Genome-Wide Identification of the Ferric Chelate Reductase (FRO) Gene Family in Peanut and Its Diploid Progenitors: Structure, Evolution, and Expression Profiles

The ferric chelate reductase (FRO) family plays a vital role in metal ion homeostasis in a variety of locations in the plants. However, little is known about this family in peanut (Arachis hypogaea). This study aimed to identify FRO genes from the genomes of peanut and the two diploid progenitors (A. duranensis and A. ipaensis) and to analyze their gene/protein structures and evolution. In addition, transcriptional responses of AhFRO genes to Fe deficiency and/or Cu exposure were investigated in two peanut cultivars with different Fe deficiency tolerance (Silihong and Fenghua 1). A total of nine, four, and three FRO genes were identified in peanut, A. duranensis, and A. ipaensis, respectively, which were divided into three groups. Most AhFRO genes underwent WGD/segmental duplication, leading to the expansion of the AhFRO gene family. In general, clustered members share similar gene/protein structures. However, significant divergences occurred in AhFRO2 genes. Three out of five AhFRO2 genes were lowly expressed in all tissues under normal conditions, which may be beneficial for avoiding gene loss. Transcription analysis revealed that AhFRO2 and AhFRO7 genes might be involved in the reduction of Fe/Cu in plasma membranes and plastids, respectively. AhFRO8 genes appear to confer Fe reduction in the mitochondria. Moreover, Fe deficiency induced an increase of Cu accumulation in peanut plants in which AhFRO2.2/2.4/2.5 and FRO7.1/7.2 might be involved. Our findings provided new clues for further understanding the roles of AhFRO genes in the Fe/Cu interaction in peanut.


Introduction
Iron (Fe) is a microelement that is essential for plant growth and development.In plants, Fe functions as a constituent of many important molecules such as Fe-sulfur (Fe-S) and heme Fe proteins, which are involved in fundamentally biological processes, including respiration, photosynthesis, chlorophyll biosynthesis, sulfur assimilation, and nitrogen fixation [1].The function of Fe is mainly based on the reversible redox reaction of ferrous (Fe 2+ ) and ferric (Fe 3+ ) ions and its ability to form octahedral complexes with various ligands.Fe deficiency not only inhibits chlorophyll synthesis and reduces photosynthetic efficiency [2] but also interrupts the respiratory electron transport and tricarboxylic acid cycle [3].Meanwhile, Fe in excess can be toxic because free Fe ions induce the formation of reactive oxygen species via the Fenton reaction [4,5].Therefore, cellular Fe levels must be strictly controlled in plants.
Although Fe is the fourth most abundant element in the earth's crust, it is not easily taken up by plants due to the predominance of insoluble ferric oxides in soils, particularly in calcareous soils that account for approximately 30% of the world's arable soils [6].Consequently, crops grown in calcareous soils often suffer from iron deficiency, limiting yield and quality.Moreover, Fe deficiency in plants also poses serious health problems because plant foods are the main source of dietary Fe for humans.It is estimated that 30%-50% of anemia in children and other groups is caused by iron deficiency [7].Thus, understanding plant iron homeostasis is essential for improving crop yield and human iron nutrition.
Plants have evolved complex mechanisms to sense and respond to iron fluctuations in the rhizosphere and prevent iron deficiency or toxicity by maintaining Fe homeostasis [4,5,8].Non-gramineous plants use the reduction strategy (strategy I) for Fe acquisition, while gramineous plants adopt the chelation strategy (strategy II).The reduction strategy includes three processes: (i) releasing protons from root cells to the rhizosphere via H + -ATPase for reducing soil pH value; (ii) reducing Fe 3+ to Fe 2+ by ferric chelate reductases (FRO) in the acidifying rhizosphere; and (iii) taking up Fe 2+ into root cells through iron-regulated transporter 1 (IRT1) in the plasma membrane.Gramineous plants can secrete the mugeneic acids (MAs) like phytosiderophores from their roots to the soil to dissolve Fe 3+ in the rhizosphere to form a Fe 3+ -MA complex and then absorb the Fe 3+ -MA complex into root cells through yellow stripe like protein (YSL) in the plasma membrane.
The FRO family plays a vital role in metal ion homeostasis in a variety of locations in plants [9].It belongs to the flavocytochrome superfamily that transfers electrons across membranes [10].FRO proteins contain eight transmembrane helices and share three typical domains, a heme-containing ferric reductase domain (PF01794) in the transmembrane region, and the FAD-binding (PF08022) and NAD-binding (PF08030) domains inside the membrane of the C-terminal region [11,12].In Arabidopsis, AtFRO2 is responsible for the reduction of solubilized Fe 3+ to Fe 2+ at the root surface, where Fe 2+ is then transported into the cytoplasm via AtIRT1 in the root plasma membrane [10,13].AtFRO6 mediates the reduction of Fe 3+ to Fe 2+ at the plasma membrane of leaf cells [9,14].AtFRO7 plays a role in chloroplast iron acquisition by reducing Fe 3+ to Fe 2+ [15,16].AtFRO3 and AtFRO8 have been predicted to localize to mitochondrial membranes and might serve an analogous function in the mitochondrial iron homeostasis [16,17].While several FRO genes were functionally characterized in Arabidopsis, little is known about the roles of this family in other plant species including peanut (Arachis hypogaea L.), a major oil-seed crop mainly grown in temperate and tropical regions of the world.
In this study, based on the whole-genome sequences [18,19], FRO family genes of the peanut (cv.Tifrunner) and the two wild ancestral species (A.duranensis and A. ipaënsis) were identified, and the structures, functions and evolutionary relationships were characterized.Moreover, the expression of AhFRO genes in response to Fe deficiency and/or Cu exposure was investigated.Our data would provide a basis for further functional characterization of AhFROs and shed new light on the possible roles of the AhFRO family in the uptake and translocation of Fe and Cu in plants.

