Some divalent metal ions, such as Zn²⁺, Fe²⁺, Mn²⁺, Cu²⁺, Co²⁺, and Ni²⁺, are essential elements in plants, playing crucial roles in numerous processes, including DNA replication, protein processing, photosynthesis, electron transport in the chloroplasts, and mitochondria. Although deficiency of these metals negatively affects plant growth and development, they can also result in toxicity at excessive levels (Bhardwaj et al., 2020). Correspondingly, plants have deployed a variety of adaptive mechanisms to precisely maintain metal homeostasis, including controlling the uptake, efflux, mobilization, translocation, trafficking, and storage (Liu et al., 2019). All these processes are mediated by numerous transporters belonging to different protein families including cation diffusion facilitators (CDFs).

The CDFs have been identified to be Me²⁺/H⁺(K⁺) antiporters that are involved in the transport of divalent cations such Fe²⁺, Zn²⁺, Cd²⁺, Mn²⁺, Ni²⁺, or Co²⁺ in both prokaryotes and eukaryotes (Singh et al., 2015). The CDF family contains three sub-families: (1) Zn-CDF, (2) Fe/Zn-CDFs, and (3) Mn-CDFs (Montanini et al., 2007). Most CDF proteins contain approximately six transmembrane domains (TMDs), a modified signature, and a C-terminal cation efflux domain (Montanini et al., 2007). The six TMDs are normally interconnected by extra-and intra-cellular loops. Among them, one cytosolic loop usually contains a histidine-rich domain, which is predicted to be potential metal binding domains (Montanini et al., 2007). Plant CDFs are usually named metal tolerance proteins (MTPs), while vertebrate CDFs are called solute carrier family 30 (SLC30) or zinc transporter (ZnT).

The MTP proteins in plants can be classified into seven phylogenetic groups: the groups 1 (MTP1–MTP4), 5 (MTP5), and 12 (MTP12) function as Zn-CDFs, the groups 6 (MTP6) and 7 (MTP7) as Fe/Zn-CDFs, and the groups 8 (MTP8) and 9 (MTP9–MTP11) as Mn-CDFs (Gustin et al., 2011). Since the first MTP protein was identified in Arabidopsis (ZAT, also named AtMTP1) (van der Zaal et al., 1999), there are large amount of MTP genes were identified in several plant species such as Arabidopsis (12 genes), rice (Oryza sativa; 10 genes), and wheat (Triticum aestivum; 20 genes) (Vatansever et al., 2017). However, most of them have not been functionally characterized in detail. In Arabidopsis, both AtMTP1 and AtMTP3 were confirmed to localize in the vacuole, conferring the Zn and/or Co tolerance by the vacuole sequestration of excess Zn²⁺ and/or Co²⁺ (Kobae et al., 2004; Arrivault et al., 2006). AtMTP12 interacts with AtMTP5 to form a functional complex, transporting Zn²⁺ from cytosol to the Golgi apparatus (Fujiwara et al., 2015). As Mn transporters, AtMTP8 and AtMTP11 protect plant cells from Mn toxicity by producing excessive endoplasmic vesicles that phagocytize and excrete Mn²⁺ (Delhaize et al., 2007; Peiter et al., 2007). Rice OsMTP1 localized in vacuole and is involved in the translocation and homeostasis of several divalent metals (Zn²⁺, Cd²⁺, Co²⁺, and Fe²⁺) (Yuan et al., 2012; Menguer et al., 2013). The five Mn-CDF members (OsMTP8.1, OsMTP8.2, OsMTP9, OsMTP11, and OsMTP11.1) were characterized to participate in the maintenance of Mn homeostasis in rice (Chen et al., 2013; Ueno et al., 2015; Takemoto et al., 2017; Ma et al., 2018; Tsunemitsu et al., 2018).

