2.1 HPLC analysis for determining isoflavone content and composition
Seventeen soybean genotypes were selected for estimation of isoflavone content using HPLC. Among these, ten genotypes had yellow-coloured seed coats, while the remaining seven possessed black-coloured seed coats. Seeds were generously procured from Dr S.K. Lal, Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi. Two contrasting varieties were selected for further molecular studies based on the total isoflavone content. Three biological replications were used throughout the experiment. Preparation of sample extract, standard curve and HPLC conditions were performed as per Kumar et al (2010) with slight modifications. The chromatography was performed in a Waters 2695 chromatograph (Waters Corp., Milford, MA, USA) with a 2998 photodiode array (PDA) detector and a C18 silica column as the stationary phase. A binary gradient of acetonitrile (ACN) and water was used as the mobile phase with a flow rate of 1.5 ml/min. 13% ACN was used for the initial 1 min of HPLC separation with the ACN concentration reaching 30% at 20 min and again reducing to 13% at the end of the run, i.e., 25 min.
2.2 Estimation of TBA number
Upon lipid peroxidation, malondialdehyde (MDA), a degradation product of lipid hydroperoxides, is formed. The level of MDA formed was measured in terms of TBA number. An adduct is formed upon the reaction of MDA with TBA. The adduct gives a pink colour whose absorbance is estimated at 532 nm (Ohkawa et al. 1979). Briefly, approximately 400 mg of overnight soaked soybean seeds was homogenised with 4.0 ml of distilled water followed by centrifugation at 13,000 rpm for 35 minutes to obtain the clear extract. Then, 0.2 ml of this extract was mixed with 0.2 ml of 8.1% sodium dodecyl sulphate (SDS), 1.5 ml of 20% acetic acid (pH 3.5) and 1.5 ml of 0.8% TBA and kept in a boiling water bath for 60 minutes. After cooling, 1.0 ml of distilled water and 5.0 ml of n-butanol-pyridine (15:1) were added, and the tubes were shaken vigorously. It was then centrifuged at 4000 rpm for 10 minutes. The pink-coloured organic layer formed at the top was separated from the aqueous phase, and absorbance was measured at 532 nm. The standard curve was prepared using tetra ethoxy propane (TEP) ranging from 10 to 70 nmoles, and the level of lipid peroxides was expressed as nmoles of MDA/g fresh weight.
2.2 Assessment of carbonyl value
The carbonyls were estimated using the method described by Henick et al (1954). The 2,4-dinitrophenyl hydrazine reacts with the aldehydes and ketones to form their 2,4-dinitrophenylhydrazone derivatives, which are converted to wine red-coloured complexes under alkaline conditions by KOH. Carbonyls were determined by measuring the absorbance of the coloured complex at 480 nm. Briefly, the sample extract (0.5 ml) prepared above was mixed with 0.5 ml of 2,4-dinitrophenylhydrazine (0.05%) and heated in a water bath at 60°C for 30 minutes. After cooling, 1.0 ml of KOH (4%) was added, and a characteristic wine-red colour appeared. Ten minutes after the appearance of the wine-red colour, absorbance was measured at 480 nm. Carbonyls were estimated by using the formula (A = E max x C x L), where A= Absorbance, E max = 2.72 x 104 (L mol-1 cm-1), C = Concentration (mol L-1), and L = Path length (cm).
2.3 Quantitative expression analysis of the isoflavone synthase gene
Using the isoflavone content data estimated above, we selected two extremely contrasting genotypes, viz., Bragg (high isoflavone) and PUSA 40 (low isoflavone). Real-time expression analysis of IFS1 and IFS2 was performed at 35, 45 and 55 days after flowering (DAF) in contrasting soybean genotypes grown in the open field at ICAR-IARI, New Delhi. RNA was isolated using the TRIzol method, and the first strand of cDNA was synthesized from it. All the primers for qPCR (~150 bp) were designed using PrimerBlast software available at NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The housekeeping gene actin 2/7 was used as an internal control for data normalisation (S1). qPCR was carried out using a BioRad CFX96 machine in a reaction volume of 20 ml containing SYBRR Green JumpStartTM Taq ReadyMixTM (Sigma), forward primer (10 mM), reverse primer (10 mM) and ten ng/ml of cDNA using conditions of 94°C for 5 min, then 40 cycles of 94°C for 15 s, 55°C for 30 s and 72°C for 45 s. The relative expression level was calculated following the 2-ΔΔCt method (Livak and Schmittgen 2001). Standard errors and standard deviations were calculated from 3 technical and biological replicates. Seed samples at 35 DAF were considered the calibrator. The resulting PCR products were also analysed by agarose gel electrophoresis to determine the specificity.
2.4 Cloning of IFS1 and IFS2 cDNA
According to the manufacturer's protocol, total RNA was isolated from soybean seeds using RNAiso plus (TAKARA). The full-length IFS1 and IFS2 CDSs were amplified from the DS2706 soybean cultivar. Both forward and reverse primers were designed using PrimerBlast. The amplified PCR products were gel eluted using a Quick Guide PCR purification kit (Solgent) and kept for ligation in a pENTR/D-TOPO vector, an entry vector for the Gateway® System, for 30 minutes at 23°C in a 2:1 molar ratio of insert and vector. Three microliters of ligation mix was transformed into TOP10 chemically competent E. coli cells. Six colonies from each clone were inoculated for plasmid isolation. The confirmation was performed by (A) PvuII digestion, (B) PCR and (C) Sanger sequencing.
2.5 In silico analysis
The nucleotide and deduced amino acid sequences of IFS1 and IFS2 were compared through NCBI BLAST. The three-dimensional structures of the IFS1 and IFS2 proteins from their amino acid sequences were generated using RoseTTAFold, a deep learning-based structure prediction tool (https://robetta.bakerlab.org). This algorithm is a nonhomology-based method that works by pattern detection in protein sequences, interaction among the amino acid residues and probable protein tertiary structure, and simultaneously determining everything ((Baek et al. 2021). These predicted models were tested using the MolProbity server (Williams et al. 2018), where the favoured rotamers and Ramachandran plot distribution with ϕ and ψ angles were analysed. After testing the predicted structures, protein‒ligand docking analysis was performed via the CB-Dock2 tool. It is based on the blind docking principle using AutoDock Vina, and the protein structure was input as a PDB file, whereas the ligand was provided in the SDF format (Liu et al. 2020). The interactions of IFS1 and IFS2 with other proteins were predicted using STRING (Szklarczyk et al. 2015).