Conserved Motifs, Domain Architectures, and Exon-intron Organization
A total of ten conserved motifs (1-10) were identified in FRO proteins, wit varying from 21 to 50 (Figure 2A and Table S1).The majority of FRO protein the ten conserved motifs.However, AhFRO2.4,AdFRO2.2, and AdFRO2.3onl four, three, and two motifs, respectively.The composition of conserved motifs within phylogenetic groups.All FRO proteins contained the typical domains duct, FAD_binding_8, and NAD_binding_6) except AhFRO2.4,AdFRO2.2, and in which only the NAD_binding_6 domain was detected (Figure 2B).
Multiple sequence alignment indicated that all AhFROs have conserved as C(L/M)AxL, YHxWLG, and HG in the Ferric_reduct domain, LQWH(P FAD_binding domain, and GGxG(I/L)(T/S)PF in the NAD_binding domain (Fi addition, some conserved motifs including LxxGL, FExFxYxHxLY, LRxx EGPY(G/E), and GV(L/F)(V/A)(C/S)GP were also detected in other regions.
To gain insight into the evolution of the FRO family in peanut, exon-int zations were examined (Figure 2C).FRO genes typically contained eight or n which were separated by seven or eight exons, while only two exons were AhFRO2.4,AdFRO2.2, and AdFRO2.3.The exon-intron organization varied am ent phylogenetic groups; however, FRO genes belonging to the same group ge similar structures.

Conserved Motifs, Domain Architectures, and Exon-Intron Organization
A total of ten conserved motifs (1-10) were identified in FRO proteins, with the length varying from 21 to 50 (Figure 2A and Table S1).The majority of FRO proteins contained the ten conserved motifs.However, AhFRO2.4,AdFRO2.2, and AdFRO2.3only contained four, three, and two motifs, respectively.The composition of conserved motifs was similar within phylogenetic groups.All FRO proteins contained the typical domains (Ferric_reduct, FAD_binding_8, and NAD_binding_6) except AhFRO2.4,AdFRO2.2, and AdFRO2.3 in which only the NAD_binding_6 domain was detected (Figure 2B).
To gain insight into the evolution of the FRO family in peanut, exon-intron organizations were examined (Figure 2C).FRO genes typically contained eight or nine introns, which were separated by seven or eight exons, while only two exons were detected in AhFRO2.4,AdFRO2.2, and AdFRO2.3.The exon-intron organization varied among different phylogenetic groups; however, FRO genes belonging to the same group generally had similar structures.
The Ka/Ks ratios of all gene duplication pairs were greatly lower than one (Table 2), indicating that AhFRO genes evolved under purifying selection [20].The divergence time of the four whole genome duplicated gene pairs ranged from 1.21 Mya to 2.38 Mya, which was considerably less than that of the two segmental duplicated gene pairs (43.14 and 44.70 Mya, respectively) (Table 2).

3D Model Predictions and Multiple Sequence Alignment
To obtain a reasonable theoretical structure of FROs, 3D model predictions were performed using the Swiss-Model server (Figure 4 and Table S2).Most of the FRO2 proteins in peanut and the progenitors were well modeled with the homologous template, 7d3f.1, which is a cryo-EM structure of human DUOX1-DUOXA1 in a high-calcium state (Figure 4 and Table S2).All FRO7 were well modeled with 6wxr.1, the cryoEM structure of mouse DUOX1-DUOXA1 complex in the absence of NADPH, while FRO8 was well modeled with 8gz3.1, the structure of human phagocyte NADPH oxidase in the resting state.Apart from three short sequence proteins (AhFRO2.4,AdFRO2.2, and AdFRO2.3),all FRO proteins from peanut and the progenitors share more than 20% sequence identity with their homologous templates, and the GMQE values ranged from 0.29 to 0.43 (Table S2), suggesting a high reliability of 3D model predictions.

