Plant Physiol. (1999) 119: 1261-1270
Molecular Cloning and Characterization of Apricot Fruit
Polyphenol Oxidase
Tony Chevalier1,
David de Rigal1,
Didier Mbéguié-A-Mbéguié1,
Frédéric Gauillard,
Florence Richard-Forget*, and
Bernard R. Fils-Lycaon
Institut National de la Recherche Agronomique, Site Agroparc,
Domaine Saint Paul, Station de Technologie des Produits
Végétaux, 84914 Avignon cedex 9, France (T.C., D.d.R.,
D.M.-A.-M., F.G., F.R.-F.); and Institut National de la Recherche
Agronomique, Domaine Duclos, Station de Technologie des Produits
Végétaux, B.P. 515, 97165 Pointe-à-Pitre cedex,
French West Indies (B.R.F.-L.)
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ABSTRACT |
A reverse transcriptase-polymerase
chain reaction experiment was done to synthesize a homologous
polyphenol oxidase (PPO) probe from apricot (Prunus
armeniaca var Bergeron) fruit. This probe was further used to
isolate a full-length PPO cDNA, PA-PPO (accession no.
AF020786), from an immature-green fruit cDNA library.
PA-PPO is 2070 bp long and contains a single open
reading frame encoding a PPO precursor peptide of 597 amino acids with a calculated molecular mass of 67.1 kD and an isoelectric point of
6.84. The mature protein has a predicted molecular mass of 56.2 kD and
an isoelectric point of 5.84. PA-PPO belongs to a multigene family. The gene is highly expressed in young, immature-green fruit and is turned off early in the ripening process. The ratio of PPO
protein to total proteins per fruit apparently remains stable
regardless of the stage of development, whereas PPO specific activity
peaks at the breaker stage. These results suggest that, in addition to
a transcriptional control of PPO expression, other regulation factors
such as translational and posttranslational controls also occur.
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INTRODUCTION |
PPOs (EC 1.14.18.1), also referred to as catecholoxidases, are
copper-containing enzymes widely distributed in the plant kingdom. PPOs
are known as the main class of enzymes involved in the browning of
damaged fruits or vegetables. PPOs catalyze the oxidation of phenols
into o-quinones with a concomitant O2
reduction. The so-formed o-quinones undergo subsequent
reactions leading to dark-colored pigments (Nicolas et al., 1994
).
Although PPOs are localized in plastids (Vaughn et al., 1988), their
phenolic substrates are mainly located in the vacuole so that enzymatic
browning occurs only when this subcellular compartmentation is lost.
Because of the considerable economic and nutritional loss induced by
enzymatic browning in the commercial production of fruits and
vegetables, numerous studies have been devoted to the biochemical and
catalytic properties of PPOs (Mayer and Harel, 1991; Zawistowski et
al., 1991). However, the physiological function of PPOs in plants
remains unclear. Three main arguments have supported the assumption
that PPOs are involved in disease resistance: (a) the impervious scab of melanin generated by the o-quinones' secondary
reactions, which prevents the spread of infection (Zawistowski et al.,
1991); (b) the ability of o-quinones to covalently bind
plant proteins, thereby decreasing the nutritive availability of
nucleophilic amino acids (Duffey and Felton, 1991); and (c) the
bacteriostatic effect of o-quinones (Mayer and Harel, 1979).
Because of their plastidic location, PPOs were also supposed to play a
role in the photosynthetic reaction of chloroplasts, more precisely in
the mediation of pseudocyclic photophosphorylation (Vaughn and Duke,
1984; Trebst and Depka, 1995).
Investigators have recently succeeded in cloning and characterizing the
multigene families encoding leaf PPOs from different plant species,
including faba bean (Cary et al., 1992), potato (Hunt et al., 1993),
tomato (Newmannn et al., 1993), and Virginian pokeweed (Joy et al.,
1995). These studies have revealed a high degree of sequence
conservation among the investigated PPOs. Leaf PPO sequences can be
roughly divided into three domains, with a central domain containing
the copper-binding sites (Van Gelder et al., 1997). All genes encode
mature proteins of 52 to 62 kD and 8- to 12-kD transit peptides
responsible for the transport of the enzyme into the thylakoid lumen.
Research on fruit PPOs is not yet quite as advanced, although two
reports showing high sequence homology with leaf PPOs have recently
been published. In 1994, Dry and Robinson described the molecular
cloning and characterization of grape berry PPO. Southern analysis
suggested the presence of only one gene in the grapevine. Moreover,
high levels of gene expression were found in young, developing berries, whereas expression in mature tissues was low. More recently,
Boss et al. (1995) isolated a full-length cDNA clone encoding apple PPO
and described it as a multigene family.
