The monolayer tapetum cells of the maturing f lowers of Brassica napus contain abundant subcellular globuli-filled plastids and special lipid particles, both enriched with lipids that are supposed to be discharged and deposited onto the surface of adjacent maturing pollen. We separated the two organelles by f lotation density gradient centrifugation and identified them by electron microscopy. The globuli-filled plastids had a morphology similar to those described in other plant species and tissues. They had an equilibrium density of 1.02 g͞cm 3 and contained neutral esters and unique polypeptides. The lipid particles contained patches of osmiophilic materials situated among densely packed vesicles and did not have an enclosing membrane. They exhibited osmotic properties, presumably exerted by the individual vesicles. They had an equilibrium density of 1.05 g͞cm 3 and possessed triacylglycerols and unique polypeptides. Several of these polypeptides were identified, by their N-terminal sequences or antibody cross-reactivity, as oleosins, proteins known to be associated with seed storage oil bodies. The morphological and biochemical characteristics of the lipid particles indicate that they are novel organelles in eukaryotes that have not been previously isolated and studied. After lysis of the tapetum cells at a late stage of f loral development, only the major plastid neutral ester was recovered, whereas the other abundant lipids and proteins of the two tapetum organelles were present in fragmented forms or absent on the pollen surface.

Full-length cDNA clones encoding deoxyhypusine synthase (DHS) and eucaryotic initiation factor 5A (eIF-5A) have been isolated from a cDNA expression library prepared from tomato leaves (Lycopersicon esculentum, cv. Match) exposed to environmental stress. DHS mediates the first of two enzymatic reactions that activate eIF-5A by converting a conserved lysine to the unusual amino acid, deoxyhypusine. Recombinant protein obtained by expressing tomato DHS cDNA in Escherichia coli proved capable of carrying out the deoxyhypusine synthase reaction in vitro in the presence of eIF-5A. Of particular interest is the finding that DHS mRNA and eIF-5A mRNA show a parallel increase in abundance in senescing tomato flowers, senescing tomato fruit, and environmentally stressed tomato leaves exhibiting programmed cell death. Western blot analyses indicated that DHS protein also increases at the onset of senescence. It is apparent from previous studies with yeast and mammalian cells that hypusine-modified eIF-5A facilitates the translation of a subset of mRNAs mediating cell division. The present study provides evidence for senescenceinduced DHS and eIF-5A in tomato tissues that may facilitate the translation of mRNA species required for programmed cell death.

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M embrane deterioration is an early and characteristic feature of plant senescence engendering increased permeability, loss of ionic gradients, and decreased function of key membrane proteins such as ion pumps (1). One of the clearest manifestations of this is the onset of membrane leakiness measurable as increased conductivity of diffusates from intact tissue. This is detectable in carnation petals, for example, well before petal inrolling, the first morphological manifestation of senescence in this tissue, and also before the climacteric-like rise in ethylene production (2). The decline in membrane structural integrity at the onset of senescence appears to be largely attributable to accelerated metabolism of membrane lipids and ensuing change in the molecular organization of the bilayer. Indeed, loss of membrane phospholipid is one of the best documented indices of membrane lipid metabolism during senescence and has been demonstrated for senescing flower petals, leaves, cotyledons and ripening fruit (3, 4).The selective depletion of phospholipid fatty acids from the membranes of senescing tissues results in an increase in the sterol:fatty acid ratio in the bilayer and a consequent decrease in bulk lipid fluidity. This has been demonstrated by fluorescence depolarization and electron spin resonance for microsomal membranes from senescing cotyledons, flowers, leaves, and ripening fruit (5-8) and for plasmalemma of ripening fruit and senescing flowers (5). The decrease in lipid fluidity is engendered by an enrichment of free sterols relative to fatty acids in the bilayer as fatty acids are cleaved from the membrane lipids and selectively removed, reflecting the fact that free sterols are known to restrict the mobility of phospholipid fatty acids (9). As well, in some senescing tissues, the decrease in bulk lipid fluidity appears to be caused in part by a selective depletion of polyunsaturated fatty acids from membranes and an ensuing increase in the saturated-to-unsaturated fatty acid ratio (6). There are also reports that the large changes in bulk membrane lipid fluidity accompanying senescence may alter the conformation of membrane proteins, rendering them prone to proteolysis (10, 11).Recent data suggest that free fatty acids arising from the metabolism of membrane lipids may be removed from the bilayer by blebbing of lipid particles highly enriched in free fatty acids from the membrane surface into the cytosol (12-14). These lipid particles appear to be structurally analogous to oil bodies. Indeed, there is growing evidence that the free fatty acids released from senescing membranes are metabolized by glyoxylate cycle enzymes also induced at the onset of senescence (15). However, it is also clear that free fatty acids accumulate in senescing membranes and induce lipid-phase separations. The resulting mixture of liquid-crystalline and gel phase lipid domains in the bilayer contributes to the leakiness of senescing membranes because of packing imperfections at the phase boundaries (16).Deesterification of membr...

