Identification and Expression Analysis of the eIF4E1 Gene Family in Maize

Identification and Expression Analysis of the eIF4E1 Gene Family in Maize | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification and Expression Analysis of the eIF4E1 Gene Family in Maize Qi Wang, Hang Zhou, Lufei Zhao, Pengfei Chu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7863718/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Nitrogen is an essential nutrient for maize growth, and nitrogen use efficiency (NUE) remains below 40%, causing considerable resource waste and environmental pollution. Although previous studies have primarily focused on transcriptional regulation and nitrogen transporters, the contribution of translational control mechanisms to nitrogen responses remains unknown. In this study, the eIF4E1 gene family was systematically identified in maize, and its expression patterns under nitrogen stress were investigated. Bioinformatic analysis revealed six ZmeIF4E1 genes, and qRT-PCR assays demonstrated their constitutive expression across diverse tissues, together with member-specific responses to both low (0.2 mM KNO 3 ) and high (10 mM KNO 3 ) nitrogen treatments. ZmeIF4E1.1.1 was specifically induced in roots under low nitrogen, whereas ZmeIF4E1.1.2 exhibited strong upregulation in leaves. These findings highlight the potential involvement of eIF4E1-mediated translational regulation in maize nitrogen adaptation and identify promising candidate targets for improving NUE through molecular breeding. Maize eIF4E1 Nitrogen use efficiency Gene expression Translational regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Nitrogen is an essential nutrient for plant growth and development and plays an irreplaceable role in maize yield formation and grain quality regulation. Statistical data indicate that producing one ton of maize grain requires 16–18 kg of nitrogen, whereas the global nitrogen fertilizer use efficiency (NUE) in maize production generally remains below 40% [1]. Such inefficient utilization not only increases production costs but also leads to serious ecological and environmental problems, including water eutrophication, soil acidification, and greenhouse gas emissions [2]. Studies have indicated that drought stress during the flowering stage can significantly impair ear morphology, reducing the cob diameter at the base (CDB) by 13–16%. However, appropriate nitrogen application (e.g., 300 kg ha⁻¹ of urea) can mitigate the inhibitory effects of drought on chlorophyll fluorescence parameters (Fv/Fm) and the harvest index (HI), which contributes to a biomass increase of 23.7% even under stress conditions [3, 4]. Therefore, the identification of genetic resources with high nitrogen use efficiency and elucidation of the regulatory mechanisms underlying carbon–nitrogen balance have become central challenges in achieving the maize breeding objective of “reducing fertilizer input while enhancing efficiency” [3]. The nitrogen response mechanism in maize involves multilayered regulatory network. At the transcriptional level, nitrogen signaling activates transcription factors that regulate the expression of downstream target genes. At the translational level, nitrogen availability influences the efficiency of translation initiation. At the metabolic level, the products of nitrogen assimilation provide feedback that controls the allocation of carbon skeletons [3, 4]. Genome-wide association studies (GWAS) have identified multiple quantitative trait loci (QTLs) associated with nitrogen uptake and metabolism in maize. For instance, a GWAS analysis of nitrate accumulation in the leaves of 350 maize inbred lines revealed 16 significant SNP loci on chromosome 4. Candidate genes such as NAC79 , GA20ox7 , and PREP2 can be upregulated 2- to 3.5-fold under low-nitrogen conditions [1, 5]. These findings provide important insights into the molecular basis of nitrogen use efficiency in maize. However, key genes within the regulatory network and their functional modules still require systematic and comprehensive elucidation. In maize, several pivotal regulatory factors involved in nitrogen signal perception and transduction have been characterized. The transcription factor ZmNLP5, a member of the NIN-like protein (NLP) family, is specifically expressed in root and vascular tissues and functions as a central regulator of nitrate signaling [6]. Under low-nitrogen stress, ZmNLP5 directly binds to the nitrogen response cis-element (NRE) in the promoter region of the nitrite reductase gene ZmNIR1.1 , thereby activating the nitrogen assimilation pathway. The zmnlp5 mutant exhibits reduced nitrate accumulation in roots and a pronounced decrease in grain nitrogen content. Functional complementation of this gene restores nitrate uptake capacity, confirming its essential role in nitrogen signal transduction [1, 7]. Additionally, THP9 , isolated from wild maize, encodes asparagine synthetase 4 (ASN4). This enzyme catalyzes the synthesis of asparagine, which serves as a nitrogen donor in transamination reactions, markedly increasing grain protein content and improving NUE [1]. Recent studies have demonstrated that transcriptional regulation is crucial for nitrogen signaling. The transcription factor PBF1 regulates carbon–nitrogen allocation during endosperm development by altering its DNA-binding specificity in response to nitrogen availability. Under nitrogen-deficient conditions, PBF1 reduces its binding to the promoters of maize zein genes, thereby suppressing the expression of sugary1 and amylase2b [1, 8]. This adjustment shifts carbon flux towards carbohydrate biosynthesis. This dynamic regulation ensures the coordinated accumulation of starch and protein in the endosperm, providing key mechanistic insights into carbon–nitrogen crosstalk. Nitrogen uptake in maize is primarily mediated by two major gene families: nitrate transporters (NRTs) and ammonium transporters (AMTs). Based on substrate affinity, nitrate transport systems are classified into high-affinity transport systems (HATS) and low-affinity transport systems (LATS) [9, 10]. As a low-affinity nitrate transporter, ZmNPF6.6 shows a significant correlation between allelic variation and nitrogen uptake efficiency in maize, rendering it a promising candidate for molecular marker-assisted breeding. As reported by Yuan et al., maize root architecture, including traits such as lateral root density and root hair length, is dynamically regulated by nitrogen availability. Auxin response factors including ZmARF34 and ZmAUX/IAA12 modulate root nitrogen-foraging capacity by integrating nitrogen signals with hormone signaling pathways [1]. At the level of nitrogen assimilation, glutamine synthetase (GS) and glutamate synthase (GOGAT) constitute the GS/GOGAT cycle, which is the central pathway for ammonia incorporation [11]. ZmGS1.3 is specifically expressed in the vascular bundles of leaves, where it assimilates reduced nitrogen products transported from