Returning ryegrass to continuous cropping soil improves soil nutrients and soil microbiome, producing good-quality flue-cured tobacco.

The widespread and continuous cultivation of tobacco has led to soil degradation and reduced crop yields and quality. Green manure is an essential organic fertilizer that alleviates obstacles to continuous cultivation. However, the plant-soil microecological effects of green manure on flue-cured tobacco cultivation remain unclear. Thus, a positioning trail including two treatments, chemical fertilizer application only (treatment NPK) and chemical fertilizer application with turning ryegrass (treatment NPKG) was conducted, and the effect of ryegrass returning on the soil physicochemical properties, soil microbiome, crop yield, and quality of flue-cured tobacco in continuous cropping soil were investigated. Results showed that returning ryegrass to the field increased the thickness of soil humus layer from 13 cm to 15 cm, reduced the humus layer soil bulk density to 1.29 cm3/g. Ryegrass tilled and returned to the field increased soil organic matter content by 6.89-7.92%, increased rhizosphere soil available phosphorus content by 2.22-17.96%, and converted the soil non-exchangeable potassium into potassium that was available for plant absorption and utilization. Ryegrass tilling and returning to the field increased the potassium content of middle leaves of flue-cured tobacco by 7.69-10.07%, the increased potassium content in flue-cured tobacco was accompanied by increased total sugar, reducing sugar, and the ratio of reducing sugar to nicotine, which facilitated the harmonization of the chemical composition of cured tobacco leaves. Moreover, the increased number of markedly improved operational taxonomic units enhanced the complexity of the soil bacterial community and its compactness after ryegrass tillage and their return to the field. The available potassium, available phosphorus, total potassium content, pH, and sampling period of the rhizosphere soil had considerable effects on the rhizosphere microbial. Ryegrass tilling and returning to the field changed the soil microbiome, which increased the abundance of bulk soil Proteobacteria, rhizosphere soil Fibrobacterota, and microbes with anti-pathogen activity (Lysobacteria, Sphingomonas, Chaetomium, and Minimedusa); and reduced the abundance of pathogenic fungi Neocosmospore genus in the soil. In brief, ryegrass returned to the field, improved soil microecology and restored soil nutrients, and established a new dynamic balance of soil ecology, thereby improving the quality of cultivated land and the quality of flue-cured tobacco.

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.

[Effect of Organic Material Amendments on Soil Respiration in Tobacco Fields of Central Henan].

A field experiment was conducted to study the effects of different organic material amendments on soil respiration in a flue-cured tobacco field. Five treatments were set up:no fertilizer (NF), chemical fertilizer (NPK), chemical fertilizer+ryegrass (NPKG), chemical fertilizer+wheat straw (NPKS), and chemical fertilizer+tobacco straw biochar (NPKB). The results showed that:① Compared with that under NPK, NPKG and NPKS decreased the temperature sensitivity (Q10) of total soil respiration and heterotrophic respiration, whereas NPKB increased the Q10 of heterotrophic respiration. The two-factor fitting model of soil respiration and soil hydrothermal factors accounted for 50%-80% of the variation in soil respiration. ② The addition of organic materials significantly increased the content of soil soluble organic carbon (DOC) and root dry matter. Soil heterotrophic respiration(Rh) was significantly positively correlated with DOC content, and soil autotrophic respiration(Ra) was significantly parabolically correlated with root biomass, with an R2 of 0.327-0.634. ③ Soil respiration increased first and then decreased during the tobacco growth period. Compared with that under the NF treatment, the NPK treatment significantly promoted soil respiration and its components. Compared with those of the NPK treatment, Rsrates were significantly increased by 20.08%, 10.32%, and 9.88% under the NPKG, NPKS, and NPKB treatments, respectively; Rh rate increased by 24.21%, 16.51%, and 11.68% respectively, and Ra rate was increased by 15.12% in the NPKG treatment. In summary, straw returning and biochar addition significantly increased Rh by increasing soil DOC, thereby promoting Rs. Incorporation of ryegrass not only increased the Rh but also increased Ra by promoting the growth and development of roots and therefore the Rs.