Peanut (Arachis hypogaea L., 2n = 4x = 40) is one of the major oilseed crops grown throughout the tropics and subtropics regions. It provides both edible oil and food protein for people all over the world and accounts for 30% of the total oilseed production in China. Like other crops, peanut often encounters various metal stresses during its life history, which not only limits yield, but also threatens human health due to the accumulation of toxic metals (Liu et al., 2017; Yu et al., 2019). We have demonstrated that peanuts show wide cultivar variation in Cd accumulation and tolerance (Su et al., 2013, 2014); however, the underlying mechanism is not fully understood. Recently, the whole-genome sequences of the cultivated peanut (cv. Tifrunner) as well as the two wild ancestral species, A. duranensis and A. ipaënsis, have been released (Bertioli et al., 2016, 2019). Based on these important resources, genome-wide analysis has been conducted on some gene families such as WRKY (Song et al., 2016), Mlo (Mildew Locus O; Traore et al., 2021), growth-regulating factors (GRFs) (Zhao et al., 2019), and monosaccharide transporter (MST) (Wan et al., 2020). However, little progress has been made in peanut MTP genes.

To fill the knowledge gap, we identified 24 AhMTPs from cultivated peanut and characterized the structure and evolutionary relationship of these genes. Furthermore, two peanut cultivars (Fenghua 1 and Silihong) differing in Cd and Fe-deficiency tolerance (Liu et al., 2017; Cao et al., 2019; Yu et al., 2019) were used for evaluating the expression of AhMTP genes in response to several divalent metals (Fe²⁺, Mn²⁺, Zn²⁺, and Cd²⁺). Our findings are expected to provide a perspective on the evolution of MTP genes in peanut and were helpful for further functional characterization of AhMTP genes, shedding some light on the molecular mechanisms of metal transport and homeostasis in peanut.

Genome-wide identification of MTP proteins has been extensively performed in diverse plant species, including Arabidopsis (Montanini et al., 2007), sweet orange (Citrus sinensis) (Fu et al., 2017), wheat (Vatansever et al., 2017), turnip (Brassica rapa var. rapa) (Li et al., 2018), tobacco (Nicotiana tabacum) (Liu et al., 2019), grape (Vitis vinifera) (Shirazi et al., 2019), and Populus trichocarpa (Gao et al., 2020). However, there is little information about MTP family in peanut that limits understanding the molecular mechanisms underlying the regulation of metal homeostasis. Herein, 24 putative AhMTP genes were identified in cultivated peanut, which were divided into seven groups (1, 5, 6, 7, 8, 9, and 12), belonging to three major substrate-specific groups (Zn-CDFs, Zn/Fe-CDFs, and Mn-CDFs) (Figure 1). Our findings concurred with the results obtained from Arabidopsis (Montanini et al., 2007), turnip (Li et al., 2018), tobacco (Liu et al., 2019), and P. trichocarpa (Gao et al., 2020), suggesting that AhMTPs may have similar functions to their homologs in these plant species.

In agreement with previous studies (Vatansever et al., 2017; Liu et al., 2019; Shirazi et al., 2019; Gao et al., 2020), most of AhMTP proteins were predicted to localize to vacuole membranes. All AhMTP proteins except AhMTP2.4 contained the typical domain of MTP, cation efflux domain. These results indicated that AhMTPs might be metal transporters playing key roles in the homeostasis or detoxification of divalent metals. Although AhMTP2.4 does not contain cation efflux domain, collinearity analysis revealed that it is a whole-genome duplicated gene of AhMTP2.3 (Figure 2B). Moreover, phylogenetic analysis indicated that AhMTP2.4 shows a close relationship with other members of group 6 (Figure 1). Therefore, it could be regarded as an AhMTP member.

