, vesicle trafficking, and cell wall biogenesis will impact plant cytokinesis. Relatively few genes involved in cytokinesis have been identified by mutation in plants. tso1, stud, tardy asynchronous meiosis, tetraspore, and sidecar pollen specifically affect cytokinesis in floral organs or during pollen development (Chen and McCormick, 1996; Hu¨lskamp et al., 1997; Spielman et al., 1997; Hauser et al., 2000; Song et al., 2000; Magnard et al., 2001). Genes required for cytokinesis in somatic plant cells seem to be partially distinct from those required during gametophytic development (Lauber et al., 1997; Otegui and Staehelin, 2000; Assaad et al., 2001) and fall into two classes. Genes in the first class are required for the proper orientation of the plane of division, and genes in the second class are required
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for the execution of cytokinesis. fas/tonneau, discordia, and tangled (for review, see Sylvester, 2000; Smith, 2001) mutants are implicated in regulating the plane of division. The cyd mutants of pea (Pisum sativum) and Arabidopsis, and the KNOLLE, KEULE, and HINKEL genes of Arabidopsis are required for the execution of cytokinesis (Liu et al., 1995; Assaad et al., 1996; Lukowitz et al., 1996; Yang et al., 1999; Strompen et al., 2002). We focus here on the second class of mutants. Light and electron microscopy showed that dividing cells in cyd, cyd1, knolle, and keule mutants are often multinucleate, with gapped or incomplete cross walls, which defines these mutants as cytokinesis-defective (Liu et al., 1995; Assaad et al., 1996; Lukowitz et al., 1996; Yang et al., 1999). The multinucleate cells are invariably enlarged (Assaad et al., 1996) and account for the rough surface and bloated appearance of these cytokinesis mutants. The KNOLLE, HINKEL, and KEULE genes have been cloned. HINKEL encodes a plant-specific kinesinrelated protein required for the reorganization of phragmoplast microtubules during cell-plate formation (Strompen et al., 2002). KNOLLE encodes a novel cytokinesis-specific syntaxin (Lukowitz et al., 1996; Lauber et al., 1997). Thus, HINKEL and KNOLLE represent two distinct subclasses of cytokinesisspecific mutants, affecting cytoskeletal and membrane dynamics, respectively. KEULE encodes a key regulator of vesicle trafficking, a Sec1 protein, that interacts genetically and biochemically with the syntaxin KNOLLE (Waizenegger et al., 2000; Assaad et al., 2001). In both keule and knolle mutants, vesicles are transported to the equator of a dividing cell but do not fuse (Waizenegger et al., 2000). A biochemical and reverse genetic approach has implicated an additional vesicle trafficking gene, SNAP33, in plant cytokinesis (Heese et al., 2001). Cell wall stubs and radial swelling are characteristic of cell wall mutants such as korrigan, procuste, rsw1/rms, and cyt1 (Nickle and Meinke, 1998; Fagard et al., 2000; Zuo et al., 2000; Lukowitz et al., 2001). Cytokinesis defects have also been described in sterol-defective fackel mutants (Schrick et al., 2000) and in titan or pilz mutants, characterized by the incidence of giant nuclei (Liu and Meinke, 1998; Mayer et al., 1999). Because mutations affecting cytokinesis, nuclear content, sterol biosynthesis, and cell wall biogenesis share a number of common phenotypic features, an important question is to what extent one can distinguish between mutations affecting these diverse pathways based on phenotypic analysis. We monitor embryo, seedling, stomatal, root hair, trichome, and postembryonic development in the novel as well as previously characterized cytokinesis-defective mutants and address the following questions: By what criteria do we define a mutant as being predominantly cytokinesis defective? Are cytokinesis-defective mutants impaired in cellular processes other than cytokinesis? What are Plant Physiol. Vol. 129, 2002
the developmental consequences of a cytokinesis defect? And what phenotypes may be used in attempting to isolate novel cytokinesis-defective loci? RESULTS AND DISCUSSION Isolation and Initial Characterization of the Novel Cytokinesis-Defective Mutants
A number of novel cytokinesis-defective mutants were uncovered in a large-scale screen for mutations affecting seedling body organization (Mayer et al., 1991, 1999; Nacry et al., 2000). Three of the loci identified in this screen, KNOLLE, KEULE, and HINKEL, have since been characterized in detail (Assaad et al., 1996, 2001; Lukowitz et al., 1996; Lauber et al., 1997; Strompen et al., 2002). The collection of cytokinesisdefective mutants includes four knolle alleles, 16 keule alleles, one hinkel allele, and one pleiade allele (see “Materials and Methods”). pleiade mutants were identified in a screen for mutations affecting root morphogenesis (M.T. Hauser, personal communication; see also Hauser et al., 1997). In addition to the above-mentioned mutants, six mutant lines were isolated that resembled knolle mutants, and seven mutant lines were isolated that resembled keule mutants but were not in the KEULE, KNOLLE, HINKEL, or PLEIADE complementation groups (U. Mayer and F.F. Aassad, unpublished data). Although keule and knolle mutants are indistinguishable at a cellular level, cytokinesis defects are more severe in knolle mutants (see Table II). As a result, knolle mutants are tuber-like and keule mutants club-like in shape, whence their names. The six knolle-like lines have been described elsewhere (Nacry et al., 2000). In this study, we focus on the seven keule-like lines and compare them with cytokinesis-specific mutants such as hinkel and knolle identified in this screen. Data on keule, knolle, hinkel, or pleiade are presented where unpublished or as reference points. Complementation analysis together with mapping showed that the seven keule-like mutant lines represent six novel loci (Table I; “Materials and MethTable I. Six novel cytokinesis-defective loci Mapping was carried out with PCR-based molecular markers, with sufficient resolution to map adjacent loci to distinct genetic intervals. Complementation tests were then performed between each of the uncharacterized mutants and any nearby keule-like mutants. The six loci are distributed on four of the five Arabidopsis chromosomes, with a cluster, including KNOLLE, KEULE, and HINKEL on the upper arm of chromosome I. See “Materials and Methods” for further details. * Corresponds to a rough map location. Locus
