Plant Physiol. (1998) 117: 363-373
Polygalacturonase Gene Expression in Ripe Melon Fruit Supports a
Role for Polygalacturonase in Ripening-Associated Pectin Disassembly
Kristen A. Hadfield,
Jocelyn K.C. Rose,
Debbie S. Yaver,
Randy M. Berka, and
Alan B. Bennett*
Mann Laboratory, Department of Vegetable Crops, University of
California, Davis, California 95616 (K.A.H., J.K.C.R., A.B.B.); and Novo Nordisk Biotech, 1445 Drew Avenue, Davis, California 95616 (D.S.Y., R.M.B.)
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ABSTRACT |
Ripening-associated pectin
disassembly in melon is characterized by a decrease in molecular mass
and an increase in the solubilization of polyuronide, modifications
that in other fruit have been attributed to the activity of
polygalacturonase (PG). Although it has been reported that PG activity
is absent during melon fruit ripening, a mechanism for PG-independent
pectin disassembly has not been positively identified. Here we provide
evidence that pectin disassembly in melon (Cucumis melo)
may be PG mediated. Three melon cDNA clones with significant homology
to other cloned PGs were isolated from the rapidly ripening cultivar
Charentais (C. melo cv Reticulatus F1 Alpha) and were
expressed at high levels during fruit ripening. The expression pattern
correlated temporally with an increase in pectin-degrading activity and
a decrease in the molecular mass of cell wall pectins, suggesting that
these genes encode functional PGs. MPG1 and MPG2 were closely related
to peach fruit and tomato abscission zone PGs, and MPG3 was closely
related to tomato fruit PG. MPG1, the most abundant melon PG mRNA, was
expressed in Aspergillus oryzae. The culture filtrate
exponentially decreased the viscosity of a pectin solution and
catalyzed the linear release of reducing groups, suggesting that MPG1
encodes an endo-PG with the potential to depolymerize melon fruit cell
wall pectin. Because MPG1 belongs to a group of PGs divergent from the
well-characterized tomato fruit PG, this supports the involvement
of a second class of PGs in fruit ripening-associated pectin
disassembly.
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INTRODUCTION |
Fruit ripening is a genetically programmed event that is
characterized by a number of biochemical and physiological processes that alter fruit color, flavor, aroma, and texture (Brady, 1987
). Extensive cell wall modifications occur during ripening and are thought
to underlie processes such as fruit softening, tissue deterioration,
and pathogen susceptibility. These modifications are regulated at least
in part by the expression of genes that encode cell wall-modifying
enzymes (Fischer and Bennett, 1991). Pectins are a major class of cell
wall polysaccharides that are degraded during ripening, undergoing both
solubilization and depolymerization. In tomato the majority of
ripening-associated pectin degradation is attributable to the cell wall
hydrolase PG. Transgenic tomato plants with altered PG gene expression
indicated that PG-dependent pectin degradation is neither required nor
sufficient for tomato fruit softening to occur (Sheehy et al., 1988;
Smith et al., 1988; Giovannoni et al., 1989). However, data from
experiments using fruit of the same transgenic lines strongly suggested
that PG-mediated pectin degradation is important in the later,
deteriorative stages of ripening and in pathogen susceptibility of
tomato fruit (Schuch et al., 1991; Kramer et al., 1992).
In melon (Cucumis melo) substantial amounts of pectin
depolymerization and solubilization take place during ripening
(McCollum et al., 1989; Ranwala et al., 1992; Rose et al., 1998),
implicating a role for PG in ripening-associated cell wall disassembly
in melons. However, melons have been reported to lack PG enzyme
activity (Hobson, 1962; Lester and Dunlap, 1985; McCollum et al., 1989; Ranwala et al., 1992). The possibility exists that PG is present in
melon but that it does not conform to the expected enzymic properties
in terms of abundance and/or lability, a point illustrated by recent
reports in apple and strawberry, which were previously reported to lack
PG activity but that do in fact accumulate low amounts of protein
and/or measurable activity (Nogata et al., 1993; Wu et al., 1993). In
light of the unexplained discrepancy between ripening-associated pectin
depolymerization and undetectable PG activity in melons, we have
undertaken a study to reexamine the status of PG in melon using the
rapidly ripening cv Charentais (C. melo cv Reticulatus F1
Alpha).
As reported for other cultivars, Charentais melons exhibit substantial
solubilization and a downshift in the molecular-mass profile of
water-soluble pectins, but this is associated with the later stages of
ripening, after softening is initiated (Rose et al., 1998). By
utilizing a molecular approach to analyze PG in melon, we have
attempted to overcome some of the potential limitations of biochemical
methods, such as low abundance of protein, reliance on other cell wall
components, and unknown cofactors for activity and/or lability during
extraction. In doing so, we have identified and characterized a
multigene family encoding putative PGs from Charentais melon, including
three PG homologs that are expressed abundantly during fruit ripening.
The pattern of PG gene expression correlates temporally with the
depolymerization of water-soluble pectins and an increase in
pectin-degrading enzyme activity. Three additional PG homologs were
also identified and shown to be expressed in mature anthers and
fruit-abscission zones, tissues that, similar to ripening fruit, are
undergoing cell separation. The most abundant ripening-associated
putative PG mRNA, MPG1, was expressed in the filamentous fungus
Aspergillus oryzae. The culture filtrate from the
transformed A. oryzae strain XMPG1 exhibited endo-PG activity, further supporting a role for
endo-PG in ripening-associated pectin disassembly in
Charentais melon fruit.
