Sequence retrieval and analysis:
Panicum miliaceum L. (Proso millet) P5CS, ProDH, and OAT amino acid sequences were retrieved from the SwissProt database (https://swissmodel.expasy.org/repository). For P5CS UniProtKB AC name A0A3L6SZ65 was selected, where as for ProDH, and OAT A0A3L6TP97 and A0A3L6TJF0 was selected respectively. ProtParam (https://web.expasy.org/protparam) was used to predict the primary structure. The physicochemical properties were calculated and included the molecular weight of the molecule, the theoretical isoelectric point (pI), the composition of amino acids and atomic composition, the total number of positive and negative residues, the extinction coefficient, the half-life estimate, the instability index, the aliphatic index, and the grand average of hydropathicity.
Secondary structural analysis:
The sequences of P5CS, ProDH, and OAT was retrieved in the form of FASTA format and used for secondary structure analysis. PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred) and the Chou-Fasman methods/tools (http://www.biogem.org/tool/chou-fasman) were used to predict the secondary structure of the protein. We calculated secondary structural features of the protein sequence, such as alpha helixes, sheets, coils, and turns.
Three dimensional Structure modeling:
Homology modeling was used to determine the three dimensional structures of the proteins P5CS, ProDH and OAT from Panicum miliaceum L. using Swiss modeling software (https://swissmodel.expasy.org). A Swiss repository was searched for the target sequence of interest (https://swissmodel.expasy.org/repository). The templates of Panicum miliaceum L. were selected. Using homology modeling, the target sequence and sequences of known structures similar to the query sequence were aligned. The sequence alignment and template structure are then used to create a structural model of the target. A sequence with the highest sequence identity score was preferred from the obtained BLAST results.
In Swiss modeling, the in-build structure assessment tool was used to evaluate the models (https://swissmodel.expasy.org/assess). For analysis of possible conformations of dihedral angles Ψ and Φ of amino acid residues in protein structures, the modeled structure was verified using the Ramachandran plot. We calculated MolProbity Score, Clash Score, Ramachandran favoured percentage, Ramachandran outliers, Rotamer outliers, C-beta deviations, bad angles and Q mean Z score. ProSA (https://prosa.services.came.sbg.ac.at/prosa.php) was used for energy criteria analysis calculations.
Enzyme (Receptor), ligand processing and Molecular docking:
All the models prepared and validated in Swiss modeling tool (https://swissmodel.expasy.org), were downloaded in .PDB format for further requirement. Ligands (NaCl and Se in the form of selenite) were obtained from NCBI Pub chem. (https://pubchem.ncbi.nlm.nih.gov). Both models and ligands were processed and repaired for errors prior to molecular docking experiments. Open Babel GUI 3.1.1 program was used to convert ligands to other formats when ever required. To determine the best orientation and energy of interaction between ligand molecules and target proteins, molecular docking studies were executed. The docking program like CBDock (http://clab.labshare.cn/cb-dock/php) and Dock Thor (http://www.dockthor.Incc.br) was used for carrying out blind docking. Several poses were generated for each ligand based on the docking calculation. Choosing of the best pose was done keeping in view the interaction energy among ligand and the protein relations with the key residues of the protein.
Plant growth and treatments:
The seeds of Panicum miliaceum L. were collected from Ladakh region (India) and identified at Centre for Biodiversity and Taxonomy, University of Kashmir. The seeds were sterilized using 70% (v/v) ethanol for 1 minute and washed with sterile distilled water. Surface sterilization of seeds was performed using 0.1% HgCl2 (Merck, India) solution (w/v) for 3 min followed by rinsing with sterilized distilled water. The seeds were sown in pots having 20 cm diameter and containing autoclaved sand. A controlled environment with a 26 ± 1°C temperature and a 16-h photoperiod was maintained as per Desoky et al. 2019; Hussain & Ashraf 2008. Three sets of plants were grown with three replicates each. Concentrations viz., 0, 150 and 200 mM of NaCl were added to the pots and 1 μM, 5 μM, 10 μM selenite (Se) was applied as Se source. All treatments were given as per Shah et al. 2020; Rasool et al. 2020. For the growth of the seedlings, Hoagland nutrient medium (pH 6.5) containing all macro and micro nutrients was used. The plants were harvested after 22 days of sowing. Plant growth-specific parameters were calculated as per Kausar et al. 2012; Singh & Kumar 2008. Total proline was calculated in control, NaCl treated and Se treated set according to Zhu et al. 2020.
Proline content estimation:
The ninhydrin reaction was used to assess the proline content of the leaves as per Zhu et al. 2020 with some modifications. Leaves and roots (0.5 g) collected were extracted in 3 percent (w/v) sulfosalicylic acid. The leaves/roots were weighed and finely grounded using liquid nitrogen. The mixture was kept as such for few minutes and was centrifuged at 12,000 g for 10 min. The supernatant obtained after centrifuge was used to estimate proline content. The supernatant was combined with 2 ml of acid ninhydrin and 2 ml of glacial acetic acid and placed in a 100oC water bath for 1 hour. The reaction was stopped by immersing the test tubes in an ice bath. Further 4 ml toluene was added to the mixture and the absorbance at 520 nm was measured with a spectrophotometer. The content of proline was measured using a proline standard curve made with different concentrations.
Determination of plant growth parameters and tolerance index (TI):
Following the experiment, 10 plants were taken at random from each treatment and gently cleaned four times with deionized water to remove adherent sand from the root surfaces. The morphological parameters were examined as per Dikobe et al. 2021; Kausar et al. 2012; Singh & Kumar 2008. To evaluate the capacity of plants to thrive under high saline environments, the tolerance index (TI) was calculated by the equation given by Wilkin’s 1957:
Tolerance Index (TI %) = MLT ∕ MLC × 100
MLT = Mean length (root, shoot) of the longest root in treated plants, MLC = Mean length (root, shoot) of longest root in control.
Gene expression studies of P5CS, ProDH and OAT genes:
Molecular characterization of these genes of proline pathway in Panicum miliaceum L. was performed. Samples in triplicates were prepared in a mortar using liquid nitrogen. Total RNA was isolated from plant material using Trizol reagent (Invitrogen) according to the modified protocol of Deepa et al. 2014. RNase free DNase (Promega) was added to remove genomic DNA contamination. The quality of RNA was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies). For validation of these genes of proline pathway, cDNA was synthesized using reverted aid cDNA synthesis kit (Thermo scientific) by manufacturer’s protocol and gene expression studies were carried out by transcriptomic analysis.
Two way analysis of variance (ANOVA) and significance of data (P < 0.0001) was calculated statically. All these tests were performed with XLSTAT 2021 and graph pad prism 6.