No abstract

Phloem isolated from canola (Brassica napus L.) stems was found to contain phospholipid, diacylglycerol, triacylglycerol, steryl and wax esters, and comparatively high concentrations of unesterified fatty acids. Indeed, the composition of phloem lipid was markedly different from that of microsomal membranes and cytosol isolated from both leaves and stems. Specifically, phloem lipid consisted predominantly of unesterified fatty acids and was enriched in medium-chain fatty acids, in particular, lauric, myristic and pentadecanoic acids. This unique composition also distinguished phloem lipid from that of well-characterized cytosolic lipid particles such as oil bodies found in plant cells. Moreover, levels of medium-chain fatty acids in the phloem increased when canola plants were stressed by exposure to sublethal doses of ultraviolet irradiation. Phloem levels of lauric acid, for example, increased by 11-fold upon treatment with sublethal ultraviolet irradiation. Spherical lipid particles were discernible in isolated phloem sap by electron microscopy, suggesting that the lipid in phloem is in the form of lipid particles. The presence of lipid in phloem may be reflective of long-distance lipid transport in plants, primarily in the form of free fatty acids.

The effects of suppressing deoxyhypusine synthase (DHS) have been examined in tomato (Solanum lycopersicum cv UCT5). DHS mediates the first of two sequential enzymatic reactions that activate eukaryotic translation initiation factor-5A (eIF-5A) by converting a conserved Lys to the unusual amino acid, deoxyhypusine. DHS protein levels were suppressed in transgenic plants by expressing the 3#-untranslated region of tomato DHS under regulation of the constitutive cauliflower mosaic virus promoter. Fruit from the transgenic plants ripened normally, but exhibited delayed postharvest softening and senescence that correlated with suppression of DHS protein levels. Northern-blot analysis indicated that all four gene family members of tomato eIF-5A are expressed in fruit, and that three are up-regulated in parallel with enhancement of DHS mRNA as the fruit begin to senesce and soften. Transgenic plants in which DHS was more strongly suppressed were male sterile, did not produce fruit, and had larger, thicker leaves with enhanced levels of chlorophyll. The activity of PSII was 2 to 3 times higher in these transgenic leaves than in corresponding leaves of wild-type plants, and there was also enhanced deposition of starch in the stems. The data collectively indicate that suppression of DHS has pleiotropic effects on growth and development of tomato. This may, in turn, reflect the fact that there is a single DHS gene in tomato and that its cognate protein is involved in the activation of four distinct isoforms of eIF-5A.

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 immunopurification 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, triacylglycerol 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 microvesiculation from the surface of the plasmalemma. Such removal may be part of normal membrane turnover.

Lipid-protein particles originating from the plasma membrane discernible in the cytosol of carnation petal cells by transmission electron microscopy, and the association of H + -ATPase were immunopurified from the cytosol of carnation petal cells catabolites with a subpopulation of these particles was con-(Dianthus caryophyllus L. cv. Improved White Sim) using firmed by immunogold labelling with H + -ATPase antiserum. antibodies raised against the central hydrophilic domain of the Cross-reaction of the H + -ATPase antiserum with elements of H + -ATPase. The immunopurified particles are enriched in lipid metabolites, in particular free fatty acids and steryl/wax the cytosol was also evident by immunofluorescent light miesters, by comparison with corresponding microsomal mem-croscopy. These observations collectively indicate that lipidbranes, and the lipids of the particles are more saturated than protein particles of plasma membrane origin are present in the cytosol of carnation petal cells and that their formation may those of microsomal membranes. Proteolytic catabolites of the serve as a means of removing lipid and protein metabolites H + -ATPase, a protein associated with the plasma membrane, but not the native H + -ATPase protein, are also present in the from the plasma membrane which would otherwise destabilize immunopurified cytosolic particles. Osmiophilic particles were its structure.about 12 min (Hager et al. 1991). It can be assumed that this degradation is mediated by the action of proteases, and this raises the question of how proteolytic catabolites of the H + -ATPase are removed from the plasma membrane bilayer. Lipid-protein particles enriched in lipid metabolites have been isolated from the cytosol of carnation petals (Hudak and Thompson 1996). Several lines of evidence indicate that these cytosolic particles originate from membranes. In particular, they contain phospholipid and can also be generated in vitro from isolated membranes under conditions in which phospholipid catabolism has been activated (Hudak and Thompson 1996). As well, pulse-chase labelling of intact carnation petals with 14 C-acetate has provided evidence for