OVATE family protein 6 controls leaf angle through dual regulation of cytokinin catabolism and leaf dorsiventrality in lettuce

Leaf angle is a key agronomic trait for improving planting density and yield in lettuce, particularly in controlled-environment agriculture and high-density field cultivation. Leaf angle regulation is well studied in monocots; however, the genetic and molecular mechanisms in dicots remain largely unknown. Here, we genetically clone and functionally characterize LsOFP6a, an OVATE family protein gene, as a key regulator of leaf angle in lettuce. A nonsense mutation in LsOFP6a in large-leaf-angle cultivars produces a truncated protein with impaired function. CRISPR/Cas9 knockout and complementary tests confirmed that LsOFP6a negatively regulates leaf angle in lettuce. LsOFP6a physically interacts with the BELL-like homeodomain transcription factor LsBLH2. Genetic analyses revealed that LsOFP6a regulates leaf angle through an LsBLH2-dependent pathway, and LsBLH2 is recessive-epistatic to LsOFP6a. LsBLH2 directly upregulates the expression of the cytokinin oxidase gene LsCKX5a. LsOFP6a represses the transcriptional activity of LsBLH2 on LsCKX5, leading to elevated cytokinin levels and small leaf angle. Furthermore, LsOFP6a inhibits the effects of LsBLH2 on repressing abaxial gene LsYAB1, leading to enhanced abaxial cell elongation and erect leaves. Loss of function of LsOFP6a decreases the cytokinin level and represses abaxial cells, resulting in large leaf angles. In summary, the LsOFP6a-LsBLH2 module orchestrates cytokinin catabolism and leaf dorsiventrality to regulate lettuce leaf angle. Our study suggests potential novel strategies for the breeding of lettuce with compact architecture and suitable for high-density planting in the open field and plant factories.