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.

Overexpression of the WRKY transcription factor gene NtWRKY65 enhances salt tolerance in tobacco (Nicotiana tabacum).

BACKGROUND: Salt stress severely inhibits plant growth, and the WRKY family transcription factors play important roles in salt stress resistance. In this study, we aimed to characterize the role of tobacco (Nicotiana tabacum) NtWRKY65 transcription factor gene in salinity tolerance. RESULTS: This study characterized the role of tobacco (Nicotiana tabacum) NtWRKY65 transcription factor gene in salinity tolerance using four NtWRKY65 overexpression lines. NtWRKY65 is localized to the nucleus, has transactivation activity, and is upregulated by NaCl treatment. Salinity treatment resulted in the overexpressing transgenic tobacco lines generating significantly longer roots, with larger leaf area, higher fresh weight, and greater chlorophyll content than those of wild type (WT) plants. Moreover, the overexpressing lines showed elevated antioxidant enzyme activity, reduced malondialdehyde content, and leaf electrolyte leakage. In addition, the Na+ content significantly decreased, and the K+/Na+ ratio was increased in the NtWRKY65 overexpression lines compared to those in the WT. These results suggest that NtWRKY65 overexpression enhances salinity tolerance in transgenic plants. RNA-Seq analysis of the NtWRKY65 overexpressing and WT plants revealed that NtWRKY65 might regulate the expression of genes involved in the salt stress response, including cell wall component metabolism, osmotic stress response, cellular oxidant detoxification, protein phosphorylation, and the auxin signaling pathway. These results were consistent with the morphological and physiological data. These findings indicate that NtWRKY65 overexpression confers enhanced salinity tolerance. CONCLUSIONS: Our results indicated that NtWRKY65 is a critical regulator of salinity tolerance in tobacco plants.

NtGCN2 confers cadmium tolerance in Nicotiana tabacum L. by regulating cadmium uptake, efflux, and subcellular distribution.

General control non-derepressible-2 (GCN2) is widely expressed in eukaryotes and responds to biotic and abiotic stressors. However, the precise function and mechanism of action of GCN2 in response to cadmium (Cd) stress in Nicotiana tabacum L. (tobacco) remains unclear. We investigated the role of NtGCN2 in Cd tolerance and explored the mechanism by which NtGCN2 responds to Cd stress in tobacco by exposing NtGCN2 transgenic tobacco lines to different concentrations of CdCl2. NtGCN2 was activated under 50 µmol·L-1 CdCl2 stress and enhanced the Cd tolerance and photosynthetic capacities of tobacco by increasing chlorophyll content and antioxidant capacity by upregulating NtSOD, NtPOD, and NtCAT expression and corresponding enzyme activities and decreasing malondialdehyde and O2·- contents. NtGCN2 enhanced the osmoregulatory capacity of tobacco by elevating proline (Pro) and soluble sugar contents and maintaining low levels of relative conductivity. Finally, NtGCN2 enhanced Cd tolerance in tobacco by reducing Cd uptake and translocation, promoting Cd efflux, and regulating Cd subcellular distribution. In conclusion, NtGCN2 improves the tolerance of tobacco to Cd through a series of mechanisms, namely, increasing antioxidant, photosynthetic, and osmoregulation capacities and regulating Cd uptake, translocation, efflux, and subcellular distribution. This study provides a scientific basis for further exploration of the role of NtGCN2 in plant responses to Cd stress and enhancement of the Cd stress signaling network in tobacco.

Characterization of the DUF868 gene family in Nicotiana and functional analysis of NtDUF868-E5 involved in pigment metabolism.

Domains of unknown function (DUF) proteins represent a large group of uncharacterized protein families. The DUF868 gene family in Nicotiana has not yet been described. In the present study, we identified 12, 11, and 25 DUF868 family members in the genome of Nicotiana sylvestris, N. tomentosiformis, and N. tabacum, respectively. Based on phylogenetic analysis, these were categorized into five groups (A-E). Within each group, the gene structures, motifs, and tertiary structures showed high similarity. NtDUF868 family expansion during evolution was mainly driven by segmental duplication events. MicroRNA (miRNA) target site prediction identified 12 miRNA members that target 16 NtDUF868 family genes. The promoters of these genes contain cis-regulatory elements responsive to light, phytohormones, and abiotic stresses. Expression profiling revealed their tissue- and stage-specific expression patterns. RNA-sequencing and quantitative reverse transcription PCR revealed that the NtDUF868 family genes are potentially involved in the response to abiotic and biotic stresses, particularly drought and hormone stresses, and in the resistance to black shank and bacterial wilt. We generated transformed plants using NtDUF868-E5 overexpression and gene-editing vectors. NtDUF868-E5 overexpression resulted in enhanced tobacco plant growth and development, leading to increased leaf photosynthetic capacity and higher chlorophyll and carotenoid contents. This study provided a comprehensive genome-wide analysis of the DUF868 gene family, shedding light on their potential roles in plant growth and stress responses.

