The potential of glycinin basic peptide derived from soybean as a promising candidate for the natural food additive and preservative: A review.

Glycinin basic peptide (GBP) is the basic polypeptide of soybean glycinin that is isolated using cheap and readily available raw materials (soybean meals). GBP can bear high-temperature processing and has good functional properties, such as emulsification and adhesion properties et al. GBP exhibits broad-spectrum antimicrobial activities against Gram-positive and Gram-negative bacteria as well as fungi. Beyond that, GBP shows enormous application potential to improve the quality and extend the shelf life of food products. This review will systematically provide information on the purification, physicochemical and functional properties of GBP. Moreover, the antimicrobial activities and multi-target antimicrobial mechanism of GBP as well as the applications of GBP in different food products are also reviewed and discussed in detail. This review aims to offer valuable insights for the applications of GBP in the food industry as a promising natural food additive and preservative.

Thymol Induces Fenton-Reaction-Dependent Ferroptosis in Vibrio parahemolyticus.

Thymol has efficient bactericidal activity against a variety of pathogenic bacteria, but the bactericidal mechanism against Vibrio parahemolyticus (V. parahemolyticus) has rarely been reported. In the current study, we investigated the bactericidal mechanism of thymol against V. parahemolyticus. The Results revealed that 150 µg/mL of thymol had 99.9% bactericidal activity on V. parahemolyticus. Intracellular bursts of reactive oxygen species (ROS), Fe2+accumulation, lipid peroxidation, and DNA breakage were checked by cell staining. The exogenous addition of H2O2 and catalase promoted and alleviated thymol-induced cell death to a certain extent, respectively, and the addition of the ferroptosis inhibitor Liproxstatin-1 also alleviated thymol-induced cell death, confirming that thymol induced Fenton-reaction-dependent ferroptosis in V. parahemolyticus. Proteomic analysis revealed that relevant proteins involved in ROS production, lipid peroxidation accumulation, and DNA repair were significantly upregulated after thymol treatment. Molecular docking revealed two potential binding sites (amino acids 46H and 42F) between thymol and ferritin, and thymol could promote the release of Fe2+ from ferritin proteins through in vitro interactions analyzed. Therefore, we hypothesized that ferritin as a potential target may mediate thymol-induced ferroptosis in V. parahemolyticus. This study provides new ideas for the development of natural inhibitors for controlling V. parahemolyticus in aquatic products.

Structure, functional and physicochemical properties of lotus seed protein under different pH environments.

BACKGROUND: The present study investigated the structure, functional and physicochemical properties of lotus seed protein (LSP) under different pH environments. The structures of LSP were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Fourier transform infrared spectroscopy (FTIR), zeta potential, particle size distributions, free sulfhydryl and rheological properties. The functional and physicochemical properties of LSP were characterized by color, foaming property, emulsification property, solubility, oil holding capacity, water holding capacity, differential scanning calorimetry analysis and surface hydrophobicity. RESULTS: LSP was mainly composed of eight subunits (18, 25, 31, 47, 51, 56, 65 and 151 kDa), in which the richest band was 25 kDa. FTIR results showed that LSP had high total contents of α-helix and ß-sheet (44.81-46.85%) in acidic environments. Meanwhile, there was more ß-structure and random structure in neutral and alkaline environments (pH 7.0 and 9.0). At pH 5.0, LSP had large particle size (1576.98 nm), high emulsion stability index (91.43 min), foaming stability (75.69%) and water holding capacity (2.21 g g-1), but low solubility (35.98%), free sulfhydryl content (1.95 µmol g-1) and surface hydrophobicity (780). DSC analysis showed the denaturation temperatures (82.23 °C) of LSP at pH 5.0 was higher than those (80.10, 80.52 and 71.82 °C) at pH 3.0, 7.0 and 9.0. The analysis of rheological properties showed that LSP gel had high stability and great strength in an alkaline environment. CONCLUSION: The findings of the present study are anticipated to serve as a valuable reference for the implementation of LSP in the food industry. © 2024 Society of Chemical Industry.

Structure, physicochemical properties, and biological activities of protein hydrolysates from Zanthoxylum seed.