The cis-Regulatory Elements (CREs) of AhFRO Genes in Peanut
A total of 1040 CREs were identified in the promoter region of AhFRO genes, and most of them are associated with gene transcription, light response, phytohormone response, and abiotic stress (Table 3).The main light-responsive CREs are TCT-motif, Box 4, ATCT-motif, GT1-motif, G-box, and AT1-motif.The main phytohormone-responsive CREs included ABRE, P-box, and TCA-element.The abiotic stress-responsive CREs are TC-rich repeats, ARE, LTR, and MBS.The promoter of all AhFRO genes contained CAATbox, TATA-box, and TCT-motif; however, the distribution of remaining CREs widely varied among AhFRO genes.AhFRO2.3contained the most light-responsive CREs, while Ah-FRO8.1 had the most phytohormone-responsive elements.AhFRO2.2 and AhFRO2.5 have the fewest types of CREs (Table 3).Light responsiveness

The Cis-Regulatory Elements (CREs) of AhFRO Genes in Peanut
A total of 1040 CREs were identified in the promoter region of AhFRO genes, and most of them are associated with gene transcription, light response, phytohormone response, and abiotic stress (Table 3).The main light-responsive CREs are TCT-motif, Box 4, ATCT-motif, GT1-motif, G-box, and AT1-motif.The main phytohormone-responsive CREs included ABRE, P-box, and TCA-element.The abiotic stress-responsive CREs are TC-rich repeats, ARE, LTR, and MBS.The promoter of all AhFRO genes contained CAAT-box, TATA-box, and TCT-motif; however, the distribution of remaining CREs widely varied among AhFRO genes.AhFRO2.3contained the most light-responsive CREs, while AhFRO8.1 had the most phytohormoneresponsive elements.AhFRO2.2 and AhFRO2.5 have the fewest types of CREs (Table 3).

Tissue-Specific Expression of AhFRO Genes in Peanut
To gain an insight into tissue-specific expression, RNA-seq data of the nine AhFROs were used for studying their expression patterns in different tissues and developmental stages (Table S3).As presented in Figure 5, nine AhFRO genes were divided into three clusters.Cluster I included AhFRO7.1 and AhFRO7.2, which show high expression and are mainly transcribed in leaves and pistils.Cluster II is composed of four genes with an intermediate level of expression that is preferentially expressed in the developing seeds, roots, vegetative shoot tip, and mainstem leaves.Cluster III consists of AhFRO2.1,AhFRO2.2, and AhFRO2.4,which show low expression and were predominantly expressed in roots.

Tissue-Specific Expression of AhFRO Genes in Peanut
To gain an insight into tissue-specific expression, RNA-seq data of the nine AhFR were used for studying their expression patterns in different tissues and developmen stages (Table S3).As presented in Figure 5, nine AhFRO genes were divided into three cl ters.Cluster I included AhFRO7.1 and AhFRO7.2, which show high expression and mainly transcribed in leaves and pistils.Cluster II is composed of four genes with an in mediate level of expression that is preferentially expressed in the developing seeds, ro vegetative shoot tip, and mainstem leaves.Cluster III consists of AhFRO2.

Transcriptional Responses of AhFROs to Fe-deficiency and Cu Exposure
To elucidate the transcriptional response of AhFROs to Fe deficiency and/or Cu posure, two contrasting peanut cultivars, Fenghua 1 (Fe deficiency sensitive cultivar) a Silihong (Fe deficiency tolerant cultivar), were used for qRT-PCR analysis.As presen in Figure 6, Cu exposure repressed the expression of AhFRO7.1/7.2 in the root for b cultivars, while AhFRO2 genes were not affected.Fe deficiency induced the expression

Transcriptional Responses of AhFROs to Fe-Deficiency and Cu Exposure
To elucidate the transcriptional response of AhFROs to Fe deficiency and/or Cu exposure, two contrasting peanut cultivars, Fenghua 1 (Fe deficiency sensitive cultivar) and Silihong (Fe deficiency tolerant cultivar), were used for qRT-PCR analysis.As presented in Figure 6, Cu exposure repressed the expression of AhFRO7.1/7.2 in the root for both cultivars, while AhFRO2 genes were not affected.Fe deficiency induced the expression of AhFRO2.1/2.2/2.3/2.5 but reduced the expression of AhFRO7.1 in the root for both cultivars.The remaining AhFRO genes responded Fe deficiency in a cultivar-specific manner.Cu exposure with Fe deficiency increased the expressions of AhFRO2.1/2.2/2.4/2.5 but repressed the expression of AhFRO7.1/7.2 in the root for both cultivars, while the expression of AhFRO8.1/8.2 was unaffected (Figure 6).
AhFRO2.1/2.2/2.3/2.5 but reduced the expression of AhFRO7.1 in the root for both cu vars.The remaining AhFRO genes responded Fe deficiency in a cultivar-specific mann Cu exposure with Fe deficiency increased the expressions of AhFRO2.1/2.2/2.4/2.5 but pressed the expression of AhFRO7.1/7.2 in the root for both cultivars, while the express of AhFRO8.1/8.2 was unaffected (Figure 6).As for the gene expression in leaves, AhFRO2.2,AhFRO7.1/7.2, and AhFRO8.1 w repressed by Cu exposure for both cultivars (Figure 7).Fe deficiency induced the exp sion of AhFRO2.1/2.2/2.4/2.5 but reduced the expression of AhFRO8.1/8.2 in the leaves both cultivars.Cu exposure with Fe deficiency up-regulated the expressions of FRO2.2/2.4/2.5 in the leaves for both cultivars (Figure 7).