Lines (Allele)
Map Location
CLUB BUBLINA MASSUE ROD BLOATED BIMS
U57 (1) R3-27 (1) G88 (1), MNN (2) S100 (1) U119 (1) S302 (1)
V/109 I/29 II/66 IV/⬃65* IV/92 I/12 679
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ods”). We have named these loci club, bublina, massue, rod, bloated, and bims, based on the bloated and rodor club-shaped appearance of mutant seedlings (Fig. 1). All of the keule-like lines depicted in Figure 1 lack functional shoot and root meristems and carry recessive, seedling-lethal mutations. Nonetheless, clearing preparations show that the apical-basal and radial organization of tissue layers is generally conserved in mutant seedlings. This is illustrated by the massue-2 mutant in Figure 1I. The vascular strands often show interruptions, misalignment and deviation from the wild type with respect to the number and width of strands per bundle, as illustrated by the bims mutant in Figure 1J. In some instances, short vascular strands are found adjacent to the centrally located vascular bundles (see bloated mutant in Fig. 1K). Thus, whereas the overall organization and differentiation of tissue layers is normal, patches of cells acquire the fate of an adjacent layer or cell row, as has been described for keule and knolle (Assaad et al., 1996; Lukowitz et al., 1996). The seedling phenotypes were variable within most lines, ranging from stout seedlings with reduced cotyledons as shown for club, bublina, and bims (Fig. 1, B, C, and G) to seedlings with well-defined cotyledons and a stunted root as shown for massue-2, rod, and bloated (Fig. 1, D–F). Cytokinesis versus Cell Wall Defects
Figure 1. Bloated seedlings lacking functional root and shoot meristems. All seedlings have a rough surface layer and consist of cotyledons and a hypocotyl. Though the basal area has root-like properties, roots are absent in keule, club, bublina, and bims mutants (A, B, C, and G); stunted in massue and bloated mutants (D and F); and reduced in rod mutants (E). At the shoot apex, true leaves are absent or stunted, though these may develop upon transfer to tissue culture (see Fig. 5, F and G). The seedling phenotype is variable in all of the lines, with the exception of club and bims mutants for which the range of phenotypes is fairly narrow. The seedlings shown represent the median within the respective ranges of phenotypes, although massue mutants (D) often have considerably stronger and bloated mutants (F) considerably weaker phenotypes than those shown here. A clearing preparation shows that the apical basal body plan and the radial organization of tissue layers are generally conserved (I). The pattern elements of the seedling are shown in I and/or H; along the apical-basal axis, these include the cotyledons, hypocotyl, and root; and along the radial axis they include the epidermis, ground tissue, and vascular bundle. The vascular strands often show interruptions, misalignment, and deviation from the wild type with respect to the number and width of strands per bundle (arrow in J). In some instances, short vascular strands are found adjacent to the centrally located vascular bundles (arrow in K). Note the long root 680
To determine whether the keule-like mutants were cytokinesis defective, we examined histochemically stained sections of mutant embryos and seedlings by light microscopy. Cytokinesis-defective mutants are typically characterized by the presence of cell wall stubs in dividing cells. Therefore, we sectioned dividing tissues from globular to heart stage embryos and/or the apical meristems of seedlings. Cell wall stubs, gapped cell walls, and multinucleate cells were found in all of the keule-like mutant lines in these tissues (Fig. 2). The stubs and incomplete walls stained with the cell wall-specific periodic acid schiff stain, as shown in Figure 2G. In the case of keule and knolle, cytokinesis defects have been shown to arise as of the first division of the zygote (Assaad et al., 1996; Lukowitz et al., 1996). An analysis of clearing preparations and histological sections of dermatogen-torpedo stage embryos (for a description of Arabidopsis embryogenesis, see Ju¨ rgens and Mayer, 1994) showed that cytokinesis defects occur before the two-cell stage in hinkel and massue embryos, before the dermatogen stage in pleiade, and before the globular stage in bublina and
hairs at the basal end of the massue-1 mutant in I. The mutant lines shown are as indicated above each panel, and massue-1 is shown in I, bims is shown in J, and bloated is shown in K. keule allele MM125 is shown as reference in A. a, Apical meristem, first true leaf primordia; c, cotyledons; e, epidermis; g, ground tissue; h, hypocotyl; rh, root hairs; r, root; and v, vascular bundle. Bars ⫽ 200 m. Plant Physiol. Vol. 129, 2002
Cytokinesis-Defective Mutants of Arabidopsis
bloated embryos (Fig. 2B; data not shown). By contrast, cytokinesis defects were rarer and appeared to occur later in bims embryos (Fig. 2K), which have a relatively narrow range of phenotypes. In club mutants, early divisions of the true leaf primordia and apical meristem are affected (Fig. 2, C and D). The first appearance of cytokinesis defects during development seems to be stochastically determined and may contribute to the great variability of embryo and
seedling phenotypes characteristic of cytokinesisdefective mutants. Cell wall-defective mutants such as procuste, knopf, rsw1/rms, and korrigan share two features in common with cytokinesis-defective mutants such as keule: cell wall stubs and radial expansion of affected tissues such as the root (Fagard et al., 2000; Peng et al., 2000; Zuo et al., 2000; Boisson et al., 2001; Gillmor et al., 2002). In cytokinesis-defective mutants, stubs are of-
