Plant Physiol. (1999) 120: 665-674
A Maize Glycine-Rich Protein Is Synthesized in the Lateral Root
Cap and Accumulates in the Mucilage1
Takashi Matsuyama,
Hidetaka Satoh,
Yasuyuki Yamada, and
Takashi Hashimoto*
Graduate School of Biological Sciences, Nara Institute of Science
and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0101, Japan
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ABSTRACT |
The root cap functions in the
perception of gravity, the protection of the root apical meristem, and
facilitation of the passage of roots through the soil, but the genes
involved in these functions are poorly understood. Here we report the
isolation of a root-specific gene from the cap of maize (Zea
mays L.) primary root by cDNA subtraction and differential
screening. The gene zmGRP4
(Z. mays glycine
rich protein 4) encodes a member of
the glycine-rich proteins with a putative signal peptide at the amino terminus. The deduced molecular mass of mature zmGRP4 is 14.4 kD. In
situ-hybridization analysis has shown zmGRP4 to be
strongly expressed in the lateral root cap and weakly expressed in the root epidermis. A polyclonal antibody raised against recombinant zmGRP4
detected a protein of 36 kD in the insoluble protein fraction extracted
from the root tip and the root proper, indicating posttranslational modification(s) of zmGRP4. Immunohistochemical analysis showed the
accumulation of zmGRP4 in the mucilage that covers the root tip. These
results indicate that lateral root-cap cells secrete modified zmGRP4
into the mucilage to which the protein may contribute to its
characteristic physical properties.
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INTRODUCTION |
The root cap covers the root meristem at the root apices of
vascular plants but is absent in nonvascular plants such as liverworts and mosses. It is proposed that the root cap perceives gravity and
protects the root apical meristem (Sievers and Braun, 1996
). The root
cap has a high regenerative capacity. When the root cap, which is
sharply delineated from the root proper, is surgically removed from a
maize (Zea mays L.) root, it regenerates completely within a
few days (Barlow, 1975).
Anatomical studies suggest that the root cap consists of several
distinct regions (Moore and McClelen, 1983; Dolan et al., 1993). The
maize root cap, for example, can be divided into three regions: the
calyptrogen, the columella root cap, and the lateral root cap. The
calyptrogen faces the distal end of the quiescent center of the root
apical meristem, is composed of approximately four cell layers, and
serves as a root-cap meristem. The columella cells are generated by
periclinal cell division from the central region of the calyptrogen.
Sedimented large amyloplasts containing well-developed starch granules
are characteristic of the columella cells. These amyloplasts function
as statoliths in root gravitropism (Sievers and Braun, 1996). The
lateral root cap surrounds the columella root cap. In maize roots with
a closed-type construction, the lateral cap cells originate from the
calyptrogen (Barlow, 1996). However, in Arabidopsis roots, which have
an open-type construction, there is no discrete boundary between the
root proper and the cap, and the lateral cap cells are derived from the
same initials as the root epidermal cells (Dolan et al., 1993). The lateral root-cap cells are rich in the hypertrophied dictyosome cisternae that form large secretory vesicles (Mollenhauer et al., 1961). These cisternae may reflect the massive secretion of mucilage from the lateral cap cells, because the vesicle content was observed to
be deposited between the plasma membrane and the outer tangential walls of the lateral cap cells (Morré et al., 1967).
In addition to these three tissues, sloughed-off cap cells and root
mucilage may also be included as components, which together make up the
cap region. Root-cap cells are continuously pushed toward the root-cap
periphery and finally slough off into the external root environment.
These detached cells are found at the root periphery, even at some
distance from the root cap (Vermeer and McCully, 1982); they are
metabolically active and have unique patterns of gene expression
(Brigham et al., 1995). Several functions of the sloughed-off cap cells
as a root-soil interface have been proposed (Hawes et al.,
1998).
The root mucilage typically covers the root apex, is an amorphous and
uneven gel, and ranges in thickness from 50 µm to 1 mm. The mucilage
is secreted largely from the root cap, but the root epidermis is also
covered by a thin film of mucilage, which is histochemically distinct
from the cap-derived mucilage (Greaves and Darbyshire, 1972; Clarke et
al., 1979; Foster, 1982; Vermeer and McCully, 1982). The matrix of
maize mucilage consists of 95% polysaccharides and 5% protein (Harris
and Northcote, 1970; Bacic et al., 1986).
To understand the functions of the root cap at the molecular level, we
have identified genes that are specifically or predominantly expressed
in maize root cap. One such gene is expressed strongly in the lateral
root cap, and its gene product is secreted into and accumulates in the
mucilage.
The accession number for the sequence described in this article is
AB014475.
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MATERIALS AND METHODS |
Plant Material
Maize (Zea mays L. cv Merit) was supplied by the Asgrow
Seed Company (Kalamazoo, MI). Seeds were soaked in tap water for
72 h in the dark at 30°C. After imbibition seeds were germinated on paper towels saturated with tap water for 1 to 2 d in the dark at 30°C. When the primary roots were 2 to 3 cm long, the cap and selected portions of the root were removed by a scalpel under a
magnifying glass and immediately frozen in liquid
N2. The tip region used in this study was the
apical 5 mm of the root and included the root apical meristem and the
whole root cap. The region of the root proper was between 1 and 3 cm
from the distal end of the root.
