Isolation and characterization of neutral-lipid-containing organelles and globuli-filled plastids from Brassica napus  tapetum

Abstract

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.

Tandem oleosin genes in a cluster acquired in Brassicaceae created tapetosomes and conferred additive benefit of pollen vigor

“…Similar findings of T3-ortholog oleosin being more abundant than other individual T-oleosins in tapetosomes and pollen coat were obtained in Brassica napus (13,18) and Brassica rapa (19). The higher amount of T3 oleosin determined visually by SDS/PAGE could be due to its high molecular mass (53 kDa) compared with T5 (10 kDa), T4 (22 kDa), and T6 (15 kDa) oleosins, as well as an 8-kDa portion of these T-oleosins being the conserved nonpolar hairpin motif that was less reactive to Coomassie blue.…”

“…This hairpin, together with the adjacent amphipathic N-and C-terminal motifs, stabilizes the hydrophobic lipid droplet in the cytoplasm. In the tapetum of Brassicaceae, T-oleosins are components of the abundant organelles called the tapetosomes (14,18,19). Each tapetosome contains numerous oleosin-coated alkane lipid droplets associated ionically with many flavonoid-containing, endoplasmic reticulumderived vesicles (14).…”

Disruption of theArabidopsisCGI-58 homologue produces Chanarin–Dorfman-like lipid droplet accumulation in plants

“…In domesticated oilseeds, these stored TAGs represent a major source of calories for human and animal nutrition, an excellent feedstock for diesel fuels, and a reservoir for the deposition of industrially important fatty acids used as chemical feedstocks (3-6). Although not commonly appreciated, TAGs also are synthesized in nonseed tissues (7,8), but their abundance in these tissues is low, in part because of the metabolism of the cell and perhaps as a consequence of the continuous recycling of fatty acids for energy and membrane synthesis. Indeed, vegetative cells can incorporate radiolabeled precursors into TAG (7, 8), they express diacylglycerol acyltransferases [the only enzyme in the "Kennedy pathway" unique to TAG production (9)], and they can accumulate TAGs in β-oxidation mutants (2) and in some floral (10) and fruit (7) tissues.…”

“…Although not commonly appreciated, TAGs also are synthesized in nonseed tissues (7,8), but their abundance in these tissues is low, in part because of the metabolism of the cell and perhaps as a consequence of the continuous recycling of fatty acids for energy and membrane synthesis. Indeed, vegetative cells can incorporate radiolabeled precursors into TAG (7,8), they express diacylglycerol acyltransferases [the only enzyme in the "Kennedy pathway" unique to TAG production (9)], and they can accumulate TAGs in β-oxidation mutants (2) and in some floral (10) and fruit (7) tissues. Thus, although plant vegetative cells appear to have the metabolic machinery to synthesize and accumulate neutral lipids, there are likely underlying regulatory mechanisms in place to minimize this process, none of which are understood.…”

Protein Profiling of Plastoglobules in Chloroplasts and Chromoplasts. A Surprising Site for Differential Accumulation of Metabolic Enzymes

“…The proteome of PGs appears to be composed of more than a dozen proteins, judging from one-dimensional (1-D) SDS-PAGE profiles (Wu et al, 1997;Kessler et al, 1999;Smith et al, 2000). Only one or two proteins belonging to the so-called fibrillin family have been identified in PGs, and these were assigned different names (Pozueta-Romero et al, 1997;Kessler et al, 1999;Vishnevetsky et al, 1999;Langenkamper et al, 2001).…”