Identiﬁcation and Expression Analysis of Stilbene Synthase Genes in Arachis hypogaea in Response to Methyl Jasmonate and Salicylic Acid Induction

: Stilbene synthase is an important enzyme of the phenylpropanoid pathway, regulating the production of several biologically active stilbenoids. These compounds have antioxidant, anti-inﬂammatory, and anti-cancer properties. However, the detailed characterization of stilbene synthase genes in Arachis hypogaea has not yet been performed. In this study, the comprehensive characterization of stilbene synthase genes in A. hypogaea was conducted, commencing with identiﬁcation, phylogenetic analysis, and study of their expression in response to exogenous hormonal treatment. We identiﬁed and isolated ﬁve AhSTSs genes and recorded their expression pattern in peanut (BARD-479) in response to methyl jasmonate (MeJA) and salicylic acid (SA) treatment. The presence of Chal_sti_synt, ACP_syn_III, and FAE1_CUT1_rppA domains in all AhSTSs indicated their role in the biosynthesis of stilbene and lipid metabolism. Cis-regulatory element analysis indicated their role in light responsiveness, defense responses, regulation of seed development, plant growth, and development. Despite close structural and functional similarities, expression and correlational analysis suggested that these genes may have a speciﬁc role in peanut, as individual AhSTS exhibited differential expression upon hormonal treatment in a genotype dependent manner. Further studies on functional characterization involving the transcriptional regulation of AhSTSs can clearly explain the differential expression of stilbene synthase genes to hormonal treatment.


Introduction
Plants have developed certain physiological processes in response to the biotic and abiotic stress they encountered in their surroundings millions of years ago. Among these physiological processes is the synthesis of secondary metabolites [1]. One of these critical pathways in secondary metabolite production is the phenylpropanoid pathway, which controls the production of vital compounds such as lignin and flavonoids [2]. Flavonoids are important secondary metabolites produced in plants in response to abiotic and biotic stress. These compounds also regulate processes such as reproduction, cell physiology, and signaling [3].
A small group of substances in the phenylpropanoid group that are specifically involved in biotic and abiotic stress responses are known as phytoalexins [4]. The term phytoalexin is derived from Greek and means "warding off agents in plants" [5]. These compounds have low molecular weight, possess antimicrobial properties, and accumulate rapidly at the site of interaction with pathogens. Moreover, they also play a variety basis for unraveling the role of transcriptional factors in the differential expression of AhSTSs genes.

Identification, Isolation and Sequence Analysis of AhSTSs Genes in Peanut
A total of nine sequences of STS, including five complete coding regions (CDS) and four partial CDS, were retrieved from the NCBI database (Table S1). A BLAST search of these sequences in the Peanut DB and domain search resulted in the identification of five nucleotide sequences. Amplified sequences of AhSTSs in peanut showed 100% similarity to their retrieved sequences, respectively. Accession numbers of five AhSTSs genes identified in peanut along with selected sequence attributes have been provided in Table S2. Nucleotide and amino acid alignment of the candidate genes through the Multialin tool showed greater than 90% sequence similarity for both nucleotide and amino acids in AhSTS1, AhSTS2, AhSTS3, AhSTS4, and AhSTS6. The alignment result indicated that AhSTS1, AhSTS3, and AhSTS6 have close resemblance in terms of amino acid sequences (Figures S1 and S2). Signal P prediction confirmed the absence of signal peptide in all the genes.
The identified full-length sequences of AhSTSs predicted that four of the proteins (AhSTS1, AhSTS2, AhSTS3, AhSTS6) have a length of 389 amino acids, whereas AhSTS4 consists of 289 amino acids. Moreover, the result predicted that AhSTSs encode for thermostable proteins with a slight acidic character determined by their aliphatic index and isoelectric point as mentioned in (Table S3). The subcellular localization indicated the presence of proteins in the cytoplasmic region. The extremely low values of GRAVY for predicted protein sequences of AhSTSs suggested that they form a stable interaction with water (Table S3). The protein function is determined by the characteristic features of domain present in the protein structure. Therefore, SMART domain analysis identified three conserved domains in AhSTSs (Figure 1a). The N-and C-terminal domains comprising a Chalcone/Stilbene synthase domain and ACP synthase III domain. FAE1_CUT1_rppA domain was also detected in the center of the putative protein sequence, which predicted their role in lipid metabolism ( Figure S3) Moreover, CDS region for AhSTSs identified through gene structure analysis has been elaborated in Figure 1b. The gene structure analysis showed the absence of introns in all sequences, which predicted that the genes consist of single intron-less coding regions. The results for the motif analysis identified six conserved motifs in the genes. Motif 5 was absent in AhSTS4 protein while motifs 1,2,3,4 and 6 were conserved across the genes (Figure 1c). The presence of similar motifs in the protein sequences predicted the similarity of the biological function of proteins. All the amino acids in the motifs are not conserved, however, most of the amino acids are conserved (Figure 1d). Sequence logo analysis revealed the conservation of amino acids from the N-to C-terminal among the putative protein sequence of AhSTSs. The most observed amino acid residues predicted were valine, glycine, aspartic acid, serine, and phenylalanine. ( Figure S4).

