Download The Arabidopsis sku6-spiral1 gene encodes a plus end

The Plant Cell, Vol. 16, 1506–1520, June 2004, www.plantcell.org ª 2004 American Society of Plant Biologists
The Arabidopsis SKU6/SPIRAL1 Gene Encodes a Plus
End–Localized Microtubule-Interacting Protein Involved
in Directional Cell Expansion
W
John C. Sedbrook,a,1 David W. Ehrhardt,b Sarah E. Fisher,b Wolf-Rüdiger Scheible,c and Chris R. Somervilleb,d
a Department
of Biological Sciences, Illinois State University, Normal, Illinois 61790
Institution, Stanford, California 94305
c Max Planck Institute of Molecular Plant Physiology, 14476 Golm, Germany
d Department of Biological Sciences, Stanford University, Stanford, California 94305
b Carnegie
The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated
hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly right
skewed. The touch-dependent sku6 root skewing phenotype is suppressed by the antimicrotubule drugs propyzamide and
oryzalin, and right skewing is exacerbated by cold treatment. Cloning revealed that sku6-1 is allelic to spiral1-1 (spr1-1).
However, modifiers in the Columbia (Col) and Landsberg erecta (Ler) ecotype backgrounds mask noncomplementation in
sku6-1 (Col)/spr1-1 (Ler) F1 plants. The SPR1 gene encodes a plant-specific 12-kD protein that is ubiquitously expressed
and belongs to a six-member gene family in Arabidopsis. An SPR1:green fluorescent protein (GFP) fusion expressed in
transgenic seedlings localized to microtubules within the cortical array, preprophase band, phragmoplast, and mitotic
spindle. SPR1:GFP was concentrated at the growing ends of cortical microtubules and was dependent on polymer growth
state; the microtubule-related fluorescence dissipated upon polymer shortening. The protein has a repeated motif at both
ends, separated by a predicted rod-like domain, suggesting that it may act as an intermolecular linker. These observations
suggest that SPR1 is involved in microtubule polymerization dynamics and/or guidance, which in turn influences touchinduced directional cell expansion and axial twisting.
INTRODUCTION
The roots of plants must maneuver through a gauntlet of soil
particles, rocks, and debris to position themselves to take up
water and nutrients and to anchor the plant. Directional growth of
the root is in large part governed by touch- and gravity-induced
cell expansion processes that occur within the root tip. The root
tip is composed of a cap, a meristematic region, and adjacent
distal and main elongation zones. Gravity sensing occurs
predominantly in collumella cells within the cap (Boonsirichai
et al., 2002). The site of touch sensing is not clear, although it
likely occurs in more than one location within the tip, including
cells within the root cap (Evans, 2003; Massa and Gilroy, 2003).
Tip direction changes are the consequence of altered patterns
of polar cell expansion in the elongation zones. These cell
expansion patterns can be studied in Arabidopsis thaliana roots
growing down an inclined and impenetrable agar surface. Okada
and Shimura (1990) found that this environment promotes a regular sinusoidal pattern of growth, termed root waving.
1 To
whom correspondence should be addressed. E-mail jcsedbr@
ilstu.edu; fax 309-438-3722.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: John C. Sedbrook
([email protected]).
W
Online version contains Web-only data.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.020644.
Analysis of time-lapse videos of waving roots (J.C. Sedbrook
and C.R. Somerville, unpublished data) coupled with data from
published reports (Okada and Shimura, 1990; Simmons et al.,
1995; Rutherford and Masson, 1996; Sedbrook et al., 2002;
Massa and Gilroy, 2003) allows for the following simplified
description of root waving. A root tip responds to gravity with
a two-dimensional downward bend, resulting in the cap pushing
against the agar surface. Sensing this obstacle, a threedimensional cell expansion process is induced within the elongation zone, thereby changing the direction of root tip growth to
either the right or left of its original trajectory (viewed from above
the agar surface). This direction change is often associated with
an axial rotation of the tip, which can be seen as a twist in the
normally straight epidermal cell files within the elongation zone. A
root tip moving to the right takes on a brief right-handed twist,
and a leftward move is accompanied by a brief left-handed twist.
Massa and Gilroy (2003) propose that the root tip touch response
downregulates the graviperception machinery.
At the end of the touch-induced lateral direction change, the
cap again presses into the agar surface as the tip bends and
grows toward the gravity vector. The obstacle avoidance
response is again triggered, but this time the cell expansion
pattern has an opposite handedness compared with the previous response, thereby completing a sinusoidal wave. It has
been postulated that circumnutation drives the frequency of the
wave (Mullen et al., 1998; Migliaccio and Piconese, 2001), yet the
basis of the handedness switching is a mystery.
SPR1 Interacts with Microtubules
Little is known about the molecular mechanisms underlying
touch-induced directional cell expansion and organ axial twisting. Garbers et al. (1996) and Deruere et al. (1999) found that a
mutation in the rcn1 gene, which encodes a protein phosphatase 2A regulatory subunit involved in polar auxin transport,
resulted in root growth that slanted to the right of the normal
downward waving direction. rcn1, but not wild-type roots, formed
coils at the bottom of horizontally positioned agar plates containing the auxin transport inhibitor naphthylphthalamic acid.
aux1 mutant roots form coils instead of growing straight on
horizontal or tilted agar surfaces, revealing an enhanced directional growth bias (Mirza, 1987; Okada and Shimura, 1990).
AUX1 encodes an auxin influx carrier necessary for polar auxin
transport (Bennett et al., 1996).
Furutani et al. (2000) reported that spr1-1 roots slanted to the
right on inclined agar surfaces and that spr1-1 roots and etiolated
hypocotyls exhibited a right-handed axial twist. These root
phenotypes, along with an accompanying defect in anisotropic
cell growth, were suppressed by the antimicrotubule drug
propyzamide and the microtubule stabilizing drug taxol. Furutani
et al. (2000) also found that the cortical microtubule arrays of
elongating spr1-1 epidermal root cells were arranged in helices
with a left-handed pitch instead of with the normal transverse
orientation. Propyzamide created right-handed helical microtubule arrays in wild-type and spr1-1 elongating root cells, which
coincided with a change to leftward slanting and left-handed
twisting roots. These results prompted them to propose that
a microtubule-dependent process and SPR1 act antagonistically
to control directional cell elongation and axial twisting.
Thitamadee et al. (2002) provided a further connection
between microtubules and growth handedness with the isolation
of two suppressors of spr1 named lefty1 and lefty2. The lefty
mutants exhibited left slanting and left-handed twisting roots,
right-handed obliquely oriented cortical microtubule arrays in
elongating epidermal root cells, and increased sensitivities to
microtubule drugs. The dominant negative lefty mutations
correspond to single amino acid changes in the a-tubulin
proteins TUA4 and TUA6, with the altered residues mapping to
the tubulin dimer interface. It was postulated that the spr1 and
lefty mutations affected microtubule stability and/or dynamics
and that the resultant twisted growth was possibly because of
obliquely oriented microtubules guiding the oblique deposition of
cellulose microfibrils (Hashimoto, 2002; Thitamadee et al., 2002).
