Positive selection driving diversification in plant secondary metabolism

Abstract

In Arabidopsis thaliana and related plants, glucosinolates are a major component in the blend of secondary metabolites and contribute to resistance against herbivorous insects. Methylthioalkylmalate synthases (MAM) encoded at the MAM gene cluster control an early step in the biosynthesis of glucosinolates and, therefore, are central to the diversification of glucosinolate metabolism. We sequenced bacterial artificial chromosomes containing the MAM cluster from several Arabidopsis relatives, conducted enzyme assays with heterologously expressed MAM genes, and analyzed MAM nucleotide variation patterns. Our results show that gene duplication, neofunctionalization, and positive selection provide the mechanism for biochemical adaptation in plant defense. These processes occur repeatedly in the history of the MAM gene family, indicating their fundamental importance for the evolution of plant metabolic diversity both within and among species.

Fig. 1.

The MAM region in A. thaliana and relatives. The diverse configuration of the MAM cluster in different A. thaliana accessions (4) is represented by Col-0, Ler-0, and Sorbo. Sorbo exemplifies the archetypical configuration of the locus in A. thaliana, consisting of a MAM2, a MAM1, and a MAM-L gene. Note that the 5′ portion of MAM1 is deleted in Ler-0 (4). Numbers refer to the Arabidopsis Information Resource identifiers (www.arabidopsis.org) for chromosome 5 of the A. thaliana genome. Arrows (>, <) indicate transcription direction, p indicates genes only partially present on the respective bacterial artificial chromosome, and ψ refers to putative pseudogenes.

Fig. 2.

Phylogenetic relationships between MAM genes, according to the neighbor-joining method. The significance of the branching order was tested by bootstrapping (2,000 replicates). Isopropylmalate synthase genes from A. thaliana (At1g18500 and At1g74040; ref. 13) and rice (GenBank accession no. AK066890) were used as references. Note that branches indicated by an asterisk were shortened to half of its original lengths. Bold lines indicate branches with evidence for positive selection in maximum likelihood analyses.

Fig. 3.

Gene duplication and speciation. Based on parsimony, we partitioned nonsynonymous (N) and synonymous changes (S) along branches of a simplified phylogeny connecting A. lyrata MAMa, A. thaliana Sorbo and Col-0 MAM1, and Sorbo and Ler-0 MAM2. We excluded two synonymous sites with complex substitution patterns from the analyses. Ratios of nonsynonymous versus synonymous changes are heterogeneous across the tree (χ² = 10.76; df = 2; P = 0.0046) because of an excess of replacement changes on branch b.

Fig. 4.

Amino acid polymorphisms between MAM synthases. Shown are polymorphic amino acids from position 82 onward, i.e., signal peptides are excluded. Amino acids shared between A. lyrata MAMa and other MAM synthases are shaded gray. Residues unique to MAM1 are boxed. Small letters indicate positively selected sites in MAM1, with P > 0.95 (a), or P > 0.80 (b). Arrows indicate the region swapped between MAM1 and MAM2 expression constructs to delimit residues that determine enzyme substrate specificity.