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MATERIALS AND METHODS |
Plant Material
The Charentais melon (Cucumis melo cv Reticulatus F1
Alpha) fruit used in this study were harvested at six distinct
developmental stages that included IG, MG, and R1 to R4. These stages
are described in detail in Rose et al. (1998). Fruit-abscission zones
were collected from the peduncle of field-grown R3 fruit. In melon the
abscission zone is located immediately adjacent to the fruit and is
characterized by the area that "slips" during the ripening of most
cultivars. Anthers were collected from field-grown male flower explants
on the day of flower opening, after they had begun to shed pollen. Pistils (consisting of ovary, style, and stigma) were collected from
female flowers attached to the plant on the day of flower opening.
Roots were collected from 9- and 10-d-old seedlings grown in
vermiculite in a growth chamber at 25°C with a 16-h day/8-h night
cycle. Stem and young leaf tissues were collected from 32-d-old greenhouse-grown plants. All tissues were frozen in liquid
N2 immediately following harvest and stored at
80°C until use.
Protein Extraction and Enzyme Assay
Frozen mesocarp tissue (10 g) from fruit at each stage of
development was homogenized in a mortar in 1 volume of low-salt extraction buffer (10 mM
NaC2H3O2,
pH 4.5, 5 mM 
-mercaptoethanol, 0.5% [w/v] PVPP, 2 mM PMSF, 40 µM leupeptin, and 20 µg/mL
chymostatin). The homogenates were centrifuged at 30,000g
for 15 min, after which time the supernatants were filtered through two
layers of Miracloth (Calbiochem) and designated the low-salt soluble
fraction. The pellets were resuspended in 2 mL of high-salt extraction
buffer (50 mM
NaC2H3O2,
pH 4.5, 1.5 M NaCl, 15 mM EDTA, 5 mM -mercaptoethanol, 2 mM PMSF, 40 µM leupeptin, and 20 µg/mL chymostatin) and shaken at
moderate speed for 2 h at 4°C. The homogenate was centrifuged at
30,000g for 15 min, after which the supernatant was filtered through two layers of Miracloth and designated the high-salt soluble fraction.
Pectin-degrading activity of melon protein extracts and culture
filtrates from Aspergillus oryzae strains XMPG1 and BANe3 (see below) were assayed viscometrically in semi-micro viscometers (Canon-Manning, State College, PA). The reactions were initiated by
adding 200 µL of melon protein extract representing 0.6 g fresh weight or 4.5 µg of culture-filtrate protein (brought to a final volume of 200 µL with 40 mM
NaC2H3O2,
pH 5.0) to 800 µL of 1.0% (w/v) pectin (Sigma, 10% esterified), 50 mM
NaC2H3O2,
pH 5.2, 100 mM EGTA, 150 mM NaCl, and 0.01%
NaN3, and the reactions were incubated at 25°C
for up to 6 h. One unit was defined as the amount of enzyme that
reduced the viscosity by 1% per hour. The assays were conducted in
triplicate. Protein extracts boiled for 30 min were also assayed in
duplicate and did not cause a significant reduction in viscosity.
The pectin-degrading activity of culture filtrates from A. oryzae XMPG1 and BANe3 were determined over a time course of
culture growth at 34°C in 100 mL of MY25 medium (Yaver et al., 1996)
by gel diffusion. An aliquot of culture was removed at the time points shown in Figure 6B, filtered through two layers of Miracloth, and 280 ng of protein was assayed in a total volume of 20 µL in gel-diffusion
plates containing 0.01% (w/v) polygalacturonic acid, 1 mM
EDTA, 100 mM
NaC2H3O2,
pH 5.0, and 1% (w/v) agarose. The plates were incubated for 10 h
at 34°C, stained with 0.05% (w/v) ruthenium red for 30 min, and
destained with water. Culture filtrates from XMPG1 and BANe3 were also
assayed for pectin-degrading activity by measuring the release of
reducing sugars from pectin substrate. The composition of the reactions
was 20 µg of ultrafiltered (YM10 membrane, Amicon, Beverly, MA)
culture filtrate protein, 50 mM EGTA, 150 mM
NaCl, 40 mM
NaC2H3O2,
pH 5.0, and 0.01% NaN3 in a total volume of 800 µL. The reactions were conducted in duplicate, and 200 µL was
removed from each reaction at 0 min, 60 min, 3 h, and 6 h,
and assayed for the presence of reducing groups using cyanoacetamide
(Gross, 1982). The final values for activity of XMPG1 culture filtrate
were calculated as the difference between the BANe3 and XMPG1 values.
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| Figure 6.
A, Gel-diffusion assay of culture filtrates from
A. oryzae transformed with MPG1 (XMPG1) or
untransformed. Aliquots were removed from cultures at 24, 34, 50, 58, and 72 h and filtered through two layers of Miracloth, and equal
amounts of protein were assayed for PG activity. B, Viscometric and
reducing sugar assays of XMPG1 and untransformed culture filtrates.
Culture filtrates from one time point were incubated for up to 6.5 h and assayed at multiple time points for the ability to decrease the
viscosity or release reducing groups of a pectin solution.  ,
Untransformed viscosity;  , XMPG1 viscosity; and  , XMPG1 reducing
sugar.
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RNA Extraction
Polysomal RNA used for RT-PCR of fruit tissue was isolated as
described by Larkins and Hurkman (1978), with some modifications: Frozen melon mesocarp tissues (200 g) were homogenized in an equal volume of ice-cold extraction buffer (0.2 M Tris, pH 8.5, 0.2 M Suc, 0.1 M KCl, 25 mM EGTA,
35 mM MgCl2, 1 mM DTT, 1 mM spermidine-HCl, 0.5% [w/v] deoxycholate, and 1%
[v/v] Triton X-100) and filtered through three layers of Miracloth,
and the cellular debris were pelleted by centrifugation at
12,000g for 10 min. Additional Triton X-100, 0.01% (v/v),
was added to the supernatant, stirred at 4°C for 15 min, and the
mixture centrifuged at 12,000g for 10 min. The polysomes
were pelleted from the supernatant through a 5-mL layer of 1.8 M Suc (in 0.2 M Tris, pH 8.5, 0.1 M
KCl, 25 mM EGTA, 35 mM
MgCl2, and 1 mM DTT) at
257,000g for 3 h. The polysome pellet was resuspended
in 1 mL of 40 mM Tris, pH 8.5, 20 mM KCl, and 10 mM MgCl2, and extracted twice with
phenol equilibrated with NaC2H3O2,
pH 4.0 and twice with chloroform:isoamyl alcohol (24:1, v/v). The RNA
in the aqueous phase was precipitated by the addition of 0.5 volume of
7.5 M NH4-acetate and 2 volumes of
100% EtOH. The RNA was collected by centrifugation at
12,000g, washed with 70% EtOH, air-dried, and resuspended
in RNase-free water.
