Insights into drought stress response mechanism of tobacco during seed germination by integrated analysis of transcriptome and metabolome.

Drought stress inhibits seed germination, plant growth and development of tobacco, and seriously affects the yield and quality of tobacco leaves. However, the molecular mechanism underlying tobacco drought stress response remains largely unknown. In this study, integrated analysis of transcriptome and metabolome was performed on the germinated seeds of a cultivated variety K326 and its EMS mutagenic mutant M28 with great drought tolerance. The result showed that drought stress inhibited seed germination of the both varieties, while the germination rate of M28 was faster than that of K326 under drought stress. Besides, the levels of phytohormone ABA, GA19, and zeatin were increased by drought stress in M28. Five vital pathways were identified through integrated transcriptomic and metabolomic analysis, including zeatin biosynthesis, aspartate and glutamate synthesis, phenylamine metabolism, glutathione metabolism, and phenylpropanoid synthesis. Furthermore, 20 key metabolites in the above pathways were selected for further analysis of gene modular-trait relationship, and then four highly correlated modules were found. Then analysis of gene expression network was carried out of Top30 hub gene of these four modules, and 9 key candidate genes were identified, including HSP70s, XTH16s, APX, PHI-1, 14-3-3, SCP, PPO. In conclusion, our study uncovered some key drought-responsive pathways and genes of tobacco during seeds germination, providing new insights into the regulatory mechanisms of tobacco drought stress response.

Differential absorption of cadmium and zinc by tobacco plants: Role of apoplastic pathway.

Cadmium (Cd) contamination presents a significant challenge in global agriculture. This study explores the efficacy of chemical induction, specifically using sodium chloride (NaCl), to limit Cd uptake in tobacco (Nicotiana tabacum) and assesses its impact on essential divalent metal ions (DMIs). We conducted a comprehensive analysis encompassing ion absorption, root histology, and biochemistry to understand the influence of this method. Our results revealed that NaCl induction led to a notable 30 % decrease in Cd absorption, while maintaining minimal impact on zinc (Zn) uptake. Intriguingly, the absence of essential DMIs, such as calcium (Ca), magnesium (Mg), and Zn, was found to diminish the plant's capacity to absorb Cd. Furthermore, moderate NaCl induction resulted in an increased diameter of the root stele and enhanced lignin content, indicating a restriction of Cd absorption through the apoplastic pathway. Conversely, a compensatory absorption mechanism via the symplastic pathway appeared to be activated in the absence of essential elements. These findings highlight the potential of chemical induction as a strategy to mitigate agricultural Cd risks, offering insights into the complex interplay between plant ion transport pathways and metal uptake regulation.

The positive impact of the NtTAS14-like1 gene on osmotic stress response in Nicotiana tabacum.

KEY MESSAGE: NtTAS14-like1 enhances osmotic tolerance through coordinately activating the expression of osmotic- and ABA-related genes. Osmotic stress is one of the most important limiting factors for tobacco (Nicotiana tabacum) growth and development. Dehydrin proteins are widely involved in plant adaptation to osmotic stress, but few of these proteins have been functionally characterized in tobacco. Here, to identify genes required for osmotic stress response in tobacco, an encoding dehydrin protein gene NtTAS14-like1 was isolated based on RNA sequence data. The expression of NtTAS14-like1 was obviously induced by mannitol and abscisic acid (ABA) treatments. Knock down of NtTAS14-like1 expression reduced osmotic tolerance, while overexpression of NtTAS14-like1 conferred tolerance to osmotic stress in transgenic tobacco plants, as determined by physiological analysis of the relative electrolyte leakage and malonaldehyde accumulation. Further expression analysis by quantitative real-time PCR indicated that NtTAS14-like1 participates in osmotic stress response possibly through coordinately activating osmotic- and ABA-related genes expression, such as late embryogenesis abundant (NtLEA5), early responsive to dehydration 10C (NtERD10C), calcium-dependent protein kinase 2 (NtCDPK2), ABA-responsive element-binding protein (NtAREB), ABA-responsive element-binding factor 1 (NtABF1), dehydration-responsive element-binding genes (NtDREB2A), xanthoxin dehydrogenase/reductase (NtABA2), ABA-aldehyde oxidase 3 (NtAAO3), 9-cis-epoxycarotenoid dioxygenase (NtNCED3). Together, this study will facilitate to improve our understandings of molecular and functional properties of plant TAS14 proteins and to improve genetic evidence on the involvement of the NtTAS14-like1 in osmotic stress response of tobacco.

