Arabidopsis thaliana Columbia-0 (Col-0) seeds were obtained from Nanjing University. To promote growth, the seeds were sterilized using 10% sodium hypochlorite for 4 min, rinsed four times with double-distilled water, and planted on solidified Murashige and Skoog (MS) or 1/4 MS medium with 1% sucrose (Aldrich-Sigma) and 1% agar (Aldrich-Sigma). The growth phenotype of Pep13 in Arabidopsis was assessed as previously described (Jing et al., 2019). The plates were initially incubated in darkness at 4°C for 2 days and then positioned vertically. On day 10 after germination, primary root lengths were measured using ImageJ software (http://rsb.info.nih.gov/ij/) and the biomass was weighed.

The potato variety Unk ‘Atlantic’ was selected to investigate the effects of Pep13. This variety was obtained from the Institute of Vegetables and Flowers of the Chinese Academy of Agricultural Sciences. After germination at 22°C, the pre-elite seeds were then transplanted into a solution containing 1/4 potato Murashige and Skoog (MS) basal salts with vitamins (Coolaber). The initial pH of this solution was 5.5. The cultivation of the potatoes took place in growth chambers set at 25°C, with a 12-h light/12-h dark photoperiod.

The potato P-type ATPase gene was identified using potato gene, transcript, and protein sequences downloaded from the Phytozome website (Solanum tuberosum v6.1, Phytozome genome ID: 686, and NCBI taxonomy ID: 4113) (Pham et al., 2020). The P-type ATPase domain (sequence numbers: PF00690, PF00122, and PF13246) was used as a reference (Zhang et al., 2020) to search for P-type ATPase in the Potato Protein Sequence Database using the Hidden Markov Model (HMM). Sequences containing the structural domain of the P-type ATPase were identified and species-specific models were created. Candidate potato P-type ATPase gene sequences were further confirmed using BLAST (1e-10) to ensure their membership in the P-type ATPase gene family. Redundant sequences were removed and the resulting P-type ATPase protein sequences were submitted to the Pfam (http://pfam.janelia.org/), NCBI Conserved Domain (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), and SMART (http://smart.em-bl-heidelberg.De) databases for additional confirmation of the P-type ATPase motifs. Finally, erroneous and repetitive sequences were eliminated to identify the members of the P-type ATPase gene family. Secondary and tertiary structures of the proteins were predicted using the SOPMA database (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_server.html) and Phyre2 software (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index), respectively (Gasteiger, 2005; Kelley et al., 2015).

Multiple sequence comparisons were performed using the ClustalW tool in MEGA 7 software on the sequences of the P-type PM H⁺-ATPase proteins of Solanum tuberosum (PHA), Solanum lycopersicum (HA), Arabidopsis thaliana (AHA), Oryza sativa (OSA), Helianthus annuus (HHA), and Nicotiana plumbaginifolia (PMA) (Stritzler et al., 2017; Xu et al., 2020). Subsequently, neighbor-joining (NJ) phylogenetic trees were constructed based on the Poisson model, and the robustness of the dendrograms was evaluated by setting bootstrap values for 1000 times. To gain a deeper understanding of the potential properties of these protein sequences, themes in the sequences were further predicted using MEME software (http://meme-suite.org/) (Lu et al., 2021).

The chromosomal distribution of gene family members was determined using the GFF file of the reference genome v6.1 and the distribution was visualized using the MG2C online web service (http://mg2c.iask.in/mg2c_v2.1/). Gene duplication and collinearity analyses were conducted and visualized using the multicollinearity scanning function module in the TBtools software, as described in our previous study (An et al., 2022).

