Plant cultivation and experimental conditions
Hydroponic experiments were performed in a greenhouse at China Agricultural University, Beijing, China. It was conducted under the following conditions: 25/15 °C (day/night), photon flux with 225–300 µmol m−2 s−1, and a 14/12 h (light/dark) photoperiod with 75–80% humidity. Pepper seeds (Capsicum annuum L.) were germinated while watered with deionized water for 7 days. Uniform and healthy seedlings were transplanted into a plastic pot (2 L) and cultured in Hoagland-Arnon nutrient solution . A half-strength nutrient solution was supplied for three days, and a full-strength solution was used until harvest. The solution was continuously refilled and renewed every two days to ensure an adequate supplementation of nutrition. The plants were pretreated in the nutrient solution for 10 days.
Se (0, 0.2, and 1 mg/L) and Cd (0 and 1 mg/L) were added to the vessel in form of Cd2+ in water/medium: 5% nitric acid (1000 mg/L) and nano-Se. The nano-Se was characterized as previously described . The experiment was established with five repetitions of the six treatments that included Cd0Se0, Cd0Se0.2, Cd0Se1, Cd1Se0, Cd1Se0.2, and Cd1Se1. Samples were collected following continual treatment for 7 days. The roots were immersed in a 20 mmol/L solution of EDTA-Na2 for 5 min to remove the residual metal ions on the root surface. These plants were divided into roots, stems, and leaves after they were washed with deionized water. Some samples were used to determine the elements and metabolites. A portion of the samples was stored at − 80 °C for transcriptomics and the determination of genes.
Analyses of saccharide compounds
The samples (20 mg) were extracted with 1 mL of ultrapure water. The mixed solution was subjected to ultrasound for 30 min and then centrifuged for 10 min at 14,000 rpm. All of the supernatant was filtered with a 0.1 μm injection syringe. The liquid was then diluted 20 times with ultrapure water. The concentration of saccharide compounds was determined using a Thermo Scientific Dionex ICS-5000+ ion chromatograph (Waltham, MA, USA). It was equipped with an SP single pump, EG eluent generator, AS-AP automatic sampler, DC electrochemical detector, and Chromeleon7.2 SR5 chromatographic data analytical software. The flow rate was 1 mL/min, and the injection volume was 10 µL. Ultrapure water and 200 mM NaOH (50% of NaOH was diluted with ultrapure water) were used as mobile phases A and B, respectively. The gradient elution procedure was as follows: 91% A, 0 min; 91% A, 18 min; 0% A, 21 min; 0% A, 31 min; 91% A, 32 min; and 91% A, 40 min.
Transmission electron microscopy
The roots were merged in a 2.5% (v/v) solution of glutaraldehyde for 2 h at 4 °C. They were washed twice with phosphate-buffered saline (PBS, pH 7.2–7.4) at 4 °C for 15 min. The samples were added to 1% (v/v) OsO4 at 4 °C for 2 h and washed three times with PBS. The solution was dehydrated in a series of acetone solutions. Concentrations of 50%, 70%, 80%, and 90% of acetone were used to treat the samples for 15 min. The samples were treated twice with 100% of acetone for 10 min. The mixed solution was immersed overnight in the resin. It was polymerized at a high temperature and cut into slices of approximately 70 nm using a Lycra slicer (EM UC6; Leica, Wetzlar, Germany), dyed with uranyl acetate for 15 min, and treated with lead citrate for 10 min. The samples were dried and then photographed using a transmission electron microscope (TEM) (H-7650; JEOL. Ltd., Tokyo, Japan).
Analysis of total selenium and cadmium
A mixed solution of HNO3 and HClO4 (9:1, v/v) was added to digest the plant root, stem, and leaf samples. They were diluted to 50 mL by adding deionized water into a volumetric flask. The supernatant was determined using atomic absorption spectrometry (HG-AFS; Haiguang, China) equipped with a hydride generation system to determine the concentration of Cd and Se in different tissues.
Analysis of selenium speciation
One gram of the pepper tissues (roots, stems, and leaves) were hydrolyzed using 2 mL of Tris-HCl (75 mmol/L, pH 7.5)and 5 mg/mL of protease XIV (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C. Finally, the supernatant was filtered through a 0.22–µm filter membrane. The samples were then subjected to high-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS; SA-50, Thermo Scientific iCAP RQ).
The HPLC system was equipped with a Hypersil Gold C8 column (4.6 × 250 mm, 5 μm; Thermo, USA). A volume of 20 mM KH2PO4 that contained 0.5% heptafluorobutyric acid and methanol were used as mobile phases A and B, respectively. The flow rate was set at 1.0 mL/min. The gradient elution procedure was as follows: 100% A, 3 min; 95% A, 3.1 min; 95% A, 14 min; 100% A, 14.1 min; and 100% A, 15.0 min. The mass spectrometer conditions were as follows: the radio-frequency power was 1550 W; the plasma gas flow rate was 14 L/min; the flow rate of carrier gas was 1.06 L/min; the flow rate of auxiliary gas flow was 0.8 L/min; the dwell time was 0.1 s, and the analysis mass number was 78 Se. Five standard selenoamino acids were used in this experiment and included the following: Se(IV) (selenite), Se(IV) (selenite), SeCys (selenocysteine), MeSeCys (methylselenocysteine), and SeMet (selenomethionine) purchased from the National Institute of Metrology for Certified Reference Materials, Beijing, China.