As another step to more fully understand the structure, regulation, and
function of fruit PPO, we report here the isolation and
characterization of an apricot (Prunus armeniaca) PPO cDNA. PPO sequence conservation was exploited in an RT-PCR strategy to
generate an apricot PPO probe. We then used the probe to isolate a
full-length cDNA clone, PA-PPO, from a ripe fruit cDNA
library and studied its differential expression during ripening. To
elucidate the PPO maturation process, we purified the enzyme and
compared its sequence with that of the immature protein.
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MATERIALS AND METHODS |
Plant Material
The apricot (Prunus armeniaca var Bergeron) was used in
all of the experiments. Fruits were harvested at the immature-green-1 and -2, mature-green, straw-yellow (breaker), light-orange-1 (breaker +), light-orange-2 (half-ripe), deep-orange-1 (fully ripe), and deep-orange-2 (overripe) stages (89, 96, 103, 110, 117, 124, 128, and
131 d respectively, after anthesis) at Gotheron, near Valence, France. At each harvest date, we formed random lots, each with 30 fruits. Immediately after picking, fruits were cut into small pieces,
frozen in liquid nitrogen, and stored at 
80°C for subsequent protein and RNA analysis. Fruits used for PPO isolation and
characterization were harvested at the deep-orange-1 (fully ripe)
stage. Immediately after picking, the unseeded cortex was cut into
small pieces, frozen in liquid nitrogen, lyophilized, and stored at
20°C until use.
Leaves and stems were harvested from the same trees and immediately
frozen in liquid nitrogen before storage at 80°C. For wounding
experiments, leaves were scarified on the tree with a sterile scalpel
blade, mixed 24 h later, and immediately frozen in
liquid nitrogen before storage at 80°C.
PPO Purification and Characterization
PPO Assay
We used the procedure of Janovitz-Klapp et al. (1989) to
polarographically assay the PPO.
Extraction and Purification Procedures
A 12-g sample of lyophilized apricot was homogenized in 120 mL of
cold McIlvaine's buffer at pH 7.0, containing 30 mM
ascorbic acid, 1% Triton X-100, and 3 g of PVPP. The homogenate
was centrifuged at 40,000g for 40 min and the supernatant
was used as crude extract. Inactive proteins were partially removed by
(NH4)2SO4
precipitation (30% saturation on a molar basis). The resulting
supernatant was dialyzed and loaded onto a Phenyl Sepharose CL4B column
(Pharmacia), according to the protocol developed by Gauillard and
Richard-Forget (1997). Active fractions were combined, dialyzed
overnight against 10 mM sodium acetate buffer, pH
5.0, and applied to a DEAE-Sepharose CL6B column (10 × 1.6 cm
i.d., 20 mL bed volume [Pharmacia]), pre-equilibrated with the same
buffer. The column was eluted with the equilibration buffer and the
eluted protein was monitored by measuring the
A280. After the absorbance returned to the
baseline, we carried out a further elution, with a linear salt gradient from 0 to 0.1 M
(NH4)2SO4
in 10 mM acetate buffer, pH 5.0. Proteins still
bound to the gel were removed with 40 mL of equilibration buffer
successively supplemented with 0.1 and 0.25 M
(NH4)2SO4. The flow rate was 60 mL h
1 and the absorbance
and PPO activity were determined in each 5-mL fraction.
Protein Characterization
IEF in liquid medium of purified PPO was done with a 110-mL column
(type 8101, LKB, Bromma, Sweden) in the pH range of 3.5 to 5.0, as described by Fils et al. (1985). To check the PPO-preparation purity, an SDS-PAGE electrophoresis experiment was performed according to the procedure of Gauillard and Richard-Forget (1997). For molecular mass determination, we used the calibration kit of SDS-PAGE standards (low range of molecular mass) from Bio-Rad. A protocol adapted from
that described by Fils-Lycaon et al. (1996) was used to determine the
N-terminal amino acid sequence of the purified apricot PPO.
Protein Extraction and Electrophoretic Experiments
We extracted total proteins from the frozen fruit pericarp and
mesocarp tissues as described by Lelièvre et al. (1995) and determined the protein concentration by the Bradford method (1976), using BSA as a standard.
SDS-PAGE was performed in a minigel apparatus (Bio-Rad) as described by
Laemmli (1970). Ten micrograms of total denatured proteins was
separated in a 12% polyacrylamide denaturing gel. Separated
polypeptides were electroblotted to a 0.45-µm nitrocellulose membrane
(Hybond-C-Pure, Amersham), using a minitrans-blot cell (Bio-Rad) and a
Towbin transfer buffer (Towbin et al., 1979).
