Genome-Wide Identification of the Pectate Lyase Gene Family in Potato and Expression Analysis under Salt Stress

Pectin is a structural polysaccharide and a major component of plant cell walls. Pectate lyases are a class of enzymes that degrade demethylated pectin by cleaving the α-1,4-glycosidic bond, and they play an important role in plant growth and development. Currently, little is known about the PL gene family members and their involvement in salt stress in potato. In this study, we utilized bioinformatics to identify members of the potato pectate lyase gene family and analyzed their gene and amino acid sequence characteristics. The results showed that a total of 27 members of the pectate lyase gene family were identified in potato. Phylogenetic tree analysis revealed that these genes were divided into eight groups. Analysis of their promoters indicated that several members’ promoter regions contained a significant number of hormone and stress response elements. Further, we found that several members responded positively to salt treatment under single salt and mixed salt stress. Since StPL18 exhibited a consistent expression pattern under both single and mixed salt stress conditions, its subcellular localization was determined. The results indicated that StPL18 is localized in the endoplasmic reticulum membrane. The results will establish a foundation for analyzing the functions of potato pectate lyase family members and their expression under salt stress.


Introduction
Potato (Solanum tuberosum L.) is an important food crop in the world, second only to rice and wheat [1].Globally, approximately 1.3 billion people consume potato as a staple food.Potato tubers contain a wide range of nutrients and are rich in potassium, iron, zinc, calcium, magnesium, and other mineral elements.The vitamin C content of potato is higher than that of wheat and other major food crops, and the protein and sugar content is higher than that of many vegetables [2].These characteristics have made the potato become the only staple crop that can be grown in many parts of the world.In Africa, the expansion of potato cultivation has enhanced food security, prompting the United Nations to declare 2008 as the International Year of the Potato [3].
During the growth and development of the potato, the organ grows from small to large, and the plant grows from low to high.The intrinsic structural changes mainly involve an increase in the size of the cells and an increase in the number of cells [4].In this process, the cell wall undergoes a process of disintegration and remodeling.The cell wall not only supports and protects plant cells, but it is also the first line of defense against adversity in plants [5].The plant cell wall is composed of pectin, hemicellulose, and cellulose.They do not function independently but are interlinked to form a highly complex and dynamic network, which collectively determine the mechanical properties of the cell wall [6].
Pectin serves as a component of the primary cell wall of higher plant cells and maintains the mechanical strength of the cell wall.It consists of homogalacturonan (HG), In summary, this research is based on the DM v6.1 Genome assembly for the doubled monoploid potato information.It identifies and analyzes the members of the StPL gene family, while also examining the expression of these genes under salt stress.These findings provide a basis for the further analysis of the differentiation process and biological function of the StPL gene family in potato.These results laid the foundation for further analyzing the differentiation process and biological functions of the StPL gene family in potato and provided a theoretical basis for the discovery and screening of stress resistance genes in potato.

Chromosomal Localization of StPL Genes
A total of 93 candidate genes with "pectate lyase" annotations were obtained through the term's search in SpudDB database (http://spuddb.uga.edu/,accessed on 10 November 2023), and 27 PL family members were ultimately identified utilizing NCBI online software (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, accessed on 10 November 2023) to exclude sequences lacking the conserve domains of Pec_lyase_C and Pec_lyase_N.Based on their locations on the chromosomes, they were named StPL1-27.As shown in Figure 1, the 27 StPL genes were unevenly distributed on chromosomes 1 to 6, 9, 11, and 12.Among them, chromosome 2 had the largest number of PL genes (eight genes), followed by chromosome 3 (five genes).There was only one gene each on chromosomes 11 and 12 (StPL26 and StPL27).StPL genes on chromosomes 4, 5, 6, 9, 11, and 12 were predominantly located at the distal ends of the chromosomes.
Plants 2024, 13, x FOR PEER REVIEW 3 of 16 and conducted salt stress treatment under tissue culture conditions.The results showed that 27 family members had different responses.In summary, this research is based on the DM v6.1 Genome assembly for the doubled monoploid potato information.It identifies and analyzes the members of the StPL gene family, while also examining the expression of these genes under salt stress.These findings provide a basis for the further analysis of the differentiation process and biological function of the StPL gene family in potato.These results laid the foundation for further analyzing the differentiation process and biological functions of the StPL gene family in potato and provided a theoretical basis for the discovery and screening of stress resistance genes in potato.

Chromosomal Localization of StPL Genes
A total of 93 candidate genes with "pectate lyase" annotations were obtained through the term's search in SpudDB database (http://spuddb.uga.edu/,accessed on 10 November 2023), and 27 PL family members were ultimately identified utilizing NCBI online software (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, accessed on 10 November 2023) to exclude sequences lacking the conserve domains of Pec_lyase_C and Pec_lyase_N.Based on their locations on the chromosomes, they were named StPL1-27.As shown in Figure 1, the 27 StPL genes were unevenly distributed on chromosomes 1 to 6, 9, 11, and 12.Among them, chromosome 2 had the largest number of PL genes (eight genes), followed by chromosome 3 (five genes).There was only one gene each on chromosomes 11 and 12 (StPL26 and StPL27).StPL genes on chromosomes 4, 5, 6, 9, 11, and 12 were predominantly located at the distal ends of the chromosomes.

