A gene cluster for secondary metabolism in oat: implications for the evolution of metabolic diversity in plants

Abstract

The evolution of the ability to synthesize specialized metabolites is likely to have been key for survival and diversification of different plant species. Oats (Avena spp.) produce antimicrobial triterpenoids (avenacins) that protect against disease. The oat beta-amyrin synthase gene AsbAS1, which encodes the first committed enzyme in the avenacin biosynthetic pathway, is clearly distinct from other plant beta-amyrin synthases. Here we show that AsbAS1 has arisen by duplication and divergence of a cycloartenol synthase-like gene, and that its properties have been refined since the divergence of oats and wheat. Strikingly, we have also found that AsbAS1 is clustered with other genes required for distinct steps in avenacin biosynthesis in a region of the genome that is not conserved in other cereals. Because the components of this gene cluster are required for at least four clearly distinct enzymatic processes (2,3-oxidosqualene cyclization, beta-amyrin oxidation, glycosylation, and acylation), it is unlikely that the cluster has arisen as a consequence of duplication of a common ancestor. Although clusters of paralogous genes are common in plants (e.g., gene clusters for rRNA and specific disease resistance), reports of clusters of genes that do not share sequence relatedness and whose products contribute to a single selectable function are rare [Gierl, A. & Frey, M. (2001) Planta 213, 493-498]. Taken together, our evidence has important implications for the generation of metabolic diversity in plants.

Fig. 1.

Synthesis of sterols and triterpenoid saponins in oat. Cyclization of 2,3-oxidosqualene to cycloartenol (the committed precursor for sterol biosynthesis) or to β-amyrin (the first committed step in the avenacin biosynthetic pathway) is catalyzed by the oxidosqualene cyclase enzymes cycloartenol synthase and β-amyrin synthase, respectively. The biochemical defects of characterized sad mutants are indicated.

Fig. 2.

Mapping of the β-amyrin synthase gene AsbAS1. AsbAS1 maps to linkage group D of a diploid oat map derived from A. strigosa CI3815 × A. wiestii CI1994 (12). This region corresponds to linkage group AswC in a second map constructed using the same parents (13). The A. atlantica × A. hirtula RFLP map was constructed by Van Deynze et al. (14). The rice chromosome 6 map was derived from the Rice Genome Research Program web site (http://rgp.dna.affrc.go.jp).

Fig. 3.

Sequences closely related to AsbAS1 are conserved in Avena species but not in other cereals. Southern blot analysis of XbaI-digested genomic DNA from Avena spp. and other cereals using AsbAS1(Left) and AsCS1 (Right) cDNA probes. The same high-stringency conditions were used for both blots.

Fig. 4.

AsbAS1 has arisen from a cycloartenol synthase-like gene. (A) Phylogenetic analysis of the coding sequences of AsbAS1and other members of the oxidosqualene cyclase superfamily from plants (see Table 2, which is published as supporting information on the PNAS web site, for further details and GenBank accession nos.). Sequences were analyzed by using the dnadist-fitch program of the phylip package (Version 6.2a) (http://evolution.genetics.washington.edu/phylip.html). The numbers indicate the numbers of bootstrap replications (of 500) in which the given branching was observed. (B) Exon–intron structures of AsbAS1and of cycloartenol synthase genes from A. strigosa S75 (AsCS1), rice (OsCS1), and A. thaliana (AtCS1). Exons are indicated by boxes; the numbers above the boxes are the exon sizes. Exons that differ in size are indicated in green for AtCS1 and in red for AsbAS1.(C) Northern blot analysis of RNA from roots of A. strigosa (GenBank accession no. S75) and T. aestivum. (Chinese Spring). The cDNA probes were AsbAS1(Top) and TaOSC1 (Middle). R, root; S, stem; L, leaf; F, flower. Twenty micrograms of RNA was loaded per lane. RNA levels were monitored with methylene blue (MB) dye (Bottom).