Hussey (2002) speculated that the lefty mutations might create
tubulin dimers that formed microtubules with an altered shape,
which was reflected in the orientation of the cortical arrays.
To learn more about directional cell expansion processes, we
used the root waving assay to identify mutants with altered
patterns of cell expansion and root growth. We identified
a mutant, sku6-1 that exhibits right skewing and right-handed
axial cell file twisting. The sku6-1 mutation, which is in the
Columbia (Col) ecotype, appeared to complement the spr1-1
mutation, which is in the Landsberg erecta (Ler) background,
because of two modifiers present in the different ecotypes.
However, cloning of the genes corresponding to sku6 and spr1
revealed that they were the same gene and we have accordingly
renamed sku6-1 as spr1-6 (there are five other spr1 alleles
besides sku6-1, hence the name spr1-6; Nakajima et al., 2004).
1507
Results presented here indicate that the SPR1 protein functions
as a microtubule interacting protein that is preferentially localized
to the plus ends of cortical microtubules. We hypothesize that
SPR1 controls certain aspects of cell expansion by regulating the
growth and/or guidance of microtubule plus ends.
RESULTS
Phenotypic Analyses of spr1-6
The spr1-6 mutant (ecotype Col) was isolated as a recessive
nuclear mutation in a T-DNA activation-tagging screen for root
waving mutants. On 1.5% agar-solidified germination medium
(GM) that was tilted 408 from the vertical, growth of spr1-6 roots
skewed in a rightward direction as viewed from above the agar
surface, whereas wild-type Col roots grew straight downward
(Figures 1A and 1B). Close examination of spr1-6 roots revealed
that the epidermal cell files twisted much more in a clockwise
direction (right-handed) compared with the wild type as the root
moved to the right during the wave motion (Figures 1C and 1D,
Table 1). spr1-6 root epidermal cell files also exhibited brief lefthanded twisting similar to the wild type as the root tip moved to
the left. During the leftward motions, a portion of the spr1-6 root
had a tendency to rise off of the agar as the tip pointed toward
and grew against the agar surface (Figure 1D).
spr1-6 roots also exhibited a right-handed twisting bias when
grown within or on vertically oriented 0.8% agar-solidified GM,
as did airborne spr1-6 etiolated hypocotyls and, at times, leaf
petioles (Figures 1E to 1J, Table 1). spr1-6 roots that penetrated
the tilted agar surface and grew within the medium, however, did
not skew but instead followed the gravity vector–like wild type
until encountering the agar/plate interface, after which they again
skewed to the right (Figure 1B).
The spr1-6 root axial rotation defect as well as the surfacedependent right slanting phenotype were much stronger when
seedlings were grown at 168C on either tilted 1.5% agarsolidified GM or vertical 0.8% agar-solidified GM, revealing the
temperature-sensitive nature of these phenotypes (Figures 1K to
1M, Table 1). In fact, spr1-6 roots had more of a tendency to loop
and coil under these conditions than when grown at 228C
(Figures 1L and 1M). Growth at 168C had little effect on wild-type
directional growth (Figure 1K, Table 1).
Effect of Antimicrotubule Drugs
Furutani et al. (2000) found that the right-skewing and righthanded twisting root growth phenotypes of spr1-1 seedlings
were suppressed by the antimicrotubule drug propyzamide.
Because spr1-6 did not complement the spr1-1 mutation (see
below), we tested the effects of antimicrotubule drugs on spr1-6
root growth, using a range of concentrations. Figures 2C and 2D
and Table 2 show that on tilted medium containing 3 mM
propyzamide, spr1-6 and wild-type roots both skewed to the
left. The dinitroanaline herbicide oryzalin affected root growth
similarly, with 0.1 mM exerting the strongest left-skewing effect
(Figures 2E and 2F, Table 2). Oryzalin concentrations higher than
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Figure 1. Growth Phenotypes of spr1-6 and Wild-Type Seedlings and Plants.
(A) and (B) Wild-type (A) and spr1-6 (B) seedlings grown 7 d on 1.5% agar-solidified GM tilted at 408 from the vertical (wave assay) and imaged from
above the agar surface. The arrow in (B) delineates a root that has grown into the agar.
(C) and (D) Enlarged view of wild-type (C) and spr1-6 (D) roots that had gone through the wave assay. In (D), note the prolonged right-handed (RH) cell
file rotation of the spr1-6 root (top left portion marked by a line) as well as a portion of the spr1-6 root that was off the agar surface (the portion of the root
out of the plane of focus and indicated by an arrow). Left-handed (LH) and right-handed (RH) cell file rotations are also indicated by lines in (C).
(E) and (F) Wild-type (E) and spr1-6 (F) roots grown 7 d in 0.8% agar-solidified GM. Note the larger amount of cell file rotation for the spr1-6 root.
(G) and (H) Confocal image projections of propidium iodide–stained wild-type (G) and spr1-6 (H) etiolated hypocotyls, showing the large amount of
spr1-6 cell file rotation.
(I) and (J) Wild-type (I) and spr1-6 (J) plants growing in the greenhouse. In (J), note the two spr1-6 leaves (indicated by arrows) that are not laying flat
because of right-handed twisted petioles; the top right leaf is sideways, whereas the bottom left leaf is upside down.
(K) and (L) Wild-type (K) and spr1-6 (L) seedlings put through the wave assay at 168C.
(M) Closer view of spr1-6 root put through the wave assay at 168C. Note the more prolonged right-handed cell file rotation than seen in (D), which was
grown at 228C.
Bars in (A), (B), (K), and (L) ¼ 10 mm; bars in (C) to (H) and (M) ¼ 0.1 mm; bars in (I) and (J) ¼ 1 cm.
SPR1 Interacts with Microtubules
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Table 1. Measurements of Wild-Type (Col) and spr1-6 Roots Grown under Various Conditions
Treatment
Tilted
1.5%
228C
Vertical
0.8%
228C
Tilted
1.5%
168C
Vertical
0.8%
168C
Wild-Type
Length
(mm)
spr1-6
Length
(mm)
Wild-Type
Anglea
spr1-6
Angle
2.28 6 6.0, 32.78 6 6.2,
n ¼ 68
n ¼ 42,
P ¼ 6.2 3 1049
21.9 6 3.5, 22.3 6 3.0, 0.28 6 6.6,
28.08 6 12.2,
agar
n ¼ 66
n ¼ 61,
n ¼ 64
n ¼ 59,
P ¼ 0.5
P ¼ 1.9 3 1030
ND
ND
3.08 6 6.0,
68.48 6 11.1,
agar
n ¼ 74
n ¼ 74,
P ¼ 4.0 3 1088
ND
ND
1.88 6 5.4,
62.18 6 11.0,
agar
n ¼ 50
n ¼ 47,
P ¼ 4.9 3 1055
ND
ND
Wild-Type
Cell File
Rotationb
spr1-6
Cell File
Rotation
Wild-Type
Cortical
Microtubule
Pitch Anglec
spr1-6
Cortical
Microtubule
Pitch Angle
ND
ND
ND
ND
agar
0.5 6 1.2, 10.4 6 5.6,
1.48 6 4.4, 0.98 6 3.8,
n ¼ 27
n ¼ 23,
n ¼ 137
n ¼ 130,
P ¼ 2.8 3 1013
P ¼ 0.46
ND
ND
ND
ND
0.5 6 2.1, 26.4 6 3.7,
1.38 6 5.0, 1.98 6 4.7,
n ¼ 32
n ¼ 30,
n ¼ 180
n ¼ 202,
P ¼ 2.1 3 1043
P ¼ 6.8 3 1010
a The
average angle that roots grew on the agar surface, where 08 is vertical, and positive numbers were to the right of vertical.