BACKGROUND: Zanthoxylum seed, as a low-cost and easily accessible plant protein resource, has good potential in the food industry. But protein and its hydrolysates from Zanthoxylum seed are underutilized due to the dearth of studies on them. This study aimed to investigate the structure and physicochemical and biological activities of Zanthoxylum seed protein (ZSP) hydrolysates prepared using Protamex®, Alcalase®, Neutrase®, trypsin, or pepsin. RESULTS: Hydrolysis using each of the five enzymes diminished average particle size and molecular weight of ZSP but increased random coil content. ZSP hydrolysate prepared using pepsin had the highest degree of hydrolysis (24.07%) and the smallest molecular weight (<13 kDa) and average particle size (129.80 nm) with the highest solubility (98.9%). In contrast, ZSP hydrolysate prepared using Alcalase had the highest surface hydrophobicity and foaming capacity (88.89%), as well as the lowest foam stability (45.00%). Moreover, ZSP hydrolysate prepared using Alcalase exhibited the best hydroxyl-radical scavenging (half maximal inhibitory concentration (IC50 ) 1.94 mg mL-1 ) and ferrous-ion chelating (IC50 0.61 mg mL-1 ) activities. Additionally, ZSP hydrolysate prepared using pepsin displayed the highest angiotensin-converting enzyme inhibition activity (IC50 0.54 mg mL-1 ). CONCLUSION: These data showed that enzyme hydrolysis improved the physicochemical properties of ZSP, and enzymatic hydrolysates of ZSP exhibited significant biological activity. These results provided validation for application of ZSP enzymatic hydrolysates as antioxidants and antihypertensive agents in the food or medicinal industries. © 2023 Society of Chemical Industry.

The structural characteristics and physicochemical properties of mung bean protein hydrolysate of protamex induced by ultrasound.

BACKGROUND: The limited physicochemical properties (such as low foaming and emulsifying capacity) of mung bean protein hydrolysate restrict its application in the food industry. Ultrasound treatment could change the structures of protein hydrolysate to accordingly affect its physicochemical properties. The aim of this study was to investigate the effects of ultrasound treatment on the structural and physicochemical properties of mung bean protein hydrolysate of protamex (MBHP). The structural characteristics of MBHP were evaluated using tricine sodium dodecylsulfate-polyacrylamide gel electrophoresis, laser scattering, fluorescence spectrometry, etc. Solubility, fat absorption capacity and foaming, emulsifying and thermal properties were determined to characterize the physicochemical properties of MBHP. RESULTS: MBHP and ultrasonicated-MBHPs (UT-MBHPs) all contained five main bands of 25.8, 12.1, 5.6, 4.8 and 3.9 kDa, illustrating that ultrasound did not change the subunits of MBHP. Ultrasound treatment increased the contents of α-helix, ß-sheet and random coil and enhanced the intrinsic fluorescence intensity of MBHP, but decreased the content of ß-turn, which demonstrated that ultrasound modified the secondary and tertiary structures of MBHP. UT-MBHPs exhibited higher solubility, foaming capacity and emulsifying properties than MBHP, among which MBHP-330 W had the highest solubility (97.32%), foaming capacity (200%), emulsification activity index (306.96 m2 g-1 ) and emulsion stability index (94.80%) at pH 9.0. CONCLUSION: Ultrasound treatment enhanced the physicochemical properties of MBHP, which could broaden its application as a vital ingredient in the food industry. © 2023 Society of Chemical Industry.

Changes in quality components and antioxidant activity of peony seed soy sauce during low-salt solid-state fermentation.

BACKGROUND: In this study, the fermentation conditions of peony seed soy sauce (PSSS) koji were optimized by response surface method, and the quality components and antioxidant activity of PSSS were investigated at different low-salt solid-state fermentation stages. RESULTS: Results of response surface method showed that the optimal fermentation conditions were 460.6 g kg-1 water content, 48.6 h culture time, 31.5 °C culture temperature and ratio 2.1:1 (w/w) of peony seed meal:wheat bran, with the highest neutral protease activity (2193.78 U g-1 ) of PSSS koji. PSSS had the highest amino acid nitrogen (7.69 g L-1 ), salt-free soluble solids (185.26 g L-1 ), total free amino acids (49.03 g L-1 ), essential free amino acids (19.58 g L-1 ) and umami free amino acids (16.64 g L-1 ) at 20 days of fermentation. The highest total phenolics were 5.414 g gallic acid equivalent L-1 and total flavonoids 0.617 g rutin equivalent L-1 , as well as the highest DPPH radical scavenging activity (86.19%) and reducing power (0.8802, A700 ) of PSSS fermented at 30 days. Sensory evaluation showed that fermentation of 20 days and 25 days could produce a better taste and aroma of PSSS than 15 days and 30 days. CONCLUSION: PSSS had the highest quality components in the middle of fermentation (20 days) and the highest antioxidant activity in the late fermentation period (30 days). These results demonstrated that peony seed meal could be used to produce high-quality soy sauce with high antioxidant activity. © 2023 Society of Chemical Industry.