Western Immunoblotting
We detected the apricot PPO using the immunoblot method described
by Fraignier et al. (1995), with a serum/anti-apple PPO (a gift
from L. Marques, Université des Sciences et Techniques du
Languedoc, Montpellier, France). The second antibody reaction was
carried out using goat anti-rabbit IgG, conjugated with alkaline phosphatase (Sigma). First and second antibodies were diluted 1000- and
7500-fold, respectively. Prestained molecular mass standards were
obtained from Bio-Rad.
Total RNA Extraction and Purification and Poly(A+)-Rich
RNA Preparation
We extracted and purified total RNAs from frozen intact leaves,
wounded leaves, and stems, according to the method of Fils-Lycaon et
al. (1996).
Total RNAs were extracted from fruit by modifying the method of Wan and
Wilkins (1994). Seven grams of frozen tissue was powdered in liquid
nitrogen with a blender (Waring) and mixed with 0.46 g of
diethyldithiocarbamic acid. The mixture was then homogenized (10 min at
65°C) in 12 mL of preheated extraction buffer (0.2 M Gly,
0.2 M boric acid, and 0.6 M sodium chloride, pH
9.6), supplemented with 5% SDS, 1% Nonidet P-40, 1 g of PVPP
(preswollen in the extraction buffer), and 0.65 mL of
2-mercaptoethanol. Twelve milliliters of phenol (pH 8.0, equilibrated
with Tris-HCl at room temperature) was then added to the extract, which
was shaken at 65°C for 10 additional minutes. After the sample was
centrifuged the aqueous phase was brought to a final concentration of
160 mM potassium chloride and left to precipitate for
1 h on ice. The nucleic acids in the aqueous phase were
precipitated overnight with ethanol at 20°C, recovered by
centrifugation, and purified on cellulose CC 41 (Whatman), according to
the method of Fils-Lycaon et al. (1996).
We used a polyATtract mRNA isolation system (Promega) to
obtain poly(A+)-rich RNAs. Total RNAs were
passed through an oligo(dT)-cellulose column, and
poly(A+)-rich RNAs were eluted as described
by the manufacturer.
Reverse Transcription, PCR Amplifications, and Cloning
Consensus sequences of the thylakoid-transfer domain (in the
transit peptide) and the copper-binding domain-B of previously published PPOs were used to design the following degenerate
oligonucleotide primers: (I, downstream)
5
-AGGAGAAA(CT)(AG)TICT(CT)IT(AT)GG(GC)(CT)T(AT)GG-3 for 5-RRN(VM)L(IL)G(LI)G-3; and (II, upstream)
5-CATCC(GT)(AG)TC(GC)AC(AG)(AT)TIG(AC)(AG)TGGTG-3 for
5-HH(AS)NVDRM-3.
We performed reverse transcription and PCR (Saiki et al., 1985) in the
same 0.5-mL tube, using an Access RT-PCR system kit (Promega), as
described by the manufacturer. Degenerate primers (1 µM
each), 1 µg of total RNA from immature-green-1 or mature-green fruit
tissue, 5 units of AMV (avian
myeloblastosis virus) RT, and 5 units of
Tfl (Thermus
flavus) DNA polymerase were used in 50 µL of a
standard reaction mixture made of supplied buffer, 1 mM magnesium sulfate, and 0.2 mM of each dNTP.
First-strand cDNA was synthesized by incubation at 50°C for 1 h.
The reaction mixture was then heated at 94°C for 2 min to denature
the RNA/cDNA hybrid and inactivate the AMV RT.
The second-strand cDNA was then produced and amplified in the following
PCR conditions: 45 cycles of template denaturation at 94°C for
30 s, primer annealing at 51°C for 1 min, and primer extension
at 68°C for 2 min.
The cDNA fragment produced by the PCR amplification was then purified
from the electrophoresis gel by digestion of agarose with AgarACE
(Promega) as described by the manufacturer. The purified cDNA fragment
was amplified again using the same PCR protocol, except that
Tfl DNA polymerase was used alone with its appropriate, supplied buffer.
For cloning, the reamplified cDNA fragment was treated with
Pfu (Pyrococcus
furiosus) DNA polymerase (Stratagene) to generate blunt ends and then ligated to the SmaI-digested pBS
SK plasmid vector (Stratagene), using a T4 DNA
ligase (Life Technologies, GIBCO-BRL). Positive clones of transformed
Easy-Pore Electro-Competent cells (Eurogentec, Seraing, Belgium) were
isolated on an X-GAL-, isopropyl-
-thiogalactopyranoside-supplemented
Luria broth agar-ampicillin medium. Eurogentec sequenced the cloned
cDNA fragment.
