Transcriptome analysis reveals the key role of overdominant expression of photosynthetic and respiration-related genes in the formation of tobacco(Nicotiana tabacum L.) biomass heterosis.

BACKGROUND: Leaves are the nutritional and economic organs of tobacco, and their biomass directly affects tobacco yield and the economic benefits of farmers. In the early stage, our research found that tobacco hybrids have more leaves and larger leaf areas, but the performance and formation reasons of biomass heterosis are not yet clear. RESULTS: This study selected 5 parents with significant differences in tobacco biomass and paired them with hybrid varieties. It was found that tobacco hybrid varieties have a common biomass heterosis, and 45 days after transplantation is the key period for the formation of tobacco biomass heterosis; By analyzing the biomass heterosis of hybrids, Va116×GDH94 and its parents were selected for transcriptome analysis. 76.69% of the differentially expressed genes between Va116×GDH94 and its parents showed overdominant expression pattern, and these overdominant expression genes were significantly enriched in the biological processes of photosynthesis and TCA cycle; During the process of photosynthesis, the overdominant up-regulation of genes such as Lhc, Psa, and rbcl promotes the progress of photosynthesis, thereby increasing the accumulation of tobacco biomass; During the respiratory process, genes such as MDH, ACO, and OGDH are overedominantly down-regulated, inhibiting the TCA cycle and reducing substrate consumption in hybrid offspring; The photosynthetic characteristics of the hybrid and its parents were measured, and the net photosynthetic capacity of the hybrid was significantly higher than that of the parents. CONCLUSION: These results indicate that the overdominant expression effect of differentially expressed genes in Va116×GDH94 and its parents plays a crucial role in the formation of tobacco biomass heterosis. The overdominant expression of genes related to photosynthesis and respiration enhances the photosynthetic ability of Va116×GDH94, reduces respiratory consumption, promotes the increase of biomass, and exhibits obvious heterosis.

Genome-wide identification and characterization of Glutathione S-Transferases (GSTs) and their expression profile under abiotic stresses in tobacco (Nicotiana tabacum L.).

BACKGROUND: Glutathione S-transferases (GSTs) are large and multifunctional proteases that play an important role in detoxification, protection against biotic and abiotic stresses, and secondary metabolite transportation which is essential for plant growth and development. However, there is limited research on the identification and function of NtGSTs. RESULTS: This study uses K326 and other six tobacco varieties (Hongda, HG, GDH11, Va116, VG, and GDH88) as materials to conduct comprehensive genome-wide identification and functional characterization of the GST gene in tobacco. A total of 59 NtGSTs were identified and classified into seven subfamilies via the whole-genome sequence analysis, with the Tau type serving as the major subfamily. The NtGSTs in the same branch of the evolutionary tree had similar exon/intron structure and motif constitution. There were more than 42 collinear blocks between tobacco and pepper, tomato, and potato, indicating high homology conservation between them. Twelve segmental duplicated gene pairs and one tandem duplication may have had a substantial impact on the evolution and expansion of the tobacco GST gene family. The RT-qPCR results showed that the expression patterns of NtGSTs varied significantly among tissues, varieties, and multiple abiotic stresses, suggesting that NtGST genes may widely respond to various abiotic stresses and hormones in tobacco, including NtGSTF4, NtGSTL1, NtGSTZ1, and NtGSTU40. CONCLUSIONS: This study provides a comprehensive analysis of the NtGST gene family, including structures and functions. Many NtGSTs play a critical regulatory role in tobacco growth and development, and responses to abiotic stresses. These findings offer novel and valuable insights for understanding the biological function of NtGSTs and the reference materials for cultivating highly resistant varieties and enhancing the yield and quality of crops.

The NtDEGP5 gene improves drought tolerance in tobacco (Nicotiana tabacum L.) by dampening plastid extracellular Ca2+ and flagellin signaling and thereby reducing ROS production.

