Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway Adam M. Takos 1 , Camilla Knudsen 1 , Daniela Lai 1 , Rubini Kannangara 1 , Lisbeth Mikkelsen 1 , Mohammed S. Motawia 1 , Carl E. Olsen 2 , Shusei Sato 3 , Satoshi Tabata 3 , Kirsten Jørgensen 1 , Birger L. Møller 1 and Fred Rook 1,* 1 Plant Biochemistry Laboratory, Department of Plant Biology and Biotechnology, University of Copenhagen, 1871 Frederiksberg, Denmark, 2 Department of Basic Sciences and Environment, University of Copenhagen, 1871 Frederiksberg, Denmark, and 3 Kazusa DNA Research Insititute, Kisarazu, Japan Received 11 May 2011; revised 20 June 2011; accepted 21 June 2011. * For correspondence (fax +45 35333300; e-mail frro@life.ku.dk). SUMMARY Cyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific b-glucosidases following tissue damage results in the release of hydrogen cyanide. The cyanogenesis deficient1 (cyd1) mutant of Lotus japonicus carries a partial deletion of the CYP79D3 gene, which encodes a cytochrome P450 enzyme that is responsible for the first step in cyanogenic glucoside biosynthesis. The genomic region surrounding CYP79D3 contains genes encoding the CYP736A2 protein and the UDP-glycosyltransferase UGT85K3. In combination with CYP79D3, these genes encode the enzymes that constitute the entire pathway for cyanogenic glucoside biosynthesis. The biosynthetic genes for cyanogenic glucoside biosynthesis are also co-localized in cassava (Manihot esculenta) and sorghum (Sorghum bicolor), but the three gene clusters show no other similarities. Although the individual enzymes encoded by the biosynthetic genes in these three plant species are related, they are not necessarily orthologous. The independent evolution of cyanogenic glucoside biosynthesis in several higher plant lineages by the repeated recruitment of members from similar gene families, such as the CYP79s, is a likely scenario. Keywords: cyanogenic glucosides, gene clustering, cytochrome P450, Lotus japonicus, Manihot esculenta, Sorghum bicolor. INTRODUCTION Plants have evolved a large spectrum of chemical defence compounds, and while some are produced by only a limited number of related species, others, such as cyanogenic glu- cosides, are more widely distributed. Over 60 different cya- nogenic glucosides, amino acid-derived a-hydroxynitrile glucoside compounds, are known from over 2600 plant species ranging from ferns to angiosperms, including a disproportionately large number of crops (Seigler, 1975; Conn, 1980; Jones, 1998). Upon tissue disruption, cyano- genic glucosides are degraded by specific b-glucosidases, resulting in the release of hydrogen cyanide, which provides a defence mechanism against generalist herbivores (Morant et al., 2008). Cyanogenesis is often regarded as an evolu- tionarily ancient plant defence mechanism with a single evolutionary origin implied due to the fact that all presently identified enzymes for the first committed step, the conver- sion of an amino acid into an oxime, are cytochrome P450s belonging to the CYP79 family (for reviews, see Bak et al., 2006; Bjarnholt and Møller, 2008). The first genes encoding enzymes for cyanogenic gluco- side biosynthesis were identified in Sorghum bicolor using biochemical approaches (Koch et al., 1995; Bak et al., 1998a; Jones et al., 1999), which provided the basis for the identi- fication of genes in other plant species by sequence homo- logy. Sorghum produces the aromatic cyanogenic glucoside dhurrin, derived from tyrosine, and its biosynthesis requires three sequential steps involving two cytochrome P450 enzymes and an UDP-glucosyltransferase. The first cyto- chrome P450, CYP79A1, converts tyrosine into an oxime intermediate, which is converted by the second cytochrome ª 2011 The Authors 1 The Plant Journal ª 2011 Blackwell Publishing Ltd The Plant Journal (2011) doi: 10.1111/j.1365-313X.2011.04685.x