Plant hormone analyses
The method is a modification of previous research . The roots and leaves were ground to a powder in liquid nitrogen. The samples (0.1 g) were then subjected to 1 mL of extraction solution that contained methanol: water: formic acid (80:19:1, v/v/v) using ultrasound for 10 min. They were centrifuged for 5 min at 12,000 rpm. These supernatants were added to 50 mg of primary secondary amine (CNW Technologies GmbH, Shanghai, China) in a 2 mL centrifuge tube. The mixed solution was dried with nitrogen, and the volume was kept constant to 100 µL with an aqueous solution of 80% methanol. All of the supernatant was filtered using a 0.22-µm organic membrane. An Agilent 6465 Triple Quadrupole UPLC−MS/MS (Ultivo; Agilent Technologies, Santa Clara, CA, USA) was equipped with an EclipsePlus C18 column (2.1 × 50 mm, 1.8 μm). The flow rate was 0.4 mL/min. Acetonitrile and 0.1% formic acid in water were used as mobile phases A and B, respectively. The gradient elution procedure was as follows: 80% A, 0 min; 5% A, 4 min; 80% A, 4.1 min, and 80% A, 5.2 min. It used positive and negative ion scanning mode and multiple reaction monitoring (MRM). All the parameters of MRM and collision energy (Additional file 1: Table S1) were optimized for maximum selectivity and sensitivity.
Determination of the metabolites related to lignin synthesis
A total of 0.1 g of roots was ground to a powder in liquid nitrogen. The samples were extracted using 1 mL of an aqueous solution of 80% methanol (1% formic acid). These mixtures were shaken for 3 min, treated with an ultrasonic instrument for 10 min and centrifuged at 10,000 rpm for 5 min. The supernatant was added to a 2 mL centrifuge tube filled with 50 mg of C18 and 5 mg of multiwalled carbon nanotubes (MWCNTs) purchased from the Sinopharm Chemical Reagent Co. (Beijing, China). They were vortexed for 2 min and centrifuged for 5 min at 10,000 rpm at 4℃. All the supernatant was filtered using a 0.22-µm organic membrane.
An Agilent 6465 Triple Quadrupole UPLC−MS/MS (Ultivo, Agilent Technologies) was equipped with a Venusil Hilic (2.1 × 50, 1.8 μm). The flow rate was 0.25 mL/ min. Acetonitrile and 0.1% formic acid water were used as mobile phases A and B, respectively. The gradient elution procedure was as follows: 5% A, 0 min; 95% A, 4 min; 5% A, 4.1 min; and 5% A, 5.2 min. It used positive and negative ion scanning mode and MRM. All the parameters of MRM and collision energy (Additional file 1: Table S2) were optimized for maximum selectivity and sensitivity.
The HPLC−UV (Agilent Technologies) was equipped with a Venusil Hilic (4.6 × 100, 5 μm). The flow rate was 1 mL/min. Acetonitrile and ultrapure water were used as mobile phases A and B, respectively. The compounds were eluted from a 5 min isocratic run at a 60:40 ratio of A to B. The UV absorption was monitored at 217 nm.
mRNA expression analysis
The roots and leaves were stored at − 80 °C and then used with an RNAprep pure Plant Kit (Tiangen Biotech, Beijing, China) to extract the total RNA following the manufacturer’s instructions. The concentration of RNA in different tissues was measured using an RNA Nano 6000 Assay Kit (Thermo Fisher Scientific, Wilmington, DE, USA). The Agilent Bioanalyzer 2100 system to assess the integrity of RNA (Agilent Technologies). A NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, Beverly, MA, USA) was used to generate sequencing libraries following the manufacturer’s recommendations, and index codes were added to the attribute. EdgeR was used to perform an analysis of the differential expression of two samples. Significant differential expression was established as the threshold for the Fold Change ≥ 1.5 and P < 0.01. The GOseq R packages based on Wallenius non-central hyper-geometric distribution implemented Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) that adjusted for gene length bias in the DEGs. A pathway enrichment analysis was performed using KOBAS software to test the statistical enrichment of DEGs in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.
Real-time quantitative PCR
Ten representative genes were selected to verify the transcriptomics results using quantitative real-time reverse transcriptase–PCR (qRT-PCR). The RNA extraction utilized an RNAprep pure Plant Kit according to the manufacturer’s instructions. The cDNA synthesis used a FastQuant RT Kit. The qRT-PCR was performed with SuperReal PreMix Plus (SYBR Green). The expression of the β-actin served as a reference. The primers for sequences (Additional file 1: Table S3) were purchased from Sangon Biotech (Shanghai, China). The specific methods used were previously described .
A difference analysis was performed by a one-way analysis of variance (ANOVA) using SPSS 26.0 (IBM, Inc., Armonk, NY, USA). The graphs were constructed in Origin 2021 (OriginLab, Northampton, MA, USA.) and GraphPad Prism Version 8.0 (San Diego, CA, USA). Tukey’s t-test was used to evaluate the separation of means, and significant differences were detected at a p < 0.05.
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