Inhibition of phospholipase D by lysophosphatidylethanolamine, a lipid-derived senescence retardant

Abstract

Phospholipid signaling mediated by lipidderived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, f lowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dosedependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphotidylserine had no significant effect on PLD activity whereas PLD was stimulated by lysophosphatidic acid and inhibited by lysophosphatidylinositol. Membrane-associated and soluble PLD, extracted from cabbage and castor bean leaf tissues, also was inhibited by LPE. Consistent with acyl-specific inhibition of PLD by LPE, senescence of cranberry fruits as measured by ethylene production was more effectively inhibited according to the increasing acyl chain length and unsaturation of LPE. There are no known specific inhibitors of PLD in plants and animals. We demonstrate specific inhibitory regulation of PLD by a lysophospholipid.Lipids previously were thought to play important roles only in membrane structure and energy reserves. It is now evident that lipids and their metabolites have many other critical cellular functions particularly as mediators in signal transduction, cell activation, and cell proliferation (1, 2). Studies in animal systems have demonstrated that phospholipases, a group of phospholipid-hydrolyzing enzymes, are key enzymes capable of generating lipids that can act as second messengers in signal transduction (2-4).We recently have found that lysophosphatidylethanolamine (LPE), a hydrolysis product of PE by phospholipase A 2 , is able to retard plant senescence. LPE-treated attached and detached leaves had a higher chlorophyll content and a lower rate of both respiration and ethylene production than the controls (5). Tomato fruits at the red stage of physiological maturity were detached with their pedicles and dipped in LPE (50 mg͞L) solution. After 5 days of treatment, LPE-treated fruits had a much lower production of ethylene as well as lower electrolyte leakage than the controls (5). In support of this, the vase-life of LPE-treated cut flowers was prolonged to 7 days compared with 2 days in the control (6). LPE-treated flowers also had lower ion leakage and lower ethylene production. When LPE was sprayed on tomato leaves together with ethephon, an ethylene-releasing compound, LPE mitiga...

Inhibition of Phospholipase Dα byN-Acylethanolamines

“…1 and 2). All NAEs were effective inhibitors at high concentrations (200 m), similar to the concentrations reported for LPE (Ryu et al, 1997). In general, the long-chain, unsaturated NAEs demonstrated less inhibitory effects on castor bean PLD␣ than did saturated or shorter chain types (Fig.…”

“…In separate control experiments, exogenous myristic acid up to 10 m did not influence the activity of the cabbage or the castor bean PLD␣ (data not shown). Other workers have demonstrated that ethanolamine itself has no inhibitory effect on PLD (Ryu et al, 1997), suggesting that the inhibitory effect of NAE is conferred by a structural specificity of the entire molecule, and may be an inherent property of plant PLD␣ isoforms.…”

“…The second notable difference is in the concentration range for inhibition. LPE 18:1 was the most potent inhibitor of cabbage PLD␣, and this was in the range of 10 to 200 m (Ryu et al, 1997). NAE 12:0 and NAE 14:0 were the most potent inhibitors of castor bean and cabbage PLD␣ activity, and this was in the range of 0.1 to 1 m (Figs.…”

“…LPE was shown recently to inhibit plant PLD␣ and to have a profound effect on the physiological symptoms associated with postharvest senescence of flowers and fruits (Ryu et al, 1997). It is interesting that there are two key differences between LPE and NAE inhibition of PLD␣.…”

“…High levels of phosphatidylethanolamine (80 mol%) are important in general for optimal PLD activity in vitro (Wang, 2001a). Furthermore, lysophosphatidylethanolamine (LPE) was recently shown to be inhib-itory toward cabbage (Brassica oleracea) PLD␣ (Ryu et al, 1997). These data, as well as the varied and potent biological activities ascribed to NAEs in animal systems (Di Marzo, 1998), prompted us to hypothesize that plant NAEs might act as lipid mediators to modulate PLD␣ activity.…”