Glyoxysomes in cotyledons of cotton (Gossypium hirsutum, L.) seedlings enlarge dramatically within 48 h after seed imbibition (Kunce, C. M., R. N. Trelease, and D. C. Doman. 1984. Planta (Berl.). 161:156-164) to effect mobilization of stored cotton-seed oil. We discovered that the membranes of enlarging glyoxysomes at all stages examined contained a large percentage (36-62% by weight) of nonpolar lipid, nearly all of which were triacylglycerols (TAGs) and TAG metabolites. Free fatty acids comprised the largest percentage of these nonpolar lipids. Six uncommon (and as yet unidentified) fatty acids constituted the majority (51%) of both the free fatty acids and the fatty acids in TAGs of glyoxysome membranes; the same six uncommon fatty acids were < 7% of the acyl constituents in TAGs extracted from cotton-seed storage lipid bodies. TAGs of lipid bodies primarily were composed of palmitic, oleic, and linoleic acids (together 70%). Together, these three major storage fatty acids were < 10% of both the free fatty acids and fatty acids in TAGs of glyoxysome membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) constituted a major portion of glyoxysome membrane phospholipids (together 61% by weight). Pulse-chase radiolabeling experiments in vivo clearly demonstrated that C-14-PC and C-14-PE were synthesized from C-14-choline and C-14-ethanolamine, respectively, in ER of cotyledons, and then transported to mitochondria; however, these lipids were not transported to enlarging glyoxysomes. The lack of ER involvement in glyoxysome membrane phospholipid synthesis, and the similarities in lipid compositions between lipid bodies and membranes of glyoxysomes, led us to formulate and test a new hypothesis whereby lipid bodies serve as the dynamic source of nonpolar lipids and phospholipids for membrane expansion of enlarging glyoxysomes. In a cell-free system, H-3-triolein (TO) and H-3-PC were indeed transferred from lipid bodies to glyoxysomes. H-3-PC, but not H-3-TO, also was transferred to mitochondria in vitro. The amount of lipid transferred increased linearly with respect to time and amount of acceptor organelle protein, and transfer occurred only when lipid body membrane proteins were associated with the donor lipid bodies. H-3-TO was transferred to and incorporated into glyoxysome membranes, and then hydrolyzed to free fatty acids. H-3-PC was transferred to and incorporated into glyoxysome and mitochondria membranes without subsequent hydrolysis. Our data are inconsistent with the hypothesis that ER contributes membrane lipids to glyoxysomes during postgerminative seedling growth. Instead, the data support a novel source for glyoxysome (peroxisome) membrane lipids; lipid bodies, which house storage lipids that are converted to carbohydrate during heterotrophic seedling growth, also provide enlarging glyoxysomes with nonpolar lipids and phospholipids to accommodate membrane expansion. A working model depicting the origin and intracellular trafficking of membrane lipids for enlarging cottonseed glyoxysomes is presented.
