We provide genetic evidence that the production
of methanol in tomato fruit is regulated by pectin methylesterase (PME,
EC 3.1.1.11), an enzyme that catalyzes demethoxylation of pectins. The
role of PME in methanol production in tomato fruit was examined by
relating the tissue methanol content to the PME enzymatic activity in
wild-type Rutgers and isogenic PME antisense fruits with lowered PME
activity. In the wild-type, fruit development and ripening were
accompanied by an increase in the abundance of PME protein and activity
and a corresponding ripening-related increase in methanol content. In
the PME antisense pericarp, the level of methanol was greatly reduced
in unripe fruit, and diminished methanol content persisted throughout
the ripening process. The close correlation between PME activity and
levels of methanol in fruit tissues from wild-type and a PME antisense
mutant indicates that PME is the primary biosynthetic pathway for
methanol production in tomato fruit. Interestingly, ethanol levels that
were low and unchanged during ripening of wild-type tomatoes increased
progressively with the ripening of PME antisense fruit. In
vitro studies indicate that methanol is a competitive inhibitor
of the tomato alcohol dehydrogenase (ADH, EC 1.1.1.1) activity
suggesting that ADH-catalyzed production of ethanol may be arrested by
methanol accumulation in the wild-type but not in the PME mutant where
methanol levels remain low.
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INTRODUCTION |
Methanol is emitted by actively growing plant tissues (1) and
ripening fruit (2). A major source of methanol may be pectin methyl
esters (3) that are de-esterified to methanol and pectic substances by
a PME1-catalyzed reaction
(4). Although methanol production is correlated with PME activity in
germinating seeds or other plant tissues (1), the role of PME in
methanol accumulation in plants has not been firmly established (4,
5).
A developmentally regulated increase in PME gene expression occurs in
developing tomato fruit (6) and may be used as a test system to relate
PME activity to methanol metabolism. We have created transgenic tomato
fruits with severely impaired expression of PME by introducing a
fruit-specific PME antisense gene under the control of cauliflower
mosaic virus 35S promoter (7). We compared methanol production in the
wild-type and the PME antisense tomato fruits to examine whether the
arrest of PME gene expression results in diminished production of
methanol. Our results show that methanol accumulation in ripening
tomato pericarp of either the wild-type or the PME antisense is related
to PME activity, suggesting that the PME activity is the primary source
of methanol production in tomato fruits. A surprising finding is that
the tissue methanol and ethanol content in ripening tomato fruit were inversely related.
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EXPERIMENTAL PROCEDURES |
Plant Material--
Wild-type tomato (Lycopersicon
esculentum cv Rutgers) and transgenic 37-81 Rutgers, transformed
with a PME antisense gene (7), tomato plants were grown at the Purdue
Agricultural Experiment Research Farm (8). Fruit from wild-type and PME
antisense tomatoes were harvested at the immature green stage (IMG),
the mature green (MG), breaker (BR), turning (TU), and red ripe (RR)
stages as described earlier (9). The pericarp was frozen in liquid
nitrogen and held at
80 °C until being used for the analysis of
methanol and ethanol content or purification of ADH.
PME Activity and Immunoblot Analysis--
Changes in PME
activity and protein were determined as described earlier (6, 7).
ADH Purification--
ADH purification (10) was done with some
modifications which consisted of the following: 2 g of frozen,
lyophilized pericarp was suspended in 5 ml of buffer consisting of 1 M Tris-HCl, pH 7.4, 10 mM dithiothreitol, 1 mM EDTA, and 1% polyvinylpyrrolidone-insoluble (w/v))
(11). The resulting slurry was centrifuged, and the resulting supernatant was supplemented with
(NH4)2SO4. The 60%
(NH4)2SO4 precipitate was
resuspended in 50 mM Tris-HCl, pH 7.4, 5 mM
dithiothreitol, and dialyzed against the same buffer overnight using
dialysis tubing with a molecular weight cutoff of 6,000 to 8,000. The
dialyzed solution was loaded onto a 1 × 14 cm column of Cibacron
blue (10), and the eluent was concentrated using an Amicon Centriprep
10 Ultrafiltration unit. ADH was purified 700-fold based on specific activity and consisted predominantly of ADH-2 isozyme and only traces
of ADH-1 isozyme (10). Protein concentration was determined using the
Bradford method (Bio-Rad), standardized against bovine serum
albumin.