Tobacco is an essential cash crop, but drought has become a major factor in the decline of global tobacco production as a result of changes in the global climate. The HtrA protease is an oligomeric serine endopeptidase that responds to stress in plants. DEGP5 is a member of the gene family that encodes HtrA protease, which promotes plant adaptation to adversity. The aim of this study was to investigate the role and mechanism employed by the DEGP5 gene in response to drought stress in tobacco. NtDEGP5-overexpression lines were obtained by genetic transformation and the phenotypes and transcriptomes of NtDEGP5-overexpression lines and wild-type (K326) tobacco seedlings were compared under drought stress. The results demonstrated that plants overexpressing NtDEGP5 exhibited greater drought tolerance. The differentially expressed genes involved in the regulation of drought tolerance by DEGP5 were enriched in metabolic pathways, such as plant-pathogen interaction and glutathione metabolism, with the plant-pathogen interaction pathway having the most differentially expressed genes. An analysis of the plant-pathogen interaction pathway revealed that these genes contributed to the suppression of plastid extracellular Ca2+ signaling and flagellin signaling to inhibit reactive oxygen species production, and that lower levels of reactive oxygen species act as a signal to regulate the activation of the antioxidant system, further balancing the production and removal of reactive oxygen species in tobacco seedlings under drought stress. These findings suggest that the NtDEGP5 gene can enhance the drought tolerance of tobacco by regulating the homeostasis of reactive oxygen species by inhibiting extracellular plastids.

Genome-wide identification and expression analysis of the ftsH protein family and its response to abiotic stress in Nicotiana tabacum L.

BACKGROUND: The filamentous temperature-sensitive H protease (ftsH) gene family plays an important role in plant growth and development. FtsH proteins belong to the AAA protease family. Studies have shown that it is a key gene for plant chloroplast development and photosynthesis regulation. In addition, the ftsH gene is also involved in plant response to stress. At present, the research and analysis of the ftsH gene family are conducted in microorganisms such as Escherichia coli and Oenococcus and various plants such as Arabidopsis, pear, rice, and corn. However, analysis reports on ftsH genes from tobacco (Nicotiana tabacum L.), an important model plant, are still lacking. Since ftsH genes regulate plant growth and development, it has become necessary to systematically study this gene in an economically important plant like tobacco. RESULTS: This is the first study to analyze the ftsH gene from Nicotiana tabacum L. K326 (NtftsH). We identified 20 ftsH genes from the whole genome sequence, renamed them according to their chromosomal locations, and divided them into eight subfamilies. These 20 NtftsH genes were unevenly distributed across the 24 chromosomes. We found four pairs of fragment duplications. We further investigated the collinearity between these genes and related genes in five other species. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis identified differential expression patterns of NtftsH in different tissues and under various abiotic stress conditions. CONCLUSIONS: This study provides a comprehensive analysis of the NtftsH gene family. The exon-intron structure and motif composition are highly similar in NtftsH genes that belong to the same evolutionary tree branch. Homology analysis and phylogenetic comparison of ftsH genes from several different plants provide valuable clues for studying the evolutionary characteristics of NtftsH genes. The NtftsH genes play important roles in plant growth and development, revealed by their expression levels in different tissues as well as under different stress conditions. Gene expression and phylogenetic analyses will provide the basis for the functional analysis of NtftsH genes. These results provide a valuable resource for a better understanding of the biological role of the ftsH genes in the tobacco plant.

Genome-wide identification and expression analysis of the bZIP transcription factor family genes in response to abiotic stress in Nicotiana tabacum L.

BACKGROUND: The basic leucine zipper (bZIP) transcription factor (TF) is one of the largest families of transcription factors (TFs). It is widely distributed and highly conserved in animals, plants, and microorganisms. Previous studies have shown that the bZIP TF family is involved in plant growth, development, and stress responses. The bZIP family has been studied in many plants; however, there is little research on the bZIP gene family in tobacco. RESULTS: In this study, 77 bZIPs were identified in tobacco and named NtbZIP01 through to NtbZIP77. These 77 genes were then divided into eleven subfamilies according to their homology with Arabidopsis thaliana. NtbZIPs were unevenly distributed across twenty-two tobacco chromosomes, and we found sixteen pairs of segmental duplication. We further studied the collinearity between these genes and related genes of six other species. Quantitative real-time polymerase chain reaction analysis identified that expression patterns of bZIPs differed, including in different organs and under various abiotic stresses. NtbZIP49 might be important in the development of flowers and fruits; NtbZIP18 might be an important regulator in abiotic stress. CONCLUSIONS: In this study, the structures and functions of the bZIP family in tobacco were systematically explored. Many bZIPs may play vital roles in the regulation of organ development, growth, and responses to abiotic stresses. This research has great significance for the functional characterisation of the tobacco bZIP family and our understanding of the bZIP family in higher plants.

Overdominant expression of related genes of ion homeostasis improves K+ content advantage in hybrid tobacco leaves.

