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The Plant Cell, Vol. 14, 101–118, January 2002, www.plantcell.org © 2002 American Society of Plant Biologists
The Arabidopsis SPIKE1 Gene Is Required for Normal Cell
Shape Control and Tissue Development
Jin-Long Qiu,a Ross Jilk,b,1 M. David Marks,b,c and Daniel B. Szymanskia,2
a Department
of Agronomy, Purdue University, 1150 Lilly Hall of Life Sciences, West Lafayette, Indiana 47907-1150
of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108
c Plant Molecular Genetics Institute, University of Minnesota, St. Paul, Minnesota 55108
b Department
Regulated growth and cell shape control are fundamentally important to the function of plant cells, tissues, and organs.
The signal transduction cascades that control localized growth and cell shape, however, are not known. To better understand the relationship between cytoskeletal organization, organelle positioning, and regulated vesicle transport, we
conducted a forward genetic screen to identify genes that regulate cytoskeletal organization in plants. Because of the
distinct requirements for microtubules and actin filaments during leaf trichome development, a trichome-based morphology screen is an efficient approach to identify genes that affect cytoplasmic organization. The seedling lethal
spike1 mutant was identified based on trichome, cotyledon, and leaf-shape defects. The predicted SPIKE1 protein
shares amino acid identity with a large family of adapter proteins present in humans, flies, and worms that integrate extracellular signals with cytoskeletal reorganization. Both the trichome phenotype and immunolocalization data suggest
that SPIKE1 also is involved in cytoskeletal reorganization. The assembly of laterally clustered foci of microtubules and
polarized growth are early events in cotyledon development, and both processes are misregulated in spike1 epidermal
cells.
INTRODUCTION
In multicellular organisms, specialized cytoplasmic organization and cell shape underlie the unique functions of cells, tissues, and organs. It is reasonable to propose that the dynamic
properties of the microtubule and actin cytoskeletons and the
proteins that bind them underlie much of the observed asymmetry in plant cells. During leaf and root trichoblast morphogenesis, organized actin filaments and microtubules are
required (Bibikova et al., 1999; Mathur et al., 1999; Szymanski
et al., 1999; Baluska et al., 2000). We are using the diagnostic
shape defects of cytoskeleton-disrupted leaf trichomes to
guide mutant screens for essential genes that participate in
cytoskeletal reorganization and morphogenesis.
Decades of cytological and biochemical research have
demonstrated the importance of the actin filament and microtubule cytoskeletons during plant morphogenesis (for reviews of the interphase microtubule and actin cytoskeletons
in plant cells, see Giddings and Staehelin, 1991; Cyr, 1994;
Staiger, 2000). In most plant cell types, the interphase mi-
1 Current
address: Department of Biology, Rockhurst University, 1100
Rockhurst Road, Kansas City, MO 64110
2 To whom correspondence should be addressed. E-mail dszyman@
purdue.edu; fax 765-496-2926.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.010346.
crotubule array is cortical. The most commonly cited function of the cortical microtubule array is the regulation of the
alignment of newly synthesized cellulose microfibrils. However, there are cases in which microtubules and cellulose microfibrils are not coaligned (Baskin et al., 1999;
Wasteneys, 2000). The interphase actin filament cytoskeleton is composed of bundled transvacuolar filaments, nucleus-associated filaments, and cortical actin filaments
(Traas et al., 1987). Actin filaments provide both a scaffolding for the relatively immobile network of the endoplasmic
reticulum and tracks for the rapid intracellular transport of
Golgi stacks (Satiat-Jeunemaitre and Hawes, 1996; Boevink
et al., 1998; Nebenfuhr et al., 1999). In growing cells, the actin cytoskeleton is not a static structure. Fine and dynamic
cortical actin filaments may define regions of high rates of
exocytosis and growth. Several studies have correlated the
presence of fine cortical actin filaments with subcellular regions of localized growth (Thimann et al., 1992; Waller and
Nick, 1997; Gibbon et al., 1999; Szymanski et al., 1999; Fu
et al., 2001).
In the context of polarized growth, the roles of the actin
and microtubule cytoskeletons vary depending on the species and cell type. An unperturbed F-actin cytoskeleton is
required for the establishment of polarity in Fucus and Pelvetia embryos (Quatrano, 1973; Alessa and Kropf, 1999).
Actin filaments also are the primary cytoskeletal determinant
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of polarized tip growth in pollen tubes (Mascarenhas and
LaFountain, 1972; Heslop-Harrison et al., 1986; Gibbon et
al., 1999; Fu et al., 2001). Much of leaf and cotton trichome
growth is caused by polarized diffuse growth, and the resulting pharmacological sensitivities are quite different from
those of pollen tubes. In general, microtubule-disrupting
drugs block the initiation of polarized growth, and actin filament inhibitors mainly affect the maintenance of polarized
growth (Tiwari and Wilkins, 1995; Mathur et al., 1999;
Szymanski, 2000). The requirements for both microtubules
and actin filaments also have been examined during the polarized growth of other leaf cell types. Lateral microtubule
association precedes localized secondary wall formation in
developing Zinnia tracheary elements and may be sufficient
to localize secondary wall formation (Falconer and Seagull,
1985b). Drugs that disrupt microtubules block localized secondary wall formation, whereas those that destabilize actin
filaments do not (Kobayashi et al., 1988). Wheat mesophyll
cells also are highly polarized, lobed cells. Constricted regions of the cell correspond to locations of increased wall
thickness (Jung and Wernicke, 1991). Pharmacological and
localization experiments suggest that lateral microtubule
clustering is an early essential event during wheat mesophyll
cell lobe initiation (Wernicke and Jung, 1992). Lateral microtubule clustering seems to be a general mechanism for polarized growth in leaf cells with a lobed morphology
(Apostolakos et al., 1991; Panteris et al., 1993a, 1994). Although a class of candidate microtubule-associated proteins that form lateral cross-links has been identified in
plants, their in vivo function is not known (Chan et al., 1999;
Smertenko et al., 2000).
A forward genetic analysis of cytoskeleton-based functions in Arabidopsis is a feasible approach to understand
how plants have solved the problem of regulated cytoskeletal organization during polarized morphogenesis. For example, the reduced trichome branch phenotypes of zwichel
(zwi) and fragile fiber2 (fra2) phenocopy the effects of microtubule-disrupting, but not actin filament–disrupting, agents
(Oppenheimer et al., 1997; Mathur et al., 1999; Szymanski et
al., 1999; Burk et al., 2001). The ZWI and FRA2 gene products share amino acid sequence identity with the known microtubule binding proteins kinesin and katanin, respectively.
In the case of ZWI (also known as KCBP), minus end–directed
motor activity has been determined experimentally (Song et
al., 1997). The “distorted” class of trichome mutants appears
to be affected in actin-dependent processes (Szymanski,
2000). Clearly, a molecular genetic approach will provide important functional information about the plant cytoskeleton.
