Download The Arabidopsis Callose Synthase Gene GSL8 Is

The Arabidopsis Callose Synthase Gene GSL8 Is
Required for Cytokinesis and Cell Patterning1[C][W]
Xiong-Yan Chen2, Lin Liu2, EunKyoung Lee, Xiao Han, Yeonggil Rim, Hyosub Chu, Seon-Won Kim,
Fred Sack, and Jae-Yean Kim*
Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Plant
Molecular Biology and Biotechnology Research Center, Environmental Biotechnology National Core Research
Center, Jinju 660–701, Korea (X.-Y.C., L.L., X.H., Y.R., H.C., S.-W.K., J.-Y.K.); and Department of Botany,
University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (E.L., F.S.)
Cytokinesis is the division of the cytoplasm and its separation into two daughter cells. Cell plate growth and cytokinesis
appear to require callose, but direct functional evidence is still lacking. To determine the role of callose and its synthesis during
cytokinesis, we identified and characterized mutants in many members of the GLUCAN SYNTHASE-LIKE (GSL; or CALLOSE
SYNTHASE) gene family in Arabidopsis (Arabidopsis thaliana). Most gsl mutants (gsl1–gsl7, gsl9, gsl11, and gsl12) exhibited
roughly normal seedling growth and development. However, mutations in GSL8, which were previously reported to be
gametophytic lethal, were found to produce seedlings with pleiotropic defects during embryogenesis and early vegetative
growth. We found cell wall stubs, two nuclei in one cell, and other defects in cell division in homozygous gsl8 insertional
alleles. In addition, gsl8 mutants and inducible RNA interference lines of GSL8 showed reduced callose deposition at cell plates
and/or new cell walls. Together, these data show that the GSL8 gene encodes a putative callose synthase required for
cytokinesis and seedling maturation. In addition, gsl8 mutants disrupt cellular and tissue-level patterning, as shown by the
presence of clusters of stomata in direct contact and by islands of excessive cell proliferation in the developing epidermis. Thus,
GSL8 is required for patterning as well as cytokinesis during Arabidopsis development.
Cytokinesis divides the cytoplasm of a plant cell by
the deposition of plasma membrane and a cell wall
during late mitosis. This process requires the phragmoplast, a dynamic, plant-specific cytoskeletal and
membranous array, which delivers vesicles containing
lipids, proteins, and cell wall components to the division plane to construct the cell plate. Cell plate forma-
1
This work was supported by the Korea Research Foundation
(grant no. C00239), by grants from Korea Science and Engineering
Foundation/Ministry of Education, Science and Technology to the
National Research Lab Program (grant no. M10600000205–06J0000–
20510), the World Class University Program (grant no. R33–2008–
000–1002–0), and the Environmental Biotechnology National Core
Research Center (grant no. R15–2003–012–01003–0), by the Natural
Sciences and Engineering Research Council of Canada (Discovery
grant to F.D.S.), and by scholarships from the BK21 Program from
the Ministry of Education, Science, and Technology, Korea, to X.-Y.C.,
X.H., Y.R., H.C., and L.L.
2
These authors contributed equally to the article.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Jae-Yean Kim ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.133918
tion involves several stages: initiation through vesicle
fusion, the formation of a tubular-vesicular network, a
transition to a solely tubular phase, and then further
fusion to form a fenestrated sheet (Samuels et al.,
1995). The outward growth of the cell plate leads to its
fusion with the parental cell wall (Jürgens, 2005a,
2005b; Backues et al., 2007).
Key regulators of cytokinesis include KNOLLE,
KEULE, KORRIGAN, and HINKEL, which when defective induce pleiotropic phenotypes and seedling
lethality (Lukowitz et al., 1996; Nicol et al., 1998; Zuo
et al., 2000; Assaad et al., 2001; Strompen et al., 2002).
KNOLLE, a syntaxin homolog, is required for the
fusion of exocytic vesicles via a SNARE/SNAP33
complex (Lukowitz et al., 1996; Heese et al., 2001).
KEULE, a homolog of yeast Sec1p, regulates syntaxin
function by interacting with KNOLLE (Waizenegger
et al., 2000; Assaad et al., 2001). KORRIGAN is an
endo-1,4-b-glucanase required for cell wall biogenesis
during cytokinesis (Zuo et al., 2000). And HINKEL is a
kinesin-related protein required for the reorganization
of phragmoplast microtubules during cytokinesis
(Strompen et al., 2002).
Additional regulators include Formin5, TWO-INONE (TIO), and Arabidopsis (Arabidopsis thaliana)
dynamin-like proteins (ADLs; Kang et al., 2001, 2003;
Hong et al., 2003; Collings et al., 2005; Ingouff et al.,
2005; Oh et al., 2005). Formin5 localizes to the cell plate
and is an actin-organizing protein involved in cytoki-
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Chen et al.
nesis and cell polarity. TIO, a Ser/Thr protein kinase,
functions in cytokinesis in plant meristems and in
gametogenesis (Oh et al., 2005). Members of the
Arabidopsis DRP family associate with the developing cell plate, whereas DRP1a (ADL1A) locally constricts tubular membranes, interacts with callose
synthase, and may facilitate callose deposition into
the lumen.
