Genome-wide identification and characterization of the RPA gene family in maize

Abstract

Background

The Replication Protein A (RPA) is a highly conserved heterotrimeric complex that binds single-stranded DNA and interacts with multiple proteins, playing essential roles in DNA replication, repair, and homologous recombination, thereby maintaining genome stability and supporting normal development. However, the specific members and functions of the RPA gene family remain uncharacterized in maize.

Results

In this study, 13 ZmRPA members in maize were identified, which exhibited a wide range of protein lengths, from 86 to 876 amino acids. A phylogenetic analysis of these ZmRPA genes revealed their classification into three distinct subgroups: RPA1, RPA2, and RPA3. Notably, within each subgroup, the motifs of ZmRPA proteins were similar, although variations were observed among different subfamilies. An analysis of gene duplication events suggests that RPA genes have remained highly conserved in maize. Furthermore, the analysis of cis-elements indicated that RPA genes are associated with light-responsive, as well as plant growth and development. RNA-seq analysis revealed that some ZmRPA genes exhibited pronounced expression changes under cold and drought stresses, while the results of tissue-specific expression analysis showed that most ZmRPA genes were highly expressed in the actively dividing meristems and reproductive primordia. Protein interaction and yeast two-hybrid assays revealed that ZmRPA family members interact with each other to perform their biological functions.

Conclusions

Collectively, these findings suggest that ZmRPA family subunits may play important roles in plant growth and development, as well as stress responses, by forming various types of heteromeric complexes through protein interactions. This study provides a comprehensive understanding of the RPA gene family in maize and will facilitate further effective characterization of the RPA genes.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08280-9.

Introduction

Replication protein A (RPA) is a heterotrimeric protein complex that is highly conserved among eukaryotes. It binds to single-stranded DNA (ssDNA) and interacts with various proteins, thereby participating in multiple DNA metabolic processes, including DNA replication, homologous recombination, and DNA repair [1]. RPA is composed of three subunits: the 70 kDa RPA1 (also referred to as RPA70), the 32 kDa RPA2 (also known as RPA32), and the 14 kDa RPA3 (also called RPA14). Each subunit contains at least one oligonucleotide/oligosaccharide-binding fold (OB-fold) DNA-binding domain, which is essential for mediating RPA’s interactions with both DNA and proteins [1].

During the process of DNA replication, the minichromosome maintenance (MCM) helicase enzyme is responsible for unwinding double-stranded DNA (dsDNA), thereby producing ssDNA [2]. The RPA promptly associates with the exposed ssDNA, safeguarding it against potential damage and inhibiting the formation of secondary structures [3]. This action is crucial for maintaining the accuracy and orderly progression of DNA replication. Beyond its role in ssDNA stabilization, RPA interacts directly with histones H3 and H4, facilitating their deposition onto adjacent dsDNA and promoting nucleosome assembly [4]. Furthermore, the RPA-ssDNA complex functions as a scaffold for histone chaperones, aiding in the transfer of histones to the replication fork and supporting the re-establishment of nucleosomes on the newly synthesized DNA strand [4]. Moreover, RPA facilitates the recruitment of the E3 ubiquitin ligase Bre1, which catalyzes H2B ubiquitination, thereby establishing a connection between histone modification and DNA replication and repair processes [5]. This linkage is crucial for the proper progression of the cell cycle and the maintenance of genome stability. Additionally, RPA has the capacity to bind RNA, forming RPA-RNA complexes that promote the formation of R-loops. These R-loops can function as primers to restart stalled DNA replication forks resulting from DNA damage [6].

In yeast and most animal species, each subunit of the RPA complex is generally encoded by a single gene. Conversely, plant species frequently exhibit multiple genes encoding homologs for each RPA subunit. In the case of Oryza sativa, three homologs for RPA1 (OsRPA1a, b, c), three for RPA2 (OsRPA2a, b, c), and one homolog for RPA3 (OsRPA3) have been identified, forming 3 different types of the RPA complexes with following subunits in combination: OsRPA1a-OsRPA2b-OsRPA3 (Type A), OsRPA1b-OsRPA2a-OsRPA3 (Type B) and OsRPA1c-OsRPA2c-OsRPA3 (Type C) [1, 7, 8]. In Arabidopsis thaliana (A. thaliana), the RPA1 subunit is represented by five homologs, including of RPA1A, RPA1B, RPA1C, RPA1D, and RPA1E, all of which maintain the essential single-stranded DNA-binding functionality. The RPA2 subunit is encoded by two homologous genes, RPA2A and RPA2B, whereas the RPA3 subunit is represented by two homologs, RPA3A and RPA3B. The RPA2 and RPA3 subunits play critical roles in stabilizing the RPA complex structure and regulating key processes such as DNA replication and repair [9, 10].

Accumulating evidence indicates that abiotic stresses, such as UV light, heavy metal stress, extreme temperatures, drought, and nutrient deprivation, often induce DNA damage in plants, thereby activating the DNA damage response (DDR) and repair pathways to maintain genome integrity [11, 12]. RPA plays crucial roles in the DDR and ssDNA-bound RPA serves as an essential platform to recruit the ataxia telangiectasia and Rad3-related (ATR) kinase, the central regulator of DDR, to the DNA damages sites and the activation of DDR [13]. In rice, OsRPA1a plays essential roles in DNA repair [14] and promotes the accurate resolution of recombination intermediates and in limits redundant chiasma formation during meiosis [15], while OsRPA2c and OsRPA1c are required to promote meiotic crossovers [16]. In A. thaliana, RPA1A and RPA1C are implicated in homologous recombination during meiosis, whereas RPA1B and RPA1D are predominantly associated with DNA replication, and RPA1C along with RPA1E are involved in DNA repair [17, 18]. Despite RPA2A and RPA2B sharing approximately 40% amino acid identity and 60% similarity, potential protein-protein interaction studies and phylogenetic analyses indicate that they fulfill distinct biological roles [1, 19]. Among them, RPA2B could interact with the DNA double strand binding protein INVOLVED IN DE NOVO2 (IDN2) to facilitate DNA double-strand break repair [19]. More research has focused on the function of RPA2A in plants. RPA2A is highly expressed in the meristematic tissues of root and shoot apices, and its mutant exhibits phenotypes akin to those observed in the rpa1b rpa1d double mutant, including defects in cell division and proliferation, plant growth, and development, suggesting that RPA2A is likely crucial for DNA replication [9, 20, 21]. Furthermore, rpa2a mutants demonstrate hypersensitivity to the DNA alkylating agent methyl methanesulfonate (MMS), indicating a significant role for RPA2A in DNA repair [9]. Additionally, RPA2A mediates the recruitment of the PRC2 complex to key flowering genes via protein interactions, promoting H3K27me3 modification, repressing their expression, and consequently delaying flowering [22]. Similarly, PLEIOTROPIC REGULATORY LOCUS 1 (PRL1), a component of the NineTeen Complex, is involved in C-deprivation-induced plant senescence by directly binding to RPA2A [13]. These studies suggest that RPA play an important regulatory role in plant development and abiotic stress responses. Further investigation into how RPA integrates DDR with stress adaptation will provide deeper insights into its mechanistic role.

