Cold stress is a major environmental factor that impairs plant growth and development, geographic distribution, and crop productivity. The C-repeat binding factor (CBF) regulatory pathway has an essential role in response to cold stress. Here, we characterized a bHLH transcription factor from Nicotiana tabacum, NtbHLH123, in response to cold stress (4°C). Overexpression of NtbHLH123 enhanced cold tolerance in transgenic tobacco plants. Based on yeast one-hybrid, chromatin immunoprecipitation PCR, and transient expression analysis assays, NtbHLH123 binds directly to the G-box/E-box motifs in the promoter of the NtCBF genes and positively regulates their expression. Furthermore, NtbHLH123-overexpressing plants showed lower electrolyte leakage, reduced malondialdehyde contents, H2O2 and reactive oxygen species (ROS) accumulation under cold stress, which contributed to alleviating oxidative damage to the cell membrane after cold stress treatment. And NtbHLH123 increased stress tolerance by improving the expression of a number of abiotic stress-responsive genes to mediate the ROS scavenging ability and other stress tolerance pathways. Taken together, we present a model suggesting that NtbHLH123 is a transcriptional activator that functions as a positive regulator of cold tolerance by activating NtCBF, ROS scavenging-related, and stress-responsive genes.

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Drought stress often limits plant growth and global crop yields. Catalase (CAT)-mediated hydrogen peroxide (H 2 O 2) 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 H 2 O 2 , 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 H 2 O 2 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 H 2 O 2. Knockdown of NtCAT in the NtERF172-overexpression lines displayed a more drought stresssensitive 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 CATmediated H 2 O 2 homeostasis.

Cigar tobacco is an important economic crop that is widely grown around the world. In recent years, varietal identification has become a frequent problem in germplasm preservation collections, which causes considerable inconvenience and uncertainty in the cataloging and preservation of cigar germplasm resources, in the selection of parental lines for breeding, and in the promotion and use of high quality varieties. Therefore, the use of DNA fingerprints to achieve rapid and accurate identification of varieties can play an important role in germplasm identification and property rights disputes. In this study, we used genotyping-by-sequencing (GBS) on 113 cigar tobacco accessions to develop SNP markers. After filtering, 580,942 high-quality SNPs were obtained. We used the 580,942 SNPs to perform principal component analysis (PCA), population structure analysis, and neighbor joining (NJ) cluster analysis on the 113 cigar tobacco accessions. The results showed that the accessions were not completely classified based on their geographical origins, and the genetic backgrounds of these cigar resources are complex and diverse. We further selected from these high-quality SNPs to obtained 163 SNP sites, 133 of which were successfully converted into KASP markers. Finally, 47 core KASP markers and 24 candidate core markers were developed. Using the core markers, we performed variety identification and fingerprinting in 216 cigar germplasm accessions. The results of SNP fingerprinting, 2D barcoding, and genetic analysis of cigar tobacco germplasm in this study provide a scientific basis for screening and identifying high-quality cigar tobacco germplasm, mining important genes, and broadening the basis of cigar tobacco genetics and subsequent breeding work at the molecular level.

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.

Many applications and protocols in wireless sensor networks need to know the locations of sensor nodes. A low-cost method to localize sensor nodes is to use received signal strength indication (RSSI) ranging technique together with the least-squares trilateration. However, the average localization error of this method is large due to the large ranging error of RSSI ranging technique. To reduce the average localization error, we propose a localization algorithm based on maximum a posteriori. This algorithm uses the Baye's formula to deduce the probability density of each sensor node's distribution in the target region from RSSI values. Then, each sensor node takes the point with the maximum probability density as its estimated location. Through simulation studies, we show that this algorithm outperforms the least-squares trilateration with respect to the average localization error.

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