cell file rotation is the number of epidermal cell files that crossed a 1-mm-long line drawn down the longitudinal axis of the root, from ;1.5 to
2.5 mm from the tip. Positive values represent right-handed cell file rotation, whereas negative values represent left-handed cell file rotation.
c The cortical microtubule pitch angle is the average angle of cortical microtubules in root cells within the elongation zone (measurements taken 0.2 to
0.6 mm from the root tip quiescent center), where 08 would be transverse, negative angles right-handed oblique, and positive angles left-handed
oblique (n ¼ number of cells sampled in 28 to 39 roots for each category).
Numbers are averages 6 SD; n ¼ sample size; P ¼ Student’s t test probability (two tailed), where <0.05 is statistically significant. ND, not determined.
b The
0.1 mM inhibited root elongation and abolished slanting and
waving.
We also quantified root axial twisting by measuring the amount
of cell file rotation seen in spr1-6 and wild-type roots grown on
vertical 0.8% agar-containing GM with or without each of these
drugs (Table 2). We found that, on average, the right-handed
axial twisting bias usually seen in spr1-6 roots switched to a lefthanded twist in the presence of 3 mM propyzamide as well as
0.1 mM oryzalin. Wild-type Col roots, which usually do not have
a twisting bias, also acquired a left-handed twist on agar media
containing these drugs (Table 2).
Cloning of the SPR1-6 Gene
The spr1-6 mutant was isolated from a T-DNA activation-tagged
population. However, the mutation segregated as a single
recessive allele, suggesting that the phenotype was because of
loss of gene function rather than activation of a gene. The original
mutant line contained several T-DNA insertions, but after a cross
to the wild type, a spr1-6 line designated JS79 that contained
a single T-DNA insertion was identified. The genomic DNA
flanking the T-DNA in JS79 was recovered by thermal asymmetric interlaced PCR (Liu et al., 1995). The resulting 1.3-kb
fragment contained a region of the T-DNA left border inserted
within the open reading frame of putative gene At2g03680.
To confirm that the disruption of At2g03680 by the T-DNA
insertion was the cause of the spr1-6 mutant phenotype, we
isolated a 2.5-kb XhoI genomic fragment from BAC F19Bll that
contained the putative SPR1 gene along with 0.8-kb upstream
and 0.7-kb downstream sequences (Figure 3A). The XhoI
fragment, which did not carry any other intact annotated genes,
was cloned into the pBIN19 binary vector and transformed into
spr1-6 plants using Agrobacterium tumefaciens (Clough and
Bent, 1998). Analysis of T1 and T2 generation transformants
revealed that this fragment was able to complement all of the
spr1-6 phenotypic abnormalities (data not shown).
SPR1 Is a Novel Plant-Specific Gene Belonging to
a Multigene Family
The T-DNA element responsible for the spr1-6 mutation was
inserted at codon 75 within the second exon of a three-exon
gene (At2g03680) predicted to encode a novel 119–amino acid
polypeptide (12 kD; Figures 3A and 3B). BLAST searches
of GenBank identified no SPR1 homologs outside of plants
but identified five other SPR1-like genes within Arabidopsis (At1g26360, At1g69230, At3g02180, At4g23496, and
At5g15600). These genes were predicted to encode proteins
sharing from 44 to 56% amino acid sequence identity with SPR1
(Figure 3B). ESTs were identified for three of the five SPR1-like
genes (At3g02180, At4g23496, and At5g15600). ESTs for
At3g02180 and At4g23496 appeared to be full length, supporting
the genome project annotations of these genes.
Twenty ESTs corresponding to the SPR1 gene were
identified in GenBank. Many appeared to be full length or
nearly full length, supporting the annotation of the SPR1 open
reading frame (ORF) by the genome project. The SPR1 gene
sequence was also annotated as interacting with nitrilase
based on a yeast two-hybrid screen. However, the two-hybrid
interaction was not confirmed by other means and may have
been artifactual. The SPR1 and SPR1-like proteins have a low
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The Plant Cell
A SPR1:Green Fluorescent Protein Fusion Localizes
to Microtubules
To determine the subcellular localization of the SPR1 protein, we
generated transgenic spr1-6 seedlings expressing a SPR1:green
fluorescent protein (GFP) fusion consisting of the ENHANCED
GREEN FLUORESCENT PROTEIN (EGFP) open reading frame
(Cutler et al., 2000) fused to the C terminus of the predicted SPR1
open reading frame. To preserve normal regulatory sequences,
the construct pJS1 contained the EGFP gene in frame with the
genomic SPR1 gene, along with SPR1 upstream and downstream genomic sequences. This construct complemented the
spr1-6 mutant phenotypes in 100 of 107 transgenic plants (data
not shown).
Table 2. Measurements of Wild-Type and spr1-6 Seedling Roots
Grown on Either 1.5% Agar-Solidified GM or 0.8% Agar-Solidified GM
with or without an Antimicrotubule Drug
Figure 2. Effects of Antimicrotubule Drugs on Root Skewing.
Sample
No Drug
Propyzamide
3 mM
Oryzalin
0.1 mM
Seedlings were grown 4 d on 1.5% agar-solidified GM tilted 408 from the
vertical and then transferred onto the same media either without ([A] and
[B]) or with 3 mM propyzamide ([C] and [D]) or 0.1 mM oryzalin ([E] and
[F]). (A), (C), and (E) are the wild type; (B), (D), and (F) are spr1-6. The bar
in (B) ¼ 10 mm, which is the same scale for all panels.
Wild type,
1.5%,
anglea
spr1-6,
1.5%,
angle
Wild type,
0.8%,
angle
spr1-6,
0.8%,
angle
Wild type,
0.8%,
lengthb
spr1-6,
0.8%,
length
Wild type,
0.8%,
CFRc
spr1-6,
0.8%,
CFR
2.2 6 6.0
28.8 6 10.9
5.3 6 6.8
complexity composition within the middle portions of the
polypeptides, with some proteins containing continuous
stretches of Thr residues. The middle portion of SPR1 is rich
in Pro and Thr residues. A highly conserved direct repeat
sequence is present at the N-terminal and C-terminal ends of
these proteins, the consensus motif being GGGSSLG/DYLFG
(Figure 3B). Database searches did not report this motif as
being present in other proteins. Comparison of the SPR1
sequence to GenBank using the MAXHOM alignment program
(Sander and Schneider, 1991) revealed significant sequence
similarity between SPR1 and a portion of the human Filamin A
protein (FLNa; Figure 3C; 28% amino acid identity and 45%
conservation over a continuous 87–amino acid stretch, smallest
sum probability of 1.1 3 106). FLNa is a 280-kD actin crosslinking and scaffolding protein consisting of an N-terminal actin
binding domain and 24 repeat sequences, each repeat being
;96 amino acids long (Tigges et al., 2003). SPR1 shares
homology with the repeats 12 and 13.
