Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

PHOSPHOLIPASE D, MEMBRANE DETERIORATION, AND SENESCENCE 229
PC hydrolysis (%)
100
90
80
70
60
50
40
30
20
10
0
Soluble PLD assay (mature-green stage)
Wild type
Anti 10-5-C
Anti 8-4-A
Boiled 5 min 10 min 20 min 40 min
Incubation time
Fig. 9.18 Assay of soluble PLD alpha activity in acetone powder prepared from outer pericarp tissue of “Rutgers”
tomato fruit harvested 40 days after pollination (mature green stage). Data are presented for fruit from three lines,
including the wild type and two LePLDα2 antisense transgenic lines numbered 10-5-C and 8-4-A. The bars indicate
the percent hydrolysis of 800 nmol of soybean PC to phosphatidic acid (PA) during incubation at 30 ◦ C for 5, 10,
20, or 40 min. Methods were the same as those used in the assays at different ripening stages (Fig. 9.17) with the
exception that soluble PLD preparations were twofold more concentrated (0.2 g acetone powder used). Boiled
control samples from each line were also included in the assay. Relative to the wild type, antisense lines 10-5-C
and 8-4-A exhibited, respectively, about 65 and 80% lower levels of LePLDα2 transcript at the mature green stage
of fruit development.
five- to tenfold higher at the mature green stage. Again, this is in accord with the LePLDα2
mRNA levels in fruit harvested at 40 DAP (mature green).
In addition to assays of PLD alpha activity in fruit of wild type and LePLDα2 antisense
lines at various stages of ripening, a time-course analysis of PLD alpha activity with incubation
times ranging from 5 to 40 min was also performed, comparing mature green fruit
of wild type and two antisense lines, 10-5-C and 8-4-A (Fig. 9.18). For these time course
assays, the concentration of the enzyme preparations was doubled such that about 73% of
the PC substrate was hydrolyzed to PA in 5 min by the wild-type PLD preparations. With
longer incubations, the percent of PC hydrolysis with the wild-type enzyme approached 100
asymptotically, reaching a value of about 96% after 40 min. PLD preparations from fruit
of the two LePLDα2 antisense lines, on the other hand, gave an essentially linear increase
in percent PC hydrolysis over the span of 5–40 min. After a 40-min incubation period, the
extent of PC hydrolysis in the assay for antisense line 10-5-C (about 76%) slightly exceeded
that observed after 5 min in the wild-type assays, indicating a roughly eightfold lower level
of PLD alpha activity in the transgenic fruit. The rate of PC hydrolysis was considerably
slower with enzyme preparations from fruit of the 8-4-A antisense line, and by extrapolation,
the level of PLD alpha activity was about 30-fold lower in 8-4-A compared with
wild-type fruit.
9.7.8 Influence of LePLDα2 antisense suppression on fruit physiology
Ripening, both on the plant and after harvest was delayed and/or protracted in LePLDα2
antisense compared with wild-type fruit. Surprisingly, however, this general effect on fruit
physiology was observed in the LePLDα2 overexpressing line, 8-2-E, as well as in the antisense
suppressed lines. Relative to the wild-type fruit, both the rise in ethylene production