Genome-Wide Identification, Characterization and Expression Profiling of Potato (Solanum tuberosum) Frataxin (FH) Gene

Frataxin (FH) plays a crucial role in the biogenesis of mitochondria and the regulation of iron in the cells of various organisms. However, there has been very little research on FH in plants. In this study, the potato FH gene (StFH) was identified and characterized using a genome-wide approach, and its sequence was compared to those of FH genes from Arabidopsis, rice, and maize. The FH genes were found to have a lineage-specific distribution and were more conserved in monocots than in dicots. While multiple copies of FH genes have been reported in some species, including plants, only one isoform of FH was found in potato. The expression of StFH in leaves and roots was analyzed under two different abiotic stress conditions, and the results showed that StFH was upregulated more in leaves and that its expression levels increased with the severity of the stress. This is the first study to examine the expression of an FH gene under abiotic stress conditions.


Introduction
Iron represents a vital nutrient for all living organisms. In fact, it functions as a cofactor in several critical cellular processes, including oxygen transport, photosynthesis, respiration, DNA replication, DNA repair, and amino acid synthesis [1]. Iron deprivation has been shown to dramatically impair these pathways, whereas iron overload has been found to result in oxidative stress as well as irreversible cell and tissue damage [1][2][3]. Therefore, iron homeostasis is essential for ensuring both optimum development and a healthy lifespan.
Frataxin (FH) is a ubiquitous mitochondrial protein known to prevent the oxidative stress that results from excess iron accumulation and iron detoxification [4][5][6]. It is very highly conserved in both prokaryotes and eukaryotes [7,8]. Moreover, FH plays an important role in the oversight of Fe storage, Fe-S cluster assembly, heme metabolism, respiration, and reactive oxygen species (ROS) regulation. FH deficiency can result in several metabolic problems, sensitivity to oxidative stress, and a decreased lifespan [9,10]. A recent study revealed that FH also serves to prevent biotic stresses in plants [11]. More specifically, having transformed to overexpress the AtFH gene, Arabidopsis (Arabidopsis thaliana) plants were found to have developed basal resistance and become resistant against Pseudomonas syringae pv. tomato DC3000, which indicated the role of FH in plant immunity [11].
To date, the research concerning FH in plants has been very limited. One FH gene in Arabidopsis [12] and two homologous FH genes in maize (Zea mays) (ZmFH1 and ZmFH2) have been identified genome-wide [4]. The Arabidopsis FH (AtFH) was the first FH gene to be identified in plants, sharing 65% homology with the human FH gene. The protein encoded by the AtFH gene contains a transit peptide for mitochondrial localization [6]. As mentioned previously, FH deficiency leads to an increase in the free-iron content within the domains server (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 1 May 2021) [45]. Moreover, the genomic, coding, and amino acid sequence data of FH genes were retrieved for sequential and structural analyses of the proteins. The coding sequences were analyzed using DnaSPv6 [46], including GC content, numbers of polymorphic (segregating) sites, parsimony-informative sites, nucleotide diversity, and Tajima's D statistics [47]. The physiochemical properties of FH proteins were identified on the ProtParam server (http://web.expasy.org/protparam) [48]. The sub-cellular localizations of FH proteins were predicted on the WoLFPSORT server [49] (https://wolfpsort.hgc.jp/, accessed on 1 May 2021). The BioEdit v7.2.5 program was employed to reveal the sequence identity matrices and the amino acid compositions of FH proteins [50]. The NetPhos 3.1 server (http://www.cbs.dtu.dk/services/NetPhos/, accessed on 1 May 2021) was used to predict phosphorylation sites in the FH proteins [51].

Phylogenetic and Gene Ontology Analyses
The phylogenetic tree was drawn using the neighbor-joining (NJ) method [52]. The evolutionary distances in the tree were assumed using the Poisson correction method. The phylogenetic analyses were performed in MEGA7 [53]. The functional annotation and gene ontology (GO) classes of StFH protein were processed using the PANNZER (Protein ANNotation with Z-scoRE) server (http://ekhidna2.biocenter.helsinki.fi/sanspanz/, accessed on 1 May 2021) [54].

