Download The Glycerophosphoryl Diester

Plant Cell Physiol. 49(10): 1522–1535 (2008)
doi:10.1093/pcp/pcn120, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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The Glycerophosphoryl Diester Phosphodiesterase-Like Proteins SHV3
and its Homologs Play Important Roles in Cell Wall Organization
Shimpei Hayashi 1, 2, Tadashi Ishii 3, Toshiro Matsunaga 4, Rumi Tominaga 2, Takashi Kuromori 2,
Takuji Wada 2, Kazuo Shinozaki 2 and Takashi Hirayama 1, 5, *
1
International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama, 230-0045 Japan
RIKEN Plant Science Center, 1-7-22 Tsurumi, Yokohama, 230-0045 Japan
3
Forestry and Forest Products Research Institute, 1 Matunosato, Tsukuba, Ibaraki, 305-8687 Japan
4
National Agricultural Research Center, National Agriculture and Food Research Organization, 3-1-1 Kannondai, Tsukuba, Ibaraki,
305-8666 Japan
5
RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan
2
Despite the importance of extracellular events in cell
wall organization and biogenesis, the mechanisms and related
factors are largely unknown. We isolated an allele of the
shaven3 (shv3) mutant of Arabidopsis thaliana, which
exhibits ruptured root hair cells during tip growth. SHV3
encodes a novel protein with two tandemly repeated
glycerophosphoryl diester phosphodiesterase-like domains
and a glycosylphosphatidylinositol anchor, and several of
its paralogs are found in Arabidopsis. Here, we report the
detailed characterization of mutants of SHV3 and one of
its paralogs, SVL1. The shv3 and svl1 double mutant
exhibited additional defects, including swollen guard cells,
aberrant expansion of the hypocotyl epidermis and ectopic
lignin deposits, suggesting decreased rigidity of the cell wall.
Fourier-transform infrared spectroscopy and measurement
of the cell wall components indicated an altered cellulose
content and pectin modification with cross-linking in the
double mutant. Furthermore, we found that the ruptured
root hair phenotype of shv3 was suppressed by increasing the
amount of borate, which is supposed to be involved in
pectic polysaccharide cross-linking, in the medium. These
findings indicate that SHV3 and its paralogs are
novel important factors involved in primary cell wall
organization.
Keywords: Arabidopsis thaliana — Cell wall — Cellulose —
GPI-anchored protein — Pectin — Root hair.
Abbreviations: AIR, alcohol-insoluble residue; AVG,
1-aminoethoxyvinyl glycine; CaMV, cauliflower mosaic
virus; FTIR, Fourier-transform infrared; GFP, green
fluorescent protein; GPD, glycerophosphoryl diester phosphodiesterase; GPDL, GPD-like; GPI, glycosylphosphatidylinositol; GUS, b-glucuronidase; PC, principal
component; RG-II, rhamnogalacturonan-II; RT–PCR,
reverse transcription–PCR; SHV3, SHAVEN3; SVL,
SHV3-like.
Introduction
Plant shape and size are determined primarily by cell
division and expansion. Cell expansion involves controlled
cell wall organization. Primary walls require both mechanical stability and extensibility to allow for cell expansion
without cell rupture due to excessive turgor pressure. Cell
walls must be able selectively to loosen, integrate newly
synthesized cell wall components, and form a new cell wall
matrix quickly and correctly during cell expansion
(Cosgrove 2005). Therefore, cell expansion requires the
organized disruption and formation of linkages among cell
wall components. Because of the complex structure of cell
walls, understanding the regulatory mechanisms of cell wall
organization during cell expansion and shape change is a
major challenge in plant science.
Primary cell walls, which are deposited during cell
expansion, are composed mainly of cellulose microfibrils,
hemicelluloses, pectins and proteins. The cellulose microfibrils, which are crystallized fibers of b-1,4-glucose chains,
are synthesized by the cellulose synthase complex at the
plasma membrane. The other components, including the
hemicelluloses and pectins, are synthesized in the Golgi
apparatus then secreted into the cell wall matrix (Scheible
and Pauly 2004, Cosgrove 2005). Cell walls have a complex
structure comprising several different types of linkages
between their components. Hemicelluloses, such as xyloglucan, form hydrogen bonds with the surfaces of cellulose
microfibrils and other polysaccharides. One of the pectic
components, rhamnogalacturonan II, forms borate diester
cross-links between apiose residues (O’Neill et al. 2004).
Another pectic component, homogalacturonan, forms
calcium cross-links between carboxyl groups in a methyl
esterification-dependent manner (Ridley et al. 2001).
Understanding cell wall organization requires identifying not only the components but also the factors that
modulate the components and comprise the network
*Corresponding author: E-mail, [email protected]; Fax, þ81-45-508-7363.
1522
SHV3 and SVLs in cell wall organization
structure of the cell wall. Several enzymes that modify the
linkages between the components of the cell wall have been
identified. Transglycosylation between free and cellulosebound xyloglucans catalyzed by xyloglucan endotransglycosylase is thought to be involved in cell wall loosening
(Vissenberg et al. 2003). Pectin methylesterases, which
remove the methyl groups of pectins in the cell wall, control
the number of calcium cross-links among the pectins. The
vgd1 mutant, an Arabidopsis line defective in pectin
methylesterase, exhibits slow-growing pollen tubes leading
to reduced male fertility in vivo and unstable pollen tubes
that are susceptible to bursting in vitro (Jiang et al. 2005).
Expansins are thought to disrupt the non-covalent interactions between cell wall polymers in acid-induced cell
growth, although their molecular functions have not yet
been elucidated (Li et al. 2003). In addition, recent studies
have revealed that a large number of proteins predicted to
be located in the cell wall have important roles in cell wall
organization. Several glycosylphosphatidylinositol (GPI)anchored proteins that attach to the outer cell surface have
been functionally characterized in cell wall organization.
COBRA is required for the deposition of cellulose and the
regulation of anisotropic expansion, and cobra mutants
have swollen roots because of abnormal radial cell
expansion (Schindelman et al. 2001, Roudier et al. 2005).
SKU5 is structurally similar to multiple copper oxidases,
and its defective mutant exhibits an altered root waving
pattern (Sedbrook et al. 2002). A mutant of SOS5, which
encodes a protein with fasciclin-like and arabinogalactan
protein-like domains, exhibits arrested root growth
and root swelling under salt stress conditions (Shi et al.
2003). It has been postulated that these factors are involved
in the construction of cell wall networks.
Root hair cells exhibit tip growth and a fast rate of
expansion. A sophisticated regulatory mechanism is
believed to maintain the organization of the cell wall to
prevent the tip from rupturing during root hair elongation.
Ruptured root hairs have been observed only in the kojak
(kjk) (Favery et al. 2001), lrx1lrx2 (Baumberger et al. 2003)
and mrh4 (Jones et al. 2006) mutants, which are thought to
have abnormal cell wall organization. To learn more about
the mechanism of cell wall organization, we isolated shaven3
(shv3) from a screen of Ds insertional mutants for lines with
abnormally shaped root hairs. SHV3 encodes a GPIanchored protein similar to glycerophosphoryl diester
phosphodiesterase (GPD) and is required for root hair
elongation (Parker et al. 2000, Jones et al. 2006).
In this study, we found that a combination of shv3 and
a disruptant mutation in a paralog conferred additional
unique phenotypes to the morphology of the epidermal cells
and a number of phenotypes common to cell wall-related
mutants. Furthermore, an analysis of the cell wall
components in the double mutant revealed an altered
1523
crystalline cellulose content with pectin modifications and
changes in the amount of cross-linking. These findings
suggest that SHV3 and its paralogs encode novel proteins
with important roles in cell wall organization.
Results
Identification of the SHV3 locus by Ds transposon tagging
We isolated a recessive mutant defective in root hair
formation from a screen of RIKEN Arabidopsis Ds
transposon insertion lines (Kuromori et al. 2004,
Kuromori et al. 2006). In the mutant line 15-1096-1, tip
growth in nearly all root hair cells was blocked due to a
rupture at the tip (Fig. 1A). However, bulges were observed
at the appropriate positions in the trichoblasts, indicating
normal root hair development. No other visible phenotypes
were observed in this mutant. The transposon element
was found to be inserted into At4g26690, which encodes a
GPD-like protein (Fig. 1C) that is predicted to have a
glycosylphosphatidylinositol (GPI) anchor sensitive to
phosphatidylinositol-specific phospholipase C (Borner
et al. 2003). This gene was recently identified as the gene
responsible for the root hair-defective mutant, shv3/mrh5
(Parker et al. 2000, Jones et al. 2006). Jones et al. (2006)
found that the expression of At4g26690 was correlated with
the root hair morphogenesis, and T-DNA insertion mutations of this gene caused a root hair-defective phenotype. All
of the F1 plants generated by crossing 15-1096-1 with shv3
had the same defective root hair phenotype as the parents,
indicating that this Ds mutation is allelic to shv3 (Fig. 1B).
