First report of Fusarium humuli causing stem spots in Nicotiana tabacum L in China.

Tobacco is one of the most important economic crops in China. Disease is one of the main factors affecting the quality of tobacco production (Cai et al. 2022). Stem spot disease of tobacco was observed in the Planting Demonstration Garden in Chang Ning (26°37N; 112° 31E), Hunan Province of China, from May to June 2023. The disease seriously retarded tobacco growth and the incidence rate was about 30-50% of the plants(Yun Yan 87). Most of infected tobacco had black spots on the stems, and the spots expanded and joined together quickly, while many stems turned black and withered. For pathogen isolation, symptomatic stem samples were collected and disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 1 minute, followed by rinsing with sterile distilled water three times. Subsequently, small pieces (5 × 5 mm) of diseased tissues were placed on potato dextrose agar (PDA) and incubated in the dark at 25 °C for 24 h to 36 h. The emerging fungal hyphal tips were transferred to PDA and purified by the single-spore method(Yu et al. 2022). In total, 16 cultures with the same appearance were isolated from 30 disease spots on the stem. Strain coded as hnxryc2 was randomly selected for identification. After culturing in PDA for 7 days, white and dense colonies wereobserved with a mean radial growth rate of 6.4 mm/day. The strain cultured 10 days on SNA. Morphological observations were made on 10-day-old culture on SNA medium, and macroconidia were sickle-shaped and slightly curved, with 3-5 septa (2.32-7.00 µm × 0.53-1.17 µm, n = 50), neither microconidia nor chlamydospores were observed. These morphological characteristics were consistent with the description of Fusarium humuli (Wang et al. 2019, Li et al. 2023). Furthermore, primers ITS1/ITS4, EF728F/EF986R, RPB1-F5/RPB1-R8 and fRPB2-5F2/fRPB2-7cR(Xie et al. 2023) were used to amplify the ITS region, EF-1α, RPB1, and RPB2 from strain hnxryc2, respectively. The sequence alignment of hnxryc2 with the NCBI database and FUSARIOID-ID shows the following results: The sequence of ITS region(GenBank accession number PP543715) was 100% identical to these of Fusarium sp. (MN428026.1), the sequences of EF-1α, RPB1, and RPB2 of strain hnxryc2(GenBank accession numbersOR257586, OR326856 and OR257587 respectively) were 99% to 100% identical to these of F. humuli (GenBank accession numbers MK289578.1, MZ824672.1 and MZ824673.1, respectively). Then a phylogenetic tree based on ITS region, EF-1α, and RPB2 sequences was constructed (Kroon et al. 2004). The strain hnxryc2 was more closely related to F. humuli (CGMCC3.19374 GenBank accession nos. MK280845.1, MK289570.1 and MK289724.1, respectively), with bootstrap values 88%. Pathogenicity tests were performed on detached stems of tobacco and potted plants. Wounded stems were inoculated with conidial suspensions (100 µL, 1×107 spores/mL), and the controls were inoculated with sterile water (Xu et al. 2023). The inoculated detached stems were kept in humid chambers (Zhong et al., 2019), each treatment was given a 12h/12h light/dark cycle at 25°C. Deep black spots were observed for 3 days after inoculation. After 9 days, typical symptoms similar to the original diseased plants in the field were found on all inoculated stems, while the control stems did not exhibit any symptoms. Pathogenicity assays were repeated thrice. The pathogen F. humuli was successfully reisolated from the stem of inoculated samples showing symptoms. To our knowledge, this is the first report of F. humuli inducing stem spot on tobacco in China. Since F. humuli is a common pathogenic fungus that infects different plant species, more attention should be paid to its prevalence in tobacco, and the potential risk of a disease outbreak in other provinces of China.

Functional and evolutionary comparative analysis of the DIR gene family in Nicotiana tabacum L. and Solanum tuberosum L.