In Silico Gene Expression and Synteny Analyses
The digital gene expression data were obtained from the RNA-Seq database of the Potato Genome Sequencing Consortium [57]. The data comprise three different expression datasets, each of which includes 39,031 genes. The first dataset includes gene expressions under Phytophthora infestans infection and some elicitors, including acibenzolar-S-methyl (BTH) and DL-b-amino-n-butyric acid (BABA) (hereafter biotic treatment or dataset). The second dataset includes gene expressions under the following abiotic treatments: salinity (150 mM NaCl), drought (mannitol 260 mM), and heat (35 • C) (hereafter abiotic treatment or dataset). Lastly, the third dataset is composed of expression data under hormone treatments (hereafter hormone dataset), such as abscisic acid (ABA; 50 µM), 6-benzylaminopurine (BAP; 10 µM), gibberellic acid (GA3; 50 µM), and indole-3-acetic acid (IAA; 10 µM). The RNA-Seq data analysis was conducted on the Galaxy platform [58] according to the Tuxedo protocol [59]. Concisely, a quality check was applied to the raw data, and the data were trimmed. The trimmed data were again quality-checked by TopHat, and Bowtie was used for the alignment against the potato genome. Cufflinks were used for transcript assembly, and Cuffdiff was used to calculate gene expressions.

Plant Materials and Stress Treatments
The potato (S. tuberosum) cultivar 'Agria' was used in this study. The plantlets were subcultured on MS-0 media (4.4 g MS salts, 3% sucrose, 0.7% agar, pH 5.7) and maintained in a growth chamber under 16 h light / 8 h dark photoperiod at 22 • C and 70% relative humidity. Five weeks later, the plantlets were transferred to the hydroponic systems in a growth chamber containing Hoagland's solution (pH 5.8). Following 2 days of acclimatization, the solutions in the hydroponic systems were replaced with fresh Hoagland's solution containing 25% PEG-6000 or 200 mM NaCl for the drought stress or salt stress treatments, respectively. The Hoagland solution of the control plants was also refreshed simultaneously. After 24 h of the drought stress and salt stress treatments, the plant leaves and roots were harvested for RNA isolation and physiological assays.

Assaying Proline and MDA (Malondialdehyde) Contents
To assay the proline content of the tissues, 0.5 g of leaf or root tissue was grounded in liquid nitrogen and suspended in 10 mL of 3% (v/v) sulfosalicylic acid. The suspensions were filtered through filter papers (Whatman, Florham Park, NJ, USA). Two ml of each filtrate were mixed with 2 mL acid ninhydrin and 2 mL glacial acetic acid and kept at 95 • C for one hour. The reaction was terminated by placing the mixes on ice, and 4 mL of toluene was added to each. The chromophore phases were transferred into quartz cuvettes, and the absorbances were measured at 520 nm using a spectrophotometer [67].
The MDA content of the plants was assayed using a method modified from that of Ohkawa et al. [68]. Grounded in liquid nitrogen, 0.2 g of leaf or root tissue was suspended in 2 mL of 5% trichloroacetic acid (TCA). The homogenates were transferred into clean 2 mL microfuge tubes and centrifuged at 12,000 rpm at room temperature. Equal amounts of lysate and freshly prepared 0.5% thiobarbituric asit (TBA) in 20% TCA were mixed and incubated at 96 • C for 25 min. The tubes were chilled on ice until they reached room temperature and centrifuged at 10,000 rpm for 5 min. The absorbances of the supernatants were measured at 532 nm and 600 nm wavelengths to clear off non-specific reflections due to turbidity. A freshly prepared 0.5% TBA in 20% TCA solution was used as blank. The MDA content of the samples was assayed using an absorbance coefficient of 155 mM −1 cm −1 .