Figure 2. Histological sections of embryos or seedlings revealing cell wall stubs and multinucleate cells. Histological sections were stained with toluidine blue, which stains the nuclei and cell wall, with the exception of that in G, which is stained with the cell wall-specific periodic acid schiff stain. Toluidine blue-stained nuclei appear as vacuolate structures with dark, round- or doughnut-shaped nucleoli. A, B, E, G, I, J, K, and L are embryo sections; C, D, F, and H are seedling sections. In C and D, an apical meristem is shown. B, This section depicts the basal area of a torpedo stage pleaide embryo, representing a severely affected hypocotyl and root primordium; in the same mutant, the cotyledons appear unaffected (not shown); the multinucleate cell shown encompasses more than one-half of the mutant embryo, as can be seen by comparison with the small outer cells in the same section, which suggests that the cytokinesis defects occurred early during embryogenesis; note the large nuclei with multiple nucleoli (black spots designated by arrowhead). F, Severely affected surface layer of a bublina cotyledon. H, Cotyledon cells adjacent to a true leaf primordium show the difference in cell size between expanded (lower cell with cytokinesis defects) and meristematic (upper left) cells in a rod seedling. Arrows point to cell wall stubs, small white arrow heads to multinucleate cells; larger, white arrow heads point to a cell wall gap in H and to a metaphase plate in J. In some instances, there are two juxtaposed wall stubs on either side of the cell (see black arrowhead in J), but for the most part, the cell wall stubs are anchored to the mother cell wall on one side of the cell, with no stub on the other side (see arrows in D, E, G, H, and K as well as text). Bars are 50 m in A, C, F, I, and L; 20 m in B, E, G, J, and K; and 10 m in D. H and K are the same scale. Plant Physiol. Vol. 129, 2002
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ten asymmetric, attached to the parent cell wall on one side of the cell, with no stub on the opposing side (Figs. 2 and 3B; Assaad et al., 1996). A four-dimensional analysis of cytokinesis shows that the cell plate often anchors on one side of the cell at an early stage of cytokinesis, then grows across the cell in a highly polarized fashion (Ehrhardt and Cutler, 2002). This provides a simple explanation for cell wall stubs as resulting from normal polar cytokinesis caught at early stages (Ehrhardt and Cutler, 2002). Cell wall gaps, consisting of two opposing stubs, as seen in procuste (Fagard et al., 2000), could arise upon cell expansion as the disruption of a mechanically compromised cell wall. Mechanically compromised cell walls are, in fact, the expected outcome of mutations at the CYT1, KORRIGAN, and KNOPF loci, which result in considerably reduced cellulose levels (Lukowitz et al., 2000; Boisson et al., 2001; His et al., 2001; Gillmor et al., 2002). Incomplete cell walls are observed in both vacuolated/expanded and nonvacuolated cells in the collection of keule-like lines presented here, and in cyd1 mutants (Figs. 2 and 3; Yang et al., 1999). By contrast, incomplete walls are observed later in development and only in vacuolated cells in cell wall mutants (Nickle and Meinke, 1998; Fagard et al., 2000; Zuo et al., 2000). We conclude that, to distinguish between cytokinesis and cell wall-defective mutants, it is helpful to monitor populations of rapidly dividing cells, as found in early embryos or in meristems.
Nuclear Defects in Cytokinesis-Defective Mutants and in titan and pilz Mutants
In histological sections of embryos, interphase nuclei appear as vacuolate structures with darkly staining, round or doughnut-shaped nucleoli (Fig. 3B). Light and electron microscopy of mutant embryos revealed enlarged nuclei, often containing multiple nucleoli (as illustrated in Figs. 2B and 3A). This has been observed in all the novel mutant lines, as well as in knolle, keule, and pleiade mutants (see Fig. 2, A, B, J, and K; Assaad et al., 1996). Mitotic figures are also more frequent in the mutant lines than in the wild type, as has been reported for keule (Fig. 2J; Assaad et al., 1996). The nuclear phenotypes observed in cytokinesis-defective mutants, large nuclei with multiple nucleoli, are distinct from the giant nuclei observed in titan/pilz mutants (Liu and Meinke, 1998; Mayer et al., 1999) and are, in all likelihood, a secondary consequence of a primary defect in cytokinesis. Presumably as a result of increased DNA content, the mutant seedlings have larger cells than the wildtype seedlings, and there are fewer cells per file in the cotyledons and hypocotyl (compare bublina and wild-type seedlings in Fig. 3, C and D). The number of cell files, however, appears roughly conserved. It is well known that disrupting the nuclear cycle blocks cytokinesis, and titan/pilz mutants could most 682
Figure 3. Cytokinesis-defective mutants have enlarged nuclei with multiple nucleoli, and enlarged cells. A and B are electron micrographs of embryos and C and D are histological seedling sections. A, Nucleus with two nucleoli (n), resembling a nuclear fusion, in a keule embryo. B, Wild-type nuclei (N) with single nucleoli (n) in a wild-type embryo. Note the difference in size between the nuclei in A and B, shown at the same scale. C, bublina seedling. D, Wild-type seedling, showing hypocotyl and apical meristem with the true leaf primordia; the cotyledons and root are not included due to the slightly tangential angle of the section. The bublina seedling shown in B has fewer cells per file than the wild type, but a roughly conserved number of cell files. The cells are larger than in the wild type (compare C and D, shown at the same scale). The arrowhead points to an irregularly shaped bloated surface cell, and the arrow to a cell wall stub. Bar is 5 m in A for A and B and 100 m in D for C and D.