To determine where the SPR1 gene is expressed, RNA gel
blots were performed on total RNA from various wild-type tissues
(Figure 4). A 0.6-kb transcript was identified in all wild-type
tissues tested, including seedling roots and shoots as well as
adult leaves, inflorescence stems, and flowers. No transcripts
were identified in RNA from the spr1-6 mutant, confirming that
the probe was specific to SPR1 transcripts and that the spr1-6
mutation was a null.
a The
32.7 6 6.2,
34.1 6 12.1,
P ¼ 6.2 3 1049 P ¼ 0.059
0.2 6 6.6
2.4 6 11.2
10.3 6 8.4,
P ¼ 7.6 3 104
7.0 6 8.3
28.0 6 12.2,
4.4 6 6.8,
1.1 6 7.3,
P ¼ 1.9 3 1030 P ¼ 2.0 3 103 P ¼ 7.4 3 105
21.9 6 3.5
16.3 6 2.4
16.3 6 2.4
22.3 6 3.0,
P ¼ 0.502
15.8 6 1.5,
P ¼ 0.283
18.6 6 2.7,
P ¼ 1.0 3 103
0.5 6 1.2
5.7 6 7.3
7.1 6 4.4
10.4 6 5.6,
9.5 6 8.5,
P ¼ 2.8 3 1013 P ¼ 0.074
5.9 6 4.6,
P ¼ 0.273
average angle that roots grew on the agar surface, where 08 is
vertical and positive numbers were to the right of vertical.
b Average root length in millimeters.
c Cell file rotation is the number of epidermal cell files that crossed a
1-mm-long line drawn down the longitudinal axis of the root, from ;1.5
to 2.5 mm from the tip. Positive values signify right-handed cell file
rotation, and negative values signify left-handed cell file rotation.
Numbers are averages 6 SD; n ¼ sample size; P ¼ Student’s t test
probability (two tailed), where <0.05 is statistically significant. The P
values correspond to a comparison between wild-type and spr1-6
samples of a given treatment. For angle and length measurements, 34 <
n < 69; for cell file rotation measurements, 22 < n < 31. ND, not
determined.
SPR1 Interacts with Microtubules
1511
Figure 3. The SPR1 Gene Structure and Amino Acid Sequence Alignments.
(A) Diagram of the SPR1 gene. Solid rectangles are the SPR1 exons, and thick connecting lines are introns. Translational start and stop are marked by
arrows. Thin broken line at the left represents 0.8 kb of sequences between the XhoI site and transcriptional start, whereas the thin broken line at the
right represents 0.7 kb of genomic DNA from transcriptional stop to the other XhoI site. The relative location of the T-DNA is shown (not drawn to scale).
(B) Amino acid sequence alignment of SPR1 and the SPR1-like predicted proteins. The two solid lines under the sequences mark the locations of
a direct repeat sequence. Solid boxes represent amino acids that match the consensus.
(C) Amino acid sequence alignment between SPR1 (bottom) and a portion of the human Filamin A protein (FLNa; top). Identical residues are represented
by letters between the two sequences, whereas conserved residues are delineated by plus signs (28% identity, 45% conserved, smallest sum
probability of 1.1 3 106). Alignment was performed by WU-BLAST 2.0 (Altschul et al., 1990) using FLNa residues 1420 to 1516 as the query.
Confocal microscopic analysis of these seedlings identified
GFP fluorescence emanating from the cytoplasm as well as from
filamentous structures around the cortex of root and shoot cells
(Figures 5A to 5C). The filamentous patterns were consistent with
cortical microtubule localization. For instance, within the root tip,
the filamentous arrays were disorganized within meristematic
cells and transversely oriented within elongation zone cells
(Figure 5A). Moreover, these arrays at times became oblique then
longitudinally oriented within root mature zone cells (data not
shown).
Because microtubules also form distinctive arrays at others
stages of the cell cycle, we examined whether SPR1:GFP
localized to preprophase bands, phragmoplasts, and mitotic
spindles in dividing cells. As was the case in the interphase cell
cortex, SPR1:GFP-related fluorescence was visible in patterns
consistent with microtubule localization within these arrays
(Figures 5D to 5F).
SPR1:GFP Is Concentrated at Microtubule Plus Ends of
Cortical Microtubules
Although the SPR1:GFP fusion protein exhibited fluorescence
patterns consistent with microtubule localization, in most
interphase cells the distribution of fluorescence was not
uniform along the cortical microtubules but instead appeared
to be more densely accumulated in specific regions (Figures 5B
and 5C). To investigate this localization further, we performed
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The Plant Cell
Figure 4. RNA Gel Blot Analysis of SPR1 Expression.
Total RNA gel blot probed with radiolabled SPR1 DNA (top panel, 0.6-kb
band) or stained with methylene blue to visualize rRNA (loading control).
The first lane is total RNA from spr1-6 seedlings, and the other lanes
contain total RNA from various wild-type tissues. Note that the rRNA
staining shows the spr1-6 seedlings, and wild-type flowers/buds
samples were loaded heavier than the others.
time-lapse imaging of SPR1:GFP using confocal microscopy.
We observed that SPR1:GFP fluorescence commonly moved in
comet-like streaks across the cell cortex (Figures 6A and 6B;
see Supplemental Movies S1 and S2 online). The fluorescence
in each streak was typically asymmetrically distributed, being of
highest intensity at the end leading the direction of travel and
diminishing exponentially to form a trailing tail (Figure 6C).
These patterns of localization and movement were consistent
with the label showing polarized accumulation at the growing
ends of microtubules. By contrast, transgenic seedlings
expressing a yellow fluorescent protein b-TUBULIN fusion
(YFP:TUBULIN) exhibited uniform fluorescence across cortical
microtubules in interphase cells (see Supplemental Movie S3
online).
We asked if the SPR1:GFP comets shared other characteristics displayed by the dynamic ends of cortical microtubules. As
predicted, SPR1:GFP microtubule localization appeared de novo
at the cell cortex, with label moving away from the site of origin,
just as observed with TUBULIN:GFP-labeled microtubules (Shaw
et al., 2003; Figure 7A and see Supplemental Movie S4 online).
The plus ends of cortical microtubules undergo frequent shifts
among growth, pause, and shrinking states, a pattern termed
dynamic instability (Mitchison and Kirschner, 1984). SPR1:GFP
comets moved relatively smoothly in one direction but often
disappeared abruptly. Upon closer examination, these episodes
of disappearance appeared to be associated with a transition to
polymer shrinkage and at times could be clearly imaged because
of less dissipation of label along the sidewalls (Figure 7B and see
Supplemental Movie S5 online).