Phylogenetic Analysis of AhSTSs Genes in Peanut
Stilbenoids are a class of polyphenolic compounds found in some plant families. To gain insight into the phylogenetic relationship among STS from other plants, phylogenetic analysis was performed ( Figure 2). The analysis showed that AhSTSs can be distributed into three monophyletic groups (I, II and, III). Group I was further subdivided into three subgroups A, B and, C. The phylogenetic tree showed multiple branches in group I. Whereas AhSTSs from peanut were distributed in group I, AhSTS3, AhSTS4 and AhSTS6 were clustered in subgroup A, and AhSTS1 and AhSTS2 were present in subgroup B. Moreover, several orthologous genes were found in group I. The subgroup C contained stilbene genes from Vitis vinifera and Vitis riparia. AhSTS4 and AhSTS6 showed a close relation to STS2 from Arachis duranensis, while AhSTS3 showed close resemblance to the stilbene synthase 3-like gene from Arachis iapensis. Furthermore, the results showed that AhSTS4 and AhSTS6   stilbene genes from Vitis vinifera and Vitis riparia. AhSTS4 and AhSTS6 showed a close relation to STS2 from Arachis duranensis, while AhSTS3 showed close resemblance to the stilbene synthase 3-like gene from Arachis iapensis. Furthermore, the results showed that AhSTS4 and AhSTS6 clustered together, hence, their function must be assessed to see whether they form a group of paralogous or orthologous genes. The phylogenetic analysis showed a close resemblance of the genes to each other, putting emphasis on the fact that STS belongs to the same group of multigene family. Figure 2. Phylogenetic analysis of AhSTSs through Neighbor Joining method using bootstrap with 1000 replicates indicated three monophyletic groups. AhSTSs are highlighted in bold, whereas pink, rust, and light violet represent group I, green represents group II, and blue represent group III.

Promoter Sequence Analysis
To understand the mechanism of the regulation of STS genes, putative cis-acting elements were identified using 1500 bp upstream promoter regions of AhSTSs in the peanut using Peanut DB ( Figure 3 and Table S4). A total of 213 cis-acting elements were predicted in the promoter region of AhSTSs. Stress-related responsive elements include light responsive elements (Box-4), (GATA-motif), (LAMP-element), (chs-CMA1a), (TCT-motif), (chs- The phylogenetic analysis showed a close resemblance of the genes to each other, putting emphasis on the fact that STS belongs to the same group of multigene family.

