The PAT Gene Family in Citrus: Genome-Wide Identification and Its Potential Implications for Organic Acid Metabolism

Abstract

Protein palmitoylation, a key post-translational modification (PTM) regulating protein transport and function, is catalyzed by palmitoyl transferases (PATs). PATs play vital roles in plant growth, development, and stress responses, yet their characterization in citrus remains limited. This study identified 23 PAT genes (CitPATs) possessing the conserved DHHC domain in the citrus genome through comprehensive genome-wide analysis. Analysis revealed that most CitPAT proteins are hydrophilic, basic, and stable, with significant variations in sequence length. Gene structure and motif analysis confirmed 10 conserved motifs, with the DHHC domain being the most conserved among all 23 members. The CitPAT genes were unevenly distributed across nine chromosomes and exhibit high evolutionary conservation. Promoter analysis identified numerous cis-acting elements associated with abiotic stress and hormone responses, including basic regulatory elements, light-responsive elements, and stress-responsive elements, with light-responsive elements being predominant. Expression profiling during fruit development revealed distinct correlation patterns with citric acid dynamics: CitPAT6, CitPAT18, and CitPAT23 showed positive correlations with acid accumulation, while CitPAT1, CitPAT10, and CitPAT13 exhibited negative correlations. Further RT-qPCR experiments revealed that CitPAT1 and CitPAT10 consistently demonstrated strong negative correlations with citrate content throughout fruit development. This functional diversification suggests roles in regulating citric acid metabolism. These findings provide novel insights into quality formation in facility-cultivated citrus and establish a foundation for understanding PAT-mediated regulation of fruit development.

1. Introduction

Protein S-acyl transferases (PATs) catalyze S-acylation, a reversible post-translational modification critical for membrane association, trafficking, and stability of substrate proteins. These transmembrane enzymes contain 4–6 transmembrane domains and feature a conserved DHHC-CRD catalytic domain where cysteine residues act as catalytic sites for palmitoylation [1,2]. Palmitoylation, a key post-translational modification (PTM), covalently attaches a C-16 fatty acyl chain to specific cysteine residues. This modification regulates protein localization and protein–protein interactions, playing essential roles in animals, plants, and microbial stress responses [3]. Furthermore, DHHC2 catalyzes its own palmitoylation at a C-terminal cysteine residue via its intrinsic PAT activity, thereby regulating its localization to the inner membrane complex (IMC) [4]. Notably, the IMC-anchored interferon-stimulated protein (ISP) interacts with the microtubule component β-tubulin to maintain the structural integrity of the subpellicular microtubules (SPM). Concurrently, a protein palmitoylation cascade links the cortical membrane to microtubules, maintaining the appropriate cytoskeletal architecture required for syncytial kinetochore assembly [5].

In plants, the first described palmitoyl transferase was Arabidopsis TIP GROWTH DEFECTIVE 1 (TIP1). As a member of the palmitoyltransferase family containing an ankyrin repeat domain, TIP1 is constitutively expressed in roots, leaves, inflorescence stems, and flowers. It modulates protein hydrophobicity to regulate protein-membrane binding, signal transduction, and intracellular vesicle trafficking [6]. AtPAT1 specifically mediates the decapping of the canonical ABA-responsive gene COR15A, thereby facilitating the decay of ABA-responsive genes and regulating stress responses [7]. Moreover, another Arabidopsis palmitoyltransferase, AtPAT10, exhibits catalytic activity and participates in regulating cell expansion, cell division, and reproductive fitness [8]. Localized to both the Golgi apparatus and tonoplast [8,9], AtPAT10 loss-of-function mutants generated through T-DNA insertions consistently display impaired cell expansion/division and hypersensitivity to salt stress. Notably, AtCBL2 and AtCBL3 were identified as potential substrates of AtPAT10 and shown to regulate salt tolerance [9].

