Genome-Wide Analysis of PIN Genes in Cultivated Peanuts (Arachis hypogaea L.): Identification, Subcellular location, Evolution, and Expression patterns

doi:10.21203/rs.3.rs-3134347/v1

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Research Article

Genome-Wide Analysis of PIN Genes in Cultivated Peanuts (Arachis hypogaea L.): Identification, Subcellular location, Evolution, and Expression patterns

https://doi.org/10.21203/rs.3.rs-3134347/v1

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published 21 Oct, 2023

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Background

Auxin is an important hormone in plants and the PIN-FORMED (PIN) genes are essential to auxin distribution in growth and developmental processes of plants. Peanut is an influential cash crop, but research into PIN genes in peanuts remains limited.

Results

In this study, 27 PIN genes were identified in the genome of cultivated peanut, resolving into four subfamilies. Most of the PIN genes were predicted to be located in the plasma membrane and a subcellular location experiment confirmed the prediction. The gene structure, cis-elements in the promoter, and evolutionary relationships were elucidated, facilitating our understanding of peanut PINs and their evolution. In addition, the expression patterns of these PINs in various tissues were analyzed according to a previously published transcriptome dataset and qRT-PCR, which gave us a clear understanding of the temporal and spatial expression of PIN genes in different growth stages and different tissues. Expression during peanut pod development helps us to study geotropism of the peanut peg. Moreover, the gene network and gene ontology enrichment of these PINs were presented.

Conclusions

This study details a comprehensive analysis of PIN genes in cultivated peanuts and lays the foundation for subsequent studies of peanut gene function and phenotype.

Peanut

PINs

Genome-wide identification

Subcellular location

Expression pattern

Plant growth and development are regulated by a variety of plant hormones, among which auxin was the first discovered to participate throughout the plant life cycle, making it indispensable [1]. Auxin is involved in many biological processes, such as embryogenesis, cell division, root growth, phototropism, and geotropism [2]. Auxin is predominantly synthesized in the apical meristem and moves among cells through a combination of membrane diffusion and carrier-mediated transport [3]. Membrane diffusion depends on the concentration gradient of auxin and requires no energy consumption and has no fixed direction. Carrier-mediated transport is directional flow that is critical to plant morphogenesis and adaptation [4]. Studies about Arabidopsis show two types of auxin carriers: influx and efflux. The former includes AUX1 (AUXIN-RESISTANT 1) and the LAX (LIKE AUXIN) family and the latter includes the PIN (PIN-FORMED) and PGP/ABCB (P-glycoprotein/ ATP-binding cassette-B) families [5]. The polar location of PIN protein is closely related to the direction of auxin flow between cells, which is dynamic and strictly controlled on certain sides of specific cells, regulating important growth and developmental processes in higher plants [6].

Tropism is an adaptive response of plants to environmental stimuli, and polar auxin movement is the main cause of organ curvature due to cell growth differences in most tropism responses [7]. For example, the downward growth of plant roots caused by gravity is mainly due to the polar distribution of auxin [8]. In Arabidopsis, many studies support an essential role of PIN-mediated directed auxin flow in the root gravity response [9]. There are eight PIN members in Arabidopsis and PIN2, PIN3, and PIN7 are all involved in root gravitropism [10]. AtPIN2 plays a critical role in mediating auxin transport in root gravitropism, which transmits gravity signals perceived in the root cap to the root elongation zone [11]. The loss of function PIN2 mutant exhibits a gravity-insensitive root growth phenotype [12]. After gravity stimulation, the abundance of PIN2 decreases in the upper root epidermis, and increases in the lower root epidermis. The asymmetric abundance of PIN2 enhances the transport of polar auxin to the root extension zone, resulting in downward root growth [13]. Moreover, PIN3 and PIN7 also change their positions after gravity stimulation through endocytosis. By default, both PIN3 and PIN7 are located on the plasma membrane of columnar cells. After gravity stimulation, they rapidly move to the new lower sides of columella cells, driving auxin flow towards the lower side of the root tip [14].