BACKGROUND: The dirigent (DIR) genes encode proteins that act as crucial regulators of plant lignin biosynthesis. In Solanaceae species, members of the DIR gene family are intricately related to plant growth and development, playing a key role in responding to various biotic and abiotic stresses. It will be of great application significance to analyze the DIR gene family and expression profile under various pathogen stresses in Solanaceae species. RESULTS: A total of 57 tobacco NtDIRs and 33 potato StDIRs were identified based on their respective genome sequences. Phylogenetic analysis of DIR genes in tobacco, potato, eggplant and Arabidopsis thaliana revealed three distinct subgroups (DIR-a, DIR-b/d and DIR-e). Gene structure and conserved motif analysis showed that a high degree of conservation in both exon/intron organization and protein motifs among tobacco and potato DIR genes, especially within members of the same subfamily. Total 8 pairs of tandem duplication genes (3 pairs in tobacco, 5 pairs in potato) and 13 pairs of segmental duplication genes (6 pairs in tobacco, 7 pairs in potato) were identified based on the analysis of gene duplication events. Cis-regulatory elements of the DIR promoters participated in hormone response, stress responses, circadian control, endosperm expression, and meristem expression. Transcriptomic data analysis under biotic stress revealed diverse response patterns among DIR gene family members to pathogens, indicating their functional divergence. After 96 h post-inoculation with Ralstonia solanacearum L. (Ras), tobacco seedlings exhibited typical symptoms of tobacco bacterial wilt. The qRT-PCR analysis of 11 selected NtDIR genes displayed differential expression pattern in response to the bacterial pathogen Ras infection. Using line 392278 of potato as material, typical symptoms of potato late blight manifested on the seedling leaves under Phytophthora infestans infection. The qRT-PCR analysis of 5 selected StDIR genes showed up-regulation in response to pathogen infection. Notably, three clustered genes (NtDIR2, NtDIR4, StDIR3) exhibited a robust response to pathogen infection, highlighting their essential roles in disease resistance. CONCLUSION: The genome-wide identification, evolutionary analysis, and expression profiling of DIR genes in response to various pathogen infection in tobacco and potato have provided valuable insights into the roles of these genes under various stress conditions. Our results could provide a basis for further functional analysis of the DIR gene family under pathogen infection conditions.

Field Examinations on the Application of Novel Biochar-Based Microbial Fertilizer on Degraded Soils and Growth Response of Flue-Cured Tobacco (Nicotiana tabacum L.).

Southwestern China is receiving excessive chemical fertilizers to meet the challenges of continuous cropping. These practices are deteriorating the soil environment and affecting tobacco (Nicotiana tabacum L.) yield and quality adversely. A novel microbially enriched biochar-based fertilizer was synthesized using effective microorganisms, tobacco stalk biochar and basal fertilizer. A field-scale study was conducted to evaluate the yield response of tobacco grown on degraded soil amended with our novel biochar-based microbial fertilizer (BF). Four treatments of BF (0%, 1.5%, 2.5% and 5%) were applied in the contaminated field to grow tobacco. The application of BF1.5, BF2.5 and BF5.0 increased the available water contents by 9.47%, 1.18% and 2.19% compared to that with BF0 respectively. Maximum growth of tobacco in terms of plant height and leaf area was recorded for BF1.5 compared to BF0. BF1.5, BF2.5 and BF5.0 increased SPAD by 13.18-40.53%, net photosynthetic rate by 5.44-60.42%, stomatal conductance by 8.33-44.44%, instantaneous water use efficiency by 55.41-93.24% and intrinsic water use efficiency by 0.09-24.11%, while they decreased the intercellular CO2 concentration and transpiration rate by 3.85-6.84% and 0.29-47.18% relative to BF0, respectively (p < 0.05). The maximum increase in tobacco yield was recorded with BF1.5 (23.81%) compared to that with BF0. The present study concludes that the application of BF1.5 improves and restores the degraded soil by improving the hydraulic conductivity and by increasing the tobacco yield.

