Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

218 POSTHARVEST BIOLOGY & TECHNOLOGY OF FRUITS, VEGETABLES, & FLOWERS
electrostatic rather than a hydrophobic interaction. It was also recently shown that the C2
domain of PLD alphas possesses binding sites that enable Ca 2+ -independent association
with an aspartic protease, cardosin A, involving RGD and KGE motifs in the protease sequence
(Simões et al., 2005). The C2 domain in plant PLD alphas comprises the range of
amino acids from about 6 to 150 (Fig. 9.12), and as in other C2 superfamily proteins is characterized
by the presence of eight beta strand motifs that form an antiparallel beta sandwich
(Nalefski et al., 1997; Rizo and Südhof, 1998). The proposed locations of the eight beta
strands in C2 of plant PLD alphas and betas appear to vary depending on whether modeling
is based on the crystal structure of rat synaptotagmin or PLC delta 1 and cPLA 2 (Pappan
et al., 1997; Rizo and Südhof, 1998; Zheng et al., 2000). Nevertheless, a common feature
in both models is the presence of several critical amino acids that participate in binding of
up to four Ca 2+ ions and are located at specific sites on exposed loops connecting the beta
sheets. In Arabidopsis PLD beta 1 and rat synaptotagmin, four acidic amino acid residues
(glutamic acid; E or aspartic acid; D) were identified as those most involved in Ca 2+ binding
(Pappan et al., 1997). A subsequent alignment of Arabidopsis PLD alpha and beta with
PLC delta 1 and cPLA 2 confirmed three of those four amino acids and identified a single
asparagine (N) residue as also being important (Zheng et al., 2000). The five corresponding
residues in LePLDα1 occur at AA 38, 49, 69, 97, and 99 (Fig. 9.12), and notably, acidic
AAs at 49 and 99 have been substituted by basic lysine (K) and polar but uncharged asparagine
(N), respectively. These, or similar substitutions seen in the seven fruit PLD alphas
were thought to account for the requirement of alpha-type PLDs for millimolar rather than
micromolar concentrations of calcium for activity at neutral pH (Wang, 2000, 2001; Zheng
et al., 2000). However, other studies (Yuan et al., 2006a; Tiwari and Paliyath, 2007) have
confirmed that strawberry PLD alpha and a tomato PLD alpha C2 domain show activation
at physiologically activated, low micromolar levels of calcium suggesting the presence of
high-affinity calcium-binding sites. It is interesting that the third of the four critical acidic
AAs, at position 95 in LePLDα2 and LePLDα3, is substituted by nonpolar and hydrophobic
alanine or phenylalanine (Fig. 9.12), raising the possibility that Ca 2+ binding is severely
compromised in these two PLD alphas. As well, LePLDα1 is the only PLD alpha of the
seven to have asparagine (AA 69) at the nonacidic Ca 2+ -binding position found in PLC
delta 1 and cPLA 2 , although all the PLD alphas have two acidic AAs (E or D) adjacent
to this residue that could take part in binding calcium ions. Conceivably the C2 domains
in the seven fruit PLD alphas could exist in different topologies, thus exposing different
sets of calcium-binding sites, but nevertheless exhibiting conformational changes required
for phospholipid (membrane) binding, some of these at physiologically activated cytosolic
calcium levels.
C2 in Arabidopsis PLD beta was shown to have three high-affinity Ca 2+ -binding sites
with dissociation constants (K d s) of 0.8, 2.4, and 24 μM, whereas calorimetry data indicated
that C2 of Arabidopsis PLD alpha has 1–3 low-affinity sites with K d in the range of 470–
590 μM (Zheng et al., 2000). However, this property in Arabidopsis PLD alpha is a unique
situation and may not be reflective of several PLD alphas that show high-affinity calciumbinding
sites and are activated at micromolar calcium levels. Aside from in the C2 domain,
Pappan et al. (2004) demonstrated that specific binding of calcium ions also occurs in the
catalytic portion of Arabidopsis PLD beta 1. A bacterially expressed PLD beta 1 lacking
the C2 region had much lower activity than the complete enzyme, required higher levels of
calcium for maximum activity, and was activated by phosphatidylserine much more than