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Plant Physiol. (1999) 120: 501-512
Barley Coleoptile Peroxidases. Purification, Molecular Cloning,
and Induction by Pathogens1
Brian Kåre Kristensen*,
Helle Bloch, and
Søren Kjærsgaard Rasmussen
Plant Biology and Biogeochemistry Department, PBK-301, Risø
National Laboratory, P.O. Box 49, DK-4000 Roskilde, Denmark (B.K.K.,
S.K.R.); and Department of Biochemistry and Nutrition, Technical
University of Denmark, DK-2800 Lyngby, Denmark (H.B.)
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ABSTRACT |
A cDNA clone encoding the Prx7
peroxidase from barley (Hordeum vulgare L.) predicted a
341-amino acid protein with a molecular weight of 36,515. N- and
C-terminal putative signal peptides were present, suggesting a vacuolar
location of the peroxidase. Immunoblotting and reverse-transcriptase
polymerase chain reaction showed that the Prx7 protein and mRNA
accumulated abundantly in barley coleoptiles and in leaf epidermis
inoculated with powdery mildew fungus (Blumeria graminis). Two isoperoxidases with isoelectric points of 9.3 and 7.3 (P9.3 and P7.3, respectively) were purified to homogeneity from
barley coleoptiles. P9.3 and P7.3 had Reinheitszahl values of 3.31 and
2.85 and specific activities (with
2,2 -azino-di-[3-ethyl-benzothiazoline-6-sulfonic acid], pH 5.5, as
the substrate) of 11 and 79 units/mg, respectively. N-terminal amino
acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry peptide analysis identified the P9.3
peroxidase activity as due to Prx7. Tissue and subcellular accumulation
of Prx7 was studied using activity-stained isoelectric focusing gels
and immunoblotting. The peroxidase activity due to Prx7 accumulated in
barley leaves 24 h after inoculation with powdery mildew spores or
by wounding of epidermal cells. Prx7 accumulated predominantly in the
epidermis, apparently in the vacuole, and appeared to be the only
pathogen-induced vacuolar peroxidase expressed in barley tissues. The
data presented here suggest that Prx7 is responsible for the
biosynthesis of antifungal compounds known as hordatines, which
accumulate abundantly in barley coleoptiles.
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INTRODUCTION |
The structurally diverse secretory plant peroxidases constitute a
class within the superfamily of peroxidases (EC 1.11.1.7; H2O2 oxidoreductase)
(Welinder, 1992 ). Each group of structural relatives in the superfamily
is likely to be involved in specific and distinct biosynthetic
pathways. Plant development and environmental changes, including biotic
stress, are often followed by dramatic changes in peroxidase activity
and in the number of isoperoxidases present in specific tissues.
Peroxidases participate in a variety of plant defense mechanisms
(Moerschbacher, 1992) in which
H2O2 is often supplied by
an oxidative burst, a common event in defense responses (Lamb and
Dixon, 1997). The cell wall appears to be a major site for
defense-related peroxidase polymerization reactions such as
lignification (Hammerschmidt and Kuc, 1982), suberization (Espelie et
al., 1986), cross-linking of structural cell wall proteins (Bradley et
al., 1992), and dimerization of ferulate esters (Ikegawa et al., 1996).
Appositions at the cell wall known as papillae are induced in the
pathogenic interaction between barley (Hordeum vulgare L.) and the biotrophic powdery mildew fungus (Blumeria [syn.
Erysiphe] graminis f.sp. hordei)
(Aist and Bushnell, 1991; Collinge et al., 1997) and are a
race-nonspecific defense component of major importance. The papilla
response is well described at the cellular and biochemical levels, with
active phenylpropanoid biosynthesis (Carver et al., 1994) and early
accumulation of peroxidase mRNA (Boyd et al., 1994), apparently
important processes for inhibition of the fungus.
Several proteins, such as peroxidases (Scott-Craig et al., 1995),
accumulate in the papillae and in the leaf (Kerby and Somerville, 1989;
Thordal-Christensen et al., 1992). The papillae are surrounded by an
autofluorescent halo, which is most likely due to the presence of
phenolic compounds. There is also evidence suggesting that these
phenolics are polymerized p-coumarylagmatine and
p-coumarylhydroxyagmatine (Wei et al., 1994; von
Röpenack et al., 1998). Hordatines, soluble dimers of the hydroxycinnamic acid amides p-coumarylagmatine
and ferulylagmatine, have antifungal activity and can be isolated from
barley coleoptiles (Stoessl, 1967) or dark-grown seedlings (Stoessl and
Unwin, 1970). Coleoptiles are resistant to invasion by
Cochliobulus sativus ([Ito and Kurib.] Drechs. ex Dastur), allowing the seedling to remain free from infection for the first few
days after emergence. There is correlative evidence that hordatines are
the mediator of this resistance (Stoessl, 1967, and refs. therein).
Hordatines also accumulate slowly but substantially in the barley leaf
in response to powdery mildew fungus attack (Smith and Best, 1978).
Stoessl (1967) showed that hordatines could be generated in vitro by
horseradish peroxidase from the appropriate
p-coumarylagmatines, suggesting that a peroxidase mediates
the last step in the biosynthetic pathway to hordatine.
The barley Prx7 peroxidase was first cloned as a partial cDNA from
dark-grown barley (Rasmussen et al., 1991a). The gene mapped tightly to
the MlLa powdery mildew resistance locus on chromosome 2 (Giese et al., 1993). Because it was the seventh peroxidase gene to be
mapped in barley, the gene was designated Prx7 and the
encoded protein Prx7. Prx7 mRNA has been shown to accumulate in
response to pathogens in barley leaves (Thordal-Christensen et al.,
1992) and roots (Valé et al., 1994) and also in the barley cell-elongation mutant Slender (Schünmann et al.,
1994). In the present study we describe the cDNA cloning, purification,
and characterization of Prx7.
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MATERIALS AND METHODS |
Plant and Fungal Material
Barley (Hordeum vulgare L. cv Pallas near-isogenic line
P-02; Kølster et al., 1986) plants were grown in a 16-h photoperiod at
220 µE m 2 s1, at
18°C day/16°C night temperatures, and at a constant (65%) RH.
Coleoptiles were cut from 7-d-old barley seedlings. The barley powdery
mildew fungus isolates C15 and A6 were used to inoculate plants and
were maintained on cv Manchuria. The primary leaves of 7-d-old barley
plants were held down on a horizontal plexiglass plate to expose the
abaxial epidermis for inoculation. Inoculation was performed in a
settling tower using pressurized air to release spores from the
conidial chains. Wounding of 7-d-old primary leaves was done by gently
rubbing powdered silicon carbide (no. 2FE coarse, Carborundum Company,
Manchester, UK) onto the leaves. Epidermal strips and the remaining
mesophyll from primary barley leaves were obtained by removing spores
from the abaxial epidermis with moist cotton balls. An incision was
then made through the adaxial epidermis and mesophyll. The cut side of
the epidermis was immobilized on electrical tape, and the intact
inoculated epidermis was stripped off. Epidermal strips and mesophyll
were immediately frozen in liquid nitrogen.
Cloning of pcD1311BE
A barley leaf cDNA library in  phage (ZAP-XR, Stratagene)
(Scott-Craig et al., 1995) was screened under low-stringency conditions with 5× SSPE (20× SSPE is 3 M NaCl, 0.2 M
NaH2PO4, and 0.02 M EDTA), by probing with the partial clone (pcD1311,
accession no. X62438) as described previously (Rasmussen et al.,
1991a). One positive plaque was purified after a wash at high
stringency with 0.1× SSPE. The 1316-bp
BlnI/EcoRI fragment of the insert was blunt-ended
and subcloned into the SmaI site of pUC13. This clone was
designated pcD1311BE (accession no. AJ003141).