Hydrogel-potassium humate composite alleviates cadmium toxicity of tobacco by regulating Cd bioavailability.

Cadmium (Cd) removal from soil to reduce Cd accumulation in plants is essential for agroecology, food safety, and human health. Cd enters plants from soil and affects plant growth and development. Hydrogels can easily combine with Cd, thereby altering its bioavailability in soil. However, few studies have evaluated the effects of hydrogel on the complex phytotoxicity caused by Cd uptake in plants and the microbial community structure. Herein, a new poly (acrylic acid)-grafted starch and potassium humate composite (S/K/AA) hydrogel was added to soil to evaluate its impact on tobacco growth and the soil microenvironment. The results indicate that the addition of S/K/AA hydrogel can significantly improve the biomass, chlorophyll (Chl) content, and photosynthetic capacity of tobacco plants during Cd stress conditions, and decrease Cd concentration, probably by affecting Cd absorption through the expression of Cd absorption transporters (e.g., NRAMP5, NRAMP3, and IRT1). Moreover, the application of S/K/AA hydrogel not only reduced the accumulation of reactive oxygen species (ROS), but also reduced the antioxidant activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), suggesting that S/K/AA hydrogel alleviates Cd toxicity via a non-antioxidant pathway. Notably, we further analyzed the effectiveness of the hydrogel on microbial communities in Cd-contaminated soil and found that it increased the Cd-tolerant microbial community (Arthrobacter, Massilia, Streptomyces), enhancing the remediation ability of Cd-contaminated soil and helping tobacco plants to alleviate Cd toxicity. Overall, our study provides primary insights into how S/K/AA hydrogel affects Cd bioavailability and alleviates Cd toxicity in plants.

NtHSP70-8b positively regulates heat tolerance and seed size in Nicotiana tabacum.

Heat stress considerably restricts the geographical distribution of crops and affects their growth, development, and productivity. HSP70 plays a critical regulatory role in plant growth response to heat stress. However, the mechanisms of this regulatory remain poorly understood. Here, an HSP70 gene, NtHSP70-8b, which is involved in the heat stress response of tobacco, was cloned and identified. The expression of NtHSP70-8b was induced by exogenous abscisic acid (ABA) treatment and abiotic stress, including heat, drought, and salt. Notably, high NtHSP70-8b expression occurred under heat stress conditions, which was consistent with the ß-glucuronidase histochemical analysis. Moreover, NtHSP70-8b overexpression markedly enhanced heat stress tolerance by changing the stomatal conductance and antioxidant capacity in tobacco leaves. qRT-PCR showed that the expression levels of ABA synthesis and response genes (NtNCED3 and NtAREB), stress defence genes (NtERD10C and NtLEA5), and other HSP genes (NtHSP90 and NtHSP26a) in NtHSP70-8b-overexpressing tobacco were high under heat stress. The interaction of NtHSP70-8b with NtHSP26a was further confirmed by a luciferase complementation imaging assay. In contrast, NtHSP70-8b knockout mutants showed significantly reduced antioxidant capacity compared to the wild type (WT) under heat stress conditions, suggesting that NtHSP70-8b acts as a positive regulator of heat stress in tobacco. Moreover, NtHSP70-8b overexpression increased the 1000-seed weight. Taken together, NtHSP70-8b is involved in the heat stress response, and NtHSP70-8b overexpression contributed to enhanced tolerance to heat stress, which is thus an essential gene with potential application value for developing heat stress-tolerant crops.

A non-specific lipid transfer protein, NtLTPI.38, positively mediates heat tolerance by regulating photosynthetic ability and antioxidant capacity in tobacco.

