TRANSPARENT TESTA 12 genes from Brassica napus and parental species: cloning, evolution, and differential involvement in yellow seed trait.

ABSTRACT

Molecular dissection of the Brassica yellow seed trait has been the subject of intense investigation. Arabidopsis thaliana TRANSPARENT TESTA 12 (AtTT12) encodes a multidrug and toxic compound extrusion (MATE) transporter involved in seed coat pigmentation. Two, one, and one full-length TT12 genes were isolated from B. napus, B. oleracea, and B. rapa, respectively, and Southern hybridization confirmed these gene numbers, implying loss of some of the triplicated TT12 genes in Brassica. BnTT12-1, BnTT12-2, BoTT12, and BrTT12 are 2,714, 3,062, 4,760, and 2,716 bp, with the longest mRNAs of 1,749, 1,711, 1,739, and 1,752 bp, respectively. All genes contained alternative transcriptional start and polyadenylation sites. BrTT12 and BoTT12 are the progenitors of BnTT12-1 and BnTT12-2, respectively, validating B. napus as an amphidiploid. All Brassica TT12 proteins displayed high levels of identity (>99%) to each other and to AtTT12 (>92%). Brassica TT12 genes resembled AtTT12 in such basic features as MatE/NorM CDs, subcellular localization, transmembrane helices, and phosphorylation sites. Plant TT12 orthologs differ from other MATE proteins by two specific motifs. Like AtTT12, all Brassica TT12 genes are most highly expressed in developing seeds. However, a range of organ specificity was observed with BnTT12 genes being less organ-specific. TT12 expression is absent in B. rapa yellow-seeded line 06K124, but not downregulated in B. oleracea yellow-seeded line 06K165. In B. napus yellow-seeded line L2, BnTT12-2 expression is absent, whereas BnTT12-1 is expressed normally. Among Brassica species, TT12 genes are differentially related to the yellow seed trait. The molecular basis for the yellow seed trait, in Brassica, and the theoretical and practical implications of the highly variable intron 1 of these TT12 genes are discussed.