Blocking the conversion of β-carotenoids into xanthophylls through HYb gene editing via CRISPR/Cas9 confers enhanced heat stress tolerance in Nicotiana tabacum.

Plants produce protective metabolites to withstand stress, and β-carotenoids act as crucial antioxidants. β-Carotene is converted into xanthophylls by β-carotene hydroxylase (BCH). In this study, the BCH gene in Nicotiana tabacum was targeted using CRISPR/Cas9 genome editing to redirect metabolic flux toward β-carotene accumulation and evaluate its effects on heat tolerance. Following Agrobacterium-mediated transformation with a BCH-specific gRNA, three mutant plants were obtained; two carrying frameshift mutations (P1 and P3) and one with mis-sense mutations (P2). A significant increase in chlorophyll contents was observed in the edited lines compared with wild-type plants. Interestingly, the frameshift mutants exhibited substantial increase in total chlorophyll (71% and 31% in P3 and P1 respectively) as compared to mis-sense mutant P2 (18%). Further, biochemical profiling using HPLC showed that the edited plants accumulated 3.74-fold increase in β-carotene than wild-type plants. Under heat stress (42 °C for 96 h), the edited plants exhibited enhanced thermotolerance by remaining greener and delayed wilting, whereas wild-type plants developed necrosis rapidly under heat stress. This enhanced thermotolerance might has resulted from the increased expression of ε-cyclase (an upstream key enzyme in β-carotene biosynthesis) as revealed by expression profiling of edited plants. In contrast, the expression of downstream β-cryptoxanthin and zeaxanthin biosynthetic genes was reduced, which seems consistent with mutation of BCH. DAB staining further confirmed reduced ROS accumulation in edited plants as compare to wild type supporting the protective role of β-carotene. In summary, the downregulation of BCH in N. tabacum boosted β-carotene levels and chlorophyll retention, enhancing heat stress tolerance. These findings demonstrate the potential of targeting carotenoid biosynthesis to improve crop resilience.