Comets of SPR1:GFP were also observed to join and travel
along common tracks, suggesting the formation and association
of individual microtubules into bundles of polymers, as also
observed with TUBULIN:GFP (Shaw et al., 2003; see Supple-
mental Movie S6 online). SPR1:GFP patches occasionally
appeared to detach from the cortex and were subjected to
buffeting by the underlying streaming cytosol (see Supplemental
Movie S7 online). This behavior would be difficult to explain if the
patches of label were not at the ends of the polymers. Finally,
previous observations revealed that the lagging ends of cortical
microtubules show slow and persistent depolymerization (Shaw
et al., 2003). We did not observe movement of the fluorescent
comets consistent with lagging end depolymerization (60
observations in nine cells and four plants). Taken together, these
observations strongly suggest that SPR1:GFP is concentrated in
gradients at the plus ends of growing cortical microtubules and
that this tip accumulation is typically sensitive to the growing
state of the microtubule.
Not all observed microtubules labeled with SPR1:GFP
displayed pronounced accumulation at the growing end. For
instance, interphase cells were sometimes observed in which
most microtubules showed a more even distribution of label
(Figures 5A and 7B). Such cells tended to be in tissue that had
been wounded or mounted for extended periods of time (data
not shown). There also was no clearly observed bias in the
distribution of label within the phragmoplast, mitotic spindle, and
preprophase band arrays (Figures 5D to 5F). In time-lapse
images of these arrays (see Supplemental Movies S8 to S10
online), short dashes of label could occasionally be seen in one to
three consecutive image frames, but this limited information
made it difficult to determine if these were end-labeled polymers
or fragmented polymers.
Modifiers of spr1 Mutations in Different Ecotypes
The spr1-6 mutation (Col ecotype background) was originally
called sku6-1. Because the sku6-1 mutant exhibited similar
phenotypes to the spr1-1 mutant (Ler ecotype background;
Furutani et al., 2000), we performed a complementation analysis.
When assayed for root skewing, the roots of F1 sku6-1/spr1-1
seedlings exhibited a definite left slanting waving pattern like that
seen with wild-type Ler roots (Figure 8). Furthermore, F2
seedlings displayed approximately a 9:7 ratio of left slanting to
right slanting roots, consistent with the independent assortment
of two loci (70 left:66 right; null hypothesis of 9:7 ratio, x2 ¼ 1.39,
P > 0.20; null hypothesis of 3:1 ratio, x2 ¼ 40.16, P < 0.0005).
Although these data suggested that sku6-1 and spr1-1 were
not allelic and were segregating independently, the cloning of the
corresponding mutations confirmed that they both disrupted the
same gene. The spr1-1 allele was the result of a deletion that
removed the beginning portion of the SPR1 gene, and the spr1-1
mutant phenotype was rescued by a genomic fragment carrying
the SPR1 gene (Nakajima et al., 2004). Moreover, Nakajima et al.
(2004) isolated five spr1 alleles, all of which contained mutations
within the SPR1 gene. We used primers flanking the spr1-1
deletion as well as primers specific to the spr1-6 T-DNA to
confirm that the spr1-6 by spr1-1 cross was a success (data not
shown).
Given that both spr1-1 and spr1-6 seedlings contained no
SPR1 transcripts (Nakajima et al., 2004; Figure 4), it is likely that
both are null alleles. Therefore, to explain the complementation
data, we hypothesize that two independent modifiers exist, one
SPR1 Interacts with Microtubules
1513
Figure 5. GFP Fluorescence from a SPR1:GFP Fusion Protein Imaged by Confocal Microscopy.
(A) Root epidermal cells within the meristem/distal elongation zone.
(B) Root epidermal cells within the main elongation zone.
(C) Hypocotyl epidermal cells.
(D) Phragmoplast localization within a dividing root epidermal cell (indicated by an arrow).
(E) Spindle localization within a dividing root cortical cell (indicated by an arrow).
(F) Pre-prophase bands in two cap cells of the root tip (indicated by arrows).
(B), (C), and (D) are brightest point projections of multiple confocal sections (21 slices and 0.5-mm steps for [B], 36 slices and 0.4-mm steps for [C], and
43 slices and 0.7-mm steps for [D]), and images [A], [E], and [F] are individual sections. Bars ¼ 20 mm.
with a dominant allele in the Col background (i.e., genotype A/A
b/b) and the other with a dominant allele in the Ler background
(i.e., genotype a/a B/B). These modifiers produce left slanting
roots when dihybrid (A/a B/b) in a spr1-6/spr1-1 null background. However, when either or both of these modifiers is
homozygous recessive (A/- b/b, a/a B/-, or a/a b/b) in a spr1-6/
spr1-1 null background, the resultant phenotype is rightskewing roots.
According to this hypothesis, one would expect that onesixteenth (6.25%) of the spr1-6 by spr1-1 F2 plants would be
double homozygous dominant for the modifiers (A/A B/B) and
would produce homozygous left slanting F3 families of plants.
Indeed, we found that five out of 89 F2 plants (5.6%) produced
F3 families that were homozygous left slanting (x2 ¼ 0.06, P >
0.8). This data does not fit the 1:3 ratio one would expect
associated with a single modifier (x2 ¼ 30.77, P < 0.0005).
Moreover, 45 of the 89 F3 families proved to be heterozygous
and, as expected, were segregating either 9:7 left:right
skewing (F2 parental genotype of A/a B/b) or 3:1 left:right
skewing (F2 parental genotype of either A/A B/b or A/a B/B).
The remaining 39 of 89 F2 plants (43.82%) that had rightskewing roots all gave rise to F3 families that were homozygous right skewing, which is very close to the expected 7/16th
ratio (43.75%).
1514
The Plant Cell
instead of left-handed oblique throughout the entire distal and
main elongation zones, even though the spr1-6 roots had been
twisting as they grew (Table 1, Figures 9A to 9C). This average
microtubule pitch angle was statistically indistinguishable from
the wild type (Table 1). When spr1-6 seedlings were grown at
168C, which causes a more severe spr1-6 right-handed root axial
twisting phenotype, the microtubule pitch angle in elongation
zone cells was, on average, slightly left-handed oblique and
statistically different from that of the wild type (Table 1, Figure
9D). However, many of the spr1-6 roots (16 out of 39) had
a majority of elongation zone cells with transverse and/or righthanded obliquely oriented arrays. Moreover, even though wildtype and spr1-6 average microtubule pitch angles were
comparably small, there was a big difference in the relative
average amounts of wild-type and spr1-6 root cell file rotation
(Table 1).
To determine whether, under our conditions, antimicrotubule
drugs induced right-handed obliquely oriented microtubule
arrays, as Furutani et al. (2000) and Thitamadee et al. (2002)
reported for propyzamide, we examined cortical microtubules in
spr1-6 and wild-type roots that were growing vertically on 0.8%
agar-solidified GM containing either 3 mM propyzamide or
0.1 mM oryzalin. Both drug treatments often created righthanded obliquely oriented microtubule arrays in spr1-6 and wildtype elongating root cells (Figures 9E and 9F; data not shown).
DISCUSSION
Figure 6. Tip Gradient of SPR1:GFP.
(A) and (B) Kymographs of SPR1:GFP gradients at the growing ends of
interphase cortical microtubules. A transition to polymer shortening
(catastrophe) and subsequent rapid loss of SPR1:GFP label is indicated
by ‘‘cat’’ in (A).