Construction of the cDNA Library of Immature-Green Fruit
A UNI-ZAP XR (Stratagene) cDNA library was prepared from 5 µg of
immature-green-1 apricot poly(A+), containing
RNAs as described by the manufacturer. The number of independent
recombinants generated was 2.3 × 107. We
estimated the average size of the cloned cDNA at 1.3 kb using PCR
analysis of individual plaques.
UNI-ZAP XR cDNA Library DNA Screening and Clone Analysis
The cDNA previously generated by RT-PCR and coding for a putative
PPO was randomly labeled to high activity with
[
-32P]dCTP, using a Ready-to-Go labeling kit
(Pharmacia) as described by the manufacturer. We used this probe to
screen 2.5 × 105 recombinant plaques of our
cDNA library by in situ plaque hybridization (Sambrook et al., 1989).
Duplicate plaque lifts were made with a Nytran+
membrane (Schleicher & Schuell, Cera-Labo, Aubervilliers, France), and
the DNA was fixed by baking at 80°C for 2 h and treatment with
UV. The membranes were prehybridized at 40°C for 2 h in a prehybridization buffer solution containing 40% formamide, 5× SSC
(20× SSC = 3 M sodium chloride and 0.3 M
sodium citrate, pH 7.0), 2× Denhardt's reagent (0.2 g of Ficoll,
0.2 g of PVPP, and 0.2 g of BSA), 0.5% SDS, and 100 µg
µL1 denatured salmon-sperm DNA. Hybridization
was performed overnight at 40°C with fresh prehybridization solution,
supplemented with the labeled probe. Hybridized membranes were washed
in 2× SSC-1% SDS, twice at 40°C for 20 min, and twice at 45°C for
20 min, before being autoradiographed overnight at 80°C, using
Kodak X-AR film and an intensifying screen.
Two positive clones were plaque purified and subcloned by the Zap
procedure (Stratagene). Eurogentec then fully sequenced both DNA
strands of each clone.
Sequence Analysis
We used the advanced BLAST program (Altschul et al., 1997) to
search the nonredundant peptide sequence database on the National Center for Biotechnology Information BLAST E-mail server (National Library of Medicine, Bethesda, MD).
We determined the molecular mass, pI, potential glycosylation sites,
and hydrophilic/hydrophobic profiles using the method of Kyte and
Doolittle (1982) and the Genetics Computer Group software of the
University of Wisconsin (Devereux et al., 1984). The sequences were
aligned with the MultAlin program of Corpet (1988).
Southern-Blot Analysis
We prepared genomic DNA from apricot leaves by modifying the
method of Bernatzky and Tanksley (1986). Approximately 20 µg of DNA
was digested with EcoRI and/or HindIII
restriction enzymes. DNA fragments were then separated on a 0.8%
agarose gel, depurinated in 0.25 N HCl for 1 h,
denatured in 0.4 N NaOH for 1 h, and blotted to a
Nytran+ membrane (Schleicher & Schuell,
Cera-Labo) by overnight capillary transfer in 0.4 N
NaOH. Prehybridization, hybridization, and autoradiography of blots
were performed as described above for cDNA library screening. Hybridized membranes were washed in 2× SSC-1% SDS, once at room temperature for 20 min, twice at 40°C for 20 min, and once at 45°C
for 10 min.
Northern-Blot Analysis
Fifteen micrograms of total RNA of intact leaves, wounded leaves,
stem, and fruit tissues taken at different ripening stages was
separated on a 1.2% agarose denaturing gel containing 10% formaldehyde. After electrophoresis, the RNA was transferred to a
Nytran+ membrane (Schleicher & Schuell,
Cera-Labo) by overnight capillary transfer in 20× SSC.
Prehybridization, hybridization, and autoradiography of the membranes
were done as described above for cDNA library screening. Hybridized
membranes were washed in 2× SSC-1% SDS, twice at 40°C for 20 min,
once at 45°C for 20 min, and once at 50°C for 20 min.
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RESULTS AND DISCUSSION |
PPO Purification and Characterization
Full extraction of apricot PPO required the use of Triton X-100.
