Nanoselenium foliar application enhances biosynthesis of tea leaves in metabolic cycles and associated responsive pathways

Dong Li, Chunran Zhou, Nan Zou, Yangliu Wu, Jingbang Zhang, Quanshun An, Jia-Qi Li, Canping Pan,
a Innovation Center of Pesticide Research, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, 100193, China
b College of Plant Protection, Shandong Agricultural University, Shandong, 271000, China

An emerging stress of pesticides in plant and soil is closely watched as it affects crop antioxidant systems, nutritional quality, and flavor. Although selenium (Se) can enhance the resistance of plants, the pro- tective mechanism of nanoselenium is still not known under the long-term pesticide stress in tea trees. In this study, we investigated the potential effects of foliar application of nanoselenium for a two-year field experiment on tea plants under pesticide-induced oxidative stress. Compared to control, nano-Se (10 mg/L) markedly enhanced the protein, soluble sugar, carotenoid, tea polyphenols, and catechins contents. High levels of theanine, glutamic acid, proline, and arginine were found to be induced most likely by adjusting the GS-GOGAT cycle. Se-supplementation may promote tea leaves’ secondary meta- bolism, thus increasing the accumulation of total phenols and flavonoids (apigenin, kaempferol, quer- cetin, myricetin, and rutin). It also minimized the accumulation of malondialdehyde, hydrogen peroxide, and superoxide anion by activating the antioxidants enzymes including in the AsA-GSH cycle. Selenium- rich tea also showed better fragrance and flavor. In summary, nano-Se can ameliorate the nutrients quality and abiotic stresses resistance of crops.

1. Introduction
Tea (Camellia sinensis L.) is one of the most popular plants in the human diet due to its health function. Tea quality is evaluated from four aspects of color, aroma, taste, and appearance (Chen et al., 2015). It depends on the comprehensive effects of variouscomponents in tea such as aroma components, tea polyphenols, amino acids, flavonoids, phenolics, and tea polysaccharides (Chen et al., 2018; Probst et al., 2018). These components exhibited anti- bacterial, antiviral, antioxidant, anti-atherosclerosis, anti-throm- bosis, anti-angiogenesis, anti-inflammatory, and anti-tumor effects (Fang et al., 2019; Owona et al., 2020). However, tea trees grow in warm temperate and the subtropical area which is easily infected by plant diseases and insect pests. The irrational use of chemical pesticides in the tea garden is prominent (Xie et al., 2019). The pesticide is important to prevent and control diseases and insects, promote the stable and high yield of food and agriculture (de et al., 2020). However, frequent and abuse use of pesticides causes adverse effects on ecology and environment (Sharma et al., 2020). Tea plantations have used many pesticides by spraying foliage or soil irrigation to ensure production and control pests. This led to an increased in soils pollution and decreased in tea growth (He et al., 2020). Su et al. found that long-term usage of chlorothalonil can disturb soil microbial-mediated nitrogen cycle by intervening N2O emission pattern and soil microbial denitrification in a tea field (Su et al., 2020). Imidacloprid was detected with high level in Chinese tea plantations of Taiwan (Nantou), Tibet (Yigong), Guangdong(Zhanjiang), and Fujian. It even was found in the organic plantation (He et al., 2020). Another similar study suggested acetamiprid and imidacloprid was more frequently detected from 726 tea samples in Chinese tea over a period of seven years. The detection frequency of acetamiprid was reached 73%. Besides, imidacloprid residues in approximately 4.6% of these samples exceeded the Chinese maximum residue limits since 2014 (Li et al., 2020b). Wang et al. found that the pesticides including the imidacloprid, acetamiprid, and difenoconazole from 97 tea samples were most frequently detected (Wang et al., 2019). Excessive application of pesticides can disrupt various metabolic processes of plants. These pesticides could induce the excessive formation of reactive oxygen species(ROS) including the hydrogen peroxide (H2O2) and superoxide (O—2 ) (He et al., 2020). They damage biological system or weaken anti-oxidant defense system. It also affects the physiological and biochemical characteristics of crops, including nutritional compo- sition, taste, color, and flavor (Parween et al., 2016). Imidacloprid, acetamiprid, and difenoconazole also cause potential toxicity of non-target organisms, including the zebrafish and honey bees (Jiang et al., 2020; Shi et al., 2020; Willis Chan et al., 2019). These chemicals hampered the sensitive ecological balance through bio- accumulation in the ecosystem (Sharma et al., 2020). However, the mechanism of oxidative stress of imidacloprid, acetamiprid, and difenoconazole in tea plants were hardly found in the literature.
At present, intervention factors such as biochar, salicylic acid, sodium selenite, nanomaterials can relieve the effects of pesticide- induced oxidative stress to improve crop quality (Khalid et al., 2020; Liu et al., 2020; Usman et al., 2020; Zhang et al., 2020). Ac- curate and rationale fertilization technique provides the nutrients in a controlled way (Mikula et al., 2020). Nanotechnology has found many applications in agriculture, such as nano-fertilizers, nano- pesticides, and nano-bio-sensors (Usman et al., 2020). By sprayed on plants or delivered to the soil for root absorption, nano- fertilizers can enhance the antioxidant capacity and secondary metabolism to promote the health and vitality of the soil, crop yield, and quality. They also reduce production costs with higher nutrient utilization efficiency, thus contributing to the sustainable development of agriculture (Usman et al., 2020). Besides, these compounds improve nitrogen utilization efficiency, reduce toxicity, and minimize potential negative effects (such as groundwater pollution) (Mikula et al., 2020). Low Se concentration can promote plant growth, increase yield, and counteract oxidative stress or pathogenic bacterial infection to improve the nutritional quality of plants (White, 2018). Selenium-enriched green tea exhibits higher antioxidant activity in linoleic acid and lard oxidation systems than ordinary green tea (Xu et al., 2003). Nano-Se with a low dosage and less toxicity has excellent dispersibility and antibacterial ability relative to inorganic Se (El-Ramady et al., 2014). Zahedi et al. showed that the treatment of sodium selenate or nano-Se enhanced maturity index and decreased fruit cracking of pome- granate. Meanwhile, nano-Se is more efficacy than sodium selenate (Zahedi et al., 2019b). To date, most of the research related to tea quality focuses on the influence of environment (Li et al., 2018), pesticides and fertilizers (Ji et al., 2020; Xie et al., 2019), and the production process (Cheng et al., 2020). However, effective exoge- nous interventions to alleviate pesticide stress still lack systematic study due to the long growth cycle and the complex environment of tea gardens. No research on the relationship between nano-Se and tea quality under long-term pesticide-induced oxidative stress.
