Genome-Wide Identification and Expression Analysis of the PEPC Gene Family in Zanthoxylum armatum Reveals Potential Roles in Environmental Adaptation

Simple Summary

This study presents the first comprehensive analysis of the PEPC (Phosphoenolpyruvate carboxylase) gene family in the Sichuan pepper plant (Zanthoxylum armatum). PEPC, a key enzyme in photosynthetic carbon fixation and stress response, was investigated using integrated bioinformatics and molecular approaches. Twelve PEPC genes were identified, exhibiting distinct expression patterns across environmental gradients. Notably, the highest expression levels were observed in high-altitude, high-light regions of Yunnan, China. This finding indicate that these genes may help Z. armatum adapt to diverse climates and resist stresses such as drought. This finding provides genetic resources and a theoretical basis for improving photosynthetic efficiency, stress response, and fruit quality in Z. armatum, offering important value for promoting the breeding and sustainable development of this economically significant crop.

Abstract

Phosphoenolpyruvate carboxylase (PEPC) is a crucial enzyme in plant photosynthesis and stress responses, yet its gene family remained uncharacterized in Zanthoxylum armatum. This study presents the first genome-wide identification and comprehensive analysis of the PEPC gene family in Z. armatum. A total of 12 ZaPEPC genes were identified and classified into plant-type (PTPC) and bacterial-type (BTPC) subfamilies based on phylogenetic analysis. These genes exhibited conserved protein domains but distinct gene structures, with evidence of gene duplication events contributing to family expansion. Promoter analysis revealed an abundance of stress- and hormone-responsive cis-elements, particularly those related to light, abscisic acid (ABA), and methyl jasmonate (MeJA). Expression profiling demonstrated that ZaPEPC genes display environment-specific expression patterns, with ZaPEPC7 and ZaPEPC11 showing significantly higher expression in high-altitude, high-light environments (Yunnan) compared to other regions (Shandong and Chongqing). Co-expression network analysis further indicated interactions between specific ZaPEPCs and stress-related transcription factors. These findings systematically reveal the molecular characteristics and potential roles of the ZaPEPC gene family in environmental adaptation, providing valuable genetic resources and a theoretical foundation for improving stress tolerance and photosynthetic efficiency in Z. armatum through molecular breeding.

1. Introduction

Zanthoxylum armatum DC. commonly known as Sichuan pepper, is an important characteristic plant in China with both medicinal and edible applications. It plays an increasingly vital role in regional economic development and rural revitalization [1]. China is rich in Z. armatum resources, with its cultivation area expanding at an annual rate of 20–30% and an annual output exceeding one million metric tons [2]. Owing to its unique numbing and aromatic flavor, along with a variety of bioactive compounds, it enjoys considerable market popularity. This crop not only generates substantial economic benefits for farmers in mountainous regions but also holds broad application potential in the food, spice, and traditional Chinese medicine industries [3]. However, climate change-induced abiotic stressors, such as extreme heat and drought, are increasingly threatening global agricultural productivity. These challenges not only jeopardize the yield and quality of staple crops like wheat [4] but also pose serious threats to the sustainable cultivation of many high-value cash crops. As a representative high-value cash crop, Z. armatum faces significant industrial challenges. Its cultivation is mainly concentrated in topographically complex mountainous and hilly areas, where it is often exposed to multiple abiotic stresses, including seasonal drought, poor soil conditions, high temperature and intense light, as well as low temperature and frost [5,6,7,8]. These factors severely constrain its growth and development, the accumulation of secondary metabolites, and ultimately the improvement of both yield and quality. Therefore, a deeper understanding of the molecular regulatory networks underlying the environmental stress responses of Z. armatum is crucial for breeding stress-resistant varieties and ensuring the stability and sustainable development of this industry.

During long-term evolution, plants have developed complex and sophisticated molecular mechanism to cope with environmental stresses [9]. These mechanisms involve the coordinated regulation of multiple key gene families, which enhance a plant’s adaptive capacity by modulating its physiological and biochemical processes. For example, in wheat, the BAG co-chaperone family has been shown to play a central role in mediating responses to both heat and cold stress through its molecular chaperone activity [10]. Among these regulatory mechanisms, phosphoenolpyruvate carboxylase (PEPC), a key node enzyme linking primary carbon and nitrogen metabolism, has functions extending far beyond its initially recognized role in photosynthesis in C4 and crassulacean acid metabolism (CAM) plants [11,12]. In C3 plants, including woody species, PEPC catalyzes the irreversible carboxylation of phosphoenolpyruvate (PEP) to generate oxaloacetate (OAA). This reaction provides substrates for replenishing carbon skeletons in the tricarboxylic acid (TCA) cycle, supports the synthesis of amino acids such as aspartate, and helps maintain intracellular pH homeostasis [13,14]. A growing body of evidence indicates that PEPC plays a central role in plant responses to various abiotic and biotic stresses, including drought, salinity, low temperature, nutrient deficiency, and pests and diseases [15,16,17,18,19]. For instance, under stress conditions, the upregulation of PEPC activity enhances the synthesis of organic acids, thereby contributing to ion balance and redox homeostasis. Concurrently, its carbon fixation function supports the reallocation of energy and carbon skeletons necessary for effective stress responses [11,20,21].

The PEPC enzyme is encoded by a multigene family. In model species such as Arabidopsis thaliana [22], Oryza sativa (rice) [23], and Setaria italica (foxtail millet) [24], the PEPC gene family members have been systematically identified and classified into distinct subfamilies—primarily plant-type (PTPC) and bacterial-type (BTPC)—based on gene structure and phylogenetic relationships [16,21,25]. These members exhibit significant functional diversification: some perform essential housekeeping roles, while others are specifically induced to participate in stress response processes [13,16,22,23]. Plant-type PTPCs are homotetramers composed of approximately 100–110 kDa polypeptides. Their encoding genes typically contain 9–10 introns. These PEPCs are characterized by a conserved serine residue at the N-terminus and a distinctive C-terminal QNTG (Gln-Asn-Thr-Gly) tetrapeptide motif. In contrast, bacterial-type BTPCs form heterooctamers consisting of 116–118 kDa polypeptides. The genes encoding BTPCs possess a greater number of introns (19–21), and the resulting proteins lack the N-terminal serine, instead featuring a C-terminal (R/K) NTG (Arg/Lys-Asn-Thr-Gly) tetrapeptide [13,23]. The presence of the N-terminal serine is a hallmark of plant-type PEPCs, allowing reversible phosphorylation by PEPC kinase—a key regulatory mechanism absent in bacterial-type enzymes [26]. This post-translational modification not only governs primary carbon fixation in C4 and CAM plants but is also widely involved in carbon metabolism regulation in non-photosynthetic tissues, playing critical roles in processes such as seed germination and storage compound synthesis in cereals [27]. Bacterial-type PEPCs, on the other hand, primarily function to replenish tricarboxylic acid cycle intermediates and recapture CO₂ released during respiration [14,23]. Beyond these hallmark N-terminal and C-terminal signatures, comparative analyses of PEPC proteins across diverse plant species have revealed a set of conserved protein motifs that are critical for their structure and function. For instance, plant-type PTPCs typically contain a set of conserved motifs that are stable in number (e.g., up to ten) and exhibit a conserved sequential order, whereas bacterial-type BTPCs, while also harboring these core motifs, may display alterations in their arrangement or the absence of specific individual motifs. Differences in the composition and configuration of these motifs serve not only as molecular hallmarks for distinguishing the PTPC and BTPC subfamilies but also suggest potential functional divergence between the two subfamilies over the course of long-term evolution.

