Genome-Wide Identification of LOX Gene Family and Its Expression Analysis under Abiotic Stress in Potato (Solanum tuberosum L.)

The lipoxygenases (LOXs) are non-heme iron-containing dioxygenases that play an important role in plant growth and defense responses. There is scarce knowledge regarding the LOX gene family members and their involvement in biotic and abiotic stresses in potato. In this study, a total of 17 gene family members (StLOXs) in potato were identified and clustered into three subfamilies: 9-LOX type I, 13-LOX type I, and 13-LOX type II, with eleven, one, and five members in each subfamily based on phylogenetic analysis. By exploiting the RNA-seq data in the Potato Genome Sequencing Consortium (PGSC) database, the tissue-specific expressed and stress-responsive StLOX genes in double-monoploid (DM) potato were obtained. Furthermore, six candidate StLOX genes that might participate in drought and salt response were determined via qPCR analysis in tetraploid potato cultivars under NaCl and PEG treatment. Finally, the involvement in salt stress response of two StLOX genes, which were significantly up-regulated in both DM and tetraploid potato under NaCl and PEG treatment, was confirmed via heterologous expression in yeast under salt treatment. Our comprehensive analysis of the StLOX family provides a theoretical basis for the potential biological functions of StLOXs in the adaptation mechanisms of potato to stress conditions.


Introduction
Lipoxygenase (LOX) is a dioxygenase containing non-heme iron [1], which can catalyze the oxygenation of unsaturated fatty acids and their corresponding esters in plants.The resultant product of the oxygenation reaction catalyzed by LOXs is hydroperoxides.The secondary metabolites of these hydroperoxides, such as jasmonic acid (JA), green leafy volatiles (GLVs), and divinyl ether, play important roles in regulating seed germination [2], root growth [3], fruit ripening [4], pathogen infection [5], pest resistance [6], and response to abiotic stress [7].
Lipoxygenase is ubiquitously present in plants, animals, algae, and fungi [8][9][10][11].Plants harbor LOX proteins that possess two conserved structural domains: the PLAT/ LH2 domain located at the N-terminus and the lipoxygenase domain situated at the Cterminus [12].The catalytic site of the enzyme resides within the lipoxygenase structural domain, which encompasses 38 amino acids [His-(X)4-His-(X)4-His-(X)17-His-(X)8-His], including 5 highly conserved histidine (His) residues [13].The LOX family can be classified into 9-LOX and 13-LOX according to the reaction position of carbon atoms when they catalyze the oxidation of substrates.In addition, the classification of the LOX family into Type I and Type II is determined by two factors: the degree of similarity in amino acid sequences and whether a chloroplast transit peptide is present at the Nterminal.Type I LOX sequences exhibit high similarity and are devoid of transit peptides, whereas Type II LOX sequences display moderate similarity and possess chloroplast transit peptides at the N-terminus [1,14].
LOX enzyme plays a pivotal role in oxidizing unsaturated fatty acids, thereby exerting a crucial influence on secondary metabolism and vital plant processes.Six LOX members have been found in Arabidopsis thaliana, of which AtLOX2, AtLOX3, AtLOX4, and AtLOX6 belong to 13-LOX and AtLOX1 and AtLOX5 belong to 9-LOX [15].The four 13-LOX enzymes are involved in jasmonate synthesis after leaf injury [16], and the 9-LOX enzymes may control the emergence of lateral roots by producing 9-Hydroxyoctadecatrienoic acid (9-HOT), thus regulating root development [17].Hou et al. [18] found that the overexpression of the persimmon (Diospyros kaki Thunb.)gene DkLOX3 increased the resistance of transgenic Arabidopsis to Botrytis cinerea.The overexpression of pepper CaLOX1 could rapidly remove ROS and induce the expression of ABA biosynthesis-related genes, which improved the tolerance of transgenic Arabidopsis to drought and salt stress [19].In tomato (Solanum lycopersicum), the overexpression of TomloxD (Solyc03g122340, 13-LOX) exhibited enhanced resistance against the Cladosporium fulvum and high-temperature stress, which was due to an increase in lipoxygenase activity, endogenous levels of JA, and the expression of defense genes such as LeHSP90, LePR1, LePR6, and LeZAT [20,21].Numerous LOXs have been shown to play a pivotal role in plants' ability to respond to various stressors, encompassing both biotic stressors like pathogens [20] and aphids [22] and abiotic stressors such as cold [23], heat [24], salt, and drought [19].Currently, surprisingly little research has focused on elucidating the LOX gene family in potato, despite the extensive identification and analysis of LOXs in diverse plants, including 8 members in pepper (Capsicum annuum) [25], 11 members in radish (Raphanus sativus) [26], 20 members in poplar (Populus trichocarpa) [27], and 11 members in tea tree (Camellia sinensis) [23].
Potato, which originates from the Andean region of Bolivia and Peru, is the third most-produced food crop, following rice (Oryza sativa) and wheat (Triticum aestivum) [28].Potato is widely cultivated worldwide because of its tolerance to barrenness, wide adaptability, and high and stable yield.Potato tuber production is impacted by various biotic and abiotic stressors, including pests and diseases, high temperature, salt stress, and drought stress, all of which significantly hinder the growth and progress of the potato industry.
In this study, 17 members of the LOX gene family were identified through comprehensive genome-wide analysis of potato.Then, the StLOX gene structure and duplication events, phylogenetic relationships, and conserved motifs of proteins were systematically investigated.Moreover, the expression of StLOX genes in different tissues of doublemonoploid (DM) potato, as well as under different stresses and hormone treatments, were analyzed based on RNA-seq data in the PGSC database.Furthermore, some candidate genes in tetraploid potato cultivars that responded to NaCl and PEG were confirmed via qPCR.Of these, two candidate genes, Soltu.DM.01G002140 and Soltu.DM.01G038840, that might be involved in the response to salt stress were verified via the yeast heterologous expression method.

