Comparative physiological and metabolomic analyses reveal that Fe3O4 and ZnO nanoparticles alleviate Cd toxicity in tobacco.

BACKGROUND: Heavy metals repress tobacco growth and quality, and engineered nanomaterials have been used for sustainable agriculture. However, the underlying mechanism of nanoparticle-mediated cadmium (Cd) toxicity in tobacco remains elusive. RESULTS: Herein, we investigated the effects of Fe3O4 and ZnO nanoparticles (NPs) on Cd stress in tobacco cultivar 'Yunyan 87' (Nicotiana tabacum). Cd severely repressed tobacco growth, whereas foliar spraying with Fe3O4 and ZnO NPs promoted plant growth, as indicated by enhancing plant height, root length, shoot and root fresh weight under Cd toxicity. Moreover, Fe3O4 and ZnO NPs increased, including Zn, K and Mn contents, in the roots and/or leaves and facilitated seedling growth under Cd stress. Metabolomics analysis showed that 150 and 76 metabolites were differentially accumulated in roots and leaves under Cd stress, respectively. These metabolites were significantly enriched in the biosynthesis of amino acids, nicotinate and nicotinamide metabolism, arginine and proline metabolism, and flavone and flavonol biosynthesis. Interestingly, Fe3O4 and ZnO NPs restored 50% and 47% in the roots, while they restored 70% and 63% in the leaves to normal levels, thereby facilitating plant growth. Correlation analysis further indicated that these metabolites, including proline, 6-hydroxynicotinic acid, farrerol and quercetin-3-O-sophoroside, were significantly correlated with plant growth. CONCLUSIONS: These results collectively indicate that metal nanoparticles can serve as plant growth regulators and provide insights into using them for improving crops in heavy metal-contaminated areas.

Biodegradation of Nicotine and TSNAs by Bacterium sp. Strain J54.

BACKGROUND: Microorganisms play an important role in reducing harmful substances in flue-cured tobacco. Numerous studies have been conducted to degrade nicotine by microorganisms. OBJECTIVES: The present research deals with the isolation of a potent bacterial strain able to efficiently degrade nicotine and tobacco-specific nitrosamines (TSNAs) in flue-cured tobacco. MATERIAL AND METHODS: Bacterial strain J54, capable of efficiently degrading nicotine and tobacco-specific nitrosamines (TSNAs), was isolated from tobacco leaves and identified. The strain J54 can use nicotine as the sole carbon and nitrogen source and could effectively degrade nicotine while growing in a nicotine isolation medium (NIM) medium. RESULTS: Compared with the control (CK), the total TSNAs content in the tobacco flue-cured eaves after being sprayed with a solution of the J54 strain was found to decrease by 26.22%. Therein, the degradation rates of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), and N'-nitrosoanabasine (NAB) were 24.01%, 26.27%, 28.6%, and 1.83%, respectively. CONCLUSIONS: Bacterial strain J54, was isolated from tobacco leaves and identified as a bacterium, which is similar to Bacillus altitudinis based on its morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. To our knowledge, this is the first report of the isolation and characterization of a Bacillus sp. strain that can efficiently degrade nicotine and TSNAs. The findings pave the way for the application of new biotechnologies for the degradation of nicotine and TSNAs by microorganisms.

The physiological response of different tobacco varieties to chilling stress during the vigorous growing period.

Tobacco is be sensitively affected by chilling injury in the vigorous growth period, which can easily lead to tobacco leaf browning during flue-curing and quality loss, however, the physiological response of tobacco in the prosperous period under low temperature stress is unclear. The physiological response parameters of two tobacco varieties to low temperature stress were determined. The main results were as follows: ① For tobacco in the vigorous growing period subjected to low-temperature stress at 4-16 °C, the tissue structure of chloroplast changed and photosynthetic pigments significantly decreased compared with each control with the increase of intensity of low-temperature stress. ② For tobacco in the vigorous growing period at 10-16 °C, antioxidant capacity of the protective enzyme system, osmotic adjustment capacity of the osmotic adjusting system and polyphenol metabolism in plants gradually increased due to induction of low temperature with the increase of intensity of low-temperature stress. ③ Under low-temperature stress at 4 °C, the protective enzyme system, osmotic adjusting system and polyphenol metabolism of the plants played an insignificant role in stress tolerance, which cannot be constantly enhanced based on low-temperature resistance at 10 °C. This study confirmed that under the temperature stress of 10-16 °C, the self-regulation ability of tobacco will be enhanced with the deepening of low temperature stress, but there is a critical temperature between 4 and 10 °C. The self-regulation ability of plants under low temperature stress will be inhibited.