RNA used for cDNA library construction was isolated from frozen melon
tissue based on the protocol of Wadsworth et al. (1988) with all
volumes scaled up to accommodate 30 g of tissue. Total RNA used
for northern-blot analyses of all tissues and RT-PCR of anthers and
abscission zones was isolated as described by Wan and Wilkins (1994).
Poly(A+) RNA was selected using latex beads
supplied in the Oligotex kit (Qiagen, Chatsworth, CA) and following the
manufacturer's instructions (with the exception that the annealing
time was increased to 45 min).
Oligonucleotide Design and RT-PCR
Degenerate oligonucleotides were designed based on regions of high
homology between aligned PG-deduced amino acid sequences from tomato
(Greirson et al., 1986), peach (Lee et al., 1990) and O. organensis (Brown and Crouch, 1990), and were synthesized by the
Protein Structure Laboratory at the University of California (Davis).
The sequence of the upstream primer, PG1.2, was 5
-ACI GGI GA(T/C)
GA(TC) TG(TC) ATI UC 3, and the sequence of the downstream primer,
PG2.2, was 5-CCA IGT (C/T)TT (A/G/T)AT IC(G/T) IAC ICC (A/G)TT-3
(where I is inosine). The two oligonucleotides correspond to the amino
acid sequences TGGDDCIS (PG1.2) and NGVRIKTW (PG2.2) at positions 267 to 274 and 323 to 330 of tomato fruit PG, respectively. These two
regions flank an additional conserved region at amino acid position 286 to 297 of the tomato fruit protein that has the sequence CGPGHGISIGSL.
The third sequence was used diagnostically to identify cDNAs that
encode putative PGs due to its high level of conservation in all plant
and microbial PGs cloned to date.
First-strand cDNAs were synthesized from 2 µg of total polysomal RNA
from stage R3 Charentais fruit in three separate experiments. The first
experiment used RNA that had not been treated with DNase. The other two
experiments used RNA that had been DNase treated (fast-protein liquid
chromatography-pure, Pharmacia) according to the manufacturer's
instructions. First-strand cDNAs were also synthesized from 2 µg of
total RNA treated with RNase-free DNase, from fruit-abscission zones,
or from 40 ng of poly(A+) RNA from anthers. RNAs
were incubated in 20 µL of 1× first-strand buffer (50 mM
Tris-HCl, pH 8.3, 75 mM KCl, and 3 mM
MgCl2; GIBCO-BRL), 0.5 mM each dATP,
dCTP, dGTP, and deoxyribothymine triphosphate (Pharmacia), 100 ng of
oligo dT12-18 (Pharmacia), 10 mM DTT
(GIBCO-BRL), and 20 units of RNasin (Promega) at 65°C for 10 min and
then placed on ice. Two microliters of Moloney murine leukemia virus RT
(200 units/µL, GIBCO-BRL) was added and the reaction was incubated at
37°C for 1 h. Reactions were then heated to 95°C for 5 min,
and then placed on ice or stored at 20°C until further use. For
each RNA sample, a control reaction with 2 µL of 1× first-strand
buffer added in place of 2 µL of RT was included.
Four microliters of each first-strand reaction or 1 ng of pBS1.9
(tomato fruit full-length PG cDNA; DellaPenna and Bennett, 1988) was
used as a template in PCR. The reaction mixtures were composed of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1 mM MgCl2, 0.01% (w/v) gelatin, 0.2 mM each dATP, dCTP, dGTP, and deoxyribothymine triphosphate
(Pharmacia), 0.8 µM each PG1.2 and PG2.2
oligonucleotides, and 0.2 µL of Taq polymerase
(Perkin-Elmer). The conditions for amplification were 25 or 40 cycles
of 94°C for 1 min, 40°C for 1 min, and then 72°C for 2 min. The
pBS1.9 amplification product served as a reference for gel purification
of melon PCR products. Products were gel purified using Sephaglas
(Pharmacia) and the products were cloned into pCRII using a cloning kit
(Invitrogen, San Diego, CA), both according to the manufacturer's
instructions. Cloned PCR products were sequenced by the
dideoxynucleotide method (Sanger et al., 1977) using
[35S]dATP and modified T7 DNA polymerase
(Sequenase, United States Biochemical), according to the
manufacturer's instructions. Sequence analysis was carried out using
the MacDNASIS Pro 3.5 software package (Hitachi, San Bruno, CA).