Identification of StLOXs
By employing HMM 3.1, we conducted a search of the potato genome sequence to identify proteins containing the lipoxygenase domain (PF00305).We utilized the SMART and NCBI Batch CD-search databases to screen for sequences that harbor the two conserved domains of PLAT/LH2 and lipoxygenase.At the end of the analysis, a complete set of 17 members belonging to the StLOX gene family was identified.They were un-evenly distributed in 6 out of 12 chromosomes.Chromosome 1 harbored the highest count of genes, encompassing eight StLOX genes, while chromosomes 3, 5, and 12 exhibited the lowest representation, with only a single StLOX gene for each chromosome (Figure 1).The physical and chemical property analyses of StLOX members showed that the lengths of StLOX proteins ranged from 794 amino acids (Soltu.DM.08G005470) to 914 amino acids (Soltu.DM.03G037120), the molecular weights ranged between 89.90 kDa (Soltu.DM.08G005470) and 103.59 kDa (Soltu.DM.03G037120), and the pI values ranged from 5.15 (Soltu.DM.01G038910) to 8.6 (Soltu.DM.05G011130) (Table S1).S1).

Phylogenetic Analysis and Classification of StLOXs
To understand the evolutionary relationship of the StLOX gene family, 60 LOX proteins from 6 species (potato, Arabidopsis, tomato, cabbage, rice, and maize) were compared, and a phylogenetic tree was constructed.The results showed that these 60 LOX proteins were classified into 3 subfamilies: 9-LOX type I, 13-LOX type I, and 13-LOX type II, in which there were 11, 1, and 5 members of the StLOX family, respectively.The three subfamilies were further divided to five subgroups: C1-C5; among them, C1 and C2 belonged to 13-LOX type II, C3 and C4 belonged to 13-LOX type I, and C5 belonged to 9-LOX subfamily.Seventeen StLOXs were characterized into four subgroups, except for the C4 subgroup (Figure 2).Most of the StLOX members were closely related to those of tomatoes, which all belonged to the Solanaceae family, but relatively distant from those of other dicotyledonous and monocotyledonous species.

Phylogenetic Analysis and Classification of StLOXs
To understand the evolutionary relationship of the StLOX gene family, 60 LOX proteins from 6 species (potato, Arabidopsis, tomato, cabbage, rice, and maize) were compared, and a phylogenetic tree was constructed.The results showed that these 60 LOX proteins were classified into 3 subfamilies: 9-LOX type I, 13-LOX type I, and 13-LOX type II, in which there were 11, 1, and 5 members of the StLOX family, respectively.The three subfamilies were further divided to five subgroups: C1-C5; among them, C1 and C2 belonged to 13-LOX type II, C3 and C4 belonged to 13-LOX type I, and C5 belonged to 9-LOX subfamily.Seventeen StLOXs were characterized into four subgroups, except for the C4 subgroup (Figure 2).Most of the StLOX members were closely related to those of tomatoes, which all belonged to the Solanaceae family, but relatively distant from those of other dicotyledonous and monocotyledonous species.

Gene Structure and Conserved Motifs of StLOXs
To gain insight into the architecture of the StLOX genes, the introns and exons of its family members were subjected to analysis and visualization (Figure 3B).Of the 17 StLOX genes, only 1 member (Soltu.DM.03G037120) contained 7 introns, 12 members contained 8 introns, and 4 members (Soltu.DM.01G002150,Soltu.DM.01G002140,Soltu.DM.12G02875, and Soltu.DM.08G005470) contained 9 introns.Additional analysis of the conserved motifs present in the StLOXs was carried out using MEME.The findings revealed the identification of ten distinct motifs, designated as Motif 1-Motif 10 (Figure 3C and Table S2).In total, 10 9-LOX members contained all 10 motifs except for Soltu.DM.01G038930, while all 6 13-LOX members lacked motif 6.The conserved motifs of potato LOX family proteins are highly conserved, as evidenced by the identical arrangement order among StLOX members.Motif 1 comprises 50 amino acids residues and