A total of 10 AhMTPs were identified to be Mn-CDF members, which were further divided into two groups: group 8 and 9. These proteins share the following characteristics: (1) containing both the cation efflux domain and ZT-dimer; (2) shared a specific motif complex composed of motifs 1, 2, 4, 5, and 9, among them motifs 4 and 5 were annotated to be the TMD and CTD of cation efflux, respectively; (3) containing four to six TMDs; (4) best modeled with 3h90.1 (Structural basis for the autoregulation of the zinc transporter YiiP); and (5) localizing to vacuolar or plasma membranes. YiiP is a dimeric Zn²⁺/H⁺ antiporter from Escherichia coli that selectively binds Zn²⁺ or Cd²⁺ to the active site and transports the bound ions across the bacterial membrane using the proton motive force (Lu et al., 2009; Lopez-Redondo et al., 2018). The presence of ZT-dimer and specific motif complex in Mn-CDFs suggested that these proteins might serve as metal transporters by forming homodimers or heterodimers functional complexes (Liu et al., 2019). These structural features indicate that Mn-CDFs might be multiple metal transporter in peanut plants.

Some proteins of group 8 have been proven to be vacuolar-or Golgi-localized Mn transporters (Delhaize et al., 2003; Chen et al., 2013; Pedas et al., 2014). Meanwhile, several members of group 9, including AtMTP11 from Arabidopsis (Delhaize et al., 2007; Peiter et al., 2007), OsMTP11 from rice (Tsunemitsu et al., 2018), and BmMTP10 and BmMTP11 from Beta vulgaris spp. maritima (Erbasol et al., 2013), were also characterized as the Golgi- and/or pre-vacuolar-localized transporters that confer Mn tolerance via intra-cellular Mn compartmentalization.

A total of eight Zn-CDF members were identified in peanut, which were further divided into three groups: group 1 (AhMTP1.1/1.2/1.3 and AhMTPB1/B2), 5 (AhMTPC2.1/C2.2), and 12 (AhMTP12). Unlike the Mn-CDF subfamily, Zn-CDF members share characteristics as follows: (1) containing cation efflux domain but not ZT-dimer; (2) containing motifs 6 and 11, which were annotated as cation efflux TMD; (3) containing five to six TMDs except AhMTP1.3 and AhMTP12, which contained 2 and 16 TMDs respectively; (4) best modeled with 6xpd.1 (Cryo-EM structure of human ZnT8 double mutant—D110N and D224N, determined in outward-facing conformation); and (5) localizing to vacuolar membranes. ZnT8 is a Zn²⁺/H⁺ antiporter that plays essential roles in regulating Zn²⁺ accumulation in the insulin secretory granules of pancreatic β cells (Xue et al., 2020). The structural features suggest that Zn-CDF members might serve as Zn transporters in peanut plants. Two Zn-CDF members from Arabidopsis, AtMTP1 and AtMTP3, belonging to group 1, have been showed to be Zn transporters that are localized to vacuolar membrane and mediate the vacuole sequestration of Zn (Kobae et al., 2004; Arrivault et al., 2006).

Interestingly, AhMTP12 contained 16 TMDs, which is higher than the homologous gene from other plant species, including BrrMTP12 from turnip (Li et al., 2018), CitMTP12 (12 TMDs) from sweet orange (Fu et al., 2017), PtrMTP12 (12 TMDs) from P. trichocarpa (Gao et al., 2020), VvMT12 (12 TMDs) from grape (Shirazi et al., 2019), and NtMTP12.1 (10 TMDs) and NtMTP12.2 (eight TMDs) from tobacco (Liu et al., 2019). It was also observed that AhMTP12 had the largest protein size (867 amino acids) and MW (97.04 kDa). The results were in accordance with the characteristics of MTP12 in other plants (Li et al., 2018; Liu et al., 2019; Gao et al., 2020). Moreover, collinearity analysis showed that AhMTP12 is the only gene in the AhMTP family that has not experienced gene duplication events. The current results indicate that AhMTP12 might be distinctive both in the evolutionary process and physiological functions in peanut plants. In Arabidopsis, AtMTP12 has been shown to forms a functional complex with AtMTP5t1, one of the splicing variants of AtMTP5, transporting Zn into the Golgi (Fujiwara et al., 2015).

Another distinctive gene of Zn-CDFs is MTP1.3, which has the shortest sequence of the gene and protein. Only two TMDs and two motifs (6 and 11) were harbored in the MTP1.3 protein. Collinearity analysis revealed that AhMTP1.3 and AhMTP1.2 might be resulted from segmental duplication within the same subgenome.