The Accumulation and Translocation of Fe and Cu in the Two Peanut Cultivars
The two peanut cultivars differed from each other in Fe accumulation, which significantly influenced by Fe deficiency and Cu exposure as well as their interactions

The Accumulation and Translocation of Fe and Cu in the Two Peanut Cultivars
The two peanut cultivars differed from each other in Fe accumulation, which was significantly influenced by Fe deficiency and Cu exposure as well as their interactions (Table 4).Under normal conditions (control), Fenghua 1 showed higher Fe concentrations in roots and shoots, and higher total amounts of Fe in plants than Silihong (Table 4).Fe deficiency significantly reduced Fe uptake and accumulation in the peanut plant depending on cultivar and Cu exposure.Cu exposure significantly increased root Fe concentrations in Fe-sufficient peanut plants, resulting in an increase in total amounts of Fe in plants and a reduction of root-to-shoot Fe translocation (Table 4).The two peanut cultivars are similar in Cu accumulation and translocation (Table 4).Cu concentrations in roots and shoots and total amounts of Cu in plants were significantly enhanced by Fe deficiency and Cu exposure, while the percentage of Cu in shoots was reduced (Table 4).There are significant Cu × Fe interactions on Cu accumulation and translocation in the two peanut cultivars (Table 4).

Relationships between AhFRO Genes and Metal Accumulation in Peanut
Pearson's correlation analysis was performed to determine relationships between AhFRO genes and the accumulation and translocation of Fe and Cu.As shown in Table 5, the expression of all AhFRO2 genes was negatively correlated with Fe concentrations in roots (p < 0.05) and shoots (p < 0.01) as well as the total Fe in plants (p < 0.05).In contrast, AhFRO7.1 and AhFRO8.2 were observed to positively correlate with Fe accumulation (p < 0.05).Cu accumulation in peanut plants was positively correlated with the expression of AhFRO2.2/2.4/2.5 (p < 0.01) but negatively correlated with the expression of AhFRO7.1/7.2 (p < 0.05).The percentage of Cu in shoots was negatively related to the expression of FRO2.2/2.5 (p < 0.01) but positively correlated with the expression of AhFRO7.1/7.2 (p < 0.01).No significant correlation was found between AhFRO genes and Fe translocation (Table 5).

