Genome-Wide Investigation of Apyrase (APY) Genes in Peanut (Arachis hypogaea L.) and Functional Characterization of a Pod-Abundant Expression Promoter AhAPY2-1p

Peanut (Arachis hypogaea L.) is an important food and feed crop worldwide and is affected by various biotic and abiotic stresses. The cellular ATP levels decrease significantly during stress as ATP molecules move to extracellular spaces, resulting in increased ROS production and cell apoptosis. Apyrases (APYs) are the nucleoside phosphatase (NPTs) superfamily members and play an important role in regulating cellular ATP levels under stress. We identified 17 APY homologs in A. hypogaea (AhAPYs), and their phylogenetic relationships, conserved motifs, putative miRNAs targeting different AhAPYs, cis-regulatory elements, etc., were studied in detail. The transcriptome expression data were used to observe the expression patterns in different tissues and under stress conditions. We found that the AhAPY2-1 gene showed abundant expression in the pericarp. As the pericarp is a key defense organ against environmental stress and promoters are the key elements regulating gene expression, we functionally characterized the AhAPY2-1 promoter for its possible use in future breeding programs. The functional characterization of AhAPY2-1P in transgenic Arabidopsis plants showed that it effectively regulated GUS gene expression in the pericarp. GUS expression was also detected in flowers of transgenic Arabidopsis plants. Overall, these results strongly suggest that APYs are an important future research subject for peanut and other crops, and AhPAY2-1P can be used to drive the resistance-related genes in a pericarp-specific manner to enhance the defensive abilities of the pericarp.


Introduction
Peanut, or groundnut (Arachis hypogaea L.), is an important food legume of tropical and subtropical countries. It is the primary source of edible oil and proteins and the staple food of many African and Asian countries [1]. Peanut is cultivated in more than 100 countries across the world, while China, India, Nigeria, the United States, and Sudan are leading peanut-producing countries [2]. Botanically, peanut is a unique plant among legumes due to the pegging phenomenon [3]. It produces flowers above ground, and after pollination, the gynophore enters the soil and produce seeds underground [3,4]. As it bore seeds beneath the soil, seeds are always prone to attack by soil-borne fungal and bacterial pathogens. These pathogens mainly deteriorate peanut yield and quality [5].
pericarp-specific expression. The pericarp is the first organ that protects edible seeds from biotic and abiotic stresses. Several studies are available for pod/pericarp development, but there is a lack of work on the resistance-related behavior of the pericarp. We selected the pericarp-specific promoter for further study. Promoters are key regions to guide and regulate a gene's expression. We cloned the promoter region of the AhAPY2-1 gene and functionally characterized it in transgenic Arabidopsis plants. This study first reported the Apyrases/Nucleotide phosphatases proteins in the genome of cultivated peanut and characterized a pericarp abundant expression promoter in peanut. We are convinced that this study will enhance the understanding of peanut apyrases and provide a base for further research.

Identification, Genomic Locations, and Physicochemical Properties of AhAPYs
The APYs/NTPs genes in the genome of cultivated peanut were identified stepwise. Protein sequences of seven Arabidopsis apyrases were used as queries to search the apyrases in diploid peanut species. The BLAST results revealed the presence of eight APYs homologs in the genomes of A. duranensis and A. ipaensis each (Table S2), and 17 genes were found in the cultivated peanut genome ( Table 1). The apyrases of A. duranensis, A. ipaensis, and A. hypogaea were named based on the phylogenetic relationships with Arabidopsis apyrases. The chromosomal distribution analysis of AhAPYs showed their uneven distribution in the genome. Chromosomes 2, 4, 7-9, 12, 14, 17-19 did not possess any apyrases. Chromosome 6 possessed four APYs, and chromosome 16 possessed three APYS. Chromosomes 1 and 5 possessed two APYs each, while all remaining chromosomes (3, 10, 11, 13, 15, and 20) possessed one APYs each ( Figure 1). The chromosomal distribution of A. duranensis and A. ipaensis are presented in Figure S1. All AhAPYs possessed varying physicochemical properties. The subcellular localization prediction showed that most proteins are located in the plasma membrane, with few in the cytoplasm, chloroplast, mitochondria, extracellular spaces, nucleus, and vacuole. The theoretical isoelectric points of AhAPYs varied from 4.69 (AhAPY2-4) to 9.94 (AhAPY7-4), and molecular weight ranged from 14.96 KDa (AhAPY2-4) to 82.39 KDa (AhAPY7-1) ( Table 1). The physicochemical properties, protein, CDS lengths, and the number of exons in AdAPYs and AiAPYs are given in Table S2.    Note. MW = molecular weight, pI = theoretical isoelectric point, -and + signs represent negative and positive strands.