Gene Structure and Conserved Motifs of StLOXs
To gain insight into the architecture of the StLOX genes, the introns and exons of its family members were subjected to analysis and visualization (Figure 3B).Of the 17 StLOX genes, only 1 member (Soltu.DM.03G037120) contained 7 introns, 12 members contained 8 introns, and 4 members (Soltu.DM.01G002150,Soltu.DM.01G002140,Soltu.DM.12G02875, and Soltu.DM.08G005470) contained 9 introns.Additional analysis of the conserved motifs present in the StLOXs was carried out using MEME.The findings revealed the identification of ten distinct motifs, designated as Motif 1-Motif 10 (Figure 3C and Table S2).In total, 10 9-LOX members contained all 10 motifs except for Soltu.DM.01G038930, while all 6 13-LOX members lacked motif 6.The conserved motifs of potato LOX family proteins are highly conserved, as evidenced by the identical arrangement order among StLOX members.Motif

Gene Duplication Events in the StLOX and Analysis of Synteny with Other Species
The duplication of genes plays a pivotal role in the enlargement of gene families and the diversification of their functions.During the current investigation, we identified four pairs of StLOX genes (6/17, 35.29%) as being tandemly duplicated.Specifically, chromosome 1 harbored three such pairs, while chromosome 8 contained one such pair (Figure 1).
The assessment of selection pressure on genes was conducted by calculating the ratio between non-synonymous mutations (Ka) and synonymous mutations (Ks).A Ka/Ks ratio equal to 1 signified neutral evolution, while a ratio less than 1 signified the influence of purification selection on the gene.Conversely, a Ka/Ks ratio exceeding 1 hinted at the presence of positive selection.For the tandemly duplicated genes, the Ka/Ks values ranged from 0.1558 to 0.3906, with a mean of 0.3078 (Table S3).These values, all being below 1, provide further evidence that the evolution of these genes was significantly influenced by purifying selection.
To elucidate the potential evolution of the LOX gene family in tomato, Arabidopsis, cabbage, rice, maize, and potato, we conducted a detailed analysis of their orthologous genes (Figure 4).Consequently, we identified 11, 3, 3, 0, and 1 pairs of orthologs, respectively.The corresponding Ka/Ks values ranged from 0.0554 to 0.4125 (Figure 4 and Table S4).Notably, all these ratios were below 1, indicating that these genes underwent evolutionary processes under purifying selection.

Gene Duplication Events in the StLOX and Analysis of Synteny with Other Species
The duplication of genes plays a pivotal role in the enlargement of gene families and the diversification of their functions.During the current investigation, we identified four pairs of StLOX genes (6/17, 35.29%) as being tandemly duplicated.Specifically, chromosome 1 harbored three such pairs, while chromosome 8 contained one such pair (Figure 1).
The assessment of selection pressure on genes was conducted by calculating the ratio between non-synonymous mutations (Ka) and synonymous mutations (Ks).A Ka/Ks ratio equal to 1 signified neutral evolution, while a ratio less than 1 signified the influence of purification selection on the gene.Conversely, a Ka/Ks ratio exceeding 1 hinted at the presence of positive selection.For the tandemly duplicated genes, the Ka/Ks values ranged from 0.1558 to 0.3906, with a mean of 0.3078 (Table S3).These values, all being below 1, provide further evidence that the evolution of these genes was significantly influenced by purifying selection.
To elucidate the potential evolution of the LOX gene family in tomato, Arabidopsis, cabbage, rice, maize, and potato, we conducted a detailed analysis of their orthologous genes (Figure 4).Consequently, we identified 11, 3, 3, 0, and 1 pairs of orthologs, respectively.The corresponding Ka/Ks values ranged from 0.0554 to 0.4125 (Figure 4 and Table S4).Notably, all these ratios were below 1, indicating that these genes underwent evolutionary processes under purifying selection.

Patterns of StLOX Gene Expression across Different Tissues in DM Potato
Leveraging RNA-seq data retrieved from the PGSC database, a study investigated the unique expression patterns of the StLOX gene across diverse tissues of DM potato, ranging from sepals and leaves to roots and shoots, as well as callus, stolons, mature flowers, tubers, petioles, petals, stamens, carpels, the insides of fruits, and both mature and immature fruits (Figure 5 and Table S5).Three StLOXs (SoltuDM.03G037120,Soltu.DM.09G024180,Soltu.DM.05G011130) had high expression levels in all tissues (FPKM > 3).Certain StLOX genes had a tissue-specific expression, such as Soltu01G038900 and Soltu01G038910, which were highly expressed in fruit, while three StLOX genes (Soltu08G010990, Soltu08G005480, and Soltu03G0317120) were highly expressed in stolons and three StLOX genes (Soltu08G005480, Soltu08G005440, and Soltu08G005470) were highly expressed in tubers.

