IDENTIFICATION, SUBCELLULAR-LOCALIZATION, AND DEVELOPMENTAL STUDIES OF OLEOSINS IN THE ANTHER OF BRASSICA-NAPUS

mRNAs encoding putative oleosins have been detected in the tapetum of developing anthers in Brassica and Arabidopsis, but the authentic prot eins have not been previously documented. Antibodies against a synthet ic 15-residue polypeptide that represents a portion of the putative ta petum oleosins encoded by two cloned Brassica napus genes were raised. Using these antibodies for immunoblotting after SDS-PAGE of the sporo phytic extracts of B. napus developing anthers, two oleosins of simila r to 48 and 45 kDa were detected. These two oleosins were judged to be the putative oleosins encoded by cloned Brassica genes because of the ir identical N-terminal sequences. The two oleosins were present in th e anthers only during the developmental stage when the tapetum cells w ere packed with organelles. A fraction of low-density organelles was i solated from the developing anthers by flotation centrifugation. The f raction contained plastoglobule-filled plastids and lipid-containing p articles. The structures of these two isolated organelles were similar to those in situ in the tapetum cells. Of subcellular fractions of th e anther homogenate, the two oleosins were present exclusively in the low density organelle fraction. They were absent in the surface fracti ons of the developing microspores and the mature pollen, although frag mented oleosin molecules were earlier reported to be present on the po llen. By immunocytochemistry, immunogold particles were found largely on the periphery of the plastoglobuli inside the plastids in the tapet um cells. The antibodies also detected oleosins on the surface of stor age oil bodies inside the maturing microspores. Apparently, the gameto phytic microspore oil-body oleosins share common epitopes at the gener ally non-conserved C-terminal domain with the sporophytic tapetum oleo sins.