The structural and regulatory distinctions between the two PEPC types underlie a clear division of labor and functional synergy in planta. Substantial evidence confirms that specific PEPC family members are selectively induced during plant stress responses, a pivotal function demonstrated across diverse species. For instance, in A. thaliana, three of its four PEPC genes belong to the plant-type subfamily, while the remaining one is a bacterial-type PEPC. A bacterial-type PEPC has likewise been identified in rice [23]. The functional significance of this gene family in stress tolerance is increasingly clear. In soybean, among its 10 GmPEPC genes, GmPEPC6, GmPEPC8, and GmPEPC9 are notably induced by aluminum toxicity, chilling, and salt stress [16]. Similarly, AtPPC4 in Arabidopsis is activated under drought stress [22]. Such abiotic adversities, including drought, salinity, and cold, are widely reported to strongly induce the expression of specific PEPC genes [22,28]. The observed decline in PEPC activity during drought stress correlates with reduced photosynthetic and electron transport rates, suggesting that PEPC contributes to plant drought resistance, potentially through the regulation of photosynthesis [29]. However, compared with annual herbaceous plants, our knowledge of the PEPC gene family in perennial woody plants remains limited, particularly for economically significant specialty tree species like Z. armatum. Key aspects such as gene family composition, expression regulation, and functional divergence are still poorly characterized. Given the long life cycle and complex habitats of Z. armatum, it is an open and pressing scientific question whether its PEPC gene family has evolved unique regulatory mechanisms to adapt to variable environments and recurrent stresses.

The rapid advancement of high-throughput sequencing technologies in recent years has enabled whole-genome sequencing for a vast number of plant species, creating unprecedented opportunities for genome-wide identification and analysis of gene families. By integrating multi-omics approaches, such as genomics, transcriptomics, and proteomics, it is now feasible to comprehensively identify members of specific gene families, elucidate their evolutionary relationships, gene structures, conserved motifs, and promoter cis-regulatory elements. When coupled with expression profiling, these strategies can effectively reveal the potential functions of gene family members in plant development and stress responses [10,30]. This genome-scale analytical framework has thus become a powerful tool for mining key stress-resistance genes and clarifying the molecular basis of plant environmental adaptation. In evolutionary genomics, syntenic blocks represent conserved ancestral genomic segments. Analyzing synteny across species provides insights into the architecture of ancestral genomes and elucidates the evolutionary forces, such as polyploidization and chromosomal rearrangements, that have driven genome diversification following species divergence [31].

The recent publication of a high-quality reference genome for Zanthoxylum armatum [32] provides an ideal foundation for such an investigation. As an autotetraploid species (2n = 4x = 132) with a contig N50 of 3.31 Mb, this genome assembly offers a continuous and reliable framework for accurate gene annotation and family-wide analysis. Therefore. using integrated bioinformatics approaches, we performed comprehensive analyses of their phylogenetic relationships, chromosomal locations, gene structures, conserved protein domains, physicochemical properties, and promoter cis-regulatory elements. Furthermore, by leveraging transcriptome data from Z. armatum under diverse abiotic stresses, we investigated the expression patterns of ZaPEPC genes. Our work aims to uncover the molecular characteristics and functional divergence of the PEPC gene family in this species, clarify its potential roles in environmental adaptation and abiotic stress responses, and provide important theoretical insights and genetic resources for further elucidating stress resistance mechanisms in woody plants. Ultimately, these findings will support molecular-assisted breeding efforts aimed at developing new, stress-resistant cultivars of Z. armatum.

2. Materials and Methods

2.1. Plant Materials

Leaf samples from three-year-old Z. armatum plants in the common garden at Chongqing University of Arts and Sciences. These plants were originally from Shandong Province (SD; 34°58′59″ N, 117°17′41″ E), Chongqing City (CQ; 29°12′50″ N, 105°50′47″ E), and Yunnan Province (YN; 25°39′54″ N, 101°54′25″ E), China. From each plant, we selected current-year shoots and collected the third compound leaf from the apex to ensure sample uniformity. Three biological replicates were established for each regional sample. After collection, the leaves were immediately put into liquid nitrogen for quick freezing treatment, and stored in ultra-low temperature freezing equipment (−80 °C) for RNA extraction. Detailed collection and processing of research materials can also be found in our previous published paper [33,34], the primary transcriptome data have been deposited in the National Genomics Data Center with the primary accession number PRJCA037036.

2.2. Identification of PEPC Gene Family Members

To identify the PEPC gene family in Z. armatum, we employed two independent searches against its published genome [32]. First, we used a Hidden Markov Model (PF00311) in the Pfam database and used HMMER3.0 software to identify the PEPC genes of Z. armatum with an E value < 10⁻⁴⁰ as the threshold. The second approach involved retrieving the protein sequences of previously reported PEPC gene family members from Arabidopsis official website TAIR (https://www.arabidopsis.org) (accessed on 15 January 2024). A BLASTP search was then performed on NCBI website (the National Center for Biotechnology Information Website: https://www.ncbi.nlm.nih.gov) (accessed on 15 January 2024) using these sequences as queries, with an E-value threshold set to 10⁻⁵, to obtain the FASTA formats of both the protein and gene sequences of the candidate ZaPEPC genes. The candidate genes common to both searches were consolidated. Subsequently, the presence of a complete PEPCase domain in each candidate was verified via the conserved domain database (CDD) at NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (accessed on 15 January 2024). Only sequences possessing the full domain were retained for further analysis.