Discussion
FRO members have been demonstrated to play crucial roles in the homeostasis of Fe and Cu [9].However, there has been little work on genome-wide identification of the FRO family in plants.In this study, we identified nine, four, and three FRO genes in peanut, A. duranensis, and A. ipaensis, respectively (Table 1).The number of AhFRO genes in peanut is higher than that in most reported plant species [9,12].The same phenomenon has been reported in other gene families of peanut [22][23][24][25].Peanut, as an allotetraploid species derived from the hybridization of diploid ancestral species, A. duranensis (AA) and A. ipaensis (BB) [19], has experienced at least three rounds of WGD events [26].Our results indicated that eight out of nine AhFRO genes have experienced WGD events.Moreover, two paralogous gene pairs (AhFRO2.1/2.2 and AhFRO2.3/2.4) were found to be segmental duplications.Expectedly, the divergence time indicates that segmental duplication events (43.14-44.70Mya) of AhFRO genes occurred dramatically earlier than WGD (1.21-2.38Mya) (Table 2).It is likely that WGD/segmental duplication contributes to the expansion of the AhFRO gene family in peanut.
Gene duplication is a major source of novel genes that contribute to the acquirement of novel functions [27].However, it results in functional redundancy [28] and, consequently, most duplicated genes quickly pseudogenize and get lost [29].In the current study, we found that the number of FRO genes differed between the two sub-genomes of peanut and between A. duranensis and A. ipaensis, which suggests an asymmetrical evolution in the family.Synteny analysis revealed that the orthologs of AhFRO2.5 and AhFRO2.3 have been lost in the genome of A. ipaensis after allopolyploidization (Figure 3).Likewise, an ortholog of AdFRO2.3 has been lost in the sub-genome A of peanut.These results, which are in agreement with our previous study [25], confirmed that gene loss is easier in A. ipaensis than A. duranensis.The number of AhFRO genes in peanut is greater than the sum of the two ancestors, suggesting that heteropolyploid is more capable of avoiding gene loss than diploid.
Another approach for avoiding gene loss of duplicated genes is the reduction of their expression compared to the ancestral gene [28].In the present study, three AhFRO2 genes showed low expression levels in all tissues of peanut (cv.Tifrunner) under normal conditions (Figure 5).The results concurred with previous studies [25,28], suggesting that the reduction of gene expression might be beneficial for the maintenance of multiple duplicated genes and avoidance of functional redundancy.
Surviving duplicated genes would be subject to purifying selection, which could lead to divergence in both the coding and regulatory regions [30].At the coding regions, AhFRO2.4 from peanut and AdFRO2.2 and AdFRO2.3 from A. duranensis only have two exons, while the remaining FRO2 genes contained eight exons.Gene/protein structures indicate that these genes appear to derive from continuous gene shortening during evolution, which may cause neofunctionalization or pseudogenization.The inducible gene expression by Fe-deficiency confirms that AhFRO2.4still has a function in the Fe-deficient response of peanut roots and leaves.
At the regulatory regions, CREs play essential roles in regulating gene expression through interacting with transcription factors and RNA polymerase [22].Our results show that, although all duplicated genes of FRO7, FRO8, and some of FRO2 (i.e., AhFRO2.1/2.3)share a similar exon-intron organization, none of them have similar CREs.The promoter of AhFRO7.1 specifically contains TCCC-motif, LTR, and GCN4_motif, while that of AhFRO7.2 specifically contains TCA-element, MBS, and TC-rich repeats.Similarly, the promoter of AhFRO8.1 specifically contains AT1-motif, chs-CMA1a, chs-CMA2a, and GARE-motif, while that of AhFRO8.2 specifically contains MRE and GCN4_motif.The differential CREs in promoters imply a divergence of transcriptional regulation between the duplicated genes.
Apart from the three short sequence proteins (AhFRO2.4,AdFRO2.2, and AdFRO2.3),all FROs contained the typical domains: Ferric_reduct, FAD_binding_8, and NAD_binding_6 (Figure 2B).Ferri_reduct domain is a ferric reductase-like transmembrane component that can transfer electrons from extracellular ferric ions to generate the reduced form of ferrous ions for transporting across the plasma membrane by specific iron transporters [12,31].NADand FAD-binding domains participate in membrane electron transfer from intracellular NADPH and FAD to extracellular oxygen for superoxide production [11].Consistent with gene structures, AhFRO2.4,AdFRO2.2, and AdFRO2.3only contain the NAD_binding_6 domain, indicating a distinct physiological function from other homologous proteins.
AhFRO proteins were well modeled with three kinds of 3D model templates such as 6wxr.1,8gz3.1, and 7d3f.1 (Table S2).The best template of FRO2 for a 3D model is 7d3f.1, a cryo-EM structure of human DUOX1-DUOXA1 in a high-calcium state [32].The best template of FRO7 is 6wxr.1, a cryo-EM structure of mouse DUOX1-DUOXA1 complex in the absence of NADPH [33].DUOX1 is an NADPH oxidase family member that catalyzes the production of hydrogen peroxide by transferring electrons from intracellular NADPH to extracellular oxygen [32,33].FRO8 is well modeled with 8gz3.1, the structure of human phagocyte NADPH oxidase in the resting state [34].Phagocyte NADPH oxidase membranebound redox enzymes transfer electrons from intracellular NADPH to extracellular oxygen for producing superoxide anions [34].Structural analysis indicates that AhFROs have redox activity and might reduce metal ions in different pathways.
