Gene duplication in the diversification of secondary metabolism: tandem 2-oxoglutarate-dependent dioxygenases control glucosinolate biosynthesis in Arabidopsis.

Secondary metabolites are a diverse set of plant compounds believed to have numerous functions in plant-environment interactions. The large chemical diversity of secondary metabolites undoubtedly arises from an equally diverse set of enzymes responsible for their biosynthesis. However, little is known about the evolution of enzymes involved in secondary metabolism. We are studying the biosynthesis of glucosinolates, a large group of secondary metabolites, in Arabidopsis to investigate the evolution of enzymes involved in secondary metabolism. Arabidopsis contains natural variations in the presence of methylsulfinylalkyl, alkenyl, and hydroxyalkyl glucosinolates. In this article, we report the identification of genes encoding two 2-oxoglutarate--dependent dioxygenases that are responsible for this variation. These genes, AOP2 and AOP3, which map to the same position on chromosome IV, result from an apparent gene duplication and control the conversion of methylsulfinylalkyl glucosinolate to either the alkenyl or the hydroxyalkyl form. By heterologous expression in Escherichia and the correlation of gene expression patterns to the glucosinolate phenotype, we show that AOP2 catalyzes the conversion of methylsulfinylalkyl glucosinolates to alkenyl glucosinolates. Conversely, AOP3 directs the formation of hydroxyalkyl glucosinolates from methylsulfinylalkyl glucosinolates. No ecotype coexpressed both genes. Furthermore, the absence of functional AOP2 and AOP3 leads to the accumulation of the precursor methylsulfinylalkyl glucosinolates. A third member of this gene family, AOP1, is present in at least two forms and found in all ecotypes examined. However, its catalytic role is still uncertain.

Destabilization of rbcS sense transcripts by antisense RNA.

Steady-state rbcS mRNA levels are drastically reduced in transgenic tobacco plants that express rbcS antisense RNAs. We have found that these reductions are not due to an effect of the antisense RNA at the level of rbcS transcription; rather, the sense mRNAs are more actively degraded in the mutant than wild-type plants. We have examined the kinetics of this turnover process by inhibiting transcription with cordycepin, and have found that rbcS sense mRNA decay is accelerated about five-fold in the antisense plants. This provides direct evidence that antisense RNAs can serve to destabilize sense transcripts in plants.