RNA Isolation and Gene Expression Analysis
RNAs were extracted from leaf and root tissues using RNA Plant Mini Kit (Qiagen, Cat No: 74904) according to the manufacturer's instructions. The samples were treated with Turbo Dnase (Thermo Fisher, San Jose, CA, USA USA). RNA intactness and possible DNA contamination in the samples were checked by gel electrophoresis. The RNA amounts were determined with Qubit (Invitrogen, Waltham, MA, USA). Real time-quantitative PCR (RT-qPCR) was carried out using Rotor-Gene Q (Qiagen, Germantown, MD, USA). The expressions of the genes were quantified in 10 ng RNA samples using Luna Universal One-Step RT-qPCR Kit (NEB, Ipswich, MA, USA). The StFH gene forward (F: CTGCTTCT-GCTTCGGAAATC) and reverse (R: AGCATCTT-GGAGAGGTGACG) primers were designed, yielding a 146 bp amplicon. The StSec3 gene was utilized as the reference control in the study (F: GCTTGCACACGCCATATCAAT; R: TGGATTTTACCACCTTCCGCA). The StSec3 amplicon size was 160 bp [69]. Gene expression was determined using the ∆∆C T method [70]. For the gene expression analysis, the C T values of the reference gene (StSec3) were subtracted from the C T of the target genes to normalize each sample. For example, The value of ∆∆C T was obtained by comparing the ∆C T value of stress applied plant to non-stressed plant: The fold change for each stressed plant based on the corresponding reference (nonstressed) was calculated using the following equation: Average C T values were obtained from at least six biological and four technical replicates for StFH.

Nucleotide and Protein Sequence Analyses
GC content is considered a significant feature since it affects various genomic regulations and determines the physical properties of a DNA molecule [71]. In the present study, the GC contents of potato, Arabidopsis, rice, and maize FH genes were found as 43.93%, 42.02%, 53.77%, and 51.98%, respectively. The monocots had higher GC contents than the dicots, and these variations were thought to be related to the FH gene's structures. Utilizing the nucleotide sequence analysis, a total of 546 sites (excluding the ones with gaps / missing data) and 324 polymorphic (segregating) sites were identified. Among the polymorphic sites, 251 (77.5%) singleton-variable and 73 (22.5%) parsimony-informative sites were identified. The nucleotide diversity (π) was found to be 0.38. Since the queried FH genes were selected from both monocots and dicots, it was expected that the FH proteins from these two distinct classes naturally had distinct specificities and selectivity. Consequently, the plants from two dissimilar classes had large differences because of the balancing selection; i.e., the present variation among genes was sustained. This situation might also be originated from the low levels of both low-and high-frequency polymorphisms [72]. Overall, the FH genes showed no rare mutation in terms of nucleotide diversity.
The sequence analyses of FH proteins from four plant species are presented in Table 1. Their protein lengths ranged from 187 to 201 amino acid residues, and their molecular weights were between 214.08 kDa and 220.28 kDa. All the FHs contained a conserved FH_Cyay domain (PF01491) structure (Figure 1), and their subcellular localizations were predicted as mitochondria. The pIs of FH proteins displayed variations, suggesting differences in the amino acid compositions. Phosphorylation events can influence the enzymatic activity of a phosphoprotein, its protein stability, and cellular localization. They may also enhance or inhibit protein binding events [73]. Phosphorylation occurs primarily at nine amino acids, including serine, threonine, and tyrosine, which are the most extensively phosphorylated. Among the different types of phosphoproteins, phosphoserines are the most prevalent, followed by phosphothreonines and phosphotyrosines. [74,75]. A total of 123 serine, threonine, or tyrosine phosphorylation sites were identified for the FH proteins, supporting their functional diversities. Five exons were identified for each FH gene, whereas their exon sizes were different per species, suggesting variations in the FH genes.
Using the identity matrix of BioEdit, potato and Arabidopsis, potato and rice, potato and maize, potato and soybean, potato and S. pombe, and potato and E. coli UMN026 identity values were found to be 55.3%, 51.4%, 47.7%, 56.3%, 24.2%, and 13.9%, respectively. The highest identity value was 71.6% for the maize and rice FHs, suggesting a more conserved FH gene structure in the monocots than in the dicots. proteins, supporting their functional diversities. Five exons were identified for each FH gene, whereas their exon sizes were different per species, suggesting variations in the FH genes.  Using the identity matrix of BioEdit, potato and Arabidopsis, potato and rice, potato and maize, potato and soybean, potato and S. pombe, and potato and E. coli UMN026 identity values were found to be 55.3%, 51.4%, 47.7%, 56.3%, 24.2%, and 13.9%, respectively. The highest identity value was 71.6% for the maize and rice FHs, suggesting a more conserved FH gene structure in the monocots than in the dicots.