Promoter Sequence Analysis
To understand the mechanism of the regulation of STS genes, putative cis-acting elements were identified using 1500 bp upstream promoter regions of AhSTSs in the peanut using Peanut DB ( Figure 3 and Table S4). A total of 213 cis-acting elements were predicted in the promoter region of AhSTSs. Stress-related responsive elements include light responsive elements (Box-4), (GATA-motif), (LAMP-element), (chs-CMA1a), (TCT-motif), (chs-Unit 1), (G-Box) and (G-box), elements involved in regulating seed development (RY-element), gibberellin-responsiveness (TATC-box), auxin responsive elements (TGAelements), salicylic acid responsiveness (TCA-elements), MeJA responsiveness (CGTAmotif/TGACG-motif), and common cis-elements in the promoter and enhancer regions (CAAT). MeJA responsive elements (12, CGTA-motif/TGACG-motif) were predicted in the promoter region of AhSTS2, AhSTS3, AhSTS4, and AhSTS6 but absent in AhSTS1. SA responsive elements (4, TCA-elements) were predicted in AhSTS1, AhSTS3, AhSTS4, and AhSTS6 while absent in AhSTS2. It was revealed that the distribution of cis-regulatory elements varied in AhSTSs. The variation in cis-regulatory elements may be responsible for the differential expression of AhSTSs in response to hormonal treatment. That can be confirmed by further experimental proofs. Moreover, the prediction of anaerobic induction elements (ARE) suggested their role in abiotic responses needs to be confirmed through further experimentation.
AhSTS6 while absent in AhSTS2. It was revealed that the distribution of cis-regulatory elements varied in AhSTSs. The variation in cis-regulatory elements may be responsible for the differential expression of AhSTSs in response to hormonal treatment. That can be confirmed by further experimental proofs. Moreover, the prediction of anaerobic induction elements (ARE) suggested their role in abiotic responses needs to be confirmed through further experimentation.

Protein Structure Analysis of AhSTSs
The secondary structure prediction analysis indicated the presence of alpha helices more than other secondary structures in the five enzymes ( Figures S5-S9). The percentage of alpha helices in AhSTS1 was 43.19%, beta turns 5.66%, and random coils were 35.8%. In AhSTS2, the percentage of alpha helices was 44.73%, while the beta turns were 6.43%. A total of 33.98% were random coils. In AhSTS3, 44.73% alpha helical structure were present, with 5.14% beta turns, and 34.19% random coils. The percentage of alpha helices in AhSTS4 was 43.19%, beta turns 5.66%, and random coils were 35.8%. Moreover, in AhSTS6, 44.73% alpha helical structure were present, with 5.14% beta turns, and 34.19% random coils. The 3-dimensional (3D) structure of AhSTS enzymes were predicted through homology modelling using SWISSMODEL (Figures S10-S14). There were no disordered arrangements or states found for all five proteins. Moreover, the homology modelling analysis predicted that the AhSTSs forms a homodimer protein, consisting of two distinct α and ß chains. The predicted structure for the enzyme was validated through Ramachandran Plot analysis ( Figures S15-19). The percentage of residues in the favored region was greater than 92% for the stilbene synthase proteins, enhancing confidence of the quality of the predicted structure.

Enzyme-Substrate Interaction Analysis
Enzyme-substrate interaction was performed to predict the activity of stilbene synthase enzyme. To determine the binding affinity of the substrate to the AhSTS enzyme, the best scoring poses with the least binding energy were examined and compared for AhSTSs enzymes, and their substrates and presented in Figures 4 and 5. The amino acid residues lining the active site showed conventional hydrogen bonding, van der Waals interaction, carbon-hydrogen bonding, and unfavorable bumps with the substrate's

Protein Structure Analysis of AhSTSs
The secondary structure prediction analysis indicated the presence of alpha helices more than other secondary structures in the five enzymes ( Figures S5-S9). The percentage of alpha helices in AhSTS1 was 43.19%, beta turns 5.66%, and random coils were 35.8%. In AhSTS2, the percentage of alpha helices was 44.73%, while the beta turns were 6.43%. A total of 33.98% were random coils. In AhSTS3, 44.73% alpha helical structure were present, with 5.14% beta turns, and 34.19% random coils. The percentage of alpha helices in AhSTS4 was 43.19%, beta turns 5.66%, and random coils were 35.8%. Moreover, in AhSTS6, 44.73% alpha helical structure were present, with 5.14% beta turns, and 34.19% random coils. The 3-dimensional (3D) structure of AhSTS enzymes were predicted through homology modelling using SWISSMODEL (Figures S10-S14). There were no disordered arrangements or states found for all five proteins. Moreover, the homology modelling analysis predicted that the AhSTSs forms a homodimer protein, consisting of two distinct α and ß chains. The predicted structure for the enzyme was validated through Ramachandran Plot analysis (Figures S15-S19). The percentage of residues in the favored region was greater than 92% for the stilbene synthase proteins, enhancing confidence of the quality of the predicted structure.