RNA Gel-Blot Hybridization
Two micrograms of poly(A+) RNA from fruit
tissues or 1 µg of poly(A+) RNA from root,
stem, leaf, pistil, anther, and fruit-abscission zones was separated by
1.1% (w/v) formaldehyde/1.2% (w/v) agarose gel electrophoresis and
transferred to nylon membranes (Hybond-N, Amersham), according to the
manufacturer's instructions. Membranes were probed with gel-purified,
[
-32P]dATP-labeled insert DNA from pMPG1,
pMPG2, and pMPG3 (full-length clones, see below) and pPG1 to pPG14
(partial-length PCR clones). Probes were labeled by random-hexamer
priming using Klenow DNA polymerase (United States Biochemical). The
hybridizations were carried out for 16 h at 65°C in 2% (w/v)
SDS, 1 M NaCl, 10% (w/v) dextran sulfate, and 100 µg
mL
1 denatured salmon-sperm DNA, with
approximately 50 ng of labeled probe. The blots were washed twice in
1× SSC (0.15 M NaCl and 15 mM sodium citrate)
and 0.1% (w/v) SDS at 65°C and twice in 0.2× SSC and 0.1% SDS at
65°C. Blots were exposed to film with one intensifying screen
(Reflection, DuPont) at 80°C. To estimate the relative abundance of
mRNA encoded by MPG1, MPG2, and MPG3 during fruit ripening, the
corresponding blots were probed with inserts that were labeled to
approximately the same specific activity and exposed to film for 4 h. All other blots were exposed to film for 2 to 4 d, with the
exception of the nonfruit blot probed with labeled insert from pPG4,
which was exposed to film for 4 h.
cDNA Library Construction and Screening
A cDNA library was constructed using 3.3 µg of
poly(A+) RNA prepared from stage R3 Charentais
melon fruit using a 
-ZAP-cDNA synthesis kit (Stratagene). cDNAs
were cloned into the Uni-XAP-XR -phage vector (Stratagene) and
packaged in Gigapack Gold (Stratagene), and the primary library was
amplified according to the manufacturer's protocols.
Duplicate plaque lifts of 1 × 106 (PG1) or
4 × 105 (PG2 and PG3) amplified
recombinants were hybridized with gel-purified, radiolabeled inserts
from pPG1, pPG2, or pPG3 using protocols described by Stratagene.
Hybridized filters were washed twice in 1× SSC and 0.1% SDS at 65°C
and twice in 0.2× SSC and 0.1% SDS at 65°C, and exposed to film
with one intensifying screen (DuPont) at 80°C. Positive plaques
were carried through two additional rounds of screening for
purification and then in vivo excised to release the phagemid DNA. Both
strands of positive cDNA clones corresponding to MPG1, MPG2, and MPG3
were sequenced in the Plant Genetics Facility at the University of
California (Davis) using an automated DNA sequencer (model ABI 377, Perkin Elmer/Applied Biosystems), and gene-specific oligonucleotides
synthesized by Genset (La Jolla, CA) or vector sequence
oligonucleotides for sequencing the ends of the cDNAs. Sequence
analyses were carried out using the MacDNASIS Pro 3.5 software package
and Sequencher 3.0 (Genecode, Madison, WI). The cleavage site of the
signal sequences were predicted using the rules of von Heijne (1983).
All three of the cDNAs contained complete open reading frames. The
deduced amino acid sequence alignments were generated using the Clustal
V multiple-alignment software package (Higgins et al., 1992).
DNA Gel-Blot Hybridization
Total genomic DNA was isolated as previously described by Murray
and Thompson (1980) as modified by Bernatzky and Tanksley (1986). Then,
5 µg was digested with the restriction enzymes EcoRI, BamHI, and HindIII (New England Biolabs),
separated on 0.8% agarose gels, and transferred to Hybond nylon
membranes according to the manufacturer's (Amersham) instructions.
Membranes were probed with gel-purified,
[-32P]dATP-labeled insert DNA from pMPG1,
pMPG2, and pMPG3 (full-length clones), and pPG4, pPG5, and pPG6
(partial-length RT-PCR clones) under the same conditions described
above for RNA-blot hybridizations, washed in 5× SSC and 0.1% SDS at
65°C (Tm 32°C), and exposed to film with one intensifying screen
(DuPont) at 80°C for 4 h. The blots were then washed twice in
0.2× SSC and 0.1% SDS at 65°C (Tm 8°C) and exposed to film as
described above.
Phylogenetic Analysis
The deduced amino acid sequences of MPG1, MPG2, and MPG3 were
aligned to 17 full-length deduced amino acid sequences of PG homologs
using Clustal V multiple-sequence alignment software (Higgins et al.,
1992). The sequences were: tomato fruit (pTOM6; Grierson et al., 1986)
and abscission zone (TAPG1, TAPG2, and TAPG4; Kalaitzis et al., 1995,
1997), peach fruit (PRF5; Lester et al., 1994) and genomic clone (Lee
et al., 1990), apple (Malus domestica) fruit (pGDP-1;
Atkinson, 1994), kiwifruit (Actinidia deliciosa) genomic
clone (Atkinson and Gardner, 1993), avocado (Persea
americana) fruit (pAVOpg; Kutsunai et al., 1993), oilseed rape
(Brassica napus) pod-dehiscence zone (SAC66; Jenkins et al., 1996) and pollen (Sta 44-4; Robert et al., 1993), maize (Zea
mays) pollen (3C12; Rogers et al., 1991), Arabidopsis
thaliana pollen (GenBank accession no. x73222), tobacco
(Nicotiana tabacum) pollen (NPG1; Tebbutt et al., 1994),
alfalfa (Medicago sativa) pollen (P73; Qiu and Erickson,
1996), cotton (Gossypium hirsutum) pollen (G9; John and
Petersen, 1994), and the fungus Aspergillus flavus (Whitehead et al., 1995). Phylogenetic trees were inferred from the
aligned sequences using the maximum parsimony algorithm of the PAUP
software package, version 3.1 (Swofford, 1990). The aligned sequences
were analyzed by a heuristic search with 100 replicates under the
random stepwise addition option and global (tree bisection and
reconnection) branch swapping, with the A. flavus sequence defined as the outgroup.