Patterns of StLOX Gene Expression across Different Tissues in DM Potato
Leveraging RNA-seq data retrieved from the PGSC database, a study investigated the unique expression patterns of the StLOX gene across diverse tissues of DM potato, ranging from sepals and leaves to roots and shoots, as well as callus, stolons, mature flowers, tubers, petioles, petals, stamens, carpels, the insides of fruits, and both mature and immature fruits (Figure 5 and Table S5).Three StLOXs (SoltuDM.03G037120,Soltu.DM.09G024180,Soltu.DM.05G011130) had high expression levels in all tissues (FPKM > 3).Certain StLOX genes had a tissue-specific expression, such as Soltu01G038900 and Soltu01G038910, which were highly expressed in fruit, while three StLOX genes (Soltu08G010990, Soltu08G005480, and Soltu03G0317120) were highly expressed in stolons and three StLOX genes (Soltu08G005480, Soltu08G005440, and Soltu08G005470) were highly expressed in tubers.
Moreover, RNA-seq data retrieved from PGSC were utilized to investigate the transcriptional patterns of StLOX genes in response to hormone treatments, encompassing ABA, IAA, GA3, and BAP.The results showed that four, one, and two StLOX genes responded to ABA, GA3, and BAP treatments (FPKM > 1, |log 2 FC| > 1), respectively (Figure 6 and Table S7).Two StLOX genes responded to two or more hormone treatments.The expression of Soltu.DM.03G037120 was up-regulated in response to ABA and GA3 treatments, while it was down-regulated under BAP treatment.Similarly, the expression of Soltu.DM.08G010990Moreover, RNA-seq data retrieved from PGSC were utilized to investigate the transcriptional patterns of StLOX genes in response to hormone treatments, encompassing ABA, IAA, GA3, and BAP.The results showed that four, one, and two StLOX genes responded to ABA, GA3, and BAP treatments (FPKM > 1, |log2FC| > 1), respectively (Figure 6 and Table S7).Two StLOX genes responded to two or more hormone treatments.The expression of Soltu.DM.03G037120 was up-regulated in response to ABA and GA3 treatments, while it was down-regulated under BAP treatment.Similarly, the expression of Soltu.DM.08G010990 was up-regulated by ABA and down-regulated by BAP.Additionally, the expressions of Soltu.DM.01G038840 and Soltu.DM.09G024180 were elevated upon ABA treatment.

Expression of Six StLOX Genes in Potato Plantlets under NaCl and PEG Treatments
To validate the responsiveness of six StLOX genes to abiotic stresses in tetraploid potato, we conducted a qPCR analysis to assess their expression patterns in potato plantlets (CIP98 clone) exposed to NaCl and PEG treatments, given that these genes exhibit differential expression in DM potato under abiotic stresses.qPCR analysis showed that four StLOX genes (Soltu.DM.08G005480,Soltu.DM.01G002140,Soltu.DM.03G037120, and Soltu.DM.01G038840) were significantly up-regulated compared with CK under NaCl treatment.Of them, Soltu03G037120 was the most significantly up-regulated gene, showing a 2.67 times higher expression level than that of the control.PEG treatment led to the up-regulation of these four genes, with the most pronounced increase observed in

Expression of Six StLOX Genes in Potato Plantlets under NaCl and PEG Treatments
To validate the responsiveness of six StLOX genes to abiotic stresses in tetraploid potato, we conducted a qPCR analysis to assess their expression patterns in potato plantlets (CIP98 clone) exposed to NaCl and PEG treatments, given that these genes exhibit differential expression in DM potato under abiotic stresses.qPCR analysis showed that four StLOX genes (Soltu.DM.08G005480,Soltu.DM.01G002140,Soltu.DM.03G037120, and Soltu.DM.01G038840) were significantly up-regulated compared with CK under NaCl treatment.Of them, Soltu03G037120 was the most significantly up-regulated gene, showing a 2.67 times higher expression level than that of the control.PEG treatment led to the up-regulation of these four genes, with the most pronounced increase observed in Soltu.DM.01G038840, which exhibited a 2.77-fold higher expression compared to the control (Figure 7 and Table S8).The results further confirmed that these three StLOX genes were involved in the responses to abiotic stress, and their functions are worthy of further study.
Soltu.DM.01G038840, which exhibited a 2.77-fold higher expression compared to the control (Figure 7 and Table S8).The results further confirmed that these three StLOX genes were involved in the responses to abiotic stress, and their functions are worthy of further study.