Enzyme-Substrate Interaction Analysis
Enzyme-substrate interaction was performed to predict the activity of stilbene synthase enzyme. To determine the binding affinity of the substrate to the AhSTS enzyme, the best scoring poses with the least binding energy were examined and compared for AhSTSs enzymes, and their substrates and presented in Figures  Glu-143. The bond length was calculated using discovery studio tool. As it formed a favorable interaction with malonyl-CoA and p-coumaroyl CoA, it predicts that the enzyme utilizes these compounds as substrates during the stilbene biosynthetic pathway.
The higher the number of hydrogen bonds, the more stable interaction.
and (e) AhSTS6 with malonyl CoA. The higher the number of conventional hydrogen bonds, the more stable the interaction is that formed among enzymes and substrates.

Expression Characterization of AhSTSs in Response to MeJA and SA
Differential expression of AhSTSs genes have been observed in the leaves of peanut (BARD-479) in response to MeJA and SA hormonal treatment ( Figure 6). All AhSTSs genes in BARD-479 were significantly up-regulated under the influence of MeJA treatment at all-time points except AhSTS2 that showed a 3.6 mean fold increase (p-value = 0.028) only at 24 h. A higher expression of AhSTSs was observed in the plants treated with hormones as compared to control plants. The results were in accordance with those found by Vannozzi et al [31] The expression of genes was higher during the initial few hours of hormonal treatment, and this declined with time. Some of the genes also showed enhanced expression after 6 h of hormonal treatment that can be linked to the transcriptional regulation of genes. The correlation analysis of AhSTSs with respect to time in BARD-479 and (e) AhSTS6 with malonyl CoA. The higher the number of conventional hydrogen bonds, the more stable the interaction is that formed among enzymes and substrates.

Expression Characterization of AhSTSs in Response to MeJA and SA
Differential expression of AhSTSs genes have been observed in the leaves of peanut (BARD-479) in response to MeJA and SA hormonal treatment ( Figure 6). All AhSTSs genes in BARD-479 were significantly up-regulated under the influence of MeJA treatment at all-time points except AhSTS2 that showed a 3.6 mean fold increase (p-value = 0.028) only at 24 h. A higher expression of AhSTSs was observed in the plants treated with hormones as compared to control plants. The results were in accordance with those found by Vannozzi et al [31] The expression of genes was higher during the initial few hours of hormonal treatment, and this declined with time. Some of the genes also showed enhanced expression after 6 h of hormonal treatment that can be linked to the transcriptional regulation of genes. The correlation analysis of AhSTSs with respect to time in BARD-479 revealed a strong   (Table S6).
Pearson's correlation coefficient revealed that MeJA affected expression of AhSTS2, AhSTS3 and AhSTS4 was negatively correlated to SA affected expression of AhSTS2, AhSTS3 and AhSTS4 in peanut (Table S6). Under SA treatment, the expression of AhSTS2 is positively correlated with the expression of AhSTS3 in peanut. The expression of AhSTS6 was negatively correlated with the expression of AhSTS1, AhSTS2, and AhSTS3 in peanut (Table S7). This showed that MeJA and SA works in antagonistic order.

Discussion
Stilbenes are well known phytoalexins, regulating plant defense responses against biotic and abiotic stresses in several plants [32]. STS is one of the vital enzymes of phenylpropanoid pathway that catalyze the biosynthesis of plant stilbenes [33]. Owing to their physiological and pharmaceutical significance, stilbenes have been subjected to intensive research. Peanut stilbenes are considered as one of the major sustaining factors of  (Table S6).
Pearson's correlation coefficient revealed that MeJA affected expression of AhSTS2, AhSTS3 and AhSTS4 was negatively correlated to SA affected expression of AhSTS2, AhSTS3 and AhSTS4 in peanut (Table S6). Under SA treatment, the expression of AhSTS2 is positively correlated with the expression of AhSTS3 in peanut. The expression of AhSTS6 was negatively correlated with the expression of AhSTS1, AhSTS2, and AhSTS3 in peanut (Table S7). This showed that MeJA and SA works in antagonistic order.

