Genome-Wide Identiﬁcation and Functional Characterization of FAR1-RELATED SEQUENCE ( FRS ) Family Members in Potato ( Solanum tuberosum )

: FAR1-RELATED SEQUENCE (FRS) transcription factors are generated by transposases and play vital roles in plant growth and development, light signaling transduction, phytohormone response, and stress resistance. FRSs have been described in various plant species. However, FRS family members and their functions remain poorly understood in vegetative crops such as potato ( Solanum tuberosum , St ). In the present study, 20 putative StFRS proteins were identiﬁed in potato via genome-wide analysis. They were non-randomly localized to eight chromosomes and phylogenetic analysis classiﬁed them into six subgroups along with FRS proteins from Arabidopsis and tomato. Conserved protein motif, protein domain, and gene structure analyses supported the evolutionary relationships among the FRS proteins. Analysis of the cis -acting elements in the promoters and the expression proﬁles of StFRSs in various plant tissues and under different stress treatments revealed the spatiotemporal expression patterns and the potential roles of StFRSs in phytohormonal and stress responses. StFRSs were differentially expressed in the cultivar “Xisen 6”, which is exposed to a variety of stresses. Hence, these genes may be critical in regulating abiotic stress. Elucidating the StFRS functions will lay theoretical and empirical foundations for the molecular breeding of potato varieties with high light use efﬁciency and stress resistance.


Introduction
All life on earth directly or indirectly depends on light for growth. Light drives photosynthesis, which provides energy and biomass for the growth of many autotrophic organisms. For plants, light is also an important signaling molecule regulating vegetative and reproductive growth, senescence, metabolism, circadian rhythms, and adaptation to dynamic changes in the environment [1,2]. The importance of transposase-generated transcription factors (TFs) FAR1-RELATED SEQUENCE (FRS) and FRS-RELATED FACTOR (FRF) in light signaling transduction has been recently established and their mechanisms are gradually being elucidated [3].
Under far-red (rather than white) light conditions, the far-red elongated hypocotyls3 (fhy3) mutant is characterized by a long hypocotyl phenotype and was first identified through forward genetics [4]. FHY3 and its homolog FAR-REDIMPAIRED RESPONSE1 (FAR1) are generated by transposases and are typical members of the FRS family [5]. In Arabidopsis thaliana (Arabidopsis), the AtFRS family comprises fourteen AtFRSs and four AtFRFs [6]. Most of these contain the N-terminal C 2 H 2 zinc-finger (or the FAR1 DNA-binding) domain, a central putative transposase domain resembling that of MULE transposase, and a C-terminal SWIM (SWI2/SNF2 and MuDR transposase) zinc-finger structure [7]. FRS TFs exert their regulatory effects through the FAR1 DNA-binding domain at the N-terminal and recognize cis-acting (FBS; CACGCGC) elements that induce or repress downstream genes [8,9].
Plants have evolved conserved mechanisms to maintain a balanced relationship between growing and stressing responses. A number of regulatory factors involved in light signaling have been shown to play important roles in plant stress responses, such as phyA, phyB, HY5 and LIGHT INSENSITIVE PERIOD1 in Arabidopsis, G-box BINDING FACTOR1 in wheat and PHYTOCHROME-INTERACTING FACTOR-LIKE14 in rice [36][37][38][39][40][41][42]. The involvement of FRS family members in tea and eucalyptus in responding to stress has been initially reported [30,31]. In contrast, little is known about the mechanisms of action of how FRS is involved in abiotic stress response in other crops.
Potato (Solanum tuberosum) is native to the Andes Mountains of South America and is now widely grown globally. It has low average water consumption, high photosynthetic efficiency, strong environmental adaptability, and substantial yield [43][44][45]. Potato is the third largest food crop and the most important tuber crop worldwide. It is a staple food for 1.3 billion individuals and an important source of raw materials for food and industrial processing [46][47][48][49]. The sequencing program conducted on the potato genome provides an asset for genome-wide analysis of the potato FRS family [50]. However, the actions and mechanism of regulation of StFRSs in potatoes have not been scientifically reported.
Here, we investigated the distribution of 20 StFRS genes in the potato genome, systematically studied their structures, chromosomal locations, conserved motifs, the cis-elements in their promoter regions, their phylogenetic classification, and their functional interaction networks. We also compared the FRS homologs in potato, Arabidopsis, tomato, eggplant, pepper, and tobacco. The expression patterns of StFRSs in different tissues and under multiple stress conditions were analyzed using transcriptome data. We further investigated the expression of StFRSs with different abiotic stresses in the "Xisen 6" cultivar. This work will help elucidate the functions of FRSs and provide the theoretical and practical basis for their use in molecular crop improvement.