Citrus ranks among the world’s most economically significant fruit crops, prized for its sensory attributes and nutritional value. The fruit provides essential nutrients including sugars, volatile compounds, organic acids, dietary metabolites, amino acids, fiber, vitamin B6, vitamin C, and macro-/micronutrients [10]. Notably, organic acids, particularly citric acid are primary determinants of fruit flavor. In commercial citrus species, citric acid constitutes 60–90% of total acidity and accumulates predominantly within juice vesicles [11]. In fruits, citric acid is primarily synthesized in mitochondria via the tricarboxylic acid (TCA) cycle and subsequently transported to vacuoles for storage through the action of proton pumps and transporters. During ripening, citrate is translocated to the cytoplasm for degradation [12,13]. This metabolic process is tightly regulated by multiple genes, including those involved in citrate synthesis (e.g., CitCS) [14], degradation (e.g., CitAco3) [15], transporters (e.g., CsCit) [16], and proton pumps (e.g., CitPH1 and CitPH5) [17]. Furthermore, regulatory factors such as transcription factors (e.g., CitPH4 and CitSAR) [18,19] and epigenetic modifiers (e.g., CitHAG29) [20] have been reported to participate in citric acid regulation. Despite these findings, whether protein S-acyl transferases (PATs) modulate citric acid metabolism remains unclear.

Recent advances in high-throughput sequencing have revolutionized transcriptomics, proteomics, and metabolomics, enabling comprehensive insights into gene expression, protein dynamics, and metabolic networks. In this context, Genome-Wide Association Studies (GWAS) have emerged as a powerful tool in plant science. By linking genetic variations to phenotypic traits and integrating multi-omics data, GWAS accelerates the discovery of key genes for crop improvement, stress resistance, and metabolic pathways. This technology provides invaluable support for addressing global challenges in food security and sustainability, driving innovation in plant research [21].

In this study, we conducted a comprehensive genome-wide analysis to identify and characterize the PAT gene family in citrus, revealing 23 CitPAT genes encoding conserved DHHC domains. Most CitPAT proteins were hydrophilic, alkaline, and structurally stable, though exhibiting substantial length variation. Gene architecture and motif analyses confirmed 10 conserved motifs, with the DHHC domain universally preserved across all members. CitPAT genes displayed uneven distribution across nine chromosomes while maintaining high phylogenetic conservation. Promoter interrogation identified abundant cis-acting elements associated with abiotic stress and hormone responses, predominantly light-responsive elements alongside core regulatory and stress-related motifs. Expression profiling during fruit development demonstrated distinct correlation patterns with citrate dynamics: CitPAT6, CitPAT11, CitPAT18, and CitPAT23 exhibited positive correlations with acid accumulation, whereas CitPAT1, CitPAT10, and CitPAT13 showed negative correlations. This study provides foundational insights into the CitPAT family and reveals its potential functional diversification in regulating a key determinant of citrus fruit quality.

2. Materials and Methods

2.1. Plant Materials

‘‘Ponkan’’ fruits (Citrus reticulata Blanco cv. ‘Ponkan’) were harvested from a commercial orchard in Quzhou, Zhejiang Province, China. Fruit sampling was conducted at 90 d, 120 d, 150 d, 180 d, and 210 d after full bloom. ‘‘Ponkan’’ trees with consistent growth vigor and fruiting status both inside and outside greenhouses were selected. Fruits with uniform size and appearance were selected and were immediately frozen in liquid nitrogen and then stored in a −80 °C freezer for subsequent research. Each sampling time point set three biological replicates.

2.2. Identification of PAT Family in Citrus Genome

To identify the PAT family members in Citrus clementina, we performed a comprehensive bioinformatics analysis using the following approach: First, 24 AtPAT protein sequences from Arabidopsis thaliana were employed as queries to search against the Citrus clementina genome database (NCBI accession: GCF_000493195.1) through the Blast function implemented in TBtools software (TBtools-II v2.356) [22,23]. Subsequently, the conserved domains of the putative PAT family members were systematically analyzed using the Pfam database (http://pfam.xfam.org) and visualized through TBtools software (TBtools-II v2.356). Following this analysis, we specifically identified protein sequences containing the characteristic DHHC domain (Pfam: PF01529) as bona fide members of the CitPAT family.

