Project description:BackgroundWD40 proteins, which are highly prevalent in eukaryotes, play important roles in plant development and stress responses. However, systematic identification and exploration of WD40 proteins in tobacco have not yet been conducted.ResultsIn this study, a total of 399 WD40 regulatory genes were identified in common tobacco (Nicotiana tabacum). Gene structure and motif analysis revealed structural and functional diversity among different clades of tobacco WD40 regulatory genes. The expansion of tobacco WD40 regulatory genes was mainly driven by segmental duplication and purifying selection. A potential regulatory network of NtWD40s suggested that NtWD40s might be regulated by miRNAs and transcription factors in various biological processes. Expression pattern analysis via transcriptome analysis and qRT-PCR revealed that many NtWD40s exhibited tissue-specific expression patterns and might be involved in various biotic and abiotic stresses. Furthermore, we have validated the critical role of NtTTG1, which was located in the nuclei of trichome cells, in enhancing the drought tolerance of tobacco plants.ConclusionsOur study provides comprehensive information to better understand the evolution of WD40 regulatory genes and their roles in different stress responses in tobacco.
Project description:BackgroundDrought stress is the most harmful one among other abiotic stresses with negative impacts on crop growth and development. Drought-hardening is a feasible and widely used method in tobacco seedlings cultivation. It has gained extensive interests due to its role in improving drought tolerance. This research aimed to investigate the role of drought-hardening and to unravel the multiple mechanisms underlying tobacco drought tolerance and adaptation.ResultsThis study was designed in which various drought-hardening treatments (CK (no drought-hardening), T1 (drought-hardening for 24 h), T2 (drought-hardening for 48 h), and T3 (drought-hardening for 72 h)) were applied to two tobacco varieties namely HongHuaDaJinYuan (H) and Yun Yan-100 (Y). The findings presented a complete framework of drought-hardening effect at physiological, biochemical, and gene expression levels of the two tobacco varieties under drought stress. The results showed that T2 and T3 significantly reduced the growth of the two varieties under drought stress. Similarly, among the various drought-hardening treatments, T3 improved both the enzymatic (POD, CAT, APX) and non-enzymatic (AsA) defense systems along with the elevated levels of proline and soluble sugar to mitigate the negative effects of oxidative damage and bringing osmoregulation in tobacco plants. Finally, the various drought-hardening treatments (T1, T2, and T3) showed differential regulation of genes expressed in the two varieties, while, particularly T3 drought-hardening treatment-induced drought tolerance via the expression of various stress-responsive genes by triggering the biosynthesis pathways of proline (P5CS1), polyamines (ADC2), ABA-dependent (SnRK2, AREB1), and independent pathways (DREB2B), and antioxidant defense-related genes (CAT, APX1, GR2) in response to drought stress.ConclusionsDrought-hardening made significant contributions to drought tolerance and adaptation in two tobacco variety seedlings by reducing its growth and, on the other hand, by activating various defense mechanisms at biochemical and molecular levels. The findings of the study pointed out that drought-hardening is a fruitful strategy for conferring drought tolerance and adaptations in tobacco. It will be served as a useful method in the future to understand the drought tolerance and adaptation mechanisms of other plant species. Drought-hardening improved drought tolerance and adaptation of the two tobacco varieties. T1 indicates drought-hardening for 24 h, T2 indicates drought-hardening for 48 h, T3 indicates drought-hardening for 72 h.
Project description:1. Coproporphyrinogenase was extracted and purified from tobacco (Nicotiana tabacum L.). Enzyme activity was mainly located in mitochondria rather than in chloroplasts. The enzyme was purified by differential centrifugation, ammonium sulphate fractionation, calcium phosphate gel adsorption and dialysis. A 69-fold final purification was obtained. 2. An apparent K(m) value of 3.6x10(-5)m was found, the value being largely dependent on the amount of coproporphyrin III recovered after reduction with sodium amalgam to coproporphyrinogen III. Protoporphyrin formation was linear up to 3h and decreased with further incubation. The enzyme activity increased with the concentration of enzyme protein up to 30mug/ml of solution. 3. Enzyme activity was greatly enhanced by increasing Fe(2+) concentrations up to 0.5mm, beyond which inhibition occurred. Co(2+) and Mn(2+) were also found to activate at low concentrations (0.1mm) and inhibit at higher concentrations (5mm). Fe(3+) and Cu(2+), both at 0.1mm, and o-phenanthroline and EDTA, each at 1mm, were found to be inhibitory.