Callose, a b-1,3-glucan polymer with b-1,6-branches
(Stone and Clarke, 1992), is synthesized in both sporophytic and gametophytic tissues and appears to play
various roles. Callose accumulates at the cell plate
during cytokinesis, in plasmodesmata, where it regulates cell-to-cell communication, and in dormant
phloem, where it seals sieve plates after mechanical
injury, pathogen attack, and metal toxicity (Stone and
Clarke, 1992; Samuels et al., 1995; Lucas and Lee,
2004).
Twelve GLUCAN SYNYHASE-LIKE (GSL) genes
(also known as CALLOSE SYNTHASE [CalS]) have
been identified in the Arabidopsis genome based on
sequence homology (Richmond and Somerville, 2000;
Hong et al., 2001; Enns et al., 2005). A GSL that
functions in callose deposition after injury and pathogen treatment is GSL5 (Jacobs et al., 2003). Five other
members of the Arabidopsis GSL family are required
for microgametogenesis. GSL1 and GSL5 act redundantly to produce a callosic wall that prevents microspore degeneration, and both are needed for
fertilization (Enns et al., 2005). GSL2 is required for
the callosic wall around pollen mother cells, for the
patterning of the pollen exine (Dong et al., 2005), and
for callose deposition in the wall and plugs of pollen
tubes (Nishikawa et al., 2005). GSL8 and GSL10 are
independently required for the asymmetric division of
microspores and for the entry of microspores into
mitosis (Töller et al., 2008; Huang et al., 2009).
Callose is a major component of the cell plate,
especially during later plate development (Kakimoto
and Shibaoka, 1992; Samuels et al., 1995; Hong et al.,
2001). Callose appears to structurally reinforce the
developing cell plate after the breakdown of the
phragmoplast microtubule array and during plate
consolidation (Samuels and Staehelin, 1996; Rensing
et al., 2002). It is likely that callose is synthesized at the
cell plate rather than in the endoplasmic reticulum and
in the Golgi (Kakimoto and Shibaoka, 1988). GSL6
(CalS1) appears to be involved in callose synthesis at
the cell plate, since a 35S::GFP-GSL6 fusion in transgenic BY-2 tobacco (Nicotiana tabacum) cells increases
callose accumulation, and GFP fluorescence was
found specifically at the cell plate (Hong et al., 2001).
However, functional and genetic data on the role of
any GSL in Arabidopsis sporophytic cytokinesis are
still lacking.
Here, we report that GSL8 (CalS10) is required for
normal cytokinesis. In addition, gsl8 mutants exhibit
excessive cell proliferation and abnormal cell patterning,
phenotypes not previously reported for cytokinesisdefective mutants.
RESULTS
Isolation of Homozygous T-DNA Insertion Lines of GSL
Family Members
We previously isolated T-DNA insertional mutants
in 11 members, GSL1 to GSL9, GSL11, and GSL12, of
the Arabidopsis GSL family and screened these primarily for gametophytic phenotypes (Huang et al.,
2009). Homozygous T-DNA-tagged lines were identified for 11 members of the GSL family except for GSL10
(Supplemental Table S1), which was gametophytic
lethal (Töller et al., 2008; Huang et al., 2009), thus
explaining why gsl10 homozygous mutants could not
be isolated. To identify the possible functions of different GSL genes in sporophytic callose formation and
in cytokinesis, we examined gross and microscopic
seedling phenotypes in homozygous gsl mutants.
We first examined gsl6 insertion lines because GSL6
has been reported to be involved in cell plate-specific
callose synthesis (Hong et al., 2001). Two independent
homozygous gsl6 insertion lines showed developmental phenotypes similar to those of wild-type plants
(Supplemental Fig. S1, A–C). These two alleles are
likely to be nulls, since no GSL6 mRNA could be
detected by reverse transcription (RT)-PCR using a
primer set corresponding to the catalytic domain of
callose synthase (Supplemental Fig. S1D). Like Hong
and Verma (2007), we were unable to detect any
cytokinesis defects in gsl6 insertion lines, such as
multiple nuclei in one cell and cell wall stubs (data
not shown). Thus, if GSL6 regulates cytokinesis, its
function might be masked by redundancy in the GSL
gene family.
We then evaluated the rosette-stage phenotypes of
mutations in the remaining 10 GSL family members.
