NtARF11 positively regulates cadmium tolerance in tobacco by inhibiting expression of the nitrate transporter NtNRT1.1.

Heavy metal cadmium (Cd) is widespread in contaminated soil and an important factor limiting plant growth. NO3- (nitrate) affects Cd uptake and thus changes Cd tolerance in plants; however, the underlying molecular regulatory mechanisms have not yet been elucidated. Here, we analyzed a novel gene, NtARF11 (auxin response factor), which regulates Cd tolerance in tobacco via the NO3- uptake pathway, through experiments with NtARF11-knockout and NtARF11-overexpression transgenic tobacco lines. NtARF11 was highly expressed under Cd stress in tobacco plants. Under Cd stress, overexpression of NtARF11 enhanced Cd tolerance in tobacco compared to that in wild-type tobacco, as shown by the low Cd concentration, high chlorophyll concentration, and low accumulation of reactive oxygen species in NtARF11-overexpressing tobacco. Moreover, low NO3- concentrations were observed in NtARF11-overexpressing tobacco plants. Further analyses revealed direct binding of NtARF11 to the promoter of the nitrate transporter NtNRT1.1, thereby negatively regulating its expression in tobacco. Notably, NtNRT1.1 knockout reduced NO3- uptake, which resulted in low Cd concentrations in tobacco. Altogether, these results demonstrate that the NtARF11-NtNRT1.1 module functions as a positive regulator of Cd tolerance by reducing the Cd uptake in tobacco, providing new insights for improving Cd tolerance of plants through genetic engineering.

Dynamic changes in fungal communities and functions in different air-curing stages of cigar tobacco leaves.

Introduction: Air curing (AC) plays a crucial role in cigar tobacco leaf production. The AC environment is relatively mild, contributing to a diverse microbiome. Fungi are important components of the tobacco and environmental microbiota. However, our understanding of the composition and function of fungal communities in AC remains limited. Methods: In this study, changes in the chemical constituents and fungal community composition of cigar tobacco leaves during AC were evaluated using flow analysis and high-throughput sequencing. Results: The moisture, water-soluble sugar, starch, total nitrogen, and protein contents of tobacco leaves exhibited decreasing trends, whereas nicotine showed an initial increase, followed by a decline. As determined by high-throughput sequencing, fungal taxa differed among all stages of AC. Functional prediction showed that saprophytic fungi were the most prevalent type during the AC process and that the chemical composition of tobacco leaves is significantly correlated with saprophytic fungi. Conclusion: This study provides a deeper understanding of the dynamic changes in fungal communities during the AC process in cigar tobacco leaves and offers theoretical guidance for the application of microorganisms during the AC process.

Melatonin alleviates cadmium toxicity and abiotic stress by promoting glandular trichome development and antioxidant capacity in Nicotiana tabacum.

Melatonin is a well-known signaling molecule that mediates a range of physiological activities and various stress reactions in plants. We comprehensively tested the effect of melatonin on the development of root hairs and glandular trichomes and found that melatonin pretreatment of tobacco seeds significantly increased the length of root hairs. Furthermore, melatonin-treated tobacco exhibited significantly higher density of trichomes and larger glandular heads on long-stalk glandular trichomes than untreated plants, which resulted in enhanced secretion in glandular trichomes. Exogenous melatonin enhanced the aphid resistance of plants by facilitating the accumulation of cembranoids in the glandular trichomes and alleviated cadmium toxicity by increasing the Cd-exudation capacity of long glandular trichomes. Metabolic analysis indicated that the contents of 108 metabolites significantly changed upon melatonin treatment, with the contents of those that are directly/indirectly involved in melatonin metabolism changing the most. Further, KEGG pathway analysis suggested that the metabolic pathways of amino acids, reducing sugar, secondary metabolites, indole alkaloid biosynthesis, purine, pyrimidine, and ABC transporters were greatly influenced by exogenous melatonin application. Moreover, metabolisms of melatonin-related antioxidants and pyrimidine nucleoside antibiotics were enhanced after melatonin treatment. Melatonin improved tobacco resistance to high salinity, drought, and extreme temperature stresses, as indicated by improved photosynthetic and antioxidant capacities in treated vs. untreated plants. This study lays a foundation for the comprehensive application of melatonin to increase the stress tolerance of plants.

Mutation of NtNRAMP3 improves cadmium tolerance and its accumulation in tobacco leaves by regulating the subcellular distribution of cadmium.

