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Plant Physiol. (1999) 120: 799-810
Molecular Cloning and Tissue-Specific Expression of an Anionic
Peroxidase in Zucchini1
Sabine Carpin,
Michèle Crèvecoeur,
Hubert Greppin, and
Claude Penel*
Laboratoire de Biochimie et Physiologie Végétales,
Université de Genève, Place de l'Université 3, CH-1211 Geneva, Switzerland
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ABSTRACT |
A calcium-pectate-binding anionic
isoperoxidase (APRX) from zucchini (Cucurbita pepo) was
purified and subjected to N-terminal amino acid microsequencing. The
cDNA encoding this enzyme was obtained by reverse transcriptase
polymerase chain reaction from a cDNA library. It encoded a mature
protein of 309 amino acids exhibiting all of the sequence
characteristics of a plant peroxidase. Despite the presence of a
C-terminal propeptide, APRX was found in the apoplast. APRX protein and
mRNA were found in the root, hypocotyls, and cotyledons. In situ
hybridization showed that the APRX-encoding gene was expressed in many
different tissues. The strongest expression was observed in root
epidermis and in some cells of the stele, in differentiating tracheary
elements of hypocotyl, in the lower and upper epidermis, in the
palisade parenchyma of cotyledons, and in lateral and adventitious root primordia. In the hypocotyl hook there was an asymmetric expression, with the inner part containing more transcripts than the outer part.
Treatment with 2,3,5-triiodobenzoic acid reduced the expression of the
APRX-encoding gene in the lower part of the hypocotyl. Our observations
suggest that APRX could be involved in lignin formation and that the
transcription of its gene was related to auxin level.
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INTRODUCTION |
Plant peroxidases (EC 1.11.1.7) exist in numerous molecular forms.
For example, more than 50 different sequences encoding peroxidase have
been identified in Arabidopsis. These enzymes are mainly located in
cell walls and vacuoles (Mäder, 1992 ) and catalyze the reduction
of hydrogen peroxide into water using electrons from various donor
molecules. This redox activity allows them to oxidize many substances,
such as polyphenols, flavonoids (Gaspar et al., 1982), and alkaloids
(Blom et al., 1991), or to promote the oxidative cross-linking of cell
wall polymers (Fry, 1986). Their substrate specificity is generally
considered to be low, except toward some electron donors such as
scopoletin (Reigh et al., 1973) and extensin (Brownleader et al.,
1995), which are oxidized by specific isoforms. In some cases, they can
also oxidize molecules such as IAA (Gazaryan et al., 1996) through
catalytic mechanisms that differ from the classical peroxidative cycle. They have also been shown to produce hydrogen peroxide in the presence
of reducers such as NADH (Elstner and Heupel, 1976) and Cys (Pichorner
et al., 1992).
The wide spectrum of biochemical reactions that peroxidases are able to
catalyze and the great number of molecular isoforms explain the
difficulty encountered in the study of plant peroxidases. Studies
combining the techniques of molecular biology and biochemistry are
necessary to get an insight into their precise function in plants. It
is also essential to identify the mechanisms that determine their
microlocalization in cells, particularly within the extracellular matrix network. Most of the known peroxidase mRNAs encode a signal peptide that targets the neosynthesized protein to the secretory pathway. Some sequences also encode a C-terminal extension thought to
direct the protein to the vacuole (Welinder, 1992; Neuhaus et al.,
1994).
All peroxidase molecules can diffuse randomly throughout the cell wall.
Once they have been released out of the cell by exocytosis, it is
possible that some of them are targeted to specific microdomains where
they fulfill their catalytic function. This implies that certain
peroxidase isoforms exhibit an affinity for particular polymers of the
extracellular matrix acting as docking structures. Such a specific
interaction has been observed between isoperoxidases from zucchini
(Cucurbita pepo) (Penel and Greppin, 1994) and horseradish (Penel et al., 1996) and the pectins in their calcium-induced conformation. In zucchini one anionic and three cationic isoperoxidases have been shown to exhibit this particular binding property (Penel and
Greppin, 1996), which has been shown to be dependent on the presence of
cationic amino acid residues exposed at the surface of the protein
(Penel and Greppin, 1996). In situ binding experiments done on
hypocotyl cross-sections have demonstrated that the APRX binds in a
calcium-dependent manner to the cell wall of several tissues, with the
strongest affinity being exhibited by the cell walls of the epidermis
(Penel et al., 1996).