Peanut is a geocarpic species that produces pods within the soil. Its special organ, the gynophore or peg, is an elongated pistil stalk with root-like gravitropism, pushing the ovary into the soil and forming the pod [15]. Roots of higher plants exhibit gravitropism, while shoots exhibit negative gravitropism [16]. Although the gynophore is attached to the aboveground part of the peanut plant, it is gravitropic like roots rather than negatively gravitropic like shoots [17]. Immunolocalization experiments showed that IAA (indoleacetic acid) was distributed in the intercalary meristem, epidermis, and cortex of the elongation region and the region near the seed of the vertically growing gynophores [18]. IAA redistributes to the upper side of the epidermis and cortical regions of peanut gynophores following horizontal reorientation, after which the growth rate of the upper side of the gynophores was faster than that of the lower side, promoting downward growth [18]. These results suggest that the asymmetric distribution of auxin is also the basis of the gravitropic response of peanut gynophores.

Interestingly, in both roots and stems, auxin responds to gravity by accumulating in the lower side of the tissue, whereas in gynophores, it accumulates in the upper side in response to gravity. In addition, although gynophores exhibit gravitropism like roots, their response to auxin is consistent with that of shoots (auxin promotes cell expansion) rather than roots (auxin inhibits cell expansion). This indicates that the gynophore is a special organ, with traits like both shoots and roots. Transcriptomics and proteomics have also been used to explore genetic and protein changes in peanut gynophores in response to gravity. Auxin related genes and proteins have been respectively identified as differentially expressed or abundant, such as auxin conjugate hydrolase, auxin efflux carrier, and ABC transporter [19–21]. However, further research on these genes and proteins is scarce.

In this study, a comprehensive analysis including identification, subcellular locations, gene structure, cis-elements, evolutionary relationships, transcriptional expression, and interaction networks of PINs in cultivated peanut was performed. The goal was to give researchers a basic understanding of these genes in peanut to facilitate subsequent research.

Identification of PIN genes in peanut

A total of 27 AhPIN genes were identified in the peanut genome, and were designated as AhPIN1 to AhPIN27 according to their chromosomal location (Table 1). As shown in Table 1, the length of AhPIN proteins ranged from 73 (AhPIN10) to 661 (AhPIN5) amino acids (aa) with an average of 377.52 aa, and corresponding MWs from 8.44 to 91.56 KDa. The pI values varied from 6.55 (AhPIN19) to 11.32 (AhPIN11). The GRAVY values of AhPIN proteins ranged from − 0.47 to 1.48, 23 of which were positive GRAVY values, indicating that these proteins shared the characteristic of hydrophobicity. The prediction results of AhPIN subcellar location showed that 81.48% of AhPINs were found in the plasma membrane.

Membrane localization of AhPINs

To detect the subcellular localization of PIN family genes in peanut, we constructed a PIN-GFP expression vector using 1300-GFP (Fig. 1a) as the fundamental skeleton. Through an Agrobacterium-mediated transient expression system, PIN-GFP was injected into tobacco leaves that were 2–3 weeks old. Compared with 1300-GFP, all PIN-GFPs were expressed in the plasma membrane (Fig. 1b), consistent with the results predicted by the software, indicating that most PIN family genes of peanut might have a regulatory role in membrane transport.

Sequence alignment, gene structure, and phylogenetic analysis of AhPINs

To further assess the evolutionary relationships of AhPIN genes, an unrooted phylogenetic tree was constructed based on an alignment of 26 AhPIN, eight AtPIN, and 12 OsPIN full-length protein sequences (Fig. 2, Supplemental Table 1). Given the criteria for wheat PIN classifications [22], the AhPIN members were categorized into four subfamilies: I, II, III, and IV. The sequence of AhPIN10 was too short to use in phylogenetic tree construction, so it was not classified into a subfamily. Subfamily I possessed 11 AhPINs and subfamily IV contained two AhPINs, which were the largest and the smallest group, respectively.

