1 Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock
Drive, Madison, WI 53706, USA
2 Department of Plant Biology, Ohio State University, 1735 Neil Avenue,
Columbus, OH 43210, USA
3 Department of Molecular, Cellular and Developmental Biology, University of
Colorado, Boulder, CO 80309, USA
* Author for correspondence (e-mail: bednarek{at}biochem.wisc.edu)
Accepted 19 May 2003
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SUMMARY |
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Key words: Cell Plate, Cell Expansion, Cytokinesis, DENN domain, Guard cell, Vesicle trafficking, Arabidopsis thaliana
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INTRODUCTION |
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Several genes involved in plant cytokinesis have been revealed recently
through the characterization of mutant plants that either display defects in
the determination of the division plane or that show aberrant cell plate
formation. Mutants that fall into the first class such as ton/fass,
tangled and discordia 1 show defects in the proper alignment of
the division machinery and thus have misoriented cell walls
(Smith, 2001). In contrast,
mutants that are defective in the construction of the cell plate have
multinucleated cells with incomplete or missing cell walls
(Nacry et al., 2000
;
Söllner et al.,
2002
).
Several embryo and seedling-defective Arabidopsis mutants with
multinucleated cells and incomplete cell walls have been described. Cloning of
these genes has revealed three classes of proteins essential for cell plate
formation. Genes in the first class include KNOLLE
(Lukowitz et al., 1996) and
KEULE (Assaad et al.,
2001
) that are required for cell plate membrane fusion.
KNOLLE encodes a homolog of syntaxin, a mammalian plasma membrane
SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptors)
required for the fusion of exocytic vesicles, whereas KEULE encodes a
homolog of yeast Sec1p that regulates syntaxin function. These factors have
been shown to interact both genetically and biochemically, indicating that
they are components of the same cell plate membrane fusion pathway
(Assaad et al., 2001
).
Mutations in a second class of genes required for cell plate biogenesis affect
the synthesis of the primary cell wall. Cell wall biosynthesis, which is
initiated within the cell plate lumen, is thought to stabilize the cell plate
as it expands outward toward the parental plasma membrane
(Verma, 2001
).
KORRIGAN (KOR) (Nicol et
al., 1998
; Zuo et al.,
2000
) and CYT1
(Lukowitz et al., 2001
) are
two genes involved in cell wall biosynthesis that encode an endo-1,
4-ß-glucanase and a mannose-1-phosphate guanylyltransferase,
respectively. HINKEL (Strompen et
al., 2002
), which encodes a kinesin-related protein, defines a
third class of genes required for phragmoplast-mediated expansion and cell
plate guidance. Recent reverse genetic studies have linked the function of
HINKEL and division plane-localized components of a mitogen-activated protein
kinase (MAPK) pathway in the control of phragmoplast expansion
(Nishihama et al., 2002
).
The identification of these genes has yielded insight into the molecular
mechanisms that govern plant cytokinesis. However, given the complexity of
this process, it is clear that genetic screens for other components of the
cytokinetic machinery are not yet saturated. For example, genes involved in
polarized trafficking of exocytic and endocytic cell plate vesicles have not
been identified to date. Severe loss-of-function mutants in such genes may be
difficult to isolate and characterize as they may encode proteins required for
both cell division and expansion. Alternatively, functionally redundant genes
involved in embryonic cell division may not be revealed in a phenotypic screen
for embryo and seedling defective cytokinesis mutants. However, it may be
possible to isolate mutants that display tissue- and/or cell-type-specific
defects in cell division when the requirement for these genes is under
developmental or cell-type-specific control. Recently, several examples of
such cytokinesis-defective mutants have been identified including
tso1 (Hauser et al.,
2000; Song et al.,
2000
), which affects floral tissue development, the root
morphogenesis mutants pleiade and hyade
(Müller et al., 2002
),
the pollen cytokinesis-defective mutant gem1/mor1
(Park and Twell, 2001
), and
cyd1, which displays variable cell-type specific defects in stomata,
roots, and other cell types (Yang et al.,
1999
).
Stomata are found in the leaf epidermis and consist of two guard cells
surrounding a pore. They function to regulate gas exchange between the
atmosphere and internal air spaces of leaves. Stomatal density is determined
by environmental and internal cues. Unlike most other leaf cell types, guard
cells are continually generated throughout leaf development by the
differentiation and subsequent division of guard mother cells
(Nadeau and Sack, 2002a;
Nadeau and Sack, 2002b
). The
stereotyped divisions of guard mother cells that produce two guard cells
arranged with mirror-like symmetry facilitates the identification of
cytological defects when using microscopy-based mutant screens for genes that
participate in cytokinesis. Cytokinesis-defective stomata have been readily
observed in plants treated with caffeine
(Galatis and Apostolakos,
1991
; Terryn et al.,
1993
), an agent that disrupts the consolidation of the cell plate
membrane system (Samuels and Staehelin,
1996
). As shown previously by Terryn et al.