Maize plantlets were grown under 18-h light/6-h dark or 24-h dark
conditions at 30°C on layered wet paper towels in plastic pots.
RNA Isolation, cDNA Synthesis, and Subtractive Hybridization
Poly(A+) RNA was extracted directly from the
root cap and the root proper of maize primary roots using
Dynabeads oligo(dT25) (Dynal, Oslo, Norway).
Several hundred nanograms of poly(A+) RNA were
used to construct double-stranded cDNA using a cDNA synthesis kit
(Pharmacia).
Subtractive hybridization was done essentially as described by Wang and
Brown (1991) and Hashimoto et al. (1993). The double-stranded cDNAs
were fragmented by AluI and RsaI and ligated to a
PCR linker. cDNA fragments of 0.2 to 2.0 kb were amplified by PCR. The
cDNA fragments from the root proper were then biotinylated with
Photoprobe biotin (Vector Laboratories, Burlingame, CA) and used as the
driver DNA.
One subtraction cycle consisted of five steps: hybridization of the
excess driver DNA to the tracer DNA from the root cap for 20 h at
68°C; removal of nonhybridizing driver DNA by binding to streptavidin
and extraction with organic solvent; another hybridization of the
excess driver DNA to the remaining tracer DNA once again for 2 h
at 68°C; removal of driver DNA as above; and PCR amplification of the
tracer DNA. This subtraction cycle was repeated twice to produce
subtracted root-cap cDNA fragments. Subtracted root-proper cDNA
fragments were also generated in the same way, except that cDNA
fragments from the root cap and the root proper were used, respectively, as the driver and the tracer DNAs.
Screening of Differentially Expressed cDNAs
The subtracted root-cap cDNA fragments were digested with
EcoRI, which cleaved the PCR linker, and inserted into the
EcoRI site of pBluescript II SK(
) (Stratagene). These
plasmids were introduced into the bacterial strain DH5
to construct
a root-cap cDNA library, and 386 independent colonies were grown
overnight in Luria-Bertani medium containing 50 µg
mL
1 ampicillin at 37°C. From each culture a
50-µL aliquot was blotted in duplicate onto a membrane (Hybond
N+, Amersham) using a filtration manifold system
(GIBCO-BRL). After denaturation and neutralization, the duplicate
filters were hybridized at 42°C for 16 h with either a
32P-labeled, subtracted root-cap cDNA probe or a
32P-labeled, nonsubtracted root-proper cDNA
probe, in a hybridization buffer containing 50% formamide, 10%
dextran sulfate, 1% SDS, 5× SSPE (1× SSPE: 180 mM NaCl, 1 mM EDTA, and 10 mM
Na2HPO4, pH 7.5), 5×
Denhardt's solution (1× Denhardt's solution: 0.02% [w/v] BSA,
0.02% [w/v] Ficoll, and 0.02% [w/v] PVP), and 100 µg
mL1 salmon testis DNA, and washed at 65°C in
0.1× SSPE and 0.1% SDS. Seventy-two positive cDNA clones, which
hybridized only to the root-cap cDNA probe, were obtained.
To group the positive clones, the 72 recombinant bacterial cultures
containing positive cDNA clones were blotted onto a Hybond N+ membrane and processed as described above. One
cDNA clone, which had hybridized specifically and strongly to the
root-cap cDNA probe, was chosen, labeled with
32P, and hybridized to the membrane. Positive
clones were regarded as members of the same group. Next, another
strongly and specifically hybridizing cDNA clone other than the members
of this group was chosen and processed as above. Four hybridizations
were done, resulting in four independent groups and 29 remaining cDNA
clones. Representative clones from the four groups and the extra 29 cDNA clones were partially sequenced by a DNA sequencer (model 373A, Perkin-Elmer), using M13 reverse and universal primers. The sequence analysis classified the root-cap-positive cDNA clones into 23 groups.
Subtracted cDNA fragments from the root cap, nonsubtracted cDNA
fragments from the root cap, and nonsubtracted cDNA fragments from the
root proper were blotted in amounts of 0.05, 0.5, and 5 µg per slot
onto a Hybond N+ membrane, as described above.
Representative cDNA fragments from the 23 groups were used as the
probes for hybridization. Ten cDNA fragments hybridized to the
subtracted and nonsubtracted root-cap cDNA pools or to the subtracted
root-cap cDNA pool, but not to the root-proper cDNA pool, and will be
referred to as "root-cap abundant." The other 13 clones either
hybridized to the root-proper cDNA pool or did not hybridize to any
cDNA pools.