Mutants in nine of the remaining family members
were indistinguishable from those of wild-type controls (Supplemental Fig. S1E).
gsl8 Mutations Affect Plant Development, Resulting in
Seedling Lethality
Mutations in the remaining locus, GSL8, have been
reported to be male gametophytic lethal, with aberrant
gsl8 pollen failing to enter pollen mitosis I (Töller et al.,
2008), leading these authors to speculate that the role
of GSL8 during pollen development is independent of
its function in callose synthesis. However, as briefly
noted by Huang et al. (2009), three homozygous
T-DNA insertions, gsl8-1, gsl8-2, and gsl8-3, still allow
some sporophytic growth (see below). Similar phenotypes were also found in three newly isolated insertional alleles (Figs. 1 and 2). Previously reported gsl8
seedling viability problems might be attributable to
poor germination and/or to the low transmission of
mutations (Supplemental Table S2), since growth on
3% Suc resulted in the recovery of homozygous mutants at a rate of 9% to 15% instead of the 25% expected
in the case of normal Mendelian segregation. The
recovery of homozygous mutants in a medium with-
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GSL8 in Cytokinesis and Cell Patterning
Figure 1. Characterization of GSL8 T-DNA insertion lines. A, The GSL8
gene contains 45 exons in the coding region. Insertion sites are
numbered for T-DNA lines: Salk_111094 (gsl8-1), SAIL_679_H10
(gsl8-6), GABI_851C04 (gsl8-2), SALK_057120 (gsl8-3), SALK_
109342 (gsl8-4), and SAIL_21_B02 (gsl8-5). B, RT-PCR of total RNA
showing the presence of a 500-bp PCR product against the 3# coding
region from wild-type (WT) plants but none from the T-DNA lines.
ACTIN2 was used as an internal control. [See online article for color
version of this figure.]
out Suc was dramatically reduced to about 2.5%. This
reduction was mainly caused by poor germination of
homozygous mutants (Supplemental Table S2). Six
independent homozygous mutant alleles (gsl8-1 to
gsl8-6) were seedling lethal and displayed almost
identical multiple phenotypes (Fig. 2).
gsl8 mutants showed a dwarf phenotype, and seedlings exhibited abnormally shaped cotyledons (Fig.
2A). The surface of the gsl8 cotyledon epidermis was
undulated, and hypocotyls and roots were thicker and
shorter than those of the wild type (Fig. 2, A–C). Darkgrown etiolated gsl8 seedlings showed open cotyledons, fat hypocotyls, and reduced height compared
with wild-type seedlings; however, hypocotyl length
was significantly longer than that of light-grown gsl8
plants (Fig. 2, C–E). Because cotyledons and hypocotyls are embryonic organs, we investigated the effect
of gsl8 on embryo development. Compared with wildtype controls (Fig. 3, A–E), gsl8 disrupted embryonic
development, with shape defects beginning at the
globular stage (Fig. 3F) and increasing in severity
over time (Fig. 3, G–I). Mature gsl8 embryos showed a
range of aberrant phenotypes, including asymmetrically shaped cotyledons and single or triple cotyledonlike organs (Fig. 3I). For example, the percentage
(12.9%) of abnormal gsl8-3 embryos was similar to
that of homozygous mutants observed at the seedling
stage (Supplemental Table S2).
gsl8 Seedlings Display Pleiotropic Phenotypes,
Including Patterning Defects
The gsl8 dwarfing could be due to a disruption in
cell division frequency and/or elongation. Normally,
the shoot apical meristem displays well arranged layers
(L1–L3) as well as cells that are small and relatively
even (Fig. 4A). By contrast, gsl8 meristem layers were
disrupted, shapes were irregular, and many cells were
enlarged (especially in L3 and in young primordia;
Fig. 4B), suggesting a loss of meristematic activity. In
addition, 3-week-old callus induced from gsl8 seedlings was much smaller than that in the wild type
(Supplemental Fig. S2), again indicating a growth
defect.
Cross sections of cotyledons revealed that gsl8 mutants showed not only bulging and giant epidermal
cells but also misarranged mesophyll cells with larger
intercellular spaces (Fig. 4, C and D). Venation patterns
were also defective in cotyledons and young leaves
(Supplemental Fig. S3, A and B). Compared with the
wild type, gsl8 hypocotyls showed excess divisions in
the pericycle, endodermis, and phloem (Fig. 4, E–J and
K). Similar phenotypes were found in roots, with extra
cells appearing in the root columella (Fig. 4, G–I) and
cortex (Fig. 4, L and M). Cell layering was also
disrupted in gsl8 roots, especially outside the stele
and the columella (Fig. 4, L and M), and many root
cells were abnormally enlarged. gsl8 roots also exhibited aberrant cell division planes (Supplemental Fig.
S3C). Thus, GSL8 is directly or indirectly required for
cell number and spatial distribution.
