Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

PHOSPHOLIPASE D, MEMBRANE DETERIORATION, AND SENESCENCE 213
and reverse primers were generated to conduct 3 ′ and 5 ′ rapid amplification of cDNA ends
(RACE) using the 883-bp fragment, and a 2,430-bp full-length cDNA was isolated encoding
a PLD alpha 809 amino acids long (LePLDα1).
In contrast with LePLDα1, the second tomato PLD alpha isogene, LePLDα2
(AF154425), was cloned by probing a fruit cDNA library (Smith et al., 1998) with a 1.6-
kb fragment of the open reading frame (ORF) from the castor bean PLD alpha cDNA,
L33686 (detailed in Whitaker et al., 2001). Ten of 11 positive clones were from the same
gene, and the longest of these was selected for sequencing. The complete cDNA was 2,863
nucleotides long, including a 2,424-bp ORF encoding a PLD alpha 807 amino acids in
length (LePLDα2). The complete cDNA of a third tomato PLD alpha isogene, LePLDα3
(AY013253), was cloned during the course of a study examining PLD gene expression patterns
in tomato plants infected with a fungal pathogen or challenged with a fungal elicitor
(Laxalt et al., 2001). A total of five tomato PLD cDNAs was isolated, including three PLD
alphas and two betas, using degenerate primers and cDNA generated from root plus stem
tissue RNA, or by probing a cDNA library produced using RNA from Fusarium-infected
root plus stem tissue. Among the five PLD clones were a full-length PLD alpha cDNA,
AY013252, nearly identical to LePLDα1 (AF201661), and a partial PLD alpha cDNA,
AY013254, which represents the same gene as LePLDα2 (AF154425). Northern blot analysis
of PLD alpha transcript levels in various tissues indicated that LePLDα2 and LePLDα3
are most abundantly expressed in flowers > fruit, and that levels of LePLDα2 and LePLDα3
mRNA increase through the orange stage of fruit ripening, remaining high in red ripe fruit.
In contrast, LePLDα1 transcript was most abundant in immature green fruit, and levels declined
thereafter in ripening fruit. Whitaker et al. (2001) performed isogene-specific DNA
and RNA gel blot analyses of LePLDα2 in “Rutgers” tomato plants using a radiolabeled
probe PCR amplified from the 3 ′ -untranslated region (3 ′ -UTR) of the cDNA. LePLDα2 was
shown to be a single copy gene that, as determined by Laxalt et al. (2001), is most abundantly
expressed in floral and fruit pericarp tissues (Fig. 9.10). The abundance of LePLDα2
transcript increased throughout fruit development from 10 days postanthesis, reaching a
maximum at the pink/orange stage of ripening (Fig. 9.10a). A cDNA encoding LePLDα3
←−
Fig. 9.10 (a) Abundance of LePLDα2 transcript in “Rutgers” tomato fruit pericarp and locular tissues over the
course of development and ripening (i) and in floral and vegetative tissues (ii). Total RNA was isolated from fruit
at 10, 20, 30, 35, and 40 days postanthesis and at the breaker (BK), turning (TN), pink (PK), red ripe (RR), and
overripe (OR) stages of ripening, as well as from stem, leaf, root, and flower tissues. About 20 μg per lane was run
on 3% agarose gels and probed with a 3 ′ -UTR fragment of the LePLDα2 cDNA. The blots were then stripped and
hybridized with a soybean 26S DNA probe to evaluate loading of total RNA (as indicated by levels of tomato 27S
rRNA). Bar graph above shows the ratio of LePLDα2 mRNA to rRNA (×100) derived from densitometry scans of
the autoradiograph bands. (b) Semiquantitative RT-PCR analysis of LePLDα3 transcript levels in (i) pericarp tissue
of wild-type (WT) “Rutgers” fruit during development and ripening; (ii) other WT plant organs including roots
(R), stems (S), leaves (L), and flowers (F); and (iii) pericarp tissue of fruit from LePLDα2 antisense lines (10-6-C,
10-5-C, 10-3-B, 8-2-E) harvested 40 days after pollination, as well as WT harvested at the mature green (MG),
breaker (BK), and pink (PK) stages. Isogene-specific primers were used to generate the 0.6-kb fragment corresponding
to nucleotides 333–908 of the LePLDα3 coding region. The QuantumRNA quantitative RT-PCR kit
(Ambion) was used to determine levels of LePLDα3 cDNA relative to the control 18S cDNA. Ethidium bromidestained
gels of the RT-PCR products were used for quantification of the amplified LePLDα3 and 18S cDNA
fragments by densitometry. The left lane in each image shows the location of a 0.5-kb DNA marker. Developmental
stages in (i) were 10, 20, and 30 days after pollination, mature green (MG), breaker (BK), turning (TN), pink
(PK), and red ripe (RR). (Reproduced with permission from Whitaker et al., 2001.)