A cDNA library of the maize primary root-tip region from within 1 mm of
the distal-tip end was made in 
ZAPII (Stratagene) (Matsuyama et al.,
1999). A total of 4 × 106 recombinants were
independently screened with the 10 root-cap-abundant cDNA fragments as
probes. Hybridization and other procedures were done as described
above. Positive plaques were identified with 3 of the 10 root-cap-abundant probes. In this report, one cDNA representing six
phage clones was analyzed. These positive recombinant phages were
converted to pBluescript SK() plasmids by in vivo excision using the
manufacturer's protocol (Stratagene). Both DNA strands of the longest
insert of the six clones were sequenced. DNA and predicted amino acid
sequences were analyzed with GeneWorks software (IntelliGenetics,
Campbell, CA).
Genomic-DNA-Hybridization Analysis
Total genomic DNA was isolated from 3-d-old etiolated maize
seedlings by cetyl-trimethyl-ammonium bromide extraction (Murray and
Thompson, 1980). Genomic DNA (30 µg) was digested with restriction enzymes, electrophoresed on a 1% agarose gel, and blotted onto a
Hybond N+ membrane. The membrane was hybridized
to the full-length zmGRP4 (Z.
mays GRP 4)
cDNA probe and washed under the conditions described above.
Northern-Hybridization and RT-PCR Analysis
Total RNA was isolated from several tissues, including the root
tip, root proper, young leaves from 2-week-old plants, and shoots from
3-d-old light-grown and etiolated plants using phenol:chloroform extraction and LiCl precipitation (Mohnen et al., 1985).
Poly(A+) RNA was purified from total RNA using
Oligotex-dT30 Super (Takara Shuzo, Tokyo, Japan).
Poly(A+) RNA (1.5 µg per lane) was
electrophoresed on a 1.2% formaldehyde agarose gel, blotted onto
a Hybond N+ membrane, and hybridized to the
full-length zmGRP4 cDNA probe under the conditions described
above. After stripping the probe from the membrane by incubating at
67°C in a buffer containing 50% formamide, 10 mM Tris-HCl, and 10 mM
EDTA, pH 8.0, the 32P-labeled
PstI-SacI fragment of a ubiquitin cDNA
(Christensen and Quail, 1989) was hybridized to the same membrane.
For RT-PCR analysis, total RNA was isolated from approximately 100 mg
of root tip, root proper, shoot, and etiolated shoot using the RNeasy
Plant Mini kit (Qiagen, Chatsworth, CA) and used to
construct first-strand cDNA using the SUPERSCRIPT preamplification system (GIBCO-BRL). PCR primers for zmGRP4 were
5
-TTGTATCTCACAATGGCAGGC and 5-GCGTTGGAATTCCAAGAACC (Fig. 1) and PCR
primers for maize -tubulin1 (Montoliu et al., 1989) were
5-CTTGATCGCATCAGGAAGC and 5-TCAGCAGAGATGACTGGAGC. PCR amplification
was carried out for 18 cycles of denaturation at 94°C for 1 min,
annealing at 60°C for 30 s, and elongation at 72°C for 2 min.
Amplified zmGRP4 fragments were electrophoresed on a 1.2%
agarose gel, blotted onto a Hybond N+ membrane,
and hybridized to the full-length zmGRP4 cDNA probe as
described above. Representative amplified DNA fragments were partially
sequenced to confirm their identity.
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| Figure 1.
Nucleotide and deduced amino acid sequences of
zmGRP4. A putative signal peptide is double underlined.
The sequence between the vertical arrowheads was used as both antisense
and sense probes for in situ-hybridization analysis. The two arrows
indicate the positions of PCR primers used for RT-PCR analysis. The
stop codon is shown by an asterisk.
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In Situ-Hybridization Analysis
Maize primary root tips were fixed in 3% paraformaldehyde and 2%
glutaraldehyde for 12 h at 4°C. After samples were dehydrated in
a graded ethanol series and cleared in a graded xylene series, they
were embedded in wax (Histoprep 580, Wako, Osaka, Japan) and sectioned
at 10 µm by using a rotary microtome. Digoxigenin-labeled antisense
and sense RNA probes were prepared from a 3-untranslated region of
zmGRP4 (see Fig. 1) using an RNA labeling kit (DIG, Boehringer Mannheim). Samples were incubated with the RNA probes at
50°C for 16 h, treated with RNase A (2.5 µg
mL1 in 0.5 M NaCl, 10 mM Tris-HCl, and 1 mM EDTA,
pH 7.5) at 37°C for 30 min, and washed with several changes of 2×
SSC (1× SSC: 150 mM NaCl and 15 mM
Na3C6H5O7)
and once with 0.1× SSC at 50°C. Signals were detected by a nucleic
acid detection kit (DIG, Boehringer Mannheim). The color reaction was
stopped with 10 mM Tris-HCl and 1 mM EDTA, pH 8.0. Sections were passed through an
ethanol series and mounted for microscopic observation.