The accumulated pesticides during the crop growing seasonaffect the flavor of agricultural products. The aroma with a large number of odor components is an important property of tea quality, which gives the tea its unique aroma. There are more than 600 aroma components identified in various tea, including alcohols, aldehydes, ketones, acids, lipids and phenols. During the processingof green tea (heating, rolling, and baking), the typical aroma types are fragrant, chestnut, and floral, which lead to various aroma characteristics of the final products (Baba and Kumazawa, 2014). In tea brewing, some amino acids emit aromas by the action of heat, such as phenylalanine (rose aroma), alanine (flower fragrance), and serine (wine aroma) (Ekborg-Ott et al., 1997). Volatile compounds in tea can be formed by a variety of pathways, such as carotenoid derivative, fatty acid derivative, terpene derivative, phenyl- propanoid/benzene derivative, glycoside hydrolysis, and maillard reaction pathway (Ravichandran, 2002; Yang et al., 2013). At pre- sent, research methods for the key aroma components in the field of food flavor components generally include aroma extraction, identification, and recombination (Yang et al., 2013). For example, the combination of gas chromatography (GC) and mass spectrom- etry (MS) is one of the most important instrumental separation techniques for the analysis of constituents of plant volatiles. However, these methods are often time-consuming and laborious and require complex sample pretreatment (high temperature and potential oxidation or thermal reactions) before GC analysis for volatile extraction (Niu et al., 2019). Ion migration spectroscopy (IMS) is an emerging technology due to its rapid response, low cost, ease of operation, and high sensitivity (Kalapothakis and Barran, 2013). Compared with the electronic nose and GC-MS, GC-IMS in trace detection of volatile organic compounds showed more ad- vantages of simple, portable, and high sensitivity (Hern´andez-Mesa et al., 2017). Besides, it does not need pretreatment and is widely used in various fields such as food flavor, edible vegetable oil, tea, and grade identification (Herna´ndez-Mesa et al., 2017; Li et al., 2019). Up to now, more research is focused on the pesticides resi- dues and risk assessment of tea. However, the long-term combined application of pesticides has less research on the volatiles of tea.
We hypothesized that long-term application of imidacloprid, acetamiprid, and difenoconazole might induce oxidative stress in tea which may lead to the decline of nutritional quality. Therefore, it was aim to explore whether nano-Se can alleviate the damage caused by these residues in tea plants by evaluating levels of an- tioxidants, nutrients, and secondary metabolites. The optimal concentration of nano-Se was selected as a potential field appli- cation and to reveal the remediation effect of nano-Se on quality improvement under pesticide-induced oxidative stress.

2. Experimental section
2.1. Materials, standards, and reagents
Pesticides standards (purities in 95.0e99.8%) were purchased from the Institute of the Control of Agrochemicals, Ministry of Agriculture People’s Republic of China. The mixed standard stocksolution (concentration in 10 mg/L) was prepared by chromato- graphic grade acetonitrile and stored at below 20 ◦C. Acetonitrilewas obtained from Fisher Chemicals (Fair Lawn, NJ, USA). The anhydrous magnesium sulfate (MgSO4) and sodium chloride (NaCl) were purchased from Sinopharm Chemical Reagent (Beijing, China). The particle range of multiwalled carbon nanotubes (MWCNTs) is 7e15 nm, which was purchased by Beijing BTMA Biotechnology Co., Ltd. (China). Octadecylsilane (C18, 40 mm) was obtained from HAMAG instrument technology Co., Ltd. (China). Catechins, tea polyphenols, theanine, and flavonoids standards (purities in 98e99%) were purchased from Shanghai Yuanye Bio Co., Ltd. (China). Selenium and amino acid mixed standard solu- tions were obtained from Tanmo quality control – Reference Ma- terial Center (Beijing, China). The chlorophyll, ascorbic acid (AsA), soluble sugar, carotenoid, protein, glutathione peroxidase (GSH- Px), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT),ascorbate peroxidase (APX), glutathione (GSH), malondialdehyde (MDA), hydrogen peroxide (H2O2), superoxide anion (O2—), dehy- droascorbic acid reductase (DHAR), glutathione reductase (GR), oxidized glutathione (GSSG), glutamine synthetase (GS), glutamate synthetase (GOGAT), flavonoids, and total phenols assay kits werepurchased from Suzhou Comin Biotechnology Co., Ltd. (China).

2.2. Tea material and field trial
The experimental tea field is located in Bihai Road, Donggang District, Rizhao City, Shandong Province, China (119◦370100 E,35◦3005100 N). The area with a temperate monsoon climate is suit-able for the growth of tea trees. The soil type is loamy clay with a pH of 7.3, cation exchange content of 9.0 cmol/kg, and organic matter content of 18.5 g/kg. This experiment uses tea trees (Camellia sen- tences (L.) O. Kuntze cv. Zhongcha 108) as the experimental mate- rial. These leaves have specific traits of the oblique, long oval, green, slight rise, flat, microwave-edged, acuminate, medium in texture, corolla diameter (3.2e3.9 cm), petals white (6e8 leaves), and hairy ovary medium (3-split plants), which are suitable for the prepara- tion of green tea.
These trees used in this experiment have a history of more than 5 years. The formulations were used the imidacloprid 70% water- dispersible granule, acetamiprid 20% wettable powder, and dife- noconazole 10% water-dispersible granule, which were widely applied by native tea farmers from march to august in 2016e2018 (Table S1). Imidacloprid and acetamiprid were used to control the tea lesser leafhopper. Difenoconazole was used to control the Col- letotorichum gloeosporioides. The management measures were consistent to reduce the influence of other factors and ensure the normal growth of tea trees. Therefore, nano-Se was selected as an external intervention factor to alleviate pesticide stress. The nano- Se (Se0 nanoparticles; mean size: 50e78 nm) was characterized in the previous article (Li et al., 2020a). The experimental area of each treated tea tree is 60 square meters. Foliar application of nano-Se was used every 10 days for a total of four applications in March of 2018 and 2019. The time was after 4 o’clock in cloudy or sunny days and ensured there no rain for 4 h after spraying. The experi- ment was set up with five treatments of nano-Se levels-0, 2.5, 5, 10, and 20 mg/L-for nano-Se levels (nano-Se0, nano-Se2.5, nano-Se5, nano-Se10, and nano-Se20, respectively). The standard one budand two leaves of tea tree germination were collected on May 5, 2019. These leaves were withered for 4 h, simmered at 270 ◦C for 15 min, and then dried (100 ◦C) to make green tea.