2.3. Physicochemical Properties and Subcellular Localization Analysis of ZaPEPC Gene Family

Isoelectric points, molecular weights, sequence lengths, instability indices, lipid coefficients, and average hydrophilicity coefficients were calculated for the ZaPEPC proteins using ExPASy (https://web.expasy.org/protparam/) (accessed on 20 January 2024), and their subcellular localization was predicted using Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) (accessed on 20 January 2024).

2.4. Phylogenetic Analysis of the PEPC Gene Family and Collinearity Analysis of ZaPEPC Genes with Other Species

To elucidate the evolutionary relationships of PEPC genes, the PEPC protein sequences of Arabidopsis thaliana (At), Oryza sativa (Os), Zea mays (Zm), Glycine max (Gm), Setaria italica (Si) and Zanthoxylum armatum (Za) were subjected to multiple sequence comparisons using MAFFT v7.0 [35], and subsequently, poorly conserved and gap-rich regions were trimmed from the alignment to generate a high-quality multiple sequence alignment for robust phylogenetic analysis. MEGA v12.0 software [36] was used to construct a multi-species phylogenetic tree using the neighbor-joining (NJ) method; the reliability of the tree topology was evaluated by bootstrap analysis with 1000 replicates and all other parameters were set as default values. Cereal protein sequences were obtained from the phytozome database (https://phytozome.jgi.doe.gov/) (accessed on 22 January 2024). The details for the remaining four crops were obtained from the maize database (https://www.soybase.org/) (accessed on 22 January 2024), the rice database (https://www.soybase.org/) (accessed on 22 January 2024), the soybean database (https://www.soybase.org/) (accessed on 24 January 2024), and the A. thaliana database (http://www.arabidopsis.org) (accessed on 22 January 2024).

For collinearity analysis, nucleic acid sequences and annotation files from the following public databases: Arabidopsis thaliana (TAIR; https://www.arabidopsis.org) (accessed on 25 January 2024), Oryza sativa (the Rice Annotation Project Database; https://rapdb.dna.affrc.go.jp) (accessed on 25 January 2024), Zea mays (MaizeGDB; https://www.maizegdb.org) (accessed on 27 January 2024), Glycine max (SoyBase; https://www.soybase.org) (accessed on 27 January 2024), and Setaria italica (phytozome; https://phytozome.jgi.doe.gov/) (accessed on 28 January 2024). These files were used as input for the “One Step MCScanX” tool in TBtools v2.056 [37]. The tool first performed an all-against-all BLASTP analysis (E-value cutoff ≤ 1×10⁻¹⁰) to identify homologous genes, and then the MCScanX algorithm was applied to detect collinear genomic blocks between Z. armatum and each reference genome. The syntenic blocks containing PEPC genes were highlighted.

2.5. Analysis of ZaPEPC Gene Structure and Conserved Motifs and Promoter Cis-Regulatory Elements

The GFF file of Z. armatum gene annotation was searched using TBtools v2.056, and the intron and exon structures of each member of the ZaPEPC family were visually analyzed. The online tool MEME v4.12.0 (http://memesuite.org/tools/meme) (accessed on 2 February 2025) was used to analyze the conserved protein motifs of PEPC family members in Z. armatum, and the motif search value was set to 10 motifs. According to the whole genome data of Z. armatum [32], the sequence of 2000 bp upstream of the start codon (ATG) was extracted as the candidate promoter sequence, and the cis-acting element analysis was performed using the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 2 February 2025) website. Finally, TBtools v2.056 were used for visual mapping.

2.6. Chromosomal Localization and Gene Duplication Analysis

The chromosomal locations of the ZaPEPC genes (including flanking genes) and the genome-wide gene density were visualized using TBtools v2.056. Gene density visualization was performed as follows: Firstly, gene location visualize from GFF/GTF function was utilized, with the complete genome annotation file (in GFF/GTF format) used as input. Subsequently, the draw gene density module was selected. This function employs a sliding window algorithm to calculate the number of genes per megabase (Mb) along each chromosome. Additionally, the Simple Ka/Ks Calculator in TBtools v2.056 was used to analyze the selection pressure on duplicated gene pairs. The analysis required three input files: the CDS sequences, protein sequences of Z. armatum, and a list of collinear ZaPEPC gene pairs. The program output the non-synonymous (Ka) and synonymous (Ks) substitution rates and their ratio (Ka/Ks) to determine the selection pressure after gene replication.

2.7. Analysis of ZaPEPC Gene Expression Pattern in Z. armatum with Different Latitudes

Transcriptome sequencing was performed following the well-established protocol detailed in our previous publications [33,34]. The expression characteristics of the PEPC gene family were analyzed by transcriptome sequencing of leaf samples collected from three-year-old Z. armatum plant from three different regions: Shandong (SD), Chongqing (CQ), and Yunnan (YN) in China. For each region, three biological replicates were sequenced. The transcriptome sequencing was performed using Illumina and compared with the reference genome (Z. armatum_genome fa.gz). After obtaining the original data, low-quality reads were eliminated, HISAT2 v2.2.2.0 software was used for annotation, and the reads mapped to each gene were analyzed using the feature counting program to calculate the number of reads per thousand base fragments (FPKM) value of each million mapping reads of the gene. Finally, the FPKM values of PEPC gene family genes in Z. armatum were extracted and an expression clustering heat map was drawn using TBtools v2.056.

2.8. RNA Extraction and RT-qPCR Expression Analysis

To validate the transcriptome data, RT-qPCR was performed using the same RNA samples described in Section 2.7. Total RNA was extracted from Z. armatum leaves using an RNA extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd., Beijing, China, DP220706). The RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was generated by reverse transcription using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Nanjing Vazyme Biotech Co., Ltd, Nanjing, China). For RT-qPCR, a CFX Connect Real-Time Fluorescent Quantitative PCR Detection System (Bio-Rad, Hercules, CA, USA) was utilized. The RT-qPCR was conducted in a 25 µL reaction mixture, which included 12.5 µL of enzyme, 1 µL forward primer (10 µmol/L), 1 µL reverse primer (10 µmol/L), 1 µL cDNA template, and 9.5 µL ddH₂O. The PCR protocol consisted of an initial pre-denaturation at 94 °C for 20 s, followed by 35 cycles of denaturation at 94 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 10 s. The relative expression levels of the candidate genes across different samples were determined using the 2^−ΔΔCT method. The ZaUBQ gene was used as an internal control for normalization, and three replicates were analyzed for each sample [38]. The expression data were then analyzed using SPSS v20.0 to assess the significance of the differences. Primers were designed using Primer v5.0 software and synthesized by Shanghai Sangon Biological Engineering Co., Ltd., Shanghai, China. The sequences of all primers used for RT-qPCR are listed in Supplementary Materials (Table S1).