zmGRP4 Antiserum
A portion of the zmGRP4 cDNA encoding the
carboxyl-terminal amino acid residues from 132 to 192 was subcloned
into the EcoRI site of pET-32b(+) (Novagen), which would
then express a fusion protein consisting of the N-terminal region of
thioredoxin and the C-terminal region of zmGRP4. This plasmid was
introduced into the BL21 (DE3) bacterial strain (Novagen), and the
expression of the fusion protein was induced by 1 mM isopropyl
-D-thiogalactopyranoside at 37°C for 3 h in Luria-Bertani medium. Bacterial cells were harvested by
centrifugation, suspended in 50 mM potassium
phosphate buffer, pH 8.0, containing 1% Triton X-100 and 1 µg
mL1 lysozyme, incubated at 30°C for 15 min,
and then ruptured by three cycles of freeze/thaw treatment and
sonication for 1 min. After centrifugation of the homogenate, the
fusion protein in the supernatant was separated by preparative SDS-PAGE
using a PrepCell (model 491, Bio-Rad). The eluate fractions containing the fusion protein were concentrated with a YM-10 membrane filter (Amicon). The buffer of the concentrated protein solution was exchanged
by using a PD-10 column (Pharmacia) equilibrated with buffer A
(20 mM Tris-HCl and 10% glycerol, pH 7.0).
The above solution was loaded onto a Mono-Q fast-protein
liquid-chromatography column (Pharmacia) previously equilibrated with
buffer A and eluted with a linear gradient of KCl from 0 to 0.5 M in buffer A. The fractions containing the
fusion protein were concentrated with a YM-10 filter and desalted using
a PD-10 column. The fusion protein had an approximate purity of 99.9%,
as determined by staining with Coomassie Brilliant Blue R-250 after
SDS-PAGE, and was used to raise antiserum in mice.
The reactivity of the antiserum against zmGRP4 was confirmed as
follows. A BamHI-SmaI fragment of the
zmGRP4 cDNA encoding the carboxyl-terminal amino acid
residues from 137 to 192 was subcloned into pGEX-2T (Pharmacia). The
resultant pGEX-zmGRP4 plasmid or pGEX-2T was introduced into the BL21
(DE3) bacterial strain, and the expression of either a chimeric protein
consisting of zmGRP4 and GST, or GST alone, respectively, was induced
at 37°C for 3 h with 1 mM isopropyl
-D-thiogalactopyranoside. Approximately 100 ng
of total protein extracts from these bacterial cells was separated on a
15% SDS-polyacrylamide gel and transferred to an Immobilon PVDF
membrane (Millipore). The membrane was blocked at room temperature in
buffer B (100 mM Tris-HCl and 150 mM NaCl, pH 7.5) containing 5% skim milk powder
for 1 h. The antiserum was diluted 1:5000 in buffer B and
incubated with the membranes at room temperature for 1 h. After
washing the antiserum several times with buffer B containing 0.2%
Tween 20, the following procedures, including secondary antibody
treatment and immunodetection, were performed by using an ECL Plus kit
(Amersham) according to the manufacturer's instructions. The antiserum
reacted strongly with the fusion protein consisting of zmGRP4 and GST,
but not with GST alone.
Immunohistochemical Analysis
Fixed sections were prepared as described for in situ
hybridization and blocked at room temperature in buffer B containing 0.2% Tween 20 and 3% BSA for 1 h. Anti-zmGRP4 serum or preimmune mouse serum was diluted 1:300 in blocking buffer and incubated with the
sections at room temperature for 1 h. After washing the primary
antibody several times in buffer B containing 0.2% Tween 20, anti-mouse IgG conjugated with alkaline phosphatase (Kirkegaard and
Perry Laboratories, Gaithersburg, MD) was diluted 1:1000 in buffer B
and incubated with the sections at room temperature for 1 h. After
briefly washing the slides with buffer B, immunodetection was performed
as described for in situ-hybridization analysis.
Immunoblot Analysis
Approximately 100 maize root tips were frozen with liquid
N2 and ground to a fine powder with a pestle. For
some sample preparations used in Figure 6B, after root mucilage
including sloughed-off cap cells was gently wiped from the root tip
with a paper towel, the root tips were immediately collected in liquid
nitrogen. The pulverized root cells were extracted with buffer (50 mM Tris-HCl, 3 mM EDTA, 3 mM DTT,
and 3 mM PMSF), and the suspension was centrifuged at
15,000g for 5 min. The supernatant was referred to as the
soluble fraction. The pellet was resuspended in sample buffer (125 mM Tris-HCl, pH 6.8, containing 1% SDS) and used
as the insoluble fraction. A total protein fraction was prepared by
directly extracting the pulverized cells with sample buffer. Protein
concentration was determined using the BCA protein assay reagent
(Pierce).
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| Figure 6.
Immunoblot analysis of zmGRP4. Each lane was
loaded with 10 µg of protein extracted from the root tip or root
proper. A, Total protein fraction (lanes T), Tris-buffer-soluble
fraction (lanes S), and Tris-buffer-insoluble but SDS-buffer-soluble
fraction (lanes I) were separated on a 15% SDS-polyacrylamide gel. B,
The Tris-buffer-soluble fraction (S) and Tris-buffer-insoluble but
SDS-buffer-soluble fraction (I) were extracted from intact root tips
(+) or root tips from which mucilage had been removed ().