Superoxide Dismutase Premodulates Oxidative Stress in Plastids for Protection of Tobacco Plants from Cold Damage Ultrastructure Damage.

ROS-dependent induction of oxidative damage can be used as a trigger initiating genetically determined non-specific protection in plant cells and tissues. Plants are potentially able to withstand various specific (toxic, osmotic) factors of abiotic effects, but do not have sufficient or specific sensitivity to form an adequate effective response. In this work, we demonstrate one of the possible approaches for successful cold acclimation through the formation of effective protection of photosynthetic structures due to the insertion of the heterologous FeSOD gene into the tobacco genome under the control of the constitutive promoter and equipped with a signal sequence targeting the protein to plastid. The increased enzymatic activity of superoxide dismutase in the plastid compartment of transgenic tobacco plants enables them to tolerate the oxidative factor of environmental stresses scavenging ROS. On the other hand, the cost of such resistance is quite high and, when grown under normal conditions, disturbs the arrangement of the intrachloroplastic subdomains leading to the modification of stromal thylakoids, probably significantly affecting the photosynthesis processes that regulate the efficiency of photosystem II. This is partially compensated for by the fact that, at the same time, under normal conditions, the production of peroxide induces the activation of ROS detoxification enzymes. However, a violation of a number of processes, such as the metabolism of accumulation, and utilization and transportation of sugars and starch, is significantly altered, which leads to a shift in metabolic chains. The expected step for further improvement of the applied technology could be both the use of inducible promoters in the expression cassette, and the addition of other genes encoding for hydrogen peroxide-scavenging enzymes in the genetic construct that are downstream in the metabolic chain.

The oxidative pentose phosphate pathway in photosynthesis: a tale of two shunts.

CO2 release in the light (RL) and its presumed source, oxidative pentose phosphate pathways, were found to be insensitive to CO2 concentration. The oxidative pentose phosphate pathways form glucose 6-phosphate (G6P) shunts that bypass the nonoxidative pentose phosphate reactions of the Calvin-Benson cycle. Using adenosine diphosphate glucose and uridine diphosphate glucose as proxies for labeling of G6P in the stroma and cytosol respectively, it was found that only the cytosolic shunt was active. Uridine diphosphate glucose, a proxy for cytosolic G6P, and 6-phosphogluconate (6PG) were significantly less labeled than Calvin-Benson cycle intermediates in the light. But ADP glucose, a proxy for stromal G6P, is labeled to the same degree as Calvin-Benson cycle intermediates and much greater than 6PG. A metabolically inert pool of sedoheptulose bisphosphate can slowly equilibrate keeping the label in sedoheptulose lower than in other stromal metabolites. Finally, phosphorylation of fructose 6-phosphate (F6P) in the cytosol can allow some unlabeled carbon in cytosolic F6P to dilute label in phosphenolpyruvate. The results clearly show that there is oxidative pentose phosphate pathway activity in the cytosol that provides a shunt around the nonoxidative pentose phosphate pathway reactions of the Calvin-Benson cycle and is not strongly CO2-sensitive.

Effects of biochar treatments on the elemental composition of tobacco (Nicotiana tabacum L.) leaves based on the priming period.