Gene Structure, Conserved Motifs, and Protein 3D Structure Simulation
Structurally, all AhAPYs were diverse, and they possessed a different number of exons. Exon numbers varied from 2 to 12 exons, while 9 exons were common among AhAPYs ( Figure 2). AhAPYs possessed varying genomic, CDS, and protein lengths, as AhAPY2-2 is the largest gene, comprising 11,962 bp long genomic sequence, 1395 bp long CDS, and 464 aa protein length, while the AhAPY2-4 is the smallest gene with genomic sequence of 2387 bp. Interestingly, AhAPY7-1 possessed the longest CDS and protein length of 2253 bp and 750 aa, respectively (Table 1). Conserved motif analysis revealed the presence of common and unique motifs in AhAPY genes. Proteins sharing common motifs tend to crowd in the same group, indicating their similar functions. The 1st, 3rd, 4th, and 10th motifs were found in most AhAPYs. AhAPY1-5 and AhAPY7-4 possessed only one motif each ( Figure 2). Gene structures and conserved motif patterns of A. duranensis APYs are given in Figure S2, and of A. ipaensis are shown in Figure S3. Simulations of 3D modules of tertiary protein structures of 17 AhAPYs showed that an extended strand linked the similar subunits that are further surrounded by an alpha helix. Overall, this arrangement represents a characteristic feature of the GDA1-CD39 NPT superfamily ( Figure S4). Previous studies on Arabidopsis APYs showed that AtAPY1 and AtAPY2 perform similar functions, and AtAPY6 and AtAPY7 perform similar functions; the 3D structures of AhAPYs represent the structural similarity of the first group with the second, and the sixth group with the seventh. This fact is also evident from the conserved motif analysis, which divides the peanut APYs into three groups.
subunits that are further surrounded by an alpha helix. Overall, this arrangement represents a characteristic feature of the GDA1-CD39 NPT superfamily ( Figure S4). Previous studies on Arabidopsis APYs showed that AtAPY1 and AtAPY2 perform similar functions, and AtAPY6 and AtAPY7 perform similar functions; the 3D structures of AhAPYs represent the structural similarity of the first group with the second, and the sixth group with the seventh. This fact is also evident from the conserved motif analysis, which divides the peanut APYs into three groups.

Phylogenetic and Gene Duplication Analysis
To study the evolutionary relationships of cultivated peanut APYs with their wild relatives and model legume crops, a phylogenetic tree was constructed among the protein sequences of A. hypogaea and their homologs in A. duranensis, A. ipaensis, A. thaliana, and G. max. The phylogenetic tree divided all the APYs into three main groups. APY1 and APY2 of all species tended to cluster in the same group. APY6 of all species clustered in a separate group, and members of APY7 from all species clustered in a separate group ( Figure 3). Phylogenetic grouping provided evidence of solid evolutionary relationships among these species. Gene duplication is a major force behind genome evolution. Duplicated gene pairs among AhAPYs were identified by their phylogenetic relationships. Among 17 AhAPYs, seven duplicated gene pairs were found (Figure 4). The Ks and Ka values for each duplicated pair were calculated by a simple Ka/Ks calculator available at TBtools. Evolutionary rates (Ka/Ks ratio) for each duplicated gene pair were calculated. The Ka/Ks = 1 was considered neutral selection pressure, Ka/Ks > 1 was regarded as positive selection pressure, and Ka/Ks < 1 was considered purifying selection pressure [46]. Ka/Ks values of all gene pairs showed that purifying selection pressure was mainly involved in the duplication process ( Table 2). The expected divergence time for duplicated pairs varied from 1.26 Mya (million years ago) for the gene pair AhAPY7-2:AhAPY7-3 to 127.405 Mya for AhAPY1-5:AhAPY1-4 ( Table 2). values for each duplicated pair were calculated by a simple Ka/Ks calculator available at TBtools. Evolutionary rates (Ka/Ks ratio) for each duplicated gene pair were calculated. The Ka/Ks = 1 was considered neutral selection pressure, Ka/Ks > 1 was regarded as positive selection pressure, and Ka/Ks < 1 was considered purifying selection pressure [46]. Ka/Ks values of all gene pairs showed that purifying selection pressure was mainly involved in the duplication process ( Table 2). The expected divergence time for duplicated pairs varied from 1.26 Mya (million years ago) for the gene pair AhAPY7-2:AhAPY7-3 to 127.405 Mya for AhAPY1-5:AhAPY1-4 ( Table 2).

Prediction of miRNAs Targeting AhAPYs and Analysis of Putative Protein-Protein Interactions
Non-coding miRNAs, as the key regulators of post-transcriptional gene regulation, have attracted the attention of many researchers. Some studies have reported their role in biotic and abiotic stress responses [47,48]. To illustrate the possible miRNAs involved in regulating the peanut APYs, we predicted the miRNAs targeting the AhAPYs through the

Prediction of miRNAs Targeting AhAPYs and Analysis of Putative Protein-Protein Interactions
Non-coding miRNAs, as the key regulators of post-transcriptional gene regulation, have attracted the attention of many researchers. Some studies have reported their role in biotic and abiotic stress responses [47,48]. To illustrate the possible miRNAs involved in regulating the peanut APYs, we predicted the miRNAs targeting the AhAPYs through the online miRNAs database, the PsRNATarget database. We found four different miRNAs targeting seven peanut APYs (Table S3). The ahy-miR3508 targeted AhAPY7-2 and AhAPY7-3; ahy-miR3513-5p targeted AhAPY2-2; ahy-miR3516 targeted AhAPY7-1 and AhAPY7-4; ahy-miR3520-5p targeted two genes, AhAPY6-1 and AhAPY6-2. These predicted miRNAs and their target sites in the CDS region are shown in Figure 5. These miRNAs provide future research dimensions for functional validation of their expression levels and role in gene regulation.
To understand the putative functions of AhAPYs, the protein interaction network analysis was performed based on APYs orthologs in Arabidopsis using the STRING database. The top 10 interactions were considered with a high threshold level (0.7). The interaction network prediction showed that AhAPY1-5 has functions related to Arabidopsis ZEU1 protein, Arabidopsis ADSS (Adenylosuccinate synthetase, chloroplastic), Fac1 (AMP deaminase), and some other proteins ( Figure S5). Protein AhAPY6-2 showed strong interactions with Arabidopsis ADSS, ZEU1, FAC1, THY1 (Bifunctional dihydrofolate reductase-thymidylate synthase 1), THY2 (Bifunctional dihydrofolate reductase-thymidylate synthase 2), and with other Arabidopsis proteins. AhAPY7-6 showed interaction with PUM10 (Putative pumilio homolog 10). Multiple sequence search methods showed that other AhAPYs did not interact with Arabidopsis proteins. Their interactions need more work, or there is a possibility that these proteins have some special functions that are not exploited well.
analysis was performed based on APYs orthologs in Arabidopsis using the STRING database. The top 10 interactions were considered with a high threshold level (0.7). The interaction network prediction showed that AhAPY1-5 has functions related to Arabidopsis ZEU1 protein, Arabidopsis ADSS (Adenylosuccinate synthetase, chloroplastic), Fac1 (AMP deaminase), and some other proteins ( Figure S5). Protein AhAPY6-2 showed strong interactions with Arabidopsis ADSS, ZEU1, FAC1, THY1 (Bifunctional dihydrofolate reductasethymidylate synthase 1), THY2 (Bifunctional dihydrofolate reductase-thymidylate synthase 2), and with other Arabidopsis proteins. AhAPY7-6 showed interaction with PUM10 (Putative pumilio homolog 10). Multiple sequence search methods showed that other AhAPYs did not interact with Arabidopsis proteins. Their interactions need more work, or there is a possibility that these proteins have some special functions that are not exploited well.