Its particular binding property makes this APRX especially interesting
for a molecular study. In this work we have purified and microsequenced
APRX, cloned its mRNA, and sequenced the resulting full-length cDNA. To
obtain information on the location of this isoperoxidase in zucchini
seedlings, we have detected its transcripts by in situ hybridization on
sections made from roots, hypocotyls, and cotyledons. The observed
expression patterns suggest a role for this peroxidase isoform in xylem
differentiation and relationships with auxin.
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MATERIALS AND METHODS |
Plant Material
Zucchini (Cucurbita pepo L. cv Black Beauty) seedlings
were grown on moist, absorbent paper at 25°C in darkness.
Four-day-old seedlings were harvested for protein and RNA extractions
and for in situ hybridization.
Adventitious root formation was studied using 6-cm-tall etiolated
seedlings whose root systems had been cut off. The seedlings were
placed in beakers with their cut ends in nutrient solution (1%
Sinesol, Iriland, Geneva, Switzerland) with or without
10 4 M TIBA and placed in the light
at 20°C. Under these conditions the first adventitious roots appeared
4 d after root excision. Two-centimeter segments were taken each
day at the bottom of the hypocotyls for RNA extraction.
Purification and Microsequencing of APRX
APRX was extracted and purified as described by Penel and Greppin
(1996). The purified protein was separated by two-dimensional PAGE
according to the method of O'Farrell (1978) and electroblotted onto a
PVDF membrane (Bio-Rad). The electroblotted protein was incubated with
pyroglutamate aminopeptidase according to the method of LeGendre et al.
(1993) to remove the pyroglutamyl group from the blocked N terminus.
The N-terminal sequence was determined by automated Edman degradation
using a DNA sequencer (model 373A, Applied Biosystems, Foster
City, CA).
cDNA Amplification, Cloning, and Sequencing of APRX cDNA
Total RNA was isolated from etiolated zucchini hypocotyls with a
phenol-SDS procedure (Dean et al., 1985).
Poly(A+) RNAs were isolated with
oligo(dT)-cellulose (Redi-Col, Pharmacia). The cDNA library was
synthesized by RT-PCR with a cDNA amplification kit (Marathon,
CLONTECH, Basel, Switzerland), which added a specific sequence
(adaptor) to the 5 and 3 ends of each cDNA.
For PCR amplification of the cDNA encoding APRX, two degenerate
oligonucleotide primers were synthesized (Microsynth, Balgach, Switzerland) whose nucleotide sequences were designed to be
complementary to the coding strand for the peptide sequences FYDQTCP
and TCPRLPNIV present in the N-terminal sequence of APRX. The sequence
of primer 1 (5-TTYTAYGAYCARACITGYCC-3; where I is inosine, R is A/G,
and Y is C/T) corresponded to the sense orientation of FYDQTCP; the sequence of primer 2 (5-ACITGYCCIMGNYTICCIA AYATHGT-3; where I and Y
are as above, and H is A/T/C, M is A/C, and N is A/C/G/T) corresponded
to the sense orientation of TCPRLPNIV. The first PCR amplification was
performed with 0.2 µM primer 1 and with 0.2 µM AP1 primer (5-CCATCCTAATACGACTCAC TATAGGGC-3
situated in the adaptor added with the cDNA amplification kit) using
the cDNA polymerase (Advantage, CLONTECH). The PCR procedure started with 1 min of denaturation at 94°C and was carried on with 30 cycles
of 30 s of denaturation at 94°C, 30 s of annealing at
54°C, and 3 min of extension at 68°C. A nested PCR was performed
with 0.2 µM primer 2 and 0.16 µM primer 3 (5-TTCTAGAATTCAGCGGCCGC[T]30N1N-3, where N1 is A/C/G and N is theA/C/G/T contained
in the adaptor of the cDNA amplification kit) using the same PCR cycles
as above, except that the annealing temperature was higher (64°C) and
a final step at 68°C for 4 min was added. The nucleotide sequence of
the 5-untranslated leader of APRX was obtained
by a 5-rapid amplification of cDNA ends (RACE) PCR
procedure with the same cDNA amplification kit using primer 4 (5-CGATGCCGGGAGCATCCTCTAGC-3) and primer AP1.
