Comprehensive analysis of the LHT gene family in tobacco and functional characterization of NtLHT22 involvement in amino acids homeostasis.

Amino acids are vital nitrogen (N) sources for plant growth, development, and yield. The uptake and translocation of amino acids are mediated by amino acid transporters (AATs). The AATs family including lysine-histidine transporters (LHTs), amino acid permeases (AAPs), and proline transporters (ProTs) subfamilies have been identified in various plants. However, little is known about these genes in tobacco. In this study, we identified 23 LHT genes, the important members of AATs, in the tobacco genome. The gene structure, phylogenetic tree, transmembrane helices, chromosomal distribution, cis-regulatory elements, and expression profiles of NtLHT genes were systematically analyzed. Phylogenetic analysis divided the 23 NtLHT genes into two conserved subgroups. Expression profiles confirmed that the NtLHT genes were differentially expressed in various tissues, indicating their potential roles in tobacco growth and development. Cis-elements analysis of promoters and expression patterns after stress treatments suggested that NtLHT genes probable participate in abiotic stress responses of tobacco. In addition, Knock out and overexpression of NtLHT22 changed the amino acids homeostasis in the transgenic plants, the contents of amino acids were significantly decreased in NtLHT22 overexpression plants than wild-type. The results from this study provide important information for further studies on the molecular functions of the NtLHT genes.

Comparative Chloroplast Genomes of Nicotiana Species (Solanaceae): Insights Into the Genetic Variation, Phylogenetic Relationship, and Polyploid Speciation.

Nicotiana L. is a genus rich in polyploidy, which represents an ideal natural system for investigating speciation, biodiversity, and phytogeography. Despite a wealth of phylogenetic work on this genus, a robust evolutionary framework with a dated molecular phylogeny for the genus is still lacking. In this study, the 19 complete chloroplast genomes of Nicotiana species were assembled, and five published chloroplast genomes of Nicotiana were retrieved for comparative analyses. The results showed that the 24 chloroplast genomes of Nicotiana, ranging from 155,327 bp (N. paniculata) to 156,142 bp (N. heterantha) in size, exhibited typical quadripartite structure. The chloroplast genomes were rather conserved in genome structure, GC content, RNA editing sites, and gene content and order. The higher GC content observed in the IR regions could be a result of the presence of abundant rRNA and tRNA genes, which contained a relatively higher GC content. A total of seven hypervariable regions, as new molecular markers for phylogenetic analysis, were uncovered. Based on 78 protein-coding genes, we constructed a well-supported phylogenetic tree, which was largely in agreement with previous studies, except for a slight conflict in several sections. Chloroplast phylogenetic results indicated that the progenitors of diploid N. sylvestris, N. knightiana, and the common ancestor of N. sylvestris and N. glauca might have donated the maternal genomes of allopolyploid N. tabacum, N. rustica, and section Repandae, respectively. Meanwhile, the diploid section Noctiflorae lineages (N. glauca) acted as the most likely maternal progenitor of section Suaveolentes. Molecular dating results show that the polyploid events range considerably in ~0.12 million (section Nicotiana) to ~5.77 million (section Repandae) years ago. The younger polyploids (N. tabacum and N. rustica) were estimated to have arisen ~0.120 and ~0.186 Mya, respectively. The older polyploids (section Repandae and Suaveolentes) were considered to have originated from a single polyploid event at ~5.77 and ~4.49 Mya, respectively. In summary, the comparative analysis of chloroplast genomes of Nicotiana species has not only revealed a series of new insights into the genetic variation and phylogenetic relationships in Nicotiana but also provided rich genetic resources for speciation and biodiversity research in the future.

NtNAC053, A Novel NAC Transcription Factor, Confers Drought and Salt Tolerances in Tobacco.

The NAC (NAM, ATAF1/2, and CUC2) family acts as one of the largest families of the transcription factor in the plant kingdom and was revealed to function as the important regulators in various environmental stresses. However, a few studies were reported about the biofunctions of the NAC transcription factor in tobacco. In the current study, we characterized a novel NAC transcription factor encoding the gene NtNAC053 in tobacco, which was significantly up-regulated when exposed to salt and drought treatments. The results of cis-acting elements analysis suggested that the promoter region of NtNAC053 gene possesses a number of stress-responsive elements, and this gene could be induced by exogenous abscisic acid (ABA) treatment. Moreover, the NtNAC053-GFP fusion protein was localized in the cell nucleus and possessed a transactivation domain in its C-terminal, implying that NtNAC053 may undertake as a transcriptional activator in tobacco. Notably, the overexpression of NtNAC053 in tobacco resulted in hypersensitivity to ABA treatment. Furthermore, these overexpression lines showed significantly enhanced tolerances to drought and salt stresses. Under salt and drought stresses, these overexpression lines possessed higher superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities. Interestingly, the expressions of putative stress-related genes, including NtCOR15A, NtRAB18, NtDREB1A, NtERF5, NtKAT2, and NtERD11, were up-regulated in these overexpression lines when subjected to salt and drought stresses. The clues provided in our study suggested that the NtNAC053 gene encodes a novel NAC transcription factor and could confer the drought and salt stress tolerances by inspiring the downstream stress-responsive genes and antioxidant system in tobacco.

Comprehensive Analysis of Long Non-coding RNA Modulates Axillary Bud Development in Tobacco (Nicotiana tabacum L.).

