Genome-Wide Characterization and Expression Analysis of CsPALs in Cucumber (Cucumis sativus L.) Reveal Their Potential Roles in Abiotic Stress and Aphid Stress Tolerance

Abstract

Phenylalanine ammonia lyase (PAL) is a pivotal enzyme in the phenylalanine metabolic pathway in plants and has a crucial role in the plant’s response to environmental stress. Although the PAL family has been widely studied in many plant species, limited is known about its particular role in cucumbers under stress. We investigated the physicochemical properties, gene structure, gene duplication events, conserved motifs, cis-acting elements, protein interaction networks, stress-related transcriptome data, and quantitatively validated key stress-related genes. The main results indicated that 15 PAL genes were grouped into four clades: I, II, and III when arranged in a phylogenetic tree of PAL genes in angiosperms. The analysis of the promoter sequence revealed the presence of multiple cis-acting elements related to hormones and stress responses in the cucumber PAL genes (CsPALs). The analysis of protein interaction networks suggested that CsPAL1 interacts with eight other members of the PAL family through CsELI5 and CsHISNA, and directly interacts with multiple proteins in the 4CL family. Further investigation into the expression patterns of CsPAL genes in different tissues and under various stress treatments (NaCl, Cu²⁺, Zn²⁺, PEG6000, aphids) demonstrated significant differential expression of CsPALs across cucumber tissues. In summary, our characterization of the CsPAL family offers valuable insights and provides important clues regarding the molecular mechanisms of CsPALs in managing abiotic and biotic stress interactions in cucumbers.

1. Introduction

Phenylalanine ammonia lyase (PAL) initiates the phenylpropanoid pathway. It acts as a catalyst for the conversion of L-phenylalanine, which is produced by the shikimate pathway, into trans-cinnamic acid [1]. This enzyme plays a crucial role in bridging primary and secondary metabolism in plants, as it provides the necessary building blocks for different branches of phenylpropanoid metabolism. The pathway produces intermediate compounds including trans-cinnamic acid, coumaric acid, ferulic acid, and salicylic acid, which are further converted into coumarins, chlorogenic acid, or CoA esters, ultimately resulting in the formation of lignin, flavonoids, and other secondary metabolites. These chemicals play a role in several plant biological processes, including morphogenesis, growth and development, resistance to pathogen invasion, and responses to environmental stimuli [2,3,4]. Alterations in PAL activity have a strong correlation with plant resistance to diseases and the ability to withstand stress, making it a reliable indicator for environmental and biological stressors.

PAL genes were first obtained from barley (Hordeum vulgare) [5], and subsequent genome sequencing has revealed that the model plant Arabidopsis thaliana contains four unique PAL genes [6,7,8,9,10], specifically AtPAL1, AtPAL2, AtPAL3, and AtPAL4 [9]. However, only AtPAL1 and AtPAL2 have been thoroughly studied in terms of their functions [8]. Expression analysis demonstrates that expression levels of PAL genes vary under different conditions and are frequently spatiotemporal and tissue-specific [11]. Therefore, it is crucial to analyze and investigate each PAL gene from a variety of perspectives to enhance plant stress resistance.

PAL genes significantly enhance plant tolerance and resistance under various abiotic and biotic stress conditions by regulating the synthesis of secondary metabolites and hormones. PAL genes are triggered in response to abiotic stressors such as heavy metals, high salt levels, drought conditions, cold temperatures, and waterlogging. The tolerance to drought stress is greatly enhanced in pal1 pal2 double mutants in Arabidopsis [12]. The accumulation of suberin polyphenolics (SPP) and suberin polyaliphatics (SPA) was facilitated by the upregulation of PAL genes in potatoes under Ca²⁺ treatment, which contributed to the plant’s resistance to heavy metal stress [13]. Cold priming primarily stimulates the synthesis of salicylic acid (SA) through the PAL pathway, and when wheat is exposed to low temperatures, the PAL genes and endogenous SA levels are greatly increased [14]. Under waterlogging stress, wheat’s PAL gene expression is increased, raising SA concentration, triggering aerenchyma formation, and stimulating the production of adventitious roots, allowing wheat plants to react to waterlogging through morphological alterations [15]. In terms of biotic stress tolerance, the PAL gene enhances plant resistance by regulating the production of secondary metabolites and hormone levels. In maize, PAL genes confer resistance to sugarcane mosaic virus by promoting the accumulation of SA [16]. Increased expression of OsPAL8 in rice stimulates the production and accumulation of SA and lignin, greatly improving rice’s ability to withstand brown planthopper infestation [17]. Furthermore, aphid infestation in susceptible wheat varieties leads to a significant increase in the expression of PAL genes. This is mainly caused by the aphid’s probing, which triggers hormone-dependent responses including SA and jasmonic acid (JA) [18].