Deciphering the roles of tobacco MYB transcription factors in environmental stress tolerance.

The MYB members play important roles in development, metabolism, and stress tolerance in plants. In the current study, a total of 246 tobacco R2R3-MYB transcription factors were identified and systemically analyzed from the latest genome annotation. The newly identified tobacco members were divided into 33 subgroups together with the Arabidopsis members. Furthermore, 44 NtMYB gene pairs were identified to arise from duplication events, which might lead to the expansion of tobacco MYB genes. The expression patterns were revealed by transcriptomic analysis. Notably, the results from phylogenetic analysis, synthetic analysis, and expression analysis were integrated to predict the potential functions of these members. Particularly, NtMYB102 was found to act as the homolog of AtMYB70 and significantly induced by drought and salt treatments. The further assays revealed that NtMYB102 had transcriptional activities, and the overexpression of the encoding gene enhanced the drought and salt stress tolerance in transgenic tobacco. The results of this study may be relevant for future functional analyses of the MYB genes in tobacco.

Transcriptome-based gene regulatory network analyses of differential cold tolerance of two tobacco cultivars.

BACKGROUND: Cold is one of the main abiotic stresses that severely affect plant growth and development, and crop productivity as well. Transcriptional changes during cold stress have already been intensively studied in various plant species. However, the gene networks involved in the regulation of differential cold tolerance between tobacco varieties with contrasting cold resistance are quite limited. RESULTS: Here, we conducted multiple time-point transcriptomic analyses using Tai tobacco (TT, cold susceptibility) and Yan tobacco (YT, cold resistance) with contrasting cold responses. We identified similar DEGs in both cultivars after comparing with the corresponding control (without cold treatment), which were mainly involved in response to abiotic stimuli, metabolic processes, kinase activities. Through comparison of the two cultivars at each time point, in contrast to TT, YT had higher expression levels of the genes responsible for environmental stresses. By applying Weighted Gene Co-Expression Network Analysis (WGCNA), we identified two main modules: the pink module was similar while the brown module was distinct between the two cultivars. Moreover, we obtained 100 hub genes, including 11 important transcription factors (TFs) potentially involved in cold stress, 3 key TFs in the brown module and 8 key TFs in the pink module. More importantly, according to the genetic regulatory networks (GRNs) between TFs and other genes or TFs by using GENIE3, we identified 3 TFs (ABI3/VP1, ARR-B and WRKY) mainly functioning in differential cold responses between two cultivars, and 3 key TFs (GRAS, AP2-EREBP and C2H2) primarily involved in cold responses. CONCLUSION: Collectively, our study provides valuable resources for transcriptome- based gene network studies of cold responses in tobacco. It helps to reveal how key cold responsive TFs or other genes are regulated through network. It also helps to identify the potential key cold responsive genes for the genetic manipulation of tobacco cultivars with enhanced cold tolerance in the future.

Comparative transcriptome and metabolome analyses reveal the methanol dissimilation pathway of Pichia pastoris.