Physicochemical Properties and Subcellular Localization of the StPL Proteins
Physicochemical property analysis and subcellular localization prediction of StPL members were performed using the online tools ProtParam and WoLF PSORT (Table 1).The results revealed that the length of the family in potato varied from 83 (StPL2) to 1332 amino acids (StPL16).Ten out of the twenty-seven members encode acidic proteins, while the remaining members encode neutral or basic proteins.Additionally, the molecular mass of the StPLs ranged from 150.18 kDa (StPL16) to 9.53 kDa (StPL2).Except for StPL1 and StPL2, whose instability index was greater than 40, the rest of the proteins had values

Physicochemical Properties and Subcellular Localization of the StPL Proteins
Physicochemical property analysis and subcellular localization prediction of StPL members were performed using the online tools ProtParam and WoLF PSORT (Table 1).The results revealed that the length of the family in potato varied from 83 (StPL2) to 1332 amino acids (StPL16).Ten out of the twenty-seven members encode acidic proteins, while the remaining members encode neutral or basic proteins.Additionally, the molecular mass of the StPLs ranged from 150.18 kDa (StPL16) to 9.53 kDa (StPL2).Except for StPL1 and StPL2, whose instability index was greater than 40, the rest of the proteins had values below 40, suggesting that members were relatively stable.The analysis based on the predicted values of the grand average of hydropathicity showed that all the values were negative, and the aliphatic index varied from 68.52 to 92.89, indicating that most of these proteins exhibit some level of hydrophilicity.Eight StPLs were localized to the extracellular matrix, five to the vacuole, four to the chloroplast, and three to the cytoskeleton.Three members were localized to the plasma membrane, two members were localized to the peroxisome, and only StPL16 was localized to the mitochondria.The above results indicate that the molecular mass of different members varied and were widely distributed.In addition, the hydrophilicity of all StPLs was less than 0. Therefore, it is hypothesized that the proteins in this family may be hydrophilic.

Gene Duplication and Collinearity Analysis of StPL Gene Family Members
The StPL family genes were analyzed for covariance using MCScanX sequencing in TBtools (Figure 2).The results revealed that 15 genes were tandemly duplicated, and there were also segmentally duplicated genes with covariance.Most of the covariate genes were located on chromosomes 2, 3, 5, and 6, with the highest number found on chromosome 2, totaling six pairs.On chromosome 1, StPL1 was identified as an independent tandem duplication gene, while the remaining genes were not only tandem duplicated but also fragment duplicated.This indicates that both tandem duplications and fragment duplications play a role in the expansion of the StPL gene family.

Gene Duplication and Collinearity Analysis of PL Gene Family Members in Potato and Arabidopsis
Co-linearity mapping of the AtPLL gene of A. thaliana and the StPL gene of potato revealed that chromosome 2 had the greatest number of co-linear genes, with 12 pairs.StPL3 was co-linear with AtPLL8, 10, and 11; StPL5 was co-linear with AtPLL4 and 6; StPL7 was co-linear with AtPLL8, 9, 10, and 11; and StPL9 was co-linear with AtPLL10, 15, and 16.It indicates that there is some similarity between the PL genes of potato and A. thaliana (Figure 3).The red lines represent genes within these two families that have collinearity relationships.The gray color represents genes with no covariate relationship.

Phylogenetic Tree Analysis of StPL Gene Family Members
Referring to the previous classification of PL genes in A. thaliana [16], rice [15], and tomato [18], we divided the 27 PL genes into eight groups, with Group 1 having the most members at 11. AtPLL12, AtPLL13, AtPLL23, and AtPLL26 in Group 1, as well as AtPLL1 in Group 5, induce cell elongation and differentiation through auxin [15,24].SlPL, which

Gene Duplication and Collinearity Analysis of PL Gene Family Members in Potato and Arabidopsis
Co-linearity mapping of the AtPLL gene of A. thaliana and the StPL gene of potato revealed that chromosome 2 had the greatest number of co-linear genes, with 12 pairs.StPL3 was co-linear with AtPLL8, 10, and 11; StPL5 was co-linear with AtPLL4 and 6; StPL7 was co-linear with AtPLL8, 9, 10, and 11; and StPL9 was co-linear with AtPLL10, 15, and 16.It indicates that there is some similarity between the PL genes of potato and A. thaliana (Figure 3).