Maize (Zea mays) is recognized as one of the most significant staple and economic crops globally, serving a crucial function in food security and various industrial applications. Although the roles of the RPA in DNA replication and genome stability are well-documented across eukaryotic organisms, research specifically addressing the functional characterization of the RPA in maize remains scarce. To elucidate the potential biological functions of the RPA gene family in maize, a comprehensive genome-wide analysis was performed. This study systematically examined the molecular characteristics of maize RPA genes, encompassing conserved domain architectures, phylogenetic relationships, chromosomal localization, and gene duplication events. This study provides foundational insights into the evolutionary dynamics and structural diversity of the RPA family in maize, derived from integrative bioinformatics analyses. These results not only establish a basis for future molecular and functional investigations of the RPA genes in maize but also present valuable resources for exploring their roles in DNA metabolism, stress responses, and developmental regulation in crop plants.

Materials and methods

Data sources and identification of the RPA genes in maize

The reference genome data of B73 maize (B73-v4) were downloaded from the MaizeGDB (https://www.maizegdb.org/). The genome data of other 9 species (A. thaliana, Brachypodium distachyon, Oryza sativa, Sorghum bicolor, Setaria italica, Triticum aestivum, Tritucum urartu, Aegilops tauschii and Hordeum vulgare) were downloaded in Phytozome database [23]. The hidden Markov Model (HMM) file of the RPA1A-E (PF01336, PF04057, PF08646, and PF16900), RPA2A-B (PF01336, PF08784) and RPA3A-B (PF08661) was acquired from InterPro database [24]. Subsequently, HMMER 3.0 was used to identify potential RPA-domain proteins in the above 10 species (E-value < = 5e⁻⁵). To enhance search proteins, a novel HMM model was constructed using the hmmbuild program [25]. Concurrently, BLASTP was conducted using AtRPA sequences as references (E-value < = 1e⁻¹⁰) [26]. Candidate RPA proteins were further validated for the presence of conserved domains using Pfam-scan (http://pfam.xfam.org/) [27], NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) [28] and InterProScan (https://www.ebi.ac.uk/interpro/about/interproscan/) [29]. Finally, the R package seqfinder (https://github.com/yueliu1115/seqfinder) was used to select and retain the longest transcript isoform. The results of the ZmRPA identification were showed in Supplementary Table S1.

Phylogenetic analysis of ZmRPA

Multiple sequence alignment of the RPA proteins was performed using Muscle software [30]. To investigate RPA in maize, phylogenetic trees for RPA across the 10 species and for ZmRPA subunits alone were constructed, using IQ-TREE (2.0.3) employing the maximum likelihood (ML) method with bootstrap values derived from 1000 replicates [31], and the tree using the R package ggtree [32]. To further investigate the evolutionary relationship of the RPA family in plants, the RPA of 27 species were used to construct a phylogenetic tree through the same method, including the 10 species mentioned above as well as the other 17 species (Solanum lycopersicum, Vitis vinifera, Brassica napus, Populus trichocarpa, Glycine max, Medicago truncatula, Amborella trichopoda, Sphagnum palustre, Chlamydomonas reinhardtii, Volvox carteri, Dunaliella salina, etc.).

Domain, gene structures, and conserved motifs analysis of ZmRPA genes

Domain and positional information were determined utilizing the Pfam-Scan software with Pfam database [27]. Conserved motifs were analyzed by MEME software with the maximum number of 10 and optimum widths of 20–150 amino acids, with subsequent extraction performed by Python scripts [33]. The exon and intron locations of ZmRPA genes were derived from GFF3 annotation files. Visualization of domains, conserved motifs and gene structures was accomplished using the R package ggtree and gggenes (https://github.com/wilkox/gggenes).

Analysis of chromosome distribution, gene duplication events, and selection pressure

The chromosomal distribution of ZmRPA genes was determined using the GFF3 annotation file. Gene duplication and collinearity analyses were performed with MCScanX [34], which identified the duplication types including tandem duplications (TD) and whole genome duplications (WGD). The protein sequences and coding sequences (CDS) of the RPA genes with gene duplication were aligned using ClustalW [35]. Furthermore, the KaKs_Calculator [36] was utilized to compute the synonymous substitution rate (Ks), nonsynonymous substitution rate (Ka), and the evolutionary ratio (Ka/Ks) between duplicate genes.

Analysis of cis-elements in the promoter of ZmRPA genes

The 2 kb promoter sequences upstream of the ZmRPA genes were extracted using a Python script and analyzed with the PlantCare database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to predict cis-regulatory elements [37]. The results were subsequently visualized using the R packages ggtree and gggenes (https://github.com/wilkox/gggenes).

ZmRPA potential protein interaction network analysis

The potential protein-protein interaction (PPI) network of ZmRPA proteins was predicted using the AraNet2 database [38], restricting the analysis to interactions with weight scores > 3. Functional annotation of all proteins within the potential PPI network was performed using the Eggnog-mapper database [39]. The resulting network was visualized using the R package ggNetView (https://github.com/yueliu1115/ ggNetView).

Expression pattern analysis of ZmRPA genes

Transcriptome data under drought and cold stress conditions of Zea mays ssp. mexicana L was retrieved from the SRA database under the accession number PRJNA309111 [40], while transcriptome data under drought and cold stress conditions of inbred line B73 (Zea mays ssp. mays), the mean of FPKM (Fragments Per Kilobase of transcript sequence per million base pairs sequenced), was retrieved from qTeller (https://qteller.maizegdb.org/) [41, 42]. The data of the tissues expression in B73 maize was also downloaded from qTeller (https://qteller.maizegdb.org/) [43]. For RNA-seq analysis, the FASTQ files were aligned using the genome alignment software Hisat2 [44]. Subsequently, expression count data were quantified utilizing the featureCounts [45], and FPKM was calculated based on gene length and the number of reads mapped to each gene. Differential expression analysis between the two groups was performed using the DESeq2 R package [46]. A corrected P-value ≤ 0.05 and |log₂(fold change)| ≥ 1 were set as thresholds for significant differential expression, with adjusted P-value using the Benjamini and Hochberg method to control the false discovery rate. The R package Pheatmap [47] was employed to visualize the resultant data. The results of the differential expression analysis were showed in Supplementary Table S2.

