278 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
was suppressed to 0.5–1% of wild-type levels by
the expression of an antisense PG transgene under
the control of the cauliflower mosaic virus 35S
(CaMV35S) promoter. Fruit with suppressed levels
of PG ripened normally as wild-type plants did. An
increase in the storage life of the fruit was observed
in the PG-suppressed fruit (Schuch et al. 1991,
Kramer et al. 1992, Langley et al. 1994). The influ-
ence of PG suppression on individual classes of cell-
wall polymers was investigated (Carrington et al.
1993, Brummell and Labavitch 1997). Sequential
extractions of cell-wall constituents revealed a de-
crease in the extent of depolymerization of CDTA-
soluble polyuronides during fruit ripening. Natur-
ally occurring mutant tomato lines exist that carry
mutations affecting the normal process of ripening.
One of these lines, the ripening-inhibitor (rin),pos-
sesses a single locus mutation that arrests the
response to ethylene. rinfruit fails to synthesize eth-
ylene and remains green and firm throughout ripen-
ing (DellaPenna et al. 1989). Transgenic rinplants
that expressed a functional PG gene driven by the
ethylene-inducible E8 promoter were obtained.
After exposure to propylene, PG mRNA accumula-
tion as well as active PG protein was attained, but
the fruit did not soften. In these fruits, depolymer-
ization and solubilization of cell-wall pectin was
at near wild-type levels (Giovannoni et al. 1989,
DellaPenna et al. 1990). Altogether, results obtained
from analyzing transgenic tomato fruit with altered
levels of PG indicate that PG-mediated depolymer-
ization of pectin does not influence softening at the
early stages of ripening, and it is not enough to
cause fruit softening. However, its outcome is mani-
fested at the later stages of ripening and in fruit
senescence.
Suppression of -subunit expression by the intro-
duction of a -subunit cDNA in the antisense orien-
tation resulted in reduction of immunodetectable -
subunit protein levels and PG1 protein levels to
1% of PG1 in control fruit. In transgenic lines,
PG2 expression remained unaltered (Watson et al.
1994). Suppression of-subunit expression result-
ed in increased CDTA polyuronide levels in trans-
genic fruit during ripening. The size distribution
profiles of CDTA-extractable polyuronides in green
fruit of transgenic and control plants were nearly
identical at the mature green stage. However, at sub-
sequent ripening stages, an increased number of
smaller size polyuronides were observed in sup-
pressed -subunit fruit compared with control fruit
(Watson et al. 1994).GENETICENGINEERING OFPG ANDFOOD
PROCESSINGApart from the influence of transgenic modification
of PG gene expression on the ripening characteris-
tics and shelf life of fresh tomato fruit, the process-
ing characteristics of the fruit were also modified by
the introduction of the PG transgene. A shift in the
partitioning of polyuronides in water and CDTA ex-
tracts of cell walls from pastes prepared from con-
trol and suppressed PG lines was observed. The
majority of polyuronides were solubilized in the
water extract in control pastes, whereas in pastes
from PG-suppressed fruit, a greater proportion was
solubilized in CDTA. Water and CDTA extracts
from control paste contained a larger amount of
smaller sized polyuronides compared with the corre-
sponding extracts from PG-suppressed fruit (Fig.
12.4). However, the molecular size profiles from
carbonate-soluble pectin were indistinguishable in
the two lines. Tomato paste prepared from PG-sup-
pressed fruit exhibited enhanced gross and serum
viscosity (Brummell and Labavitch 1997, Kalamaki
2003). The enhancement of viscosity characteristics
of pastes from transgenic PG fruit was attributed to
the presence of pectin polymers of higher molecular
size and wider size distribution in the serum. Al-
though PG seems to play only a small role in fruit
softening, the viscosity of juice and reconstituted
paste prepared from antisense PG lines is substan-
tially increased, largely due to reduced breakdown
of soluble pectin during processing (Schuch et al.
1991, Kramer et al. 1992, Brummell and Labavitch
1997, Kalamaki et al. 2003a).GENETICENGINEERING OFPME INTOMATO
FRUITPectin methylesterase (PME) protein is accumulated
during tomato fruit development, and protein levels
increase with the onset of ripening. Production of
transgenic tomato lines in which the expression of
PME has been suppressed via the introduction of an
antisense PME gene driven by the CaMV35S pro-
moter has been reported (Tieman et al. 1992, Hall et
al. 1993). PME activity was reduced to less than 10%
of wild-type levels, and yet fruit ripened normally.