Non-specific lipid transfer proteins (nsLTPs) play an important role in plant growth and stress resistance; however, their function in tobacco remains poorly understood. Therefore, to explore the function of NtLTP in response to high temperature, we identified an NtLTPI.38 from tobacco, obtained its overexpression and knockout transgenic plants, and further studied their response to heat stress (42 °C). The results showed that NtLTPI.38 overexpression in tobacco reduced chlorophyll degradation, alleviated the high temperature damage to photosynthetic organs, and enhanced the photosynthetic capacity of tobacco under heat stress. NtLTPI.38 overexpression in heat-stressed tobacco increased the contents of soluble sugar and protein, proline, and flavonoid substances, reduced the relative conductivity, and decreased H2O2, O2•-, and MDA accumulation, and increased the enzymatic antioxidant activities, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), compared to wild type (WT) and knockout mutant plants. RT-PCR confirmed that the expression levels of antioxidant enzymes and thermal stress-related genes were significantly upregulated under thermal stress in overexpression plants. Therefore, NtLTPI.38 enhanced heat tolerance in tobacco by mitigating photosynthetic damage and improving osmoregulation and antioxidant capacity. These results provided the theoretical basis and a potential resource for further breeding projects to improve heat tolerance in plants.

NtLTPI.38, a plasma membrane-localized protein, mediates lipid metabolism and salt tolerance in Nicotiana tabacum.

Non-specific lipid transfer proteins (nsLTPs) typically have conserved structural resemblance, low sequence identity, and broad biological functions in plant growth and stress resistance. Here, a plasma membrane-localized nsLTP, NtLTPI.38, was identified in tobacco plants. Multi-omics integrated analysis revealed that NtLTPI.38 overexpression or knock out significantly changed glycerophospholipid and glycerolipid metabolism pathways. NtLTPI.38 overexpression remarkably increased phosphatidylcholine, phosphatidylethanolamine, triacylglycerol, and flavonoid levels, but decreased ceramides compared to wild type and mutant lines. Differentially expressed genes were associated with lipid metabolite and flavonoid synthesis. Many genes related to Ca2+ channels, abscisic acid (ABA) signal transduction, and ion transport pathways were upregulated in overexpressing plants. NtLTPI.38 overexpression in salt-stressed tobacco triggered a Ca2+ and K+ influx in leaves, increased the contents of chlorophyll, proline, flavonoids, and osmotic tolerance, and raised enzymatic antioxidant activities as well as the expression level of related genes. However, mutants accumulated more O2- and H2O2, exhibited ionic imbalance, gathered excess Na+, Cl-, and malondialdehyde, with more severe ion leakage. Therefore, NtLTPI.38 enhanced salt tolerance in tobacco by regulating lipid and flavonoid synthesis, antioxidant activity, ion homeostasis, and ABA signaling pathways.

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.

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.

Selenium Modulates the Level of Auxin to Alleviate the Toxicity of Cadmium in Tobacco.

Cadmium (Cd) is an environmental pollutant that potentially threatens human health worldwide. Developing approaches for efficiently treating environmental Cd is a priority. Selenium (Se) plays important role in the protection of plants against various abiotic stresses, including heavy metals. Previous research has shown that Se can alleviate Cd toxicity, but the molecular mechanism is still not clear. In this study, we explore the function of auxin and phosphate (P) in tobacco (Nicotiana tabacum), with particular focus on their interaction with Se and Cd. Under Cd stress conditions, low Se (10 µM) significantly increased the biomass and antioxidant capacity of tobacco plants and reduced uptake of Cd. We also measured the auxin concentration and expression of auxin-relative genes in tobacco and found that plants treated with low Se (10 µM) had higher auxin concentrations at different Cd supply levels (0 µM, 20 µM, 50 µM) compared with no Se treatment, probably due to increased expression of auxin synthesis genes and auxin efflux carriers. Overexpression of a high affinity phosphate transporter NtPT2 enhanced the tolerance of tobacco to Cd stress, possibly by increasing the total P and Se content and decreasing Cd accumulation compared to that in the wild type (WT). Our results show that there is an interactive mechanism among P, Se, Cd, and auxin that affects plant growth and may provide a new approach for relieving Cd toxicity in plants.

Expression, purification, and in vitro characterization of kinase domain of NtGCN2 from tobacco.

General control nonderepressible 2 (GCN2) can phosphorylate the α subunit of eukaryotic initiation factor eIF2 (eukaryotic translation initiation factor 2) to down-regulateprotein synthesis in response to various biotic and abiotic stresses. However, the kinase activity of plant GCN2 has not been well-characterized in vitro. In this study, the kinase domain of Nicotiana tabacum GCN2 (NtGCN2) was inserted into the pET15b vector for prokaryotic expressionin Escherichia coli BL21-CodonPlus-(DE3)-RIPL after induction by 0.5 mmol L-1 IPTG for 13 h at 16 °C. The soluble protein was collected and purified by Ni2+-NTA agarose column, anion exchange, and molecular sieve, and the purified proteinwas used for kinase assays and the preparation of a polyclonal antibody. Enzyme-linked immunosorbent assay results showed that the titer of the antiserum was 1:520K. Western blot analysis showed that the prepared antibody reacted with GCN2 in tobacco. Additionally, the kinase activity of NtGCN2 was characterized by using recombinant NteIF2α protein as a substrate in vitro. The results showed that NtGCN2 phosphorylated NteIF2α in vitro, with the level of phosphorylation positively correlated with the NtGCN2 concentration and reaction time. Our study has prepared a specific antibody, and proves NtGCN2 can phosphorylate NteIF2α in vitro, which lays a foundation for further study of the function and interaction network of NtGCN2.