BACKGROUND: Pichia pastoris (Komagataella phaffii) is a model organism widely used for the recombinant expression of eukaryotic proteins, and it can metabolize methanol as its sole carbon and energy source. Methanol is oxidized to formaldehyde by alcohol oxidase (AOX). In the dissimilation pathway, formaldehyde is oxidized to CO2 by formaldehyde dehydrogenase (FLD), S-hydroxymethyl glutathione hydrolase (FGH) and formate dehydrogenase (FDH). RESULTS: The transcriptome and metabolome of P. pastoris were determined under methanol cultivation when its dissimilation pathway cut off. Firstly, Δfld and Δfgh were significantly different compared to the wild type (GS115), with a 60.98% and 23.66% reduction in biomass, respectively. The differential metabolites between GS115 and Δfld were mainly enriched in ABC transporters, amino acid biosynthesis, and protein digestion and absorption. Secondly, comparative transcriptome between knockout and wild type strains showed that oxidative phosphorylation, glycolysis and the TCA cycle were downregulated, while alcohol metabolism, proteasomes, autophagy and peroxisomes were upregulated. Interestingly, the down-regulation of the oxidative phosphorylation pathway was positively correlated with the gene order of dissimilation pathway knockdown. In addition, there were significant differences in amino acid metabolism and glutathione redox cycling that raised our concerns about formaldehyde sorption in cells. CONCLUSIONS: This is the first time that integrity of dissimilation pathway analysis based on transcriptomics and metabolomics was carried out in Pichia pastoris. The blockage of dissimilation pathway significantly down-regulates the level of oxidative phosphorylation and weakens the methanol assimilation pathway to the point where deficiencies in energy supply and carbon fixation result in inefficient biomass accumulation and genetic replication. In addition, transcriptional upregulation of the proteasome and autophagy may be a stress response to resolve formaldehyde-induced DNA-protein crosslinking.

Transcriptomic and metabolic regulatory network characterization of drought responses in tobacco.

Drought stress usually causes huge economic losses for tobacco industries. Drought stress exhibits multifaceted impacts on tobacco systems through inducing changes at different levels, such as physiological and chemical changes, changes of gene transcription and metabolic changes. Understanding how plants respond and adapt to drought stress helps generate engineered plants with enhanced drought resistance. In this study, we conducted multiple time point-related physiological, biochemical,transcriptomic and metabolic assays using K326 and its derived mutant 28 (M28) with contrasting drought tolerance. Through integrative analyses of transcriptome and metabolome,we observed dramatic changes of gene expression and metabolic profiles between M28 and K326 before and after drought treatment. we found that some of DEGs function as key enzymes responsible for ABA biosynthesis and metabolic pathway, thereby mitigating impairment of drought stress through ABA signaling dependent pathways. Four DEGs were involved in nitrogen metabolism, leading to synthesis of glutamate (Glu) starting from NO-3 /NO-2 that serves as an indicator for stress responses. Importantly, through regulatory network analyses, we detected several drought induced TFs that regulate expression of genes responsible for ABA biosynthesis through network, indicating direct and indirect involvement of TFs in drought responses in tobacco. Thus, our study sheds some mechanistic insights into how plant responding to drought stress through transcriptomic and metabolic changes in tobacco. It also provides some key TF or non-TF gene candidates for engineering manipulation for breeding new tobacco varieties with enhanced drought tolerance.

Quantitative Relationship Between Paddy Soil Properties and Cadmium Content in Tobacco leaves.

Tobacco readily accumulates cadmium (Cd), an unnecessary and poisonous element. A total of 107 soil and tobacco leaf samples were collected from South China, to clarify the quantitative relationship between soil properties and Cd content in tobacco leaves. The results showed that 86.9% of the total sampling points had soil cadmium in excess of standard value, and the ratio of active Cd content to total soil Cd content was 24.0%. The enrichment factor of tobacco Cd was 3.43. There was a significant positive correlation between Cd concentration in tobacco leaves and soil Cd content. Soil pH, organic matter and cation exchange amount were negatively correlated with the Cd enrichment factor of tobacco. This present study has provided a regression model of tobacco Cd content based on soil factors, which could accurately predict Cd content in different parts of tobacco.

Quantitative source apportionment, risk assessment and distribution of heavy metals in agricultural soils from southern Shandong Peninsula of China.