Figure 2. Phenotypes of gsl8 mutant seedlings and roots. A, Ten-dayold light-grown seedlings on MS plates containing 3% Suc. Mutants are
seedling lethal and display short, wide, and serrated roots and irregular
cotyledon margins. Numbers in A and C denote gsl8 mutant alleles. B,
Higher magnification of gsl8 mutant cotyledons. C, gsl8 mutant seedlings grown in the dark (7 d) have short, wide, and deformed hypocotyls
and roots. D and E, Higher magnifications of dark-grown cotyledons
from wild-type (D) and gsl8 mutant (E) plants. Bars = 5 mm in A and C
and 1 mm in B, D, and E. WT, Wild type. [See online article for color
version of this figure.]
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Chen et al.
Figure 3. Embryo development in wild-type and
gsl8 mutant plants. Seeds were harvested from
wild-type (A–D) and gsl8-4 homozygous mutant
(E–I) plants at different developmental stages and
then cleared and viewed using Nomarski optics.
Abnormal phenotypes are detectable at the globular (F) and heart (H) stages as well as in mature
embryos (I and J). The other five gsl8 mutant
alleles showed similar phenotypes to those of
gsl8-4. Embryos in E and I were cleared from the
same gsl8-4 heterozygous siliques. Embryos in H
and J were dissected away from surrounding seed
tissues. Bars = 50 mm in A to D, F, and G and 100
mm in E and H to J.
GSL8 Is Required for Epidermal Patterning
The epidermis of young gsl8 cotyledons and leaves
was highly disorganized (Fig. 5, B–E). Nonstomatal
cells showed a much wider range in size and in aspect
ratio than in the wild type (e.g. some cells were
exceptionally long and narrow). In addition, cell outlines were often less sinuous (Fig. 5, B–E).
Stomata also showed extreme variations in size as
well as defects in pore formation and bilateral symmetry (Fig. 5, C–E). In all six gsl8 mutant alleles, many
gsl8 stomata formed in contact in clusters, whereas
normally stomata were spaced at least one cell apart
(Fig. 5A; Supplemental Fig. S4). Clusters in gsl8 varied
in the number and arrangement of stomata as well as
in overall cluster shape and outline. Strikingly, the gsl8
epidermis showed regions with an excessive number
of small cells, many of which divided asymmetrically
to produce stomatal precursor cells (Fig. 5, C and D).
The regions with small cells were often adjacent to
groups of larger and more mature epidermal cells
(including stomata). By contrast, in the developing
epidermis of wild-type leaves, small cells and asymmetric divisions were much more distributed and
interspersed with larger cells. Thus, GSL8 appeared
to be required for correctly distributing asymmetric
divisions in the stomatal cell lineage throughout the
epidermis.
Each asymmetric division in Arabidopsis stomatal
development generates a meristemoid, a precursor cell
that ultimately produces a stoma. Mutants of gsl8
appeared to produce many more meristemoids in
contact than wild-type plants (Fig. 5B). Normally,
meristemoids themselves divide asymmetrically, divisions that can restore a one-celled spacing pattern
between stomatal precursor cells in wild-type plants
(Supplemental Fig. S5). However, gsl8 meristemoids
seemed to divide fewer times than in the wild type;
this defect, along with the abundance of meristemoids
in islands of small cells, appeared to induce pattern
violations. Because stomatal patterning might involve
cell-cell signaling via the apoplast, we asked whether
spacing defects were only found in cells whose apoplast was abnormal. As in other gsl8 epidermal cells
(see below), those in the stomatal lineage often showed
gaps in their walls. However, many stomata and
meristemoids in contact had apparently intact cell
walls throughout their cell depth (data not shown),
suggesting that local cytokinesis defects were not
directly responsible for stomatal pattern violations.
GSL8 Is Widely Expressed
Data from the AtGenExpress expression atlas
(Schmid et al., 2005) show that GSL8 is widely expressed in most organs throughout plant development. To determine the organ-level expression pattern
of GSL8, we performed RT-PCR using RNA extracted
from various organs. GSL8 transcripts were detected
in all organs and regions tested (Supplemental Fig. S6).
GSL8 expression was relatively higher in actively
dividing cells, such as in root tips and floral buds,
than in fully expanded or expanding tissues, such as
rosette and cauline leaves, cotyledons, and flowers.
Expression was also detected in mature organs, such
as rosette leaves of 6-week-old plants.
gsl8 Mutants Exhibit Cell Wall Stubs and
Incomplete Cytokinesis
GSL8 encodes a putative callose synthase, and callose is the major component of developing cell plates
during plant cytokinesis. We thus examined whether
gsl8 mutants disrupt cytokinesis, as evidenced by
defects such as cell wall stubs and two or multiple
nuclei in one cell that have previously been described
for other genes (Söllner et al., 2002). We first examined
the epidermal cells of gsl8 mutant cotyledons. As
shown in Figure 6, A to C, cell wall stubs were
observed throughout the depth of cotyledon epidermal cells stained with propidium iodide. Transmission
electron microscopy (TEM) analysis confirmed the
presence of binucleated cells with or without cell
wall stubs in gsl8 cotyledons and roots (Fig. 6, D and
E). To determine whether embryo abnormality in gsl8
mutants might result in part from defective cytokinesis, we used TEM to examine abnormal embryos at
various developmental stages from siliques produced
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GSL8 in Cytokinesis and Cell Patterning
these results show that gsl8 mutations disrupt cell
division and that GSL8 is required for Arabidopsis
cytokinesis starting in the early embryo.