(C) Mean intensity of SPR1:GFP in a 7-pixel-wide lane calculated at each
axial position along the end of the labeled microtubule. The image of the
labeled polymer end is depicted below the graph, with lines indicating
the mapping of the image to the plotted values. The arrow indicates the
direction of growth of the labeled end. The curve is an exponential
regression to the tailing values only [y ¼ 49 e^ ð0:025 xÞ; R2 ¼ 0:92].
Cortical Microtubules in spr1-6 Root Elongation Zone
Cells Remain Transverse Even Though Root Axial
Rotation Occurs
Previously, Furutani et al. (2000) reported that cortical microtubules in spr1-1 root elongation zone cells were oriented in lefthanded oblique arrays instead of being transversely oriented like
in the wild type. They hypothesized that the orientations of
cortical arrays govern the direction of cell expansion; thus,
oblique arrays caused the spr1-1 root axial twisting phenotype.
To determine if spr1-6 elongating root cells also had left-handed
obliquely oriented microtubule arrays, we grew spr1-6 and wildtype seedlings on vertical 0.8% agar-solidified GM for 5 d at
228C, fixed the tissue, and stained for tubulin using whole-mount
immunofluorescence. Under our conditions, the cortical microtubule arrays within spr1-6 elongating root cells were mostly
transverse and on average were slightly right-handed oblique
The spr1-6 null mutation results in roots that respond to surfaceinduced touch stimulation on vertically oriented agar media by
growing to the right of the gravity vector (viewed from above the
agar surface). spr1-6 organs also exhibit predominantly righthanded twisting about their longitudinal growth axes because
of an underlying directional cell expansion bias. These observations implicate SPR1 as being an important regulator of directional cell expansion.
A wild-type Col root usually forms waves straight down an
inclined hard-agar surface because of the root tip making
rightward direction changes that equal leftward direction
changes (Figures 1A to 1C). Moreover, when a Col root changes
direction to the right or left, the elongation zone briefly displays
a right-handed or left-handed twist, respectively, each being of
equal duration (Figure 1C). An spr1-6 root, on the other hand,
exhibits more of a rightward change in root tip direction
coinciding with a prolonged right-handed twist (Figures 1B and
1D). It also makes the periodic leftward lateral movement and
brief left-handed twist to complete the wave, but the leftward
motion is accompanied and followed by the tip bending and
growing toward the gravity vector and against the agar surface
more than normal. This appears to coincide with less differential
cell expansion on the upper and lower flanks of the elongation
zone, resulting in a portion of the root growing off of the agar
surface (Figure 1D). Therefore, the spr1-6 defect alters the overall
cell expansion pattern in such a way as to enhance rightward
lateral direction changes and inhibit leftward movements.
Part of the spr1-6 bias in waving direction may be caused by
the prolonged right-handed axial twisting of the root tip. Axial
twisting likely enhances lateral direction changes when the root
SPR1 Interacts with Microtubules
1515
Figure 7. Dynamic Behavior of SPR1:GFP.
(A) Time-lapse confocal images showing a SPR1:GFP-labeled structure (green arrowheads) appearing in a location at the cell cortex that previously
contained no detectable organized label (yellow arrowheads), suggesting a de novo origin at the cortex for the labeled structures. The numbers indicate
time in seconds before and after the appearance of the structure.
(B) Time-lapse confocal images showing a single SPR1:GFP-labeled structure exhibiting episodes of dynamic growth (green arrowheads) and
shortening behavior (red arrowheads) similar to microtubule catastrophe and rescue. The numbers indicate time in seconds. Scale bars ¼ 5 mm.
cap and meristem are not aligned with the elongation zone. This
is the case most of the time on a tilted agar surface because of
the root tip bending toward the gravity vector. On the other hand,
spr1-6 roots grow relatively straight when embedded within an
agar medium because the tip is straight as the root twists.
Whereas root axial twisting may amplify directional movement
of a bent root tip, differential cell expansion on the right and left
flanks likely also contributes to root waving and skewing. Buer
et al. (2003) found that wild-type Ler roots waved and skewed in
the absence of twisting on zero-nutrient agar medium. We also
observed both wild-type and spr1-6 roots at times waving
without twisting on our nutrient agar medium. In fact, the spr1-6
root tip in Figure 1D can be seen bent to the right as it begins to
form a wave even though axial twisting has not yet begun.
Therefore, touch-induced differential flank cell expansion may
also be affected in spr1-6 plants.
We observed that the anti-microtubule drug propyzamide
made spr1-6 and Col roots skew to the left on inclined agar
surfaces, consistent with what was seen by Furutani et al. (2000)
with spr1-1 and Ler. Additionally, we found that like propyza-
mide, oryzalin also suppressed spr1-6 right skewing (Figures 2C
to 2F). Given that propyzamide and oryzalin are chemically
distinct drugs that are thought to target microtubules differently
(Anthony and Hussey, 1999; Young and Lewandowski, 2000), it
appears that these drugs affect root skewing by altering
microtubule growth and/or organization without specifically
binding to the site of SPR1 action.
Somewhat surprisingly, the microtubule stabilizing drug taxol
also causes left skewing of wild-type and spr1-1 roots (Furutani
et al., 2000; Hashimoto, 2002), whereas propyzamide and
oryzalin make right skewing wvd2 roots skew even more strongly
to the right (Yuen et al., 2003). Therefore, the cause of root
skewing is more complicated than a simple stabilization or
destabilization of microtubules.
The SPR1 gene encodes a small plant-specific protein and
belongs to a six-member gene family. A short sequence near the
SPR1 N terminus (GGGQSSLDYLF) is repeated at its C terminus
(GGGSSLDYLF). This direct repeat, which flanks a region of low
complexity, is highly conserved within the SPR1 gene family
(Figure 3B). The PROF secondary structure prediction program
1516
The Plant Cell
Figure 8. Seedlings Grown on Tilted 1.5% Agar-Solidified GM.
Top row, F1 seedlings originating from a spr1-6 by spr1-1 cross; bottom
row left of line, spr1-1 seedlings; bottom row right of line, spr1-6
seedlings. Note F1 seedlings wave to the left like wild-type Ler (data not
shown) and not to the right like spr1-1 and spr1-6. spr1-1 is in the Ler
background. Scale bar ¼ 10 mm.
(Rost and Sander, 1993; http://www.embl-heidelberg.de/
predictprotein/submit_def.html) predicts that 85% of SPR1
amino acid residues are solvent exposed, suggesting the protein
forms a rod-like structure. Consistent with this prediction is the
observation that an 87–amino acid stretch of SPR1 shares 28%
identity and 45% conservation with a rod-forming repeat
sequence present in the human FLNa protein (Figure 3C). The
FLNa protein reorganizes the actin cytoskeleton by interacting
with integrins, transmembrane receptor complexes, and second
messengers (Tigges et al., 2003). Thus, the SPR1 protein may
have a rod-like structure with nearly identical structural features
at each end, suggestive of a function as a linker protein.
Our analyses of transgenic plants expressing a SPR1:GFP
fusion protein indicate that SPR1 interacts with microtubules.