Analysis of Cis-Regulatory Elements
Analyzing transcription factor binding sites or cis-regulatory elements is key for functional genomic studies. We searched the 2 kb upstream regions of promoters to find out the cis-regulatory elements to predict the possible functions of AhAPYs. Aside from the core promoter elements (TATA box, CAAT box), other important regulatory elements were also found in the promoter regions. Other important cis-elements mainly included lightresponsive elements (G-box, Box 4, GATA-motif), hormone-responsive elements (abscisic acid, ABRE; salicylic acid, SARE; methyl jasmonate, MeJA; gibberellin, GBRE), growthand development-related (anaerobic induction, ARE; zein metabolism, O2-site; meristem expression; CAT-box), and stress-responsive elements (wound responsive elements, WUN motif; low temperature responsive, LTR; defense responsive; TC-rich repeats). The detail of cis-regulatory elements and their positions in the promoter regions are shown in Figure 6.
(abscisic acid, ABRE; salicylic acid, SARE; methyl jasmonate, MeJA; gibberellin, GBRE), growth-and development-related (anaerobic induction, ARE; zein metabolism, O2-site; meristem expression; CAT-box), and stress-responsive elements (wound responsive elements, WUN motif; low temperature responsive, LTR; defense responsive; TC-rich repeats). The detail of cis-regulatory elements and their positions in the promoter regions are shown in Figure 6. . Cis-regulatory elements in the promoter regions (2 kb upstream of the start codon) of AhAPYs. Cis-elements analysis revealed that promoter regions of AhAPYs possessed important elements responsive to light, phytohormones, defense and stress, low temperature, and wound responsiveness. Figure 6. Cis-regulatory elements in the promoter regions (2 kb upstream of the start codon) of AhAPYs. Cis-elements analysis revealed that promoter regions of AhAPYs possessed important elements responsive to light, phytohormones, defense and stress, low temperature, and wound responsiveness.

Synteny and Functional Annotation Analysis
The collinearity analysis was performed among A. hypogaea, A. duranensis, A. ipaensis, and A. thaliana to assess their syntenic relationships. Collinearity analysis showed strong evolutionary relationships of APYs among these species. A. hypogaea showed strong syntenic relationships with its wild parents compared to Arabidopsis (Figure 7). The functional annotation analysis was performed to prophesize the potential functions of AhAPYs. Gene ontology (GO) enrichment analysis revealed that AhAPYs are involved in important GO categories, including molecular functions (MF), cellular components (CC), and biological processes (BP). For the MF category, AhAPYs were involved in hydrolase catalytic activity; for the CC category, the AhGLPs were mainly found as a component of the nucleus; and for the BP category, AhAPYs were involved in a large number of subcategories, including reproduction, catabolic processes, stress responses, and many developmental processes ( Figure 8a). These results depict the importance of AhAPYs in different metabolic, cellular, and biological processes. The KEGG pathway is a computerized representation of a biological system through which we can infer the role of a protein/gene [49]. We also performed the KEGG enrichment analysis of AhAPYs to infer their metabolic roles. AhAPYs were involved in key metabolic pathways, including 00230 purine metabolism, B09104 Nucleotide metabolism, 00240 Pyrimidine metabolism, and A09100 metabolism pathways. AhAPYs were also enriched in signaling and cellular processes and as 04090 CD molecules (Figure 8b).
important GO categories, including molecular functions (MF), cellular components (CC), and biological processes (BP). For the MF category, AhAPYs were involved in hydrolase catalytic activity; for the CC category, the AhGLPs were mainly found as a component of the nucleus; and for the BP category, AhAPYs were involved in a large number of subcategories, including reproduction, catabolic processes, stress responses, and many developmental processes (Figure 8a). These results depict the importance of AhAPYs in different metabolic, cellular, and biological processes. The KEGG pathway is a computerized representation of a biological system through which we can infer the role of a protein/gene [49]. We also performed the KEGG enrichment analysis of AhAPYs to infer their metabolic roles. AhAPYs were involved in key metabolic pathways, including 00230 purine metabolism, B09104 Nucleotide metabolism, 00240 Pyrimidine metabolism, and A09100 metabolism pathways. AhAPYs were also enriched in signaling and cellular processes and as 04090 CD molecules (Figure 8b).