Cloning and Functional Analysis of StLOX Gene
To further verify whether the three candidate StLOX genes (Soltu.DM.01G002140,Soltu.DM.03G037120 and Soltu.DM.01G038840) were involved in abiotic stresses, the CDS fragments of Soltu.DM.01G002140 (2700bp), Soltu.DM.03G037120 (2745bp), and Soltu.DM.01G038840 (2589bp) were cloned by using the cDNA of potato clone "CIP98" as a template, then ligated into the yeast expression vector pYES2-NTB and treated with NaCl of various concentrations for 7 days.The yeast pYES2 was used as a control.Under normal conditions, the growth statuses of three LOX-overexpressing transgenic yeasts were indistinguishable from that of pYES2 yeast cells.The results indicated that there were no differences in the growth statuses of three LOX-overexpressing transgenic yeast or pYES2 yeast cells under normal conditions.However, under 0.5 M and 1.0 M NaCl treatments, the yeast cells overexpressing Soltu.DM.01G002140 exhibited optimal growth activity, followed by the yeast cells overexpressing Soltu.DM.01G038840.When under 1.5 M NaCl stress, the yeast cells overexpressing Soltu.DM.01G002140 could still grow properly even when diluted 10 3 and 10 4 times (Figure 8).The findings demonstrated that Soltu.DM.01G002140 and Soltu.DM.01G038840 genes enhance salt stress tolerance in transgenic yeast cells, further confirming their involvement in abiotic stress responses.

Cloning and Functional Analysis of StLOX Gene
To further verify whether the three candidate StLOX genes (Soltu.DM.01G002140,Soltu.DM.03G037120 and Soltu.DM.01G038840) were involved in abiotic stresses, the CDS fragments of Soltu.DM.01G002140 (2700bp), Soltu.DM.03G037120 (2745bp), and Soltu.DM.01G038840 (2589bp) were cloned by using the cDNA of potato clone "CIP98" as a template, then ligated into the yeast expression vector pYES2-NTB and treated with NaCl of various concentrations for 7 days.The yeast pYES2 was used as a control.Under normal conditions, the growth statuses of three LOX-overexpressing transgenic yeasts were indistinguishable from that of pYES2 yeast cells.The results indicated that there were no differences in the growth statuses of three LOX-overexpressing transgenic yeast or pYES2 yeast cells under normal conditions.However, under 0.5 M and 1.0 M NaCl treatments, the yeast cells overexpressing Soltu.DM.01G002140 exhibited optimal growth activity, followed by the yeast cells overexpressing Soltu.DM.01G038840.When under 1.5 M NaCl stress, the yeast cells overexpressing Soltu.DM.01G002140 could still grow properly even when diluted 10 3 and 10 4 times (Figure 8).The findings demonstrated that Soltu.DM.01G002140 and Soltu.DM.01G038840 genes enhance salt stress tolerance in transgenic yeast cells, further confirming their involvement in abiotic stress responses.

Discussion
Numerous studies have been carried out on the LOX gene families in various plant species, revealing their possible functions in promoting plant growth, development, and defensive responses.The present study identified and analyzed 17 StLOXs in terms of phylogenetic relationships, gene and protein structures and expression profiles.The results showed that StLOX genes play a subtle tissue-specific role during development and are essential in plant stress response.Thus, the data presented herein will provide a useful basis for the functional characterization of StLOX genes.
The primary forces driving the emergence of new family members and novel functions in plant evolution are undergoing tandem duplication and segmental duplication [29].A total of four pairs of tandemly duplicated genes were identified in potato, all of which belonged to the 9-LOX type Ⅰ.This implied that the evolution of StLOX genes was dominated by tandem replication, which plays an especially important role in the expansion of the 9-LOX gene family.Additionally, analysis of multi-species collinearity revealed that certain LOX genes are highly conserved during the evolution of plants.For example, Soltu.DM.03G037120 is homologous with tomato Solyc03g122340, Arabidopsis At1G17420, cabbage Bo8g104870, and corn Zm00001d027893; Soltu.DM.09G024180 is homologous with tomato Solyc09g075860, Arabidopsis At3G22400, and cabbage Bo1g105160.These LOX genes may perform similar functions in different plants.
LOX proteins play a crucial role in plant growth, development, and resilience against adverse environmental conditions [30][31][32].Solt.DM.08G005440 and Soltu.DM.08G005470 are specifically expressed in tubers but not in stolons, and they may be involved in the development processes of tubers.In response to stress, three 9-LOX genes (Soltu.DM.08G010990,Soltu.DM.01G038930, and Soltu.DM.08G005480) exhibited differential expression patterns, analogous to those observed in radish upon exposure to NaCl and PEG stress [26].Through their study, Hu et al. revealed that overexpressing TomloxD (Solyc03g122340) in tomato plants significantly boosts the production of endogenous Jasmonic acid, subsequently improving the plant's tolerance of both biotic and abiotic stress [20].As a homologous gene of Solyc03g122340, Soltu.DM.03G037120 showed differential expression under the treatment of mannitol, multiple hormones, and BTH, which may have potential applications in crop resistance to biotic and abiotic stress.ZS-3, an ecofriendly salt tolerance inductor, augments the growth of plants under salt stress.AtLOX2 is induced by ZS-3 and enhances the salt tolerance capacity of plants through the jasmonic acid pathway [33].We found that Soltu.DM.01G002140 was in the same subgroup as At-LOX2, which was differentially up-regulated under NaCl and ABA treatment and differentially down-regulated under heat stress and BABA treatment.The overexpression of Soltu.DM.01G002140 significantly improved the salt (NaCl) tolerance of yeast (INVsc1).Despite their role in catalyzing the oxygenation of polyunsaturated fatty acids in vivo, it remains to be experimentally confirmed in future investigations whether LOXs are activated by distinct external signals to maintain the metabolite balance in potato.