Heterologous Expression of MPG1
Gene-specific oligonucleotides designed to introduce a
SwaI restriction site at the 5 end and a PacI
restriction site at the 3end of the MPG1-coding region were used to
amplify this region from pMPG1. The reaction mixtures were composed of
10 mM Tris-HCl, pH 8.85, 25 mM KCl, 5 mM
(NH4)2SO4,
2 mM MgSO4, 0.5 mM each
dATP, dCTP, dGTP, and deoxyribothymine triphosphate (Pharmacia), 0.4 µM each oligonucleotide, and 2.5 units of Pwo
DNA polymerase (Boehringer Mannheim). The conditions for amplification
were one cycle of 94°C for 3 min; 25 cycles of 94°C for 1 min,
50°C for 1 min, and 72°C for 1.5 min; and one cycle of 72°C for 5 min. The product was gel purified using the Qiaquick gel-extraction kit
(Qiagen) and cloned into pCR-Blunt using a PCR-cloning kit (Zero Blunt,
Invitrogen).
Both strands of the cloned PCR product in the resulting plasmid, pP1,
were sequenced using Taq polymerase cycle-sequencing and an
automatic DNA sequencer (model 373A, version 1.2.0, Applied Biosystems). The SwaI/PacI insert of pP1 was
ligated to SwaI/PacI digested pBANe15 to obtain
the A. oryzae expression vector pXMPG1. The pBANe15 vector
includes the promoter sequence from the A. oryzae
-amylase gene, the termination sequence from the Aspergillus niger glucoamylase gene and the Aspergillus nidulans
acetamidase (amdS) gene used as a selectable marker
(Christensen et al., 1988; Nelson et al., 1997). Protoplasts from
A. oryzae strain BANe3 (A1560 [Christensen et al., 1988]
and 
amdS, amyA, amyB, and amyC [Nelson et al., 1997])
were prepared and transformed with 10 µg of pXMPG1 DNA as previously
described (Christensen et al., 1988), and transformants were selected
on minimal-medium plates with acetamide as the sole N source (Cove,
1966). Culture filtrates of the primary transformants were screened for
PG activity by gel-diffusion assays (described above), and
transformants expressing high levels of activity were spore purified
and rescreened. One strain expressing a high level of activity, XMPG1,
was selected for further analysis.
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RESULTS |
Ethylene Production, Textural Changes, and PG Activity during
Melon Fruit Ripening
Charentais melon fruit undergo a ripening-associated decrease in
fruit firmness, which is accompanied by a dramatic increase in internal
ethylene concentration (Rose et al., 1998). In the ripening fruit used
in the present study, softening occurred rapidly between stages R1 and
R2 and continued through R4. Softening beyond stage R3 represented
overripe deterioration of the fruit. Protein was extracted from IG, MG,
and R1 to R4 fruit and assayed for pectin-degrading activity
viscometrically using commercially available citrus pectin. When
high-salt extracts were assayed, a reduction in viscosity was detected
at all time points but was highest at stage R3, after softening was
initiated and during the later stages of ripening (Fig.
1). Low-salt extracts did not cause a
significant reduction in viscosity (data not shown). The highest level
of activity was correlated with the period of depolymerization of water-soluble pectins (Rose et al., 1998).
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| Figure 1.
Pectin-degrading activity in high-salt protein
extracts from developing melon fruit. Extracts from fruit at six
developmental stages of ripening (IG, MG, and R1-R4) were assayed
viscometrically using 10% esterified citrus pectin. One unit was
defined as the amount of enzyme that reduced the viscosity by 1% per
hour. Each value is an average ± SD of three
independent measurements. Low-salt and boiled (30 min) high-salt
protein extracts did not cause a significant reduction in viscosity
(data not shown). gfw, Grams fresh weight.
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Cloning of a Gene Family Encoding Putative PGs
Degenerate oligonucleotides were designed based on regions of
homology between aligned PG deduced amino acid sequences from tomato,
peach, and O. organensis and were used to amplify
partial-length melon cDNAs from reverse-transcribed RNA of ripe fruit
mesocarp, ripe fruit-abscission zones, and mature anthers. The
amplified product was 190 bp, as predicted from the sequences of known
PGs.
A total of 27 individual RT-PCR clones were sequenced from ripe fruit,
resulting in the identification of 12 unique sequences, designated pPG1
to pPG3, pPG5 to pPG6, and pPG8 to pPG14. All of the sequences were
represented only once except pPG1 (seven times), pPG5 (five times),
pPG8 (two times), pPG9 (three times), and pPG10 (three times).
Ten individual PG cDNA clones were sequenced from anthers and found to
be identical. This sequence was unique compared with the melon fruit
clones and was designated pPG4. Seven individual RT-PCR clones from
fruit-abscission zones were sequenced, five of which were identical to
pPG5 and two of which defined a second gene, designated pPG7. In
total, 14 distinct, partial-length cDNAs encoding putative
PGs were identified in melon.
Genome Structure of PGs
The full-length cDNAs MPG1, MPG2, and MPG3 and the partial length
cDNAs pPG4, pPG5, and pPG6 were used to probe genomic DNA blots to
determine the organization of these genes in the melon genome. MPG1,
MPG2, MPG3, and pPG5 all hybridized to a small number of distinct
genomic fragments at both low stringency (Tm 32°C; data not shown)
and high stringency (Tm 8°C; Fig. 2),
indicating that they are transcribed from divergent genes of low copy
number. pPG4 and pPG6, however, hybridized to a common set of genomic fragments at low stringency (Tm 33°C; data not shown) but each to a
distinct subset at high stringency (Tm 8°C; Fig. 2), indicating that they belong to the same genomic subfamily but that they are transcribed from distinct genes.
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| Figure 2.
Melon genomic DNA gel-blot analysis of PG.
Genomic DNA (5 µg/lane) was digested with EcoRI (E),
BamHI (B), or HindIII (H). The blots were
probed with the MPG1, MPG2, or MPG3 full-length cDNA or the PG4, PG5,
or PG6 PCR fragments, and washed at a stringency of Tm 8°C.
