Insect odorant binding proteins (OBPs) and chemosensory proteins (CSPs) play an important role in chemical communication of insects. Gene discovery of these proteins is a time-consuming task. In recent years, expressed sequence tags (ESTs) of many insect species have accumulated, thus providing a useful resource for gene discovery.

CB-1 and K326 are closely related tobacco cultivars; however, their cold tolerance capacities are different. K326 is much more cold tolerant than CB-1. We studied the transcriptomes and metabolomes of CB-1 and K326 leaf samples treated with cold stress. Totally, we have identified 14,590 differentially expressed genes (DEGs) in CB-1 and 14,605 DEGs in K326; there was also 200 differentially expressed metabolites in CB-1 and 194 in K326. Moreover, there were many overlapping genes (around 50%) that were cold-responsive in both plant cultivars, although there were also many differences in the cold responsive genes between the two cultivars. Importantly, for most of the overlapping cold responsive genes, the extent of the changes in expression were typically much more pronounced in K326 than in CB-1, which may help explain the superior cold tolerance of K326. Similar results were found in the metabolome analysis, particularly with the analysis of primary metabolites, including amino acids, organic acids, and sugars. The large number of specific responsive genes and metabolites highlight the complex regulatory mechanisms associated with cold stress in tobacco. In addition, our work implies that the energy metabolism and hormones may function distinctly between CB-1 and K326. Differences in gene expression and metabolite levels following cold stress treatment seem likely to have contributed to the observed difference in the cold tolerance phenotype of these two tobacco cultivars.

Tobacco plants (Nicotiana tabacum L.) exhibit considerable potential for phytoremediation of soil cadmium (Cd) pollutants, owing to their substantial biomass and efficient metal accumulation capabilities. The reduction of Cd accumulation in tobacco holds promise for minimizing Cd intake in individuals exposed to cigar smoking. NRAMP transporters are pivotal in the processes of Cd accumulation and resistance in plants; however, limited research has explored the functions of NRAMPs in tobacco plants. In this investigation, we focused on NtNRAMP6c, one of the three homologs of NRAMP6 in tobacco. We observed a robust induction of NtNRAMP6c expression in response to both Cd toxicity and iron (Fe) deficiency, with the highest expression levels detected in the roots. Subsequent subcellular localization and heterologous expression analyses disclosed that NtNRAMP6c functions as a plasma membrane-localized Cd transporter. Moreover, its overexpression significantly heightened the sensitivity of yeast cells to Cd toxicity. Through CRISPR-Cas9-mediated knockout of NtNRAMP6c, we achieved a reduction in Cd accumulation and an enhancement in Cd resistance in tobacco plants. Comparative transcriptomic analysis unveiled substantial alterations in the transcriptional profiles of genes associated with metal ion transport, photosynthesis, and macromolecule catabolism upon NtNRAMP6c knockout. Furthermore, our study employed plant metabolomics and rhizosphere metagenomics to demonstrate that NtNRAMP6c knockout led to changes in phytohormone homeostasis, as well as shifts in the composition and abundance of microbial communities. These findings bear significant biological implications for the utilization of tobacco in phytoremediation strategies targeting Cd pollutants in contaminated soils, and concurrently, in mitigating Cd accumulation in tobacco production destined for cigar consumption.