Discussion
Numerous studies have been carried out on the LOX gene families in various plant species, revealing their possible functions in promoting plant growth, development, and defensive responses.The present study identified and analyzed 17 StLOXs in terms of phylogenetic relationships, gene and protein structures and expression profiles.The results showed that StLOX genes play a subtle tissue-specific role during development and are essential in plant stress response.Thus, the data presented herein will provide a useful basis for the functional characterization of StLOX genes.
The primary forces driving the emergence of new family members and novel functions in plant evolution are undergoing tandem duplication and segmental duplication [29].A total of four pairs of tandemly duplicated genes were identified in potato, all of which belonged to the 9-LOX type I.This implied that the evolution of StLOX genes was dominated by tandem replication, which plays an especially important role in the expansion of the 9-LOX gene family.Additionally, analysis of multi-species collinearity revealed that certain LOX genes are highly conserved during the evolution of plants.For example, Soltu.DM.03G037120 is homologous with tomato Solyc03g122340, Arabidopsis At1G17420, cabbage Bo8g104870, and corn Zm00001d027893; Soltu.DM.09G024180 is homologous with tomato Solyc09g075860, Arabidopsis At3G22400, and cabbage Bo1g105160.These LOX genes may perform similar functions in different plants.
LOX proteins play a crucial role in plant growth, development, and resilience against adverse environmental conditions [30][31][32].Solt.DM.08G005440 and Soltu.DM.08G005470 are specifically expressed in tubers but not in stolons, and they may be involved in the development processes of tubers.In response to stress, three 9-LOX genes (Soltu.DM.08G010990,Soltu.DM.01G038930, and Soltu.DM.08G005480) exhibited differential expression patterns, analogous to those observed in radish upon exposure to NaCl and PEG stress [26].Through their study, Hu et al. revealed that overexpressing TomloxD (Solyc03g122340) in tomato plants significantly boosts the production of endogenous Jasmonic acid, subsequently improving the plant's tolerance of both biotic and abiotic stress [20].As a homologous gene of Solyc03g122340, Soltu.DM.03G037120 showed differential expression under the treatment of mannitol, multiple hormones, and BTH, which may have potential applications in crop resistance to biotic and abiotic stress.ZS-3, an eco-friendly salt tolerance inductor, augments the growth of plants under salt stress.AtLOX2 is induced by ZS-3 and enhances the salt tolerance capacity of plants through the jasmonic acid pathway [33].We found that Soltu.DM.01G002140 was in the same subgroup as AtLOX2, which was differentially up-regulated under NaCl and ABA treatment and differentially down-regulated under heat stress and BABA treatment.The overexpression of Soltu.DM.01G002140 significantly improved the salt (NaCl) tolerance of yeast (INVsc1).Despite their role in catalyzing the oxygenation of polyunsaturated fatty acids in vivo, it remains to be experimentally confirmed in future investigations whether LOXs are activated by distinct external signals to maintain the metabolite balance in potato.

Conserved Motif and Gene Structural Characterization of StLOXs
The motifs present in StLOX proteins were analyzed via the MEME Version 5.5.5 program (https://meme-suite.org/meme/tools/meme accessed on 20 October 2023).A maximum of 10 motifs were allowed, with lengths varying from 6 to 50 amino acids [40].Furthermore, the GSDS 2.0 (http://gsds.gao-lab.orgaccessed on 21 October 2023) tool was used to offer a graphical depiction of the gene structures of the StLOXs [41].

Duplication Events and Syntenic Patterns of LOX Genes
The MCScanX v1.5.1 [42] was used to identify StLOX duplication and collinearity analysis, and Circos v0.69 [43] was applied to provide a visual representation of the syntenic relationships between LOX genes in various species, including Solanum tuberosum, Arabidopsis thaliana, Solanum lycopersicum, Brassica oleracea, Oryza sativa, and Zea mays.Following that, the KaKs Calculator 2.0 [44] was employed to determine the non-synonymous (Ka) and synonymous (Ks) substitution rates for each duplicated pair of LOX genes.