First, SPR1:GFP localized to filamentous structures resembling
microtubules in the four major microtubule structures: the
interphase cortical array, the preprophase band, the phragmoplast, and the mitotic spindle. Second, the patterns of these
filaments changed in the same way that microtubule patterns
change as cells go through various developmental stages. Third,
time-lapse imaging of SPR1:GFP revealed a variety of dynamic
behaviors also observed in cortical microtubules: filamentous
growth and shrinkage consistent with polymer dynamic instability
(Dhonukshe and Gadella, 2003; Shaw et al., 2003), de novo
origination of filaments at the cell cortex (Shaw et al., 2003), and
occasional episodes of rapid and erratic movement consistent
with polymer detachment from the cell cortex (Shaw et al., 2003).
We found SPR1:GFP to be concentrated at the growing and
presumed plus ends of cortical microtubules, having the
appearance of passing comets in time-lapse movies (Figures
6A to 6C and see Supplemental Movies S1 and S2 online). This
SPR1:GFP plus end labeling was noticeably different from the
uniform labeling observed with a YFP:TUBULIN marker in
interphase cells (see Supplemental Movie S3 online). The plus
ends of microtubules are the primary sites of growth and
shortening, the cycling of which is termed dynamic instability
Figure 9. Confocal Microscopic Projection Images of Anti-TUBULIN
Whole-Mount Immunolocalization in Roots.
(A) A montage of images depicting a spr1-6 root. Note the cell file
rotation.
(B) Closer view of cells in (A) within spr1-6 root main elongation zone
(cells’ location marked by the arrow in [A]).
(C) to (F) Close-up views of main elongation zone cells in a wild-type root
(C), a spr1-6 root grown at 168C (D), and spr1-6 roots grown on 3 mM
propyzamide (E) or 0.1 mM oryzalin (F).
All roots were grown at 228C (except [D]) on vertical 0.8% agar-solidified
GM with or without the drug before fixation. Note the transverse
microtubule arrays in (C), the left-handed oblique arrays in (D), and the
right-handed oblique arrays in (B), (E), and (F). Scale bar in (A) ¼ 0.1 mm;
scale bars in (B) to (F) ¼ 10 mm.
SPR1 Interacts with Microtubules
(Desai and Mitchison, 1997). There is growing evidence that
a large protein complex resides at microtubule plus ends and
regulates dynamic instability (Schroer, 2001; Carvalho et al.,
2003; Howard and Hyman, 2003). For instance, the microtubule
plus end binding EB1 protein increases microtubule rescues and
decreases catastrophes (shortening) and pausing, resulting in an
overall increase in polymerization (Tirnauer et al., 2002). EB1
binds directly to a subunit of dynactin, which appears to have
a potent microtubule nucleation effect (Ligon et al., 2003). A
dominant negative form of CLIP-170, which is a plus end binding
microtubule–associated protein that acts as a linker between
membranes and microtubules (Howard and Hyman, 2003), did
not affect microtubule growth or shortening but, instead,
significantly reduced the rescue frequency (Komarova et al.,
2002). Removal of CLIP-170 in Schizosaccharomyces pombe
caused an increased rate of catastrophes (Brunner and Nurse,
2000).
The labeling pattern of SPR1:GFP on interphase cortical
microtubules is similar to the localization patterns of EB1 and
CLIP-170, although EB1 shows pronounced plus end gradients
in mitotically arrested cell extracts and uniform labeling of
microtubule walls in interphase extracts (Tirnauer et al., 2002).
Moreover, Chan et al. (2003) recently showed that a GFP fusion
to the Arabidopsis EB1 protein may also be concentrated at the
minus ends of microtubules, which we do not observe with
SPR1:GFP. Like EB1 and CLIP-170, SPR1:GFP plus end labeling
typically disappears upon microtubule shortening, suggesting
that SPR1 is important for microtubule polymerization or the
inhibition of depolymerization.
EB1 and CLIP-170 have been shown to bind directly to
microtubules (Pierre et al., 1992; Berrueta et al., 1998; Hayashi
and Ikura, 2003). By contrast, we were unable to pull down
detectable quantities of GST:SPR1 fusion protein in vitro with
polymerized tubulin (see Supplemental Figure 1 online). Therefore, the SPR1 protein may associate with microtubules indirectly by binding to another protein or a protein complex that
binds tubulin.
It is unclear if SPR1:GFP forms microtubule plus end gradients
in the spindle, phragmoplast, and preprophase band (see
Supplemental Movies S8 to S10 online). If most microtubules in
the spindle extend from the poles and overlap at their plus ends,
one would expect a pronounced bias in label distribution in the
center of the spindle. Because this is not observed, either
SPR1:GFP labels these polymers more uniformly than it does
cortical microtubules or there are enough plus ends distributed at
other locations within the array to obscure a centralized bias in
label distribution. A similar argument could be made about
SPR1:GFP localization in the phragmoplast. The high concentration of microtubules in the preprophase band makes it difficult
to determine localization of SPR1:GFP along individual polymers.
SPR1 function appears not to be essential for the mitotic
spindle and phragmoplast arrays, based on normally oriented
cell divisions and normal root elongation rates in spr1-6 plants
(data not shown; Tables 1 and 2). Its dispensability in these
processes may be because of redundant functions of one or
more of the SPR1 family members. Consistent with this idea, the
SPR1-like gene At3g02180 appears to be ubiquitously ex-
1517
pressed at levels comparable to SPR1, based on microarray
analysis of various tissues (see Supplemental Table 1 online).
Microtubule arrays are commonly transversely oriented within
elongating epidermal root cells before at times becoming
obliquely or longitudinally oriented upon cessation of expansion.
Several mutations in Arabidopsis that cause root skewing and
axial twisting exhibit obliquely oriented cortical microtubule
arrays within elongating root cells. (Furutani et al., 2000;
Thitamadee et al., 2002; Yuen et al., 2003). Left-handed oblique
microtubule arrays were correlated with right-handed axial
twisting, and right-handed oblique arrays were found with lefthanded root twisting mutants. The treatment of seedlings with
the microtubule drugs propyzamide, oryzalin, and taxol also
resulted in a correlation between microtubule orientation and
helical growth, with right-handed oblique arrays occurring with
left-handed twisting (Furutani et al., 2000; Figures 9E and 9F,
Table 2). Furutani et al. (2000) postulated that the orientation
of cortical microtubules within elongating cells directed cell
elongation, perhaps by directing cellulose deposition. However,
it is not clear that cortical microtubules direct the deposition of
cellulose microfibrils (Baskin, 2001; Himmelspach et al., 2003;
Sugimoto et al., 2003).
Our data shines another light on directional cell expansion by
showing an uncoupling of microtubule pitch and root axial
twisting within elongating root cells. We found that many spr1-6
roots twisted even though the cortical microtubules were
transversely oriented in cells throughout the entire lengths of
their elongation zones (Table 1, Figures 9A and 9B). Moreover,
we found no correlation between the average amounts of cell
file rotation and the average microtubule pitch angles in wildtype and spr1-6 roots (Table 1). Indeed, wild-type roots twisted
very little even though the average cortical microtubule pitch
angles in their cells were of a similar degree as those in spr1-6
elongating cells. This result was somewhat surprising because
Furutani et al. (2000) observed obvious left-handed oblique
microtubule arrays in right-handed twisting spr1-1 cells. We
attribute the discrepancy between our results and theirs to
growth condition and/or ecotype differences. For instance,
under our root waving conditions, spr1-1 roots barely skewed
compared with what they found. Additionally, we identified two
modifiers affecting root skewing in the Col and Ler backgrounds
(Figure 8). These or other modifiers could potentially affect
microtubule pitch.