Total RNA was extracted from potato and Arabidopsis roots using the TRIzol reagent (Invitrogen). The extracted RNA was used to synthesize poly(dT)-complementary DNA using M-MLV reverse transcriptase (Promega). For reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the SYBR Green I Master Kit (Roche Diagnostics) was used on a CFX Connect Real-Time System (Bio-Rad), following the manufacturer’s instructions. RT-qPCR primers for eight members of the PM H⁺-ATPase gene family were designed using Primer Premier 5.0. ACTIN (Soltu.DM.03G011750.2) was used as an internal reference gene (Zhang et al., 2022). The primers used are listed in Supplementary Table S1 . Each sample was replicated three times and differential expression was determined using the 2 [- Delta C(T)] method (Livak and Schmittgen, 2001).

The Pep13 small peptide (sequence VWNQPVRGFKVYE) utilized in this study was synthesized by Sangon Biotech (Shanghai, China). To meet the experimental requirements, the peptide was dissolved in water to prepare a stock solution at a concentration of 1 mM.

The Atlantic root was chosen as the source for RNA extraction, and total RNA was isolated using an extraction kit from Tiangen Biochemical Technology Co., Ltd. (Beijing, China). Reverse transcription was used to synthesize cDNA. PCR amplification was conducted using the cDNA as a template with the primers PHA2-F1 and PHA2-R1 ( Supplementary Table S1 ). The amplified products were separated by 1% agarose gel electrophoresis. The concentration of the PHA2 fragment was determined after recovery using a DNA Gel Extraction Kit. The recovered products were polyadenylated using the Taq enzyme and ligated into the pMD 19-T vector. The ligated vector was transformed into E.coli DH 5α competent cells and the cells were cultured on a screening medium containing antibiotics. Finally, monoclonal colonies were selected for PCR and their accuracy was confirmed by sequencing.

To create expression vectors for the PHA2 transgenic expression vector, the PHA2 coding sequence (CDS) was amplified from cDNA obtained from the Atlantic. For Arabidopsis transformation, the PHA2 sequence was cloned into the SpeI/NheI sites of the plant expression vector pCAMBIA1302, resulting in the generation of the plant overexpression vector pCAMBIA1302–35S-PHA2. The resulting vector was then transferred into Agrobacterium tumefaciens strain GV3101, and Arabidopsis Col-0 plants were transformed using the floral dip method (Clough and Bent, 1998). The seeds were sterilized and plated on a solid 1/2 MS medium containing 35 μg mL^–1 of kanamycin. After 4 days of cultivation at 4°C, the seeds were transferred to a growth chamber set to a light cycle of 16 h of light and 8 h of dark at 25°C for 8 days. The plants were then transplanted into the soil. To screen for positive plants, specific primers ( Supplementary Table S1 ) were designed for the PCR identification of transgenic Arabidopsis. Three heterologously overexpressing homozygous T3 transgenic lines (OE strains) were obtained for each gene and the best OE-expressing strain (OE–PHA2-#5) was selected for subsequent Pep13 experiments ( Supplementary Figure S1 ).

To investigate the effects of Pep13 treatment on Atlantic roots, a 1/6 MS solution without sucrose (pH 5.8) was used as the growth medium. Three-week-old Atlantic roots were transferred to medium with or without 20 nM Pep13. After 3 d of treatment, the roots were collected for further analysis. Plasma membrane vesicles were prepared from the collected roots following a previously described protocol (Shen et al., 2005). To determine the feasibility of the assay, 10 µM vanadate (VA) was used. The vanadate-sensitive ATPase accounted for 85% of the total activity in the plasma membrane fraction. The activity of the PM H⁺-ATPase was measured at a wavelength of 700 nm using a U-2910 spectrophotometer (HITACHI, Tokyo, Japan).

Statistical analyses were conducted using Student’s t-test (P<0.05) on at least three independent replicates for all experiments.