Our objective is to use genetic screens based on trichome
shape to identify additional genes that affect cell signaling,
cytoskeletal reorganization, and polarized growth. Unlike
most of the reduced trichome branch mutants reported to
date, the spike1 (spk1) mutant displays defects in polarized
growth in all cotyledon and leaf epidermal cell types as well
as seedling viability. The SPK1 gene encodes a 207-kD
modular protein that shares amino acid identity with the
CDM family of adapter proteins present in humans, flies, and
worms (Hasegawa et al., 1996; Nolan et al., 1998; Wu and
Horvitz, 1998). CDM proteins are thought to integrate extracellular signals and cytoskeletal reorganization. Consistent
with the notion that a cytoskeletal defect is associated with
the spk1 phenotype, localized lateral microtubule clustering,
which usually precedes lobe formation in developing epidermal pavement cells, is not observed in the mutant.
RESULTS
Characterization of the spk1 Phenotype
To improve our chances of finding genes that affect cytoskeletal organization during seedling development, we used
the diagnostic cell shape defects of trichomes to suggest
whether microtubule- or actin filament–dependent activities
may be affected (Szymanski, 2000). After family screens of
pooled lines of T-DNA–mutagenized plants, the seedling lethal spk1-1 allele was recovered in a heterozygous plant. In
segregating F2 populations, spk1-1 behaved as a simple recessive mutation (3 wild-type:1 mutant). spk1-1 displayed
a reduction in trichome branching and stalk elongation: its
phenotype was similar to that of microtubule-disrupted cells
and the trichomes on zwi and fra2 leaves. At 30 to 50% humidity, the spk1-1 allele caused seedling lethality of soilgrown plants, usually within 2 to 3 days after germination.
When grown at 100% humidity, soil-grown spk1-1 plants
survived for longer periods of time but were dwarfed compared with those of the wild type, and the cotyledons and
leaves of spk1-1 were very narrow (Figure 1A). When cultured on agar-based media, spk1-1 fresh weight was similar
to that of the wild type after 12 days of growth, but the organ shape and trichome defects persisted (Figures 1B and
1C). Although an inflorescence formed occasionally, the
mutant plants were completely sterile. The sterility defect was
sporophytic, because in reciprocal crosses the mutant allele
was transmitted efficiently through both the male and female gametophytes of heterozygous plants.
Scanning electron micrographs of the normal and mutant
leaf adaxial epidermal phenotypes are shown in Figures 1D
to 1H. Compared with the normal trichomes of the wild type
with three branches (Figure 1D), those of spk1-1 leaves displayed two types of aberrant morphologies. Type 1 trichomes were highly elongated, unbranched, and often
abnormally swollen or blebbed at various positions along
the stalk (Figure 1E). Type 2 spk1-1 trichomes had a short
stalk and two branches roughly parallel to the leaf surface
(Figure 1F). The wild-type leaf epidermis is composed of a
two-dimensional array of interdigitated lobed pavement
cells (Figure 1G). The pavement cells of spk1-1 leaves were
simple in shape; lobes were either absent or severely reduced in size, and most cell expansion occurred along a
single axis (Figure 1H).
Cell Shape Control in Arabidopsis
Figure 1. Arabidopsis Wild-Type and spk1-1 Seedling Phenotypes Grown under Different Conditions.
(A) One-week-old seedlings grown on soil at 100% humidity. The wild type is at left, spk1-1 mutant at right.
(B) Wild-type seedling at 9 DAG on agar-based medium.
(C) spk1-1 seedling at 9 DAG on agar-based medium.
(D) Scanning electron microscopy of wild-type leaf trichomes.
(E) Scanning electron microscopy of spk1-1 leaf trichomes with aborted branches.
(F) Scanning electron microscopy of spk1-1 leaf trichomes with two branches.
(G) Leaf epidermal pavement cells of the wild type.
(H) Leaf epidermal pavement cells of spk1-1.
Bar in (A) 0.5 mm; bars in (B) and (C) 1 mm; bars in (D) to (F) 100 m; bars in (G) and (H) 10 m.
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Figure 2. Cell and Organ Shape Defects during spk1-1 Cotyledon Development.
(A) to (D) Scanning electron microscopy of the adaxial surface of wild-type cotyledons.
(A) Wild-type cotyledon at 2 DAG.
(B) Higher magnification of wild-type cotyledon epidermal cells at 2 DAG.
(C) Wild-type cotyledon at 5 DAG.
(D) Higher magnification of wild-type cotyledon epidermal cells at 5 DAG.
(E) to (H) Scanning electron microscopy of the adaxial surface of spk1-1 cotyledons.
(E) spk1-1 cotyledon at 2 DAG.
(F) Higher magnification of spk1-1 cotyledon epidermal cells at 2 DAG.
(G) spk1-1 cotyledon at 5 DAG.
(H) Higher magnification of spk1-1 cotyledon epidermal cells at 5 DAG.
Bars in (A), (C), (E), and (G) 100 m; bars in (B), (D), (F), and (H) 10 m.
Given the interesting cell shape defects of the spk1-1 mutant, an experimental system was developed to examine the
cell and tissue level functions of SPK1. We focused on cotyledon epidermal development for several reasons. First,
postgermination cotyledon growth is the first recognizable
spk1-1 phenotype in the shoot, and therefore it is the stage
at which we are most likely to identify the direct effects of
the mutation. Second, cotyledon epidermal cell growth is
both highly polarized and synchronous, and it involves the
coordinated growth of a population of cells. Third, we have
Cell Shape Control in Arabidopsis
sensitive methods to analyze the organization of microtubules and actin filaments in epidermal cells at several
developmental stages. Our current inability to visualize
the cytoskeletons of intact spk1-1 trichomes has hindered
our studies of SPK1 function in this cell type.
The shape of the cotyledon was affected severely in the
spk1-1 mutant. At 2 days after germination (DAG), wild-type
cotyledons were roughly circular (Figure 2A). Their epidermal pavement cell shapes were variable; some cells initiated
several lobes, whereas others had a simple shape with either no clear indication of polarized growth or a rudimentary
bulge within the plane of the epidermis (Figure 2B). In this
article, the extended regions of the lobe are referred to as
protrusions and the lateral flanks of the lobe are referred to
as indentations. At 1 DAG, spk1-1 cotyledon shape was indistinguishable from that of the wild type, but at 2 DAG, cotyledons were slightly more narrow (Figures 2E and 3, Table
1). Some of the spk1-1 epidermal cells were slightly irregular
in shape, and there was no clear indication of lobe initiation
along the cell perimeter (Figure 2F). At 5 DAG, wild-type cotyledons retained a circular shape, and almost all cells contained multiple, well-elaborated protrusions (Figures 2C and
2D). At the same time, the spk1-1 cotyledons were narrow,
and the epidermal cells lacked protrusions and were quite
small compared with those of the wild type (Figures 2G and
2H, Table 1). Gaps were evident between mutant epidermal
cells (Figure 2H). At 8 to 10 DAG, the cotyledon continued to
expand, and the defects in cell-to-cell adhesion became
quite severe. These large gaps in the epidermis explain
the developmental stage–specific seedling lethality of the
mutant at low humidity. A thorough analysis of the carbohydrate composition and ultrastructure of cell walls of
mutant and wild-type plants failed to reveal any significant differences (D. Szymanski and N. Carpita, unpublished results).
