Agriculture Reference
In-Depth Information
nearly constant throughout the developmental stages studied. Cytosolic PLD activity started
to increase at the mature green stage, peaked at the turning orange stage, and started to de-
cline at the orange and red stages. PLD activity associated with microsomal membranes,
which remained nearly unchanged until the mature green stage, started to increase at the
turning orange stage and peaked at the orange stage, temporally coinciding with the decline
in cytosolic PLD. Thus, increased membrane association appears to be a major mode of
developmental regulation of PLD activity in tomato fruit.
By contrast to tomato, which is a climacteric fruit, strawberry fruits are nonclimacteric
and largely unresponsive to ethylene.
Fruit softening in strawberries has been primarily coined to the activities of polygalac-
turonases that result in cell separation, although recent reports have shown that
-xylosidase
(Bustamante et al., 2006) and expansins (Dotto et al., 2006) could also be important in cell
wall modification. To evaluate the potential role of PLD activity in fruit development and
ripening, PLD activity was analyzed at various stages of development in two strawberry
cultivars, Aromas and Seascape (Fig. 9.6). PLD activity was analyzed by monitoring the
liberation of radiolabeled choline from dipalmitoyl phosphatidylcholine provided as an
external substrate. The strawberry fruits were homogenized and subjected to differential
centrifugation to separate mitochondrial membrane, microsomal membrane, and soluble
fractions. The soluble fraction from strawberry fruit contained very little protein and PLD
activity and was not used for further analyses. By contrast, the mitochondrial fraction
(15,000
β
g
) consistently showed detectable
levels of activity. Various stages of strawberry fruit development are designated as 1—young
immature (G1); 2—young expanding (G2); 3—mature white (W); 4—turning orange stage
(T); and 5—firm ripened (R) stages. In general, PLD activity was higher in “Seascape” at
all stages except the mature white stage (W). There was very little change in mitochondrial
PLD activity during development of “Aromas” fruit. The microsomal PLD activity increased
during fruit development and reached a maximum level of 28 nmol choline released per mg
protein in 30 min at the mature white stage (W). PLD activity declined further during fruit
development and ripening. PLD-specific activity in the microsomal fraction was the highest
at the young immature stage (G1) in “Seascape,” after which it declined to nearly half of its
original specific activity. Mitochondrial PLD activity also showed a decline from G1 to G2;
however, it continued to increase during further development and reached a maximal value
of nearly 29 nmol choline released per mg protein at the turning orange stage (T; Fig. 9.6b).
These results suggested that PLD activity increased during strawberry fruit development
and ripening and thus may play a key role in fruit softening, as in tomato.
×
g
) and the microsomal fraction (105,000
×
9.3.2 Properties of strawberry PLD
Properties of strawberry PLD were analyzed using mitochondrial and microsomal mem-
branes isolated from the fruit. The release of radiolabeled choline from dipalmitoyl phos-
phatidylcholine (DPPC) in the presence of strawberry microsomal and mitochondrial mem-
brane under various conditions was quantified.
9.3.2.1 Regulation of PLD by pH
Several previous studies have indicated that PLD is stimulated under acidic conditions
(Galliard, 1980), and in particular, the relatively abundant alpha-type PLDs are activated by
Search WWH ::
Custom Search