the roots. In contrast, ZmGS1.4 is expressed predominantly in root tips, where it facilitates the direct assimilation of soil ammonium [12, 13]. This tissue-specific expression pattern enables efficient spatial partitioning of nitrogen metabolism. Furthermore, ZmGDH1 (glutamate dehydrogenase) displays markedly enhanced activity under nitrogen-deficient conditions. By catalyzing both the reductive amination and deamination of α-ketoglutarate, it functions as a metabolic safety valve that helps maintain carbon–nitrogen balance [14]. Kernel development in maize depends on the precise coordination between photosynthetic carbon fixation and nitrogen assimilation. From 7 to 49 d after silking, the sucrose content in the kernels exhibits a single-peaked pattern correlated with sucrose synthase (SS) activity. Meanwhile, the peak activity of ADP-glucose pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is strongly influenced by nitrogen supply [15, 16]. Under sufficient nitrogen application (200 kg ha − 1 ), the peak in AGPase activity occurred 7–10 d earlier, promoting more efficient starch accumulation. In contrast, excessive nitrogen fertilization (> 300 kg ha − 1 ) constrains AGPase activity and reduces the rate of starch synthesis [17]. This non-linear response highlights the complex interactions within the carbon–nitrogen metabolic network. Root–microbe interactions also play a critical role in maintaining carbon–nitrogen balance. Mycorrhizal fungi enhance nitrogen acquisition through extensive extraradical mycelial networks, whereas maize roots secrete flavonoids (e.g., coumarins) to attract nitrogen-fixing microbes in the rhizosphere, thereby establishing a mutualistic carbon-for-nitrogen exchange [18, 19]. Yuan Lixing et al. reported that the symbiosis between maize and the beneficial bacterium Bacillus velezensis SQR9 improved NUE by 15–20%, which was the particularly significant effect under nitrogen-deficient conditions [1]. The eukaryotic translation initiation factor eIF4E1 functions as a cap-binding protein and plays a central role in the initiation of mRNA translation [20]. By recognizing the 5′ cap structure (m 7 GpppX) of mRNAs, it associates with eIF4G and eIF4A to form the eIF4F translation initiation complex, thereby mediating the assembly of the 40S ribosomal subunit [21, 22]. Although eIF4E1 is evolutionarily highly conserved, its biological functions exhibit substantial diversification across species. In Arabidopsis, eIF4E1 ( At4g18040 ) has been identified as a key regulator of nitrate signaling. The research group led by Wang Yong at Shandong Agricultural University isolated the nitrate response-deficient mutant Mut36 through EMS mutagenesis screening. This mutant carries a G→A point mutation in the second exon of the eIF4E1 gene, resulting in impaired translation initiation [22, 23]. Notably, the phosphorylation of eIF4E1 serves as a critical switch for its functional regulation. The laboratory of Yu Feng at Hunan University discovered that the Arabidopsis receptor kinase FERONIA, in response to the small peptide signal RALF1, phosphorylates eIF4E1 at Tyr118 and Thr140 [24]. Phosphorylated eIF4E1 exhibited a 3.5-fold increase in binding affinity for mRNAs encoding root hair growth-related genes (such as ROP2 and RSL4), thereby promoting polar root hair development through spatially restricted protein synthesis. This finding establishes a direct molecular link between nitrogen signaling and the regulation of cellular growth patterns [25]. To address the research gap in translational initiation regulation within maize nitrogen response mechanisms, this study performed the first systematic analysis of the eIF4E1 gene family at the genome-wide level. Using bioinformatics approaches, six maize ZmeIF4E1 family members were identified and comprehensively characterized in terms of their phylogenetic relationships, gene structures, conserved motifs, chromosomal localizations, and promoter cis-acting elements. In addition, by integrating public transcriptomic data with qRT-PCR experiments, the spatiotemporal expression specificity of these family members across multiple tissues and different maize developmental stages was examined. To directly evaluate their involvement in the nitrogen response, the ZmeIF4E1 gene expression changes in the roots and leaves of seedlings were analyzed under low-nitrogen (0.2 mM KNO 3 ) and high-nitrogen (10 mM KNO 3 ) stress conditions. This study aimed to determine whether the ZmeIF4E1 gene could be considered a potential target for genetic improvement. Materials and Methods Identification of eIF4E1 in the Maize Genome The eIF4E1 sequence was retrieved from the maize genome (Zm-B73-REFERENCE-NAM-5.0.55) using the Basic Local Alignment Search Tool (BLASTP; https://blast.ncbi.nlm.nih.gov/Blast.cgi ). Maize gff3, protein, coding sequence, and genome files were downloaded from the Plant Genome Database ( https://phytozome-next.jgi.doe.gov/ ). The Hidden Markov Model (HMM) profile for the eIF4E1 protein domain, IF4E (Pfam: pfam01652), was obtained from the Pfam database ( http://pfam.xfam.org/ ). The Arabidopsis AT4g18040 protein sequence was downloaded from the Arabidopsis Information Resource (TAIR) database ( https://www.arabidopsis.org/ ) and used for BLASTP alignment with the maize protein database. After integrating the HMMER and BLASTP search results(HMMER search (Pfam: pfam01652) with E-value ≤ 1e-5; BLASTP search against maize protein database with E-value ≤ 1e-10 and identity ≥ 50%), non-redundant protein sequences were submitted to the NCBI CD-search ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ) and SMART server ( https://smart.embl-heidelberg.de/ ) to confirm the presence of this conserved domain. Proteins containing the IF4E domain were classified as members of the eIF4E1 gene family in maize and designated based on their chromosomal locations. Chromosomal Localization of the ZmeIF4E1 Gene The chromosomal location of the ZmeIF4E1 gene was determined using the annotated maize genome. For synteny analysis, pairwise genome comparisons were performed using BLASTP searches (E-value < 10 − 10 ) with the NCBI BLAST suite to identify putative homologous gene pairs. Chromosomal mapping was visualized using TBtools (Figure S1 ). Phylogenetic Analysis of eIF4E1 Protein Using MEGA 11.0 software ( https://www.megasoftware.net/ ), eIF4E1 protein sequences from Arabidopsis, wheat, maize, rice, and sorghum were aligned and subjected to phylogenetic analysis. The aligned sequences were processed using the NJ method with the Poisson model and pairwise deletion, and a phylogenetic tree was constructed using 1000 bootstrap replicates. Gene Structure and Conserved Motif Analysis of ZmeIF4E1 The nucleotide sequence structure of the ZmeIF4E1 gene was analyzed using maize gff3 files. TBtools software was used to generate UTR-CDS distribution diagrams. The upstream 2.0 kb sequence of each ZmeIF4E1 gene was selected as the promoter region and extracted from