We examined the nucleotide sequence of this gene in the shv3
mutant and identified a C-to-T transversion mutation
causing an amino acid substitution Thr(364) to Ile,
consistent with the effect of ethylmethanesulfonate, which
was used for mutagenesis (data not shown) (Parker et al.
2000). We obtained a T-DNA insertion mutant
(SALK_024208) of this gene and confirmed that it conferred
the same phenotype. In the previous study, SALK_024208
was named mrh5-3 (Jones et al. 2006). To avoid confusion, in
this study, we refer to the mutant isolated by Parker et al.
(2000) as shv3-1, the T-DNA insertion line as shv3-2, and the
Ds insertion line 15-1096-1 as shv3-3; shv3-2 was used in the
following experiments. In shv3-2 and shv3-3, transcript
fragments were detected by reverse transcription–PCR
(RT–PCR) (Supplementary Fig. S1). We assumed that the
shv3-2 and shv3-3 products were not functional, however,
because the predicted polypeptides lack the N-terminal
signal sequence or the C-terminal region required for correct
protein localization (see below).
Localization of SHV3
The predicted SHV3 protein contains a putative signal
sequence at the N-terminus and a putative GPI anchor
1524
SHV3 and SVLs in cell wall organization
A
B
Ds15-1096-1
x shv3-1 F1
Ds15-1096-1
x WT F1
C
shv3-3
Ds15-1096-1
shv 3-1
C>T
shv3-2
SALK024208
TGA
2280
ATG
1
GPD-like
GPD-like
AGP
GPD-like
AGP
759aa
signal
sequence
GPD-like
GPI-anchore
D
Fig. 1 Identification of the shv3 mutant allele and SHV3
localization. (A) Phenotype of Ds15-01096-1. The primary root
(left and middle) and root hair (right) of 4-day-old Ds5 (control) and
Ds15-1096-1 seedlings. The root hair is ruptured and tip growth is
blocked in Ds15-1096-1. (B) Allelism between Ds15-1096-1 and
shv3 (Parker et al. 2000). The F1 progeny of a test cross between
Ds15-1096-1 and shv3 exhibited the parental phenotype.
(C) Structure of the SHV3 gene and its translation product.
addition site (omega site) near the C-terminus, indicating its
localization at the plasma membrane (Borner et al. 2002).
To confirm this assumption, we generated transgenic plants
expressing a chimeric green fluorescence protein (GFP)–
SHV3 protein with GFP attached to the C-terminal end of
the SHV3 signal sequence. The root hair phenotype of shv3
was completely restored by transformation with this
construct (data not shown), indicating that the fusion
protein was fully functional. GFP fluorescence was
observed at the plasma membrane (Fig. 1D, upper
panels), suggesting its function at the cell surface. In
addition to the plasma membrane, GFP fluorescence was
often observed in some intracellular structures (Fig. 1D,
lower panels and Supplementary Fig. S2). This intracellular
fluorescence may be derived from the GPI-anchored SHV3
protein on the membrane trafficking pathway. In the root
hair cell, the fluorescence was not polarized at the tip where
SHV3 is supposed to function and was observed in a wide
area of cell surface and intracellular structures. The
fluorescence intensity varied among the cells but was low
in most cases, despite the fact that expression of the
transgene was driven by the cauliflower mosaic virus
(CaMV) 35S promoter. Exceptionally strong and stable
fluorescence was observed in the trichomes; however, little
fluorescence was detected at the basal surface of the
trichomes, indicating the polarized localization of SHV3
(Fig. 1D and Supplementary Fig. S2).
Glycerophosphoryldiester phosphodiesterase-like genes
SHV3 has two tandemly repeated GPD-like domains,
though the biochemical function of these domains is
unknown (Fig. 2A). Using the MIPS database (http://
mips.gsf.de/), we identified six genes from Arabidopsis that
are closely related to SHV3 [GPDL1 (glycerophosphodiesterase-like) to GPDL6]. Other genes encoding GPD-like
proteins exist in the Arabidopsis genome, however. Thus,
to avoid confusion, in this study we refer to GPDL1
(At5g55480), 3 (At1g66970), 4 (At3g20520), 5 (At5g58050)
and 6 (At5g58170) as SVL (SHV3-like) 1, 2, 3, 4 and 5,
respectively (Supplementary Table S1). A BLAST search
identified putative orthologs of SHV3 only in plants (e.g.
rice). A transcript encoding a polypeptide with high
similarity to SHV3 was also found in Physcomitrella
patens (Physcobase, http://moss.nibb.ac.jp). In addition to
SHV3 (At4g26690) encodes a protein containing tandemly
repeated glycerophosphoryl diester phosphodiesterase (GPD)-like
domains and an arabinogalactan protein (AGP)-like domain. The
mature protein is presumably modified with a glycosylphosphatidylinositol (GPI) anchor. (D) Confocal imaging of cells expressing
the GFP–SHV3 fusion protein. GFP fluorescence (green) and chlorophyll autofluorescence (red) in the hypocotyl (upper and lower
left), and a root hair (lower right) of a transgenic plant expressing
GFP::SHV3 under control of the CaMV 35S promoter is shown.
An arrowhead indicates intracellular structures. Bar ¼ 20 mm.
SHV3 and SVLs in cell wall organization
A SHV3 & SVLs
GPD-like N
1525
GPD-like C
At1g74210
C
E. coli UgpQ
At5g08030
E. coli GlpQ
E. coli GlpQ
B
shv3-1, T>I
SVL5-C
SVL4-C
SVL2-C
At1g74210
At5g08030
SHV3-C
SVL1-C
SVL3-N
SVL3-C
SVL1-N
SVL5-N
SVL4-N SVL2-N SHV3-N
0.1
Fig. 2 Comparison of the primary sequences of SHV3, SVLs and GPDs. (A) Schematic representation of SHV3, SVLs, two Arabidopsis
candidate GPDs (At1g74210 and At5g08030) and an E. coli GPD (GlpQ). The gray portions indicate the most conserved parts of the GPDlike domains (i.e. the residues that were used for alignment in B). (B) Alignment of the residues from the most conserved parts of the GPDlike domains and GPDs. Those residues thought to interact with the substrate or calcium ions are indicated by open and filled triangles,
respectively. The shv3-1 transversion was observed in the C-terminal GPD-like domain. Clustal X (Thompson et al. 1997) was used to
create the alignment. Amino acids identical in all sequences are highlighted in black, while those identical in at least eight sequences are
highlighted in gray. (C) Phylogenic tree of the GPD-like domains and GPDs. The phylogenic distances were calculated by comparing the
primary sequences of the GPD-like domains and GDPs using Clustal X. The phylogenic tree was drawn using TreeView (Page 1996). The
bar indicates 0.1 substitutions per site.
SHV3 and SVL genes, a gene (At1g66980) encoding a
protein with a similar domain organization and an
additional Ser/Thr kinase domain was found in
Arabidopsis. Because this gene has 480% identity to the
flanking gene (SVL2), and as no other similar gene was
found in any other plant species, it may be an Arabidopsisspecific gene produced by chromosomal rearrangement. We
examined a disruptant mutant of At1g66980 (Ds 12-1469-1),
but failed to detect any visible phenotype (data not shown).
GPD catalyzes the hydrolysis of glycerophosphoryl
diester to a glycerol 3-phosphate and alcohol. Carrot GPD
activity was detected in cell wall fractions (Van Der Rest
et al. 2004). In Arabidopsis, At1g74210 and At5g08030 are
thought to be equivalent to the carrot GPD, based on their
peptide sequences. To compare the GPD-like domains of
SHV3 and SVLs, their primary sequences were aligned with
those of the putative Arabidopsis GPDs and a known GPD
in Escherichia coli, GlpQ (Larson et al. 1983, Tommassen
et al. 1991) (Fig. 2). The crystal structure of E. coli GlpQ
and glycerol complex (PDB: 1ydy) revealed a number of
residues that are probably essential for interactions with the
substrate and calcium ions. These residues are conserved in
At1g74210 and At5g08030, but not in SHV3 or SVLs,
suggesting that the biochemical function of the GPD-like
domains in SHV3 and SVLs is distinct from that in typical
GPD (Fig. 2). A comparison of the N- and C-terminal
GPD-like domains indicated that these two domains have
distinct sequences, suggesting that they have different
biochemical functions. The recessive phenotype caused by
the shv3-1 missense mutation in the C-terminal GPD-like
domain indicates that this domain is necessary for SHV3
function. We attempted to determine whether recombinant
SHV3 produced in E. coli has GPD activity using
glycerophosphocholine as a substrate, but we were unable
to detect any such activity (Supplementary Fig. S6).