StFRS Identification and Analysis
In this study, a total of 20 StFRS genes were identified in the potato genome, and these genes have been named according to the chromosomal loci where they are located (Table 1; Figure 1). The CDS and protein sequences are listed in Table S1. As shown in Table 1, the StFRS proteins are between 89 aa (StFRS18) and 597 aa (StFRS1) long. The MWs of the StFRSs are between 9.99 kDa (StFRS18) and 66.50 kDa (StFRS1); the predicted pI values for the StFRSs are between 4.38 (StFRS18) and 9.39 (StFRS13); most StFRSs are localized to the cytosols or the nuclei; however, StFRS2 and StFRS12 are localized to the mitochondria while StFRS4 and StFRS17 are localized to the plastids. Hence, the genes at these locations might have undergone novel functional evolution.

Chromosomal Location and Duplication of StFRSs
To understand the distribution of StFRS genes in the potato genome, we noted chromosomal location based on their information from the genome database. As sho in Figure 1, twenty genes were localized on eight chromosomes, including five on ch mosome 6, four on chromosome 9, three on chromosome 3, two each on chromosomes 2, and 8, one each on chromosomes 4 and 7, and none on chromosomes 5, 10, 11, or 12

Chromosomal Location and Duplication of StFRSs
To understand the distribution of StFRS genes in the potato genome, we noted the chromosomal location based on their information from the genome database. As shown in Figure 1, twenty genes were localized on eight chromosomes, including five on chromosome 6, four on chromosome 9, three on chromosome 3, two each on chromosomes 1, 2, and 8, one each on chromosomes 4 and 7, and none on chromosomes 5, 10, 11, or 12.
Tandem and segmental duplications contribute to gene family generation during evolution [51]. There are two clusters of tandem StFRS on the potato chromosome, including StFRS10 and StFRS11, and StFRS12 and StFRS13 (Figure 1), and these two pairs of gene clusters with tandem duplication belong to the following two subgroups: subgroup II and V, respectively ( Figure 3). Segmental duplications are fragments of DNA with nearly identical sequences that are larger than 1 kb [52]. We analyzed synteny blocks among the potato chromosomes to clarify the evolution of potato StFRSs through a Multiple Collinearity Scan toolkit X version (MCScanX). Figure S1 shows that one pair of segmental duplications (StFRS9/StFRS10) was identified and their Ka/Ks < 1 (Table S2). Thus, they might have undergone purifying selection during evolution. Tandem and segmental duplications may play important roles in the evolutionary expansion of StFRSs.

Synteny Analysis of FRS in Different Plant Species
To investigate the molecular evolutionary relationships of FRS in plants, we performed synteny analysis between StFRSs and homologs in other species. Figure 2 showed that the numbers of collinear FRS pairs between potato and Arabidopsis, tomato, pepper, eggplant, and tobacco were 8, 11, 5, 8, and 0, respectively. The FRS orthologs syntenic with the StFRSs in other plant species are listed in Table S2.
II and V, respectively ( Figure 3). Segmental duplications are fragments of DNA w nearly identical sequences that are larger than 1 kb [52]. We analyzed synteny blo among the potato chromosomes to clarify the evolution of potato StFRSs through a M tiple Collinearity Scan toolkit X version (MCScanX). Figure S1 shows that one pair of s mental duplications (StFRS9/StFRS10) was identified and their Ka/Ks < 1 (Table S2 Thus, they might have undergone purifying selection during evolution. Tandem and s mental duplications may play important roles in the evolutionary expansion of StFRS

