Pergamon 0031-9422(94)00878-7 Phytochemistry, Vol. 39, No. 2, pp. 323-326, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/95 $9.50 + 0.00 THE BIOSYNTHESIS OF CYANOGENIC GLUCOSIDES IN ROOTS OF CASSAVA LIANGCHENGDU, MPOKO BOKANGA,* BIRGER LINDBERGMOLLER and BARBARA ANN HALKIERt Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark; *International Institute of Tropical Agriculture, PMB 5320 Oyo Road, Ibadan, Nigeria (Received in revised form 21 September 1994) Key Word Index--Manihot esculenta; Euphorbiaceae; cassava root; cyanogenic glucosides; biosynth- esis. Abstraet--Linamarin is the main cyanogenic glucoside of cassava. De novo synthesis of linamarin in cassava roots was demonstrated in vivo by feeding [14C]valine to excited segments of phelloderm. In vitro, a microsomal enzyme system isolated from cassava roots was shown to catalyse the conversion of valine to acetone cyanohydrin, the aglucone of linamarin. An antibody raised against cytochrome P450TW, the enzyme which catalyses the initial step in the biosynthesis of the cyanogenic glucoside dhurrin in sorghum, cross-reacts with a major polypeptide of similar molecular mass in cassava microsomes. Cyanogenic glucosides are known to accumulate in cassava roots, but hitherto de novo synthesis has only been demonstrated in the leaves, suggesting translocation of cyanogenic glucosides from leaves to roots. Our results show that at least part of the cyanogenic glucosides are synthesized in the roots. The data demonstrate that acyanogenic cassava roots cannot be obtained solely by blocking the transport of cyanogenic glucosides to the roots from other parts of the cassava plant. INTRODUCTION Cyanogenic glucosides are amino acid-derived plant con- stituents present in more than 2500 plant species. Cas- sava contains two major cyanogenic glucosides linamarin (2-fl-o-glucopyranosyloxy-2-methylpropiono- nitrile) and lotaustralin [.(2R)-2-fl-D-glucopyranosyloxy- 2-methylbutyronitrile] derived from valine and isoleucine, respectively. The ratio of linamarin and lotaustralin in leaves and roots is about 93 : 7. Cassava is an important crop in tropical and subtropical regions where its large tuberous and starchy roots form the major staple food for over 300 million people [1]. In cassava, cyanogenic glucosides are known to accu- mulate in all parts of the plant, but de novo synthesis of cyanogenic glucosides has only been demonstrated in vivo in the primary leaves of young seedlings [1, 2]. In Hevea brasiliensis, the translocation of cyanogenic gluco- sides are suggested to proceed by the 'linustatin pathway' [3, 4]. According to this pathway linamarin and lotaus- tralin are initially glucosylated to produce the digluco- sides linustatin and neolinustatin which then serve as transient forms resistant to the hydrolysis by fl- glucosidases present in apoplastic spaces l-3, 4]. Based on isolation of trace amounts of linustatin and neolinustatin from cassava seedlings, Lykkesfeldt and Moiler have fAuthor to whom correspondence should be addressed. suggested that the linustatin pathway is also involved in the transiocation of cyanogenic glucosides from the leaves to the roots in cassava plant [2]. In vitro biosynthesis of cyanogenic glucosides in cas- sava has been demonstrated by Koch et al. using a micro- somal enzyme system isolated from etiolated cassava seedlings [5]. This microsomal enzyme system catalyses the conversion of valine and isoleucine to the corres- ponding cyanohydrins which dissociate into the corres- ponding aldehydes or ketones and HCN, the end prod- ucts obtained in vitro. In vivo, the cyanohydrins are glucosylated into linamarin and lotaustralin by a soluble UDP-glucose:glucosyltransferase I-1]. The involvement of cytochrome P450 in biosynthesis of cyanogenic gluco- sides has been demonstrated in the microsomal enzyme systems isolated from etiolated seedlings of sorghum and cassava [5, 6]. In the present paper, we demonstrate de novo synthesis of linamarin in cassava roots both in vivo and in vitro. Our results show that at least part of the cyanogenic glucosides are synthesized in the roots. RESULTS AND DISCUSSION Cassava roots consist of three distinct regions: the phelloderm which is the I-4 mm thick readily removable peel, the cortex which consists of parenchyma cells and constitutes the region of carbohydrate storage, and the inner core which is the central vascular cylinder [I]. In 323