Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

PHOSPHOLIPASE D, MEMBRANE DETERIORATION, AND SENESCENCE 211
strawberry fruit membranes may also be due to PLD alpha. Western blot analysis of strawberry
membrane proteins using polyclonal antibodies raised against rapeseed PLD alpha
(Novotna et al., 1999) showed a predominant cross reactivity with a 92-kDa protein (Yuan
et al., 2005), and nothing above this molecular mass, suggesting that PLD beta (112 kDa)
and gamma (98 kDa) isoforms may occur below detectable levels, or do not react with
the PLD alpha antibodies. As well, the PLD alpha 1 encoded by FaPLDα1 from strawberry
fruit (Yuan et al., 2005) is very similar to that encoded by LePLDα1 from tomato.
In general, strawberry PLD alpha is also similar to tomato PLD alpha in its properties and
regulation by calcium (Tiwari et al., unpublished). Despite differences in kinetic properties,
strawberry fruit mitochondrial PLD appears to be similar to microsomal PLD in its molecular
mass, about 92 kDa, which is indicative of an alpha-type PLD (Yuan et al., 2006a).
The different kinetic properties of mitochondrial versus microsomal PLD may arise from
posttranslational modifications, or the presence/absence of particular modulatory lipids or
other effectors. Irrespective of whether a fruit is climacteric (tomato), weakly climacteric
(hybrid honeydew melon), or nonclimacteric (strawberry), PLD may play a key role in the
initiation and progression of ripening and senescence.
Three isogenes encoding PLD alphas (referred to here as LePLDα1, LePLDα2, and
LePLDα3) have been cloned from tomato and further studied (Table 9.2). LePLDα1
(Almquist and Paliyath, 2000; GenBank accession number AF201661) was cloned using
a novel strategy for the design of primers known as the CODEHOP program (Rose
et al., 1998), by which PCR primers were designed based on highly conserved regions of
Arabidopsis thaliana PLD alpha-, beta-, and gamma-encoded amino acid sequences aligned
using the Clustal W program (Altschul et al., 1997). A CODEHOP primer is degenerated at
the 3 ′ core region, with a length of 11–12 bp spanning four codons representing highly conserved
amino acids, and nondegenerate at the 5 ′ consensus clamp region, which is variable
in length. The hybrid structure (5 ′ consensus and 3 ′ degenerate) of CODEHOP primers has
been reported to result in specific amplification of cDNA during early cycles of PCR, and
selective amplification of products during later cycles. Forward and reverse primers were
generated and PCR performed using tomato root cDNA (reported in Pinhero et al., 2003).
Initially this step amplified two cDNA fragments: one 883-bp long corresponding to a PLD
alpha and another 936-bp long corresponding to a PLD beta or second PLD alpha. Forward
Table 9.2 Percent deduced amino acid sequence identity/similarity among seven PLD alpha full-length
cDNAs from tomato (LePLDα1, LePLDα2, and LePLDα3), strawberry (FaPLDα1), honeydew melon
(CmPLDα1 and CmPLDα2), and cucumber (CsPLDα1)
LePLDα1 LePLDα2 LePLDα3 FaPLDα1 CmPLDα1 CmPLDα2 CsPLDα1
LePLDα1 — 76/87 74/86 81/89 82/92 75/98 81/91
LePLDα2 76/87 — 87/94 75/87 76/87 74/87 76/87
LePLDα3 74/86 87/94 — 73/86 74/86 71/86 74/86
FaPLDα1 81/89 75/87 73/86 — 82/91 76/88 82/91
CmPLDα1 82/92 76/87 74/86 82/91 — 78/90 97/98
CmPLDα2 75/88 74/87 71/86 76/88 78/90 — 77/89
CsPLDα1 81/91 76/87 74/86 82/91 97/98 77/89 —
The corresponding GenBank protein accession numbers are as follows: LePLDα1 = AAF17557, LePLDα2
= AAG48162, LePLDα3 = AAG45486, FaPLDα1 = AAW83125, CmPLDα1 = ABB82551, CmPLDα2 =
ABS86615, CsPLDα1 = ABN13537.