This study determined the effects of different doses of biochars (B) on Virginia tobacco (Nicotiana tabacum L.) cultivar, on first and second harvest dependent change in plant nutrients (N, P, K, Ca, Mg, Cl, Zn, Fe, Mn, Cu, and B), leaf color parameters (L*, a*, and b*), chlorophyll value (SPAD), electrolyte leakage (EL), crude ash, number of leaves, and plant height. Pot experiments were conducted with biochar treatments of 10 tons ha-1 (B1), 20 tons ha-1 (B2), 40 tons ha-1 (B3), and 80 tons ha-1 (B4). Tobacco leaf macroelement (N, P, K, Ca, and Mg) levels increased with increasing biochar doses. The highest values were obtained for B4 treatments (80 tons ha-1) and the lowest for control (B0) treatments. Microelements (Fe, Zn, Mn, and B) exhibited a non linear change, while Cl and Cu exhibited a linear change. Color parameters (L*, a*, and b*) for the first and second priming showed the highest L* and b* values for B2 and B3 treatments, respectively, and the highest a* values for the B2 treatment. Leaf SPAD values increased with increasing biochar doses; further, the obtained SPAD values were ordered as B4 > B3 > B2 > B1 > B0. Leaf electrolyte leakage values were 25.90 %-37.25 % in the first priming and 26.90 %-40.59 % in the second priming. For both the primings, the highest crude ash values (21.94 % and 19.05 %) were observed for the B4 treatments, whereas the lowest values (17.89 % and 17.01 %) were observed for the B0 treatments. the tallest plant height (121.9 cm) and the highest number of leaves (45.3) were determined in B4 applications. Overall, considering the nutrition and quality of tobacco, B2 application is recommended.

The transporter PHO84/NtPT1 is a target of aluminum to affect phosphorus absorption in Saccharomyces cerevisiae and Nicotiana tabacum L.

The molecular mechanism of aluminum toxicity in biological systems is not completely understood. Saccharomyces cerevisiae is one of the most used model organisms in the study of environmental metal toxicity. Using an unbiased metallomic approach in yeast, we found that aluminum treatment caused phosphorus deprivation, and the lack of phosphorus increased as the pH of the environment decreased compared to the control strain. By screening the phosphate signaling and response pathway (PHO pathway) in yeast with the synthetic lethality of a new phosphorus-restricted aluminum-sensitive gene, we observed that pho84Δ mutation conferred severe growth defect to aluminum under low-phosphorus conditions, and the addition of phosphate alleviated this sensitivity. Subsequently, the data showed that PHO84 determined the intracellular aluminum-induced phosphorus deficiency, and the expression of PHO84 was positively correlated with aluminum stress, which was mediated by phosphorus through the coordinated regulation of PHO4/PHO2. Moreover, aluminum reduced phosphorus absorption and inhibited tobacco plant growth in acidic media. In addition, the high-affinity phosphate transporter NtPT1 in tobacco exhibited similar effects to PHO84, and overexpression of NtPT1 conferred aluminum resistance in yeast cells. Taken together, positive feedback regulation of the PHO pathway centered on the high-affinity phosphate transporters is a highly conservative mechanism in response to aluminum toxicity. The results may provide a basis for aluminum-resistant microorganisms or plant engineering and acidic soil treatment.

Comprehensive transcriptome and WGCNA analysis reveals the potential function of anthocyanins in low-temperature resistance of a red flower mutant tobacco.

The anthocyanin is a protective substance in various plants, and plays important roles in resisting to low-temperature. Here, we explored transcriptome analysis of pink flower (as CK) and the natural mutant red flower (as research objects) under low-temperature conditions, and aimed to reveal the potential functions of anthocyanins and anthocyanin-related regulatory factors in resistance to low-temperature. Our results showed that most of the differentially expressed genes (DEGs) encoding key enzymes in the late stage of anthocyanin metabolism in the mutant were significantly up-regulated. Meanwhile, several genes significantly differentially expressed in CK or mutant were obtained by classification and analysis of transcription factors (TFs), phytohormones and osmoregulators. Additionally, WGCNA was carried out to mine hub genes resistanted to low-temperature stress in flavonoid pathway. Finally, one UFGT family gene, three MYB and one bHLH were obtained as the future hub genes of this study. Combined with the above information, we concluded that the ability of the red flower mutant to grow and develop normally at low-temperatures was the result of a combination of flavonoids and cold resistance genes.

Integrated physiological and omics analyses reveal the mechanism of beneficial fungal Trichoderma sp. alleviating cadmium toxicity in tobacco (Nicotiana tabacum L.).

