BLBS102-c12 BLBS102-Simpson March 21, 2012 13:11 Trim: 276mm X 219mm Printer Name: Yet to Come
238 Part 2: Biotechnology and Enzymologyto be of microbial origin and were commonly isolated from mac-
erated plant tissue infected with fungal or bacterial pathogens
(Yoder et al. 1993, Barras et al. 1994, Mayans et al. 1997, Yadav
et al. 2009). In plants, presence of PL-like sequences was first
identified in mature tomato flowers, anthers, and pollen (Wing
et al. 1989). Presence of PL in pollen of other species (Albani
et al. 1991, Taniguchi et al. 1995, Wu et al. 1996, Kulikauskas
and McCormick 1997) as well as in other tissues (Domingo
et al. 1998, Milioni et al. 2001, Pilatzke-Wunderlich and Nessler
2001) has been well documented. Recently, PL-like sequences
were identified in ripening banana (Pua et al. 2001, Mar ́ın-
Rodr ́ıguez et al. 2003) and strawberry fruit (Medina-Escobar
et al. 1997, Ben ́ıtez-Burraco et al. 2003). Transgenic strawberry
plants that expressed a pectin lyase antisense construct were
firmer than controls, with the level of PL suppression being cor-
related to internal fruit firmness (Jim ́enez-Bermudez et al. 2002). ́
Additional studies using transgenic strawberries suppressed in
the expression of PL support a key role of this enzyme in straw-
berry fruit softening (Santiago-Domenech et al. 2008, Youssef ́
et al. 2009) and strawberry juice viscosity (Sesmero et al. 2009).
PL activity has been detected in ripening cherry tomato fruit
grown under different environmental conditions (Rosales et al.
2009) and several putative PL sequences exist in the tomato ex-
pressed sequence tag databases (Mar ́ın-Rodr ́ıguez et al. 2002),
suggesting that these enzymes may also contribute to cell wall
disassembly during tomato fruit ripening.GENETIC ENGINEERING OF PECTIC
ENZYMES IN TOMATOCell wall disassembly in ripening fruit is an important contrib-
utor to the texture of fresh fruit. Modification of cell wall en-
zymatic activity during ripening, using genetic engineering, can
impact cell wall polysaccharide metabolism, which in turn can
influence texture. Texture of fresh fruit, in turn, influences pro-
cessing characteristics and final viscosity of processed tomato
products. The development of transgenic tomato lines in which
the expression of single or multiple genes is altered has allowed
the role of specific enzymes to be evaluated both in fresh fruit
and processed products.Single Transgenic Tomato LinesPolygalacturonasePG was the first target of genetic modification aiming to retard
softening in tomatoes (Sheehy et al. 1988, Smith et al. 1988).
Transgenic plants were constructed in which expression of PG
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 influence 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 decrease in the extent of
depolymerization of CDTA soluble polyuronides during fruit
ripening. Naturally occurring mutant tomato lines exist, which
carry mutations, affecting the normal process of ripening. One
of these lines, theripening-inhibitor(rin), possesses a single
locus mutation that arrests the response to ethylene.rinfruit fail
to synthesize ethylene and remain green and firm throughout
ripening (DellaPenna et al. 1989). Transgenicrinplants that
expressed a functional PG gene driven by the ethylene-inducible
E8 promoter were obtained. After exposure to propylene, PG
mRNA accumulation as well as active PG protein was attained,
but the fruit did not soften. In these fruit, depolymerization 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 depolymerization
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 manifested at the later stages of ripening and in
fruit senescence.
Suppression ofβ-subunit expression by the introduction of
aβ-subunit cDNA in the antisense orientation resulted in re-
duction of immunodetectableβ-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). Sup-
pression ofβ-subunit expression resulted in increased CDTA-
extractible polyuronide levels in transgenic fruit during ripening.
The size distribution profiles of CDTA-extractable polyuronides
of transgenic and control fruit were nearly identical at the ma-
ture green stage. However, at subsequent ripening stages, an
increased number of smaller size polyuronides were observed
in suppressedβ-subunit fruit compared to control fruit (Watson
et al. 1994).
Apart from the influence of transgenic modification of PG
gene expression on the ripening characteristics and shelf life
of fresh tomato fruit, the processing characteristics of these
fruit were also modified by the introduction of the PG trans-
gene. A shift in the partitioning of polyuronides in water and
CDTA extracts 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 corresponding 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-suppressed 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. Although 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 break down of soluble