2.3. Phylogenetic Analysis and Chromosomal Location of PAT Family

Phylogenetic analysis of PAT family proteins in Citrus clementina, Arabidopsis, and rice was conducted using MEGA11 software [24]. Multiple sequence alignment was performed using Clustal W, and the phylogenetic tree was constructed employing the maximum likelihood method with 1000 bootstrap replicates [25]. The final tree was visualized and annotated using iTOL (https://itoleditor.letunic.com/) [26]. For chromosomal localization analysis of CitPATs, the genome annotation file (GFF format) of Citrus clementina was downloaded from its genome database (NCBI accession: GCF_000493195.1) and visualized using TBtools software (TBtools-II v2.356).

To elucidate the evolutionary relationships and genomic distribution of PAT family proteins, we performed comprehensive phylogenetic and chromosomal localization analyses. The phylogenetic reconstruction was conducted using MEGA11 software, incorporating PAT family proteins from Citrus clementina, Arabidopsis thaliana, and Oryza sativa. Multiple sequence alignment was executed using the Clustal W algorithm, followed by phylogenetic tree construction through the maximum likelihood method with 1000 bootstrap replicates to ensure statistical robustness. The resulting phylogenetic tree was subsequently visualized and annotated using the Interactive Tree of Life (iTOL) platform (https://itoleditor.letunic.com/). For chromosomal localization analysis of CitPATs, we retrieved the annotation file (GFF format) of Citrus clementina from the NCBI genome database (accession: GCF_000493195.1) and performed visualization using TBtools software (TBtools-II v2.356) to determine the genomic distribution patterns of PAT family members across chromosomes. Furthermore, we systematically designated the nomenclature of PAT family members based on their sequential chromosomal localization patterns.

2.4. Gene Structure, Conserved Motif and Domain Analysis of CitPATs

The gene structure information of PATs in Citrus clementina was obtained from its genome annotation files. For conserved motif analysis of CitPATs, we utilized the MEME Suite 5.5.2 tool (https://memesuite.org/meme/tools/meme) with protein sequences as input [27]. All visualization and data integration were performed using TBtools,

2.5. Promoter Cis-Regulatory Elements Analysis of CitPATs

The promoter cis-regulatory elements of CitPATs were systematically identified using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Following the standard protocol described in original PlantCARE publication, we analyzed 2 kb genomic sequences upstream of the transcription start sites, which were precisely extracted from the Citrus clementina genome using the Phytozome 13 database (https://phytozome-next.jgi.doe.gov) [28,29]. The identified cis-elements, including both enhancers and repressors, were annotated based on their positional matrices and consensus sequences, and subsequently visualized using TBtools software (TBtools-II v2.356) for comprehensive comparative analysis.

2.6. Organic Acid Measurement

Organic acid content in citrus fruits was measured according to Liu et al. [19]. A ground sample (0.1 g) was extracted with 1.4 mL of chromatographic-grade methanol at 70 °C for 15 min, followed by centrifugation at 12,000× g and 4 °C for 10 min. To the supernatant, 0.75 mL of trichloromethane and 1.5 mL of purified water were added. The mixture was vortexed thoroughly for 30 s (repeated 3 times) and centrifuged. A 100 μL aliquot of the resulting upper phase was collected, dried under vacuum, and spiked with 20 μL of ribitol (0.2 mg/mL) as an internal standard. The dried residue was derivatized by first dissolving it in 60 μL of pyridine containing 20 mg/mL methoxyamine hydrochloride, followed by incubation at 37 °C for 1.5 h. Subsequently, 40 μL of N, O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) was added, and the mixture was incubated at 37 °C for 30 min. A 1 μL aliquot of the derivatized sample was injected in split mode (10:1 ratio) into a GC-MS system equipped with a fused-silica capillary column. The injector temperature was 250 °C, and helium carrier gas flow was maintained at 1.0 mL/min. The oven temperature program was: 100 °C (hold 1 min), ramp to 185 °C at 3 °C/min, then ramp to 250 °C at 15 °C/min (hold 2 min). Mass spectrometry parameters were: ionization voltage 70 eV, ion source temperature 230 °C, and transfer line temperature 280 °C.

2.7. RNA Extraction and cDNA Synthesis

Total RNA was isolated using the RNAiso Plus kit (Takara, Beijing, China) according to the manufacturer’s protocol. Subsequently, genomic DNA (gDNA) was removed from 1 µg of total RNA using gDNA wiper (Vazyme, Nanjing, China). First-strand cDNA synthesis was then performed using HiScript^® II qRT SuperMix (Vazyme).