The PCR products were cloned into vector (pGEM-T Easy,
Promega), and the resulting ligation product was transformed into
Escherichia coli strain XL-1-Blue (Stratagene) according to
the method of Sambrook et al. (1989). The dideoxy method was used for
sequencing the double-stranded DNA using T7 Sequenase (version 2.0, Amersham).
RNA and DNA Extraction and Hybridization
Total RNA was extracted from 4-d-old etiolated seedlings (Dean et
al., 1985) and DNA from 2-week-old plant leaves according to the method
of Rogers and Bendich (1994). Northern and Southern analyses were
performed according to the method of Sambrook et al. (1989). After
blotting onto membranes (Hybond-N, Amersham), RNA and DNA were
hybridized using as a probe the clone obtained by RT-PCR labeled with
DNA- or RNA-labeling kits (DIG, Boehringer Mannheim). For control of
RNA loading, membranes were rehybridized with a tobacco cDNA probe
complementary to 18S rRNA.
In Situ Hybridization
In situ hybridization was performed following the procedure
described below, which was modified from that of Komminoth (1996). A
300-bp untranslated 3-end APRX-specific probe was obtained from the RT-PCR clone with PstI. This 300-bp clone was
linearized with PstI to produce the antisense probe, and
with EcoRI to produce the sense probe. Digoxigenin-labeled
sense and antisense specific probes were synthesized according to the
instructions of manufacturer. For the preparation of paraffin sections,
pieces from different parts of seedlings were fixed with 3%
formaldehyde and 0.25% glutaraldehyde in 100 mM
phosphate buffer, pH 7.2, at 4°C overnight. After fixation, the
samples were rinsed with the same phosphate buffer, dehydrated in a
graded-alcohol series, and embedded in paraffin. Sections (10 µm)
were attached on poly-L-Lys-coated slides
(Polysine, Menzel-Glaser, Germany).
The sections were deparaffinized by soaking the slides in 100% xylene
followed by several washes in xylene:ethanol (50:50, v/v), rehydrated
by transferring them through a graded ethanol series (90%, 70%, 50%,
and 25%), and briefly rinsed with DEPC-treated water. The sections
were then incubated for 30 min at 37°C with 1 µg/mL proteinase K in
100 mM Tris/HCl, pH 7.5, containing 5 mM EDTA.
The proteinase K was removed by rinsing three times with DEPC-treated
water. The sections were then treated for 10 min at room temperature
with 100 mM triethanolamine, pH 8.0, containing 0.25%
acetic anhydride, followed by 5 min in 2× SSC, and twice for 5 min in
DEPC-treated water. Finally, the sections were dehydrated through an
ethanol series ranging from 25% to 100% ethanol, dried, and
prehybridized for 2 h at 44°C in 4× SSC, 50% formamide, 1× Denhardt's solution, 5% dextran sulfate, 1 µg/mL salmon-sperm DNA,
and 0.25 µg/mL yeast tRNA, and hybridized overnight in the same
buffer containing 10 ng/µL sense or antisense probes.
After hybridization the slides were washed at 50°C with 2× SSC twice
for 45 min, with 1× SSC for 45 min, and with 0.5× SSC for 15 min.
Subsequently, an RNaseA treatment (0.5 µg/mL in 500 mM
NaCl, 10 mM Tris/HCl, pH 7.5, and 1 mM EDTA)
was performed at room temperature for 30 min followed by a 15-min rinse
in 0.5× SSC, then with HIS 1 buffer (100 mM Tris/HCl, pH
7.5, and 150 mM NaCl). After blocking for 1 h in 2%
BSA (w/v), 0.3% Triton X-100 in 100 mM Tris/HCl, pH 7.5, and 150 mM NaCl, the sections were incubated with
antidigoxygenin antibody-alkaline phosphatase conjugate (dilution
1:500, Boehringer Mannheim) at room temperature for 1 h, and
washed two times in HIS 1 buffer for 10 min. They were then stained
with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate
according to the instructions of the manufacturer. The reaction was
stopped by placing the slides into 10 mM Tris-HCl, pH 7.5, containing 1 mM EDTA. For each tissue, sections were
incubated with an antisense probe to detect the APRX
transcripts and with a sense probe as a control.
