The experiment of this study consisted of six treatments, which was set in a complete randomized block design with two factors below: (1) AMF treatments, i.e., R. intraradices and non-AMF inoculated control; (2) three available As concentrations in soils, i.e., 0, 50, and 100 mg As/kg dry soil. By complying with the established standard for the grade of As pollution in Risk Control Standard of Soil Pollution in Agricultural Land in China (GB 15618-2018), when the As content in farmland soil exceeded 100 mg/kg, it would be considered high risk, and the soil was forbidden to plant agricultural products. Besides, when the 50 mg/kg As content in soil was identified as a middle-level pollution, agricultural products would be controlled by Food and Drug Administration. Each of the six treatments contained three replicates, so there were a total of 18 pots (one seedling per pot).
Farmland topsoil (5–20 cm) was collected from the campus of the Henan University of Science and Technology (HAUST), Henan Province, China. Subsequently, the soils were mixed with the sand and organic matter (soil, sand, and organic matter at 3:1:1, v/v/v). The physicochemical properties of mixed soil were as follows:, organic matter 50.52 g kg−1, available potassium 83.35 mg kg−1, available nitrogen 40.52 mg kg−1, Olsen phosphorus 8.17 mg kg−1, and pH 7.8 (1:5 soil:water ratio), and the extractable metal concentrations in soils were as follows: As 5.34, Fe 3.16, Mn 2.17, Cu 0.14, and Zn 0.98 mgkg−1. 5.65 g Na3AsO4 •12 H2O dissolved in 1 L pure water, and then 0, 50 and 100 mL arsenic solutions were respectively introduced into per 1 kg dry soil mixture. Then, the mixture was stirred completely with the blender. Lastly, three available As concentrations in soils (0, 50, and 100 mg As per kg dry soil) were prepared. For the mentioned potted experiment, the soil mixture was autoclaved for 2 h at 121 °C and 0.11 MPa prior to the application.
Plant material and growth conditions
Sophora viciifolia seeds were collected in November 2016 from Shimen Realgar Mine (N 29°38′32″, E 111°2′17″), Hunan Province. This mine acted as the largest producer of realgar in China. Plump S. viciifolia seeds were treated with 75% ethanol for 15 min, washed with purified water, and then sowed in autoclaved wet sand at 28 °C. In the previous study of the authors, the root morphological characteristics of S. viciifolia seedlings in conical frustum plastic containers containing 2 kg of soil mixture were suggested to have no obvious difference with those grown in field for three months (Zhang et al. 2020). After growing for 20 days, healthy seedlings were transplanted into conical frustum plastic containers with 2 kg of soil mixture contained. S. viciifolia received the cultivation in a solar greenhouse, the average temperature ranged from 15 to 25 °C, and the temperature was regulated by using the ventilation system and the thermal insulation quilt. The relative humidity of the growth chamber ranged from 50% to 80% from April to June 2016. All the treatments received a nutrient supplement of 500 mL Hoagland’s solution (1.0 mmol/L NaH2PO4) (Hoagland and Arnon 1950). To avoid the inhibition of AMF symbiosis by excessive P in soils, 50 mL modified Hoagland solution (containing only 25% of P, 0.25 mmol/L NaH2PO4) was supplemented weekly, and the soil moisture was maintained at a field capacity of 70% by adding a certain amount of deionized water.
AMF strain Rhizophagus intraradices (BGC BJ09) (N.C. Schenck & G.S. Sm.) C. Walker and A. Schüßler originated from the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China. This AMF strain was proved to facilitate the plant growth, root morphology and phytohormone balance of Robinia Pseudoacacia in arsenic-contaminated soils (Zhang et al. 2020). Mycorrhizal inocula comprised a mixture of AMF spores (the spore density of 350 per 10 g dry sand soil), mycorrhizal hyphae, R. intraradices-infected clover root segments (average 73% AMF colonization rate), as well as sandy soil. During the seedling transplantation, the respective pot in the mycorrhizal treatment was inoculated with 30 g R. intraradices inoculumat a soil depth of 3–4 cm, and the control non-inoculated plants received 30 g of heat-sterilized inoculum (autoclaved at 121 °C for 30 min) plus 50 ml of microbial filtrate (0.45 μm pore size) to provide a similar microflora except for AM fungus.
AMF colonization rate
The AMF colonization in plant roots was detected by using 1-cm-long root fragments. The collected root fragments were washed with deionized water, soaked in 10% KOH at 90 °C for 15 min, decolorized in alkaline hydrogen peroxide (3 mL NH4OH, 30 mL 10% H2O2, 60 mL H2O) for 20 min, and then acidified in 1% HCl and stained with 0.05% (w/v) trypan blue in lactophenol (Phillips and Hayman 1970). They were observed under the Microscope, and the AMF and arbuscules colonization rate was determined by using the grid-line intersect with the method presented by Giovannetti and Mosse (Giovannetti and Mosse 1980).
