ABSTRACrDuring senescence of cut canation flowers, there is extensive breakdown of microsomal phospholipid. This is attributable, at least in part, to lipolytic activity associated directly with the microsomal membranes. Evidence indicating that one or more of the lipid-degrading enzymes in these membranes preferentially degrade phospholipid molecular species containing two diunsaturated acyl chais or at least one polyunsaturated acyl chain has been obtained by using radiolabeled phosphatidylcholine the lipids more susceptible to attack by lipases.In the present study, we describe evidence indicating that the phospholipid-degrading enzymes associated with microsomal membranes of senescing carnation flowers exhibit specificity for phospholipid molecular species containing two diunsaturated fatty acids or at least one polyunsaturated fatty acid. This finding is discussed in the context that provision of specific molecular species of phospholipids, which serve as preferred substrates for membrane-associated lipases, may regulate the progression and rate of phospholipid degradation in senescing membranes. MATERIALS AND METHODSPlant Material and Membrane Isolation. Carnation flowers (Dianthus caryophyllus L. cv White Sim) were grown in a commercial greenhouse (Unsworth and Sons, Burlington, Ontario). They were cut at a young stage when the petals had expanded approximately 2 cm beyond the sepals. The stems were trimmed to a length of 22 cm, and the flowers were placed individually in glass culture tubes containing deionized water. They were maintained at 22°C under continuous illumination (240 ft-c) until they had reached specific stages of senescence, viz., stage II, flowers that still possessed yellowish-tinted centers, but were fully expanded; stage III, flowers that were completely white in color but were not yet showing petal-inrolling; stage IV, senescent flowers showing petal-inrolling.Microsomal membranes were isolated from stage II, III, and IV flowers in 10 mm Epps3 (pH 8.5), as described previously (25) and washed once by resuspension in the same buffer and centrifugation at 131,000g for 1 h. The resulting pellet of membranes was resuspended in 3 ml 70 mM Epps (pH 8.5), and dialyzed at 4°C against 3 changes of 600 ml of 2 mm Epps (pH 8.5), for 15 h. After dialysis, the protein concentration was adjusted to 1 mg ml-' with 70 mM Epps (pH 8.5). Protein was measured as described by Bradford (5).Phospholipid Degradation. The capability of isolated microsomal membranes to degrade various molecular species of phospholipids was determined by using radiolabeled substrates. An aliquot (1.8 ml) of washed, dialyzed membrane suspension (I mg protein ml-') was vortexed for 3 min with 0.

Microsomal membranes from the petals of senescing carnation (Dianthus caryophyllus L.) flowers contain phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol. These phospholipid classes decline essentially in parallel during natural senescence of the flower and when microsomal membranes isolated from young flowers are aged in vitro. However, measurements of changes in the endogenous molecular species composition of microsomal phospholipids during natural senescence of the flower petals and during in vitro aging of isolated membranes have indicated that the various molecular species of phospholipids have quite different susceptibilities to catabolism. Acyl chain composition and the nature of the head group are both determinants of their susceptibility to catabolism. As well, a comparison of the phospholipid catabolism data for naturally senesced membranes and for membranes aged in vitro suggests that the phospholipid composition of membranes is continuously altered during senescence by acyl chain desaturation and possibly retailoring so as to generate molecular species that are more prone to catabolism. The results collectively indicate that provision of particular molecular species of phospholipids with increased susceptibility to degradation contributes to enhanced phospholipid catabolism in the senescing carnation petal.Loss of membrane integrity is an early and fundamental feature of senescence in plant tissues, and perhaps the clearest manifestation of this is the onset of leakiness attributable to increased membrane permeability. In petals of cut carnation flowers, for example, solute leakage is detectable before petal inrolling and the climacteric-like rise in ethylene production, symptoms that are thought to denote the initiation of petal senescence (7). Studies with various techniques including electron spin resonance (20), fluorescence depolarization (9), freeze-fracture electron microscopy (24), and x-ray diffraction ( 19) (18) and results in a substantial increase in the sterol:phospholipid ratio of membranes as senescence progress (9, 29). Several phospholipid degrading enzymes have been identified in plant tissues including phospholipase D (13), lipolytic acyl hydrolase (10), and phospholipase C ( 14), and recent evidence suggests that phospholipase D may initiate phospholipid catabolism in senescing membranes (5). Although there is clear evidence for a net decline in phospholipid levels with advancing senescence, the extent to which this reflects decreased synthesis, enhanced catabolism, or possibly both has not been resolved. An age-related reduction in phospholipid synthesizing ability has been demonstrated in a number of plant tissues (3). Increased lipid catabolism during senescence has also been observed, but under conditions in which there is no absolute increase in phospholipase activity (27). This suggests that in at least some senescing tissues enhanced degradation of phospholipid may be attributable to alterations in membrane lipids that make...

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