Constituents of the tapetosomes and elaioplasts inBrassica campestristapetum and their degradation and retention during microsporogenesis

Abstract

SummaryIn Brassica anthers during microsporogenesis, the tapetum cells contain two abundant lipid-rich organelles, the tapetosomes possessing oleosins and triacylglycerols (TAGs), and the elaioplasts having unique polypeptides and neutral esters. B. campestris, for its simplicity of possessing only the AA genome and one predominant oleosin of 45 kDa, was studied. In the developing anthers, the lipids and proteins of the tapetosomes and elaioplasts were concomitantly accumulated but selectively degraded or retained. Upon incubation of isolated tapetosomes in a pH-5 medium, the predominant 45 kDa oleosin underwent selective enzymatic proteolysis to a 37 kDa fragment, which was not further hydrolyzed upon prolonged incubation. The unreacted 45 kDa oleosin was retained in the organelles, whereas the 37 kDa fragment was released to the exterior. The fragment would become the predominant 37 kDa polypeptide in the pollen coat. Isolated tapetosomes did not undergo hydrolysis of the TAGs upon incubation in media of diverse pHs. An alkaline lipase in the soluble fraction of the anther extract was presumed to be the enzyme that would hydrolyze the tapetosome TAGs, which disappeared in the anthers during development. The tapetum elaioplasts contained several unique polypeptides of 31-36 kDa. The gene encoding a 32 kDa polypeptide was cloned, and its deduced amino acid sequence was homologous to those of two proteins known to be present on the surface of fibrils in chromoplasts. Upon incubation of isolated elaioplasts in media of diverse pHs, the organelle polypeptides were degraded completely and most rapidly at pH 5, whereas the neutral esters remained unchanged; these neutral esters would become the major lipid components of the pollen coat. The findings show that the constituents of the two major tapetum organelles underwent very different paths of degradation, or modification, and transfer to 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).…”

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.…”

“…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.…”

“…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.…”