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Full-length cDNA corresponding to Arabidopsis (Arabidopsis thaliana) gene At2g31690, which has been annotated in GenBank as a putative triacylglycerol (TAG) lipase, was obtained by reverse transcription-polymerase chain reaction using RNA from senescing rosette leaves of Arabidopsis as a template. The cognate protein was found to contain the lipase active site sequence, and corresponding recombinant protein proved capable of deesterifying TAG. In vitro chloroplast import assays indicated that the lipase is targeted to chloroplasts. This was confirmed by confocal microscopy of rosette leaf tissue treated with fluorescein isocyanate-labeled, lipase-specific antibody, which revealed that lipase protein colocalizes with plastoglobular neutral lipids. Western-blot analysis indicated that the lipase is expressed in roots, inflorescence stems, flowers, siliques, and leaves and that it is strongly up-regulated in senescing rosette leaf tissue. Transgenic plants with suppressed lipase protein levels were obtained by expressing At2g31690 cDNA in antisense orientation under the regulation of a constitutive promoter. Transgenic plants bolted and flowered at the same time as wild-type plants, but were severely stunted and exhibited delayed rosette senescence. Moreover, the stunted growth phenotype correlated with irregular chloroplast morphology. The chloroplasts of transgenic plants were structurally deformed, had reduced abundance of thylakoids that were abnormally stacked, and contained more plastoglobular neutral lipids than chloroplasts of wild-type plants. These observations collectively indicate that this TAG lipase plays a role in maintaining the structural integrity of chloroplasts, possibly by mobilizing the fatty acids of plastoglobular TAG.

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A full-length cDNA clone encoding deoxyhypusine synthase (DHS) was isolated from a cDNA expression library prepared from senescing leaves of Arabidopsis thaliana. Southern blot analysis indicated that DHS is encoded by a single-copy gene in Arabidopsis. During leaf development, the abundance of DHS mRNA in the third pair of rosette leaves peaked at days 14 and 35 after emergence coincident with the initiation of bolting and the later stages of leaf senescence, respectively. These changes in DHS expression were paralleled by corresponding changes in transcript abundance for eIF-5A1, one of three isoforms of eIF-5A in Arabidopsis. Levels of DHS transcript also increased in detached leaves coincident with post-harvest senescence. DHS was suppressed in transgenic plants by introducing antisense full-length or 3'-untranslated Arabidopsis DHS cDNA under the regulation of the constitutive cauliflower mosaic virus (CaMV-35S) promoter. Plants expressing the antisense transgenes had reduced levels of leaf DHS protein and, depending on the level of DHS suppression, exhibited delayed natural leaf senescence, delayed bolting, increased rosette leaf and root biomass, and enhanced seed yield. Suppression of DHS also delayed premature leaf senescence induced by drought stress resulting in enhanced survival in comparison with wild-type plants. In addition, detached leaves from DHS-suppressed plants exhibited delayed post-harvest senescence. These pleiotropic effects of DHS suppression indicate that the protein plays a central role in plant development and senescence.