DNA Sequencing and RNA and DNA Manipulations
Sequencing was carried out on a DNA sequencer (model ABI 373, Perkin-Elmer) using a dye-terminator cycle-sequencing kit (Thermo Sequenase, Amersham). Both strands were sequenced using internal primers. Sequences were proofread using Sequencher software (version 3.0, Gene Codes Corporation, Ann Arbor, MI). Plasmids for sequencing were prepared using the Wizard kit (Promega). Restriction digests and
other standard procedures were as instructed by the manufacturers or by
using the method of Sambrook et al. (1989). Total RNA was prepared from
frozen tissue samples by the method of Chirgwin et al. (1979). RT-PCR
using beads (Ready-to-Go, Amersham-Pharmacia Biotech, Allerød,
Denmark) was essentially as described by the manufacturer.
Before reverse transcription, 0.8 µg of total RNA was denatured in
the presence of 0.5 µg of oligo-T and 1 unit/µL RNasin (Promega) in
a volume of 33 µL for 5 min at 95°C. The RNA/oligo-T mixture was
then divided and transferred to three PCR tubes for separate
amplification of GAPDH, Prx7, and Prx8 mRNA, each in a final volume of
50 µL. Negative controls in which the murine leukemia virus RT
had been heat inactivated were used to check for DNA contamination, and
no fragments were amplified in these controls. Reverse transcription
was carried out without amplification primers at 42°C for 20 min.
After the addition of the relevant primers and initial denaturing at
95°C for 5 min, PCR was carried out for 25 cycles using the following
protocol: denaturing at 94°C for 45 s, annealing at 58°C for
45 s, and elongation at 72°C for 1 min. The primers TH1
(5-TATCTCTCACATGTCAGCGGC-3) and TH2 (5-TACTACTTCGACCTGATCGCG-3)
were used to amplify a 277-bp fragment from Prx7 mRNA. The primers
RT-Prx8F (5-TGTTCAACAACGACACCACC-3) and RT-Prx8R
(5-CATTCACGTGTCGTGCTAGC-3) were used for Prx8 mRNA to generate a
222-bp fragment. GAPDH1 (5-CAAGGACTGGAGRGGTGG-3) and GAPDH2
(5-CCCACTCGTTGTCRTACC-3) were used to amplify a 376-bp fragment from
mRNA corresponding to the barley GAPDH gene as an internal positive
control.
Construction of Recombinant Prx7 Plasmid and Expression in
Escherichia coli
The blunt-ended BglI-DraI fragment of
pcD1311BE was ligated into the blunt-ended BamHI site of the
pET15b expression vector (Novagen, Madison, WI), and the orientation
was confirmed by sequencing. E. coli BL21 (DE3) pLysS
harboring the recombinant Prx7 (rPrx7) construct was grown in
superbroth (32 g of tryptone, 20 g of yeast extract, and 5 g/L
NaCl, pH 7.0) at 37°C. Induction of rPrx7 expression involved the
addition of isopropyl- -D-thiogalactoside to
0.5 mM at an A600 of
10 to 12. Cells were harvested after an additional 4 h of
incubation and lysed by two freeze-thaw cycles, followed by sonication
in the presence of denaturing buffer I (8 M urea, 100 mM
NaH2PO4, and 10 mM Tris-HCl, pH 8.0). Solubilized His-tagged rPrx7 was purified from the supernatant by
Ni+-nitrilotriacetic acid agarose (Qiagen,
Hilden, Germany) affinity chromatography as described by the
manufacturer. His-tagged protein was desalted on a P-6 Bio-Gel column
(Bio-Rad) and bound on a Resource-Q column (Amersham-Pharmacia
Biotech). Protein was eluted with a linear gradient of 20 mM Tris-HCl, pH 8.6, 4 M
urea, and 1 M NaCl in the loading buffer. The
purity of the rPrx7 fractions was verified by SDS-PAGE.
Production of Polyclonal Antibodies
Anti-rPrx7 antibodies were raised in Danish White rabbits (Dako,
Glostrup, Denmark). Immunoglobulins in blood serum were purified on
protein A-agarose beads (KemEnTec, Copenhagen, Denmark) as described by
Harlow and Lane (1988).
Purification of Peroxidases from Coleoptiles
Coleoptiles (75 g fresh weight) were ground to a fine powder in
liquid nitrogen using a mortar and pestle. The powder was extracted for
2 h with 500 mL of buffer II (50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, and 0.2 mM PMSF) at 4°C,
filtered, and centrifuged for 30 min at 16,000g. Protein in
the cleared crude extract was precipitated by the addition of saturated
(NH4)2SO4
to 60% saturation and incubated for 4 h. The precipitate was
collected by centrifugation at 15,000g for 40 min,
redissolved in 60 mL of deionized water, and dialyzed overnight against
2× 5 L of dialysis buffer C (10 mM Na-acetate,
pH 5.2, and 1 mM CaCl2). The
remaining peroxidase in the
(NH4)2SO4
supernatant was batch adsorbed to 20 mL of Phenyl-Sepharose CL4B medium
(Amersham-Pharmacia Biotech). Bound protein was eluted stepwise using
buffer III (15 mM Na-acetate, pH 4.75). The first 120 mL eluted was dialyzed against 10 mM
Na-acetate, pH 5.2, and 1 mM CaCl2,
and the volume was adjusted to 200 mL. All steps were performed at
4°C or on ice unless indicated otherwise.
Column-chromatography purification steps were performed on a
fast-protein liquid chromatography system (Amersham-Pharmacia Biotech)
at room temperature. Dialyzed protein was loaded separately onto a 6-mL
Resource-S column, and bound proteins were eluted with a linear
gradient of buffer IV (15 mM Na-acetate, pH 4.75, and 1.0 M NaCl). Unbound activities were purified on a 1-mL
Resource-Q column in 50 mM Tris-HCl, pH 8.0, and eluted
with a linear gradient of buffer V (50 mM Tris-HCl, pH 8.0, and 1.0 M NaCl). Fractions with peroxidase activity were
pooled and prepared for HIC by adding (NH4)2SO4
to a final concentration of 1.5 M. HIC was performed on a
1.7-mL phenyl ether column (POROS-PE, PerSeptive Biosystems, Framingham, MA) equilibrated in 50 mM Tris-HCl, pH 8.0, and
1.5 M
(NH4)2SO4,
and the bound peroxidases were eluted with a linear gradient of 50 mM Tris-HCl, pH 8.0. Fractions were analyzed by electrophoresis (IEF, SDS-PAGE, and immunoblotting).
N-Terminal Amino Acid Sequencing
Purified P9.3 and P7.3 were run on SDS-PAGE, blotted onto a PVDF
membrane, and stained with Coomassie Brilliant Blue R-250. Bands were
cut from the membrane and the proteins were deblocked by cleavage of
the N-terminal pyroglutamic acid with pyroglutamyl amino peptidase (EC
3.4.19.3) (Unizyme Laboratories, Hørsholm, Denmark) as described
previously (Hirano et al., 1993). Amino acid sequencing was performed
on a sequencer (model 477A, Applied Biosystems) with an on-line
phenylthiohydantoin amino acid analyzer.