the maize genome. The conserved motifs of the ZmeIF4E1 protein were identified using the Motif Elicitation (Multiple Expectation Maximization for Motif Elicitation, MEME) online platform ( http://meme-suite.org/ ). Conserved motifs were visualized using TBtools software. Maize Seedling Growth and Nitrogen Treatment Seeds of maize variety B73 were first subjected to flotation in sterile water to remove non-viable or shriveled kernels. One subset of seeds was sown in experimental fields for stage-specific sampling, while another subset was surface sterilized by soaking in a 2.6% sodium hypochlorite solution for 30 min. After five rinses with sterile water, the sterilized seeds were germinated for 48 h in a constant-temperature incubator at 28°C. Germinated seeds were then transferred to PhytoTC seed germination bags containing half-strength MS medium and cultured for two weeks in a plant growth chamber,Plant growth chamber conditions: 16 h light (300 µmol m − 2 s − 1 )/8 h dark photoperiod, day/night temperature 27℃/22℃, relative humidity 60%. Following the removal of endosperm tissue, seedlings were transplanted into PhytoTC bags filled with 2.5 mM ammonium succinate (NH 4 Suc) medium and grown for an additional 2 d. Plants with stable growth were treated with 0.2 mM KCl, 0.2 mM KNO 3 , 10 mM KCl, or 10 mM KNO 3 , respectively, for 2 h. RNA Extraction, cDNA Synthesis, and qRT-PCR Analysis Total RNA was extracted from maize tissues at various growth stages, as well as from the leaves and roots of nitrogen-treated controls, using an RNA Plant Extraction Kit (CWBIO, Cat. CW0581S, China). cDNA was synthesized using the HiFiScript All-in-One RT Master Mix for qPCR Kit (CWBIO, Cat. CW3371). All samples were stored at − 20°C until further use. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the SuperStar Universal SYBR Master Mix Kit (CWBIO, Cat. CW3360). Relative gene expression was quantified using a LightCycler® 480 instrument (Roche) with a reference housekeeping gene as the internal control, following the 2 −∆Ct method [26]. Data analysis and visualization were performed with GraphPad Prism 8.0 ( https://www.graphpad.com ). Data are presented as the mean of three technical replicates ± standard error (SE). Gene-specific primers are listed in Supplementary Dataset 1. Statistical significance was assessed using independent-samples t-tests. Error bars represent SE, and p-values < 0.05 (*) or < 0.01 (**) were considered statistically significant. Results Identification of the maize eIF4E1 gene and characterization of its protein physicochemical properties After integrating search results from HMMER and BLASTP, nine non-redundant proteins were identified and screened for conserved domains. Among these, six proteins contained the IF4E domain (Pfam: pfam01652) and were classified as members of the maize eIF4E1 gene family. Based on their chromosomal locations (Figure S1 ), the corresponding genes were designated ZmeIF4E1.1.1 to ZmeIF4E1.5 . The nucleotide sequences of these genes ranged from 827 bp (ZmeIF4E1.3.2) to 1243 bp ( ZmeIF4E1.3.1 ). Further analysis revealed the physicochemical properties of the eIF4E1 proteins (Table 1). ZmeIF4E1.1.2 and ZmeIF4E1.1.1 represented the shortest and longest proteins, consisting of 179 and 229 amino acids, respectively. Their molecular weights ranged from 19,936.53 Da ( ZmeIF4E1.1.2 ) to 26,541.70 Da ( ZmeIF4E1.1.1 ), with theoretical isoelectric points (pI) ranging from 5.66 ( ZmeIF4E1.5 ) to 6.32 ( ZmeIF4E1.1.1 ), indicating that all ZmeIF4E1 proteins were weakly acidic. Based on the instability index, two proteins were predicted to be unstable (> 40), whereas the remaining four were classified as stable (ranging from 29.98 to 38.31). The aliphatic index values ranged from 61.11 ( ZmeIF4E1.5 ) to 74.98 ( ZmeIF4E1.1.1 ), suggesting moderate thermal stability. The grand average of hydropathicity (GRAVY) values ranged from − 0.477 ( ZmeIF4E1.3.2 ) to − 0.79 ( ZmeIF4E1.1.1 ), indicating that all ZmeIF4E1 proteins are hydrophilic (Table 1). Phylogenetic Analysis of the eIF4E1 Gene To elucidate the evolutionary relationships of the eIF4E1 gene, a phylogenetic tree was constructed using a total of 33 eIF4E1 protein sequences from five species, including 7 from Arabidopsis, 3 from sorghum, 3 from rice, 14 from wheat, and 6 from maize (Figs. 1 and S2). Based on this analysis, the six ZmeIF4E1 proteins were grouped into three clusters: Clusters I–III. Cluster II contained the largest number of members, with 14 protein sequences, including 2 ZmeIF4E1 proteins and 7 wheat protein sequences. Group III included 12 members, consisting of 3 ZmeIF4E1 proteins and 4 wheat sequences. Group I contained the fewest members, with only 7 protein sequences, including 1 ZmeIF4E1 sequence. Structure and Motif Composition of the ZmeIF4E1 Gene To analyze the structural characteristics of the ZmeIF4E1 gene family, the coding sequences (CDS) of each member were systematically compared with their genomic DNA sequences using bioinformatics methods, thereby precisely determining the arrangement patterns of coding and non-coding regions (Fig. 2 ). The results indicated that most genes in this family exhibited structural conservation, containing 4 to 5 introns, and demonstrated subfamily specificity. Specifically, ZmeIF4E1.1.1 , belonging to Group I, contained five introns, whereas other genes in Groups II and III each harbored four introns. Correspondingly, ZmeIF4E1.1.1 possessed six exons, whereas the remaining genes each contained five exons. Further structural comparisons revealed that closely related members, such as ZmeIF4E1.1.3 and ZmeIF4E1.1.2, not only shared identical numbers of coding elements but also exhibited highly similar distribution patterns of untranslated regions (UTRs, green boxes in Fig. 2 b) and CDSs (yellow boxes). The CDS regions of these subtypes were more compactly arranged, suggesting strong functional constraints during evolution and reflecting branch specificity. This finding provides critical evidence for the classification and functional differentiation of the ZmeIF4E1 gene family at the structural level, laying the groundwork for further studies on transcriptional regulation and alternative splicing mechanisms. To investigate the conserved sequence features of ZmeIF4E1 family proteins, the MEME online tool was used to identify conserved motifs, resulting in the identification of 10 motifs(MEME parameters: minimum motif width = 6, maximum motif width = 50, maximum number of motifs = 10). As shown in Fig. 2 c, all ZmeIF4E1 proteins contained Motifs 1, 2, and 3, indicating that these motifs represent the core functional elements of the family. The closely related members ZmeIF4E1.3.1 and ZmeIF4E1.3.2 exhibited highly similar motif compositions and arrangements, each containing Motifs 1, 2, 3, 4, 5, 6, 7, and 10. This sequence-level result supported the phylogenetic grouping. Notably, ZmeIF4E1.1.1 displayed the simplest motif composition, containing only three core