Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

208 POSTHARVEST BIOLOGY & TECHNOLOGY OF FRUITS, VEGETABLES, & FLOWERS
calcium (Fig. 9.8, bottom panel), which provides free calcium in the range of ≤1to40μM.
Nearly threefold stimulation in PLD activity is observed at free calcium in the range of
1 μM (0.2-mM added calcium and 0.2-mM EGTA), which is in the range of physiologically
activated levels of calcium (Felle, 1988; Yuan et al., 2006b). Thus, microsomal PLD
is stimulated at low micromolar levels of calcium, implying a potential role in signal transduction
processes. By contrast, mitochondrial PLD activity did not show any significant
promotion at these levels of calcium (Fig. 9.8). Maximal stimulation of mitochondrial PLD
was observed between 1 and 3 mM free calcium.
9.4 Subcellular localization of phospholipase D
PLD is a soluble enzyme. However, analysis of PLD activity in subcellular fractions revealed
the presence of PLD activity in mitochondrial, endoplasmic reticulum, and plasma
membrane fractions. Membrane-associated PLD activity increases during ripening and after
wounding. PLD was localized by immunoelectron microscopy in germinating castor bean
tissue, and was shown to be associated with the plasma membrane and the vacuole (Xu et al.,
1996). After wounding of castor bean leaves, lipolysis by PLD increased sharply following
calcium-mediated translocation from the cytoplasm to microsomal membranes (Ryu and
Wang, 1996). In rice, PLD was localized in cell walls, membranes, and chloroplasts (McGee
et al., 2003), and was recruited to the plasma membrane at the point of adhesion of a bacterial
pathogen (Young et al., 1996). In tomato fruits, PLD was localized in cytoplasm, plasma
membrane, endoplasmic reticulum, mitochondria, and nuclear membrane (Pinhero et al.,
2003). During early stages of development, PLD was primarily localized in the cytoplasm.
As development progressed, a relatively large number of PLD polypeptides were observed
in the endoplasmic reticulum (Fig. 9.9). In ripened strawberry fruit, a large number of PLD
molecules were localized in the cell wall space (Yuan et al., 2006a). This may represent
an advanced stage where cell permeability is compromised and the PLD molecules have
leaked out of the cell or possibly were transported by exocytosis.
9.5 Characteristics of phospholipase D
PLD (EC 3.1.4.4) is a key enzyme that catalyzes the hydrolysis of membrane phospholipids
yielding PA and a hydrophilic head group (Galliard, 1980). PLD in plants was originally proposed
to be important in phospholipid catabolism, initiating a lipolytic cascade in membrane
deterioration during senescence and stress (Paliyath and Droillard, 1992). PLD could also
be involved in phospholipid turnover that maintains cell viability and homeostasis (Dawidowicz,
1987). Recent studies in plants indicate that PLD action plays an important role in
transmembrane signaling and cellular regulation (Wang, 2002, 2005). Activation of PLD
generates lipid messengers, most importantly PA, which mediate an array of physiological
responses (Wang, 2005; Bargmann and Munnik, 2006; Wang et al., 2006).
The role of phospholipase D in the initiation of membrane deterioration during ripening
and senescence has been well recognized (Paliyath and Droillard, 1992). About 15–25%
loss of total phospholipids occurs during full ripening of tomato fruit pericarp tissue, with
a coincident increase in PA, suggesting that PLD is involved in membrane degradation
(Güçlü et al., 1989; Whitaker, 1994). Moreover, all the enzyme activities involved in the
senescence cascade, including PLD, have been demonstrated in tomato fruit microsomal