All experiments were conducted at the Plant Growth Facility of Anhui Science and Technology University (32°55’N, 117°23’E), located in Chuzhou, Anhui Province, China. The facility maintains controlled-environment chambers with standardized conditions for photoperiod, temperature, and humidity control. The maize inbred line B73 was cultivated in a controlled-environment growth chamber using a specific substrate composed of a sterilized potting mixture with a ratio of peat:vermiculite:perlite of 3:1:1. Regular irrigation and fertilization were conducted to maintain optimal growth conditions. Fertilization was performed with 5 grams per pot of NPK 15-15–15 compound fertilizer, applied once every 8 days after seedling emergence. Plants were grown in plastic pots with a diameter of 15 centimeters, filled with the sterilized potting mixture. The cultivation was carried out under controlled conditions: 28°C, a photoperiod of 16 hours of light/8 hours of darkness, and relative humidity of 50-60%. The substrate was sterilized prior to use to prevent microbial contamination.

Experimental treatments commenced at the three-leaf developmental stage, with triplicate biological replicates implemented throughout. Lepidopteran test species (Spodoptera frugiperda, Ostrinia furnacalis, and Spodoptera exigua) were obtained as laboratory-adapted colonies from the Engineering Technology Institute of Maize Breeding in Anhui Province. Larvae were maintained in climate-controlled chambers under standardized rearing conditions (25 ± 1°C, 60 ± 5% RH, 16h light cycle), fed artificial diet until reaching uniform 2nd-3rd instar developmental stages. Adult specimens received nutritional supplementation via 10% honey solution. All insect bioassays included triplicate biological replicates to ensure experimental robustness. The study employed a completely randomized design with three biological replicates per treatment. For lepidopteran infestation experiments, three-leaf stage maize plants were randomly assigned to four treatment groups: (1) control (no infestation), (2) Spodoptera frugiperda infestation, (3) Ostrinia furnacalis infestation, and (4) Spodoptera exigua infestation. Each treatment group contained 15 plants (3 plants per replicate). Infestation was performed using 2nd-instar larvae at a density of 5 larvae per plant.

The 26 maize pangenome datasets were retrieved from MaizeGDB following Hufford’s genomic framework (Hufford et al., 2021). The PAL-specific HMM profile (PF00320) was acquired from the InterPro database (Paysan-Lafosse et al., 2023). A dual-algorithm approach employing HMMER v3.3.2 and BLASTP identified candidate sequences containing PAL domains, with candidates selected using an E-value threshold <1e-10. Following sequence extraction and redundancy elimination, structural validation through SMART and functional annotation via UniProt confirmed definitive PAL family members across all pangenome assemblies (Bateman et al., 2021; Letunic et al., 2021) (Please refer to Supplementary Table S1 for the URLs and access times of all the websites in Section 2).

Phylogenetic relationships among ZmPALs proteins were reconstructed through comparative analysis with Arabidopsis and rice homologs. Reference PAL protein sequences from Arabidopsis thaliana and Oryza sativa were retrieved from NCBI and Phytozome13 databases (Pruitt et al., 2011; Goodstein et al., 2012). Multiple sequence alignment of maize, rice, and Arabidopsis PAL homologs was performed using the ClustalW algorithm, followed by phylogenetic tree construction via the neighbor-joining method in MEGA11 with 1000 bootstrap replicates. The resultant tree topology was visualized and annotated using Evolview to enhance interpretative resolution of evolutionary relationships (Subramanian et al., 2019).

Presence-absence variation (PAV) profiling of ZmPALs was conducted using genomic data from Hufford’s pangenome study. A custom script generated binary PAV matrices, which were subsequently processed for phylogenomic representations of 26 genotypes through the ggplot2 package in R. Presence/absence patterns across genotypes were visualized as heatmaps via the ComplexHeatmap R package, enabling systematic evaluation of PAL family genomic architecture heterogeneity.