Reduced Callose Deposition at Cell Plates and New Cell
Walls in gsl8
Callose localization by aniline blue staining or immunolocalization in root tips has been used frequently
to localize cell plates and/or young daughter cell
walls (Samuels et al., 1995; Frantzios et al., 2001). To
test whether the loss of GSL8 activity in gsl8 mutants
correlated with reduced callose deposition at cell
plates, root tips were stained with aniline blue. In
wild-type roots, we detected prominent aniline blue
staining at positions that mark a cell plate (Fig. 7, A–C).
Callose is also assumed to be deposited at new cell
walls, since we sometimes observed callose bands on
two opposite sides of cells, suggesting that at least one
of them is new cell wall (Fig. 7, B and C). In gsl8
mutants, we failed to find clear callose bands at these
positions, although we sometimes found ectopic callose deposition (Fig. 7, D–F). This failure to detect
callose might be caused by a low frequency of cell
divisions. To produce plants with a range of cytokinesis defects, we constructed inducible RNA interference
(RNAi) lines for GSL8. A dexamethasone-induced
reduction in GSL8 mRNA levels produced seedlings
that resembled gsl8 homozygotes in their dwarfism
and defective cell patterning (Supplemental Fig. S7).
These lines also resembled severe gsl8 alleles in the
absence of callose staining at root cell walls. Some
induced plants showed relatively mild developmental
phenotypes and the presence of callose at cell plates
and/or the new cell walls, but their frequency and
intensities were less than in noninduced lines (Fig. 7,
G–I). Taken together, these observations suggest that
GSL8 is involved in the deposition of callose at cell
plates during cytokinesis.
Figure 4. Tissue phenotypes in gsl8 mutant seedlings. A to D, Light
micrographs of sections of shoot apical meristems (A and B) and
cotyledons (C and D). Compared with the wild type (A and C), gsl8-4
meristem (B) and mesophyll (D) tissues are disorganized. The arrow in D
shows a large intercellular space. E to H, Cross sections of hypocotyls (E
and F) and longitudinal sections of roots (G and H) from the wild type (E
and G) and the gsl8-4 mutant (F and H). I, TEM of columella cell layers in
gsl8-4. The arrowhead indicates the quiescent center. J and K, TEM of
hypocotyls in cross section of the wild type (J) and gsl8-4 (K) with
endodermal cells numbered. L and M, TEM of the root tip in the wild type
(L) and gsl8-4 (M). Co, Columella cells; CR, cortex; CRi, internal layer of
cortex; CRo, outer layer of cortex; EN, endodermis; EP, epidermis; PE,
pericycle; QC, quiescent center; S, sieve elements; T, tracheary elements.
Similar patterns were observed from gsl8-1 and gsl8-2 mutants. Bars = 20
mm in A to D and I, 30 mm in E to H, and 10 mm in J to M.
by heterozygous gsl8 plants. Perturbations similar to
those found in the seedling stage were found in
embryos that presumably were homozygous for gsl8.
These defects included disorganized cell files, cell wall
stubs, and binucleated embryo cells (Fig. 6F). Together,
DISCUSSION
To obtain genetic data on the role of callose in
cytokinesis, we identified T-DNA insertional mutants
for most GSL callose synthase genes in Arabidopsis.
We have shown that GSL8 is required for cytokinesis,
as evidenced by the presence of incomplete cell walls
and two nuclei in a single cell in seedling tissues and
by reduced callose in cell plates and in new cell walls
of gsl8 mutants or inducible RNAi lines of GSL8. In
addition, GSL8 was found to play a novel and pervasive role in cell and tissue patterning.
GSL8 Is Critical for Cytokinesis in the GSL Gene Family
Callose deposition has been extensively studied in
plants. Green algae, bryophytes, ferns, and seed plants
are all capable of producing wound-induced callose,
and all plant taxa investigated, from multicellular
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Chen et al.
Figure 5. GSL8 is required for stomatal and epidermal patterning. A, Developing wild-type epidermis showing distributed
asymmetric divisions and relative uniformity in cell size. B to E, Developing gsl8 epidermis of leaves and cotyledons. Stars
indicate clusters of developing or mature stomata or of three adjacent meristemoids in the lower part of B. Arrows in D and E
indicate regions containing abnormally high numbers of small cells. Ten-day-old plants were used. All images show cell walls
stained with propidium iodide and visualized by confocal microscopy. All propidium iodide images have inverted gray scales.