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Tissue-Specific Expression of PG Genes in Melon
Gel blots of RNA from preripe and ripening fruit and other
nonfruit tissues were probed with the 14 partial length cDNAs described above to determine their patterns of expression. The expression of six
of the corresponding PG genes was detected in a variety of tissues;
three of the PG genes, MPG1, MPG2, and
MPG3, were expressed in ripening fruit. The full-length
clones corresponding to these genes were isolated from a ripe-fruit
cDNA library (see below). Figure 3
shows the results of hybridization with labeled inserts from the
full-length clones MPG1, MPG2, and MPG3 and labeled inserts from the
partial length clones pPG4, pPG5, and pPG6. pPG4 hybridized strongly to
a 1.7-kb mRNA present in anthers, and was not detected in any other
tissue examined. pPG6 also hybridized to a mRNA from anthers but was
larger, 1.9 kb, and much less abundant compared with pPG4. The
sequences of these two cDNAs were also very similar to each other
compared with other melon PGs over the same region. pPG5 hybridized to
a 1.7-kb mRNA in fruit-abscission zones, but did not hybridize to RNA
from any other tissue.
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| Figure 3.
RNA-blot analysis of PG RNA in developing melon
fruit and nonfruit tissues. Each lane was loaded with 2 µg of
poly(A+) RNA isolated from fruit tissues at six stages of
development (IG, MG, and R1-R4) or 1 µg of poly(A+) RNA
from roots (R), young leaves (L), stems (S), pistils (P), anthers (A),
and fruit-abscission zones (AZ). The blots were probed with the MPG1,
MPG2, or MPG3 full-length cDNA or the PG4, PG5, or PG6 PCR fragments,
and washed at a stringency of Tm 8°C.
|
|
To estimate the relative abundance of MPG1, MPG2, and MPG3 mRNA during
fruit ripening, the full-length clones were labeled to the same
specific activity and the fruit RNA blots were exposed to film for an
equal length of time. The genes corresponding to MPG1, MPG2, and MPG3
were all expressed during fruit ripening but differed in the relative
abundance of mRNA accumulation, the temporal pattern of expression, and
in the size of mRNAs they encode. MPG1 hybridized predominantly to a
1.5-kb mRNA but also detected a 1.9- and a 2.6-kb mRNA. The abundance
of the predominant 1.5-kb mRNA was highest at stage R2 and then
decreased slightly at stages R3 and R4. The largest transcript was
least abundant at stage R2, increased at stage R3, and remained at the
same level at stage R4. The intermediate-sized transcript appeared at
stage R3 and then increased. The relative abundance of the two larger transcripts hybridizing to MPG1 were approximately the same at stage
R4, each about one-half as abundant as the 1.5-kb transcript. MPG2
hybridized to a 1.6-kb transcript and was expressed very abundantly at
stage R2 and at much reduced levels at stages R3 and R4. When the MPG2
blot was exposed to film for a longer time (data not shown), a 3.0-kb
transcript became evident at stage R2. MPG3 hybridized to a 1.8-kb
transcript and was highest at stages R2 and R3, but the overall
abundance of this transcript was much lower in fruit compared with MPG1
and MPG2.
After extended exposure times, hybridization of MPG1, MPG2, and MPG3 to
mRNA from nonfruit tissues was also detected at moderate levels. MPG1
hybridized to a 1.5-kb mRNA from fruit-abscission zones, but was not
detected in any other tissue examined. MPG2 hybridized moderately to a
1.6-kb transcript in fruit-abscission zones, and MPG3 hybridized
moderately to 1.8-kb transcripts in pistil and anther RNA and very
weakly to transcripts in all of the other tissues examined.
Isolation and Characterization of cDNAs Encoding MPG1, MPG2, and
MPG3
A cDNA library was constructed from ripe melon fruit RNA and
screened with inserts from pPG1, pPG2, and pPG3. The resulting cDNA
clones MPG1, MPG2, and MPG3 were 1521, 1641, and 1767 bp in length,
respectively, and each contained a complete open reading frame (Fig.
4). The length of the cDNA clones
corresponded to the size of the most abundant corresponding mRNA, and
it is assumed that they represent full-length mRNAs.
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| Figure 4.
Sequence analysis of the MPG1, MPG2, and MPG3
cDNAs and alignment of their deduced amino acid sequences. Asterisks
and dots indicate identical and conserved amino acid residues,
respectively, between the MPG1, MPG2, and MPG3 sequences. Arrows
indicate predicted signal sequence cleavage sites. Potential Asn
glycosylation sites (N-X-S/T) in each sequence are underlined.
|
|
Analysis of the deduced amino acid sequences revealed several
structural features of interest (Fig. 4). MPG1, MPG2, and MPG3 encoded
predicted mature proteins of 40, 43, and 47 kD, respectively, with
basic pIs of 7.9, 8.5, and 7.5, respectively. All three contained an
N-terminal hydrophobic signal sequence characteristic of proteins that
are translocated into the lumen of the ER, the point of entry into the
secretory system for proteins targeted to various cellular compartments, including the cell wall. Additionally, the three mature proteins contained potential N-glycosylation sites (N-X-S/T); one, located at amino acid position 259 to 261 in MPG1, is
conserved between MPG1, MPG2, and many other plant PGs. There was a
high level of conservation of Cys residues and short domains between the melon sequences and other plant PGs, suggesting that these regions
may be critical to activity. A Gly-rich region in the carboxyl-half of
the sequences (position 237-249 in MPG1) is highly conserved, and a
His residue (position 241 in MPG1) found in this region is present in
all PGs sequenced to date and has been ascribed a catalytic function
(Scott-Craig et al., 1990; Caprari et al., 1996). In addition, a Tyr
residue at amino acid position 310 in MPG1 is strictly conserved and
has been shown to be essential for the activity of PG from
Aspergillus spp. (Stratilova et al., 1996). The three
deduced sequences differed in their N termini of the predicted mature
(after cleavage of the signal peptide) protein. MPG3 encoded for an
additional 47 amino acids and MPG2 for an additional 16 compared with
MPG1. This region of MPG3 is reminiscent of the 47-amino acid
prosequence that is present in tomato fruit PG and cleaved during
transport through the secretory system (DellaPenna and Bennett, 1988).