Discussion
Stilbenes are well known phytoalexins, regulating plant defense responses against biotic and abiotic stresses in several plants [32]. STS is one of the vital enzymes of phenylpropanoid pathway that catalyze the biosynthesis of plant stilbenes [33]. Owing to their physiological and pharmaceutical significance, stilbenes have been subjected to intensive research. Peanut stilbenes are considered as one of the major sustaining factors of the plant's resistance to diseases and have proven to be beneficial for human health [34,35]. Genome-wide identification and characterization of stilbene synthase genes have been performed in some plants but the comprehensive characterization of STS in peanut has not been reported. In the present study, the five AhSTSs genes were identified and isolated from peanut, followed by molecular characterization. Although the number of STS genes in peanut is less than compared to other plants in higher taxonomic status, it shows that STS belongs to a multigene family with significant importance in peanut. Moreover, the role of AhSTSs against biotic and abiotic stress was evaluated through the expression analysis under the influence of MeJA and SA treatment in peanut (BARD-479).
The phylogenetic analysis showed the independent evolution of AhSTSs in Arachis hypogaea from other plant families and close sequence similarity to parent species, i.e., A. iapensis and A. duranensis. According to phylogeny, AhSTS1, AhSTS2 and, AhSTS3 evolved earlier while AhSTS4 and AhSTS6 evolved later. Furthermore, sequence analysis predicted that AhSTS1, AhSTS3, and AhSTS6 showed greater sequence similarity as compared to AhSTS4 and AhSTS2. Based on phylogenetic analysis, STSs can be distributed into three monophyletic groups (I, II and, III). Among, the stilbenes, AhSTSs were clustered in Group I, which was further subdivided into three subgroups A, B, and C based on their close evolutionary relation. Moreover, based on sequence similarities, AhSTS3, AhSTS4, and AhSTS6 were clustered in subgroup A, and AhSTS1 and AhSTS2 were present in subgroup B. Thus, it is predicted that AhSTS2 might be closely related to the gene of origin, while AhSTS4 and AhSTS6 are recently evolved genes, however, AhSTS4 is more diverse than AhSTS6. The analysis also predicted that the AhSTS genes belong to a multigene family closely related to each other based on their distance from each other in the phylogenetic tree.
The presence of Chal_sti_synt and ACP_syn_III domains in all the AhSTSs predicted their role in the biosynthesis of stilbenes as they are involved in condensation of p-coumaroyl CoA [18] and malonyl CoA to resveratrol, while the presence of the FAE1_CUT1_RppA domain predicted their role in lipid metabolism [24]. Gene structure and protein motif analysis predicted the sequence conservation in peanut stilbene genes. The results for motif analysis identified six conserved motifs in the genes. Motif 5 was absent in AhSTS4 protein while motifs 1,2,3,4 and 6 were conserved, which predicted that AhSTS4 is advanced among all AhSTSs. Secondary structure predicted more alpha helices than other secondary structures in AhSTSs, while homology modelling analysis predicted that the AhSTSs form a homodimer protein consisting of two distinct chains. SeqLogo analysis demonstrated the conservation of amino acids from N-to C-terminal, among AhSTSs proteins, and showed a slightly acidic character of protein due to the presence of aspartic acid and glutamic acid. Moreover, this analysis also predicted that the most observed amino acid residues were valine, glycine, aspartic acid, serine, and phenylalanine.
The docking of protein with substrates can be used to predict the binding affinity of enzyme to possible substrates. Our docking results predicted that AhSTS enzymes displayed high binding affinity for malonyl CoA and p-coumaroyl CoA, which are initial substrate for stilbene synthesis [36] as all AhSTSs formed favorable interactions with both substrates, respectively, indicating their role in stilbene biosynthesis. Promoter region analysis predicted the role of AhSTSs in light responsiveness, defense responses, regulation of seed development, regulation plant growth, and development. Moreover, presence of hormone responsive cis-regulatory elements predicted their responsiveness for gibberellic acid, auxin, ABA, SA, and MeJA, which can be confirmed through further analysis.
The expression patterns of the gene during various environmental conditions helps to determine the role of the genes in plant growth and development. So, we examined the expression of stilbene synthase in response to MeJA and SA treatment. Despite close structural and functional similarities, expression and correlational analysis suggested that these genes might have a specific role in peanut, as individual AhSTS exhibited differential expression upon hormonal treatment in a genotype dependent manner. MeJA mostly up-regulated the expression of AhSTSs genes ascompared to SA, which suggested these genes mostly participate in abiotic responses because MeJA is an essential regulator of abiotic stress responses [37]. On the contrary, SA, a crucial regulator of biotic stress responses [37], down-regulated the expression of AhSTSs at various time points in peanut but time-dependent up-regulation has also been observed in all AhSTSs, which indicated that the expression of AhSTSs might be tightly regulated during biotic stress. Moreover, correlation analysis revealed that the expression of AhSTS1, AhSTS4, AhSTS2, and AhSTS6 is positively correlated with time after MEJA treatment while no such correlation was observed in case of SA treatment. On the other hand, moderate degree negative correlation has been observed for AhSTS1, AhSTS2, and AhSTS3 in BARD-479 with respect to time after SA treatment, which indicated that AhSTSs are early responsive genes against biotic stresses.
In addition, Pearson's correlation coefficient indicated the antagonistic regulatory behavior of MeJA and SA, as moderate level negative correlations have been observed when the MeJA-affected expression of AhSTSs is compared to the SA-affected expression of AhSTSs. AhSTSs exhibited differential expression in genotypes under both hormonal treatments, which was also confirmed by the study in [31]. Additionally, correlation among the expression of AhSTSs in peanut suggested a coordinated response of AhSTSs in genotype and stimuli-dependent manner. Diversity in AhSTSs response to hormonal treatment in different genotypes also provides a hint for co-regulation to create specificity in the response. Further studies on the regulation and functional analysis of AhSTSs may reveal more interesting knowledge of these genes, reveal their use as a potential target for metabolic engineering for higher production of stilbenes at industrial scale, and help to develop disease-resistant crop plants.