Cucumber (Cucumis sativus L.) is an important vegetable crop that is widely cultivated globally and is highly valued for its nutritional value and economic significance [19]. Cucumbers are rich in vitamins and minerals, making them a major component of many diets [20]. Additionally, this crop serves as an important model system for studying plant sex determination and vascular tissue formation [21,22]. The growth and development of cucumbers are susceptible to various adverse stresses, such as heavy metals [23], waterlogging [24], dehydration [25], heat stress [26], and aphids [27], which can lead to reduced yield and quality. Utilizing biotechnological breeding techniques is an important approach to improve crop yield, quality, and stress resistance. This study aims to comprehensively explore the function of the PAL genes in cucumber in response to abiotic and aphid-induced stress by identifying and analyzing their expression profiles. The findings are intended to provide theoretical support for the green and efficient production of cucumber.

2. Results

2.1. Identification of PAL Genes in Cucumber

To identify PAL genes in cucumber, a Blastp and HMM search was performed on the Cucumber Chinese Long V3 genome using four AtPAL protein sequences [28]. Through this process, 15 CsPAL genes with PAL domains were successfully identified and named CsPAL1 to CsPAL15 based on their chromosomal positions. The open reading frame lengths of the PAL family ranged from 927 to 2154 bp, corresponding to protein lengths of 308 to 717 amino acids. Except for CsPAL3, the isoelectric points (PI) of the other CsPAL proteins ranged from 5.33 to 6.37. Additionally, except for CsPAL2 and CsPAL3, the molecular weights of the other CsPAL proteins ranged from 77.17 to 78.37 KDa (Table S1).

To study the evolutionary relationships within the cucumber PAL gene family, a phylogenetic tree was constructed among cucumber, Arabidopsis, tomato, melon, watermelon, grape, corn, wheat, and rice (Figure 1A). According to the classification results from the phylogenetic tree, CsPAL genes can be divided into three subfamilies: one gene in subfamily I, twelve genes in subfamily II, and two genes in subfamily III. The PAL family proteins of monocotyledonous and dicotyledonous plants show clear boundaries in the clustering of the evolutionary tree, suggesting that during plant evolution, PAL proteins in monocots and dicots have evolved in relatively independent directions based on their actual needs for growth, development, or stress response. To better understand the gene evolutionary process, the exon-intron structure of the cucumber PAL genes was further analyzed. The analysis showed that the number of exons in CsPAL genes ranged from one to two, indicating high conservation (Figure 1B). The conserved motifs were analyzed using the MEME online tool, identifying 10 different motifs (named motifs 1–10). Except for CsPAL2 and CsPAL3, CsPAL genes contained motifs 1–10, with CsPAL4–CsPAL15 showing the same motif distribution, demonstrating conservation and similar functions. CsPAL2 contained only motifs 1, 2, 3, 5, and 7, while CsPAL3 contained motifs 3, 6, 8, 9, and 10 (Figure 1C). Multiple sequence alignment results (Figure 2) demonstrated that, except for CsPAL2 and CsPAL3, most CsPAL domains are highly conserved.

