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12 Pectic Enzymes in Tomatoes 237gene.β-subunit mRNA levels increase during fruit development
and reach a maximum at 30 days after pollination (i.e., just be-
fore the onset of ripening), then decrease to undetectable levels
during ripening, whereas immunodetectableβ-subunit protein
persists throughout fruit development and ripening (Zheng et al.
1992, 1994). The presence of the PGβ-subunit alters the physic-
ochemical properties of PG2. Although the PGβ-subunit does
not possess any glycolytic activity, binding to PG2 leads to mod-
ification of PG2 activity with respect to pH optima, heat stability,
and Ca++requirements (Knegt et al. 1991, Zheng et al. 1992).
Since theβ-subunit is localized at the cell wall long before PG2
starts accumulating, it may serve as an anchor to localize PG2 to
certain areas of the cell wall (Moore and Bennett 1994, Watson
et al. 1994). Another proposed action for theβ-subunit is that of
limiting access of PG to its substrate or restricting PG activity
by binding to the PG protein (Hadfield and Bennett 1998).
Generally, PG-mediated pectin disassembly contributes to
fruit softening at the later stages of ripening and during fruit
deterioration. Overall, PG activity is neither sufficient nor neces-
sary for fruit softening, as it is evident from data using transgenic
tomato lines with suppressed levels of PG mRNA accumulation
(discussed later in this chapter) and studies on ripening-impaired
tomato mutants. It is evident from data in other fruit, espe-
cially melon (Hadfield and Bennett 1998), persimmon (Cutillas-
Iturralde et al. 1993), and apple (Wu et al. 1993), that very low
levels of PG may be sufficient to catalyze pectin depolymeriza-
tioninvivo.Pectin MethylesterasePME is a de-esterifying enzyme (EC 3.1.1.11) catalyzing the re-
moval of methyl ester groups from GalA residues of pectin, thus
leaving negatively charged carboxylic residues on the pectin
backbone (Fig. 12.3). Demethylesterification of galacturonan
residues leads to a change in the pH and charge density on
the HGA backbone. Free carboxyl groups from adjacent poly-
galacturonan chains can then associate with calcium or other
divalent ions to form gels (Fig. 12.2A). PMEs are encoded by
large multigene families in many plant species (Pelloux et al.
2007). In tomato, PME protein is encoded by at least four genes
(Turner et al. 1996, Gaffe et al. 1997). The PME polypeptide is
540–580 amino acids long and contains a signal sequence target-
ing it to the apoplast. The mature protein has a molecular mass
of 34–37 kDa and is produced by cleaving an amino-terminal
prosequence of approximately 22 kDa (Gaffe et al. 1997). PME
is found in multiple isoforms in fruit and other plant tissues
(Gaffe et al. 1994). PME isoforms have pIs in the range of 8–8.5
in fruit and around 9.0 in vegetative tissues (Gaffe et al. 1994).
Tomato fruit PME is active throughout fruit development and
influences accessibility of PG to its substrate. PME transcript
accumulates early in tomato fruit development and peaks at the
mature green fruit stage, followed by a decline in transcript
levels. In contrast, PME protein levels increase in developing
fruit at the early stages of ripening and then decline (Harriman
et al. 1991, Tieman et al. 1992). Pectin is synthesized in highly
methylated form, which is then demethylated by the action of
PME. In ripening tomato fruit, the methylester content of pectinis reduced from an initial 90% at the mature green stage to about
35% in red ripe fruit (Koch and Nevins 1989). Evidence sup-
ports the hypothesis that demethylesterification of pectin, which
allows Ca++cross-linking to occur, may restrict cell expansion.
Constitutive expression of a petunia PME gene in potato resulted
in diminished PME activity in some plants. A decrease in PME
activity in young stems of these transgenic potato plants cor-
related with an increased growth rate (Pilling et al. 2000). The
action of PME is required for PG action on the pectin backbone
(Wakabayashi et al. 2003), indicating a major role of PME in
pectin remodeling during ripening.β-Galactosidaseβ-Galactosidase (EC 3.2.1.23) is an exo-acting enzyme that cat-
alyzes the cleavage of terminal galactose residues from pectin
β-(1,4)-d-galactan side chains (Fig. 12.2B). Loss of galactose
from wall polysaccharides occurs throughout fruit develop-
ment, and it accelerates with the onset of ripening (Gross 1984,
Seymour et al. 1990). In tomato fruit,β-galactosidase is encoded
by a small multigene family of at least seven members,TBG1
through 7 (Smith et al. 1998, Smith and Gross 2000). On the
basis of sequence analysis, it was found thatβ-galactosidase
genes encode putative polypeptides with a predicted molecu-
lar 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 target-
ing these proteins to the apoplast was only identified inTBG4,
TBG5,andTBG6,whereasTBG7is predicted to be targeted to
the chloroplast. These seven genes showed distinct expression
patterns during fruit ripening, and transcripts of all clones ex-
ceptTBG2were detected in other tomato plant tissues (Smith
and Gross 2000).β-galactosidase transcripts were also detected
in the ripening impairedripening-inhibitor(rin),nonripening
(nor),andnever-ripe(nr) mutant tomato lines, which do not
soften during ripening; however, the expression profile differed
from that in wild-type fruit.TGB4mRNA accumulation was im-
paired in comparison to wild-type levels, whereas accumulation
ofTBG6transcript persisted up to 50 days after pollination in
fruit from the three mutant lines (Smith and Gross 2000). Pres-
ence ofβ-galactosidase transcripts in these lines suggests that
β-galactosidase activity alone cannot lead to fruit softening. Ex-
pression of theTBG4clone, which encodes theβ-galactosidase
isoform II, in yeast resulted in production of active protein. This
recombinant protein was able to hydrolyze synthetic substrates
(p-nitro-phenyl-β-d-galactopyranoside) and lactose, as well as
galactose-containing wall polymers (Smith and Gross 2000).
Heterologous expression ofTBG1in yeast also resulted in the
production of a protein with exo-galactanase/β-galactosidase
activity, also active in cell wall substrates (Carey et al. 2001).Pectate LyasePLs (pectate transeliminase, EC 4.2.2.2) are a family of enzymes
that catalyze the random cleavage of demethylesterified poly-
galacturonate byβ-elimination generating oligomers with 4,5-
unsaturated reducing ends (Yoder et al. 1993). PLs were thought