Coding sequences (CDS) and corresponding protein sequences of PAL family members were extracted from 26 maize genomes. Evolutionary selection pressures were quantified using KaKs_Calculator v2.0 to compute Ka/Ks ratios for each PAL homolog. A visualization pipeline integrating three R packages (ggridges, ggplot2, and pheatmap) generated comparative evolutionary profiles - ridgeline plots for full Ka/Ks distribution analysis and heatmaps specifically highlighting positive selection candidates (Ka/Ks>1). The evolutionary framework defined neutral mutations at Ka/Ks=1, positive selection at ratios >1, and purifying selection at ratios <1, enabling systematic detection of selection signatures across phylogenetic contexts (Li et al., 2023b).

SV data and corresponding gene expression profiles across cultivars were retrieved from Hufford’s genomic resource, with B73 serving as the reference genome for structural variant calling. SV annotations were performed using ANNOVAR, followed by targeted extraction of PAL-associated structural variants. Correlation analyses between SV status and transcriptional activity of PAL members were conducted, with differentially expressed candidates visualized through histogram representations. Genome annotation files (GFF3 format) for all 26 maize lines were acquired from MaizeGDB to resolve structural configurations of significant PAL variants. Motif discovery in ZmPAL protein sequences was executed via the MEME Suite (v5.5.5) using default parameters except for specifying 10 motifs (Bailey et al., 2015). Final gene structure diagrams integrating motif patterns and genomic architectures were generated using TBtools, leveraging GFF annotations and motif prediction outputs for comparative visualization (Chen et al., 2023).

ZmPAL protein sequences from insect-resistant accession CML333 and reference genome B73 were submitted to the MEME Suite for comparative motif analysis, specifying 10 conserved motifs and generating WebLogo outputs to visualize sequence conservation patterns. Promoter regions (-2000 bp upstream) of ZmPALs were extracted from both genotypes for cis-element identification using PlantCARE database, prioritizing elements associated with stress responsiveness, developmental regulation, and hormonal signaling pathways (Rombauts et al., 1999).

Publicly available RNA-seq datasets documenting maize responses to lepidopteran infestations (Spodoptera frugiperda: PRJNA675077, Ostrinia furnacalis: PRJNA772910, Spodoptera exigua: PRJNA625224) were retrieved from NCBI for expression profiling of ZmPALs pan-genes (Tzin et al., 2017; Tang et al., 2021; Ye et al., 2022). Raw sequence quality was assessed using FastQC v0.12.1, followed by adapter removal and quality filtering through Trimmomatic v0.40. Processed reads were aligned to the B73_V5 reference genome using TBtools’ One-Step RNAseq plugin, implementing HISAT2 for alignment and StringTie for transcript quantification to generate FPKM expression matrices. Differential expression patterns were visualized as log₂(RPKM+1)-transformed heatmaps using the ComplexHeatmap package (v2.6.2) in R v4.0.3, enabling cross-infestation comparative analysis of PAL transcriptional dynamics.

Lepidopteran infestation was performed by placing five 2nd-instar larvae (starved for 4 hr) on the abaxial surface of the third fully expanded leaf using a fine brush. Larvae were confined to individual plants using mesh bags (30×40 cm, 120-mesh) to prevent escape and ensure controlled feeding. Infestation duration was standardized to 24 hours for all experiments, after which larvae were removed and leaf damage was quantified. Leaf samples were collected from maize plants at 0h (uninfested control), 4h, 12h, and 24h post-infestation by lepidopteran larvae (Spodoptera frugiperda, Ostrinia furnacalis, Spodoptera exigua). Total RNA was isolated using the RNAprep Pure Plant Kit (TIANGEN, Beijing, China) following manufacturer protocols, with subsequent cDNA synthesis performed via HiscriptII Reverse Transcriptase (Vazyme, Nanjing, China). qRT-PCR primers targeting ZmPALs were designed using Primer 6 software, with maize elongation factor 1-alpha (EF-1α) serving as the endogenous control (Ye et al., 2012) ( Supplementary Table S2 ). Amplification reactions (20μL) containing 5μL cDNA template, 0.1μM primers, and SYBR Green Master Mix were conducted in triplicate on an ABI ViiA 7 system under standardized thermal cycling conditions: 95°C for 30 sec, 40 cycles of 95°C/10 sec and 60°C/30 sec, followed by melt curve analysis (95°C/15 sec, 60°C/60 sec, 95°C/15 sec). Melting curve profiles and cycle threshold (Ct) values were acquired using QuantStudio™ Software v1.6.1, with relative expression quantified through the 2^−ΔΔCt method (Livak and Schmittgen, 2001). All data were analyzed using SPSS 26.0 (IBM Corp.). One-way ANOVA followed by Tukey’s HSD test was employed for multiple comparisons between treatment groups. For temporal expression patterns, two-way ANOVA was used to examine the effects of time and treatment. Data are presented as mean ± standard error (SE) of three biological replicates, with statistical significance set at p < 0.05. Data visualization was performed using GraphPad Prism 8 software (Berkman et al., 2018). All qRT-PCR reactions were performed in triplicate for each sample.