Phylogenetic Analyses
To understand the evolutionary history of FH genes, phylogenetic analyses were performed, which resulted in the formation of two major groups, group A and group B (Figure 2). The main group A was divided into three subgroups: subgroup A1 of dicot plants, subgroup A2 of monocot plants, and subgroup A3 of fungi. The main group B was composed of bacterial species, and it was separated from the other main group (100%). When the phylogenetic tree was examined in general, it was observed that the separation between the monocots and dicots was very clear (100%). Eventually, it can be proposed that the FH genes showed a lineage-specific distribution.

Phylogenetic Analyses
To understand the evolutionary history of FH genes, phylogenetic analyses were performed, which resulted in the formation of two major groups, group A and group B (Figure 2). The main group A was divided into three subgroups: subgroup A1 of dicot plants, subgroup A2 of monocot plants, and subgroup A3 of fungi. The main group B was composed of bacterial species, and it was separated from the other main group (100%). When the phylogenetic tree was examined in general, it was observed that the separation between the monocots and dicots was very clear (100%). Eventually, it can be proposed that the FH genes showed a lineage-specific distribution.

Digital Gene Expression and Syntenic Analyses
To provide insights into the regulation of the StFH gene, its expression profiles under hormone, abiotic, and biotic treatments deposited in the Potato RNA-Seq Database were examined (Figure 3). The analyses showed that the expression of the StFH gene increased in all the treatments except for 6-benzylaminopurine (BAP as synthetic cytokinin) and P.

Digital Gene Expression and Syntenic Analyses
To provide insights into the regulation of the StFH gene, its expression profiles under hormone, abiotic, and biotic treatments deposited in the Potato RNA-Seq Database were examined (Figure 3). The analyses showed that the expression of the StFH gene increased in all the treatments except for 6-benzylaminopurine (BAP as synthetic cytokinin) and P. infestans elicitors DL-β-amino-n-butyric acid (BABA) applications. Ranging from 0.07to 0.62-fold, StFH was mainly upregulated in the majority of treatments. However, the downregulations, ranging from 0.44-to 2.11-fold, were more stringent. Particularly, all of the abiotic treatments upregulated StFH, suggesting that it is mainly involved in abiotic stress response. Frataxin, a nuclear-encoded mitochondrial protein, has been shown to participate in Fe-S cluster biosynthesis, defend against oxidative stress, and regulate iron metabolism [13,76], supporting our findings.

Promotor Sequence Analyses
First, predicted transcription factor binding sites were determined using the PlantPAN database, and the information on TF members that were particularly high in number is shown in Table 2. In particular, WRKY, bZIP, Dof, bHLH, C2H2, NAC, Trihelix, and HD-ZIP were detected as high in the number of TFs. Transcription factors (TFs) play a crucial role in regulating gene expression by binding to specific DNA sequences at the promoters of target genes. In plants, organ development is a continuous process that extends beyond the embryonic phase, and as sessile organisms, plants must adapt to a diverse array of environmental stressors. Signaling pathways controlling both development and stress response converge at the level of gene expression. Given the high proportion of TF-coding genes in plant genomes, ranging from 6-10%, transcriptional regulation may play an even more critical role in plants than in animals [77]. The obtained data from this study may support the large-scale activity of FT genes in cellular metabolic pathways. To obtain more insights about cis-elements, stress-related cis-elements in the promoter regions of the FT genes were analyzed, as shown in Table 3. A total of seven types of stressrelated cis-elements were checked, such as ABRE, SARE, G-box, W-box, P1BS, SURE, and CG-box. Notably, W-box (WRKY binding site) cis-element was found in the highest number in all four plant species. WRKY transcription factors (TFs) play a key role in regulating plant tolerance to both abiotic and biotic stresses. Cis-regulatory elements, such as the W-box (with the core sequence C/TTGACT/C), are essential for controlling gene expression in a spatiotemporal manner. Studies have shown that the tetramer sequence TGAC within the W-box is highly conserved among different plant species [78]. Considering the data obtained, it can be proposed that especially the WRKY TF family plays an active role in the regulation of FT genes in cell metabolism.