(Terryn et al., 1993
), guard
cells from caffeine-treated Arabidopsis plants
(Fig. 1A-C) resemble the guard
cells observed in mutants that are defective in components of the general
cytokinetic machinery, including the cell plate-associated mitogen-activated
protein kinase (MAPK) kinase kinase, NPK1
(Jin et al., 2002
;
Nishihama et al., 2001
), its
associated kinesin-like protein, NACK1
(Nishihama et al., 2002
) and
the secretory membrane fusion factor, KEULE
(Söllner et al., 2002
).
Similar abnormal stomata are present in cyd1
(Yang et al., 1999
) and
several other recently identified Arabidopsis cytokinesis-defective
mutants (Söllner et al.,
2002
) whose genes have not yet been cloned. Because of their
sensitivity to defects in cell division, we reasoned that additional genes
required for general plant cytokinesis would be revealed through the
identification of weaker non-lethal mutations that mainly disrupt stomatal
development. Here we report the identification of a conditional mutant allele
that severely disrupts the formation of the cell plate during guard mother
cell cytokinesis, and the cloning of its associated gene, SCD1
(stomatal cytokinesis-defective 1). We have analyzed T-DNA::scd1
insertion alleles and demonstrated that SCD1 is also critical for
polarized cell expansion and most likely functions in regulating exocytic
vesicle trafficking.
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MATERIALS AND METHODS |
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Inhibitor studies
Seeds from segregating wild-type and heterozygous scd1-1 and
scd1-2 plants were germinated on MS-Suc plates at 22°C under
continuous light. After 3-4 days, phenotypically wild-type and mutant
seedlings were transplanted and grown vertically for an additional 4-5 days on
freshly prepared MS-Suc 0.8% w/v agar plates in the presence and absence of
various concentrations of inhibitors. The initial position of transplanted
seedling root tips was marked with a pen on the back of the plates and the
root length of 5-15 plants per treatment was subsequently measured at 2-day
intervals for an additional 4-5 days. For imaging, roots grown in the presence
and absence of inhibitors for 4-5 days were stained with propidium iodide and
analyzed by laser scanning confocal microscopy as described below. Inhibitor
stocks were prepared as follows. 9 mM brefeldin A (Molecular Probes, Eugene,
OR) in dimethylsulfoxide (DMSO); 100 mM caffeine in distilled water
(dH2O); 1 mM latrunculin B in DMSO; 100 mM propyzamide (Chem
Service, West Chester, PA) in isopropanol.
Microscopic analysis
For epidermal peels, leaves were attached to microscope slides using
double-sided adhesive tape and nonadherent cell layers were scraped away. The
remaining epidermal cell layer was washed with dH2O and incubated
with 0.05% w/v Toluidine Blue, 50 mM citrate buffer pH 4.6 with gentle warming
(50°C-60°C for about 10-20 seconds).
Transgenic plants expressing the Arabidopsis guard cell-specific
potassium channel KAT1 promoter ß-glucuronidase (GUS)
gene fusion (KAT1::GUS) (Nakamura
et al., 1995) were kindly provided by M. Sussman (UW-Madison) and
were crossed with scd1-1 plants grown at 16°C. Histochemical
assays for GUS activity in the F2 progeny were conducted as
described previously (An et al.,
1996
).
Arabidopsis lines expressing the nuclear-localized N7 and plasma
membrane 29-1 GFP-tagged marker proteins
(Cutler et al., 2000) were
obtained from the Arabidopsis Biological Resource Center (Ohio State
University, Columbus OH) and were crossed with scd1-1 plants grown at
16°C. To visualize guard cell and pavement cell walls, seedlings were
stained with 0.1 mg/ml propidium iodide in dH20 for 5-15 minutes at
room temperature prior to imaging. The length of wild-type, scd1-1 and
scd1-2 epidermal root cells was determined from confocal laser scanning
microscopic images of roots that were briefly stained with 0.1 mg/ml propidium
iodide for 30 seconds to 1 minute.
Microscopy
Low magnification images were taken with a Nikon CoolPix 950 digital
camera. Flower bud and root pictures were obtained with a Leica MZ6 stereo
microscope, equipped with a SPOT digital camera (Diagnostic Instruments,
Sterling Heights, MI). Higher magnification bright-field images were obtained
as described previously (Kang et al.,
2001). Confocal laser scanning microscopic (CLSM) images were
acquired on a Biorad 1024 confocal microscope (Hercules, CA) equipped with an
argon laser using a 63x objective lens. 15-20 frames were averaged for
each image. For cryo-scanning electron microscopy, samples were plunge frozen
in liquid N2 transferred to a cryoprechamber (Emitech K-1150,
Houston TX) and sputter coated with gold. Imaging was performed at the CBS
Imaging Center (University of Minnesota, St. Paul, MN) in a Hitachi S3500N
scanning electron microscope under high vacuum using 5 kV accelerating
voltage. For transmission electron microscopy, leaf samples were processed by
high-pressure freezing and freeze substitution at the Department of Molecular,
Cellular, and Developmental Biology Electron Microscope Service Facility
(University of Colorado, Boulder, CO). For improved preservation of
Arabidopsis shoots, plants were grown on MS-Suc for 4 days and then
acclimated over a 2-day period with increasing sucrose concentrations
according to the following schedule: 5 ml 3% (w/v) sucrose in dH2O
was added to the MS-Suc plate for 24 hours, followed by addition of 5 ml 5.4%
(w/v) (150 mM) sucrose in dH2O for 24 hours prior to cryofixation.
