Genome-wide analysis of HAK potassium transporter gene family reveals asymmetrical evolution in tobacco (Nicotiana tabacum)

Being an essential mineral nutrient, potassium (K+) plays numerous important roles in plant growth and development and determines the yield and quality of crop products. The cellular level of K+ is controlled to a large extent by the potassium transporter which belongs to the KT/HAK/KUP (HAK) family. However, little is known about these genes in tobacco. In this study, we surveyed the tobacco genome and identified forty-one putative NtHAK genes (NtHAKS1-NtHAKS21 and NtHAKT1-NtHAKT20). Investigation of the cis-elements in upstream regions of these NtHAKs suggests that members of this family respond to environmental clues and phytohormones. Expression data mining reveals that NtHAKs showed clear sub-genome dominance. In all, these results will provide molecular insights into the potassium transporter research in tobacco.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Characterization of Nicotiana tabacum genotypes possessing deletion mutations that affect potyvirus resistance and the production of trichome exudates

Abstract Background Advances in genomics technologies are making it increasingly feasible to characterize breeding lines that carry traits of agronomic interest. Tobacco germplasm lines that carry loci designated VAM and va have been extensively investigated due to their association with potyvirus resistance (both VAM and va) and defects in leaf surface compounds originating from glandular trichomes (VAM only). Molecular studies and classical genetic analyses are consistent with the model that VAM and va represent deletion mutations in the same chromosomal region. In this study, we used RNA-seq analysis, together with emerging tobacco reference genome sequence information to characterize the genomic regions deleted in tobacco lines containing VAM and va. Results Tobacco genotypes TI 1406 (VAM), K326-va and K326 (wild type) were analyzed using RNA-seq to generate a list of genes differentially expressed in TI 1406 and K326-va, versus the K326 control. Candidate genes were localized onto tobacco genome scaffolds and validated as being absent in only VAM, or missing in both VAM and va, through PCR analysis. These results enabled the construction of a map that predicted the relative extent of the VAM and va mutations on the distal end of chromosome 21. The RNA-seq analyses lead to the discovery that members of the cembratrienol synthase gene family are deleted in TI 1406. Transformation of TI 1406 with a cembratrienol synthase cDNA, however, did not recover the leaf chemistry phenotype. Common to both TI 1406 and K326-va was the absence of a gene encoding a specific isoform of a eukaryotic translation initiation factor (eiF4E1.S). Transformation experiments showed that ectopic expression of eiF4E1.S is sufficient to restore potyvirus susceptibility in plants possessing either the va or VAM mutant loci. Conclusions We have demonstrated the feasibility of using RNA-seq and emerging whole genome sequence resources in tobacco to characterize the VAM and va deletion mutants. These results lead to the discovery of genes underlying some of the phenotypic traits associated with these historically important loci. Additionally, initial size estimations were made for the deleted regions, and dominant markers were developed that are very close to one of the deletion junctions that defines va

Systematic Identification of Methyl Jasmonate-Responsive Long Noncoding RNAs and Their Nearby Coding Genes Unveils Their Potential Defence Roles in Tobacco BY-2 Cells

Long noncoding RNAs (lncRNAs) are distributed in various species and play critical roles in plant growth, development, and defence against stimuli. However, the lncRNA response to methyl jasmonate (MeJA) treatment has not been well characterized in Nicotiana tabacum Bright Yellow-2 (BY-2) cells, and their roles in plant defence remain elusive. Here, 7848 reliably expressed lncRNAs were identified in BY-2 cells, of which 629 differentially expressed (DE) lncRNAs were characterized as MeJA-responsive lncRNAs. The lncRNAs in BY-2 cells had a strong genus specificity in Nicotiana. The combined analysis of the cis-regulated lncRNAs and their target genes revealed the potential up- and downregulated target genes that are responsible for different biological functions and metabolic patterns. In addition, some lncRNAs for response-associated target genes might be involved in plant defence and stress resistance via their MeJA- and defence-related cis-regulatory elements. Moreover, some MeJA-responsive lncRNA target genes were related to quinolinate phosphoribosyltransferase, lipoxygenases, and endopeptidase inhibitors, which may contribute to nicotine synthesis and disease and insect resistance, indicating that MeJA-responsive lncRNAs regulate nicotine biosynthesis and disease resistance by regulating their potential target genes in BY-2 cells. Therefore, our results provide more targets for genetically engineering the nicotine content and plant defence in tobacco plants

Transcriptomic profile of tobacco in response to Tomato zonate spot orthotospovirus infection