This study successfully identified 48 members of the potato P-type ATPase gene family from the potato genome database using Biastp online comparison and an HMMER search. This is similar to Arabidopsis and rice, which have 46 and 43 P-type ATPase genes, respectively (Palmgren, 2001; Baxter et al., 2003). To better understand the chromosomal distribution of P-type ATPase genes, we used the potato genome database to create a chromosomal localization map ( Figure 1 ). The findings revealed that these genes were unevenly distributed across the 12 chromosomes. Chromosome chr09 exhibited the highest number of markers, encompassing eight genes. In contrast, chromosome chr10 had fewer markers, with only one gene identified as Soltu.DM.10G028130.1. The 48 identified PTAP protein sequences were used to construct a phylogenetic tree to determine the subfamily classification of potato P-type ATPase genes. The results showed that the potato P-type ATPase genes could be categorized into five subfamilies ( Supplementary Figure S2 ), which is consistent with findings in other plants (Palmgren and Nissen, 2011; Pedersen et al., 2012). Among these, the number of genes in the P4 subfamily was low, whereas the number of genes in the other subfamilies was high. Interestingly, the eight PM H⁺-ATPase members of the P3A subfamily have relatively similar evolutionary homology.

Gene duplication, tandem repeats, and whole-genome duplication are important factors driving the expansion of plant gene families. To elucidate the expansion and evolutionary characteristics of the P-type ATPase gene family in potatoes, we conducted whole-genome segmental duplication and collinearity analyses. Among all members of the gene family, only seven gene pairs (comprising 12 genes) were involved in the formation of segmental duplications ( Figure 2A ; Supplementary Table S2 ). The results indicate that tandem repeats of the P-type ATPase gene family mainly occur on chromosomes 7, 8, and 9. In particular, eight tandem gene family members on chromosome 9 formed a gene cluster ( Figure 2A ), which may have distinct functional characteristics compared to other family members. Collectively, these results indicate that both segmental and tandem duplications play roles in the expansion of the P-type ATPase gene family. As is well known, members of the same gene family in different species share a common ancestral origin, and collinearity analysis can effectively reveal the evolutionary relationships of specific gene families among different species. In the present study, we conducted a whole-genome collinearity analysis of potatoes with Arabidopsis, rice, and tomatoes ( Figures 2B-D ). These results indicate that compared to rice, potato and tomato genomes are highly syntonic, with more collinear gene pairs present. Additionally, many collinear gene pairs have been identified between potato and Arabidopsis, suggesting that the P-type ATPase gene family may have undergone a similar evolutionary history in potato, Arabidopsis, and tomato plants.

A motif is a short sequence in a protein with specific functional or structural features that can affect its structure, stability, and activity. Motifs can also serve as signal sequences that guide protein localization and transport within cells. For the potato P-type ATPase gene family, we identified ten conserved motifs, with each protein sequence containing between one and eight motifs ( Figure 3 ). For the potato P-type ATPase gene family, we identified 10 conserved motifs, with each protein sequence containing between one and eight motifs. Among these, some highly conserved motifs (such as motif 5) were identified in every protein sequence, whereas motifs 9 and 10 were found only in Class III protein sequences. Cis-acting elements in promoter regions regulate gene transcription. In addition to the identification of a large number of light-responsive elements, many hormone-related elements, such as ABA and GA response elements, were found in the promoter region of the P-type ATPase gene family ( Figure 4A ). Furthermore, many promoters contain cis-acting elements that respond to stress such as low temperature and drought, suggesting that members of the P-type ATPase gene family may play diverse functional roles in potatoes. Additionally, the results of the gene structure, phylogenetic tree, and protein motif analyses were highly consistent and all members of the P-type ATPase gene family were classified into three major classes. Overall, compared with Classes I and III, Class II genes contained fewer CDS, whereas Class I sequences contained many longer intron sequences than Class III ( Figure 4B ).