2.2. Analysis of Cis-Regulatory Elements in CsPAL

To gain a better understanding of the cis-acting elements present in the promoter regions of the cucumber PAL gene family and investigate how the family responds to stressors, we conducted a prediction of the cis-acting elements within the 2000 base pairs upstream of the cucumber PAL gene family. The prediction results revealed numerous elements involved in light, hormone, and abiotic stress responses in the cucumber PAL gene family (Figure 3). Seven types of hormone response elements and five types of abiotic stress response elements were identified. All CsPAL genes contained MYB and MYC elements, totaling 58 and 49, respectively; fourteen CsPAL genes contained AAGAA-motif (abscisic acid response element) and ERE (ethylene response element), with totals of 35 and 37, respectively; CsPAL11 showed a more significant response to the AAGAA-motif; twelve CsPAL genes contained ARE elements (anaerobic induction element), totaling 29; eleven CsPAL genes contained ABRE elements (abscisic acid response element); nine CsPAL genes contained TCA-element (salicylic acid response element) and WUN-motif (wound response element), with totals of 11 and 19, respectively; seven CsPAL genes contained as-1 element (salicylic acid response element), STRE (salt stress response element), TGACG-motif (jasmonic acid response element), CGTCA-motif (methyl jasmonate response element), and WRE3 element (wound-induced element), with totals of 13, 16, 13, 13, and 12, respectively; six CsPAL genes contained LTR (low temperature response element), totaling six; five CsPAL genes contained TCA element (salicylic acid response element) and TGA-element (auxin response element); four CsPAL genes contained Myb binding sites and P-box (gibberellin response element), with totals of seven and four, respectively. Only three CsPAL genes contained AuxRR-core (auxin response element), CCAAT-box (MYBHv1 binding site), and MBS (MYB binding site induced by drought). The light-responsive elements exhibited the highest diversity and abundance, encompassing several types such as Box4, G-box, GATA-motif, I-box, MRE, TCT-motif, ATC-motif, GT1-motif, Gap-box, etc. It is worth noting that all members of these elements comprise Box4. The findings indicate that the majority of cucumber CsPAL family members possess hormone response elements and stress response elements, indicating that the CsPAL family may participate in various environmental stress responses through these cis-acting elements.

2.3. Chromosomal Location, Tandem Duplication, and Ka/Ks Ratio of CsPAL Genes

Using the annotated cucumber genome data, the locations of CsPAL genes were mapped onto the cucumber chromosomes. The results showed an uneven distribution of all genes across three chromosomes: CsPAL1 is located on chromosome 1 alone, while the rest of the CsPAL genes are concentrated on chromosomes 4 and 6 (Figure 4). Studies suggest that tandem duplications tend to amplify genes related to membrane protein functions and those closely associated with biotic and abiotic stresses [28]. To explore the biased retention and selective pressures on the cucumber PAL gene family due to tandem duplication during evolution, we performed a duplication analysis on its 15 family members using MCScanX and conducted a Ka/Ks analysis. The duplication analysis revealed that among the 15 cucumber CsPAL gene family members, there are nine pairs of genes with tandem duplication relationships: CsPAL3 and CsPAL4, CsPAL4 and CsPAL5, CsPAL5 and CsPAL6, CsPAL9 and CsPAL10, CsPAL10 and CsPAL11, CsPAL11 and CsPAL12, CsPAL12 and CsPAL13, CsPAL13 and CsPAL14, and CsPAL14 and CsPAL15 (Table S2). The tandemly repeated CsPAL genes may have similar functions. This similarity allows other duplicated CsPAL genes to compensate for the function of a mutated or non-functional PAL gene, ensuring the plant’s adaptability to environmental stresses. Ka/Ks analysis showed that the Ka/Ks ratios for all nine gene pairs were less than one, indicating that these gene pairs primarily underwent synonymous substitutions during evolution, maintaining the stability of the protein amino acid sequences, with a relatively low rate of non-synonymous substitutions. This indicates that these gene pairs were under strong negative selective pressure during evolution, maintaining high conservation, and preserving the stability of gene function and structure. Specifically, tandemly duplicated CsPAL genes have experienced negative selection pressure, which has helped them retain essential functions. These tandemly duplicated CsPAL genes have similar sets of upstream regulatory elements. This similarity suggests that these genes might be co-regulated, maintaining conserved functions despite undergoing duplication. Additionally, the presence of multiple gene copies allows some of these copies to accumulate mutations without compromising the overall biological function, providing new possibilities for evolutionary adaptation.

2.4. Protein-Protein Interaction Network Analysis

Using the online tool STRING, the protein interaction network of the cucumber PAL family members was predicted with the cucumber Chinese Long V2 protein database as a reference, identifying interactions among nine family members (Figure 5). No predicted interaction relationships were found among the other 6 cucumber PAL family member proteins. The results obtained from this Protein-Protein Interaction (PPI) analysis suggest that cucumber PAL family members are interconnected through complex multigenic interactions rather than direct interactions. The identification of 28 nodes in the CsPAL protein interaction network highlights the intricate relationships among the proteins involved, with CsPAL1 emerging as a central figure due to its more interactions with other members in the network. This finding indicates that CsPAL1 may play a crucial role in mediating various functions within the PAL family, potentially influencing the phenylpropanoid metabolic pathway, which is vital for plant defense and development. The strong interactions between CsPAL1 and other significant proteins, such as CsELI5, CsHISNA, and Csa_2G070200 (cytochrome P450 cinnamate 4-hydroxylase), are particularly noteworthy. CsELI5 is implicated in ethylene signaling, suggesting that there could be a regulatory link between the PAL family and ethylene response pathways. The interaction with CsHISNA, a gene involved in histidine biosynthesis, points to potential metabolic connections that could affect overall plant growth and stress responses. Additionally, the interaction with cytochrome P450, a key enzyme in the phenylalanine metabolic pathway, reinforces the idea that the PAL family is integral to the biosynthesis of secondary metabolites.