Gene Duplication and Collinearity Analysis of PL Gene Family Members in Potato and Arabidopsis
Co-linearity mapping of the AtPLL gene of A. thaliana and the StPL gene of potato revealed that chromosome 2 had the greatest number of co-linear genes, with 12 pairs.StPL3 was co-linear with AtPLL8, 10, and 11; StPL5 was co-linear with AtPLL4 and 6; StPL7 was co-linear with AtPLL8, 9, 10, and 11; and StPL9 was co-linear with AtPLL10, 15, and 16.It indicates that there is some similarity between the PL genes of potato and A. thaliana (Figure 3).The red lines represent genes within these two families that have collinearity relationships.The gray color represents genes with no covariate relationship.

Phylogenetic Tree Analysis of StPL Gene Family Members
Referring to the previous classification of PL genes in A. thaliana [16], rice [15], and tomato [18], we divided the 27 PL genes into eight groups, with Group 1 having the most members at 11. AtPLL12, AtPLL13, AtPLL23, and AtPLL26 in Group 1, as well as AtPLL1 in Group 5, induce cell elongation and differentiation through auxin [15,24].SlPL, which The red lines represent genes within these two families that have collinearity relationships.The gray color represents genes with no covariate relationship.

Phylogenetic Tree Analysis of StPL Gene Family Members
Referring to the previous classification of PL genes in A. thaliana [16], rice [15], and tomato [18], we divided the 27 PL genes into eight groups, with Group 1 having the most members at 11. AtPLL12, AtPLL13, AtPLL23, and AtPLL26 in Group 1, as well as AtPLL1 in Group 5, induce cell elongation and differentiation through auxin [15,24].SlPL, which belongs to the same branch as StPL13 and StPL20, can be induced by auxin, abscisic acid, and ethylene.Inhibition of its expression will increase the pectin content of tomato fruits, delay fruit rot, and extend the storage period.Therefore, it is hypothesized that StPL13 and StPL20 may be associated with potato fruit hardness [22].AtPLL13 (PMR6) is in the same group as StPL18.The PMR6 mutation causes plants to dwarf, leaves to become small and rounded, and results in increased resistance to powdery mildew in pmr6 plants [21].The knockout of StPL18 causes morphological changes in potato plants [25].Many PL genes are related to flower development.Silencing OsPLL4 results in abnormal pollen microspores, while StPL21, StPL24, and StPL25 belong to the same evolutionary branch as OsPLL4.1 and OsPLL4.2.Mutations in StPL21, StPL24, and StPL25 are speculated to affect the floral organ morphology of potato.Additionally, silencing OsPLL3 in Group 6 leads to pollen degradation, which affects the development of floral organs and spikelets [15].StPL5 and StPL6 were assigned to Group 3, which consisted of seven members.AtPLL4 and AtPLL7, also part of this group, showed high expression in the floral organs.It was speculated that StPL5 and StPL6 were associated with flower development [19], while the remaining members in the evolutionary tree had not been functionally characterized.The above results suggest that StPL genes are more closely related to PL genes in other species (Figure 4).belongs to the same branch as StPL13 and StPL20, can be induced by auxin, abscisic acid, and ethylene.Inhibition of its expression will increase the pectin content of tomato fruits, delay fruit rot, and extend the storage period.Therefore, it is hypothesized that StPL13 and StPL20 may be associated with potato fruit hardness [22].AtPLL13 (PMR6) is in the same group as StPL18.The PMR6 mutation causes plants to dwarf, leaves to become small and rounded, and results in increased resistance to powdery mildew in pmr6 plants [21].
The knockout of StPL18 causes morphological changes in potato plants [25].while the remaining members in the evolutionary tree had not been functionally characterized.The above results suggest that StPL genes are more closely related to PL genes in other species (Figure 4).The different colors on the outside of the tree represent the corresponding groups, where green, yellow, orange, red, purple, blue, light purple, and cyan represent Group 1, Group 2, Group 3, Group 4, Group 5, Group 6, Group 7, and Group 8, respectively.

StPL Gene Family Members' Gene Structure and Conserved Motif Analysis
The gene structure and conserved motif analysis of StPL members were conducted using TBtools, revealing differences in the number and types of motifs present in StPL.5a).Screening of the NCBI-CDD [26] conserved domains revealed that all StPLs, except StPL2, have the complete Pec_lyase_C domain.StPL2 has the Pec_lyase_N domain, further identifying all members as belonging to the pectate lyase family (Figure 5b).StPL1, StPL3, StPL4, StPL5, StPL10, StPL11, StPL14, StPL19, and StPL27 have no UTRs, which may have implications for mRNA transport, stability, translational regulation, and incomplete gene annotation [27].

StPL Gene Family Members' Gene Structure and Conserved Motif Analysis
The gene structure and conserved motif analysis of StPL members were conducted using TBtools, revealing differences in the number and types of motifs present in StPL.Six differently positioned and relatively conserved motifs were screened.Most members contain six motifs.StPL2 does not contain any motifs.StPL7 has only one motif.StPL15 has two motifs.StPL23 has three motifs, and StPL11 has five motifs.Taken together, these motifs and their arrangements show a high degree of conservation in the StPL family (Figure 5a).Screening of the NCBI-CDD [26] conserved domains revealed that all StPLs, except StPL2, have the complete Pec_lyase_C domain.StPL2 has the Pec_lyase_N domain, further identifying all members as belonging to the pectate lyase family (Figure 5b).StPL1, StPL3, StPL4, StPL5, StPL10, StPL11, StPL14, StPL19, and StPL27 have no UTRs, which may have implications for mRNA transport, stability, translational regulation, and incomplete gene annotation [27].

