The NtBAG5-CaM complex integrates Ca ²⁺ signals to regulate leaf senescence via the antioxidant system in tobacco

Leaf yellowing critically impacts tobacco quality and economic value. The Bcl-2-associated athanogene (BAG) gene family regulates plant development and senescence, yet the role of NtBAG5 in tobacco remains poorly understood. Here, we demonstrate that NtBAG5 acts as a key promoter of leaf senescence. CRISPR/Cas9-generated <i>NtBAG5</i> mutants exhibited delayed senescence, enhanced activities of antioxidant enzymes, and reduced malondialdehyde (MDA) content, whereas <i>NtBAG5</i>-overexpressing plants showed the opposite effects. Promoter-GUS analysis revealed high <i>NtBAG5</i> expression in roots and stems. Hormone treatments indicated that <i>NtBAG5</i> expression is upregulated by ABA, ETH, IAA, and GA (at late stage) but downregulated by MeJA. Mechanistically, NtBAG5 interacts with calmodulin (CaM) via its BAG domain and IQ motif, as confirmed by yeast two-hybrid and BiFC assays. Ca^{2 +} was found to modulate CaM conformation and strengthen the NtBAG5-CaM interaction. Silencing <i>NtCaM</i> via VIGS induced severe leaf yellowing and growth defects. Our results reveal that the NtBAG5-CaM complex, regulated by Ca^{2 +} and hormones, modulates leaf senescence through the antioxidant system, providing new insights for improving tobacco quality.