BACKGROUND: Potassium(K+) plays a vital role in improving the quality of tobacco leaves. However, how to improve the potassium content of tobacco leaves has always been a difficult problem in tobacco planting. K+ content in tobacco hybrid is characterized by heterosis, which can improve the quality of tobacco leaves, but its underlying molecular genetic mechanisms remain unclear. RESULTS: Through a two-year field experiment, G70×GDH11 with strong heterosis and K326×GDH11 with weak heterosis were screened out. Transcriptome analyses revealed that 80.89% and 57.28% of the differentially expressed genes (DEGs) in the strong and weak heterosis combinations exhibited an overdominant expression pattern, respectively. The genes that up-regulated the overdominant expression in the strong heterosis hybrids were significantly enriched in the ion homeostasis. Genes involved in K+ transport (KAT1/2, GORK, AKT2, and KEA3), activity regulation complex (CBL-CIPK5/6), and vacuole (TPKs) genes were overdominant expressed in strong heterosis hybrids, which contributed to K+ homeostasis and heterosis in tobacco leaves. CONCLUSIONS: K+ homeostasis and accumulation in tobacco hybrids were collectively improved. The overdominant expression of K+ transport and homeostasis-related genes conducted a crucial role in the heterosis of K+ content in tobacco leaves.

Coumarins ameliorate diabetogenic action of dexamethasone via Akt activation and AMPK signaling in skeletal muscle.

Glucocorticoids are widely prescribed for lots of pathological conditions, however, can produce 'Cushingoid' side effects including central obesity, glucose intolerance, insulin resistance and so forth. Our study is intended to investigate the improving effects of coumarins on diabetogenic action of dexamethasone in vivo and in vitro and elucidate potential mechanisms. ICR mice treated with dexamethasone for 21 days exhibited decreased body weight, increased blood glucose and impaired glucose tolerance, which were prevented by fraxetin (40 mg/kg/day), esculin (40 mg/kg/day) and osthole (20 mg/kg/day), respectively. Esculin, fraxetin and osthole also could promote glucose uptake in normal C2C12 myotubes, and improve insulin resistance in myotubes induced by dexamethasone. Western blotting results indicated that esculin, fraxetin and osthole could boost Akt activation, stimulate GLUT4 translocation, thus alleviate insulin resistance. Esculin and osthole also could activate AMPK, thereby phosphorylate TBC1D1 at Ser237, and consequently ameliorate diabetogenic action of dexamethasone. Our study indicates coumarins as potential anti-diabetic candidates or leading compounds for drug development.

Overdominant expression of genes plays a key role in root growth of tobacco hybrids.

Heterosis has greatly improved the yield and quality of crops. However, previous studies often focused on improving the yield and quality of the shoot system, while research on the root system was neglected. We determined the root numbers of 12 F1 hybrids, all of which showed strong heterosis, indicating that tobacco F1 hybrids have general heterosis. To understand its molecular mechanism, we selected two hybrids with strong heterosis, GJ (G70 × Jiucaiping No.2) and KJ (K326 × Jiucaiping No.2), and their parents for transcriptome analysis. There were 84.22% and 90.25% of the differentially expressed genes were overdominantly expressed. The enrichment analysis of these overdominantly expressed genes showed that "Plant hormone signal transduction", "Phenylpropanoid biosynthesis", "MAPK signaling pathway - plant", and "Starch and sucrose metabolism" pathways were associated with root development. We focused on the analysis of the biosynthetic pathways of auxin(AUX), cytokinins(CTK), abscisic acid(ABA), ethylene(ET), and salicylic acid(SA), suggesting that overdominant expression of these hormone signaling pathway genes may enhance root development in hybrids. In addition, Nitab4.5_0011528g0020、Nitab4.5_0003282g0020、Nitab4.5_0004384g0070 may be the genes involved in root growth. Genome-wide comparative transcriptome analysis enhanced our understanding of the regulatory network of tobacco root development and provided new ideas for studying the molecular mechanisms of tobacco root development.

Integrated transcriptome and proteome analysis provides insights into the mechanism of cytoplasmic male sterility (CMS) in tobacco (Nicotiana tabacum L.).

