Structural Deterioration in Produce: Phospholipase D, Membrane Deterioration, and Senescence - Postharvest Biology and Technology of Fruits, Vegetables, and Flowers - page 227

LePLD α 2

18S
rRNA

(a) WT 8-2-E 10-3-B 8-4-A 10-5-C 10-6-C

LePLD α 2

18S

(b) WT 8-2-E 10-3-B 8-4-A 10-5-C 10-6-C

LePLD α 2 transcript levels

200

Northern
RT-PCR

150

100

50

0

WT 8-2-E 10-3-B 8-4-A 10-5-C 10-6-C

(c) Rutgers tomato line

Fig. 9.16 Comparison of RNA gel blot and semiquantitative RT-PCR analyses of LePLD α 2 transcript levels in

outer pericarp tissue of mature green fruit from wild type (WT) and five LePLD 2 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

α

μ α

2 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 LePLD

g per lane total RNA probed with a 645-bp fragment of the LePLD

2 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 LePLD

α

α 2 and 18S cDNA fragments. The bar graph (c) shows the

relative levels of LePLD α 2 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
LePLD

α 1 expression is altered in the LePLD α 2 antisense lines, but this was not tested.

9.7.7 Total PLD alpha activity in fruit of LePLD α 2 antisense lines

α

Pericarp tissue from fruit of “Rutgers” wild type and LePLD 2 antisense transgenic lines

harvested at four ripening stages (mature green, breaker, turning, and pink/orange) was