Cinnamyl alcohol dehydrogenase (CAD) underlies the orange lemma trait and lignin reduction in barley

Barley (Hordeum vulgare L.) is one of the world’s major cereal crops and is widely used for feed, brewing, and food. Reducing lignin content in barley grain and straw is beneficial for improving forage digestibility and enhancing the efficiency of bioethanol production. The orange lemma mutation—characterized by orange pigmentation of husks and nodes—has long been associated with altered lignin metabolism; however, its genetic basis has remained unclear. We show that Cinnamyl Alcohol Dehydrogenase (CAD) is the causal gene underlying the orange lemma phenotype in barley. Genetic mapping using a ‘Haruna Nijo’ × ‘L053’ F₂ population and the Barley 50 K SNP array defined a candidate region on chromosome 6H containing the CAD gene. Sequence analysis revealed that all orange lemma mutants, including both naturally occurring and EMS-induced lines, carried nucleotide changes in the CAD gene, resulting in amino acid substitutions or premature stop codons. Furthermore, CRISPR/Cas9-mediated knockout of CAD in the cultivar ‘Golden Promise’ reproduced the orange lemma phenotype, providing functional validation. The knockout plants also showed significantly lower lignin content than wild-type plants. These findings resolve a long-standing question regarding the genetic basis of the orange lemma trait and demonstrate that disruption of CAD alters lignin accumulation in barley. This work provides a valuable genetic resource for breeding barley cultivars with improved biomass utilization for feed and bioenergy applications.