2.5. Expression Patterns of CsPAL in Different Tissues

To study the expression of CsPAL in different tissues, we quantitatively analyzed the expression levels of 15 CsPAL genes in female flowers, male flowers, leaves, roots, petioles, tendrils, and stems by qRT-PCR. Figure 6 shows that there are differences in gene expression patterns among different tissues. CsPAL2, CsPAL3, CsPAL4, CsPAL5, CsPAL6, CsPAL12, and CsPAL14 exhibit higher expression in roots and leaves. CsPAL9 and CsPAL13 are expressed at higher levels in both female and male flowers. CsPAL7 is highly expressed in tendrils, stems, and petioles, while CsPAL15 has relatively lower expression across all tissues (Figure 6).

2.6. Expression Patterns of CsPAL under Abiotic Stress Conditions

Phenylalanine ammonia lyase (PAL) plays a crucial role in the plant response to abiotic stresses. We further investigated the response of CsPAL to heavy metal stress, drought, and salt stress. Cucumber seedlings were treated with Cu²⁺, Zn²⁺, PEG6000, and NaCl to simulate heavy metal stress, drought stress, and salt stress, respectively, and gene expression was measured. Based on the expression patterns in different tissues (Figure 6), we selected seven CsPAL genes that are highly expressed in roots for quantitative analysis: CsPAL2, CsPAL3, CsPAL4, CsPAL5, CsPAL6, CsPAL12, and CsPAL14.

When compared to untreated controls, the majority of CsPAL genes, with the exception of CsPAL3, exhibited a pattern of initially increasing and then reducing expression within 12 h after being treated with Cu²⁺. CsPAL3 exhibited a trend of increasing, then decreasing, and then increasing again (Figure 7A). CsPAL2, CsPAL3, CsPAL4, and CsPAL6 showed a generally increasing trend within 12 h of Zn²⁺ treatment, reaching their highest expression levels at 12 h; CsPAL5 and CsPAL12 showed a trend of increasing, then decreasing, and then increasing again within 12 h of Zn²⁺ treatment, reaching their highest levels at 12 h; CsPAL14 showed the most complex pattern under Zn²⁺ treatment, with an increasing, then decreasing, then increasing, and again decreasing trend, reaching its peak at 2 h of treatment (Figure 7B). Overall, compared to Zn²⁺ treatment, CsPAL showed a more pronounced response to Cu²⁺ treatment. CsPAL3, CsPAL4, CsPAL5, and CsPAL6 notably responded to both Zn²⁺ and Cu²⁺.

Under treatment with PEG6000, the expression levels of CsPAL significantly increased compared to controls, showing the same trend where the most notable change in expression occurred 2 h after treatment. CsPAL2 and CsPAL3 showed more than a tenfold increase in expression compared to controls at this time point. After exceeding 2 h of treatment, the expression levels of CsPAL generally showed a decreasing trend, with some genes’ expression levels falling below those of the control group. Most genes returned to control levels at 12 h post-treatment. These results indicate that CsPAL genes respond rapidly to drought stress, particularly CsPAL2 and CsPAL3 (Figure 7C).

Under salt treatment, compared to the control group, the relative expression levels of CsPAL5 and CsPAL6 significantly increased by more than threefold, while CsPAL3 and CsPAL4 demonstrated a twofold increase. The expression of CsPAL12 only showed significant differences at 12 h during salt stress treatment. However, the expression changes in CsPAL2 and CsPAL14 were minimal. Overall, under salt treatment, the expression levels of CsPAL were higher than those of the control group but generally lower than under heavy metal stress and PEG6000 treatment (Figure 7D).