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Protein preparations (10 µg per lane) were separated on a 15%
SDS-polyacrylamide gel. The remaining steps were performed as described
above, except that 3% BSA was substituted for 5% skim milk powder in
the blocking buffer.
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RESULTS |
Isolation of a Maize GRP cDNA That Is Highly Expressed in Root
Cap
The root cap and the root proper are sharply delineated in the
maize primary root, which has a closed-type construction. This anatomical feature is used to facilitate excision of maize root-cap tissues from the root proper using a scalpel (Barlow, 1975). We collected approximately 500 root caps and extracted
poly(A+) RNA directly from the root cap and also
from the root proper. The cDNAs specifically present in the root cap
were enriched by subtracting the root-proper cDNA fragment pool from
the root-cap cDNA fragment pool. Subsequently, the subtracted root-cap
cDNA fragment library was duplicated and hybridized independently with the above root-proper cDNA fragment pool or the root-cap cDNA fragment
pool as the probes. This differential screening recovered 72 cDNA
fragments that hybridized specifically to the root-cap cDNA fragment
pool, and these clones were classified into 23 groups by
cross-hybridization and partial DNA sequencing. Further slot-blot hybridization with the above two probes confirmed that 10 cDNA groups
were much more abundant in the root cap than in the root proper.
Representative cDNA fragments from these 10 clones were used as the
probes to screen a maize root-tip cDNA library, and three distinct cDNA
clones were obtained. One of the three cDNA clones is reported here.
The other two clones encoded a novel protein (Matsuyama et al., 1999)
and a maize expansin.
Figure 1 shows the nucleotide and deduced
amino acid sequences of a cDNA clone. The cDNA was 821 bp long and
contained an open reading frame encoding a 16.9-kD polypeptide of 192 amino acids. The predicted protein had a hydrophobic putative signal peptide with a potential cleavage site between 22 and 23 amino acid
residues (von Heijne, 1985) and was a member of the cell wall GRPs
(Showalter, 1993). We will refer to this protein as zmGRP4. zmGRP4,
excluding the putative signal peptide, was rich in Gly (40%), Ser
(19%), Asn (7%), Ala (7%), and Tyr (6%). The high content of
Gly, Ser, and Ala of zmGRP4 is consistent with the general
characteristics of Gly-rich cell wall proteins.
Genomic DNA blot-hybridization analysis was done with a full-length
zmGRP4 cDNA probe at high-stringency conditions (Fig. 2). BamHI and EcoRI
digested the zmGRP4 cDNA once, whereas BglII and
XhoI did not digest the cDNA. Two to three strong bands and two to four weak bands were detected when the maize genome was digested
with BamHI, BglII, EcoRI, or
XhoI. Therefore, a small number of genes homologous to
zmGRP4 are likely to exist in maize. In support of this, the
amino acid sequence of another maize GRP cDNA (accession no.
AF031083) and zmGRP4 share an 82% identical region of approximately
90 amino acid residues (data not shown). At the nucleotide
sequence level, this region is 83% identical between these two
GRPs.
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| Figure 2.
Genomic DNA-hybridization analysis of
zmGRP4. Full-length zmGRP4 cDNA was used
as a probe. Maize genomic DNA (20 µg) was digested with
BamHI, BglII, EcoRI, or
XhoI. The positions of the molecular markers are shown
on the left.
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zmGRP4 Is Expressed Strongly in the Lateral Root Cap and Weakly in
the Epidermis of the Root Proper
RNA blot-hybridization analysis with the full-length cDNA of
zmGRP4 as the probe detected zmGRP4 expression in
the root tip but not in the root proper, etiolated shoot, shoot, or
mature leaf (Fig. 3A). Since RNA-blot
hybridization may not detect low levels of zmGRP4 expression
and may detect expression of zmGRP4-related gene(s) as well,
RT-PCR was done to specifically amplify zmGRP4 RNA (Fig.
3B). Expression of zmGRP4 was detected in the root tip and
root proper but not in the shoot and etiolated shoot. Although quantitative analysis is often difficult with RT-PCR, repeated RT-PCR
analyses in which different amplification cycles were used (data not
shown) confirmed that zmGRP4 is expressed more strongly in
the root tip than in the root proper. Amplification of maize -tubulin RNA indicated that approximately equal amounts of cDNA were
used for each tissue sample.
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| Figure 3.
Expression of zmGRP4 in maize
tissues. A, Northern-hybridization analysis. Poly(A+) RNA
(1.5 µg) was isolated from 2- to 3-cm-long roots, 3-d-old etiolated
shoots, and 3-d-old shoots, and leaves of 2-week-old plants. A maize
ubiquitin probe served as a control to estimate the relative loading of
RNA in each lane. B, RT-PCR analysis. S, Shoot; ES, etiolated shoot;
root proper, 0- to 0.5-, 0.5- to 1.0-, and 1.0- to 1.5-cm regions
distal from the excision site; RT, root tip; NT, negative control
without reverse transcription. A maize -tublin gene
(TUA) served as a positive control.
