BLBS102-c27 BLBS102-Simpson March 21, 2012 13:25 Trim: 276mm X 219mm Printer Name: Yet to Come
27 Biochemistry of Fruits 545fraction, which can account for nearly 95% of the total lipids.
Palmitic (16:0), palmitoleic (16:1), oleic (18:1) and linoleic
(18:2) acids are the major fatty acids of triglycerides. The oil
content progressively increases during maturation of the fruit,
and the oils are compartmentalised in oil bodies or oleosomes.
The biosynthesis of fatty acids occurs in the plastids, and the fatty
acids are exported into the endoplasmic reticulum where they are
esterified with glycerol-3-phosphate by the action of a number
of enzymes to form the triglyceride. The triglyceride-enriched
regions then are believed to bud off from the endoplasmic retic-
ulum as the oil body. The oil body membranes are different
from other cellular membranes, since they are made up of only a
single layer of phospholipids. The triglycerides are catabolised
by the action of triacylglycerol lipases with the release of fatty
acids. The fatty acids are then broken down into acety CoA units
throughβ-oxidation.
Even though phospholipids constitute a small fraction of the
lipids in fruits, the degradation of phospholipids is a key fac-
tor that controls the progression of senescence. As in several
senescing systems, there is a decline in phospholipids as the
fruit undergoes senescence. With the decline in phospholipid
content, there is a progressive increase in the levels of neu-
tral lipids, primarily diacylglycerols, free fatty acids and fatty
aldehydes. In addition, the levels of sterols may also increase.
Thus, there is an increase in the ratio of sterol:phospholipids.
Such changes in the composition of membrane can cause the
formation of gel phase or non-bilayer lipid structures (micelles).
These changes can make the membranes leaky, thus resulting
in the loss of compartmentalisation, and ultimately, senescence
(Paliyath and Droillard 1992).
Membrane lipid degradation occurs by the tandem action of
several enzymes, one enzyme acting on the product released by
the previous enzyme in the sequence. Phospholipase D (PLD)
is the first enzyme of the pathway which initiates phospholipids
catabolism and is a key enzyme of the pathway (Fig. 27.6).
PLD acts on phospholipids liberating phosphatidic acid and the
respective headgroup (choline, ethanolamine, glycerol, inosi-
tol). Phosphatidic acid, in turn, is acted upon by phosphatidate
phosphatase which removes the phosphate group from phospha-
tidic acid with the liberation of diacylglycerols (diglycerides).
The acyl chains of diacylglycerols are then de-esterified by the
enzyme lipolytic acyl hydrolase liberating free fatty acids. Un-
saturated fatty acids with acis-1,4- pentadiene structure (linoleic
acid, linolenic acid) are acted upon by lipoxygenase (LOX) caus-
ing the peroxidation of fatty acids. This step may also cause the
production of activated oxygen species such as singlet oxygen,
superoxide and peroxy radicals and so on. The peroxidation
products of linolenic acid can be 9-hydroperoxy linoleic acid
or 13-hydroperoxy linoleic acid. The hydroperoxylinoleic acids
undergo cleavage by hydroperoxide lyase resulting in several
products including hexanal, hexenal andω-keto fatty acids (keto
group towards the methyl end of the molecule). For example,
hydroperoxide lyase action on 13-hydroperoxylinolenic acid re-
sults in the formation ofcis-3-hexenal and 12-keto-cis-9- dode-
cenoic acid. Hexanal and hexenal are important fruit volatiles.
The short-chain fatty acids may feed into catabolic pathway
(β-oxidation) that results in the formation of short-chain acylCoAs, ranging from acetyl CoA to dodecanoyl CoA. The short-
chain acyl CoAs and alcohols (ethanol, propanol, butanol, pen-
tanol, hexanol, etc.) are esterified to form a variety of esters
that constitute components of flavour volatiles that are charac-
teristic to fruits. The free fatty acids and their catabolites (fatty
aldehydes, fatty alcohols, alkanes, etc.) can accumulate in the
membrane causing membrane destabilisation (formation of gel
phase, non-bilayer structures, etc.). An interesting regulatory
feature of this pathway is the very low substrate specifity of
enzymes that act downstream from PLD for the phospholipids.
Thus, phosphatidate phosphatase, lipolytic acyl hydrolase and
LOX do not directly act on phospholipids, though there are ex-
ceptions to this rule. Therefore, the degree of membrane lipid
catabolism will be determined by the extent of activation of PLD
(Fig. 27.5).
The membrane lipid catabolic pathway is considered as an
autocatalytic pathway (Fig. 27.5). The destabilisation of the
membrane can cause the leakage of calcium and hydrogen ions
from the cell wall space, as well as the inhibition of calcium-
and proton ATPases, the enzymes responsible for maintaining a
physiological calcium and proton concentration within the cy-
toplasm (calcium concentration below micromolar range, pH in
the 6–6.5 range). Under conditions of normal growth and devel-
opment, these enzymes pump the extra calcium- and hydrogen
ions that enter the cytoplasm from storage areas such as apoplast
and the ER lumen in response to hormonal and environmental
stimulation using ATP as the energy source. The activities of
calcium- and proton ATPases localised on the plasma membrane,
the endoplasmic reticulum and the tonoplast are responsible for
pumping the ions back into the storage compartments. In fruits
(and other senescing systems), with the advancement in ripen-
ing and senescence, there is a progressive increase in leakage of
calcium and hydrogen ions. PLD is stimulated by low pH and
calcium concentration over 10μM. Thus, if the cytosolic con-
centrations of these ions progressively increase during ripening
or senescence, the membranes are damaged as a consequence.
However, this is an inherent feature of the ripening process
in fruits, and results in the development of ideal organoleptic
qualities that makes them edible. The uncontrolled membrane
deterioration can result in the loss of shelf life and quality in
fruits (Paliyath et al. 2008).
The properties and regulation of the membrane degradation
pathway are increasingly becoming clear. Enzymes such as PLD
and LOX are very well studied. There are several isoforms of
PLD designated as PLD alpha, PLD beta, PLD gamma and so
on. The expression and activity levels of PLD alpha are much
higher than that of the other PLD isoforms. Thus, PLD alpha is
considered as a housekeeping enzyme; however, it is also de-
velopmentally regulated (Pinhero et al. 2003). The regulation of
PLD activity is an interesting feature. PLD is normally a soluble
enzyme. The secondary structure of PLD shows the presence of a
segment of around 130 amino acids at theN-terminal end, desig-
nated as the C2 domain. This domain is characteristic of several
enzymes and proteins that are integral components of the hor-
mone signal transduction system. In response to hormonal and
environmental stimulation and the resulting increase in cytosolic
calcium concentration, C2 domain binds calcium and transports