Abstract Background Tomato zonate spot virus (TZSV), a dominant species of thrips-transmitted orthotospoviruses in Yunnan and Guangxi provinces in China, causes significant loss of yield in lots of crops and is a major threat to incomes of rural families. However, the detailed molecular mechanism of crop disease caused by TZSV remains obscure. Methods Next-generation sequencing (NGS)-based transcriptome analysis (RNA-seq) was performed to investigate and compare the gene expression changes in systemic leaves of tobacco upon infection with TZSV and mock-inoculated plants as a control. Results De novo assembly and analysis of tobacco transcriptome data by RNA-Seq identified 135,395 unigenes. 2102 differentially expressed genes (DEGs) were obtained in tobacco with TZSV infection, among which 1518 DEGs were induced and 584 were repressed. Gene Ontology enrichment analysis revealed that these DEGs were associated with multiple biological functions, including metabolic process, oxidation-reduction process, photosynthesis process, protein kinase activity. The KEGG pathway analysis of these DEGs indicated that pathogenesis caused by TZSV may affect multiple processes including primary and secondary metabolism, photosynthesis and plant-pathogen interactions. Conclusion Our global survey of transcriptional changes in TZSV infected tobacco provides crucial information into the precise molecular mechanisms underlying pathogenesis and symptom development. This is the first report on the relationships in the TZSV-plant interaction using transcriptome analysis. Findings of present study will significantly help enhance our understanding of the complicated mechanisms of plant responses to orthotospoviral infection

Development of a PVY Resistant Flue-Cured Tobacco Line via EMS Mutagenesis of eIF4E

Recessive resistance against potyviruses, such as Potato virus Y (PVY), relies on mutations in the eukaryotic translation initiation factor 4E (eIF4E) or one of its isoforms. The eIF4E1-S mutants of burley tobacco (Nicotiana tabacum L.) exhibit recessive resistance against PVY strains. Here, we developed a TILLING population of flue-cured tobacco (N. tabacum cv. Yunyan87) using ethyl methanesulfonate (EMS) to identify eIF4E1-S mutants. M3 plants homozygous for a nonsense mutation in exon 1 of the eIF4E1-S gene demonstrated resistance against PVYMN. These M3 plants were backcrossed to 'Yunyan87', and BC4F3 plants were screened using derived cleaved amplified polymorphic sequence (dCAPS) markers. BC(4)F(3)( )plants showing agronomic traits comparable to the recurrent parent 'Yunyan87' and resistance against PVYO, PVYN, and PVYNTN strains were identified. These genotypes would provide useful germplasm for future tobacco improvement and would aid in basic research on PVY resistance in flue-cured tobacco

Development of a nornicotine-reduced flue-cured tobacco line via EMS mutagenesis of nicotine N-demethylase genes

Substantial progress had been made in reducing nornicotine accumulation in burley tobacco, as nornicotine is a precursor of the carcinogen N-nitrosonornicotine (NNN). Three members of the CYP82E2 family encoding nicotine N-demethylase (NND) have been reported to be responsible for the majority of nicotine demethylation that forms nornicotine in burley tobacco. We had obtained a nonsense mutant of each NND member in flue-cured tobacco from an ethyl methanesulfonate (EMS)-mutagenized population. In this study, we developed dCAPS markers for each nonsense mutation. Using marker-assisted selection, NND mutants were crossed with each other to generate a triple mutant GP449. In line with previous reports, the triple knockout caused significantly decreased levels of nornicotine and NNN in flue-cured tobacco. With the decreased nornicotine, the nicotine level was expected to accumulate. However, the nicotine level in GP449 was significantly decreased to 72.80% of wild type. Realtime RT-PCR analysis showed that the nicotine reduction was correlated with inhibited expression of nicotine biosynthetic pathway genes. The triple mutant and dCAPS markers can be utilized to develop new flue-cured tobacco varieties with lower levels of nornicotine and NNN

Enhanced rutin accumulation in tobacco leaves by overexpressing the <i>NtFLS2</i> gene

<p>Rutin, one of the metabolites of the flavonoid pathway, shows great potential in industrial applications as a key component in pharmaceutical medicines and biological pesticides. Although the genetic manipulation of transcription factors (TFs) could increase rutin levels in plants, the accompanying accumulation of structurally similar chemicals complicates industrial rutin extraction. In this study, we demonstrated remarkably elevated rutin content (3.5-4.4-fold relative to controls) in transgenic tobacco plants by overexpressing <i>NtFLS2</i>. The levels of other intermediates in the branch pathway did not change much except for a moderate increase of kaempferol-3-<i>O</i>-rutinoside. Furthermore, the transcript levels of pathway genes in transgenic lines were comparable with controls, indicating genetic engineering did not significantly alter the branch pathway. Additionally, the transgenic tobacco plants appeared normal except for a flower color change from light red to white suggesting that it could be a valuable material for industrial extraction of rutin.</p

Additional file 4: of Characterization of Nicotiana tabacum genotypes possessing deletion mutations that affect potyvirus resistance and the production of trichome exudates

Figure S1. Typical examples of potyvirus infection of TI 1406 and K326-va plants transformed with 35S::eiF4E1.S construct or vector control (VC). Pictures were taken 14 days post-infection with PVYNN or TEV. (DOCX 54 kb

Additional file 1: of Characterization of Nicotiana tabacum genotypes possessing deletion mutations that affect potyvirus resistance and the production of trichome exudates

Table S1. Primer sequences. (DOCX 551 kb