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The 3-untranslated region of the zmGRP4 cDNA was used as a
probe for in situ-hybridization analysis to study the detailed expression pattern of zmGRP4 in maize primary root. The
antisense probe detected strong zmGRP4 expression in the
lateral root-cap cells and rather weak expression in epidermal cells of
the root proper (Fig. 4A). The sense
probe did not detect any hybridization signals (Fig. 4B). Peripheral
cells that had been or were being detached from the lateral root cap
showed little zmGRP4 expression (Fig. 4C, arrowheads),
whereas weak zmGRP4 expression extended to several
peripheral cells toward the central region of the root cap (Fig. 4D).
zmGRP4 expression in epidermal cells of the root proper
terminated in the region where zmGRP4 expression in the lateral root cap ended (Fig. 4C, arrow).
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| Figure 4.
In situ-hybridization analysis of
zmGRP4. Longitudinal sections of maize primary root tip
were hybridized with antisense (A, C, and D) or sense (B)
digoxigenin-labeled zmGRP4-specific probes. The images
in C and D were enlarged from the rectangles in A. An arrow shows the
end of zmGRP4 expression in the epidermis, whereas
arrowheads indicate sloughed-off cap cells. Co, Columella; LRC, lateral
root cap; Ep, epidermis; RAM, root apical meristem. Scale bars = 100 µm.
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zmGRP4 Accumulates in Root Mucilage
A polyclonal antibody was raised against a truncated zmGRP4
protein that contained amino acid residues 132 to 192. This
carboxy-terminal region of zmGRP4 includes amino acid stretches of low
Gly abundance and is expected to be specific for zmGRP4. The closest
homolog of zmGRP, encoded by a maize expressed sequence tag (AF031083), is 58% identical in this region (data not shown).
Immunohistochemical analysis using this antiserum showed that zmGRP4 is
present specifically in the mucilage that covers the root tip (Fig.
5A). A preimmune mouse antiserum detected
no signals (Fig. 5B). Longer exposure detected a relatively small
amount of zmGRP4 in the lateral root-cap cells (Fig. 5C). Sloughed-off cap cells appeared to contain little zmGRP4 (Fig. 5C, red arrowheads). A weak signal was also observed in epidermal cells of the root proper
in the distal 1 cm of the root tip (Fig. 5D). The presence of mucilage
at the periphery of the root epidermis was not apparent because the
layer of root epidermal mucilage is expected to be very thin (Foster,
1982).
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| Figure 5.
Immunolocalization of zmGRP4. Longitudinal
sections of maize primary root tip were incubated with anti-zmGRP4
serum (A, C, and D) or preimmune serum (B). The lateral root-cap region
adjacent to the root proper is shown with a higher magnification in C,
whereas the root proper region 1 cm distal to the cap is shown in D. Alkaline-phosphatase reactions were done for 1 h in A and B, and
for 3 h in C and D. Red arrowheads indicate sloughed-off cap
cells. RC, Root cap; RAM, root apical meristem; M, mucilage; RP, root
proper; LRC, lateral root cap; Cx, cortex; Ep, epidermis. Scale
bars = 100 µm.
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zmGRP4 May Be Posttranslationally Modified
Immunoblot analysis using the anti-zmGRP4 serum revealed that
zmGRP4 exists in the maize root as a major band with an apparent molecular mass of 36 kD and a minor band with an apparent molecular mass of 34 kD in the insoluble fraction that was extracted with the
SDS-containing buffer (Fig. 6A).
Extraction with Tris buffer without SDS did not recover any zmGRP4
protein. The 36-kD form was much more abundant in the root tip than in
the root proper. A few faint bands of 27 and 25 kD were also detected
in the insoluble fraction from the root tip. Since the deduced
molecular mass of the mature zmGRP4 is 14.4 kD, posttranslational
modifications of zmGRP4 in maize root are suspected. Manual removal of
root mucilage and detached cap cells from the root tip markedly reduced the abundance of the 36-kD zmGRP4 in the preparation (Fig. 6B). This
strongly suggests that zmGRP4 accumulates in root mucilage mainly as a
modified 36-kD protein.
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DISCUSSION |
zmGRP4 Is a New Member of Maize GRPs
Many structural cell wall proteins that have putative signal
peptides and no catalytic domains have been reported in various plants
(Showalter, 1993).These cell wall proteins are characterized by a high
abundance of a single amino acid, repetitive sequence motifs, and a
tendency to become insolubilized within the cell wall. The three major
plant cell wall protein classes include HRGPs, PRPs, and GRPs. GRP
cDNAs have been isolated from several plants, including three from
maize. zmGRP is expressed in the epidermal cells of embryo,
scutellar tissue, and young leaf, and induced by ABA, water stress, and
wounding in leaves (Gómez et al., 1988). Since zmGRP does not
have an amino-terminal signal peptide, it may be a cytosolic protein.