Total RNA extraction and qRT-PCR analysis

In this study, specific primers for ZmRPA genes were designed using Primer 5.0 software (Supplementary Table S3). At the six-leaf stage (V6) of maize, samples were collected from the 3rd (L3), 4th (L4), 5th (L5), 6th (L6), and 7th (L7) leaves that had grown, as well as from the stem tip, root tip, and roots. And the tassel, ear, and core bract were collected shortly after the male spikelets had fully emerged but before they began to flower and shed pollen, and the ears had not yet emerged with silks at this time. Total RNA was isolated using the FastPure Plant Total RNA Isolation Kit (Vazyme, RC401). Subsequent synthesis of complementary DNA (cDNA) was conducted employing the HiScript II 1 st Strand cDNA Synthesis Kit (Vazyme, R211-01). Each reaction mixture for reverse transcription quantitative polymerase chain reaction (qRT-PCR) comprised 5 µL of 2× ChamQ SYBR qPCR Green Master Mix (Vazyme, Q311-02), 4 µL of cDNA template, 0.5 µL of forward primer and reverse primer each (final concentration of 10 µM), culminating in a total volume of 10 µL. The qRT-PCR amplification protocol entailed an initial denaturation at 95 °C for 3 min, followed by 50 cycles of 95 °C for 10 s and 60 °C for 30 s, with a melting curve 65℃ to 95℃, incrementing 0.5℃ for 5 s. Independent biological replicates of plants were harvested for each condition, with three replicates for each treatment. Expression was determined by Livak (2^-ΔΔCt) method [48], with sample L3 as the reference sample and ZmGAPC2 (Zm00001d035156) as the internal control [49].

Yeast two-hybrid assays

The CDS of ZmRPA1A, ZmRPA1B1, ZmRPA1B2, ZmRPA2A1, ZmRPA2B, ZmRPA2C1, ZmRPA2C2, and ZmRPA3A3, were cloned into prey vector pGADT7 (AD-RPAs) respectively, by using DNA seamless cloning technique (ClonExpress II One Step Cloning Kit, Vazyme, C112-01). The results of the RPA gene amplification and PCR detection of the AD-RPAs vector are shown in Supplementary Fig. S1A and B. Following construction, the accuracy of the AD-RPAs vectors was confirmed by Sanger sequencing, ensuring the complete fidelity of the inserted sequence. Then the CDS of the target genes were subcloned from the vectors of AD-RPAs into the bait vector pGBKT7 via restriction digestion and ligation to construct the vectors of BD-RPAs (Supplementary Fig. S1C, D). Sanger sequencing was also used to verify the orientation and sequence of the insert within the bait vector pGBKT7. The primers, used in the PCR amplification of the gene inserts destined for vector construction, and PCR identification of the constructed vector, as well as for sanger sequencing, are listed in Supplementary Table S3. Bait and prey vectors were cotransformed into the yeast strain Y2H Gold according to the Yeast Protocols Handbook. The combination of AD-T and BD-p53 served as positive control, while the AD-T and BD-Lam served as negative control. Yeast transformants harboring AD-RPAs and BD-RPAs were employed to test the interactions among the various subunits of ZmRPA. Medium DDO (lacking Trp and Leu), TDO (lacking Trp, Leu, and His), and QDO + 10 mM 3-AT (lacking Trp, Leu, His, and Ade, with 10 mM 3-Amino-1,2,4-triazole), were used for selection. For BD-RPAs that show no autoactivation, protein-protein interactions were screened on TDO, while those exhibiting autoactivation, screening was performed on QDO + 10 mM 3-AT. For spotting on the plates, 5 µL aliquots of yeast cell suspensions (OD₆₀₀=1, 0.1, and 0.01) were applied.

Results

Genome-wide identification of the ZmRPA genes

In this study, to identify and characterize the main members of the RPA in maize (Zea mays), a total of 141 RPA genes were identified in nine monocots and one dicot model plant (Supplementary Table S4). Among them, the genome-specific copy numbers ranged from 7 to 35. This variation underscores both lineage-specific expansion and conservation of the RPA gene family among different plant species. Notably, Zea mays contained 13 genes, Sorghum bicolor 12, Setaria italica 11, Oryza sativa 7, Triticum urartu 21, and Triticum aestivum exhibited the highest number with 35 genes; additionally, Hordeum vulgare possessed 17 genes, Brachypodium distachyon 7, and Aegilops tauschii 9 (Supplementary Table S1, S4). The observed differences in copy number suggest that certain species, such as Triticum aestivum, have experienced multiple rounds of whole-genome duplication or segmental duplication events, leading to a significant expansion of the RPA family, whereas others have maintained relatively conserved copy numbers. Upon the identification of candidate genes, a comprehensive characterization of sequence features was performed, including coding sequence length, molecular weight, theoretical isoelectric point (pI), and hydropathicity (Fig. 1, Supplementary Table S4). The predicted RPA proteins demonstrated considerable variation, with amino acid lengths ranging from 86 to 1111 residues, molecular weights spanning 9.3 to 119.5 kDa, and theoretical pI values between 4.46 and 9.90, thereby indicating substantial physicochemical diversity within this protein family. Further analysis of hydropathicity yielded values ranging from − 0.57 to 0.19, with the majority of proteins exhibiting negative scores, suggesting that RPA proteins are predominantly hydrophilic in nature. This characteristic aligns with their functional necessity to engage with nucleic acids and various protein partners during DNA replication and repair processes [4, 21]. These findings offer essential insights into the structural diversity of the RPA proteins and establish a foundation for future phylogenetic and functional analyses.

Fig. 1.

Characteristics of the RPA family members in the 10 species investigated in this study, including protein length, molecular weight, isoelectric point and hydrophilicity

Phylogenetic analysis of the ZmRPA

To analyze the evolutionary relationship of the RPA in plants, a comprehensive genome-wide analysis was conducted across 27 representative plant species, comprising nine dicotyledons, 11 monocotyledons, one fern, three bryophytes, and three algal species (Supplementary Fig. S2). A comparative analysis of the RPA gene family across 27 plant species revealed that it is highly conserved overall during plant evolution, yet has undergone marked lineage-specific expansion. In green algae such as Chlamydomonas reinhardtii, Volvox carteri, and Dunaliella salina, only a few RPA members were detected, suggesting that the family originated from a minimal and highly conserved core set of genes, which providing a support for a pre-land-plant origin of this gene family, as suggested by prior work [18]. During the transition from aquatic to terrestrial environments, bryophytes and early land plants showed a moderate increase in RPA copy number, indicating stronger selective pressure on DNA replication and repair functions in early land colonization. In the basal angiosperm Amborella trichopoda, the RPA family composition was largely complete but not significantly expanded, representing the ancestral state of angiosperms. By contrast, monocots, particularly the Poaceae lineages, exhibited pronounced copy-number expansion in multiple RPA subunits, with polyploid species, such as Triticum aestivum and durum Triticum turgidum, showing the most notable increases. This expansion pattern corresponds closely with known whole-genome duplication and polyploidization events in grass family, highlighting the important role of genomic duplication in shaping the RPA gene family [50]. In summary, despite the high evolutionary conservation of the RPA gene family in plants, lineage-specific gene duplication events, especially in polyploid grasses, likely facilitated functional differentiation to meet the increased demand for genome stability maintenance in large and complex genomes.