simply be classified as having a perturbed nuclear cycle resulting in aborted cytokinesis (Mayer et al., 1999; McElver et al., 2000; Nacry et al., 2000; Nigg, 2001). It is less clear, however, what role aborted cytokinesis has on the nuclear cycle (Balasubramanian et al., 2000). The incidence in cytokinesis-defective mutants of multiple nuclei within a single cell shows that new nuclear cycles are initiated even if cytokinesis is incomplete. Whereas multinucleate cells are found early on in development, mature cells often have single, enlarged nuclei with high DNA content Plant Physiol. Vol. 129, 2002
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(Assaad et al., 1996). It is not clear whether these enlarged nuclei arise by endoreduplication or by the fusion of multiple nuclei present in the same cell. Cytokinesis-Defective versus Sterol-Deficient or Membrane Mutants
There is considerable overlap between the ranges of phenotypes observed in the sterol-deficient fackel mutants and in those seen here. keule mutants are typically club-like in shape, with two reduced cotyledons and a bloated hypocotyl. Strong alleles of keule segregate rod, oval, or ball-shaped seedlings. fackel seedlings are stout, often lacking a hypocotyl, and have a highly disorganized apex with supernumerary apices and cotyledons. Histological sections of fackel mutants reveal cytokinesis defects, namely, the presence of cell wall stubs and multinucleate cells in dividing embryonic cells (Schrick et al., 2000). Cytokinesis-defective mutant lines conversely segregate a small percentage of fackel-like seedlings, which may have twinned as opposed to single vascular bundles (Table II; Fig. 4, E and F), as described for fackel (Jang et al., 2000; Schrick et al., 2000). In addition to these defects, the keule-like lines segregate a small percentage of cup-shaped, pin-shaped, or fused cotyledons, as well as mono- or tricotyledonous seedlings (Table II; Fig. 4, A–D), as described in fackel (Jang et al., 2000; Schrick et al., 2000). Whereas 2% to 9% of cytokinesis-defective mutant seedlings have a grossly aberrant body organization (Table II), 73% to 100% of fackel seedlings are severely perturbed in their body organization, depending on allele strengths. Thus, mutations at the FACKEL locus have a strong effect on body organization but a weak effect on cytokinesis (see also Schrick et al., 2000). The cytokinesis mutants described above conversely have a strong effect on cytokinesis without grossly affecting the organization of the body plan. Sterols determine the fluidity and permeability of plant membranes. By altering the sterol metabolism of the plant cell, mutations at the FACKEL locus may impact membrane dynamics, and, thereby, have an effect on cytokinesis in much the same way as mutations at the knolle and keule loci. Sterols are also the precursors of steroid hormones such as brassinosteroids. Thus, it is not surprising that mutations at the FACKEL locus are more pleiotropic in their effects than the cytokinesis defectives. We conclude that the difference between fackel mutants and cytokinesis defectives can be recognized by virtue of the differential effect of such mutations on body organization as compared with cytokinesis. Defining Cytokinesis-Defective Mutants
Cytokinesis-defective lines have a number of additional phenotypes such as organ fusions, anomalies in cellular differentiation, anomalies in organ numPlant Physiol. Vol. 129, 2002
ber and/or in body organization, and perturbations of the nuclear cycle. We infer that most of these gross phenotypes are secondary consequences (direct or indirect) of the cytokinesis defect. We have provisionally defined mutants as being cytokinesis defective if they have cell wall stubs, gapped walls, and multiple nuclei in dividing as opposed to expanding cells. In contrast to cell wall-defective mutants, these defects tend to occur early during embryo development, as of the first division of the zygote. Furthermore, the body plan is in general conserved. By these criteria, all of the keule-like loci described here seem to be predominantly defective in cytokinesis, with the possible exception of BIMS. BIMS mutants have four features in common with fackel mutants: a reduced hypocotyl, the production of stout and reduced rosette leaves in tissue culture, a weak cytokinesis defect that may occur later in development, and a severely reduced etiolation response (F.F. Assaad, unpublished data; Schrick et al., 2000; Assaad et al., 2001). Yet bims mutants do not have the gross aberrations in body organization that characterize fackel mutants. It may be useful to examine whether mutations at the BIMS locus affect membrane dynamics, as in fackel mutants, yet without impacting the biosynthesis of crucial signaling and growth molecules such as brassinosteroids.