Overlapping and distinct distributions of the expression of
SHV3 and SVLs in tissues
To determine the tissue-specific expression pattern of
SHV3 and SVL genes, RT–PCR analysis was performed
using total RNA isolated from the aerial parts or roots of
7-d-old seedlings, and dissected tissues from 6-week-old
plants (Fig. 3). In 7-d-old seedlings, only SHV3, SVL1 and
SVL2 transcripts were detected. SHV3 and SVL1 mRNA
was detected in both the aerial parts and roots, whereas
SVL2 mRNA was detected only in the aerial parts. SHV3,
SVL1 and SVL2 mRNA was detected in various 6-week-old
vegetative tissues. SVL3 mRNA was detected in the siliques
1526
SHV3 and SVLs in cell wall organization
7-day-old
6-week-old
Aerial Root
Rosette Cauline
Root Leaf
Leaf Stem Flower Silique
SHV3
SVL1
SVL2
SVL3
A number of intriguing novel phenotypes were observed
in shv3-2svl1-1, but not in shv3-2svl2-1 or svl1-1svl2-1.
Additionally, we generated an shv3-2svl1-1svl2-1 triple
mutant, but its phenotype did not differ from those of the
double mutants. We also tried unsuccessfully to generate an
svl4svl5 double mutant presumably due to small genetic
distance between them.
SVL4
SVL5
rRNA
Fig. 3 RT–PCR analysis of the expression SHV3 and SVL genes.
The expression of SHV3 and SVL genes in 7-d-old seedlings and
6-week-old plants was analyzed by semi-quantitative RT–PCR
using gene-specific primers. 18S rRNA was used as an internal
control.
and, to a lesser extent, flowers. SVL4 and SVL5 mRNA
was detected predominantly in the flowers, consistent with
data showing that SVL4 and SVL5 are expressed specifically in pollen (Lalanne et al. 2004).
To examine the detailed expression patterns of SHV3,
SVL1 and SVL2, we performed a histochemical analysis of
transgenic plants harboring the b-glucuronidase (GUS) gene
fused to the putative promoter regions of each of these genes
(Fig. 4). In the PSHV3::GUS transgenic plants, GUS activity
was strongest in the root hair cells (Fig. 4A, J, K). The
appearance of GUS activity in the root hair cells appeared to
coincide with the initiation of root hair formation (Fig. 4J),
consistent with previous data (Jones et al. 2006). GUS
activity was also detected in the petioles, hypocotyls (Fig. 4L)
and young leaves (Fig. 4D, M). PSVL1::GUS activity was
strongest in the vascular tissues (Fig. 4B, E, P, Q, T) and root
meristems (Fig. 4B, N, O). Additionally, GUS activity was
observed in the hypocotyl epidermis (Fig. 4P), young leaf
guard cells (Fig. 4S) and trichomes (Fig. 4R). PSVL2::GUS
activity was strongest in the leaves (Fig. 4C, F, V). GUS
activity was also detected in the hypocotyls of dark-grown
PSHV3::GUS and PSVL1::GUS transgenic plants (Fig. 4G,
H). GUS activity in PSVL2::GUS was detected in light-grown
hypocotyls (Fig. 4U), but not in dark-grown hypocotyls
(Fig. 4I). These results demonstrate that the expression of
SHV3 and SVL genes is strictly regulated and that they have
overlapping and distinct tissue expression patterns.
Characterization of the SVL disruptant mutants
To investigate whether the disruption of SVL genes
would produce an shv3-like phenotype, we screened SVL
T-DNA insertion lines (Fig. 5A). Presumably because of
functional redundancy among these gene products, no
visible phenotype was observed. Therefore, we generated
mutants of SHV3, SVL1 and SVL2 because our earlier data
indicated that these genes are expressed in various tissues.
The shv3-2svl1-1 double mutant exhibits altered cell wall
organization and cell expansion
The shv3-2svl1-1 double mutant seedlings exhibited
increased anthocyanin accumulation (data not shown) and
frequently collapsed trichomes (Supplementary Fig. S3). In
addition, some brown discoloration was observed around
the endodermis in the hypocotyl and root (Fig. 5B). This
phenotype was initially observed in the lower part of the
hypocotyl then gradually spread to the upper hypocotyl and
root (data not shown). Histochemical analysis using
phloroglucinol-HCl revealed ectopic lignification at the
discolored sites in the double mutant, but not in any of the
monogenic mutants (Fig. 5B). Ectopic lignification was
previously observed in several mutants showing altered cell
wall organization and cell expansion regulation with
shortened dark-grown hypocotyls, including eli1/cev1
(Cano-Delgado et al. 2000, Ellis et al. 2002), kor1 (Nicol
et al. 1998), elp1/ctl1 (Zhong et al. 2002) and kob1/abi8
(Pagant et al. 2002, Brocard-Gifford et al. 2004). Therefore,
we investigated whether the shv3-2svl1-1 double mutant had
a similar phenotype. Four-day-old dark-grown shv3-2svl1-1
seedlings displayed shorter and thicker hypocotyls than the
wild type (Fig. 5C, D), which is consistent with the
hypocotyl expression of SHV3 and SVL1 as indicated by
GUS staining in the promoter–GUS transgenic lines
(Fig. 4P, Q).
The eli1/cev1 mutant, which is defective in a cellulose
synthase subunit (CESA3), overproduces ethylene and
jasmonic acid, and accumulates transcripts of their responsive genes. Similar phenotypes have been reported in the
elp1/ctl1 and kob1/abi8 mutants, and it has been proposed
that ethylene and jasmonic acid signaling is activated by
alterations in cell wall organization. To determine whether
such signaling is activated in shv3-2svl1-1, the transcript
levels of PDF1.2 and VSP1 were examined by RT–PCR
using total RNA extracted from 7-d-old plants. PDF1.2
expression is induced by jasmonic acid and ethylene
(Penninckx et al. 1998), whereas VSP1 is induced by
jasmonic acid but not ethylene (Rojo et al. 1999). Strong
mRNA expression of PDF1.2 and VSP1 was detected in the
double mutant, whereas that in the wild type was relatively
weak (Fig. 5E). The shortened dark-grown hypocotyls of
eli1/cev1 and elp1/ctl1 are restored by etr1, a strong
ethylene-insensitive mutation, and partially by inhibitors
of ethylene production [e.g. 1-aminoethoxyvinyl glycine
SHV3 and SVLs in cell wall organization
A
B
C
J
K
L
O
P
Q
T
U
1527
D
E
F
G
H
I
N
M
R
S
V
Fig. 4 Promoter activity of SHV3, SVL1 and SVL2. Histochemical analysis of transgenic plants harboring the GUS gene under control of
the putative SHV3 (A, D, G, J–M), SVL1 (B, E, H, N–T) and SVL2 (C, F, I, U, V) promoters. (A–C) Seven-day-old seedlings. (D–F) Aerial parts
of 3-week-old seedlings. (G–I) Four-day-old dark-grown seedlings. (J) Root of a 7-d-old seedling. (K) Root cross-section. Arrowheads
indicate the root hair cells. (L) Upper portion of the hypocotyl from an 8-d-old seedling. Arrowheads indicate the petioles. (M) Leaf at an
early developmental stage (indicated by a broken line). (N) Root tip. (O) Lateral root primordium (arrowhead). (P) Lower hypocotyl and
root. (Q) Root cross-section. (R) Leaves at an early developmental stage. (S) Guard cells. (T) Leaf cross-section. Arrowheads indicate the
vascular tissues. (U) Hypocotyl of a 3-d-old seedling. (V) Leaf cross-section.
(AVG)] or binding (Agþ), respectively. Unlike the elp1/ctl1
mutants, AVG did not restore the shortened dark-grown
hypocotyls of shv3-2svl1-1 (Supplementary Fig. S4), which
suggests that the shv3-2svl1-1 mutation directly affects cell
wall organization and cell expansion.