Expression Analysis
The transcriptome expression pattern of AhAPYs in various tissues and under different hormones/stress treatments was assessed from the peanut genome resource database. All AhAPYs showed expression in studied tissues, including leaf, stem, root, flower, peg,

Expression Analysis
The transcriptome expression pattern of AhAPYs in various tissues and under different hormones/stress treatments was assessed from the peanut genome resource database. All AhAPYs showed expression in studied tissues, including leaf, stem, root, flower, peg, pericarp, testa, cotyledons, and embryo. AhAPY7-1 to AhAPY7-6 did not show any remarkable expression in the studied tissue. AhAPY6-1 and AhAPY6-2 showed uniform expression in all tissues, but expression level was low, and AhAPY1-1 and AhAPY1-2 showed relatively higher expression levels in all tissues. AhAPY2-1 and AhAPY2-3 showed abundant expression in the pericarp compared to other tissues, and AhAPY2-2 showed abundant expression in the stem, root, and cotyledons. AhAPY2-4 showed decreased expression in the stem tip Further, the expression of all AhAPYs genes was assessed under abscisic acid treatment to check whether their expression corresponds to the transcriptome expression. Peanut plants were treated with ABA (10 μg/mL), and quantitative expression was assessed at different time points. The qRT-PCR results validated the transcriptome expression data under ABA treatment. The qRT-PCR-based expression of 17 AhAPYs genes is shown in Figure 10. Genes of group 1 (AhAPY1-1 to AhAPY1-4), group 6 (AhAPY6-), and group 7 (AhAPY7-) showed upregulated transcriptome expression, and the qRT-PCR results also found similar expression pattern. AhAPY1-5, and all genes of group 2 (AhAPY2-), did not show any change in transcriptome expression, and real-time expression results found a similar expression pattern.  AhAPYs showed varying expression levels under different hormones and stress treatments. AhAPY2-1 and AhAPY2-2 showed decreased expression under ABA treatment. In contrast, AhAPY1-1 and AhAPY1-2 showed higher expression under all stress conditions, including ABA, SA, Brassinolide, paclobutrazol, ethephon treatments, drought, normal irrigation, ddH 2 O spray, low temperature, and room temperature. AhAPY1-5 and AhAPY2-4 did not respond to the hormones, water, and temperature treatments. The expression matrix of AhAPYs in response to different hormones and stress agents is shown in Figure 9b Figure 10. Genes of group 1 (AhAPY1-1 to AhAPY1-4), group 6 (AhAPY6-), and group 7 (AhAPY7-) showed upregulated transcriptome expression, and the qRT-PCR results also found similar expression pattern. AhAPY1-5, and all genes of group 2 (AhAPY2-), did not show any change in transcriptome expression, and real-time expression results found a similar expression pattern.

Selection of Gene for Promoter Cloning and In Silico Analysis of Promoter
In the previous section, the expression profiles showed that the AhAPY2-1 gene was more highly expressed in the pod or pericarp than in any other tissue; we considered it a pericarp-abundant gene. Based on this consideration, it can be assumed that the promoter of the pericarp-abundant gene can be used to drive a foreign gene in a podspecific manner. The RNA seq-and gene-chip expression data of AhAPY2-1 is given in File S1. qPCR was performed to verify the expression of the AhAPY2-1 gene in different tissues. Results of the qRT-PCR analysis showed a high number of transcripts of AhAPY2-1 in pod/pericarp tissues compared to all other tissues, indicating its pod-abundant expression pattern Figure 11a. Based on these results, we hypothesized that the promoter of the AhAPY2-1 gene could be used to drive a foreign gene in a pod-abundant manner.
For cloning of the AhAPY2-1 promoter, the 2044 bp upstream region was selected and scanned through online promoter analysis databases, the PlantCARE database. It was found that the promoter region contained core promoter elements, including the TATA Box and CAAT box, both of which are required for precise initiation of transcription and tissuespecific activity [50,51]. Aside from these core promoter elements, several other important cis-elements were also present in the AhAPY2-1 promoter. These elements include the hormone-responsive elements auxin (TGA-elements), gibberellin (TATC-box), salicylic acid (TCA-element), abscisic acid (ABRE), methyl jasmonate (TGACG-motif, CGTCAmotif), ethylene responsiveness (ERE), and light-responsive elements (GT1-motif, G-Box, GATA-motif, Box 4, AT-1 motif). Moreover, wound-responsive elements (WUN-motif), defense-related elements (MYB sites), anaerobic induction (ARE), and zein metabolism regulatory element O2-site were also present. Additionally, some elements with unknown functions were also present. Detailed information on the AhAPY2-1 promoter is provided in Figure S6. The new PLACE database also predicted several key elements in the promoter region, such as seed-specific elements (RY-element) and binding sites for WRKY and MYB transcription factors. Table S4 contains information on cis-elements, their position, sequence, and functions predicted by the PLACE database. These elements suggest that the AhAPY2-1 promoter could be used to replace the native promoter of a gene. The CDS, protein, and promoter sequences of the AhAPY2-1 gene are given in File S2.

