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Mechanisms underlying the depletion of phospholipid in senescing membranes have been examined using microsomes isolated from bean cotyledons (Phaseolus vulgaris) at various stages of development. As the cotyledons age, microsomal phospholipid levels relative to protein decrease by 93% indicating that phospholipids are selectively depleted from senescing membranes. This reflects active phospholipid catabolism, but can also be attributed to a reduction in phospholipid synthesis. Specifically, the activities of choline phosphotransferase and ethanolamine phosphotransferase, enzymes mediating the terminal step in the synthesis of phosphatidylcholine and phosphatidylethanolamine, respectively, decrease dramatically as the cotyledons senesce

Fluorescent peroxidized lipids are present in lipid extracts of microsomal membranes and cytosol from young and senescing bean (Phaseolus vulgaris) cotyledon tissue. In young tissue, the peroxidized membrane lipids are mainly phospholipids, whereas those in the cytosol are primarily free fatty acids. With advancing senescence, microsomal peroxidized lipids increase by 200% relative to membrane protein and by 50% on a per cotyledon basis, and the increase is mainly attributable to enhanced levels of peroxidized free fatty acids. Cytosolic peroxidized lipids expressed on a per cotyledon basis decline by 55% over the same period. Fractionation of the cytosol revealed that, for both young and senescing tissue, about 50% of the cytosolic fluorescent peroxidized lipids are associated with non-sedimentable microvesicles

The effects of lipid degradation on proteins of smooth microsomal membranes isolated from young bean cotyledons have been examined by three techniques, viz. fluorescence energy transfer from tryptophan to cis-parinaric acid; protein spin-labelling with 3-maleimido PROXYL; and SDS-PAGE. Lipid degradation was induced in isolated membranes by activiting phospholipase D and phosphatidic acid phosphatase through the addition of Ca 2+ , by treatment with exogenous phospholipase C to simulate the concerted actions of phospholipase D and phosphatidic acid phosphatase or by treatment with exogenous phospholipase A 2 to generate endogenous substrate for lipoxygenase. All of the treatments induced time-dependent changes in lipid-protein interaction and in protein conformation, and the treatment with phospholipase A 2 also engendered proteolysis

Eukaryotic translation initiation factor 5A (eIF5A) is thought to facilitate protein synthesis by participating in the nuclear export of specific mRNAs. In Arabidopsis, there are three isoforms of eIF5A. One of them, AteIF5A1, has been shown to be expressed in vascular tissue, specifically developing vessel members, using GUS as a reporter. In order to determine whether AteIF5A1 plays a role in xylem formation, its full-length cDNA was constitutively over-expressed in transgenic Arabidopsis plants. Microscopic analysis revealed that the cross-sectional area of the xylem in the main inflorescence stems of transgenic plants was 1.9-fold higher than those of corresponding inflorescence stems of wild-type plants. In wild-type stems, the primary xylem typically comprised six cell layers and was approximately 105 mum thick, but increased to 9-11 cell layers, 140-155 mum thick, in transgenic stems. Similarly, the secondary xylem increased from six cell layers, approximately 70 mum thick, in control stems to approximately 9 cell layers, 95-105 mum thick, in transgenic stems. Moreover, constitutive down-regulation of AteIF5A1 using antisense technology resulted in the major suppression of xylem formation compared with control plants, and the antisense transgenic plants were also stunted. These data collectively indicate that eIF5A1 plays a role in xylogenesis.

Cet article porte sur l'etude de la degradation des phospholipides membranaires par les enzymes lipolytiques des membranes microsomales de petales de fleur d'œillet dans le cadre de la senescence. La preference de la phospholipase-D pour certains types de phosphotidylcholine est etudiee

A subpopulation of plasma membrane vesicles enriched in membrane lipid metabolites has been isolated from petals of carnation flowers and leaves of canola seedlings. This was achieved by immuno-purification from a microsomal membrane preparation using region-specific antibodies raised against a recombinant polypeptide of the plasma membrane H + -ATPase. The properties of this subpopulation of vesicles were compared with those of purified plasma membrane isolated by partitioning in an aqueous dextran-polyethylene glycol two-phase system. The lipid composition of the immunopurified vesicles proved to be clearly distinguishable from that of phase-purified plasma membrane, indicating that they represent a unique subpopulation of plasma membrane vesicles. Specifically, the immunopurified vesicles are highly enriched in lipid metabolites, including free fatty acids, diacylglycerol, triacyl-glycerol and steryl and wax esters, by comparison with the phase-purified plasma membrane. These findings can be interpreted as indicating that lipid metabolites generated within the plasma membrane effectively phase-separate by moving laterally through the plane of the membrane to form discrete domains within the bilayer. It is also apparent that these domains, once formed, are released as vesicles into the cytosol, presumably by micro-vesiculation from the surface of the plasmalemma. Such removal may be part of normal membrane turnover.

Liposomes containing 79 mM Tris-acetate and 50 mM KC1 were prepared from the total lipid extracts of smooth microsomal membranes isolated from 2, 4, 7 and 9 d old bean cotyledons. Permeability to glycerol was determined by spectrophotometric measurements of osmotic swelling when the liposomes were placed in either isotonic or slightly hypotonic glycerol. For liposomes from 2 and 4 d old membrane there was a time-dependent decrease in absorbance at 450 nm from which initial swelling rates reflecting the influx of glycerol and water were calculated. At 25 °C these rates were not significantly different. For liposomes from 7 and 9 d old membrane there was no change in absorbance with time at 450 nm signifying that these older liposomes were equally permeable to electrolytes and non-electrolytes, and therefore incapable of swelling. Permeability to glucose was determined by preparing the liposomes in a solution of the sugar, passing them through a Sephadex column to eliminate unsequestered glucose, and quantifying sugar leaked from the liposomes over time by measuring NADPH formation through the tandem actions of hexokinase and glucose-6-phosphate dehydrogenase. The rate constants for glucose leakage from 2 and 7 d old liposomes were 0-55 and 3-94 respectively, again indicating a dramatic increase in permeability with advancing age. These changes in permeability correlate temporally with the appearance of gel phase lipid in both liposomes and the membranes from which they were derived, suggesting that the coexistence of discrete liquid-crystalline and gel phase lipid domains renders membranes leaky and contributes to loss of intracellular compartmentation in senescing tissue.