2.3. Sample preparation and pesticide residues of tea
To investigate the use of pesticides in the experiment base, the registered pesticides were tested on tea. The samples were randomly collected at 13 locations in the test site. Pesticide residues were analyzed by the optional QuEChERS method (quick, easy, cheap, effective, rugged, and safe) with liquid chromatography- tandem mass spectrometry (LC-MS/MS) detection. An amount (2.0 ± 0.1 g) of tea was weighed into a 50 mL centrifuge tube. It was added to 5 mL water and 10 mL acetonitrile, and then vortexed for 2min. Four grams of MgSO4 and 1 g of NaCl were added in the tube. The mixture was shaken for 2min and cooled to room temperature in an ice water bath immediately. After that, the tube was centrifuged at 3800 rpm for 5 min. One milliliter of supernatant was transferred to a 2 mL tube which contained the 10 mg MWCNTs 150 mg anhydrous MgSO4. Then, the tube was shaken for 2 min and centrifuged for 1 min at 10,000 rpm. Finally, the supernatant was filtered through a 0.22-mm nylon syringe filter into an autosampler vial for analysis (Wu et al., 2020).
The chromatographic system was the Agilent 1200 series HPLC system. It was set a flow rate of 0.3 mL/min. These pesticides were separated by Hypersil GOLD-C18 analytical column (100 mm × 2.1 mm × 1.8 mm) from Thermo Scientific (USA). Thecolumn temperature was maintained at 30 ◦C and the injectionvolume was 5 mL. Gradient elution was performed with acetonitrile as mobile phase A and 0.1% formic acid in ultrapure water as mobile phase B. Gradient elution was performed with acetonitrile as mo- bile phase A and 0.1% formic acid in ultrapure water as mobile phase B. The gradient elution was 0e3 min, 20e50% A; 3e6 min, 50e70% A; 6e15 min, 70e95% A; 15e17 min, 20% A; 17e20, 20% A.
The MS conditions were as follows: spray voltage, 3500 V; vapor- izer temperature, 300 ◦C; capillary temperature, 350 ◦C; sheath gas(N) pressure, 35 Arb; auxiliary gas pressure, 15 Arb; collision gas (Ar) pressure, 1.5 mTorr. Selective reaction monitoring (SRM) mode was used to detected pesticide compounds. The ion pair, collision energy (CE), and retention time of each pesticide were shown in Table S2.

2.4. Oxidative stress indices and ROS-detoxification test
Fresh leaves of tea were ground into powder in liquid nitrogen, and then 0.1 g of powder was added to 1 mL 0.1 mol/L phosphate buffer solution (PBS, pH 7.0e7.4) and vortexed for 3 min. The ho-mogenate was centrifuged at 5000 rpm for 10 min at 4 ◦C. It wastaken to test oxidative stress indices (MDA, H2O2, and O—2 ) and antioxidants (GSH-Px, SOD, POD, CAT, APX, DHAR, GR, GSSG, AsA,and GSH) quantified by corresponding kits.

2.5. Physiological and biochemical test
The Se content in tea leaves was determined using an atomic absorption spectrometer (HG-AFS, Haiguang, China) equipped with a hydride generation system. The tea powder was made into a 10% homogenate solution by mixing 0.1 mol/L PBS (pH 7.0e7.4). The homogenate was shaken for 5min and centrifuged at 5000 rpm for10 min at 4 ◦C. It was measured by the specific kit of chlorophyll,carotenoid, protein, and soluble sugar.
The green tea was ground into a powder. Then, 0.1 g of thesample was added to 10 mL of a 70% methanol solution (v/v) pre- heated in a 70 ◦C water bath for 10 min. After that, the tube was shaken for 2 min, and put in water with 70 ◦C for 10 min. The su-pernatant was centrifuged at 4500 rpm for 10 min. Re-extracting the extraction once using the same method, and then all super- natants were combined. They were passed through a 0.22-mm nylon filter into an autosampler vial for liquid chromatography analysis of catechins (HPLC, Agilent 1200).
Supernatant preparation was consistent with the catechins pretreatment method. Then, 1 mL of gallic acid, water, supernatant, and 5 mL folin reagent were combined and shaken for 3e8min. The mixed solution was added to 4 mL of a 7.5% sodium carbonate so- lution reacting for 1 h. They were passed through a 0.22-mm nylon filter into an autosampler vial for HPLC analysis of tea polyphenols (HPLC, Agilent 1200).
The green tea was ground into a powder. Then, 0.1 g of the tea powder was added to 50 mL of water at 100 ◦C for 30min. Then, itwas replenished the water to 50 mL at room temperature. They were passed through a 0.45-mm water film into an autosampler vial for HPLC analysis of theanine (HPLC, Agilent 1200).

2.6. GS-GOGAT cycle and amino acid analysis
Tea was ground into a powder and made a 10% homogenized solution with 0.1 mol/L PBS (pH 7.0e7.4). The mixture was extrac- ted with a boiling water bath for 15 min and centrifuged at8000 rpm for 10min. The supernatant was measured for GS and GOGAT activities by their corresponding kit.
Briefly, the amino acid was formed by hydrolysis of peptides by 6 M hydrochloric acid at 110 ◦C for 6 h. The color reaction wasreacted with ninhydrin solution after separation by the ion- exchange column (sulfonic cation exchange resin). The amino acid level was determined by a visible light spectrophotometer at wavelength 440 and 570 nm (Xu et al., 2020).

2.7. Flavonoids compounds test
The tea leaves powder was weighed 2 g and added 5 mL of the 60% ethanol solutions in 10 mL tube. It was ultrasonic at 30 ◦C for 30min. These mixed solutions were shaken for 5min and centri-fuged at 3800 rpm for 5min. All supernatant was combined by repeating the above steps. The 5 mL of the mixed solution was taken and then blew dry with nitrogen. After that, the extraction solution was added into the 2 mL tube to make the final sample volume 1 mL. Finally, it was used 100 mg C18 purification and then filtered through a 0.22-mm nylon syringe filter into an autosampler vial for analysis. The Agilent 6410 B Triple Quadrupole LC MS/MS (Agilent Technologies, USA) was equipped with an HPLC reverse phase C18 column (Athena C18-WP 2.1 50 mm, 3 mm). The flow rate was 0.3 mL/min. Acetonitrile and formic acid water were used as mobile phases A and B, respectively. The isocratic elution was carried out for 5 min at an 80 (A):20 (B) ratio. MS was performed using negative electrospray ionization and multiple reaction monitoring modes (MRM). The specific instrument parameter for flavonoids compounds of LC-MS/MS was shown in Table S3.

2.8. Tea flavor analysis
Compared with other groups, nano-Se10 had the best effect on quality improvement. It was further selected to identify flavor dif-ferences by GC-IMS. Sample processing: 1 g of the green tea powder was added to a 20 mL headspace vial and then incubated at 80 ◦C for 20 min before injecting. Table S4 (gas chromatographic condi-tions) and Table S5 (GC-IMS unit) have detailed parameters.
