Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

228 POSTHARVEST BIOLOGY & TECHNOLOGY OF FRUITS, VEGETABLES, & FLOWERS
lyophilized and then used to prepare acetone powders as described by Jandus et al. (1997).
Aliquots of the soluble supernatant (200 μL 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 thinlayer
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 LePLDα2 cDNA open reading frame in E. coli (Whitaker et al., 2001).
The results of a series of assays with the soluble PLD preparations from fruit of wild-type
and LePLDα2 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 LePLDα2 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 LePLDα2
antisense-suppressed lines tested, each showed a reduced level of activity commensurate
with the reduction in LePLDα2 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
PC hydrolysis (%)
90
80
70
60
50
40
30
20
10
Tomato fruit pericarp PLD assay
MG BK TN PK
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 LePLDα2 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 LePLDα2 expression at the mature green stage of fruit development, whereas line
8-2-E showed an unexpected 50% increase in LePLDα2 transcript.