Cotyledon, root tissue and most of the hypocotyl were removed from plants,
then the meristematic region, including leaf primordia, was transferred to
aluminum sample holders in 150 mM sucrose and frozen in a Baltec HPM 010 high
pressure freezer (Technotrade, Manchester, NH), and then transferred to liquid
nitrogen. For freeze substitution the samples were incubated in 4%
OsO4 in anhydrous acetone at -80°C for 5 days, followed by slow
warming to room temperature over a period of 2 days. The samples were rinsed
in several acetone washes, incubated in propylene oxide for 30 minutes, rinsed
again in acetone and infiltrated with increasing concentrations of Epon
812-Araldite resin (EM Sciences, Fort Washington, PA) over a period of 8 days
and polymerized in 100% resin at 60°C for 2 days. Silver-gold (
80 nm)
thin sections were mounted on uncoated copper 200 mesh grids, stained with 8%
uranyl acetate in 50% (v/v) ethanol (15 minutes at 60°C) and Reynold's
lead citrate (10 minutes at 60°C) and viewed at 80 kV using a Phillips
CM120 transmission electron microscope (Eindoven, Netherlands) at the Medical
School Electron Microscope Facility (University of Wisconsin, Madison, WI).
All images were processed for publication using Adobe Photoshop 7.0 and
Illustrator 10.0 (Adobe Systems, San Jose, CA) software.
Carbon isotope ratios
For isotope ratio measurements of total fixed carbon, plants were grown
both under continuous illumination and long days. Analysis was performed in
triplicate on dried leaf tissue samples by the University of Utah SIRFER
facility
(http://www.ecophys.biology.utah.edu/sirfer.html).
Oligonucleotides
All oligonucleotides used in this study
(Table 1) were designed using
the Primer3 software web interface (Rozen
and Skaletsky, 2000)
(http://www-genome.wi.mit.edu/genome_software/other/primer3.html)
and synthesized by Integrated DNA Technologies Inc. (Coralville, IA). Lower
case letters in the oligonucleotide sequence indicate added restriction
sites.
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Molecular complementation
A 12.8 kb region of genomic DNA containing the entire coding region of
At1g49040, 2.3 kb of 5' putative promoter DNA, and 863 bp of 3'
flanking untranslated DNA was cloned from the F27J15 BAC DNA into the binary
plant transformation vector (pPZP211)
(Hajdukiewicz et al., 1994)
and transformed using the floral dip method
(Clough and Bent, 1998
) into
homozygous scd1-1 plants grown at 16°C. Transgenic
(T1) plants were identified on MS-Suc nutrient agar in the absence
and presence of 40 µg/ml kanamycin. Upon transfer to soil these plants
displayed normal stomatal and floral development when grown at 22°C.
T2 progeny from five independent self-fertilized complemented lines
segregated 3:1 [(wild type (scd1-1/scd1-1::SCD1):
scd1-1/scd1-1], indicating that the transformants contained a single
T-DNA insert that complemented the scd1-1 phenotype.
RT-PCR analysis and cloning of the SCD1 cDNA
Total RNA (3 µg) was isolated from various tissues and actively dividing
suspension-cultured cells (T87) (Axelos et
al., 1992) using TRI reagent (Sigma Chemical Co., St Louis, MO),
reverse transcribed as described (Kang et
al., 2001
) and diluted 20 fold before PCR amplification. For
analysis of SCD1 expression, a 740 bp product was amplified from the
first strand cDNA from each tissue sample using primer pairs P3 + P4,
corresponding to a 2270 bp genomic region spanning 9 introns. Genomic DNA and
no DNA templates were included in control reactions. Ubiquitin control primers
amplify a 484 bp cDNA fragment from nt 1083 to 1567 of the UBQ10 mRNA
(At4g05320). DNA sequence analysis confirmed the presence of the C
T
transition in the scd1-1 RT-PCR product amplified with primers P1 and
P2, and sequenced with P2.
Full-length SCD1 cDNA was amplified from first strand cDNA isolated from T87 cells using Proofstart (Qiagen) polymerase (primers P1 and P7) and cloned into the pGEMTeasy vector (Promega, Madison, WI). The 3.5 kb cDNA was sequenced to determine the gene structure and deposited in GenBank (Accession no. AY082605). Three splice site adjustments to the original At1g49040 annotated sequence were made.
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RESULTS |
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Defective scd1-1 guard cells were found to be binucleate,
confirming that the scd1-1 phenotype is manifest during cytokinesis.
