Many plants produce cyanogenic glucosides as part of their chemical defense. They are α-hydroxynitrile glucosides, which release toxic hydrogen cyanide (HCN) upon cleavage by endogenous plant β-glucosidases. In addition to cyanogenic glucosides, several plant species produce β- and γ-hydroxynitrile glucosides. These do not release HCN upon hydrolysis by β-glucosidases and little is known about their biosynthesis and biological significance. We have isolated three β-hydroxynitrile glucosides, namely (2Z)-2-(β-d-glucopyranosyloxy)but-2-enenitrile and (2R,3R)- and (2R,3S)-2-methyl-3-(β-d-glucopyranosyloxy)butanenitrile, from leaves of Ribes uva-crispa. These compounds have not been identified previously. We show that in several species of the genera Ribes, Rhodiola and Lotus, these β-hydroxynitrile glucosides co-occur with the l-isoleucine-derived hydroxynitrile glucosides, lotaustralin (α-hydroxynitrile glucoside), rhodiocyanosides A (γ-hydroxynitrile glucoside) and D (β-hydroxynitrile glucoside) and in some cases with sarmentosin (a hydroxylated rhodiocyanoside A). Radiolabelling experiments demonstrated that the hydroxynitrile glucosides in R. uva-crispa and Hordeum vulgare are derived from l-isoleucine and l-leucine, respectively. Metabolite profiling of the natural variation in the content of cyanogenic glucosides and β- and γ-hydroxynitrile glucosides in wild accessions of Lotus japonicus in combination with genetic crosses and analyses of the metabolite profile of the F2 population provided evidence that a single recessive genetic trait is most likely responsible for the presence or absence of β- and γ-hydroxynitrile glucosides in L. japonicus. Our findings strongly support the notion that the β- and γ-hydroxynitrile glucosides are produced by diversification of the cyanogenic glucoside biosynthetic pathway at the level of the nitrile intermediate. ; Nanna Bjarnholt, Fred Rook, Mohammed Saddik Motawia, Claus Cornett, Charlotte Jørgensen, Carl Erik Olsen, Jerzy W. Jaroszewski, Søren Bak and Birger Lindberg Møller ; © 2008 Published by Elsevier