No PPO activation (in the presence of SDS, trypsin, or fatty acids) was
observed to take place in our crude extract. This suggests either that
apricot fruit does not contain latent PPO forms or that full activation
was achieved during extraction. Fraignier et al. (1995) have also
reported the absence of latent forms in PPO crude extracts from several
Prunus sp., including apricot. Moreover, if latent PPO forms
have been frequently described in plant leaves, latency of fruit PPO
was seldom reported. To our knowledge, the existence of latent PPO
forms has been shown in only four species: avocado (Kahn, 1977), grape
(Rathjen and Robinson, 1992), mango (Robinson et al., 1993), and pear
(Gauillard and Richard-Forget, 1997). Several factors may be involved
in activation: (a) the existence of a proenzyme (Rathjen and Robinson,
1992; Söderhall, 1995), (b) the removal of a PPO-bound inhibitor
(Sanchez-Ferrer et al., 1993), and (c) a conformational change
(Gauillard and Richard-Forget, 1997).
The results of the PPO purification procedure are summarized in Table
I. Precipitation with ammonium sulfate
resulted in more than 90% recovery of activity with a 4.2-fold
purification. All PPO activity was eluted from the Phenyl Sepharose
CL4B column in a single peak, representing more than 80% of the loaded
activity with less than 30% of the applied protein, indicating an
overall purification factor greater than 13. Further purification was done using ion-exchange chromatography on a DEAE-Sepharose CL6B (Pharmacia). The eluted activity representing 92% of the loaded activity was recovered in only one peak. The purification factor of the most active fraction was close to 38. The homogeneity and purity
of the former fraction were checked by SDS-PAGE analysis (Fig.
1). A single band at 60 kD was revealed
by Coomassie brilliant blue staining. Antibodies raised against an
apple PPO interacted strongly with this 60-kD protein after its
transfer on nitrocellulose (data not shown). Antibodies did not react
with any fractions eluted from ion-exchange chromatography other than
that containing the PPO activity. Mature and active apricot PPO was
therefore characterized by a 60-kD molecular mass. This value is higher than that previously reported (43 kD) by Fraignier et al. (1995) for
different Prunus sp., including apricot. This difference may be due to the occurrence during the extraction and purification procedure of a proteolytic cleavage at the C-terminal end of the protein, as suggested by Robinson and Dry (1992).
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| Figure 1.
SDS-PAGE of purified apricot PPO. MW, Molecular
size markers; F IE, most active fraction eluted from the DEAE-Sepharose
CL6B ionic exchange column.
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A second hypothesis may be an inadequate reduction of the protein
sample before electrophoresis, leaving presumed intramolecular disulfide bridges intact and thereby preventing an accurate
electrophoretic molecular mass estimation. Thus, Cary et al. (1992),
who observed a 45-kD faba bean PPO form under partially denatured
conditions, reported that this form was converted to a 63-kD one with
full denaturation. Isoenzyme composition of the purified PPO extract was determined by IEF in a liquid medium with a 3.5 to 5.0 pH gradient.
The profile obtained (Fig. 2) shows the
presence of a main peak with a maximum at pH 4.6 and three minor peaks
at pH 3.8, 4.3, and 4.9. The former pI values are consistent with data
available in the literature for other PPO species (Zawistowski et al.,
1991). At this step of our investigation it is difficult to conclude
whether we are in the presence of isoforms and/or forms resulting from
interactions of the protein with phenolic compounds, as reported by
Smith and Montgomery (1985). The 18 N-terminal residues of the purified
PPO were
N-Asp-Pro-Ile-Ala-Pro-Pro-Asp-Leu-Thr-Thr-Cys-Lys-Pro-Ala-Glu-Ile-Thr-Pro.
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| Figure 2.
IEF in liquid medium of apricot PPO. IEF was
performed at 4°C for 3 d in a pH gradient (3.5-5.0). On each
fraction of 1.5 mL collected, pH was measured and PPO activity was
assayed.
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Isolation of a PPO-Related cDNA
PCR amplification of an apricot fruit cDNA generated by RT-PCR led
to the production of one cDNA fragment of 915 bp, whose amino acid
sequence presented a very high homology with that of Malus
domestica PPO cDNA (Boss et al., 1995). This clone was therefore used to screen the cDNA library. Two apricot cDNAs that strongly hybridized were isolated and found to be identical after sequencing. The longer cDNA clone was 2070 bp long and presented a complete coding
sequence. It was deposited in GenBank database, given the accession no.
AF020786, and labeled PA-PPO (for Prunus
armeniaca polyphenol oxidase).
This cDNA contained 3 bp of 5-untranslated region, an open reading
frame of 1794 nucleotides encoding for 597 amino acids, and 273 nucleotides of the 3-untranslated region. The 3-untranslated region
contained three AATAA polyadenylation signals (Joshi, 1987).
