CRISPRa-driven activation of IbbZIP11 boosts saline-alkali tolerance in sweet potato by reinforcing antioxidant defense
Haoqiang Zhao, Zheng Fu, Ruitao Liu, Yi Sun, Kunfeng Xing, Hong Zhai, Shaozhen He, Huan Zhang, Ning Zhao, Qingchang Liu, Shaopei GaoAbstract
Saline-alkali stress limits the cultivation of sweet potato (Ipomoea batatas) on marginal lands, yet the key tolerance genes remain poorly characterized. This knowledge gap is compounded by the crop's heterozygous hexaploid genome, where allelic diversity and genetic redundancy complicate functional genomics studies. Given the conserved role of S1-group bZIPs in stress signaling, the robust induction of IbbZIP11 by multiple stresses, and its high expression in roots, we selected this candidate to decipher its function in saline-alkali tolerance. To dissect its function in this polyploid context, we applied a streamlined CRISPR activation/interference (CRISPRa/i) toolkit using dCas9-6×TAL-2×VP64 (TV) for activation and dCas9-SRDX for repression. By targeting the conserved promoter region of IbbZIP11, we achieved transcriptional modulation of its homologous alleles. Under saline-alkali stress, CRISPRa-mediated activation of IbbZIP11 enhanced growth and biomass accumulation in both hydroponic and soil-based assays, and improved photosynthesis in soil-based assays. In contrast, CRISPRi-mediated repression increased stress sensitivity, causing severe leaf senescence and growth inhibition. Mechanistically, we identified catalase gene IbCAT1 as a direct transcriptional target of IbbZIP11. Notably, IbCAT1-overexpressing lines phenocopied the enhanced tolerance of CRISPRa lines, whereas IbCAT1-RNAi lines displayed hypersensitivity similar to CRISPRi lines, indicating that IbbZIP11-mediated stress resilience is dependent on IbCAT1 activation. Among the tested genes, IbSOD, IbPOD, IbP5CS, and IbP5CR were indirectly regulated. This IbbZIP11-IbCAT1 axis mitigates oxidative damage via reinforced ROS scavenging and improved ROS homeostasis. In summary, our study reveals the IbbZIP11-IbCAT1 module as an important downstream regulatory component and positions IbbZIP11 as a promising target for engineering saline-alkali tolerance.