Besides being rich in Gly, zmGRP has a putative RNA-binding motif
(Gómez et al., 1988). Therefore, zmGRP and zmGRP4 belong to
different subclasses of the GRP family. zmGRP3 (Goddemeier et
al., 1998) has an N-terminal signal peptide but shows no significant
homology to zmGRP4, except for abundant Gly residues. The expression of
zmGRP3 was root specific, with the highest expression level
in the meristematic and elongation regions (Goddemeier et al.,
1998). RNA-blot analysis of zmGRP3 indicates that
zmGRP3 and zmGRP4 are expressed in different
regions of the maize root.
zmGRP4 Expression in the Root
The expression of cell wall proteins depends on cell
type, developmental stage, and stress responses (Showalter, 1993).
zmGRP4 is expressed strongly in the lateral root cap and
weakly in root epidermis but scarcely in sloughed-off cap cells (Fig.
4). The immunohistochemical localization of zmGRP4 (Fig. 5) strongly
indicates that zmGRP4 is synthesized in lateral root-cap and
root-epidermal cells and then secreted into the mucilage. Lateral
root-cap cells develop considerable hypertrophied Golgi cisternae and
are the main site of mucilage secretion. Maize root epidermal cells are also reported to contain hypertrophied dictyosome cisternae and release
mucilage (Clarke et al., 1979; Foster, 1982). Detached cap cells,
however, have dictyosomes that are no longer hypertrophied (Clowes and
Juniper, 1968). This close correlation between zmGRP4 expression and differentiation of secretion machinery suggests that
zmGRP4 is secreted via hypertrophied Golgi cisternae into the mucilage.
Likewise, bean GRP 1.8 was localized to dictyosomes of xylem parenchyma
cells and was suggested to be exported into the walls of neighboring
protoxylem vessels (Ryser and Keller, 1992).
Sloughed-off cells did not express zmGRP4, whereas the
outermost cap periphery cells did express zmGRP4 (Fig. 4, C
and D). A notable switch in gene expression was also reported to occur upon cap-border cell differentiation in pea (Brigham et al., 1995).
zmGRP4 Is Posttranslationally Modified
Many cell wall proteins are modified posttranslationally. For
example, Pro residues of HRGP are enzymatically converted into Hyp
residues, which are then glycosylated to various degrees (Cassab, 1998). zmGRP4 mainly existed as a 36-kD protein, whereas the deduced molecular mass of mature zmGRP4 is 14.4 kD. The high Gly content in
GRPs may cause aberrant electrophoretic migration on SDS gels. When
zmGRP4 was expressed in Escherichia coli as a GST-fusion protein, the recombinant fusion protein detected was approximately 2-kD
larger than expected by SDS-PAGE analysis (T. Matsuyama and T. Hashimoto, unpublished results). However, this aberrant
migration alone does not explain the more than 20-kD difference between the expected and observed size of zmGRP4 extracted from maize root
tips.
Insolubilization of cell wall proteins has been observed in various
developmental or stress-responsive processes (Cassab, 1998).
Insolubilization of bean GRP 1.8 occurs during hypocotyl development
(Keller et al., 1989). H2O2
generated by fungal elicitor or glutathione treatment of bean or
soybean cells causes oxidative cross-linking and therefore the
insolubilization of PRP (Bradley et al., 1992). Recovery of
isodityrosine after hydrolysis of cross-linked HRGP indicates that the
Tyr hydroxy groups in HRGP undergo intermolecular condensation via
H2O2 (Fry, 1986). zmGRP4
contains a relatively high percentage of Tyr residues. Oxidative
cross-linking between zmGRP4s themselves or between zmGRP4 and other
proteins via Tyr residues might result in insolubilization and
increased molecular mass of zmGRP4. It should also be noted that PRPs
insolubilized by H2O2 were
not extracted even in SDS-containing buffer (Brisson et al., 1994), and
potential cross-linking of xylem GRPs with the aromatic residues of
lignin has also been proposed (Showalter, 1993; Cassab, 1998). The
absence of lignin and polyphenolics in root mucilage suggests that the
cross-linking partners of zmGRP4 may be at least partly different from
those of previously reported cell wall proteins.
Glycosylation is a common posttranslational modification found in
secreted proteins. However, there are few reports of the potential
glycosylation of GRPs (Showalter, 1993). Exceptions include a 30-kD GRP
purified from soybean aleurone layers, which was reported to contain
approximately 9% (w/w) sugars, including Man, Ara, Glc, Xyl, and Gal
(Matsui et al., 1995). Purified soybean GRP showed a broad band after
SDS-PAGE separation, indicating a microheterogeneity in the sugar
component (Matsui et al., 1995). On the other hand, zmGRP4 extracted
from maize root tips migrated as discrete bands on SDS-PAGE (Fig. 6).