A total of 29 ZmPALs family members were identified across 26 maize pan-genome assemblies. Comparative analysis using B73 as the reference genome revealed marked disparities in both gene count and protein sequence length among different maize lines ( Figure 1a ). The Ki3 and MS71 lines exhibited the highest PALs content with 16 members each, while M162W showed the minimal count of 11 members. Structural classification of ZmPALs demonstrated a composition of 7 core genes, 2 near-core genes, 12 non-core genes, and 8 line-specific private genes ( Figure 1b ). The identification of 29 ZmPALs across 26 maize genomes revealed four distinct categories: (1) 7 core genes present in all accessions, likely maintaining essential functions in phenylpropanoid metabolism; (2) 2 near-core genes absent in only 1–2 lines, suggesting conserved but potentially specialized roles; (3) 12 non-core genes showing intermediate frequencies (present in 3–24 genomes), which may contribute to lineage-specific adaptations; and (4) 8 private genes unique to single accessions, representing recent evolutionary innovations or deletions in other lines. This distribution pattern reflects the dynamic evolutionary history of the ZmPAL family, where core genes preserve fundamental functions while non-core and private genes may enable metabolic diversification and environmental adaptation. Particularly, the substantial proportion of non-core and private genes (20/29, ~69%) highlights the remarkable genetic plasticity of phenylpropanoid pathways in maize, potentially facilitating rapid responses to biotic stresses like lepidopteran herbivory across different environments.

Protein length comparisons revealed that ZmPAL8 (core) displayed the longest sequence, followed by ZmPAL7. The near-core ZmPAL9 maintained consistent protein length in 25 lines despite its absence in CML228. Among non-core genes, ZmPAL21 exhibited the longest average protein length compared to the shortest ZmPAL12. Substantial variation in protein lengths was observed across other family members ( Figure 1d ). Comprehensive data for all PAL family members are provided in Supplementary Table S3 .

Phylogenetic analysis utilizing protein sequences from maize, rice, and Arabidopsis established three distinct subfamilies following the classification by Wu et al. ( Figure 1c ). Subfamily III contained the highest number of ZmPALs (17 members), followed by Subfamily II with 7 members, while Subfamily I exhibited the smallest contingent (5 members). Core, non-core, and private genes predominantly accumulated in Subfamily III, though near-core genes were notably absent from this subfamily. Arabidopsis PALs were exclusively clustered in Subfamily I, whereas rice PALs distributed across both Subfamilies II and III. This evolutionary pattern demonstrates closer phylogenetic relationship between maize and rice compared to their divergence from Arabidopsis.

