Characterization of anther‐expressed genes encoding a major class of extracellular oleosin‐like proteins in the pollen coat of Brassicaceae^†

Abstract

SummaryA large, heterogeneous, highly expressed gene family encoding oleosin-like proteins is described in the Brassicaceae. Seven related cDNA sequences were isolated from Brassica napus anther mRNA using RACE-PCR and compared with other recently described anther-specific oleosin-like genes from B. napus. The expression patterns of four representative members of this diverse gene family were analyzed by Northern blotting and in situ hybridization. In all cases, the genes were expressed specifically in the tapatum of 3-5 mm B. napus buds, which contained microspores 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 acid motifs or repeated residues. Tryphine (pollen coat) fractions from mature B. napus pollen were found to be particularly enriched in polypeptides of apparent molecular weights 32-38 kDa, plus numerous less abundant 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 microsequencing to be identical to parts of the predicted amino acid sequences of three of the tapetal-expressed oleosin-like genes. This indicates the possibility of posttranslational modification of these proteins resulting in a cleavage of the primary translation products in order to generate the mature tryphine polypeptides. These data implythat a large and diverse group of oleosin-like proteins is synthesized in the tapatum of B. napus anthers and that

Comparisons of pollen coat genes across Brassicaceae species reveal rapid evolution by repeat expansion and diversification

“…Thus, the high levels of change observed in the GRPs are unusual and do not reflect a genomic region with an elevated divergence rate. The most variable feature of these genes is exon 2, which contains abundant overlapping repetitive motifs (Tables 1 and 4); the domain encoded by this exon resides in the pollen coat (16)(17)(18) and likely contacts the stigma.…”

“…Moreover, it was not clear whether these rapid changes were a consequence of a particularly dynamic genomic region or instead were specific to GRP genes. We address these questions, assessing changes in the GRPs relative to their genomic neighbors in near (5-8 MY diverged) and more distant relatives (15)(16)(17)(18)(19)(20). This work provides insight into the molecular genetic mechanisms that drive the evolution of genes required for compatible mating and shows that rapid evolution of the GRPs is driven by their repetitive nature.…”

“…The first exon encodes a lipid-binding oleosin domain, and the second exon encodes a glycine-rich repetitive domain (15). Before the coat is deposited on the pollen surface, the oleosin domain is cleaved, leaving the repetitive domain available for interaction with the stigma (16)(17)(18). Five of the eight A. thaliana GRPs have been detected in the pollen coat (GRP14 and GRP16-GRP19; ref.…”

Pollen and Stigma Structure and Function: The Role of Diversity in Pollination

“…These cells secrete oil droplets and proteins and subsequently degenerate, filling the cavities of the exine with their cytoplasmic contents. Compared with sporopollenin-rich exines, pollen coats are better characterized; they can be extracted and their contents can be purified in sufficient quantity for identification (Doughty et al, 1993;Ross and Murphy, 1996;Ruiter et al, 1997;Murphy and Ross, 1998;Mayfield et al, 2001). The first biochemical isolations were in Liliacea, in which pollen coats were found to be largely lipidic, with carotenoids as major pigments (Heslop-Harrison, 1968).…”

“…In B. oleracea, >100 pollen coat proteins, ranging in size from 5 to 50 kD, have been separated on denaturing gels. These include proteins involved in pollen-stigma adhesion or self-incompatibility responses (Preuss et al, 1993;Ross and Murphy, 1996;Ruiter et al, 1997;Murphy and Ross, 1998). Analysis of higher molecular mass proteins from Arabidopsis yielded a simpler mixture, including lipases, Gly-rich oleosins, calcium binding proteins, and proteins with similarity to the extracellular domains of receptor kinases, although lacking the kinase domain (Mayfield and Preuss, 2000;Mayfield et al, 2001).…”