The exon-intron structure of subfamilies exhibited significant diversity (Fig. 3a and b). Exon numbers of these AhPINs ranged from 1 (AhPIN10) to 9 (AhPIN15) and the lengths of exons varied from 2 to 1,662 bp with an average of 281.47 bp. The length of introns varied from 47 to 30,482 bp with an average of 618.07 bp, displaying larger variation than exons. AhPINs clustered in the same group shared similar exon-intron structures, and exon numbers tended to be consistent within a group. Group III had complex gene structures, indicating that they may have versatile functions in growth and development. Domain analysis showed that all AhPINs had the membrane transport domain (PF03547) and three of them (AhPIN14, 23, and 24) had the PHD-zinc-finger like domain (Supplemental Table 2). These PINs belonged to group III and were in the same evolutionary branch, and presumably have similar functions (Fig. 3a).

MEME software was used to explore conserved motifs of AhPIN protein sequences, and ten conserved motifs were predicted and annotated (Fig. 3c and d). The most conserved motifs were 3 and 4, predicted in 21 AhPIN genes (Fig. 3c). Notably, some conserved motifs were identified in specific subgroups. For example, motif 7 was uniquely located in subfamily I and II, motif 1 was located in the N-terminal region, and motif 2 was exclusively distributed in the C-terminal region. AhPIN27, 13, and 5 contain all ten motifs, and AhPIN10 contains only motif 9. Overall, conserved motif arrangements and gene structure differed greatly in subfamilies I and III, and were relatively conserved in subfamilies II and IV.

Cis-regulatory elements analysis

As the region of the transcription factor binding site that initiates transcription, the promoter is essential in controlling the tissue-specific or stress responses of gene expression. To further infer the regulatory mechanisms and potential functions of AhPINs, the upstream 1.5 kb promoter region sequences from the initiation codon were extracted to explore cis-regulatory elements (Fig. 4 and Supplemental Table 3).

Four types of cis-regulatory elements (development/tissue specificity, light responsiveness, stress, and hormone response) were detected (Fig. 4a). The majority of AhPINs possessed hormone- or stress-response related cis-elements, except for AhPIN26. In total, 20 gibberellin (GARE-motif, P-box, TATC-box), 52 MeJA (CGTCA-motif, TGACG-motif), 29 ABA (ABRE), 12 auxin (TGA-element, AuxRR-core), 61 ethylene (ERE), and 15 salicylic acid (TCA-element) cis-elements were detected in all AhPINs (Fig. 4b and Supplemental Fig. 1). There were four abiotic stress cis-elements detected, including Wun-motif (wound-responsive element, 15 genes), ARE (cis-acting regulatory element essential for the anaerobic induction, 31 genes), and LTR (low-temperature responsiveness, ten genes).

Other cis-elements were also found, including 28 development/tissue specificity-related elements and 92 light-responsive elements (Supplemental Table 3). In total, 55.56% of the AhPINs contained development/tissue specificity-related elements, including AC-II, CCGTCC-box, dOCT, GCN4_motif, circadian, and O2-site. There were 14 types of light-responsive elements observed in the promoter regions of AhPIN family genes, with the largest being I-box, having 13 in total (Fig. 4 and Supplemental Table 3).

Chromosomal distribution and gene duplications

The chromosome location information of 27 AhPIN genes in peanut was obtained from PeanutBase (https://www.peanutbase.org/home). AhPIN genes were distributed on all chromosomes except 7, 8, 17, and 19 (Supplemental Fig. 2). Chromosomes 4, 11, 14, and 20 contain three genes, while chromosomes 3, 5, and 10 contain two genes and the others contain only one gene.

In plant genome evolution, gene duplications (tandem and segmental duplication) lead to gene family expansion. All the identified AhPIN gene members were used to ascertain gene duplication in the peanut genome. A total of 17 gene pairs consisting of 24 genes were identified as duplicated, which were associated with segmental duplications (Fig. 5a). Replication events mainly occurred on chromosomes except chromosomes 6, 7, 8, 17, and 19. As no tandem duplication was detected, segmental duplication events played a predominant role in the expansion of the PIN gene family. The Ka/Ks ratios for 17 AhPIN paralogous gene pairs (Supplemental Table 4) varied from 0.0046 to 1.2188 with an average of 0.2088, suggesting that the PIN gene family has mainly undergone strong purifying selection pressure during the gene evolution process with limited functional divergence occurring after segmental duplication. The divergence time of paralogous genes ranged from 7.42 to 4,821.29 million years ago (Mya) with an average of 1,515.12 Mya.

