OVATE family protein 6 interacts with CONSTANS-LIKE 3 to regulate bolting by transcriptional control of FLOWERING LOCUS T / TERMINAL FLOWER 1 family genes in lettuce

As one of the most globally cultivated vegetables, lettuce’s commercial yield is severely compromised by premature bolting, an undesirable agronomic trait that diminishes crop quality and market value. In this study, we genetically cloned the OVATE family protein 6 (LsOFP6) controlling bolting time in lettuce. LsOFP6 had a nonsense mutation in the late bolting parent. CRISPR/Cas9 knockout and complementary tests confirmed that LsOFP6 positively regulates bolting in lettuce. LsOFP6 physically interacts with the CONSTANS-LIKE 3 (LsCOL3). Genetic analyses suggest that LsOFP6 regulates bolting through an LsCOL3-dependent pathway in the Lsblh2 Lskn1 genotype background, where we have previously established that LsBLH2 and LsKN1, the positive and negative transcriptional regulators of bolting, respectively, both interact with LsOFP6 and exert their functions via the gibberellin pathway. LsCOL3 represses bolting development by directly binding to the promoters of TWIN SISTER OF FLOWERING LOCUS T (LsTSF1) and TERMINAL FLOWER 1 (LsTFL1), downregulating the former while upregulating the latter. These regulations are antagonized by LsOFP6, which promotes bolting by compromising the effects of LsCOL3 on LsTSF1 and LsTFL1. Therefore, loss-of-function of Lsofp6 decreases LsTSF1 and increases LsTFL1 expression, resulting in late bolting. In summary, the LsOFP6–LsCOL3 module regulates lettuce bolting development by controlling the transcript levels of LsTSF1 and LsTFL1 genes. This study deepens the molecular understanding of bolting development and provides valuable genetic resources and novel strategies for developing lettuce with improved resistance to premature bolting, highlighting the power of natural variation to address agricultural challenges.