BLBS102-c27 BLBS102-Simpson March 21, 2012 13:25 Trim: 276mm X 219mm Printer Name: Yet to Come
27 Biochemistry of Fruits 537Methyl thioribose1-Aminocyclopropane-
1-carboxylic acidACC synthaseS-Adenosyl methionine (SAM)Methionine adenosyl transferaseMethionineEthylene biosynthetic pathwayACC oxidaseEthyleneEthylene receptorFruit ripening/senescenceFigure 27.1.Summary of ethylene biosynthesis and action during
fruit ripening. ACC, 1-Aminocyclopropane 1- Carboxylic Acid.Ethylene is biosynthesised through a common pathway
that uses the amino acid methionine as the precursor (Yang
1981, Fluhr and Mattoo 1996) (Fig. 27.1). The first reac-
tion of the pathway involves the conversion of methionine to
S-adenosyl methionine (SAM) mediated by the enzyme me-
thionine adenosyl transferase. SAM is further converted into
1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme
ACC synthase. The sulphur moiety of methylthioribose gener-
ated during this reaction is recycled back to methionine by the
action of a number of enzymes. ACC is the immediate precur-
sor of ethylene and is acted upon by ACC oxidase to generate
ethylene. ACC synthase and ACC oxidase are the key control
points in the biosynthesis of ethylene. ACC synthase is a soluble
enzyme located in the cytoplasm, with a relative molecular mass
of 50 kDa (kiloDalton). ACC oxidase is found to be associated
with the vacuolar or mitochondrial membrane. Using molecular
biology tools, a cDNA (complementary DNA representing the
coding sequences of a gene) for ACC oxidase was isolated from
tomato (Hamilton et al. 1991) and is found to encode a protein
with a relative molecular mass of 35 kDa. There are several
isoforms of ACC-synthase. These are differentially expressed in
response to wounding, other stress factors and at the initiation
of ripening. ACC oxidase reaction requires Fe^2 +, ascorbate and
oxygen.
Regulation of the activities of ACC synthase and ACC oxidase
is extremely important for the preservation of shelf life and qual-ity in fruits. Inhibition of the ACC synthase and ACC oxidase
gene expression by the introduction of their respective antisense
cDNAs resulted in delayed ripening and better preservation of
the quality of tomato (Hamilton et al. 1990, Oeller et al. 1991)
and apple (Hrazdina et al. 2000) fruits. ACC synthase, which
is the rate-limiting enzyme of the pathway, requires pyridoxal-
5-phosphate as a cofactor, and is inhibited by pyridoxal phos-
phate inhibitors such as aminoethoxyvinylglycine (AVG) and
aminooxy acetic acid (AOA). Field application of AVG as a
growth regulator (RetainTM, Valent Biosciences, Chicago) has
been used to delay ripening in fruits such as apples, peaches and
pears. Also, commercial storage operations employ 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 biochemi-
cal changes are induced in the fruit, which ultimately results
in the development of ideal texture, taste, colour and flavour.
Several biochemical pathways are involved in these processes
as described in the subsequent text.Carbohydrate MetabolismCell Wall DegradationCell wall degradation is the major factor that causes soften-
ing of several fruits. This involves the degradation of cellulose
components, pectin components or both. Cellulose is degraded
by the enzyme cellulase orβ-1,4-glucanase. Pectin degradation
involves the enzymes pectin methylesterase, polygalacuronase
(pectinase) andβ-galactosidase (Negi and Handa 2008). The
degradation of cell wall can be reduced by the application of
calcium as a spray or drench in apple fruits. Calcium binds
and cross-links the free carboxylic groups of polygalacturonic
acid components in pectin. Calcium treatment, therefore, also
enhances the firmness of the fruits.
The activities of both cellulase and pectinase have been ob-
served to increase during ripening of avocado fruits and result in
their softening. Cellulase is an enzyme with a relative molecular
mass of 54.2 kDa and formed by extensive post-translational pro-
cessing of a native 54 kDa protein involving proteolytic cleavage
of the signal peptide and glycosylation (Bennet and Christo-
pherson 1986). Further studies have shown three isoforms of
cellulose ranging in molecular masses between 50 and 55 kDa.
These forms are associated with the endoplasmic reticulum,
the plasma membrane and the cell wall (Dallman et al. 1989).
The cellulase isoforms are initially synthesised at the style end
of the fruit at the initiation of ripening, and the biosynthesis
progressively increases towards the stalk end of the fruit with
the advancement of ripening. Degradation of hemicelluloses
(xyloglucans, glucomannans and galactoglucomannans) is also
considered as an important feature that leads to fruit softening.
Degradation of these polymers could be achieved by cellulases
and galactosidases.
Loss of pectic polymers through the activity of polygalactur-
onases (PG) is a major factor involved in the softening of fruits