Project description:Lipid transfer proteins (LTPs), a class of small, ubiquitous proteins, play critical roles in various environmental stresses. However, their precise biological functions remain unknown. Here we isolated an extracellular matrix-localised LTP, NtLTP4, from Nicotiana tabacum. The overexpression of NtLTP4 in N. tabacum enhanced resistance to salt and drought stresses. Upon exposure to high salinity, NtLTP4-overexpressing lines (OE lines) accumulated low Na+ levels. Salt-responsive genes, including Na+/H+ exchangers (NHX1) and high-affinity K+ transporter1 (HKT1), were dramatically higher in OE lines than in wild-type lines. NtLTP4 might regulate transcription levels of NHX1 and HKT1 to alleviate the toxicity of Na+. Interestingly, OE lines enhanced the tolerance of N. tabacum to drought stress by reducing the transpiration rate. Moreover, NtLTP4 could increase reactive oxygen species (ROS)-scavenging enzyme activity and expression levels to scavenge excess ROS under drought and high salinity conditions. We used a two-hybrid yeast system and screened seven putative proteins that interact with NtLTP4 in tobacco. An MAPK member, wound-induced protein kinase, was confirmed to interact with NtLTP4 via co-immunoprecipitation and a firefly luciferase complementation imaging assay. Taken together, this is the first functional analysis of NtLTP4, and proves that NtLTP4 positively regulates salt and drought stresses in N. tabacum.
Project description:Drought stress is one of the main factors limiting crop production, which provokes a number of changes in plants at physiological, anatomical, biochemical and molecular level. To unravel the various mechanisms underpinning tobacco (Nicotiana tabacum L.) drought stress tolerance, we conducted a comprehensive physiological, anatomical, biochemical and transcriptome analyses of three tobacco cultivars (i.e., HongHuaDaJinYuan (H), NC55 (N) and Yun Yan-100 (Y)) seedlings that had been exposed to drought stress. As a result, H maintained higher growth in term of less reduction in plant fresh weight, dry weight and chlorophyll content as compared with N and Y. Anatomical studies unveiled that drought stress had little effect on H by maintaining proper leaf anatomy while there were significant changes in the leaf anatomy of N and Y. Similarly, H among the three varieties was the least affected variety under drought stress, with more proline content accumulation and a powerful antioxidant defense system, which mitigates the negative impacts of reactive oxygen species. The transcriptomic analysis showed that the differential genes expression between HongHuaDaJinYuan, NC55 and Yun Yan-100 were enriched in the functions of plant hormone signal transduction, starch and sucrose metabolism, and arginine and proline metabolism. Compared to N and Y, the differentially expressed genes of H displayed enhanced expression in the corresponding pathways under drought stress. Together, our findings offer insights that H was more tolerant than the other two varieties, as evidenced at physiological, biochemical, anatomical and molecular level. These findings can help us to enhance our understanding of the molecular mechanisms through the networks of various metabolic pathways mediating drought stress adaptation in tobacco.
Project description:Tobacco mosaic virus (TMV) is a common source of biological stress that significantly affects plant growth and development. It is also useful as a model in studies designed to clarify the mechanisms involved in plant viral disease. Plant responses to abiotic stress were recently reported to be regulated by complex mechanisms at the post-translational modification (PTM) level. Protein phosphorylation is one of the most widespread and major PTMs in organisms. Using immobilized metal ion affinity chromatography (IMAC) enrichment, high-pH C18 chromatography fraction, and high-accuracy mass spectrometry (MS), a set of proteins and phosphopeptides in both TMV-infected tobacco and control tobacco were identified. A total of 4905 proteins and 3998 phosphopeptides with 3063 phosphorylation sites were identified. These 3998 phosphopeptides were assigned to 1311 phosphoproteins, as some proteins carried multiple phosphorylation sites. Among them, 530 proteins and 337 phosphopeptides corresponding to 277 phosphoproteins differed between the two groups. There were 43 upregulated phosphoproteins, including phosphoglycerate kinase, pyruvate phosphate dikinase, protein phosphatase 2C, and serine/threonine protein kinase. To the best of our knowledge, this is the first phosphoproteomic analysis of leaves from a tobacco cultivar, K326. The results of this study advance our understanding of tobacco development and TMV action at the protein phosphorylation level.