Synteny Analysis of FRS in Different Plant Species
To investigate the molecular evolutionary relationships of FRS in plants, we p formed synteny analysis between StFRSs and homologs in other species. Figure 2 show that the numbers of collinear FRS pairs between potato and Arabidopsis, tomato, pepp eggplant, and tobacco were 8, 11, 5, 8, and 0, respectively. The FRS orthologs syntenic w the StFRSs in other plant species are listed in Table S2. The gene pairs most homologous to other species were StFRS9, StFRS10 and StFRS on chromosome 6 ( Figure 2). Certain StFRSs were syntenic with > 2 genes in the Arabid sis and tomato genomes. Potato StFRS10, StFRS12, and StFRS14 were syntenic with t The gene pairs most homologous to other species were StFRS9, StFRS10 and StFRS12 on chromosome 6 ( Figure 2). Certain StFRSs were syntenic with >2 genes in the Arabidopsis and tomato genomes. Potato StFRS10, StFRS12, and StFRS14 were syntenic with two AtFRSs from Arabidopsis. Solyc03g117520.2.1, Solyc06g068210.2.1, and Solyc06g065940.3.1 in tomato were syntenic with StFRS9. For all direct homolog pairs, Ka/Ks is <1 (Table S2). Therefore, these genes evolved under the influence of negative or purifying selection. The FRS orthologs in various plants may have facilitated evolution of the FRS family. The FRS orthologs between potato and other plants were anchored in conserved syntenic blocks. In addition, no pairs of collinear FRSs were shared between potato and tobacco, suggesting a long-distance phylogenetic relationship between the two species.

Phylogenetic, Gene Structure, Conserved Domain, and Motif Analyses
To investigate the evolutionary relationships of FRSs between potato and the other species, we plotted a maximum likelihood (ML) phylogenetic tree according to the multiple protein sequence alignments of 65 FRSs, including 20 StFRSs from potato, 27 SlFRSs from tomato [53], and 18 AtFRSs from Arabidopsis [6,54]. The phylogenetic distribution indicated that the StFRSs were divided into six groups designated I-VI ( Figure 3). Except for subgroup IV, which contained only 1 gene, the other StFRS transcription factor subfamilies contained 3-5 genes in varying numbers ( Figure 4A). With the exception of subgroup VI, which consisted of StFRS5, StFRS16, and StFRS20, the subfamily classification of the StFRS proteins closely resembles that of the Arabidopsis and tomato FRS subfamilies, making the results of this classification similar to those of other reported species, including maize, tea, eucalyptus and cotton [30,31,55]. Therefore, investigating the function of StFRS may benefit from looking at other reported species.
Ka/Ks is < 1 (Table S2). Therefore, these genes evolved under the influence o purifying selection. The FRS orthologs in various plants may have facilitated the FRS family. The FRS orthologs between potato and other plants were anc served syntenic blocks. In addition, no pairs of collinear FRSs were shared be and tobacco, suggesting a long-distance phylogenetic relationship between the

Phylogenetic, Gene Structure, Conserved Domain, and Motif Analyses
To investigate the evolutionary relationships of FRSs between potato species, we plotted a maximum likelihood (ML) phylogenetic tree according ple protein sequence alignments of 65 FRSs, including 20 StFRSs from pota from tomato [53], and 18 AtFRSs from Arabidopsis [6,54]. The phylogeneti indicated that the StFRSs were divided into six groups designated I-VI (Fig for subgroup IV, which contained only 1 gene, the other StFRS transcriptio families contained 3-5 genes in varying numbers ( Figure 4A). With the exce group VI, which consisted of StFRS5, StFRS16, and StFRS20, the subfamily of the StFRS proteins closely resembles that of the Arabidopsis and tomato lies, making the results of this classification similar to those of other reporte cluding maize, tea, eucalyptus and cotton [30,31,55]. Therefore, investigating of StFRS may benefit from looking at other reported species.   We further investigated the StFRS motif distribution, and ten putative conserved motifs among the 20 StFRS members were identified ( Figure 4B). Details of these motifs are illustrated in Figure S2. Only the first subgroup comprising StFRS4 and StFRS15 contained all ten motifs, whereas the other subgroups had two to nine motifs. All members of the same phylogenetic group had similar motifs numbers and positions, which was consistent with the numbers and positions of conserved domains ( Figure 4C). All StFRSs contained the FHY3 or FAR1 superfamily domains, while the Znf_PMZ domain was identified in FRS2/4/7/9/10/11/15 and the SWIM domain was identified in FRS2/4/7/11/15 ( Figure 4C). The protein structures were generally uniform among members of the same subgroup. This observation suggested that they might have similar biological functions.
We examined the gene intron and exon compositions and exhibited the StFRS structures by comparing genomic DNA sequences. Figure 4D shows that three StFRSs (StFRS3, StFRS16, and StFRS20) had one exon, six StFRSs (StFRS5, StFRS9, StFRS11, StFRS12, StFRS13, and StFRS18) had two exons, and all other StFRSs had three to eleven exons. The foregoing results combined with those of the StFRS phylogenetic tree analysis ( Figure 4A) suggested a similar gene structure within the same subgroup. Hence, the observed differences in gene structure might be related to the biological functions. We further investigated the StFRS motif distribution, and ten putative conserved motifs among the 20 StFRS members were identified ( Figure 4B). Details of these motifs are illustrated in Figure S2. Only the first subgroup comprising StFRS4 and StFRS15 contained all ten motifs, whereas the other subgroups had two to nine motifs. All members of the same phylogenetic group had similar motifs numbers and positions, which was consistent with the numbers and positions of conserved domains ( Figure 4C). All StFRSs contained the FHY3 or FAR1 superfamily domains, while the Znf_PMZ domain was identified in FRS2/4/7/9/10/11/15 and the SWIM domain was identified in FRS2/4/7/11/15 ( Figure 4C). The protein structures were generally uniform among members of the same subgroup. This observation suggested that they might have similar biological functions.
We examined the gene intron and exon compositions and exhibited the StFRS structures by comparing genomic DNA sequences. Figure 4D shows that three StFRSs (StFRS3, StFRS16, and StFRS20) had one exon, six StFRSs (StFRS5, StFRS9, StFRS11, StFRS12, StFRS13, and StFRS18) had two exons, and all other StFRSs had three to eleven exons. The foregoing results combined with those of the StFRS phylogenetic tree analysis ( Figure 4A) suggested a similar gene structure within the same subgroup. Hence, the observed differences in gene structure might be related to the biological functions.