MS and Tryptic in-Gel Digestion
After SDS-PAGE, the protein band from the Coomassie Blue-stained
gel was excised, washed in 200 µL of 100 mM
NH4HCO3, and treated with 3 mM DTT (Sigma) and 100 mM
NH4HCO3 in 150 µL at 60°C for 30 min. The protein was alkylated by the addition of 10 µL
of 100 mM iodoacetamide (Merck, Darmstadt, Germany) and incubation for 30 min in the dark at room temperature. The solvent was
discarded and the gel piece was washed in 500 µL of 50% (v/v) CH3CN (Merck) and 100 mM
NH4HCO3 for 1 h. The
gel pieces were dehydrated in 50 µL of CH3CN
for 10 min, dried under vacuum, and reswelled for 10 min with 10 µL
of 25 mM
NH4HCO3 containing 0.2 µg
of trypsin (EC 3.4.21.4) (Sigma). After the addition of 20 µL of 25 mM NH4HCO3, the
digestion was carried out overnight at 37°C. The supernatant was
saved and the peptides were extracted from the gel slices twice with 50 µL of 60% CH3CN and 0.1% (v/v) trifluoroacetic acid (Sigma) for 20 min. The supernatant and extracts were combined and dried under vacuum. Peptides were reconstituted in
0.1% (v/v) trifluoroacetic acid and mixed 1:1 (v/v) with the matrix
(33 mM  -cyano-4-hydroxy-trans-cinnamic acid
[MALDI grade] from Hewlett-Packard). The mass spectra of the peptides
were recorded in the range up to 104 D in a
linear positive mode with an acceleration voltage of 28 kV using a
MALDI-TOF MS system (model G2025A, Hewlett-Packard) with a sampling
rate of 200 MHz. All spectra were recorded at a tube pressure of less
than 9 × 106 torr. To analyze the whole
protein, sinapinic acid (Hewlett-Packard) was used as the matrix
and the mass spectra were recorded in the range up to
105 D.
Enzyme Assays
A rapid spot test for peroxidase activity based on a guaiacol
substrate (80 mM guaiacol [Sigma], 80 mM
H2O2 [Merck], and 50 mM Na-acetate, pH 5.5) was used for visual monitoring of
column fractions. Substrate (10 µL) was spotted onto Parafilm
(American National Can, Greenwich, CT), followed by 5 µL of the
sample fraction. Activity was estimated visually. Specific activities
were measured at 20°C on an ELISA plate scanner (Titertek Multiscan
MKII, Flow Laboratories, Lugano, Switzerland) equipped with a 405-nm
filter using 100 mM Na-acetate, pH 5.5, 0.36 mM
ABTS (Sigma), and 6 mM H2O2 as substrate. The
increase in absorbance was followed for 90 s at 10-s intervals. In
each reaction, 0.035 to 4.5 µg of protein (based on
A280) was analyzed. The absorption
coefficient for the ABTS product used was 36.0 mM1
cm1 (Childs and Bardsley, 1975). Absorption
spectra and Reinheitszahl (A403-408/A280)
values were determined using a spectrophotometer (model UV2101PC,
Shimadzu Scientific Instruments, Columbia, MD, or model M4QII, Zeiss).
Protein Sample Preparation
Total protein was prepared from barley tissues by homogenizing
frozen samples in liquid nitrogen using a mortar and pestle. The frozen
tissue powder was extracted with 2 volumes (v/w) of ice-cold buffer (50 mM K-acetate, pH 5.2, 100 mM KCl, 1 M NaCl, 1 mM CaCl2, 1 mM ascorbic acid, 0.1% Triton X-100, and 0.2 mM PMSF) followed by centrifugation at 15,000g
for 10 min at 4°C and measurement of protein concentration. For IEF
the supernatant was dialyzed against 3 mM
Na-acetate, pH 5.2, containing 1 mM CaCl2 followed by lyophilization and measurement of protein
concentration. Extraction of extracellular proteins was performed by
vacuum infiltration and low-speed centrifugation as described
previously (Kerby and Somerville, 1989) using 50 mM K-acetate, pH 5.2, and 100 mM KCl as an infiltration buffer. This extraction
method does not lead to leakage of intracellular protein into the
washing fluid (Kristensen et al., 1997). The extracted washing fluid
and all other protein samples were adjusted to 1 mM CaCl2 and 1 mM ascorbic acid before storage at  20°C.
Protein Determination
Protein was quantified in duplicate by spectrophotometry using
A2800.1% (w/v) = 1 cm1 or according to the method of Bradford
(1976), with IgG as the standard protein (Bio-Rad).
Electrophoretic Protein Analyses and Immunoblotting
Proteins separated by SDS-PAGE (Laemmli, 1970) were stained with
Coomassie Blue G-250 or electroblotted using a semidry apparatus (Kyhse-Andersen, 1984) onto 0.45-µm PVDF membranes (Millipore) for
sequencing or onto nitrocellulose membranes (Amersham) for immunodetection. Membranes were probed with anti-rPrx7 polyclonal rabbit antibodies diluted 1:10,000 in TBST (50 mM Tris-HCl,
pH 7.4, 150 mM NaCl, and 0.05% [v/v] Tween 20). The
secondary antibody was alkaline phosphatase-conjugated anti-rabbit goat
antibody (Stratagene) diluted 1:2,000 in TBST. The blots were washed
and developed (Harlow and Lane, 1988) with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates (Sigma). IEF was
performed as described previously (Kristensen et al., 1997) and stained
for peroxidase activity using 3-amino-9-ethylcarbazole (Sigma) as the
substrate (Kerby and Somerville, 1989).
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RESULTS |
Cloning and Sequencing of a Full-Length Prx7 cDNA
A partial cDNA clone, pcD1311, had previously been obtained by
heterologous probing with the cDNA clone pcR7 corresponding to barley
grain peroxidase BP1 (Rasmussen et al., 1991a). In the present study we
used this partial clone as a probe on a -phage cDNA library prepared
from barley leaves inoculated with powdery mildew (Scott-Craig et al.,
1995). One full-length clone, pcD1311BE, was isolated and the 700-bp
sequence in the 3 end of the DNA was found to be identical to pcD1311.
The open reading frame starts 93 bases from the 5 end of the cDNA
clone and encodes a 341-amino acid protein with a calculated
Mr of 36,515. We designated this protein
Prx7 in accordance with the restriction fragment-length polymorphism
mapping history of the partial clone (Giese et al., 1993). The
C-terminal amino acid sequence of the protein derived from pcD1311
(Rasmussen et al., 1991a) does not correspond to the C-terminal
sequence of Prx7 derived from pcD1311BE because of an artifact in the
sequencing of the pcD1311 clone, in which a base was inserted,
resulting in a +1 reading frame shift.
All of the domains characteristic of secretory plant peroxidases
(Welinder, 1992) are highly conserved in Prx7. The functions of these
domains are consistent with the properties of purified Prx7. In
addition to these domains, there are two consensus putative Asn-glycosylation sites at residues 91 and 176 (Fig.
1). Like all plant peroxidase precursors,
the uncleaved form of Prx7 has an N-terminal putative ER-targeting
peptide. The cleavage site between residue 24 (G) and 25 (Q) can be
deduced from alignment with HRP-C (Welinder, 1979) and Prx8 (Kristensen
et al., 1997), for which the first N-terminal residues in the mature
protein are known (Fig. 1). Aligning Prx7 with HRP-C (Fujiyama et al., 1988), the barley peroxidases Prx8 (Thordal-Christensen et al., 1992),
BP1 (Rasmussen et al., 1991b), and BP2 (Theilade and Rasmussen, 1992)
revealed that Prx7 carries a C-terminal elongation similar to those in
HRP-C, BP1, and BP2, although there was low sequence similarity among
these elongations.
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| Figure 1.
Amino acid sequence of Prx7 and alignment to
selected peroxidases. Alignment to peroxidases from barley: Prx8
(pBH6-301; Thordal-Christensen et al., 1992), BP1 (Rasmussen et al.,
1991b), and horseradish HRP-C gene A (Welinder, 1979; Fujiyama et al.,
1988). The lower line shows invariant residues, and variable residues
are indicated by dots. The eight Cys residues forming disulfide bridges
and the two His residues essential for catalysis are indicated in bold
and are underlined. Gaps (-) are introduced to maximize the alignment.
N- and C-terminal sequences shown to be absent from the mature proteins
are in lowercase italics. Residues shown in bold in Prx7 were
determined by amino acid sequencing. Underlined regions in the Prx7
sequence correspond to peptides identified by MALDI-TOF MS tryptic
fingerprinting. The underlined and bold Asn residues at positions 91 and 176 in the Prx7 sequence are putative
N-glycosylation sites.