2.8. RNA-seq

To understand the transcriptional process in Ponkan fruits developed inside and outside the greenhouse during 120 and 150 DAFB, we performed RNA-seq for 12 different samples. Total RNA was extracted from 12 different fruit samples using Trizol reagent (Ambion, Austin, TX, USA, #15596018) according to the manufacturer’s instructions. RNA integrity was determined using regular agarose gel electrophoresis, Nanodrop (ThermoFisher Scientific, Waltham, MA, USA), and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA sample of high quality (OD260/280 within the range [1.8, 2.2], OD260/230 ≥ 2.0, RIN ≥ 8) was used to construct the sequencing library. Library construction and sequencing were performed by Novogene Technology Inc. (Tianjin, China) with Hiseq platform (Illumina Inc., San Diego, CA, USA) using the paired-end sequencing strategy (150 bp for each end). The RNA-seq raw reads files has been deposited at the NCBI Sequence Read Archive (SRA) under the accession number PRJNA1333619.

2.9. Expression Analysis of CitPATs

Gene-specific primers for RT-qPCR were designed using the Primer-BLAST tool on the NCBI website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ accessed on 1 September 2025). cDNA template concentrations were normalized based on the threshold cycle (CT) value of the actin reference gene (Ciclev10025866m). Each 20 μL RT-qPCR reaction contained: 10 μL ChamQ Universal SYBR qPCR Master Mix, 2 μL diluted cDNA, 0.4 μL of each gene-specific primer (10 μM), and 7.2 μL DEPC-treated water. Reactions were performed using the Ssofast™ EvaGreen^® Supermix Kit on a CFX96 instrument (Bio-Rad, Hercules, CA, USA) with the following thermal profile: initial denaturation at 95 °C for 5 min; 50 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and extension at 75 °C for 15 s. Melting curve analysis was performed at the end of each run to verify amplicon specificity, using the citrus actin gene (XM_006464503) as the internal reference control. The 2^−ΔCT method was used to analyze the gene expression levels. Primers used for RT-qPCR were listed in Supplemental Table S4.

2.10. Statistical Analysis

Data analysis was conducted using Microsoft Excel and TBtools 9. All experiments incorporated at least three independent biological replicates. Error bars represent the standard error (SE). Differences between developmental stages were assessed using Fisher’s Least Significant Difference (LSD) test at a significance level of p < 0.05. Figures were generated using GraphPad Prism 9 and finalized in Microsoft PowerPoint.

3. Results

3.1. Identification of CitPATs Family in C. clementina Genome

A total of 23 CitPATs genes were identified from the Citrus clementina genome by homologous sequence alignment with the AtPAT genes in Arabidopsis. All of the CitPAT proteins contain DHHC domain (Pfam: PF01529) which have been reported in Arabidopsis before [30].

3.2. Chromosomal Location and Syntenic Relationships of CitPATs

The 23 CitPAT genes are unevenly distributed across all nine scaffolds of the citrus genome, systematically designated as CitPAT1-23 based on their chromosomal locations (Figure 1A). The distribution pattern reveals 1, 2, 4, 4, 3, 4, 2, 2, and 1 PAT genes per chromosome, respectively. Genomic evolution in plants is primarily driven by gene duplication events, including tandem and segmental duplications, which represent fundamental mechanisms for gene family expansion. Our analysis identified two tandem duplication events encompassing multiple genes across all nine scaffolds (Figure 1B). These findings demonstrate that both tandem and segmental duplication events have significantly contributed to the expansion of the PAT gene family in the citrus genome, with tandem duplication emerging as the predominant evolutionary force. The interspecies collinearity analysis of the PAT gene family in Citrus clementina revealed 16 syntenic relationships with the PAT gene family in Arabidopsis and 14 syntenic pairs with the DHHC gene family in Oryzaiva (Figure 1C). The comparative analysis demonstrated that the evolutionary relationship of PAT genes in Citrus clementina exhibits a closer phylogenetic affinity to Arabidopsis thaliana than to Oryza sativa (rice).