Separation of Isoperoxidases by IEF
Soluble proteins were extracted from roots, hypocotyls, and
cotyledons by grinding in 20 mM Hepes, pH 7.0, containing 1 mM EGTA (1 mL for each 1 g fresh weight). The extract
was filtrated and centrifuged for 10 min at 10,000g.
Proteins were assayed with Coomassie Blue reagent (Spector, 1978).
Extracellular proteins were obtained by the following procedure.
Segments of 1 cm taken throughout hypocotyls were placed in 20 mM Hepes, pH 7.0, containing 5 mM EGTA or 2 mM
CaCl2 and subjected to four 1-min periods of vacuum. The segments were then collected, dried on absorbing paper, and
centrifuged for 5 min at 1,000g in a swingout rotor to
collect the intercellular fluid, which was used directly for IEF
separation performed as described previously (Penel and Greppin, 1996).
Peroxidase bands were visualized with o-dianisidine/hydrogen
peroxide.
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RESULTS |
APRX Purification and Localization
APRX was purified from whole etiolated seedlings as described
previously (Penel and Greppin, 1996). The procedure yielded one major
acidic peroxidase band (Fig. 1, lane a)
that was found in extracts from roots, hypocotyls, and cotyledons. As
cotyledons contain many proteins, a larger quantity of proteins had to
be electrophoresed on the gel to detect APRX. In hypocotyls APRX could
be released from extracellular spaces after vacuum infiltration, showing that it was an apoplastic isoperoxidase (Fig. 1). In addition, the amount of APRX that could be recovered by this technique was higher
when vacuum infiltration was performed in the presence of EGTA,
suggesting the calcium-dependent binding of this enzyme in the
apoplast. A cationic isoperoxidase was found to exhibit the same
calcium-dependent behavior, as shown previously (Penel and Greppin,
1994, 1996). In contrast, a moderately cationic band was recovered only
when calcium was present in the infiltration buffer (Fig. 1).
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| Figure 1.
Peroxidase isoenzymes from various fractions of
zucchini seedlings separated on acrylamide gel by IEF. Extracts are
from hypocotyls (H) (10 µg of proteins); cotyledons (C) (100 µg);
roots (R) (10 µg); purified APRX (a) (28 ng); and extracellular fluid
obtained in the presence of 5 mM EGTA (E) or 2 mM CaCl2 (C). Arrowheads indicate the positions
of APRX on the gel.
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The N-terminal amino acid sequence of APRX was determined by subjecting
purified enzyme to two-dimensional electrophoresis and transferring it
onto a PVDF membrane. Microsequencing was then carried out after
deblocking of the N terminus. The sequence obtained,
TETFYDQTCPRLPNIVRQ, was 64% identical to a poplar (accession no.
X97348) and a flax (accession no. L07554) peroxidase.
Isolation and Sequencing of the APRX cDNA Clone
The cDNA encoding APRX was obtained using a cDNA library prepared
from etiolated zucchini hypocotyls. PCR reactions using the specific
primers 1, 2, and 3 generated a 1,145-bp cDNA clone that encoded a
plant peroxidase. To obtain the complete sequence, including the signal
peptide and the 5-untranslated sequences, we used the 5-RACE-PCR
procedure, with the specific primer 4 and primer AP1 from the cDNA
amplification kit. The resulting 238-bp clone was sequenced and
exhibited an overlap of 157 bp with the first 1,145-bp clone without
any sequence discrepancy. APRX was therefore encoded by a 1,226-bp cDNA
(Fig. 2). The open reading frame
corresponded to a deduced protein of 309 amino acids with a signal
peptide of 16 amino acids. It exhibited a calculated Mr of 33,894 and a pI of 5.1. The N
terminus of the mature protein was blocked by a pyroglutamyl residue.
Like other peroxidases, this protein contained two putative
N-glycosylation sites (Asn-X-Thr/Ser). In addition, the cDNA
contained a 5-untranslated leader sequence of 6 bp and a 242-bp
sequence in the 3-untranslated region, without the animal consensus
polyadenylation signal or a known plant polyadenylation signal.
Southern analysis performed on zucchini leaf DNA digested with several
restriction enzymes showed that APRX was encoded by a single gene (data
not shown).
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| Figure 2.