2.9. Co-Expression Relationship Network and Kyoto Encyclopedia of Genes and Genomes (KEGG) Analysis of ZaPEPC Genes

To explore the regulatory networks of the ZaPEPC genes, a co-expression analysis was performed using transcriptome data (FPKM) from Z. armatum leaves sampled from three latitudinal regions with three biological replicates each. Genes with low expression (FPKM < 1 in all samples) were first filtered out. Subsequently, the Pearson Correlation Coefficient (R) between each ZaPEPC gene and all other genes was calculated using the R rsgcc package v4.5.0 [39]. Gene pairs with an absolute R value ≥ 0.98 and a statistically significant p-value (p ≤ 0.05) were identified as significantly co-expressed, yielding a high-confidence expression matrix of 3746 genes for subsequent analysis. Finally, a co-expression network was visualized with Cytoscape v3.5.1 [40]. All genes significantly co-expressed with ZaPEPCs were compiled and subjected to KEGG pathway enrichment analysis in TBtools v2.056 [41], with a significance threshold of p-value < 0.05.

2.10. Statistical Analysis

Statistical significance (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001) was assessed by Student’s t-test and one-way ANOVA. Data are expressed as mean ± SD. Analyses were performed using GraphPad Prism v9. In the co-expression network, gene pairs with a Pearson correlation coefficient |R| > 0.98 and a p-value ≤ 0.05 were deemed significant.

3. Results

3.1. Identification Analysis of PEPC Gene Members in Z. armatum

Twelve PEPC family members were identified in Z. armatum and named as ZaPEPC1-ZaPEPC12 according to their physical locations (

Table 1. Basic physical and chemical properties of ZaPEPC proteins.

Gene ID	Rename	Size (Amino Acids)	Molecular Weight (kDa)	Aliphatic Index	Instability Index	Isoelectric Point (pI)	Grand Average of Hydropathicity
ZaA2_C01.Contig2868.3	ZaPEPC1	1039	116.85	88.4	50.99	7.33	−0.411
ZaA1_C01.Contig900.7	ZaPEPC2	1057	118.67	88.3	51.69	6.15	−0.412
ZaA2_C03.Contig267.3	ZaPEPC3	969	110.99	92.12	47.93	6	−0.375
ZaA1_C03.Contig793.20	ZaPEPC4	960	109.93	91.76	46.79	5.78	−0.361
ZaA1_C03.Contig1140.18	ZaPEPC5	867	99.63	89.67	48.91	6.51	−0.384
ZaA1_C03.Contig2837.3	ZaPEPC6	969	110.96	90.41	49.15	6	−0.395
ZaA1_C14.Contig1122.26	ZaPEPC7	969	110.82	91.52	46.83	5.96	−0.385
ZaA2_C14.Contig391.47	ZaPEPC8	1115	127.93	94.91	49.00	6.45	−0.307
ZaA2_C05.Contig15.2.10	ZaPEPC9	1034	115.77	89.69	48.89	6.16	−0.366
ZaA2_C23.Contig1171.3	ZaPEPC10	1034	115.71	90.07	49.78	6.12	−0.356
ZaA1_C28.Contig69.318	ZaPEPC11	965	110.37	90.25	44.48	5.98	−0.393
ZaA2_C10.Contig6.1.47	ZaPEPC12	965	110.27	90.05	44.15	5.90	−0.391

and Table S2). Analysis of the candidate ZaPEPC protein parameters showed that the number of amino acids encoded by ZaPEPC members was between 867 and 1115. The molecular weight of the members ranged from 99.63 kDa to 127.93 kDa, the isoelectric point of the protein ranged from 5.78 to 7.33, the instability index ranged from 44.15 to 51.69, the aliphatic index ranged from 88.3 to 94.1, and the average hydrophobic index ranged from −0.412 to −0.307 (Table 1), indicating that all ZaPEPC proteins are hydrophilic. Analysis of secondary metabolic structures of the PEPC proteins indicates that the proportion of α-helix of ZaPEPC members was 56.09–61.87%, and the proportion of random coil was 27.86–33.5% (

Table 2. Secondary structure of PEPC family proteins.

Gene	Alpha Helix (%)	Extended Strand (%)	Beta Turn (%)	Random Coil (%)	Subcellular Localization
ZaPEPC1	56.69%	6.06%	4.43%	32.82%	Cytoplasm
ZaPEPC2	57.62%	6.62%	4.16%	31.60%	Cytoplasm
ZaPEPC3	61.71%	5.88%	3.92%	28.48%	Cytoplasm
ZaPEPC4	60.94%	5.94%	4.17%	28.96%	Cytoplasm
ZaPEPC5	59.98%	6.46%	4.27%	29.30%	Cytoplasm
ZaPEPC6	61.71%	6.19%	4.23%	27.86%	Cytoplasm
ZaPEPC7	59.96%	6.19%	4.13%	29.72%	Cytoplasm
ZaPEPC8	57.94%	8.79%	4.75%	28.52%	Cytoplasm
ZaPEPC9	56.09%	6.19%	4.16%	33.56%	Cytoplasm
ZaPEPC10	57.35%	6.00%	3.97%	32.69%	Cytoplasm
ZaPEPC11	61.87%	6.01%	4.04%	28.08%	Cytoplasm
ZaPEPC12	61.66%	6.22%	4.15%	27.98%	Cytoplasm

), indicating that α-helix and random coil played a major role in the formation of tertiary structure.

