Agriculture Reference
In-Depth Information
LePLD
3 (AA 738-742). The degree of conservation declines in the other
fruit PLD alphas. It should be noted that the PIP
2
-binding domains in PLD alphas are, in
comparison with PLD beta and gamma, deficient in one or more positively charged, basic
AA, and consequently binding of PIP
2
may be reduced as noted in
Arabidopsis
PLD alpha
(Qin et al., 1997; Wang, 2000).
Differential expression and subcellular distribution of various PLDs have been studied
during plant development. In a study that explored these aspects using
Arabidopsis
, Fan et al.
(1999) indeed observed a differential expression and subcellular distribution of various PLD
classes. However, the relative roles of these classes are less clear since the PIP
2
-independent
activity (PLD alpha) is nearly a 1,000-fold higher than the PIP
2
-dependent activity (PLDs
beta and gamma). The categorization of PLD on the basis of PIP
2
dependence may be
somewhat misleading since an earlier report (Qin et al., 1997) showed that all the PLD
isoforms were stimulated by PIP
2
, and in the order alpha > beta > gamma, when the reaction
conditions for PLD alpha included 5-mM calcium and no detergent. As well, PIP
2
binding
to PLD isoforms is not a reliable criterion since this binding could be highly influenced by
the presence of calcium ions (Zheng et al., 2000). With the sequence and structural analogy
to
Arabidopsis
PLD beta and gamma at the active site and the C2 domain, PLD alphas
from tomato, strawberry, and melon may play a universal role during fruit development and
ripening.
α
2 and LEPLD
α
9.7 Antisense suppression of Le PLD
α
1 and its influence
on developmental events
The role of PLD action in membrane deterioration during senescence has been studied
through antisense suppression of PLD expression. Antisense suppression of PLD alpha in
Arabidopsis
resulted in retardation of ABA- and ethylene-promoted leaf senescence (Fan
et al., 1997) without affecting the natural senescence of leaves. ABA and ethylene also
enhanced the expression of PLD alpha. From these results, it was concluded that PLD
alpha was not a direct promoter of natural senescence. However, several studies show that
PLD alpha has a direct role in promoting fruit senescence. Antisense suppression of PLD
alpha in Celebrity tomato fruit resulted in a decrease in PLD expression and activity in fruit
during development. Even though very little transcript was detected at the mature green,
orange, and red stages in the antisense PLD Celebrity fruit, phospholipase D activity was
still present at these stages. This suggests that PLD may have a very low turnover rate when
it is bound to the membrane, and PLD synthesized at young/intermediate stages remain
functional even at the red stage. Alternatively, because the coding region of
LePLD
1
used
in the antisense construct shares only 73% nucleotide identity with the coding regions of
LePLD
α
3
, expression of these other two PLD alpha isogenes may not be
completely suppressed in the Celebrity antisense lines, and their transcripts would not have
been detected in Northern blots using the 309-bp
LePLD
α
2
and
LePLD
α
1
probe at high stringency. If in
fact PLD alpha enzyme has a low rate of turnover, PLD expression has to be reduced at an
early stage of fruit development for effective inhibition of PLD via antisense suppression.
During ripening, a significantly lower level of PLD activity was maintained in the transgenic
Celebrity fruit, showing that natural senescence process was retarded, which was translated
into increased firmness in these fruits. Thus, fruits may differ in their pattern of senescence,
and the relative role of PLD may differ between fruits and leaves. As well, the observation
α
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