Leucine-Derived Cyano Glucosides in Barley

Kirsten Annette Nielsen ^*, Carl Erik Olsen , Katrine Pontoppidan , and Birger Lindberg Møller

Plant Biochemistry Laboratory, Department of Plant Biology (K.A.N., K.P., B.L.M.), Center for Molecular Plant Physiology (K.A.N., C.E.O., K.P., B.L.M.), and Department of Chemistry (C.E.O.), 40 Thorvaldsensvej, Royal Veterinary and Agricultural University, DK--1871 Frederiksberg C, Copenhagen, Denmark

^* Corresponding author; email: kani{at}kvl.dk.

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 ${alpha}$ -, as well as ß- and ${gamma}$ -, 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-[¹⁴C]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 ß-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.

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