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.

Summary: Drought stress often limits plant growth and global crop yields. Catalase (CAT)‐mediated hydrogen peroxide (H₂O₂) scavenging plays an important role in the adaptation of plant stress responses, but the transcriptional regulation of the CAT gene in response to drought stress is not well understood. Here, we isolated an APETALA2/ETHYLENE‐RESPONSIVE FACTOR (AP2/ERF) domain‐containing transcription factor (TF), NtERF172, which was strongly induced by drought, abscisic acid (ABA) and H₂O₂, from tobacco (Nicotiana tabacum) by yeast one‐hybrid screening. NtERF172 localized to the nucleus and acted as a transcriptional activator. Chromatin immunoprecipitation, yeast one‐hybrid assays, electrophoretic mobility shift assays and transient expression analysis assays showed that NtERF172 directly bound to the promoter region of the NtCAT gene and positively regulated its expression. Transgenic plants overexpressing NtERF172 displayed enhanced tolerance to drought stress, whereas suppression of NtERF172 decreased drought tolerance. Under drought stress conditions, the NtERF172‐overexpressed lines showed higher catalase activity and lower accumulation of H₂O₂ compared with wild‐type (WT) plants, while the NtERF172‐silenced plants showed the inverse correlation. Exogenous application of amino‐1,2,4‐triazole (3‐AT), an irreversible CAT inhibitor, to the NtERF172‐overexpression lines showed decreased catalase activity and drought tolerance, and increased levels of cellular H₂O₂. Knockdown of NtCAT in the NtERF172‐overexpression lines displayed a more drought stress‐sensitive phenotype than NtERF172‐overexpression lines. We propose that NtERF172 acts as a positive factor in drought stress tolerance, at least in part through the regulation of CAT‐mediated H₂O₂ homeostasis.

Glandular trichomes of tobacco (Nicotiana tabacum) produce blends of acylsucroses that contribute to defence against pathogens and herbivorous insects, but the mechanism of assembly of these acylsugars has not yet been determined. In this study, we isolated and characterized two trichome-specific acylsugar acyltransferases that are localized in the endoplasmic reticulum, NtASAT1 and NtASAT2. They sequentially catalyse two additive steps of acyl donors to sucrose to produce di-acylsucrose. Knocking out of NtASAT1 or NtASAT2 resulted in deficiency of acylsucrose; however, there was no effect on acylsugar accumulation in plants overexpressing NtASAT1 or NtASAT2. Genomic analysis and profiling revealed that NtASATs originated from the T subgenome, which is derived from the acylsugar-producing diploid ancestor N. tomentosiformis. Our identification of NtASAT1 and NtASAT2 as enzymes involved in acylsugar assembly in tobacco potentially provides a new approach and target genes for improving crop resistance against pathogens and insects.