3.3. Phylogenetic Analysis of PAT Family

To elucidate the evolutionary relationships among CitPATs and their orthologs in other species, we constructed a comprehensive phylogenetic tree incorporating AtPATs from Arabidopsis thaliana and OsPATs from Oryza sativa (Figure 2). The phylogenetic analysis revealed that the CitPAT family members cluster into five distinct groups, mirroring the classification pattern observed in the Arabidopsis PAT family. Specifically, Group I comprises seven members (CitPAT2, CitPAT3, CitPAT9, CitPAT17, CitPAT18, CitPAT19, and CitPAT20); Group II contains two members (CitPAT1 and CitPAT21); Group III includes two members (CitPAT12 CitPAT22); Group IV encompasses six members (CitPAT4, CitPAT6, CitPAT8, CitPAT11, CitPAT14, and CitPAT15); and Group V consists of two members (CitPAT5 and CitPAT7). This classification provides valuable insights into the evolutionary conservation and diversification of the PAT gene family across plant species.

3.4. Gene Structure, Conserved Motif and Domain Analysis of CitPATs

Conserved domain analysis of CitPAT proteins identified 10 distinct motifs, with Motif1 and Motif7 exhibiting the highest conservation across the majority of proteins (Figure 3A). Proteins within the same phylogenetic clade displayed identical motif compositions, indicating functional similarity and close evolutionary relationships. For example, Group I members (CitPAT2, CitPAT3, CitPAT9, CitPAT17, CitPAT18, and CitPAT20) consistently contained Motif1, Motif2, Motif3, Motif4, Motif5, Motif6, and Motif7, with the exception of CitPAT19, which lacked Motif3 and Motif7. Similarly, Group II members (CitPAT1 and CitPAT21) and Group III member CitPAT22 shared Motif3, Motif4, Motif5, and Motif7. The positional arrangement and sequential order of these motifs were highly conserved across most proteins, except for specific absent motifs. Notably, the majority of CitPAT family members harbored the DHHC (Asp-His-His-Cys) domain, a signature sequence crucial for palmitoyl acyltransferase activity, which is essential for protein palmitoylation (Figure 3B). However, CitPAT4, CitPAT8, CitPAT21, and CitPAT22 contained the DHHC superfamily domain, suggesting potential functional or structural divergence within these proteins.

3.5. Promoter Cis-Regulatory Elements Analysis of CitPATs

In the promoter region of the CitPAT genes, a variety of basic cis-acting elements, light-responsive elements, hormone-responsive elements (such as gibberellin, abscisic acid, etc.), stress-responsive elements, and plant growth and development-related elements were predicted (Figure 4). The basic cis-acting elements include TATA-box, CAAT-box, and AT~TATA-box promoter elements. Specifically, there are 22 auxin-responsive elements, 5 ATBP-1 binding sites, 45 salicylic acid-responsive elements, 49 abscisic acid-responsive elements, 144 light signal-responsive elements, 80 jasmonic acid signal-responsive elements, 14 MYB binding sites involved in drought induction, 14 gibberellin-responsive elements, 11 zein metabolism regulatory elements, and 2 seed-specific regulatory elements. It is speculated that the CitPAT genes play an important role in the growth and development, hormone regulation, stress response, and light regulation of Citrus clementina.

3.6. Changes in Organic Acid During ‘Ponkan’ Fruit Development

The content of organic acids in ‘Ponkan’ fruit exhibits a characteristic pattern of initial increase followed by a decrease during its developmental stages (Figure 5). Malic acid demonstrates a rapid ascent in the early phase, peaking approximately 90 d post-flowering, and then gradually declines throughout the observed period up to 210 days, with no significant difference observed between the final two time points (180 and 210 days). The peak concentration of malic acid in greenhouse-cultivated ‘Ponkan’ is 8.70 mg/g, compared to 6.84 mg/g in open-field conditions, with no statistically significant difference observed between the two cultivation environments. In contrast, citric acid, the predominant organic acid in citrus fruits also experiences a swift increase in the initial stages, reaching its zenith around 120 d after flowering. The peak citric acid level in ‘Ponkan’ under controlled facility cultivation is notably higher at 81.48 mg/g, significantly surpassing the control group’s 58.62 mg/g. Following this peak, citric acid levels gradually diminish, throughout the observed period up to 210 days. Upon reaching maturity, the organic acid content in ‘Ponkan’ from both cultivation methods shows no significant variation.