The Ka/Ks ratio served as a critical evolutionary indicator to investigate selection pressures on PAL family members across 26 maize genomes. Comprehensive calculation of Ka/Ks values revealed that except for private genes (uncalculable ratios), only ZmPAL8 exhibited a ratio exceeding 1, while most members maintained ratios below this threshold ( Figure 2a ). Distinct positive selection signals were detected for ZmPAL8 in Ki3 and Tx303 lines, contrasting with purifying selection patterns in other genes. Heatmap analysis of ratios >1 confirmed ZmPAL8 as the sole gene demonstrating elevated Ka/Ks values, indicative of sustained selective pressure during maize evolution ( Figure 2b ).

Structural variation (SV) analysis of PAL family members revealed distinct expression patterns through Pearson correlation coefficients comparing SV-overlapping and non-overlapping genes. Expression profiling demonstrated elevated transcript levels for ZmPAL3, ZmPAL7, and ZmPAL10, while ZmPAL1 and ZmPAL13 showed complete transcriptional silence ( Figure 3a ). Notably, ZmPAL5 exhibited significant differential expression between SV-present and SV-absent conditions (p<0.05), demonstrating substantial SV-mediated regulation of its transcriptional activity ( Figure 3b ).

Genomic analysis identified 196 structural variations (SVs) overlapping with 29 ZmPALs loci and their flanking 2-kb regulatory regions. Comparative profiling against the B73 reference genome revealed SVs predominantly manifested as insertions ( Figure 3c ) and deletions ( Figure 3d ). Notably, line CML228 exhibited a 131-bp upstream insertion, while Ki3 displayed a 12-bp intragenic deletion. Comparative structural mapping of ZmPAL5 across 26 genomes demonstrated conserved gene architecture and conserved domains identical to the B73 reference, with no substantial structural divergences observed ( Figure 3e ).

Comparative analysis of cis-acting elements in ZmPALs promoters was conducted between CML333 and B73, focusing on hormone responsiveness, plant growth/development, and stress-related regulatory motifs ( Figure 4a ). The B73 genome exhibited greater cis-element diversity compared to CML333. Hormonal response elements for abscisic acid, gibberellin, and salicylic acid were prevalent across most ZmPAL promoters. Light-responsive elements were ubiquitously present except in ZmPAL9_CML333. Stress-inducible motifs including low-temperature responsiveness, anaerobic induction, and drought-inducibility were systematically identified. These findings suggest that ZmPALs may orchestrate maize growth, development, and stress adaptation through combinatorial regulation by diverse promoter cis-elements.

Based on the conserved motifs of ZmPALs in the reference genomes B73 and CML333, a conserved domain alignment diagram was generated ( Figure 4b ). The results indicated that all ten conserved domains identified in the CML333 genome corresponded to those in the B73 reference genome. However, the amino acids within the corresponding conserved domains of each group did not exhibit complete alignment, a phenomenon potentially attributable to selective pressures affecting the conserved domains of ZmPALs.

Transcriptomic re-analysis of published datasets (PRJNA675077: Spodoptera frugiperda; PRJNA772910: Ostrinia furnacalis; PRJNA625224: Spodoptera exigua) using the B73_V5 reference genome revealed conserved expression patterns among ZmPALs pan-family members under Lepidopteran herbivory ( Figure 5 ). Two members (ZmPAL4 and ZmPAL13) exhibited transcriptional silence across all stress conditions, while five genes (ZmPAL2, ZmPAL3, ZmPAL7, ZmPAL10 and ZmPAL23) demonstrated elevated expression profiles. The pan-family displayed coordinated transcriptional responses to these biotic stressors, suggesting functional conservation in insect defense mechanisms.

Lepidopteran-specific induction patterns revealed distinct regulatory responses among ZmPALs members. Spodoptera frugiperda infestation ( Figure 5a ) triggered significant upregulation of five genes (ZmPAL5, ZmPAL6, ZmPAL7, ZmPAL18 and ZmPAL23), with ZmPAL6 showing the most pronounced induction. Ostrinia furnacalis challenge ( Figure 5b ) upregulated eight ZmPALs (ZmPAL1, ZmPAL2, ZmPAL3, ZmPAL5, ZmPAL6, ZmPAL7, ZmPAL10 and ZmPAL23), particularly ZmPAL6 and ZmPAL23, while downregulating ZmPAL8 and ZmPAL9. Spodoptera exigua exposure ( Figure 5c ) elevated seven transcripts (ZmPAL3, ZmPAL5, ZmPAL7, ZmPAL8, ZmPAL9, ZmPAL10 and ZmPAL23), with ZmPAL23 demonstrating the highest induction magnitude.