Physicochemical, functional and antioxidant properties of mung bean protein enzymatic hydrolysates.

This study aimed to investigate physicochemical, functional and antioxidant properties of mung bean protein (MBP) enzymatic hydrolysates (MBPEHs) by alcalase, neutrase, protamex, flavourzyme and papain. Physicochemical properties were evaluated by SDS-PAGE, particle size distribution, FTIR, ultraviolet visible and fluorescence spectrophotometries. ABTS, hydroxyl scavenging, Fe2+ chelating activity were used to evaluate antioxidant activity. Enzymolysis with five proteases decreased average particle size, α-helix, ß-sheet, surface hydrophobicity of hydrolysates. Alcalase hydrolysate had the highest degree of hydrolysis (23.55%), absolute zeta potential (33.73 mV) and the lowest molecular weight (<10 kDa). Protamex and papain hydrolysates had higher foaming capacities, emulsification activity indexes, emulsion stability indexes (235.00%, 123.07 m2/g, 132.54 min; 200.10%, 105.39 m2/g, 190.67 min) than MBP (135.03%, 20.03 m2/g, 30.88 min). Alcalase hydrolysate demonstrated the lowest IC50 (mg/mL) in ABTS (0.12), hydroxyl (2.98), Fe2+ chelating (0.22). These results provide support for application of MBPEHs as foaming agent, emulsifier and antioxidant in food industry.

Influence of ultrasound treatment on the physicochemical and antioxidant properties of mung bean protein hydrolysate.

This study aimed to investigate influence of ultrasonic treatment on physicochemical and antioxidant properties of mung bean protein hydrolysate (MPH). Physicochemical properties of MPH were evaluated by Tricine-SDS-PAGE, particle size distribution, fourier transform infrared spectroscopy (FTIR) and fluorescence spectroscopy, among others. Radicals scavenging activities of ABTS, hydroxyl, superoxide anion, Fe2+ chelating ability and reducing power characterized antioxidant activities of MPH. MPH contained four bands of 25.6, 12.8, 10.6 and 4.9 kDa, in which 4.9 kDa was the most abundant. Ultrasonic treatment increased the contents of aromatic and hydrophobic amino acids in MPH. Ultrasonic treatment decreased the content of α-helix of MPH and increased ß-sheet and ß-turn compared to MPH. MPH-546 W (ultrasonic treatment 546 W, 20 min) had the lowest average particle size (290.13 nm), zeta potential (-36.37 mV) and surface hydrophobicity (367.95 A.U.). Antioxidant activities of ultrasonicated-MPH increased with the ultrasonic power, achieving the lowest IC50 (mg/mL) of 0.1087 (ABTS), 1.796 (hydroxyl), 1.003 (superoxide anion) and 0.185 (Fe2+ chelating ability) in 546 W power. These results indicated ultrasonic treatment would be a promising method to improve the antioxidant properties of MPH, which would broaden the application scope of MPH as bioactive components in the food industry.

Physicochemical properties and antioxidant activities of tree peony (Paeonia suffruticosa Andr.) seed protein hydrolysates obtained with different proteases.

The physicochemical and antioxidant properties of tree peony seed protein (TPSP) hydrolysates by Alcalase, Neutrase, Papain, Protamex, and Flavourzyme were investigated in this study. The physicochemical properties were characterized by SDS-PAGE, particle size distribution, fourier transform infrared and fluorescence spectroscopy etc. The antioxidant activities were determined by DPPH radical, ABTS radical, Fe2+ chelating, and reducing power. The results showed five proteases produced hydrolysates with a significantly reduced average particle size, α-helices, and surface hydrophobicity compared to TPSP. Alcalase and Neutrase hydrolysis enhanced the nutritional value of the hydrolysates. Alcalase hydrolysates possessed the highest degree of hydrolysis (27.97%) and lowest molecular weight (<13 kDa) with average particle size (231.33 nm). Alcalase hydrolysate displayed the highest radical scavenging (DPPH IC50 = 0.18 mg/mL, ABTS IC50 = 1.57 mg/mL), Fe2+ chelating activity (IC50 = 0.99 mg/mL), and reducing power (0.594). These results provide the fundamentals for TPSP hydrolysates as antioxidants to be employed in food industry or pharmaceutical industry.