As a superfamily of membrane proteins, P-type ATPases play important roles in plants and are involved in several fundamental physiological processes (Zhang et al., 2020). Particularly noteworthy are the PM H⁺-ATPase family members of the P3 subfamily, which play key roles in plant growth, development, and stress resistance (Morth et al., 2011; Falhof et al., 2016; Li et al., 2022). These enzymes maintain the stability of the intra- and extracellular environments by hydrolyzing ATP to drive ion transport and maintain electrochemical equilibrium across the membrane (Morth et al., 2011). In this study, we used 58 PM H⁺-ATPase protein sequences from six different species (Solanum tuberosum, Solanum lycopersicum, Arabidopsis thaliana, Oryza sativa, Helianthus annuus, and Nicotiana plumbaginifolia) to construct maximum likelihood trees to explore possible functional clustering relationships among PM H⁺-ATPase family member genes ( Figure 5 ). The results showed that PM H⁺-ATPases could be classified into five subfamilies and PM H⁺-ATPase genes from species with close relatives tended to cluster. For example, PMA3 and PHA3 in sub-cluster I clustered with a bootstrap value of 100. AHA1, AHA1, OSA7, HHA9, AHA10, and PMA9 also formed a preferred cluster in Subcluster II. The PM H⁺-ATPase genes of Helianthus annuus were mainly clustered together in sub-cluster III. Interestingly, the PM H⁺-ATPase genes of potato and tomato showed closer affinity in the phylogenetic tree, whereas AHA7 formed a separate branch and was related to rice OSA10 and Arabidopsis AHA7. In conclusion, this study provides an in-depth analysis of the functional clustering relationships among PM H⁺-ATPase family member genes and lays the groundwork for further research on the functions of these genes.

To further investigate potato PM H⁺-ATPase family proteins, their spatial structures were predicted using an online software. An analysis of the secondary structures of the PM H⁺-ATPase family proteins revealed that all proteins consist of three different structural units, including α-helices, random coils, and extended chains. However, the proportion and sequential distribution of these structural units vary among proteins. Random coils had the highest proportion, ranging from 50.22% to 55.24%, followed by α-helices, ranging from 35.95% to 39.43%. The proportion of extended chains varied less among different proteins, ranging from 8.10% to 10.71% ( Supplementary Figure S3 ). Interestingly, among all PHA family proteins, PHA8 had the highest percentage of α-helical structures. PHA6, on the other hand, had the highest percentage of random coil structures and lowest percentage of extended strands, while the opposite was true for PHA5. Based on the publicly reported cryo-electron microscopy structure of AHA2, the tertiary structure of the PHA protein family was predicted using Phyre2 online software. The results showed that the tertiary structures of PHA2, PHA3, and PHA5 were similar to that of AHA2 in Arabidopsis ( Figure 6 ). These findings provide important clues for exploring the function and mechanism of action of PM H⁺-ATPases and lay the foundation for a deeper understanding of the biological functions of the PM H⁺-ATPase protein family and their roles in resistance to late blight.

Examining the differences in the expression of PHAs in various organs provides insights into the functional roles of PM H⁺-ATPase family members in different organs. The expression levels of different PHAs vary across roots, stems, leaves, sprouts, and tubers of potatoes ( Figure 7 ). In the roots and tubers, PHA2, PHA3, and PHA7 exhibited the highest expression levels (10-fold, 7.8-fold, and 3.8-fold, respectively), followed by leaves. The flowers exhibited the lowest expression levels. In the leaves, PHA6 showed relatively high expression levels, which were 7.5-fold higher than those in the roots, followed by the stems, where the expression levels were the lowest among the organs. Additionally, PHA6 exhibited higher expression levels in flowers than in other organs but was lower overall than in other genes. In summary, there were differences in expression among PHA family members in different organs, with significantly higher expression of PHA2 and PHA3 in the roots and high expression of PHA5 and PHA6 in flower petals. Interestingly, there was no detectable expression of potato PHA8 in any of the tissues, similar to the results in tomato (Liu et al., 2016). These findings provide significant clues for understanding the functional characteristics of PHA gene family members in different organs.