The complete nucleotide and deduced amino acid
sequences of APRX (accession no. Y17192). The signal peptide is shown
in italics and the C-terminal propeptide is underlined. The four
primers used to produce the full cDNA are shown. The two boxes
designate the putative glycosylation sites. The stop codon is marked
with an asterisk.
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A comparison of APRX mature protein with the peroxidase sequences
available in databases was made using the Gap program (Genetics Computer Group, Madison, WI). The best similarity scores were with two
cucumber anionic peroxidases: 74% with accession no. M91373 and 88%
with accession no. M91374 (Rasmussen et al., 1995) and 70% with
tobacco anionic peroxidase (accession no. J02979; Lagrimini et al.,
1987) (Fig. 3). Like other plant
peroxidases, APRX contained eight Cys residues at positions 11, 44, 49, 90, 96, 174, 206, and 294, allowing the formation of four disulfide bridges (Kjærsgård et al., 1997). The amino acid sequence of mature APRX exhibited the conserved distal catalytic residues Arg-38 and
His-42 and the proximal His-167 and Asp-241, which characterize plant
peroxidases (Welinder, 1992).
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| Figure 3.
Mature peroxidase protein sequence comparison. The
predicted protein sequences encoded by zucchini APRX
clone (this study), two cucumber peroxidase cDNAs, Csprepera and
Cspreper (Rasmussen et al., 1995), and a tobacco peroxidase cDNA,
Ntpxdlf (Lagrimini et al., 1987), are shown. The alignment was created
with the PileUp program and sequence homology was determined with the
PrettyPlot program (both programs from Genetics Computer Group).
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APRX Expression in Zucchini Seedlings
The level of expression of APRX was assessed in various
organs of zucchini seedlings after RNA separation by electrophoresis and transfer onto a membrane. As shown in Figure
4, the mRNA encoding APRX was detected
in hypocotyls, in roots, and, to a much lesser extent, in
cotyledons.
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| Figure 4.
Northern-blot analysis using total RNA (10 µg)
from root (R), hypocotyls (H), and cotyledons (C). APRX
mRNA was detected with the 1145-bp cDNA as a specific probe. Detection
of ribosomal 18S RNA was performed as a control.
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In situ hybridization was used to identify the tissues or cells
expressing APRX using a 300-bp specific probe corresponding to the 3-flanking untranslated region (Figs.
5-10). The mRNA encoding APRX was
present in almost all cell types, but with great differences in the
level of expression. Figure 5 shows root cross-sections taken 1 mm
above the tip. At this level the differentiation of vascular tissues
within the stele was not achieved. Only some protoxylem vessels could
be distinguished. Control sections incubated with the sense probe did
not show any staining (Fig. 5A) in any tissue observed in this work
(Figs. 5-10). With the antisense probe the strongest staining was
observed in the stele and in the epidermis (Fig. 5B). Epidermal cells
exhibited an asymmetric distribution of transcripts that accumulated in
the part of the cytoplasm facing the exterior of the root (Fig. 5C).
This polarized distribution of APRX mRNA was not observed in
other cell types. Lateral root primordia appeared in longitudinal
sections made 2 or 3 mm above the tip (Fig.
6) and were heavily stained, with all
cells containing a large amount of APRX-encoding mRNA.
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| Figure 5.
Detection of APRX mRNA by in situ
hybridization of root cross-sections. Sections were hybridized with
sense (A) or antisense (B and C) probe. co, Cortex; ep, epidermis; st,
stele; rc, root cap. Arrows indicate the accumulation of transcripts in
epidermal cells. Scale bars are in µm.
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| Figure 6.
Detection of APRX mRNA by in situ
hybridization in root longitudinal sections showing lateral root
primordia. Sections were hybridized with sense (A) or antisense
(B) probes. Scale bars are in µm.
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APRX expression varied in the various tissues of the
hypocotyl. To study this expression, in situ hybridization was
performed on longitudinal sections taken at different levels.
