Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

PHOSPHOLIPASE D, MEMBRANE DETERIORATION, AND SENESCENCE 227
LePLDα2
18S
rRNA
(a)
WT
8-2-E 10-3-B 8-4-A 10-5-C 10-6-C
LePLDα2
18S
(b)
Percent (relative to WT)
200
150
100
50
WT 8-2-E 10-3-B 8-4-A 10-5-C 10-6-C
LePLDα2 transcript levels
Northern
RT-PCR
(c)
0
WT 8-2-E 10-3-B 8-4-A 10-5-C 10-6-C
Rutgers tomato line
Fig. 9.16 Comparison of RNA gel blot and semiquantitative RT-PCR analyses of LePLDα2 transcript levels in
outer pericarp tissue of mature green fruit from wild type (WT) and five LePLDα2 antisense transgenic lines (8-
2-E, 10-3-B, 8-4-A, 10-5-C, and 10-6-C) of “Rutgers” tomato. RNA gel blot analysis (a) was performed using ca.
20 μg per lane total RNA probed with a 645-bp fragment of the LePLDα2 cDNA open reading frame (nucleotides
1,080–1,725). The blot was then stripped and hybridized with a probe for 18S rRNA to evaluate loading of total
RNA. The QuantumRNA quantitative RT-PCR kit (Ambion) was used to determine levels of LePLDα2 cDNA
relative to the control 18S cDNA (b). Ethidium bromide-stained gels of the RT-PCR products were used for
densitometric measurements of the amplified LePLDα2 and 18S cDNA fragments. The bar graph (c) shows the
relative levels of LePLDα2 transcript in the six tomato lines determined by the two methods, with WT values set
at 100%.
antisense transgenic lines (Fig. 9.10b). Considering these results, it is very unlikely that
LePLDα1 expression is altered in the LePLDα2 antisense lines, but this was not tested.
9.7.7 Total PLD alpha activity in fruit of LePLDα2 antisense lines
Pericarp tissue from fruit of “Rutgers” wild type and LePLDα2 antisense transgenic lines
harvested at four ripening stages (mature green, breaker, turning, and pink/orange) was