The six Zn/Fe-CDF members were divided into two significantly different groups: Group 6 (AhMTP2.1/2.2/2.3/2.4) and 7 (AhMTPC4.1/C4.2). The two members of group 7 are WGD genes, which have the following common characteristics: (1) containing cation efflux domain but not ZT-dimer; (2) only containing two motifs (11 and 15), of which motif 11 were annotated as cation efflux TMD; and (3) localizing to vacuolar membranes. In contrast, the group 6 is composed of two pairs of WGD genes (AhMTP2.1/2.2 and AhMTP2.3/2.4). The pair of AhMTP2.1/2.2 is similar in all structural characteristics both at protein and gene levels. However, there are significant differences in the structure of the gene and protein between AhMTP2.3 and AhMTP2.4. For instances, while AhMTP2.3 contained six TMDs, AhMTP2.4 only contained three TMDs. Unlike AhMTP2.1/2.2 that contained both cation efflux domain and ZT-dimer, AhMTP2.3 contained cation efflux domain but not ZT-dimer, whereas AhMTP2.4 contained ZT-dimer but not cation efflux domain. A 3D-model prediction revealed that all Zn/Fe-CDFs were commendably modeled to 5vrf.1 except AhMTP2.4, which preferentially modeled with 3h90.1. The large divergence in the protein structure suggested that AhMTP2.3 and AhMTP2.4 might have distinctive physiological functions in peanut plants.

Gene duplication as a major source of novel genes contributes to the acquirement of novel functions (Panchy et al., 2016). Peanut is an allotetraploid species whose genome contains essentially complete sets of chromosomes (AABB) from two diploid ancestral species, A. duranensis (AA) and A. ipaensis (BB) (Bertioli et al., 2019). Expectedly, our results indicate that almost all AhMTP genes experienced gene duplication events except AhMTP12, resulting in 13 gene pairs (Figure 2B). Among them, 11 gene pairs were evolved from WGDs, and AhMTP1.3 and AhMTP1.2 might be segmentally duplicated genes. Gene pairs of duplication were consistent with the results obtained from phylogenetic analysis.

Duplicated genes, if they survive, would diverge in both the regulatory and coding regions during evolution, resulting in distinct functions (Xu et al., 2012). Thus, exon-intron structure can provide additional evidence to support phylogenetic analysis of gene families (Zhang et al., 2012). In the present study, we found that AhMTP genes within the same phylogenetic group show highly similar exon-intron structures, despite there are large variations among groups even in the same subfamily (Figure 2A). While most of the WGD gene pairs shared a similar exon-intron structure, divergences were also found within three gene pairs (AhMTPC4.1/C4.2, AhMTP2.3/2.4, and AhMTP9.1/9.2). Divergences of duplicate genes in exon-intron structure mainly resulted from three mechanisms: exon-intron gain/loss, exonization/pseudoexonization, and insertion/deletion (Xu et al., 2012). Although all the gene pairs experienced exon-intron gain/loss events, exonization/pseudoexonization also occurred in the evolutionary process of AhMTP2.3/2.4 and AhMTP9.1/9.2, which might contribute to functionally distinct paralogues.

It is noteworthy that six Zn-CDF genes belonging to group 1 (AhMTP1.1/1.2/1.3 and AhMTPB1/B2) and 12 (AhMTP12) have one exon without intron in CDS, which is called single exon genes (SEG) (Sakharkar et al., 2004). The presence of SEG in multi-cellular eukaryotic genomes intriguing since such kind of genes are archetypal of prokaryotes (Sakharkar et al., 2004). SEGs can be divided into two groups: (i) UTR intron-containing SEGs (uiSEGs) that have introns in their untranslated region (UTR) and (ii) intronless genes (IGs) lacking introns in the entire gene (Jorquera et al., 2018). Herein, AhMTP1.3 having an intron in the UTR belong to uiSEGs while AhMTP1.1/1.2, AhMTPB1/B2, and AhMTP12 are IGs. SEGs have been reported in MTP family from tobacco (Liu et al., 2019) and tomato (Solanum lycopersicum) (El-Sappah et al., 2021).