After growing for three months, the physiological and biochemical parameters of S. viciifolia were analyzed. S. viciifolia seedlings were harvested. Soil which adhered to the root surface was removed with deionized water. A ruler was adopted to measure the plant height and the root length. Shoots and roots were divided to determine the separate fresh weights, and then they were weighed after receiving the oven drying at 70 °C for 48 h to obtain the dry weight.
As and P content
As and P content in the dry roots and leaves of S. viciifolia seedlings were extracted through the nitric acid digestion at 270 °C, which were determined with a graphite furnace atomic absorption spectrophotometer (Perkin-ElmerAnalyst400, Norwalk, CT, USA) based on USEPA Method 7060A (Li et al. 2018).
The total chlorophyll content was analyzed by complying with the method of Srivastava and Sharma (Ahmed et al. 2006). One gram of fresh leaves were crushed in 100 mL 80% acetone in ice-bath. The extracted solution was centrifuged at 2000×g for 10 min. The absorbance of supernatant was measured spectrophotometrically at 645 nm and 663 nm with a spectrophotometer (Shanghai Jinghua 752). The chlorophyll content was expressed in terms of mg chlorophyll present/g fresh weight of tissues.
Gas exchange and chlorophyll fluorescence
Using a portable photosynthesis system LI-6400 (LI-COR, Lincoln, NE, USA), the net photosynthetic rate (Pn), the stomatal conductance (gs), the intercellular CO2 concentration (Ci), and the transpiration rate (E) were determined on the fifth expanded leaf of the respective plant. The analysis was performed at 2000 μmol m−2 s−1 active radiation, 350 cm3 m−3 CO2 concentration, 25.0 °C leaf temperature and 0.5 dm3 min−1 atmospheric flow rate between 9:30 and 11:00 a.m. during the data acquisition (Zhang et al. 2017).
The chlorophyll fluorescence parameters were determined with a modulated PAM-2000 portable fluorometer (Imaging-PAM, Walz, Germany) on the fifth expanded leaves of S. viciifolia. The leaves were adapted in dark for 1 h, and then the measurements were performed between 9:30 and 11:00 a.m. at ambient temperatures. The leaves were saturated with pulse flashes of white light (2000 μmol m−2 s−1 for 3 s), and for dark-adapted leaves, the Fo (minimum fluorescence) and Fm (maximal fluorescence) were measured. Besides, for light-adapted leaves, the Fs (steady-state) and Fm′ (maximal) fluorescence were obtained. The Fo′ (minimal fluorescence level in the light-adapted state) was achieved by illuminating the leaves with a 3-s flash of far-infrared light (5 μmol m−2 s−1). By complying with the method of Genty et al. (1989), the maximum quantum yield of the PSII photochemistry ((Fm−Fo)/Fm) and the actual quantum yield of PSII electron transport ((Fm′-Fs)/Fm′) were determined (Genty et al. 1989). The quenching due to non-photochemical dissipation (NPQ=(Fm-Fm′)/Fm′) and the coefficient of photochemical quenching (qP=(Fm′-Fs)/(Fm′-Fo′)) were calculated based on the methods described previously (Maxwell and Johnson 2000).
Measurement of oxidative damage
Fresh leaves or roots (1 g) were homogenized in 10 mL 10 mM sodium phosphate buffer (pH 7.4) on an ice bath, and then the homogenate was centrifuged at 4000×g for 10 min. The malondialdehyde (MDA) content was determined with the method presented by Janero (1990). The rates of H2O2 and O2•- productions were determined with the method previously published by Wang and Luo (1990). The absorbance of H2O2 in the assay mixture was spectrophotometrically determined at 390 nm. To analyze the O2•− content, 1 mL 17 mM sulfanilic acid and 1 mL 7 mM α-naphthylamine were introduced in 1 mL of the mixture for 20 min at 25 °C, and then 3 mL anhydrous was adopted to leach chlorophyll. The concentrations of O2•− in the assay mixture were spectrophotometrically measured at 530 nm (Elavarthi and Martin 2010).
Determination of antioxidant enzymes
To extract the antioxidant enzymes, the following steps were performed under ice-cold conditions. One gram of fresh leaves or roots was homogenized in 5 mL 0.1 M cold Tris-HCl buffer (pH 7.6), and the supernatant fraction was employed after being centrifugated at 10,000×g for 20 min. The SOD activity (EC 22.214.171.124) was assayed spectrophotometrically at 560 nm with the method of Giannopolitis and Ries (Giannopolitis and Ries 1977). The amount of enzymes causing a 50% decrease in SOD-inhibitable photochemical reduction of nitroblue tetrazolium (NBT) was defined as 1 U SOD activity. With the method of Aebi (Aebi 1984), CAT (EC 126.96.36.199) activity was measured spectrophotometrically at 240 nm. A unit of CAT enzyme activity was expressed as the extinction coefficient of 1 μmol H2O2 oxidized mg−1 protein min−1. POD (EC 188.8.131.52) activity was assessed based on the method of guaiacol oxidation (Britton and Maehly 1955). POD was quantified spectrophotometrically at 470 nm, in which 1 U POD enzyme activity was the number of grams of tetraguaiacol formed per min (Zhang et al. 2017).