The shv3-2svl1-1 double mutant has abnormally shaped
epidermal cells
Besides the phenotypes observed in other cell wall
mutants, a number of unique phenotypes were observed
in the shv3-2svl1-1 double mutant. Interestingly, the guard
cells in shv3-2svl1-1 were larger than those in the wild type
(Fig. 6A, B), although the stomatal response to ABA
and light was normal (data not shown). When fixed with
ethanol and cleared with chloral hydrate, the ventral sides
of the guard cells in shv3-2svl1-1 were obviously swollen
(Fig. 6C). Another visible phenotype in shv3-2svl1-1 was
aberrant swelling of the hypocotyl epidermis (Fig. 6D).
Electron microscopic observations revealed that the peak
of the swollen site had a rough surface, suggesting
1528
SHV3 and SVLs in cell wall organization
B
shv3
WT
SHV3
SVL1
rRNA
rRNA
WT
B
A
shv3svl1
shv3svl1
svl1
Area of stomata (µm2)
WT
A
WT
shv3svl1
+ SVL1
600
500
WT
shv3svl1
400
300
200
100
0
shv3svl1
C
WT
shv3svl1
Phloroglucinol stain
D
WT
shv3svl1
Hypocotyl length (mm)
C
WT
shv3svl1
16
12
8
D
4
0
WT
E
shv3svl1
VSP1
PDF1.2
5 mm
5 mm
rRNA
Fig. 5 shv3-2svl1-1 shows features of altered cell wall and cell
expansion mutants. (A) RT–PCR analysis of SHV3 and SVL1
expression in each disruptant mutant. Total RNA was extracted
from 7-d-old seedlings. Each cDNA fragment was amplified with
specific primers. 18S rRNA was used as an internal control.
(B) Photographs of the root–shoot junction in wild-type, shv3-2svl11, phloroglucinol-stained shv3-2svl1-1 and phloroglucinol-stained
transgenic shv3-2svl1-1 plants with a wild-type SVL1 transgene.
Lignified cell walls were stained red by phloroglucinol-HCl in 3-dold shv3-2svl1-1 light-grown seedlings. (C) Dark-grown hypocotyl
of 4-d-old wild-type (WT) and shv3-2svl1-1 seedlings.
(D) Hypocotyl length of dark-grown 4-d-old WT and shv3-2svl1-1
seedlings. The means SD from 20 samples are shown.
(E) Accumulation of transcripts of the jasmonic acid- and
ethylene-responsive genes VSP1 and PDF1.2 in shv3-2svl1-1
seedlings. Total RNA was extracted from 7-d-old light-grown
seedlings, and each gene was amplified by PCR from an equal
quantity of the reverse-transcribed products. 18S rRNA was used as
an internal control.
an abnormal outer cell surface. These observations
suggest that SHV3 and SVL1 play important roles in
determining epidermal cell morphology by conferring
cell wall stability.
Fig. 6 Abnormal epidermal cells in the shv3-2svl1-1 double
mutant. (A) Stomata from shv3-2svl1-1 and wild-type plants.
Bar ¼ 10 mm. (B) Size of the opened stomata in shv3-2svl1-1,
calculated as the product of the long and short axes. Twenty
stomata from a single leaf were used in each experiment. The data
are the means SD of three independent experiments. The P-value
calculated by Student’s t-test was 50.01. (C) Aberrant shape of the
shv3-2svl1-1 stomata. An shv3-2svl1-1 leaf fixed with ethanol and
cleaned with chloral hydrate showing swelling on the ventral sides
of the guard cells. Bar ¼ 20 mm. (D) Aberrant swelling of the shv32svl1-1 hypocotyl epidermis. Electron micrographs of hypocotyls
from 7-d-old wild-type (upper left) and shv3-2svl1-1 (upper right
and lower) seedlings. Bars ¼ 50 mm (upper left), 100 mm (upper
right, bottom left) and 10 mm (bottom right).
Characterization of the shv3-2svl1-1 cell wall
To characterize the shv3-2svl1-1 cell wall, we isolated
and analyzed alcohol-insoluble residues (AIRs) from 4-dold dark-grown seedlings (Table 1). First, we measured the
SHV3 and SVLs in cell wall organization
Table 1 Composition of the AIR from 4-d-old darkgrown seedlings
0.005
−0.008
−0.004
0
0.004
0.008
PC1
PC1 loading
B
0.20
1061
1037
953
1105
0.10
1165
0
−0.10
1661
900
−0.20
1549
1000
crystalline cellulose content. The AIRs of the dark-grown
shv3-2svl1-1 seedlings contained about 45% less crystalline
cellulose, highlighting the requirement for SHV3 and SVL1
for crystalline cellulose. Secondly, we analyzed the neutral
sugar composition of the AIRs. Since excess starch-derived
glucose was detected in shv3-2svl1-1, the neutral sugar
compositions were compared excluding glucose. Indeed, the
AIRs of the dark-grown shv3-2svl1-1 seedlings contained a
larger amount of starch. No drastic changes in neutral sugar
composition were found, although slightly higher arabinose
and slightly lower xylose contents were detected in shv32svl1-1.
A shorter dark-grown hypocotyl increased accumulation of ethylene/jasmonic acid-responsive gene transcripts
and ectopic lignification were also observed in the cellulosedeficient mutant cev1/eli1. Therefore, it is possible that a
reduced cellulose content is the major cause of the
phenotypes observed in the shv3-2svl1-1 double mutant.
On the other hand, some phenotypes unique to shv3-2svl1-1,
such as ruptured root hair cells and swollen guard cells,
have not been observed in cellulose-deficient mutants.
Hence, the presence of other changes in the cell wall was
suspected. To investigate whether shv3-2svl1-1 has an
aberrant cell wall composition, we performed Fouriertransform infrared (FTIR) analysis, which provides information on cell wall composition and architecture (McCann
et al. 1992). FTIR absorption spectra of the double mutant
and wild type were obtained using 4-d-old dark-grown
hypocotyls. Principal components analysis using these
−0.010
1100
Expressed as mg mg–1 AIR.
b
Degree of methylesterified uronic acids.
c
The amount of each neutral sugar is presented as the percentage of
the total neutral sugars after deduction of the glucose content.
The data are the means SD of three experiments. Indicates a
significant difference compared with the wild type (Student’s t-test,
P50.05). yIndicates a marginally significant difference from the
wild type (Student’s t-test, P50.1).
a
−0.005
1200
11.5 0.7
4.5 0.2
26.2 2.6
21.5 0.5
4.4 0.4
31.9 1.4
1300
11.3 0.7
4.2 0.2
20.9 1.2
23.8 1.2
4.4 1.3
35.5 1.7
0
1400
84 6
53.6 1.0
85 14y
52 6
1500
154 6
5.8 0.3
63 11
71 6
1600
Cellulose
Starcha
Uronic acidsa
DM%b
Neutral sugarsc
Rhamnose
Fucose
Arabinose
Xylose
Mannose
Galactose
shv3-2svl1-1
WT
shv3svl1
1700
a
Wild type
A 0.010
PC2
Components
1529
Wave numbers (cm−1)
Fig. 7 FTIR analysis of the shv3-2svl1-1 cell wall. (A) FTIR
analysis of dark-grown hypocotyl cell walls. Principal components
analysis was performed using spectra from 14 shv3-2svl1-1 plants
and 12 wild-type plants. The shv3-2svl1-1 and wild-type data were
separated using the first principal component (PC1) score, which
explains 38.35% of the variance. (B) PC1 loading. Positive peaks
characteristic of cellulose (1,037, 1,061, 1,105 and 1,165 cm–1)
indicate that the shv3-2svl1-1 cell walls are poorer in cellulose
than those of the wild type. The peak at 953 cm–1 corresponds to
unesterified pectin. Negative peaks at 1,549 and 1,661 cm–1
indicate that the shv3-2svl1-1 cell walls are enriched in protein
relative to the wild type.
spectral data revealed a clear separation of shv3-2svl1-1
and wild type in principal component 1 (PC1) (Fig. 7A).
