Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

214 POSTHARVEST BIOLOGY & TECHNOLOGY OF FRUITS, VEGETABLES, & FLOWERS
(806 amino acids) was subsequently cloned using RNA from “Rutgers” tomato fruit and
gene-specific primers based on GenBank sequence AY013253 (Whitaker et al., unpublished).
The nucleotide sequence of the LePLDα3 open reading frame was identical to that
of AY013253 reported by Laxalt et al. (2001). However, in contrast with their Northern blot
results, semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) using
a pair of isogene-specific primers indicated that LePLDα3 transcript is most abundant in
fruit pericarp tissue from early development (10 days after pollination) through the breaker
stage of ripening, declining substantially thereafter (Fig. 9.10b). LePLDα3 expression in
organs other than fruit (Fig. 9.10b) was found to be similar to that determined for LePLDα2
(Fig. 9.10a), with the greatest abundance in floral tissues.
More recently, our studies on the role of PLD in fruit ripening and senescence have
included cloning of full-length PLD alpha cDNAs from cultivated strawberry (Fragaria ×
ananassa) and “Honey Brew” hybrid honeydew melon (Cucumis melo var. inodorus). The
strawberry cDNA (AY758359), hereafter referred to as FaPLDα1, was cloned using cDNA
reverse transcribed from fruit tissue RNA and degenerate primers based on two highly
conserved regions of plant PLDs, followed by 3 ′ and 5 ′ RACE (Yuan et al., 2005). The ORF
of FaPLDα1 is 2,433 bp encoding a PLD alpha 810 amino acids in length. similar strategy
and methodology were utilized to clone CmPLDα1 (DQ267933) from honeydew melon,
with the exception that leaf tissue RNA was used initially to isolate 5 ′ - and 3 ′ -end cDNA
fragments (Whitaker and Lester, 2006). Eventually, the entire 2,427-bp ORF, encoding
a PLD alpha 808 amino acids long, was amplified from cDNA reverse transcribed from
fruit mesocarp tissue RNA using gene-specific primers. During cloning of CmPLDα1,
a cDNA fragment representing a second C. melo PLD alpha isogene was also isolated.
Quite recently the complete cDNA, CmPLDα2 (EF543155), was cloned and reported in
GenBank (Whitaker et al., unpublished). CmPLDα2 includes a 2,424-bp ORF encoding an
807-amino acid PLD alpha. One additional, related PLD alpha cDNA worthy of mention
was cloned from cucumber (Cucumis sativus) fruit and utilized in recent studies by Mao et
al. (2007a, b) on the role of PLD and lipoxygenase (LOX) in development of chilling injury.
The complete cDNA (EF363796), hereafter referred to as CsPLDα1, is 2,756-bp long and
includes a 2,427-bp ORF encoding a PLD alpha 808 amino acids in length.
A series of BLASTP searches on the NCBI website (www.ncbi.nlm.nih.gov) was conducted
to establish the encoded amino acid sequence identity and similarity among the
seven PLD alpha genes described above from tomato, strawberry, honeydew melon, and
cucumber. The results, presented in Table 9.3, show that the encoded proteins are all highly
conserved, sharing a minimum of 71% amino acid identity and 86% similarity (LePLDα3vs
CmPLDα2). Notably, LePLDα1 segregates from LePLDα2 and LePLDα3, which are 87%
identical, but LePLDα1 is more closely similar to FaPLDα1, CmPLDα1, and CsPLDα1.
Not surprisingly, CmPLDα1 and CsPLDα1, representing the same genus, are 97% identical.
Overall, CmPLDα2 was most dissimilar from the other six PLD alphas, sharing the closest
identity with the honeydew melon isozyme CmPLDα1. Figure 9.11 presents a phylogenetic
tree indicating primary structural relatedness of the seven encoded PLD alphas from tomato,
strawberry, honeydew melon, and cucumber, as well as 18 additional PLD alphas from 13
other dicot species. It is interesting that similarity of PLD alpha primary structure is often
in accord with taxonomic proximity, for example, LePLDα1 and tobacco PLDα1 (Nicotiana
tabacum; CAB06620), and CmPLDα1 and CsPLDα1, yet isozymes from the same
species can also differ substantially. Possibly, this is an indication of separate functions