APRX transcripts were much more abundant in the upper part
of the hypocotyl than at its base. Some cells of the vascular bundles
were particularly heavily stained throughout the hypocotyl, but mainly
in the upper part, including the hypocotyl hook and the elongation zone
(Fig. 7). They were frequently aligned
one below the other (Fig. 7, B-D), which could mean that they
correspond to the formation of vascular structures. Some large cells
adjacent to the characteristic tracheary elements exhibited a strong
accumulation of transcripts at their two extremities (Fig. 7C). These
were identified as differentiating xylem elements because of their
close association with typical xylem structures and because of their
secondary wall thickenings. In the hook the distribution of transcripts
was uneven (Fig. 8). Cells of the inner
epidermis and cortical cells that were situated just below contained
more transcripts than cells of the outer part. These latter cells were
also much smaller than cells forming the outer epidermis.
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| Figure 7.
Detection of APRX mRNA by in situ
hybridization in hypocotyl longitudinal sections taken 1 cm beneath the
hook (A-D) or at the hook level (E). A, General view of a section
hybridized with the antisense probe. B and C, Detailed views showing
differentiating tracheary elements. D, Control section hybridized with
the sense probe. Arrows in C indicate the accumulation of transcripts
in differentiating xylem elements. Scale bars are in µm.
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| Figure 8.
Detection of APRX mRNA by in situ
hybridization in longitudinal sections of the hypocotyl hook. Sections
were hybridized with antisense (A and B) or sense (C) probes. ie, Inner
epidermis; oe, outer epidermis; vt, vascular tissues. Scale bars are in
µm.
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In cotyledons the presence of APRX transcripts was detected
in lower and upper epidermis, in the two ranges of palisade parenchyma cells, and in the vascular system (Fig.
9). In some palisade cells, the nucleus
was strongly labeled, indicating the presence of pre-mRNA-encoding APRX. Some cells of the vascular system exhibited strong
APRX mRNA expression.
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| Figure 9.
Detection of APRX mRNA by in situ
hybridization in transversal sections of cotyledons. Sections were
hybridized with sense (A) or antisense (B) probes. Upper (C) and lower
(D) parts of the cotyledon were hybridized with the antisense probe.
le, Lower epidermis; pp, palisade parenchyma; ue, upper epidermis; VT,
vascular tissues. Scale bars are in µm.
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APRX mRNA Expression during Adventitious Root
Formation
Root regeneration on zucchini hypocotyls occurred spontaneously 4 to 5 d after the ablation of their root system. Many root primordia appeared at the border of a vascular bundle or near the
interfascicular cambium (Fig.
10). In
situ hybridization revealed strong expression of APRX gene
in the young root primordia, which contrasted with the slight staining
of the surrounding hypocotyl cells (Fig. 10B). This massive expression
decreased as the primordia became longer, which was particularly
evident in roots emerging from the hypocotyl (Fig. 10D). Cells
belonging to the quiescent center of a young root were not stained
(Fig. 10C). Adventitious root formation on hypocotyls was prevented by
the addition of 104 M
TIBA, an anti-auxin substance known to inhibit root formation (Fujita
and Syôno, 1996) (data not shown). Figure 11 shows that the
relatively high level of APRX transcripts that could be
detected in the lower part of hypocotyls by northern analysis was
maintained during the days after root excision. In contrast, in
hypocotyls maintained in the presence of TIBA, the level of transcripts
that could be detected in the lower part of the hypocotyls was already substantially lower 1 d after the beginning of TIBA application.
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| Figure 10.
Detection of APRX mRNA by in situ
hybridization in cross-sections of hypocotyls. Shown are general views
of adventitious root primordia in sections hybridized with sense (A) or
antisense (B) probes, a young adventitious root (C), and an
adventitious root emerging out of the hypocotyl (D) hybridized with the
antisense probe. qc, Quiescent center; rp, root primordium; vb,
vascular bundle; yar, young adventitious root. Scale bars are in
µm.
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| Figure 11.
Detection of mRNA encoding APRX in the lower part
of hypocotyl of zucchini seedlings 1 to 4 d after excision of
their root system. Rootless seedlings were kept without ( ) or with
(+) 104 M TIBA. Northern analysis was
performed with 10 µg of total RNA. The detection of ribosomal 18S RNA
was performed as a control.
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DISCUSSION |
The zucchini APRX purified and cloned in the present study is an
apoplastic enzyme that was previously shown to have a strong affinity
for pectin in the presence of calcium ions in cell walls both in vitro
and in situ (Penel and Greppin, 1994, 1996; Penel et al., 1996). The
larger amount of APRX that could be recovered from hypocotyl segments
when EGTA was present in the buffer used for the vacuum infiltration
(Fig. 1) confirmed the importance of calcium for the binding of this
enzyme within the apoplast.