The data for PC1 indicated that the cell walls of shv3-2svl1-1
are poorer in cellulose (corresponding to peaks at 1,037,
1,061, 1,105 and 1,165 cm–1) but richer in protein (corresponding to peaks at 1,550 and 1,650 cm–1) than those of the
wild type (Wilson et al. 2000) (Fig. 7B), consistent with the
results of our AIR analysis. Another remarkable difference
was the absorption at 953 cm–1, which reflects the amount
of polygalacturonic acid (Synytsya et al. 2003). Because this
1530
SHV3 and SVLs in cell wall organization
absorption is diminished by methyl esterification of the
carboxyl group (Synytsya et al. 2003), the total uronic acid
content in shv3-2svl1-1 may have decreased or the degree of
methyl esterification may have increased. To confirm these
quantitative changes, the total uronic acid and methyl ester
contents were measured. Contrary to our expectations, the
cell walls of the dark-grown shv3-2svl1-1 seedlings had an
increased amount of uronic acid and a decreased amount of
methyl esterification when compared with the wild type
(Table 1). The uronic acid content and degree of methyl
esterification were not responsible for the change in
absorption at 953 cm–1 in our FTIR analysis, suggesting
that some other modification or altered interaction may
affect the infrared absorption of the pectic polysaccharides
in shv3-2svl1-1. These results suggest that the shv3-2svl1-1
mutation affects the properties and amount of the pectins
that are present.
Increasing the borate concentration partially suppresses the
shv3svl1 and shv3 phenotypes
We hypothesized that pectin cross-linking is affected by
the condition of the pectin. To examine whether promoting
pectin cross-linking would suppress the mutant phenotype,
shv3-2svl1-1 and shv3 were grown on medium containing an
increased concentration of borate, which is supposed to be
involved in the cross-linking of pectic domain rhamnogalacturonan-II (RG-II) and other cell wall carbohydrates
(Blevins and Lukaszewski 1998). Four-day-old seedlings
grown on medium containing 0.1 mM borate were transferred to medium containing a higher concentration
of borate and grown for 3 d. A significant increase in
the number of unruptured root hairs was observed in both
shv3-2svl1-1 and shv3; however, the unruptured root hairs in
shv3 were normal (i.e. elongated) in shape (Fig. 8), whereas
those in shv3-2svl1-1 were irregular (Supplementary
Fig. S5). Under the growth conditions used, 2.5 mM
borate was more effective than 0.5 mM borate, indicating
dose dependency. A borate concentration of 3.5 mM
reduced root hair elongation, presumably due to toxicity.
On the other hand, increasing the concentration of calcium,
which is involved in the cross-linking of homogalacturonan,
did not suppress any of the observed phenotypes (data not
shown). These results imply that the stability of RG-II
cross-linking is reduced in shv3-2svl1-1 and shv3. Increased
borate concentrations did not suppress any of the other
phenotypes in the shv3-2svl1-1 double mutant under the
same conditions.
To confirm whether RG-II cross-linking is altered in
the mutant, we assessed the proportion of borate estercross-linked RG-II dimers in shv3-2svl1-1 and the wild type.
AIRs purified from seedlings grown on 0.1 mM borate were
analyzed. As shown in Fig. 9, the proportion of RG-II
dimers was slightly but significantly reduced in shv3-2svl1-1.
Borate (mM)
0.1
0.5
2.5
3.5
shv3-2
WT
Fig. 8 Suppression of the shv3 phenotype by an increased
concentration of borate. Four-day-old seedlings grown on normal
medium containing 0.1 mM borate were transferred to medium
containing various concentrations (0.1, 0.5, 2.5 or 3.5 mM) of
borate and grown for 3 d. Bar ¼ 0.4 mm.
Discussion
To date, only three mutants with ruptured root hairs
have been reported: kjk, lrx1lrx2 and mrh4 (Baumberger
et al. 2001, Favery et al. 2001, Jones et al. 2006). KJK
encodes a cellulose synthase-like D3 protein predicted to be
involved in the synthesis of polysaccharides other than
cellulose (Dhugga et al. 2004, Liepman et al. 2005). LRX1
and LRX2, which have leucine-rich repeats and extensinlike domains, regulate cell wall organization in root hairs,
although their biochemical functions are unknown
(Baumberger et al. 2001, Baumberger et al. 2003, Diet
et al. 2006). MRH4 encodes the COBRA-like protein
COBL9 (Jones et al. 2006). The ruptured root hair
phenotype indicates severe cell wall defects, suggesting
that the responsible genes have important roles in cell wall
organization. However, to our knowledge, there are no
reports of other phenotypes in these mutants, and these
genes have not been evaluated for their contributions in
other tissues. In this study, we demonstrated that SHV3 has
critical roles not only in root hair cells but also in other
tissues.
Cell wall analysis revealed distinct characteristics of the
cell wall components in the shv3-2svl1-1 double mutant,
including decreased crystalline cellulose accumulation
(Table 1, Fig. 7). This result indicates that SHV3 and
SVL1 are novel factors required for the accumulation of
crystalline cellulose. A reduced cellulose content has also
been reported in mutants of ELI1/CEV1 and PRC1, which
are involved in primary cell wall synthesis (Fagard et al.
2000, Cano-Delgado et al. 2003). Although eli1/cev1 and
prc1 show more severe growth defects than the shv3-2svl1-1
SHV3 and SVLs in cell wall organization
dimer
(496s)
monomer
(536s)
W#1
RI (mV)
W#2
m#1
m#2
300
500
700
Retention time (s)
dimer / (dimer + monomer)
wild type
0.908 ± 0.015
shv3svl1
0.859 ± 0.009
Fig. 9 shv3svl1 affects the proportion of cross-linked RG-II dimers.
RG-II dimers and monomers solubilized from the AIRs by
endopolygalacturonase were separated by size-exclusion HPLC
with a refractive index detector. The proportion of RG-II dimers and
monomers is expressed as RG-II dimer/(RG-II dimer þ monomer).
The data are the means SD of three independent experiments.
A representative chromatogram of wild type (W) and shv3svl1 (m)
(in duplicate) is shown. The P-value calculated by Student’s t-test
was 50.01.
double mutant, they do not have ruptured root hairs
(Desnos et al. 1996, Cano-Delgado et al. 2000). Therefore, it
is likely that abnormalities other than the reduced cellulose
content contribute to root hair rupture in the shv3-2svl1-1
mutant.
Our methyl esterified pectin assay and FTIR spectroscopic results indicate a reduced rate of methyl esterified
pectin formation and other alterations in pectin structure
or interactions in the shv3-2svl1-1 double mutant (Fig. 7).
We also demonstrated that the mutant had a lower rate of
1531
RG-II dimer formation. Additionally, GFP–SHV3 was
localized mainly at the plasma membrane (Fig. 1). These
observations suggest that SHV3 and related proteins are
involved in pectin network formation at the plasma
membrane, rather than biosynthesis. The pectin matrix is
essential for mechanical stability of the cell wall during
rapid tip growth in pollen tubes (Jiang et al. 2005, Iwai et al.
2006). The many mechanisms common to tip growth in root
hairs and pollen tubes suggest that pectins are also essential
for tip growth in root hairs (Cole and Fowler 2006).
In this study, we demonstrated that the shv3 and shv32svl1-1 root hair phenotype could be suppressed by an
increased concentration of borate. Similar characteristics
were previously observed in mur1, a fucose-deficient
mutant; a high borate concentration (2.6 mM) successfully
rescued the reduced tensile strength of the mutant
hypocotyls and the reduced formation rate of cross-linked
borate dimers (O’Neill et al. 2001, Ryden et al. 2003). In the
mur1 mutant, it is reasonable that the altered RG-II is the
cause of the mutant phenotype and the effect of borate on
RG-II contributes significantly to the rescue of the
phenotype, because the effect of mur1 mutation on RG-II
is defined and a high concentration of borate is sufficient to
rescue the mutant phenotype. In contrast, in the case of the
shv3 and shv3svl1 double mutant, the sites of action of
borate contributing to the suppression of the root hair
phenotype were obscure, because the mutations affect not
only RG-II formation, and increasing the borate content
was not sufficient to reverse the shv3 and shv3svl1
phenotypes completely. However, there are only a few cell
wall-related mutants whose phenotypes are suppressed by
an increased concentration of borate, suggesting that this
observed borate dependency is an important character of
shv3 and shv3svl1.