motifs and Motif 4, suggesting potential functional divergence from other family members. Distribution of cis-acting elements in the ZmeIF4E1 gene promoter To clarify the transcriptional regulatory pathways associated with the ZmeIF4E1 gene, cis-acting regulatory elements in the promoter regions were analyzed using PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) within the 2000-bp upstream sequence. In total, 71 cis-acting elements were identified in the promoters of the six ZmeIF4E1 genes. Visualization of the 32 explicitly annotated cis-elements revealed five major categories (Fig. 3 ), including 8 nitrogen-responsive elements, 13 light-responsive elements, 9 anaerobic-responsive elements, and two maize protein regulatory elements. In addition to these named cis-elements, the predictions indicated that the remaining 39 sequences contained 28 putative functional elements associated with light response, hormone response, and other regulatory functions (Table S4 ). Nitrogen- and anaerobic-response elements were identified in ZmeIF4E1.1.1 , ZmeIF4E1.1.2 , ZmeIF4E1.1.3 , and ZmeIF4E1.3.2 . Light-responsive elements included both G-box and I-box types. ZmeIF4E1.1.1 contained both types, whereas ZmeIF4E1.1.2 , ZmeIF4E1.1.3 , and ZmeIF4E1.3.2 harbored only the G-box type, suggesting the potential functional differentiation among these members within light regulatory networks. Notably, only six putative hormone-responsive elements were detected in the promoter region of ZmeIF4E1.5 . Expression of the ZmeIF4E1 Gene in Different Maize Tissues To further elucidate the role of the ZmeIF4E1 gene in maize development, its expression profiles across six tissues/organs were analyzed using published transcriptome data obtained from the Phytozome database. Gene expression levels were quantified in FPKM. Genes with FPKM values below 1.00 were classified as non-expressed, while those above this threshold were categorized as follows: low expression (1.00 ≤ FPKM < 5.00), medium expression (5.00 ≤ FPKM < 15.00), and high expression (FPKM ≥ 15.00) [27]. qRT-PCR analysis was conducted on the roots and leaves of seedlings at the three-leaf stage. The results (Figs. 4 and S3a) demonstrated significant differences in the expression of ZmeIF4E1 family members between roots and leaves. Among them, ZmeIF4E1.5 exhibited the highest expression, markedly exceeding that of the other homologs in both tissues. ZmeIF4E1.1.1 ranked the second, whereas ZmeIF4E1.1.3 displayed the lowest expression level. As illustrated in Figs. 5 and S3b, qRT-PCR profiling further revealed distinct tissue-specific expression patterns across four tissues (leaves, roots, leaf sheaths, and internodes) at the jointing stage. ZmeIF4E1.1.2 displayed exceptionally high expression in leaves, significantly surpassing other homologs, while ZmeIF4E1.1.3 also presented elevated expression in leaves, second only to ZmeIF4E1.1.2 . In contrast, ZmeIF4E1.3.2 exhibited relatively low expression across all internode-stage tissues examined. qRT-PCR expression analysis was performed on four tissues (leaves, roots, leaf sheaths, and internodes) of maize during the big-flare period. The results (Figs. 6 and S3c) indicated that members of the ZmeIF4E1 gene family displayed distinct tissue-specific expression patterns. In leaves, ZmeIF4E1.1.1 and ZmeIF4E1.5 exhibited the highest expression levels, significantly exceeding those of the other homologs. ZmeIF4E1.1.3 and ZmeIF4E1.1.2 demonstrated the next highest levels. In roots, ZmeIF4E1.1.1 had the highest expression, followed by ZmeIF4E1.1.2 and ZmeIF4E1.1.3 . In leaf sheaths, ZmeIF4E1.1.2 exhibited the most pronounced expression, followed by ZmeIF4E1.1.3 . ZmeIF4E1.3.2 consistently exhibited the lowest expression in all four tissues. At the tasseling stage, qRT-PCR analysis of six tissues (leaves, roots, leaf sheaths, internodes, male flowers, and female flowers) revealed further spatial variation (Figs. 7 and S3d). ZmeIF4E1.1.1 exhibited the highest expression in internodes, followed by ZmeIF4E1.1.2 . In leaves and leaf sheaths, ZmeIF4E1.1.2 exhibited relatively higher expression than other homologs. ZmeIF4E1.5 displayed the highest expression in pistils, whereas ZmeIF4E1.3.2 exhibited the lowest expression levels across all six tissues. During the grain-filling stage, qRT-PCR analysis of four tissues (leaves, roots, leaf sheaths, and internodes) confirmed distinct expression profiles (Figs. 8 and S3e). In leaf sheaths, ZmeIF4E1.1.3 exhibited the highest expression, followed by ZmeIF4E1.1.2 . In roots, ZmeIF4E1.5 and ZmeIF4E1.1.1 showed elevated expression, whereas ZmeIF4E1.3.2 had the lowest expression across all four tissues. Expression of ZmeIF4E1 in Response to Nitrogen To further explore the potential role of the ZmeIF4E1 gene in nitrogen response, we analyzed the expression patterns of the six ZmeIF4E1 genes in maize leaves and roots subjected to low-nitrogen (0.2 mM KNO 3 ) and high-nitrogen (10 mM KNO 3 ) treatments using qRT-PCR. As shown in Fig. 9 , the expression level of ZmeIF4E1.1.2 in leaves under low-nitrogen treatment (0.2 mM KNO 3 ) was significantly higher than that in the corresponding KCl control (0.2 mM). Similarly, ZmeIF4E1.1.1 expression in roots under low-nitrogen treatment was markedly elevated compared with the KCl control. In contrast, the expression of all six ZmeIF4E1 genes in leaves was significantly repressed under high-nitrogen treatment (10 mM KNO 3 ) relative to the 10 mM KCl control, indicating that high nitrogen availability negatively affected leaf development. Notably, in roots treated with 10 mM KNO 3 , the expression levels of ZmeIF4E1.1.1 , ZmeIF4E1.1.2 , ZmeIF4E1.1.3 , and ZmeIF4E1.3.2 were significantly higher than the 0.2 mM KCl control. Discussion Efficient nitrogen utilization is a central goal of the genetic improvement of maize. Previous studies have primarily focused on transcriptional regulators, such as NLP and BZIP transcription factors, and nitrogen transporters, including NRT and AMT families [28, 29]. However, the role of translational regulation, which is a crucial step in gene expression in plant nitrogen response, has only recently begun to be elucidated in Arabidopsis [22, 23] and remains largely unexplored in maize. This study presents the first systematic identification of the eIF4E1 gene family in the maize genome and a comprehensive analysis of the expression patterns of its members under nitrogen stress, thereby offering important insights into the translational regulatory mechanisms that may affect NUE in maize. This study successfully identified six members of the maize eIF4E1 gene family, each containing the characteristic IF4E (Pfam: pfam01652) cap-binding domain, thereby confirming the evolutionary conservation of their molecular function. Phylogenetic analysis (Figs. 1 and S2) demonstrated that maize eIF4E1 proteins formed a well-supported clade with homologs from Arabidopsis and rice, reflecting the high degree of conservation of eukaryotic