Analysis of the Cis-Acting Elements in the Promoter of StPL Gene Family Members
The cis-acting element analysis of StPL promoter regions by the PlantCARE database revealed the presence of numerous hormone response elements in 27 StPL promoter regions (Figure 6).These elements include the auxin response element, abscisic acid response element, methyl jasmonate (MeJA) response element, and gibberellin response element.MeJA is a significant phytohormone abundant in plants, playing a crucial role in various physiological processes like flowering, fruiting, and senescence [28].It has been found that the exogenous application of MeJA elicits a defense response in plants, suggesting that StPL may be involved in the plant's response to adversity stress through

Analysis of the Cis-Acting Elements in the Promoter of StPL Gene Family Members
The cis-acting element analysis of StPL promoter regions by the PlantCARE database revealed the presence of numerous hormone response elements in 27 StPL promoter regions (Figure 6).These elements include the auxin response element, abscisic acid response element, methyl jasmonate (MeJA) response element, and gibberellin response element.MeJA is a significant phytohormone abundant in plants, playing a crucial role in various physiological processes like flowering, fruiting, and senescence [28].It has been found that the exogenous application of MeJA elicits a defense response in plants, suggesting that StPL may be involved in the plant's response to adversity stress through MeJA and wound response elements [29].In addition, defense and stress response elements, low-temperature response elements, and MYB binding sites involved in drought inducibility response elements were present in the promoter regions of the StPL genes.Many light-responsive elements were also found on the promoters of all the StPL genes, further suggesting that the StPL genes are involved in the plant's stress response.
MeJA and wound response elements [29].In addition, defense and stress response elements, low-temperature response elements, and MYB binding sites involved in drought inducibility response elements were present in the promoter regions of the StPL genes.Many light-responsive elements were also found on the promoters of all the StPL genes, further suggesting that the StPL genes are involved in the plant's stress response.

Expression Pattern Analysis of StPL Gene Family Members under Salt Stress
To investigate the response of StPL genes when plants encountered salt stress, we treated potato seedlings under single (Figure 7a) and mixed salt (Figure 7b) conditions.Then, we analyzed the expression of each gene in the transcriptome data and utilized TBtools to generate a differential expression heatmap.The results indicated that most of the members were up-regulated under salt stress, suggesting that members of the family play a role in the potato's response to salt.Under single salt stress, StPL3, StPL4, StPL5, and StPL10 were not expressed.Except for StPL1, StPL7, and StPL23, whose expression decreased under single salt stress, the expression of the other members increased to varying degrees.StPL10 and StPL11 were not expressed under mixed salt stress, while the expression of StPL2, StPL12, StPL13, StPL17, StPL20, StPL21, StPL22, StPL23, StPL24, StPL25, and StPL26 was up-regulated with the duration of treatment under mixed salt stress.This suggests that although the StPL family members play a role in salt stress, there may be a division of labor among them.
In addition, we observed that the expression of StPL18 increased with treatment under both single and mixed salt stress conditions.Its expression was notably distinct from that of other members.Yang discovered that the knockout of the StPL18 gene led to potato plants with more numerous dwarfed branches and yellowing leaf margins, ultimately impacting the yield [25].Therefore, the StPL18 gene will be selected for further study.

Expression Pattern Analysis of StPL Gene Family Members under Salt Stress
To investigate the response of StPL genes when plants encountered salt stress, we treated potato seedlings under single (Figure 7a) and mixed salt (Figure 7b) conditions.Then, we analyzed the expression of each gene in the transcriptome data and utilized TBtools to generate a differential expression heatmap.The results indicated that most of the members were up-regulated under salt stress, suggesting that members of the family play a role in the potato's response to salt.

Construction of Overexpression Vector of Potato StPL18 Gene and Subcellular Localization in Tobacco
To gain a deeper understanding of the StPL18 gene function, both upstream and downstream primers were designed.The reverse primer was designed to exclude the termination codon.PCR amplification was performed using the constructed pEASYT1-StPL18 as a template to produce electrophoretic bands of the expected size.The resulting

Construction of Overexpression Vector of Potato StPL18 Gene and Subcellular Localization in Tobacco
To gain a deeper understanding of the StPL18 gene function, both upstream and downstream primers were designed.The reverse primer was designed to exclude the termination codon.PCR amplification was performed using the constructed pEASYT1-StPL18 as a template to produce electrophoretic bands of the expected size.The resulting products were purified, recovered, and subsequently used for ligation and transformation through homologous recombination with the purified and recovered super1300, utilizing XbaI and KpnI double digestion.Linear vectors were ligated for transformation, and positive colonies were screened, identified by PCR, and sent for sequencing after proper digestion and identification (Figure S1).Under confocal microscopy, the target gene was found to overlap with the endoplasmic reticulum marker in tobacco leaves, indicating that StPL18 is localized in the endoplasmic reticulum (Figure 8).