APRX is the first isoperoxidase from zucchini to be sequenced. The
strongest homology it exhibits is with two APRXs from cucumber and one
from tobacco. None of the numerous Arabidopsis peroxidase sequences
that are known exhibits a homology higher than 58%. The
Mr calculated from the deduced amino acid
sequence was 33,894, whereas it was previously estimated to be
approximately 41,400 by SDS-PAGE (Penel and Greppin, 1994). The
difference could be explained by the presence of carbohydrate chains,
since it appears from the sequence analysis that APRX contains two
putative glycosylation sites. There is also a difference between the
calculated pI (5.1) and the pI that was determined experimentally (4.3)
(Penel and Greppin, 1994). APRX sequence analysis also
showed the presence of a C-terminal propeptide that is generally
supposed to target the protein toward the vacuole (Johansson et al.,
1992; Neuhaus et al., 1994; Omann and Tyson, 1996). In spite of that,
APRX was found at least partly in apoplastic spaces (Fig. 1). Other
proteins containing a C-terminal extension, such as  -mannosidase,
chitinase, and glucanase, were also reported to have an extracellular
localization, and the hypothesis was put forward that these enzymes
were secreted into the medium via a pathway in which the proteins pass
through the vacuolar compartment (Kunze et al., 1998).
The APRX gene appears to be expressed in all parts of
zucchini seedlings. Roots, hypocotyls, and cotyledons contain the mRNA encoding the isoperoxidase and the isoperoxidase itself. Both are also
present in leaves and other organs of light-grown plants (data not
shown), probably indicating an essential role for this isoperoxidase. A
similar wide expression was reported for some isoperoxidases
(Kjærsgård et al., 1997; Teichmann et al., 1997; Klotz et al., 1998),
whereas others exhibit an organ-specific expression or are synthesized
only after a specific treatment (Morgens et al., 1990; Mohan et al.,
1993; Rasmussen et al., 1995; Omann and Tyson, 1996). The northern
analyses done in this work (Fig. 4) could suggest that hypocotyl cells
contain a higher amount of transcripts than root and cotyledon cells.
The detection of transcripts on sections does not completely confirm
such a conclusion (Figs. 5-10). Actually, the three organs contain
cells that express APRX quite strongly.
The data obtained by in situ hybridization showed that APRX
expression is tissue-specific and developmentally regulated, as was
reported for other peroxidases such as the anionic peroxidase of
tobacco (Klotz et al., 1998). In the hypocotyl there is a gradient of
expression, with the upper part being more active than the lower part.
Root primordia also exhibited a strong expression. Therefore, like many
other peroxidases, the presence of APRX must not be
correlated with cell growth cessation (Gaspar et al., 1982; Zheng and
Van Huystee, 1992; Lagrimini et al., 1997). In the hypocotyl hook,
however, small cells from the inner zone contained more transcripts
than large cells situated in the outer part (Fig. 8). As seen on
longitudinal sections from upper hypocotyls (Fig. 7), the transcripts
accumulated mainly in vascular bundles in files of cells that most
likely correspond to differentiating vessels.
Elongated cells exhibiting secondary thickenings of their walls were
also heavily labeled at their two extremities, indicating that APRX
could be involved in lignin deposition during the formation of xylem
vessels. Such a function has been repeatedly attributed to peroxidases
and has been confirmed by cytochemical and biochemical studies
(Catesson et al., 1986; Ros Barceló, 1995; Sato et al., 1995;
Christensen et al., 1998) and by immunolocalization (Smith et al.,
1994). It is shown here, for the first time to our knowledge, that one
particular peroxidase gene is expressed in xylem cells at the beginning
of their differentiation.
APRX transcripts were also abundant in the epidermis of
cotyledons and in the inner part of the hypocotyl hook and roots. The
reason for this presence in dermal tissues has not been elucidated. The
epidermis was often shown to contain a strong peroxidase activity (Gaspar et al., 1982), which could be involved in the polymerization of
phenolics to produce the lignin-like material found in cutin and
suberin (Riley and Kolattukudy, 1975; Hendricks and van Loon, 1990).
