Isolation of fulvic acid-like compounds from black liquor
Spent liquor was obtained from the Shandong Tranlin Group (Liaocheng, Shandong, China), a wheat-straw processing company. The straw is digested with (NH4)2SO3 and ammonia−water at 240 °C and 0.2 mPa for 400 min. PFAs were isolated as described [39], focusing on PFAs with molecular weights below 10 kDa. PFA concentrations (mg-C/L) were expressed by the organic carbon concentration measured by potassium dichromate oxidation [40].
Characterization of PFAs
The elemental contents were measured using a Vario EL III (Elementar, Germany) [41]. Fourier-transform infrared spectroscopy (FTIR) was performed on a Nicolet iS10 spectrometer (Thermo Fisher, Waltham, MA, USA) at 400–4000 cm− 1 [42].
Plant materials and treatments
Selected healthy peanut seeds (Yunnan colorful wild ground peanuts) were sterilized in 5% NaClO for 20 min, followed by three to five rinses with sterile water. The seeds were germinated in culture dishes at 20 °C with a sprouting machine (DYJ-S6365, Bear). They were divided into six groups (60 seeds in each group) and sprayed for 1 min once per hour with different concentrations of Na2SeO3 (0, 5, 15, and 25 mg·L− 1 Na2SeO3; 15 mg·L− 1 Na2SeO3 solution containing 60 mg-C/L PFA; 25 mg·L− 1 Na2SeO3 containing 60 mg-C/L PFA). Following a 6-day growth period, seedlings were sampled to assess relevant changes in physiology or biochemistry.
Measurement of MDA
After 6 days of seed germination, the MDA content in hypocotyls was determined by the method of Ding et al. (2018) using the formula [43]:
$$\textrm{MDA}\left(\textrm{mmol}\cdot {\textrm{kg}}^{-1}\right)=\frac{\left[6.45\times \left({OD}_{532}-{OD}_{600}\right)-0.56\times {OD}_{450}\right]}{V_s\times m}\times {V}_t\times {V}_r$$
where m is the sample mass, Vr, Vs, and Vt are the total volume of the reaction solution, the volume of the extract in the solution, and the complete volume of the extract, respectively.
Enzyme isolation and measurement
One gram of hypocotyls from each treatment condition was homogenized in 3 mL of chilled 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, 3 mM β-mercaptoethanol, and 2% (w/v) polyvinylpyrrolidone. After centrifugation (10,000 g, 15 min, 4 °C), the enzyme activities in the supernatants were measured.
Ascorbate peroxidase (APX) was measured as described by Nakano and Asada (1981) using 0.5 mM ascorbate, 0.6 mM H2O2, and 0.1 mL of enzyme extract in 50 mM potassium phosphate buffer (pH 7.0) in a volume of 3.0 mL. After initiation of the reaction by adding H2O2, the reduction in absorbance at 290 nm was determined from 1 to 60 s [44].
Glutathione reductase (GR) activity was determined as described by Knörzer (1996) using a 3 mL reaction solution containing 5 mM MgCl2, 0.2 mM NADPH, 1 mM oxidized glutathione (GSSG), and 0.1 mL PFA in 50 mM Tris-HCl, pH 7.5. GSSG was added to initiate the reaction and absorbances at 340 nm were monitored over 60 s [45].
Glutathione S-transferase (GST) activities were determined as described by Habig et al. (1974) [46], Mauch and Dudler (1993) [47]. The 2 mL reaction solution contained 100 μL of glutathione, 800 μL of 1.25 Mm 1-chloro-2,4-dinitrobenzene, and 200 μL PFA in 100 mM PBS, pH 6.5. Increases in absorbance at 340 nm were measured at 30 s intervals and GST activity was calculated as an increase in absorbance of 0.001 per milligram of protein within 1 min.
Peroxidase (POD) activity was assessed as the change in absorbance at 470 nm resulting from the catalysis of H2O2, according to Wang (2017) [13]. The reaction solution was prepared by the addition of 112 μL ml guaiacol to 200 ml of PBS (50 mM, pH 6.0), followed by heating and stirring to dissolve it. After cooling to room temperature, 112 μL of 30% H2O2 was added and mixed well. One hundred microliters of the enzyme extract were added to 2.9 mL of the solution and changes in absorbance at 470 nm were recorded immediately and at 15 s intervals thereafter for 3 min. The enzyme activity was calculated from the absorbance change of 0.01 per milligram of protein within 1 min.