In Silico Analysis of Cis-Elements in StFRS Promoters
To provide insight into the regulatory mechanisms, metabolic networks, and functions of the StFRSs, the 2 kb upstream sequences of the StFRS start codons were used to scan for cis-elements. We identified six classes of cis-elements involved in plant growth and development of the StFRS promoters ( Figure 5). These included the GCN4-motif, 3-AF1 binding site, CAT-box, CCGTCC-box, circadian element, and O 2 -site. The promoters of 19/20 StFRSs each contained ≥2 phytohormone response elements responding to various signals such as gibberellins, auxins, ethylene, abscisic acid, jasmonic acid, and salicylic acid. Only StFRS6 lacked any phytohormone response element. Moreover, we identified 16 classes of light response elements among the StFRS promoters. In total, 16 of the 20 StFRSs contained a total of 43 Box-4 elements and the latter were the most abundant in the present study. Several abiotic and biotic stress response elements such as AREs, MBSs, TC-rich repeats, LTRs, and WUN-motifs were identified among the StFRS promoters. Thus, certain StFRSs may be implicated in potato responses to drought, hypoxia, low temperatures, and pathogen and predator attack. The preceding findings suggest that StFRSs have a wide range of biological functions in potato growth and development in addition to light signaling response.

In Silico Analysis of Cis-Elements in StFRS Promoters
To provide insight into the regulatory mechanisms, metabolic networks, and functions of the StFRSs, the 2 kb upstream sequences of the StFRS start codons were used to scan for cis-elements. We identified six classes of cis-elements involved in plant growth and development of the StFRS promoters ( Figure 5). These included the GCN4-motif, 3-AF1 binding site, CAT-box, CCGTCC-box, circadian element, and O2-site. The promoters of 19/20 StFRSs each contained ≥2 phytohormone response elements responding to various signals such as gibberellins, auxins, ethylene, abscisic acid, jasmonic acid, and salicylic acid. Only StFRS6 lacked any phytohormone response element. Moreover, we identified 16 classes of light response elements among the StFRS promoters. In total, 16 of the 20 StFRSs contained a total of 43 Box-4 elements and the latter were the most abundant in the present study. Several abiotic and biotic stress response elements such as AREs, MBSs, TC-rich repeats, LTRs, and WUN-motifs were identified among the StFRS promoters. Thus, certain StFRSs may be implicated in potato responses to drought, hypoxia, low temperatures, and pathogen and predator attack. The preceding findings suggest that StFRSs have a wide range of biological functions in potato growth and development in addition to light signaling response.