The phylogenetic tree revealed that FRO members are grouped into three groups (I, II, and III), which is consistent with previous results [9,12].Group I is composed of five paralogs of AhFRO2 (AhFRO2.1-2.5), which exhibited considerable differences in the sequence and gene/protein structure.AhFRO2.4 is a short sequence gene encoding 233 aa, with two TMDs, while other members contained ten TMDs.AhFRO2 is closely related to AtFRO1-3 from Arabidopsis.AtFRO2 is responsible for the reduction of solubilized Fe 3+ to Fe 2+ at the root surface in Arabidopsis, where Fe 2+ is then transported into the cytoplasm via IRT1 in the root plasma membrane [10,13].AtFRO3 localizes to mitochondrial membranes and might serve an analogous function in the mitochondrial iron homeostasis [16,17].In this study, AhFRO2 proteins were predicted to be localized in plasma membranes, and most of AhFRO2 genes were predominantly expressed in roots.Moreover, the expression of AhFRO2 genes was strongly induced by Fe deficiency in both the roots and leaves of peanut seedlings.Similar results have been extensively reported in AtFRO2 and AtFRO3 of Arabidopsis [35,36].The expression of AhFRO2 genes in roots was significantly correlated with Fe concentrations in roots and shoots as well as the total Fe in plants, suggesting that AhFRO2 genes might be involved in the reduction of Fe in peanut roots.
Group II contained two paralogs of AhFRO7 (AhFRO7.1/7.2), which resulted from WGD events.The two paralogs are very similar in their sequence, physicochemical properties, and gene/protein structure, suggesting the same role in peanut.Phylogenetic analysis indicates that AhFRO7 is closely clustered with AtFRO6/7 from Arabidopsis and OsFRO1 from rice.AtFRO6 has been proven to mediate the reduction of Fe 3+ to Fe 2+ at the plasma membrane of leaf cells [9,14], while AtFRO7 plays a role in chloroplast iron acquisition by reducing Fe 3+ to Fe 2+ [15,16].In the current study, AhFRO7 proteins were predicted to be localized in chloroplast, which is consistent with AtFRO7 in Arabidopsis [15,16].Concurrent with Mukherjee et al. [9], who found that AtFRO6 and AtFRO7 show high expression in all the green parts of Arabidopsis plants, RNA-seq data showed that AhFRO7.1/7.2 are highly expressed in leaves and pistils.The findings indicate a possible role for AhFRO7.1/7.2 in regulating chloroplast iron acquisition.Additionally, it is thought that Fe is mainly stored in plastids of plant cells as ferritin [9].Thus, the repression of AhFRO7.1/7.2 expression in the roots under Fe deficiency might contribute to Fe translocation to leaves by reducing Fe storage in the plastids of root cells.This is illustrated by the positive correlation between the expression of AhFRO7.1/7.2 and shoot Fe concentration.In contrast to roots, the expression of AhFRO7.1/7.2 was induced or unaffected in the leaves.This could maintain or improve Fe reduction ability for importing into chloroplasts in leaves.
Group III included two paralogs of AhFRO8 derived from WGD, which share the same physicochemical properties and gene/protein structure.Phylogenetic analysis indicates that AhFRO8 is closely clustered with AtFRO8 from Arabidopsis.Similar to AtFRO8 [16,17], AhFRO8.1/8.2 were predicted to localize to mitochondrial membranes.Unlike AtFRO8 which is highly expressed in Arabidopsis shoots [9], our results show that AhFRO8.1/8.2 are primarily expressed in seeds and roots (AhFRO8.1) of peanut.Previous studies showed that AtFRO8 is not regulated by Fe availability [9].However, our results show that Fe deficiency reduces the expression of AhFRO8.1/8.2 in the roots of Silihong and in the leaves of both cultivars.In addition, the expression of AhFRO8.1/8.2 in roots was observed to be positively correlated with shoot Fe concentrations.Although the functions of FRO8 are yet uncharacterized even in Arabidopsis, our data implies AhFRO8.1/8.2 might be involved in mitochondrial iron homeostasis.The reduction of AhFRO8.1/8.2 under Fe deficiency could reduce Fe storage in the mitochondria, leading to more Fe allocation to chloroplasts.
FRO genes are also assumed to be involved in copper reduction [9,10].Arabidopsis AtFRO2 has been shown to take a role in the reduction of Cu 2+ to Cu + at the root surface [10].Although AhFRO2 genes are not regulated by Cu in peanut roots, down-regulation of AhFRO2.2/2.4 was observed in the leaves under Cu exposure.Moreover, the expression of AhFRO2.2/2.4/2.5 positively correlated with Cu concentrations in roots and shoots as well as total Cu in plants, indicating a possible role in Cu reduction at the plasma membrane for the uptake of Cu into cells.In addition, we found that excess Cu considerably represses the expression of AhFRO7.1/7.2 in the roots and leaves for both cultivars.The expression of AhFRO7.1/7.2 in the roots negatively correlated with Cu concentrations in roots and shoots but positively correlated with root-to-shoot Cu translocation in peanut plants.These data suggest that AhFRO7.1/7.2 might be involved in Cu homeostasis in peanut plants.
Interestingly, Cu and Fe could interact with each other in their accumulation and translocation in the two peanut cultivars (Table 4).Consistent with previous studies [37], we found that Fe deficiency significantly enhanced Cu concentrations in roots and shoots, and total amounts of Cu in plants, but reduced the percentage of Cu in shoots.As Fe deficiency can induce the expression of AhFRO2.2/2.4/2.5 in roots, which positively correlated with Cu concentrations in roots and shoots as well as total Cu in plants, we assumed that AhFRO2.2/2.4/2.5 might be responsible for higher Cu accumulation in Fe-deficient peanut plants.Similarly, the reduction of AhFRO7.1/7.2 expression under Fe deficiency appears to decrease Cu storage in plastids of root cells and, consequently, contribute to a higher capability of Cu translocation from roots to shoots.Although Cu exposure significantly increased root Fe concentrations in Fe-sufficient peanut plants, none of the AhFRO genes could well explain the phenomenon.
As for the two cultivars, Silihong (Fe-deficiency tolerant cultivar) showed higher expressions of AhFRO2.1/2.3 than Fenghua 1 (Fe-deficiency sensitive cultivar) under Fedeficiency.Higher expressions of AhFRO2.1/2.3 indicate a higher capacity for the reduction of Fe 3+ to Fe 2+ or Cu 2+ to Cu + .This is in accordance with the higher concentrations of Fe and Cu in the root of Silihong under Fe deficiency compared with Fenghua 1.It is likely that higher expressions of AhFRO2.1/2.3 contribute to Fe-deficiency tolerance in Silihong.