The shv3-2svl1-1 double mutant had abnormal epidermal cells (Fig. 6). Root hair-like cell expansion in the
hypocotyl epidermis was previously observed in transgenic
plants expressing recombinant GLABRA2 (GL2), which
was modified to activate the expression of genes involved in
root hair cell differentiation (Ohashi et al. 2003). This
implies that Arabidopsis hypocotyl epidermal cells have the
ability to form root hair-like structures. SHV3 and SVL1
may negatively regulate such structures by modulating cell
wall organization. The shape of the guard cells in the
mutants was different from that in the wild type,
presumably due to loose ventral walls (Fig. 6C). Adequate
guard cell movement is thought to be conferred primarily by
guard cell-specific cell wall organization (Majewska-Sawka
et al. 2002, Jones et al. 2003). It is likely that SHV3 and
SVL1 play some role in the development of the guard cell
wall. Consistent with this, considerably stronger SVL1
expression was observed in the guard cells (Fig. 4S). To our
knowledge, this guard cell phenotype has not been reported
1532
SHV3 and SVLs in cell wall organization
previously. It would be interesting to examine this
phenotype in other cell wall-related mutants.
Taken together, the defects in SHV3 and SVL1 include
abnormal cellulose deposition and pectin network formation, suggesting the function of these proteins in cell wall
organization. Given the complex composition of the cell
wall and our current lack of understanding of how defects
in individual cell wall components affect cell wall organization, it is difficult to determine how SHV3 and SVLs are
directly involved in cellulose deposition or pectin network
formation without precise demonstrations of their biochemical activities. Several GPI-anchored proteins are
essential for cell wall organization in plants (Gillmor et al.
2005). The biochemical and molecular functions of individual GPI-anchored proteins, however, are largely unknown.
The GPD-like domain in SHV3 and its related proteins
implies their functions in catabolism of glycerophosphodiesters such as glycerophosphocholine. Two types of GPD
have been reported in carrot and Arabidopsis. They are
vacuolar GPD and cell wall GPD, which are assumed to be
involved in membrane degradation and uptake of nutrients,
respectively (Van der Rest et al. 2002). If this is the case, the
absence of SHV3 and SVLs may initially cause the
compositional changes in plasma membrane lipid.
Disturbance of the lipid composition of the membrane
would affect the membrane transport system and the
cellulose biosynthesis, and they in turn would cause
abnormal cell wall organization, although there is no
report demonstrating the direct effect of the phospholipid
composition on the cell wall organization. Alternatively,
SHV3 and related proteins may possess completely different
enzymatic activity from that of GPD, as suggested by the
different domain organization and the distinct amino acid
residues at the active center. In addition, if the biochemical
activity of SHV3 and its paralogs is the same as that of
GPDs, the presence of typical GPDs in the cell wall (Van
der Rest et al. 2004) would mask the phenotype of the defect
of the SHV3 family. A large number of modifications are
required for the arrangement of and interactions between
cell wall components. It is possible that SHV3 and its
related proteins are involved in their regulation. This idea
may be consistent with the notion that homologs of these
genes are found only in plants which have developed the
unique process of cell wall organization. Determining the
exact activity of these proteins will not only allow us to
characterize their biochemical properties but will also
enhance our understanding of cell wall organization.
background) mutant line was obtained from a collection of Ds
transposon-tagged lines (Kuromori et al. 2004). The shv3-1 seeds
were kindly provided by Dr. Claire Grierson (University of Bristol,
UK). The T-DNA insertion lines shv3-2 (SALK_024208), svl1-1
(SALK_064539) and svl2-1 (SALK_057865) (Alonso et al. 2003)
were obtained from the Arabidopsis Biological Resource Center.
The plants were grown as described previously (Nishimura et al.
2005).
The point mutation in shv3-1 was detected by DNA
sequencing of the At4g26690 gene in the shv3-1 mutant. To
generate the double and triple mutants, single mutants were
crossed and the presence of T-DNA insertions in the F2 progeny
was confirmed by PCR (Supplementary Table S2). The methods
used to isolate genomic DNA and the PCR conditions were
described previously (Nishimura et al. 2005).
RT–PCR analysis
First-strand cDNA was synthesized from 0.8 mg of total RNA
pre-treated with RQ1 RNase-free DNase (Promega, Madison, WI,
USA) using a ReverTra Ace RT–PCR Kit (Toyobo, Tokyo,
Japan) with random hexamers according to the manufacturer’s
instructions. Semi-quantitative RT–PCR was performed with 1/40
of the first-strand reaction mixture using gene-specific primers
(Supplementary Table S2). The PCR conditions were 958C for
90 s followed by 16 or 32 cycles of 958C for 15 s, 558C for 20 s and
728C for 60 s, and a final hold at 728C for 4 min. In total, 32 cycles
were used to amplify SHV3, SVL1, SVL2, SVL3, SVL4, SVL5,
VSP1 and PDF1.2. 18S rRNA was used as an internal control
(16 cycles).
Generation and analysis of the transgenic plants
To construct GFP::SHV3, sGFP (S65T) was amplified by
PCR and inserted in-frame into the NcoI site of SHV3 (RAFL0816-D08, obtained from the RIKEN BioResource Center), and then
the fusion gene was inserted downstream of the CaMV 35S
promoter in the T-DNA region of the binary vector pMSH1
(Kawasaki et al. 1999). Imaging of the transgenic plants was
performed using an LSM510 confocal microscope (Carl Zeiss,
Jena, Germany). For the promoter::GUS constructs, 1.9, 1.5 and
1.5 kb of the putative promoter regions of SHV3, SVL1 and SVL2,
respectively, were amplified by PCR using the primers listed in
Supplementary Table S2, and inserted upstream of the GUS gene
in the T-DNA region of the binary vector pBI101. Thin sections of
stained tissues were prepared using a Technovit 7100 Plastic
Embedding Kit (Kulzer, Wehrheim, Germany). For complementation of the shv3-2svl1-1 mutation, a genomic DNA fragment
containing the SVL1 coding region (1.5 kb upstream and 0.5 kb
downstream) was amplified by PCR and cloned into the T-DNA
region of the binary vector pGreenII, and then introduced into the
shv3-2svl1-1 double mutant. The M2 and M3 progeny were used for
characterization.
Materials and Methods
Phloroglucinol staining
Phloroglucinol staining was performed as described by CanoDelgado et al. (2000). The plants were cleaned with ethanol then
mounted in a 2% phloroglucinol-HCl solution. Lignin staining was
observed in seedlings embedded in chloral hydrate : glycerol : water
(8 : 1 : 2).
Plant materials and growth conditions
Arabidopsis thaliana (L) Heynh. ecotypes Columbia (Col) and
Nossen (Nos) were used in this study. The shv3-3 (15-1096-1, Nos
Cell wall characterization
FTIR analysis of dark-grown hypocotyls was performed as
described by Fagard et al. (2000). Four-day-old dark-grown
SHV3 and SVLs in cell wall organization
seedlings were pressed onto a barium fluoride window then rinsed
with water. The samples were subsequently dried at room
temperature for 3 h. Infrared spectra were collected using a
PerkinElmer AutoIMAGE FT-IR Microscope System attached
to a Spectrum One FT-IR Spectrometer (PerkinElmer, Shelton,
CT, USA) from the middle region of the hypocotyl, avoiding the
central cylinder, with a 20 mm 40 mm aperture. All data sets were
corrected for the baseline and normalized. Principal components
analysis was performed using Win-Discrim software (E. K.
Kemsley, Institute of Food Research, Norwich, UK).
To prepare the AIRs, plant tissues were collected and ground
in liquid nitrogen then washed with 80% ethanol, 95% ethanol,
99.5% ethanol, chloroform : methanol (1 : 1) and acetone, and airdried. To measure the crystalline cellulose content, the AIRs were
treated with acetic acid : nitric acid : water (8 : 1 : 2) for 1 h at 1008C,
then the sugar content of the insoluble materials was measured
with anthrone reagent as described by Updegraff (1969). Avicel
PH-101 (Fluka, Bruchs, Switzerland) was used to generate a
standard curve. The neutral sugar composition of the AIRs was
determined by gas chromatography–mass spectrometry (GC-MS)
of the alditol acetate derivatives (York et al. 1985). To measure the
total uronic acid content, the AIRs were treated with trifluoroacetic acid and the soluble fraction was used. Determination of
the uronic acid content was performed using sulfamic acid and
m-hydroxybiphenyl reagent as described by Filisetti-Cozzi and
Carpita (1991). D-Glucuronic acid was used to generate a standard
curve. The presence of uronosyl methyl esters was determined by
saponification according to the method of Wood and Siddiqui
(1971). The degree of methyl esterification was calculated from
paired assays of the total uronic acids. Starch in the AIRs was
detected enzymatically using an F-Kit (Boehringer Mannheim,
Mannheim, Germany) according to the manufacturer’s instructions. Determination of the ratio of borate ester cross-linked RG-II
dimers to RG-II monomers was performed as described by
Matsunaga and Ishii (2006).
