Physiological, biochemical and transcriptomic insights into the mechanisms by which molybdenum mitigates cadmium toxicity in Triticum aestivum L.

There are many heavy metal stresses in agricultural biological systems, especially cadmium (Cd) stress, which prevent the full growth of plants, lead to a serious decline in crop yield, and endanger human health. Molybdenum (Mo), an essential nutrient element for plants, regulates plant growth mainly by reducing the absorption of heavy metals and protecting plants from oxidative damage. The aim of this study was to determine the protective effect of Mo (1 µM) application on wheat plants under conditions of Cd (10 µM) toxicity. The biomass, Cd and Mo contents, photosynthesis, leaf and root ultrastructure, antioxidant system, and active oxygen content of the wheat plants were determined. Mo increased the total chlorophyll content of wheat leaves by 43.02% and the net photosynthetic rate by 38.67%, and ameliorated the inhibitory effect of cadmium on photosynthesis by up-regulating photosynthesis-related genes and light-trapping genes. In addition, Mo reduced the content of superoxide anion (O2•-) by 16.55% and 31.12%, malondialdehyde (MDA) by 20.75% and 7.17%, hydrogen peroxide (H2O2) by 24.69% and 8.17%, and electrolyte leakage (EL) by 27.59% and 16.82% in wheat leaves and roots, respectively, and enhanced the antioxidant system to reduce the burst of reactive oxygen species and alleviate the damage of Cd stress on wheat. According to the above results, Mo is considered a plant essential nutrient that enhances Cd tolerance in wheat by limiting the absorption, accumulation and transport of Cd and by regulating antioxidant defence mechanisms. ENVIRONMENTAL IMPLICATION: Cadmium (Cd)，is one of the most toxic heavy metals in the environment, and Cd pollution is a global environmental problem that threatens food security and human health. Molybdenum (Mo), as an essential plant nutrient, is often used to resist environmental stress. However, the mechanism of Mo treatment on wheat subjected to Cd stress has not been reported. In this study, we systematically analysed the effects of Mo on the phenotype, physiology, biochemistry, ultrastructure and Cd content of wheat subjected to Cd stress, and comprehensively analysed the transcriptomics. It not only reveals the mechanism of Mo tolerance to Cd stress in wheat, but also provides new insights into phytoremediation and plant growth in Cd-contaminated soil.

Identifying ADHD-Related Abnormal Functional Connectivity with a Graph Convolutional Neural Network.

Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder that is characterized by inattention, hyperactivity, and impulsivity. The neural mechanisms underlying ADHD remain inadequately understood, and current approaches do not well link neural networks and attention networks within brain networks. Our objective is to investigate the neural mechanisms related to attention and explore neuroimaging biological tags that can be generalized within the attention networks. In this paper, we utilized resting-state functional magnetic resonance imaging data to examine the differential functional connectivity network between ADHD and typically developing individuals. We employed a graph convolutional neural network model to identify individuals with ADHD. After classification, we visualized brain regions with significant contributions to the classification results. Our results suggest that the frontal, temporal, parietal, and cerebellar regions are likely the primary areas of dysfunction in individuals with ADHD. We also explored the relationship between regions of interest and attention networks, as well as the connection between crucial nodes and the distribution of positively and negatively correlated connections. This analysis allowed us to pinpoint the most discriminative brain regions, including the right orbitofrontal gyrus, the left rectus gyrus and bilateral insula, the right inferior temporal gyrus and bilateral transverse temporal gyrus in the temporal region, and the lingual gyrus of the occipital lobe, multiple regions of the basal ganglia and the upper cerebellum. These regions are primarily involved in the attention executive control network and the attention orientation network. Dysfunction in the functional connectivity of these regions may contribute to the underlying causes of ADHD.

Raman Flow Cytometry and Its Biomedical Applications.