The amino acid permease (AAP) protein family is an important class of amino acid transporter. However, little information about this gene family is available in tobacco. Here, a total number of 15, 7, and 6 full-length putative AAP genes have been identified in the genomes of Nicotiana tabacum and progenitors of modern tobacco, Nicotiana sylvestris and Nicotiana tomentosiformis, respectively. We performed a multiple analyses of the AAP gene family in tobacco and reported data including phylogenetic relationships, gene structures, predicted protein structures and information about conserved motifs. These tobacco AAP family members shared high levels of similarity in their nucleotide and amino acid sequences. The gene structures of the NtAAP loci were all separated by introns. Phylogenetic analyses indicated that these AAP genes were clustered into three groups. We also performed expression profiling analyses of the NtAAP genes in various tobacco tissues/organs using quantitative PCR. The differential expression patterns of the NtAAP genes suggested that the family members might have different biological functions in tobacco plants. To study the function of NtAAP2-2, its coding sequence was cloned and overexpression/RNA interference vectors were constructed and these vectors were transformed into tobacco. Compared with wild-type tobacco, the contents of Glu, Gln, Asp and Asn were found to change in the transgenic tobacco leaves, indicating that NtAAP2-2 was involved in the transportation of Glu, Gln, Asp and Asn in tobacco. Our results can serve as fundamental research for future evolutionary and functional characterization studies of the AAP genes in tobacco.

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 bp with low or no protein-coding ability, which play essential roles in various biological processes in plants. Tobacco is an ideal model plant for studying nicotine biosynthesis and metabolism, and there is little research on lncRNAs in this field. Therefore, how to take advantage of the mature tobacco system to profoundly investigate the lncRNAs involved in the nicotine pathway is intriguing. By exploiting 549 public RNA-Seq datasets of tobacco, 30,212 lncRNA candidates were identified, including 24,084 large intervening non-coding RNAs (lincRNAs), 5,778 natural antisense transcripts (NATs) and 350 intronic non-coding RNAs (incRNAs). Compared with protein-coding genes, lncRNAs have distinct properties in terms of exon number, sequence length, A/U content, and tissue-specific expression pattern. lincRNAs showed an asymmetric evolutionary pattern, with a higher proportion (68.71%) expressed from the Nicotiana sylvestris (S) subgenome. We predicted the potential cis/trans-regulatory effects on protein-coding genes. One hundred four lncRNAs were detected as precursors of 30 known microRNA (miRNA) family members, and 110 lncRNAs were expected to be the potential endogenous target mimics for 39 miRNAs. By combining the results of weighted gene co-expression network analysis with the differentially expressed gene analysis of topping RNA-seq data, we constructed a sub-network containing eight lncRNAs and 25 nicotine-related coding genes. We confirmed that the expression of seven lncRNAs could be affected by MeJA treatment and may be controlled by the transcription factor NtMYC2 using a quantitative PCR assay and gene editing. The results suggested that lncRNAs are involved in the nicotine pathway. Our findings further deepened the understanding of the features and functions of lncRNAs and provided new candidates for regulating nicotine biosynthesis in tobacco.

Nanomaterials have been shown to promote crop growth, yield and stress resistance. Carbon nanosol (CNS), a type of nanomaterial, is used to regulate tobacco shoot and root growth. However, information about the application of CNS to crop plants, especially tobacco, is still limited. Based on differential expression analysis and trend analysis, several miRNAs (miRN21-Novel-5p-mature, miR319b-Probable-5p-mature, miR160a-c-Known/Probable-5p-mature and miR156c-e-Known-5p-mature/star) and their target genes, including transcription factors (TFs), are likely responsible for the effect of CNS on promoting the growth of tobacco plants. In addition, we characterized nine TFs [Nitab4.5_00001789g0110 (NbbZIP), Nitab4.5_00001176g0010 (NbMYB), Nitab4.5_0001366g0010 (NbNAC), Nitab4.5_00000895g013 (NbMYB), Nitab4.5_0001225g0120 (NbNAC), Nitab4.5_0000202g0230 (NbDof), Nitab4.5_0002241g0010 (NbMYB-related), Nitab4.5_0000410g0060 (NbTCP), and Nitab4.5_0000159g0180 (NbC2H2)] associated with the response of tobacco to CNS according to the differential expression analysis, TF-gene interaction network analysis and weighted correlation network analysis (WGCNA). Taken together, the findings of our study help understand CNS-mediated growth promotion in tobacco plants. The identification of candidate miRNAs and genes will provide potential support for the use of CNS in tobacco.