Protein Interaction Networks of StFRS Proteins
To investigate StFRS potential interactions and characterize their biological functions, the STRING server was used to integrate StFRS into the Arabidopsis association model. As illustrated in Figure 6, the interaction network of StFRS was mapped to the Arabidopsis protein interaction network. There is an existing interaction between the subgroup I of StFRS15 (corresponding to AtFAR1 in Arabidopsis) and StFRS14 (corresponding to AtFHY3), both of which can potentially interact with several other protein types, including phyA, FHY1, FHL and HY5, further demonstrating the important role of StFRS in light signaling. In addition, StFRS proteins can potentially interact with CCA1, ABA3, JAZ3, HDA15 and SPCH among others, suggesting a multifaceted function of StFRS proteins.

Protein Interaction Networks of StFRS Proteins
To investigate StFRS potential interactions and characterize their biological functions, the STRING server was used to integrate StFRS into the Arabidopsis association model. As illustrated in Figure 6, the interaction network of StFRS was mapped to the Arabidopsis protein interaction network. There is an existing interaction between the subgroup I of StFRS15 (corresponding to AtFAR1 in Arabidopsis) and StFRS14 (corresponding to AtFHY3), both of which can potentially interact with several other protein types, including phyA, FHY1, FHL and HY5, further demonstrating the important role of StFRS in light signaling. In addition, StFRS proteins can potentially interact with CCA1, ABA3, JAZ3, HDA15 and SPCH among others, suggesting a multifaceted function of StFRS proteins.

StFRS Expression Patterns
The potato transcriptome data from Spud DB were used to analyze StFRS expression in different potato tissues. As shown in Figure 7, no reads were detected for StFRS3, StFRS5, or StFRS20. However, the other 17 StFRSs were expressed with varying degrees in the roots, stems, leaves, tubers, sepals, carpels, stamens, petals, whole mature flowers, and fruits. StFRS14 was highly expressed in all tissues tested. By contrast, StFRS4, StFRS7 and StFRS15 within the same subfamily were expressed at relatively low levels in all

StFRS Expression Patterns
The potato transcriptome data from Spud DB were used to analyze StFRS expression in different potato tissues. As shown in Figure 7, no reads were detected for StFRS3, StFRS5, or StFRS20. However, the other 17 StFRSs were expressed with varying degrees in the roots, stems, leaves, tubers, sepals, carpels, stamens, petals, whole mature flowers, and fruits. StFRS14 was highly expressed in all tissues tested. By contrast, StFRS4, StFRS7 and StFRS15 within the same subfamily were expressed at relatively low levels in all tissues   We also analyzed StFRSs expression in DM potato subjected to various phy mones. Figure 8B suggested that abscisic acid (ABA) not only induced most of the S but also strongly modulated StFRS4 and StFRS6 expression (Log2 FoldChange > 1.5). Ind acetic acid (IAA) could induce StFRS4/11/13/15, with the strongest ability to i  Table S3.