To determine if spk1-1 differentially affected cotyledon
length and width, we measured the cotyledon shape of wildtype and spk1-1 cotyledons. At the earliest time point (1
DAG), the mean length and width measurements of wild-
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type and mutant cotyledons were nearly identical (Figure 3).
In addition, the duration and rate of cotyledon elongation of
the mutant were not appreciably different from those in the
wild type (Figure 3, Table 1). However, the lateral growth
rate of spk1-1 cotyledons was reduced. Qualitative differences in cotyledon diameter were apparent at 2 DAG and
were statistically significant at 12 DAG (Figure 3, Table 1).
The mean cell surface area was reduced 29 and 38% in
2- and 5-DAG mutant cotyledons, respectively (Table 1).
The contribution of cell number and cell size to the spk1-1
cotyledon shape defects also was determined. Consistent
with the observation that very little cell division occurs during Arabidopsis cotyledon development (Tsukaya et al.,
1994), the mean cotyledon cell number in the wild type increased from 382 cells at 2 DAG to 409 cells at 5 DAG (Table 1). Although this is not a substantial difference, using
scanning electron microscopy, we routinely observed recently divided cells in wild-type cotyledons from 5 to 8 DAG.
At 2 DAG, the spk1-1 mean cotyledon cell number was not
significantly different from that of the wild type, although
some individual spk1-1 cotyledons had increased cell numbers that were not observed in the wild type. At 5 DAG, the
spk1-1 epidermis contained 75% more cells than the wild
type (Table 1). Within the midblade clusters of dividing cells,
neither the cell division planes nor the direction of cell expansion was oriented with respect to the long axis of the
leaf (Figures 2G and 2H). Therefore, the additional cells in
spk1-1 cotyledons do not contribute preferentially to cotyledon elongation. In most spk1-1 cotyledons, a subset of epidermal cells near the tip and along the perimeter expanded
preferentially along the long axis of the cotyledon (Figures
2E and 2G).
Cloning the SPK1 Gene
To initiate a functional analysis of the control of cell shape in
the epidermis, we cloned the SPK1 gene. In a segregating
population, we failed to detect any recombination between
Table 1. Cotyledon Size and Cell Number of spk1 and Wild-Type Cotyledons at Different Times
2 DAG
5 DAG
Size and Number
spk1a
Wild Type
spk1
Wild Type
Length (mm)b
Width (mm)b
Area (mm2)c
Cell Numberd
0.80 0.26
0.47 0.14
0.25 0.08
456 151
0.82 0.29
0.67 0.24
0.35 0.08
382 161
1.89 0.57
1.07 0.18
1.37 0.55
715 174
1.93 0.57
1.60 0.45
2.20 0.73
409 82
measurements are expressed as the mean 1.96 SD, which defines the 95% confidence level for significance.
length and width values were measured for each cotyledon.
c Adaxial surface area.
d Measurements were taken from scanning electron microscopy images of the adaxial epidermis. Cell number is total cotyledon area/mean cell
area (see Methods).
a All
b Maximum
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Figure 3. Growth Rates of Wild-Type and spk1-1 Cotyledons.
Maximum cotyledon lengths and widths were measured from 1 to
12 DAG. Closed squares, wild-type cotyledon length; open squares,
spk1-1 cotyledon length; closed triangles, wild-type cotyledon
width; open triangles, spk1-1 cotyledon width. Error bars indicate
SD 1.96 to reflect a 95% confidence interval for significance.
the kanamycin resistance gene (Kn r) of the T-DNA element
and the spk1-1 mutation (n 88). Therefore, the spk1-1 allele is within 1.2 centimorgans of the T-DNA insertion. Sequence data from the left border of the T-DNA located the
insertion to the 18th intron of a large open reading frame on
chromosome 4 (Figure 4A). DNA gel blots of genomic DNA
from wild-type and mutant plants confirmed that the flanking genomic sequences are linked to the T-DNA and defined an 81-bp interval within which the effects of the
insertion were limited (data not shown). We used a polymerase chain reaction (PCR)–based reverse genetic approach to identify an independent heterozygous line (spk12) that segregated 3:1 for both Knr and the spk1 phenotype (McKinney et al., 1995; Krysan et al., 1996). The mutation was linked to the insertion; of 102 spk1-2 plants
examined, all harbored the selectable marker associated
with the T-DNA. Sequence information from the plant DNA
that flanked the left border of the T-DNA located the insertion to the seventh intron of the SPK1 gene (Figure 4A). The
gross cotyledon, leaf, and trichome phenotypes of the
spk1-1 and spk1-2 alleles were indistinguishable (data not
shown).
If the insertions into the SPK1 gene cause the mutant
phenotype, then the expression of the gene may be altered
in mutant plants. To address this question, we assayed
SPK1 gene expression in mutant and wild-type plants using
reverse transcription (RT)–PCR. SPK1-specific primers upstream (Figure 4B, lanes 1 to 4) and downstream (Figure 4B,
lanes 5 to 8) from the spk1-1 T-DNA insertion detected
SPK1 message in wild-type plants (lanes 1 and 5). In all
cases, the signal was derived from mRNA because no amplified product was obtained in the absence of RT (lanes 2,
4, 6, 8, 10, and 12). In RNA samples derived from spk1-1
plants, accumulation of SPK1 mRNA was either reduced
greatly (lane 3) or undetectable (lane 7). The failure to detect
SPK1 mRNA was not the result of a problem with the RNA
sample because the ACTIN7 (ACT7) gene was detected
easily (lanes 11 and 12). SPK1 message was not detectable
in the spk1-2 background using PCR primers that flanked
the T-DNA insertion (data not shown).
To confirm that disruption of the SPK1 gene caused the
spk1 phenotype, we transformed wild-type plants with a
transgene that transcribes double-stranded RNA corresponding to the SPK1 gene. Control plants transformed with
either the empty vector (Figure 5A) or a double-stranded
RNA construct corresponding to an intron of SPK1 (Figure
5B) had no detectable trichome, pavement cell, or cotyledon shape phenotype. However, plants transformed with
double-stranded RNA–expressing constructs corresponding to 1089 bp (DS-1; Figure 5C) and 677 bp (DS-2; Figure
5D) of the SPK1 cDNA sequence displayed all aspects of
the spk1 phenotype: 88.5% of DS-1 and 93.2% of DS-2
transformed plants displayed the narrow cotyledon phenotype. For both DS-1 and DS-2 transformed lines, trichome
branching was reduced noticeably in 5% of the viable T1
plants. Many of the severely affected transformed plants
that displayed a trichome phenotype were seedling lethal.
Together, these results prove that we identified the SPK1
gene correctly.