Synteny analysis for PINs in peanut and its relatives

To further examine the evolutionary relationships of AhPIN genes, synteny analysis was performed between peanut and other species, namely A. ipaensis, A. duranensis, and A. thaliana. We found 13, 11, and five orthologous pairs between AhPINs and other PIN genes in A. ipaensis, A. duranensis, and A. thaliana (Fig. 5b, c and d). Moreover, 20 AhPINs were mapped to A. ipaensis and A. duranensis chromosomes with one on Adchr1, three on Adchr3, three on Adchr4, one on Adchr5, one on Adchr9, two on Adchr10, one on Aichr1, one on Aichr2, one on Aichr3, four on Aichr4, one on Aichr5, one on Aichr6, one on Aichr8, and three on Aichr10.

To provide a basic framework of orthologous relationships of PIN genes among peanut and its relatives, the Ka/Ks was also calculated (Supplemental Table 5). A. ipaensis and A. duranensis, the progenitors of cultivated peanuts, had 21 of 24 orthologous pairs with Ka/Ks values less than 1, suggesting that these genes have experienced intense purifying selection during evolution. Interestingly, the Ka/Ks values of A. thaliana and peanut homologous genes were both greater than 1.

Expression profiling of peanut PIN genes in various tissues

Based on the peanut RNA-seq data from publicly available databases, the expression profiles of AhPINs in 22 tissues were investigated (accession numbers and sample information of RNA-seq data are listed in Supplemental Fig. 3). Supplemental Table 6 shows the FPKM values of all expressed AhPIN genes.

The results revealed that most of the 27 AhPINs genes had distinct tissue-specific expression patterns based on the log-2-transformed (FPKM + 1) (Fig. 6a); AhPIN9, AhPIN22, and AhPIN24 were expressed in all 22 tissues. AhPIN5 and AhPIN19 had extremely high levels in leaf, especially AhPIN5, whose FPKM value in the three leaf tissues (seedling leaf ten days post-emergence, main stem leaf, and lateral stem leaf) were all greater than 18. AhPIN5 and AhPIN19 also exhibited significantly high expression levels in flower and pistils. Six AhPIN genes (AhPIN5, 9, 13, 19, 22, and 27) were expressed at a high level with FPKM > 5 in shoot, and AhPIN22 had the highest expression in reproductive shoot. AhPIN5, 9, 13, 19, 22, and 27 showed the highest expression in stamen. AhPIN5, 9, 19, and 22 showed the highest expression in tip.

Furthermore, we used qRT-PCR to detect the expression levels of nine AhPIN genes in roots, hypocotyls, stems, leaves, and pegs at different periods (Fig. 6b and c). Eight AhPIN genes were highly expressed in stems and leaves, except AhPIN22 (Ah72M57I.1), which was only highly expressed in stems. On the second day after penetration into the soil, AhPIN6 (AhK2GLG.1) and AhPIN22 (Ah72M57I.1) were highly expressed, while AhPIN24 (AhI2RKDG.1) and AhPIN13 (Ah66N4V9.1) were highly expressed on the sixth day after penetration. There was no significant difference in the expression of other AhPIN genes. The results indicated that AhPINs may function in the growth of peanut seedlings and the development of peanut gynophores.

Protein interaction analysis of AhPIN genes

To investigate protein-protein interactions between AhPINs and AtPINs, the STRING database was used to construct a protein interaction network (Fig. 7a, Supplemental Table 7). The network was formed by 29 nodes and 129 protein interactions, including nine AhPINs and 20 other genes. Among them, eight genes (AhPIN1, 6, 9, 10, 11, 12, 15, and 27) can interact with ABCB19 and AUX1. AhPIN24 exhibited an interactive relationship with HTA8 (Probable histone H2A variant 2), HTA9 (Probable histone H2A variant 3), PIE1 (SNF2 domain-containing protein/helicase domain-containing protein), TRO (Set1/Ash2 histone methyltransferase complex subunit ASH2), and AT3G21060 (Transducin/WD40 repeat-like superfamily protein).