The heavy metals, including cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), nickel (Ni), lead (Pb), zinc (Zn), and the metalloid arsenic (As) were detected in surface and core soil samples collected from a tobacco growing region in Shandong Peninsula on the east coast of China to evaluate their pollution levels, ecological and health risks, and to analyze their spatial and vertical distributions. The heavy metal sources were identified quantitatively using the positive matrix factorization (PMF) receptor model. In accordance, most of the soils did not have accumulations and were not contaminated by As, Cr, Cu, Ni, Pb, and Zn. High accumulations of Cd and Hg occurred in the soils, posing an ecological risk to the local agricultural environment, while Cr and Ni levels presented a carcinogenic health risk to humans. Four main sources of heavy metals in the soils were identified. Correspondingly Ni and Cr were mainly originated from natural sources, Hg from coal combustion, Cd from agricultural practices, Cu, Pb, and Zn from agricultural practices and industrial activities, and As from industrial activities.

Reduced cadmium accumulation in tobacco by sodium chloride priming.

Cadmium (Cd) pollution threatens agricultural security worldwide. This study tested the efficacy of priming chemicals to decrease Cd uptake by tobacco plants (Nicotiana tabacum). After initial screening from nine different chemicals (NaCl, Cd(CH3COO)2, Cd(NO3)2, CdCl2, KHNO3, polyethylene glycol 6000 (PEG-6000), indole-3-acetic acid (IAA), ß-aminobutyric acid (BABA), and glutathione (GSH)), NaCl and PEG-6000 were further investigated because of their low risks to plant growth and efficiency to Cd reduction. Priming procedures (concentrations) were optimized for both chemicals and the best one (100 mM NaCl) was used to test both soil and hydroponic media. The results showed 31.3% lower Cd concentrations in shoots after priming with 100 mM NaCl. Phenotype parameters of the plants were also measured and showed no significant impacts of the priming procedures on the shoot biomass and the uptakes of nitrogen (N), phosphorus (P), and potassium (K), nor the photosynthetic capacity (net photosynthesis rate (Pn) and chlorophyll concentration (SPAD)). Histological observations of the roots showed a significant increase of the stele diameter after NaCl priming and a subsequent negative correlation between shoot Cd concentration and stele diameter was found after NaCl priming at different levels. This study confirmed 100 mM NaCl as an efficient priming treatment to decrease Cd uptake and the coarsening of the root stele was identified as a potential explanation for the observed decrease of Cd in tobacco shoots.

Properties of silver contained coatings on CoCr alloys prepared by vacuum plasma spraying.

The silver contained coatings on cast Cobalt Chrome (CoCr) alloys were prepared by vacuum plasma spraying technique. The Scanning Electron Microscopy (SEM), X-ray photoelectron spectroscopy (XPS), X-ray Diffraction (XRD), energy dispersive spectrometry (EDS), properties of corrosion resistance, wear resistance and effect of vitro antibacterial on the surface of silver contained coating were investigated. The cytotoxicity of the coatings was performed with L-929 fibroblasts by MTT assay. SEM showed that the surfaces of the coatings were dense, smooth, no obvious cracks except only a few pores. XRD analysis indicated that the contents of the surface were mainly Ag and Cr except a small amount of Ag2O, Cr2O3. EDS analysis indicated that the distributions of Cr and Ag were uniform without any large-scale clustering. The wear resistance of silver coatings is similar to that of CoCr alloys, and the corrosion resistance is slightly better than that of CoCr alloys. The Ag coating had no significant effect on the proliferation of L929 cells. The antibacterial results indicated that the number of S. mutans and C. albicans were significantly reduced on the surface of silver contained coating than that of CoCr alloys. All the results indicated that the silver contained coatings can be achieved by vacuum plasma spraying technique with good surface characteristic and antibacterial properties and have promising applications in biomedical area.

A novel bHLH transcription factor, NtbHLH1, modulates iron homeostasis in tobacco (Nicotiana tabacum L.).