Selection of Gene for Promoter Cloning and in Silico Analysis of Promoter
In the previous section, the expression profiles showed that the AhAPY2-1 gene wa more highly expressed in the pod or pericarp than in any other tissue; we considered it

Functional Study of Promoter Activity in Transgenic Plants
Samples from different tissues of transgenic plants were used to check the activity of the AhAPY2-1-controlled GUS gene. Leaf, roots, stem, seedlings, flowers, siliques, and seeds were incubated in the GUS solution. The GUS staining showed that the siliques outer covering/pod has a dense blue color. Flowers of transgenic Arabidopsis plants also showed some blue color after GUS staining, while in all other tissues the GUS staining was very low or absent, Figure 12a. Arabidopsis wild plants (Col-0) were also used for GUS staining to compare the results. To assure the proper staining of cotyledons and embryos, seeds were ruptured and incubated in the staining solution. Cryostat sectioning of those seeds was performed to check whether staining was present in cotyledons and embryos. Staining was not detected in any seed tissues, including seed coat/testa, cotyledons, or

Cloning and Genetic Transformation
The promoter region of the AhAPY2-1 gene was amplified from the DNA template of the Xinhuixiaoli (XHXL) cultivar with the specific primers (Table S1). Clones having >99% sequence similarity with the original sequence were used to construct the vector. Twostep Gateway cloning was used in which the promoter was first ligated into entry vector pDONR207 by BP reaction. The promoter fragment was ligated into the expression vector pMDC164 by LR reaction in the second step. The complete procedure of amplifying the AhAPY2-1 promoter and vector construction is shown in Figure 11b The expression vector was transformed into A. tumefaciens, and (GV3101) cells were used for the genetic transformation of Arabidopsis plants by the floral dip method. Positively transformed T0 seeds were screened on Hygromycin (50 mg mL −1 ) selection medium, and eight HygR resistant plants were verified by PCR amplification, Figure 11e. Hygromycin and PCR screening were performed in each generation, and homozygous T3 generation was obtained.

Functional Study of Promoter Activity in Transgenic Plants
Samples from different tissues of transgenic plants were used to check the activity of the AhAPY2-1-controlled GUS gene. Leaf, roots, stem, seedlings, flowers, siliques, and seeds were incubated in the GUS solution. The GUS staining showed that the siliques outer covering/pod has a dense blue color. Flowers of transgenic Arabidopsis plants also showed some blue color after GUS staining, while in all other tissues the GUS staining was very low or absent, Figure 12a. Arabidopsis wild plants (Col-0) were also used for GUS staining to compare the results. To assure the proper staining of cotyledons and embryos, seeds were ruptured and incubated in the staining solution. Cryostat sectioning of those seeds was performed to check whether staining was present in cotyledons and embryos. Staining was not detected in any seed tissues, including seed coat/testa, cotyledons, or embryos, Figure 12a. Arabidopsis wild type (Col-0) did not show any blue color. The staining results indicated that the AhAPY2-1 promoter has successfully regulated the GUS gene in a pod/pericarp-abundant manner in transgenic Arabidopsis plants. Int Figure 12a. Arabidopsis wild type (Col-0) did not show any blue color. The staining results indicated that the AhAPY2-1 promoter has successfully regulated the GUS gene in a pod/pericarp-abundant manner in transgenic Arabidopsis plants.
The qRT-PCR analysis was performed to check the quantitative expression of the GUS gene in different tissues of transgenic Arabidopsis plants. The qRT-PCR analysis showed that the GUS gene was highly expressed in silique outer coverings (pod/pericarp) of transgenic seedlings compared to other tissues, Figure 12b. In contrast, the expression level in all other tissues was very low. Collectively, these results showed that the AhAPY2-1 promoter effectively drove the expression of the GUS gene in the pericarp-abundant manner; hence, this promoter could be a suitable candidate for pod/pericarp-abundant expression of a transgene.

Response of AhAPY2-1 Promoter to Hormones
Expression responses of AhAPY2-1 promoter under different phytohormones treatment were studied by exposing the transgenic Arabidopsis plants to different hormones The qRT-PCR analysis was performed to check the quantitative expression of the GUS gene in different tissues of transgenic Arabidopsis plants. The qRT-PCR analysis showed that the GUS gene was highly expressed in silique outer coverings (pod/pericarp) of transgenic seedlings compared to other tissues, Figure 12b. In contrast, the expression level in all other tissues was very low. Collectively, these results showed that the AhAPY2-1 promoter effectively drove the expression of the GUS gene in the pericarp-abundant manner; hence, this promoter could be a suitable candidate for pod/pericarp-abundant expression of a transgene.

Response of AhAPY2-1 Promoter to Hormones
Expression responses of AhAPY2-1 promoter under different phytohormones treatment were studied by exposing the transgenic Arabidopsis plants to different hormones stress. The expression of the GUS gene controlled by the AhAPY2-1 promoter was studied to check the activity of the promoter in response to these hormones. The GUS gene showed no remarkable expression under all hormonal/ddH 2 O treatments at all time points. The expression of the GUS gene was decreased as compared to the control. Under ABA treatment, expression level decreased at 3 h post-treatment and showed a decreasing trend until 24 h of ABA treatment. Under BR and SA treatment, a similar expression pattern was observed. In response to ethephon and paclobutrazol treatment, the expression level was decreased compared to the control. Still, there was a gradual increase in expression at different time points, but the expression was less than the controlled one. Overall, the expression of the GUS gene was decreased under all treatments than in control plants ( Figure 13). These results indicated that the AhAPY2-1 promoter did not induce GUS gene expression under hormone treatments. In other words, we can say that AhAPY2-1 is a pericarp-specific gene that is not influenced by other stress conditions. In response to ethephon and paclobutrazol treatment, the expression level was decreased compared to the control. Still, there was a gradual increase in expression at different time points, but the expression was less than the controlled one. Overall, the expression of the GUS gene was decreased under all treatments than in control plants ( Figure 13). These results indicated that the AhAPY2-1 promoter did not induce GUS gene expression under hormone treatments. In other words, we can say that AhAPY2-1 is a pericarp-specific gene that is not influenced by other stress conditions.