Nuclei were visualized by confocal microscopy in wild type and homozygous
scd1-1 plants expressing a nuclear-localized green fluorescent
protein (GFP)-fusion protein marker
(Cutler et al., 2000)
(Fig. 2). Multinucleated
epidermal cells with cell wall stubs were also observed in the leaves of young
scd1-1 seedlings stained with propidium iodide
(Fig. 2C) or labeled with the
plasma membrane GFP-marker 29-1 (Cutler et
al., 2000
) (Fig.
2D) demonstrating that the SCD1 gene product is a general
component of the plant cytokinetic machinery. However, no
cytokinesis-defective cells were observed in scd1-1 roots suggesting
that requirement for SCD1 in cytokinesis is likely to vary between
plant tissues and cell types.
Although the effects of the scd1-1 mutation on cytokinesis were
only observed in developing guard cells and leaf epidermal cells, the overall
growth and development of scd1-1 mutant plants was severely affected.
scd1-1 plants were smaller than wild type, and displayed defects in
seedling development, leaf expansion and flower morphology, which rendered the
mutant conditionally sterile (see below). When grown on a sucrose-containing
nutrient agar medium (MS-Suc; see Materials and Methods), the dwarf phenotype
of scd1-1 seedlings was more enhanced
(Fig. 3A). Growth of
scd1-1 seedlings on MS-Suc was stunted and the scd1-1 shoots
were smaller and darker green than wild-type plants
(Fig. 3B). scd1-1
roots were also shorter than wild type
(Fig. 3A), and root hairs did
not typically elongate. The scd1-1 mutation resulted in a decrease in
the number of cells in the root meristem and an approximately 40% inhibition
in the length of cells in the root epidermis and cortex
(Fig. 3C,
Fig. 8A). scd1-1
roots, however, did not display a radial swelling phenotype
(Fig. 8A) as described for a
number of other recently identified mutants with reduced cell elongation
(Baskin et al., 1995;
Bichet et al., 2001
;
Nicol et al., 1998
;
Wiedemeier et al., 2002
;
Williamson et al., 2001
).
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The reduction in the number of functional stomata limits gas exchange
in the leaves of scd1-1 mutant plants
The mutant plants are viable most probably owing to the presence of a small
population of functional stomata that had undergone normal cytokinesis. To
assess how a defect in stomatal formation affects the growth and development
of the whole plant, we compared the efficiency of gas exchange in wild type
and mutant leaves by analyzing the 12C/13C isotope ratio
of total fixed carbon in mutant versus wild-type plants as described
previously (Farquhar et al.,
1982). As expected, the [CO2] inside the leaf was
severely reduced in scd1-1 mutant plants. The isotopic composition,
, for wild-type plants was -30.1 (±0.32, n=6) while for
scd1-1 it was -24.2 (±1.04, n=6) corresponding to a
[CO2] inside the leaf of 259 ppm for wild type and 173 ppm for
scd1-1. This much lower [CO2] inside the leaf will
severely reduce photosynthesis in the mutant thereby restricting growth.
scd1-1 is a temperature-sensitive allele
Growth and development of scd1-1 plants was found to be
temperature sensitive. All facets of the mutant phenotype described above were
observed in plants grown at 22°C, but were completely absent in plants
grown at 16°C (Fig. 4A).
When grown at 16°C, all scd1-1 guard cells appeared normal
(Fig. 4E), and the flowers were
fully self-fertile (Fig. 4C),
making it possible to obtain seed from homozygous scd1-1 plants.
Growth at temperatures higher than 22°C did not result in a more severe
phenotype. To address whether the conditional flower morphology defect
associated with scd1-1 plants grown at 22°C was directly related
to the scd1-1 mutation or was an indirect physiological consequence
of the lack of functional stomata in the mutant plants, we performed a
temperature shift experiment: flowering scd1-1 plants grown at
22°C were transferred to 16°C for 1 week and then back to 22°C.
Morphologically normal self-fertile flowers developed only during the period
of growth at the cooler permissive temperature
(Fig. 4F), strongly suggesting
a direct requirement for SCD1 function during flower development.
Positional cloning of SCD1
Using a map-based approach, we cloned SCD1
(Fig. 5). A single missense
mutation (CT), consistent with the mutagenic properties of EMS, was
revealed in the gene At1g49040 by sequencing of genomic DNA from homozygous
scd1-1. The mutation resulted in a serine (TCT) to
phenylalanine (TTT) change at codon 131 in the predicted open
reading frame. We confirmed that At1g49040 corresponds to SCD1 by
molecular complementation as described in Materials and Methods. To determine
the genomic structure and organization of the SCD1 gene, we cloned
and sequenced a 3.5 kb cDNA (GenBank Accession no. AY082605). The gene
structure of the 9.4 kb SCD1 gene, which contains 31 exons, is shown
in Fig. 5B.