3.2. Phylogenetic Relationship, Analysis of Domains and Conserved Motifs of ZaPEPC Gene Family Members

To elucidate the evolutionary relationships among the ZaPEPC proteins, a phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates. Phylogenetic classification of the 12 ZaPEPC members revealed two subfamilies: the bacterial-type (BTPC: ZaPEPC1, 2, 9, 10) and the plant-type (PTPC: the remaining eight members) (Figure 1A). This evolutionary divergence was further reflected in their protein motifs. We firstly examined the conserved protein domains to establish the fundamental functional units. Conserved domain analysis confirmed that all ZaPEPC proteins possess complete PEPCase domains (Figure 1B). We identified finer sequence features by analyzing conserved motifs using MEME, which revealed ten predominant motifs (Figure 1C,D). ZaPEPC members contain 10 conserved motifs except for PEPC5. Moreover, except for PEPC5, which was missing motif10, the position and sequence of plant-type PEPC (PTPC) were similar, both containing motif1–10. However, the order of the bacterial BTPC and plant PTPC domains differed slightly, which manifested in the fact that motif10 was located in the third position in bacterial PEPC and the first position in plant PEPC (Figure 1C). This indicates that differences in these conserved motifs may affect the function of ZaPEPCs. To further characterize their functional motifs, we performed multiple sequence alignment (Supplementary Figure S1). This analysis revealed that all plant-type PTPCs possess a conserved serine residue at the N-terminus and a characteristic C-terminal QNTG (Gln-Asn-Thr-Gly) tetrapeptide. In contrast, all bacterial-type BTPCs lack the N-terminal serine and instead feature an (R/K) NTG (Arg/Lys-Asn-Thr-Gly) tetrapeptide at their C-terminus.

3.3. Chromosomal Localization, Gene Structure and Collinearity Analysis of ZaPEPC Gene

To elucidate the genomic organization of the ZaPEPC gene family, we integrated an analysis of their chromosomal locations with their exon–intron structures (Figure 2). Members of the plant-type (PTPC) subfamily, including ZaPEPC3, ZaPEPC4, ZaPEPC6, ZaPEPC7, ZaPEPC11, and ZaPEPC12 exhibited a relatively compact structure with 9–10 exons. In contrast, genes belonging to the bacterial-type (BTPC) subfamily (ZaPEPC1, ZaPEPC2, ZaPEPC9, and ZaPEPC10) possessed a more complex architecture characterized by 19–20 exons (Figure 2A). This pronounced divergence in gene structure underscores distinct evolutionary trajectories for the PTPC and BTPC subfamilies.

This structural divergence was further reflected in their genomic organization across the genome. The 12 ZaPEPC genes were unevenly distributed across five chromosomes of Z. armatum (Figure 2B). A notable observation was the physical adjacency of ZaPEPC4 and ZaPEPC5 on chromosome 3 (their distance was 48,722 bp), forming a typical tandem gene cluster, which suggests a recent gene duplication event.

To study the evolutionary process of the PEPC family genes in Z. armatum, we analyzed the collinearity of the PEPC gene family. We found that eight pairs of ZaPEPC genes were collinear, and ZaPEPC4 and ZaPEPC5 were tandem gene clusters on CHR3 (Figure 3). Among them, ZaPEPC1, ZaPEPC2, ZaPEPC9, and ZaPEPC10 showed collinear relationships with each other. ZaPEPC4 was collinear with ZaPEPC7, and ZaPEPC3, ZaPEPC7, and ZaPEPC8 were also collinear. These findings indicate that the expansion of the ZaPEPC gene family was driven by multiple gene duplication events, including both tandem (e.g., ZaPEPC4 and ZaPEPC5) and segmental (e.g., ZaPEPC1 and ZaPEPC2) duplications, illustrating its complex evolutionary history.

The evolutionary selection pressure analysis showed that the Ka/Ks ratios of the nine gene pairs were greater than zero and lower than one (

Table 3. Analysis of duplication events of PEPC gene pairs.

Gene 1	Gene 2	Ka	Ks	Ka/Ks
ZaPEPC9	ZaPEPC2	0.0273	0.1763	0.1550
ZaPEPC9	ZaPEPC10	0.0025	0.0193	0.1315
ZaPEPC10	ZaPEPC1	0.0452	0.1996	0.2266
ZaPEPC10	ZaPEPC2	0.0282	0.1867	0.1511
ZaPEPC1	ZaPEPC2	0.0174	0.0278	0.6234
ZaPEPC1	ZaPEPC9	0.0438	0.1907	0.2299
ZaPEPC7	ZaPEPC8	0.0069	0.0189	0.3656
ZaPEPC7	ZaPEPC4	0.0176	0.2593	0.0679
ZaPEPC3	ZaPEPC4	0.0054	0.0167	0.3249

), indicating that they have undergone strong purifying selection. This evolutionary pressure preserves essential functional domains and implies that these genes have maintained their ancestral biological functions. A few gene pairs (ZaPEPC1 and ZaPEPC2, ZaPEPC7 and ZaPEPC8) showed a trend of close to neutral evolution, which may indicate certain functional flexibility or adaptability.

3.4. The Multi-Species Phylogenetic Analysis of PEPC Genes

To study the evolutionary relationship between the ZaPEPC protein and other known species, we constructed a neighbor-joining (NJ) phylogenetic tree using 44 PEPC proteins from 6 different plant species, including 4 from A. thaliana, 6 from Oryza sativa, 6 from Zea mays, 10 from Glycine max, and 6 from Setaria italica (Table S2). The phylogenetic tree (Figure 4) shows that these PEPC-containing ZaPEPC genes could be divided into two subfamilies (PTPC and BTPC) based on their sequence similarity. The PTPC subfamily contained 33 members that were further divided into four groups (PTPC I–IV). The BTPC subfamily was further divided into two groups (BTPC I and BTPC II). Twelve ZaPEPCs were assigned to the following groups with high bootstrap values: ZaPEPC3, ZaPEPC4, ZaPEPC6, ZaPEPC5, ZaPEPC7, ZaPEPC8 in the PTPC I group. ZaPEPC11 and ZaPEPC12 were included in the PTPC II group. ZaPEPC1 and ZaPEPC2 were also identified in the BTPC I group. ZaPEPC9 and ZaPEPC10 were included in BTPC II group (Table S4). Similar distribution patterns of PEPC components were found in G. max (Table S5). Phylogenetic analysis also revealed that the PTPC I and PTPC II family members of Z. armatum were closer to GmPEPC3, GmPEPC6, GmPEPC8, GmPEPC9, GmPEPC4, GmPEPC7, and GmPEPC10 in the soybean homologous series. In addition, the bacterial-type PEPC of Z. armatum was closer to the homologous series of soybeans. This suggests that a set of ancestral PEPCs existed prior to the divergence of soybean and Z. armatum. This close phylogenetic relationship with soybean PEPCs, particularly within specific subfamilies, provides crucial evolutionary context and supports the use of soybean as a model for inferring the potential functions of the newly identified ZaPEPC genes.