Systematic analysis of the non-specific lipid transfer protein gene family in Nicotiana tabacum reveal its potential roles in stress responses.

Plant non-specific lipid transfer proteins (nsLTPs) are characterized by an eight-cysteine motif backbone stabilized by four disulfide bonds; these proteins can bind or transfer lipids. NsLTPs play important roles in plant growth and development, and in the responses to abiotic and biotic stresses. In this study, 50, 51, and 100 nsLTPs from Nicotiana sylvestris, N. tomentosiformis, and their descendant N. tabacum, respectively, were identified and classified into six types (I, II, IV, V, VII, and VIII). The phylogeny, gene structures, motifs, tertiary structures, gene duplications and expression patterns were systematically analyzed. The intron/exon patterns and the conserved motifs were highly similar among the same types of nsLTP genes. Purifying selection and segmental duplication dominated the expansion of the nsLTPs family during evolution. Cis-regulatory elements of the NtLTP promoters were involved in light responsiveness, abiotic stress, and phytohormone responsiveness. Expression pattern analysis using RNA-seq and qPCR revealed that NtLTP family genes exhibited tissue-specific expression patterns and they have potential roles in response to abiotic and biotic stresses, especially drought stress, and resistance to black shank and bacterial wilt. Furthermore, overexpression of NtLTPI.38 in tobacco increased drought tolerance by improving the antioxidant defense ability, through reducing O2•- and H2O2 accumulation and increasing the number of lateral roots. These results provide a comprehensive overview of this gene family and provide valuable insights for the functional characterization of nsLTP family genes.

Thymol improves salinity tolerance of tobacco by increasing the sodium ion efflux and enhancing the content of nitric oxide and glutathione.

BACKGROUND AND OBJECTIVE: Salt stress is one of the most important abiotic stresses affecting the yield and quality of tobacco (Nicotiana tabacum). Thymol (a natural medicine) has been widely used in medical research because of its antibacterial and anti-inflammatory activities. However, the influence of thymol on the root growth of tobacco is not fully elucidated. In this study, the regulatory effects of different concentrations of thymol were investigated. METHODOLOGY: Here, histochemical staining and biochemical methods, non-invasive micro-test technology (NMT), and qPCR assay were performed to investigate the effect of thymol and mechanism of it improving salinity tolerance in tobacco seedlings. RESULTS: In this study, our results showed that thymol rescued root growth from salt stress by ameliorating ROS accumulation, lipid peroxidation, and cell death. Furthermore, thymol enhanced contents of NO and GSH to repress ROS accumulation, further protecting the stability of the cell membrane. And, thymol improved Na+ efflux and the expression of SOS1, HKT1, and NHX1, thus protecting the stability of Na+ and K+. CONCLUSION: Our study confirmed the protecting effect of thymol in tobacco under salt stress, and we also identified the mechanism of it, involving dynamic regulation of antioxidant system and the maintenance of Na+ homeostasis. It can be a new method to improve salinity tolerance in plants.

From hazardous agriculture waste to hazardous metal scavenger: Tobacco stalk biochar-mediated sequestration of Cd leads to enhanced tobacco productivity.

Tobacco is a super-enriched plant for heavy metals, and its productivity is sensitively affected by Cd. In this study, tobacco stalk was converted to biochar (TS-biochar) for the sequestration of Cd in soils to enhance the productivity of tobacco. FTIR, SEM-EDX, and XPS characterizations of TS-biochar together with Cd2+ adsorption kinetics revealed that oxy-containing functional groups (‒OH, CË­O, and ‒COOH) in TS-biochar played a crucial role on Cd2+ adsorption. The changes of soil property and Cd speciation by adding TS-biochar in red (acidic) and cinnamon (alkaline) soils was evaluated. Effects of TS-biochar on tobacco growth and development under Cd stress was also investigated. Results indicated that a 2 wt% addition of TS-biochar in red soil could significantly increase the soil pH value (from 5.21 to 7.39) and reduce exchangeable Cd fractions (from 40% to 23%), but those were not obvious in cinnamon soil. Under the stress of Cd, TS-biochar could obviously improve the tobacco dry biomass, and decrease the accumulation of Cd in the middle and upper leaves, thus reducing economic loss. Overall, the application of TS-biochar on Cd contaminated soil can transform bioavailable Cd into low hazardous forms, so as to repair soils and improve the productivity of tobacco.