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Nakamura and Matsuoka (1993) suggested that a hydrophobic/acidic motif
structure, rather than any specific amino acid sequence, forms the core
of the C-terminal vacuolar sorting signal. The hydrophobic/acidic motif
typically consists of three to four hydrophobic amino acids followed by
one or two acidic residues. Such a motif is also present in the
peroxidase HRP-C and in the vacuolar BP2 (Theilade et al., 1993). The
motif is repeated once in the C terminus of the Prx7 preprotein,
AVAGDEGIAADM, suggesting that Prx7
is also located in the vacuole. For both vacuolar HRP-C (Welinder, 1979; Fujiyama et al., 1988) and BP1 (Rasmussen et al., 1991b), it has
been shown that these C-terminal domains are absent from the mature
protein, whereas BP2 has been localized by immunogold electron
microscopy to the vacuole of scutellum cells in barley grains (Theilade
et al., 1993). These results suggest that the C terminus of Prx7 is a
vacuole-targeting signal and that mature Prx7 is located in the
vacuole.
Purification of Peroxidases from Barley Coleoptiles
Seven-day-old coleoptiles were chosen as the starting material for
the purification of Prx7, because anti-rPrx7 antibodies reacted with
substantial amounts of the protein in this tissue (see below). Initial
trials using variations on the purification method showed a substantial
peroxidase precipitation in coleoptile extracts at pH 8.0 and 60%
(NH4)2SO4
saturation. Approximately one-fourth of the peroxidase activity in the
coleoptile extract (Table I) was
precipitated by
(NH4)2SO4.
Figure 2 provides an overview of the
purification scheme. The remaining peroxidase activity in the
(NH4)2SO4
supernatant was completely adsorbed to HIC medium.
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Table I.
Purification of barley coleoptile peroxidases
Specific activity was measured as ABTS oxidation at pH 5.5. Total
activity is given as units of ABTS oxidation. The Reinheitszahl unit
(RZ) is the ratio of the absorbance of the heme group
(A403-408) to the absorbance of the aromatic
amino acids (A280) and is a measure of
peroxidase purity.
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| Figure 2.
Flow chart for purification of barley coleoptile
peroxidases P7.3, P8.8, P9.3, and P9.6. ResS, Cation-exchange
chromatography; ResQ, anion-exchange chromatography. Details of the
purification scheme are described in ``Materials and Methods''.
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Because it was not determined at this point whether an isoform-specific
or a nonspecific, incomplete precipitation had taken place, the two
protein pools were kept separate for cation-exchange purification. Two
distinct peaks of peroxidase activity were eluted by cation-exchange
chromatography of the extracts from both the (NH4)2SO4
precipitate and the HIC-captured
(NH4)2SO4
supernatant, one at approximately 15% and the other at
approximately 30% elution buffer. Activity-stained IEF
showed that the approximately 15% elution-buffer activity
from the
(NH4)2SO4
precipitate and the HIC-captured
(NH4)2SO4
supernatant was a mixture of P6.3 and P9.6. The peroxidase eluted at a
gradient buffer strength of approximately 30% was P8.8 and P9.3. Most
of the peroxidase that did not bind to the cation exchanger
also did not bind to the anion exchanger.
One peak of activity was eluted from the anion-exchange column, but
this peroxidase was too diluted for further analysis and was discarded.
The peroxidase in the anion-exchange unbound fraction was bound on the
phenyl ether-HIC column and was characterized as a P7.3 (Fig.
3A). P7.3 is immunologically unrelated to
Prx7 (Fig. 3B) and has several molecular forms with apparent molecular masses of about 53 to 55 kD (Fig. 3C). The diffuse bands on the SDS-PAGE gel indicate a highly glycosylated peroxidase. The high Reinheitszahl value (2.85) of the P7.3 preparation makes it unlikely that the heterogeneity was the result of contamination by other proteins. P9.6 and P6.3 activities were lost on the phenyl ether-HIC column under the conditions used; the activity from P8.8 and P9.3 was
partially resolved.
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| Figure 3.
Analysis of peroxidase purification fractions. A,
Activity staining of purified peroxidases P7.3, P8.8, and P9.3 after
IEF. pH values calculated using pI markers are shown on the left. B,
Immunoblot analysis. Protein was detected using polyclonal anti-rPrx7
antibodies. Analysis of rPrx7. Lane 1, Protein not bound to
cation-exchange chromatography (ResS) or anion-exchange chromatography
(ResQ), captured on phenyl ether-HIC, and containing P7.3; lane 2, pooled 30% elution buffer eluate from ResS; lane 3, pooled 15%
elution buffer eluate from ResS; and lane 4, activity bound on and
eluted from ResQ. C, SDS-PAGE of P9.3 and P7.3. The gel was stained
with Coomassie Blue. Molecular mass markers (×103) are
shown on the right.
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IEF analysis of the fractions revealed that four fractions (PE16-PE19)
contained pure P9.3, whereas the next four fractions (PE20-PE23)
contained predominantly P8.8 (Fig. 3A). SDS-PAGE analysis showed that
the P8.8-containing fractions contained several polypeptides (not
shown), but only a trace amount of protein with a size identical to
P9.3. None of the dominant polypeptides, which were all larger than
P9.3, cross-reacted with the antibodies (Fig. 3B, lane 2), showing that
P8.8 is immunologically unrelated to Prx7. P9.3 was judged to be highly
pure on the basis of its Reinheitszahl value (3.31). The protein
responsible for P9.3 activity cross-reacted strongly with anti-rPrx7
antibodies (Fig. 3B). The apparent molecular mass was about 33 kD (Fig.
3C), and this plus the sharpness of the P9.3 band indicated that P9.3
was nonglycosylated. Immunoblotting of crude coleoptile extract (see
below) and pooled activities before phenyl ether-HIC revealed that only
P9.3 cross-reacted with the anti-rPrx7 antibodies (Fig. 3B), indicating
that P9.3 is the only product of the mRNAs corresponding to pcD1311BE.
For the oxidation of ABTS, P7.3 had a specific activity of 78.9 units/mg, whereas P9.3 had a specific activity of 11.1 units/mg (Table
I).
N-Terminal Sequencing of P9.3 and P7.3
To verify the identity of P9.3 and its N-terminal
signal-peptide cleavage site, P9.3 was blotted onto PVDF membranes and
subjected to Edman degradation. Unexpectedly, the peroxidase yielded a
sequence without pyroglutamyl deblocking. The 10-residue sequence
ATXPDLERIV, which was identical to residues 8 to 18 in the
deduced mature protein (residues 33-43 using pre-Prx7 numbering; Fig.
1), verified the identity of P9.3 as Prx7. No alternative sequence was
obtained after treatment with pyroglutamyl amino peptidase, showing
that mature Prx7 starts at residue 33. The mobility of Prx7 on
immunoblots was identical when freshly prepared coleoptile extract (see
below) and purified Prx7 were analyzed. This shows that the
N-terminal sequence determined here was truly for native Prx7 and
was not the result of contaminating proteases. The deblocking yield
from P7.3 was very low, and no sequence was obtained.
MALDI-TOF MS Analysis of P9.3 and P7.3
The barley peroxidases P9.3 and P7.3 were digested by trypsin in
both polyacrylamide gel pieces and solution. Self-digested trypsin and
trypsin-digested recombinant Prx7 from E. coli were used as
internal and positive controls for the analyses of P9.3 and P7.3. The
digests were analyzed by MALDI-TOF MS. For each run, 20 to 40 spectra
were summarized into one and the peptide masses were determined.
Only peptides detected in two or more individual runs were considered
for analysis. Peaks from self-digested trypsin were subtracted from the
spectra manually and mean peptide masses were calculated for the
remaining peaks. Seventeen peptides were found for the P9.3 sample,
whereas 13 peptides were detected from P7.3 (Table
II) and 11 peptides were detected from
rPrx7. The calculated average peptide masses were entered in the
PeptideSearch
program(www.mann.embl-heidelberg.de/Services/PeptideSearch/FR_PeptideSearchForm.html).Thedatabase searches were performed with the following restrictions: (a) calculated masses should be average masses; (b) the scored protein should be in
the 30- to 38-kD range; (c) Met residues were unmodified and Cys
residues were carbamidomethylated; (d) peptides were protonated; and
(e) the accuracy of the peptide masses should be 0.1%. This accuracy
was the same as the SD among the measured masses.