Cadmium (Cd) is a highly toxic heavy metal and readily accumulates in tobacco, which imperils public health via Cd exposure from smoking. Beneficial microbes have a pivotal role in promoting plant growth, especially under environmental stresses such as heavy metal stresses. In this study, we introduced a novel fungal strain Trichoderma nigricans T32781, and investigated its capacity to alleviate Cd-induced stress in tobacco plants through comprehensive physiological and omics analyses. Our findings revealed that T32781 inoculation in soil leads to a substantial reduction in Cd-induced growth inhibition. This was evidenced by increased plant height, enhanced biomass accumulation, and improved photosynthesis, as indicated by higher values of key photosynthetic parameters, including the maximum quantum yield of photosystem Ⅱ (Fv/Fm), stomatal conductance (Gs), photosynthetic rate (Pn) and transpiration rate (Tr). Furthermore, element analysis demonstrated that T. nigricans T32781 inoculation resulted in a remarkable reduction of Cd uptake by 62.2% and a 37.8% decrease in available soil Cd compared to Cd-stressed plants without inoculation. The protective role of T32781 extended to mitigating Cd-induced oxidative stress by improving antioxidant enzyme activities of superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX). Metabolic profiling of tobacco roots identified 43 key metabolites, with notable contributions from compounds like nicotinic acid, succinic acid, and fumaric acid in reducing Cd toxicity in T32781-inoculated plants. Additionally, rhizosphere microbiome analysis highlighted the promotion of beneficial microbes, including Gemmatimonas and Sphingomonas, by T32781 inoculation, which potentially contributed to the restoration of plant growth under Cd exposure. In summary, our study demonstrated that T. nigricans T32781 effectively alleviated Cd stress in tobacco plants by reducing Cd uptake, alleviating Cd-induced oxidative stress, influencing plant metabolite and modulating the microbial composition in the rhizosphere. These findings offer a novel perspective and a promising candidate strain for enhancing Cd tolerance and prohibiting its accumulation in plants to reduce health risks associated with exposure to Cd-contaminated plants.

Biochemical Responses to the Long-Term Impact of Copper Sulfate (CuSO4) in Tobacco Plants.

Metabolic changes under stress are often studied in short-term experiments, revealing rapid responses in gene expression, enzyme activity, and the amount of antioxidants. In a long-term experiment, it is possible to identify adaptive changes in both primary and secondary metabolism. In this study, we characterized the physiological state of tobacco plants and assessed the amount and spectrum of phenolic compounds and the lignification of axial organs under excess copper stress in a long-term experiment (40 days). Plants were treated with 100 and 300 µM CuSO4, as well as a control (Knop solution). Copper accumulation, the size and anatomical structure of organs, stress markers, and the activity of antioxidant enzymes were studied. Lignin content was determined with the cysteine-assisted sulfuric method (CASA), and the metabolite profile and phenolic spectrum were determined with UHPLC-MS and thin-layer chromatography (TLC). Cu2+ mainly accumulated in the roots and, to a lesser extent, in the shoots. Copper sulfate (100 µM) slightly stimulated stem and leaf growth. A higher concentration (300 µM) caused oxidative stress; H2O2 content, superoxide dismutase (SOD), and guaiacol peroxidase (GPOX) activity increased in roots, and malondialdehyde (MDA) increased in all organs. The deposition of lignin increased in the roots and stems compared with the control. The content of free phenolics, which could be used as substrates for lignification, declined. The proportions of ferulic, cinnamic, and p-coumaric acids in the hydrolysate of bound phenolics were higher, and they tended toward additional lignification. The metabolic profile changed in both roots and stems at both concentrations, and changed in leaves only at a concentration of 300 µM. Thus, changes in the phenolic spectrum and the enhanced lignification of cell walls in the metaxylem of axial (root and stem) organs in tobacco can be considered important metabolic responses to stress caused by excess CuSO4.