Endophytic Fungal Community of Tobacco Leaves and Their Potential Role in the Formation of "Cherry-Red" Tobacco.

"Cherry-red" tobacco is the superior variant of tobacco, appearing with the apperance of red dapples on cured leaves due to the demethylation of nicotine to nornicotine during maturation and curing. Fungi are known to have the capacity to convert nicotine to nornicotine. However, an endophytic fungal community of "cherry-red" tobacco has never been reported to our best knowledge. Here, we sampled mature leaves from both "cherry-red" and ordinary tobacco at lower, center, and upper plant sections, and we analyzed the ITS diversity using high-throughput sequencing. Results revealed a significantly different fungal community of foliar endophyte in "cherry-red" and ordinary tobacco. In comparison to the ordinary control, higher diversity and a co-occurrence network complex were found in "cherry-red" samples, especially in the center and upper leaves, where the red dapples mainly emerged. More taxa were enriched in the "cherry-red" than ordinary tobacco leaves at all plant sections. In particular, Aspergillus, some strains of which are reported capable of converting nicotine to nornicotine, was specifically enriched in upper "cherry-red" tobacco leaves, which showed most red dapples after curing. A less robust network structure was detected in the "cherry-red" tobacco compared to ordinary tobacco. The nearest taxon index (NTI) and ß NTI indicated that the local community structuration of tobacco endophytic fungi mainly driven by deterministic process, while the community turnover among plant sections was stochastic. In conclusion, our study provides the earliest information of endophytic fungal community in "cherry-red" tobacco leaf, and the community diversity, composition, and network features are synchronously varied with the appearance of red dapples, which is suggestive of their relationship to the formation of "cherry-red" tobacco.

Discrimination of Fresh Tobacco Leaves with Different Maturity Levels by Near-Infrared (NIR) Spectroscopy and Deep Learning.

The maturity affects the yield, quality, and economic value of tobacco leaves. Leaf maturity level discrimination is an important step in manual harvesting. However, the maturity judgment of fresh tobacco leaves by grower visual evaluation is subjective, which may lead to quality loss and low prices. Therefore, an objective and reliable discriminant technique for tobacco leaf maturity level based on near-infrared (NIR) spectroscopy combined with a deep learning approach of convolutional neural networks (CNNs) is proposed in this study. To assess the performance of the proposed maturity discriminant model, four conventional multiclass classification approaches-K-nearest neighbor (KNN), backpropagation neural network (BPNN), support vector machine (SVM), and extreme learning machine (ELM)-were employed for a comparative analysis of three categories (upper, middle, and lower position) of tobacco leaves. Experimental results showed that the CNN discriminant models were able to precisely classify the maturity level of tobacco leaves for the above three data sets with accuracies of 96.18%, 95.2%, and 97.31%, respectively. Moreover, the CNN models with strong feature extraction and learning ability were superior to the KNN, BPNN, SVM, and ELM models. Thus, NIR spectroscopy combined with CNN is a promising alternative to overcome the limitations of sensory assessment for tobacco leaf maturity level recognition. The development of a maturity-distinguishing model can provide an accurate, reliable, and scientific auxiliary means for tobacco leaf harvesting.

Transcriptomic analysis provides insights into the AUXIN RESPONSE FACTOR 6-mediated repression of nicotine biosynthesis in tobacco (Nicotiana tabacum L.).

KEY MESSAGE: NtARF6 overexpression represses nicotine biosynthesis in tobacco. Transcriptome analysis suggests that NtARF6 acts as a regulatory hub that connect different phytohormone signaling pathways to antagonize the jasmonic acid-induced nicotine biosynthesis. Plant specialized metabolic pathways are regulated by a plethora of molecular regulators that form complex networks. In Nicotiana tabacum, nicotine biosynthesis is regulated by transcriptional activators, such as NtMYC2 and the NIC2-locus ERFs. However, the underlying molecular mechanism of the regulatory feedback is largely unknown. Previous research has shown that NbARF1, a nicotine synthesis repressor, reduces nicotine accumulation in N. benthamiana. In this study, we demonstrated that overexpression of NtARF6, an ortholog of NbARF1, was able to reduce pyridine alkaloid accumulation in tobacco. We found that NtARF6 could not directly repress the transcriptional activities of the key nicotine pathway structural gene promoters. Transcriptomic analysis suggested that this NtARF6-induced deactivation of alkaloid biosynthesis might be achieved by the antagonistic effect between jasmonic acid (JA) and other plant hormone signaling pathways, such as ethylene (ETH), salicylic acid (SA), abscisic acid (ABA). The repression of JA biosynthesis is accompanied by the induction of ETH, ABA, and SA signaling and pathogenic infection defensive responses, resulting in counteracting JA-induced metabolic reprogramming and decreasing the expression of nicotine biosynthetic genes in vivo. This study provides transcriptomic evidence for the regulatory mechanism of the NtARF6-mediated repression of alkaloid biosynthesis and indicates that this ARF transcription factor might act as a regulatory hub to connect different hormone signaling pathways in tobacco.