3.7. Expression Pattern of CitPATs During ‘Ponkan’ Fruit Development

Utilizing RNA-seq data, we systematically analyzed the expression profiles of the PAT gene family in Citrus clementina (Figure 6A, Supplemental Table S2). The analysis revealed distinct expression patterns among PAT family members: CitPAT16, CitPAT1, CitPAT11, CitPAT13, CitPAT2, CitPAT9, CitPAT6, and CitPAT15 demonstrated high expression levels, while CitPAT4, CitPAT21, CitPAT7, CitPAT20, CitPAT19, CitPAT12, and CitPAT22 maintained moderate expression levels without significant developmental changes. Notably, CitPAT23, CitPAT8, CitPAT14, CitPAT10, CitPAT18, PAT17, CitPAT5, and CitPAT3 showed minimal or undetectable expression levels. Correlation analysis between citrate content and CitPAT gene expression at 120 and 150 days after full bloom (DAFB) identified six genes significantly associated with citrate dynamics: CitPAT1, CitPAT6, CitPAT10, CitPAT13, CitPAT18, and CitPAT23 (Figure 6B, Supplemental Table S3). Among these, CitPAT6, CitPAT18, and CitPAT23 exhibited positive correlations, with their expression levels decreasing in parallel with citrate content. In contrast, CitPAT1, CitPAT10, and CitPAT13 showed negative correlations, as their expression increased with declining citrate levels. These six candidate genes were subsequently selected for RT-qPCR validation (Figure 7). Further analysis of citrate content and CitPATs expression patterns in both inside and outside greenhouse ‘Ponkan’ fruits at 90, 120, and 150 DAFB revealed that CitPAT1 and CitPAT10 consistently demonstrated strong correlations with citrate content throughout fruit development (Supplemental Table S3). This observation aligns with our previous findings and suggests their potential involvement in citrate degradation during ‘Ponkan’ fruit development. Our comprehensive analysis provides valuable insights into the regulatory roles of specific PAT genes in citrate metabolism during ‘Ponkan’ fruit development.

4. Discussion

Protein palmitoylation is a post-translational lipid modification that occurs in eukaryotic organisms and plays critical roles in regulating protein transport, signaling, membrane association, and subcellular targeting [31]. Initial research on palmitoyltransferases (PATs) was conducted in yeast, leading to the subsequent identification of PAT homologs across nearly all species. However, studies on the PAT gene family in plants remain relatively limited. To date, PAT genes have been characterized in several plant species, including Arabidopsis [29], rice [32], and apple [33]. In this study, we identified 23 putative PAT protein sequences from the Citrus reticulata genome. The quantitative variation in PAT gene family members among different species suggests that this gene family has undergone differentiation and expansion during plant evolution [34].

Phylogenetic analysis revealed that the PAT gene family in ‘Ponkan’, along with those in Arabidopsis [29] and rice [32], can be classified into five distinct clades (Figure 2). Closely related members were observed to share similar gene structures, supporting the notion that evolutionary proximity correlates with functional similarity. Among these, Group I contained the highest number of members, implying that this clade may perform more diverse functions or exhibit functional redundancy [35]. With the exception of CitPAT22, CitPAT4, CitPAT21, and CitPAT8, all members of CitPAT family were identified to contain the DHHC (Asp-His-His-Cys) domain (Figure 3), a highly conserved motif critical for palmitoyl acyltransferase activity and essential for protein palmitoylation. The absence of this domain in a subset of genes suggests that non-DHHC domains may also contribute to the regulation of palmitoylation [36]. Furthermore, interspecies collinearity analysis of the CitPAT gene family with those of Arabidopsis and rice indicated a high degree of conservation throughout evolutionary divergence (Figure 1). Additionally, the cis-regulatory elements within the promoter regions of each CitPAT gene were predicted. As observed in PATs from other plant species [35,37], these promoters harbor varying numbers and types of regulatory elements, including core regulatory elements, light-responsive elements, hormone-responsive elements, and stress-responsive elements (Figure 4). This finding suggests their potential involvement in environmental adaptation and developmental regulation.