Metagenomic insights into effects of wheat straw compost fertiliser application on microbial community composition and function in tobacco rhizosphere soil.

The application of fertilisers incorporated with plant residues improves nutrient availability in soils, which shifts the microbial community structure and favours plant growth. To understand the impact of wheat straw compost fertiliser on soil properties and microbial community structure, tobacco planting soils were treated with four different fertilisers using varied amounts of straw compost fertiliser and a no fertiliser control (CK). Results showed that different fertilisers affected available soil nutrient contents differently. Treatment of tobacco soil with application of combined chemical fertiliser/wheat straw compost led to improved soil chemical properties, and increased soil organic matter and available phosphorus and potassium content. Treatment with FT1 200 kg/mu straw was found to be superior in improving soil fertility. Metagenomic DNA sequencing revealed that different fertiliser treatments resulted in changes in the microbial community composition. In soil treated with FT2 300 kg/mu straw for 60 days, the predominant bacterial phyla were Proteobacteria, Actinobacteria, and Verrucomicrobia, whereas Cyanobacteria, Basidiomycota, and Chlorophyta were found in high abundance in soil samples treated with FT1 200 kg/mu straw for 30 days. Functional annotation of metagenomic sequences revealed that genes involved in metabolic pathways were among the most abundant type. PCoA analysis clearly separated the samples containing straw compost fertiliser and chemical fertiliser. A significant correlation between soil properties and the dominant phyla was identified.

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.

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.

Characterization of wheat TaSnRK2.7 promoter in Arabidopsis.

MAIN CONCLUSION: Expression of TaSnRK2.7 promoter is strongly induced under abiotic stress and could be used as a valuable tool for improving plant stress resistance via transgenic techniques. The sucrose non-fermenting 1-related protein kinase 2 (SnRK2) gene family plays pivotal roles in response to abiotic stresses (drought, salinity and cold). Here, we studied the expression of five wheat TaSnRK2.7 promoter-5'-deletion constructs (- 2547, - 1621, - 806, - 599, and - 254) fused to beta-glucuronidase (GUS) in Arabidopsis. Tissue-expression analysis revealed that the - 254 to ATG fragment was sufficient for inducing GUS expression in hypocotyls. Additionally, the - 806 to - 599 and - 2547 to - 1621 fragments contained leaf- and root-specific elements, respectively. Deletion analysis showed that these fragments were unresponsive to ABA treatment, suggesting that TaSnRK2.7 participates in an ABA-independent signaling pathway. Assays examining stress responses of constructs demonstrated that the - 599 to - 254 and - 806 to - 599 fragments contained elements responsive to abiotic and osmotic stress, respectively. The TaSnRK2.7 promoter contained enhancers from - 806 to - 254 and - 2547 to - 1621, while the - 1621 to - 806 fragment contained negative regulatory elements that restrict root and leaf gene expression in response to abiotic stress. Furthermore, under drought and salt stress, the TaSnRK2.7 promoter conferred greater gene expression in leaves than the rd29A promoter, even though both were induced by abiotic stress. These findings enhance our understanding of the molecular mechanisms behind TaSnRK2.7 action, which should prove useful in transgenic studies investigating stress-induced gene expression.

Overexpression of Tobacco GCN2 Stimulates Multiple Physiological Changes Associated With Stress Tolerance.

