Lf2 is a knotted homeobox regulator that modulates leaflet number in soybean

Abstract Variation in leaf complexity modulates light capture and is a target for crop enhancement. Soybean typically has compound leaves with three leaflets each, but a spontaneous mutation, designated lf2, possesses seven leaflets, offering a means to dissect the molecular mechanisms specifying leaflet number and assess its potential for soybean improvement. However, the developmental and genetic bases of the lf2 mutation remain unknown. Here, we characterize the seven-leaflet phenotype and identify the mutation responsible for the phenotypic changes. Microscopic examination of leaf emergence sites revealed that the seven-leaflet phenotype arises in a two-step process: five leaflets form initially followed by secondary leaflet initiation at the margins of the central leaflet. Genetic mapping delineated lf2 to a ∼2.5 Mb region at the start of chromosome 11. Fortuitously, integration of pedigree analysis with comparative analysis of genomic sequences from the region pinpointed a 2-bp deletion in the coding sequence of a gene, which is homologous to the Arabidopsis KNAT7 encoding a KNOTTED1-LIKE HOMEOBOX 2 transcription factor, as the sole candidate for Lf2. The deletion is predicted to result in disruption of the putative DNA-binding homeodomain. Expression of the wild-type allele of the candidate gene in the seven-leaflet lf2 mutant restored the three-leaflet phenotype, while disruption of the wild-type allele through CRISPR-Cas9 editing induced extra leaflet formation. This study advances our understanding of leaflet formation in legumes and provides a template for utilizing compound leaf architecture to optimize photosynthetic efficiency and yield in soybean.