MPG1, MPG2, and MPG3 show differences at the level of amino acid
identity as well. MPG3 was less than 20% identical to MPG1 and MPG2,
but was 40% identical to tomato fruit PG. MPG1 and MPG2 shared 40%
identity with each other, and were also similar to peach fruit and
tomato leaf-abscission zone (TAPG1) PGs, sharing 60 and 48% identity,
respectively.
A phylogram was generated using an alignment of the deduced amino acid
sequences of MPG1, MPG2, and MPG3 and of 17 PG homologs from other
plant and fungal species, described in ``Materials and Methods''
(Fig. 5). The phylogram groups the PGs
into three major clades. PG gene family members from a single species
segregate into different clades, suggesting that the structural
divergence of plant PGs occurred prior to the divergence of the major
angiosperm families. Clade A includes PGs that are expressed in fruit
and/or abscission zones and includes the peach fruit-specific PG,
tomato abscission zone-specific PGs, and MPG1 and MPG2. Clade B
includes PGs that are expressed in fruit or dehiscence zones and
includes the tomato fruit-specific and ripening-regulated PG and MPG3.
Clade C is comprised primarily of PGs that are expressed in pollen or
anthers, tissues in which exo-PG enzyme activity is
prevalent, suggesting that the clade C PGs are likely to encode
exo-PGs. When a phylogram was generated from an alignment of
the region encoded by the partial-length melon PGs, pPG4 and pPG6 were
placed in clade C and pPG5 was placed in clade A (data not shown), as
expected based on their pattern of expression.
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| Figure 5.
Phylogram for 19 plant and 1 fungal PG cDNA and
genomic clones. The phylogram was generated from the alignment of the
full-length deduced amino acid sequences described in ``Materials and Methods'' and was created using the PAUP software package (Swofford et
al., 1990). The PG sequences segregate into three major clades that we
have designated A, B, and C.
|
|
Heterologous Expression of MPG1
The most abundant putative PG mRNA in ripening melon fruit was
MPG1, whereas mRNA corresponding to the apparent homolog of the tomato
fruit-ripening PG, MPG3, was expressed at much lower levels. To test
whether MPG1 encoded a functional PG enzyme with the potential to
catalyze pectin disassembly in ripening melon fruit, the cDNA was
expressed in A. oryzae and the resulting enzyme was tested
for PG activity (Fig. 6). Several strains
of A. oryzae were tested for production of PG activity, and
one strain (XMPG1) was selected for further characterization. The XMPG1
strain was shown to have the MPG1 cDNA integrated into the A. oryzae genome and was shown to accumulate full-length MPG1
transcript. Samples of the culture filtrate from XMPG1 or an
untransformed control were collected over a 72-h culture period and
assayed for PG activity using a gel-diffusion assay to determine the
optimal growth period for heterologous protein production and to test
for the presence of endogenous PGs expressed in A. oryzae.
There was no detectable PG activity in culture filtrates of the
untransformed strain of A. oryzae under the growth
conditions used, whereas strain XMPG1 produced high levels of PG
activity that were detectable after 34 h of growth and accumulated
to high levels throughout the culture period (Fig. 6A). To determine
whether MPG1 encoded an endo- or exo-acting PG,
the XMPG1 culture filtrate was further characterized for pectinase
activity by both viscometric and reducing sugar assays (Fig. 6B).
Compared with the untransformed control, the PG present in the culture
filtrate of XMPG1 exponentially reduced the viscosity of a pectin
solution but resulted in the linear production of reducing groups (Fig.
6B), indicating that MPG1 encodes an endo-PG with the
potential to depolymerize pectin substrates in muro.
| |
DISCUSSION |
Charentais melon is a typical climacteric fruit, exhibiting an
increase in respiration and autocatalytic ethylene production that
accompanies ripening (Hadfield et al., 1995). During ripening there is
a shift to a lower-molecular-mass distribution of hemicellulose polymers and a substantial solubilization and depolymerization of
pectins, particularly the water-soluble pectins (Rose et al., 1998).
Previous attempts to detect PG activity in ripening melon have been
unsuccessful, and it has been suggested that ripening-associated pectin
degradation is PG independent (Hobson, 1962; Lester and Dunlap, 1985;
McCollum et al., 1989; Ranwala et al., 1992) and that pectin
depolymerization may result primarily from the activity of
-galactosidase in melon (Ranwala et al., 1992). We present data here
that support a role for PG in pectin depolymerization in melon.
Pectin-degrading enzyme activity was measured viscometrically in tissue
extracts from all stages of fruit maturity. The highest level of
activity was at R3 and correlates with data from cell wall analysis
showing the greatest reduction in the molecular size of water-soluble
pectins occurring at the same developmental time (Rose et al., 1998).
The activity observed in the earlier time points was largely unexpected
based on the cell wall analysis, but suggests that other constraints on
PG activity are operative in vivo. In tomato there is ample evidence
that PG-dependent pectin degradation may be limited by factors such as
Ca2+ or substrate accessibility (Brady et al.,
1987). Alternatively, the pectin-degrading activity observed in preripe
fruit may have resulted from glycosidase activity. It has been
previously reported that partially purified extracts of
-galactosidase from melon are able to cause a decrease in the
molecular mass of pectins, although this decrease does not closely
resemble that occurring endogenously during ripening (Ranwala et al.,
1992).