Young lateral roots (Fig. 6) and adventitious root primordia (Fig. 10)
also exhibited a strong expression in every cell. Peroxidases have
often been used as marker enzymes in the rooting process (Gaspar et
al., 1992). The characteristic evolution of peroxidases during
rhizogenesis includes always an increase in the activity of APRXs
during the late phase of this process, namely the appearance of root
primordia (Gaspar et al., 1992). The function of peroxidase in these
rapidly growing tissues remains to be discovered. APRX mRNA
was still produced near the tips of fully developed roots and was
expressed in almost every cell, but at different levels. The stronger
expression was observed in the stele and in epidermal cells, mainly in
the part of cytoplasm facing the root surface. One possible
interpretation of this uneven distribution could be that the
neo-synthesized APRX molecules are synthesized and secreted mainly or
exclusively toward the root surface. In addition to the transport of
proteins, the control of mRNA localization within the cytoplasm
constitutes an important mechanism to orientate the distribution of
newly synthesized proteins (Okita et al., 1998).
This work provides some indications about a possible correlation
between the level of auxin and the rate of expression of the
APRX gene. Both differentiating tracheary cells and cells forming young root primordia contain high amounts of transcripts. These
two processes, vascular differentiation and root initiation and growth,
are known to be promoted by auxin (Davies, 1987). In addition,
treatment with the anti-auxin TIBA drastically lowered the level of
transcripts that could be extracted from the bottom of rootless
hypocotyls (Fig. 11). As an auxin transport inhibitor, TIBA acts most
likely by preventing auxin accumulation in the lower part of hypocotyls
(Depta et al., 1983), inhibiting adventitious root formation (Fujita
and Syôno, 1996). This positive correlation with auxin could also
explain the high amount of APRX transcripts found in
hypocotyl hook inner epidermis (Fig. 8), which was shown to contain up
to 10-fold more IAA than the outer epidermis (Schwark and Bopp, 1993).
According to the results presented here, the main sites of expression
of the gene encoding APRX are the dermal tissues and xylem elements
during their differentiation. This expression pattern suggests that
APRX could be responsible for the formation of lignin or lignin-like
material found in cutin and suberin, as was suggested for other APRXs
(Kolattukudy, 1987; Hendricks and van Loon, 1990; Teichmann et al.,
1997). APRX could use its pectin-binding property to associate the
newly synthesized polymers to the cell wall. It has been reported that
the phenolic domain of the cuticle or the suberized layers is probably
attached to the cell wall through linkages with pectinaceous domains
(Kolattukudy, 1987).
Cells of very young roots and of cotyledon palisade parenchyma also
exhibit a strong expression. At this point it is difficult to put
forward a hypothesis concerning the possible function of APRX in these
cells. Both root primordia and palisade parenchyma are rather compact
tissues, consisting of cells that seem to be tightly associated. APRX
could have a role in the establishment of the close association
existing between these cells at the cell wall level.
Many plant functions are commonly attributed to peroxidases, with most
occurring in the apoplast. This study and other works cited herein
demonstrate that each particular peroxidase isoenzyme exhibits a
specific expression pattern within the plant. The detailed study of
this expression, combined with a precise knowledge concerning both the
catalytic characteristics of the isoperoxidase and the structural
features that could determine its localization within the extracellular
matrix, should allow for progress in the understanding of the real role
of peroxidases in plants. APRX, which is preferentially expressed in
particular tissues of zucchini seedlings and which binds specifically
to pectins in the presence of calcium, is likely to provide a good
model for this kind of study.
| |
FOOTNOTES |
1
This work was partly supported by the Federal
Office of Education and Science (grant no. 93-0090). S.C. received a
grant from the Agence Universitaire de la Francophonie.
*
Corresponding author: e-mail Claude.Penel{at}bota.unige.ch; fax
41-22-329-7795.
Received January 28, 1999;
accepted March 23, 1999.
| |
ABBREVIATIONS |
Abbreviations:
APRX, anionic isoperoxidase.
RT, reverse
transcriptase.
TIBA, 2,3,5-triiodobenzoic acid.
| |
ACKNOWLEDGMENTS |
We thank Evelyne Vasquez for her excellent technical assistance
and Dr. S. Hamdi (University of Tours, France) for generously providing
the ribosomal 18S cDNA clone.
| |
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Top
Abstract
Introduction