Full-Length SPK1 cDNA Sequence and Expression
The spk1-1 insertion event occurred within a predicted long
open reading frame (ORF 4200c) on the south arm of chromosome 4 (Bevan et al., 1998). Because of an extra T in the
genome sequence (see below), a deduced reading frame error occurred and the annotation of the genomic sequence
did not identify a candidate start codon. RNA gel blot experiments conducted using poly(A) mRNA indicated that the
SPK1 message is nearly 6 kb, twice as long as the 3.3-kb
predicted coding region of ORF 4200c (data not shown).
Using a 3-kb partial-length cDNA obtained from a seedling
library (Kieber et al., 1993) as a starting point, one 3 rapid
amplification of cDNA ends (RACE) step and three 5 RACE
steps were required to identify both the poly(A) site and an
initiation codon that contained an upstream stop codon in
the SPK1 reading frame. The complete 5.8-kb cDNA sequence and the deduced amino acid sequence are available
in GenBank. A scheme of the SPK1 protein and its con-
Cell Shape Control in Arabidopsis
107
Figure 4. Structure of the SPK1 Gene and Nature of spk1 Mutant Alleles.
(A) Exon and intron organization of the SPK1 gene. The positions and lengths of exons and introns are indicated by closed rectangles and lines,
respectively. The positions of T-DNA inserts associated with spk1-1 and spk1-2 are shown above the gene diagram.
(B) Altered expression of the SPK1 gene in spk1-1 plants. RT-PCR analysis shows SPK1 mRNA accumulation in spk1-1 and the wild type. The
locations of the upstream (SPK1 RT2) and downstream (SPK1 RT1) primer pairs are labeled. Gene-specific primers for the ACT7 gene were used
as a control. (), with reverse transcriptase (RT); (), without reverse transcriptase.
served domains is shown in Figure 6. There were 30 exons
and 29 introns in the SPK1 gene. All splice donor and acceptor sites conformed to observed Arabidopsis splice
junction border sequences. We identified 6 bp in the fulllength SPK1 cDNA that differed from the published genome
sequence. We confirmed that the cDNA sequence was correct by sequencing both strands of the cosmid clone g5845
at each ambiguous base.
The SPK1 cDNA encodes a predicted protein of 1830
amino acids and 207 kD whose mRNA is present in all major
organs (data not shown). The predicted protein contained
several 21–amino acid hydrophobic regions that are candidate membrane-spanning segments (Kyte and Doolittle,
1982). However, the protein lacked an N-terminal signal
sequence, and the TMHMM algorithm, which is the most
accurate method for identifying membrane-spanning segments, predicted SPK1 to be a soluble protein (Krogh et al.,
2001). The solubility of SPK1 in cells remains to be determined. The predicted protein contained at least three conserved regions. The most C-terminal is 370 amino acids
long and shares at least 20% pairwise identity with the animal proteins Caenorhabditis elegans CED-5, human DOCK180,
and Drosophila MYOBLAST CITY, which constitute the
CDM family of proteins (Hasegawa et al., 1996; Nolan et al.,
1998; Wu and Horvitz, 1998). Although the pairwise identity
is not immediately impressive, nearly 25% of the conserved
residues that show pairwise identity with animal proteins are
identical across several phylogenetic groups (Figure 7). This
region of the SPK1 protein is defined as the CED-5,
DOCK180, MBC, SPK1 (CDMS) domain. N-terminal to the
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Figure 5. Expression of Double-Stranded SPK1 RNA Causes a spk1 Phenotype.
(A) Wild-type plants transformed with the empty binary vector.
(B) Wild-type plants transformed with DSCK. DSCK expresses double-stranded RNA corresponding to the 27th intron of SPK1.
(C) Wild-type plants transformed with DS1. DS1 corresponds to 1089 bp of the SPK1 coding sequence.
(D) Wild-type plants transformed with DS2. DS2 corresponds to 677 bp of the SPK1 coding sequence.
Insets in each panel show scanning electron micrographs of the adaxial cotyledon epidermal cells of similarly staged plants. Bars 1 mm; inset
bars 100 m.
CDMS domain, the SPK1 gene product contained two additional conserved regions of 130 and 510 amino acids
(Figure 6). Because these domains are found only in the genomes of multicellular organisms, we refer to the 130– and
510–amino acid domains as multicellular organism domain 1
(MOD1) and MOD2, respectively. The CDM family proteins
do not encode conserved MOD1 and MOD2 domains.
Cytoskeletal Localization in Developing Cotyledon
Epidermal Cells
In each of the experimental systems in which the function of
CDM proteins has been examined, a role in integrating extracellular information with cytoskeletal reorganization has
been hypothesized. To begin to test for a role for SPK1 in
cytoskeletal organization, we compared the mutant and
wild-type actin and microtubule cytoskeletons in developing
cotyledon epidermal cells. We double labeled actin and tubulin with antibodies at three stages of development: (1)
symmetrical, cuboidal cells common in 1-DAG cotyledons;
(2) cells with newly formed lobes common in 2-DAG cotyledons; and (3) cells with well-defined lobes common in 5-DAG
cotyledons. Confocal images of double-labeled wild-type
and mutant cells are displayed in Figure 8. Each image is a
maximum projection of the tubulin (green channel) and actin
(red channel) signals from the outer halves of epidermal
cells. In wild-type epidermal cells that either had divided recently or had yet to initiate clear lobe-like structures, foci of
laterally associated cortical microtubules were seen frequently on anticlinal walls (walls perpendicular to the leaf
surface). Near the junction of the periclinal walls (walls parallel to the leaf surface) and anticlinal walls, the cortical microtubules splayed out along the cell cortex. A representative
Cell Shape Control in Arabidopsis
cell displaying this sort of microtubule organization is shown
in Figure 8A.
At these early stages of development, the actin cytoskeleton often was distributed between association with putative
provacuoles and the cell cortex. Actin filaments were excluded largely from the plasma membrane in regions of the
cell in which cortical microtubules were present. In microtubule-free zones of the cell, some actin filaments were associated closely with the plasma membrane (Figure 8A).
Throughout epidermal development, this predominantly
nonoverlapping pattern of actin and tubulin signal is observed consistently (Figure 8). Almost all of the yellow color
seen in Figure 8 is the result of compressing three-dimensional localization signal into two dimensions. However, isolated puncta of colocalized actin and tubulin within a single
image plane occasionally was detected at indentations near
the flanks of laterally associated microtubules. At 2 DAG, laterally associated microtubules were observed consistently in
close association with the plasma membrane along cell indentations (Figure 8C). Fine actin filaments or bundles within
actively growing protrusions were both cortical and transvacuolar; many terminated in very close proximity to the plasma
membrane (Figure 8C). In protrusions, much of the actin signal was either diffuse or punctate, and we do not know if this
is a fixation artifact or the real actin organization in vivo.
Cortical microtubules were not excluded from protrusions, and the pattern of nonoverlapping cortical microtubules and actin filaments often was observed within a
growing protrusion. For example, in Figure 8C, three repeating units of alternating microtubule- and actin filament–populated plasma membrane zones are clearly defined within a
single protrusion. The inset shows a single optical section
that includes a plane of transvacuolar cytoplasm that is enriched in actin filaments; the reticulate network of actin bundles clearly terminates near the plasma membrane between
two sets of laterally associated microtubules. At 5 DAG, laterally associated cortical microtubules populated the anticlinal wall along the indentations, but many of the tips of
protrusions were dominated by actin signal (Figure 8E).