StFRS Expression Patterns under Abiotic Stresses in "Xisen 6" Potato Cultivar
It has been shown that the light-signaling regulatory proteins play an irreplaceable role in the salt tolerance of plants [40][41][42]. To investigate the role of FRS genes in salt tolerance in potato, we selected seven genes with significant changes (Log 2 FoldChange > 1) based on the transcriptomic results of salt-treated DM potatoes (Table S3), and further analyzed the virus-free seedlings of the "Xisen 6" cultivar. "Xisen 6", a sexual hybrid selected from "Shepody" and "XS9304", has shown good yield performance in several parts of China [56], including the saline Yellow River Delta. When treated with different concentrations of NaCl for 12 h, the reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays showed that the transcript level of StFRS genes were less affected by 50 mM NaCl, and the transcript levels of the other five genes tested, except StFRS13 and StFRS19, showed differences under different concentrations of salt treatment. For example, both 100 mM and 200 mM NaCl induced an increase in StFRS15 transcript level, but no significant difference was observed under 150 mM NaCl treatment (Figure 9). These results indicate that StFRS genes exhibit differential performance in response to salt stress treatment. REVIEW 11 qPCR) assays showed that the transcript level of StFRS genes were less affected by 50 NaCl, and the transcript levels of the other five genes tested, except StFRS13 and StFR showed differences under different concentrations of salt treatment. For example, 100 mM and 200 mM NaCl induced an increase in StFRS15 transcript level, but no si icant difference was observed under 150 mM NaCl treatment (Figure 9). These result dicate that StFRS genes exhibit differential performance in response to salt stress t ment. To further investigate the function of StFRSs in stress response, transcript levels w analyzed by RT-qPCR under low temperature (4 °C), 20% polyethylene glycol (PEG) NaHCO3 treatment. As illustrated in Figure 10, the 16 StFRSs tested showed differen sponses to these stresses. Low temperature treatment resulted in significant down-r lation of the expression of ten StFRSs, while the expression of StFRS14 and StFRS15 up-regulated; PEG treatment resulted in an up-regulation of the expression of StF while the expression of another four StFRSs was down-regulated; NaHCO3 treatment resulted in a high expression of StFRS6, StFRS10 and StFRS13, while the expression le of five StFRSs were down-regulated. The above results suggest that these family mem may have different functions in potato growth and development, as StFRSs show diffe expression responses to the various stresses. To further investigate the function of StFRSs in stress response, transcript levels were analyzed by RT-qPCR under low temperature (4 • C), 20% polyethylene glycol (PEG) and NaHCO 3 treatment. As illustrated in Figure 10, the 16 StFRSs tested showed different responses to these stresses. Low temperature treatment resulted in significant downregulation of the expression of ten StFRSs, while the expression of StFRS14 and StFRS15 was up-regulated; PEG treatment resulted in an up-regulation of the expression of StFRS9, while the expression of another four StFRSs was down-regulated; NaHCO 3 treatment also resulted in a high expression of StFRS6, StFRS10 and StFRS13, while the expression levels of five StFRSs were down-regulated. The above results suggest that these family members may have different functions in potato growth and development, as StFRSs show different expression responses to the various stresses.

023, 12, x FOR PEER
Plants 2023, 12, x FOR PEER REVIEW Figure 10. The expression pattern of StFRS genes under abiotic stresses. RT-qPCR assays expressions of StFRS genes in Xisen 6 after 12 h of treatment with different treatments (low ature, PEG, and NaHCO3), and seedlings with no treatment as control. Mean ± SD (n = 3). * ** p < 0.001.

Discussion
FRS TFs are generated by transposases [7,8]. They have been domesticat adapted for an extended period of time and play important functions in sever plant biological development processes besides light signaling transduction [3]. P search on Arabidopsis FHY3 and FAR1 has clarified the vital roles of FRSs in plant and development [54]. There are ongoing studies on the FRSs in several species, rice, wheat, tomato, cotton, tea, eucalyptus, silver birch (Betula pendula) and Tibetan [30][31][32]53,55,[57][58][59]. In the present study, we identified StFRS family members in for the first time, identified the biological pathways in which they might participa focused on their roles in the abiotic stress response.

Phylogenetic Analysis of the FRS Genes in Potato
We identified twenty StFRS genes distributed over eight chromosomes, con three tandem duplicated gene clusters with a total of seven genes found on chrom 6 and 9, which are shared by two subgroups (Figures 1 and 3). The genes derive the tandem duplications and segmental duplication constituted the StFRS fam might play similar roles in various biological processes [60]. Genome-wide dup Figure 10. The expression pattern of StFRS genes under abiotic stresses. RT-qPCR assays showing expressions of StFRS genes in Xisen 6 after 12 h of treatment with different treatments (low temperature, PEG, and NaHCO 3 ), and seedlings with no treatment as control. Mean ± SD (n = 3). * p < 0.05, ** p < 0.001.

Discussion
FRS TFs are generated by transposases [7,8]. They have been domesticated and adapted for an extended period of time and play important functions in several other plant biological development processes besides light signaling transduction [3]. Prior research on Arabidopsis FHY3 and FAR1 has clarified the vital roles of FRSs in plant growth and development [54]. There are ongoing studies on the FRSs in several species, such as rice, wheat, tomato, cotton, tea, eucalyptus, silver birch (Betula pendula) and Tibetan Prunus [30][31][32]53,55,[57][58][59]. In the present study, we identified StFRS family members in potato for the first time, identified the biological pathways in which they might participate, and focused on their roles in the abiotic stress response.