The comparison between ‘Ponkan’ cultivated outside and inside greenhouse was conducted to assess the impact of environmental factors—such as temperature, humidity, and light—on the fruit’s biochemical composition, particularly citric acid levels. Greenhouse offers a controlled and stable environment, whereas outdoor growth exposes the fruit to natural fluctuations in weather and external conditions. This comparison allowed us to explore how environmental variability influences metabolic processes and quality attributes during the fruit’s developmental stages. The observed differences in citric acid levels between 90 and 150 days under the two conditions are likely driven by variations in environmental factors and relevant genes conducted by distinct environment. Conversely, the regulated greenhouse environment may promote a more gradual and consistent metabolic progression. These findings underscore the critical role of environmental conditions in shaping the biochemical profile of Ponkan fruits, providing valuable insights into the interplay between cultivation practices and fruit quality.

Accumulating evidence indicates that PAT plays an essential role in plant growth and development, including fruit quality and stress responses [37,38]. Citric acid is one of the main factors affecting fruit quality and functions in plants in response to stress [39,40]. Therefore, the relationships between CitPATs and citric acid were analyzed. Through transcriptome data analysis, we screened and identified six CitPATs genes highly correlated with citric acid. Further RT-qPCR experiments confirmed that CitPAT1 and CitPAT10 were significantly associated with citric acid, suggesting their potential involvement in the citric acid metabolism process. Currently, the functions of PAT genes in plant growth and development have been widely reported, such as AtPAT1 and AtPAT21 in Arabidopsis [41]. However, research on whether PATs participate in fruit quality regulation remains relatively scarce. A study by Jiang et al. demonstrated that apple MdPAT16 can influence fruit sugar accumulation by regulating the MdCBL1-MdCIPK13-MdSUT2.2 pathway, indicating the potential role of PATs in fruit quality control [38]. This study shows that CitPAT1 and CitPAT10 are significantly correlated with citric acid, leading us to speculate that they may participate in the regulation of citric acid in citrus fruits. Nevertheless, the specific regulatory mechanisms still require further investigation.

In this study, we conducted a systematic bioinformatics analysis of a specific gene family in citrus, revealing its distribution, evolutionary relationships, and functional characteristics within the citrus genome. Building on existing research, our findings provide valuable insights into the molecular mechanisms underlying the roles of this gene family in citrus growth, development, and stress responses. In recent years, the rapid advancement of genome sequencing technologies has significantly supported the functional study of gene families through whole-genome sequencing and annotation. For instance, Lodi et al. performed whole-genome sequencing and annotation of Trametes sanguinea ZHSJ uncovering the distribution and functional features of key gene families in its genome, which offered critical references for understanding the biosynthetic mechanisms of fungal secondary metabolites [42]. Similarly, our systematic analysis of the citrus gene family not only identified the genomic distribution of its members but also revealed its potential functions in citrus through evolutionary analysis and functional prediction. These results align with the methodologies employed by Lodi et al., further underscoring the importance of whole-genome sequencing and bioinformatics analysis in gene family research. Moreover, our study identified significant differences in the expression patterns of this gene family across various citrus tissues and under stress conditions, suggesting its potential roles in citrus growth, development, and stress responses. Future research could integrate transcriptomics and functional genomics approaches to further validate the functions of this gene family and explore its potential applications in citrus breeding.

5. Conclusions

In this study, we identified 23 PAT genes (CitPATs) harboring the conserved DHHC domain in the citrus genome. These genes demonstrated high evolutionary conservation, an uneven chromosomal distribution, and diverse cis-regulatory elements associated with stress and hormone responses. Expression profiling during fruit development unveiled distinct correlation patterns with citric acid accumulation: CitPAT6, CitPAT18, and CitPAT23 were positively correlated with acid content, whereas CitPAT1, CitPAT10, and CitPAT13 were negatively correlated. These results indicate a functional diversification among CitPATs in regulating citric acid metabolism. Our findings provide novel insights into the molecular mechanisms of citrus fruit quality formation and establish a foundation for future research on PAT-mediated regulation of fruit development.