BLBS102-c27 BLBS102-Simpson March 21, 2012 13:25 Trim: 276mm X 219mm Printer Name: Yet to Come
538 Part 5: Fruits, Vegetables, and Cerealssuch as tomato. There are three major isoforms of PG responsible
for pectin degradation in tomato, designated as PG1, PG2a and
PG2b (Fischer and Bennet 1991). PG1 has a relative molecular
mass of 100 kDa, and is the predominant form at the initia-
tion of ripening. With the advancement of ripening, PG2a and
PG2b isoforms increase, becoming the predominant isoforms
in ripe fruit. The different molecular masses of the isozymes
result from the post-translational processing and glycosylation
of the polypeptides. PG2a (43 kDa) and PG2b (45 kDa) appear
to be the same polypeptide with different degrees of glycosyla-
tion. PG1 is a complex of three polypeptides, PG2a, PG2b and
a 38kDa subunit known as theβ-subunit. The 38 kDa subunit is
believed to exist in the cell wall space where it combines with
PG2a and PG2b forming the PG1 isoform of PG. The increase
in activity of PG1 is related to the rate of pectin solubilisation
and tomato fruit softening during the ripening process.
Research into the understanding of the regulation of biosyn-
thesis and activity of PG using molecular biology tools has
resulted in the development of strategies for enhancing the shelf
life and quality of tomatoes. PG mRNA was one of the first
ripening-related mRNAs isolated from tomato fruits. All the
different isoforms of PGs are encoded by a single gene. The
PG cDNA which has an open reading frame of 1371 bases en-
codes a polypeptide having 457 amino acids, which includes
a 24 amino acid signal sequence (for targeting to the cell wall
space) and a 47 amino acid pro-sequence at theN-terminal end,
which are proteolytically removed during the formation of the
active PG isoforms. A 13 amino acid longC-terminal peptide
is also removed resulting in a 373 amino acid long polypeptide,
which undergoes different degrees of glycosylation resulting in
the PG2a and PG2b isozymes. Complex formation among PG2a,
PG2b and the 38-kDa subunit in the apoplast results in the PG1
isozyme (Grierson et al. 1986, Bird et al. 1988). In response to
ethylene treatment of mature green tomato fruits which stim-
ulates ripening, the levels of PG mRNA and PG are found to
increase. These changes can be inhibited by treating tomatoes
with silver ions, which interfere with the binding of ethylene to
its receptor and initiation of ethylene action (Davies et al. 1988).
Thus, there is a link between ethylene, PG synthesis and fruit
softening.
Genetic engineering of tomato with the objective of regulat-
ing PG activity has yielded complex results. In the rin mutant
of tomato which lacks PG and does not soften, introduction of
a PG gene resulted in the synthesis of an active enzyme; how-
ever, this did not cause fruit softening (Giovannoni et al. 1989).
As a corollary to this, introduction of the PG gene in the anti-
sense orientation resulted in near total inhibition of PG activity
(Smith et al. 1988). In both these cases, there was very little ef-
fect on fruit softening, suggesting that factors other than pectin
de-polymerisation may play an integral role in fruit softening.
Further studies using a tomato cultivar such as UC82B (Kramer
et al. 1992) showed that antisense inhibition of ethylene biosyn-
thesis or PG did indeed result in lowered PG activity, improved
integrity of cell wall and increased fruit firmness during fruit
ripening. As well, increased activity of pectin methylesterase,
which removes the methyl groups from esterified galacturonic
acid moieties, may contribute to the fruit softening process.The activities of pectin degrading enzymes have been related
to the incidence of physiological disorders such as “mealiness”
or “wooliness” in mature unripened peaches that are stored at a
low temperature. The fruits with such a disorder show a lack of
juice and a dry texture. De-esterification of pectin by the activity
of pectin methyl esterase is thought to be responsible for the
development of this disorder. Pectin methyl esterase isozymes
with relative molecular masses in the range of 32 kDa have been
observed in peaches, and their activity increases after 2 weeks
of low temperature storage. Polygalacuronase activity increases
as the fruit ripens. The ripening fruits which possess both poly-
galacturonase and pectin methyl esterase do not develop mealy
symptoms when stored at low temperature implicating the poten-
tial role of pectin degradation in the development of mealiness
in peaches.
There are two forms of PG in peaches, the exo- and endo-PG.
The endo-PG are the predominant forms in the freestone type
of peaches, whereas the exo-PG are observed in the mesocarp
of both freestone and clingstone varieties of peaches. As the
name implies, exo-PG remove galacturonic acid moieties of
pectin from the terminal reducing end of the chain, whereas the
endo-PG can cleave the pectin chain at random within the chain.
The activities of these enzymes increase during the ripening
and softening of the fruit. Two exo-PG isozymes have been
identified in peach, having a relative molecular mass of near
66 kDa. The exo-acting enzymes are activated by calcium. Peach
endo-PG is observed to be similar to the tomato endo-PG. The
peach endo-PG is inhibited by calcium. The freestone peaches
possess enhanced activities of both exo-PG and endo-PG leading
to a high degree of fruit softening. However, the clingstone
varieties with low levels of endo-PG activity do not soften as the
freestone varieties. In general, fruits such as peaches, tomatoes,
strawberries, pears and so on, which soften extensively, possess
high levels of endo-PG activity. Apple fruits which remain firm
lack endo-PG activity.Starch DegradationStarch is the major storage form of carbohydrates. During ripen-
ing, starch is catabolised into glucose and fructose, which enters
the metabolic pool where they are used as respiratory substrates
or further converted to other metabolites (Fig. 27.2). In fruits
such as banana, the breakdown of starch into simple sugars is
associated with fruit softening. There are several enzymes in-
volved in the catabolism of starch.α-amylase hydrolyses amy-
lose molecules by cleaving theα-1,4-linkages between sug-
ars providing smaller chains of amylose termed as dextrins.
β-amylase is another enzyme that acts upon the glucan chain
releasing maltose, which is a diglucoside. The dextrins as well
as maltose can be further catabolised to simple glucose units
by the action of glucosidases. Starch phosphorylase is another
enzyme, which mediates the phosphorylytic cleavage of termi-
nal glucose units at the non-reducing end of the starch molecule
using inorganic phosphate, thus releasing glucose-1-phosphate.
The amylopectin molecule is not only degraded in a similar
manner to amylose but also involves the action of de-branching