Postembryonic Processes in the Cytokinesis-Defective Lines
We monitored two postembryonic processes particularly sensitive to perturbations in the execution of cytokinesis: stomatal development and the ability of organ primordia to develop in tissue culture. Stomatal development requires a series of well-defined divisions. Asymmetric divisions in the epidermis define the guard mother cell, which divides symmetrically to form a ventral wall. Thereafter, a pore (Fig. 5D) is formed by a separation of the two guard cells at the middle lamella, and through extensive and localized remodeling of the ventral wall (Zhao and Sack, 1999). As judged by light and confocal microscopy, the cytokinesis-defective lines displayed a gradient of stomatal phenotypes, correlating roughly with the severity of their cytokinesis defect. The epidermis of knolle seedlings was so severely perturbed that we could not recognize stomatal precursors. In keule mutants, guard mother cells could be recognized, but they fail to form a ventral wall (Fig. 5C). In massue and bublina mutants, some stomata resemble those seen in keule, in others ventral wall formation was initiated but incomplete (data not shown). Incomplete ventral wall formation was also observed in bims mutants (data not shown). In bublina and pleiade mutants, some stomata had pores attached to a single side of the mother cell, suggesting that ventral wall formation might have been nearly but not fully com683
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Table II. A comparison of 10 cytokinesis-defective loci Strength of cytokinesis defecta
knolle
pleiade
⫹⫹⫹⫹⫹⫹
⫹⫹⫹⫹⫹⫹ Root
keule
⫹⫹⫹⫹⫹
hinkel
club
⫹⫹⫹⫹
⫹⫹⫹⫹
bublina
⫹⫹⫹
massue
⫹⫹⫹
⫹⫹ Shoota Extent of growth in cultureb
None
None Root
⫹ Root
bloated
bims
⫹⫹
⫹
⫹⫹⫹⫹⫹
⫹⫹⫹⫹ Stout leaves as in fackel
⫹⫹ Shoota None
None
None
⫹
⫹⫹
⫹⫹⫹ Shoot Stomata Guard mother cellc Ventral walld Pore formatione
rod
⫹⫹⫹⫹⫹ Root ⫹⫹⫹⫹ Shoot
⫺
⫹ ⫹, i ⫺/⫹
⫹ ⫺
⫹ ⫹ ⫹
⫹ ⫹
⫹ ⫺/⫹, i ⫺/⫹
⫹ ⫺/⫹, i ⫺/⫹
⫹ ⫹ ⫺/⫹
⫹ ⫺/⫹ ⫺/⫹
⫹ ⫹, i ⫺/⫹
Long root hairs Trichomesf
Yes nd
Yes Yes
No nd
Yes Yes
No nd
Yes Yes
Yes Yes
Yes Yes
Yes Yes
Yes Yes
Segregation ratio % Mutantg
25%
25%
20%–24%h
20%
20%
15%
24%
16%
20%
19%
3% 4%
3% 0%
4%–9%g 22%–23%
4% 5%
0% 1%
6% 2%
2%–7% 4%
0% 2%
3% 3%
3% 0%
4% 145
5% 81
0 112
12% 105
5% 79
4% 51
1% 250
6% 122
31% 120
5% 109
Body organization % Fackligi % Monocot or fused cotyledons % Tricot Total no. of mutants a
The strength of the cytokinesis defect was measured by the frequency with which cell wall stubs and multinucleate cells occurred in histological sections and by the size of multinucleate cells. For pleiade, strong cytokinesis defect and no growth in culture on hypocotyl and root, b weak cytokinesis defect and good growth on RIM for shoot (as described by Hauser et al. [1997]); converse for rod. Ability to produce true leaves on root-inducing medium. Lines such as bublina and massue, which died on RIM after elaborating their first true leaves, were nonetheless c d able to develop bloated, reduced leaves on SIM. Presence (⫹) or absence (⫺) of guard mother cells. Ventral wall formation (⫹); i, e incomplete wall formation; ⫺/⫹, some guard mother cells form ventral wall, others not. Presence (⫹) or absence (⫺) of well defined f stomatal pores. Trichomes on true leaves in seedlings grown on root-inducing (or shoot-inducing) medium. Some have abnormal branching g patterns. nd, Not determined, because no true leaves developed. These percentages do not take aborted embryos/seeds into account, even though the cytokinesis defects could cause a small percentage of severe embryo lethality. Non-germinated seedlings were dissected out of their seed coats for phenotyping. Percentages based on at least 100 seedlings total. The reduced segregation ratios in bublina, rod, and bims mutants h i may be indicative of a gametophytic effect. Strong alleles versus weak alleles, or vice versa. fackel-like in appearance, abnormal body organization.
plete, as has been shown in cyd1 mutants (Yang et al., 1999). Nonetheless, club, hinkel, bublina, pleiade, rod, bims, and bloated seedlings were capable of forming stomata with well-developed pores, attached to the mother cell by the ventral wall at both ends (e.g. Fig. 5, A and B). The gradient of stomatal phenotypes described here has been well documented in cyd1 mutants (Yang et al., 1999). Some lines (bloated, rod, hinkel, massue, bims, and bublina) showed variable degrees of growth in tissue culture (Table II; Fig. 5, F and G), whereas others including keule, knolle, and club could only be propagated as calli (Table II; Fig. 5E). On hormonesupplemented tissue culture media, some mutant seedlings produced true leaves, which were often bloated and irregular in appearance (e.g. Fig. 5G), or had irregular margins with more pronounced dentation as seen in cyd1 (Yang et al., 1999). In all instances, 684
the shoots that developed were abnormal. The results suggest that the KEULE-like loci are required not only during embryogenesis, but for cytokinesis in somatic cells throughout the vegetative life cycle. Whether or not the novel loci are required for cytokinesis in reproductive organs or during gametophytic development remains to be determined. In general, the ability to grow in tissue culture appeared to be correlated with the strength of the cytokinesis defect. Thus, lines that had severe cytokinesis defects were incapable of growth in tissue culture and failed to develop normal stomata. Tip Growth Processes in Cytokinesis-Defective Mutants
We considered the possibility that, in addition to their role in cytokinesis, the keule-like loci might be perturbed in other functions requiring extensive vesPlant Physiol. Vol. 129, 2002
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Figure 4. Abnormal body organization in keulelike mutants. A, Pin-shaped cotyledons. B, Cupshaped cotyledons. C, Monocotyledonous seedling. D, Tricotyledonous seedling. E, keule mutant that looks like fackel. F, Clearing preparation of keule mutant with hypocotyl deletion, with two vascular bundles (v) emerging from the basal area, as in fackel mutants. G, fackel mutant with a hypocotyl, somewhat like keule in appearance. H, Classical fackel mutant, showing central deletion and abnormal body organization. Bars ⫽ 200 m.