2.7. Expression Patterns of CsPAL under Aphid Stress

Previous studies have shown that PAL is involved in plant biotic stress responses. To further investigate the response of CsPAL to aphid stress, we selected CsPAL2, CsPAL3, CsPAL4, CsPAL5, CsPAL6, CsPAL12, and CsPAL14, which are highly expressed in leaves, and measured their expression changes within 120 h of aphid stress. The results showed that CsPAL genes exhibited an initial increase followed by a decrease in expression after aphid treatment. CsPAL5 and CsPAL6 reached their peak expression at 72 h under aphid stress, while the other genes showed peak expression after 24 h of aphid stress. After prolonged treatment, the expression levels of all these genes declined but remained significantly higher than the control group. Among them, CsPAL2 showed the most significant response to aphid stress, with its expression increasing nearly 69 times after 24 h of aphid stress. These results conclude that cucumber CsPAL genes exhibit a significant response to aphid stress (Figure 8).

3. Discussion

Phenylalanine ammonia lyase (PAL) is a crucial enzyme in the phenylpropanoid system and has been found to have a substantial impact in defending against several environmental stressors, including pathogenic attacks, low temperatures, and heavy metals [29,30,31]. Phylogenetic tree analysis is crucial for studying the evolution of species since it enables the examination of the origins and development of gene families, hence permitting additional investigation into the functionality of genes. This study developed a phylogenetic tree encompassing Arabidopsis, cucumber, melon, tomato, watermelon, grape, maize, wheat, and rice, revealing that the CsPAL genes are unequally distributed among three subgroups, which were classified based on the three branches and evolutionary relationships of the phylogenetic tree, suggesting distinct functional roles for these CsPAL genes (Figure 1A). Identification of the cucumber PAL family genes indicated that cucumber has seven genes distributed over two chromosomes within a single cluster [32]. This contrasts with our findings, where 15 genes identified were distributed across three chromosomes (Figure 4) and located in three subgroups (Figure 1). This discrepancy could be attributed to amendments in the cucumber reference genome and the ongoing progress in research, which consistently uncovers new PAL genes. For instance, a study conducted in 2019 on the cucumber PAL family uncovered 12 PAL family genes [33]. With the advancement of species genomics, processes such as genome recombination, gene duplication, deletion, and mutation may lead to the expansion or contraction of gene families in the future.

Protein sequence alignment of the CsPAL family genes in cucumber shows that CsPAL2 and CsPAL3 exhibit the most significant sequence differences compared to other genes. Recent studies in cucumbers have indicated that treatment with PGPR (Pseudomonas spp.) and Ag-nanoparticles results in increased PAL activity [34]. This is linked to the presence of hormone-responsive cis-acting elements (such as SA and JA) identified in this paper (Figure 3). In cucumbers, PAL family genes respond to UV-B irradiation, with transcription levels of CsPAL4 and CsPAL10 increasing by 30-fold compared to the control [33]. This response is associated with the light-responsive elements found among the cis-acting elements (Figure 3). Beyond these studies, there are few recent reports specifically focusing on the cucumber PAL family genes. Our paper provides a thorough analysis of the cucumber PAL family, including gene comparison results combined with gene motifs that reveal CsPAL contains a tripeptide active center composed of Ala-Ser-Gly [35]. The absence of this tripeptide active center can lead to reduced or lost PAL enzymatic activity. All 15 genes identified in the CsPAL family have this ASG tripeptide active center, therefore possessing PAL activity (Figure 1 and Figure 2). Considering the homology between cucumber PAL genes and Arabidopsis genes, it is speculated that genes with high homology have similar functions. PAL genes with high homology within the same subfamily are likely to have similar functions. In Arabidopsis, AtPAL1 and AtPAL2 regulate flavonoid biosynthesis, while AtPAL4 is involved in lignin biosynthesis [12,36]. Phylogenetic tree analysis indicates that cucumber PAL family subfamily II (Figure 1A) genes, which are highly homologous to AtPAL1 and AtPAL2, may be involved in the regulation of flavonoid synthesis. Meanwhile, CsPAL subfamily III (Figure 1A) genes, which are highly homologous to AtPAL4, may be involved in the regulation of lignin synthesis, consistent with the results of expression analysis under stress conditions. The protein interaction network diagram for cucumber CsPAL indicates that the CsPAL gene family generally interacts with CHS, 4CL, and PAT02 (Figure 5). CHS, as the first rate-limiting enzyme in the flavonoid synthesis pathway, directs the phenylpropane metabolic pathway towards the synthesis of flavonoid compounds. It plays a crucial role in plant growth and development and also serves a significant regulatory function in plant responses to external environmental stresses [37]. Studies have shown that CHS can respond to high-temperature stress and also to MeJA treatment [38], suggesting CHS plays a significant role in plant responses to biotic and abiotic stresses [39]. 4CL is a key enzyme in flavonoid metabolism and also regulates lignin metabolism. Studies in Arabidopsis indicate that At4CL1, At4CL2, and At4CL4 are involved in lignin synthesis [40], while Arabidopsis At4CL3 and rice Os4CL2 are involved in flavonoid biosynthesis [41,42]. Both lignin and flavonoids are crucial metabolic substances that enable plants to resist stress. Lignin enhances cell wall strength to resist pathogen or insect invasion, while flavonoids, as vital chemical defense substances in plants, mitigate pathogen or pest-induced stress by reducing oxidative reactions in plants [43]. Further research into the interaction mechanisms of PAL and 4CL enzyme genes will facilitate our understanding of the regulatory mechanisms of the phenylalanine metabolic pathway.