Taken together, our results strongly suggest that the pitch of
cortical microtubules in epidermal cells does not guide epidermal
cell file twisting, although it may influence the handedness of the
twist. Instead, the degree of epidermal cell file twisting may be
driven in large part by a difference in the overall elongation
duration and/or rate of epidermal cells compared with that of
ground tissue cells. For instance, if these cell types elongate
equally, there will be no cell file rotation. However, if ground
tissue cells elongate less than epidermal cells, there will be
twisting because of a torsional strain placed on the root. Given
this model, the spr1-6 mutation likely affects the balance
between ground tissue and epidermal cell elongation rates
or duration and therefore may affect the microtubule arrays
differently in these cells. We should note that Furutani et al. (2000)
proposed a similar model of differential cell expansion for spr1-1,
1518
The Plant Cell
although they emphasized a role for the pitch of microtubule
helical arrays in driving directional growth.
Our working hypothesis is that SPR1 affects microtubule
dynamic instability by promoting polymerization and/or discouraging pausing and catastrophe events. Based on the predicted
SPR1 protein structure, SPR1 could be acting to stabilize
microtubule polymers by cross-linking proteins in the plus end
complex. Alternatively, SPR1 may recruit growth machinery to
the plus end complex, stabilize or guide microtubules by linking
them to the cell cortex, or aid in the bundling of polymers. It is not
clear how changes in microtubule dynamic instability may affect
directional cell expansion. One possibility is that stabilizing
microtubules allows them to reside in a given location for longer
periods of time, which in turn prolongs related cell expansion
and/or cell wall deposition processes.
METHODS
Growth and Analysis of Plants
An activation tagging collection of 62,000 lines in the Col-0 ecotype was
generated using the SKI15 vector as described by Detlef Weigel (Weigel
et al., 2000) and has been deposited in the ABRC as accession number
CS31100. The root waving assay and quantification of organ growth were
performed as described by Rutherford and Masson (1996) and Sedbrook
et al. (2002). Propyzamide and oryzalin were obtained from Chem Service
(West Chester, PA).
Cloning and Analysis of the SPR1 Gene
Molecular and bacterial clone manipulations were performed using
standard procedures (Ausubel et al., 1994). DNA fragments flanking the
T-DNA were cloned as described by Siebert et al. (1995) and Liu et al.
(1995). RNA gel blots were probed with the SPR1 open reading frame
amplified from the EST clone 139B17 as described by Sedbrook et al.
(1999).
Generation of Transgenic Plants Carrying a SPR1:GFP
Reporter Construct
A 2.5-kb XhoI fragment from the BAC clone F19B11 (obtained from the
ABRC) carrying the SPR1 gene was cloned into the pBluescript KSþ
vector using standard procedures. The SPR1:GFP construct pJS1 was
made as follows. A fragment containing SPR1 coding sequences and 0.8kb upstream sequences was PCR amplified using the XhoI pBluescript
KSþ clone as a template and primer T7 along with a primer located at the
end of the SPR1 ORF (59-GGGGGATCCCGGGGCTAGCTTGCCACCAGTGAAGAGATAATC-39). This PCR product was digested with EcoRI
and BamHI and cloned into pZero-2.1 (Invitrogen, Carlsbad, CA; clone A).
The T3 primer along with a SPR1-specific primer located at the end of
the SPR1 ORF (59-CCCTGGATCCAAGTAAAATAATTGCAAAGACCT-39)
were used to amplify 0.7-kb downstream sequences from the XhoI
pBluescript clone. This PCR product was digested with BamHI and
Asp718 and cloned into pZero-2.1 (clone B). The BamHI/Asp718
fragment B was then introduced into clone A to make clone AB. An
NheI/BamHI EGFP fragment from the pEGAD vector (Cutler et al., 2000)
was next introduced into clone AB to make clone ABC, and the ABC
fragment was cut out of pZero-2.1 with EcoRV and Asp718, then cloned
into the SmaI and Asp718 sites of the binary vector pBIB (Becker, 1990) to
make pJS1. Clone pJS1 was introduced into spr1-6 plants using
Agrobacterium tumefaciens (Clough and Bent, 1998).
Imaging of Cortical Microtubules
Four-day-old wild-type and spr1-6 seedlings that had been growing on
a vertical 0.8% agar-solidified GM in Petri dishes within a Percival
CU36L5 incubator (228C, 16 h light; Percival, Perry, IA) were transferred to
the same medium with or without a particular antimicrotubule drug and
allowed to grow vertically for an additional 3 d before being fixed and
subjected to the whole-mount immunofluorescence protocol described
by Sugimoto et al. (2000). Cold treatment was performed by growing
seedlings on vertical 0.8% agar-solidified GM for 8 d within a Percival
E54B incubator (168C, 24 h light) before fixation. A mouse anti-a-tubulin
monoclonal antibody B-5-1-2 (Sigma T5168, 1:1000 dilution; St. Louis,
MO) and a rabbit anti-mouse IgG-fluorescein isothiocyanate secondary
antibody (Sigma F9137, 1:200 dilution) were used as probes. Confocal
images of root tips were acquired with a Zeiss LSM 510 confocal
microscope attached to a Zeiss Axiovert 100M inverted microscope
(Jena, Germany) equipped with 403 and 633 oil immersion objectives.
Image SXM imaging and analysis software was specially adapted by
Steve Barrett (http://www.ImageSXM.org.uk/) to handle the LSM 510
images on a Macintosh G4 computer.
Confocal imaging of living plants was performed essentially as
described by Sedbrook et al. (2002) and by Shaw et al. (2003). Image
analysis and quantitation was performed in NIH Image (Wayne Rasband)
and in customized software developed in the Matlab computing environment (Shaw et al., 2003). Movies of image time series were created in
Adobe After Affects software (Mountain View, CA).
Sequence data for this article has been deposited with the EMBL/
GenBank data libraries under accession number AY464947 (SPR1).
ACKNOWLEDGMENTS
We thank Takashi Hashimoto for kindly providing the spr1-1 allele for
complementation analysis and for sharing details of the spr1-1 mutation,
Detlef Weigel for providing the activation tagging vector SKI15, Steve
Barrett for kindly modifying the Image SXM code, and the ABRC for
providing wild-type seeds as well as genomic and EST clones. We also
thank the Department of Cell and Structural Biology at the University of
Illinois for the use of their confocal microscope. This work was
supported by grants from the National Institutes of Health (1 R15
GM068489) to J.C.S., the U.S. Department of Energy (DE-FG0203ER20133) to C.R.S., and the Carnegie Institution of Washington to
D.W.E.
Received January 2, 2004; accepted March 11, 2004.
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