Eugenol Confers Cadmium Tolerance via Intensifying Endogenous Hydrogen Sulfide Signaling in Brassica rapa.

Eugenol, a plant-derived small compound, shows great medicinal potential. However, whether and how eugenol regulates crop physiology remains elusive. Here we reported that eugenol induced Cd (cadmium) tolerance in the root of Brassica rapa. Roots were treated with eugenol and CdCl2 simultaneously (eugenol + Cd) or pretreated with eugenol followed by CdCl2 treatment (eugenol → Cd). Eugenol significantly attenuated Cd-induced growth inhibition, ROS accumulation, oxidative injury, and cell death, which were confirmed by in vivo histochemical analysis. Eugenol remarkably decreased free Cd2+ accumulation in root. Eugenol intensified GSH (glutathione) accumulation in roots upon CdCl2 exposure, which explained the decrease in free Cd2+ and attenuation of oxidative injury. Eugenol stimulated endogenous H2S (hydrogen sulfide) generation by upregulating the expression of BrLCD ( l-cysteine desulfhydrase) and BrDCD ( d-cysteine desulfhydrase) as well as their enzymatic activities in CdCl2-treated root. Application of H2S biosynthesis inhibitor or H2S scavenger led to the decrease in endogenous H2S level in Cd-treated root, which further compromised all the above effects of eugenol. These findings suggested that eugenol triggered H2S → GSH signaling cassette in plants to combat Cd stress, which shed new light on eugenol-modulated plant physiology and the interaction between eugenol and H2S.

Thymol Induces Conidial Apoptosis in Aspergillus flavus via Stimulating K+ Eruption.

Aspergillus flavus is a notorious foodborne fungus, posing a significant risk to humans in the form of hepatocellular carcinoma or aspergillosis. Thymol, as a food preservative, could efficiently kill conidia of A. flavus. However, the underlying mechanisms by which thymol kills A. flavus are not completely understood. With specific fluorescent dyes, we detected several apoptotic hallmarks, including chromatin condensation, phosphatidylserine externalization, DNA damage, mitochondrial depolarization, and caspase 9 activation in conidia exposed to 200 µg/mL of thymol, indicating that thymol induced a caspase-dependent conidial apoptosis in A. flavus. Chemical-protein interactome (CPI) and autodock analyses showed that KCNAB, homologue to the ß-subunit of the voltage-gated potassium channel (Kv) and aldo-keto reductase, was the potential target of thymol. Following studies demonstrated that thymol could activate the aldo-keto reductase activity of KCNAB in vitro and stimulate a transient K+ efflux in conidia, as determined using a Port-a-Patch. Blocking K+ eruption by 4-aminopyridine (a universal inhibitor of Kv) could significantly alleviate thymol-mediated conidial apoptosis, indicating that activation of Kv was responsible for the apoptosis. Taken together, our results revealed a K+ efflux-mediated apoptotic pathway in A. flavus, which greatly contributed to the development of an alternative strategy to control this pathogen.

Physicochemical characteristics and functionality of tree peony (Paeonia suffruticosa Andr.) seed protein.

The physicochemical and functional properties of tree peony seed protein were investigated. Tree peony seed protein with a favourable amino acid profile was composed of a 60kDa protein with two subunits of 38 and 23kDa. The isoelectric points of the two subunits were 3.6 and 9.0. Moreover, acid-Schiff staining indicated both of them were glycoproteins. Diagonal and 2-D electrophoresis data indicated the 38kDa subunit included three types, which two types had inter-disulphide bonds and one type had no-disulphide bonds. So did the 23kDa subunit. Circular dichroism spectra indicated the tree peony seed protein had predominantly a ß-sheet structure. Differential scanning calorimetry analysis indicated the denaturation temperatures of the tree peony seed protein at pH 5.0, 7.0 and 9.0 were 92.0, 97.1 and 95.2°C, respectively. Tree peony seed protein could be a food ingredient in the food industry due to its desirable physicochemical and functional properties.

Antibacterial Actions of Glycinin Basic Peptide against Escherichia coli.