Gene ontology enrichment

GO enrichment analyses was performed to understand the function of AhPIN genes using AgriGO (Fig. 7b, Supplemental Table 8). The results showed eight GO terms, divided into biological process and cellular component. AhPIN genes were enriched in transmembrane transport (GO:0055085), transport (GO:0006810), localization (GO:0051179), and establishment of localization (GO:0051234). The cellular component category showed similar results with subcellular localization signifying that AhPINs were enriched in the membrane (GO:0005575).

Genetic evolution of PIN genes in cultivated peanut

PINs are plant-specific transmembrane proteins found only in the genomes of land plants [23]. PIN genes in higher plants have relatively high sequence similarity with each other, so they are thought to have evolved from a single ancestor gene. The homolog PIN gene can be found in Streptophyte algae, the ancestors of land plants [24]. Because of the endoplasmic reticulum (ER) location in Arabidopsis of AtPIN5, AtPIN8, and PIN proteins from the moss Physcomitrella patens [25–26], ER-located PIN proteins were hypothesized to be ancestral PINs of land plants, while PM (plasma membrane)-located PINs are thought to have been acquired later in evolutionary time [25].

In our previous prediction and experiment on subcellular localization, PIN genes in cultivated peanuts were mostly localized in the PM (Table 1 and Fig. 1b), so, by assumption, the PINs in cultivated peanuts should have been acquired during evolution. Moreover, Ka/Ks values of PIN genes between A. thaliana and A. hypogaea were greater than 1. That is, the substitution times of non-synonymous substitution sites are greater than the substitution times of synonymous substitution sites, which means that selection has changed the protein. Therefore, we speculate that PIN proteins evolved in peanuts may have acquired new functions by modifying certain protein structures compared to model plants.

PIN genes have different roles in plants

Auxin is a hormone that regulates nearly all growth and development processes in plants. It is usually synthesized in juvenile tissues such as the tips of roots and stems, and transported to target tissues via vascular bundle tissue or polar transport [27]. A variety of auxin transporters are involved in auxin polar transport, such as AUXIN1/LIKE-AUX1 (AUX/LAX), PIN, ATP-binding cassette (ABC) transporter, nitric acid transporter 1.1 (NRT1.1), PIN-like transporter (PIL), and WALLS ARE THIN 1 (WAT1) [28]. In Arabidopsis, the function of the PIN gene has been well studied and eight PIN genes have different functions during auxin signaling mediated development and growth. PIN1 is involved in vascular development [29]; PIN2 is mainly expressed in the elongation region of root tips and is involved in root geotropism [12]; PIN3 and PIN4 have similar functions and act in phototaxis and root development [30–32]; PIN5 mediates subcellular compartmentalization of auxin localized in the ER [25]; PIN6 has dual localization in the PM and ER to regulate auxin transport in the PM and intracellular auxin homeostasis [33]; PIN7 negatively controls radial root growth [34]; and PIN8 is an ER-localized protein, which is mainly involved in reproductive development [26, 35].

In addition to Arabidopsis, some progress has been made in determining the role of PIN in other plants. In maize, ZmPIN1 protein is required for embryogenesis [36]. In cotton, PIN protein functions in regulating fiber growth [37], and in rice, OsPIN2 is expressed in root epidermis and cortex cells to regulate geotropic response and root structure [38]. In soybean, GmPIN is involved in nodule development [39]. In peanut, some PIN genes associated with peanut peg geotropism and pod development were also identified from our unpublished studies. Compared to Arabidopsis, cultivated peanuts have more than three times the number of PINs (Table 1), and their function is most likely enriched by evolution. Also, peanut has the special feature of underground pods. Therefore, additional studies of PIN function in peanuts will likely provide new insights to reveal more fascinating and novel functions of PINs.