The instrument’s analysis software includes a laboratory analytical viewer (LAV), three plug-ins, and GC IMS library search, which are used for sample analysis from different angles. (1) LAV was used to view the analytical spectrum; each point in the figure represents a volatile organic compound. (2) Reporter plug-in: direct comparison of spectral differences between samples (two-dimen- sional top and three-dimensional view). (3) Gallery Plot plug-in: fingerprint comparison, visual and quantitative comparison of volatile organic compounds between different samples. (4) Dy- namic principal component analysis (PCA) plug-in: dynamic PCA for cluster analysis of samples and rapid determination of unknown sample types. (5) GC IMS Library Search: the National Institute of Science and Technology (NIST) database and IMS database can qualitatively analyze the substance by builting into the application software.

2.9. Statistical analysis
Data analysis was performed with SPSS 26.0 (mean values and standard deviation). The graphs were constructed using GraphPad Prism Version 8.0 (San Diego, CA, USA). Separation of means was evaluated by Tukey’s t-test, and significant differences were accepted at the level p < 0.05. Multivariate statistical analysis was performed using SIMCA 13.0 software. PCA was used to aroma components data. The metaboanalyst 4.0 (http://www. was further used to analyze differential metabolites. 3. Results 3.1. The residue of pesticides and determination of oxidative stress indices in green tea Three pesticides were detected in the leaves of tea and soil at very low concentrations. These pesticides had not been detected in the soil of the tea garden. The concentrations of imidacloprid, acetamiprid, and difenoconazole were 0.022 ± 0.010, 0.088 ± 0.015, and 0.10 ± 0.018 mg/kg in green tea (Table S6). It was noted that imidacloprid was found below the maximum residue limits (MRLs) in China (0.5 mg/kg) and Codex Alimentarius Commission (50 mg/ kg) on tea. Acetamiprid was found below the established MRLs in China (10 mg/kg). Referring to the MRLs of China, the residue of difenoconazole was detected below the MRLs (10 mg/kg). Although the residue of these pesticides did not exceed the standard, the potential impact on tea trees was unknown. To compare the effects of long-term pesticide stress on tea trees, fresh leaves sprayed withand without pesticide treatment were collected. H2O2, O—2 , andMDA contents had no substantial alteration in the tea leaves under non-stress conditions of pesticides treatments (CK). They were significantly accumulated in the tea leaves with long-term appli- cation of pesticides (PT). However, foliar nano-Se observably reduced the pesticides-induced oxidative damages (Figure S1). 3.2. Nano-Se mitigates pesticides-induced oxidative damage by strengthening the antioxidants of green tea The effect of the application of nano-Se on the antioxidant ca- pacity in tea leaves mainly due to the different types of enzymes and substances under pesticides-induced oxidative damages (Fig. 1). Nano-Se treatments significantly increase the AsA-GSH cycle (AsA, APX, DHAR, GSH, and GR) and antioxidant enzyme (GSH-Px, SOD, POD, and CAT) in parallel to control and pesticides treatment. The control and pesticides treatment had no distinct alterations in antioxidants levels. The concentration of nano-Se was increased to 10 mg/L with the maximum beneficial effect and then decreased by increasing the Se-level up to 20 mg/L. Under 10 mg/L nano-Se, GSH-Px, POD, SOD, and CAT increased by 203.6%, 149.8%, 21.0%, and 13.4% respectively in comparison with their control. The APX (26.2%), DHAR (50.8%), GR (58.1%), AsA (18.4%), and GSH(38.0%) levels induced by nano-Se biofortification. Conversely,GSSG in tea leaves showed a decreasing trend with an increase of nano-Se concentration. GSSG had a larger decline at nano-Se10, which decreased by 47.2%, respectively. Overall, foliar spray nano- Se (10 mg/L) is better than other treatment groups, which can significantly improve the antioxidant capacity of green tea. 3.3. Nano-Se enhances photosynthesis and biomass accumulation of green tea under pesticides stress Pesticides treatment reduced the content of chlorophyll, which weakened the absorption of nutrients in the tea leaves compared with the control. Foliar application of nano-Se has a significant effect on the improvement of photosynthesis and biomass accu- mulation, including tea polyphenols, Se, theanine, protein, caffeine, carotenoid, soluble sugar, chlorophyll, and catechins under pesticides-induced oxidative damage (Fig. 2). The best compre- hensive effect in these nutrients was found at nano-Se10. It increased the tea polyphenols (33.1%), theanine (16.4%), protein (19.5%), caffeine (22.3%), carotenoid (19.8%), soluble sugar (32.6%), and chlorophyll (42.9%) levels. Besides, epigallocatechin (EGC), catechin (C), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG) in catechins increased by 14.1%, 21.5%, 11.0%, and 168.9%, respectively. In particular, the total Se in green tea enhanceswith the concentration of nano-Se (2.5, 5, 10, and 20 mg/L)increased by 6.1, 8.4, 12.8, and 25.4 times, respectively. 3.4. Amino acid level and GS-GOGAT cycle in green tea with nano- Se under pesticides stress Amino acids are essential aroma and flavor compounds in tea (Ye et al., 2018). Compared with the control, pesticides treatment inhibited the activity of GS and GOGAT, thereby decreasing the amino acids levels. However, the some amino acid levels have a greater overall performance at nano-Se10 (Fig. 3). The glutamic acid (55.4%), aspartic acid (45.5%), serine (37.9%) increased by foliar spraying nano-Se (10 mg/L). It also promoted the activity of GS and GOGAT increased by 54.4% and 37.9%, respectively. Besides, levels of proline (41.7%) and arginine (78.8%) directly synthesized by the GS- GOGAT cycle downstream were higher than in corresponding control leaves. 3.5. Nano-Se enhances resistance by inducing the secondary metabolites synthesis of green tea under pesticides stress Secondary metabolites enhance plant defense against biological and non-biological stress conditions and benefit human health and nutrition. As shown in Fig. 4A, E, flavonoids and total phenols levels were decreased by pesticides treatment compared with the controlgroup. Selenium-rich tea had higher flavonoids and total phenols contents compared with their control. At nano-Se10, secondary metabolites show greatest increase, including the apigenin (15.0%), kaempferol (23.0%), quercetin (36.8%), myricetin (49.3%), and rutin(19.3%). 