translation initiation mechanisms [30, 31]. Notably, substantial variation was observed among eIF4E1 family members in physicochemical properties, such as isoelectric point and instability index, which suggests potential functional divergence within the family that may enable adaptation to diverse cellular environments in maize (Table 1). Most ZmeIF4E1 genes lacked introns within their coding regions, which served as structural features shared with the Arabidopsis At4g18040 ( eIF4E1 ) homolog. This intronless architecture may facilitate rapid and constitutive expression, supporting the sustained demand for core translation initiation factors under basal conditions and enabling prompt responses to environmental changes. To thoroughly analyze the transcriptional regulatory mechanisms by which the ZmeIF4E1 gene family responds to external environmental signals, this study adopted a systematic analysis of cis-acting elements in the promoter regions of the six family members (Fig. 3 ). The diverse composition of these elements suggests that the ZmeIF4E1 family may integrate multiple environmental and endogenous signals through complex transcriptional regulatory networks, thereby playing a crucial role in maize growth, development, and stress responses. Notably, both nitrogen response elements and anaerobic response elements co-occurred in the promoter regions of ZmeIF4E1.1.1, ZmeIF4E1.1.2, ZmeIF4E1.1.3, and ZmeIF4E1.3.2. This co-occurrence suggests the potential collaborative participation of these genes in nitrogen signal perception and hypoxic environment responses, reflecting the cross-talk between signaling pathways. This finding is consistent with mechanisms previously reported in Arabidopsis [10]. Previous research has indicated that At4g18040 directly participates in nitrogen response regulation via the nitrate signaling pathway, with its functional knockout mutant exhibiting significant nitrate utilization defects [22]. These findings suggest that maize may achieve the functional specificity of eIF4E1 in nitrogen signaling pathways through conserved transcriptional regulatory mechanisms, offering new insights into nitrogen signal transduction in monocotyledons. Among the light-responsive elements, two distinct types were identified: G-box and I-box. Notably, significant differences were identified among family members. ZmeIF4E1.1.1 contained both light-responsive elements, whereas ZmeIF4E1.1.2, ZmeIF4E1.1.3, and ZmeIF4E1.3.2 contained only the G-box element. This differential distribution suggests potential functional specialization among members of the photoperiod regulation network. ZmeIF4E1.1.1 may integrate more complex light signals through multiple light-responsive elements, whereas other members primarily rely on G-box-mediated photoregulatory pathways [23]. This finding was consistent with studies in higher plants, where light signals frequently regulated gene expression through G-box elements, providing clues for investigating the coordinated regulation between maize photosynthetic products and nitrogen utilization. The promoter region of ZmeIF4E1.5 contained only six putative hormone-responsive elements, exhibiting a regulatory pattern markedly different from that of other family members. We hypothesized that this gene could not directly respond to nitrogen, light, or hypoxia signals, but could achieve transcriptional regulation through rare cis-acting modules or hormone-related pathways. Furthermore, its expression may be strongly influenced by distant enhancers and three-dimensional chromatin architecture. This unique property suggests that ZmeIF4E1.5 may perform specialized functions within the family, warranting further experimental investigation, such as promoter deletion analysis, EMSA, or ChIP-seq, to elucidate its precise regulatory mechanisms. In summary, this study revealed significant structural differences in the promoters of ZmeIF4E1 gene family members at the cis-acting element level, providing theoretical support for their functional differentiation in response to nitrogen, light, and hypoxic stress. These findings not only offer new evidence for refining the molecular regulatory network of nitrogen efficiency in maize but also identify potential cis-regulatory targets for improving crop nitrogen use efficiency through molecular design breeding. This study revealed that the ZmeIF4E1 gene was constitutively expressed across multiple maize tissues, including roots, stems, leaves, leaf sheaths, internodes, stamens, and pistils, although its expression levels varied considerably, demonstrating clear spatiotemporal specificity (Figs. 4 – 8 and S3). These findings indicate that members of this gene family could play extensive and finely tuned roles in the post-transcriptional regulation of diverse growth and developmental processes in maize. By integrating public transcriptomic data from the Phytozome database with qRT-PCR validation results, we further elucidated the dynamic expression patterns of this gene family across different developmental stages. At the three-leaf stage (Figs. 4 and S3a), ZmeIF4E1.5 emerged as the predominant subtype expressed in both roots and leaves, followed by ZmeIF4E1.1.1, whereas ZmeIF4E1.1.3 exhibited the lowest expression levels. This suggests that ZmeIF4E1.5 may play a dominant role in establishing foundational translational mechanisms during early seedling development. By the jointing stage (Figs. 5 and S3b), the expression profiles shifted markedly, where ZmeIF4E1.1.2 exhibited strong leaf-specific accumulation, ZmeIF4E1.1.3 was also elevated in leaves, and ZmeIF4E1.3.2 remained low across all tissues. This suggests a strong reliance on specific eIF4E1 subtypes to support leaf development during this phase. At the big-flare stage (Figs. 6 and S3c), the expression profiles displayed a pronounced tissue-specific divergence. ZmeIF4E1.1.1 and ZmeIF4E1.5 were strongly expressed in leaves, with ZmeIF4E1.1.1 also demonstrating preferential accumulation in roots. In contrast, ZmeIF4E1.1.2 was highly abundant in leaf sheaths, indicating that different family members achieve complementary functions in processes such as photosynthesis, nutrient uptake, and structural maintenance. During reproductive growth, ZmeIF4E1.1.1 reached peak expression in internodes at the tasseling stage (Figs. 7 and S3d), whereas ZmeIF4E1.5 was specifically enriched in pistils, suggesting critical roles in floral organ development and post-pollination translational control. At the grain-filling stage (Figs. 8 and S3e), ZmeIF4E1.1.3 and ZmeIF4E1.1.2 presented the elevated transcript levels in leaf sheaths, while both ZmeIF4E1.5 and ZmeIF4E1.1.1 were upregulated in roots. These expression patterns imply enhanced capacities for resource transport and nitrogen assimilation to meet the high metabolic demands of grain filling. Overall, the ZmeIF4E1 gene family was constitutively expressed across a broad range of maize tissues, including roots, stems, leaves, and grains, although