The biosynthetic genes for cyanogenic glucosides in Phaseolus lunatus reveal evolutionary dynamics in oxime-based defences in the Phaseolus genus. Cyanogenesis in Phaseolus lunatus

Lima bean, Phaseolus lunatus, is a crop legume that produces the cyanogenic glucosides linamarin and lotaustralin. In the legumes Lotus japonicus and Trifolium repens, the biosynthesis of these two a-hydroxynitrile glucosides involves cytochrome P450s enzymes of the CYP79 and CYP736 families and a UDP-glucosyltransferase. Here we identify CYP79D71 as the first enzyme of the pathway in P. lunatus, producing oximes from valine and isoleucine. Organisation of the biosynthetic genes in a gene cluster aided their identification in L. japonicus. In the available genome sequence of P. vulgaris, the gene orthologous to CYP79D71 is adjacent to a member of the CYP83 family. Although P. vulgaris is not cyanogenic, it does produce oximes and hydroxynitriles as volatile defence compounds. We cloned the genes encoding two CYP83s (CYP83E46 and CYP83E47) and a UDP-glucosyltransferase from P. lunatus (UGT85K31), and these completed the biosynthetic pathway for linamarin and lotaustralin in lima bean. Within the genus Phaseolus, the occurrence of linamarin and lotaustralin as functional chemical defence compounds appears restricted to species belonging to the closely related Polystachios and Lunatus groups. A pre-existing ability to produce volatile oximes and hydroxynitriles likely facilitated evolution of cyanogenesis within the Phaseolus genus.