Phylogenetic Analysis of the FRS Genes in Potato
We identified twenty StFRS genes distributed over eight chromosomes, containing three tandem duplicated gene clusters with a total of seven genes found on chromosomes 6 and 9, which are shared by two subgroups (Figures 1 and 3). The genes derived from the tandem duplications and segmental duplication constituted the StFRS family and might play similar roles in various biological processes [60]. Genome-wide duplication events play a role in maintaining the function of gene families as well as functional expansion, such as the FRS family in other species [6,30,53], and other gene families in potato [61][62][63]. Gene structure and motif composition varied widely between subgroups. Hence, the members of StFRS family are distinct. Thus, further experiments, such as genetic transformation or gene knockout, are required to complete the functional verification of this family.
A phylogenetic analysis revealed that the StFRS subfamily classification of potato resembled that of Arabidopsis and tomato ( Figure 3). Thus, the FRS family members have been highly conserved throughout evolution and may have functions similar to those of Arabidopsis or tomato. For these reasons, it may be possible to predict the functions of potato FRS family members based on those determined for Arabidopsis and tomato in prior studies. However, the three genes in the StFRS subfamily VI have undergone substantial evolutionary changes. Analyses of tissue expression patterns through RNA-seq indicated FPKM = 0 for both StFRS5 and StFRS20 (Table S3). We speculate that they may evolve into pseudogenes for undergoing deletion events or functional changes during evolution.

StFRS Protein Interaction Network
In addition to structural diversity, the StFRS family genes may have other functional diversity. Significantly, StFRS promoters contain several types of cis-elements, and the functional diversity to which they respond has been demonstrated in other species ( Figure 5) [6,30,53]. In addition, StFRS forms functional complexes with other proteins ( Figure 6). For example, interactions with HDA15 may contribute to seed germination, hypocotyl elongation and other processes in light signals [64][65][66]. Other potential StFRSprotein interactions exist in potato. However, these require further experimental validation.

StFRS Response to Abiotic Stresses
Although FRS was originally reported to be involved in light signaling, recent studies of the FRS gene family have shown that FRS proteins can directly bind to multiple genes (e.g., FHY1, FHL, COP1, HEMB1, EARLY-FLOWERING4, CLAVATA3, myo-INOSITOL-1-PHOSPHATE SYNTHASE, ISOAMYLASE2, PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and PAR2, and WRKY28, etc.) in vivo and are involved in regulating multiple physiological processes including phytohormone signaling, biotic/abiotic stress response, flowering and senescence in plants [12,16,17,19,[23][24][25][26][27]33,67]. Hormones trigger the regulation of plant growth adaptations in response to drought, salt and other stresses [68]. We found that abiotic and biotic stress can regulate StFRSs expression (Figures 8-10), but it is unclear that how FRS regulate hormonal signals in response to stresses. One possibility is the activation of the ABA signal pathway, as demonstrated in Arabidopsis [13,69]; other stress-related hormones such as ethylene, salicylic acid, and growth-related hormones such as auxin and gibberellin may also play a role in the regulation of stress via StFRS (Figure 8). The involvement of FRS in plant energy supply is another possibility for its involvement in stress response [29,70], as prolonged abiotic stress inhibits the activity of photosystem II to reduce the energy supply of plant [71].
Recent studies have shown that light is involved in regulating the response of plants to salt stress, and that the translocation of the photoreceptors phyA and phyB into the nucleus is important for salt tolerance in plants [40][41][42], and that the proteins HY5 and PIF4, which interact with FHY3 in Arabidopsis, are also involved in the salt tolerance process [37,40]. Since AtFHY3 is an important factor in allowing the translocation of phytochrome into the nucleus [8], do StFRSs play a role in salt tolerance in potato by affecting the translocation of phytochrome into the nucleus? Alternatively, is it possible that StFRSs regulate salt tolerance via direct binding to the FBS elements of salt response genes? In the present study, we examined StFRSs expression patterns in the cultivar potato subjected to salt and alkali stress (NaCl and NaHCO 3 treatments, respectively) (Figures 9 and 10). SlFHY3 enhances chilling tolerance in tomato [32], and a similar mechanism may be conserved in potato, where StFRS14/StFRS15 cold induction regulate chilling tolerance by integrating environmental light and internal (inositol synthesis) signals. Although preliminary results of the experiments indicate the transcript levels of StFRSs are affected by stress, the main question that needs to be addressed in the future is how StFRSs are involved in balancing plant growth and stress resistance.