Secondary and Tertiary Structure Analyses
The biological functions, gene sequences, and protein structures proved the presence of similarities as well as variations among the FH gene. Protein structure and function can be good resources for neutral and adaptive mechanisms [79]. In the present study, α-helices, extended strands, beta turns, and random coils ranged from 30.35% to 43.81%, 15.98% to 21.39%, 5.67% to 6.97%, and 34.54% to 41.29%, respectively (Table 4). Therefore, the secondary structures of the proteins indicate the presence of variations among them. In addition, the 3D structures of FT proteins appear to be reliable based on the Ramachandran plot and Z-score values. The predicted 3D structures of FH proteins were constructed using the Phyre 2 server (Figure 4). The comparison of 3D structures of FH proteins revealed their structural variations. Analyzed on the CLICK server, the 3D structure of StFH protein overlapped by 59.89%, 64.43%, and 64.95% with Arabidopsis, maize, and rice FH proteins at the tertiary level, respectively. To provide better insight into the 3D structure evolution of FH proteins, the StFH protein was compared to the FH of E. coli, S. cerevisiae, and H. sapiens. The FH structure overlap values were found as 83.96% between potato and E. coli, 86.18% between potato and S. cerevisiae, and 91.6% between potato and H. sapiens at the tertiary level. Surprisingly, the StFH protein was found to be more similar to the bacteria, yeast, and human FH proteins than the plant FH proteins. These results may be related to the high level of genetic variation of FH genes during evolutionary history in plant genomes. The neo-functionalization or sub-functionalization of duplicated FH genes may also cause those variations at the tertiary level in plants [80]. When the sequence identities were taken into consideration, the 3D structural similarities between StFH and the other FH proteins were more prevalent than their genomic sequence similarities. The sufficient conservation of FH protein can be attributed to its vital role in plants.
ations. Analyzed on the CLICK server, the 3D structure of StFH protein overlapped by 59.89%, 64.43%, and 64.95% with Arabidopsis, maize, and rice FH proteins at the tertiary level, respectively. To provide better insight into the 3D structure evolution of FH proteins, the StFH protein was compared to the FH of E. coli, S. cerevisiae, and H. sapiens. The FH structure overlap values were found as 83.96% between potato and E. coli, 86.18% between potato and S. cerevisiae, and 91.6% between potato and H. sapiens at the tertiary level. Surprisingly, the StFH protein was found to be more similar to the bacteria, yeast, and human FH proteins than the plant FH proteins. These results may be related to the high level of genetic variation of FH genes during evolutionary history in plant genomes. The neo-functionalization or sub-functionalization of duplicated FH genes may also cause those variations at the tertiary level in plants [80]. When the sequence identities were taken into consideration, the 3D structural similarities between StFH and the other FH proteins were more prevalent than their genomic sequence similarities. The sufficient conservation of FH protein can be attributed to its vital role in plants.

