Food Biochemistry and Food Processing

12 Pectic Enzymes in Tomatoes 277

ment, and it accelerates with the onset of ripening
(Gross 1984, Seymour et al. 1990). In tomato fruit,

-galactosidase is encoded by a small multigene
family of at least seven members, TBG1–TBG7
(Smith et al. 1998, Smith and Gross 2000). Based on
sequence analysis, it was found that
-galactosidase
genes encode putative polypeptides with a predicted
molecular mass between 89.8 and 97 kDa, except
for TBG4, which is predicted to be shorter by 100
amino acids at its carboxyl terminus (Smith and
Gross 2000). A signal sequence targeting these pro-
teins to the apoplast was only identified in TBG4,
TBG5,and TBG6,whereas TBG7is predicted to be
targeted to the chloroplast. These seven genes
showed distinct expression patterns during fruit
ripening, and transcripts of all clones except TBG2
were detected in other tomato plant tissues (Smith
and Gross 2000).
-galactosidase transcripts were
also detected in the ripening-impaired ripening-
inhibitor(rin), nonripening(nor),and never-ripe
(nr) mutant tomato lines, which do not soften during
ripening; however, the expression profile differed
from that in wild-type fruit. TGB4mRNA accumu-
lation was impaired in comparison with wild-type
levels, whereas accumulation of TBG6transcript
persisted up to 50 days after pollination in fruit from
the three mutant lines (Smith and Gross 2000).
Presence of
-galactosidase transcripts in these lines
suggests that
-galactosidase activity alone cannot
lead to fruit softening. Expression of the TBG4
clone, which encodes
-galactosidase isoform II, in
yeast resulted in production of active protein. This
recombinant protein was able to hydrolyze synthetic
substrates (p-nitrophenyl-
-D-galactopyranoside)
and lactose as well as galactose-containing wall poly-
mers (Smith and Gross 2000). Heterologous expres-
sion ofTBG1in yeast also resulted in the production
of a protein with exo-galactanase/
-galactosidase
activity, also active in cell-wall substrates (Carey et
al. 2001).

PECTATELYASE

Pectate lyases (pectate transeliminase, EC 4.2.2.2)
are a family of enzymes that catalyze the random
cleavage of demethylesterified polygalacturonate
by
-elimination, generating oligomers with 4,5-
unsaturated reducing ends (Yoder et al. 1993). Pec-
tate lyases (PLs) were thought to be of microbial
origin and were commonly isolated from macerated
plant tissue infected with fungal or bacterial path-

ogens (Yoder et al. 1993, Barras et al. 1994, Mayans

et al. 1997, and others). In plants, presence of PL-

like sequences was first identified in mature tomato

flowers, anthers, and pollen (Wing et al. 1989). Pres-

ence of PL in pollen of other species (Albani et al.

1991, Taniguchi et al. 1995, Wu et al. 1996, Kuli-

kauskas 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,

Marín-Rodríguez et al. 2003) and strawberry fruit

(Medina-Escobar et al. 1997, Benítez-Burraco et al.

2003). Transgenic strawberry plants that expressed a

pectin lyase antisense construct driven by the cauli-

flower mosaic virus 35S (CaMV35S) promoter were

produced and evaluated (Jiménez-Bermúdez et al.

2002). Fruit from PL-suppressed lines were firmer

than controls, with the level of PL suppression be-

ing correlated to internal fruit firmness (Jiménez-

Bermúdez et al. 2002). Although pectate lyase activ-

ity in ripening tomato fruit has not been detected,

several putative pectin lyase sequences exist in the

tomato expressed sequence tag (EST) databases

(Marín-Rodríguez et al. 2002), suggesting that these

enzymes may contribute to cell-wall disassembly

during tomato fruit ripening.

GENETIC ENGINEERING OF

PECTIC ENZYMES IN TOMATO

Cell-wall disassembly in ripening fruit is an impor-

tant contributor to the texture of fresh fruit. Modif-

ication of cell-wall enzymatic activity during ripen-

ing, using genetic engineering, can impact cell-wall

polysaccharide metabolism, which in turn can influ-

ence texture. Texture of fresh fruit, in turn, influ-

ences processing 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.

GENETICENGINEERING OFPG INTOMATO

FRUIT

Polygalactronase (PG) 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