Cyanogenic Eucalyptus nobilis is polymorphic for both prunasin and specific b-glucosidases Roslyn M. Gleadow*, Anita C. Vecchies, Ian E. Woodrow School of Botany, The University of Melbourne, Victoria, 3010, Australia Received 22 October 2002; received in revised form 2 April 2003 Abstract Cyanogenesis (i.e. the evolution of HCN from damaged plant tissue) requires the presence of two biochemical pathways, one controlling synthesis of the cyanogenic glycoside and the other controlling the production of a specific degradative b-glucosidase. The sole cyanogenic glycoside in Eucalyptus nobilis was identified as prunasin (d-mandelonitrile b-d-glucoside) using HPLC and GC–MS. Seedlings from three populations of E. nobilis were grown under controlled conditions and 38% were found to be acya- nogenic, a proportion far greater than reported for any other cyanogenic eucalypt. A detailed study of the acyanogenic progeny from a single open-pollinated parent found that 23% lacked a cyanogenic b-glucosidase, 32% lacked prunasin and 9% lacked both. Of the remaining seedlings initially identified as acyanogenics, 27% contained either trace amounts of b-glucosidase or prunasin, while 9% contained trace amounts of both. Results support the hypothesis that the two components necessary for cyanogenesis are inherited independently. Trace amounts are likely to result from the presence of non-specific b-glucosidases or the glycosylation of the cyanohydrin intermediate by non-specific UDP glycosyl transferases. # 2003 Elsevier Ltd. All rights reserved. Keywords: Eucalyptus nobilis (Myrtaceae); White gum; Chemical ecology; Chemotaxonomy; Polymorphism; Cyanogenic glycoside; Prunasin; b-glucosidase; Cyanogenesis; Defence 1. Introduction Cyanogenesis is the process by which plants and other living organisms liberate hydrogen cyanide (HCN) upon tissue disruption (Møller and Seigler, 1999). When cya- nogenic plants are chewed by herbivores, an endogen- ous cyanogen (usually a cyanogenic glycoside) is brought into in contact with a b-glucosidase, hydrolys- ing the molecule and ultimately liberating toxic HCN (Møller and Seigler, 1999). Cyanide, a respiratory toxin, can cause acute poisoning, but in cyanogenic plants it functions primarily as a feeding deterrent against gen- eralist herbivores (Gleadow and Woodrow, 2002a). In the important Australian genus Eucalyptus, there is evidence that up to 30 of the 600 species are cyanogenic (Finnemore et al., 1935; Gleadow and Woodrow, 2000b; Goodger et al., 2002; Goodger and Woodrow, 2002; E.E. Conn, personal communication), although only three species have been studied in detail (E. clado- calyx, E. polyantemos and E. yarraensis). Two impor- tant findings emerged from these latter studies. First, prunasin [(R)-mandelonitrile b-d-glucoside] was the only cyanogen found in the three species, and in mature trees it was almost entirely located in the foliage. Second, similar to many other cyanogenic species (e.g. Aikman et al., 1996; Schappert and Shore, 2000), the concentration of cyanogen and the capacity for cyanogenesis was highly variable between individual trees, even within single populations (Gleadow and Woodrow, 2000b; Goodger et al., 2002; Goodger and Woodrow, 2002). What is different between these eucalypts and many other cyanogenic species, however, is the rarity of acyanogenic individuals. This may indicate a difference in the genetic mechanism controlling cyanogenic capacity or, under field conditions, strong selection against acyanogenic individuals, or a combination of both. Here we describe cyanogenesis in Eucalyptus nobilis L. Johnson and K. Hill and show that it is unique amongst cyanogenic eucalypts in having a relatively high inci- 0031-9422/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0031-9422(03)00245-0 Phytochemistry 63 (2003) 699–704 www.elsevier.com/locate/phytochem * Corresponding author. Tel.: +61-3-8344-5545; fax: +61-3-9347- 5460. E-mail address: r.gleadow@unimelb.edu.au (R.M. Gleadow).