Analysis of Pectate Lyase Gene Family Members
Potato is the most important tuber crop.Members of the pectate lyase gene family play crucial roles in the growth and development of potato.In this study, 27 members were identified in potato.The physicochemical properties of the StPL indicate that the proteins encoded by most members are hydrophilic.Fragment duplication and tandem duplication are present within the StPL gene family.A total of 15 genes are tandem duplicated and fragment duplicated genes.A comparison with A. thaliana genes reveals that not only fragment duplication but also rearrangement of some chromosomes at different positions may occur among different chromosomes.Six motifs were present in most StPLs, suggesting that StPLs may exhibit relatively conserved functions during potato growth.Evolutionary tree analysis showed that StPL is closely related to PL from other species; this indicates that the PL gene is functionally conservative in different species.In addition, gene structure analysis revealed a high diversity of exon numbers in StPLs, which may be related to their functional diversity.

Expression Pattern of Pectate Lyase Gene Family Members under Salt Stress
Pectin in plant cell walls carries a negative charge and can bind to cations such as Ca 2+  and Na + , enhancing the hardness of the cell wall [30].It has been shown that in high-salt environments, excess Na + may disrupt the normal cross-linking of pectin and inhibit cell

Analysis of Pectate Lyase Gene Family Members
Potato is the most important tuber crop.Members of the pectate lyase gene family play crucial roles in the growth and development of potato.In this study, 27 members were identified in potato.The physicochemical properties of the StPL indicate that the proteins encoded by most members are hydrophilic.Fragment duplication and tandem duplication are present within the StPL gene family.A total of 15 genes are tandem duplicated and fragment duplicated genes.A comparison with A. thaliana genes reveals that not only fragment duplication but also rearrangement of some chromosomes at different positions may occur among different chromosomes.Six motifs were present in most StPLs, suggesting that StPLs may exhibit relatively conserved functions during potato growth.Evolutionary tree analysis showed that StPL is closely related to PL from other species; this indicates that the PL gene is functionally conservative in different species.In addition, gene structure analysis revealed a high diversity of exon numbers in StPLs, which may be related to their functional diversity.

Expression Pattern of Pectate Lyase Gene Family Members under Salt Stress
Pectin in plant cell walls carries a negative charge and can bind to cations such as Ca 2+ and Na + , enhancing the hardness of the cell wall [30].It has been shown that in high-salt environments, excess Na + may disrupt the normal cross-linking of pectin and inhibit cell elongation by replacing Ca 2+ bound to pectin [31].Pectin synthesis and degradation in plants are correlated with salt tolerance.Sos6 mutants, defective in the A. thaliana CSLD5 gene involved in pectin biosynthesis, impede xylan and pectin synthesis in the cell wall.This leads to the increased susceptibility of the mutants to drought and salt stress [32].Meanwhile, the degree of pectin methyl esterification is closely related to the hardness of plant cell walls.Plants regulate the level of pectin methyl esterification primarily through pectin methyl ester transferases (PMEs) and pectin methyl esterase inhibitors (PMEIs) [33].Meanwhile, salt stress can activate PMEs and promote the demethylation of pectin in the cell wall, thereby initiating downstream salt stress mechanisms [34].It has been shown that the SlPL-RNAi fruits of tomato have increased firmness, higher hemicellulose content, and greater resistance to rot and disease [18].In this study, we analyzed the promoter region of StPL genes and found that most of the genes were up-regulated in single and mixed salt stress, indicating that these genes may be involved in the response of potato to salt stress.Yang demonstrated that stpl18 is a yellow margin (YM) gene through knockout.Its mutation resulted in the yellowing of the edges of potato leaves, a decrease in single potato weight, an increase in branching, and an increase in the number of potato tubers [25].From these results, the expression of StPL18 increased with the prolongation of salt stress.During previous research, the mutation of PMR6, the homologous gene of that in A. thaliana, resulted in smaller plants, increased pectin content, enhanced resistance to powdery mildew, and affected stomatal movement [21].It was found that PMR6 influenced the size of A. thaliana's rosette by impacting cell expansion and proliferation.The homologous gene of StPL18 in rice, DEL1, serves the same function in cell cycle progression and leaf growth.The DEL1 mutation led to a reduction in the number of cell cycles, ultimately resulting in dwarf plants due to the blockage of the G1 to S phases of the cell cycle [35].It is hypothesized that the stpl18 mutant plants may have increased salt tolerance due to decreased gene expression causing increased pectin content.
The subcellular localization of StPL18 indicates that it may be located in the endoplasmic reticulum.The endoplasmic reticulum is where all secreted proteins and most membrane proteins are synthesized and folded.When plants face adversity, misfolded and unfolded proteins accumulate in large quantities in the endoplasmic reticulum, causing endoplasmic reticulum stress [36].Elucidating the endoplasmic reticulum stress response signaling pathway in plants provides a basis for understanding the mechanisms involved in responding to plant adversity.Endoplasmic reticulum stress is closely related to various environmental stresses.The up-regulated expression of the StPL18 gene may be subject to the endoplasmic reticulum stress response.