Cytoplasmic male sterility (CMS) is critical in maximizing crop yield and quality by utilizing tobacco heterosis. However, the mechanism of tobacco CMS formation remains unknown. Using paraffin section observation, transcriptome sequencing, and TMT proteomic analysis, this study describes the differences in expression profiles in morphology, transcription, and translation between the sua-CMS tobacco line (MSYY87) and its corresponding maintainer line (YY87). According to the microspore morphology, MSYY87 began to exhibit abnormal microspore development during the early stages of germination and differentiation (androgynous primordium differentiation stage). According to transcriptomic and proteomic analyses, 17 genes/proteins involved in lipid transport/binding and phenylpropane metabolism were significantly down-regulated at both the mRNA and protein levels. Through further analysis, we identified some key genes that may be involved in tobacco male sterility, including ß-GLU related to energy metabolism, 4CL and bHLHs related to anther wall formation, nsLTPs related to pollen germination and anther cuticle, and bHLHs related to pollen tapetum degradation. We speculate that the down-regulation of these genes affects the normal physiological metabolism, making tobacco plants show male sterility. SIGNIFICANCE: Cytoplasmic male sterility (CMS) plays a vital role in utilizing tobacco heterosis and enhancing crop yield and quality. We observed paraffin sections and conducted transcriptome sequencing and mitochondrial proteomics to examine the tobacco CMS line Yunyan 87 (MSYY87) and its maintainer line Yunyan 87 (YY87). The down-regulation expression of ß-GLU resulted in insufficient ATP supply, which resulted in disordered energy metabolism. The down-regulation expression of 4CL, nsLTPs and bHLHs may affect the formation of anther wall and anther cuticle, pollen germination, as well as the degradation of pollen tapetum. These various abnormal physiological processes, the male sterility of tobacco is finally caused. The findings shed light on the molecular mechanisms of tobacco CMS and serve as a model for fertility research in other flowering plants.

Negative regulation of tobacco cold stress tolerance by NtPhyA.

Cold stress is a non-biological stressor that adversely affects tobacco yield and leaf quality. Plant photoreceptor proteins, which function as dual light-temperature sensors, play a vital role in temperature changes, making them crucial for responses to non-biological stressors. However, the regulatory mechanisms of PhyA in tobacco remain poorly understood. Therefore, in this study, we aimed to clone the NtPhyA gene from tobacco and generate overexpression (OE-NtPhyA) and mutant (KO-NtPhyA) constructs of NtPhyA. By assessing the physiological and biochemical responses of the mutants under cold stress and performing transcriptome sequencing, we determined the signalling mechanism of NtPhyA under cold stress. Comparative analysis with wild-type (WT) NtPhyA revealed that KO-NtPhyA exhibited increased seed germination rates and reduced wilting under cold stress. In additional, the degree of damage to leaf cells, cell membranes, and stomatal structures was mitigated, and the levels of reactive oxygen species (ROS) were significantly decreased. Antioxidant enzyme activity, net photosynthetic rate, and Fv/Fm were significantly enhanced in KO-NtPhyA, whereas the opposite effects were observed in OE-NtPhyA. These findings indicate that KO-NtPhyA augments tobacco tolerance to cold stress, implying a negative regulatory role of NtPhyA in tobacco during cold stress. Transcriptome analysis revealed that NtPhyA governs the expression of a cascade of genes involved in the response to oxygen-containing compounds, hydrogen peroxide (H2O2), ROS, temperature stimuli, photosystem II oxygen-evolving complex assembly, water channel activity, calcium channel activity, and carbohydrate transport. Collectively, our findings indicate that NtPhyA activates downstream gene expression to enhance the resilience of tobacco to cold stress.

Comparative transcriptome analysis between inbred lines and hybrids provides molecular insights into K+ content heterosis of tobacco (Nicotiana tabacum L.).

Potassium (K+) is essential for crop growth. Increasing the K+ content can often directly promote the improvement of crop yield and quality. Heterosis plays an important role in genetic improvement and leads to genetic gains. We found that the K+ content of tobacco showed significant heterosis, which is highly significant for cultivating tobacco varieties with high K+ content. However, the mechanism by which K+ content heterosis occurs in tobacco leaves is not clear. In this study, a comprehensive comparative transcriptome sequencing analysis of root samples from the hybrid G70 × GDH11 and its parental inbred lines G70 and GDH11 was performed to elucidate the importance of the root uptake capacity of K+ in the formation of heterosis. The results showed that 29.53% and 60.49% of the differentially expressed genes (DEGs) exhibited dominant and over-dominant expression patterns, respectively. These non-additive upregulated DEGs were significantly enriched in GO terms, such as metal ion transport and reaction, ion balance and homeostasis, ion channel activity, root meristem growth, and regulation of root hairs. The KEGG annotation results indicated that these genes were mainly involved in the pathways such as energy metabolism, carbohydrate formation, amino acid metabolism, and signal transduction. Further analysis showed that probable potassium transporter 17 (NtKT17) and potassium transporter 5-like (NtKT5), associated with potassium ion absorption, glutamate receptor 2.2-like and glutamate receptor 2.8-like, associated with ion channel activity, LOC107782957, protein detoxification 42-like, and probable glutamate carboxypeptidase 2, associated with root configuration, showed a significantly higher expression in the hybrids. These results indicated that the over-dominant expression pattern of DEGs played a key role in the heterosis of K+ content in tobacco leaves, and the overexpression of the genes related to K+ uptake, transport, and root development in hybrids helped to improve the K+ content of plants, thus showing the phenomenon of heterosis.