Bars = 25 mm.
green algae to land plants, accumulate callose during
cell division (Scherp et al., 2001). At least some callose
deposition at the cell plate is not wound induced, since
rapidly frozen cells assayed using callose-specific antibodies show an increase in staining during cell plate
development (Samuels et al., 1995). Callose deposition
can be indirectly inhibited by caffeine, which destabilizes the developing cell plate and generates cytokinesis defects (Samuels and Staehelin, 1996; Yasuhara,
2005). However, while many mutants have been described that affect cytokinesis, no mutant in a callose
synthase gene has been shown to control cytokinesis
(Hong and Verma, 2007). Our findings that gsl8 alleles
and knockdown lines display severe cell division
defects and reduced callose deposition at cell plates
and new cell walls provide direct functional genetic
evidence for the importance of callose synthesis in
cytokinesis by GSL members. Although these gsl8
alleles are nulls and induce cytokinesis defects, cell
divisions still continue in these mutants, raising the
possibility that other GSL genes might be functionally
redundant. Two candidates are GSL10 and GSL6.
GSL10 encodes a callose synthase that is most similar
to that of GSL8. gsl10 mutants are gametophytic lethal,
and they perturb the symmetry of microspore division
and induce irregular callose deposition during microgametogenesis (Töller et al., 2008; Huang et al., 2009).
In addition, the silencing of GSL10 in transgenic RNAi
lines resulted in a dwarfed growth habit, suggesting
additional functions for GSL10 in normal plant growth
(Töller et al., 2008).
Although it remains to be seen if GSL6 localizes to
the cell plate in Arabidopsis, it was previously shown
that a GFP-GSL6 fusion protein expressed under the
control of a 35S promoter localizes to the cell plate in
tobacco BY-2 cells (Hong et al., 2001). However, GSL6
does not appear to be essential for cytokinesis, since
our null gsl6 mutants did not exhibit the seedling-level
phenotypes found in gsl8 (Supplemental Fig. S1B) and
since cytokinesis defects appear to be absent from gsl6
Figure 6. Aberrant cell walls and two nuclei in
one cell in gsl8-4 mutants. A to C, Cell wall stubs
in epidermal cells visualized using propidium
iodide staining and confocal microscopy. Optical
sections from outer (A), middle (B), and bottom
(C) parts of the same cells are shown. All propidium iodide images have inverted gray scales. The
other five gsl8 mutant alleles showed similar
results to those of gsl8-4. D to F, TEM of cotyledons (D) and root tips (E) from a 3-d-old gsl8-4
mutant and of a gsl8-4 embryo (F). Arrowheads
and arrows indicate cell wall stubs. N, Nucleus.
Bars = 10 mm in A to C and 2 mm in D to F. [See
online article for color version of this figure.]
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GSL8 in Cytokinesis and Cell Patterning
Figure 7. Callose deposition at cell plates
and new cell walls. A, Comparison of
images of the same roots using fluorescence optics from aniline blue staining of
the wild type (bottom root) and a dexamethasone-treated GSL8 RNAi line (top
root). Bar = 30 mm. B to I, Confocal images
following aniline blue and FM4-64 staining from root tips of 1-week-old plants of
the wild type (B and C), the gsl8 mutant
(D–F), and a dexamethasone-induced
GSL8 RNAi line (G–I). Merged images of
aniline blue (blue) and FM4-64 fluorescence (red) are shown in B, E, and H. Grayscale images of the blue channel are
shown in C, F, and I. Bright-field images
are shown in D and G. Arrowheads indicate representative aniline blue staining.
Boxes indicate callose bands on two opposite sides of a cell, suggesting that at
least one of them is a new cell wall that
originated from the cell plate. Bars =
20 mm.
alleles (Hong and Verma, 2007; this study). Similarly,
gsl6 does not appear to disrupt callose accumulation at
the cell plate (Hong and Verma, 2007). These data
suggest that GSL6 is either not required for cytokinesis
or is redundant with GSL8.
Tissue Organization in gsl8
Cytokinesis mutants often display defects in tissue
organization, in the range of cell sizes, and in division
plane placement (Lukowitz et al., 1996; Nicol et al.,
1998; Zuo et al., 2000; Assaad et al., 2001; Strompen
et al., 2002). Such defects were common in gsl8 mutants
(e.g. cells and intercellular spaces in the shoot apex and
in cotyledons were enlarged abnormally). However,
gsl8 also exhibits abnormal cell overproliferation, such
as an increased number of cells in specific tissues and
layers in the root cap columella, in the hypocotyl cortex
and endodermis, and in the phloem. This overproliferation phenotype is especially severe in the epidermis
(see below). Since an overproliferation phenotype does
not appear to have been reported for any cytokinesis
mutants (Lukowitz et al., 1996; Nicol et al., 1998; Zuo
et al., 2000; Assaad et al., 2001; Strompen et al., 2002),
these cytokinesis and division restriction functions of
GSL8 might be due to different underlying processes.