Raman flow cytometry (RFC) uniquely integrates the "label-free" capability of Raman spectroscopy with the "high-throughput" attribute of traditional flow cytometry (FCM), offering exceptional performance in cell characterization and sorting. Unlike conventional FCM, RFC stands out for its elimination of the dependency on fluorescent labels, thereby reducing interference with the natural state of cells. Furthermore, it significantly enhances the detection information, providing a more comprehensive chemical fingerprint of cells. This review thoroughly discusses the fundamental principles and technological advantages of RFC and elaborates on its various applications in the biomedical field, from identifying and characterizing cancer cells for in vivo cancer detection and surveillance to sorting stem cells, paving the way for cell therapy, and identifying metabolic products of microbial cells, enabling the differentiation of microbial subgroups. Moreover, we delve into the current challenges and future directions regarding the improvement in sensitivity and throughput. This holds significant implications for the field of cell analysis, especially for the advancement of metabolomics.

Interbrain substrates of role switching during mother-child interaction.

Mother-child interaction is highly dynamic and reciprocal. Switching roles in these back-and-forth interactions serves as a crucial feature of reciprocal behaviors while the underlying neural entrainment is still not well-studied. Here, we designed a role-controlled cooperative task with dual EEG recording to explore how differently two brains interact when mothers and children hold different roles. When children were actors and mothers were observers, mother-child interbrain synchrony emerged primarily within the theta oscillations and the frontal lobe, which highly correlated with children's attachment to their mothers (self-reported by mothers). When their roles were reversed, this synchrony was shifted to the alpha oscillations and the central area and associated with mothers' perception of their relationship with their children. The results suggested an observer-actor neural alignment within the actor's oscillations, which was related to the actor-toward-observer emotional bonding. Our findings contribute to the understanding of how interbrain synchrony is established and dynamically changed during mother-child reciprocal interaction.

Biological Interactions Mediate Soil Functions by Altering Rare Microbial Communities.

Soil microbes, the main driving force of terrestrial biogeochemical cycles, facilitate soil organic matter turnover. However, the influence of the soil fauna on microbial communities remains poorly understood. We investigated soil microbiota dynamics by introducing competition and predation among fauna into two soil ecosystems with different fertilization histories. The interactions significantly affected rare microbial communities including bacteria and fungi. Predation enhanced the abundance of C/N cycle-related genes. Rare microbial communities are important drivers of soil functional gene enrichment. Key rare microbial taxa, including SM1A02, Gammaproteobacteria, and HSB_OF53-F07, were identified. Metabolomics analysis suggested that increased functional gene abundance may be due to specific microbial metabolic activity mediated by soil fauna interactions. Predation had a stronger effect on rare microbes, functional genes, and microbial metabolism compared to competition. Long-term organic fertilizer application increased the soil resistance to animal interactions. These findings provide a comprehensive understanding of microbial community dynamics under soil biological interactions, emphasizing the roles of competition and predation among soil fauna in terrestrial ecosystems.

Molybdenum inhibited the growth of Phytophthora nicotiana and improved the resistance of Nicotiana tabacum L. against tobacco black shank.

Tobacco black shank (TBS) is a soil-borne fungal disease caused by Phytophthora nicotiana (P. nicotianae), significantly impeding the production of high-quality tobacco. Molybdenum (Mo), a crucial trace element for both plants and animals, plays a vital role in promoting plant growth, enhancing photosynthesis, bolstering antioxidant capacity, and maintaining ultrastructural integrity. However, the positive effect of Mo on plant biotic stress is little understood. This study delves into the inhibitory effects of Mo on P. nicotianae and seeks to unravel the underlying mechanisms. The results showed that 16.32 mg/L of Mo significantly inhibited mycelial growth, altered mycelial morphological structure, damaged mycelial cell membrane, and ultimately led to the leakage of cell inclusions. In addition, 0.6 mg/kg Mo applied in soil significantly reduced the severity of TBS. Mo increased photosynthetic parameters and photosynthetic pigment contents of tobacco leaves, upregulated expression of NtPAL and NtPPO resistance genes, as well as improved activities of SOD, POD, CAT, PPO, and PAL in tobacco plants. Furthermore, Mo could regulate nitrogen metabolism and amino acids metabolism to protect tobacco plants against P. nicotianae infection. These findings not only present an ecologically sound approach to control TBS but also contribute valuable insights to the broader exploration of the role of microelements in plant disease management.