To further systematically elucidate the evolutionary relationships of the maize ZmRPA gene, a phylogenetic tree was constructed based on RPA protein sequences from 10 representative species (Fig. 2). Phylogenetic analysis indicated that RPA proteins could be categorized into three principal subfamilies: RPA1, RPA2, and RPA3, a classification that aligns with the reported domain conservation in A. thaliana and other model plants (Fig. 2) [1, 8]. Further resolution of these clades demonstrated that RPA1 could be further divided into five subgroups (RPA1A-RPA1E), RPA2 into three subgroups (RPA2A-RPA2C), and RPA3 into two subgroups (RPA3A and RPA3B). This subgroup diversification implies that the RPA family has undergone multiple rounds of gene duplication and functional divergence throughout its evolutionary history.

Fig. 2.

The evolutionary examination of the 141 RPA protein sequences identified from the 10 species were conducted. These 141 RPA proteins were categorized into three distinct subgroups, labeled RPA1, RPA2, and RPA3. The evolutionary phylogeny was analyzed utilizing IQ-TREE software (2.0.3), employing the maximum likelihood (ML) method with bootstrap values derived from 1000 replicates. The phylogenetic tree was visualized through the use of the R package ggtree (3.10.0)

Regarding gene copy number, RPA1 exhibited relative conservation across most diploid species, typically comprising approximately 2 to 5 members (Fig. 2; Supplementary Fig. S2). Conversely, typical C4 photosynthetic plants, including maize, Sorghum bicolor, and Setaria italica possessed 4, 5, and 5 members, respectively (Fig. 2; Supplementary Fig. S2). This suggests potential functional redundancy or diversification linked to the evolution of C4 photosynthesis. However, the number of the RPA1 members increased significantly to 12 in hexaploid wheat, indicative of extensive gene duplication resulting from whole genome polyploidization (Fig. 2; Supplementary Fig. S2). In the Triticum species, a significant expansion of RPA2 was observed, with 10 copies identified in Hordeum vulgare, 17 in Triticum aestivum, and 13 in Triticum Urartu (Fig. 2; Supplementary Fig. S2). In contrast, other monocot and dicot species exhibited relatively consistent copy numbers, indicating a lineage-specific expansion of RPA2 in grasses, attributable to genome duplication and species-specific diversification. RPA3 exhibited general stability across most species, maintaining 2–4 copies, except in Triticum aestivum, which contained 6 copies. Notably, RPA1D and RPA1E, along with RPA3B, were exclusively found in dicots such as A. thaliana and were absent in all surveyed grasses. Conversely, RPA2C was retained solely in grasses and was lost in dicots (Fig. 2; Supplementary Fig. S2). This contrasting pattern of lineage-specific gene retention and loss underscores the evolutionary divergence between monocots and dicots. It suggests that distinct RPA subgroups may have developed specialized functions, contributing to functional differentiation and ecological adaptation across different plant lineages.

Conserved motifs, conserved domain and gene structure of ZmRPA

The diversity of protein structural features and gene architectures offers essential insights into the evolutionary dynamics of gene families. To further elucidate the ZmRPA gene family, we conducted a comprehensive analysis of conserved domains, protein motifs, and gene structures (Fig. 3). Domain annotation revealed distinct patterns among the three RPA subfamilies, indicative of their functional divergence. Notably, RPA1 contained four conserved domains: Rep-A-N, tRNA_anti-codon, REPA_OB_2, and Rep_fac_A_C, which suggests a complex role in DNA binding and replication. In contrast, RPA2 comprised two domains, tRNA_anti-codon and RPA_C, indicating a more specialized function, while RPA3 contained only a single Rep_fac-A_3 domain, potentially representing a more ancestral functional state (Fig. 3B, Supplementary Table S1).

Fig. 3.

Analysis of phylogenetic relationships, conserved domains, motifs, and gene structures related to RPA inMaize. A Phylogenetic tree of the RPA proteins using IQ-TREE (method: maximum likelihood; bootstrap values: 1000 iterations); B Conserved domains in RPA protein sequences; C Distribution of 10 conserved motifs in RPA proteins; D Gene structure of the RPA genes

The examination of conserved motifs has uncovered further divergence among subfamilies, acting as crucial indicators for differentiating RPA members (Fig. 3 C). The RPA1 subfamily is distinguished by the presence of motif-2, motif-3, and motif-6, which are highly conserved among its various members. In contrast, the RPA2 subfamily predominantly contains motif-5, while the RPA3 subfamily is characterized by a distinct motif-1. These motifs not only serve as structural markers for subfamily classification but may also be linked to specific biological functions, such as the initiation of DNA replication, the assembly of repair complexes, and regulatory signaling processes.

The analysis of gene structures further elucidated significant differences among ZmRPA members, underscoring their complex evolutionary pathways (Fig. 3D). Within the RPA1B subgroup, both ZmRPA1B1 and ZmRPA1B2 comprised 12 exons and exhibited nearly identical domain and motif configurations, suggesting strong evolutionary conservation likely attributable to recent duplication events. In contrast, ZmRPA1A and ZmRPA1C each contained two exons but demonstrated distinct exon arrangements, indicative of functional divergence. Members of the RPA2 and RPA3 subfamilies displayed even greater variability in exon number and distribution, suggesting extensive structural diversification that may facilitate their subfamily-specific functional specialization throughout plant evolution.

Gene location and gene duplication events analysis of ZmRPA genes

To identify the chromosomal distribution of ZmRPA genes within the maize genome, their physical locations were extracted from the GFF annotation file. A total of 13 ZmRPA genes were identified and mapped to chromosomes 1, 3, 4, 5, 6, and 9, with an average of 2 to 3 members per chromosome, indicating a relatively dispersed distribution pattern (Fig. 4). This evenly distributed arrangement, as opposed to a clustered one, suggests that the ZmRPA gene family has not experienced significant tandem expansion in maize. Subsequent duplication analysis revealed that only two pairs of ZmRPA genes originated from whole-genome duplication (WGD) events, with no other substantial duplication events detected (Fig. 4). These findings suggest that, despite multiple rounds of polyploidization in maize, the expansion of the ZmRPA family has been relatively constrained, maintaining a stable number of members. To elucidate the evolutionary characteristics of the RPA genes across various plant species, a comprehensive analysis of the RPA gene families in Setaria italic (Supplementary Fig. S3A), Sorghum bicolor (Supplementary Fig. S3B), Oryza sativa (Supplementary Fig. S3C), and A. thaliana (Supplementary Fig. S3D), was conducted. Within these species, RPA genes exhibited relatively consistent chromosomal distributions, with 1 to 4 members per chromosome and no significant evidence of large-scale clustering. Similar to the observations in maize, gene duplication events in these species were infrequent: only one tandem duplication and one proximal duplication were identified in Setaria italica (Supplementary Fig. S3A), two tandem duplications in Sorghum bicolor (Supplementary Fig. S3B), one tandem duplication in rice (Supplementary Fig. S3C), and two tandem duplications in A. thaliana (Supplementary Fig. S3D). These results collectively suggest that the RPA gene family across different species has maintained a relatively stable size and dispersed distribution, contrasting sharply with other gene families, such as those associated with disease resistance, which have experienced rapid expansion through frequent duplication events [51].