Scanning electron microscopy
Seedlings (7-d-old), frozen in liquid nitrogen, were attached to
the stage of a JSM5610-LV electron microscope (JEOL, Tokyo,
Japan) and observed under high-vacuum conditions according to
the manufacturer’s instructions.
Supplementary material
Supplementary material are available at PCP Online.
Funding
Grant-in-Aid from the Ministry of Education, Sports,
Culture, Science and Technology (15570045, 20570050
to T.H.), Japan; RIKEN President’s Special Research
Grant (T.H.).
Acknowledgments
We thank Drs. Claire Grierson and Miki Fujita for providing
the shv3-1 seeds and plasmids, respectively, and Dr. Chieko Saito
for helping in microscopic analysis using the confocal microscope.
We also thank the Arabidopsis Biological Resource Center and
1533
RIKEN BioResource Center for the T-DNA insertion lines and a
cDNA clone.
References
Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H., et al.
(2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana.
Science 301: 653–657.
Baumberger, N., Ringli, C. and Keller, B. (2001) The chimeric leucine-rich
repeat/extensin cell wall protein LRX1 is required for root hair
morphogenesis in Arabidopsis thaliana. Genes Dev. 15: 1128–1139.
Baumberger, N., Steiner, M., Ryser, U., Keller, B. and Ringli, C. (2003)
Synergistic interaction of the two paralogous Arabidopsis genes LRX1
and LRX2 in cell wall formation during root hair development. Plant J.
35: 71–81.
Blevins, D.G. and Lukaszewski, K.M. (1998) Boron in plant structure and
function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 481–500.
Borner, G.H., Lilley, K.S., Stevens, T.J. and Dupree, P. (2003)
Identification of glycosylphosphatidylinositol-anchored proteins in
Arabidopsis. A proteomic and genomic analysis. Plant Physiol. 132:
568–577.
Borner, G.H., Sherrier, D.J., Stevens, T.J., Arkin, I.T. and Dupree, P.
(2002) Prediction of glycosylphosphatidylinositol-anchored proteins in
Arabidopsis. A genomic analysis. Plant Physiol. 129: 486–499.
Brocard-Gifford, I., Lynch, T.J., Garcia, M.E., Malhotra, B. and
Finkelstein, R.R. (2004) The Arabidopsis thaliana ABSCISIC ACIDINSENSITIVE8 encodes a novel protein mediating abscisic acid and
sugar responses essential for growth. Plant Cell 16: 406–421.
Cano-Delgado, A.I., Metzlaff, K. and Bevan, M.W. (2000) The eli1
mutation reveals a link between cell expansion and secondary cell wall
formation in Arabidopsis thaliana. Development 127: 3395–3405.
Cano-Delgado, A., Penfield, S., Smith, C., Catley, M. and Bevan, M. (2003)
Reduced cellulose synthesis invokes lignification and defense responses in
Arabidopsis thaliana. Plant J. 34: 351–362.
Cole, R.A. and Fowler, J.E. (2006) Polarized growth: maintaining focus on
the tip. Curr. Opin. Plant Biol. 9: 579–588.
Cosgrove, D.J. (2005) Growth of the plant cell wall. Nat. Rev. Mol. Cell
Biol. 6: 850–861.
Desnos, T., Orbovic, V., Bellini, C., Kronenberger, J., Caboche, M.,
Traas, J. and Hofte, H. (1996) Procuste1 mutants identify two distinct
genetic pathways controlling hypocotyl cell elongation, respectively
in dark- and light-grown Arabidopsis seedlings. Development 122:
683–693.
Dhugga, K.S., Barreiro, R., Whitten, B., Stecca, K., Hazebroek, J.,
Randhawa, G.S., Dolan, M., Kinney, A.J., Tomes, D., Nichols, S. and
Anderson, P. (2004) Guar seed b-mannan synthase is a member of the
cellulose synthase super gene family. Science 303: 363–366.
Diet, A., Link, B., Seifert, G.J., Schellenberg, B., Wagner, U., Pauly, M.,
Reiter, W.D. and Ringli, C. (2006) The Arabidopsis root hair cell wall
formation mutant lrx1 is suppressed by mutations in the RHM1 gene
encoding a UDP-L-rhamnose synthase. Plant Cell 18: 1630–1641.
Ellis, C., Karafyllidis, I., Wasternack, C. and Turner, J.G. (2002) The
Arabidopsis mutant cev1 links cell wall signaling to jasmonate and
ethylene responses. Plant Cell 14: 1557–1566.
Fagard, M., Desnos, T., Desprez, T., Goubet, F., Refregier, G.,
Mouille, G., McCann, M., Rayon, C., Vernhettes, S. and Höfte, H.
(2000) PROCUSTE1 encodes a cellulose synthase required for normal
cell elongation specifically in roots and dark-grown hypocotyls of
Arabidopsis. Plant Cell 12: 2409–2424.
Favery, B., Ryan, E., Foreman, J., Linstead, P., Boudonck, K., Steer, M.,
Shaw, P. and Dolan, L. (2001) KOJAK encodes a cellulose synthase-like
protein required for root hair cell morphogenesis in Arabidopsis. Genes
Dev. 15: 79–89.
Filisetti-Cozzi, T.M. and Carpita, N.C. (1991) Measurement of uronic acids
without interference from neutral sugars. Anal. Biochem. 197: 157–162.
Gillmor, C.S., Lukowitz, W., Brininstool, G., Sedbrook, J.C., Hamann, T.,
Poindexter, P. and Somerville, C. (2005) Glycosylphosphatidylinositolanchored proteins are required for cell wall synthesis and morphogenesis
in Arabidopsis. Plant Cell 17: 1128–1140.
1534
SHV3 and SVLs in cell wall organization
Iwai, H., Hokura, A., Oishi, M., Chida, H., Ishii, T., Sakai, S. and Satoh, S.
(2006) The gene responsible for borate cross-linking of pectin rhamnogalacturonan-II is required for plant reproductive tissue development and
fertilization. Proc. Natl Acad. Sci. USA 103: 16592–16597.
Jiang, L., Yang, S.L., Xie, L.F., Puah, C.S., Zhang, X.Q., Yang, W.C.,
Sundaresan, V. and Ye, D. (2005) VANGUARD1 encodes a pectin
methylesterase that enhances pollen tube growth in the Arabidopsis style
and transmitting tract. Plant Cell 17: 584–596.
Jones, L., Milne, J.L., Ashford, D. and McQueen-Mason, S.J. (2003) Cell
wall arabinan is essential for guard cell function. Proc. Natl Acad. Sci.
USA 100: 11783–11788.
Jones, M.A., Raymond, M.J. and Smirnoff, N. (2006) Analysis of the root
hair morphogenesis transcriptome reveals the molecular identity of six
genes with roles in root hair development in Arabidopsis. Plant J. 45:
83–100.
Kawasaki, T., Henmi, K., Ono, E., Hatakeyama, S., Iwano, M., Satoh, H.
and Shimamoto, K. (1999) The small GTP-binding protein rac is a
regulator of cell death in plants. Proc. Natl Acad. Sci. USA 96:
10922–10926.
Kuromori, T., Hirayama, T., Kiyosue, Y., Takabe, H., Mizukado, S.,
Sakurai, T., Akiyama, K., Kamiya, A., Ito, T. and Shinozaki, K. (2004)
A collection of 11 800 single-copy Ds transposon insertion lines in
Arabidopsis. Plant J. 37: 897–905.
Kuromori, T., Wada, T., Kamiya, A., Yuguchi, M., Yokouchi, T., et al.
(2006) A trial of phenome analysis using 4000 Ds-insertional mutants in
gene-coding regions of Arabidopsis. Plant J. 47: 640–651.
Lalanne, E., Honys, D., Johnson, A., Borner, G.H., Lilley, K.S.,
Dupree, P., Grossnicklaus, U. and Twell, D. (2004) SETH1 and
SETH2, two components of the glycosylphosphatidylinositol anchor
biosynthetic pathway, are required for pollen germination and tube
growth in Arabidopsis. Plant Cell 16: 229–240.
Larson, T.J., Ehrmann, M. and Boos, W. (1983) Periplasmic glycerophosphodiester phosphodiesterase of Escherichia coli, a new enzyme of the
glp regulon. J. Biol. Chem. 258: 5428–5432.
Li, Y., Jones, L. and McQueen-Mason, S. (2003) Expansins and cell growth.