Most of the actin signal in Figure 8E was derived from the
cortical cytoskeleton, with very little contribution from transvacuolar strands.
The cytoskeletal organization of spk1 epidermal cells was
altered at the onset of the mutant cell shape phenotype. At 1
DAG, there were no significant differences in overall organ
shape (Figure 3), but at the cellular level, spk1 epidermal
cells were more rounded and lacked the small protrusions
that were observed along wild-type anticlinal walls (Figure
109
8B). Populations of actin filaments and microtubules were
present in mutant cells. However, evenly distributed cortical
microtubules were detected only faintly in mutant cells, and
no pattern of alternating foci of laterally associated cortical
microtubules and cortical actin filaments along the perimeter of the anticlinal walls was observed. Instead, putative
membrane-associated actin filaments were detected most
often at cell corners or at the ends of elongated cells (Figure
8B). At 2 DAG, cortical microtubules were aligned in an extended radial pattern and often formed evenly spaced circumferential hoops around elongated cells (Figure 8D). The
absence of regions of highly clustered microtubules at 2 DAG
was not caused by a complete lack of lateral cross-linking
activity, because pairs of radial microtubules could be found
in electron micrographs taken from ultrathin paradermal
sections (D.B. Szymanski, unpublished results). However,
distinct foci of laterally associated microtubules were not
detected along the anticlinal walls of mutant cells during
early developmental stages. In expanding mutant cells at 2
and 5 DAG, cortical actin filaments in close proximity to the
plasma membrane often were detected at seemingly random positions along the perimeter of the anticlinal cell wall,
but their cortical localization was not correlated with any
particular cell shape (Figures 8D and 8F).
DISCUSSION
Forward genetic approaches using Arabidopsis will be extremely useful for understanding the signaling pathways that
affect cytoskeletal reorganization during plant development
(Oppenheimer et al., 1997; Burk et al., 2001; Whittington et
al., 2001). Our long-term goal is to understand how the organization of the cytoskeleton is regulated and its function during epidermal cell and tissue morphogenesis. To achieve this
goal, we have initiated a functional analysis of SPK1. The
ubiquitously expressed SPK1 gene described in this article is
required for normal cytoskeletal organization, polarized cell
expansion, and tissue development in the leaf and cotyledon.
A Complex Cotyledon spk1 Phenotype
At 1 DAG, the organ shape and cell number of the mutant
were not appreciably different from those of the wild type.
However, at 2 DAG, the trend toward decreased cell size,
decreased cotyledon width, and increased cell number was
Figure 6. Scheme of the SPK1 Coding Sequence.
The MOD1, MOD2, and CDMS domains are labeled to scale according to their positions in the predicted protein.
110
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Figure 7. Alignment of the CDMS Domain of SPK1 with Homo sapiens DOCK180, Caenorhabditis elegans CED-5, Drosophila melanogaster
MYOBLAST CITY (MBC), Saccharomyces cerevisiae Ylr422wp, and Dictyostelium discoideum DocA.
Amino acid residues in black indicate identity, and those in gray indicate conserved substitutions. Residues that are identical or similar in four of
seven sequences are highlighted. The numerical amino acid scale for each sequence is labeled at right.
Cell Shape Control in Arabidopsis
apparent (Table 1). It is possible that SPK1 has multiple
functions in the cell, but we suspect that some of these
pleiotropic cotyledon phenotypes are secondary effects of
the failure to organize the cytoplasm in spk1 cells properly
at a previous stage of development. For example, the adhesion defects between mutant epidermal cells commonly
seen between 2 and 5 DAG also are observed in regions of
high mechanical strain, such as leaf margins and the apical
hook of dark-grown hypocotyls (data not shown). It is possible that both the reduction in the interdigitation of the unlobed cells and subtle wall defects contribute to the
widespread failure of spk1 epidermal cells to withstand the
forces of organ expansion. The increased cell number phenotype also may be a secondary response to defective cell
expansion or adhesion. The ability of adjacent cells to
“sense” and compensate for the reduced growth of slowgrowing sectors has been observed in maize leaves (Freeling,
1992). In any case, increased cell division is not sufficient to
restore wild-type width to spk1 cotyledons. It is not clear
why cotyledon width is affected more severely than length.
Similar defects in cotyledon shape and trichome branching
have been reported for the angustifolia (an) mutant (Rédei,
1962; Hülskamp et al., 1994). It has been proposed that genetically distinct pathways control cotyledon elongation and
lateral expansion (Tsuge et al., 1996). In the spk1 background, the elongation of a subpopulation of cells along the
cotyledon margin and near the tip may play a major role in
determining organ length.
SPK1 Gene Product
A single copy of the SPK1 gene is present in the Arabidopsis genome. It is a large gene consisting of 30 exons that
span 12.7 kb of genomic DNA. The reported 5.8-kb cDNA
sequence agrees well with the size of the SPK1 mRNA detected in RNA gel blots analysis. The predicted protein contains a conserved CDMS domain at the C terminus that
provides some clues to its function. The known CDMSencoding gene family members in animal cells are hypothesized adapter proteins that mediate cytoskeletal reorganization in response to diverse extracellular signals (Hasegawa
et al., 1996; Erickson et al., 1997). For each of the CDM family members, there is genetic and biochemical evidence for
the involvement of RAC GTPases during cell signaling
(Kiyokawa et al., 1998; Nolan et al., 1998; Reddien and Horvitz,
2000). RAC GTPases are nucleotide-gated switches that affect the cytoskeleton indirectly by modulating the activity of
effector proteins. In the case of human DOCK180, the
founding member of the family, the CDMS domain is required to coimmunoprecipitate DOCK180 and RAC in vivo,
but it is not known if the interaction is direct (Kobayashi et
al., 2001). Mutations within the CDMS domains of MBC and
CED5 cause severe loss-of-function phenotypes (Erickson
et al., 1997; Wu and Horvitz, 1998). The details of how the
proposed RAC-CDM protein complex regulates cytoskeletal
111
organization are not known. In Arabidopsis, there are 11 AtROP genes whose encoded proteins share a high level of
amino acid sequence identity with animal RAC gene products (Li and Yang, 2000). Perhaps AtROPs are involved in
SPK1-dependent signaling processes.
It is unlikely that the CDMS domain of SPK1 is the only region of functional significance. There are at least two additional regions in the protein that are highly conserved in a
large family of animal genes with no known function. Many
of the MOD1- and MOD2-encoding genes also encode a
C-terminal CDMS domain, but the overall domain organization of this class of genes is diverse. For example, in the
current database of full-length gene sequences, four of the
seven possible combinations of MOD1, MOD2, and CDMS
are present. One Drosophila gene and one C. elegans gene
encode ordered MOD1, MOD2, and CDMS domains. Perhaps these conserved regions are additional domains within
SPK1 that either recruit additional proteins and/or autoregulate the activity of other domains.