Fig. 4.

The chromosome location and duplicated gene pair of the RPA genes in Maize. The duplicate gene types were displayed in different color. WGD and TD events are shown in orange and blue, respectively

Furthermore, the Ka, Ks, and Ka/Ks ratios were computed for all duplicated RPA gene pairs in maize, Sorghum bicolor, Setaria italica, Oryza sativa, and A. thaliana utilizing the KaKs_Calculator (Supplementary Table S5). Across all species analyzed, the duplicated RPA gene pairs displayed Ka/Ks ratios of less than 1, suggesting that strong purifying selection has been the predominant evolutionary force influencing this gene family [52]. These findings indicate that the RPA gene family has remained highly conserved throughout plant evolution, preserving essential roles in genome stability and fundamental cellular processes under stringent selective pressures.

Analysis of promoter elements

Gene transcription is modulated by the interplay between cis-acting elements and transcription factors, with the distribution and nature of these elements within promoter regions often dictating the spatial and temporal expression patterns of genes during development and in response to environmental stimuli [53–55]. To investigate the potential regulatory functions of the maize ZmRPA gene family in growth, development, and responses to hormonal, biotic, and abiotic stimuli, the cis-acting elements of each RPA gene were analyzed. This analysis identified a total of 22 distinct cis-elements (Fig. 5), which were classified into four primary categories: light-responsive, growth and development-related, hormone-responsive, and stress-responsive elements. The light-responsive elements identified were CAAT-box, G-box, GC-motif, G-Box, and GATA-motif. Elements associated with growth and development included TATA-box, CAT-box, CCAAT-box, CCGTCC-motif, CCGTCC-box, and O2-site. The hormone-responsive elements comprised ABRE, CGTCA-motif, TGACG-motif, GARE-motif, ABRE3a, and ABRE4. Additionally, stress-responsive elements included as-1, ARE, LTR, DRE core, and MBS.

Fig. 5.

Cis-elements analysis of the RPA genes in Maize. A Phylogenetic tree of the RPA proteins using IQ-TREE (method: maximum likelihood; bootstrap values: 1000 iterations); B Bar chart illustrating the summary of cis-acting elements, with colors denoting various functional categories; C Distribution of cis-acting element counts, with colors indicating different count ranges

Quantitative analysis indicated that light-responsive and growth/development-related elements were the most prevalent in ZmRPA promoters, succeeded by hormone-responsive elements, with stress-responsive elements being the least common (Fig. 5B). Within the category of light-responsive elements, the CAAT-box was the most frequently observed, followed by the G-box. In terms of growth and development-related elements, the TATA-box was the most common, followed by the CAT-box. This distribution pattern was consistently observed across all members of the RPA family (Fig. 5 C), suggesting that the RPA family has maintained a conserved regulatory framework throughout evolution to fulfill its roles in essential growth and light-dependent developmental processes. Furthermore, analysis of the stress-responsive elements revealed distinct distribution patterns among subunits of ZmRPA (Fig. 5 C). as-1, which activating plant protective genes under xenobiotic chemical stress, was ubiquitous across all members of ZmRPA [56, 57]. ARE, as the regulator of redox homeostasis during oxidative stress, was more prevalent in ZmRPA2B, ZmRPA2C1 and ZmRPA2C2 [58]. For other cis elements participating abiotic stress, LTR was most abundant in ZmRPA2C1, while DRE core was primarily enriched in ZmRPA1B1 and ZmRPA1B2, and MBS showed the most limited distribution [59–63]. These results suggest that ZmRPA may be involved in responding to various abiotic stresses through the regulation of these cis-acting elements.

Collectively, these findings indicate that the ZmRPA promoters are evolutionarily enriched with cis-elements associated with light and developmental regulation. This suggests that RPA proteins may play critical roles in light-dependent and growth-related regulatory processes in maize. Additionally, these results offer potential insights into the functions of these proteins in hormone signaling and environmental adaptation.

Expression pattern analysis of ZmRPA genes in stress conditions and different tissues

To investigate the stress responsiveness of ZmRPA genes, their expression profiles were examined under control, cold, and drought conditions (Fig. 6 A; Supplementary Table S2). Distinct expression patterns were observed among different family members. In Zea mays ssp. mexicana L, a wild relative of Zea mays ssp. mays, except for ZmRPA3A4 and ZmRPA3A2, the expression of all other ZmRPA genes is inhibited by cold, suggesting a high sensitivity of this gene family to low temperatures. Among them, the expressions of ZmRPA1B2 and ZmRPA2C1 were significantly downregulated, indicating their high sensitivity to cold stress. Under drought stress, except for ZmRPA1A, most genes of the RPA family showed a slightly downregulated expression, but the changes were not significant. Similarly, the expression profiling of the ZmRPA gene family in inbred line B73 (Zea mays ssp. mays) revealed a repression pattern under abiotic stress conditions (Supplementary Table S2). A substantial portion of these genes were suppressed following exposure to both cold and drought treatments, indicating a potential common regulatory mechanism in response to these stresses. In particular, the transcripts of ZmRPA2A1, ZmRPA2A2, ZmRPA2C1, and ZmRPA2C2, were consistently and significantly reduced under both conditions, suggesting they may function as core responsive members to general stress signals. Conversely, the expression of ZmRPA1B1, ZmRPA1B2, and ZmRPA3A3, was specifically and markedly repressed only by drought stress, implying a more specialized role in the plant’s adaptation to water deficit. Taken together, the expression of the ZmRPA genes is commonly repressed under various abiotic stresses. Furthermore, the comparative analysis demonstrates that this gene family exhibits higher sensitivity to adverse conditions in the inbred line B73, mounting a more active transcriptional response, compared to Zea mays ssp. mexicana L, which may reflect adaptation to distinct genetic backgrounds, and further imply a sophisticated and adaptable transcriptional regulatory network governing this family’s stress responses.

Fig. 6.