In this study, potatoes were treated with Pep13, and the expression of the PM H⁺-ATPase subfamily genes PHA1, PHA2, PHA3, PHA4, PHA5, PHA6, PHA7, and PHA8 was analyzed in the roots, stems, and leaves ( Figure 8 ). The results showed that the expression levels of PHA1, PHA2, PHA3, and PHA7 decreased in Pep13-treated roots. Specifically, PHA2 expression was significantly down-regulated after Pep13 treatment. However, in the leaves, only PHA1 showed some degree of up-regulation. In contrast, the expression levels of seven of the eight genes remained unchanged in the stems. The highest expression level of the PHA8 gene was found in the Pep13-treated potato roots, stems, and leaves. This suggests that Pep13 treatment may affect extracellular alkalinization in potato roots and modulate resistance to late blight pathogens by regulating the expression of the PHA2 gene. Notably, PHA8 was expressed only under Pep13 treatment conditions and its expression could not be detected in the absence of stress. This finding is similar to that previously reported for tomatoes (Liu et al., 2016, 2020). Taken together, these findings indicate that Pep13 treatment significantly affects the expression levels of the PM H⁺-ATPase subfamily genes PHA1, PHA2, PHA3, and PHA8 in potato roots and leaves. These findings provide important clues for further studies on the mechanisms underlying the role of PM H⁺-ATPases in potato late blight resistance.

To investigate the role of PHA2 in the development of potato late blight, we conducted experiments using wild-type (Col-0) and heterologously overexpressing plants (OE-PHA2) plants treated with different concentrations of Pep13 (0, 5, 10, and 20 nM). The results revealed no significant differences in the growth phenotypes between Col-0 and OE-PHA2 without Pep13 treatment ( Figure 9 ). However, upon treatment with Pep13, the growth phenotype of OE-PHA2 changed significantly. In particular, root growth was severely inhibited and the degree of inhibition increased at higher Pep13 concentrations ( Figure 9A ). In contrast, Col-0 exhibited better growth after Pep13 treatment, with less severe inhibition of root growth than OE-PHA2. Biomass relative to root length and fresh weight displayed similar trends ( Figures 9B, C ). These findings suggest that the overexpression of PHA2 weakens Arabidopsis resistance to Pep13 treatment, consequently reducing its resistance to late blight pathogens. Interestingly, AHA2 activity in Arabidopsis has been detected using a pH dye (bromocresol violet) (Wang et al., 2022). The results demonstrated that acidification near the roots was affected by Pep13-treated conditions in both Col-0 and OE-PHA2 plants but the acidification levels were higher in OE-PHA2 plants than in Col-0 plants ( Figure 9D ). These observations indicated that PHA2 plays a crucial role in late blight development and may be involved in the regulation of plant resistance to pathogens.

To investigate the effect of Pep13 on the enzymatic activity of potato PM H⁺-ATPase, a series of experiments were conducted. The medium was supplemented with VA, Pep13, and their combination and the growth of Atlantic potato roots was observed. The results demonstrated that Pep13 significantly inhibited the growth of potato roots. However, when VA was added, the extent of the inhibition caused by Pep13 was alleviated ( Figure 10A ). Analysis of relative root length supported these findings ( Figure 10B ). To further investigate the enzymatic activity of PM H⁺-ATPase in the root system, PM H⁺-ATPase was extracted from Atlantic potato roots subjected to various treatments. Enzyme activity was then measured. After 3 days of treatment with 10 µM VA, PM H⁺-ATPase activity in potato Atlantic roots was inhibited by approximately 85% compared to the control ( Figure 10C ). Interestingly, PM H⁺-ATPase activity in potato Atlantic roots was lower when both VA and Pep13 were present than when only Pep13 was present ( Figure 10C ). These observations suggest that invasion by potato late-blight pathogens can inhibit the activity of PM H⁺-ATPase, which in turn leads to extracellular alkalinization as a signaling molecule. This signaling molecule regulates processes such as growth inhibition, increased immunity, and improved environmental adaptation.