Analysis of the Amino Acid Sequence Deduced from the Isolated cDNA
A search of the nonredundant peptide sequence database on the
National Center for Biotechnology Information BLAST server using the
BLAST program has pointed out a high homology of the isolated apricot
cDNA with PPOs from various sources. The deduced amino acid sequence of
the isolated clone was compared with the sequences of PPOs from other
plant species (Fig. 3). A 67.7% homology
with the PPO of apple (Boss et al., 1995) was found, which was the highest homology among the aligned sequence. This high homology is in
accordance with the crossed reaction of apple PPO antibodies with
apricot PPO. The identity and similarity scores shared by apricot PPO
with the proteins of tomato (Newmann et al., 1993), Virginian pokeweed
(Joy et al., 1995), faba bean (Cary et al., 1992), and grape berry (Dry
and Robinson, 1994) ranged from 43.0% to 53.3% and 57.1% to 65.7%.
These data confirm the high level of PPO conservation in higher plants.
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| Figure 3.
Optimal alignment of PPOs from several plant
species. Accession nos.: apricot, no. AF020786; apple, no. P43309 (Boss
et al., 1995); grape, no. P43311 (Dry and Robinson, 1994); fava bean,
no. 418754 (Cary et al., 1992); Virginian pokeweed, no. D45385 (Joy et
al., 1995); tomato, no. Q08296 (Newmann et al., 1993). A dot refers to
identity with apricot. A space denotes a gap introduced for improved
alignment. Single underlined amino acid residues correspond to the
transit peptide. Domain I of transit peptide is marked by  . Domain
II of transit peptide is marked by  . The "n-region" of domain II
of transit peptide is shaded in blue. The thylakoid transfer domain of
the domain II of transit peptide is shaded in green. Shown as bold
letters are the hydrophobic amino acids of thylakoid transfer domain
and the precleavage site. The first amino acid residue of the mature
protein is shaded in black. Double underlined amino acid residues
correspond to the sequence obtained from N-terminal sequencing of the
purified protein. Copper domains A and B of the mature protein are
shaded in yellow. His residues predicted to be copper-binding ligands
are boxed.
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Comparison of the sequence deduced from the isolated PA-PPO
cDNA with that of the purified protein showed a 100% homology, which
started with the Asp in position 102. This result suggests that the
apricot PPO is synthesized as a precursor protein. The predicted
molecular mass of the preprotein was 67.1 kD and its pI was 6.84. A
long sequence of 101 amino acids (single underlined residues in Fig.
3), with a predicted molecular mass of 10.9 kD and a pI of 10.91 preceded the mature protein and presented the structure of a
chloroplast transit peptide. Following the model given by De Boer and
Weisbeek (1991) and recently completed by Joy et al. (1995), transit
peptides of lumen-targeted proteins contain two main domains for a
two-step process to a mature protein (Sommer et al., 1994). Domain I
targets the protein to the translocation complex, where it enters the
stroma and is subsequently cleaved. Domain II targets the thylakoid
membrane or lumen. This second domain preceded the cleavage site where
the mature protein is processed from the transit peptide. In the
PA-PPO sequence a putative cleavage site can be predicted
between Ala-101 and Asp-102, which is in accordance with results
obtained from the N-terminal sequencing of the purified protein.
The mature PPO protein was composed of 496 residues, with a predicted
molecular mass of 56.2 kD and a pI of 5.84. The apricot cDNA clone
contains the two copper-binding domains typical of PPOs (Fig. 3). Both
domains are highly conserved through aligned PPO sequences, have high
homology among different species (Shahar et al., 1992), and contain
typical His residues thought to be involved in copper binding. The
amino acid sequence deduced from the PA-PPO clone does not
contain the His-rich region present in the C-terminal part of other
aligned sequences and described as a putative third
copper-binding domain by Hunt et al. (1993).
The predicted values of molecular mass and pIs of the precursor, the
peptide signal, and the mature protein are very close to those
calculated from the cDNA sequences of apple (Boss et al.,
1995), grape (Dry and Robinson, 1994), faba bean (Cary et al., 1992),
Virginian pokeweed (Joy et al., 1995), potato (Hunt et al., 1993), and
tomato (Newmann et al., 1993) PPOs.
The slight difference of 1.24 pH unit, observed between the pI
calculated from the cDNA and the value obtained for the major PPO peak
in the IEF experiment of the purified protein, may be ascribed to
phenolic compounds bound to the protein leading to a modification of
its net charge. However, charge masking may also result from the final
three-dimensional conformation of the protein, which is not entirely
taken into account in the pI prediction program.