To elucidate ZmPALs pan-family functions in maize defense against fall armyworm (FAW), a time-course induction experiment was conducted in B73 plants. Quantitative PCR analysis revealed dynamic temporal expression patterns of six ZmPALs (ZmPAL3, ZmPAL5, ZmPAL6, ZmPAL7, ZmPAL10 and ZmPAL23) at 0, 4, 12, and 24 hours post-infection (hpi) ( Figure 6 ). All examined genes exhibited FAW-responsive regulation, with significant upregulation peaking during early to mid-stages (4–12 hpi). Transcript levels subsequently declined to basal levels by 24 hpi, indicating transient induction kinetics characteristic of biotic stress responses.

Five ZmPALs members (excluding ZmPAL3) exhibited significant induction across FAW infestation stages. ZmPAL5 and ZmPAL6 peaked at 12 hpi, whereas ZmPAL23 showed an early expression surge at 4 hpi ( Figure 6 ). The transient ZmPAL3 upregulation at 12 hpi coincided with defense signaling activation, while its downregulation at 4 hpi and 24 hpi suggests dynamic transcriptional reprogramming coordinating anti-herbivore responses. These differential patterns implicate ZmPALs in FAW defense through temporal-specific regulation, potentially mediated by phased induction of defense-related signaling cascades. The observed temporal divergence in gene activation (early vs mid-phase peaks) reflects functional specialization within the PAL regulatory network during biotic stress adaptation.

Functional investigation of ZmPALs pan-family members under Asian corn borer (ACB) infestation revealed temporally differentiated expression dynamics in B73 ( Figure 7 ). ZmPAL5 and ZmPAL10 showed no significant expression alterations, suggesting limited involvement in ACB response. Notably, ZmPAL7 exhibited significant upregulation at 4 hpi, indicating potential early defense activation, while ZmPAL3 demonstrated delayed induction peaking at 24 hpi, possibly mediating late-phase resistance mechanisms. ZmPAL6 displayed consistent downregulation across all timepoints, suggesting suppression during ACB challenge. In contrast, ZmPAL23 demonstrated sustained induction throughout infestation stages, highlighting its central regulatory role. These divergent expression profiles reflect functional specialization within the ZmPALs network during ACB defense, with specific members orchestrating phase-specific resistance strategies. The identification of temporally regulated PAL isoforms provides both mechanistic insights into maize-insect interactions and potential genetic targets for breeding ACB-resistant varieties.

qRT-PCR analysis of ZmPALs expression in B73 following beet armyworm (BAW) infestation revealed temporally stratified transcriptional dynamics across four timepoints (0, 2, 12, 24 hpi) ( Figure 8 ). All responsive ZmPALs exhibited phased induction patterns: ZmPAL23 showed marked upregulation at 4 hpi, while ZmPAL7 peaked at 24 hpi. Early response genes (ZmPAL10 and ZmPAL23) demonstrated significant induction by 4 hpi, suggesting initiation of primary defense mechanisms. Mid-phase induction (12 hpi) involved ZmPAL5, ZmPAL10, and ZmPAL23, potentially coordinating intermediate defense signaling. Late-stage upregulation (24 hpi) characterized ZmPAL3 and ZmPAL7, with ZmPAL10 maintaining sustained 2–3 fold induction post-4 hpi. Contrastingly, ZmPAL6 displayed consistent downregulation, indicating negative regulatory functions. The observed temporal divergence in PAL activation suggests stage-specific defensive roles, particularly mid-phase induction potentially mediating defense pathway modulation. This dynamic transcriptional reprogramming reflects ongoing plant-insect interplay through successive defense phases.