General control non-derepressible-2 (GCN2) is a ubiquitous protein kinase that phosphorylates the α subunit of the eukaryotic initiation factor, eIF2, preventing the initiation of a new cycle of protein synthesis, subsequently reducing the global protein biosynthesis. GCN2 can also regulate the response of plants to biotic and abiotic stresses. In this study, two GCN2 homologs, NtGCN2-1 and NtGCN2-2, were cloned from Nicotiana tabacum, and were predicted to have been derived from their progenitors in N. tomentosiformis and N. sylvestris, respectively. The phosphorylation of NteIF2α could be activated by promoting the expression of NtGCN2 with plant hormones, including salicylic acid (SA), azelaic acid (AZA), methyl jasmonate (MeJA), and by imposition of different stresses (Bemisia tabaci infection, drought, and cold), indicating that NtGCN2 is involved in the response of plants to multiple biotic and abiotic stresses. We also observed that the overexpression of NtGCN2-1 significantly influenced different physiological processes. It promoted seed germination and root elongation. The content of total soluble sugars and reducing sugars were decreased, whereas those of chlorophyll a and b were increased in the GCN2 overexpressing plants. In addition, the overexpressing plants had lower content of reactive oxygen species and exhibited higher antioxidant activities. These physiological alterations could be attributed to the changes in the endogenous phytohormones, decrease in the SA and abscisic acid content, and accumulation of MeJA and AZA. It indicated that the overexpression of NtGCN2 in tobacco, stimulated the plant defense responses via phosphorylation of NteIF2α and regulation of plant hormones, and changes in the antioxidant ability and plant nutrient status.

Metabolic Flux Engineering of Cembratrien-ol Production in Both the Glandular Trichome and Leaf Mesophyll in Nicotiana tabacum.

Cembratrien-ol synthase (CBTS) catalyzes the first step in cembranoid biosynthesis, producing cembratrien-ols in plant trichomes. In our previous study, microarray transcriptomes between leaves with trichomes and leaves without trichomes showed that an NtCBTS2 gene was expressed exclusively and abundantly in trichomes. Here, two NtCBTS2 isogenes (NtCBTS2a and NtCBTS2b), derived from a diploid genome donor, Nicotiana sylvestris, were identified from N. tabacum. Both genes were expressed primarily in trichomes, with relatively decreased transcription in flowers and stems, and faint expression in roots, and no expression was detected in leaves lacking trichomes. To demonstrate the feasibility of producing natural product cembratrien-ols in tobacco mesophylls, the mesophylls of 35S:NtCBTS2b transgenic tobacco plants were used in the analysis, suggesting that constitutive expression of NtCBTS2b led to the cembratrien-ol production in mesophylls. Overexpression of NtCBTS2b using either Cauliflower mosaic virus (CaMV) 35S or trichome-specific Cyt P450 oxygenase (CYP) promoters greatly increased aphid resistance by promoting the accumulation of CBT-ols, increased the secretory cell growth in glandular trichomes and increased the levels of various physiological measures, including sugar esters, gibberellins, and cembranoid production. Meanwhile, specifically overexpressing NtCBTS2b in glandular trichomes could most efficiently promote aphid resistance in tobacco plants. Notably, our results indicate the feasibility of utilizing bio-engineering to produce large amounts of CBT-ols, and modify significantly the composition of naturally produced CBT-ols and CBT-diols, thereby promoting aphid resistance in plants.

Liquid-liquid microextraction of synthetic pigments in beverages using a hydrophobic deep eutectic solvent.

A method was developed for the determination of eight synthetic pigments in beverage samples by liquid-liquid microextraction followed by high performance liquid chromatography. Using hydrophobic deep eutectic solvent (DES) as the microextraction solvent, several key parameters were optimized, including the type and volume of the hydrophobic DES, pH value, vortex time and salt content. Detection limits were in the range 0.016-1.12 ng/mL, recoveries were in the range 74.5-102.5% and relative standard deviations were <5.4%. The method is simple, green and practical, and could be applied to the extraction and determination of synthetic pigments in beverages.

OsPht1;8, a phosphate transporter, is involved in auxin and phosphate starvation response in rice.

The responses of plants to auxin and phosphate (Pi) starvation are closely linked. However, the underlying mechanisms connecting the Pi starvation (-Pi) responses to auxin are largely unclear. Here, we show that OsPht1;8 (OsPT8), a phosphate transporter, functions in both the auxin and -Pi responses in rice (Oryza sativa L.) and tobacco (Nicotiana tabacum). The overexpression of OsPT8 (OsPT8-Oe) led to the loss of sensitivity to auxin and -Pi in adventitious roots, lateral roots, and root hairs in rice. The expression levels of OsPT8 and pOsPT8::GUS staining in roots, root-shoot junctions and leaves of rice were induced by IAA treatments. The number of young lateral roots in the OsPT8-Oe transgenic rice, which had higher auxin concentrations, was distinctly more than that in the wild-type, possibly as a result of increased expression of auxin-related genes under normal Pi condition. Moreover, tobacco overexpressing OsPT8 had a similar root phenotype to OsPT8-Oe rice. These data reveal a novel biological function of OsPT8 in the cross-talk between Pi and auxin signaling, and provide new evidence for the linkage between auxin and -Pi responses.