Southern-Blot Analysis
Previous works have shown that PPO genes isolated from
faba bean (Cary et al., 1992), potato (Hunt et al., 1993), tomato
(Newmann et al., 1993), Virginian pokeweed (Joy et al., 1995), and
apple (Boss et al., 1995) belong to a multigene family. In contrast, Southern analysis has demonstrated the presence of only one gene in
grapevine (Dry and Robinson, 1994). To determine whether more than one
gene was related to PA-PPO, the labeled PPO RT-PCR fragment was hybridized to apricot genomic DNA cut with EcoRI and/or
HindIII restriction endonucleases (Fig.
4). Digestion of apricot DNA with EcoRI (Fig. 4, lane 1) produced one major band and one faint
band. Digestion of DNA with HindIII (Fig. 4, lane 2)
produced one major band and two faint bands. Digestion of DNA with
EcoRI and HindIII (Fig. 4, lane 3) produced one
major band. Because PA-PPO does not contain an
EcoRI or a HindIII restriction site, and PPO
genes do not have introns (Newmann et al., 1993; Dry and Robinson,
1994), the number and size of the hybridizing genomic fragments
indicate that, as in other fruits, there may be more than one closely
related PPO gene in the apricot genome. However, the cloned
PA-PPO seems to be present in a single copy.
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| Figure 4.
Genomic DNA analysis of PA-PPO.
Genomic DNA (20 µg per lane) was digested with EcoRI
(lane 1), HindIII (lane 2), and EcoRI and
HindIII (lane 3), hybridized with an RT-PCR PPO fragment
from apricot and washed at low stringency.  DNA codigested with
EcoRI and HindIII (Promega) was used as a
molecular mass marker.
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Northern-Blot analysis: Expression of the PA-PPO Gene during
Apricot Ripening
The expression of the PA-PPO gene was examined during
fruit development and in vegetative tissues by northern analysis. A labeled PPO RT-PCR fragment was used as a probe in hybridization of
equal amounts of total RNAs of each tissue. A single 2.2-kb RNA
transcript accumulated at the immature-green stage of fruit development
(Fig. 5). The gene was further
transcriptionally down regulated with fruit aging (breaker stage) and
totally turned off early in the ripening process. Autoradiography
exposure for 1 week confirmed the lack of expression after the breaker
stage in fruit and also in leaf, wounded leaf, and stem (data not
shown). The early expression of PA-PPO in fruit is
consistent with results obtained in grape berry (Dry and Robinson,
1994) and apple fruit (Boss et al., 1995) and is more generally in
accordance with a higher expression of PPO genes in young tissues
(Rathjen and Robinson, 1992; Shahar et al., 1992; Thygesen et al.,
1995). In contrast, the expression of Virginian pokeweed PPO
specifically localized in ripened fruits (Joy et al., 1995) may be
highly related to the accumulation of betalains.
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| Figure 5.
Expression of PA-PPO gene during
ripening of apricot fruit. Fifteen micrograms of total RNA from apricot
fruit was used at five stages of development: IM1 (immature green 1),
BR (breaker), BR+ (breaker +), HR (half-ripe), and OR
(overripe). The blot was hybridized to an RT-PCR PPO fragment from
apricot and washed at high stringency.
|
|
Activity Assay and Protein Content
A significant PPO activity (close to 200 nkat g1
fresh weight) was found in intact and wounded leaves and in stems. In
fruit, when expressed per gram of fresh weight, PPO activity slightly decreased from 3.1 µkat at the immature-green stage to 2.2 µkat at
the overripe stage (data not shown). Similar results have already been
reported for apple fruit (Janovitz-Klapp et al., 1989). The amount of
fresh weight of apricot fruit was, however, increased from 30.8 g
at the immature-green stage to 98.5 g at the overripe stage (data
not shown). Such an effect of fruit growth has been taken into account
in Figure 6 by expressing PPO activity
per fruit. PPO activity per fruit was found to increase from 96 µkat at the immature-green stage to 188 µkat at the breaker stage. A
slight increase was then observed as PPO activity reached a value close
to 220 µkat at the overripe stage of fruit development.
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| Figure 6.
Changes in PPO activity per fruit, protein per
fruit, and PPO specific activity during ripening of var Bergeron
apricot. Six stages of development were considered: IMG 1 (immature
green 1), MG (mature green), BR (breaker), HR (half-ripe), RI (fully
ripe), and OR (overripe).
|
|
In Figure 6 the amount of total extracted proteins is also expressed
per fruit. A value close to 55 mg was determined for the immature-green
stage. The protein amount slightly decreased until the breaker stage
and then sharply increased to reach 210 mg at the half-ripe stage and
243 mg at the ripe stage.