Decoding emotion with phase-amplitude fusion features of EEG functional connectivity network.

Decoding emotional neural representations from the electroencephalographic (EEG)-based functional connectivity network (FCN) is of great scientific importance for uncovering emotional cognition mechanisms and developing harmonious human-computer interactions. However, existing methods mainly rely on phase-based FCN measures (e.g., phase locking value [PLV]) to capture dynamic interactions between brain oscillations in emotional states, which fail to reflect the energy fluctuation of cortical oscillations over time. In this study, we initially examined the efficacy of amplitude-based functional networks (e.g., amplitude envelope correlation [AEC]) in representing emotional states. Subsequently, we proposed an efficient phase-amplitude fusion framework (PAF) to fuse PLV and AEC and used common spatial pattern (CSP) to extract fused spatial topological features from PAF for multi-class emotion recognition. We conducted extensive experiments on the DEAP and MAHNOB-HCI datasets. The results showed that: (1) AEC-derived discriminative spatial network topological features possess the ability to characterize emotional states, and the differential network patterns of AEC reflect dynamic interactions in brain regions associated with emotional cognition. (2) The proposed fusion features outperformed other state-of-the-art methods in terms of classification accuracy for both datasets. Moreover, the spatial filter learned from PAF is separable and interpretable, enabling a description of affective activation patterns from both phase and amplitude perspectives.

Selenium Improves the Control Efficacy of Phytophthora nicotianae by Damaging the Cell Membrane System and Promoting Plant Energy Metabolism.

Tobacco black shank (TBS), caused by Phytophthora nicotianae, poses a significant threat to tobacco plants. Selenium (Se), recognized as a beneficial trace element for plant growth, exhibited inhibitory effects on P. nicotianae proliferation, disrupting the cell membrane integrity. This action reduced the energy supply and hindered hyphal transport through membrane proteins, ultimately inducing hyphal apoptosis. Application of 8 mg/L Se through leaf spraying resulted in a notable decrease in TBS incidence. Moreover, Se treatment preserved chloroplast structure, elevated chitinase activities, ß-1,3-GA, polyphenol oxidase, phenylalanine ammonia-lyase, and increased hormonal content. Furthermore, Se enhanced flavonoid and sugar alcohol metabolite levels while diminishing amino acid and organic acid content. This shift promoted amino acid degradation and flavonoid synthesis. These findings underscore the potential efficacy of Se in safeguarding tobacco and potentially other plants against P. nicotianae.

Long-term application of organic fertilizer prompting the dispersal of antibiotic resistance genes and their health risks in the soil plastisphere.

Microplastic (MP) pollution is a rapidly growing global environmental concern that has led to the emergence of a new environmental compartment, the plastisphere, which is a hotspot for the accumulation of antibiotic resistance genes (ARGs) and human bacterial pathogens (HBPs). However, studies on the effects of long-term organic fertilizer application on the dispersal of ARGs and virulence factor genes (VFGs) in the plastisphere of farmland soil have been limited. Here, we performed a field culture experiment by burying nylon bags filled with MPs in paddy soil that had been treated with different fertilizers for over 30 years to explore the changes of ARGs and VFGs in soil plastisphere. Our results show that the soil plastisphere amplified the ARG and VFG pollution caused by organic fertilization by 1.5 and 1.4 times, respectively. And it also led to a 2.7-fold increase in the risk of horizontal gene transfer. Meanwhile, the plastisphere tended to promote deterministic process in the community assembly of HBPs, with an increase of 1.4 times. Network analysis found a significant correlation between ARGs, VFGs, and bacteria in plastisphere. Correlation analysis highlight the important role of mobile genetic elements (MGEs) and bacterial communities in shaping the abundance of ARGs and VFGs, respectively. Our findings provide new insights into the health risk associated with the soil plastisphere due ARGs and VFGs derived from organic fertilizers.