Cytoskeletal Basis of the Epidermal Shape Defects
Based on the reduced trichome branching phenotype and
the observed differences in lateral microtubule association
at the early stages of pavement cell development, it is possible that SPK1 regulates the organization of the interphase
microtubule array. In differentiating cells, the postmitotic
cortical microtubule array is organized randomly at first;
however, depending on the cell type, it eventually adopts a
more ordered symmetrical pattern (Flanders et al., 1990;
Granger and Cyr, 2000; Bichet et al., 2001). In Arabidopsis,
the transition to an ordered interphase microtubule array is a
crucially important step in regulating cell and tissue organization (Bichet et al., 2001; Burk et al., 2001). In the leaf,
microtubule clustering and localized wall deposition are
evolutionarily conserved processes in many leaf cell types
that display a lobed cell morphology (Panteris et al., 1994;
Wasteneys et al., 1997). In many cases, microtubule bundling is an early differentiation event that leads to localized
cell wall synthesis (Falconer and Seagull, 1985a; Kobayashi
et al., 1988; Wernicke and Jung, 1992; Panteris et al.,
1993b). In Vigna sinensis, the deposition of newly synthesized wall material in the leaf epidermal cells is aligned with
clustered microtubules and is concentrated near the junction of the anticlinal and periclinal walls (Panteris et al.,
1993b). This region of the cell, in which the transition from
laterally associated to isolated microtubules at the outer
periclinal membrane occurs, may define specialized domains for the motor-dependent transport of vesicles and
cellulose synthase complexes.
At all stages of pavement cell morphogenesis examined,
actin filaments are largely excluded from the plasma membrane in regions of the cell where clustered microtubules are
present. During polarized tracheary element and wheat mesophyll cell morphogenesis, inhibitor and localization studies
112
The Plant Cell
Figure 8. Immunolocalization and Confocal Micrographs of the Cytoskeleton in Developing Wild-Type and spk1-1 Cotyledon Epidermal Cells.
Epidermal cells were double labeled with antibodies directed against -tubulin (green channel) or actin (red channel). Each image is a maximum
projection of the upper half of the cells.
(A), (C), and (E) Wild-type epidermal cells.
(A) Cuboidal wild-type epidermal cell at 1 DAG.
(B) Cuboidal spk1-1 epidermal cell at 1 DAG.
Cell Shape Control in Arabidopsis
suggest that microtubule clustering precedes cortical actin
filament deposition (Kobayashi et al., 1988; Jung and
Wernicke, 1991; Wernicke and Jung, 1992). Although there
certainly are other possibilities, the simplest explanation is
that cortical microtubule clustering occurs first and locally
excludes actin filament–associated structures from the
plasma membrane. By default, cortical actin filaments and
bundles would be capable of populating plasma membrane
domains in which clustered microtubules are absent. Although nonoverlapping in space, the organization of the
microtubule and actin filament cytoskeletons may be interdependent. For example, the extremely fine actin filaments
and punctate signal detected in epidermal lobes may have
unique functional properties and may be required to maintain localized regions of laterally associated microtubules
during development. Therefore, at this time, we cannot exclude the possibility that SPK1 affects the organization of
the actin cytoskeleton and that the microtubule phenotype
is a secondary effect.
If microtubule clustering is the first event in organizing the
cytoplasm during pavement cell morphogenesis, the means
by which this activity is regulated is not known. Certainly, in
the well-defined lobes of Arabidopsis, microtubule clustering corresponds to cell indentations (Figure 8E) (Wasteneys
et al., 1997). At early stages of pavement cell development,
microtubule clustering often is correlated with the presence
of a small indentation (Figure 8A). The protrusions of neighboring cells may initiate the morphogenesis process. Either
a slow-acting force or a specific cell wall component could
contain the spatial information for microtubule clustering. A
mechanism for repetitive lobe initiation events must be considered: as the pavement cells expand, new lobes are
formed, presumably through an iterative process. Although
the lobes of wild-type cells are enriched for actin-containing
structures, microtubules also are present (Figures 8C and
8E). Perhaps as the protrusion expands, several cortical microtubules invade the lobe and become stabilized laterally
along the anticlinal wall. Alternatively, an actin-containing
membrane-associated cortical complex may recruit microtubules or promote microtubule polymerization.
Lobe formation does not occur in spk1 epidermal cells,
and foci of laterally associated microtubules along the anticlinal walls are not observed at early developmental stages.
113
Cortical microtubules are present, but most often they
adopt an extended radial arrangement perpendicular to the
long axis of the cell. Pairs of laterally associated cortical microtubules are observed in ultrathin sections of both wildtype and spk1 pavement cells (D.B. Szymanski, unpublished results). Therefore, it is more likely that the position,
timing, or extent of lateral association is affected in the mutant. Actin filaments, many of them cortical, also are widespread in developing spk1 epidermal cells at 1 and 2 DAG.
Although the organization of the actin cytoskeleton is similar
in mutant and wild-type cells, the location of cortical actin
filaments along spk1-1 anticlinal walls does not reflect cell
shape (Figures 8D and 8E). It appears that actin filament access to the plasma membrane is not sufficient for localized
cell expansion in spk1 epidermal cells.
Several pieces of circumstantial evidence lead us to suspect that SPK1 is involved in cytoskeletal reorganization.
However, at this time, we do not understand how SPK1 participates in this process. Current experiments are focused
on understanding how far SPK1 function is removed from
cytoskeletal reorganization. If AtROP binding and microtubule organization are primary effects of SPK1 signaling, this
differs from the commonly observed effects of RAC on the
actin cytoskeleton. Almost all publications in the animal and
plant literature cite the actin cytoskeleton as the downstream target of RAC-like GTPase signaling events (reviewed by Hall, 1998; Li and Yang, 2000). However,
dominant negative forms of RAC affect both the microtubule
and actin filament cytoskeletons during Drosophila wing
development (Eaton et al., 1996; Turner and Adler, 1998).
Furthermore, RAC-dependent signaling affects the microtubule-destabilizing activity of stathmin (Daub et al., 2001).
Considering the unique roles of actin and microtubules in
plant cells and the presence of only a single family of RAClike GTPases, AtROP-dependent signaling in Arabidopsis
may not conform strictly to commonly observed functional
boundaries.
Conclusion
The SPK1 gene is a regulator of cell shape and tissue organization in plant cells. Both the trichome phenotype and the
Figure 8. (continued).
(C) A wild-type epidermal cell at 2 DAG that has initiated lobe formation. The inset shows a single optical section through a plane of the transvacuolar cytoplasm.
(B), (D), and (F) spk1-1 epidermal cells.
(D) spk1-1 epidermal cell at 2 DAG.
(E) A rapidly growing wild-type epidermal cell at 5 DAG.
(F) An elongated spk1-1 epidermal cell at 5 DAG.
White arrows, regions containing cortical microtubules that are in close proximity to the plasma membrane; black arrows, regions of the cell
containing actin filaments that are in close proximity to the plasma membrane. Bars 10 m.
