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MYB transcription factors (TFs) play important roles in plant growth and development, metabolic regulation, and stress responses. In the current study, 258 2R‐MYB TFs were identified in Nicotiana tabacum and systemically analyzed. The 2R‐NtMYB TFs were unevenly distributed on 23 chromosomes and were classified into 33 subgroups according to the Arabidopsis thaliana 2R‐MYB classification. The promoters of the 2R‐NtMYB harbor numerous potential cis‐acting elements involved in plant growth, metabolism, hormone signaling, and stress responsiveness. Chip‐based expression profiling of different tissues and growth stages revealed that the 2R‐NtMYBs showed constitutive or spatiotemporally specific expressions. RNA‐sequencing data indicated that some 2R‐NtMYBs, including NtMYB16, NtMYB27, NtMYB35, and NtMYB170, are involved in the response to one or several abiotic and biotic stresses, namely drought and resistance to black shank and bacterial wilt. Quantitative PCR results further revealed that NtMYBs respond to different hormones (abscisic acid, indole acetic acid, gibberellic acid, methyl jasmonate, and salicylic acid) and confirmed their involvement in abiotic stress responses like heat, drought, and salt. NtMYB35 overexpression and mutant lines generated using CRISPR‐Cas9 gene editing were used to verify the important role of NtMYB35 in regulating tobacco growth, pigment metabolism, and aphid resistance. These results provide a detailed overview of this TF family in N. tabacum and valuable insights for future functional analyses of NtMYB TFs. [ABSTRACT FROM AUTHOR]

Heavy metal cadmium (Cd) is widespread in contaminated soil and an important factor limiting plant growth. NO 3 - (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 NO 3 - 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 NO 3 - 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 NO 3 - 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. [Display omitted] • NtARF11 is highly expressed in tobacco roots under Cd stress and low N stress. • Overexpression of NtARF11 reduces Cd uptake by inhibiting the expression of nitrate transporter NtNRT1.1. • Overexpression of NtARF11 increases total Cd accumulation in tobacco shoots and roots. • The NtARF11- NtNRT1.1 module acts as a positive regulator in tobacco Cd tolerance. [ABSTRACT FROM AUTHOR]

In order to study the role of nitrate transporter (NRT) in the uptake and transport of nitrate in tobacco, a nitrate transporter gene encoding 612 amino acids with a total length of 1 839 bp was cloned using the cDNA of tobacco cultivar K326 as a template and named as NtNRT1.7. Bioinformatics analysis indicated that NtNRT1.7 had typical transmembrane domains. The homology analysis showed that the homology of NtNRT1.7 and Arabidopsis thaliana AtNRT1.7 was 57.23%. Subcellular localization results showed that this protein was located on the cell membrane and was a typical membrane protein. Promoter analysis and QRT-PCR results of different stress treatments indicated that NtNRT1.7 gene was highly expressed in mature leaves, its expression was suppressed by low nitrogen and enhanced by drought, low temperature, high salt and other stresses, which suggested that NtNRT1.7 gene might be involved in nitrate recycle under abiotic stresses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • A newly characterized hydrogel-potassium humate composite (S/K/AA) has been synthesized. • S/K/AA reduce Cd accumulation in tobacco by reducing soil bioavailable Cd concentration and soil bacterial community structure. • S/K/AA affect the expression of tobacco Cd uptake and transport genes under Cd stress conditions. • S/K/AA can improve photosynthetic capacity and affect the antioxidant system of tobacco under Cd stress conditions. [ABSTRACT FROM AUTHOR]

Selenite is the predominant form of selenium (Se) available to plants in soils, which is uptake by inorganic phosphate (Pi) transporters. Previous studies have suggested that selenite might affect plants’ growth and development, but the molecular mechanisms connecting Pi, Se, and auxin functions are still unclear. Here, we show that Se is involved in auxin and phosphate starvation responses in tobacco ( Nicotiana tabacum ). Low Se (1 mg L −1 ) addition increased the biomass and changed the architecture of tobacco root (increased primary root length and the number of lateral roots) compared to control treatments without adding Se, and Se treated plants also had higher auxin concentrations, possibly as a result of the increased expression of auxin synthesis genes and auxin efflux carriers. Moreover, low Se (1 mg L −1 ) enhanced tolerance to low Pi stress, and the overexpression of NtPT2 in tobacco increased its total P and Se contents under Pi-sufficient conditions. These data revealed there is an interactive mechanism among Pi, Se and auxin acting upon plant growth, and provide a potential candidate gene ( NtPT2 ) for breeding Se-enriched tobacco plants. [ABSTRACT FROM AUTHOR]