Selenium-molybdenum interactions reduce chromium toxicity in Nicotiana tabacum L. by promoting chromium chelation on the cell wall.

Chromium (Cr) is a hazardous heavy metal that negatively affects animals and plants. The micronutrients selenium (Se) and molybdenum (Mo) have been widely shown to alleviate heavy metal toxicity in plants. However, the molecular mechanism of Cr chelation on the cell wall by combined treatment with Se and Mo has not been reported. Therefore, this study aimed to explore the effects of Se-Mo interactions on the subcellular distribution of Cr (50 µM) and on cell wall composition, structure, functional groups and Cr content, in addition to performing a comprehensive analysis of the transcriptome. Our results showed that the cell walls of shoots and roots accumulated 51.0% and 65.0% of the Cr, respectively. Furthermore, pectin in the cell wall bound 69.5%/90.2% of the Cr in the shoots/roots. Se-Mo interactions upregulated the expression levels of related genes encoding galacturonosyltransferase (GAUT), UTP-glucose-1-phosphate uridylyltransferase (UGP), and UDP-glucose-4-epimerase (GALE), involved in polysaccharide biosynthesis, thereby increasing pectin and cellulose levels. Moreover, combined treatment with Se and Mo increased the lignin content and cell wall thickness by upregulating the expression levels of genes encoding cinnamyl alcohol dehydrogenase (CAD), peroxidase (POX) and phenylalanine amino-lyase (PAL), involved in lignin biosynthesis. Fourier-transform infrared (FTIR) spectroscopy results showed that Se + Mo treatment (in combination) increased the number of carboxylic acid groups (-COOH) groups, thereby enhancing the Cr chelation ability. The results not only elucidate the molecular mechanism of action of Se-Mo interactions in mitigating Cr toxicity but also provide new insights for phytoremediation and food safety.

Tire particles and its leachates: Impact on antibiotic resistance genes in coastal sediments.

Tire particles (TPs), a significant group of microplastics, can be discharged into the coastal environments in various ways. However, our understanding of how TPs impact the antibiotic resistance and pathogenic risks of microorganisms in coastal sediments remains limited. In this study, we used metagenomics to investigate how TPs and their leachates could affect the prevalence of antibiotic resistance genes (ARGs), virulence factor genes (VFGs), and their potential risks to the living creatures such as soil invertebrates and microorganisms in the coastal sediments. We discovered that TP addition significantly increased the abundance and diversity of ARGs and VFGs in coastal sediments, with raw TPs displayed higher impacts than TP leachates and TPs after leaching on ARGs and VFGs. With increasing TP exposure concentrations, the co-occurrence frequency of ARGs and mobile genetic elements (MGEs) in the same contig also increased, suggesting that TPs could enhance the dispersal risk of ARGs. Our metagenome-based binning analysis further revealed that exposure to TPs increased the abundance of potentially pathogenic antibiotic-resistant bacteria (PARB). In addition, chemical additives of TP leachates (e.g., Zn and N-cyclohexylformamide) significantly affected the changes of ARGs in the pore water. In summary, our study provides novel insights into the adverse effects of TP pollutions on aggravating the dissemination and pathogenic risks of ARGs and PARB in the coastal environment.

Probe-type all-fiber tiny-displacement sensor based on orbital-angular-momentum interferometry.