icle traffic. We investigated root hair growth and trichome morphogenesis whenever true leaves developed upon transfer to tissue culture medium. These are tip growth processes that, like cytokinesis, result in a rapid deposition of new cell walls via polarized secretion. An analysis of the root hairs in mutant seedlings shows that wherever the basal part of the seedling is strongly affected, as occurs in knolle, hinkel, bublina, and pleaide mutants, the root hairs are often multinucleate, swollen and even branched (Fig. 6E). The variability of the root hair phenotype is evident in a comparison of two bublina seedlings (Fig. 6, E and F). These defects have not been observed in fackel mutants, and may be indirect consequences of the nuclear and cell differentiation defects seen in the mutants (see above), with a variable environmental
component related to germination and humidity. Although the majority of the lines, including knolle, hinkel, pleiade, bims, and bublina are capable of growing long root hairs (Figs. 1I and 6, A and C and F–I), keule and club mutants appear incapable of doing so. Thus, unless these mutants are kept for weeks on tissue culture medium, their root hairs are invariably stunted, radially swollen, and branched (Fig. 6, D and E). The defects seen in keule and club mutants, including bulbous bases and a crooked appearance, are typical of those seen in root hair mutants defective in tip growth, such as cow1, cen1, and cen3 (Fig. 6, J and K; Parker et al., 2000), and are, therefore, likely to represent a tip growth defect. A number of mutants affected in root hair morphogenesis also affect trichome morphogenesis. We were Figure 5. A gradient of stomatal and postembryonic phenotypes. A through D, surface cells or seedling cotyledons. A and B, Histological sections. C and D, Scanning electron micrographs. Despite their heavily perturbed surface layer (A), club mutants are capable of complete ventral wall formation and form stomata with welldefined pores (B). B depicts the stomatal complex in A. By contrast, guard mother cells in keule mutants are incapable of ventral wall formation (C). E and F, Growth in tissue culture. E, keule explant on shoot-inducing medium (SIM), showing callus-like outgrowth. F, A bublina seedling transferred to SIM develops some true leaves, which show signs of being cytokinesis defective. G, rod seedling transferred to rootinducing medium shows near wild-type development, yet the leaves are irregular in shape and margins and have bloated cells. The trichomes (arrow head) have abnormal branching patterns. H, wild-type explant on SIM. Bar in A is 50 m.
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Figure 6. Root hair defects in keule-like mutants. Long root hairs are seen in knolle, hinkel, bublina, bloated, and bims mutants (A, C, F, G, H, and I). C and H, Long root hairs on one side of a hinkel or bloated mutant, stunted ones on the other side in contact with the agar on the plate. F, Long root hairs in bublina mutants (compare with E). E, Binucleate, bloated root hair in bublina seedling. B and D, stunted, bloated, crooked, and/or branched root hairs in keule and club mutants. Note the bulbous bases and crooked root hairs in club mutants, which resemble the defects seen in root hair mutants affected in tip growth, such as cen3 (J and K). n, Nucleus. Root hairs in rod mutants are often somewhat shorter than in the wild type (G), yet rod mutants are capable of growing long root hairs. Arrows designate branched root hair in B, bulbous bases in J, and crooked hairs in K. Bars are 100 m. With the exception of E, all panels are the same scale as in B.