GSL8 was expressed in all organs tested (Supplemental
Fig. S6). Its expression was not restricted to actively
dividing tissues and was also detected in mature rosette
leaves that were no longer dividing. These data raise
the possibility that GSL8 is involved in cellular processes in addition to cytokinesis.
GSL8 in Stomatal and Epidermal Development
To our knowledge, GSL8 is the only locus identified
to date that is strongly involved in both cytokinesis
and stomatal patterning. Other mutants that disrupt
cytokinesis show a more normal stomatal distribution
(Söllner et al., 2002; Falbel et al., 2003), suggesting that
patterning and cytokinesis are mostly independent
processes.
Normally, stomata follow the “one-cell-spacing”
rule, in which two stomata are separated by at least
one intervening nonstomatal cell (Geisler et al., 2000;
Bergmann and Sack, 2007). In Arabidopsis, this spacing pattern is primarily established when an asymmetric division places a new stomatal precursor (a
meristemoid) away from a preexisting stoma and only
secondarily when the meristemoids in contact divide
away from each other (Supplemental Fig. S5; Geisler
et al., 2000; Lucas et al., 2006). Several genes control
these events, including those encoding likely receptors
and ligands, such as TOO MANY MOUTHS, ERECTA,
and EPIDERMAL PATTERNING FACTOR1. Mutations
in these genes induce the formation of clusters of
stomata in contact (Nadeau and Sack, 2002; Shpak
et al., 2005; Bergmann and Sack, 2007; Hara et al.,
2007). These clusters tend to be distributed similarly
throughout the epidermis, and nonstomatal epidermal
cells mostly resemble those in wild-type plants. However, unlike the above mutants, gsl8 clusters vary
greatly in the arrangement and size of stomata. In
addition, the gsl8 epidermis displays global disruptions in the distribution of epidermal cells, such as
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Chen et al.
islands of small cells that harbor excess asymmetric
divisions and meristemoids in contact. Perhaps this
dense packing of small cells inhibits division and the
correction of pattern violations.
Clearly, GSL8 is also required for properly organizing the distribution, planes, and number of other
tissues and cell types consistent with a required and
ubiquitous role in cytokinesis throughout the plant.
However, it remains to be determined how likely
defects in callose deposition might induce these abnormal morphologies.
In summary, GSL8 encodes a putative callose synthase
required for cell plate formation during sporophytic
cytokinesis. In addition to typical cytokinesis-defective
phenotypes, the loss of function of GSL8 induces or
results in novel developmental phenotypes, including
defects in epidermal planarity and patterning, stomatal spacing, and cell type overproliferation.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
All Arabidopsis (Arabidopsis thaliana) mutant lines and wild-type plants
used in this study are in the Columbia background. Unless otherwise
indicated, seeds were surface sterilized (20% commercial bleach and 0.1%
Triton X-100) for 5 min, washed three times in sterile water, and stored at 4°C
for 3 d. The seeds were sprinkled on 0.6% agar-solidified medium containing
13 Murashige and Skoog (MS) salts and 0% to 3% Suc (pH 5.8). Growth
conditions were at 25°C with a 16-h-light/8-h-dark cycle, either in soil or
on MS plates. gsl8-1 (SALK_111094), gsl8-2 (GABI_851C04), gsl8-3 (SALK_
057120), gsl8-4 (SALK_109342), gsl8-5 (SAIL_21_B02), and gsl8-6 (SAIL_679_
H10) were obtained from the Arabidopsis Biological Resource Center or the
Nottingham Arabidopsis Stock Centre. To determine the site of T-DNA
insertion in all of these alleles, we sequenced the PCR products amplified
using the appropriate left border primer and gene-specific primer. Primer
sequences for all PCR-based genotyping are listed in Supplemental Tables S3
and S4. The homozygosity of all mutant alleles of GSL8 was verified by PCR
genotyping and RT-PCR. Calli were induced from cotyledons excised from
1-week-old wild-type and gsl8 seedlings and were grown on medium described
previously (Encina et al., 2001).
were then sequentially dehydrated through an ethanol series, embedded in
Epon-812 resin, and polymerized at 60°C for 48 h. Both semithin (1 mm) and
ultrathin (70 nm) sections were cut using an Ultracut microtome (EM UC6;
Leica). Semithin sections stained with toluidine blue O were examined with a
light microscope (Axioplan 2; Zeiss), and photographs were taken with a
digital camera (AxioCam HRc; Zeiss). Ultrathin sections were mounted on
slots and then stained with uranyl acetate followed by lead citrate for imaging
with a Philips TECNAI 12 transmission electron microscope.