Cold stress in the harvest period: effects on tobacco leaf quality and curing characteristics.

BACKGROUND: Weather change in high-altitude areas subjects mature tobacco (Nicotiana tabacum L.) to cold stress, which damages tobacco leaf yield and quality. A brupt diurnal temperature differences (the daily temperature dropping more than 20 °C) along with rainfall in tobacco-growing areas at an altitude above 2450 m, caused cold stress to field-grown tobacco. RESULTS: After the flue-cured tobacco suffered cold stress in the field, the surface color of tobacco leaves changed and obvious large browning areas were appeared, and the curing availability was extremely poor. Further research found the quality of fresh tobacco leaves, the content of key chemical components, and the production quality were greatly reduced by cold stress. We hypothesize that cold stress in high altitude environments destroyed the antioxidant enzyme system of mature flue-cured tobacco. Therefore, the quality of fresh tobacco leaves, the content of key chemical components, and the production quality were greatly reduced by cold stress. CONCLUSION: This study confirmed that cold stress in high-altitude tobacco areas was the main reason for the browning of tobacco leaves during the tobacco curing process. This adverse environment seriously damaged the quality of tobacco leaves, but can be mitigated by pay attention to the weather forecast and pick tobacco leaves in advance.

The effect of flue-curing procedure on the dynamic change of microbial diversity of tobaccos.

The purpose of the study is to explore the effect of flue-curing procedure on the diversity of microbial communities in tobaccos and the dynamic change of compositions of microbial communities in the flue-curing process. It expects to provide a theoretical basis for the application of microbes in tobacco leaves and a theoretical basis and idea for optimization of the flue-curing technologies. By investigating tobacco variety K326, the tests were carried out for comparing the conventional flue-curing procedure and dry-ball temperature set and wet-ball temperature degradation flue-curing procedure. Based on the culture-independent approach and high-throughput sequencing procedure, the relationship between the flue-curing procedure for tobaccos and microbial communities in tobaccos was revealed by measuring the dynamic change of microbial communities. The results indicated that:(1) Relative to surface wiping method, washing method was more suitable for the sampling of microbes on the surface of tobacco leaves; (2) Dry-ball temperature set and wet-ball temperature degradation flue-curing procedure was more favorable for maintaining the microbial diversity of tobaccos; (3) Relative to bacteria of the tobaccos, the succession rule of the fungal communities in tobaccos was relatively steady; (4)Compared with bacterial community diversity, the fungal community diversity presented an obvious negative correlation with temperature and humidity during the flue-curing process. (5) The function of bacterial communities in tobaccos matched with the material transformation law of tobaccos, having a direct correlation on the flue-curing process. In short, Dry-ball temperature set and wet-ball temperature degradation flue-curing procedure can more favorably maintain the microbial diversity of tobaccos; moreover, the function of the tobacco system involved in microbes in tobaccos was closely related to the material transformation law of tobaccos in the flue-curing process. It validated that the bacteria in tobaccos play an important role in the flue-curing process of tobaccos.

Salicylic Acid Effects on Flue-Cured Tobacco Quality and Curing Characteristics During Harvesting and Curing in Cold-Stressed Fields.

Salicylic acid (SA) can induce plants to actively enhance abiotic stress resistance. Spraying SA to prevent cold stress in flue-cured tobacco fields can provide theoretical support and practical guidance for the actual protection from cold stress in fields at high altitude in Yunnan. The experiment was performed in Jianchuan County Yunnan Province, China. Honghuadajinyuan, a flue-cured tobacco variety with cold resistance, was used as the research object. SA was tested at two concentrations (0.05 [SA-1] and 0.1 [SA-1] mol L-1) relative to an untreated control (Control) to compare the quality of fresh tobacco leaves, curing characteristics, enzyme activity of antioxidants, and quality of the first-cured tobacco leaves. The tissue structure thickness, SPAD, and plastid pigment content of fresh tobacco leaves were least in the control; there was no significant difference between SA-1 and SA-2. The change of moisture content during curing was SA-1 > SA-2 > Control, and the water loss rate was Control > SA-2 > SA-1, and both varied greatly at 38-48°C. In each curing stage, the carbon and nitrogen metabolites and polyphenols changed most rapidly at 38°C, and the sugar metabolites changed as follows: Control > SA-1 > SA-2. The activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase in fresh tobacco leaves were SA-1 > SA-2 > Control. Malondialdehyde content and the inactivation rate of antioxidant enzymes during curing was Control > SA-2 > SA-1. The economic character and sensory smoking quality of flue-cured tobacco leaves were SA-1 > SA-2 > Control. In high-altitude tobacco planting areas prone to cold stress in the field, early warning weather forecast and field spraying 0.05 mol L-1 SA are beneficial to protect and improve the quality of fresh tobacco leaves, curing characteristics, antioxidant system enzyme activities, and the quality of flue-cured tobacco leaves.