Extraction and Assay of Methanol and Ethanol--
Fifty g of
frozen tomato fruit tissue were homogenized for 1 min in 200 mL of
ice-cold glass-distilled water containing 1 mM
NaN3. The homogenate was filtered once through Whatman No. 1 filter paper, and the entire filtrate was transferred to a 2-liter round bottom retort-flask and heated. A Thermo-O-Watch L7 SS regulator (Instruments for Research and Industry, Cheltenham, PA) was used to
control heat output and to vaporize volatiles from the filtrate at a
rate of 50 ml/h. Water vapors and volatile compounds were fluxed
through a spiral glass condenser and collected in ice-cold 200-ml round
bottom flasks. Distillation was maintained until 100 to 120 ml of the
filtrate were recovered. Recovery of ethanol, methanol, and
acetaldehyde from test solutions containing 100 mg ml
1 of
the volatile was 100% ± 1.5 suggesting that the employed protocol led
to virtually complete recovery of the compound. The residue from tissue
homogenate filtration was suspended in water and subjected to
distillation (as mentioned above) but did not yield methanol suggesting
that the distillation process is not accompanied by heat-induced
release of methanol from the cell wall residue. The distillate samples
were stored at 2 °C and used for gas chromatography analysis as
outlined by Kimmerer and Kozlowski (12). Quantification of resolved
methanol, ethanol, or acetaldehyde was done by comparison against
standard curves made of aqueous solutions containing defined levels of
the compounds.
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RESULTS |
Relationship between PME Activity and Methanol Accumulation in
Tomato Fruit--
Development of tomato fruit was accompanied by a
progressive increase in the PME activity (Fig.
1A). The low levels of PME activity at the IMG stage increased substantially at the MG stage and
continued to increase as ripening progressed before declining at the RR
stage. In the PME antisense mutant, the PME activity at the IMG stage
was lower than wild-type and remained unchanged throughout fruit
development and ripening. The steady state PME protein levels showed a
pattern similar to PME activity during the ripening and development of
wild-type fruit (Fig. 1B). In PME antisense pericarp, the
34-kDa PME isoform was undetectable whereas the 37-kDa polypeptide,
which cross-reacts with polyclonal fruit PME antibodies (7), remained
unchanged, at all stages of fruit development and ripening. These
results are consistent with the earlier report showing that the
expression of a fruit-specific PME antisense gene impairs accumulation
of the major PME isoforms (7, 13).

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Fig. 1.
Effect of the introduced PME antisense gene
on the levels of PME specific activity (A) and PME protein
(B) in developing and ripening tomato fruit. Immature
green (IMG), mature green (MG), breaker
(Br), turning (TU), and red ripe (RR)
fruits were from field-grown wild-type Rutgers (filled bars)
and transgenic 37-81 (open bars) expressing a PME antisense
gene tomato plants. PME enzyme activity was determined titrimetrically
in the total salt-extractable protein. For PME protein, 10 mg of total
salt-extractable protein from pooled samples for each stage was
electrophoresed on a 12% SDS-PAGE, blotted onto nitrocellulose, and
visualized using polyclonal fruit PME antibodies. The lower polypeptide
bands in B represent the PME molecular forms of 34 kDa in
ripening tomato fruit, while the upper 37-kDa polypeptide band
cross-reacts with polyclonal antibodies raised to fruit PME. The
results are means of data values ± S.E. Filled and
open bars represent PME activity in wild-type and transgenic
37-81.
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We used the pronounced changes in PME activity during development and
ripening of wild-type fruits and the absence of these changes in the
PME antisense fruits to assess the role of PME in methanol accumulation
in tomato fruit. In the wild-type fruits, a basal level of methanol
content in the unripe pericarp increased progressively through the
succeeding ripening stages in a manner similar to the changes in PME
activity (Fig. 2A). However,
methanol remained at a basal level while PME activity increased between the IMG and MG ripening stages. Also, PME activity decreased whereas methanol increased between the TU and RR stages of fruit development. In the PME antisense tomato, the methanol levels in the unripe pericarp
(IMG and MG stages) were 5- to 7-fold lower than in unripe wild-type
pericarp, and fruit ripening was not associated with an increase in
methanol content. Collectively, these results show a correlation
between PME activity and methanol accumulation in tomato fruit
suggesting that methanol production in tomato fruit is regulated by PME
activity.

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Fig. 2.
Content of methanol (A) and
ethanol (B) in developing wild-type Rutgers tomato and PME
mutant (37-81) expressing a PME antisense gene. The results are
means of data values ± S.E.
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Relationship between Methanol and Ethanol Content in Ripening
Tomato Fruit--
Analyses of distillates from tomato pericarp
homogenates revealed that changes in methanol and ethanol levels showed
opposing trends (Fig. 2). In the wild-type, low ethanol levels were
found at the IMG stage, and ripening-related increases in methanol were accompanied by further reduction in ethanol content. In the PME antisense tomato pericarp, the arrest in methanol accumulation was
associated with an increase in ethanol, especially in the RR fruit.