RNA extraction and cDNA synthesis
The total RNA was extracted from the fresh leaves and roots with Plant Total RNA Isolation Kit (Sangon Biotech, Shanghai, China) by complying with the manufacturer's instructions. To remove the residual genomic DNA, the TURBO DNA-free kit (Applied Biosystems/Ambion) was applied, and the RNA quantity was detected with a NanoDrop 2000 (Thermo Scientific, Pittsburgh, PA, USA). The complementary DNA (cDNA) was reversely transcribed by employing a PrimeScript RT reagent kit with gDNA eraser (Takara Bio, Dalian, China).
Cloning of partial coding sequences (CDSs) of SvPCS1 and SvActin
Based on the method of Li et al. (2010), Sophora viciifolia cDNA acted as the template to amplify the conserved sequences of SvPCS1 and SvActin. Two pairs of degenerate primers included PCS1S (5′-GAAAGGGCCTTGGAGRTGG-3′)/PCS1A(5′-GATATDAGCATRAACCCYCT-3′) and ACTS(5′-CTCCCAGGGCTGTGTTTCCT-3′)/ACTA(5′-CTCCATGTCATC CCAGTTGCT-3′). Twenty-five milliliters of reaction system of PCR amplification contained 12.5 μl Premix Taq, one microliter S. viciifolia cDNA templates, 1 μl of each primer, and 9.5 μl RNase-Free ddH2O. The PCR reactions were performed with a C1000 Thermal cycler (Bio-Rad, Hercules, CA, USA) through the procedure below: a 5-min denaturation at 94 °C, followed by 35 cycles of denaturation at 94 °C for 30 s, a 1-min annealing at 54 or 55 °C (54 °C for SvPCS1 conserved fragment and 55 °C for SvActin conserved fragment), a 1-min extension at 72 °C, followed by a 10-min final extension at 72 °C. Subsequently, PCR products were inserted into a pGEM-T vector (Tiangen Biotech, Beijing, China) and then transformed into Escherichia coli (strain DH5α) (Tiangen Biotech, Beijing, China). Luria–Bertani (LB) medium was adopted to select the transformants. To confirm the presence of inserts, 1 μl cultured bacteria solution acted as the template DNA for PCR with primers PCSS/PCSA and ACTS/ACTA. The solutions tested to be positive were applied for sequencing (Sangon Biotech, Shanghai, China).
Analysis of gene expression
Two micrograms of RNA was exploited to synthesize the first-strand cDNA. The complementary DNA (cDNA) was reversely transcribed by applying a PrimeScript RT reagent kit with gDNA eraser (Takara Bio, Dalian, China). Specific to the qRT-PCR assay, 1 μg of total RNA was employed for the reverse-transcription, and 1 μL of the product was applied in the PCR amplification. The reaction system employed 20 μL for the qRT-PCR assay included 10 μL of SYBR Premix Ex Taq (Takara Bio, Dalian, China), and the RT-PCR was performed based on CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The primers for qRT-PCR here were QSvPCS1S (5′-TTGTTGCCAAGGAGCAGATA-3′)/QSvPCS1A (5′-CCTGTTTCAATACCTCTTCCTT-3′) and QSvACTS(5-GATGCTGAGGATATTCAACCC-3′)/QSvACTA(5′-TTTGA CCCATCCCAACCATAA-3′) (Xu et al. 2014). The RT-PCR amplification program of SvPCS1 and SvActin gene was initiated at 95 °C for 3 min to activate the polymerase, and then 40 cycles were performed at 95 °C for 5 s and 57 °C for 30 s. Three biological replicates were used for all genetic analyses, and relative quantification values of SvPCS1 gene were determined based on the 2−△△Ct method (Livak and Schmittgen 2001). All samples were technically replicated three times. Negative controls without cDNA were run within the respective analysis. In order to verify the specificity of these amplified product after the qPCR run, the analysis of melting curve was performed under the following conditions: 95 °C for 15 s, 57 °C for 15 s, 95 °C for 10 min, and then maintained at 95 °C for 15 s.
All experimental results received a two-way analysis of variance (ANOVA) to compare As treatments and AMF inoculation as the major factors. The respective experimental treatment contained three replicates. Noticeable differences among the mentioned treatments were assessed by performing Tukey’s multiple range test. The statistical analyses were conducted by using SAS Software. Figures were generated with SigmaPlot 10.0 (Systat Software Inc., San Jose, CA, USA. https://systatsoftware.com) and the package “pheatmap” in R.