Since the deduced zmGRP4 amino acid sequence has no canonical
N-glycosylation sites, the modification could be
O-glycosylation with homogeneous sugar side chains, if
zmGRP4 were to be glycosylated.
Possible Functions of zmGRP4 in Root Mucilage
Soil and sand sheaths usually cling tightly to the roots of
field-grown grasses such as maize root. The sheath is thought to be
formed by the binding of soil particles in mucilage originating from
the root (Vermeer and McCully, 1982, and the refs. therein). Root hairs
are probably not primarily responsible for the adhesion of soil
aggregates. Mucilage, soil particles, sloughed-off root-cap cells, and
some soil bacteria form the rhizosphere, and the chemical and physical
properties of the mucilage should be very important in determining the
nature of the rhizosphere.
Root mucilage is composed of 99.9% water (Guinel and McGully, 1986).
The dry mass of mucilage consists mainly of polysaccharides and
polyuronic acids (Jones and Morré, 1967; Floyd and Ohlrogge, 1970; Paull et al., 1975). Although proteins have been detected in
maize mucilage (Chaboud, 1983), their properties and possible roles have attracted little attention. Previous chemical analysis of
maize mucilage, from which detached cap cells have been mostly removed,
showed that the amino acid composition is rich in Gly (13.8% of total
amino acids) (Bacic et al., 1986). We have shown here that zmGRP4 is a
mucilage protein and possibly the major component of the protein
fraction. Other well-characterized GRPs are localized in the vascular
system, and in xylem in particular (Ryser and Keller, 1992, and the
refs. therein). Ultrastructural localization, however, has demonstrated
that bean GRP 1.8 is localized to unlignified primary walls of
protoxylem cells, and a correlation between GRP 1.8 deposition and
lignification was evidently lacking in bean hypocotyls (Ryser and
Keller, 1992; Ryser et al., 1997). An apparent positive correlation of
GRP deposition with expansive growth and an inverse correlation with
lignification have been reported for petunia ptGRP1, which is deposited
at the cell wall/membrane interface, rather than within the cell wall
(Condit, 1993). Thus, these GRPs may provide elasticity to the
stretching wall or some protective environment to cells under
frictional stress. Some GRP sequences are predicted to adopt
-pleated sheets composed of varying numbers of antiparallel
strands; such a structure could provide elasticity and tensile strength
during vascular development (Showalter, 1993).
The soil sheath adhering along the entire length of field-grown maize
roots is mostly permeated by mucilage, which is histochemically similar
to that produced by the root cap (Vermeer and McCully, 1982). An
experiment designed to measure the penetration resistance showed that
maize roots receive much less frictional resistance than metal probes
when growing into the soil (Bengough and McKenzie, 1997). One function
of root mucilage, working together with sloughing root-cap cells, may
be to decrease the frictional resistance during growth in the soil and
to protect growing roots from abrasion by soil particles. If zmGRP4 has
physical properties similar to other GRPs, it may provide elasticity to
the root mucilage and may complement other mucilage components (e.g.
polysaccharides and pectin) for a lubricant function.
Large amounts of fixed C are secreted into the rhizosphere from the
surface of grass roots (Russell, 1977). The secreted C is mostly in the
form of sugar, but a wide range of amino acids, organic acids,
vitamins, and auxins are either released from the roots or synthesized
by microorganisms in the root environment (Bar-Yosef, 1996). These
organic compounds may support survival and growth of detached cap cells
and soil bacteria. Some of the compounds even may be involved in
interactions between particular plant genotypes and soil
microorganisms. Secreted proteins in the rhizosphere may play similar
roles. In this regard, distribution of GRPs in root mucilages of other
maize genotypes and other plant species, and the stability of zmGRP4 in
the rhizosphere should be interesting to examine in the future.
| |
FOOTNOTES |
1
This work was supported in part by a
Grant-in-Aid for Scientific Research on Priority Areas ("The
Molecular Basis of Flexible Organ Plans in Plants," no. 06278103)
from the Ministry of Education, Science, Sports and Culture of Japan to
T.H. T.M. was supported by a Japan Society for the Promotion of
Science Research Fellowship for Young Scientists (no.
5487).
*
Corresponding author; e-mail
hasimoto{at}mailgate.aist-nara.ac.jp; fax 81-743-72-5489.
Received October 9, 1998;
accepted March 2, 1999.
| |
ABBREVIATIONS |
Abbreviations:
GRP, Gly-rich protein.
GST, glutathione
S-transferase.
HRGP, Hyp-rich glycoprotein.
PRP, Pro-rich protein.
RT, reverse transcriptase.
| |
ACKNOWLEDGMENTS |
We thank Dr. Katsumi Ueda for valuable advice on in
situ-hybridization techniques and Dr. Robert Winz for critical reading of the manuscript. Dr. Peter Quail of the University of California, Berkeley, and Naoki Yasumura are acknowledged for the maize ubiquitin cDNA and the cDNA library from maize root tip, respectively.
| |
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Abstract
Introduction