3.6. Nano-Se improves the aroma components of green tea under pesticides stress Based on the above results, the flavor of green tea treated with nano-Se (10 mg/L) was detected by GC-IMS. The two main factors of the PCA (Fig. 5A) explained 69% of the variance in green tea (54.1% PC1 and 14.9% PC2). The two processing data collection points are located in the PCA area without overlapping. The model parameters (Fig. 5A) and the permutation test (Fig. 5B) indicate the difference in metabolic profile between the treatment group and control. Besides, the distance between the samples can reflect the size of the odor difference. Therefore, the PCA analysis method in this experiment can effectively distinguish the overall aroma of green tea treated with nano-Se. GC-IMS spectrum projected on a three/two-dimensional plane in Fig. 5CeD, which can directly compare the differences of flavor substances of green tea treated with nano-Se. The background of the whole picture is blue, and the red vertical line at abscissa 1.0 is the reaction ion peak (RIP peak). The ordinate represents the retention time (s) of gas chromatography, and the abscissa repre- sents the ion migration time. Each point on both sides of the RIP peak is a volatile organic compound. The color of white indicates the concentration is low but red indicates the concentration is high. It can be seen that the volatile substances of treatment and control have different characteristic spectrum information. Through the Library Search plug-in, the qualitative analysis of the flavor substances in green tea was carried out. The specific in- formation about the aroma substances identified in Fig. 6A was shown in Table S7. The identified volatile substances have various aroma components, including alcohol (6), terpene (5), aldehydes (6), esters (4), ketones (2), acids (2), aromatic hydrocarbon (3),furan (2), thiol (1), pyrazine (1), and sulfide (1). To compare the differences of flavor components among sam- ples more comprehensively, the Gallery Plot plug-in of LAV soft- ware was used to automatically generate fingerprints of all chromatographic peaks to be analyzed in the two-dimensional chromatogram (Fig. 6B). It can see the overall composition infor- mation and changes of flavor substances among samples of different treatment groups. In the fingerprint, the right Y-axis is the sample number, and each horizontal line represents the signal peak of all flavor substances in the green tea sample; the X-axis is the qualitative result of volatile substances, and each column repre- sents the signal peak of the same flavor ingredient in different green tea samples. According to the intensity value of characteristic peak, the differences of flavor components of samples between different treatments were analyzed. The changes in flavor compo- nent levels with the foliar application of nano-Se were summarized in Fig. 6C. 4. Discussion Current research focuses on the impact of short-term field application of different types of high-concentration pesticides on crop quality. Little attention has been paid to the impact of long- term stress of pesticides on tea quality in real scenarios. In this study, three representative pesticides have tested the residues used in tea gardens. Imidacloprid, acetamiprid, and difenoconazole were detected at low concentrations in tea samples, while they had not been detected in the soil (Table S6). They were detected below the corresponding MRLs of China and Codex Alimentarius Commission. Although the MRLs was used as a standard for food quality, the differences of global pesticide legislation cannot guarantee the safety of consumers (de et al., 2020). Long-term foliar application ofimidacloprid, acetamiprid, and difenoconazole significantly accu- mulated the ROS levels (H2O2 and O2—) in leaves of tea (Figure S1). ROS excessively produced in plant cells can quickly cause perox- idative damage, thereby disturbing the homeostasis of antioxidant system (Pisoschi and Pop, 2015). It also causes the lipid peroxida-tion by increasing the MDA level (Figure S1). These pesticides harm the steady state of antioxidant system, primary, and secondary metabolism. Foliar spray nano-Se can significantly reduce the H2O2, O2—, and MDA levels. The mechanism was likely proposed that nano- Se mitigated the effects of oxidative stress by enhancing the anti-oxidant capacity (AsA-GSH cycle) and promoting the secondary metabolism (GS-GOGAT cycle) to improve the production of nu- trients and aroma components (Fig. 7). In this experiment, the foliar application of nano-Se10 on the leaves increased the chlorophyll, protein, carotenoid, and soluble sugar in tea leaves under pesticide-induced oxidative stress (Fig. 2C, F-H). Consistent with our previous results, foliar applica- tion of nano-Se5 on celery significantly enhanced the chlorophyll (26.1%), soluble sugar (70.4%), protein (37.1%), and b-carotene (61.4%) contents (Li et al., 2020a). Sodium selenite (1 mg/kg) increased nutrients quality of Codonopsis lanceolata, including the polysaccharide, total flavonoid, total saponin, protein, total amino acid, and essential amino acid (Zhu et al., 2017). It may be involved in the processes of carbohydrate metabolism, lipid metabolic, oxidation-reduction, and protein catabolism by Se biofortification, thus enhancing nutrients absorption of crops (Tian et al., 2018). At the same time, nano-Se (10 mg/L) significantly increased the levels of tea polyphenols, EGC, C, EGCG, and ECG (Fig. 2A, I-N). Tea poly- phenols and catechins form the color and aroma of tea and directly related to the sensory quality and physiological health functions of tea (Yan et al., 2020). Hu et al. found that total amino acid and vitamin C levels enhanced in green tea, while tea polyphenol levelssignificantly decreased by fertilization with selenite or selenate (Hu et al., 2003). These results showed that different types and con- centrations of Se such as nano and inorganic Se, which have different effects on the regulation of nutrients components in tea. At present, it is generally believed that plants can promote the absorption of Se within a specific concentration range (El-Ramady et al., 2015). This experiment manifested that nano-Se effectively increased the Se content in tea (Fig. 2E). They all reached the agricultural industry standard of the People’s Republic of China of NY/T 600e2002 (0.25e4.00 mg/kg). This nano-Se biofortification can be used as a source of dietary supplement for people with Se deficiency. Pesticides exposure was remarkably disturbed the amino acid metabolism, ammonia recycling, and porphyrin metabolism (Liu and Zhu, 2020). The