Assuming that tomato pericarp contains 95% H2O (w/w), our
results indicate that levels of methanol increased from 2.5 mM to 6.5 mM during development and ripening of
the wild-type tomato tissue while methanol levels in the PME antisense
pericarp ranged between 0.3 and 0.5 mM (Fig. 2A)
and remained unchanged during fruit development and ripening. Ethanol
levels in the wild-type ranged from 0.48 mM at the MG stage
down to 0.075 mM at the RR stage while ethanol levels in
the PME antisense tissue ranged from 0.715 mM in the MG
stage to 3.98 mM in the RR stage (Fig. 2B).
ADH-catalyzed reduction of acetaldehyde to ethanol is the only known
pathway for ethanol biosynthesis (14) and thus may be a putative target
for methanol-regulated ethanol production. Table
I supports the data in Fig. 2 that
methanol is an inhibitor in vivo since the transgenic fruit
had an overall higher ADH activity than did the wild-type fruit
indicating that methanol does possibly regulate ADH. Also, there are
indications (Table I) that the ADH activity in the PME antisense
pericarp was overall higher than in the fruit tissue of wild-type fruit
suggesting that ADH activity in the mutant was not a limitation to the
enzyme-catalyzed conversion of acetaldehyde to ethanol. In a
Lineweaver-Burk plot (Fig. 3) the
apparent Km for ethanol was determined to be 0.38 mM whereas the apparent Ki for methanol
was 5.1 mM. Methanol inhibition seems to be competitive
much like pyrazole, a known inhibitor of ADH (10). Methanol was found
to have no effect on tomato ADH activity in the absence of ethanol
(data no shown) and has been found not to be a substrate for ADH
in vitro in tomato or other plants (10). An apparent
Km value of 1.74 mM for acetaldehyde for
tomato ADH (10) suggest that methanol levels in normally ripening fruit
may not be sufficiently high to completely arrest acetaldehyde
conversion to ethanol production (Fig. 2B). In the PME
antisense pericarp, the methanol levels, approximately an order of
magnitude lower than the wild-type, may have been sufficiently
low as to be apparently not competitive with ADH-catalyzed reduction of
acetaldehyde to ethanol thus resulting in ethanol accumulation.
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Table I
Comparison of ADH activity between wild-type Rutgers and transgenic
37-81
Pericarp tissue from different developmental stages was frozen and then
pulverized by mortar and pestle. The tissue was then homogenized in 1 M Tris-Cl, pH 7.4, 10 mM dithiothreitol, and 1 mM EDTA, and ADH activity was measured as described in Fig. 3. These results are the average ± S.E. of one experiment with four replications.
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Fig. 3.
A Lineweaver-Burk plot of ADH activity at
different ethanol concentrations as influenced by various levels of
methanol. ADH activity was assayed spectrophotometrically as
described previously (12). The reaction was initiated by the addition
of ethanol, and the rate of NADH formation without ethanol
was subtracted to give the ethanol-dependent rate. Results
are representative of 4 experiments with 4 replicates per
experiment.
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DISCUSSION |
Our results provide evidence that methanol production in tomato
fruit is primarily regulated by PME. PME is a ubiquitous enzyme present
in higher plants (15), and PME activity may partially account for
methanol accumulation and emission in plant tissues (1, 2, 16). The
present results showing that methanol levels are related to PME
activity further support the hypothesis that demethoxylation of pectins
is, at least in part, a metabolic origin of methanol. The lag in
methanol accumulation (Fig. 2A) compared with the increase
in PME activity between IMG and MG stages in wild-type fruit may
reflect the inaccessibility of cell wall pectin methyl esters to PME
action at early ripening stages. Production of several cell wall
hydrolases, including polygalacturonase, lead to a change in the cell
wall structure (17) and may influence the availability of pectinic
substrate for PME activity and in consequence, accumulation of methanol
during ripening of wild-type fruits. The low level of methanol
production in PME antisense fruit pericarp may have resulted from the
low PME activity which probably arises from the group II PME isoforms,
whose synthesis is not impaired by the introduced PME antisense gene
(13). Since methanol production does not increase during ripening of
the PME antisense fruit, these results suggest that the activity of
group II PME isoforms may contribute marginally to methanol
accumulation.
The results also suggest that methanol may regulate ethanol metabolism
in ripening fruits. The decline in methanol may have created metabolic
conditions for accumulation of ethanol in the PME antisense tissue. An
increase in ADH activity during tomato fruit ripening (Table I (11))
suggests that the capacity of fruit tissue for alcoholic fermentation
is not a limitation to the production of ethanol although ethanol
accumulation may be restricted by trace amounts of acetaldehyde in
ripening tomato (results not shown) However, in the PME antisense
tissue the decline in methanol may have created metabolic conditions
for accumulation of ethanol.