Predicted Binding Sites and Channels in FH Proteins
Proteins are smart biomolecules involved in many intra-and inter-cellular activities, such as signaling within and between cells, cellular defenses, the maintenance of the structural integrity of cells, metabolism, and catabolism. The understanding of protein-ligand interactions is important to completely understand the cellular mechanisms [81]. In the present study, the predicted binding sites of FH proteins from four plant species were identified using the InterProv75 server ( Table 5). The catalytical residues in putative binding sites were distinguished with a ball-and-stick model in the 3D structures ( Figure 4). Glutamic acid (E), histidine (H), and aspartic acid (D) residues were commonly found in

Predicted Binding Sites and Channels in FH Proteins
Proteins are smart biomolecules involved in many intra-and inter-cellular activities, such as signaling within and between cells, cellular defenses, the maintenance of the structural integrity of cells, metabolism, and catabolism. The understanding of proteinligand interactions is important to completely understand the cellular mechanisms [81]. In the present study, the predicted binding sites of FH proteins from four plant species were identified using the InterProv75 server ( Table 5). The catalytical residues in putative binding sites were distinguished with a ball-and-stick model in the 3D structures ( Figure 4). Glutamic acid (E), histidine (H), and aspartic acid (D) residues were commonly found in the predicted binding sites of all FH proteins, suggesting significant contributions to the catalytic activities of plant FHs. Moreover, the residues in the catalytic sites of each protein showed variation. For instance, the 97th residue, Glu in Arabidopsis, was replaced by His and Asp in StFH and OsFH, respectively. On the other hand, Glu seemed to be strictly conserved in the 105 th positions of StFH and OsFH and 108th positions of OsFH and ZmFH (Table 5). In yeast, the conserved acidic residues of helix 1-strand 1 protein region may play roles in the iron-binding site of FH [82]. Dhe-Paganon et al. [83] reported that a cluster of 12 acidic residues was found in the first helix and the first strand of human FH at 1.8 Å resolution. In a bacterial FH ortholog (CyaY), several E(Glu) and D (Asp) clusters in α1 and β1 regions were identified as highly negatively charged ridges [84]. These results were in agreement with our findings. The molecular functions of proteins are determined by their molecular structures, such as voids, channels, and pockets interacting with other molecules in their environment [66]. Identifying protein cavities, channels, and pockets that are accessible to ligands is a crucial step in predicting protein-ligand interactions [85]. In our study, varying numbers of channels were identified in the FH proteins of potato (6), maize (8), Arabidopsis (9), and rice (13), demonstrating diversity in the FH gene family (Table 6). These structural diversities may also contribute to providing functional diversities in the cellular iron metabolism in plants.

Physiological Analyses
To confirm the efficacy of drought and salt stresses on the plants, morphological observations, as well as physiological assays, were utilized. After 24 h of stress treatment, the effects of the stresses on the seedlings became morphologically apparent (data not shown). Afterward, the proline and MDA contents of the plants were assayed ( Figure 5A,B) to evaluate if the morphological observations of the plants matched the physiological data. Proline is involved in various mechanisms to avoid the maleficent effects of abiotic stresses in plants [86]. These mechanisms vary from the adjustment of osmotic balance to the formation of antioxidants based on the stress type [87]. Proline accumulation in plants is associated with stress tolerance [88][89][90]. Research revealed that proline production was promoted and its degradation was suppressed under drought and salt stresses [91]. In the present study, it was found that proline levels in the root and leaf tissues increased under both salt and drought stresses ( Figure 5A). The increases were statistically significant and confirmed the fact that proline production was induced in response to abiotic stresses. Lipid membrane peroxidation by ROS (indicated as MDA content) is a reflection of stressinduced damage at the cellular level [92]. In the present study, it was found that the MDA content was increased between 1.2-and 1.6-fold in all tissues under both stress types ( Figure 5B). Although both the drought and salt stresses induced oxidative damage and lipid peroxidation in the potato cells, the effect of the drought stress was more severe than that of the salt stress, and the leaves became more affected by the stresses compared to the roots. both stress types ( Figure 5B). Although both the drought and salt stresses induced oxidative damage and lipid peroxidation in the potato cells, the effect of the drought stress was more severe than that of the salt stress, and the leaves became more affected by the stresses compared to the roots.