PIN genes involved in nitrogen fixation by nodules

Legumes can interact with rhizobia to form nodules. Using nodules, legumes can convert nitrogen from the air into ammonium, which can be taken up by plants. This mode of nitrogen uptake is known as biological nitrogen fixation [40]. On an average, 50–60% of soybean N demand is met by biological N2 fixation [41]. In peanut, root nodules are the greatest N source, accounting for about 50.0%, and the N supply proportion from root nodule, soil, and N fertilizer was 5:3:2 [42]. The formation and development of root nodules involves complex regulatory mechanisms in which auxin, as well as PINs, are suspected to have an essential role [43–44]. In soybeans, GmPIN1 is specifically expressed in the root nodule primordium region, and root nodule primordium development is inhibited in GmPIN1 overexpressed plants. GmPIN9d protein accumulates in the vascular bundle between roots and nodules, and synergizes with GmPIN1 to regulate auxin polar transport to promote the growth of nodules [39]. In Medicago truncatula, the number of root nodules in MtPIN2, MtPIN3, and MtPIN4-silenced plants (via RNA interference) decreased significantly [45]. In our study, there were ten AhPIN genes expressed in 25-day nodules (Fig. 6 and Supplemental Table 6). Their expression levels were not high, possibly because the root nodules had formed and thus auxin transport activity was slowed. It is possible that the detection of newly developed nodules will result in higher expression. However, the relationship between PIN genes and nodule formation is unclear, and there are numerous gaps to be filled. In the last few years, gene editing technology has profoundly changed the course of breeding, and perhaps in the near future, we may be able to improve the nitrogen fixing ability of legumes by altering, for example, the expression of PIN genes.

In this study, 27 PIN genes in cultivated peanuts were identified and their subcellular locations, gene structure, cis-elements, evolutionary relationships, transcriptional expression, and interaction networks were analyzed to give a comprehensive understanding of PINs in peanuts. This work lays a foundation for investigating auxin transport and PIN related gene function in peanuts.

Identification and prediction of AhPINs genes in Arachis hypogaea

Protein sequences of A. hypogaea cv. Tifrunner were acquired from PeanutBase (https://www.peanutbase.org/home). Protein sequences of Arabidopsis PIN protein sequences downloaded from the Arabidopsis information resource (TAIR, http://www.arabidopsis.org) were used as query sequences for BLASTP searches against A. hypogaea proteins to obtain the AhPIN genes. The reliability of these potential proteins was confirmed by CDD (Conserved Domain Database: http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) analysis. The isoelectric point (pI), molecular weight (MW), subcellular location, and GRAVY (grand average of hydropathy) of AhPIN proteins were predicted by the ProtParam online tool (https://web.expasy.org/protparam/).

Phylogenetic, protein structure, and promoter analysis of the AhPINs

Protein sequences of AtPIN and OsPIN were downloaded from TAIR and https://www.ricedata.cn/gene/. A phylogenetic tree was constructed using the maximum-likelihood (ML) method in MEGA V6.0. The gene structure of AhPINs was revealed by comparing coding domain sequences (CDS) and DNA sequences through the GSDS online tool (http://gsds.cbi.pku.edu.cn). Domain organization of AhPIN genes was predicted using HMMER (http://www.hmmer.org) with default parameters. Conserved motifs were determined using MEME (http://meme.nbcr.net/meme/intro.html). PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to identify putative cis- regulatory elements.

Chromosomal mapping and gene duplication analysis

Mapchart (https://www.wur.nl/en/show/Mapchart.htm) was used to map the genes to chromosomes. Genomic data of Arachis duranensis and Arachis ipaensis was downloaded from PeanutBase. Interspecific and intraspecific syntenic analyses were performed using Multiple Collinearity Scan toolkit (MCScanX) with default parameters. The nonsynonymous substitution rate (Ka) to synonymous substitution rate (Ks) ratios was calculated by Kakas Calculator 2.0 [46]. The divergence times in millions of years ago (Mya) were estimated by the formula: T = Ks/ (2 × 6.1 × 10 − 9) ×10 − 6 [47].

