Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

PHOSPHOLIPASE D, MEMBRANE DETERIORATION, AND SENESCENCE 233
originating from different receptors (ETR1, ETR2) and potentially interacting with one
another, affecting internodal elongation and abscission independently from fruit ripening
(Whitelaw et al., 2002).
Results from several years of study in different laboratories enable us to propose a
model for the signal transduction pathway between ethylene perception and the induction
of senescence at the membrane level. Since calcium and calmodulin appeared to be involved
in enhancing membrane deterioration (Paliyath et al., 1987; Paliyath and Thompson, 1987),
the possible link between ethylene perception and phosphatidylinositol metabolism was investigated
in carnation flowers based on the hypothesis that phosphorylation of PI to PIP 2 ,
phospholipase C action on PIP 2 -generating inositol trisphosphate (IP 3 ), and IP 3 -mediated
calcium release from the endoplasmic reticulum, were potentially the sequence of events
in the signal transduction pathway. When exposed to ethylene, fully open carnation flowers
showed visible symptoms of senescence within 6 h of treatment. Carnation flowers were
incubated with radiolabeled phosphorus and treated with ethylene. The incorporation of
radiolabel into the phospholipid fraction was followed soon after treatment. If there were a
specific turnover in any phospholipids in response to ethylene, then there would be a specific
enrichment of the radiolabel in that phospholipid (based on phospholipid phosphate). None
of the phospholipids including phosphatidylinositol showed any significant enrichment in
response to ethylene treatment, suggesting that phospholipid turnover, if it occurred, was
restricted to sites close to the ethylene receptor, and may occur in very small amounts not
quantifiable by the available techniques. As well, with a relatively low efficiency of calcium
release by IP 3 and a very low phospholipase C activity, the proposed hypothesis was not
supported. The recent results on the molecular properties of PLD enable us to propose
an alternate mechanism that may mediate the ethylene signal transduction pathway (Fig.
9.21). Studies on animal systems show that PI-3-kinase is activated by receptor tyrosine kinases
in response to primary stimuli, which leads to the production of PI (3,4)-bisphosphate
and PI (3,4,5)-trisphosphate on the inner leaflet of the plasma membrane (Blomberg et al.,
1999). These anionic domains are believed to be the anchoring sites for enzymes in the
signal transduction pathway, such as phospholipase C, which possesses a PH superfold
(plextrin homology domain). The presence of the C2 domain in PLD (analogous to the PH
superfold) provides the structural feature necessary for the electrostatic binding of PLD
to anionic sites created in the membrane in response to primary stimuli. Recent evidences
suggest that PI is converted to its phosphorylated forms in response to primary stimuli
through PI kinases (Heilman et al., 2001). Such anionic microdomains in the membrane
could provide the anchoring region for PLD even in the absence of an increase in cytosolic
calcium (Zheng et al., 2000). Generation of anionic microdomains specifically in the inner
leaflet of the plasma membrane could generate a voltage across a localized region of the
plasma membrane (hyperpolarization), and may open voltage-sensitive calcium channels,
thus increasing the cytosolic calcium levels (Roberts and Tyerman, 2002). Since the binding
of PLD to anionic domains is reversible and dependent on calcium (high micromolar
calcium reverses binding) (Zheng et al., 2000), such binding may serve as an on/off switch
regulating calcium release, through catabolism of phosphorylated phosphatidylinositols
(depolarization) (Paliyath et al., 1995). A recent report also suggests that cyclic nucleotidegated
nonselective cation channels (voltage independent) may be involved in programmed
cell death in Arabidopsis (Kohler et al., 2001). PLD may bind to other domains of the
membrane enriched in phosphatidylcholine/phosphatidylethanolamine, since this binding