Searching the database with the 17-peptide masses from P9.3 gave 47 matches. The best of these matches involved the recognition of three
peptides more than the second-best match. The best match was the cDNA
clone pcD1311BE encoding Prx7, which we had previously deposited in the
database. Nine of the 17 peptides matched tryptic fragments derived
from pcD1311BE. A second-pass search on the cDNA revealed that one
peptide matched Prx7 more closely if it was assumed to be modified
covalently by acrylamide. In total, these 10 peptides covered 147 of
the 341 amino acids in the preprotein. These peptides are marked in
Figure 1, showing the amino acid sequence of Prx7. The C-terminal
peptide (residues 311-323 using pre-Prx7 numbering) extends precisely
to the deduced cleavage site for the C-terminal putative signal
peptide. The C-terminal cleavage site was estimated by alignment with
other peroxidase proteins with known C termini and by comparison with
the empirical pI of the mature peroxidase. One of the detected peptides
carried one of the two N-glycosylation sites. The mass of
this peptide confirmed that at least some of the Prx7 molecules were
not glycosylated at this residue, supporting our previous notion that
Prx7 from coleoptiles does not appear to be glycosylated, based on the
sharpness of the Prx7 band obtained by SDS-PAGE.
Eleven tryptic peptide masses were determined from rPrx7, and seven of
these had a counterpart in the P9.3 digest (overlapping SD
values), showing the identity between the two proteins.
As expected, the search with the 13-peptide masses from P7.3 did not
give any matches. Although P7.3 has been characterized as being
constitutively expressed in the epidermis (Kogel et al., 1994), it has
not yet been cloned.
The molecular mass of nondigested P9.3 was determined with the aim of
discovering whether the protein was glycosylated and whether P9.3 was
also processed in the C terminus. The determined mass did not include
the heme group and structural Ca2+ ions that are
lost during desorption. From seven individual spectra the mean
Mr was determined to be 31,521 D
(SD = 374 D), which is in close correspondence to
the calculated mass of Prx7-containing residues 33 to 323 (31,587 D),
as indicated by peptide fingerprinting and N-terminal protein
sequencing (Table III). From this
analysis we conclude that the C terminus is processed, that Prx7 is
nonglycosylated in coleoptile tissue, and that residue 323 is the
C-terminal residue in the mature protein (because the tryptic peptide
covering residues 311 to 323 was found in the peptide analysis). On the
31,587 m/z peak in one of the seven mass spectra, weak
shoulders on the higher m/z side could be seen (not shown),
indicating the presence of small amounts of alternatively processed
Prx7 in the sample. Wobble in the processing of C-terminal peptides has
been described for BP1 (Rasmussen et al., 1991b), and this may have
been responsible for the microheterogeneity seen here.
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Table III.
Posttranslational modification of Prx7
Calculated masses using the cDNA and measured mass of P9.3 (Prx7).
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Induction of Peroxidase by Powdery Mildew Inoculation and Wounding
Time-course studies using barley plants inoculated with powdery
mildew isolates were designed to investigate the accumulation pattern
of P9.3 in response to inoculation with a pathogen and in relation to
other isoperoxidases (Fig. 4).
Accumulation of P6.3, P8.3 (Prx8), and P9.6 in total leaf extract was
evident from 9 h after inoculation. Both P9.3 and P4.8 had
accumulated significantly at 24 h after inoculation.
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| Figure 4.
Time course (h.a.i, h after inoculation) of
peroxidase accumulation in barley leaves after inoculation with powdery
mildew. IEF total protein was activity stained for peroxidase. Shown
are protein extracts (20 µg) from P-02 leaves after inoculation with
powdery mildew fungus A6 spores (incompatible interaction) (+) and from
noninoculated control leaves (). The pI values of the isozymes are
shown on the left.
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The race-specific resistance in the isogenic line used, cv Pallas P-02
(mla3), manifested itself as a multicell hypersensitive response at approximately 72 h after inoculation against the
incompatible powdery mildew fungus A6 isolate. From 24 h after
inoculation until the end of the time course (5 d after inoculation),
no new peroxidase isoforms accumulated and there appeared to be no
difference in the timing of accumulation of isoperoxidases between the
compatible (cv Pallas P-02/powdery mildew fungus C15) and the
incompatible (cv Pallas P-02/powdery mildew fungus A6) interaction
(data not shown). This is in agreement with a study by Kerby and
Somerville (1989) using a different combination of powdery mildew
isolates and barley isolines. Wounding of epidermal cells by rubbing
with silicon carbide powder induced the same peroxidases and with the same timing of accumulation as powdery mildew inoculation (data not
shown).
Tissue-Specific Peroxidase Expression
Each of the four tested organs had its own characteristic
isoperoxidase profile (Fig. 5). A total
of 12 isoenzymes could be distinguished in the various organs. On the
basis of peroxidase-band intensity, primary leaves contained the least
peroxidase on a fresh-weight basis. The coleoptile contained the most
peroxidase and also had the largest abundance of each of the peroxidase
isoenzymes among the four tissues. P9.3 was particularly abundant in
the epidermis-rich coleoptile, whereas it was present in low amounts in
the other tissues. This difference was also suggested by immunoblotting (see below). Activity due to P7.3 was the most dominant peroxidase activity in all tissues of the seedling. A similar constitutive expression has also been noted for the Arabidopsis ATP1 and ATP2 peroxidases (Kjærsgård et al., 1997), suggesting basic
metabolic functions for these peroxidases.
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| Figure 5.
Isoperoxidase expression in various barley
tissues. IEF and peroxidase activity staining of total protein extracts
(20 µg) derived from the basal part of the primary leaf sheath
covered by the coleoptile from the apical meristem (a), root (b),
coleoptile (c), and primary leaf (d) of 7-d-old P-02 seedlings. The pI
values of the isozymes are shown on the left.
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Intracellular Versus Extracellular Accumulation of Barley
Peroxidases
IEF and activity staining of peroxidases in extracellular fluid
from leaves showed that three isoperoxidases, P6.3, P8.3, and P9.6,
accumulated in the extracellular space 24 h after inoculation in
both the compatible and the incompatible interaction (Fig. 6). Analysis at later times (72 h after
inoculation) showed that P3.8, P4.8, and P8.8 also accumulated in the
extracellular space after inoculation.
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| Figure 6.
Peroxidase accumulation in total protein extracts,
intercellular washing fluid, and residual protein extracted from leaves
after extraction of intercellular proteins. Activity-stained IEF of
protein corresponding to extracts from a half leaf. Total protein
(lanes 1), residual protein (lanes 2), and intercellular protein (lanes
3) were analyzed. Protein was extracted from P-02 leaves 24 and 72 h after inoculation with powdery mildew fungus A6 spores (+), and from
noninoculated control leaves () at the same times.
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Four isoperoxidases, P6.6, P7.3, P9.2, and P9.3, were never detected in
the extracellular fluid, indicating their intracellular localization.
None of the intracellular isoperoxidases appeared to increase in
abundance as strongly as the extracellular isoperoxidases in response
to inoculation or infection.
Accumulation of Peroxidases and Peroxidase Gene Transcripts in
Epidermis and Mesophyll in Response to Powdery Mildew Inoculation
Immunoblotting of SDS-PAGE-separated proteins using antibodies
raised against rPrx7 showed the presence of Prx7 in noninoculated epidermis and a slight enhancement upon inoculation (Fig.
7B). No immunologically reacting
peroxidases could be detected in either inoculated or noninoculated
mesophyll. The immunoblot of epidermal extract had an extra band
compared with that for the coleoptile extract. This extra band may have
represented a glycosylated form of Prx7, as is the case with Prx8
(Kerby and Somerville, 1992), or it may have been a distinct but
immunologically related peroxidase present in the leaf but not in the
coleoptile (Fig. 7B, lane 8). However, all of the isoforms present in
the leaf were also present in the coleoptile (Fig. 5), and because
anti-rPrx7 antibodies did not recognize proteins in fractions from the
purification other than those containing P9.3, the antibodies were
specific for Prx7 and the alternative explanation given above is
unlikely. We also observed that storage of the epidermal samples at
4°C followed by western analysis at later times led to increasing abundance of the low-Mr band (not shown).