Propidium iodide staining was performed as described previously (Lucas
et al., 2006) using a Nikon C1 Plus confocal laser scanning microscope. Root tips
were stained with callose staining buffer for 3 to 5 min, which is a mixture of
0.1% aniline blue in autoclaved triple-distilled water and 1 M Gly, pH 9.5, at a
volume ratio of 2:3. Aniline blue was examined using a fluorescence microscope
(Axioplan 2; Zeiss). For costaining, root tips were stained with the dye FM4-64
(Molecular Probes) according to a previous study (Fujimoto et al., 2008) before
aniline blue staining. Aniline blue and FM4-64 fluorescence was examined with
405- and 523-nm lasers using a fluorescence confocal microscope (LSM5; Zeiss).
Constructs and Plant Transformation
To generate the dexamethasone-inducible GSL8 RNAi construct, the entire
GSL8 RNAi cassette comprising sense and antisense arms of GSL8 interspersed by two introns (PDK and CAT) was amplified from Agrikola
GSL8-sepcific hairpin RNA expression plasmids (CATMA 2a35110) using
the primers dsGSL8-SalI-d1 (5#-GGGCGTCGACCGCAAGACCCTTCCTCTATAT-3#; the SalI site was introduced) and dsGSL8-SpeI-r1 (5#-CCCGACTAGTCGCATATCTCATTAAAGCAGGA-3#; the SpeI site was introduced).
Sequence-confirmed PCR product was digested with SalI and SpeI and cloned
into a dexamethasone-inducible binary transformation vector, pTA7002, to
produce pTA7002-GSL8-RNAi. Dexamethasone treatment was carried out
according to Binarovaı̀ et al. (2006).
The pTA7002-GSL8-RNAi vector were transferred into Agrobacterium
tumefaciens strain GV3101 and used to transform Arabidopsis ecotype Columbia by dipping. Seeds harvested from mature transformed plants were
sterilized, stratified for 3 d, and sown on MS agar plates containing
hygromycin (50 mg L21). Positive lines were selected after growing for 10 d
on hygromycin plates. Putative transgenic plants were transplanted onto soil
and grown in a growth chamber.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Loss-of-function mutations in the GSL gene
family.
Supplemental Figure S2. Calli induction from gsl8 mutant seedlings.
Supplemental Figure S3. Patterning phenotypes of gsl8 mutants.
RNA Extraction and RT-PCR
Plant tissues were harvested and total RNA was isolated using TRIzol
(Invitrogen) according to the manufacturer’s instructions. A total of 1 mg of
DNA-free RNA was reverse transcribed using an oligo(dT) primer and reverse
transcriptase (M-MLV RTase; SolGent). The PCR amplification protocol was
96°C for 3 min followed by 30 cycles of 94°C for 30 s, 55°C for 45 s, and 72°C for
30 s. The ACTIN2 transcript was used as an internal control. The primer
sequences were as follows: AtGSL8-d1 (5#-CCCTCTGGATTTGAATGGCA-3#)
and AtGSL8-r1 (5#-GGCTAAAGAGAGTAGAGCCC-3#) for GSL8 and
ACTIN2-d1 (5#-TCAATCATGAAGTGTGATGTGG-3#) and ACTIN2-r1 (5#-TTAGAAACATTTTCTGTGAACGAT-3#) for ACTIN2.
Supplemental Figure S4. Distribution of normal stomata and of clustered
stomata in the epidermis of cotyledons in the wild type, tmm, and gsl8-4.
Supplemental Figure S5. Primary and secondary stomatal patterning
mechanisms in wild-type Arabidopsis.
Supplemental Figure S6. GSL8 is expressed in all organs tested using
RT-PCR.
Supplemental Figure S7. Dexamethasone-inducible RNAi line of GSL8
phenocopies T-DNA insertional mutants.
Supplemental Table S1. Identity of T-DNA insertion lines in GSL family
genes (excluding GSL8 and GSL10).
Microscopy
Supplemental Table S2. Segregation analysis of GSL8/gsl8.
For phenotypic analysis of the mutant embryos, developing seeds were
collected from siliques at different developmental stages from wild-type and
heterozygous gsl8 plants. They were cleared with a drop of Hoyer’s solution
(50 g of chloral hydrate, 3.75 g of gum arabic, and 2.5 mL of 100% glycerol in 15
mL of distilled water) for at least 30 min at room temperature. Cleared seeds
were examined with an Olympus FV1000 confocal microscope equipped with
Nomarski differential interference contrast optics.
For histological and TEM examination, intact tissue samples were fixed in
2% glutaraldehyde in 50 mM phosphate-buffered saline (pH 6.8) at 4°C for 12
to 24 h, followed by postfixation in 1% OsO4 buffer at 4°C overnight. Samples
Supplemental Table S3. Primers used to isolate homozygous gsl family
mutants.
Supplemental Table S4. Primers used to isolate homozygous gsl8 mutants.
ACKNOWLEDGMENT
Fluorescence imaging was partially performed by the Live Molecular Imaging
Center of the Korea Research Institute of Bioscience and Biotechnology, Korea.
112
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
GSL8 in Cytokinesis and Cell Patterning
Received December 9, 2008; accepted March 10, 2009; published March 13,
2009.
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