In this study, a probe-type all-fiber tiny-displacement sensor is proposed and experimentally demonstrated, which is realized by using an all-fiber orbital-angular-momentum (OAM) interferometer, where a probe is especially adopted and inserted into the testing arm of the OAM interferometer. The proposed device takes full advantages of the OAM interferometer and the probe-type fiber sensor, making it completely available to the tiny-displacement measurement. As a result, changes in displacement (ranging from 0 nm to 750 nm) with a real resolution of ∼8.81 nm have been successfully measured. To our knowledge, this is the first demonstration of an all-fiber probe-type OAM interferometer, which may find potential application to high-precision tiny displacement in a small confined space.

Music-emotion EEG coupling effects based on representational similarity.

BACKGROUND: Music can evoke intense emotions and music emotion is a complex cognitive process. However, we know little about the cognitive mechanisms underlying these processes, and there are significant individual differences in the emotional responses to the same musical stimuli. NEW METHOD: We used the inter-subject representational similarity analysis (IS-RSA) method to investigate the shared music emotion responses across multiple participants. In addition, we extended IS-RSA to estimate the group cross-frequency coupling effects of music emotion. Based on the cross-frequency coupling IS-RSA, we analyzed the differences in cross-frequency coupling patterns under different music emotions using MI. Comparison of existing methods: most current IS-RSA analyses focus on within-frequency band analysis. However, the cognitive processing of music emotion involves not only activation and brain network connections differences within frequency bands but also information communication between frequency bands. RESULTS: The results of the within-frequency band IS-RSA analysis showed that the theta and gamma frequency bands play important roles in the inter-participant consistency of music emotion. The inter-frequency band IS-RSA analysis showed that the theta-beta coupling pattern exhibited stronger inter-participant consistency compared to the theta-gamma coupling pattern, and the theta-beta coupling had significant consistent representation across various music conditions. Through the significant regions of cross-frequency coupling representation similarity analysis, we performed phase-amplitude coupling analysis on FC4-C6 and FC4-Pz connections. For the theta-beta coupling pattern, we found that the MI of these two connections exhibited different coupling patterns under different music conditions, and they showed a significant decrease compared to the baseline period.

Recognition of the orbital-angular-momentum spectrum for hybrid modes existing in a few-mode fiber via a deep learning method.

In this study, we theoretically and experimentally demonstrate that the convolutional neural network (CNN) in combination with the residual blocks and the regression methods can be used to precisely and quickly reconstruct the OAM spectrum of a hybrid OAM mode no matter how the consistent OAM modes have the same or different order indices in both the azimuthal and the radial direction. For cases of the simulation testing, the mean errors of all recognized parameters for hybrid OAM modes in a four-mode fiber (4MF) and a six-mode fiber (6MF) are smaller than 0.003 and 0.008, respectively. To the best of our knowledge, this is the first time that all the OAM modes, probably existing in the core of 4MFs or 6MFs, can be precisely and quickly recognized from intensity distribution of the hybrid OAM mode itself via the deep learning method.

Optimal government and manufacturer incentive contracts for green production with asymmetric information.

Governments commonly utilize subsidy policy to incentivize manufacturers to produce green products, promoting sustainable development. However, in the presence of information asymmetry, some manufacturers may dishonestly misrepresent the green degree of their products to secure higher subsidies. This study examines different incentive contracts between the government and a green product manufacturer who keeps private information of a product's green-degree in a principal-agent model. Lump-sum transfer and fixed- and flexible-proportion benefit-sharing contracts are proposed to investigate screening and improving green-degree issues. To further enhance the flexible-proportion benefit-sharing contract, we construct a non-linear coordinated contract based on the Nash bargaining solution. The revelation principle and Nash bargaining are performed for comparison and analysis of the contracts. We find that the lump-sum contract reveals true green-degree information but fails to impel manufacturers to improve product's green-degree in developing countries where green product development is in initial stages. In contrast, both fixed- and flexible- proportion benefit-sharing contracts are effective in reveling and enhancing green-degree. The non-linear coordination contract optimizes resource allocation and achieves Pareto improvement. An applied case study for inkjet printer operations and numerical experiments corroborate our model findings.