Proteomics and Co-expression Network Analysis Reveal the Importance of Hub Proteins and Metabolic Pathways in Nicotine Synthesis and Accumulation in Tobacco (Nicotiana tabacum L.).

Nicotine is a unique alkaloid present in tobacco that is widely used in cigarettes and in the agricultural, chemical, and pharmaceutical industries. However, the research on nicotine is mostly limited to its synthesis pathways, and only a few studies have explored the effects of other metabolic pathways on nicotine precursors. Regulating the nicotine content in tobacco can greatly promoting the application of nicotine in other fields. In this study, we performed global data-independent acquisition proteomics analysis of four tobacco varieties. Of the four varieties, one had high nicotine content and three had a low nicotine content. A total of 31,259 distinct peptides and 6,018 proteins across two samples were identified. A total of 45 differentially expressed proteins (DEPs) co-existed in the three comparison groups and were mainly involved in the transport and metallic processes of the substances. Most DEPs were enriched in the biosynthesis of secondary metals, glutathione metabolism, carbon metabolism, and glycolysis/gluconeogenesis. In addition, the weighted gene co-expression network analysis identified an expression module closely related to the nicotine content (Brown, r = 0.74, P = 0.006). Gene Ontology annotation and Kyoto Encyclopaedia of Genes and Genomes enrichment analysis showed that the module proteins were mainly involved in the synthesis and metabolism of nicotine precursors such as arginine, ornithine aspartate, proline, and glutathione. The increased levels of these precursors lead to the synthesis and accumulation of nicotine in plants. More importantly, these proteins regulate nicotine synthesis by affecting the formation of putrescine, which is the core intermediate product in nicotine anabolism. Our results provide a reference for tobacco variety selection with a suitable nicotine content and regulation of the nicotine content. Additionally, the results highlight the importance of other precursor metabolism in nicotine synthesis.

Effect of the over-dominant expression of proteins on nicotine heterosis via proteomic analysis.

Heterosis is a common biological phenomenon that can be used to optimize yield and quality of crops. Using heterosis breeding, hybrids with suitable nicotine content have been applied to tobacco leaf production. However, the molecular mechanism of the formation of nicotine heterosis has never been explained from the perspective of protein. The DIA proteomics technique was used to compare the differential proteomics of the hybrid Va116 × Basma, showing strong heterosis in nicotine content from its parent lines Va116 and Basma. Proteomics analysis indicated that 65.2% of DEPs showed over-dominant expression patterns, and these DEPs included QS, BBL, GS, ARAF and RFC1 which related to nicotine synthesis. In addition, some DEPs (including GST, ABCE2 and ABCF1 and SLY1) that may be associated with nicotinic transport exhibited significant heterosis over the parental lines. These findings demonstrated that the efficiency of the synthesis and transport of nicotine in hybrids was significantly higher than that in the parent lines, and the accumulation of over-dominant expression proteins may be the cause of heterosis of nicotinic content in hybrids.

Role of MS1 homolog Ntms1 gene of tobacco infertility.

The sterile line is the basis of crop heterosis utilization. To broaden the sources of male sterility in tobacco, the Ntms1 (Nicotiana tabacum L. ms1) gene was cloned from the tobacco variety K326 by homologous cloning based on the Cams1 (Capsicum annuum L. ms1) gene sequence of male-sterility genes in pepper. The protein structure and physicochemical properties of the two genes were determined by bioinformatics analysis, and the function of the Ntms1 gene was verified by the CRISPR/Cas9 system. The results showed that the sequences of Ntms1 and Cams1 were 85.25% similar, and plant homeodomains were found in both genes; the physical and chemical properties were also very similar. It is speculated that the Ntms1 gene had the same function as the Cams1 gene in controlling male sterility. Compared to the wild-type plants, the filaments of the Ntms1 knockout mutant plants were shorter, and the stamen was shorter than the pistil. The anthers did not develop fully and had few viable pollen grains; the tapetum and the anther wall had developed abnormally, and the anther chamber was severely squeezed. The malondialdehyde content in the mutant plants was significantly higher than that in the wild-type plants, while self-fertility was significantly lower in the mutant plants. The results showed that the Ntms1 gene plays an important role in regulating fertility in tobacco.