Iron (Fe) is a major micronutrient which influences plant growth, development, quality and yield. Although basic helix-loop-helix (bHLH) transcription factors (TFs) which respond to iron deficiency have been identified, the molecular mechanisms have not been fully elucidated. In this study, a novel bHLH TF, NtbHLH1, was found to be induced by iron deficiency. Further analysis indicated that NtbHLH1 is localized to the nucleus and functions as a transcriptional activator. Moreover, overexpression of NtbHLH1 resulted in longer roots, altered rhizosphere pH and increased ferric-chelate reductase activity in iron deficient conditions. Overall these changes resulted in increased iron uptake relative to wild type plants. NtbHLH1 mutants, on the other hand, had an opposite phenotype. In addition, transcript levels of seven genes associated with iron deficiency response were higher in the NtbHLH1 overexpression transgenic plants and lower in ntbhlh1 relative to the WT under iron deficiency treatment. Taken together, these results demonstrated that NtbHLH1 plays a key role in iron deficiency response and they provide new insights into the molecular basis of iron homeostasis in tobacco.

Comparison of heavy metal accumulation and cadmium phytoextraction rates among ten leading tobacco (Nicotiana tabacum L.) cultivars in China.

Cadmium (Cd) contamination is one of the most serious global environmental problems, and phytoremediation, which uses Cd-accumulator plants, is potentially one of the sustainable solutions. Pot experiments with natural and Cd-amended soils were conducted to investigate the accumulation of heavy metals in 10 leading cultivars of tobacco in China. The extraction ability and profiles of Cd accumulation among plant organs were also analyzed. The tobacco roots accumulated cobalt, nickel, and Cd, while the leaf highly bioaccumulated Cd and lowly accumulated zinc, selenium and mercury. The transport from the tobacco stem to the leaf plays a critical role in the accumulation of these elements. The ratios of Cd concentration in the leaves at lower, middle and upper positions were comparatively stable. The high Cd-extracting cultivars were "Hongda", "NC89" and "Zhongyan 100" when grown in normal soils, "CuiBi 1" and "Hongda" in moderately contaminated soils, and "YuYan 87", "LongJiang 851" and "K326" in severely contaminated soils. Tobacco leaves could accumulate about 80% of the total Cd extracted from the soil by the plant. Considering the Cd-extraction limitations exhibited by leading tobacco cultivars, screening of germplasm resources for high or low levels of Cd-accumulation is still an important target for the future.

Simulative Global Warming Negatively Affects Cotton Fiber Length through Shortening Fiber Rapid Elongation Duration.

Global warming could possibly increase the air temperature by 1.8-4.0 °C in the coming decade. Cotton fiber is an essential raw material for the textile industry. Fiber length, which was found negatively related to the excessively high temperature, determines yarn quality to a great extent. To investigate the effects of global warming on cotton fiber length and its mechaism, cottons grown in artificially elevated temperature (34.6/30.5 °C, Tday/Tnight) and ambient temperature (31.6/27.3 °C) regions have been investigated. Becaused of the high sensitivities of enzymes V-ATPase, PEPC, and genes GhXTH1 and GhXTH2 during fiber elongation when responding to high temperature stress, the fiber rapid elongation duration (FRED) has been shortened, which led to a significant suppression on final fiber length. Through comprehensive analysis, Tnight had a great influence on fiber elongation, which means Tn could be deemed as an ideal index for forecasting the degree of high temperature stress would happen to cotton fiber property in future. Therefore, we speculate the global warming would bring unfavorable effects on cotton fiber length, which needs to take actions in advance for minimizing the loss in cotton production.

Soil Potassium Deficiency Reduces Cotton Fiber Strength by Accelerating and Shortening Fiber Development.