114
The Plant Cell
defects of lateral microtubule association during the early
stages of pavement cell development suggest that SPK1dependent processes alter the microtubule cytoskeleton.
SPK1 encodes the conserved CDMS domain, an essential
region of the animal CDM family of adapter proteins. SPK1
contains additional regions of conserved amino acid sequence that may regulate the localization or activity of
SPK1-containing complexes. Our working hypothesis is that
SPK1 integrates developmental cues with cytoskeletal rearrangement during cell and tissue development. If the primary
consequence of SPK1-dependent signaling is cytoskeletal
reorganization, much work remains to elucidate the control
mechanisms of this important signal transduction pathway.
METHODS
Plant Materials and Growth Conditions
For all phenotypic characterizations, Arabidopsis thaliana ecotype
Columbia seed were grown as described by Szymanski et al. (1999).
For preparation of genomic DNA and RNA from spk1 plants, seed
were germinated and grown in liquid medium containing Murashige
and Skoog (1962) salts and vitamin mixture (Gibco BRL, Gaithersburg, MD) with 1% sucrose with shaking (150 to 200 rpm) at room
temperature under continuous illumination at 23C.
ples then were extracted in PBS containing 1% Triton X-100 and 25
mM glycine. For immunodetection, monoclonal clone DM1A (Sigma,
St. Louis, MO) IgG1 directed against chicken brain tubulin was used
at a 1:100 dilution. Monoclonal clone JLA20 (Oncogene Research
Products, Boston, MA) IgM directed against chicken gizzard actin
was used at a 1:100 dilution. Conjugated secondary antibodies and
F(ab)2 fragments were obtained from Jackson Immunochemicals
(West Grove, PA).
Cotyledon Measurements
Cotyledons were dissected from seedlings to remove the petiole.
Cotyledons were placed in a drop of water and mounted on a glass
slide. Calibrated digital images were imported into NIH Image 1.61
(National Institutes of Health, Bethesda, MD; ftp://codon.nih.gov/
pub/nih-image/). The cotyledon length, width, and area were measured according to the magnification. Adaxial epidermal cell numbers were counted using scanning electron microscopy images. Cell
number was estimated by dividing the total cotyledon area by the
mean cell area. Total cotyledon area was measured at low magnification. Single-cell area was estimated by measuring the number of
cells within three or four different subregions of known area within
the same cotyledon. Four different cotyledons of each sample were
counted. The accuracy of measurement was confirmed by hand
counting all of the cells in three different cotyledons at 2 days after
germination (DAG).
DNA and RNA Analyses
Microscopy
Low-magnification light micrographs of plants were obtained using a
Leica MZ12.5 stereomicroscope (Leica Microsystems, Heerbrugg,
Switzerland) with a SPOT RT camera (Diagnostic Instruments, Sterling Heights, MI). Confocal images were collected using an MRC BioRad 1024 confocal microscope mounted on an inverted Optiphot-2
microscope and a 60 plan apo 1.4 numerical aperture objective (Nikon, Tokyo, Japan). Fluorescein isothiocyanate fluorescence was
detected with excitation at 488 nm and a 522 DF 32 bandpass filter.
Rhodamine fluorescence was detected with excitation at 568 nm and
a 585 longpass filter. In most cases, both channels were imaged simultaneously, and 14% of the fluorescein isothiocyanate channel
was subtracted from the rhodamine channel to remove bleedthrough signal. For scanning electron microscopy, samples were frozen by plunging them into liquid N2. They were transferred to the cryo
prechamber (GATAN, Inc., Pleasanton, CA) and sputter coated with
gold for 4 sec at approximately 160C. Samples were transferred to
the main cryo stage and imaged at 140C in a JEOL JSM-840
scanning electron microscope using 4- or 5-kV accelerating voltage.
Cytoskeleton Localization
The fixation and localization protocols used here closely followed the
procedures described by Szymanski et al. (1999). Briefly, cotyledons
were dissected and immersed rapidly in microtubule stabilization
buffer (100 mM Pipes-KOH, pH 6.9, 5 mM EGTA, 2 mM MgCl2, and
0.05% Triton X-100) supplemented with 2% formaldehyde and 0.5%
glutaraldehyde for 60 min. The cell walls of fixed tissue were cracked
using the freeze shattering procedure (Wasteneys et al., 1997). Sam-
DNA and RNA gel blot analyses were performed according to standard protocols (Ausubel et al., 1994) except for RNA gel blotting, for
which ULTRAhyb (Ambion, Austin, TX) was used for hybridization.
For DNA gel blotting, DNA was hybridized with digoxigenin-labeled
probe (DIG Prime DNA Labeling and Detection Starter Kit II; Roche).
RNA was isolated using GTC buffer (Newman et al., 1993). Poly(A)
RNA was purified with Oligotex (Qiagen, Valencia, CA). For reverse
transcriptase–mediated polymerase chain reaction (RT-PCR), 100 ng
of mRNA was reverse transcribed with 500 ng of random hexanucleotides (Random Primer6; New England Biolabs, Beverly, MA) and
SuperScript II RNase H Reverse Transcriptase (Gibco BRL). Synthesized cDNA was purified with the Qiagen PCR Purification Kit. The
PCR primers for amplifying SPK1 were RT1 (5-CGGTACCAGAAAGCTGAAGATAA-3; 5-CCCAAAGTTCCAGTTTTTGC-3) and
RT2 (5-CTCCAGACAAAGCTTGAAGCT-3; 5-TATCCACCATGGCTCTGAATG-3). PCR conditions were as follows: preincubation at
94C for 2.5 min, then 30 cycles at 94C for 30 sec, 52C for 30 sec,
and 72C for 1 min. The primers for ACT7 were 5-TGCTCTTCCTCATGCTATCCTTC-3 and 5-CCACGAACCAGATAAGACAAGAC-3.
For RT-PCR analysis of SPK1 expression in different organs, primers
RT2 were used. The primers used to amplify control glyceraldehyde3-phosphate dehydrogenase were 5-CACTTGAAGGGTGGTGCCAAG-3 and 5-CCTGTTGTCGCCAACGAAGTC-3 (Ohad et al.,
1999).
