Overexpression of the Transcription Factor CbbHLH96 Improves Alkali Stress Tolerance in Cinnamomun bodinieri

Soil salinization severely impairs plant growth and development, limiting crop yield and reducing quality. The transcription factor CbbHLH96 has been implicated in the alkali stress response of Cinnamomun bodinieri. In this study, we constructed a CbbHLH96 expression vector and genetically transformed Arabidopsis thaliana to investigate the functional role of CbbHLH96 under alkali stress, using transcriptomic sequencing for further analysis. Our results demonstrate the successful cloning of a 951-bp coding sequence (CDS) for the CbbHLH96 gene. Physicochemical characterization revealed that CbbHLH96 protein is a weakly acidic, unstable, and lipophilic. Subcellular localization prediction revealed that CbbHLH96 is localized in the nucleus. Structural domain identified the presence of bHLH_AtFAMA_like domain, classifying it within the bHLH-SF superfamily. Phylogenetic analysis confirmed high conservation of the CbbHLH96 protein among Lauraceae species. Transgenic Arabidopsis thaliana lines overexpressing CbbHLH96 (designated T96) were successfully generated. Phenotypic assessment under alkali stress revealed significantly enhanced tolerance in T96 plants compared to wild-type (WT) controls. Transcriptome analysis of the alkali stress-induced (WT).V.(T96) combination showed that the up-regulated differentially expressed genes were mainly enriched in Photosynthesis-antenna proteins, Photosynthesis, Porphyrin and chlorophyll metabolism, Plant-pathogen interaction, alpha-Linolenic acid metabolism, and Cyanoamino acid metabolism. Conversely, down-regulated genes were mainly enriched in Phenylpropanoid biosynthesis, Nitrogen metabolism, Flavonoid biosynthesis, Ubiquinone and other terpenoid-quinone biosynthesis, ABC transporters, etc. Notably, the key expression genes in alpha-linolenic acid metabolism pathway and cyanogenic amino acid metabolism pathway in T96 was significantly higher than in WT. Collectively, overexpression of CbbHLH96 confers enhanced alkali stress tolerance in Arabidopsis thaliana. This improved tolerance is mechanistically associated with the upregulation of key genes involved in the alpha-linolenic acid metabolism and cyanoamino acid metabolism pathways.