Norisoboldine, a Natural Isoquinoline Alkaloid, Inhibits Diaphyseal Fracture Healing in Mice by Alleviating Cartilage Formation.

Norisoboldine (NOR), the major isoquinoline alkaloid constituent of a Chinese traditional medicine Radix Linderae, has been demonstrated to inhibit osteoclast differentiation and improve arthritis. The aim of this study is to examine the effect of NOR on bone fracture healing and the underlying mechanisms correlated with bone marrow stromal cells (BMSCs) differentiation to chondrocytes. Our results showed that NOR inhibits the tibia fracture healing process by suppressing cartilage formation, which leads to less endochondral ossification, indicated by less osterix and collage I signaling at the fracture site. Moreover, NOR significantly reduced the differentiation of primary BMSCs to chondrocytes in vitro by reducing the bone morphogenetic protein 2 (BMP2) signaling. These findings imply that NOR negatively regulates the healing of the tibial midshaft fracture, which might delay the union of the fractures and should be noticed when used in other treatments.

Selenium in soil-plant system: Transport, detoxification and bioremediation.

Selenium (Se) is an essential micronutrient for humans and a beneficial element for plants. However, high Se doses always exhibit hazardous effects. Recently, Se toxicity in plant-soil system has received increasing attention. This review will summarize (1) Se concentration in soils and its sources, (2) Se bioavailability in soils and influencing factors, (3) mechanisms on Se uptake and translocation in plants, (4) toxicity and detoxification of Se in plants and (5) strategies to remediate Se pollution. High Se concentration mainly results from wastewater discharge and industrial waste dumping. Selenate (Se [VI]) and selenite (Se [IV]) are the two primary forms absorbed by plants. Soil conditions such as pH, redox potential, organic matter and microorganisms will influence Se bioavailability. In plants, excessive Se will interfere with element uptake, depress photosynthetic pigment biosynthesis, generate oxidative damages and cause genotoxicity. Plants employ a series of strategies to detoxify Se, such as activating antioxidant defense systems and sequestrating excessive Se in the vacuole. In order to alleviate Se toxicity to plants, some strategies can be applied, including phytoremediation, OM remediation, microbial remediation, adsorption technique, chemical reduction technology and exogenous substances (such as Methyl jasmonate, Nitric oxide and Melatonin). This review is expected to expand the knowledge of Se toxicity/detoxicity in soil-plant system and offer valuable insights into soils Se pollution remediation strategies.

Selenium and molybdenum synergistically alleviate chromium toxicity by modulating Cr uptake and subcellular distribution in Nicotiana tabacum L.

Chromium (Cr) is a harmful heavy metal that poses a serious threat to plants and animals. Selenium (Se) and molybdenum (Mo) are two beneficial elements for plant growth and resistance. However, their interactive effects on Cr uptake and distribution are poorly understood. Therefore, a hydroponics experiment was conducted to explore the effects of the use of Se and Mo alone and simultaneously on mitigating Cr toxicity. In this study, Nicotiana tabacum L. seedlings were exposed to control, 50 µM Cr, 50 µM Cr + 2 µM Se, 50 µM Cr + 1 µM Mo, or 50 µM Cr + 2 µM Se + 1 µM Mo in Hoagland solution. After 2 weeks, the plant biomass, Cr, Se and Mo contents, photosynthesis, leaf ultrastructure, antioxidant system, subcellular distribution and associated gene expression in Nicotiana tabacum L. were determined. The results showed that simultaneous use of Se and Mo promoted tobacco growth under Cr stress, as evidenced by reducing reactive oxygen species (ROS) content and reducing Cr translocation factor (TF) and inducing a 51.3% reduction in Cr content in shoots. Additionally, Se-Mo interactions increased the levels of glutathione (GSH) and phytochelatin (PC) and the distribution of Cr in the cell walls and organelles. Furthermore, the relative expression of PCS1 was upregulated, while those of NtST1 and MSN1 were downregulated. The results concluded that the simultaneous use of Se and Mo effectively alleviated Cr toxicity in Nicotiana tabacum L., which not only offers an efficient way for crops to resist Cr toxicity but also provides evidence for the benefit of Se combined with Mo.