Analysis of the expression patterns of ZmRPA genes. A The expression analysis of ZmRPA genes under cold and drought stresses via RNA-seq. The data are represented as a heatmap with scaling based on FPKM values. The color scale is shown on the right. B Tissue-specific expression profiling analysis of ZmRPA genes by RNA-seq. 1-23 represent different maize tissues, namely: 1- Internode 6 and 7, 2-Internode 7 and 8, 3-Mature pollen, 4-Ear Primordium 2-4 mm, 5-Ear Primordium 6-8 mm, 6-Embryos 20 DAP, 7-Embryo 38 DAP, 8-Endosperm 12 DAP, 9-Endosperm Crown 27 DAP, 10-Female spikelet collected day of silking, 11-Germination Kernels 2 DAI, 12-Leaf Zone 1 (Symmetrical), 13-Leaf Zone 2 (Stomatal), 14-Leaf Zone 3 (Growth), 15-Mature Leaf 8, 16-Pericarp/Aleurone 27 DAP, 17-Primary Root 5 Days, 18-Root - Cortex 5 Days, 19-Root Elongation Zone 5 Days, 20-Root Meristem Zone 5 Days, 21-Secondary Root 7-8 Days, 22-Silks, 23-Vegetative Meristem 16-19 days. The data are represented as a heatmap with scaling based on FPKM values. The color scale is shown on the right. C-J The expression levels of ZmRPA genes in various tissues of maize B73 were analyzed by RT-qPCR. Values are mean ± SD (n = 3)

To explore the tissue expression characteristics of ZmRPA genes, it was found through mining of public data from qTeller that ZmRPA genes were highly expressed in the vegetative meristem, root meristem, ear primordium, and germination kernels, as well as in the developing leaves, where cell division is vigorous (Fig. 6B). Among them, ZmRPA1A, ZmRPA2A2 and ZmRPA2C1, showed high expression at internode 7 and 8; ZmRPA1B2, ZmRPA1B1, ZmRPA2A1, and ZmRPA2C2, enriched in the developing leaves; ZmRPA1C and ZmRPA3A3 are highly expressed in root meristem. The expression trends of ZmRPA1B1 and ZmRPA1B2 in different tissues were similar, as well as ZmRPA2C1 and ZmRPA2C2, which might be related to their more closely related evolutionary relationship (Figs. 4 and 5), suggesting that there may be some functional redundancy between them. Furthermore, the similar expression patterns of ZmRPA1C and ZmRPA3A3 suggest that they may act synergistically. Meanwhile, RT-qPCR was used to analyze the expression levels of ZmRPA genes (Fig. 6C-J), and similar results were obtained. The young leaves, root tips, shoot tips, young tassels and young ears, were identified as the primary sites of ZmRPA gene expression, all of which are areas where cell division is vigorous. To be more specific, within the ZmRPA1 subfamily, ZmRPA1A and ZmRPA1B1 were predominantly expressed in young tassels and young ears, suggesting their potential fundamental functions in the formation and development of male and female reproductive organs (Fig. 6 C, D). ZmRPA1B2 exhibited increased expression in stem tips and young leaves, suggesting its involvement not only in meristematic activity but also in leaf development (Fig. 6E). Members of the ZmRPA2 subfamily typically demonstrated with a relative enrichment in the stem tips and root tips, and all of them exhibited a gradual increase in expression from old to new leaves, reaching its peak in meristematic tissues, indicative of a “broad-spectrum + meristem-preferential” expression pattern, which suggests its role in the development of leaves and rapid cell division (Fig. 6F-I). Within the ZmRPA3 subfamily, ZmRPA3A3 exhibited a pronounced peak in young tassels, with relatively higher level of expression in young ears, stem tips, and root tips (Fig. 6 J), and lower expression levels in the leaves, which similar to the expression pattern of ZmRPA1A and ZmRPA1B1 (Fig. 6 C, D), pointing to a potential cooperative role of these genes. Collectively, ZmRPA genes are broadly expressed in multiple tissue parts of the maize, but mainly highly expressed in the actively dividing meristems and reproductive primordia. These expression characteristics underscore the functional diversification among subfamily members and suggest potential complementary or cooperative roles in the regulation of maize growth, development, and reproduction.

Interaction network of the RPA proteins

Given that prior studies indicate that the RPA complex typically functions as heterotrimer and exerts its biological roles through interactions with other functional proteins in A. thaliana [1, 13, 22], we therefore conducted a protein-protein interaction (PPI) analysis of ZmRPA to further elucidate its functional network. This investigation included both in computational prediction and experimental validation via yeast two-hybrid assays (Fig. 7).

Fig. 7.

Protein-protein interaction (PPI) network of the RPA proteins in Maize. A The PPI network prediction analysis of ZmRPA proteins. The colors of the node indicate different domain, while the colors of edges correspond to the interaction weights. B-D Protein interaction of ZmRPA subunits in yeast two-hybrid assays. B and C Yeast cells co-transformed with empty AD/AD fused ZmRPA proteins and BD/BD-fused ZmRPA proteins were grown on DDO, TDO, and QDO + 10 mM 3-AT media, in 1 dilution, respectively. D Yeast cells harboring pGBKT7-p53 (p53) and pGBKT7-Lam (Lam), co-transformed with pGADT7-T (T), and were used as positive and negative controls respectively, and grown on DDO, TDO, and QDO + 10 mM 3-AT media, in 1, 10^–1, 10^–2 dilutions (from left to right in each panel), respectively

The PPI network of computational prediction encompassed 158 proteins, including six RPA proteins and 152 interacting partners (Fig. 7 A; Supplementary Table S6). RPA proteins were observed to interact with several distinct protein families, including AAA proteins, MCM proteins, Rhomboid proteins, and DEAD-box proteins. The AAA (ATPases Associated with Diverse Cellular Activities) protein family comprises ATP-dependent molecular machines that participate in a variety of cellular processes, such as protein folding and unfolding, assembly and disassembly of protein complexes, DNA replication, and chromatin remodeling [64, 65]. Their interaction with RPA proteins is likely to play a role in maintaining replication fork stability and ensuring genome integrity. The MCM protein family forms the core of the DNA helicase complex and is crucial for the initiation and elongation phases of DNA replication [66]. Their association with RPA proteins may facilitate the precise activation of replication origins and the proper progression of replication forks. The Rhomboid protein family, an evolutionarily conserved group of intramembrane serine proteases, is extensively involved in membrane protein cleavage and signal transduction [67]. Their association with RPA suggests a potential linkage between DNA metabolism and membrane-associated signaling pathways during cellular responses to environmental stimuli. Similarly, the DEAD-box protein family, which comprises ATP-dependent RNA helicases, is engaged in RNA splicing, translation, degradation, and the remodeling of RNA-protein complexes [68]. Their interaction with RPA proteins indicates possible crosstalk between DNA and RNA regulatory networks, proposing that RPA may play a role not only in DNA metabolism but also in post-transcriptional regulation and the control of gene expression. Collectively, these interactions reveal a complex associative network between RPA proteins and various protein families, offering novel insights into the multifaceted roles of the RPA in maintaining genomic stability, coordinating cellular signaling, and regulating gene expression in maize. This study establishes a theoretical foundation for subsequent functional validation and mechanistic investigations.