Effect of biochar on the accumulation and distribution of cadmium in tobacco (Yunyan 87) at different developmental stages.

Cadmium (Cd) easily accumulates in tobacco, which endangers public health through Cd exposure from smoking. However, its uptake, translocation, and distribution in tobacco plants during plant development or its response to biochar application are poorly understood. A pot experiment was conducted with tobacco (Yunyan 87) grown in soil severely contaminated with Cd (30 mg kg-1) amended with 0, 1, and 2% (w/w) tobacco stem-derived biochar (BC). The absorption and accumulation of Cd in all parts of the tobacco plants were most active from the rosette stage to the fast growing stage, during which approximately 90% of the Cd deposited in the tobacco leaves occurred, especially in the lower leaves. The Cd concentrations in most plant parts without added biochar decreased significantly by 52.61-78.30% due to the rapid increase in biomass (dilution effect), although the Cd concentration in the lower leaves increased by 48.89% (P < 0.05). However, with the slowdown of the growth rate of tobacco at the maturity stage, the proportion of Cd accumulation in roots and stems without biochar addition increased by 29.01%, resulting in an increased Cd concentration in roots and stems by 63.29-86.80% (P < 0.05). In the different growth stages, the application of biochar reduced the contents of DTPA-extractable and exchangeable Cd in the soil by 5.11-35.14% and 9.20-54.05%, respectively, thus reducing the absorption, accumulation and concentration of Cd in all parts of the tobacco plant. In addition, the inhibitive effect of biochar on the Cd concentration in the leaves was weak at the rosette stage (22.17-53.72%) compared with the other stages (46.14-78.88%), and the degree of inhibition of biochar on the Cd concentration in the middle leaves (37.94-59.24%) was lower than that in the upper and lower leaves (49.04-73.54%) at all developmental stages. However, the long-term remediation effect of biochar on soil Cd contamination needs to be further verified, and the combination of biochar and other technologies should receive additional attention.

Characterization, expression, and functional analysis of polyamine oxidases and their role in selenium-induced hydrogen peroxide production in Brassica rapa.

BACKGROUND: Selenium (Se)-induced phytotoxicity has been linked to oxidative injury triggered by the accumulation of reactive oxygen species (ROS) due to the disturbance of anti-oxidative systems. However, the way Se stress induces hydrogen peroxide (H2 O2 ) production in plants is a long-standing question. Here we identified the role of polyamine oxidase (PAO) in H2 O2 production in the root of Brassica rapa upon Se stress. RESULTS: Studying Se-induced growth inhibition, H2 O2 accumulation, and oxidative injury in the root of Brassica rapa, we found that excessive Se exposure resulted in a remarkable increase in PAO activity. Inhibition of PAO activity led to decreased H2 O2 content and alleviated oxidative injury in the Se-treated root. These results indicated that Se stress induced PAO-dependent H2 O2 production. A total of six BrPAO family members were discovered in the genome of B. rapa by in silico analysis. Se stress pronouncedly upregulated the expression of most BrPAOs and further transient expression analysis proved that it could lead to H2 O2 production. CONCLUSION: These results suggest that Se stress upregulates the expression of a set of BrPAOs which further enhances PAO activity, contributing to H2 O2 generation in roots. © 2019 Society of Chemical Industry.

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.

Cinnamaldehyde Ameliorates Cadmium-Inhibited Root Elongation in Tobacco Seedlings via Decreasing Endogenous Hydrogen Sulfide Production.