Identification of Stilbene Synthase Genes in Peanut
National Center for Biotechnology Information (NCBI) database was searched for Stilbene synthase (STS) of peanut. The retrieved sequences were further observed in the SMART database (http://smart.emblheidelberg.de/, accessed on 20 August 2019) to confirm the presence of STS domain [38]. A total of 9 sequences of STS, including 5 complete coding region (CDS) and 4 partial CDS, were retrieved from NCBI database. FASTA sequence of all the nucleotide sequences from NCBI were BLAST in Peanut DB (http://bioinfolab.muohio.edu/txid3818v1, accessed on 20 August 2019) [39], which resulted in long list of contigs. Among all these contigs, contigs 2394 and 20,452 showed the presence of complete conserved domains for STS, which were further used to complete partial contigs through alignment. A total of 5 candidate genes were identified in peanut. Sequence specific primers were designed to confirm these sequences in peanut genome. Primer sequences have been given in Supplementary Table S5 for the reference.

Sequence Analysis
The nucleotide sequences of AhSTSs were further analyzed to predict intron/exon junction, cis-regulatory elements, and motifs. Gene structure display server (http://gsds. gao-lab.org/, accessed on 20 July 2020) was used to predict CDS and gene structure of AhSTSs display using default parameters [40]. ExPASY translate tool was used to get the open reading frame (ORF) of nucleotide sequence of AhSTSs. ExPASY (https://web. expasy.org/protparam/, accessed on 20 July 2020) protparam tool was used to calculate the molecular weight, amino acid length, isoelectric point (pI), aliphatic index (AI), GRAVY, and half-life of proteins [41]. The transmembrane region for AhSTS1, AhSTS2, AhSTS3, AhSTS4, and AhSTS6 was predicted using SOSUIsignal (http://harrier.nagahama-i-bio. ac.jp/sosui/sosuisignal/sosuisignal_submit.html, accessed on 20 July 2020), respectively. SignalP 5.0 server was used to predict the presence of signal peptide cleavage sites for AhSTS1, AhSTS2 and AhSTS3, AhSTS4, AhSTS6 under the default settings, respectively.
Peanut database was used to extract the upstream promoter region of the genes comprising of 1500 bp. The promoter region was analyzed in PlantCare database (http: //bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 21 February 2021) for predicting cis-regulatory elements. MEME software (http://meme-suite.org/, accessed on 21 February 2021) was used to predict the motifs present at default parameters. TB tools software was used to draw the motif pattern.