Rapid Amplification of cDNA Ends and Construction of the
Full-Length cDNA
Partial SPK1 cDNA clones was isolated from the Arabidopsis
ecotype Columbia cDNA library (Kieber et al., 1993). The 5 and 3
Cell Shape Control in Arabidopsis
ends of SPK1 cDNA were obtained by 5 and 3 rapid amplification of
cDNA ends (RACE) according to the manufacturer’s directions
(Gibco BRL) with some modifications. For 5 RACE, first-strand synthesis was primed using a gene-specific primer (GSP) and poly(A)
RNA as a template. PCR was performed first using the upstream anchor primer AAP and gene-specific RACE primer; subsequently,
nested amplification using AUAP and gene-specific nested primer
was conducted when necessary. Four rounds of 5 RACE were required to obtain the 5 end of SPK1. The first-round GSP is 5-GCTTCATCAGCCATTTCG-3, RACE primer 5-CCGTGCTTCCCACTCTCTTCT-3, and nested primer 5-GGTGATCCAGCTCCTTCACTAACA-3. The second-round GSP is 5-GCATGATACATCGGAAAACA-3, RACE primer 5-GTGCTTGCTCCAATGCCATTGAT-3,
and nested primer 5-GCAAGGCAGTAGAATCAACGTGCT-3. The
third-round GSP is 5-ATGTGGCAGGAAGAGAGA-3, RACE primer
5-CCCTAGCACCAACAGCGACCTGTGT-3, and nested primer 5TCGGATATCTGTGTCATCCTTTCTCA-3. The fourth-round GSP is
5-CCAGTGTTTGTGGTGAGAT-3, RACE primer 5-CTCTCAGTTAAGTGCACAGGCTCTT-3, and nested primer 5-AAGCAGGCAAACTGATGCTGATG-3. For 3 RACE, oligo(dT) primer (5-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT[ACG][ACGT]-3) was
used for first-strand synthesis. PCR was performed using AUAP and
gene-specific RACE primer 5-AGGAATTCCATACTCAGCTT-3.
Based on the 5 RACE sequences, we constructed the full-length
cDNA of SPK1 in two steps. First, for RT-PCR amplification of an 2.2kb fragment with primers 5-GCGTCGACCATCACATCATCTGTTATTCACT-3 and 5-CCGTGCTTCTCCACTCTCTTC-3, the fragment was
cloned to SalI and NdeI sites of a partial SPK1 cDNA clone in pBluescript SK. Second, for RT-PCR amplification of an 2.3-kb fragment
with primers 5-GCGTCGACTTACAGCTCCAGCTCGACTTTG-3 and
5-CCACCATGGCTCTGAATGCTGCAAC-3, the fragment was cloned
to SalI and NcoI sites of the recombinant partial SPK1 cDNA in the first
step. PCR was performed with Pfu DNA polymerase (Promega, Madison, WI), and the conditions were as follows: preincubation at 95C for
2 min, then 35 cycles of 95C for 30 sec, 58C for 30 sec, and 72C for
5-min. The PCR products were cloned into pCRII-TOPO (Invitrogen,
Carlsbad, CA) and sequenced before subcloning.
Reverse Genetic Screen
Pooled genomic DNA from 6500 T-DNA insertion lines in a
Wassilewskija background (Arabidopsis Biological Resource Center
[Columbus, OH] stock CS6502 [Feldmann, 1991]) were screened as
described by Krysan (1996). Pools of DNA were screened by PCR
using T-DNA left and right border-specific primers and primers specific for SPK1 every 2 kb in both directions. PCR was performed with
increasingly deconvoluted DNA template pools until individual plants
containing the insertion were identified. Cosegregation of the spk1
phenotype and the kanamycin resistance gene T-DNA marker was
tested by plating seed from selfed heterozygous plants. The location
of the T-DNA insertion was confirmed by amplifying genomic DNA by
PCR with SPK1 and left border primers from the heterozygous plant
and sequencing the PCR product.
Double-Stranded RNA Constructs and Agrobacterium
tumefaciens–Mediated Transformation
Two constructs, DS1 corresponding to 1089 bp and DS2 corresponding to 677 bp of the SPK1 cDNA, were made, along with con-
115
trol construct DSCK with sequence (419 bp) against the 27th intron
of the SPK1 gene. All of the fragments were amplified by PCR and inserted into XhoI and EcoRI sites in the sense direction and BamHI
and XbaI sites in the antisense direction of pRNA69; then they were
digested with NotI and inserted into the binary vector pJL10. For
DS1, sense direction primers 5-GCCTCGAGCGCCAAGAAGTCAGGCTAGA-3 and 5-GCGAATTCCTTGCTCCACACACCATCAT-3
and antisense direction primers 5-GCGGATCCCTTGCTCCACACACCATCAT-3 and 5-GCTCTAGACGCCAAGAAGTCAGGCTAGA-3
were used. For DS2, sense direction primers 5-GCCTCGAGGCAGCTGAAGTCGAGGGTTA-3 and 5-GCGAATTCCCTCAGTCTGCAGCACTGTT-3 and antisense direction primers 5-GCGGATCCCCTCAGTCTGCAGCACTGTT-3 and 5-GCTCTAGAGCAGCTGAAGTCGAGGGTTA-3 were used. For DSCK, sense direction primers 5-TTGCTGGTGTCAGTGACGGAAAA-3 and 5-GCGAATTCCGTGACAGGAAATGGTGCAAACA-3 and antisense direction primers
5-GCGGATCCCGTGACAGGAAATGGTGCAAACA-3 and 5-GCTCTAGATTCCTCCACGTCGACACCTATT-3 were used.
A. tumefaciens strain C58/pGV90 carrying double-stranded RNA
constructs in pJL10 was used to transform Arabidopsis ecotype Columbia by floral dip (Clough and Bent, 1998). Transformed Arabidopsis lines were selected on soil by spraying a 1:100 dilution of Finale
(AgrEvo Environmental Health, Montvale, NJ).
Sequence Analysis
Sequence alignments and presentation were made using ClustalW
(Higgins et al., 1996) (http://www.ch.embnet.org/software/ClustalW.
html) and BOXSHADE (http://www.ch.embnet.org/software/BOX_
form.html), respectively.
Accession Numbers
The GenBank accession numbers for the sequences mentioned in
this article are AF465831 (SPK1), A71430 (Arabidopsis ORF 4200c),
AAF51561 (Drosophila unknown MOD1, MOD2, CDMS-domain
gene), T34274 (C. elegans unknown MOD1, MOD2, CDMS-domain
gene), BAA09454 (Homo sapiens DOCK180), AAC38973 (C. elegans
CED-5), AAB69648 (Drosophila MBC), AAB70856 (Dictyostelium discoideum DocA), AAB67508 (Saccharomyces cerevisiae Ylr422wp).
ACKNOWLEDGMENTS
We thank Eileen Mallery for excellent technical assistance and the Purdue Cytoskeleton Group for useful discussions. The Arabidopsis
Biological Resource Center provided several strains and clones. Vector pRNA69 was a generous gift from John Bowman and John Emery.
We also thank Stan Gelvin and colleagues for help in identifying the
spk1-2 allele. This research was supported by Purdue University and
Cooperative Research, Education, and Extension Service United
States Department of Agriculture Grant No. 99-35304-8525 to D.B.S
and National Science Foundation Integrative Biology and Neuroscience Grant No. 9506192 to M.D.M. The original spk1-1 allele was
generated by Neil Olszewski, who is supported by National Science
Foundation Molecular and Cellular Biosciences Grant No. 9604126.
116
The Plant Cell
Received August 13, 2001; accepted October 15, 2001.
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The Arabidopsis SPIKE1 Gene Is Required for Normal Cell Shape Control and Tissue
Development
Jin-Long Qiu, Ross Jilk, M. David Marks and Daniel B. Szymanski
Plant Cell 2002;14;101-118
DOI 10.1105/tpc.010346
This information is current as of August 3, 2017
References
This article cites 67 articles, 25 of which can be accessed free at:
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