280 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
compared with controls (Thakur et al. 1996a). Juice
and paste prepared from antisense PME fruit had
higher viscosity and lower serum separation (Tha-
kur et al. 1996b).
GENETICENGINEERING OF-GALACTOSIDASE
INTOMATOFRUIT
In tomato fruit, a considerable loss of galactose is
observed during the course of ripening. This is attri-
buted to the action of enzymes with exo-galactanase/
-galactosidase activity. In order to further investi-
gate their role in ripening, -galactosidase TBG1
gene expression was suppressed in transgenic toma-
to by the expression of a sense construct of 376 bp
of the TBG1gene driven by the CaMV35S promoter
(Carey et al. 2001). Although plants with different
expression levels of the transgene were obtained,
there was no effect on -galactosidase protein activ-
ity and fruit softening. The results suggest that the
TBG1gene product may not be readily involved in
fruit softening but may act on a specific cell-wall
substrate (Carey et al. 2001). The -galactosidase
isoform II, encoded by TBG4,has been found to
accumulate in ripening tomato fruit (Smith et al.
1998). The activity of TBG4 has been suppressed by
the expression of a 1.5 kb antisense construct of
TBG4 (Smith et al. 2002).
DOUBLETRANSGENICTOMATOLINES INFOOD
PROCESSING
As reported above, modifying the expression of
individual cell-wall hydrolases does not alter fruit
softening substantially, suggesting that cell-wall
modifying enzymes may act as a consortium to bring
about fruit softening during ripening. Generation of
double transgenic lines further aids in understanding
the influence of red ripe fruit characteristics on the
physicochemical properties of processed products.
SUPPRESSEDPG ANDSUPPRESSEDEXP 1
The influence of the suppression of the expression
of two enzymes in the same transgenic line has been
reported (Powell et al. 2003, Kalamaki et al. 2003a).
Tomato lines with simultaneous suppression of the
expression of both PG and ripening-associated ex-
pansin have been produced and their chemical and
physical properties characterized (Kalamaki et al.
2003a, Powell et al. 2003). Expansins are proteins
that lack wall hydrolytic activity and are proposed to
act by disrupting the hydrogen bonding between cel-
lulose microfibrils and the cross-linking glycan
(xyloglucan) matrix (see Fig. 12.1). Expansins are
involved in cell-wall disassembly during ripening
(Brummell et al. 1999a). Their role in ripening is
proposed to be that of loosening the wall and in-
creasing the accessibility of wall polymers to hydro-
lytic enzymes (Rose and Bennett 1999). In tomato,
the product of one expansin gene (Exp1)is accumu-
lated exclusively during tomato fruit ripening (Rose
et al. 1997, Brummell et al. 1999b). Juices and
pastes prepared from fruit with modified levels of
Exp1 exhibit enhanced viscosity attributes (Kala-
maki et al. 2003b).
Double transgenic lines were generated by cross-
ing single transgenic homozygous lines. Fruit was
harvested at the mature green stage and allowed to
ripen at 20°C, and texture was determined as the
force required to compress the blossom end of the
tomato by 2 mm. Fruits from the double suppressed
line were firmer than controls and single transgenic
lines at all ripening stages. The same results were
observed in fruit ripened on vine. At the red ripe
stage, fruits from the double suppressed line were
20% firmer than controls. The flow properties of
juice prepared from control, single transgenic, and
double transgenic lines at the mature green/breaker,
pink, and red ripe stages were evaluated using a
Bostwick consistometer. Average juice viscosity
decreased as ripening progressed in all genotypes.
Juice prepared from fruit of the suppressed PG and
the double suppressed lines at the red ripe stage
exhibited higher viscosity than the control (Powell
et al. 2003).
In another experiment, an elite processing variety
was used as the wild-type background to suppress
the expression of PG, Exp1, and both PG and Exp1
in the same line (Kalamaki et al. 2003a). Juice and
paste were prepared from control, single suppressed,
and double suppressed fruit, and their flow proper-
ties were characterized. In paste diluted to 5° Brix,
an increase in Bostwick consistency of about 18%
was observed for the PG-suppressed and Exp1-
suppressed genotypes. Diluted paste from the dou-
ble suppressed genotype exhibited the highest vis-
cosity; however, this increase in consistency of the
double transgenic line was only an additional 4%
compared with the single transgenic lines. Analysis
of particle size distributions at 5° Brix in juices and