Erratum to: Kinetic Characterization of Tyrosinase-catalyzed Oxidation of Four Polyphenols.

The article "Kinetic Characterization of Tyrosinase-catalyzed Oxidation of Four Polyphenols", written by Wan-yu LIU, Congming ZOU, Jian-hua HU, Zi-jun XU, Lu-qin SI, Jun-jun LIU, Jian-geng HUANG, was originally published electronically on the publisher's internet portal on May 2020 without open access. With the author(s)' decision to opt for Open Choice, the copyright of the article is changed to © The Author(s) 2020 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ ), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The original article has been corrected.

Kinetic Characterization of Tyrosinase-catalyzed Oxidation of Four Polyphenols.

Phenolic compounds such as chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid and caffeic acid are widely distributed in fruits, vegetables and traditional Chinese medicines with a wide range of biological activities. Tyrosinase plays a critical role in the food industry, but recent studies have proposed unexplored aspects of clinical application. Tyrosinase-catalyzed oxidation of four polyphenols as well as its underlying mechanism remains unclear. In the current work, we investigated the kinetic properties of tyrosinase-catalyzed oxidation of the four polyphenols of interest. To measure the unstable o-quinone products, an analytical method using 3-methyl-2-benzothiazolinone hydrazone (MBTH) was established. The optimal incubation time, buffer pH, temperature and enzyme concentration for the enzyme activity in the presence of each polyphenol of interest were investigated. Under the final optimized conditions, the kinetics and substrate specificity of four polyphenols were examined. Kinetic data showed that tyrosinase had the greatest substrate affnity to chlorogenic acid compared with its isomers and caffeic acid. The catalytic effciency with chlorogenic acid was 8- to 15-fold higher than that with the other 3 polyphenols. Molecular docking study demonstrated that the tight binding of chlorogenic acid at the peripheral site should be the major reason for the specifcity to chlorogenic acid. In light of this, the rational design of high-affnity inhibitors against tyrosinase may focus on the binding of both the Cu site and peripheral site. This study will supply a basis for the selection of phenolic acids in food industry and health care.

Lanostane triterpene glycosides from the flowers of Lyonia ovalifolia var. hebecarpa and their antiproliferative activities.

Sixteen lanostane-type triterpene glycosides including eight new ones, named lyonicarposides A-H (1-8), were isolated from the flowers of Lyonia ovalifolia var. hebecarpa (Franch. ex F.B. Forbes & Hemsl.) Chun (Ericaceae). The chemical structures of the new compounds were elucidated by the comprehensive spectroscopic techniques and chemical methods. The Mo2(OAc)4-induced electronic circular dichroism method was used to determine the absolute configurations of C-24 in lyonicarposides A (1), C (3), and E (5). This is the first phytochemical study on the flowers of L. ovalifolia var. hebecarpa. All the isolates were evaluated for their antiproliferative activities against SMMC-7721, HL-60, SW480, MCF-7, and A-549 cell lines. Lyonicarposides A (1) and B (2) showed moderate antiproliferative activities against five cancer cell lines with IC50 values ranging from 12.39 to 28.71 µM. Lyonicarposides C (3) and G (7) and lyonifoloside M (12) selectively inhibited the proliferation of HL-60 and MCF-7 cell lines with IC50 values ranging from 13.03 to 17.71 µM. Interestingly, lyonifoloside L (13) selectively inhibited the proliferation of MCF-7 cell line with an IC50 value of 16.27 µM. Their structure-activity-relationships were discussed.

Dynamic changes in physiological and biochemical properties of flue-cured tobacco of different leaf ages during flue-curing and their effects on yield and quality.