Valorisation of hydrothermal liquefaction wastewater in agriculture: effects on tobacco plants and rhizosphere microbiota.

Industrial wastewater obtained from hydrothermal liquefaction (HTL-WW) of food wastes for biofuels production could represent a source of crop nutrients since it is characterized by a high amount of organic and inorganic compounds. In the present work, the potential use of HTL-WW as irrigation water for industrial crops was investigated. The composition of the HTL-WW was rich in nitrogen, phosphorus, and potassium with high level of organic carbon. A pot experiment with Nicotiana tabacum L. plants was conducted using diluted wastewater to reduce the concentration of some chemical elements below the official accepted threshold values. Plants were grown in the greenhouse under controlled conditions for 21 days and irrigated with diluted HTL-WW every 24 hours. Soils and plants were sampled every seven days to evaluate, over time, the effect of wastewater irrigation both on soil microbial populations, through high-throughput sequencing, and plant growth parameters, through the measurement of different biometric indices. Metagenomic results highlighted that, in the HTL-WW treated rhizosphere, the microbial populations shifted via their mechanisms of adaptation to the new environmental conditions, establishing a new balance among bacterial and fungal communities. Identification of microbial taxa occurring in the rhizosphere of tobacco plants during the experiment highlighted that the HTL-WW application improved the growth of Micrococcaceae, Nocardiaceae and Nectriaceae, which included key species for denitrification, organic compounds degradation and plant growth promotion. As a result, irrigation with HTL-WW improved the overall performance of tobacco plants which showed higher leaf greenness and increased number of flowers compared to irrigated control plants. Overall, these results demonstrate the potential feasibility of using of HTL-WW in irrigated agriculture.

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.

Temporal transcriptome analysis reveals several key pathways involve in cadmium stress response in Nicotiana tabacum L.

Tobacco has a strong cadmium (Cd) enrichment capacity, meaning that it can absorb large quantities from the environment, but too much Cd will cause damage to the plant. It is not yet clear how the plant can dynamically respond to Cd stress. Here, we performed a temporal transcriptome analysis of tobacco roots under Cd treatment from 0 to 48 h. The number of differentially expressed genes (DEGs) was found to change significantly at 3 h of Cd treatment, which we used to define the early and middle stages of the Cd stress response. The gene ontology (GO) term analysis indicates that genes related to photosynthesis and fatty acid synthesis were enriched during the early phases of the stress response, and in the middle phase biological process related to metal ion transport, DNA damage repair, and metabolism were enriched. It was also found that plants use precursor mRNA (pre-mRNA) processes to first resist Cd stress, and with the increasing of Cd treatment time, the overlapped genes number of DEGs and DAS increased, suggesting the transcriptional levels and post-transcriptional level might influence each other. This study allowed us to better understand how plants dynamically respond to cadmium stress at the transcriptional and post-transcriptional levels and provided a reference for the screening of Cd-tolerant genes in the future.

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.

Evaluation of reference genes for miRNA and mRNA normalization in tobacco infected with PVYNTN, PVYN-Wi and PVYZ-NTN strains.

This is the first report on identification of the most suitable reference genes for RT-qPCR quantification of miRNA and mRNA in tobacco response to the prevalent recombinant potato virus Y (PVY) strains PVYNTN, PVYN-Wi and the newly identified PVYZ-NTN. Of 10 tested genes, the expression levels of neIF5C, nU2af and nPP2A were the most stable in samples taken from non-inoculated, mock-inoculated, and infected plants at three days post-inoculation (dpi) and 14 dpi. While the homologues of eIF5 were most stably expressed in tobacco in this study and in potato in our previous study (Yin et al., 2021) following inoculation with the same three PVY strains, the homologues of other two genes PP2A and U2af were stably expressed only in tobacco but unstable in potato. The tobacco homologue of PP2A, which was the most stably expressed one in tobacco interaction with PVYNTN, PVYN-Wi and PVYZ-NTN strains in this study, was the least stable one in tobacco interaction with the non-recombinant PVYO strain in a previous study (Baek et al., 2017). This study provides evidence on the influence of host species on expression of housekeeping genes and points out virus strain as a new factor influencing expression stability of reference gene. Caution should be taken when choosing reference genes in gene expression study in Solanaceae hosts response to different PVY strains.