Long non-coding RNAs (lncRNAs) regulate gene expression and are crucial for plant growth and development. However, the mechanisms underlying the effects of activated lncRNAs on axillary bud development remain largely unknown. By lncRNA transcriptomes of axillary buds in topped and untopped tobacco plants, we identified a total of 13,694 lncRNAs. LncRNA analysis indicated that the promoted growth of axillary bud by topping might be partially ascribed to the genes related to hormone signal transduction and glycometabolism, trans-regulated by differentially expressed lncRNAs, such as MSTRG.52498.1, MSTRG.60026.1, MSTRG.17770.1, and MSTRG.32431.1. Metabolite profiling indicated that auxin, abscisic acid and gibberellin were decreased in axillary buds of topped tobacco lines, while cytokinin was increased, consistent with the expression levels of related lncRNAs. MSTRG.52498.1, MSTRG.60026.1, MSTRG.17770.1, and MSTRG.32431.1 were shown to be influenced by hormones and sucrose treatments, and were associated with changes of axillary bud growth in the overexpression of NtCCD8 plants (with reduced axillary buds) and RNA interference of NtTB1 plants (with increased axillary buds). Moreover, MSTRG.28151.1 was identified as the antisense lncRNA of NtTB1. Silencing of MSTRG.28151.1 in tobacco significantly attenuated the expression of NtTB1 and resulted in larger axillary buds, suggesting the vital function of MSTRG.28151.1 axillary bud developmen by NtTB1. Our findings shed light on lncRNA-mRNA interactions and their functional roles in axillary bud growth, which would improve our understanding of lncRNAs as important regulators of axillary bud development and plant architecture.

Genome-wide characterization and expression profiling of the PDR gene family in tobacco (Nicotiana tabacum).

The pleiotropic drug resistance (PDR) proteins of the ATP-binding cassette (ABC) family play essential roles in physiological processes and have been characterized in many plant species. However, no comprehensive investigation of tobacco (Nicotiana tabacum), an important economic crop and a useful model plant for scientific research, has been presented. We identified 32 PDR genes in the tobacco genome and explored their domain organization, chromosomal distribution and evolution, promoter cis-elements, and expression profiles. A phylogenetic analysis revealed that tobacco has a significantly expanded number of PDR genes involved in plant defense. It also revealed that two tobacco PDR proteins may function as strigolactone transporters to regulate shoot branching, and several NtPDR genes may be involved in cadmium transport. Moreover, tissue expression profiles of NtPDR genes and their responses to several hormones and abiotic stresses were assessed using quantitative real-time PCR. Most of the NtPDR genes were regulated by jasmonate or salicylic acid, suggesting the important regulatory roles of NtPDRs in plant defense and secondary metabolism. They were also responsive to abiotic stresses, like drought and cold, and there was a strong correlation between the presence of promoter cis-elements and abiotic/biotic stress responses. These results provide useful clues for further in-depth studies on the functions of the tobacco PDR genes.

Genome-Wide Identification and Characterization of GASA Gene Family in Nicotiana tabacum.

The gibberellic acid stimulated Arabidopsis (GASA) gene family is critical for plant growth, development, and stress response. GASA gene family has been studied in various plant species, however, the GASA gene family in tobacco (Nicotiana tabacum) have not been characterized in detail. In this study, we identified 18 GASA genes in the tobacco genome, which were distributed to 13 chromosomes. All the proteins contained a conserved GASA domain and highly specific 12-cysteine residues at the C-terminus. Phylogenetic analysis divided the NtGASA genes into three well-conserved subfamilies. Synteny analysis suggested that tandem and segmental duplications played an important role in the expansion of the NtGASA gene family. Cis-elements analysis showed that NtGASA genes might influence different phytohormone and stress responses. Tissue expression analysis revealed that NtGASA genes displayed unique or distinct expression patterns in different tissues, suggesting their potential roles in plant growth and development. We also found that the expression of NtGASA genes were mostly regulated by abscisic and gibberellic acid, signifying their roles in the two phytohormone signaling pathways. Overall, these findings improve our understanding of NtGASA genes and provided useful information for further studies on their molecular functions.

[Ecological reasons for the difference in nicotine content of flue-cured tobacco planted in different regions of Hunan Province].

Aimed to explore the ecological reasons for the difference in nicotine content of flue-cured tobacco planted in different regions of Hunan Province, field experiments were conducted in Sangzhi, Liuyang, and Yongzhou counties, the three typical tobacco regions of Hunan Province, taking tobacco variety K326 as the test object. Simultaneously, pot experiments with local soils and guest soils were carried out. The nicotine content of mid position tobacco leaves was analyzed at harvest time. Field experiments showed that the average nicotine content of tobacco leaves differed significantly among test sites, with that in Sangzhi being the highest, followed by Liuyang, and Yongzhou. Pot experiments showed that climate had significant effects on the average nicotine content of tobacco leaves, while soil and its interaction with climate had less effects. The contribution rate of climate, soil, and their interaction on the variance of the average nicotine content was 60.0%, 12.8% and 27.2%, respectively. The main sub-ecological factors closely related to the average nicotine content of flue-cured tobacco planted in different regions of Hunan Province were in turn the cloud cover at maturing stage, the relative humidity, sunshine hours, diurnal temperature variance, and rainfall at root-extending stage, and the average air temperature at vigorous growth stage. Generally, climate was the main ecological factor that led to the nicotine content difference of flue-cured tobacco planted in different regions of Hunan Province.