unable to monitor trichome morphogenesis in keule and club mutants because these do not develop true leaves upon transfer to tissue-culture medium. In the majority of the other lines, which did develop true leaves, we noticed that the leaves developed trichomes but with abnormal branching patterns (Table II). Thus, whereas a wild-type trichome has three branches, rod and bloated trichomes often had one to five branches, as has been described in cyd1 mutants (Yang et al., 1999). These branching defects are likely to be a secondary consequence of the cytokinesis defect. Absent, stunted, radially swollen, and branched root hairs are invariably seen in keule and club mu686
tants, yet all of the other cytokinesis-defective mutants are capable of growing long root hairs (Table II; Fig. 6). We conclude that the root hair defect is not a secondary consequence of the cytokinesis defect. A detailed analysis of keule mutants at the electron microscope level has not shown morphological defects in any of the endomembrane systems such as the endoplasmic reticulum or Golgi, nor does secretion seem to be impaired (Assaad et al., 1996). Also, pollen tubes, which require tip growth, appear unaffected in KEULE mutants (Assaad et al., 1996). Similarly, club mutants do not appear to affect gametophytic development (Table II). Thus, it seems that KEULE and CLUB are not pleiotropic in their Plant Physiol. Vol. 129, 2002
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effects, but are required specifically for two distinct yet related processes. Both cytokinesis and root hair morphogenesis involve the rapid deposition of new walls via polarized secretion (Miller et al., 1997; Assaad, 2001a; Ryan et al., 2001). In both instances, vesicle trafficking is tightly regulated, with respect to cell cycle cues in the case of cytokinesis and of developmental, hormonal, and environmental signals in the case of root hair morphogenesis. In the average cell, new cell walls are laid down in roughly 50 min (Ehrhardt and Cutler, 2002), and root hairs grow at a rate of 100 m h⫺1 (Schiefelbein et al., 1992). Thus, during both cytokinesis and root hair morphogenesis, a very large amount of cell wall material and new membrane must be brought to a specific site in a short amount of time. KEULE encodes a key regulator of vesicle trafficking and has been shown to affect vesicle fusion at the cell plate (Waizenegger et al., 2000; Assaad et al., 2001). In light of the phenotypic similarity between KEULE and CLUB, it will be interesting to see if CLUB also plays a role in polarized secretion during cytokinesis and root hair morphogenesis.
Identifying New Cytokinesis Mutants of Arabidopsis
Our mechanistic understanding of plant cytokinesis has been limited by the paucity of genes that have been implicated in this process. It is apparent from the fact that we have only single alleles for five of the keule-like loci that saturation mutagenesis has not been achieved, and further searches for keule-like mutants may well uncover additional loci. Given the complexity of cytokinesis in plants and the large and ever increasing number of genes implicated in polarized secretion in yeast and animal cells, one would expect at least 100 genes to be implicated in this process in the plant cell. Yet, we estimate by extrapolation from the available mutant collections (see also Nacry et al., 2000) that the number of loci mutating to knolle or keule-like phenotypes will not exceed 20 to 30 loci in total. Based on the severe embryo lethality of knolle keule double mutants (as opposed to the seedling lethality of keule and knolle; Waizenegger et al., 2000), it has been assumed that cytokinesis genes might mutate to phenotypes more severe than observed in either keule or knolle. Yet few cytokinesis genes have been shown to mutate to embryo lethality (Nacry et al., 2000). The possibility that a subset of the genes required for cytokinesis in somatic plant cells might mutate to gametophytic lethality remains to be explored. The novel mutant loci described here all have phenotypes weaker than those seen in keule, and reverse genetic analyses as well as analysis of the cyd1 mutant suggest that cytokinesis-defective loci of Arabidopsis can be viable and have subtle phenotypes (Yang et al., 1999; Heese et al., 2001). Taken together, these considerations point to the importance of looking not for more severe embryo-lethal Plant Physiol. Vol. 129, 2002
phenotypes, but for more subtle cytokinesis defects in viable mutants. Viable mutants will be easier to isolate, but more difficult to recognize as cytokinesis defective. In this respect, it is noteworthy that, upon propagation in tissue culture, keule-like mutants share a number of features with the viable cyd1 mutant, including leaves with irregular margins, organ fusions, abnormal trichome branching patterns, anomalies in organ number, and stomata in which ventral wall formation is not always complete (Yang et al., 1999). These common features may facilitate the identification of novel cytokinesis-defective mutants. CONCLUSION
We have provisionally defined mutants as being cytokinesis defective if they have cell wall stubs, gapped walls, and multiple nuclei in dividing as opposed to expanding cells. These defects tend to occur early during embryo development and the body plan is in general conserved. In addition, cytokinesis-defective mutants are characterized by the incidence of enlarged nuclei with multiple nucleoli and enlarged cells. A phenotypic analysis of the novel and previously characterized cytokinesis-defective mutants has lead to three conclusions. First, developmental consequences of a defect in cytokinesis include anomalies in cell differentiation, organ number, and body organization, as well as organ fusions. Second, of the 10 cytokinesis-defective loci compared in this study, two were found to be required for root hair morphogenesis. Thus, certain aspects of cytokinesis and root hair morphogenesis, two processes that result in a rapid deposition of new cell walls via polarized secretion, may be regulated by the same molecules. Third, by monitoring embryo, seedling, stomatal, and postembryonic development, we found that the cytokinesis-defective mutants present a continuous range of phenotypes linking seedling-lethal mutations to viable ones. All six loci described here have phenotypes weaker than those observed in keule mutants, which in turn have weaker phenotypes than seen in knolle mutants. The results stress the importance of looking for more subtle rather than more severe phenotypes in further screens for novel cytokinesis-defective loci. MATERIALS AND METHODS Lines and Genetic Methods Lines G67, G88, G235, R3-27, T286, S100, S302, U1-15, U57, and U119 were identified in a screen of ethyl methanesulfonate-mutagenized Landsberg erecta described by Mayer et al. (1991) and grouped as keule-like based on their seedling phenotypes (U. Mayer, personal communication). Line G235 is in the HINKEL complementation group (W. Lukowitz, personal communication) and line 687