The NtSPL Gene Family in Nicotiana tabacum: Genome-Wide Investigation and Expression Analysis in Response to Cadmium Stress.

The SQUAMOSA promoter binding protein-like (SPL)SPL family genes play an important role in regulating plant growth and development, synthesis of secondary metabolites, and resistance to stress. Understanding of the role of the SPL family in tobacco is still limited. In this study, 42 NtSPL genes were identified from the genome of the tobacco variety TN90. According to the results of the conserved motif and phylogenetic tree, the NtSPL genes were divided into eight subgroups, and the genes in the same subgroup showed similar gene structures and conserved domains. The cis-acting element analysis of the NtSPL promoters showed that the NtSPL genes were regulated by plant hormones and stresses. Twenty-eight of the 42 NtSPL genes can be targeted by miR156. Transcriptome data and qPCR results indicated that the expression pattern of miR156-targeted NtSPL genes was usually tissue specific. The expression level of miR156 in tobacco was induced by Cd stress, and the expression pattern of NtSPL4a showed a significant negative correlation with that of miR156. These results suggest that miR156-NtSPL4a may mediate the tobacco response to Cd stress. This study lays a foundation for further research on the function of the NtSPL gene and provides new insights into the involvement of NtSPL genes in the plant response to heavy metal stress.

Transcriptional multiomics reveals the mechanism of seed deterioration in Nicotiana tabacum L. and Oryza sativa L.

INTRODUCTION: Mature seeds deteriorate gradually and die eventually during long-term storage. Controlled deterioration is often used to accelerate the seed deterioration rate to assess the seed vigor and physiological quality of seed lots. OBJECTIVES: Although it is well known that the process of seed deterioration produced by controlled deterioration is distinct from that caused by long-term storage, the differences in transcriptional levels have not been reported. Clarifying the mechanism of seed deterioration is critical for identifying, conserving and utilizing germplasm resources. METHODS: Tobacco (Nicotiana tabacum L.) seeds were studied thoroughly using transcriptome, small RNA, and degradome sequencing after long-term storage (LS) and controlled deterioration (CD). Co-expression trend analysis identified transcripts involved in tobacco seed deterioration, while phylogenetic analysis helped to uncover comparable targets in rice (Oryza sativa L.) for further verification and utilization. RESULTS: In LS and CD, a total of 2,112 genes and 164 miRNAs were differentially expressed, including 20 interaction miRNA-mRNA pairs with contrasting expression. Transcriptional multiomics found that the main causes of LS were plant hormone signal transduction and protein processing in the endoplasmic reticulum, whereas the primary cause of CD was nucleotide excision repair dysfunction. The homeostatic balance of RNA degradation and the spliceosome occurred in both modes of seed deterioration. Additionally, co-expression trend analysis identified two coherent pairs, nta-miR160b-NtARF18 and nta-miR396c-NtMBD10, as being significant in LS and CD, respectively. For utilization, rice homologous targets OsARF18 and OsMBD707 were verified to play similar roles in LS and CD, respectively. CONCLUSION: This study demonstrated the transcriptional mechanism of tobacco and key genes in seed deterioration. And the application of key genes in rice also verified the feasibility of the multiomics method, guiding the identification of candidate genes to precisely delay seed deterioration in other species of seed research.

The antifeedant and antifungal cryptic metabolites isolated from tobacco endophytes induced by host medium and coculture.