Cinnamaldehyde (CA) is natural plant-derived compound that has been highly appreciated for its medicinal properties. However, little information is known about the regulation of plant intrinsic physiology by CA. To address these gaps, physiological, histochemical, and biochemical approaches were applied to investigate CA-facilitated cadmium (Cd) tolerance in the roots of tobacco (Nicotiana tabacum) seedlings. Treatment with CdCl2 at 20 µM for 72 h resulted in the significant decrease in root elongation by 40.39% as compared to control. CA alleviated Cd-inhibited root elongation in dose- and time-dependent manners. The addition of CA at 20 µM induced significant increase in root elongation by 42.58% as compared to Cd treatment alone. CA abolished Cd-induced ROS (reactive oxygen species) accumulation, lipid peroxidation, loss of membrane integrity, cell death, and free Cd2+ accumulation in roots. CA blocked the Cd-induced increase in the endogenous H2S level through the down-regulation of d-cysteine desulfhydrase (DCD) expression. H2S scavenger hypotaurine (HT) or potent H2S-biosynthetic inhibitor dl-propargylglicine (PAG) were able mimic the action of CA on the blockade of Cd-induced H2S accumulation, cell death, and growth inhibition. Enhancement of the endogenous H2S level with NaHS (H2S donor) abrogated all the beneficial capabilities of CA, HT, and PAG. Collectively, these results suggest that CA has great potential to confer plant tolerance against Cd stress, which is closely associated with its capability to inhibit Cd-induced H2S production. This study not only provides evidences for the regulation of plant physiology by CA but also sheds new light on the cross-talk between CA and H2S in physiological modulations.

Thymol Mitigates Cadmium Stress by Regulating Glutathione Levels and Reactive Oxygen Species Homeostasis in Tobacco Seedlings.

Thymol is a famous plant-derived compound that has been widely used in pharmacy due to its antioxidant and antimicrobial properties. However, the modulation of intrinsic plant physiology by thymol remains unclear. It is a significant challenge to confer plant tolerance to Cd (cadmium) stress. In the present study physiological, histochemical, and biochemical methods were applied to investigate thymol-induced Cd tolerance in tobacco (Nicotiana tabacum) seedlings. Thymol was able to alleviate Cd-induced growth inhibition of tobacco seedlings in both dose- and time-dependent manners. Both histochemical detection and in-tube assays suggested that thymol treatment blocked Cd-induced over-generation of reactive oxygen species (ROS), lipid peroxidation, and loss of membrane integrity in both leaves and roots. Thymol decreased Cd-induced cell death that was indicated in vivo by propidium iodide (PI) and trypan blue, respectively. Thymol stimulated glutathione (GSH) biosynthesis by upregulating the expression of γ-glutamylcysteine synthetase 1 (GSH1) in Cd-treated seedlings, which may contribute to the alleviation of Cd-induced oxidative injury. In situ fluorescent detection of intracellular Cd2+ revealed that thymol significantly decreased free Cd2+ in roots, which could be explained by the thymol-stimulated GSH biosynthesis and upregulation of the expression of phyochelatin synthase 1 (PCS1). Taken together, these results suggested that thymol has great potential to trigger plant resistant responses to combat heavy metal toxicity, which may help our understanding of the mechanism for thymol-modulated cell metabolic pathways in response to environmental stimuli.

[Effects of light intensity on morphological and physiological characteristics of tobacco seedlings].

Through shading with white gauze to simulate different light habitats (100%, 68.2% 35.4% and 16.7% of full sunlight), this paper studied the effects of light intensity on the morphological and physiological characteristics of tobacco seedlings. The results showed that with the decrease of relative light intensity, seedling height increased, while stem circumference, dry weight/fresh weight ratio, leaf thickness, specific leaf weight, and matter accumulation decreased. Less effect of light intensity was observed on the number of seedling leaves. Under weak light condition, the contents of free water, chlorophyll, total nitrogen, and protein in leaves increased, while bound water content, chlorophyll a/b ratio, and invertase activity decreased. The root/shoot ratio, root biomass, and root vitality also decreased. All of these suggested that weak light was unfavorable to the cultivation of strong tobacco seedlings, and the light conditions of seedbed should be improved in tobacco production.