276 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
possibly two PG2A or PG2B subunits and the PG -
subunit (also called a converter).
The PG-subunit is an acidic, heavily glycosy-
lated protein. The precursor protein contains a 30
amino acid signal peptide, an N-terminus propeptide
of 78 amino acids, the mature protein domain, and a
large C-terminus propeptide. The mature protein
presents a repeating motif of 14 amino acids and has
a size of approximately 37 to 39 kDa. This difference
in size results from different glycosylation patterns
or different posttranslational processing at the car-
boxyl terminus of the protein (Zheng et al. 1992).
The PG-subunit protein is encoded by a single
gene.-subunit mRNA levels increase during fruit
development and reach a maximum at 30 days after
pollination (i.e., just before the onset of ripening),
then decrease to undetectable levels during ripening,
whereas immunodetectable-subunit protein per-
sists throughout fruit development and ripening
(Zheng et al. 1992, 1994). The presence of the PG-
subunit alters the physicochemical properties of
PG2. Although the PG-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 Carequirements (Knegt et al.
1991, Zheng et al. 1992). Since the-subunit is
localized at the cell wall long before PG2 starts accu-
mulating, 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
of the-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 con-
tributes to fruit softening at the later stages of ripen-
ing and during fruit deterioration. Overall, PG ac-
tivity is neither sufficient nor necessary for fruit
softening, as 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, especially 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 depolymerization in vivo.
PECTINMETHYLESTERASE
Pectin methylesterase is a deesterifying enzyme (EC
3.1.1.11) catalyzing the removal of methyl ester
groups from galacturonic acid residues of pectin,
thus leaving negatively charged carboxylic residues
on the pectin backbone (Fig. 12.3). Demethyles-
terification 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).
PME protein is encoded by at least four genes in
tomato (Turner et al. 1996, Gaffe et al. 1997). The
PME polypeptide is 540–580 amino acids long and
contains a signal sequence targeting it to the ap-
oplast. 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 iso-
forms 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 a highly methylated
form, which is then demethylated by the action of
PME. In ripening tomato fruit, the methylester con-
tent of pectin is reduced from an initial 90% at the
mature green stage to about 35% in red ripe fruit
(Koch and Nevins 1989). Evidence supports the
hypothesis that demethylesterification of pectin,
which allows Cacross-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 transgenic potato plants
correlated 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).-GALACTOSIDASE-galactosidase (EC 3.2.1.23) is an exo-acting
enzyme that catalyzes cleavage of terminal galac-
tose residues from pectin -(1,4)-D-galactan side
chains (Fig. 12.2B). Loss of galactose from wall
polysaccharides occurs throughout fruit develop-