This band likely represented a nonglycosylated protein. Van Huystee and
McManus (1998) made similar observations by showing that secreted
-galactosidase present in crude extract was able to alter the size
and lectin-binding properties of the glycosylated anionic peanut
peroxidase.
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| Figure 7.
Peroxidase accumulation in coleoptiles, leaf
epidermis, and leaf mesophyll. A, IEF of protein extracts from 7-d-old
barley leaves (P-02) stained for peroxidase activity. Shown are
extracts from mesophyll (lane 1) and epidermis (lane 2) obtained from
leaves 24 h after inoculation with powdery mildew fungus A6
spores. Total protein (20 µg) was applied in each lane. pI values
calculated using markers are shown on the left. B, Immunoblot of rPrx7
and protein extracts obtained as in A using polyclonal anti-rPrx7
antibodies for detection of Prx7. Extracts from epidermis (lanes 4 and
5) and mesophyll (lanes 6 and 7) were obtained from barley leaves
inoculated with powdery mildew fungus A6 spores 24 h after
inoculation (lanes 4 and 6) and noninoculated leaves (lanes 5 and 7).
Extracts from 7-d-old coleoptiles (lane 8) were also analyzed. In lanes
4 to 8, approximately 20 µg of total protein was analyzed. In lanes 3 and 9, 200 ng of rPrx7 was analyzed. Protein was separated on 12%
SDS-PAGE gels.
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Isoperoxidase analysis of protein from epidermis obtained 24 h
after inoculation showed the location of six isoenzymes (Fig. 7A):
P6.3, P7.3, P8.3, P8.8, P9.3, and P9.6. In mesophyll, P9.3 and P8.8
were not detected, whereas P6.3, P8.3, P9.2, and P9.6 were detected.
RT-PCR analyses of mRNA from epidermis and mesophyll using
Prx7-specific primers showed enhancement in inoculated epidermis and
mesophyll at 24 h after inoculation, but less accumulation in the
mesophyll than in the epidermis (Fig. 8).
These results indicated that Prx7 was expressed preferentially in
epidermal cells and was present in the leaf as two molecular forms, one of which was likely to be glycosylated.
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| Figure 8.
RT-PCR analysis of transcript accumulation in
epidermis and mesophyll in response to powdery mildew inoculation. RNA
was extracted from 8-d-old barley (P-02) primary leaves 24 h after
inoculation with powdery mildew fungus A6 spores or from noninoculated
control leaves. Prx7 primers were used in lanes 1 to 5, Prx8 primers in
lanes 6 to 10, and GAPDH primers in lanes 11 to 15. RNA was obtained
from inoculated epidermis (lanes 1, 6, and 11), noninoculated epidermis
(lanes 2, 7, and 12), inoculated mesophyll (lanes 3, 8, and 13),
noninoculated mesophyll (lanes 4, 9, and 14), and 7-d-old P-02
coleoptiles (lanes 5, 10, and 15). A 100-bp DNA ladder is shown in the
far left lane. Two independent inoculations were performed, and
extracted RNA was analyzed twice. All four PCR experiments gave the
same banding-intensity pattern.
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RT-PCR analysis of mRNA from stripped mesophyll and epidermis showed
that the Prx8 messenger accumulated preferentially in the mesophyll
(Fig. 8). Gregersen et al. (1997) obtained similar results. Prx8
protein can be obtained from extracellular spaces in barley after
inoculation with powdery mildew by a gentle vacuum-infiltration procedure (Kerby and Somerville, 1989). These data indicate that Prx8 was transcribed preferentially in mesophyll cells and
that Prx8 accumulated in the extracellular space surrounding mesophyll cells and in vascular bundles, from which it was easily extracted.
| |
DISCUSSION |
The coleoptile is a rapidly growing protective tissue that shields
the emerging primary leaf (and later the basal part of the primary leaf
sheath) from both physical damage and infection by antagonistic soil
organisms. The coleoptile is rich in epidermal tissue and contains no
chlorophyll. Because of their rapid growth and linear cell pattern,
coleoptiles from monocots are one of the preferred experimental tissues
for studies of cell elongation and auxin action. For more than 30 years, coleoptiles have also been used widely in microscopy-based
physiological studies of the barley-powdery mildew fungus interaction
(Bushnell et al., 1967).
We chose barley coleoptiles as the starting material for Prx7
purification because immunoblotting of coleoptile proteins and RT-PCR
showed substantial accumulation of Prx7 in this tissue. After
purification of several isoperoxidases from coleoptiles, we were able
to identify the P9.3 isozyme as Prx7, and were then able to follow the
accumulation of Prx7 in barley leaves in response to powdery mildew
inoculation and to investigate the tissue-specific and subcellular
accumulation pattern of Prx7.
The C-terminal vacuolar-targeting hydrophobic/acidic motif (Nakamura
and Matsuoka, 1993) is repeated once in the C terminus of the Prx7
preprotein, suggesting that Prx7 is located in the vacuole or in
endosomes. We showed that Prx7 is targeted to the secretory pathway,
because the putative N-terminal targeting peptide is absent from the
mature protein. However, in spite of its abundance in total extracts
the protein was never recovered in the extracellular washing fluid.
Furthermore, the molecular mass determined by MALDI-TOF MS of pure Prx7
(31.5 kD) showed that the C terminus was processed like that of
vacuolar BP1 (Rasmussen et al., 1991b). These results strongly suggest
that Prx7 is a vacuolar peroxidase. The unusual processing of the Prx7
N terminus may not be related to a distinct intracellular route, but
could be the result of nonspecific carboxypeptidase activity in the
final destination of the putative vacuolar peroxidase.
Prx7 was expressed preferentially in epidermal cells and was present in
the leaf as two molecular forms, one of which was likely to be
glycosylated. In the coleoptile only one nonglycosylated form of Prx7
was present, as shown by MS of purified Prx7 and by immunoblotting of
crude extract.
The accumulation patterns of Prx7 and Prx8 mRNAs in barley leaves
inoculated with powdery mildew fungus were first reported by
Thordal-Christensen et al. (1992). The mRNA corresponding to Prx8
showed a two-peak accumulation pattern at the time of penetration attempts of the fungus, but the Prx7 mRNA always accumulated later and
to a much lower extent. The delay in the appearance of Prx7 compared
with Prx8 seen in this study corresponded to the delay in the increase
of the transcript, suggesting that translational regulation is not
important in the regulation of these genes.
The general notion that the cell wall is the major target for
peroxidase-mediated modifications is also supported by our data, because only the levels of Prx7 and P7.3 were enhanced intracellularly, whereas the levels of five isoenzymes were enhanced in the apoplast after inoculation with powdery mildew. This finding suggests a special
role of the putative vacuolar Prx7 in biochemical pathways involving
peroxidases.
Hordatines are antifungal compounds found in the young barley seedling.
Hordatine A is a dimer of two p-coumarylagmatines, and
hordatine B is a dimer of a p-coumarylagmatine and a
ferulylagmatine (Stoessl, 1967).
Stoessl (1967) showed that hordatines could be generated in vitro from
the appropriate hydroxycinnamic acid amides by horseradish peroxidase,
suggesting that a peroxidase may mediate the last step in the
biosynthetic pathway to hordatine. Hordatine M, a mixture of
glycoconjugates of hordatine A and B, was found by Stoessl (1967) to be
present in substantial amounts (277 mg/kg fresh weight) in 6-d-old
barley coleoptiles, along with small amounts of hordatine A and B. Transfer of dark-grown barley seedlings to the light rapidly reduced
the biosynthesis of the hordatines compared with that in etiolated
seedlings, especially that of hordatine M (Smith and Best, 1978). The
subcellular locations of the production and storage of these compounds
are unknown, but like the majority of glycosylated secondary plant
metabolites (Boller and Wiemken, 1986), hordatine M is likely to
accumulate in the vacuole. Stoessl (1967) showed that hordatines could
be generated in vitro by horseradish peroxidase; however, unlike the
hordatines isolated from coleoptiles, the in vitro products were not
optically active.