3.5. Multi-Species Collinearity Analysis of PEPC Gene Family

The inter-species collinearity analysis of ZaPEPC genes revealed distinct evolutionary trajectories between Z. armatum and the five representative plant species (Figure 5). Most notably, Z. armatum shared the highest number of collinear regions (15) with soybean. This finding is consistent with their shared evolutionary status as dicots and suggests a high degree of conservation in their PEPC genomic regions. In contrast, fewer collinear regions were detected with the monocot species O. sativa (3) and S. italica (3), consistent with the greater evolutionary distance and the extensive genome rearrangements known to have occurred since the monocot–dicot divergence. Notably, no collinear regions were identified with Z. mays. This absence may be attributed to more genomic rearrangements and variations during evolution. The maize genome underwent a more rapid evolution, including additional whole-genome duplications and chromosomal rearrangements [42]. These results collectively highlight a closer evolutionary relationship between the PEPC genes of Z. armatum and soybean compared to the monocots, providing a phylogenetic context for future functional studies of these genes (Table S6).

3.6. Cis-Elements in the Promoter Regions of ZaPEPC Genes

To study the possible role of PEPCs identified in the genome of Z. armatum [32], PlantCARE was used to analyze the cis-elements in the promoter region of the 2000 bp sequence upstream of the start codon of ZaPEPC genes (Figure 6A). These elements were major categorized into four major groups (Figure 6B; Table S7): light responsiveness, phytohormone response, growth and development, abiotic stress response. Promoter analysis revealed an abundance of light-responsive elements. Notably, the G-box was the most prevalent type (n = 46). We also identified 20 anaerobic-inducible elements, including GC-motif and ARE (Anaerobic Response Element). All ZaPEPC promoters contained elements responsive to abscisic acid (ABA) and methyl jasmonate (MeJA). Other phytohormone-related elements, such as those for gibberellin (GA), salicylic acid (SA), and auxin, were also detected, suggesting that ZaPEPC genes may integrate light and hormonal signals to coordinate photosynthesis and abiotic stress adaptation. Multiple stress-related elements were identified, including TC-rich repeats (defense and stress), LTR (low-temperature), and MBS (drought). Furthermore, elements linked to growth and development were present, such as the O2-site (found in ZaPEPC3, ZaPEPC4, ZaPEPC5, ZaPEPC6), CAT-box (meristem expression, in ZaPEPC1, ZaPEPC2, ZaPEPC12), GCN4_motif (endosperm expression, in ZaPEPC4, ZaPEPC9), RY-element (seed-specific, ZaPEPC1), and circadian regulatory elements (ZaPEPC6, ZaPEPC7, ZaPEPC8, ZaPEPC9).

3.7. Expression Patterns of the PEPC Family in Z. armatum from Three Different Latitudinal Regions

To analyze the expression patterns of the ZaPEPC1–12 genes in leaves, we utilized RNA-Seq data and calculated the corresponding FPKM values (Figure 7). The heat map shows the differential expression patterns of the 12 ZaPEPC genes in Z. armatum at three latitudes in China (Shandong Province, SD; Chongqing Province, CQ; and Yunnan Province, YN) (Figure 7A). Except for ZaPEPC9 and ZaPEPC12 from the YN group, which had FPKM values < 1, all other genes exhibited FPKM values > 1 (Table S8), demonstrating the expression of these genes. Notably, ZaPEPC7 and ZaPEPC11—whose promoters are predicted to be enriched in light-responsive cis-elements—showed significantly higher expression in the YN group, consistent with the high-light conditions of this region. We then validated these patterns by RT-qPCR. The results of RT-qPCR showed that the relative expression levels of ZaPEPC7 and ZaPEPC11 genes at the three different latitudes were consistent with the transcriptome data (Figure 7B), among which the expression level in the YN group was the highest, followed by the SD group, and the CQ group was the lowest. This strong correlation between in the silico prediction of light responsiveness and the observed environment-specific expression provided experimental support for the proposed regulatory model. The high light intensity and unique climatic conditions in the YN region (a high-altitude tropical area) may activate the light-responsive and stress-responsive elements in the ZaPEPC7/11 promoters, thereby inducing their high expression levels. This mechanism likely represents a molecular strategy employed by Z. armatum to adapt to high-latitude environments.

3.8. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Co-Expression Relationship Network Analysis of ZaPEPC Genes

To elucidate the potential biological functions and regulatory networks associated with the ZaPEPC genes, we performed a comprehensive co-expression and pathway enrichment analysis. The co-expression network analysis identified a total of 3746 significant co-expressed gene pairs, including 223 transcription factors (Table S9). KEGG pathway enrichment analysis was performed this gene set of 3746 genes and revealed significant associations with photosynthesis and stress-related processes (Figure 8A). Specifically, the pathways for photosynthesis (map00195), photosynthesis–antenna proteins (map00196), and glutathione metabolism (map00480) were significantly enriched. The photosynthesis–antenna proteins pathway showed the highest enrichment factor (approximately 3.5). This implies that certain ZaPEPC genes may enhance light energy capture and utilization efficiency while reducing photodamage under high-light conditions. Concurrently, significant enrichment was observed in the core photosynthesis pathway, which provides essential carbon skeletons for secondary metabolism, and in glutathione metabolism (p-value = 0.01), indicating a potential role in oxidative stress responses.

The co-expression network further elucidated how these functional associations might be regulated (Figure 8B). Eight ZaPEPC genes (ZaPEPC2, ZaPEPC4, ZaPEPC5, ZaPEPC7, ZaPEPC8, ZaPEPC10, ZaPEPC11, ZaPEPC12) was associated with photosynthesis and stress response related regulatory factors (bHLH, MYB-related, WRKY, C₃H, ERF, NAC, C₂H₂, HD-ZIP) (Figure 8B). Among them, ZaPEPC4 interacts most frequently with other genes, and interacts with ZaPEPC11, ZaPEPC5, ZaPEPC8, ZaPEPC7, respectively. ZaPEPC5 and ZaPEPC11 interact most closely with each other, and they are mostly positively correlated with TFs. ZaPEPC10 showed the highest number of TF connections, primarily positive correlations, followed by ZaPEPC8. In contrast, ZaPEPC2 and ZaPEPC12 had minimal network interactions (Table S9).