Plant material and gene expression analysis

Peanut pegs were collected at different developmental stages: 0 d (aerial peg), 2 d, 4 d, and 6 d before and after penetration into soil. Roots, hypocotyls, stems, and leaves were taken from 2-week-old peanut seedlings. Total RNA was isolated using the RNAprep Pure (TIANGEN, DP441). EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, AE311-02) were used for cDNA synthesis. For qRT-PCR, SuperReal Premix PLUS-SYBR GREEN (TIANGEN, FP205) was used to perform the reaction on an Applied Biosystems QuantStudio 5, with the following conditions: initial denaturation at 95℃ for 30 s, 40 cycles of 95℃ for 10 s, 56℃ for 20 s, and 72℃ for 20 s, then a melting curve cycle from 65–95℃. AhACTIN11 (arahy.79FMKA.1) was used as an internal control gene and qRT-PCR was conducted with three biological replicates for each sample. All primers are presented in the Supplemental Table 9. The RNA-seq data of 22 tissues from a previous study [48] were used to investigate the expression profiles of AhPIN genes.

AhPIN gene cloning and subcellular localization

We selected five AhPIN genes from PeanutBase and cloned the coding regions. The AhPINs were inserted into an overexpression vector (pCAMBIA1300-GFP). The fusion vector (pCAMBIA1300-AhPINS-GFP) and the negative control (pCAMBIA1300-GFP) were transformed into Agrobacterium tumefaciens strain GV3101 and then injected into tobacco leaves via an Agrobacterium-mediated transformation system [49]. The GFP fluorescence signal was acquired using a Nikon ECLIPSE Ti2 confocal microscope. We used NIS-Elements Viewer 5.21 to process the images. Primer sequences of the 1300-GFP are shown in Supplemental Table 10.

Protein interaction prediction and gene ontology enrichment analysis

The STRING database (https://cn.string-db.org) was used to construct the protein interaction network and the GO enrichment analyses was performed in AgriGO [50].

Ethical approval and consent to participate: All methods were in accordance with relevant institutional, national, and international guidelines.

Consent to publication: Not applicable.

Data availability statement: The RNA-Seq data used in this study derived from NCBI (http://www.ncbi.nlm.nih.gov/) under BioProject PRJNA291488. Accession number: SRR2135539-SRR2135548, SRR2135552, SRR2135554-SRR2135557, SRR2135560, SRR2135586, SRR2135589, SRR2135593, SRR2135597, SRR2135601, SRR2140727-SRR2140728, SRR2140738-SRR2140745, SRR2140748-SRR2140751, SRR2140804-SRR2140805, SRR2141559, SRR2141618-SRR2141620, SRR2141679, SRR2141695-SRR2141705).

Conflict of interest: There is no conflict of interest.

Funding: This study was supported by the grants from Taishan Scholar Foundation of Shandong Province (tsqn202103161) and the Natural Science Foundation of Shandong Province (ZR202103010405) for XL.

Acknowledgment: Not applicable.

Author contributions: XL, JB, and YC conceived this study. XL supervised the research. JB, YC, JL, YG and ST performed the experiments. XL, JB, YC, JL, YG, and ST analyzed the data and wrote the manuscript.