Gene expression was generally regulated at two different levels: transcriptional and post-transcriptional. CRE play essential roles in the transcriptional regulation of gene expression by interacting with specific transcription factors and RNA polymerase. In the current study, a large number of CAAT-box and TATA-box were detected in the promoters of AhMTP genes; this is expectedly because the two elements are involved in the regulation of expression frequency and initiation of transcription (Laloum et al., 2013). Besides, the wide distribution of light-responsive elements, phytohormonal responsive elements, and abiotic stress in the promoters indicate that expression of AhMTP genes could be regulated by many factors in multiple pathways. MYB-binding sites (MBS) were proposed to be involved in metal tolerance by driving MTP1 expression in Arabidopsis helleri (Fasani et al., 2017). The location of MBS in the promoters of AhMTP1.1/1.2, AhMTPC2.1, AhMTP12, and AhMTP4.1 suggests that these genes might be regulated at the MYB-binding sites.

MicroRNAs are generally believed to downregulate the expression of target genes by cleaving mRNA or inhibiting the translation of target genes (Bartel, 2009). Four pairs of AhMTP genes (AhMTP9.1/9.2, AhMTPC2.1/C2.2, AhMTP4.1/4.2, and AhMTP11.2/11.3) were predicted as targets of five miRNAs including ahy-miR167-5p, ahy-miR156a, ahy-miR156c, ahy-miR3511-3p, and ahy-miR3519. Although the function of miR3511-3p and miR3519 is unknown, the regulation of miR167 and miR156 on their targets has been extensively studied. The miR167 has been reported to target the mRNAs encoding the ARF6, ARF8, and IAR3, regulating auxin signaling and homeostasis in Arabidopsis (Wu et al., 2006; Kinoshita et al., 2012; Yao et al., 2019). Another study demonstrated that BnNRAMP1b is a target of miR167 in Brassica napus (Meng et al., 2017). MiR156a and miR156c play dominant roles in regulating abiotic stress resistance through a miR156-SPL regulatory pathway (Cui et al., 2014; Wang et al., 2021). The expression of miR167 and miR156 was downregulated by Ca deficiency in peanut embryos (Chen et al., 2019). Similar results were reported in the roots and shoots of high-Fe rice line under Fe deficiency (Agarwal et al., 2015). Together, it can be inferred that AhMTP9.1/9.2 and AhMTPC2.1/C2.2 might be post-transcriptionally repressed by ahy-miR167-5p and ahy-miR156(a/c), respectively, which deserves to be experimentally tested.

Tissue expression profiles indicate that half of AhMTP genes showed low expression in the 22 tissues, among them, six genes (AhMTP2.3/2.4, AhMTPB1/B2, AhMTP11.4, and AhMTP12) were not or rarely expressed in any tissues. The reduced gene expression of these genes might be beneficial for maintenance of their biological functions and avoiding gene loss during evolution processes (Qian et al., 2010; Liu et al., 2019). With respect for the remaining 18 AhMTP genes, most of them preferentially expressed in reproductive tissues such as stamens, peg tips, fruit, peg tip to fruit, pericarp, and seed. These genes might play roles in the development of pods or seeds by mediating the transport of divalent metals. High expression levels of AhMTP10.1/10.2 in leaves, perianth, and pistil indicate they might be involved in metal homeostasis in leaves and flower.