the expression intensity varied among members, reflecting their diverse contributions to growth and development. Notably, ZmeIF4E1.1.1 and ZmeIF4E1.1.2 exhibited tissue-specific responses to nitrogen stress (Fig. 9 ). Under low-nitrogen conditions (0.2 mM KNO 3 ), ZmeIF4E1.1.1 was significantly induced in roots, whereas ZmeIF4E1.1.2 was strongly upregulated in leaves. These patterns were consistent with previous findings in Arabidopsis. For instance, research from Yong Wang’s group at Shandong Agricultural University demonstrated that a loss-of-function mutant of Arabidopsis eIF4E1 ( At4g18040 ; Mut36 ) caused severe nitrate uptake defects [22]. It is plausible that ZmeIF4E1.1.1 in maize functions in sensing low-nitrogen signals in roots through a conserved pathway, thereby enhancing the translational efficiency of specific mRNAs encoding nitrate transporters (NRTs) or key enzymes in nitrogen assimilation. This mechanism could contribute to improved nitrogen uptake and NUE in maize. We observed a pronounced tissue-specific response to high-nitrogen treatment (10 mM KNO 3 ). The expression of nearly all ZmeIF4E1 genes was strongly repressed in leaves, implying the existence of a feedback mechanism to prevent nitrogen overload. In contrast, the expressions of ZmeIF4E1.1.1 , ZmeIF4E1.1.2 , ZmeIF4E1.1.3 , and ZmeIF4E1.3.2 were markedly induced in roots. This sharp divergence between roots and leaves suggests a spatial functional division of labor among eIF4E1 family members in regulating nitrogen responses. As the primary site for nitrogen uptake and initial assimilation, roots may require translational reprogramming under high-nitrogen conditions to efficiently process incoming nitrogen sources. Conversely, leaves, as the central organs for assimilation, may downregulate global translational activity to avoid excessive carbon skeleton consumption, thereby maintaining carbon-nitrogen balance [32–34]. Such organ-specific regulation can represent an adaptive strategy that enables maize to sustain high-yield traits by optimizing NUE while preserving overall metabolic homeostasis. Although this study revealed the potential roles of the ZmeIF4E1 gene family in nitrogen signaling and highlighted its spatiotemporal expression patterns through bioinformatics and expression profiling, certain limitations remain. These findings require direct functional validation through genetic experiments. Generating targeted knockout mutants of ZmeIF4E1 genes using CRISPR/Cas9 or creating overexpression lines via transgenic approaches would provide more definitive evidence of their roles in nitrogen uptake, assimilation, and plant growth. Such genetic evidence is critical for establishing precise gene functions. Similar to how the function of eIF4E1 in Arabidopsis was conclusively demonstrated through the T-DNA insertion mutant mut36 [22], functional analyses of homologous genes in maize should require analogous loss-of-function and gain-of-function studies to validate their contributions to nitrogen response and utilization. More critically, as a canonical cap-binding protein, the essential function of eIF4E1 lies in regulating translation initiation. Hence, it is crucial to employ ribosome profiling (Ribo-seq) to comprehensively identify the mRNA populations preferentially bound and translationally regulated by each ZmeIF4E1 protein, thereby delineating their downstream targets with precision. For instance, studies in Arabidopsis have indicated that eIF4E1 not only governs global translational activity but also selectively enhances the translation of key genes, such as nitrate transporters (e.g., NRT1.1) and auxin response factors (e.g., ARFs) [22, 24]. Whether maize homologs regulate specific targets, including auxin-related transcription factors (e.g., ZmARF34) or nitrogen metabolic enzymes, through analogous mechanisms remains to be determined [24]. Such validation will require integrated multi-omics strategies that combine Ribo-seq with RNA-seq, RNA immunoprecipitation (RIP), and related molecular techniques[35]. Of particular importance is the translational regulation of Arabidopsis eIF4E1 by phosphorylation. Determining whether maize homologs undergo comparable post-translational modifications is a promising direction for future research. The focused development of specialized genetic resources and in-depth mechanistic analyses of these molecular interactions are essential. Such efforts will advance a more comprehensive understanding of ZmeIF4E1-mediated regulation at the translational level during nitrogen signaling. This knowledge will not only identify potential molecular targets for enhancing NUE in crops via molecular breeding but also broaden comparative insights into the evolutionary conservation and divergence of eIF4E1 function across species. Furthermore, nitrate plays a pivotal role not only as a nutrient but also as an environmental signal that extends beyond plant physiology to influence broader ecosystems. Recent studies on sludge composting have suggested that nitrate availability can reshape microbial communities, enriching aromatic-ring-degrading microbes that drive complex organic matter breakdown [34, 36]. This parallel highlights the fundamental importance of nitrate as both a metabolic substrate and a signaling molecule, orchestrating biological processes from microbial decomposition in the environment to translational control within plants. The discovery that ZmeIF4E1 responds to nitrate stress underscores this conserved principle across kingdoms, where nitrogen serves not only as a nutrient but also as a signaling hub governing metabolic and developmental outcomes. In conclusion, this study systematically identified and characterized the maize eIF4E1 gene family, revealing its distinct expression patterns and member-specific responses to nitrogen signaling. These findings extend the current knowledge of NUE beyond transcriptional and transporter-level regulation into the domain of translational control. Our study establishes a new framework for understanding the molecular mechanisms underlying nitrogen efficiency in maize and provides promising genetic targets, such as ZmeIF4E1.1.1 and ZmeIF4E1.1.2 , for crop improvement. Overall, this study offers valuable genetic resources and strategic insights for breeding nitrogen-efficient maize varieties using molecular design approaches. Declarations Acknowledgements This research was supported by the Horizontal Research Project “Key Engineering Technologies for Smart Agriculture” (K23LD90), “Technical Services for the Development of Modern Agricultural Industries” (K24LD179) and the Open Project Program of State Key Laboratory of Crop Biology (2021KF04). Author Contributions Statement Q. W. and L. Z. designed the project. Q. W. conducted the experiments. The data were analyzed by L. Z., Q. W., and H. Z. The manuscript was written by Q. W., H. Z., L. Z., and P. C. All authors read and approved the final manuscript. Additional Information Supplementary information can be found in the online version of this article. Competing interests: The authors declare that they have no competing interests. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Funding Horizontal Research Project “Key Engineering Technologies for Smart Agriculture” (K23LD90), “Technical Services for the Development of Modern Agricultural Industries” (K24LD179) and the Open Project Program of State Key Laboratory of Crop Biology (2021KF04). Authors' information Qi Wang, [email protected] , Liaocheng University CN and Hang Zhou, [email protected] , Shandong Shennong Zhiyi Intelligent Technology Co., Ltd CN. Corresponding author: Pengfei Chu, [email protected] , Liaocheng University CN, and Lufei Zhao, [email protected] , Liaocheng University CN. Ethics Statement Maize ( Zea mays L. ) inbred line B73 is a widely used public genetic resource with no endangered or protected status. No specific ethical approval or permits were required for the collection, cultivation, and experimental use of this material in this study, which complies with the national agricultural research policies of the People’s Republic of China.” Data Availability All raw gene sequences of ZmeIF4E1 family members, and promoter cis-acting element analysis results are included in the Supplementary Information (Datasets S1-S4, Figures S1-S3). Public transcriptome data used in this study were retrieved from Phytozome (https://phytozome-next.jgi.doe.gov/) under accession number: PRJNA10769 (Maize), PRJNA13876 (Sorghum), and EnsemblPlants (https://plants.ensembl.org/) under accession number: GCA_900519105 (Wheat), GCA_001433935 (Rice), and the data of Arabidopsis thaliana is from TAIR (https://www.arabidopsis.org/) . 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Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files TABLE1.xlsx SupplementaryDataset1.xlsx SupplementaryDataset2.xlsx SupplementaryDataset3.xlsx SupplementaryDataset4.xlsx Supplementaryfigure.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":8523682,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of ZmeIF4E1 protein. A neighbor-joining phylogenetic tree was constructed using MEGA 11.0 software with 1000 bootstrap replicates.\u003c/p\u003e","description":"","filename":"FIGURE1.png","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/9a1c9b2e829e9362d7d804e6.png"},{"id":96349112,"identity":"590cedf8-f879-4abc-a386-f54504baec35","added_by":"auto","created_at":"2025-11-20 06:58:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4032813,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Unrooted phylogenic tree of six ZmeIF4E1 proteins constructed using MEGA11.0 software based on the complete sequences. (b) Structural analysis of the coding and non-coding regions of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene, with yellow and green boxes representing the coding and non-coding regions, respectively. (c) Distribution of conserved motifs in ZmeIF4E1 protein. Ten putative motifs and the IF4E domain are depicted in differently colored boxes.\u003c/p\u003e","description":"","filename":"FIGURE2.png","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/cfdb71cae067f17bc3911d02.png"},{"id":96367197,"identity":"76371813-f4ce-4c34-a13c-28a1f974a337","added_by":"auto","created_at":"2025-11-20 10:12:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":905655,"visible":true,"origin":"","legend":"\u003cp\u003eCis-acting elements in the promoter regions of the six \u003cem\u003eZmeIF4E1\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/2dfe125026ab06db5b91dcef.png"},{"id":96366032,"identity":"8dcc2da0-9430-4a59-a7d6-67b47df77065","added_by":"auto","created_at":"2025-11-20 10:11:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":717402,"visible":true,"origin":"","legend":"\u003cp\u003eExpression heatmap of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene family in different maize tissues at the three-leaf stage.\u003c/p\u003e","description":"","filename":"FIGURE4.png","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/f44f27ed850d70ff05be31a5.png"},{"id":96366736,"identity":"49e6d6cd-b107-4841-adbd-377eebf0facc","added_by":"auto","created_at":"2025-11-20 10:11:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":903666,"visible":true,"origin":"","legend":"\u003cp\u003eExpression heatmap of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene family in different maize tissues at the jointing stage.\u003c/p\u003e","description":"","filename":"FIGURE5.png","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/5ffb880bdf27a3d718774957.png"},{"id":96349130,"identity":"513f5254-0953-41bc-92af-6e677899b3bf","added_by":"auto","created_at":"2025-11-20 06:58:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":867644,"visible":true,"origin":"","legend":"\u003cp\u003eExpression heatmap of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene family in different maize tissues during the big flare period.\u003c/p\u003e","description":"","filename":"FIGURE6.png","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/48b8fbab4fd0ae7704a6559b.png"},{"id":96367049,"identity":"de247022-58d8-4a9f-8977-2b64bafbf783","added_by":"auto","created_at":"2025-11-20 10:12:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":961251,"visible":true,"origin":"","legend":"\u003cp\u003eExpression heatmap of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene family in different maize tissues during the tasseling period.\u003c/p\u003e","description":"","filename":"FIGURE7.png","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/37d9c7cac3c96fbc26c0af6d.png"},{"id":96349127,"identity":"dca82012-ff3f-49f1-84f7-517083c45494","added_by":"auto","created_at":"2025-11-20 06:58:14","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":851242,"visible":true,"origin":"","legend":"\u003cp\u003eExpression heatmap of the \u003cem\u003eZmeIF4E1 \u003c/em\u003egene family in different maize tissues during the pustulation period.\u003c/p\u003e","description":"","filename":"FIGURE8.png","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/c50658b31dbf5c5bccb2669b.png"},{"id":96367301,"identity":"0c6d4133-1114-441b-8e7a-2455c268173f","added_by":"auto","created_at":"2025-11-20 10:12:30","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4343139,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of \u003cem\u003eZmeIF4E1\u003c/em\u003e genes in maize leaves and roots under low-nitrogen (0.2 mM KNO\u003csub\u003e3\u003c/sub\u003e) and high-nitrogen (10 mM KNO\u003csub\u003e3\u003c/sub\u003e) conditions. Expression levels were determined using qRT-PCR from three independent experiments. Expression of ZmeIF4E1 genes in maize leaves and roots under low-nitrogen (0.2 mM KNO\u003csub\u003e3\u003c/sub\u003e) and high-nitrogen (10 mM KNO\u003csub\u003e3\u003c/sub\u003e) conditions (0.2 mM KCl and 10 mM KCl as respective controls).Error bars indicate the standard error (SE). Statistical significance is shown as p-values \u0026lt; 0.05 (*) , p-values \u0026lt; 0.01 (**) and p-values\u0026lt; 0.001(***),independent-samples t-test\u0026nbsp;.