Discussions
Extracellular ATPs, the energy currency of all organisms, play key regulatory roles in plants and animals. The signaling roles of extracellular ATPs in mammals have been under investigation for the last 40 years [52], but these investigations are at an early stage in plants. Recent studies have demonstrated that extracellular ATP levels play critical roles in regulating various cellular and stress responses [53,54]. Apyrases (APYS)/Nucleoside phosphatases (NTPs) are key regulators of cell growth and stress responses by maintaining extracellular ATP levels [55]. Several studies have shown that cellular ATP levels decrease under stressed conditions, which ultimately results in increased cellular ROS levels causing cell apoptosis [56,57]. Thus, APYs are critical in cleaving the extracellular ATP in the stressed environment to maintain cellular ATP levels and avoid the adverse effects of ROS accumulation. Overexpression of APYs has been reported to inhibit ROS accumulation and increase stress tolerance [36]. During the past decade, some studies have shown that APYs play fundamental roles against cold stress [36], salt stress [58], and drought stress [21]. Their roles in biotic stress tolerance have recently been reported, including powdery mildew response in wheat [59]. These findings provide the future scope of APYs in managing the stress induced by external agents in plants. In Arabidopsis, seven apyrases have been reported, and some of them have been functionally characterized [55]. In 2019, Liu and his team investigated apyrases in wheat at a genome-wide scale and reported the defense roles of APYs against powdery mildew [59].
Peanut is an important legume crop worldwide and a livelihood source for many people. At present, peanut is a resource-rich legume with a large amount of data available on yield quality traits [60][61][62][63], diseases/pathogens resistance [5,6,64], seed dormancy [61,65], etc. With the availability of the genome sequence of tetraploid peanut (Arachis hypogaea) [10,11], it becomes easy to investigate the apyrases in peanut and study their function. In this study, we used seven Arabidopsis APYs and identified 17 APY homologs in the cultivated peanut genome (Table 1). These 17 peanut APYs were divided into three phylogenetic groups based on their evolutionary relationships with Arabidopsis and soybean. We also identified eight APYs in each A. duranensis and A. ipaensis (Table S2). The larger numbers of APYs in the genome of cultivated peanut than its diploid parents are due to its tetraploid nature and larger genome size. The evolutionary process has resulted in the structural diversity of genes/gene families in crops; similarly, the APYS of wild and cultivated peanuts possessed large structural diversity. As the number of exons is important in determining gene expression patterns and levels [66], the cultivated peanut also possessed varying exons ranging from 2 to 12 ( Figure 2).
The phylogenetic analysis divided the AhAPYs into three groups; mainly, these groups correlated to their functions. As previous studies have reported the similar functions of AtAPY1 and AtAPY2, their homologs in peanut tend to cluster in one group. AtAPY7 homologs in peanuts clustered into a separate group. Although AtAPY6 and AtAPY7 possessed almost the same functions, the AtAPY6 homologs clustered into separate groups (Figure 3). A similar phylogenetic grouping has been reported for wheat apyrases [59]. Gene duplication and variations in genome size are key features of genetic diversity. Gene duplication events are important for gene expression diversification and neofunctionalization. We performed the gene duplication analysis for AhAPYs. Gene duplication analysis revealed eight duplicated gene pairs (Table 2). Mainly purifying selection pressure was involved in the duplication process, and all of the genes were segmentally duplicated ( Figure 4).
Non-coding micro-RNAs are key regulatory elements that play multiple roles in growth, regulation, and defense responses [67]. miRNAs have been a research hotspot for the last few years, and more studies are becoming available on their regulatory roles [47,68,69]. miRNAs are important in managing stress responses. We predicted the putative miRNAs targeting the AhAPYs through the published miRNAs data [70]. We found four miRNAs targeting seven AhAPYs (Table S3, Figure 5). Cis-regulatory elements are of key importance as they determine a gene's expression pattern, regulatory roles, and stress responses [71,72]. Cis-regulatory elements analysis showed important elements involved in light, hormones, growth and development, stress, and defense responses ( Figure 6). Genome collinearity and syntenic relations are important evolutionary events that lead to understanding genome duplication and neofunctionalization [73]. The collinearity analysis revealed key syntenic relations of AhAPYs with their diploid parents and Arabidopsis (Figure 7). Similarly, functional annotation is an important tool to emphasize the functions of a gene/gene family. The gene ontology (GO) analysis showed that AhAPYs are part of molecular functions (MF), Biological processes (BP), and cellular components (CC), and (Figure 8a). The KEGG pathway enrichment analysis showed that AhAPYs were also involved in key metabolic pathways (Figure 8b).
Owing to their geocarpic nature, peanut seeds are constantly attacked by soil-and seed-borne diseases. Different fungal, bacterial, and viral pathogens attack peanut plants at different growth stages and deteriorate yield and quality [45,74]. The pod/pericarp is an inedible part of peanut seed and a vital defense organ against various biotic and abiotic stress agents. The promoter is a part of a non-coding DNA sequence present upstream of a gene and regulates the expression of the downstream gene. Previous transgenic projects mainly use constitutive promoters to drive the desired gene(s). The constitutive expression of a foreign gene can have adverse effects on plant growth and development, as the constitutive expression imposes an extra metabolic burden by expressing the gene in tissues/organs where it is not required. Tissue-specific/abundant promoters are suitable alternatives to constitutive promoters as they express the desired gene in a tissue-specific manner. If a tissue-specific promoter with a resistant gene can successfully be transformed into peanut, it can improve the defense capabilities of the plant. We hypothesized that a pericarp-specific promoter would be a good choice to express resistant genes in a pericarpspecific manner to improve the defensive abilities of the pericarp. From the transcriptome expression data (Figure 9), we found that AhAPY2-1 is abundantly expressed in the pericarp without showing any remarkable expression in other tissues. We selected AhAPY2-1 for promoter cloning and functional characterization.
Genetic transformation in peanut is a challenging task. There are some reports on successful genetic transformation in peanut [75,76], but not a single well-established protocol exists for transformation. Due to these bottlenecks, Arabidopsis becomes a better alternative for the functional characterization of genes and promoters. So, we selected Arabidopsis for the functional characterization of the AhAPY2-1 promoter. We cloned a 2044 bp upstream region of the AhAPY2-1 gene and analyzed the key cis-regulatory elements ( Figure S6). Cis-regulatory elements clearly showed that AhAPY2-1P could be a suitable promoter. The qRT-PCR results validated the transcriptome expression data and clearly showed that this gene expresses in the pericarp-abundant manner (Figure 11a).
The promoter was cloned from peanut variety XHXL, and the plant expression vector was constructed using the backbone of the pMDC164 vector following the gateway cloning system. The floral-dip method was used for the genetic transformation of Arabidopsis plants, and positively transformed plants were grown to T3 homozygous generation. GUS staining assay showed strong blue color in the silique's outer coverings/pericarp; some staining was also present in the flowers. These minute changes in AhAPY2-1 expression in peanut and Arabidopsis are possibly due to the species change. Staining was absent in all other tissues. The cryostat sectioning of dissected seeds showed that the GUS gene was not expressed in seed coat/testa, cotyledons, and embryo. Expression of the GUS gene determined by qRT-PCR analysis also revealed significantly high expression of the GUS gene in the pericarp compared to other tissues. The literature shows that a set of phytohormones plays a key role in regulating several physiological, biochemical, and molecular processes under normal and stressed conditions [77]. Therefore, to check whether the expression of the GUS gene under the control of the AhAPY2-1 promoter in transgenic Arabidopsis corresponds to the transcriptome expression of the AhAPY2-1 gene in response to different hormones or not, transgenic plants were treated with phytohormones, and qPCR was performed. Although there were some deviations in the expression patterns, overall, we can conclude that it followed a similar expression pattern. These findings strongly suggest that AhAPY2-1P can regulate a foreign gene's expression in a pericarp-abundant manner.