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 bioengineering 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. [ABSTRACT FROM AUTHOR]

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 (β-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. [Display omitted] • A novel Cd transporter gene NtNRAMP3 was identified in Nicotiana tabacum. • NtNRAMP3 was confirmed to modulate Cd-tolerance in tobacco. • Knockout of NtNRAMP3 improves Cd-tolerance by reducing the Cd transport from vacuole to cystsol in tobacco leaves. • NtNRAMP3 effects leaf chlorophyll degradation and ROS accumulation in tobacco under Cd stress conditions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • 12, 11, and 25 DUF868 were identified from Nicotiana sylvestris , N. tomentosiformis , and N. tabacum , respectively, and systematically analyzed. • NtDUF868 gene family show their tissue and stage-specific expression patterns and play potential roles in response to abiotic and biotic stresses. • NtDUF868-E5 overexpression can promote tobacco growth and development, improve leaf photosynthetic capacity, and increase chlorophyll and carotenoids contents. [ABSTRACT FROM AUTHOR]

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 β-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. • NtHSP70-8b was high expression under heat stress condition. • NtHSP70-8b is involved in the regulation of heat tolerance in tobacco. • NtHSP70-8b overexpression tobacco could increase antioxidant capacity and seed size. • NtHSP70-8b interacted with NtHSP26a. [ABSTRACT FROM AUTHOR]

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 O 2 − and H 2 O 2, 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. • NtLTPI.38 is a plasma membrane - localized protein • Metabolomics, lipidomics, and transcriptomics revealed that NtLTPI.38 overexpression in tobacco significantly increased phosphatidylethanolamine, triacylglycerol, phosphatidylcholine, and flavonoid, but decreased ceramides levels. • NtLTPI.38 overexpression changed the gene expression levels related to Ca2+ channels, ABA signal transduction, and ion transport pathways, triggered a Ca2+ and K+ influx in tobacco leaves. • NtLTPI.38 enhances salt tolerance in tobacco by regulating lipid and flavonoid synthesis, antioxidant activity, ion homeostasis, and ABA signaling pathways. [ABSTRACT FROM AUTHOR]

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 H 2 O 2 , O 2 •−, 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. [Display omitted] • NtLTPI.38 overexpression and knockout transgenic plants were established. • NtLTPI.38 overexpression in tobacco reduced the chlorophyll degradation, alleviated the high temperature damage to photosynthetic organs and enhanced the photosynthesis capacity under heat stress. • NtLTPI.38 overexpression reduced tobacco oxidative damage, enhanced heat tolerance through increasing the contents of proline and flavonoid substances, decreasing ROS and MDA accumulation, and increasing the enzymatic antioxidant activities. [ABSTRACT FROM AUTHOR]

Selenite is the predominant form of selenium (Se) available to plants in soils. Recent studies have suggested that selenite uptake is probably mediated by Pi transporters. Research on the molecular mechanism of Pi and Se uptake by plants is important for the development of Se-enriched agricultural products. In this study, we report the function of a Pi transporter, OsPT8, which was not only involved in Pi uptake but also in Se uptake in tobacco. Overexpression of OsPT8 in tobacco significantly increased the total P concentration under Pi-sufficient conditions, and the biomass of OsPT8-Oe lines was higher than that of the wild type under Pi-deficient conditions. OsPT8 overexpression not only increased the Pi concentration, but also resulted in greater Se accumulation in transgenic tobacco compared with the wild type under a high Pi concentration. Expression analysis showed that the Pi transporter genes NtPT1 and NtPT2 were strongly upregulated in transgenic tobacco under high Pi conditions. Together, these results suggest that Pi and Se uptake and translocation by the Pi transporter OsPT8 might function in a complex combinational way in tobacco, and that OsPT8 might be a potential candidate gene for breeding Se-enriched tobacco. [ABSTRACT FROM AUTHOR]

