21 Biochemistry of Fruits 493Abbot Laboratories) has been used to delay ripening
in fruits such as apples, peaches, and pears. Also,
commercial storage operations employ a controlled
atmosphere with very low oxygen levels (1–3%) for
long-term storage of fruits such as apples to reduce
the production of ethylene, as oxygen is required for
the conversion of ACC to ethylene.
In response to the initiation of ripening, several
biochemical changes are induced in the fruit, which
ultimately results in the development of ideal tex-
ture, taste, color, and flavor. Several biochemical
pathways are involved in these processes, as de-
scribed below.
CARBOHYDRATEMETABOLISM
Cell Wall Degradation
Cell wall degradation is the major factor that causes
softening of several fruits. This involves the degra-
dation of cellulose components, pectin components,
or both. Cellulose is degraded by the enzyme cellu-
lase or -1,4-glucanase. Pectin degradation involves
the enzymes pectin methylesterase, polygalactur-
onase (pectinase), and -galactosidase. The degra-
dation of cell wall can be reduced by the application
of calcium as a spray or drench in apple fruits. Cal-
cium binds and cross-links the free carboxylic groups
of polygalacturonic acid components in pectin. Cal-
cium treatment therefore also enhances the firmness
of the fruits.
The activities of both cellulase and pectinase have
been observed to increase during ripening of avoca-
do fruits and to result in their softening. Cellulase is
an enzyme with a relative molecular mass of 54.2
kDa and is formed by extensive posttranslational
processing of a native 54 kDa protein involving pro-
teolytic cleavage of the signal peptide and glycosy-
lation (Bennet and Christofferson 1986). Further
studies have shown three isoforms of cellulose,
ranging in molecular mass between 50 and 55 kDa.
These forms are associated with the endoplasmic re-
ticulum, the plasma membrane, and the cell wall
(Dallman et al. 1989). The cellulase isoforms are
initially synthesized at the style end of the fruit at
the initiation of ripening, and the biosynthesis
moves towards the stalk end of the fruit with the
advancement of ripening. Degradation of hemicellu-
loses (xyloglucans, glucomannans, and galactoglu-
comannans) is also considered as an important fea-
ture that leads to fruit softening. Degradation of
these polymers could be achieved by cellulases and
galactosidases.
Loss of pectic polymers through the activity of
polygalacturonases (PG) is a major factor involved
in the softening of fruits such as tomato. There are
three major isoforms of polygalacturonases respon-
sible 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 initiation of ripening.
With the advancement of ripening, PG2a and PG2b
isoforms increase, becoming the predominant iso-
forms in ripe fruit. The different molecular masses
of the isozymes result from the posttranslational pro-
cessing and glycosylation of the polypeptides. PG2a
(43 kDa) and PG2b (45 kDa) appear to be the same
polypeptide with different degrees of glycosylation.
PG1 is a complex of three polypeptides, PG2a,
PG2b, and a 38 kDa subunit known as the-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. The increase in activity of
PG1 is related to the rate of pectin solubilization and
tomato fruit softening during the ripening process.
Research into the understanding of the regulation
of biosynthesis and activity of PG using molecular
biology tools has resulted in the development of
strategies for enhancing the shelf life and quality of
tomatoes. Polygalacturonase mRNA was one of the
first ripening-related mRNAs isolated from tomato
fruits. All the different PG isoforms are encoded by
a single gene. The PG cDNA, which has an open
reading frame of 1371 bases, encodes a polypeptide
having 457 amino acids, including a 24 amino acid
signal sequence (for targeting to the cell wall space)
and a 47 amino acid prosequence at the N-terminal
end, which are proteolytically removed during the
formation of the active PG isoforms. A 13 amino
acid C-terminal peptide is also removed, resulting
in a 373 amino acid long polypeptide that under-
goes different degrees of glycosylation to result 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 treat-
ment of mature green tomato fruits that stimulates
ripening, the levels of PG mRNA and PG are found
to increase. These changes can be inhibited by treat-
ing tomatoes with silver ions, which interfere with