Four new cryptic metabolites including one fumagillol derivative (2), one cyclohexenone derivative (4), one 10-membered lactone (5), and one natural 4-epi-brefeldin C (8), along with seven known compounds were found from isogenesis endophytes Aspergillus fumigatus, Penicillium janthinellum, Nigrospora sp., and Stagonosporopsis sp. induced by host Nicotiana tabacum medium and co-culture. The structures were determined mainly by spectroscopic methods, including extensive 1D, 2D NMR, MS techniques, ECD calculation, and Mosher's method. Compound 2 possessed a novel 1, 3-dioxetane residue and cyclohexane-containing terpenoid skeleton. Compounds 2, 4-7 and 10 showed significant antifungal activities against the plant pathogen Nigrospora sp. with MICs of 1 µg/mL. 2, 4, 5-7, and 10 indicated antifungal activities against Penicillium janthinellum, Aspergillus fumigatus, Phomopsis sp., and Alternaria sp. with MICs ≤8 µg/mL. Compounds 2, 6-8, and 10 (50 µg/cm2) and microbial fermentation extracts (100 µg/cm2) showed antifeedant activities against silkworms with feeding deterrence indices of 21-100%.

Silver Nanoparticle Effects on Antioxidant Response in Tobacco Are Modulated by Surface Coating.

The antimicrobial properties of silver and enhanced reactivity when applied in a nanoparticle form (AgNPs) led to their growing utilization in industry and various consumer products, which raises concerns about their environmental impact. Since AgNPs are prone to transformation, surface coatings are added to enhance their stability. AgNP phytotoxicity has been mainly attributed to the excess generation of reactive oxygen species (ROS), leading to the induction of oxidative stress. Herein, in vitro-grown tobacco (Nicotiana tabacum) plants were exposed to AgNPs stabilized with either polyvinylpyrrolidone (PVP) or cetyltrimethylammonium bromide (CTAB) as well as to ionic silver (AgNO3), applied in the same concentrations, either alone or in combination with cysteine, a strong silver ligand. The results show a higher accumulation of Ag in roots and leaves after exposure to AgNPs compared to AgNO3. This was correlated with a predominantly higher impact of nanoparticle than ionic silver form on parameters of oxidative stress, although no severe damage to important biomolecules was observed. Nevertheless, all types of treatments caused mobilization of antioxidant machinery, especially in leaves, although surface coatings modulated the activation of its specific components. Most effects induced by AgNPs or AgNO3 were alleviated with addition of cysteine.

The Role of Alternative Oxidase in the Interplay between Nitric Oxide, Reactive Oxygen Species, and Ethylene in Tobacco (Nicotiana tabacum L.) Plants Incubated under Normoxic and Hypoxic Conditions.

The transgenic tobacco (Nicotiana tabacum L.) plants with the modified levels of alternative oxidase (AOX) were used to evaluate the physiological roles of AOX in regulating nitro-oxidative stress and metabolic changes after exposing plants to hypoxia for 6 h. Under normoxia, AOX expression resulted in the decrease of nitric oxide (NO) levels and of the rate of protein S-nitrosylation, while under hypoxia, AOX overexpressors exhibited higher NO and S-nitrosylation levels than knockdowns. AOX expression was essential in avoiding hypoxia-induced superoxide and H2O2 levels, and this was achieved via higher activities of catalase and glutathione reductase and the reduced expression of respiratory burst oxidase homolog (Rboh) in overexpressors as compared to knockdowns. The AOX overexpressing lines accumulated less pyruvate and exhibited the increased transcript and activity levels of pyruvate decarboxylase and alcohol dehydrogenase under hypoxia. This suggests that AOX contributes to the energy state of hypoxic tissues by stimulating the increase of pyruvate flow into fermentation pathways. Ethylene biosynthesis genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and ethylene-responsive factors (ERFs) were induced during hypoxia and correlated with AOX and NO levels. We conclude that AOX controls the interaction of NO, reactive oxygen species, and ethylene, triggering a coordinated downstream defensive response against hypoxia.