Plant Materials and Treatments
The experimental material comprised a tetraploid potato plantlet named "CIP397098.12"(CIP98), which was obtained from the International Potato Center (CIP).The tissue cultured seedlings were placed in Murashige and Skoog (MS) liquid media containing 3% sucrose (w/v, pH 6.0 ± 0.5) and incubated in a light incubator for three weeks (23 ± 1 • C, 12 h light/12 h dark photoperiod).For the simulated drought and salinity stress treatments, these plantlets were treated with 10% PEG 6000 and 100 mM NaCl for 24 h, respectively.Each triangular flask contained five plantlets, and each of the three replicates contain three triangular flasks.The sample was immediately frozen in liquid nitrogen and stored at −80 • C for RNA extraction.

Extraction of RNA and qPCR
RNA extraction was carried out using the RNA extraction kit (TaKaRa MiniBEST Plant RNA Extraction Kit, Code No. 9769, TaKaRa, Kusatsu, Japan) from all samples, and the integrity and concentration of the RNA were subsequently assessed via agarose gel electrophoresis and Nanodrop ND-2000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) measurements, respectively.The FastKing gDNA Dispelling RT SuperMix (Tiangen KR118, Beijing, China) was used to synthesize the first-strand cDNA, with the RNA serving as the template.The qPCR experiments were conducted on the Bio-Rad CFX96 (Bio-Rad, Hercules, CA, USA), employing the SuperReal PreMix Plus kit (SYBRGreen FP217, Tiangen, Beijing, China).Each assay was carried out with three technical replicates.The PCR program was as follows: 95 • C for 2 min, followed by 40 cycles of 95 • C for 5 s and 60 • C for 15 s and dissociation curve detection under 65-95 • C. Additionally, the internal control gene StEF-1α (AB061263) was employed [47].The quantification of LOX gene expression was determined via the 2 −∆∆Ct method [48].The primers are shown in Table S8.

Cloning of the 3 StLOX Genes
The CDS sequences of the candidate genes Soltu.DM.01G002140,Soltu.DM.03G037120, and Soltu.DM.01G038840 were amplified by using "CIP98" cDNA as a template.The primers were listed in Table S8, and the amplified sequences are shown in Figure S1.The gene amplification process was carried out in a 50 µL reaction mixture by utilizing PrimeSTAR Max DNA Polymerase (TaKaRa R045Q, Japan).The linearization of the pYES2 vector was performed using EcoRI and BamHI (TaKaRa 1040S/1010S, Japan), and the target gene was cloned into the pYES2 vector by using In-Fusion Snap Assembly cloning kits (TaKaRa 638947, Japan).

Salt Stress Tolerance Verification of Candidate Genes in Yeast
Yeast has been proven to be an effective system for evaluating gene tolerance [49].Three candidate genes, Soltu.DM.01G002140,Soltu.DM.03G037120, and Soltu.DM.01G038840, in the pYES2 vector were transformed into yeast strain INVsc1 and coated on the corresponding yeast medium (SD-Ura).A single colony was picked up after incubation at 30 • C for 3 days, and positive colonies were identified using PCR.For the salt stress assay, transformed yeast cells were incubated in SG/-Ura liquid medium at 30 • C for 16 h (OD 600 ≈ 0.6); then, these yeast cells were serially diluted 10-fold (10 0 , 10 −1 , 10 −2 , 10 −3 , and 10 −4 ) with sterile water and dripped in the SG/-Ura medium treated with different concentrations of NaCl (0 M, 0.5 M, 1.0 M, 1.5 M).

Conclusions
This study provides a thorough analysis of the StLOX gene family, encompassing its phylogeny, gene structure, motif arrangement, evolution, and patterns of gene expression under various abiotic and biotic stresses.This study found that the distribution of StLOX members on the six chromosomes was uneven.The primary cause of the enlargement of the StLOX gene family was tandem duplication occurrences.Furthermore, qPCR analysis of StLOX expression in DM and tetraploid potato under abiotic stresses indicated that three StLOX genes were involved in responses to salt and drought stresses, and the overexpression of Soltu01G002140 and Soltu01G038840 enhanced the salt tolerance of yeast cells.These results provide a theoretical basis for further understanding the function of the StLOX gene family in potato.

Figure 1 .
Figure 1.Seventeen StLOX genes were distributed in six chromosomes.The blue area marks tandem duplicated genes.The transcript IDs are translated into their respective gene IDs, and 'Soltu.DM.' is abbreviated to 'Soltu' for brevity's sake.

Figure 1 .
Figure 1.Seventeen StLOX genes were distributed in six chromosomes.The blue area marks tandem duplicated genes.The transcript IDs are translated into their respective gene IDs, and 'Soltu.DM.' is abbreviated to 'Soltu' for brevity's sake.

Figure 2 .
Figure 2. Phylogenetic tree of LOX proteins of six species.Five subgroups (C1-C5) are represented by five distinct colored lines (red, orange, blue, green, and purple).The varying colored circles signify members of different species: red circles represent Arabidopsis, blue circles represent potato, light blue circles represent tomato, green circles represent cabbage, orange circles represent rice, and purple circles represent maize.