Due to most of AhMTP proteins were predicted to localize to vacuole membranes, we speculated that changing expressions of AhMTP genes in roots might alter the root-to-shoot translocation of divalent metals. To test this hypothesis, the expression of AhMTP genes in roots of two peanut cultivars in response to metal stresses and its relationship to metal translocation was investigated. AhMTP genes showed wide differences in response to various metal stresses depending on cultivars and metal exposure. Generally, 10 AhMTP genes tested in the current study were sensitive to excess metal exposure and most of them showed higher expressions under metal stress (particularly in Fe, Cd, and Zn treatments). In agreement with previous studies (Liu et al., 2017; Yu et al., 2019), we found that Silihong showed higher capacity for the translocation of Cd from roots to shoots than Fenghua 1. Cd translocation was also reported to be decreased by increasing Cd concentration in rhizosphere (Yu et al., 2019). Most of AhMTP genes were induced by Cd in a cultivar-dependent manner, and the increase of gene expression was more pronounced in Silihong than in Fenghua 1. These results indicate that AhMTP genes, particularly AhMTP1.1, AhMTPB1, AhMTPC2.1, and AhMTP10.1, might be responsible for the cultivar difference of Cd translocation in peanuts.

Stepwise linear regression analysis revealed Fe translocation from roots to shoots might be associate to AhMTP4.1, AhMTP9.1, AhMTPC4.1, AhMTPC2.1, and AhMTP12. The upregulation of AhMTPC2.1 and AhMTP12 might contribute to the reduction of Fe translocation via sequestration of Fe in vacuoles or other vesicles under excess metal exposure. More sensitive response of AhMTP4.1 and AhMTPC4.1 to metal exposure in the roots might be responsible for the higher translocation of Fe in plants of Fenghua 1.

The significant and negative correlation of the expression of AhMTP1.1 and the percentage of Mn in shoots indicates that AhMTP1.1 might be involved in Mn translocation. The downregulation of AhMTP1.1 in the root under Fe, Mn, and Zn stresses might contribute to increased Mn translocation in Silihong. Higher Mn translocation in Fenghua 1 under metal exposure might result from higher expression of AhMTP1.1 in the root. The translocation of Zn was not affected by Cd and Fe exposure, while excess Zn reduced its translocation in Silihong, and excess Mn largely decreased Zn translocation in Silihong but increased that in Fenghua 1. Stepwise linear regression analysis revealed AhMTP2.1 and AhMTPC2.1 might be associate with Zn translocation in peanut plants. Thus, the reverse change of percentages of Zn in shoots between Silihong and Fenghua 1 under Mn exposure might also be in part a consequence of the reverse response of AhMTP2.1 and AhMTPC2.1 to Mn exposure in the root of the two cultivars.

A total of 24 AhMTP genes were identified in peanut, which were divided into seven groups belonging to three substrate-specific clusters (Zn-CDFs, Zn/Fe-CDFs, and Mn-CDFs). All AhMTP genes underwent whole genome or segmental gene duplication events except AhMTP12. Most AhMTP members within the same subfamily or group generally have similar gene and protein structural characteristics. However, some genes, such as AhMTP1.3, AhMTP2.4, and AhMTP12, showed wide divergences. Most of AhMTP genes preferentially expressed in reproductive tissues, suggesting that these genes might play roles in metal transport during the pod and seed development stages. Excess metal exposure induced expressions for most of AhMTP genes in peanut roots depending on cultivars. By contrast, AhMTP genes in the root of Fenghua 1 were more sensitive to excess Fe, Cd, and Zn exposure than that of Silihong. The differential responses of AhMTP genes to metal exposure might be, at least partially, responsible for the different metal translocation from roots to shoots between Fenghua 1 and Silihong. The findings provide clues to further characterize the functions of AhMTP proteins in the uptake and translocation of metal ions in peanut plants, which is great of importance for screening or breeding cultivars for peanut safe production in heavy metal contaminated soil.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: NCBI’s GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE71357), accession number: GSE71357.

XW and CW carried out most of the experimental work with assistance from GS and ZZ. GS and ZZ were responsible for experimental design. GS and XW carried out data analyses and wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the Natural Science Foundation of Anhui Province (grant number 2108085MC83), the Natural Science Foundation for Colleges and Universities of Anhui Province (grant numbers KJ2020ZD83 and KJ2019A0587), and the Innovation Team of Scientific Research Platform of Anhui Province (grant number KJ2015TD001).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.791200/full#supplementary-material