Identification of Members of the Potato PL Gene Family
A search for the keyword "Pectate lyase" in the potato genomics resources SpudDB database (http://spuddb.uga.edu,accessed on 10 November 2023), which identified 93 genes annotated with pectate lyase, and their gene IDs were obtained.Then, download the genome assembly for the doubled monoploid potato DM 1-3 516 R44-v6.1 and genome annotation: DM_1-3_516_R44_potato.v6.1.hc_gene_models.gff3 files from the database.Then, utilize TBtools software V2.086 [37].Within the software, navigate to the Sequence Toolkit, click on GFF3/GTF Manipulate, select GXF Sequence Extract, and proceed to insert the GFF3 file.Finally, extract all gene CDS sequences of potato.Open Fasta tools and then Fasta Extract (recommended).Input the gene ID and CDS file to extract the CDS of StPL.The extracted CDS is translated into protein using the Sequence Toolkit, ORF Prediction, and Batch Translate CDS to Protein.Upload the protein sequence to the NCBI Batch Web CD-Search Tool [38], screen for the structural domains, and only keep proteins containing the Pec_lyase_C and Pec_lyase_N structural domains with Pfam accession numbers PF00544 and PF04431.After screening, a total of 27 StPL genes were obtained.

Chromosomal Localization of Potato StPL Genes
In TBtools, open the "Graphics" tab, select "Show Genes on Chromosome", choose "Gene Location Visualize from GTF/GFF", import the GFF3 file of the potato and the ID of the StPL, enter the "Rename ID" and the "Chromosome Sort ID", and then click "Start".

Analysis of Physicochemical Properties of Potato StPL Protein and Prediction of Subcellular Localization
Protein sequences in FASTA format were uploaded using the online websites Prot-Param (https://web.expasy.org/protparam,accessed on 10 November 2023) [39] and WoLF PSORT (https://wolfpsort.hgc.jp/,accessed on 10 November 2023) [40] to obtain amino acid length, molecular mass, theoretical isoelectric point, instability index, lipid solute number, hydrophilicity results, and predictions of subcellular localization.

Phylogenetic Tree and Gene Structure Analysis of the Potato StPL Gene
The PL gene sequences of tomato, A. thaliana, and rice were obtained from the articles by Lu Yang [18], Lingxia Sun [16], and Yinzhen Zheng [15].The MEGAX software V10.2.6 was used to construct the evolutionary tree.Firstly, the FASTA file was imported, and the meg format file was saved after sequence comparison.The meg file was then imported, and the "Construct/Test Neighbor-Joining Tree" option under PHYLOGENY was selected with the following parameter settings: the Bootstrap value was set to 500, and the model for genetic distance calculation was set to P-distance.Partition deletion was set to 50%, while the rest of the parameters were kept as default.The evolutionary tree was further enhanced using ITOL v6 (https://itol.embl.de,accessed on 10 November 2023), online software [41].
Use TBtools, click on "Others", then select "MEME Suite Wrapper", and finally choose "Simple MEME Wrapper".Upload protein sequences from StPLs and set the output path.The number of motifs to find is set to 6, and the maximum E-value is set to 0.0001.The mode selection is set to zero or one occurrence per sequence to obtain meme xml file.
Use NCBI-CDD to obtain the prediction result of the conservative domain.Download Excel sheet file.
Using TBtools, select Gene Structure View, insert the protein ID, potato GFF3 file, motif xml file and NCBI-CDD excel sheet file, then click Start to analyze, and obtain the results.

Gene Collinearity Analysis
Open TBtools, select "Others", then choose "Plugin", followed by "Plugin Store".Check "One Step MCScanX [Super-Fast] by CJ" in the menu bar and click the "Install" button to install the selected plugin.Select "Others" > "Plugin" > "Plugin Store", then choose "One Step MCScanX [Super-Fast] by CJ" from the menu bar and click on the "Install Selected Plugin" button to install it.The genome files of A. thaliana and potato were imported using the One Step MCScanX plugin, and the gene covariance schematic was generated.

Analysis of Potato StPL Promoter Cis-Acting Elements
Open TBtools, go to Graphics, select Gene Structure View (Advanced), import StPL gene ID, DM V6.1 GFF3 file, ID rename file, and click on start.Use TBtools and the GTF/GFF3 Sequence Extract tool to extract the 2000 bp upstream of all coding sequences (CDS) of potato.Utilize Quick Fasta Extractor or Filter to extract the 2000 bp upstream of the CDS of the StPL gene.Then, open Fasta Sequence Manipulator to convert the sequence to uppercase.Convert the sequence to uppercase.The extracted files were submitted to the PlantCare website (http://bioinformatics.psb.ugent.be/webtools/plantcare,accessed on 10 November 2023) [42].For predicting the cis-acting elements.The predicted table files were analyzed using Excel, then imported into TBtools.Subsequently, the Graphics option was selected, followed by the Simple BioSequence Viewer to draw the schematic diagram of promoter cis-acting elements.

