BLBS102-c12 BLBS102-Simpson March 21, 2012 13:11 Trim: 276mm X 219mm Printer Name: Yet to Come
12 Pectic Enzymes in Tomatoes 239UA equivalents,μg/mL total UA5101520253035404550
Control -P G05101520253035404550(A)(B)19 29 39 49 59 69
Fraction numberFigure 12.4.Size exclusion chromatography of CDTA-soluble
pectin fractions from juice (A) and paste (B) prepared from control
(-) and suppressed PG (.) fruit. Larger polysaccharide molecules
elute close to the void volume of the column (fraction 20), whereas
small molecules elute at the total column volume (fraction 75).
(Based on data presented by Kalamaki 2003.)pectin during processing (Schuch et al. 1991, Kramer et al. 1992,
Brummell and Labavitch 1997, Kalamaki et al. 2003a).Pectin MethylesterasePME protein is accumulated during tomato fruit development,
and protein levels increase with the onset of ripening. Pro-
duction 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 promoter has been reported
(Tieman et al. 1992, Hall et al. 1993). PME activity was re-
duced to less than 10% of wild-type levels, and yet fruit ripened
normally. The degree of methylesterification increased signifi-
cantly in antisense PME fruit by 20–40%, compared to wild-type
fruit. Additionally, the amount of chelator-soluble polyuronides
decreased in transgenic lines, a result that was expected since
demethylesterification is necessary for calcium-mediated cross-
linking of pectin. Furthermore, chelator-soluble polyuronides
from red ripe tomato pericarp exhibited a larger amount of inter-
mediate size polymers in transgenic lines than controls, indicat-
ing a decrease in pectin depolymerization of the chelator-soluble
fraction. As described above, PME action precedes that of PG.
An increase of about 15% in soluble solids was also observed in
transgenic fruit pericarp cell walls (Tieman et al. 1992). Expres-
sion of the antisense PME construct also influenced the abilityof the fruit tissue to bind divalent cations. Calcium accumulation
in the wall was not influenced; however, two-thirds of the total
calcium in transgenic fruit was present as soluble calcium, com-
pared to one-third in wild-type fruit (Tieman and Handa 1994).
Pectin with a higher degree of methylesterification was found in
PME-suppressed fruit, thus presenting fewer sites for calcium
binding in the cell wall.
From a food-processing standpoint, the effect of these mod-
ifications in PME activity in tomato cell walls during ripening
was further investigated. Juice prepared from transgenic anti-
sense PME fruit had about 35–50% higher amount of total UAs
than control fruit. Pectin extracted from juice processed either
by a hot break, microwave break, or a cold-break method was
methylesterified at a higher degree in PME-suppressed juices.
In addition, pectin from transgenic fruit was of larger molecu-
lar mass compared to controls (Thakur et al. 1996a). Juice and
paste prepared from antisense PME fruit had higher viscosity
and lower serum separation (Thakur et al. 1996b).β-GalactosidaseIn tomato fruit, a considerable loss of galactose is observed dur-
ing the course of ripening. This is attributed to the action of en-
zymes with exo-galactanase/β-galactosidase activity. In order to
further investigate their role in ripening,β-galactosidaseTBG1
gene expression was suppressed in transgenic tomato by the ex-
pression of a sense construct of 376 bp of theTBG1gene driven
by the CaMV35S promoter (Carey et al. 2001). Although plants
with different expression levels of the transgene were obtained,
there was no effect onβ-galactosidase protein activity and fruit
softening. The results suggest that theTBG1gene product may
not be readily involved in fruit softening but may act on a spe-
cific cell wall substrate (Carey et al. 2001). Theβ-galactosidase
isoform II, encoded byTBG4, has been found to accumulate in
ripening tomato fruit (Smith et al. 1998). The activity of TBG4,
has been suppressed by the expression of an 1.5 kb antisense
construct ofTBG4(Smith et al. 2002). Antisense lines exhibited
various degrees ofTBG4mRNA suppression that was correlated
to reduced extractable exo-galactanase activity. In one antisense
line, a 40% increase in fruit firmness was observed, which corre-
lated with the highest—among transgenic lines—TBG4mRNA
suppression, lowest exo-galactanase activity levels, and highest
galactosyl content during the early stages of ripening, suggesting
a possible role forTBG4in the early events of fruit softening.
(Smith et al. 2002).Double Transgenic Tomato LinesAs reported above, modifying the expression of individual cell
wall hydrolases did not alter fruit softening substantially, sug-
gesting that cell-wall-modifying enzymes may act as a consor-
tium to bring about fruit softening during ripening. Generation
of double transgenic lines further aids in understanding the in-
fluence of cell wall deconstruction during ripening to the texture
of ripe fruit and on the physicochemical properties of processed
tomato products.