Homeodomain leucine zipper protein controls the lobed leaf formation by modulating auxin distribution in watermelon

The homeodomain leucine zipper protein ClLMI1 plays a crucial role in the development of lobed leaf morphology in watermelon by influencing the auxin distribution. Lobed leaf is a critical phenotypic trait that influences light penetration and resultant canopy photosynthesis for potential productivity, which plays an important role in horizontal growing crops like watermelon. However, molecular mechanisms underlying the genetic variations of lobed leaves in watermelon remain poorly understood. In this study, we identified ClLMI1 encoding homeodomain leucine zipper protein as the causal gene for lobed leaf formation via BSA-Seq and subsequent fine mapping approaches. A splice-site SNP in an intron caused a 24 bp deletion in ClLMI1 coding region, leading to a deletion of eight amino acids in the leucine zipper domain of the mutant protein cllmi1 for lobe-free leaf phenotype. Additionally, CRISPR/Cas9-mediated knockout of ClLMI1 in watermelon validated its essential role in lobed leaf formation. Expression analysis revealed that ClLMI1 expression peaks at the tips of lobes, consistent with auxin accumulation patterns. Exogenous application of auxin and the auxin polar transport inhibitor N-1-naphthylphthalamic acid (NPA) inhibited lobes development in lobed leaf watermelon. Integrative yeast one-hybrid (Y1H) assay, electrophoresis mobility shift assay (EMSA) and dual-luciferase assay demonstrated that ClLMI1 can directly bind to the promoters of ClPIN1 and ClCUC2, activating their transcriptions to mediate auxin gradient distribution along leaf margin. Collectively, our findings elucidate ClLMI1 as a key regulator of leaf morphogenesis in watermelon and enhance the understanding of the regulatory mechanism of plant lobed leaf formation, facilitating the improvement of canopy photosynthesis by molecular design breeding in economically important watermelon crop.