Phosphorus (P) is a critical nutrient for plants, and inorganic orthophosphate (Pi) deficiency results in declining crop quality and yields. Auxin plays a key regulatory effect on plant growth in response to Pi deficiency, yet the regulatory mechanisms remain poorly understood. In this study, we cloned and identified an auxin efflux transporter, NtPIN3 , involved in the low phosphate response of tobacco (Nicotiana tabacum). NtPIN3 is located on the cytoplasmic membrane, and high expression levels of NtPIN3 were observed under phosphorus deficiency conditions. Moreover, NtPIN3 overexpression in tobacco could dramatically enhance low Pi tolerance by increasing the root length, which was consistent with the GUS activity and the auxin concentration in the root. Notably, Pi concentration and antioxidant capacity in overexpression transgenic tobacco were significantly increased compared with the wild type (WT) under low Pi stress conditions due to Pi uptake (NtPT1 and NtPT2) and antioxidant capacity (superoxide dismutase, peroxidase, and catalase). In contrast, compared with WT, the ntpin3 mutant significantly reduced root length, auxin concentration, Pi concentration, and antioxidant capacity under low Pi stress conditions, suggesting that NtPIN3 is a positive regulator of low phosphate in tobacco. In addition, the yeast-two-hybrid and luciferase complementation imaging assay confirmed that NtPIN3 interacted with NtPIN2. Altogether, the NtPIN3 involved in low Pi response and overexpression of NtPIN3 contributed to enhanced tolerance to low Pi stress, which proved an underlying gene for breeding low Pi-stress tolerant plants. • NtPIN3 was high expression under phosphorus deficiency condition. • NtPIN3 is involved in the regulation of low Pi tolerance in tobacco. • NtPIN3 overexpression transgenic tobacco could increase the root length, Pi concentration and antioxidant capacity. • NtPIN3 interacted with NtPIN2. [ABSTRACT FROM AUTHOR]

• NtHSP70-8 involvement in drought, ABA and auxin responses in tobacco. • NtHSP70-8 is an endoplasmic reticulum-localized protein and overexpression of NtHSP70-8 enhance the drought tolerance in tobacco. • Overexpression of NtHSP70-8 reduce the water loss of leaves by regulating ABA and IAA signaling pathway. • NtHSP70-8 is involved regulating the antioxidant capacity, possibly caused by the expression of antioxidant-related genes. The 70-kDa heat shock protein (HSP70) is an abiotic stress response factor in plants. However, the functions and underlying molecular mechanisms employed by HSP70 in tobacco have not been studied thoroughly. In this study, we cloned and identified a novel tobacco (Nicotiana tabacum) HSP70 gene, NtHSP70-8 , which was upregulated in response to drought stress and treatment with exogenous abscisic acid (ABA) and auxin (IAA). NtHSP70-8 was exclusively localized in the endoplasmic reticulum. NtHSP70-8 overexpression substantially elevated the expression level of NtNCED3 , NtNCED 5 (ABA-synthesis gene) and increased the ABA concentration, which caused stomatal closing to prevent water loss under drought conditions. Moreover, NtHSP70-8 overexpression caused a significant reduction in the expression levels of auxin efflux carriers, such as NtPIN1a , NtPIN1c , NtPIN3 , NtPIN5 , and NtPIN7 , and an increase in the IAA concentration, which lowered the stomata density to prevent water loss. Under drought conditions, the NtHSP70-8 -overexpressing plants had a substantially higher antioxidant capacity than wild-type tobacco plants, possibly caused by the increased expression of NtSOD , NtPOD , and NtCAT. In contrast, nthsp70-8 tobacco significantly reduced the antioxidant capacity compared with wild-type. Thus, our findings suggest that NtHSP70-8 is an endoplasmic reticulum-localized protein that confers drought tolerance in tobacco by regulating water loss and antioxidant capacity. Furthermore, we demonstrate the involvement of NtHSP70-8 in the interaction between ABA signaling and auxin signaling under drought stress. [ABSTRACT FROM AUTHOR]