Agriculture Reference
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lyophilized and then used to prepare acetone powders as described by Jandus et al. (1997).
Aliquots of the soluble supernatant (200
L of 3 mL) derived from a homogenate of 0.1 g of
the acetone powder were used in assays of total PLD alpha activity as reported by Whitaker
et al. (2001), with the exception that 800 nmol of soybean PC was included as substrate in
each assay. At the end of incubation (5-40 min) with gentle shaking at 30 C, the reaction was
terminated by addition of chloroform/methanol, 2:1 and vortexing. The remaining substrate
(PC) and hydrolysis product (PA) were extracted in the chloroform phase, separated by thin-
layer chromatography, and quantified by ashing and assay of phosphate (Ames, 1966). This
method was used previously to demonstrate PLD alpha activity of the enzyme produced by
expression of the LePLD
μ
coli (Whitaker et al., 2001).
The results of a series of assays with the soluble PLD preparations from fruit of wild-type
and LePLD
α
2 cDNA open reading frame in E
.
α
2 antisense lines 8-2-E, 10-6-C, 10-5-C, and 10-3-B at four ripening stages
are shown in Fig. 9.17. Values in the bar graph are expressed as percent hydrolysis of the
PC substrate during a 15-min incubation. As expected, wild-type control samples boiled
for 5 min and then cooled prior to assay were devoid of PLD activity. For wild-type fruit,
PC hydrolysis nearly doubled between the mature green and turning stages, then dropped
off somewhat by the pink stage. Antisense line 8-2-E, which exhibited substantially higher
levels of LePLD
2 transcript than WT when mature green (Fig. 9.17), also showed a faster
rate of PC hydrolysis at this early stage of ripening. Thereafter (breaker through pink), the
percent PC hydrolysis in 15 min was roughly equivalent in 8-2-E and wild type. Although
PLD alpha activity at the four ripening stages was rather variable among the three LePLD
α
2
antisense-suppressed lines tested, each showed a reduced level of activity commensurate
with the reduction in LePLD
α
α
2 transcript (Fig. 9.16). It is notable that PLD alpha activity
was almost nil in mature green fruit of 10-6-C and 10-5-C, whereas in 10-3-B activity was
Tomato fruit pericarp PLD assay
90
80
MG
BK
TN
PK
70
60
50
40
30
20
10
0
WT
8-2-E
10-6-C
10-5-C
10-3-B
WT-boil
“Rutgers” tomato line
Fig. 9.17 Assay of soluble PLD alpha activity in acetone powder prepared from outer pericarp tissue of “Rutgers”
tomato fruit harvested at four ripening stages (MG, mature green; BK, breaker; TN, turning; PK, pink/orange). Data
are presented for fruit from five lines, including the wild type (WT) and four LePLD
2 antisense transgenic lines
numbered 8-2-E, 10-6-C, 10-5-C, and 10-3-B. The bars indicate the percent hydrolysis of 800 nmol of soybean
PC to phosphatidic acid during incubation for 15 min at 30 C. Boiled wild-type control samples (WT boil) were
also included in the assay. Relative to the wild type, antisense lines 10-6-C, 10-5-C, and 10-3-B each exhibited
about 50-80% suppression of LePLD
α
2 expression at the mature green stage of fruit development, whereas line
8-2-E showed an unexpected 50% increase in LePLD
α
α
2 transcript.
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