Conclusions
We identified 20 putative StFRSs in potato by genome-wide analysis and characterized them according to their chromosomal locations, phylogenetic relationships, gene structures, conserved motifs, duplication events, synteny, promoter cis-elements, and expression profiles. The StFRSs were divided into five subfamilies that were evolutionarily related to the tomato FRSs. StFRS expression profiles revealed that they occur in many potato tissues and are involved not only in light signaling but also in response to environmental stresses, where they may play an irreplaceable role. These findings provided theoretical and empirical bases for improving the agronomic traits of potato and other solanaceous crops such as tomato, eggplant, tobacco, and pepper. Furthermore, with the deciphering of the genome of the highly complex tetraploid potato cultivar [72,73], we will gain a deeper understanding of the mechanisms about StFRSs in potato growth, development and stress resistance, which will provide theoretical guidance for the future development of new potato cultivars with high yield and resistance to climate change.
The conserved motifs of the StFRS proteins were analyzed using the Simple MEME Wrapper in TBtools, with a maximum of 10 misfits and an optimal motif width of 6-50 amino acid residues, with any number of repetitions [80]. The StFRS structures and chromosomal locations were analyzed and displayed using Gene Structure View and Gene Location Visualize in TBtools, respectively [80].

Prediction of Cis-Elements and Protein Interaction Networks
The sequence 2 kb upstream of the StFRSs was extracted from Spud DB. The Plant-CARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 19 January 2023)) was consulted to screen potential cis-elements in the StFRS promoters [81]. The functional interaction networks of StFRS family proteins were predicted by STRING (https://string-db.org/ (accessed on 16 June 2023)), using the protein sequences of the StFRSs to search the potato and Arabidopsis databases.

Transcriptome Analysis
The publicly available transcriptome dataset was obtained from Spud DB to analyze the expression patterns of 20 StFRSs in various tissues of doubled monoploid (DM) potato [50,90]. The StFRS expression patterns were analyzed in whole potato plants subjected for 24 h to various abiotic stress such as 150 mM NaCl (salt), 260 µM mannitol (drought), 35 • C (heat), and different phytohormones including 50 µM ABA (abscisic acid), 10 µM IAA (auxin), and 50 µM GA 3 (gibberellic acid). The biotic stress treatments comprised Phytophthora infestans (P. infestans) infection and the elicitors acibenzolar-S-methyl (BTH), and DL-β-amino-N-butyric acid (BABA) for 24 h, 48 h, and 72 h, respectively. Accession numbers of transcriptomic data from Spud DB in the present study are listed in Table S4.

Plant Materials and Growth Conditions
Freshly cut single-node potato plants (Solanum tuberosum "Xisen 6" cultivar) were placed into a culture bottle containing 1 2 Murashige and Skoog (MS) medium with 1% (w/v) sucrose at 25 • C under a 16 h light/8 h dark photoperiod. Plant material treatments from previously reported studies with slight modifications were used [62,91]. For the salt, alkali and drought treatments, the 14-day-old seedlings roots were exposed to the liquid medium containing 0 mM, 50 mM, 100 mM, 150 mM or 200 mM NaCl, 50 mM NaHCO 3 , and 20% (w/v) PEG 6000, respectively. For the cold treatment, the 14-day-old seedlings grown under normal conditions were transferred to an artificial climate chamber maintained at 4°C. All seedlings were harvested at 12 h by liquid nitrogen, with three biological replicates per treatment, and stored at −80 • C until further analysis.

Total RNA Isolation and Expression Analysis
Whole potato seedlings subjected to the NaCl treatment were ground in liquid nitrogen and their total RNA was isolated with a Biospin Plant Total RNA Extraction Kit (BSC65S1B, Hangzhou Bioer Technology, Hangzhou, China). Then, 1 µg of RNA per sample was reverse-transcribed into cDNA with an All-in-One First-Strand Synthesis MasterMix Kit (EG15133S; Yugong Biolabs, Lianyungang, China). The real-time fluorescence quantitative PCR (RT-qPCR) protocol used was described in previous works [92]. All primers used in the present study are listed in Table S5.

Statistical Analysis
All samples were analyzed in triplicate. Data are presented as mean ± standard deviation (SD), unless otherwise specified. Statistical analyses were performed using Excel 2019 (Microsoft Corp., Redmond, Washington, USA). Statistically significant differences between treatment means were determined by Student's t-test. ns was considered no significant, * p < 0.05 was considered significant and ** p < 0.001 was considered extremely significant.