The gene evolution tree showed that CsPALs and AtPALs were closely related, suggesting that cucumber CsPALs and Arabidopsis thaliana AtPALs had similar functions, and AtPAL genes were mostly related to stress responses. Gene structure and cis-acting element analysis also indicated that CsPAL genes might be involved in some biotic and abiotic stress responses. To verify the above hypothesis, we treated cucumber with aphid and abiotic stress, and the results also showed that CsPALs responded to aphid and abiotic stress. The protein interaction network shows that CsPALs interact with some proteins involved in stress resistance, so it is predicted that CsPALs may not only respond to stress but also participate in the stress response as resistance genes.

4. Materials and Methods

4.1. Plant Materials and Growing Conditions

The cucumber variety ‘JinYan 4’ seeds were germinated on filter paper soaked in distilled water for 24 h and then transferred to a growth substrate composed of peat, vermiculite, and perlite (volume ratio 3:1:1). Seedlings at the two-true leaf stage were selected for all the stress treatment experiments. Seedlings were grown in an environment conducive to the growth of both cucumber and aphids, with a light period of 16 h and a dark period of 8 h, at a temperature of 26 °C and 60% humidity.

4.2. Aphid and Abiotic Stress Treatment

Aphids (Aphis gossypii Glover) were collected from the cucumber fields of Yangzhou University and self-bred for at least six generations before the experiment. Cucumber and aphids were kept in an insect-proof net to ensure a consistent background. For aphid stress treatment, 2–3 instar apterous aphids were starved for 2 h. Then, 40 apterous aphids were evenly inoculated on the first true leaf. Samples were taken at 6 h, 24 h, 72 h, and 120 h after aphid inoculation. Before sampling, aphids were gently removed, and honeydew secretions were wiped off with tissue paper. The central part of each sample leaf was cut out with alcohol-disinfected scissors and used for RNA extraction. For heavy metal stress treatments, 100 µM CuSO₄ and 500 µM ZnSO₄ were used. For salinity stress, plant roots were subjected to 150 mM NaCl to simulate salt stress, and 15% PEG6000 to simulate drought stress [44,45]. Control plants were treated with double distilled water. All plants were grown hydroponically, and samples were collected at 2 h, 4 h, 6 h, and 12 h after treatment. Each sample consisted of three biological replicates from three different plants. After cutting, leaf samples were quickly frozen in liquid nitrogen, ground into a fine powder, and stored at −80 °C.

4.3. Identification of CsPAL Gene Family Members in Cucumber

The protein sequences of Arabidopsis thaliana were obtained from the Arabidopsis Information Resource (TAIR) database (http://www.arabidopsis.org/ (accessed on 10 February 2024)), and PAL homologous sequences in the cucumber whole genome database (http://cucurbitgenomics.org/organism/20 (accessed on 10 February 2024)) were identified through BLAST alignment and filtering (with an E-value threshold set at <10⁻¹⁰). The Hidden Markov Model (PF00221) of the PAL protein characteristic domain was downloaded from the Pfam database (https://pfam.xfam.org (accessed on 10 February 2024)) to obtain the conserved domain of PAL. HMMER 3.0 HMM search was used to screen candidate sequences PAL genes from the cucumber whole genome database (http://cucurbitgenomics.org/organism/20 (accessed on 10 February 2024)), with an expectation value (e-value) of 1 × 10⁻⁵. The sequence information of all PAL proteins identified by the above results was submitted to the SMART website (http://smart.embl-heidelberg.de/ (accessed on 10 February 2024)) for cross-validation to confirm the presence of specific domains in the proteins. This validation ensured the reliability of CsPAL in cucumber [46].