Project description:Drought stress is one of the primary environmental stress factors that gravely threaten crop growth, development, and yields. After drought stress, plants can regulate the content and proportion of various hormones to adjust their growth and development, and in some cases to minimize the adverse effects of drought stress. In our previous study, the tobacco cis-abienol synthesis gene (NtCPS2) was found to affect hormone synthesis in tobacco plants. Unfortunately, the role of NtCPS2 genes in the response to abiotic stress has not yet been investigated. Here, we present data supporting the role of NtCPS2 genes in drought stress and the possible underlying molecular mechanisms. NtCPS2 gene expression was induced by polyethylene glycol, high-temperature, and virus treatments. The results of subcellular localization showed that NtCPS2 was localized in the cell membrane. The NtCPS2-knockdown plants exhibited higher levels of gibberellin (GA) content and synthesis pathway genes expression but lower abscisic acid (ABA) content and synthesis pathway genes expression in response to drought stress. In addition, the transgenic tobacco lines showed higher leaf water loss and electrolyte loss, lower soluble protein and reactive oxygen species content (ROS), and lower antioxidant enzyme activity after drought treatment compared to wild type plants (WT). In summary, NtCPS2 positively regulates drought stress tolerance possibly by modulating the ratio of GA to ABA, which was confirmed by evidence of related phenotypic and physiological indicators. This study may provide evidence for the feedback regulation of hormone to abiotic and biotic stresses.
Project description:Cadmium (Cd)-induced oxidative stress detrimentally affects hyperaccumulator growth, thereby diminishing the efficacy of phytoremediation technology aimed at Cd pollution abatement. In the domain of plant antioxidant mechanisms, the role of glutathione peroxidase (GPX) in conferring Cd tolerance to tobacco (Nicotiana tabacum) remained unclear. Our investigation employed genome-wide analysis to identify 14 NtGPX genes in tobacco, revealing their organization into seven subgroups characterized by analogous conserved domain patterns. Notably, qPCR analysis highlighted NtGPX8a as markedly responsive to Cd2+ stress. Subsequent exploration through yeast two-hybridization unveiled NtGPX8a's utilization of thioredoxins AtTrxZ and AtTrxm2 as electron donors, and without interaction with AtTrx5. Introduction of NtGPX8a into Escherichia coli significantly ameliorated Cd-induced adverse effects on bacterial growth. Transgenic tobacco overexpressing NtGPX8a demonstrated significantly augmented activities of GPX, SOD, POD, and CAT under Cd2+ stress compared to the wild type (WT). Conversely, these transgenic plants exhibited markedly reduced levels of MDA, H2O2, and proline. Intriguingly, the expression of NtGPX8a in both E. coli and transgenic tobacco led to increased Cd accumulation, confirming its dual role in enhancing Cd tolerance and accumulation. Consequently, NtGPX8a emerges as a promising candidate gene for engineering transgenic hyperaccumulators endowed with robust tolerance for Cd-contaminated phytoremediation.
Project description:The overall metabolism of purines was studied in tobacco (Nicotiana tabacum) mesophyll protoplasts. Metabolic pathways were studied by measuring the conversion of radioactive adenine, adenosine, hypoxanthine and guanine into purine ribonucleotides, ribonucleosides, bases and nucleic acid constituents. Adenine was extensively deaminated to hypoxanthine, whereupon it was also converted into AMP and incorporated into nucleic acids. Adenosine was mainly hydrolysed to adenine. Inosinate formed from hypoxanthine was converted into AMP and GMP, which were then catabolized to adenine and guanosine respectively. Guanine was mainly deaminated to xanthine and also incorporated into nucleic acids via GTP. Increased RNA synthesis in the protoplasts resulted in enhanced incorporation of adenine and guanine, but not of hypoxanthine and adenosine, into the nucleic acid fraction. The overall pattern of purine-nucleotide metabolic pathways in protoplasts of tobacco leaf mesophyll is proposed.