BACKGROUND: The leaf age for harvesting flue-cured tobacco leaves is closely related to the quality of tobacco leaves, so an appropriate leaf age for harvesting is important for improving yield and quality of flue-cured tobacco, however, at present, there are few studies on effects of leaf age on physiological and biochemical changes during flue-curing and there is no clear standard of proper leaf ages for harvesting in production. RESULTS: In the Yunnan tobacco-growing area, an experiment was carried from 2016 to 2017 and different leaf ages were set. The results demonstrate that leaf age has a significant on tissue cell gap, leaf age and flue-curing stages exert significant effects on upper epidermis, palisade and spongy tissue, and leaf thickness of tobacco leaves. The thicknesses of upper and lower epidermis as well as palisade and spongy tissues at different ages show an approximately W-shaped change trend during flue-curing. With the advance of flue-curing stages, contents of starch, chlorophyll, carotenoid, and water in tobacco leaves at different leaf ages decrease, while polyphenol and malondialdehyde (MDA) contents increase. The older the leaf, the faster the chlorophyll, carotenoid, and water contents reduce, while the faster the polyphenol and MDA content rise during flue-curing. The flue-cured tobacco leaves at 116 DAT (days after transplanting) show the highest contents of total nitrogen and nicotine, followed by 123 DAT and those at 130 DAT are the lowest; however, the contents of total sugar and reducing sugar demonstrate a contrary tendency, and the starch content at 116 DAT is much lower than those in the other two treatments. The proportion of superior tobacco, average price, yield, and output value of upper tobacco leaves at different leaf ages are the highest at 123 DAT. The highest sensory evaluation score is found at 123 DAT, while that at 130 DAT is significantly lower in comparison with the other two treatments. CONCLUSIONS: Tobacco leaves harvested at 123 DAT are mature and exhibit a low degree of membrane lipid peroxidation, moderate chemical compositions, and high economic value. 123 DAT improves availability of tobacco leaves.

Effects of enzymatic browning reaction on the usability of tobacco leaves and identification of components of reaction products.

The enzyme browning reaction results in grey speckles on tobacco leaves, which impairs the value and industrial usability of tobacco leaves. To demonstrate the influences of different browning degrees (BDs) of tobacco leaves on the usability of different cultivars and positions and identified structure of brown (grey) matter, we selected three flue-cured tobacco cultivars (K326, Yunyan87, and Honghuadajinyuan (Hongda)) and set four different BDs (<25%, 25% to 50%, 50% to 75%, and >75%). Indices related to: economic traits, chemical components, physical properties, and sensory quality of tobacco leaves with different cultivars were evaluated. Moreover, by utilising thin-layer chromatography and high-performance liquid chromatography, we analysed and identified the structure of the grey matter in terms of chemical composition. The experimental results show that the main component of grey speckles on tobacco leaves is 3-acetyl-6,7-dimethoxycoumarin (YC-ZJF). With the increase of BD, the amount of total sugar and reducing sugar, output value, the proportion of superior tobacco, shatter resistance index, and sensory evaluation score of the three cultivars significantly decrease, while the starch content increases significantly. The changes in protein, total nitrogen, and nicotine are insignificant with changing BD. In addition, other indices show different trends for different cultivars of flue-cured tobacco. After separation and identification of the components of grey speckled leaves, it is proved that the substance derived from grey speckles on tobacco leaves is YC-ZJF. The research is important to the study of browning mechanisms in tobacco leaves and provides corresponding targets for strategies to reduce browning thereof.

Determination of the Bridging Ligand in the Active Site of Tyrosinase.

Tyrosinase is a type-3 copper enzyme that is widely distributed in plants, fungi, insects, and mammals. Developing high potent inhibitors against tyrosinase is of great interest in diverse fields including tobacco curing, food processing, bio-insecticides development, cosmetic development, and human healthcare-related research. In the crystal structure of Agaricus bisporus mushroom tyrosinase, there is an oxygen atom bridging the two copper ions in the active site. It is unclear whether the identity of this bridging oxygen is a water molecule or a hydroxide anion. In the present study, we theoretically determine the identity of this critical bridging oxygen by performing first-principles hybrid quantum mechanics/molecular mechanics/Poisson-Boltzmann-surface area (QM/MM-PBSA) calculations along with a thermodynamic cycle that aim to improve the accuracy. Our results show that the binding with water molecule is energy favored and the QM/MM-optimized structure is very close to the crystal structure, whereas the binding with hydroxide anions causes the increase of energy and significant structural changes of the active site, indicating that the identity of the bridging oxygen must be a water molecule rather than a hydroxide anion. The different binding behavior between water and hydroxide anions may explain why molecules with a carboxyl group or too many negative charges have lower inhibitory activity. In light of this, the design of high potent active inhibitors against tyrosinase should satisfy both the affinity to the copper ions and the charge neutrality of the entire molecule.