Tobacco transcription factor bHLH123 improves salt tolerance by activating NADPH oxidase NtRbohE expression

In plants, reactive oxygen species (ROS) produced following the expression of the respiratory burst oxidase homolog (Rboh) gene are important regulators of stress responses. However, little is known about how plants acclimate to salt stress through the Rboh-derived ROS signaling pathway. Here, we showed that a 400-bp fragment of the tobacco (Nicotiana tabacum) NtRbohE promoter played a critical role in the salt response. Using yeast one-hybrid (Y1H) screens, NtbHLH123, a bHLH transcription factor, was identified as an upstream partner of the NtRbohE promoter. These interactions were confirmed by Y1H, electrophoretic mobility assay, and chromatin immunoprecipitation assays. Overexpression of NtbHLH123 resulted in greater resistance to salt stress, while NtbHLH123-silenced plants had reduced resistance to salt stress. We also found that NtbHLH123 positively regulates the expression of NtRbohE and ROS production soon after salt stress treatment. Moreover, knockout of NtRbohE in the 35S::NtbHLH123 background resulted in reduced expression of ROS-scavenging and salt stress-related genes and salt tolerance, suggesting that NtbHLH123-regulated salt tolerance is dependent on the NtbHLH123-NtRbohE signaling pathway. Our data show that NtbHLH123 is a positive regulator and acts as a molecular switch to control a Rboh-dependent mechanism in response to salt stress in plants.