Barley (Hordeum vulgare) seedlings contain five cyano glucosides derived from the amino acid l-leucine (Leu). The chemical structure and the relative abundance of the cyano glucosides were investigated by liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses using spring barley cultivars with high, medium, and low cyanide potential. The barley cultivars showed a 10-fold difference in their cyano glucoside content, but the relative content of the individual cyano glucosides remained constant. Epiheterodendrin, the only cyanogenic glucoside present, comprised 12% to 18% of the total content of cyano glucosides. It is proposed that the aglycones of all five cyano glucosides are formed by the initial action of a cytochrome P450 enzyme of the CYP79 family converting l-Leu into Z-3-methylbutanal oxime and subsequent action of a less specific CYP71E enzyme converting the oxime into 3-methylbutyro nitrile and mediating subsequent hydroxylations at the ␣-, as well as -and ␥-, carbon atoms. Presence of cyano glucosides in the barley seedlings was restricted to leaf tissue, with 99% confined to the epidermis cell layers of the leaf blade. Microsomal preparations from epidermal cells were not able to convert l-[14 C]Leu into the biosynthetic intermediate, Z-3-methylbutanal-oxime. This was only achieved using microsomal preparations from other cell types in the basal leaf segment, demonstrating translocation of the cyano glucosides to the epidermal cell layers after biosynthesis. A -glucosidase able to degrade epiheterodendrin was detected exclusively in yet a third compartment, the endosperm of the germinating seed. Therefore, in barley, a putative function of cyano glucosides in plant defense is not linked to cyanide release.Cyanogenesis, i.e. the ability of living cells to release cyanide under certain biotic and/or abiotic stress conditions, is an old trait (Lechtenberg and Nahrstedt, 1999). In most cases, cyanide release reflects cleavage of a cyanogenic glucoside into the corresponding cyanohydrin and Glc by the initial action of a -glucosidase. Subsequently, the cyanohydrin is cleaved into a ketone or aldehyde and hydrogen cyanide, either catalyzed by an ␣-hydroxy nitrilase or nonenzymatically. Cyanogenic glucosides have been found in over 3,000 plant species and may exert a role in plant defense reactions (Tattersall et al., 2001).Cyanogenic glucosides are derived from the amino acids l-Val, l-Ile, l-Leu, l-Phe, or l-Tyr and the nonprotein amino acid cyclopentenyl-Gly. The biosynthetic pathway is initiated by conversion of the amino acid into an aldoxime by a multifunctional P450 monooxygenase belonging to the CYP79 family. A second P450 monooxygenase belonging to the CYP71E family converts the oxime into a cyanohydrin with a nitrile as an intermediate. The cyanohydrin is finally glucosylated to produce the cyanogenic glucoside by a UDP-Glc-glucosyl transferase (for review, see Jones et al., 2000). In sorghum (Sorghum bicolor), cyanogenic glucoside synthesis proceeds in the etiolated se...