Lignans are optically active dimers of hydroxycinnamic acids and are
likely to be synthesized by peroxidases (Nose et al., 1995;
Dinkova-Kostova et al., 1996) and the novel stereospecific dirigent
protein in concert (Davin et al., 1997). Lignans are believed to be
stored as glycoconjugates in the vacuole (Lewis and Yamamoto, 1990;
Lewis and Davin, 1994) and to have antimicrobial properties, in
addition to having a role as lignification nuclei in the wall (Nose et
al., 1995). Stoessl (1967) also speculated that hordatine A and B have
a role in the biogenesis of the barley cell wall. The enhanced
expression of Prx7 in the leaf-elongation zone in the
Slender mutant of barley (Schünmann et al., 1994) may
indicate that this peroxidase does not restrain cell wall expansion,
but rather provides building blocks for the wall.
Prx7 is abundant in the coleoptile, where hordatine M also is present,
and the slow accumulation of Prx7 in response to inoculation with
powdery mildew fungus parallels the accumulation of hordatine (Smith
and Best, 1978). Likewise, Prx7 is present at low levels in the
noninoculated, light-grown, green primary leaf, where the biosynthesis
of hordatine is down-regulated (Smith and Best, 1978). These
observations strongly suggest that Prx7 is the peroxidase responsible
for the last step in the biosynthesis of hordatine. A protein similar
to the dirigent protein may also be involved.
Because Prx7 and the constitutive P7.3 differ significantly in their
specific activities, as measured by ABTS oxidation at pH 5.5, Prx7 is
likely to have a function distinct from that of the constitutive
putative vacuolar peroxidase P7.3. Even though both enzymes appear to
be vacuolar or endosomal, their preferred substrates, pH optima, and
functions in vivo are likely to be different. It must be noted that the
artificial substrate ABTS has very little similarity to
p-coumarylagmatine or hydroxycinnamic acids and alcohols, so
the specific activities determined in the present study may not reflect
activities measured with more physiologically relevant substrates.
| |
FOOTNOTES |
1
This study was supported by the Danish Agricultural
and Veterinary Research Council (grant no. 5.23.26.10), Molecular
Strategies for Crop Improvements, the Danish Research Academy, and the
Danish Cereal Network, which is supported by the Ministry of Food,
Agriculture, and Fisheries.
*
Corresponding author; e-mail brian.kristensen{at}risoe.dk; fax
45-46-77-4122.
Received November 30, 1998;
accepted March 10, 1999.
| |
ABBREVIATIONS |
Abbreviations:
ABTS, 2,2-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid).
GAPDH, glyceraldehyde-3-P dehydrogenase.
HIC, hydrophobic interaction
chromatography.
MALDI-TOF, matrix-assisted laser desorption/ionization
time of flight.
P#, peroxidase with a pI of #.
RT, reverse
transcriptase.
| |
ACKNOWLEDGMENTS |
We thank Dr. J.S. Scott-Craig and Dr. S. Somerville for the
barley leaf cDNA library and Dr. Thomas H. Roberts for constructive reading of the manuscript. Charlotte Koutras and Stanko Djudjevic are
thanked for skilled technical assistance. Yvonne Berger and Arne Jensen
of the University of Copenhagen kindly performed the amino acid
sequencing. Unizyme A/S (Hørsholm, Denmark) generously donated
pyroglutamyl amino peptidase.
| |
LITERATURE CITED |
Aist JR, Bushnell WR (1991) Invasion of plants by powdery
mildew fungi, and cellular mechanisms of the resistance. In
GT Cole, HC Hoch, eds, The Fungal Spore and Disease Initiation in
Plants and Animals. Plenum Press, New York, pp 299-321
Boller T,
Wiemken A
(1986)
Dynamics of vacuolar compartmentation.
Annu Rev Plant Physiol
37:
137-164
[CrossRef][Web of Science]
Boyd LA,
Smith PH,
Brown JKM
(1994)
Molecular and cellular expression of quantitative resistance in barley to powdery mildew.
Physiol Mol Plant Pathol
45:
47-58
Bradford MM
(1976)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254
[CrossRef][Web of Science][Medline]
Bradley DJ,
Kjellborn P,
Lamb CJ
(1992)
Elicitor- and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defense response.
Cell
70:
21-30
[CrossRef][Web of Science][Medline]
Bushnell WR,
Dueck J,
Rowell JB
(1967)
Living haustoria and hyphae of Erysiphe graminis f.sp. hordei with intact and partly dissected host cells of Hordeum vulgare.
Can J Bot
45:
1719-1732
Carver TLW,
Zeyen RJ,
Bushnell WR,
Robbins MP
(1994)
Inhibition of phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase increases quantitative susceptibility of barley to powdery mildew (Erysiphe graminis DC.).
Physiol Mol Plant Pathol
44:
261-272
Childs RE,
Bardsley WG
(1975)
The steady-state kinetics of peroxidase with 2,2-azino-di-(3-ethyl-benzthiazole-6-sulfonic acid) as chromogen.
Biochem J
145:
93-103
[Medline]
Chirgwin JJ,
Przbyla AE,
MacDonald RJ,
Rutter WJ
(1979)
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18:
5294-5299
[CrossRef][Medline]
Collinge DB, Bryngelsson T, Gregersen PL, Smedegaard-Petersen V,
Thordal-Christensen H (1997) Resistance against fungal pathogens:
its nature and regulation. In AS Basra, RK Basra, eds,
Mechanisms of Environmental Stress Resistance in Plants. Harwood
Academic Publishers, London, pp 335-372
Davin LB,
Wang HB,
Crowell AL,
Bedgar DL,
Martin DM,
Sarkanen S,
Lewis NG
(1997)
Stereoselective bimolecular phenoxy radical coupling by an auxiliary (dirigent) protein without an active center.
Science
275:
362-366
[Abstract/Free Full Text]
Dinkova-Kostova AT,
Gang DR,
Davin LB,
Bedgar DL,
Chu A,
Lewis NG
(1996)
(+)-Pinoresinol/(+)-lariciresinol reductase from Forsythia intermedia: protein purification, cDNA cloning, heterologous expression and comparison to isoflavone reductase.
J Biol Chem
271:
29473-29482
[Abstract/Free Full Text]
Espelie KE,
Franceschi VR,
Kolattukudy PE
(1986)
Immunocytochemical localization and time course of appearance of an anionic peroxidase associated with suberization in wound-healing potato tuber tissue.
Plant Physiol
81:
487-492
[Abstract/Free Full Text]
Fujiyama K,
Takemura H,
Shibayama S,
Kobayashi K,
Choi J-K,
Shinmyo A,
Takano M,
Yamada Y,
Okada H
(1988)
Structure of the horseradish peroxidase C genes.
Eur J Biochem
173:
681-687
[Web of Science][Medline]
Giese H,
Holm-Jensen AG,
Jensen HP,
Jensen J
(1993)
Localization of the Laevigatum powdery mildew resistance gene to barley chromosome 2 by the use of RFLP markers.
Theor Appl Genet
85:
897-900
Gregersen PL,
Thordal-Christensen H,
Forster H,
Collinge DB
(1997)
Differential gene transcript accumulation in barley leaf epidermis and mesophyll in response to attack by Blumeria graminis f.sp. hordei (syn. Erysiphe graminis f.sp. hordei).
Physiol Mol Plant Pathol
51:
85-97
[CrossRef]
Hammerschmidt R,
Kuc J
(1982)
Lignification as a mechanism for induced systemic resistance in cucumber.
Physiol Plant Pathol
20:
61-71
Harlow E,
Lane D
(1988)
Antibodies: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Hirano H,
Komatsu S,
Kajiwara H,
Takagi Y,
Tsunasawa S
(1993)
Microsequence analysis of the N-terminally blocked proteins immobilized on polyvinylidene difluoride membrane by western blotting.
