Antagonism and convergence of MiCOL14B-GQ and MiCOL14B-JH in mango (Mangifera indica) flowering and abiotic stress

The CONSTANS/CONSTANS-LIKE (CO/COL) gene family plays important roles in plants flowering and stress response. In this study, two variants of the MiCOL14B gene were identified from two different mango cultivars; they were designated as MiCOL14B-GQ and MiCOL14B-JH, which exhibited significant differences in sequence and B-box domain. Both genes were expressed in various tissues of mango, localized in the nucleus, and responsive to drought and salt stress. In transgenic Arabidopsis thaliana, MiCOL14B-GQ delayed flowering, while MiCOL14B-JH promoted flowering. This phenotypic divergence stemmed from their molecular regulatory specificity. Yeast one-hybrid (Y1H) and dual-luciferase reporter assays demonstrated that both variants directly bind to the promoters of florigen genes (MiFTs), with MiCOL14B-GQ repressing their transcription and MiCOL14B-JH enhancing it. Altered expression levels of MiFTs in the roots of transgenic mango further validated this mechanism. Moreover, both MiCOL14B-GQ and MiCOL14B-JH improved stress tolerance under drought and salt conditions in transgenic A. thaliana as well as in transgenic mango roots. These variants significantly increased stress tolerance by increasing proline (Pro) content and superoxide dismutase (SOD) activity, while reducing malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) accumulation. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays revealed that MiCOL14B-GQ and MiCOL14B-JH interact with several stress-related proteins. This study demonstrates for the first time the functional effects of sequence variation in the MiCOL14B gene on flowering and stress responses, providing valuable genetic resources for mango molecular breeding.