Cadmium (Cd) is a harmful element that affects plant growth and development. Genetic improvements could be applied for enhancing Cd tolerance and accumulation in plants. Here, a novel Cd stress-induced gene, NtNRAMP3, was identified in tobacco. We constructed two NtNRAMP3-knockout (KO) tobacco lines using the CRISPR/Cas9 system, which enhanced Cd tolerance and Cd accumulation in tobacco leaves compared with those in the wildtype (WT). Subcellular localization analysis suggested that NtNRAMP3 is a tonoplast protein and GUS (ß-glucuronidase) histochemical analysis showed that NtNRAMP3 is highly expressed in the conductive tissue of leaves. NtNRAMP3-KO tobacco showed reduced Cd translation from vacuole to cytosol in leaves compared with the WT, and its vacuolar Cd concentration was significantly higher (20.78-22.81%) than that in the WT; in contrast, Cd concentration in the cytosol was reduced by 13.72-20.15%, preventing chlorophyll degradation and reducing reactive oxygen species accumulation in the leaves. Our findings demonstrate that NtNRAMP3 is involved in regulating Cd subcellular distribution (controlling Cd transport from vacuoles to the cytosol) and affects Cd tolerance and its accumulation in tobacco. This provides a key candidate gene to improve the phytoremediation efficiency of plants via genetic engineering.

Changes of lignin biosynthesis in tobacco leaves during maturation.

Lignin is one of the most valuable renewable industrial materials. To elucidate the mechanism via which lignin is synthesised, we compared the lignin content, leaf hardness, cell wall thickness of palisade tissue, and gene expression patterns of lignin biosynthetic enzymes in three tobacco (Nicotiana tabacum L.) varieties during maturation. The results consistently showed that during maturation, the accumulation of lignin gradually increased in tobacco leaves, reaching a peak at full maturity (45 days after topping), and then gradually decreased. Similarly, the transcript level analysis revealed that the gene expression pattern of NtPAL, NtC4H, NtCCoAOMT and NtCOMT were relatively high, and consistent with the lignin content changes. Thus, the four genes may play regulatory roles in the synthesis of tobacco lignin. Analysis of tissue expression patterns of the lignin synthesis-related gene showed that the NtPAL, NtC4H, Nt4CL, NtHCT, NtCCoAOMT, NtCOMT, NtCCR, NtCAD, and NtPAO were all expressed in stems, roots, and leaves. NtC3H and NtF5H were specifically expressed in stems and roots, and not in leaves. Consistently, the NtC3H promoter induced high GUS expression in stems and petioles, marginal in roots, and no GUS activity in leaves. These results provide insights into molecular regulation of lignin biosynthesis in tobacco.

Overexpression of OsPT8 Increases Auxin Content and Enhances Tolerance to High-Temperature Stress in Nicotiana tabacum.

Temperature is a primary factor affecting the rate of plant development; as the climate warms, extreme temperature events are likely to increasingly affect agriculture. Understanding how to improve crop tolerance to heat stress is a key concern. Wild plants have evolved numerous strategies to tolerate environmental conditions, notably the regulation of root architecture by phytohormones, but the molecular mechanisms of stress resistance are unclear. In this study, we showed that high temperatures could significantly reduce tobacco biomass and change its root architecture, probably through changes in auxin content and distribution. Overexpression of the OsPT8 phosphate transporter enhanced tobacco tolerance to high-temperature stress by changing the root architecture and increased the antioxidant ability. Molecular assays suggested that overexpression of OsPT8 in tobacco significantly increased the expression of auxin synthesis genes NtYUCCA 6, 8 and auxin efflux carriers genes NtPIN 1,2 under high-temperature stress. We also found that the expression levels of auxin response factors NtARF1 and NtARF2 were increased in OsPT8 transgenic tobacco under high-temperature stress, suggesting that OsPT8 regulates auxin signaling in response to high-temperature conditions. Our findings provided new insights into the molecular mechanisms of plant stress signaling and showed that OsPT8 plays a key role in regulating plant tolerance to stress conditions.

Genome-wide identification and expression analysis of HSP90 gene family in Nicotiana tabacum.