4. Discussion

4.1. Genome-Wide Identification and Evolutionary Analysis of the PEPC Family in Z. armatum

Z. armatum is an economically important crop with significant medicinal value [1]. This study conducted a genome-wide identification of the ZaPEPC gene family and analyzed its expression differences across three regions (Shandong, Yunnan, and Chongqing province) to elucidate its potential role in adversity resistance. This research holds great importance for the conservation and utilization of Z. armatum germplasm resources. We identified 12 PEPC genes in Z. armatum, and the number of ZaPEPC genes was significantly higher than that of PEPC members in foxtail millet (6 PEPC genes) [24], A. thaliana (4 PEPC genes) [22], rice (6 PEPC genes) [23], maize (6 PEPC genes) [43]. This expansion can likely be attributed to the two ancient polyploidization events documented in Z. armatum [32], which often lead to gene family duplication. Previous studies have found that PEPC members are divided into two subfamilies: PTPC and BTPC [22]. In this study, eight ZaPEPCs were clustered in subfamily I (PTPC) and four were clustered in subfamily II (BTPC). Except for Z. armatum (4) and soybean (3), there was only one BTPC in each species (Table S5). The phylogenetic tree showed that Z. armatum clusters closely with soybean within the dicot-specific clades, which are distinct from the monocot clades including rice. This clear phylogenetic separation indicates that the PEPC gene family diversified before the monocot–dicot split, an event estimated to have occurred around 200 million years ago [44]. The presence of the bacterial-type PEPC (BTPC) in both lineages further supports this ancient origin and the hypothesis of a nuclear ancestry for the PEPC family [45]. Consequently, this nuclear-derived PEPC gene family has gradually differentiated during the evolution of plants, forming a variety of isozymes that adapt to different physiological functions and metabolic needs, thus playing an important role in photosynthesis, stress response, and metabolic regulation in plants [46]. Moreover, collinearity analysis indicated that PEPC gene family members showed certain functional conservation during evolution, highlighting that similar biological functions may exist between species [47].

4.2. Structural Features and Duplication Events of ZaPEPCs

The composition of introns and exons is closely linked to the evolutionary history of gene families and is often regarded as an evolutionary relic [48]. Our analysis of the gene structure of ZaPEPCs revealed clear distinctions between plant-type (PTPC) and bacterial-type (BTPC) members, consistent with patterns reported in other species [49]. Specifically, ZaPTPC genes contain 8–10 introns and 9–11 exons, whereas bacterial-type ZaBTPC genes possess 19 introns and 20 exons, indicating that the PEPC gene family in Z. armatum has undergone an evolutionary trajectory similar to that in other plants. In plant evolution, genes often favor shorter introns or even intron loss, which facilitates rapid transcription and enables quicker responses to external environmental stresses [50]. In this context, ZaPEPC3/4/6/7, with their relatively compact gene structures, are likely transcribed more rapidly than ZaPEPC1/2/9/10, which exhibit more complex architectures. On the other hand, larger introns can promote genetic diversity and functional evolution by providing more sites for alternative splicing and sequence variation [51]. The comparatively long introns in ZaPEPC1/2/9/10 may thus offer greater potential for structural diversification of PEPC, potentially enhancing the adaptive capacity of Z. armatum to environmental challenges over evolutionary time.

Furthermore, systematic analysis revealed distinct correlations between gene structures, protein domain architectures, and the phylogenetic distribution of ZaPEPCs. In plants, exon–intron organization is closely associated with alternative splicing, a mechanism that generates diverse transcript isoforms and protein variants, thereby increasing functional diversity and facilitating rapid adaptation to environmental changes [52]. In this study, we found that the plant-type ZaPEPCs (PTPCs; including ZaPEPC3/4/5/6/7/8/11/12) typically contain 8–10 introns and 9–11 exons, and encode proteins with 10 conserved motifs arranged in a highly consistent order. This relatively simple and conserved gene architecture may facilitate rapid transcription and translation [52], potentially enabling Z. armatum to efficiently respond to common environmental fluctuations. For instance, in high-altitude, high-light regions such as Yunnan, plant-type PTPCs may help enhance photosynthetic efficiency and support basic stress acclimation [16]. In contrast, four bacterial-type ZaPEPCs (BTPCs; ZaPEPC1/2/9/10) exhibit more complex gene structures, with 19 introns and 20 exons, along with a distinct arrangement of conserved motifs—most notably, a shifted position of Motif10. This structural complexity and domain reorganization suggest that BTPCs may play a role in adapting to more severe or sustained environmental stresses. Under extensive stress conditions, complex gene structures can promote alternative splicing, enabling these genes to acquire specialized metabolic functions and improve evolutionary adaptability [53,54]. In summary, the ZaPEPC gene family exhibits coordinated evolution of gene and protein structures. The two subfamilies, BTPC and PTPC, have evolved distinct structural and functional characteristics, working in concert to enhance the resilience of Z. armatum to both common and extreme environmental challenges.

Gene duplication events are fundamental drivers of gene family expansion and functional diversification [55]. These events are broadly classified into two categories: whole-genome duplication (WGD), which can lead to genome doubling and species differentiation, and regional duplications (such as segmental or tandem duplications), which primarily contribute to the expansion of specific gene families [56]. The PEPC gene family in Z. armatum has undergone notable expansion, with its member count being two to three times that of rice, A. thaliana, maize, and foxtail millet. This observation suggests that Z. armatum may have experienced one or more whole-genome duplication events. Synteny analysis further classified the ZaPEPC genes into two major segmental duplication groups: one comprising ZaPEPC1/2/910 (bacterial-type, BTPC), and the other consisting of ZaPEPC3/5/7/8 (plant-type, PTPC). This grouping indicates that BTPC and PTPC represent two distinct evolutionary branches that emerged during the expansion of the PEPC gene family in this species. To understand the evolutionary pressures acting on these genes, we calculated the Ka/Ks ratios for nine duplicated gene pairs. All pairs showed values greater than 0 but less than 1 (Table 3), indicating that they have undergone strong purifying selection. This pattern, which is consistent with observations in other plant species such as foxtail millet and bamboo [24,47], underscores the essential and evolutionarily conserved role of the PEPC enzyme in plant metabolism.