Low potassium (K)-induced premature senescence in cotton has been observed worldwide, but how it affects cotton fiber properties remain unclear. We hypothesized that K deficiency affects cotton fiber properties by causing disordered fiber development, which may in turn be caused by the induction of a carbohydrate acquisition difficulty. To investigate this issue, we employed a low-K-sensitive cotton cultivar Siza 3 and a low-K-tolerant cultivar Simian 3 and planted them in three regions of different K supply. Data concerning lint yield, Pn and main fiber properties were collected from three years of testing. Soil K deficiency significantly accelerated fiber cellulose accumulation and dehydration processes, which, together with previous findings, suggests that the low-K induced carbohydrate acquisition difficulty could cause disordered fiber development by stimulating the expression of functional proteins such as CDKA (cyclin-dependent kinase). As a result, fiber strength and lint weight were reduced by up to 7.8% and 2.1%, respectively. Additional quantitative analysis revealed that the degree of accelerated fiber development negatively correlated with fiber strength. According to the results of this study, it is feasible to address the effects of soil K deficiency on fiber properties using existing cultivation strategies to prevent premature senescence of cotton plants.

Fruiting Branch K(+) Level Affects Cotton Fiber Elongation Through Osmoregulation.

Potassium (K) deficiency in cotton plants results in reduced fiber length. As one of the primary osmotica, K(+) contributes to an increase in cell turgor pressure during fiber elongation. Therefore, it is hypothesized that fiber length is affected by K deficiency through an osmotic pathway, so in 2012 and 2013, an experiment was conducted to test this hypothesis by imposing three potassium supply regimes (0, 125, 250 kg K ha(-1)) on a low-K-sensitive cultivar, Siza 3, and a low-K-tolerant cultivar, Simian 3. We found that fibers were longer in the later season bolls than in the earlier ones in cotton plants grown under normal growth conditions, but later season bolls showed a greater sensitivity to low-K stress, especially the low-K sensitive genotype. We also found that the maximum velocity of fibre elongation (V max) is the parameter that best reflects the change in fiber elongation under K deficiency. This parameter mostly depends on cell turgor, so the content of the osmotically active solutes was analyzed accordingly. Statistical analysis showed that K(+) was the major osmotic factor affecting fiber length, and malate was likely facilitating K(+) accumulation into fibers, which enabled the low-K-tolerant genotype to cope with low-K stress. Moreover, the low-K-tolerant genotype tended to have greater K(+) absorptive capacities in the upper fruiting branches. Based on our findings, we suggest a fertilization scheme for Gossypium hirsutum that adds extra potash fertilizer or distributes it during the development of late season bolls to mitigate K deficiency in the second half of the growth season and to enhance fiber length in late season bolls.

Relationship between potassium fertilization and nitrogen metabolism in the leaf subtending the cotton (Gossypium hirsutum L.) boll during the boll development stage.

The nitrogen (N) metabolism of the leaf subtending the cotton boll (LSCB) was studied with two cotton (Gossypium hirsutum L.) cultivars (Simian 3, low-K tolerant; Siza 3, low-K sensitive) under three levels of potassium (K) fertilization (K0: 0 g K2O plant(-1), K1: 4.5 K2O plant(-1) and K2: 9.0 g K2O plant(-1)). The results showed that total dry matter increased by 13.1-27.4% and 11.2-18.5% under K supply for Simian 3 and Siza 3. Boll biomass and boll weight also increased significantly in K1 and K2 treatments. Leaf K content, leaf N content and nitrate (NO3(-)) content increased with increasing K rates, and leaf N content or NO3(-) content had a significant positive correlation with leaf K content. Free amino acid content increased in the K0 treatment for both cultivars, due to increased protein degradation caused by higher protease and peptidase activities, resulting in lower protein content in the K0 treatment. The critical leaf K content for free amino acid and soluble protein content were 14 mg g(-1) and 15 mg g(-1) in Simian 3, and 17 mg g(-1) and 18 mg g(-1) in Siza 3, respectively. Nitrate reductase (NR), glutamic-oxaloace transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities increased in the K1 and K2 treatments for both cultivars, while glutamine synthetase (GS) and glutamate synthase (GOGAT) activities increased under K supply treatments only for Siza 3, and were not affected in Simian 3, indicating that this was the primary difference in nitrogen-metabolizing enzymes activities for the two cultivars with different sensitivity to low-K.