Phylogenetic Analysis
Nucleotide sequence of AhSTSs were then aligned with sequences from A. duranensis, A. iapensis, O. sativa, M. trunculata, V. vinifera, V. riparia, and A. thaliana using MUSCLE alignment at default settings. An unrooted phylogenetic tree was constructed using the Neighbor-joining method at a bootstrap value of 1000 in MEGA X. Seq2 logo was used to generate the position specificity of amino acids in AhSTSs under default parameters through sequence alignment (http://www.cbs.dtu.dk/biotools/Seq2Logo, accessed on 1 July 2021).

Enzyme-Substrate Interaction
Interaction between AhSTS enzyme and their substrate was predicted using molecular docking in pyrx with Autodock Vina algorithm. Based on literature, p-coumaroyl CoA and malonyl CoA were selected as the ligand of AhSTSs enzyme for analysis. The interaction of the ligand with active site residues and bond length was analyzed through Discovery studio.

Plant Material and Hormonal Treatment
The seeds of registered commercial peanut variety (BARD-479) developed by Oilseed department of National Agricultural Research Centre (NARC), Pakistan were obtained after permission from authorized person. All the experiments were performed according to the guidelines provided by Coordinated Framework for Regulation of Biotechnology and institutional review board (IRB), ASAB, NUST, Pakistan. Seeds were de-husked and surface sterilized using 70% ethanol followed by washing with autoclaved distilled water. To break seed dormancy, seeds were kept at 4 • C for two days. Seeds were kept in dark for germination in petri plate provided with moist filter paper. Equal sized seedlings were transferred in autoclaved mixture of soil and peat moss in 1:1 ratio. Seedlings were grown at temperature of 28

RNA Isolation, cDNA Synthesis and RT-PCR
Total RNA was extracted from leaves sample of both control and treated peanut plants (BARD-479) through the Trizol reagent method [47]. Integrity and purity of RNA was tested by agarose gel electrophoresis and NanoDrop™ 2000/2000 c Spectrophotometers. An amount of 1 µg of total RNA was converted into cDNA using Thermo Scientific™ RevertAid First Strand cDNA Synthesis Kit using oligo dT primers. Reverse transcriptasepolymerase chain reaction (RT-PCR) was performed to confirm the presence of candidate genes in the peanut genome.

Cloning and Sanger Sequencing
Amplicons were cleaned with EXoSAP-IT TM PCR product Cleanup Reagent (Thermo ScientificWILM, Wilmington, NC, USA) and cloned in DH5α of E. coli through electroporation. Clones were sequenced to confirm amplified gene product. Obtained sequences were submitted in NCBI to get accession number and termed as AhSTS1, AhSTS2, AhSTS3, AhSTS4, and AhSTS6.

Relative Gene Expression Analysis
Relative gene expression of AhSTSs was determined in the leaves sample of selected peanut variety at 0.5 h, 3 h, 6 h, 8 h, and 24 h in relation to control plant samples. The 7500 Fast Real-Time PCR system (Applied Biosystem, WLM, MA, USA) was used for real-time PCR analysis, according to instructions given in Maxima SYBR Green/ROX qPCR Master Mix by Thermo Fisher Scientific. Fold changes were calculated using Livak method (2-∆∆Ct method) [48]. Actin was used as housekeeping gene. Primer sequences used in the analysis has been given in Supplementary Table S5. Each experiment was replicated three times by taking individual leaves from three plants at the different time points.

Statistical Analysis
Microsoft Excel 365 and GraphPad Prism ® version 9 was used to organize and analyze data. Two-way ANOVA (analysis of variance) was performed with Bonferroni post hoc test to measure significant variation in control vs. treated samples. Correlation analysis was performed using Pearson's correlation coefficient. p-value 0.05 was considered to be statistically significant.