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T286 was shown to be allelic to pleiade; both lines were used for phenotypic analysis in this study. In addition, lines U1-15 and G67 were shown to be allelic to keule, validating the keule-like designation for the mutant collection. The hinkel and pleiade alleles described here have the same overall appearance as the alleles found in other screens for embryo or seedling lethal mutations (Strompen et al., 2002; W. Lukowitz, personal communication). It does not seem, therefore, that our screening criteria have selected for rare alleles of known loci such as keule, hinkel, and pleaide. One should note, however, that the pleaide alleles uncovered in seedling or embryo-lethal screens are stronger than the viable alleles of pleaide isolated in root morphogenesis screens (Hauser et al., 1997). Line AP297, identified by W. Lukowitz and U. Mayer in a screen of x-ray mutagenized Landsberg erecta (Mayer et al., 1999), was shown to be allelic to knolle and was used in this study in light of its relatively high germination efficiency; AP297 corresponds to a weak allele of knolle, with a phenotype intermediate between stereotypical knolle and keule mutants. The massue-2 allele, line MNN, was isolated by M. Hu¨ lskamp from an ethyl methanesulfonate population of Landsberg erecta. Previously described keule alleles MM125, T282, and R227 and knolle were used in this study (Assaad et al., 1996; Lukowitz et al., 1996). Alleles of pleiade and fackel and the root hair-defective mutants cen3-1 and cen3-2 were kindly provided by W. Lukowitz and C. Grierson, respectively. To eliminate potential second-site mutations, the mutant lines were self-fertilized four to 10 times and outcrossed to wild type (Landsberg erecta and/or Columbia). Phenotypes were scored in the progeny of healthy, fully fertile, greenhouse-grown plants. Seedling Phenotype and Tissue Culture To observe mutant seedlings, seeds were surface sterilized with 5% (w/v) calcium hypochlorite for 20 min at room temperature, rinsed, sown on Murashige and Skoog medium, and placed at 4°C for 3 to 4 d and then at 22°C at 16-h light/8-h night cycles for 5 to 6 d. Dark-germinated seedlings were treated in the same way but petri dishes were wrapped with aluminum foil. To test the ability to grow in tissue culture, seedlings were transferred to SIM (0.15 mg L⫺1 indole-3-acetic acid; 6-(␥,␥-dimethylallylamino) purine 5 mg L⫺1) or root-inducing medium (1 mg L⫺1 indolebutyric acid). Tissue culture media contained 0.2% (w/v) phytagel (gellum gum), Murashige and Skoog salts, 0.5 g L⫺1 MES, B5 vitamins, and 0.4% (w/v) Glc, and the final pH was adjusted to 5.7 with KOH. For dark-field and phase contrast microscopy, seedlings were cleared as described (Mayer et al., 1993). Light and Electron Microscopy and Image Analysis Samples for light and electron microscopy were prepared as described (Assaad et al., 1996). The cell wallspecific periodic acid schiff staining method for histological sections was as described by Nickle and Meinke (1998). The light microscopes used were an Axiophot (Zeiss, Jena, 688
Germany) and a DMB fluorescence, phase contrast and DIC microscope (Leica Microsystems, Wetzlar, Germany). The electron microscopes were a Zeiss EM912 and an S-4100 field emission scanning electron microscope (Hitachi Software Engineering, Yokohama, Japan). All images were processed with Photoshop and/or Illustrator software (Adobe Systems, Mountain View, CA). Root Hair Analysis and Confocal Imaging Root hairs were stained with propidium iodide at a concentration of 10 g mL⫺1 in Murashige and Skoog salts and mounted on cover slips. Confocal imaging was performed using an MRC1024 laser scanning confocal head (Bio-Rad, Hercules, CA) mounted on a Diaphot 200 inverted microscope (Nikon, Tokyo). The objective used was a 20⫻ Nikon PlanApo water immersion (Technical Instruments, San Francisco); excitation, 568 nm; emission, 585 nm long pass filter. Three-dimensional reconstructions of image stacks were generated with Lasersharp software (Bio-Rad) or NIH Image (Wayne Rasband, National Institute of Mental Health, Bethesda, MD). Mapping and Complementation For mapping, the mutant lines in the Landsberg background were crossed to wild-type Columbia. The lines were mapped with respect to the simple sequence length polymorphisms and to the cleaved amplified polymorphisms PHYA, NCC1, m59, and g2395. Primer sequences, polymorphisms, and precise map locations for all the molecular markers can be found at http://www.Arabidopsis. org. With the exception of the ROD locus, bulk segregant analysis on pools of mutant seedlings was followed by the phenotyping and genotyping of 48 to 300 individual F2 plants for linked markers flanking the mutant locus, as described (Lukowitz et al., 2000). Complementation analysis between the mutants presented here as well as to known cytokinesis-defective mutants was carried out based on similarity of map location. ACKNOWLEDGMENTS F.F.A. is especially grateful to Ulli Mayer for making the mutant lines available for this analysis and thanks Regine Kahmann and August Bo¨ ck for their support. Many thanks to Ramon Torres Ruiz, Thomas Berleth, and Simon Misera for their contribution to the seedling-lethal screen and to Martin Hu¨ lskamp for the massue-2 allele. Claire Grierson kindly provided root hair mutants affected in tip growth. Steffi Cubash, Heddy Bendjaballah, Yoann Huet, Frank Coutand, Eric Le Gouille, and Natalia Kalinina helped with sectioning or mapping. Thanks to Wolfgang Lukowitz for stimulating discussions, for sharing unpublished information and mutant lines, and for useful suggestions on the manuscript. Thanks to Dave Ehrhardt for help with imaging, useful discussions, and critical evaluation of the manuscript. Received February 11, 2002; accepted March 18, 2002. Plant Physiol. Vol. 129, 2002
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