Expression and sequence analysis of SCD1
Reverse transcriptase PCR analysis revealed that the SCD1
transcript was expressed in all organs of the adult wild-type plant tested and
in actively dividing Arabidopsis suspension-cultured cells (T87)
(Fig. 6). scd1-1
transcript was also found to be expressed in the scd1-1 mutant
suggesting that the missense mutation does not affect scd1-1
steady-state mRNA levels. RNA samples prepared from 3-day-old T87 cells
yielded reproducibly stronger SCD1 signals both in RT-PCR experiments
and RNA blots (data not shown). Although these experiments were not
quantitative, they suggest that SCD1 may be more highly expressed in
actively dividing tissue.
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(MAPKs) (Levivier et al.,
2001). Most DENN domains are flanked by two additional elements,
upstream (u)DENN and downstream (d)DENN, which span several hundred amino
acids (Levivier et al., 2001
).
SCD1 was found to be the only gene in the Arabidopsis genome
predicted to encode all three DENN sub-elements. Two other
Arabidopsis genes of unknown function, At5g35560 and At2g20320,
encode proteins containing the uDENN and DENN sub-domains but lacking the
dDENN domain which has been suggested to be critical for binding to target
proteins, including Rab GTPases (Levivier
et al., 2001
). The putative At5g35560 and At2g20320 encoded
proteins share very limited amino acid sequence identity (<40%) over a
short (
65 amino acid) stretch of the SCD1 DENN domain. The
scd1-1 mutation disrupts a highly conserved serine within the DENN
domain (Fig. 5D). Rice
(Oryza sativa) has a putative SCD1 ortholog (GenBank Accession no.
BAB90268] that is 68% identical and 81% similar to SCD1 over the entire length
of the protein including the N-terminal DENN and C-terminal WD-40 domains,
suggesting that the function of these proteins has been conserved in flowering
plant evolution.
scd1::T-DNA mutants have severe defects in polarized cell
expansion
To further analyze the function of SCD1 we isolated and characterized two
additional scd1 alleles. Two independent TDNA insertion alleles,
scd1-2 and scd1-3 (Fig.
5B) were identified in the SIGnAL (SALK Institute Genomic Analysis
Laboratory) collection of individual sequence indexed T-DNA mutagenized
Arabidopsis lines
(http://signal.salk.edu/cgi-bin/tdnaexpress).
The T-DNA was present in the 23rd exon for scd1-2,
disrupting the predicted WD-40 repeats of SCD1, and in the 6th exon
for scd1-3, disrupting the predicted DENN domain. However,
scd1-2 and scd1-3 plants displayed identical phenotypes,
suggesting that these are complete loss-of-function alleles. When germinated
on MS-Suc, scd1-2 and scd1-3 mutant seedlings were smaller
than scd1-1 seedlings (Fig.
3A), with more severely stunted roots and reduced leaf expansion.
In soil, the scd1-2 and scd1-3 seedlings produced additional
leaves, however the rosettes were more dwarfed than scd1-1. In
contrast to scd1-1, the T-DNA alleles were not temperature sensitive
and after a period of three months at 18oC the tiny plants
generated defective flowers (Fig.
4G) with buds arresting at similar developmental stage as observed
in scd1-1 plants grown at 22°C
(Fig. 4B).
Further morphological and molecular characterization was performed on the scd1-2 T-DNA-induced mutation. scd1-2 segregated as a single recessive gene (a heterozygous scd1-2 plant segregated 384 wild type:121 dwarf). PCR genotype analysis of segregating scd1-2 seedlings confirmed that the dwarfed plants (Fig. 3) were homozygous for the T-DNA insert (data not shown). To test for allelism, fertile homozygous scd1-1 plants were crossed with scd1-2 heterozygotes. Progeny of several independent reciprocal crosses segregated approximately 1:1 for scd1 and wild-type phenotype seedlings when grown on MS-Suc medium, confirming that scd1-1 and scd1-2 are allelic.
Stomatal development was severely disrupted in scd1-2. Morphologically identifiable guard cells or guard mother cells were virtually absent in the leaf epidermis (Fig. 7B,F,G). Therefore the defect in guard cell development in the scd1-2 mutant is more severe than in the weaker scd1-1 allele. Leaf epidermal pavement cell shape was also altered in scd1-2 seedlings (Fig. 7A,B). In wild-type leaves and scd1-1 mutants these cells have sinuous cross-walls with interdigitating lobes and valleys giving a `jigsaw puzzle'-type appearance to the leaf surface (Figs 1, 2 and Fig. 7A). In contrast, scd1-2 (Fig. 7B) and scd1-3 (data not shown) pavement cells were less expanded in all directions than wild-type cells, and the cell walls were less lobed giving the mutant cells a more rectangular appearance. Small gaps were also observed between mutant pavement cells (Fig. 7F).