Insight into Lotusine and Puerarin in Repairing Alcohol-Induced Metabolic Disorder Based on UPLC-MS/MS.

Alcohol is an essential element in human culture. However, alcoholism has contributed to numerous health issues, including alcoholic fatty liver and sudden death. We found that the alkaloid lotusine possessed hepato- and neuroprotection against alcohol injuries. Lotusine showed comparable protective effects to puerarin, a widely recognized antagonist against alcohol damage. To better understand the metabolic response to alcohol injury and antagonist molecules, we applied sensitive zebrafish and LC-ESI-MS to collect metabolites related to alcohol, puerarin and lotusine exposure. LC-MS identified 119 metabolites with important physiological roles. Differential metabolomic analysis showed that alcohol caused abnormal expression of 82 metabolites (60 up-regulated and 22 down-regulated). These differential metabolites involved 18 metabolic pathways and modules, including apoptosis, necroptosis, nucleotide and fatty acid metabolism. Puerarin reversed seven metabolite variations induced by alcohol, which were related to necroptosis and sphingolipid metabolism. Lotusine was found to repair five metabolites disorders invoked by alcohol, mainly through nucleotide metabolism and glutathione metabolism. In phenotypic bioassay, lotusine showed similar activities to puerarin in alleviating behavioral abnormalities, neuroapoptosis and hepatic lipid accumulation induced by alcohol exposure. Our findings provided a new antagonist, lotusine, for alcohol-induced damage and explored the roles in repairing abnormal metabolism.

NtRAV4 negatively regulates drought tolerance in Nicotiana tabacum by enhancing antioxidant capacity and defence system.

KEY MESSAGE: NtRAV4 is a nucleus-localised protein and no self-activation effect. ntrav4 mutants maintain the steady state of the ROS system under drought stress by enhancing antioxidant capacity and defence system. The APETALA2/ethylene response factor (AP2/ERF) transcription factor (TF) family plays an important role in plant responses to environmental stresses. In this study, we identified a novel NtRAV4 TF, a member of RAV subfamily among AP2/ERF gene family, which have AP2 and B3 domain in its N- and C-terminus, respectively. Subcellular localisation and self-activation activity analysis revealed that NtRAV4 localised in the nucleus and had no self-activation effect. The overexpression and gene editing vectors of NtRAV4 were constructed by homologous recombination and CRISPR/Cas9 gene editing methods, and transformed into tobacco by agrobacterium-mediated method. ntrav4 led to the appearance of termination codon in advance and lacked the unique B3 domain of RAV subfamily protein. Further analysis displayed that knockout of the NtRAV4 in tobacco increased drought tolerance with high relative water content, accompanied by reduced stomatal aperture, density, and stomatal opening ratio compared to overexpression lines and WT. Moreover, ntrav4 knockout plants also exhibited increased osmotic tolerance with low malondialdehyde (MDA) and ion leakage (EL), less accumulation of O2•- and H2O2, and high enzymatic antioxidant (SOD, POD, CAT) activities, non-enzymatic antioxidant (AsA-GSH cycle) contents and hormone (IAA, ABA, GA3, and ZR) levels under drought stress. Furthermore, ntrav4 mutants in tobacco improved the expression levels of ROS-related proline synthesis and stress-responsive genes under osmotic stress. Our results indicate that NtRAV4 negatively regulates plant tolerance to drought stress by reducing water loss and activating the antioxidant system and stress-related gene expression to maintain the steady state of the ROS system.