Identification of FRO Proteins in the Three Arachis Species
Protein sequences of Arabidopsis AtFROs (AtFRO1-8) were retrieved from a phytozome database (https://phytozome-next.jgi.doe.gov/,accessed on 2 May 2022).Using these sequences as queries, BLASTp was carried out against protein databases of A. hypogaea cv.Tifrunner, A. duranensis, and A. ipaënsis, which was retrieved from NCBI (https:// github.com/ncbi,accessed on 10 May 2022).Non-redundant putative candidates were examined for the presence of typical conserved domains of FROs, Ferric_reduct (PF01794), FAD_binding_8 (PF08022), and NAD_binding_6 (PF08030), using the hmmscan tool (https: //www.ebi.ac.uk/Tools/hmmer/search/hmmscan, accessed on 12 June 2023).Sequences containing conserved domains were used for the ClustalW alignment and phylogenetic analysis using the MEGA-X program (v. 10.2.6) together with the eight AtFROs.The phylogenetic trees were built using the neighbor-joining (NJ) method based on the Poisson model with 1000 bootstrap replicates.The proteins clustered with AtFROs were assigned as putative FRO proteins.

Tissue-specific Expression Profiles of AhFRO Genes in Peanut
RNA-seq data of 22 different tissues in peanut (cv.Tifrunner) were obtained from PeanutBase (https://www.peanutbase.org/,accessed on 15 June 2022) [21].After being transformed from read counts, TPMs (Transcripts Per Kilobase of exon model per Million mapped reads) were used as lg(TPM + 1) for constructing a heatmap diagram by Origin 2021 (v 9.8.0.200,OriginLab Corp., Northampton, MA, USA).4.5.Plant Growth, Treatment, Metal Determination, and RT-qPCR Analysis Two contrasting peanut cultivars, Fenghua 1 (Fe deficiency sensitive cultivar) and Silihong (Fe deficiency tolerant cultivar), were used for hydroponic experiments [37].After the surface was sterilized with 5% sodium hypochlorite solution, seeds were rinsed in deionized water for 24 h at room temperature and then sown in sand for germination.Three-day-old seedlings with uniform sizes were transplanted into polyethylene pots for hydroponic culture.The culture conditions and nutrient solutions were followed as described previously by Lu et al. [45].Ten-day-old seedlings were exposed to 0 or 10 µM CuSO 4 under Fe-sufficient (+Fe, 50 µM Fe-EDTA) or Fe-deficient (−Fe, 0 µM Fe-EDTA) conditions, respectively.Each treatment per cultivar was repeated three times (biological replicates) with three plants per replication.Nutrient solutions were renewed twice a week during the growing period.After 14 days of treatment, plants were harvested for metal determination and RT-qPCR analysis.
The harvested roots were rinsed with 20 mM Na 2 EDTA for 15 min to remove the surface-bound metal ions and then oven-dried together with shoots.After being weighed and ground, tissue powders were digested with HNO 3 -HClO 4 (3:1, v/v).Cu and Fe concentrations in the samples were determined by flame atomic absorbance spectrometry (WFX-110, Beijing Rayleigh Analytical Instrument Company, Beijing, China).The total Fe/Cu in plants and the percentage of Fe/Cu in shoots were calculated using the equations reported by Liu et al. [46].
Frozen tissues were used for total RNA extraction, cDNA strand synthesis, and RT-qPCR analysis, which were strictly followed according to the methods described by Tan et al. [25].The relative mRNA abundance was normalized using the endogenous reference gene (60S, NCBI Entrez gene ID:112697914).The primers of AhFROs and 60S are listed in Table S4.The relative gene expression was calculated with three biological replicates using the 2 −∆∆CT method [47].Each biological replication was technically replicated three times.

Statistical Analysis
1 and Duncan's Multiple Range Test (p < 0.05) was used for detecting differences among group means.Pearson's correlation analysis was used to determine the relationship between gene expression and Fe/Cu accumulation.All statistical analyses were conducted using IBM SPSS Statistics v.22 (IBM, New York, NY, USA).