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The epidermal and cortical cells of scd1-2 mutant roots were
approximately 1/3 the length of wild type cells and like scd1-1,
there were fewer cells within the root division zone
(Fig. 3C,
Fig. 8A). Root hair formation
was usually arrested in all scd1 mutants at the transition to tip
growth stage of development (Parker et
al., 2000), resulting in short bulges in the basal end of
trichoblasts (Fig. 3D-F,
scd1-2 shown). Under certain conditions, however, all scd1
alleles formed root hairs that were significantly shorter (
150 µm in
length) than those of the wild type (Fig.
3F-H). These root hairs appeared to form under growth conditions
that have previously been reported to cause a stress-induced pathway of root
hair development (Okada and Shimura,
1994
). In particular, scd1 root hair expansion was
observed when seedlings were grown on MSSuc containing high agar
concentrations (1 to 1.5% w/v), which prevented adequate root contact with the
surface of the growth medium. In contrast to fully expanded wild-type root
hairs (Fig. 3H), abnormal
bulges and branches were observed on stress-induced scd1-2 root hairs
(Fig. 3F,G).
Electron microscopic analysis of scd1 mutant cells
To address whether a loss of the SCD1 protein affected membrane trafficking
we analyzed cytokinesis-defective scd1 leaf epidermal cells by
transmission electron microscopy. For studying cell plate biogenesis,
ultrarapid cryofixation is superior to chemical fixation, which may cause
artifactual vesiculation of this dynamic membrane system
(Otegui and Staehelin, 2000;
Otegui et al., 2001
;
Samuels et al., 1995
;
Samuels and Staehelin, 1996
).
Thin sections of wild-type and scd1-2 leaf primordia (<0.5 mm in
length) were prepared from high-pressure frozen, freeze substituted material.
Fig. 7H shows a dividing
wild-type cell with an expanding cell plate at the fenestrated sheet stage.
Several vesicles can be observed near the leading edge of the developing cell
plate as it grows toward the cell cortex. In contrast, a cluster of secretory
vesicles containing electron dense cell wall material was observed near the
tip of the cell wall stub of a cytokinesis-defective scd1-2 cell
(Fig. 7I). Similar defects in
vesicle accumulation and cell plate maturation have been observed in
cytokinesis-defective cells of knolle and keule embryos
(Lauber et al., 1997
;
Waizenegger et al., 2000
).
Inhibitor studies
To further analyze the function of SCD1 we examined the effects of several
membrane trafficking inhibitors and cytoskeletal antagonists on cell expansion
and cytokinesis in scd1 mutant and wild-type root cells. The
inhibitors used in these studies include caffeine (blocks cell plate
consolidation), brefeldin A (BFA; inhibits vesicle secretion), latrunculin B
(Lat B; microfilament (MF)-depolymerization), and propyzamide (microtubule
(MT)-destabilization). We used roots for these studies because they can be
grown in direct contact with the inhibitors and because their very regular
pattern of cell files make them highly amenable to imaging. To compare the
effects of the various inhibitors on root growth, we first grew the
Arabidopsis seedlings for several days on MS-Suc in the absence of
the inhibitors, transplanted them to medium containing the drugs and then
measured the extent of root growth over a 5-day period. Shown in
Fig. 8 are representative
wild-type and scd1 root tips from the various inhibitor treatments
and root length dose-response curves for each set of drug treatments. Although
the untreated scd1 root tips (Fig.
8A) appear slightly narrower than wild-type ones, no significant
differences in the overall diameter of wild type, scd1-1 and
scd1-2 roots were routinely observed
(Fig. 3D-G).
The difference observed in scd1 versus wild-type root length (Fig. 3A) is due not only to an inhibition in anisotropic cell expansion (Fig. 3C) but also to a reduction in cell production. Analysis of the growing tips of wild-type, scd1-1 and scd1-2 roots revealed that there are fewer cells in the division and transition zones of both mutants compared to wild type (Fig. 8A). In wild-type roots the division and transition zones (from the meristem to the start of the elongation zone) contained approximately 30-50 cells in the epidermal or cortical cell files, whereas scd1-1 roots contained 10-15 and scd1-2 roots contained 7-10 cells, respectively. All of the drug treatments we tested reduced the size of the transition zone in both wild type and mutants, resulting in the initiation of cell expansion immediately adjacent to the meristem in the scd1 roots.
Wild-type root cells were extremely swollen and disorganized when grown in the presence of 8 µM propyzamide (Fig. 8E). The mutant root cells in comparison were moderately swollen, and the roots less affected than wild type with respect to changes in their general shape, organization and diameter. The MT-stabilizing drug taxol (paclitaxel) caused similar effects on root elongation and cell expansion as observed for propyzamide (data not shown).
scd1 root cell morphology was more sensitive to the effects of caffeine, BFA and Lat B than wild type. No differences in the degree of root growth inhibition, relative to the no drug control, were observed between mutants and wild type. Rather, the cells in scd1 root tips became more disorganized than in wild type in the presence of caffeine, BFA and Lat B (Fig. 8B,C). As described above, cytokinesis-defective cells were not detected in scd1-1 and scd1-2 roots. However, in the presence 1.2 mM caffeine we observed an increase in the number of scd1 root cells containing one or more cell wall stubs relative to wild type (Fig. 8B). Cell wall stubs were also occasionally observed in mutant roots treated with BFA and Lat B. Lat B caused a slight increase in radial growth of elongating scd1 root cells. In untreated scd1-2 roots we have occasionally observed swollen epidermal cells. The frequency of these abnormal cells increased in both scd1-1 and scd1-2 roots grown in the presence of Lat B.