The PPI network analysis showed that members of the RPA family also exhibit intra-family protein-protein interactions (Fig. 7 A; Supplementary Table S6), and the yeast two-hybrid assays were conducted to validate the interactions among ZmRPA proteins (Fig. 7B-D; Supplementary Fig. S4). The findings revealed that members of the ZmRPA family are capable of forming multiple interaction combinations within the family. Within the ZmRPA1 subfamily, interaction signals were observed among ZmRPA1A, ZmRPA1B1 and ZmRPA1B2 (Fig. 7 A, C; Supplementary Table S6), all of them exhibiting similar expression patterns, with high concurrent expression in shoot apical meristems, root tips, and young ears, as well as under drought stress (Fig. 6). This suggests that subfamily members may form functional complexes through homomeric or closely related protein interactions. In terms of cross-subfamily interactions, ZmRPA1A, ZmRPA1B1 and ZmRPA1B2 were all found to interact with ZmRPA2A1, ZmRPA2B, ZmRPA2C1 and ZmRPA2C2 (Fig. 7 C). These genes exhibit comparable expression profiles, with elevated levels in actively dividing tissues (Fig. 6). Additionally, ZmRPA1A and ZmRPA1B1 also formed complexes with ZmRPA3A3 (Fig. 7 C). Consistent with this, these three genes exhibit remarkably similar tissue-specific expression patterns (Fig. 6 C, D, J). These results imply that ZmRPA1 members may function as central nodes linking different subfamilies. Intra-subfamily interactions were also identified among members of the ZmRPA2 subfamily. Specifically, ZmRPA2A1 was shown to interact with ZmRPA2B, while ZmRPA2B was also found to associate with ZmRPA3A3 (Fig. 7 C). These three genes are all highly expressed in the stem tip (Fig. 6 F, G, J). Moreover, ZmRPA2C1 was shown to slightly interact with ZmRPA2C2, and ZmRPA2C2 was also associate with itself (Fig. 7 C). These two genes arise from a gene duplication event and consequently exhibit highly similar expression patterns (Figs. 4 and 6B, H and I; Supplementary Table S5). These results indicate that ZmRPA members not only form internal networks but also extend their connectivity to other subfamilies, implying that these proteins may function as heterodimers or heterotrimers in biological processes. Collectively, these findings suggest that the ZmRPA family establishes interaction networks both within and across subfamilies, resulting in complex protein assemblies. These interaction patterns imply that ZmRPA proteins may do not function independently but rather operate through cooperative networks to perform their roles in DNA replication, repair, and stress responses in maize.

GO and KEGG enrichment analysis of ZmRPA genes

GO and KEGG enrichment analyses were conducted to investigate the potential biological functions of the ZmRPA gene family (Fig. 8; Supplementary Table S7, S8). In the Biological Process (BP) category, genes related to ZmRPA were significantly enriched in pathways linked to genome stability and cell cycle regulation, such as DNA repair, DNA recombination, DNA replication, nuclear DNA replication, mitotic cell cycle process, and telomere organization and maintenance. In the Cellular Component (CC) category, these genes were mainly associated with nuclear structures, especially chromatin, nuclear chromosome, protein-DNA complexes, replication fork, and replisome, indicating that their products primarily operate within chromatin-related complexes. For the Molecular Function (MF) category, enrichment was noted in ssDNA binding, telomerase RNA binding, and sequence-specific ssDNA binding, suggesting a direct role of ZmRPA proteins in DNA-RNA interactions and telomere stability.

Fig. 8.

The enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for RPA genes in Maize. The GO terms are systematically classified into three categories: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The vertical axis illustrates the annotated GO and KEGG terms, whereas the horizontal axis indicates the number of genes associated with each GO and KEGG term. The categories BP, CC, and MF are depicted in purple, red, and, blue respectively, while KEGG pathways are represented in green

Furthermore, the KEGG pathway analysis indicated enrichment in glycosphingolipid biosynthesis, specifically within the globo and isoglobo series, suggesting that certain RPA members may also be involved in cell membrane metabolism and signaling. Collectively, these findings underscore that ZmRPA genes are predominantly engaged in DNA replication, repair, recombination, and telomere maintenance, thereby playing crucial roles in preserving genomic stability. Additionally, they may serve as a potential nexus between DNA metabolism and broader cellular processes, including membrane biosynthesis and signaling.

Discussion

The RPA gene family, known for its role as a highly conserved ssDNA-binding complex in eukaryotes, exhibits significant evolutionary conservation, as demonstrated in this study focusing on maize [8]. A total of 13 ZmRPA genes was identified, which are distributed across six chromosomes in a dispersed rather than clustered manner (Figs. 2 and 3). Gene duplication analyses revealed that only two gene pairs originated from whole-genome duplication (WGD) events, with no evidence of significant tandem or segmental duplications (Fig. 4). This limited expansion is in stark contrast to gene families such as resistance (R) genes, which frequently undergo rapid duplication and diversification [69]. Comparative analyses across Sorghum bicolor, Setaria italica, Oryza sativa, and A. thaliana corroborated this pattern, as RPA gene families in these species also displayed relatively uniform chromosomal distributions and restricted duplication events (Supplementary Fig. S3). Calculations of the Ka/Ks ratio demonstrated that duplicated RPA gene pairs across all examined species are subject to strong purifying selection (Supplementary Table S5). This finding supports the hypothesis that the RPA gene family is evolutionarily constrained to maintain its essential functions. The observed stability suggests that the critical role of the RPA in DNA replication and genome integrity has exerted significant evolutionary pressure to preserve a limited and functionally conserved gene repertoire [5, 8, 70, 71]. Despite the limited overall expansion, diversification at the subfamily level offers insights into functional specialization. Phylogenetic and motif analyses have categorized ZmRPA genes into three principal subfamilies: RPA1, RPA2, and RPA3 (Figs. 2 and 3). These analyses reveal motif conservation within subfamilies and divergence among them. This pattern indicates that while the structural requirements for ssDNA binding are highly conserved, subfamily-specific motifs may facilitate specialized interactions with various protein partners, enabling functional differentiation within the family [70]. In A. thaliana, for example, RPA1A and RPA1C are essential for meiotic homologous recombination, RPA1B and RPA1D are primarily associated with DNA replication, and RPA1C and RPA1E are involved in DNA repair [17]. A similar pattern may be observed in maize, where distinct ZmRPA subfamilies orchestrate unique yet complementary roles in genome maintenance. Additionally, the presence of cis-regulatory elements related to light responsiveness, growth, and development in promoter regions suggests that ZmRPA genes integrate environmental and developmental signals, highlighting the complex nature of their regulation (Fig. 5). Therefore, future investigations into ZmRPA function under abiotic stress are warranted. Stress treatment experiments should be designed to assess its performance in complex, physiologically relevant or field environments, as suggested by its promoter architecture.