Expression Profiles of StFH under Drought and Salt Stresses
Fe, an essential element in the plant system, plays a crucial role in regulating lifesustaining processes such as respiration, photosynthesis, chloroplast development, and

Expression Profiles of StFH under Drought and Salt Stresses
Fe, an essential element in the plant system, plays a crucial role in regulating lifesustaining processes such as respiration, photosynthesis, chloroplast development, and chlorophyll biosynthesis by participating in electron transportation. Maintaining Fe homeostasis is critical for the plant life cycle under stress conditions [93]. Frataxin, a highly conserved protein from both prokaryotes and eukaryotes, has been suggested to play multiple roles in iron metabolism, including Fe-S cluster and heme synthesis, response to oxidative damage, and oxidative phosphorylation. Plants deficient in AtFH have been found to have slower growth, increased reactive oxygen species (ROS), and oxidative stress markers [13,76]. Copper is also necessary for the assembly and function of cytochrome c oxidase, a key enzyme in the respiratory chain, in plant mitochondria [94]. Frataxins are involved in copper control in mitochondria and chloroplasts [76]. They have the ability to react with free cupric ions to form cuprous ions in vitro [95]. Chloroplasts contain over 50% of the Cu present in plants, highlighting the significance of Cu in photosynthesis [96].
To obtain further insights into the regulation of StFH in response to drought and salt stresses, the gene expressions in a potato variety were analyzed by means of RT-qPCR ( Figure 6). The StFH was found to be 0.13-to 0.53-fold upregulated under salt stress, while it was 0.43-to 0.95-fold upregulated under drought stress. The StFH upregulation under both stresses was detected in both the leaves and the roots. The expressions in the leaves and under drought stress were determined to be higher than those in the roots and under salt stress, respectively. Buchensky et al. [4] reported that the expressions of the ZmFH-1 and ZmFH-2 genes were very limited in the roots when compared with the leaves under even regular conditions. The tissue-specific expression patterns of the ZmFH genes in their study correlate with our findings. Fonseca et al. [11] indicated that the Arabidopsis lines overexpressing the AtFH gene developed basal immunity and gained the ability to combat both host and non-host pathogens. Despite being limited in terms of their number, the prior studies conducted on plant FH genes, including the present study, have verified that the FH gene plays an important role in response to both biotic and abiotic stresses in plants.

Conclusions
Iron is known to be a vital nutrient for plants, and it has been shown to be involved in the structures of many biomolecules. In the present study, the FH gene was identified in the potato genome, and comparative bioinformatics, physiological, and expression analyses were performed. Our analyses revealed the FH genes to be involved in diverse biological processes, owing to the gene-duplication event stemming from evolutionary processes. We also found that the properties of the FH gene/protein varied, while the gene expression levels increased, especially under abiotic stress conditions. The level of nucleotide diversity (π) was identified as 0.38, indicating sequence variations. Despite the differences identified in their 3D protein structures, such as channel number, the active sites (particularly glutamic acid (E), histidine (H), and aspartic acid (D) residues) and domain structures (FH_Cyay domain (PF01491)) within all the FH genes were well conserved. The

Conclusions
Iron is known to be a vital nutrient for plants, and it has been shown to be involved in the structures of many biomolecules. In the present study, the FH gene was identified in the potato genome, and comparative bioinformatics, physiological, and expression analyses were performed. Our analyses revealed the FH genes to be involved in diverse biological processes, owing to the gene-duplication event stemming from evolutionary processes. We also found that the properties of the FH gene/protein varied, while the gene expression levels increased, especially under abiotic stress conditions. The level of nucleotide diversity (π) was identified as 0.38, indicating sequence variations. Despite the differences identified in their 3D protein structures, such as channel number, the active sites (particularly glutamic acid (E), histidine (H), and aspartic acid (D) residues) and domain structures (FH_Cyay domain (PF01491)) within all the FH genes were well conserved. The expression analyses performed in this study resulted in the upregulation of the StFH gene in the root and leaf tissues under both drought and salt stresses. The findings presented here are particularly important in terms of understanding the uptake mechanisms of metals such as iron and sulfur under environmental stresses in plants. They will also contribute to the understanding of the molecular role of the FH gene in response to abiotic stress in plants. Funding: This research received no external funding. The APC was waived by MDPI.

Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.