CHARACTERIZATION OF ANTHER-EXPRESSED GENES ENCODING A MAJOR CLASS OF EXTRACELLULAR OLEOSIN-LIKE PROTEINS IN THE POLLEN COAT OF BRASSICACEAE

A large, heterogeneous, highly expressed gene family encoding oleosin- like proteins is described in the Brassicaceae. Seven related cDNA seq uences were isolated from Brassica napus anther mRNA using RACE-PCR an d compared with other recently described anther-specific oleosin-like genes from B. napus. The expression patterns of four representative me mbers of this diverse gene family were analyzed by Northern blotting a nd in site hybridization. In all cases, the genes were expressed speci fically in the tapetum of 3-5 mm B. napus buds, which contained micros pores at the late-vacuolate and bicellular stages of development. The predicted protein products are ordered into subclasses, each of which has a characteristic C-terminal domain, containing different amino aci d motifs or repeated residues. Tryphine (pollen coat) fractions from m ature B. napus pollen were found to be particularly enriched in polype ptides of apparent molecular weights 32-38 kDa, plus numerous less abu ndant polypeptides of less than 15 kDa. The N-terminal 15-20 residues of three of these polypeptides (12, 32 and 38 kDa) were found by micro sequencing to be identical to parts of the predicted amino acid sequen ces of three of the tapetal-expressed oleosin-like genes. This indicat es the possibility of posttranslational modification of these proteins resulting ire a cleavage of the primary translation products in order to generate the mature tryphine polypeptides. These data imply that a large and diverse group of oleosin-like proteins is synthesized in th e tapetum of B. napus anthers and that following tapetal degradation, these proteins, possibly in modified form, then relocate to the develo ping microspores where they eventually constitute some of the major co mponents of the extracellular tryphine of mature pollen grains. These proteins share a conserved 70 amino acid residue hydrophobic domain an d are related structurally to the seed-specific intracellular oleosins , although their biological function may be different.