BACKGROUND: Heat shock proteins 90 (HSP90s) are a highly conserved protein family of cellular chaperones widely found in plants; they play a fundamental role in response to biotic and abiotic stresses. The genome-wide analysis of HSP90 gene family has been completed for some species; however, it has been rarely reported for the tobacco HSP90 genes. RESULTS: In this study, we systematically conducted genome-wide identification and expression analysis of the tobacco HSP90 gene family, including gene structures, evolutionary relationships, chromosomal locations, conserved domains, and expression patterns. Twenty-one NtHSP90s were identified and classified into eleven categories (NtHSP90-1 to NtHSP90-11) based on phylogenetic analysis. The conserved structures and motifs of NtHSP90 proteins in the same subfamily were highly consistent. Most NtHSP90 proteins contained the ATPase domain, which was closely related to conserved motif 2. Motif 5 was a low complexity sequence and had the function of signal peptide. At least 6 pairs of NtHSP90 genes underwent gene duplication, which arose from segment duplication and tandem duplication events. Phylogenetic analysis showed that most species expanded according to their own species-specific approach during the evolution of HSP90s. Dynamic expression analysis indicated that some NtHSP90 genes may play fundamental roles in regulation of abiotic stress response. The expression of NtHSP90-4, NtHSP90-5, and NtHSP90-9 were up-regulated, while NtHSP90-6, and NtHSP90-7 were not induced by ABA, drought, salt, cold and heat stresses. Among the five treatments, NtHSP90s were most strongly induced by heat stress, and weakly activated by ABA treatment. There was a similar response pattern of NtHSP90s under osmotic stress, or extreme temperature stress. CONCLUSIONS: This is the first genome-wide analysis of Hsp90 in N. tabacum. These results indicate that each NtHSP90 member fulfilled distinct functions in response to various abiotic stresses.

Genome-wide identification and characterization of Hsp70 gene family in Nicotiana tabacum.

Heat shock proteins 70 (Hsp70) constitute a highly conserved protein family of cellular chaperones widely distributed in plants, where they play a fundamental role in response to biotic and abiotic stress. Until now, genome-wide analyses of the Hsp70 gene family have been conducted for some species. However, reports about Hsp70 genes in Nicotiana tabacum are scarce. In this study, we systematically conducted genome-wide identification and expression analysis of the Hsp70 gene family in tobacco, including gene structure, classification, evolutionary relationships, promoters, and transcript levels in response to abiotic stress treatments. In all, 61 Hsp70 members were identified and classified into six groups that were mapped onto 18 chromosomes, where most were distributed on both ends of the chromosome. The conserved structures and motifs of NtHsp70 proteins in the same subfamily were highly consistent. At least 15 pairs of NtHsp70 genes underwent gene duplication by segment and tandem duplications. Most NtHsp70 proteins contained N-terminal hexokinase conserved motifs. Phylogenetic analysis showed that most species expanded according to their own species-specific approach during the evolution of Hsp70s. Tissue-specific expression analysis indicated that all NtHsp70 genes were involved in at least one or more abiotic stress responses, highlighting the wide participation of NtHsp70 genes in environmental adaptation. This is the first genome-wide analysis of Hsp70 in N. tabacum. These results indicate that each NtHsp70 member fulfilled distinct functions in response to various abiotic stresses.

NtMYB12 Positively Regulates Flavonol Biosynthesis and Enhances Tolerance to Low Pi Stress in Nicotiana tabacum.

Phosphorus (P) is an essential macronutrient for plant growth and development. The concentration of flavonol, a natural plant antioxidant, is closely related to phosphorus nutritional status. However, the regulatory networks of flavonol biosynthesis under low Pi stress are still unclear. In this study, we identified a PFG-type MYB gene, NtMYB12, whose expression was significantly up-regulated under low Pi conditions. Overexpression of NtMYB12 dramatically increased flavonol concentration and the expression of certain flavonol biosynthetic genes (NtCHS, NtCHI, and NtFLS) in transgenic tobacco. Moreover, overexpression of NtMYB12 also increased the total P concentration and enhanced tobacco tolerance of low Pi stress by increasing the expression of Pht1-family genes (NtPT1 and NtPT2). We further demonstrated that NtCHS-overexpressing plants and NtPT2-overexpressing plants also had increased flavonol and P accumulation and higher tolerance to low Pi stress, showing a similar phenotype to NtMYB12-overexpressing transgenic tobacco under low Pi stress. These results suggested that tobacco NtMYB12 acts as a phosphorus starvation response enhancement factor and regulates NtCHS and NtPT2 expression, which results in increased flavonol and P accumulation and enhances tolerance to low Pi stress.