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Table 1. Characteristic features of PIN gene family in Peanut.
No.	PIN names	Peanut gene ID	Start	End	Chromosome	Amino acid length	pI	Mw(kDa)	Subcellular location	GRAVY
1	AhPIN1	Arahy.Y9MC4Z.1	95315993	95318730	1	549	9.44	60.4903	PM	0.108
2	AhPIN2	Arahy.L8N3TB.1	71463740	71465378	2	124	7.95	12.9188	PM	1.372
3	AhPIN3	Arahy.KHTN7F.1	39010286	39014376	3	524	9.22	57.8506	PM	0.237
4	AhPIN4	Arahy.F7NT5K.1	70192219	70193755	3	124	7.95	13.009	PM	1.48
5	AhPIN5	Arahy.FCRD5F.1	52897656	52901853	4	661	6.93	71.5555	PM	0.125
6	AhPIN6	Arahy.K2GLG1.1	1.19E+08	1.19E+08	4	373	8.44	40.8305	PM	0.692
7	AhPIN7	Arahy.13W9YS.1	1.22E+08	1.22E+08	4	370	9.3	40.6013	PM	0.504
8	AhPIN8	Arahy.B3BVVR.1	99872642	99874280	5	124	7.93	12.8998	PM	1.377
9	AhPIN9	Arahy.L76SLB.1	1.02E+08	1.02E+08	5	616	8.78	67.3518	PM	0.095
10	AhPIN10	Arahy.B6RTIS.1	1.07E+08	1.07E+08	6	73	7.98	8.44304	Extracellular	0.856
11	AhPIN11	Arahy.YV5TH4.1	1.08E+08	1.08E+08	9	170	11.3	18.6888	PM	0.72
12	AhPIN12	Arahy.V3Y7Y0.1	1.08E+08	1.08E+08	10	368	8.63	40.2731	PM	0.561
13	AhPIN13	Arahy.66N4V9.1	1.12E+08	1.12E+08	10	593	8.98	63.7347	PM	0.267
14	AhPIN14	Arahy.DEWH52.1	39216071	39218110	11	221	8.75	24.7467	Nuclear	-0.163
15	AhPIN15	Arahy.J0955H.1	81897023	81904963	11	417	9.01	46.4634	Mitochondrial	-0.222
16	AhPIN16	Arahy.B9QDDU.1	1.49E+08	1.49E+08	11	496	8.93	54.7493	PM	0.016
17	AhPIN17	Arahy.HJG3BW.1	60240847	60242316	12	179	9.94	19.0661	PM	0.93
18	AhPIN18	Arahy.HCQJ24.1	41011682	41015263	13	524	9.22	57.9156	PM	0.233
19	AhPIN19	Arahy.2QV4RS.1	40338987	40343406	14	647	6.55	70.0827	PM	0.104
20	AhPIN20	Arahy.953E9B.1	1.33E+08	1.33E+08	14	381	7.56	41.5382	PM	0.691
21	AhPIN21	Arahy.LAPE95.1	1.36E+08	1.36E+08	14	328	9.7	36.0163	PM	0.636
22	AhPIN22	Arahy.72M57I.1	1.42E+08	1.42E+08	15	621	8.89	67.6211	PM	0.091
23	AhPIN23	Arahy.FM31KL.1	41812464	41846154	16	213	9.59	23.9715	Mitochondrial	-0.468
24	AhPIN24	Arahy.I2RKDG.1	51883385	51886580	18	279	8.65	31.2511	Extracellular	-0.251
25	AhPIN25	Arahy.2QW795.1	54532245	54535674	20	257	9.24	29.2531	PM	0.06
26	AhPIN26	Arahy.FS1I4N.1	1.33E+08	1.33E+08	20	368	8.31	40.3452	PM	0.553
27	AhPIN27	Arahy.E6QCJN.1	1.39E+08	1.39E+08	20	593	8.98	63.7617	PM	0.268

PM: plasma membrane

No competing interests reported.

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Genome-Wide Analysis of PIN Genes in Cultivated Peanuts (Arachis hypogaea L.): Identification, Subcellular location, Evolution, and Expression patterns

Status:

Journal Publication

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Abstract

Background

Results

Conclusions

Figures

Background

Results

Identification of PIN genes in peanut

Membrane localization of AhPINs

Sequence alignment, gene structure, and phylogenetic analysis of AhPINs

Cis-regulatory elements analysis

Chromosomal distribution and gene duplications

Synteny analysis for PINs in peanut and its relatives

Expression profiling of peanut PIN genes in various tissues

Protein interaction analysis of AhPIN genes

Gene ontology enrichment

Discussion

Genetic evolution of PIN genes in cultivated peanut

PIN genes have different roles in plants

PIN genes involved in nitrogen fixation by nodules

Conclusion

Methods

Phylogenetic, protein structure, and promoter analysis of the AhPINs

Chromosomal mapping and gene duplication analysis

Plant material and gene expression analysis

AhPIN gene cloning and subcellular localization

Protein interaction prediction and gene ontology enrichment analysis

Declarations

References

Table 1

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1