Conclusions
In the present study, we have identified five AhSTS genes in peanut. These AhSTS genes were subdivided into three subgroups based on sequence identity and evolutionary relativeness. Several cis-regulatory elements were identified, which showed their role in light responsiveness, defense responses, the regulation of seed development, plant growth and development. Most of the genes were differentially expressed in response to MeJA and SA treatment in a time-dependent manner, indicating the regulation of plant defense responses. Our findings pave way for exploring the potential of AhSTS genes to improve the resistance of peanut to biotic and abiotic stress through genetic engineering. The study on transcriptional regulation of these genes would help us better understand the differential expression and how to get increased stilbene production in plants.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11131776/s1, Table S1: Table displaying the conservation and non-conservation of significant contigs in 9 major nucleotide sequences. Table S2: Attributes of five Stilbene synthase (AhSTSs) genes identified in peanut, Table S3: Physiochemical properties of Stilbene synthase (AhSTSs) genes identified in peanut, Table S4: Predicted cis-acting elements in the promoter region of stilbene synthase (AhSTSs) genes, Table S5: Primers identity and sequences used for Stilbene synthase (AhSTSs) gene amplification and RT-PCR analysis, Table S6: Correlation among the expression of stilbene synthase genes in Peanut. Figure S1: Multiple sequence alignment of nucleotide sequence of the AhSTS1, AhSTS2, AhSTS3, AhSTS4, AhSTS6. The sequences conserved across all genes are highlighted in red while non-conserved sequences are shown in black. The sequences highlighted in blue shows sequences conserved in some genes. Figure S2: Multiple sequence alignment of amino acid sequences of AhSTS1, AhSTS2, AhSTS3, AhSTS4, and AhSTS6. The sequences conserved across all genes are highlighted in red while non-conserved sequences are shown in black. The sequences highlighted in blue shows sequences conserved in some genes. Figure S3: Showing the conserved N-terminal Chal_sti_synt domain and C-terminal ACP_syn_III FAE1_CUT1_rppA, in the sequences of AhSTS1, AhSTS2, AhSTS3, AhSTS4 and AhSTS6, Figure S4: Sequence logo of AhSTSs. X-axis showing the position of amino acid whereas stack of symbols representing the amino acids (big stacks represent conserved positions and small stacks represents variable position). Figure S5: secondary structure of Arachis hypogaea STS1 showing alpha helices and beta sheets. Loops are also observed in the structure. Figure S6: Predicted secondary structure of Arachis hypogaea STS2 showing alpha helices and beta sheets. Loops are also observed in the structure. Figure S7: Predicted secondary structure of Arachis hypogaea STS3 showing alpha helices and beta sheets. Loops are also observed in the structure. Figure S8: Predicted secondary structure of Arachis hypogaea STS4 showing alpha helices and beta sheets. Loops are also observed in the structure. Figure  S9: Predicted secondary structure of Arachis hypogaea STS6 showing alpha helices and beta sheets. Loops are also observed in the structure. Figure S10: 3D structure of AhSTS1predicted from the SWISS-MODEL. The red color indicates helices, green color represents loops and cyan color represent β-sheets. Figure S11: 3D structure of AhSTS2 predicted from the SWISS-MODEL. The red color indicates helices, green color represents loops and cyan color represent β-sheets. Figure S12: 3D structure of AhSTS3 predicted from the SWISS-MODEL. The red color indicates helices, green color represents loops and cyan color represent β-sheets. Figure S13: 3D structure of AhSTS4 predicted from the SWISS-MODEL. The red color indicates helices, green color represents loops and cyan color represent β-sheets. Figure S14: 3D structure of AhSTS6 predicted from the SWISS-MODEL. The red color indicates helices, green color represents loops and cyan color represent β-sheets; Figure S15: Ramachandran Plot of stilbene synthase protein in Arachis hypogaea. The graph shows the location of amino acid residues in the favored region of protein. For AhSTS1, the location of amino acids in the favored region 92.9% forming a stable protein, Figure S16: Ramachandran Plot of stilbene synthase protein in Arachis hypogaea. The graph shows the location of amino acid residues in the favored region of protein. For AhSTS2, the location of amino acids in the favored region 93.8% forming a stable protein, Figure S17: Ramachandran Plot of stilbene synthase protein in Arachis hypogaea. The graph shows the location of amino acid residues in the favored region of protein. For AhSTS3, the location of amino acids in the favored region 92% forming a stable protein, Figure S18: Ramachandran Plot of stilbene synthase protein in Arachis hypogaea. The graph shows the location of amino acid residues in the favored region of protein. For AhSTS4, the location of amino acids in the favored region 92.3% forming a stable protein, Figure S19: Ramachandran Plot of stilbene synthase protein in Arachis hypogaea. The graph shows the location of amino acid residues in the favored region of protein. For AhSTS6, the location of amino acids in the favored region 92.3% forming a stable protein.