GSH-Px activity was assessed as described by Flohe and Gunzler (1984) [48], Zhang and Wu (2004) [49]. Four hundred microliters of 1.0 mM GSH (containing 2.5 mM NaN3) were placed in a 10 mL tube, followed by 400 μL of enzyme solution (blank control was water), and the reaction was initiated by the addition of 200 μL 1.5 mM pre-warmed H2O2 at 37 °C. After 3 min, 4 mL 1.67% metaphosphoric acid precipitant was added to precipitate the protein and the solution was centrifuged at 3000 rpm for 10 min. Two milliliters of the supernatant were aspirated (blank tube contained 0.4 mL double-distilled water and 1.6 mL 1.67% metaphosphoric acid precipitant) and 2.5 mL of 0.32 M Na2HPO4 and 0.5 mL DTNB (5,5 ‘- disulfide-p-dinitrobenzoic acid) were added for color development, and the absorbance was measured at 423 nm within 5 min. GSH-Px activity was calculated based on the change of absorbance value of 0.001 per gram of material fresh weight within 1 min (excluding the GSH of non-enzymatic reaction).
Determination of total soluble sugar content
The total sugar content was determined by the phenol sulfuric acid method described by Dubois (1956) [50]. One gram of the powdered hypocotyls from the different treatments was weighed into a centrifuge tube and 20 mL of double-distilled water was added. The tube was vortexed and placed in a water bath in boiling water for 10 min to fully destroy the cell wall and release the soluble sugars. After centrifugation at 10000 g for 10 min, the supernatant containing the sugar extract was collected, diluted threefold, and 100 μL of the diluent was added to 1 mL of water. Phenol solution (0.5 mL of 6% phenol solution) and concentrated sulfuric acid (2.5 mL) were then added. The solution was mixed well, allowed to stand for 30 min at room temperature, and the absorbance at 490 nm was measured in a spectrophotometer. Standard glucose solutions of different concentrations were prepared in double-distilled water using anhydrous glucose as the reference, and absorbances were read as above. A standard curve was drawn and the total soluble sugar content of the sample was calculated.
Determination of total soluble protein content
One gram of powdered hypocotyls was added to 3 mL of pre-chilled extraction solution (50 mM potassium phosphate buffer, pH 7.0, containing 1 mm EDTA, 3 mm β- mercaptoethanol, and 2% polyvinylpyrrolidone), vortexed for 1 min, and allowed to stand at 4 °C for 2 h. After centrifugation at 10000 g for 14 min at 4 °C, the total protein content in the supernatant was measured by the Bradford method using bovine serum albumin as the standard [51].
Determination of organic and inorganic selenium
Six days after the start of selenium enrichment treatment, hypocotyls and roofs were collected from the peanut seedlings, rinsed three to five times with tap water, and blotted to remove the excess water. One gram of sample was used to determine the total and inorganic Se concentrations, as below.
The total Se content was determined according to the national food safety standard of the People’s Republic of China (GB 5009.93–2010, China) for measuring Se in food products. The total Se content was calculated by plotting a standard curve from the data obtained by the Se standard detection.
Measurement of inorganic Se requires sample pretreatment with the addition of 10 mL of 50% hydrochloric acid to the samples in a stoppered test tube. The material was mixed well, ultrasonicated for 30 min, and placed in boiling water for 30 min. After cooling naturally, the material was filtered through absorbent cotton, and the Se-containing filtrate was collected. The Se concentration was measured as described above for the total Se content and was calculated from the standard curve. The organic Se content was calculated as the difference between the total Se content and the inorganic Se content.
Determination of amino acid content
Two hundred micrograms of hypocotyls were weighed into a hydrolysis tube to which was added 10 mL of 6 M hydrochloric acid. The tube was degassed ultrasonically for 10 min, evaporated under nitrogen, sealed, and placed in an oven at 111 °C for 23 h. The material was then cooled to room temperature, diluted to 100 mL with deionized water, shaken well, and filtered through a 0.45 μm filter. One milliliter was then pipetted into a 10 mL volumetric flask and an aliquot was filtered through a 0.45 μm filter into the injection bottle and analyzed on an L-8900 automatic amino acid analyzer (Hitachi High-Technologies Corp., Tokyo, Japan) [52].
Statistical analyses
Data were analyzed by SPSS v10.0 using one-way ANOVA with Duncan’s test and significance levels set at P < 0.05.