Analysis of the Expression Pattern of StPL Family Genes in Potato Seedlings under Single and Mixed Salts
The plant material used in this study is diploid potato 320-02.This material is closely related to the reported diploid potato RH, both belonging to the Tuberrosum subspecies of potato.A total of 320-02 were subjected to different time-lengths of NaCl (100 mmol/L) single salt and equal volume mixed-salt treatments of NaCl + Na 2 SO 4 (100 mmol/L), respectively; the processing time points were 0, 6, 24, and 48 h, with three replicates per time point.
Take samples for transcriptome sequencing.
The resulting transcriptome data were compared to the potato reference genome DM (V6.1) using HISAT2 (V2.2.1) to construct an index.HISAT2 compared the reads of each sample to the genome to obtain SAM files, which were then sorted and converted to BAM files using Samtools [43].Splicing transcripts used StringTie software (V2.2.3) and conducted a reference transcriptome analysis to estimate transcript expression and use R language script Ballgown for further analysis [44,45].The gene expression levels were calculated according to the FPKM method.The heatmap of gene expression of pectate lyase gene family was produced using TBtools.

Subcellular Localization of StPL Genes
To obtain the subcellular localization information of StPL proteins, a fusion protein expression vector of StPL18 and GFP was constructed.GFP was utilized as a reporter gene, and the position of GFP was observed to determine the location of StPL proteins.The recombinant protein, with GFP fused to the coding region of the StPL gene and the stop codon removed, was expressed using the 35S promoter.The subcellular localization of the protein was analyzed later.Sper was employed as an endoplasmic reticulum localization signaling protein to construct a fusion protein with the red fluorescent protein mKATE [46].
Experimental Steps (1) Pick Agrobacterium containing the corresponding vectors and single colonies of Agrobacterium containing the P19 plasmid.Inoculate them into 10 mL of LB liquid medium with the corresponding resistance and incubate them for 24 h at 28 • C and 220 rpm until the OD600 value greater than 0.6.(2) Centrifuge at 5000 rpm for 2 min and discard the liquid medium.
(3) Resuspend the organisms by adding 700 µL of tobacco infestation buffer, mix well, and take 200 µL of the bacterial solution to measure the OD600 value.(4) Mix Agrobacterium with different vectors in a 2 mL centrifuge tube so that the OD600 value of Agrobacterium containing the P19 plasmid is adjusted to 0.3, and other Agrobacterium is adjusted to 0.5.Leave the mixture at room temperature for about 3 h.Afterward, carefully pipette the well-grown Agrobacterium from the center of the centrifuge tube and discard the rest.(5) Take the tobacco leaves (tobacco has about four true leaves and is 10 cm tall) with good growth conditions and inject Agrobacterium from the back of the leaves using a 1 mL syringe until the leaves are completely wet.(6) Incubate in the dark for 24 h and then transfer to dim light culture for 36 h.(7) For co-localization, the marker plasmid was transformed into Agrobacterium, operating in suspension with the constructed vector plasmid Agrobacterium, and mixed at an equivalent ratio before injection into tobacco leaves.

Conclusions
By analyzing the PL gene family in potato, StPL genes were classified into eight subfamilies based on phylogeny and chromosomal localization.All StPLs had a conserved Pec_lyase_C domain and diverse gene structures.Expression analysis revealed that StPLs exhibited different expression patterns under various salt treatments.These results suggest that StPLs play important roles in plant growth, development, and stress response.
In this study, 27 StPL genes in potato were screened using salt treatments.Eventually, the StPL18 protein, located in the endoplasmic reticulum, garnered attention.It was discovered to regulate the morphogenesis of potato, as previously researched, and StPL18 was up-regulated under different salt treatments.The elevated expression of pectin cleavage enzymes destabilizes the cell wall structure, compromising its protective function.This allows harmful ions like Na + to penetrate the cell, increasing the plant's salt sensitivity.This sensitivity can be heightened by acquiring material from the pl mutant.Mutations in the PL gene impact the weight of potato tubers but result in more branches and tubers, which is advantageous for certain consumers preferring smaller tubers.Therefore, this gene may hold significant value in enhancing potato salt tolerance and cultivating new varieties.

Figure 1 .
Figure 1.Genomic distribution of StPLs on potato chromosomes.Chromosome numbers are on the left and StPLs are on the right of chromosomes.Scale bar on the left indicates chromosome length.

Figure 1 .
Figure 1.Genomic distribution of StPLs on potato chromosomes.Chromosome numbers are on the left and StPLs are on the right of chromosomes.Scale bar on the left indicates chromosome length.