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DISCUSSION |
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A novel feature of the scd1-1 plants is their
temperature-sensitive phenotype. At the restrictive temperature of 22°C,
scd1-1 mutants display obvious defects in leaf cell cytokinesis. In
addition, mutant plants are dwarfed and show defects in floral organ and root
morphogenesis. Previous studies have shown that defects in cell division and
expansion affect normal floral organogenesis
(Hauser et al., 1998);
however, the cellular basis for the abnormal flowers in the scd1-1
plants remains to be characterized. At lower (i.e. permissive) temperature,
the scd1-1 gene product appears to retain greater activity, as
scd1-1 plants were fully fertile and stomata were completely normal.
The results of our temperature-shift experiments
(Fig. 4) suggest that the
missense mutation in the DENN domain of scd1-1 is a temperature
sensitive partial loss-of-function mutation. scd1-2 and
scd1-3 plants, which are not temperature-sensitive, displayed more
severe phenotypes than scd1-1 under all growth conditions and are
thus likely to be complete loss-of-function mutants. The temperature-sensitive
nature of the scd1-1 mutation should aid the molecular
characterization of SCD1 function.
Conditions that promote rapid growth (e.g. the presence of exogenous sucrose) enhanced the severity of the scd1 phenotype. During rapid growth, scd1-1 stomata were primarily the oblate type, which lack cell wall stubs and pores, suggesting that disruption of cell plate assembly in the mutant guard mother cells had occurred very early during cytokinesis.
Role of SCD1 in cytokinesis and polarized cell expansion
During cytokinesis, exocytic vesicles carrying membrane and cell wall
material are targeted toward the division plane to assemble the cell plate.
Likewise, plant cell expansion, which controls cell shape and ultimately plant
morphology, is accomplished by polarized membrane trafficking and localized
release of secretory pathway-derived membrane and cell wall material at
specific sites on the plasma membrane.
Characterization of two membrane-trafficking factors, KEULE
(Söllner et al., 2002)
and the Arabidopsis dynamin-related protein, ADL1A
(Kang et al., 2003
) has
recently shown that common molecular components of the membrane-trafficking
machinery are used during polarized cell expansion and cytokinesis. Similarly,
sequence analysis and phenotypic characterization of the scd1 mutants
suggest that SCD1 is required for vesicular trafficking during cell plate
formation as well as for the polar growth of various plant cell types.
SCD1 encodes a unique Arabidopsis protein containing two
domains that are likely to be critical for function, an N-terminal DENN domain
and a series of C-terminal WD-40 repeats that may mediate protein-protein
interaction. DENN domains have been identified in several proteins that
associate with members of the Rab family of small GTP-binding proteins, which
regulate membrane trafficking. Although the precise function of DENN domains
remains to be defined, its presence in the Rab3 guanine nucleotide exchange
factor (GEF) from Drosophila
(Shirataki et al., 1994), rat
(Oishi et al., 1998
) and
C. elegans (Iwasaki and Toyonaga,
2000
) and the mammalian Rab6 interacting protein, Rab6IP1
(Janoueix-Lerosey et al.,
1995
) has led to the suggestion that DENN domain-containing
proteins may regulate Rab activity
(Levivier et al., 2001
).
Each branch of the secretory pathway is thought to contain a specific Rab
that functions to coordinate targeting and docking of transport vesicles to
their appropriate acceptor membranes
(Rutherford and Moore, 2002).
Inhibition of Rabs and their accessory proteins, including GEFs that function
in Golgi to plasma membrane transport, has been shown to lead to the
accumulation of unfused secretory vesicles in yeast
(Novick et al., 1981
;
Novick et al., 1980
;
Novick and Schekman, 1979
).
Similarly we have observed the accumulation of unfused vesicles in the
cytokinesis-defective scd1-2 leaf cell shown in
Fig. 7I. Consistent with its
potential role in regulating membrane trafficking to the cell plate and cell
surface, our preliminary localization and subcellular fractionation studies
indicate that SCD1 is a tightly-associated peripheral membrane protein
associated with the plasma membrane and vesicular structures (T.G.F., L.M.K.
and S.Y.B., unpublished data).
As shown in yeast and mammalian cells, it is likely that multiple exocytic
routes mediate the polarized trafficking of proteins and cell wall
polysaccharides to the plant cell surface and division plane during
cytokinesis (for review see Bednarek and
Falbel, 2002). Yeast mutants lacking only one of two exocytic
pathways remain viable but grow more slowly
(Gurunathan et al., 2002
).