4.4. Physicochemical Properties of CsPAL Family Proteins

The online tool Expasy (http://web.expasy.org/compute_pi/ (accessed on 14 February 2024)) was used to predict the amino acid number, isoelectric point, molecular weight, and hydrophilicity index of CsPAL family members.

4.5. Analysis of Conserved Motifs, Gene Structure, Cis-Acting Elements, Multiple Sequence Alignment, and Phylogenetic Analysis

From the TAIR database, the Sol Genomics Network, the Rice Genome Database, and the Cucumber Genome Database, full-length amino acid sequences of PAL proteins for the respective species were obtained. Multiple sequence alignments were performed using the MUSCLE function in MEGA11.0 software, and the output data was stored in both MEGA and fasta formats. The resulting fasta files were submitted to GeneDoc to create multiple sequence alignment diagrams; the MEGA files were used to construct phylogenetic trees using the Maximum-Likelihood (ML) method, with a bootstrap value set at 1000. Based on the gene location on the chromosomes, the 15 candidate PAL genes in cucumber were named. Using the cucumber genome gff3 file and amino acid sequence information, gene structure analysis of the CsPAL family members was performed, with the Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/index.php (accessed on 20 February 2024)) used for visualizing the gene structure analysis. The online tool MEME (http://meme-suite.org/tools/meme (accessed on 20 February 2024)) was employed to analyze the conserved motifs, with a set prediction value of 10 motifs, and TBtools was used for visual motif plotting. Additionally, the nucleotide sequences of 2000 bp upstream of the start codon of each cucumber PAL gene were submitted to PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 20 February 2024)) for predicting cis-acting elements. The obtained results were filtered and categorized based on their regulatory functions.

4.6. Chromosomal Localization and Ka/Ks Analysis

Using the distribution information of CsPAL family members on chromosomes from the cucumber genome gff3 format file, visual mapping was performed using Mapchart (https://www.omicsclass.com/article/397 (accessed on 22 February 2024)). Ka, Ks, and Ka/Ks of tandemly duplicated genes were calculated using ParaAT (https://ngdc.cncb.ac.cn/tools/paraat (accessed on 22 February 2024)).

4.7. Protein-Protein Interaction Prediction

Protein interaction predictions for the CsPAL family members were performed using the online tool STRING (https://string-db.org (accessed on 25 February 2024)) with Cucumber Chinese Long V2 as the reference. The maximum number of 1st shell interacting proteins was set to 20, and for the 2nd shell, it was set to 10, with a minimum required interaction confidence of 0.9. Other parameters were set to default values. The protein interaction networks were visualized using Cytoscape (version 3.9.1).

4.8. RT-qPCR Expression Analysis

Total RNA from cucumber root and leaf tissues was extracted using the SteadyPure Plant Total RNA Extraction Kit (Hunan Accurate Biotechnology Co., Ltd., Changsha, China). cDNA was synthesized with HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The expression levels of the selected genes were detected by RT-qPCR using the ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Gene-specific primers were designed using the online software Prime5.0 Each treatment was set with three technical replicates. The cucumber actin gene (CsActin) was used as the reference gene to calculate the relative expressions via the 2^−∆∆Ct method. Graphs were plotted and significant differences analyzed using GraphPad Prism v10.0. The sequence of primers used in this paper is shown in Table S3.

5. Conclusions

Through bioinformatics analysis of cucumber CsPAL, this study identified 15 CsPAL genes and conducted a comprehensive analysis of this family, including sequence features, cis-acting elements, tissue specificity, protein interaction network prediction, and expression profiles under abiotic and aphid stresses. The expression profiles showed that CsPAL2, CsPAL3, CsPAL4, CsPAL5, CsPAL6, CsPAL12, and CsPAL14 all respond to stress to varying degrees, with CsPAL3, CsPAL4, CsPAL5, and CsPAL6 potentially regulating cucumber’s tolerance to heavy metals and salt stress; CsPAL2 and CsPAL3 potentially involved in drought stress; and CsPAL2 likely playing a significant regulatory role in aphid stress. The findings of this study offer a solid foundation for the selection of CsPAL genes in cucumber breeding programs aimed at enhancing resistance to various stressors.