Electrophoresis
14:
839-846
[CrossRef][Web of Science][Medline]
Ikegawa T,
Mayama S,
Nakayashiki H,
Kato H
(1996)
Accumulation of diferulic acid during the hypersensitive response of oat leaves to Puccinia coronata f. sp. avenae and its role in the resistance of oat tissues to cell wall degrading enzymes.
Physiol Mol Plant Pathol
48:
245-255
[CrossRef]
Kerby K,
Somerville S
(1989)
Enhancement of specific intercellular peroxidases following inoculation of barley with Erysiphe graminis f. sp. hordei.
Physiol Mol Plant Pathol
35:
323-337
Kerby K,
Somerville SC
(1992)
Purification of an infection-related, extracellular peroxidase from barley.
Plant Physiol
100:
397-402
[Abstract/Free Full Text]
Kjærsgård IV,
Jespersen HM,
Rasmussen SK,
Welinder KG
(1997)
Sequence and RT-PCR expression analysis of two peroxidases from Arabidopsis thaliana belonging to a novel evolutionary branch of plant peroxidases.
Plant Mol Biol
33:
699-708
[CrossRef][Web of Science][Medline]
Kogel KH,
Beckhove U,
Dreschers J,
Munch S,
Romme Y
(1994)
Acquired resistance in barley. The resistance mechanism induced by 2,6-dichloroisonicotinic acid is a phenocopy of a genetically based mechanism governing race-specific powdery mildew resistance.
Plant Physiol
106:
1269-1277
[Abstract]
Kølster P,
Munk L,
Stølen O,
Løhde J
(1986)
Near-isogenic barley lines with genes for resistance to powdery mildew.
Crop Sci
26:
903-907
[Abstract/Free Full Text]
Kristensen BK,
Brandt J,
Bojsen K,
Thordal-Christensen H,
Kerby KB,
Collinge DB,
Mikkelsen JD,
Rasmussen SK
(1997)
Expression of a defense-related intercellular barley peroxidase in transgenic tobacco.
Plant Sci
122:
173-182
[CrossRef]
Kyhse-Andersen J
(1984)
Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose.
J Biochem Biophys Methods
10:
203-209
[CrossRef][Web of Science][Medline]
Laemmli JJ
(1970)
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685
[CrossRef][Medline]
Lamb C,
Dixon RA
(1997)
The oxidative burst in plant disease resistance.
Annu Rev Plant Physiol Plant Mol Biol
48:
251-275
[CrossRef][Web of Science]
Lewis NG,
Davin LB
(1994)
Evolution of lignan and neolignan biochemical pathways.
In
WD Nes,
eds, Isopentenoids and Other Natural Products: Evolution and Function. Symposium Series 562.
American Chemical Society, Washington, DC, pp 202-246
Lewis NG,
Yamamoto E
(1990)
Lignin: occurrence, biogenesis and biodegradation.
Annu Rev Plant Physiol Plant Mol Biol
41:
455-496
[CrossRef][Web of Science][Medline]
Moerschbacher BM (1992) Plant peroxidases: involvement in response
to pathogens. In C Penel, T Gaspar, H Greppin, eds, Plant
Peroxidases 1980-1990: Topics and Detailed Literature on Molecular,
Biochemical, and Physiological Aspects. University of Geneva,
Switzerland, pp 91-99
Nakamura K,
Matsuoka K
(1993)
Protein targeting to the vacuole in plant cells.
Plant Physiol
101:
1-5
[CrossRef][Web of Science][Medline]
Nose M,
Bernards MA,
Furlan M,
Zajicek J,
Eberhardt TL,
Lewis NG
(1995)
Towards the specification of consecutive steps in macromolecular lignin assembly.
Phytochemistry
39:
71-79
[CrossRef][Web of Science][Medline]
Rasmussen SK, Johansson A, Rasmussen HN, Theilade B (1991a)
Molecular analysis and cloning of barley peroxidase genes.
In J Lobarzewski, H Greppin, C Penel, T Gaspar, eds,
Biochemical, Molecular and Physiological Aspects of Plant Peroxidases.
University of Geneva, Switzerland, pp 21-29
Rasmussen SK,
Welinder KG,
Hejgaard J
(1991b)
cDNA cloning, characterization and expression of an endosperm-specific barley peroxidase.
Plant Mol Biol
16:
317-327
[CrossRef][Web of Science][Medline]
Sambrook J,
Fritsch EF,
Maniatis T
(1989)
Molecular Cloning: A Laboratory Manual, Ed 2.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Schünmann PHD,
Harrison J,
Ougham HJ
(1994)
Slender barley, an extension growth mutant.
J Exp Bot
45:
1753-1760
Scott-Craig JS,
Kerby K,
Stein BD,
Somerville SC
(1995)
Expression of an extracellular peroxidase that is induced in barley (Hordeum vulgare) by the powdery mildew pathogen (Erysiphe graminis f.sp. hordei).
Physiol Mol Plant Pathol
47:
407-418
[CrossRef]
Smith TA,
Best G
(1978)
Distribution of the hordatines in barley.
Phytochemistry
17:
1093-1098
[CrossRef]
Stoessl A
(1967)
The antifungal factors in barley. IV. Isolation, structure, and synthesis of the hordatines.
Can J Chem
45:
1745-1760
[CrossRef]
Stoessl A,
Unwin CH
(1970)
The antifungal factors in barley. V. Antifungal activity of the hordatines.
Can J Bot
48:
465-470
Theilade B,
Rasmussen SK
(1992)
Structure and chromosomal localization of the gene encoding barley seed peroxidase BP 2A.
Gene
118:
261-266
[CrossRef][Medline]
Theilade B, Rasmussen SK, Rosenkrands I, Frøkier H, Hejgaard J,
Theilade J, Pihakaski-Maunsbach K, Maunsbach AB (1993) Subcellular
localization of barley grain peroxidase by immuno-electron microscopy.
In KG Welinder, SK Rasmussen, C Penel, H Greppin, eds, Plant
Peroxidases: Biochemistry and Physiology. University of Geneva,
Switzerland, pp 321-324
Thordal-Christensen H,
Brandt J,
Cho BH,
Rasmussen SK,
Gregersen PL,
Smedegaard-Petersen V,
Collinge DB
(1992)
cDNA cloning and characterization of two barley peroxidase transcripts induced differentially by the powdery mildew fungus Erysiphe graminis.
Physiol Mol Plant Pathol
40:
395-409
[CrossRef]
Valé GP,
Torrigiani E,
Gatti A,
Delogu G,
Potta-Puglia A,
Vannacci G,
Cattivelli L
(1994)
Activation of genes in barley roots in response to infection by two Drechslera graminea isolates.
Physiol Mol Plant Pathol
44:
207-215
[CrossRef]
Van Huystee RB,
McManus MT
(1998)
Glycans of higher plant peroxidases: recent observations and future speculations.
Glycoconjugate J
15:
101-106
[Medline]
von Röpenack E,
Parr A,
Schulze-Lefert P
(1998)
Structural analyses and dynamics of soluble and cell wall-bound phenolics in a broad spectrum resistance to the powdery mildew fungus in barley.
J Biol Chem
273:
9013-9022
[Abstract/Free Full Text]
Wei YD,
de Neergaard E,
Thordal-Christensen H,
Collinge DB,
Smedegaard-Petersen V
(1994)
Accumulation of a putative guanidine compound in relation to other early defense reactions in epidermal cells of barley and wheat exhibiting resistance to Erysiphe graminis f.sp. hordei.
Physiol Mol Plant Pathol
45:
469-484
[CrossRef]
Welinder KG
(1979)
Amino acid sequence studies of horseradish peroxidase: amino and carboxyl termini, cyanogen bromide and tryptic fragments, the complete sequence, and some structural characteristics of horseradish peroxidase C.
Eur J Biochem
96:
483-502
[Web of Science][Medline]
Welinder KG
(1992)
Superfamily of plant, fungal and bacterial peroxidases.
Curr Opin Struct Biol
2:
388-393
[CrossRef]
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Abstract
Introduction