Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

226 POSTHARVEST BIOLOGY & TECHNOLOGY OF FRUITS, VEGETABLES, & FLOWERS
“Celebrity” tomatoes. In particular, with the aim of minimizing possible effects of PLD alpha
gene disruption on plant growth and development, the fruit-specific E8 promoter was used
instead of the constitutive CaMV35S promoter. The E8 promoter is responsive to ethylene,
but high-level expression in tomato fruit appears to be regulated by an ethylene-independent,
ripening-related cis element (Deikman et al., 1998). Aside from fully developed fruit tissues,
E8 is expressed mainly in anthers of mature, stage 4 flowers. An antisense construct of
LePLDα2 was cloned into the binary vector pBI121 (with 35S and GUS excised) for use in
Agrobacterium-mediated transformation of tomato cotyledon explants. The entire construct
included a copy of the npt II (neomycin phosphotransferase) antibiotic-resistance gene under
control of the NOS1 (nopaline synthase) promoter and terminator, and a 2.0-kb fragment of
the LePLDα2 cDNA (nucleotides 880–2,846, constituting 70% of the coding region plus
the 3 ′ -UTR) in antisense orientation regulated by the E8 promoter and NOS1 terminator.
Following selection based on kanamycin resistance, 25 putative antisense LePLDα2
transformants were identified by DNA gel blot (Southern) analysis using leaf tissue genomic
DNA and probes for npt II and E8. Of these, plants representing 16 lines were raised
to maturity, and pericarp tissue from mature green fruit (40 days after pollination) was tested
for suppression of LePLDα2 by semiquantitative RT-PCR using isolated total RNA. The
Ambion QuantumRNA TM quantitative RT-PCR kit was used to determine levels of LePLDα2
cDNA relative to control 18S cDNA. The amplified LePLDα2 and 18S cDNA fragments
produced by RT-PCR were separated on agarose gels and quantified by densitometry measurements
after staining with ethidium bromide. Among the sixteen lines tested, four showed
clear suppression of LePLDα2, whereas three surprisingly exhibited overexpression of the
gene. The four LePLDα2 suppressed lines (designated 8-4-A, 10-3-B, 10-5-C, and 10-6-C),
plus line 8-2-E that showed the highest level of LePLDα2 overexpression, were propagated
through two subsequent generations by self-fertilization for further analyses of PLD alpha
gene expression and activity, as well as fruit physiology and quality attributes.
9.7.6 Levels of LePLDα transcripts in fruit of LePLDα2 antisense lines
LePLDα2 transcript abundance in pericarp of wild type and antisense transgenic fruit harvested
40 days after pollination (DAP) was determined by both RNA gel blots (Northern
blots) and the semiquantitative RT-PCR 18S competimer system (Ambion QuantumRNA)
described earlier (Fig. 9.16). For the Northern blots, a 646-bp segment of the LePLDα2
cDNA coding region (nucleotides 1,080–1,725) was used as the radiolabeled probe, whereas
in the RT-PCR experiments a 0.6-kb cDNA fragment of LePLDα2 (coding region nucleotides
735–1,323) was amplified. The two methods gave similar results, and averaging
the two values, transcript levels in the four suppressed lines ranged from about 20 to 45%
of that in wild-type controls, and transcript in line 8-2-E was roughly 75% higher than that
in wild type.
Concerning possible suppression or overexpression of the other two tomato PLD alpha
isogenes, because of the high percentage of sequence identity between LePLDα2 and
LePLDα3 (91% in the open reading frame), it seemed likely that LePLDα2 antisense would
also affect expression of the LePLDα3 isogene. However, perhaps because the 3 ′ -UTR was
included in the 2-kb antisense LePLDα2 fragment, this does not appear to be the case. Semiquantitative
RT-PCR analysis indicated that in pericarp tissue of fruit harvested at 40 DAP
there was little if any reduction (or increase) in LePLDα3 transcript in the five LePLDα2