Using sensitive molecular techniques we have identified three genes
that are expressed abundantly during melon fruit ripening and that
encode putative PGs. The similarity of the deduced amino acid sequences
to other plant PGs and the temporal correlation between the appearance
of their mRNAs, in vitro pectin-degrading activity, and the decrease in
molecular mass of cell wall pectins suggests that the proteins encoded
by these genes are PGs. Expression of the most abundant fruit-ripening
PG, MPG1, in the heterologous host A. oryzae and analysis of
culture filtrate from strain XMPG1 showed that MPG1 encodes an
endo-PG and has the potential to depolymerize pectin in
muro. The overlapping expression of MPG1, MPG2, and MPG3 suggests a
potential cooperative role of these enzymes in degrading pectin,
perhaps reflecting their mode of action as endo- or
exo-PGs. A similar case exists in peach, in which three
separate PG activities are present, two of which are exo-
and one of which is endo-acting (Lester et al., 1994).
The patterns of hybridization shown on genomic Southern analysis
indicate that the melon PG gene family is composed of members that are
highly divergent. This has been shown in other species such as tomato,
in which the fruit-ripening PG is encoded by a single gene that does
not hybridize to any other genomic fragment, even at low stringency
(DellaPenna et al., 1986). Similarly, in melon, MPG1 and MPG2 each
hybridized strongly to a single genomic fragment, and MPG3 to a small
number of genomic fragments at low and high stringency, indicating that
they are encoded by single genes that are divergent from each other.
(The presence of multiple fragments that hybridize to MPG3 at high
stringency can be partially accounted for by restriction sites known to
exist in the cDNA sequence.)
pPG5 hybridizes to a unique set of fragments at low stringency and to a
subset of these fragments at high stringency, indicating that pPG5
belongs to a small subset of closely related PGs that does not include
the fruit-ripening PGs. A similar result was reported for a PG gene
expressed in tomato-abscission zones, which hybridizes to a set of
genomic fragments distinct from that of the fruit-ripening PG
(Kalaitzis et al., 1995). pPG4 and pPG6 hybridize to a common set of
genomic restriction fragments at low stringency but to distinct subsets
at high stringency, indicating that they are members of yet another
subfamily of PG genes, this one possibly encoding PGs that are all
expressed in pollen. It has been shown in maize that pollen PG is
encoded by a small family of closely related genes that differ in
nucleotide sequence by only 1% (Niogret et al., 1991).
Evolutionary relationships were inferred from a phylogenetic tree
generated from an alignment of the deduced amino acid sequences of
MPG1, MPG2, and MPG3, with one fungal homolog and 16 plant PG homologs.
The PGs group into three major families, which we designated clades A,
B, and C. The PGs in clade C are probably related in terms of function
and tissue specificity because they are all found in pollen or anthers
and are presumably exo-PGs. Exo-PGs do not,
however, appear to group with clade C exclusively. A gene expressed
during tomato seed germination that encodes a putative
exo-PG is a member of clade B (B. Downie, Y. Sitrit, K.A.
Hadfield, A.B. Bennett, and K.J. Bradford, personal communication).
The two most abundant ripening-induced PG genes in melon, MPG1 and
MPG2, are members of clade A, and the least abundant, MPG3, is a member
of clade B. A distinction based on mode of action or tissue specificity
between members of clade A and B is not evident, but one feature that
distinguishes clade B from the other two clades is the presence of a
predicted prosequence immediately following the N-terminal hydrophobic
signal sequence. In tomato fruit PG, the propeptide is cleaved from the
mature protein during transport through the secretory system en route
to the cell wall (DellaPenna and Bennett, 1988). The purpose of the
prosequence is not known, but it has been hypothesized that it
functions to keep proteins inactive as they travel to their ultimate
destination in the cell, or that it is involved in subcellular
localization. The same three clades were formed when a phylogenetic
tree was generated using the 190-bp conserved region defined by the
RT-PCR primers, indicating that the presence of the prosequence is not the basis of the divergence of the members of clade B. The members of
clades A and B appear to share a common ancestor and are more closely
related to each other than to members of clade C. It remains to be seen
whether detailed analyses of a variety of PGs will eventually reveal a
biochemical and/or tissue-specific basis for their divergence into
three major clades.
As more sensitive techniques are used to detect PGs in tissues
undergoing pectin degradation, it is becoming evident that PG is
consistently associated with this process, not only during fruit
maturation, but in other tissues where cell separation is taking place,
such as abscission and pod-dehiscence zones, germinating pollen, and
growing pollen tubes (Brown and Crouch, 1990; Taylor et al., 1990;
Bonghi et al., 1992; Allen and Lonsdale, 1993; Kalaitzis et al., 1995,
1997; Petersen et al., 1996). Because of its high abundance,
tomato-fruit PG is the most widely studied in relation to pectin
disassembly. However, experiments with transgenic plants indicate that
suppression of PG gene expression by 80% has little effect on pectin
disassembly (Smith et al., 1990), suggesting that the enzyme is present
in at least 5-fold excess. Low PG levels, such as those found in apple
and melon, may be more typical in fruit undergoing ripening-associated
pectin disassembly. In addition, PGs divergent from tomato-fruit PG,
such as MPG1, may also be important in the ripening process of other
fruit. Suppression of MPG1 gene expression in transgenic melons will
provide a critical assessment of this possibility.
| |
FOOTNOTES |
*
Corresponding author; e-mail abbennett{at}ucdavis.edu; fax
1-530-752-4554.
Received July 2, 1997;
accepted January 1, 1998.
1
This research was supported in part by a grant
from Zeneca Plant Science, Jealotts' Hill, UK.
| |
ABBREVIATIONS |
Abbreviations:
IG, immature-green ripening stage.
MG, mature-green ripening stage.
PG, polygalacturonase.
R1 to R4, ripening
stages 1 to 4.
RT, reverse transcriptase.
| |
ACKNOWLEDGMENTS |
We would like to thank Dr. John Labavitch for his careful
reading of this manuscript and Aubrey Jones for providing A. oryzae protoplasts.
| |
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Abstract
Introduction