title: Three genes encoding AOP2, a protein involved in aliphatic glucosinolate biosynthesis, are differentially expressed in Brassica rapa.
abstract: The glucosinolate biosynthetic gene AOP2 encodes an enzyme that plays a crucial role in catalysing the conversion of beneficial glucosinolates into anti-nutritional ones. In Brassica rapa, three copies of BrAOP2 have been identified, but their function in establishing the glucosinolate content of B. rapa is poorly understood. Here, we used phylogenetic and gene structure analyses to show that BrAOP2 proteins have evolved via a duplication process retaining two highly conserved domains at the N-terminal and C-terminal regions, while the middle part has experienced structural divergence. Heterologous expression and in vitro enzyme assays and Arabidopsis mutant complementation studies showed that all three BrAOP2 genes encode functional BrAOP2 proteins that convert the precursor methylsulfinyl alkyl glucosinolate to the alkenyl form. Site-directed mutagenesis showed that His356, Asp310, and Arg376 residues are required for the catalytic activity of one of the BrAOP2 proteins (BrAOP2.1). Promoter-β-glucuronidase lines revealed that the BrAOP2.3 gene displayed an overlapping but distinct tissue- and cell-specific expression profile compared with that of the BrAOP2.1 and BrAOP2.2 genes. Quantitative real-time reverse transcription-PCR assays demonstrated that BrAOP2.1 showed a slightly different pattern of expression in below-ground tissue at the seedling stage and in the silique at the reproductive stage compared with BrAOP2.2 and BrAOP2.3 genes in B. rapa. Taken together, our results revealed that all three BrAOP2 paralogues are active in B. rapa but have functionally diverged.
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Zhongguancun Nandajie No. 12, Haidian District, Beijing 100081, PR China.
authors.forename: Jifang
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Zhongguancun Nandajie No. 12, Haidian District, Beijing 100081, PR China.
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Zhongguancun Nandajie No. 12, Haidian District, Beijing 100081, PR China.
authors.forename: Jianli
authors.initials: J
authors.lastname: Liang
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Zhongguancun Nandajie No. 12, Haidian District, Beijing 100081, PR China.
authors.forename: Jian
authors.initials: J
authors.lastname: Wu
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Zhongguancun Nandajie No. 12, Haidian District, Beijing 100081, PR China.
authors.forename: Feng
authors.initials: F
authors.lastname: Cheng
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Zhongguancun Nandajie No. 12, Haidian District, Beijing 100081, PR China wangxiaowu@caas.cn.
authors.forename: Xiaowu
authors.initials: X
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029741:Brassica rapa / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011123:Polyploidy
chemical_list: D010940:Plant Proteins
keywords: BrAOP2; Brassica rapa; expression pattern; functional divergence; glucosinolates; paralogues.