content and composition of free amino acids have obvious effects on the taste, color, aroma, and freshness of tea soup. Theanine as a special chemical component of tea has a high sweet and delicious taste, which not only constitutes the fresh taste of tea soup but also reduces the bitter and astringent taste of tea (Mu et al., 2015). GS-GOGAT pathway can adjust N assimilation, and its products are used as signals or precursors, which further regu- late the primary and secondary metabolism of plants (Liu et al., 2019). It was shown that GOGAT and GS activities enhanced by nano-Se treatment (Fig. 3A and B). Arginine (Arg), glutamic acid (Glu), proline (Pro), and theanine are synthesized by the GS-GOGAT cycle. Besides, aspartic acid (Asp) and serine (Ser) also higher in Se- treated samples than in the control (Fig. 3D). The results indicated that nano-Se is more conducive to the formation of amino acids in tea, which could involve amino acids, carbohydrates, and secondary metabolism (Zhu et al., 2018b). Ulhassan et al. found that sodium selenite (5 mM) alleviated the chromium (Cr) induced phytotoxicity by ameliorating nutrients uptake and amino acid metabolism of Brassica napus (oilseed rape) (Ulhassan et al., 2019b). Sodiumselenate treatments (9 mM) also decreased nitrogen losses by hin- dering arsenic (As) - and cadmium (Cd) -induced changes in the N- metabolism of potatoes (Solanum tuberosum L. cv. Sante) (Shahid et al., 2019). Those amino acids can be transformed into each other by degrading to precursors or intermediates of the tricar- boxylic acid cycle. Zhang et al. reported that the application of Arg (0.2 mM) increased the accumulation of polyamines, especially putrescine and Pro in tomato fruits, which reduced the chilling injury of tomato fruit after harvest (Zhang et al., 2013). The increase of Pro content can enhance plant defense capabilities (Ashraf and Foolad, 2007; Haudecoeur et al., 2009). Synergistic effect of Arg and Pro markedly enhances plant growth and improves plant environmental stress tolerance. The stress of pesticides on plants promotes oxidative damage of plants by inducing the excessive production of reactive oxygen species (ROS) in cells. The plants equipped with enzymatic (GSH- Px, SOD, POD, CAT, GR, APX, and DHAR) and non-enzymatic (GSH and AsA) to scavenge the ROS-induced oxidative damages (Ulhassan et al., 2019a). Nano-Se supplement mitigates the pesticides-induced oxidative injuries to maintain cellular integrityby reducing the H2O2, O2—, and MDA content (Figure S1). The currentstudy revealed that nano-Se in tea increased the GSH-Px, POD, SOD, and CAT levels (Fig. 1AeD). It also significantly activated the AsA- GSH cycle, thus enhanced the APX, DHAR, GR, ASA, and GSH levels while GSSG content decreased with pesticide stress in tea leaves (Fig. 1EeJ). These enzymes suggested the nano-Se has greater potential in enhancing plant tolerance against pesticide stress and maintaining ROS homeostasis. Previous studies are consistent with our results that Se-biofortification activated the AsA-GSH cycle and antioxidant enzymes under Cr and salinity stress (Ulhassan et al., 2019b; Zahedi et al., 2019a). Se-supplement also enhanced secondary metabolites (total phenols, flavonoids, and anthocyanins) and the relative gene expression of chalconesynthase and phenylalanine ammonialyase levels under Cr stress (Handa et al., 2019). Our study found that total phenols and flavo- noids (apigenin, kaempferol, quercetin, myricetin, and rutin) by foliar application of nano-Se significantly enhanced in response to pesticides stress (Fig. 4). Similarly, sodium selenite (80 mg/kg) alleviated the damage in garlic plants exposed to imidacloprid (1.2 mg/kg) by increasing the absorption of mineral elements to enhance the antioxidant enzymes and chlorophyll levels. Besides, it also enhanced the indole and nitrogen metabolism, which acti- vated the secondary metabolites and maintained the balance of the plant energy metabolism (Zhang et al., 2020). These results shown that nano-Se can improve protective ability of crops against stress. Further, our study found that the improvement of the flavor of green tea by nano-Se application under pesticide stress. In this study, GC-IMS was used to determine the volatile aroma compo- nents of green tea in different treatment groups. Methyl salicylate, acetone, ethyl acetate, toluene, dimethyl disulfide, 3- methylbutanoic acid, ethanol, propanoic acid, 2-heptanone, myr- cene, benzaldehyde-M, and p-xylene enhanced in Se-treated samples, while butanal, isobutyl acetate, 3-methylbutanal, 3- methylbutanol, linalool, and 2-ethylfuran decreased (Fig. 6C). Myrcene generally had a flowery and sweet aroma. Ethyl acetate and 2-heptanone with strong fruit aroma contribute to the unique flavor of green tea. The low concentration of acetone gives off a light fragrance, which helps to promote the coordination of ester aromas. Methyl salicylate and dimethyl disulfide are an important contributor to the chestnut-like aroma of green tea (Ho et al., 2015). Benzaldehyde is a volatile substance derived from phenylalanine in tea and has an odor characteristic of bitter almond (Yang et al., 2013). However, foliar spray nano-Se decreased linalool and 3- methylbutanal content. They are also the common aroma of chestnut in green tea (Zhu et al., 2018a). Consequently, the aroma of tea treated with nano-Se is sharp and strong, but the control is lighter. Besides, the synergy between the key odorants promotes the appearance of chestnut aroma in the finished tea throughsynergistic and indirect effects. 5. Conclusion In conclusion, two-year field studies on tea plants revealed that long-term use of pesticides (imidacloprid, acetamiprid, and dife- noconazole) in tea gardens extensively accumulated ROS levels, which caused oxidative damage and affected the nutritional quality of tea. Nano-Se foliar spray could enhance tea tolerance to pesticide-induced oxidative stress. The remediation mechanism involved is mainly that nano-Se application improved the anti- oxidative defense system (AsA-GSH cycle) and enhanced secondary metabolite levels, reducing the ROS accumulation and MDA content in green tea. It is helpful to minimize the toxicity of pesticide stress. Nutrients such as tea polyphenols and catechins were positively influenced by Se-biofortification. The levels of the amino acid further strengthened by regulating the GS-GOGAT cycle. These nutrients help improve the flavor and nutritional value of green tea. By GC-IMS detection, foliar spray of nano-Se increased the key aroma components which contribute to the aroma of green tea stronger. Therefore, our study can provide insights that the role of nano-Se in ameliorating the plants ability to mitigate the effects of pesticides-induced oxidative stress by regulating the nutrients levels, secondary metabolism, and antioxidant ability. References Ashraf, M., Foolad, M.R., 2007. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ. Exp. Bot. 59, 206e216. Baba, R., Kumazawa, K., 2014. Characterization of the potent odorants contributing to the characteristic aroma of Chinese green tea infusions by aroma extract dilution analysis. J. Agric. Food Chem. 62, 8308e8313. Chen, Q., Zhang, D., Pan, W., Ouyang, Q., Li, H., Urmila, K., Zhao, J., 2015. Recent developments of green analytical techniques in analysis of tea’s quality and nutrition. Trends Food Sci. Technol. 43, 63e82. Chen, Y., Zhou, Y., Zeng, L., Dong, F., Tu, Y., Yang, Z., 2018. Occurrence of functional molecules in the flowers of tea (camellia sinensis) plants: evidence for a second resource. Molecules 23, 790. Cheng, L., Yang, Q., Chen, Z., Zhang, J., Chen, Q., Wang, Y., Wei, X., 2020. Distinct changes of metabolic profile and sensory quality during qingzhuan tea pro- cessing revealed by LC-MS-based metabolomics. J. Agric. Food Chem. 68, 4955e4965. de, O.G.H., Menezes, J.M.C., da Costa, J.G.M., Coutinho, H.D.M., Teixeira, R.N.P., do Nascimento, R.F., 2020. A socio-environmental perspective on pesticide use and food production. Ecotoxicol. Environ. Saf. 197, 110627. Ekborg-Ott, K.H., Taylor, A., Armstrong, D.W., 1997. Varietal differences in the total and enantiomeric composition of theanine in tea. J. Agric. Food Chem. 45, 353e363. El-Ramady, H.R., Domokos-Szabolcsy, E´., Abdalla, N.A., Alshaal, T.A., Shalaby, T.A.,Sztrik, A., Prokisch, J., Fa´ri, M., 2014. Selenium and nano-selenium in agro- ecosystems. Environ. Chem. Lett. 12, 495e510. El-Ramady, H.R., Domokos-Szabolcsy, E´., Shalaby, T.A., Prokisch, J., F´ari, M., 2015.Selenium in agriculture: water, air, soil, plants, food, animals and nanoselenium. In: Lichtfouse, E., Schwarzbauer, J., Robert, D. (Eds.), CO2 Sequestration, Biofuels and Depollution. Springer International Publishing, Cham, pp. 153e232. Fang, J., Sureda, A., Silva, A.S., Khan, F., Xu, S., Nabavi, S.M., 2019. Trends of tea in cardiovascular health and disease: a critical review. Trends Food Sci. Technol. 88, 385e396. Handa, N., Kohli, S.K., Sharma, A., Thukral, A.K., Bhardwaj, R., Abd_Allah, E.F., Alqarawi, A.A., Ahmad, P., 2019. Selenium modulates dynamics of antioxidative defence expression, photosynthetic attributes and secondary metabolites to mitigate chromium toxicity in Brassica juncea L. plants. Environ. Exp. Bot. 161, 180e192. Haudecoeur, E., Planamente, S., Cirou, A., Tannie`res, M., Shelp, B.J., More´ra, S., Faure, D., 2009. Proline antagonizes GABA-induced quenching of quorum- sensing in Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. Unit. States Am. 106, 14587e14592. He, H., Shi, L., Yang, G., You, M., Vasseur, L., 2020. Ecological risk assessment of soil heavy metals and pesticide residues in tea plantations. Agriculture 10, 47. Herna´ndez-Mesa, M., Escourrou, A., Monteau, F., Le Bizec, B., Dervilly-Pinel, G., 2017. Current applications and perspectives of ion mobility spectrometry to answer chemical food safety issues. Trac. Trends Anal. Chem. 94, 39e53. Ho, C.-T., Zheng, X., Li, S., 2015. Tea aroma formation. Food Science and Human Wellness 4, 9e27. Hu, Q., Xu, J., Pang, G., 2003. Effect of selenium on the yield and quality of green tea leaves harvested in early spring. J. Agric. Food Chem. 51, 3379e3381. Ji, L., Ni, K., Wu, Z., Zhang, J., Yi, X., Yang, X., Ling, N., You, Z., Guo, S., Ruan, J., 2020.Effect of organic substitution rates on soil quality and fungal community composition in a tea plantation with long-term fertilization. Biol. Fertil. Soils 1e14. Jiang, J., Chen, L., Wu, S., Lv, L., Liu, X., Wang, Q., Zhao, X., 2020. Effects of difeno- conazole on hepatotoxicity, lipid metabolism and gut microbiota in zebrafish (Danio rerio). Environ. Pollut. 265, 114844. Kalapothakis, J.M.D., Barran, P.E., 2013. Ion mobility mass spectrometry e principles. In: Roberts, G.C.K. (Ed.), Encyclopedia of Biophysics. Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 1142e1148. Natasha Khalid, S., Shahid, M., Murtaza, B., Bibi, I., Asif Naeem, M., Niazi, N.K., 2020. A critical review of different factors governing the fate of pesticides in soil under biochar application. Sci. Total Environ. 711, 134645. Li, X., Ahammed, G.J., Zhang, L., Yan, P., Zhang, L., Han, W.-Y., 2018. Elevated Carbon Dioxide-Induced Perturbations in Metabolism of Tea Plants, pp. 135e155. Li, J., Yuan, H., Yao, Y., Hua, J., Yang, Y., Dong, C., Deng, Y., Wang, J., Li, H., Jiang, Y., Zhou, Q., 2019. Rapid volatiles fingerprinting by dopant-assisted positive photoionization ion mobility spectrometry for discrimination and character- ization of Green Tea aromas. Talanta 191, 39e45. Li, D., An, Q., Wu, Y., Li, J.-Q., Pan, C., 2020a. Foliar application of selenium nano- particles on celery stimulates several nutrient component levels by regulating the a-linolenic acid pathway. ACS Sustain. Chem. Eng. 8, 10502e10510. Li, S., Ren, J., Li, L., Chen, R., Li, J., Zhao, Y., Chen, D., Wu, Y., 2020b. Temporal variation analysis and risk assessment of neonicotinoid residues from tea in China. En- viron. Pollut. 266, 115119. Liu, N., Zhu, L., 2020. Metabolomic and transcriptomic investigation of metabolic perturbations in oryza sativa L. Triggered by three pesticides. Environ. Sci. Technol. 54, 6115e6124. Liu, M.Y., Tang, D., Shi, Y., Ma, L., Li, Y., Zhang, Q., Ruan, J., 2019. Short-term inhibition of glutamine synthetase leads to reprogramming of amino acid and lipid metabolism in roots and leaves of tea plant (Camellia sinensis L.). BMC Plant Biol. 19, 425. Liu, T., Yuan, C., Gao, Y., Luo, J., Yang, S., Liu, S., Zhang, R., Zou, N., 2020. Exogenous salicylic acid mitigates the accumulation of some pesticides in cucumber seedlings under different cultivation methods. Ecotoxicol. Environ. Saf. 198, 110680. Mikula, K., Izydorczyk, G., Skrzypczak, D., Mironiuk, M., Moustakas, K., Witek- Krowiak, A., Chojnacka, K., 2020. Controlled release micronutrient fertilizers for precision agriculture - a review. Sci. Total Environ. 712, 136365. Mu, W., Zhang, T., Jiang, B., 2015. An overview of biological production of L-the- anine. Biotechnol. Adv. 33, 335e342. Niu, Y., Wang, P., Xiao, Z., Zhu, J., Sun, X., Wang, R., 2019. Evaluation of the perceptual interaction among ester aroma compounds in cherry wines by GC- MS, GC-O, odor threshold and sensory analysis: an insight at the molecular level. Food Chem. 275, 143e153. Owona, B.A., Abia, W.A., Moundipa, P.F., 2020. Natural compounds flavonoids as modulators of inflammasomes in chronic diseases. Int. Immunopharm. 