Genomic Positions, Structure, and Physicochemical Properties of APYs
The genomic positions of AhAPYs were determined from the PGR database [10]. Genes were mapped out using the TBtools software [79]. The information regarding the gene structure was also retrieved from the PGR database. The conserved motifs in the protein sequences were elucidated by the MEME suite while setting the maximum number of motifs as ten with other default parameters [80]. Protein tertiary structures were predicted by the ExPASy server, while 3D models were drawn by Swiss-Model (https://www. swissmodel.expasy.org/; accessed on 4 August 2022) following the default parameters. The physicochemical properties, such as theoretical molecular weight (MW) and isoelectric points (pI), were determined by the ProtParam tool (https://web.expasy.org/protparam/; accessed on 3 August 2022) [81], and subcellular localizations were predicted by CELLO v2.5 tool (http://cello.life.nctu.edu.tw/; accessed on 4 August 2022) [82].

Phylogeny and Duplication Analysis
The phylogenetic tree was constructed among the APYs of A. hypogaea, G. max, A. thaliana, A. duranensis, and A. ipaensis to study their evolutionary relationships. MUSCLE program was used to align the protein sequences, and an ML tree with 1000 bootstrap repeats was constructed by MEGA-X. The genome and GFF3 files were run through MCScanX to identify the duplicated gene pairs. A simple Ka/Ks calculator was used to calculate the expected synonymous (Ks) and non-synonymous (Ka) substitution rates. Divergence time (million years ago, MYA) for duplicated gene pairs was calculated as 't = Ks/2r' while using the neutral substitution rate for peanut r = 8.12 × 10 −9 [12].

Protein-Protein Interaction Analysis and Putative miRNAs Targeting AhAPYs
Possible interactions of AhAPYs with other proteins were elucidated by constructing their protein-protein interaction network based on their homologs in Arabidopsis using the STRING 11.5 tool (https://www.string-db.org/cgi/; accessed on 5 August 2022). Interacting proteins with 100% similarity and <10 −5 were considered. The top 10 interactions with a high threshold (0.7) were considered. MCL clustering with inflation parameter 3 was used, and dotted lines were used between cluster edges. The putative miRNAs targeting the peanut APYs were predicted through the psRNATarget database (https://www.zhaolab.org/psRNATarget/analysis?function=2; accessed on 7 August 2022) [70]. The CDS sequences of peanut apyrases were scanned through the psRNATarget database for the prediction of putative miRNAs with default settings.

Promoter Cis-Elements Analysis
Online database available for the promoter elements prediction, viz., PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/; accessed on 7 August 2022) [83] was used to identify the cis-elements in APY promoters. The promoter sequences of AhAPYs were obtained from the PGR database. For the identification of cis-elements, 2 kb upstream sequences were used.

Comparative Genome Synteny and Functional Annotation Analysis
The syntenic relationships of three peanut species and Arabidopsis were developed to view the comparative genomic conservations. For this purpose, the genome and GFF3 files of all these species were scanned against each other through One Step MCScanX. The resulting Collinearity, GFF3, and CTL files were merged and used for multiple synteny plots with the help of TBtools [79]. We performed the functional annotation analysis to understand the possible functions/regulatory roles of AhAPYs. Whole-genome protein sequences were scanned for functional annotation analysis at the EggNOG server (http: //eggnog-mapper.embl.de/; accessed on 8 August 2022). The resulting annotation files were used to perform the GO and KEGG enrichment analysis.