4.3. Regulatory Mechanisms and Expression Patterns of ZaPEPCs

To elucidate the transcriptional regulation of ZaPEPC genes, we performed a systematic analysis of predicted cis-acting elements in their promoter regions. Studies have shown that genes involved in multiple stress responses are often closely related to cis-acting elements in their promoter region [57]. The primary function of cis-regulatory elements in gene upstream regions is to serve as transcription factor (TF) binding sites, enabling their crucial role in controlling gene expression [58]. Promoter analysis revealed an abundance of putative light-responsive elements, consistent with a potential role for light in regulating PEPC function. More importantly, the promoter contains multiple predicted hormone-responsive elements, especially ABA and MeJA. This suggests that ZaPEPC expression might be induced by ABA and MeJA, potentially integrating these signals into the plant’s stress response and signaling pathways [59]. The presence of these predicted elements also implies a potential role in helping plants cope with environmental challenges such as drought [60]. Gibberellin (GA), a phytohormone known to regulate processes such as cell division, seed germination, and fruit development [61], may exert its effects through specific downstream components. Notably, the identification of predicted GA-responsive elements suggests that PEPC could be one such component within the GA signaling pathway, which coordinately regulates plant growth and stress resistance [62]. Although PEPC genes exhibit a high degree of conservation, this conserved gene family performs diverse functions, including photosynthetic and non-photosynthetic functions, for survival in terrestrial environments [13]. The diverse predicted cis-regulatory landscape of the ZaPEPC family offers valuable insights into how hormones and environmental cues modulate its activity. These results advance our understanding of the intricate control mechanisms governing plant adaptation, with implications for engineering stress-resilient crops.

KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis indicated that the ZaPEPC genes are predominantly enriched in pathways related to photosynthesis and stress-related metabolism. Notably, arginine and proline metabolism (map00330) and glutathione metabolism (map00480), both critical for osmotic adjustment and oxidative defense, respectively [63,64], were significantly enriched. As a key anaplerotic enzyme, PEPC replenishes the tricarboxylic acid (TCA) cycle by catalyzing the conversion of phosphoenolpyruvate (PEP) to oxaloacetate (OAA), thereby supplying essential carbon skeletons for biosynthesis and energy metabolism. It is noteworthy that intermediates from arginine and proline metabolism (e.g., glutamate) directly feed into the TCA cycle, thereby linking carbon and nitrogen metabolism [65]. Thus, under favorable growth conditions such as those in Yunnan, high ZaPEPC expression may bolster the cellular pool of carbon skeletons, priming the plant for rapid synthesis of osmolytes like proline upon stress onset [66]. Furthermore, co-expression network analysis revealed that eight ZaPEPC genes exhibit positive or negative correlations with eight transcription factors (TFs), including bHLH, MYB-related, WRKY, C₃H, ERF, NAC, C₂H₂, and HD-ZIP. Most of these TFs are well-documented regulators of environmental stress responses [67,68,69]. For instance, in Iris laevigata, IlWRKY22 enhances total chlorophyll content, reduces stomatal aperture, boosts antioxidant enzyme activities, and improves photosynthetic efficiency under stress. Similarly, NAC TFs help maintain photosynthetic performance and modulate hormone signaling during drought [67]. Additionally, certain bHLH members mediate light signaling pathways [69]. These co-expression relationships suggest that ZaPEPCs operate within a coordinated regulatory network, reinforcing the functional link between photosynthetic carbon metabolism and stress adaptation.

Our expression analysis revealed a distinct spatial pattern for ZaPEPC7 and ZaPEPC11, with transcript levels highest in Yunnan (YN), intermediate in Shandong (SD), and lowest in Chongqing (CQ). This pattern, consistently observed in both RNA-seq and qRT-PCR data, correlates with the contrasting environmental conditions across these regions: YN (plateau, temperate climate, ample light and rainfall) > SD (high-latitude, cold-dry, high light intensity) > CQ (lowland, humid-hot, low light). Phylogenetically, ZaPEPC7 clusters closely with soybean genes GmPEPC6, GmPEPC8, and GmPEPC9, which were established regulators of diverse abiotic stresses, including aluminum toxicity, chilling, and salinity [16]. This strong phylogenetic conservation suggests that ZaPEPC7 may fulfills a similar role in stress adaptation in Z. armatum. The significant variation in PEPC gene expression across latitudes implies a substantial influence of environmental factors such as light, temperature, and humidity. This regulatory potential is further supported by the presence of numerous light-, hormone-, and stress-responsive cis-elements in the promoters of Z. armatum PEPC genes. We hypothesize that in the high-light environment of Yunnan, light-signaling pathways activate specific transcription factors that bind to these promoter elements, thereby initiating the elevated expression of ZaPEPC7 and ZaPEPC11 [70]. This hypothesis is reinforced by our co-expression network analysis, which revealed significant correlations between ZaPEPCs and stress-associated transcription factors (e.g., NAC, WRKY). These TFs may constitute a regulatory module that coordinately controls PEPC expression alongside other stress-responsive genes, forming an integrated adaptive network. Moreover, light is a key inducer of PEPC kinase (PPCK) expression and activity [71]. PPCK phosphorylates PEPC at a conserved serine residue, alleviating feedback inhibition by malate and thereby enhancing enzyme activity [26,72]. As a central enzyme in plant carbon metabolism, the differential expression of PEPC across latitudes underscores its role in modulating carbon metabolic strategies in Z. armatum in response to environmental gradients. This study establishes a foundation for elucidating the regulatory mechanisms of ZaPEPC7 and ZaPEPC11. Future work should employ functional genetic tools, such as the CRISPR-Cas9 system [73] and RNA interference (RNAi) [74], to precisely manipulate these genes and investigate their molecular functions. Such insights will be of considerable theoretical importance for developing new, superior cultivars of Sichuan pepper with enhanced stress resilience.

5. Conclusions

In summary, this study presents a systematic identification and characterization of 12 ZaPEPC genes in Z. armatum, revealing their structural conservation, evolutionary divergence, and potential roles in environmental adaptation. The ZaPEPC family members are classified into plant-type (PTPC) and bacterial-type (BTPC) subfamilies, which exhibit conserved functional domains and distinct gene structures. Gene duplication events have contributed to the expansion of this gene family. Phylogenetic analysis revealed a closer homology between ZaPEPCs and soybean PEPCs, suggesting shared evolutionary origins that predate the monocot–dicot divergence. Promoter cis-element analysis identified an abundance of stress-responsive motifs, such as those associated with light, ABA, and MeJA, indicating the potential involvement of ZaPEPCs in abiotic stress regulation. Expression profiling further demonstrated environment-specific expression patterns, with notably elevated transcript levels of ZaPEPC7 and ZaPEPC11 in the Yunnan group, underscoring their likely role in local environmental adaptation. Collectively, these findings provide fundamental insights into the functional diversity and stress response mechanisms of the ZaPEPC gene family, and highlight promising targets for improving photosynthetic efficiency and stress tolerance in Z. armatum. Future research should prioritize functional validation of key genes such as ZaPEPC7 and ZaPEPC11 using reverse genetics tools like CRISPR-Cas9 and RNAi, and further explore their regulatory networks and metabolic contributions under diverse stress conditions to support molecular breeding efforts.