84, 106498. Parween, T., Jan, S., Mahmooduzzafar, S., Fatma, T., Siddiqui, Z.H., 2016. Selective effect of pesticides on plant–A review. Crit. Rev. Food Sci. Nutr. 56, 160e179. Pisoschi, A.M., Pop, A., 2015. The role of antioxidants in the chemistry of oxidative stress: a review. Eur. J. Med. Chem. 97, 55e74. Probst, Y., Guan, V., Kent, K., 2018. A systematic review of food composition tools used for determining dietary polyphenol intake in estimated intake studies. Food Chem. 238, 146e152. Ravichandran, R., 2002. Carotenoid composition, distribution and degradation to flavour volatiles during black tea manufacture and the effect of carotenoid supplementation on tea quality and aroma. Food Chem. 78, 23e28. Shahid, M.A., Balal, R.M., Khan, N., Zotarelli, L., Liu, G.D., Sarkhosh, A., Fern´andez- Zapata, J.C., Martínez Nicola´s, J.J., Garcia-Sanchez, F., 2019. Selenium impedes cadmium and arsenic toxicity in potato by modulating carbohydrate and ni- trogen metabolism. Ecotoxicol. Environ. Saf. 180, 588e599. Sharma, A., Shukla, A., Attri, K., Kumar, M., Kumar, P., Suttee, A., Singh, G., Barnwal, R.P., Singla, N., 2020. Global trends in pesticides: a looming threat and viable alternatives. Ecotoxicol. Environ. Saf. 201, 110812. Shi, J., Zhang, R., Pei, Y., Liao, C., Wu, X., 2020. Exposure to acetamiprid influences the development and survival ability of worker bees (Apis mellifera L.) from larvae to adults. Environ. Pollut. 266, 115345. Su, X., Wang, Y., Peng, G., He, Q., 2020. Long-term effects of chlorothalonil on mi- crobial denitrification and N2O emission in a tea field soil. Environ. Sci. Pollut. Res. Int. 27, 17370e17381. Tian, M., Xu, X., Liu, F., Fan, X., Pan, S., 2018. Untargeted metabolomics reveals predominant alterations in primary metabolites of broccoli sprouts in response to pre-harvest selenium treatment. Food Res. Int. 111, 205e211. Ulhassan, Z., Gill, R.A., Ali, S., Mwamba, T.M., Ali, B., Wang, J., Huang, Q., Aziz, R., Zhou, W., 2019a. Dual behavior of selenium: insights into physio-biochemical, anatomical and molecular analyses of four Brassica napus cultivars. Chemo- sphere 225, 329e341. Ulhassan, Z., Gill, R.A., Huang, H., Ali, S., Mwamba, T.M., Ali, B., Huang, Q., Hamid, Y.,Khan, A.R., Wang, J., Zhou, W., 2019b. Selenium mitigates the chromium toxicity in Brassicca napus L. by ameliorating nutrients uptake, amino acids metabolism and antioxidant defense system. Plant Physiol. Biochem. 145, 142e152. Usman, M., Farooq, M., Wakeel, A., Nawaz, A., Cheema, S.A., Rehman, H.U., Ashraf, I., Sanaullah, M., 2020. Nanotechnology in agriculture: current status, challenges and future opportunities. Sci. Total Environ. 721, 137778. Wang, F., Li, S., Feng, H., Yang, Y., Xiao, B., Chen, D., 2019. An enhanced sensitivity and cleanup strategy for the nontargeted screening and targeted determination of pesticides in tea using modified dispersive solid-phase extraction and cold- induced acetonitrile aqueous two-phase systems coupled with liquid chromatography-high resolution mass spectrometry. Food Chem. 275, 530e538. White, P.J., 2018. Selenium metabolism in plants. Biochim. Biophys. Acta Gen. Subj.1862, 2333e2342. Willis Chan, D.S., Prosser, R.S., Rodriguez-Gil, J.L., Raine, N.E., 2019. Assessment of risk to hoary squash bees (Peponapis pruinosa) and other ground-nesting bees from systemic insecticides in agricultural soil. Sci. Rep. 9, 11870. Wu, Y., An, Q., Wu, J., Li, P., He, J., Pan, C., 2020. Development and evaluation of an automated multi-channel multiplug filtration cleanup device for pesticide residue analysis on mulberry leaves and processed tea. RSC Adv. 10, 2589e2597. Xie, S., Feng, H., Yang, F., Zhao, Z., Hu, X., Wei, C., Liang, T., Li, H., Geng, Y., 2019. Does dual reduction in chemical fertilizer and pesticides improve nutrient loss and tea yield and quality? A pilot study in a green tea garden in Shaoxing, Zhejiang Province, China. Environ. Sci. Pollut. Res. Int. 26, 2464e2476. Xu, J., Zhu, S., Yang, F., Cheng, L., Hu, Y., Pan, G., Hu, Q., 2003. The influence of se- lenium on the antioxidant activity of green tea. J. Sci. Food Agric. 83, 451e455. Xu, X.H., Lv, P.F., Wang, T.X., Wang, B.X., Shi, Y., Wang, B.X., Meng, Z.R., Chen, Q.X., Zhuang, J.X., Wang, Y.Y., 2020. Bone-strengthening effects and safety of com- pound peptides from skin of Chiloscyllium plagiosum and Mustelus griseus. Food Sci. Nutr. 8, 1522e1533. Yan, Z., Zhong, Y., Duan, Y., Chen, Q., Li, F., 2020. Antioxidant mechanism of tea polyphenols and its impact on health benefits. Animal Nutrition 6, 115e123. Yang, Z., Baldermann, S., Watanabe, N., 2013. Recent studies of the volatile com- pounds in tea. Food Res. Int. 53, 585e599. Ye, Y., Yan, J., Cui, J., Mao, S., Li, M., Liao, X., Tong, H., 2018. Dynamic changes in amino acids, catechins, caffeine and gallic acid in green tea during withering.J. Food Compos. Anal. 66, 98e108. Zahedi, S.M., Abdelrahman, M., Hosseini, M.S., Hoveizeh, N.F., Tran, L.P., 2019a. Alleviation of the effect of salinity on growth and yield of strawberry by foliar spray of selenium-nanoparticles. Environ. Pollut. 253, 246e258. Zahedi, S.M., Hosseini, M.S., Daneshvar Hakimi Meybodi, N., Teixeira da Silva, J.A., 2019b. Foliar application of selenium and nano-selenium affects pomegranate (Punica granatum cv. Malase Saveh) fruit yield and quality. South Afr. J. Bot. 124, 350e358. Zhang, X., Shen, L., Li, F., Meng, D., Sheng, J., 2013. Amelioration of chilling stress by arginine in tomato fruit: changes in endogenous arginine catabolism. Post- harvest Biol. Technol. 76, 106e111. Zhang, X., Chen, L., Leng, R., Zhang, J., Zhou, Y., Zhang, Y., Yang, S., He, K., Huang, B., 2020. Mechanism study of the beneficial effect of sodium selenite on metabolic disorders in imidacloprid-treated garlic plants. Ecotoxicol. Environ. Saf. 200, 110736. Zhu, L., Wang, P., Zhang, W., Hui, F., Chen, X., 2017. Effects of selenium application on nutrient uptake and nutritional quality of Codonopsis lanceolata. Sci. Hortic. 225, 574e580. Zhu, Y., Lv, H.P., Shao, C.Y., Kang, S., Zhang, Y., Guo, L., Dai, W.D., Tan, J.F., Peng, Q.H., Lin, Z., 2018a. Identification of key odorants responsible for chestnut-like aroma quality of green teas. Food Res. Int. 108, 74e82. Zhu, Z., Zhang, Y., Liu, J., Chen, Y., Zhang, X., 2018b. Exploring the effects of Myricetin selenium treatment on the nutritional quality of tomato fruit. Food Chem. 252, 9e15.