Glycinin basic peptide (GBP) is an antibacterial ingredient that occurs naturally in the basic parts of soybean glycinin. The antibacterial actions of GBP against Escherichia coli ATCC 8739 were investigated in this study. The minimum inhibitory concentration of GBP against E. coli was 200 µg/mL. The exposure of E. coli cells to GBP induced significant cell damage and inactivated intracellular esterases (stressed and dead cells, 70.9% ± 0.04 for 200 µg/mL of GBP and 91.9% ± 0.06 for 400 µg/mL of GBP), as determined through dual staining in flow cytometry. GBP resulted in the exposure of phosphatidylserine in E. coli cells. The analyses of flow cytometry-manifested GBP treatment led to the shrinkage of the cell surface and the complication of cell granularity. The observations in transmission electron microscopy demonstrated that 400 µg/mL of GBP severely disrupted the membrane integrity, resulting in ruptures or pores in the membrane, outflows of intracellular contents, or aggregation of the cytoplasm. Release of alkaline phosphatase, lipopolysaccharide, and reducing sugar further verified that the membrane damage was due to GBP. In addition, GBP treatment changed the helicity and base staking of DNA, as determined by circular dichroism spectroscopy. These results showed that GBP had strong antibacterial activity against E. coli via membrane damage and DNA perturbation. Additionally, GBP exhibited no cytotoxicity on the viability of human embryonic kidney cells. Thus, GBP may be a promising candidate as a natural antibacterial agent.

GSK-3ß inhibitor attenuates urinary albumin excretion in type 2 diabetic db/db mice, and delays epithelial-to-mesenchymal transition in mouse kidneys and podocytes.

The mechanism underlying epithelial­to­mesenchymal transition (EMT) caused by high glucose (HG) stimulation in diabetic nephropathy (DN) remains to be fully elucidated. The present study investigated the effects of HG on EMT and the activity of glycogen synthase kinase 3ß (GSK­3ß) in podocytes and the kidneys of db/db mice, and assessed the effects of (2'Z, 3'E)­6­bromoindirubin­3'­oxime (BIO), an inhibitor of GSK­3ß, on EMT and glomerular injury. The resulting data showed that the activity of GSK­3ß was upregulated by HG and downregulated by BIO in the podocytes and the renal cortex. The expression levels of epithelial markers, including nephrin, podocin and synaptopodin, were decreased by HG and increased by BIO, whereas the reverse were true for mesenchymal markers, including α­smooth muscle actin (α­SMA) and fibronectin. The expression levels of ß­catenin and Snail, in contrast to current understanding of the Wnt signaling pathway, were increased by HG and decreased by BIO. In addition, expression of the vitamin D receptor (VDR) was decreased by HG and increased by BIO. In conclusion, the present study revealed that the mechanism by which BIO inhibited HG­mediated EMT in podocytes and the renal cortex was primarily due to the VDR. Treatment with BIO protected renal function by maintaining the integrity of the filtration membrane and decreasing UAE, but not by regulating blood glucose. Therefore, GSK­3ß may be used as a sensitive biomarker of DN, and its inhibition by BIO may be effective in the treatment of DN.

Effects of glycinin basic peptide on physicochemical characteristics and microbial inactivation of pasteurized milk.

The effects of glycinin basic peptide (GBP) on physicochemical characteristics and microbial inactivation of pasteurized milk were investigated over 21d of storage at 4°C. Sensory properties, total bacterial count, pH, alcohol levels, lactose content, and protein changes of pasteurized milk differentially treated with GBP were analyzed periodically during refrigerated storage. Compared with the control, reductions for total bacterial count and specific bacterium (Staphylococcus aureus) in pasteurized milk treated with GBP during storage were found. However, sensory scores, pH, lactose, and protein contents of pasteurized milk treated with GBP were much higher than those of the control. A concentration of 0.015% (wt/vol) GBP could effectively inhibit the growth and reproduction of bacteria in pasteurized milk, enhance its sensory and physicochemical properties, and extend its shelf life to 15d. Thus, GBP has good potential to be a natural milk preservative.

Effects of glycinin basic polypeptide on sensory and physicochemical properties of chilled pork.