Conclusions
A total of nine, four, and three FRO genes were identified in peanut, A. duranensis, and A. ipaensis, respectively, which were divided into three groups (I to III).Most of the AhFRO genes underwent WGD/segmental duplication, leading to the expansion of the AhFRO gene family.Clustered members generally share similar gene/protein structures.However, structural or CRE divergences and reduced expression existed in AhFRO genes, which may be beneficial for the maintenance of duplicate genes.AhFRO2 and AhFRO7 genes might be involved in the reduction of Fe/Cu in plasma membranes and chloroplast (or plastids in root cells), while AhFRO8 genes appear to confer Fe reduction in the mitochondria.Fe deficiency-induced Cu accumulation in both cultivars, which might be associated with AhFRO2.2/2.4/2.5 and FRO7.1/7.2.Our findings provide a basis for further functional characterization of AhFRO genes and shed new light on the possible roles of the AhFRO family in the Fe/Cu interaction in plants.

Plants 2024 , 18 Figure 2 .
Figure 2. Conserved motifs (A) and domains (B) in FRO proteins and exon-intron organization of FRO genes (C) from the three Arachis species.UTR and CDS represent untranslated regions and coding sequences, respectively.

Figure 2 .
Figure 2. Conserved motifs (A) and domains (B) in FRO proteins and exon-intron organization of FRO genes (C) from the three Arachis species.UTR and CDS represent untranslated regions and coding sequences, respectively.

Figure 2 .
Figure 2. Conserved motifs (A) and domains (B) in FRO proteins and exon-intron organization of FRO genes (C) from the three Arachis species.UTR and CDS represent untranslated regions and coding sequences, respectively.

Figure 3 .
Figure 3. Synteny relationship of FRO gene pairs in the three Arachis species.(A) Synteny relationship of AhFRO gene pairs in A. hypogaea.(B) Synteny relationship of FRO gene pairs between A. duranensis and A. ipaensis.(C) Synteny relationship of FRO gene pairs among the three Arachis

Figure 3 .
Figure 3. Synteny relationship of FRO gene pairs in the three Arachis species.(A) Synteny relationship of AhFRO gene pairs in A. hypogaea.(B) Synteny relationship of FRO gene pairs between A. duranensis and A. ipaensis.(C) Synteny relationship of FRO gene pairs among the three Arachis species.The red and blue lines represent segmental duplicated genes and synteny genes, respectively.The gray lines show the collinear blocks of the plant genomes.

Figure 4 .
Figure 4. Predicted 3D structure of peanut AhFRO proteins by Swiss-Model.Models were visualized with rainbow colors from N to C terminus.

Figure 4 .
Figure 4. Predicted 3D structure of peanut AhFRO proteins by Swiss-Model.Models were visualized with rainbow colors from N to C terminus.

Figure 6 .
Figure 6.Expression levels of AhFRO genes in the root of two peanut cultivars in response to deficiency and/or Cu exposure.Data (means ± SE, n = 3) sharing the same letter(s) above the e bars are not significantly different at the 0.05 level according to the Duncan multiple range test.

Figure 6 .Figure 7 .
Figure 6.Expression levels of AhFRO genes in the root of two peanut cultivars in response to Fe deficiency and/or Cu exposure.Data (means ± SE, n = 3) sharing the same letter(s) above the error bars are not significantly different at the 0.05 level according to the Duncan multiple range test.Plants 2024, 13, x FOR PEER REVIEW 10

Figure 7 .
Figure 7. Expression levels of AhFRO genes in the leaves of two peanut cultivars in response to Fe deficiency and/or Cu exposure.Data (means ± SE, n = 3) sharing the same letter(s) above the error bars are not significantly different at the 0.05 level based on the Duncan multiple range test.

Table 1 .
Molecular characterization of FRO genes and corresponding proteins identified in A. hypogaea, A. duranensis, and A. ipaënsis.
a Molecular weight, b amino acid number, c grand average of hydropathicity, d isoelectric points, e transmembrane domain, f plasma membrane, g chloroplast, h mitochondria.

Table 2 .
Ka/Ks analysis of all gene duplication pairs for AhFRO genes.
a The number of nonsynonymous substitutions per nonsynonymous site, b the number of synonymous substitutions per synonymous site, c Ka/Ks ratios.

Table 3 .
The cis-regulatory elements in the promoter regions of AhFRO genes in peanut.

Table 3 .
The cis-regulatory elements in the promoter regions of AhFRO genes in peanut.

Table 4 .
The accumulation and translocation of Fe and Cu in two peanut cultivars exposed to Fe-deficiency and/or Cu for 14 days.
a Fe concentration in roots, b Fe concentration in shoots, c Cu concentration in roots, d Cu concentration in shoots.Data (means ± SE, n = 3) sharing the same letter(s) in the same column are not significantly different at the 0.05 level based on the Duncan multiple range test.* p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Table 5 .
Pearson's correlation analysis (r value, n = 24) of metal accumulation and the expression of AhFRO genes in the roots and leaves of Fenghua 1 and Silihong.
a Fe concentration in roots, b Fe concentration in shoots, c Cu concentration in roots, d Cu concentration in shoots, * p < 0.05, ** p < 0.01.