Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

210 POSTHARVEST BIOLOGY & TECHNOLOGY OF FRUITS, VEGETABLES, & FLOWERS
melon, and others. In recent years, multiple functional roles for PLD in plants have been
demonstrated, including cell signaling in response to molecules such as abscisic acid (ABA)
and fungal elicitors, programmed cell death, root hair development, and stress responses
such as development of freezing or drought tolerance. Various plant PLDs have highly
conserved regions that contribute to their enzyme function, as well as regions that differ
from one another that provide the diverse functional characteristics in response to cellular
and environmental signals. Plants possess a highly diverse PLD gene family. Arabidopsis,
which has served as a model plant system, contains 12 PLD genes. By contrast, there are
2 PLD genes in mammalian systems and 1 in yeast. The 12 PLD genes from Arabidopsis
are grouped into the alpha (3), beta (2), gamma (3), delta (1), epsilon (1), and zeta (2) types.
Of these, only the two zeta-type PLDs contain plextrin homology (PH) plus phox homology
(PX) domains in the N-terminal region, which are conserved in PLDs from other kingdoms.
The remaining 10 Arabidopsis PLDs contain a C2 domain at the N-terminus, which is a
calcium-dependent phospholipid-binding structural fold present in a number of lipid signaling
and metabolic proteins, but unique to plant PLDs (Wang et al., 2006). These differences,
along with differences in internal motifs, create the diversity in plant PLD structure and
function. In addition to the conserved pair of PLD active site (HxKxxxxD) domains, different
PLDs possess motifs that interact with calcium, polyphosphoinositides, G-proteins,
and other factors. Phospholipases are soluble proteins, and their translocation to the membrane
on physiological stimulation may be achieved through conformational changes in the
C2 domain on binding to calcium released into the cytosol, or by other changes such as
the biosynthesis of phosphorylated inositol phospholipids on the membrane that serve as
binding sites for the PH/PX super-fold in zeta-type PLDs. In addition, the concentration of
calcium ions required for activation varies widely with the type of plant PLD, due at least
in part to modifications in amino acid sequences in the C2 domain, and there can also be a
marked influence of pH on activity and calcium binding.
9.6 Cloning and homology of tomato, strawberry, and
melon PLD alpha cDNAs
Following cloning of the first phospholipase D (PLD) gene from castor bean (Wang et al.,
1994), there has been substantial progress in determining physiological roles of members
of the plant PLD gene family, now known to comprise six classes: alpha, beta, gamma,
delta, epsilon, and zeta (Bargmann and Munnik, 2006; Wang et al., 2006). Most notably,
phosphatidic acid derived from PLD hydrolysis of PC and other phospholipids is an important
signaling molecule that mediates responses to various types of biotic and abiotic
stress (Wang, 2002, 2005; Bargmann and Munnik, 2006; Wang et al., 2006). Despite this
progress, however, one of the earliest roles ascribed to PLD, that is, initiator of the cascade
of phospholipid catabolism in senescing plant tissues (Paliyath and Droillard, 1992), has
received relatively little attention. PLDs of the alpha class, typically the most abundantly
expressed and accounting for most of the total activity (Fan et al., 1999), are the best candidates
to perform this function. Although antisense knockout of AtPLDα1 did not alter
natural senescence, however, it did delay ethylene- and abscisic acid–induced senescence
of detached leaves of Arabidopsis (Fan et al., 1997). This finding indicates a likely role of
PLD in postharvest senescence of fresh fruits and vegetables, particularly in climacteric
fruits such as tomato, which produce high levels of ethylene. PLD activity estimated in