Patterns of evolutionary conservation of ascorbic acid-related genes following whole-genome triplication in Brassica rapa

Abstract

Ascorbic acid (AsA) is an important antioxidant in plants and an essential vitamin for humans. Extending the study of AsA-related genes from Arabidopsis thaliana to Brassica rapa could shed light on the evolution of AsA in plants and inform crop breeding. In this study, we conducted whole-genome annotation, molecular-evolution and gene-expression analyses of all known AsA-related genes in B. rapa. The nucleobase-ascorbate transporter (NAT) gene family and AsA l-galactose pathway genes were also compared among plant species. Four important insights gained are that: 1) 102 AsA-related gene were identified in B. rapa and they mainly diverged 12-18 Ma accompanied by the Brassica-specific genome triplication event; 2) during their evolution, these AsA-related genes were preferentially retained, consistent with the gene dosage hypothesis; 3) the putative proteins were highly conserved, but their expression patterns varied; and 4) although the number of AsA-related genes is higher in B. rapa than in A. thaliana, the AsA contents and the numbers of expressed genes in leaves of both species are similar, the genes that are not generally expressed may serve as substitutes during emergencies. In summary, this study provides genome-wide insights into evolutionary history and mechanisms of AsA-related genes following whole-genome triplication in B. rapa.

Keywords: AsA-related genes; Brassica rapa; evolutionary conservation; expression pattern; gene dosage hypothesis; synteny analysis.

Fig. 1.—

Proposed model for AsA biosynthesis and recycling pathways in plants. Four possible pathways produce AsA: the l-galactose (Smirnoff–Wheeler) (I), galacturonate (II), l-gulose (III), and myo-inositol (IV) pathways. Gray lines connect metabolites of substrates to products with the corresponding enzymes (named in boxes), and red lines indicate hypothetical reactions. Arrowheads denote directionality. Known enzymes are highlighted in blue boxes, and the color intensity reflects the corresponding enzyme gene numbers.

Fig. 2.—

AsA-related homologous genes in segmental syntenic regions of the genomes of Brassica rapa and Arabidopsis thaliana. Conserved collinear blocks of genes (blue irregular lines) are shown between the ten B. rapa chromosomes (horizontal axis) and the five A. thaliana chromosomes (vertical axis). Red dots indicate AsA-related homologs in the two species. The colored horizontal lines denote copy number in B. rapa. The A. thaliana AsA genes are shown on their respective chromosomes.

Fig. 3.—

Retention of homologous copies in the syntenic region. (A) Collinear correlations of genes surrounding AsA genes in the Arabidopsis thaliana and Brassica rapa genomes. The B. rapa and A. thaliana chromosomes are colored according to the inferred ancestral chromosomes following an established convention. The lines representing AsA-related genes are red, those for 458 randomly selected genes are yellow, those for 458 core eukaryotic genes in the syntenic region are blue, and those for AsA-neighboring genes are gray. The figure was created using Circos software. (B) Retention of AsA-related genes and of neighboring, randomly selected, and core eukaryotic genes in the syntenic region after genome triplication and fractionation in Brassica rapa. (C) Retention rates of ascorbate transporter genes, AsA biosynthesis genes, AsA recycling pathway genes, and all AsA-related genes together. (D) Retention rates of genes in four possible AsA biosynthesis pathways, the l-galactose, galacturonate, l-gulose, and myo-inositol pathways.

Fig. 4.—

Pairwise comparison of K_s values for AsA-related genes. (A) The distribution of K_s values for AsA-related genes between Arabidopsis thaliana and Brassica rapa. The blue line indicates the divergence time (15 Ma). (B) The distribution of K_s values for AsA-related genes between each of the three B. rapa subgenomes and A. thaliana. (C) The distribution of K_s values for AsA transporter, biosynthetic pathway, and recycling pathway. The blue line indicates the main concentrated area of the K_s value (0.4–0.5).

Fig. 5.—

Copy number variation in the NAT family in eudicots. The phylogenetic tree of NAT genes is shown on the left, and the species tree is shown at the top. The α, β, γ, and salicoid duplications and the Brassica-specific triplication are indicated on the branches of the trees according to the Plant Genome Duplication Database. The NAT-family phylogenetic tree was constructed from protein sequences using maximum likelihood in MEGA5. Numbers are copy numbers of each gene in Brassica rapa (Bra), Arabidopsis thaliana (Ath), Carica papaya (Cpa), Populus trichocarpa (Ptr), Vitis vinifera (Vvi), and Amborella trichopoda (Atr).

Fig. 6.—

Deeply conserved AsA l-galactose pathway genes. l-Galactose pathway genes (rows) are conserved among plant families (columns), as indicated by species represented in Plant Genome Duplication Database. Boxes are highlighted if the enzyme-related genes were identified, and the color intensity reflects gene number according to KEGG.

Fig. 7.—

Characteristics of the AsA recycling pathway genes and their expression patterns in Arabidopsis thaliana and Brassica rapa. The recycling pathway can be represented as a triangular loop. Gray arrows (reactions) connect metabolites of substrates to products via the corresponding enzymes. Maximum likelihood trees of each of four multigene families (APX, AO, DHAR, and MDAR) were built. Multiple sequence alignment of full-length proteins was performed using ClustalW2, and the phylogenetic trees were constructed using the MEGA5.2. Expression levels of these genes were determined in four tissues (root, stem, leaf, and flower).

Fig. 8.—

Expression patterns analysis of all AsA-related genes in Arabidopsis thaliana and Brassica rapa. Expression levels were analyzed in root, stem, leaf, and flower tissues. (A) The A. thaliana expression profiling was analyzed using the AtGenExpress Visualization Tool with mean-normalized values (supplementary table S10, Supplementary Material online). (B) Heat map of RNA-Seq data for Brassica rapa AsA-related genes. Gene expression FPKM values were analyzed. The bar at the bottom of each heat map represents relative expression values (supplementary table S11, Supplementary Material online). (C) Venn diagram showing the numbers of AsA-related genes with similar and different expression patterns in A. thaliana and B. rapa; those gene names are colored red in (A) and (B).

Fig. 9.—

Interaction network of AsA-related genes in Arabidopsis thaliana and Brassica rapa. (A) Specific protein interactions of AsA-related genes in A. thaliana were constructed using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins; http://string-db.org/, last accessed January 8, 2015). (B) The interaction network of AsA-related genes in B. rapa was based on the orthologs in A. thaliana. Ellipses represent AsA-related genes; green indicates genes with high expression levels in leaves, and white indicates those with no or low expression in leaves.