Tissue-specific expression of HCN and its metabolic precursors in <i>Trifolium repens</i>

Cyanogenesis, the release of hydrogen cyanide (HCN), is governed by two loci, Ac and Li, in white clover ( Trifolium repens L.). Ac encodes a three gene region that regulates cyanogenic glucoside production, while Li encodes linamarase that hydrolyzes cyanogenic glucosides to produce HCN. We examined Ac and Li expression in leaf, floral, and root tissues of cyanogenic and acyanogenic plants, complemented by gene expression analysis, to understand tissue-specific expression of HCN metabolism. Ac metabolic expression was present in leaves, absent in roots, and rare in flowers. Li metabolic expression in leaves perfectly correlated with that in flowers but was present in roots regardless of linamarase presence in leaves, suggesting spatial enzyme expression variation due to a homologous gene copy. HCN expression in flowers was infrequently expressed at low concentrations in plants with functional Ac and Li. Gene expression analysis confirmed some tissue-specific expression patterns and also suggested more complex molecular regulation than previously described. This study provides the first detailed examination of cyanogenesis tissue specificity in white clover, a plant of agriculture significance. The varied expression of cyanogenic secondary metabolites across tissues indicates the need for research into how white clover regulates its defenses to conserve resources.