Figure 2 .
Figure 2. The collinearity analysis of StPL genes is represented via a Circos map, exhibiting the relationships among the gene pairs.Gene pairs are represented by red lines.

Figure 3 .
Figure 3. Collinearity analysis of the PL gene family in potato and the PL gene family in Arabidopsis.The red lines represent genes within these two families that have collinearity relationships.The gray color represents genes with no covariate relationship.

Figure 2 .
Figure 2. The collinearity analysis of StPL genes is represented via a Circos map, exhibiting the relationships among the gene pairs.Gene pairs are represented by red lines.

Plants 2024 , 16 Figure 2 .
Figure 2. The collinearity analysis of StPL genes is represented via a Circos map, exhibiting the relationships among the gene pairs.Gene pairs are represented by red lines.

Figure 3 .
Figure 3. Collinearity analysis of the PL gene family in potato and the PL gene family in Arabidopsis.The red lines represent genes within these two families that have collinearity relationships.The gray color represents genes with no covariate relationship.

Figure 3 .
Figure 3. Collinearity analysis of the PL gene family in potato and the PL gene family in Arabidopsis.The red lines represent genes within these two families that have collinearity relationships.The gray color represents genes with no covariate relationship.
Many PL genes are related to flower development.Silencing OsPLL4 results in abnormal pollen microspores, while StPL21, StPL24, and StPL25 belong to the same evolutionary branch as OsPLL4.1 and OsPLL4.2.Mutations in StPL21, StPL24, and StPL25 are speculated to affect the floral organ morphology of potato.Additionally, silencing OsPLL3 in Group 6 leads to pollen degradation, which affects the development of floral organs and spikelets [15].StPL5 and StPL6 were assigned to Group 3, which consisted of seven members.AtPLL4 and AtPLL7, also part of this group, showed high expression in the floral organs.It was speculated that StPL5 and StPL6 were associated with flower development [19],

Figure 4 .
Figure 4. Neighbor joining phylogenetic tree of potato (St), Arabidopsis (At), tomato (Sl), and rice (Os), where StPL in potato is highlighted.The different colors on the outside of the tree represent the corresponding groups, where green, yellow, orange, red, purple, blue, light purple, and cyan

Figure 4 .
Figure 4.Neighbor joining phylogenetic tree of potato (St), Arabidopsis (At), tomato (Sl), and rice (Os), where StPL in potato is highlighted.The different colors on the outside of the tree represent the corresponding groups, where green, yellow, orange, red, purple, blue, light purple, and cyan represent Group 1, Group 2, Group 3, Group 4, Group 5, Group 6, Group 7, and Group 8, respectively.
Plants 2024, 13, 1322 7 of 15 Six differently positioned and relatively conserved motifs were screened.Most members contain six motifs.StPL2 does not contain any motifs.StPL7 has only one motif.StPL15 has two motifs.StPL23 has three motifs, and StPL11 has five motifs.Taken together, these motifs and their arrangements show a high degree of conservation in the StPL family (Figure

Figure 5 .
Figure 5. Gene structure diagram of StPL gene family.(a) Conserved motifs in the family, where different colors represent different motifs.A maximum of 6 motifs were searched with motif length between 10 and 50 residues.Motif occurrence was set to zero or one occurrence per sequence.(b) Conserved domains possessed by members of the family, where different colors represent different conserved domains; (c) Gene structure of StPLs, where blue square represents UTR and yellow square represents CDS, the line represents Intron.

Figure 5 .
Figure 5. Gene structure diagram of StPL gene family.(a) Conserved motifs in the family, where different colors represent different motifs.A maximum of 6 motifs were searched with motif length between 10 and 50 residues.Motif occurrence was set to zero or one occurrence per sequence.(b) Conserved domains possessed by members of the family, where different colors represent different conserved domains; (c) Gene structure of StPLs, where blue square represents UTR and yellow square represents CDS, the line represents Intron.

Figure 6 .
Figure 6.Cis-acting elements predicted in 2000 bp sequence upstream of the StPL gene.Different colored squares represent various types of cis-acting elements.

Figure 6 .
Figure 6.Cis-acting elements predicted in 2000 bp sequence upstream of the StPL gene.Different colored squares represent various types of cis-acting elements.

16 Figure 7 .
Figure 7. Expression patterns of the members of the potato StPL gene family under single salt and mixed salt stress.(a) Expression levels of the members of the family under single salt; (b) expression levels of the members of the StPL family under mixed salt.The color bar in the figure represents the log2 expression values, and the expression values were normalized using TPM + 1.

Figure 7 .
Figure 7. Expression patterns of the members of the potato StPL gene family under single salt and mixed salt stress.(a) Expression levels of the members of the family under single salt; (b) expression levels of the members of the StPL family under mixed salt.The color bar in the figure represents the log2 expression values, and the expression values were normalized using TPM + 1.

Table 1 .
Physiological properties and subcellular localization of the StPL gene family.