Likewise, cell plate biogenesis and cell expansion may not be fully blocked in
the scd1 mutants because other SCD1-independent trafficking routes
may partially compensate for the loss of SCD1. Consistent with a role for SCD1
in membrane trafficking, scd1 root cell cytokinesis was more
sensitive to low doses of caffeine, an inhibitor of cell plate consolidation
(Samuels and Staehelin, 1996
)
and to BFA, a general inhibitor of vesicle formation. At the concentrations of
BFA used in these experiments plant Golgi remain intact but secretion is
inhibited (Geldner et al.,
2001
; Staehelin and Driouich,
1997
). However, we cannot exclude the possibility that changes in
drug uptake as well as changes in the overall physiology of scd1
plants may affect their response to the various inhibitors we have tested.
Therefore it will be necessary to further test the role of SCD1 in membrane
transport through additional biochemical and genetic experiments.
We hypothesize that SCD1 regulates the function of a Rab GTPase required
for targeting and fusion of one class of secretory vesicles during cell plate
formation and cell expansion. The loss of SCD1 therefore reduces the
efficiency of membrane and cell wall material secretion necessary for rapid
cell expansion and could, in certain cells, cause defects in cell plate
assembly. Because the precise role of only a few plant Rabs have been
experimentally defined to date, it is premature to speculate which of the
fifty-seven Rabs encoded in the Arabidopsis genome
(Rutherford and Moore, 2002)
could potentially be the target of SCD1. The enhanced radial cell expansion
defects observed upon treating scd1 root cells with BFA or Lat B
could result from reduced secretion of cell wall precursors and biosynthetic
enzymes necessary to maintain cell wall integrity and constrain the
anisotropic direction of cell growth. As shown in
Fig. 7, expanding
scd1-2 trichomes are indeed mechanically unstable and rupture prior
to the initiation of branching. This phenomenon may be similar to that
observed in the root hairs of the defective cellulose synthase-like protein
mutant, kojak (Favery et al.,
2001
), which burst after initiation.
Vesicle trafficking and cytoskeletal dynamics are clearly interrelated
processes whose activity must be coordinated to establish proper cell shape.
For example, BFA has been shown to inhibit the proper localization of ROP
GTPases, which regulate actin dynamics during cell expansion
(Molendijk et al., 2001).
Interestingly, the leaf pavement cells of transgenic plants expressing a
dominant-negative GDP-bound mutant form of ROP2 were smaller and had fewer
lobes (Fu et al., 2002
)
similar to scd1-2. Although our data suggest that SCD1 functions
primarily in membrane trafficking, loss of SCD1 may also have an effect on the
plant cytoskeleton. We have found by immunofluoresence microscopy, using
anti-tubulin antibodies, that the cortical, mitotic spindle and phragmoplast
MTs were organized normally in scd1 root cells (data not shown). Our
inhibitor studies, however, indicated that scd1 root cell expansion
was less sensitive than wild type to MT antagonists. One possible explanation
for this is that MT dynamics may be affected by the rate of cell plate and
cell surface expansion.
In addition to its role in membrane trafficking, SCD1 may also be involved
in other signaling pathways required for cell expansion and cytokinesis. For
example, proteins containing DENN-domains such as the mammalian
mitogen-activated kinase activating death domain protein (MADD) are also
involved in the regulation of MAPK signaling pathways in addition to their
function in Rab-mediated processes (Majidi
et al., 1998; Schievella et
al., 1997
). Evidence for a role of DENN-containing proteins in
both signaling pathways also comes from the study of the C. elegans
Rab3 GEF, AEX-3, which is highly related to the human MAPK interacting
DENN-protein, MADD. aex-3 mutants display additional defects not
observed in rab-3 mutants, indicating that this Rab3 GEF functions in
both vesicular trafficking and other signaling pathways
(Iwasaki et al., 1997
;
Iwasaki and Toyonaga,
2000
).
MAPK signaling pathways are involved in plant cytokinesis and cell
expansion (Nishihama et al.,
2002; Samaj et al.,
2002
). Interestingly, defects in the tobacco MAPK kinase kinase
(MAPKKK), NPK1, and the Arabidopsis NPK1 orthologs, ANP2, ANP3
(Jin et al., 2002
;
Krysan et al., 2002
;
Nishihama et al., 2001
)
resulted in the development of dwarfed plants that display stomatal
cytokinesis defects similar to scd1-1
(Nishihama et al., 2001
);
T.G.F., P. J. Krysan and S.Y.B., unpublished data). In addition, the
stress-induced MAPK (SIMK in Medicago and Nicotiana,
homologous to MPK6 in Arabidopsis) has recently been shown to play an
important role in actin-dependent root hair elongation
(Samaj et al., 2002
). Analysis
of the phenotypes of scd1 anp2 anp3 triple and scd1 mpk6
double mutants will help to determine if SCD1 and the MAPKs function in the
same or in parallel signaling pathways to regulate cytokinesis and cell
expansion.
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ACKNOWLEDGMENTS |
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