Membrane Deterioration in Senescing Carnation Flowers

Abstract

The lipid fluidity of microsomal membranes from the petals of cut carnation flowers decreases as the flowers senesce. A comparable chane in fluidity was induced by in vitro aging of microsomal membranes from youn flowers under conditions in which membranous lipoxygenase-like activity was active. There was no change in fluidity when the membranes were aged in the presence of inhibitors of lipoxygenase or were heatdenatured prior to aging. Membranes from naturally senesced flowers and membranes that had been aged in vitro both sustained an increase in saturated.unsaturated fatty acid ratio that accounted for the decrease in lipid fluidity, and in both i s there was evidence for depletion of the unsaturated fatty acids, linoleic acid, and linolenic acid, which are substrates for lipoxygenase. Loss of lipid phosphate reflecdtng breakdown of membrane phospholipids preceded the depletion of unsaturated fatty acids attributable to the lipoxygenase-like activity. The data have been interpreted as indicating that fatty acid substrates for membrane-associated lipoxygenase-like activity are made available by the initiation of phospholipid degrdation, and that the utilization of these substrates results in a selective depletion of unsaturated fatty adds from the membrane and an ensuing decrease in bulk lipid fluidity.Membrane deterioration attributable to lipid degradation and ensuing destabilization ofthe bilayer appears to be a fundamental feature of senescence (28). During the early stages of senescence, there is a pronounced decline in membrane phospholipid that becomes manifested as an increased membrane sterol:fatty acid ratio (2,19,26 lipid fluidity (1,4,19,26). There are also large changes in the phase properties of senescing membranes that have been attributed to lipid peroxidation (24,27).Lipoxygenase, an enzyme that is capable of forming activated oxygen and initiating lipid peroxidation, is also associated with senescing membranes (16, 17). As senescence progresses, linoleic acid and linolenic acid, which are substrates for lipoxygenase, would be among the fatty acids released from membrane phospholipids. In the present study, we have examined the extent to which membrane deterioration in senescing carnation flowers can be attributed to the concerted effects of phospholipid degradation and the action of membrane-associated lipoxygenase-like activity. Epps3 (pH 8.5), and centrifugation at 131,000g for 1 h. The resulting pellet was suspended in 2 mm Epps (pH 8.5), to a concentration of 2 mg protein ml-' and either used directly or dialyzed. In the latter case, 6 ml of membrane suspension (2 mg protein ml-') were dialyzed against 1,000 ml of 10 mm Epps (pH 8.5), at 5°C. The buffer was changed after 1 and 2 h, and dialysis was then continued for an additional 12 h. MATERIALS AND METHODS PlantFor in vitro aging experiments, the membrane suspension was diluted to 0.1 mg protein ml-' in 2 mM Epps adjusted to pH 7.3 or 8.5, and 60 ml of the diluted suspension were incubated in a 50 ml Erlenmeyer flask at 24°C. ...

Subcellular Localization of Secondary Lipid Metabolites Including Fragrance Volatiles in Carnation Petals

“…Lipoxygenase and hydroperoxide lyase, which form fatty acid hydroperoxides and C6-aldehydes, respectively, are known to be membrane-associated (Fobel et al, 1987;Hatanaka et al, 1987;Matsui et al, 1991;Vianello et al, 1995). This, together with the finding in the present study that lipoxygenase pathway-derived C6-aldehydes and alcohols are present in microsomal membranes, suggests that volatile lipid metabolites originate from membranes.…”

“…This enzyme appears to be associated with microsomal membranes of carnation petal tissue (Fobel et al, 1987). To test this hypothesis the effect of n-propyl gallate on the release of polar lipid mefabolites from microsomal membranes was determined.…”

“…Lipid-protein particles generated in vitro from microsomal membranes, which had been labeled in situ with [14C]acetate or in vitro with [14C]linoleic acid, were also enriched in radiolabeled polar lipid metabolites compared with their membranes of origin. These observations collectively indicate that at least some of the polar lipid metabolites detectable in the lipid-protein particles were formed by membranous lipoxygenase, which utilizes free linoleic and linolenic acids as substrates (Fobel et al, 1987). Moreover, >60% of the [14C]acetate-derived polar lipid metabolites in lipid-protein particles proved to be, and several metabolites of the lipoxygenase pathway are known to be, volatile (Hildebrand, 1989;Gardner, 1995).…”

“…n-Propyl gallate at a concentration of 2 mM inhibits carnation petal microsomal lipoxygenase by >90% (Fobel et al, 1987). One possible interpretation of the finding that the appearance of radiolabeled polar lipid metabolites in the lipid-protein particles was only reduced by approximately 40% is that preformed radiolabeled metabolites are released under these conditions.…”

“…In animal systems, for example, Ca2+ appears to mediate the association of lipoxygenase with membranes, which then acts on arachidonic acid to form fatty acid hydroperoxides that lead to the formation of prostaglandins (Axen et al, 1973). In plant cells there are both cytosolic and membranous forms of lipoxygenase (Fobel et al, 1987;Hildebrand et al, 1988;Vianello et al, 1995) that form fatty acid hydroperoxides from free linolenic and linoleic acids. Fatty acid hydroperoxides in turn serve as substrates for both hydroperoxide lyase and allene oxide synthase.…”