Effects of glycinin basic polypeptide (GBP) on sensory and physicochemical properties of pork during chilled storage were investigated. Pork treated with GBP was analyzed periodically for sensory properties, pH, total volatile base nitrogen (TVB-N), α-thiobarbituric acid (TBA), and total viable count (TVC) values. Compared with controls, TBA values of pork treated with GBP did not change. TVB-N, pH, and TVC values of pork showed reductions with increasing concentrations of GBP during 8 days of storage. However, there were increases in sensory scores. TVC values of treated pork showed a positive linear relationship with both pH and TVB-N values. GBP at 0.16 and 0.20% efficiently inhibited bacterial growth, and enhanced chilled pork sensory scores. Therefore, GBP has potential as a pork biological preservative for extension of shelf life during chilled storage.

Antibacterial characteristics of glycinin basic polypeptide against Staphylococcus aureus.

This paper aims to study the antibacterial action of glycinin basic polypeptide (GBP) on Staphylococcus aureus (S. aureus). Herein, the minimum inhibitory concentration (MIC) of GBP against S. aureus was 0.2 mg/mL. Atomic force microscopy (AFM) imaging showed that GBP seriously damaged the morphology of the S. aureus cells. GBP (0.8 mg/mL) enhanced the relative release of ß-galactosidase to 25.48% when compared to the control. The activity of the respiratory-chain dehydrogenase of S. aureus decreased with increasing GBP concentration. GBP could cause a leakage of intracellular substances. Additionally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that S. aureus bacterial proteins decreased in response to the time period of treating the bacterial cells with GBP. These results indicate that GBP could remarkably inhibit S. aureus and is, therefore, a potential food preservative.

Selenium inhibits root elongation by repressing the generation of endogenous hydrogen sulfide in Brassica rapa.

Selenium (Se) has been becoming an emerging pollutant causing severe phytotoxicity, which the biochemical mechanism is rarely known. Although hydrogen sulfide (H2S) has been suggested as an important exogenous regulator modulating plant physiological adaptions in response to heavy metal stress, whether and how the endogenous H2S regulates Se-induce phytotoxicity remains unclear. In this work, a self-developed specific fluorescent probe (WSP-1) was applied to track endogenous H2S in situ in the roots of Brassica rapa under Se(IV) stress. Se(IV)-induced root growth stunt was closely correlated with the inhibition of endogenous H2S generation in root tips. Se(IV) stress dampened the expression of most LCD and DCD homologues in the roots of B. rapa. By using various specific fluorescent probes for bio-imaging root tips in situ, we found that the increase in endogenous H2S by the application of H2S donor NaHS could significantly alleviate Se(IV)-induced reactive oxygen species (ROS) over-accumulation, oxidative impairment, and cell death in root tips, which further resulted in the recovery of root growth under Se(IV) stress. However, dampening the endogenous H2S could block the alleviated effect of NaHS on Se(IV)-induced phytotoxicity. Finally, the increase in endogenous H2S resulted in the enhancement of glutathione (GSH) in Se(IV)-treated roots, which may share the similar molecular mechanism for the dominant role of H2S in removing ROS by activating GSH biosynthesis in mammals. Altogether, these data provide the first direct evidences confirming the pivotal role of endogenous H2S in modulating Se(IV)-induced phytotoxicity in roots.

The endogenous nitric oxide mediates selenium-induced phytotoxicity by promoting ROS generation in Brassica rapa.

Selenium (Se) is suggested as an emerging pollutant in agricultural environment because of the increasing anthropogenic release of Se, which in turn results in phytotoxicity. The most common consequence of Se-induced toxicity in plants is oxidative injury, but how Se induces reactive oxygen species (ROS) burst remains unclear. In this work, histofluorescent staining was applied to monitor the dynamics of ROS and nitric oxide (NO) in the root of Brassica rapa under Se(IV) stress. Se(IV)-induced faster accumulation of NO than ROS. Both NO and ROS accumulation were positively correlated with Se(IV)-induced inhibition of root growth. The NO accumulation was nitrate reductase (NR)- and nitric oxide synthase (NOS)-dependent while ROS accumulation was NADPH oxidase-dependent. The removal of NO by NR inhibitor, NOS inhibitor, and NO scavenger could alleviate Se(IV)-induced expression of Br_Rbohs coding for NADPH oxidase and the following ROS accumulation in roots, which further resulted in the amelioration of Se(IV)-induced oxidative injury and growth inhibition. Thus, we proposed that the endogenous NO played a toxic role in B. rapa under Se(IV) stress by triggering ROS burst. Such findings can be used to evaluate the toxic effects of Se contamination on crop plants.