Expression Analysis of AhAPYs
To understand the expression matrices of AhAPYs in different tissues and under different hormones/stress agents, the transcriptome expression data were accessed from the peanut genome resource database [10]. The log10 normalized FPKM (fragments per kilobase million) values were used to construct the expression heatmaps for different tissues and under different stress conditions. Further, the transcriptome expression was verified by treating the peanut plants with abscisic acid (ABA) and performing the qRT-PCR analysis. The peanut seeds were grown in small plastic pots, and plants were grown in the greenhouse at 26 • C and 16 h/86 day/night photoperiod. Four-leaf-old plants were treated with ABA (10 µg/mL), and samples were taken at different time points. Total RNA was extracted by the CTAB method with some modification, and the first strand cDNA was synthesized by the PrimeScript 1st strand cDNA Synthesis Kit (Takara, Dalian, China). qRT-PCR analysis was performed to compare the transcriptome and real-time expression of all APY genes. The qRT-PCR reaction was performed by the Applied Biosystems 7500 realtime PCR system (Thermofisher Scientific, Waltham, MA, USA) with the cycling program: 94 • C (1 min), 60 • C (1 min), and 72 • C (1 min) for 40 cycles. Peanut Actin gene was used as an internal control. Primers used for qRT-PCR are given in Table S1.

Plant Materials and Cloning of AhAPY2-1 Promoter
The seeds of peanut cultivar Xinhuixiaoli (XHXL) and Arabidopsis thaliana Col-0, used in this study, were grown in a greenhouse at the Institute of Oil Crops, Fujian Agriculture and Forestry University, Fuzhou, China. Based on the transcriptome expression data, we found that AhAPY2-1 was abundantly expressed in the pericarp. We selected a 2044 bp upstream region of the AhAPY2-1 gene for promoter cloning and characterization. The expression of the AhAPY2-1 gene in different tissues of peanut was checked by the qRT-PCR analyses. The promoter region of the AhAPY2-1 gene was cloned from the DNA template of peanut variety XHXL, and binary plasmid pMDC164 was used to construct the plant expression vector by the Gateway cloning method. Expression vectors were transformed into A. tumefaciens (GV3101) competent cells through the heat shock method. The floral dipping method was used to transform young Arabidopsis plants [84]. Positive transgenic plants were identified on a Hygromycin (HygR) selection medium (50 mg mL −1 ), and further verified by PCR amplification. Primers used in this study are given in Table S1. Our previous study gives a detailed procedure for qPCR and promoter cloning and transforma-tion [85]. Eight positive plants (verified by HygR resistance and PCR amplification in every generation) were grown to T3 generation for functional characterization.

Study of Promoter Activity in Transgenic Plants
Different tissues of transgenic Arabidopsis plants were used for GUS staining [86] to check the activity of the GUS gene under the control of the AhAPY2-1 promoter. Leaf, stem, flower, seedlings, siliques, and seeds were incubated in GUS staining solution for 12 h and then washed and decolorized with 75% ethanol. To achieve better staining results in embryo and cotyledons, the seeds of transgenic plants were ruptured before incubation. After that, cryostat sectioning was performed with the help of Leica CM1950 Cryostat Microtome (Leica Biosystems, Wetzlar, Germany). The images of stained tissues/organs were taken by Olympus microscope (BX3-CBH) (Olympus, Tokyo, Japan). The quantitative expression of the GUS gene in different tissues of transgenic plants was checked by qRT-PCR. Transgenic plants were also treated with different phytohormones; abscisic acid (ABA, 10 µg/mL), Brassinolide (BR, 0.1 mg/L), ethephon (ETH, 1 mg/mL), paclobutrazol (PAC, 150 mg/L), salicylic acid (SA, 3 mmol/L), and low temperature (4 • C). The expression of the GUS gene in response to these hormones and low temperature was analyzed by qRT-PCR at different time points.

Statistical Data Analysis
The qRT-PCR was performed for three biological replicates, and data were normalized by the 2 −∆∆CT method. The expression levels were expressed as mean ± standard errors. Statistical significance of expression levels at different time points was assessed by analysis of variance (ANOVA) with significance level α = 0.005 followed by LSD test.

Conclusions
We investigated the Apyrase (APYs)/Nucleosied phosphatase (NTPs) family in cultivated peanut. Seventeen apyrase homologs were found in the genome of cultivated peanut, while eight homologs were also found in diploid peanut species (A. duranensis and A. ipaensis each). The genome-wide identification, phylogenetic analysis, structural and expression analysis, GO, and KEGG enrichment, and miRNAs prediction provided a theoretical base for future functional studies. This study highlighted some key genes that effectively respond to different stress agents. AhAPY1-1, AhAPY1-2, AhAPY6-1, and AhAPY6-2 showed upregulated expression against all hormone, water, and temperature treatments. These genes could be suitable candidates for drought and low-temperature resistance. AhAPY7-4 also showed increased expression against all treatments, while its expression was specifically high against drought treatment. This gene can also be a suitable candidate for drought resistance. Further, we functionally characterized a pericarp/podabundant expression promoter. The quantitative PCR analysis validated the transcriptome expression analysis. The quantitative and qualitative expression analysis of the GUS gene under AhAPY2-1P in transgenic Arabidopsis plants provided the practical significance of this promoter in deriving a gene in a pericarp-abundant manner

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The datasets presented in this study can be found in the online PGR database (http://peanutgr.fafu.edu.cn/; accessed on 2 August 2022). Moreover, the datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. However, most of the data is shown in the Supplementary files.