We also report in Figure 6 the PPO specific activity during fruit
development. Starting from 1.75 µkat mg1 at
the immature-green stage, specific activity reached a peak (close to
4.5 µkat mg1) at the breaker stage. A sharp
decrease was then observed; a value close to 0.95 µkat
mg1 was determined for the half-ripe stage.
Specific activity remained stable during the following development
stages.
Western-Blot Analysis
An immunoassay analysis was also performed on a crude protein
extract of fruit with antibodies raised against apple PPO. The specificity of the antibodies was examined using total proteins of
fruit taken from the immature-green to the overripe stages. One band
was visible on the immunoblot (Fig. 7) at
approximately 63 kD, whatever the fruit age, which is in accordance
with the measurable activity during the same period. Moreover, the
apparent constant intensity of the band suggested that the ratio of PPO protein to total proteins remained stable whatever the development stage. The size of the band was similar to that deduced from SDS-PAGE analysis of the purified protein. The slight difference between molecular mass calculated from the PA-PPO cDNA (56.2 kD) and
the experimental molecular mass of apricot PPO under denaturing
conditions in electrophoresis experiments is thus confirmed. Such a
difference, already reported for faba bean PPO by Cary et al. (1992),
and in a less extensive report by Robinson and Dry (1992), was
ascribed to the glycosylation of the protein by the former authors. The mature apricot PA-PPO clone contains effectively one
putative glycosylation site. The PPO may also be artifactually bound by carbohydrates such as phenolic glucosides, which could partially explain its higher-than-56.2-kD size. The results obtained from this
western-blot experiment performed with proteins from a crude extract
have also indicated that no inactive PPO form putatively different from
63 kD had been lost during the purification procedure.
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| Figure 7.
Amounts of PPO protein during ripening of apricot
fruit. Total proteins from apricot fruit at six stages of development:
IM1 (immature green 1), MG (mature green), BR (Breaker), HR
(half-ripe), RI (fully ripe), OR (overripe) were used. Proteins (10 µg per lane) of each stage were separated on SDS-PAGE, transferred
and immunoblotted with anti-apple PPO crude serum. Molecular size
markers are in lane 1.
|
|
| |
CONCLUSIONS |
We have demonstrated that apricot PPO is still active and present
at an advanced stage of the fruit development, whereas its mRNA cannot
be detected. PPO appears therefore as a rather stable protein. A
similar stability of the PPO protein has also been described in potato
tissues (Hunt et al., 1993) and in the young, developing tissues of
grape berries (Dry and Robinson, 1994).
There was no transcript of PA-PPO in leaves, wounded leaves,
and stems that have been characterized by a significant PPO activity. These data may be explained by the expression of other forms of PPO in
these tissues, as reported by Newmann et al. (1993) for tomato tissues.
However, since the RT-PCR probe used for northern hybridization
contains the thylakoid and copper A- and B-binding domains, which are
highly conserved in all PPOs, this probe should have detected other
forms of PPO if they were present. This led us to conclude that,
whatever the PPO gene expressed, the leaves and stems may have been
harvested at a stage too advanced to present a detectable amount of
transcript, even after stimulation by wounding (Boss et al., 1995).
Results obtained from western-blot analysis (Fig. 7) have suggested
that the ratio of PPO protein to total protein remained stable
throughout ripening. We have also demonstrated that specific activity
reached a peak at the breaker stage (Fig. 6). These two different
trends led us to conclude that, in addition to the transcriptional control of PPO expression demonstrated in this work, other regulation factors such as posttranslational controls also occur in the appearance and amount of apricot PPO activity.
This work has allowed real progress in the characterization of a new
fruit PPO. However, the physiological role of the apricot fruit protein
remains to be elucidated. The presence of the protein at all stages of
fruit development may argue for an active role in disease resistance.
| |
FOOTNOTES |
1
These authors contributed equally to this work.
*
Corresponding author; e-mail forget{at}avignon.inra.fr; fax
33-04-90-31-62-58.
Received June 16, 1998;
accepted January 15, 1999.
| |
ABBREVIATIONS |
Abbreviations:
PPO, polyphenol oxidase.
PVPP, polyvinylpolypyrrolidone.
RT, reverse transcriptase.
| |
ACKNOWLEDGMENTS |
The authors express their gratitude to Dr. Laurence
Marquès from Université de Montpellier II for providing
them with PPO antibodies and to Dr. P. Sautière from Centre
National de la Recherche Scientifique, Institut Pasteur, for the
N-terminal sequencing of the purified PPO protein.
| |
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Top
Abstract
Introduction