Figure 2 .
Figure 2. Phylogenetic tree of LOX proteins of six species.Five subgroups (C1-C5) are represented by five distinct colored lines (red, orange, blue, green, and purple).The varying colored circles signify members of different species: red circles represent Arabidopsis, blue circles represent potato, light blue circles represent tomato, green circles represent cabbage, orange circles represent rice, and purple circles represent maize.

Figure 3 .
Figure 3. Phylogenetic, gene structure, and motif analysis of the StLOXs.(A) Evolutionary tree comprising StLOX proteins.The purple box, blue box, and pink box were used to denote the subfamilies 13-LOX Type Ⅰ, 13-LOX Type Ⅱ, and 9-LOX Type Ⅰ, respectively.(B) Exon/intron structure in StLOX genes.Blue boxes represent exons, while black lines represent introns.Similarly, red boxes denote 5′UTR and 3′UTR.The numbers 0, 1, and 2 signify the splicing phases of the introns.(C) Conserved motif distribution of StLOX.The 10 different colored boxes represented 10 specific motifs.

Figure 3 .
Figure 3. Phylogenetic, gene structure, and motif analysis of the StLOXs.(A) Evolutionary tree comprising StLOX proteins.The purple box, blue box, and pink box were used to denote the subfamilies 13-LOX Type I, 13-LOX Type II, and 9-LOX Type I, respectively.(B) Exon/intron structure in StLOX genes.Blue boxes represent exons, while black lines represent introns.Similarly, red boxes denote 5 ′ UTR and 3 ′ UTR.The numbers 0, 1, and 2 signify the splicing phases of the introns.(C) Conserved motif distribution of StLOX.The 10 different colored boxes represented 10 specific motifs.

Figure 4 .
Figure 4.The orthologous relations of the StLOX genes among tomato, Arabidopsis, cabbage, rice, and maize.A visual representation of the orthology between the StLOX gene and multiple plant species, including tomato, Arabidopsis, cabbage, rice, and maize, is indicated by a red line.The average values of Ka, Ks, and Ka/Ks for the duplicated genes are presented.The horizontal axis represents tandem duplication in potato (Tandem) and homologous genes between potato and Arabidopsis (St-At), cabbage (St-Bo), tomato (St-Sl), and maize (St-Zm), respectively.

Figure 4 .
Figure 4.The orthologous relations of the StLOX genes among tomato, Arabidopsis, cabbage, rice, and maize.A visual representation of the orthology between the StLOX gene and multiple plant species, including tomato, Arabidopsis, cabbage, rice, and maize, is indicated by a red line.The average values of Ka, Ks, and Ka/Ks for the duplicated genes are presented.The horizontal axis represents tandem duplication in potato (Tandem) and homologous genes between potato and Arabidopsis (St-At), cabbage (St-Bo), tomato (St-Sl), and maize (St-Zm), respectively.

Figure 5 .
Figure 5. Visual representation of StLOX expression across multiple tissues in DM potato.The expression levels of genes are normalized using log2FPKM and denoted by colored blocks varying from blue to red.

Figure 5 .
Figure 5. Visual representation of StLOX expression across multiple tissues in DM potato.The expression levels of genes are normalized using log 2 FPKM and denoted by colored blocks varying from blue to red.

Figure 6 .
Figure 6.The transcriptional patterns of StLOX genes in DM potato, subjected to multiple stresses and treated with hormones including IAA, ABA, GA3, and BAP, are demonstrated.The colored blocks ranging from blue to red indicate the expression levels of these genes, which are normalized using log2FC.

Figure 6 .
Figure 6.The transcriptional patterns of StLOX genes in DM potato, subjected to multiple stresses and treated with hormones including IAA, ABA, GA3, and BAP, are demonstrated.The colored blocks ranging from blue to red indicate the expression levels of these genes, which are normalized using log 2 FC.

Figure 7 .
Figure 7. Relative expression levels of StLOX genes in potato plantlet leaves under NaCl and PEG treatments for 24 h.Data represent the mean ± standard error of three biological replicates.Error bars represent standard errors.Different letters on the top of the bars indicate significant differences (p < 0.05).

Figure 7 .
Figure 7. Relative expression levels of StLOX genes in potato plantlet leaves under NaCl and PEG treatments for 24 h.Data represent the mean ± standard error of three biological replicates.Error bars represent standard errors.Different letters on the top of the bars indicate significant differences (p < 0.05).

Figure 8 .
Figure 8.The growth phenotype of three StLOX-overexpressing transgenic yeasts under NaCl treatments with concentrations of 0 M, 0.5 M, 1 M, and 1.5 M, respectively.

Figure 8 .
Figure 8.The growth phenotype of three StLOX-overexpressing transgenic yeasts under NaCl treatments with concentrations of 0 M, 0.5 M, 1 M, and 1.5 M, respectively.