Curr. Opin. Plant Biol. 6: 603–610.
Liepman, A.H., Wilkerson, C.G. and Keegstra, K. (2005) Expression of
cellulose synthase-like (Csl) genes in insect cells reveals that CslA family
members encode mannan synthases. Proc. Natl Acad. Sci. USA 102:
2221–2226.
Majewska-Sawka, A., Munster, A. and Rodriguez-Garcia, M.I. (2002)
Guard cell wall: immunocytochemical detection of polysaccharide
components. J. Exp. Bot. 53: 1067–1079.
Matsunaga, T. and Ishii, T. (2006) Borate cross-linked/total rhamnogalacturonan II ratio in cell walls for the biochemical diagnosis of
boron deficiency in hydroponically grown pumpkin. Anal. Sci. 22:
1125–1127.
McCann, M.C., Hammouri, M., Wilson, R., Belton, P. and Roberts, K.
(1992) Fourier transform infrared microspectroscopy is a new way to
look at plant cell walls. Plant Physiol. 100: 1940–1947.
Nicol, F., His, I., Jauneau, A., Vernhettes, S., Canut, H. and Hofte, H.
(1998) A plasma membrane-bound putative endo-1,4-b-D-glucanase is
required for normal wall assembly and cell elongation in Arabidopsis.
EMBO J. 17: 5563–5576.
Nishimura, N., Kitahata, N., Seki, M., Narusaka, Y., Narusaka, M.,
Kuromori, T., Asami, T., Shinozaki, K. and Hirayama, T. (2005)
Analysis of ABA hypersensitive germination2 revealed the pivotal
functions of PARN in stress response in Arabidopsis. Plant J. 44:
972–984.
O’Neill, M.A., Eberhard, S., Albersheim, P. and Darvill, A.G. (2001)
Requirement of borate cross-linking of cell wall rhamnogalacturonan II
for Arabidopsis growth. Science 294: 846–849.
Ohashi, Y., Oka, A., Rodrigues-Pousada, R., Possenti, M., Ruberti, I.,
Morelli, G. and Aoyama, T. (2003) Modulation of phospholipid
signaling by GLABRA2 in root-hair pattern formation. Science 300:
1427–1430.
O’Neill, M.A., Ishii, T., Albersheim, P. and Darvill, A.G. (2004)
Rhamnogalacturonan II: structure and function of a borate
cross-linked cell wall pectic polysaccharide. Annu. Rev. Plant Biol. 55:
109–139.
Pagant, S., Bichet, A., Sugimoto, K., Lerouxel, O., Desprez, T.,
McCann, M., Lerouge, P., Vernhettes, S. and Höfte, H. (2002)
KOBITO1 encodes a novel plasma membrane protein necessary for
normal synthesis of cellulose during cell expansion in Arabidopsis. Plant
Cell 14: 2001–2013.
Page, R.D. (1996) TreeView: an application to display phylogenetic trees on
personal computers. Comput. Appl. Biosci. 12: 357–358.
Parker, J.S., Cavell, A.C., Dolan, L., Roberts, K. and Grierson, C.S. (2000)
Genetic interactions during root hair morphogenesis in Arabidopsis. Plant
Cell 12: 1961–1974.
Penninckx, I.A., Thomma, B.P., Buchala, A., Metraux, J.P. and
Broekaert, W.F. (1998) Concomitant activation of jasmonate and
ethylene response pathways is required for induction of a plant defensin
gene in Arabidopsis. Plant Cell 10: 2103–2113.
Ridley, B.L., O’Neill, M.A. and Mohnen, D. (2001) Pectins: structure,
biosynthesis, and oligogalacturonide-related signaling. Phytochemistry
57: 929–967.
Rojo, E., Leon, J. and Sanchez-Serrano, J.J. (1999) Cross-talk between
wound signaling pathways determines local versus systemic gene
expression in Arabidopsis thaliana. Plant J. 20: 135–142.
Roudier, F., Fernandez, A.G., Fujita, M., Himmelspach, R., Borner, G.H.,
Schindelman, G., Song, S., Baskin, T.I., Dupree, P., Wasteneys, G.O. and
Benfey, P.N. (2005) COBRA, an Arabidopsis extracellular glycosylphosphatidyl inositol-anchored protein, specifically controls highly
anisotropic expansion through its involvement in cellulose microfibril
orientation. Plant Cell 17: 1749–1763.
Ryden, P., Sugimoto-Shirasu, K., Smith, A.C., Findlay, K., Reiter, W.D.
and McCann, M.C. (2003) Tensile properties of Arabidopsis cell walls
depend on both a xyloglucan cross-linked microfibrillar network and
rhamnogalacturonan II–borate complexes. Plant Physiol. 132:
1033–1040.
Scheible, W.R. and Pauly, M. (2004) Glycosyltransferases and cell wall
biosynthesis: novel players and insights. Curr. Opin. Plant Biol. 7:
285–295.
Schindelman, G., Morikami, A., Jung, J., Baskin, T.I., Carpita, N.C.,
Derbyshire, P., McCann, M.C. and Benfey, P.N. (2001) COBRA encodes
a putative GPI-anchored protein, which is polarly localized and
necessary for oriented cell expansion in Arabidopsis. Genes Dev. 15:
1115–1127.
Sedbrook, J.C., Carroll, K.L., Hung, K.F., Masson, P.H. and
Somerville, C.R. (2002) The Arabidopsis SKU5 gene encodes an
extracellular glycosyl phosphatidylinositol-anchored glycoprotein
involved in directional root growth. Plant Cell 14: 1635–1648.
Shi, H., Kim, Y., Guo, Y., Stevenson, B. and Zhu, J.K. (2003) The
Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein
and is required for normal cell expansion. Plant Cell 15: 19–32.
Synytsya, A., Copikova, J., Matejka, P. and Machovic, V. (2003) Fourier
transform Raman and infrared spectroscopy of pectins. Carbohyd.
Polym. 54: 97–106.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and
Higgins, D.G. (1997) The CLUSTAL_X windows interface: flexible
strategies for multiple sequence alignment aided by quality analysis tools.
Nucleic Acids Res. 25: 4876–4882.
Tommassen, J., Eiglmeier, K., Cole, S.T., Overduin, P., Larson, T.J. and
Boos, W. (1991) Characterization of two genes, glpQ and ugpQ, encoding
glycerophosphoryl diester phosphodiesterases of Escherichia coli. Mol.
Gen. Genet. 226: 321–327.
Updegraff, D.M. (1969) Semi-micro determination of cellulose in biological
materials. Anal. Biochem. 32: 420–424.
Van Der Rest, B., Boisson, A.M., Gout, E., Bligny, R. and Douce, R. (2002)
Glycerophosphocholine metabolism in higher plant cells. Evidence of a
new glyceryl-phosphodiester phosphodiesterase. Plant Physiol. 130:
244–255.
Van Der Rest, B., Rolland, N., Boisson, A.M., Ferro, M., Bligny, R. and
Douce, R. (2004) Identification and characterization of plant glycerophosphodiester phosphodiesterase. Biochem. J. 379: 601–607.
Vissenberg, K., Van Sandt, V., Fry, S.C. and Verbelen, J.P. (2003)
Xyloglucan endotransglucosylase action is high in the root elongation
zone and in the trichoblasts of all vascular plants from Selaginella to Zea
mays. J. Exp. Bot. 54: 335–344.
SHV3 and SVLs in cell wall organization
Wilson, R.H., Smith, A.C., Kacurakova, M., Saunders, P.K., Wellner, N.
and Waldron, K.W. (2000) The mechanical properties and molecular
dynamics of plant cell wall polysaccharides studied by Fourier-transform
infrared spectroscopy. Plant Physiol. 124: 397–405.
Wood, P.J. and Siddiqui, I.R. (1971) Determination of methanol and its
application to measurement of pectin ester content and pectin methyl
esterase activity. Anal. Biochem. 39: 418–428.
1535
York, W.S., Darvill, A.G., McNeil, M., Stevenson, T.T. and Albersheim, P.
(1985) Isolation and characterization of plant cell walls and cell wall
constituents. Methods Enzymol. 118: 3–40.
Zhong, R., Kays, S.J., Schroeder, B.P. and Ye, Z.H. (2002) Mutation
of a chitinase-like gene causes ectopic deposition of lignin,
aberrant cell shapes, and overproduction of ethylene. Plant Cell 14:
165–179.
(Received June 6, 2008; Accepted August 15, 2008)