Expression profiling has indicated that ZmRPA genes exhibit differential regulation in response to developmental signals and environmental stressors (Fig. 6). RNA sequencing data revealed significant transcriptional alterations under cold and drought conditions, implying a potential role for ZmRPA genes in stabilizing replication forks and facilitating DNA repair during stress [72, 73]. Tissue expression profile analysis of ZmRPA genes through RNA-seq and qRT-PCR further demonstrated that the majority of ZmRPA genes are highly expressed in actively dividing meristematic tissues and floral development, especially in the stem tips and young tassels (Fig. 6B-J). Stem tips contain apical meristems, where active cell proliferation and organ initiation occur, while tassels are reproductive structures critical for male inflorescence development [74, 75]. The elevated expression of ZmRPA genes in these tissues may imply roles for these proteins in regulating cell division, meristem maintenance, or hormonal signaling processes essential for both vegetative growth and reproductive development, which is consistent with the findings regarding cis-acting elements and the results of GO and KEGG enrichment analyses (Figs. 5, 6 and 8). These findings are consistent with previous research in A. thaliana, where mutations in specific RPA members have been shown to disrupt meiosis, DNA repair, meristem function, floral transition, and senescence [13, 17, 22]. Overall, these findings suggest that the ZmRPA gene family plays a crucial role in maize stress responses and adaptation, potentially through a division of labor among its members under stress conditions. Although further functional validation is required, the expression pattern aligns with the possibility that these putative ZmRPA proteins participate in signaling or transcriptional networks that modulate shoot architecture and reproductive organ formation. Future studies focusing on protein localization, knockout phenotypes, and transgenic complementation, will help clarify their precise functions in these specific tissues.

In addition to transcriptional regulation, the analyses of protein-protein interactions and yeast two-hybrid assays have demonstrated that members of the ZmRPA family are capable of forming interaction networks both within and across subfamilies (Fig. 7; Supplementary Table S6). These cooperative associations align with the trimeric structure of the RPA and imply that functional complexes in maize may be constituted from various combinations of RPA1, RPA2, and RPA3 subunits [73]. The capacity of family members to interact with each other likely facilitates the fine-tuned regulation of DNA metabolic processes, thereby ensuring flexibility and redundancy in critical pathways such as replication, recombination, and repair. This underscores the significance of subunit coordination and supports the hypothesis that the functional diversity of the RPA is achieved not only through gene divergence but also through the dynamic assembly of protein complexes. According to the results of Y2H analysis, the speculation was gained that the ZmRPA protein likely functions within a heteromeric complex, potentially a heterodimer or heterotrimer, to exert biological function, which is consistent with the known structures of the homologous proteins of RPA in other species [1, 4, 7]. However, as a binary interaction assay, Y2H primarily indicates direct binding and does not definitively prove the simultaneous formation of a higher-order complex in vivo. Therefore, while this data provides a strong foundation, the precise stoichiometry and structural assembly of this complex remain to be elucidated. To unequivocally establish the oligomeric state of ZmRPA proteins, future work should employ rigorous biochemical and biophysical techniques. These would include co-purification of the candidate partners followed by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine the absolute molecular weight and composition of the native complex [76]. Additionally, structural approaches such as X-ray crystallography or cryo-electron microscopy would be invaluable for visualizing the detailed interaction interfaces [40]. Confirming the complex architecture will be crucial for a mechanistic understanding of ZmRPA function and for guiding subsequent functional studies.

In summary, these findings offer the first comprehensive characterization of the RPA gene family in maize, elucidating its evolutionary conservation, limited duplication history, and functional diversification. The integration of phylogenetic, structural, and expression data underscores a gene family constrained by purifying selection, yet capable of fine-scale specialization. The ZmRPA genes are implicated in maintaining genome stability across developmental stages and in response to environmental stress, with protein-protein interactions facilitating cooperative functionality within the family. These insights advance our understanding of the evolutionary dynamics and functional significance of the RPA family in plants, laying the groundwork for future research, aimed at elucidating their specific roles in maize growth, reproduction, and stress tolerance.

Conclusions

RPA complex is a highly conserved heterotrimer that binds single-stranded DNA and interacts with multiple proteins, playing essential roles in DNA replication, repair, and homologous recombination, thereby maintaining genome stability and supporting normal development. Despite its importance, the specific members and functions of the RPA gene family in maize remain largely unexplored. In this study, 13 ZmRPA genes in maize were identified, with protein lengths ranging from 86 to 876 amino acids. Phylogenetic analysis classified these genes into three distinct subgroups: RPA1, RPA2, and RPA3. Within each subgroup, ZmRPA proteins displayed conserved motif architectures, although differences were observed among subfamilies. Gene duplication analysis indicated that the RPA family in maize has remained highly conserved. Cis-element analysis further revealed that ZmRPA genes are associated with light responsiveness and plant growth and development. Expression profiling showed that ZmRPA genes undergo marked transcriptional changes under cold and drought stresses, while tissue expression profile analysis confirmed that most ZmRPA genes are highly expressed in the meristem and reproductive primordia. These results suggest potential roles of ZmRPA genes in vegetative growth, flower development and stress responses. In addition, protein interaction and yeast two-hybrid assays demonstrated that certain members of the maize RPA family are capable of interacting with each other, which likely underpins their biological function. Collectively, these findings provide the first comprehensive characterization of the RPA gene family in maize and lay a foundation for future functional studies. Furthermore, considering that the present study relies heavily on computational analysis, expression profiling, and yeast two-hybrid assays, these inferences necessitate further direct experimental corroboration and additional validation through biochemical and structural approaches.

Authors’ contributions

X.Z. and X.S. offered the conceptualization, designed the work, and wrote the main manuscript text; C.S., and B.L. acquired and analyzed the data; Q.Y., Huanli. L., and H.Z. interpreted the data; Hongli. L., Y.Z., and J.L. participated in experiments. All authors reviewed and approved the final manuscript.

Funding

This work was supported by Research Start-up Fund for Talented Scholars — Outstanding Young Doctor Award at Shanxi Agricultural University (No. 2023BQ22), Shanxi Provincial Basic Research Project (No. 202303021212083), Doctoral Research Grant Program from the Cotton Research Institute of Shanxi Agricultural University (No. SBSJJ2023-01), Shanxi Province doctoral graduates, PhD After the researchers to Jin work award funding research project (No. SXBYKY2023024), Subproject of Shanxi Science and Technology Major Program-Challenge-based Research Initiative (No. 202201140601025-5-04), Shanxi Agricultural University Science and Technology Innovation Promotion Project (No. CXGC2025040), and Shanxi Agricultural University Cotton Research Institute Innovation Development project (No. SJJCX2023-04).

Data availability

Data are contained within the article or supplementary material.

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Competing interests

The authors declare no competing interests.