\u003c/p\u003e","description":"","filename":"FIGURE9.png","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/ba2baf5540dd62d0b65667fd.png"},{"id":108805741,"identity":"42df8227-4ca4-4a67-af3e-78ab085b2f58","added_by":"auto","created_at":"2026-05-08 15:26:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18172953,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/22303d73-7d2d-4ec7-a849-e67c92c93549.pdf"},{"id":96349117,"identity":"ddaf554f-b0da-4fd7-95ae-50205deb0329","added_by":"auto","created_at":"2025-11-20 06:58:14","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11506,"visible":true,"origin":"","legend":"","description":"","filename":"TABLE1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/e80e851bb21fae416a33e9ce.xlsx"},{"id":96349163,"identity":"3537e3f4-998c-4244-8c30-0eaceefed4df","added_by":"auto","created_at":"2025-11-20 06:58:56","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11704,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDataset1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/0beb2563eb0a8af2fc67d178.xlsx"},{"id":96367080,"identity":"b427d0d6-2024-42b9-8c74-3d2c649a974e","added_by":"auto","created_at":"2025-11-20 10:12:09","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13517,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDataset2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/37d304fa9b4ef0dc08103fbd.xlsx"},{"id":96349115,"identity":"dcb4db27-d63a-444e-90ef-c8a29218bfd4","added_by":"auto","created_at":"2025-11-20 06:58:13","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11086,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDataset3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/9ea29d4513d2c2734e92e7ca.xlsx"},{"id":96367085,"identity":"e1d9eb50-05cd-4ac6-9ab0-fd7e6c114425","added_by":"auto","created_at":"2025-11-20 10:12:09","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11114,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDataset4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/33ce80da881a44f6d484eeee.xlsx"},{"id":96367426,"identity":"fbdd4afb-a0c7-476d-8772-2bc4712f5281","added_by":"auto","created_at":"2025-11-20 10:12:45","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1750014,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7863718/v1/e2f86b12c69194ba2a6ee197.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification and Expression Analysis of the eIF4E1 Gene Family in Maize","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNitrogen is an essential nutrient for plant growth and development and plays an irreplaceable role in maize yield formation and grain quality regulation. Statistical data indicate that producing one ton of maize grain requires 16\u0026ndash;18 kg of nitrogen, whereas the global nitrogen fertilizer use efficiency (NUE) in maize production generally remains below 40% [1]. Such inefficient utilization not only increases production costs but also leads to serious ecological and environmental problems, including water eutrophication, soil acidification, and greenhouse gas emissions [2]. Studies have indicated that drought stress during the flowering stage can significantly impair ear morphology, reducing the cob diameter at the base (CDB) by 13\u0026ndash;16%. However, appropriate nitrogen application (e.g., 300 kg ha⁻\u0026sup1; of urea) can mitigate the inhibitory effects of drought on chlorophyll fluorescence parameters (Fv/Fm) and the harvest index (HI), which contributes to a biomass increase of 23.7% even under stress conditions [3, 4]. Therefore, the identification of genetic resources with high nitrogen use efficiency and elucidation of the regulatory mechanisms underlying carbon\u0026ndash;nitrogen balance have become central challenges in achieving the maize breeding objective of \u0026ldquo;reducing fertilizer input while enhancing efficiency\u0026rdquo; [3].\u003c/p\u003e\u003cp\u003eThe nitrogen response mechanism in maize involves multilayered regulatory network. At the transcriptional level, nitrogen signaling activates transcription factors that regulate the expression of downstream target genes. At the translational level, nitrogen availability influences the efficiency of translation initiation. At the metabolic level, the products of nitrogen assimilation provide feedback that controls the allocation of carbon skeletons [3, 4]. Genome-wide association studies (GWAS) have identified multiple quantitative trait loci (QTLs) associated with nitrogen uptake and metabolism in maize. For instance, a GWAS analysis of nitrate accumulation in the leaves of 350 maize inbred lines revealed 16 significant SNP loci on chromosome 4. Candidate genes such as \u003cem\u003eNAC79\u003c/em\u003e, \u003cem\u003eGA20ox7\u003c/em\u003e, and \u003cem\u003ePREP2\u003c/em\u003e can be upregulated 2- to 3.5-fold under low-nitrogen conditions [1, 5]. These findings provide important insights into the molecular basis of nitrogen use efficiency in maize. However, key genes within the regulatory network and their functional modules still require systematic and comprehensive elucidation.\u003c/p\u003e\u003cp\u003eIn maize, several pivotal regulatory factors involved in nitrogen signal perception and transduction have been characterized. The transcription factor ZmNLP5, a member of the NIN-like protein (NLP) family, is specifically expressed in root and vascular tissues and functions as a central regulator of nitrate signaling [6]. Under low-nitrogen stress, ZmNLP5 directly binds to the nitrogen response cis-element (NRE) in the promoter region of the nitrite reductase gene \u003cem\u003eZmNIR1.1\u003c/em\u003e, thereby activating the nitrogen assimilation pathway. The zmnlp5 mutant exhibits reduced nitrate accumulation in roots and a pronounced decrease in grain nitrogen content. Functional complementation of this gene restores nitrate uptake capacity, confirming its essential role in nitrogen signal transduction [1, 7]. Additionally, \u003cem\u003eTHP9\u003c/em\u003e, isolated from wild maize, encodes asparagine synthetase 4 (ASN4). This enzyme catalyzes the synthesis of asparagine, which serves as a nitrogen donor in transamination reactions, markedly increasing grain protein content and improving NUE [1].\u003c/p\u003e\u003cp\u003eRecent studies have demonstrated that transcriptional regulation is crucial for nitrogen signaling. The transcription factor PBF1 regulates carbon\u0026ndash;nitrogen allocation during endosperm development by altering its DNA-binding specificity in response to nitrogen availability. Under nitrogen-deficient conditions, PBF1 reduces its binding to the promoters of maize zein genes, thereby suppressing the expression of sugary1 and amylase2b [1, 8]. This adjustment shifts carbon flux towards carbohydrate biosynthesis. This dynamic regulation ensures the coordinated accumulation of starch and protein in the endosperm, providing key mechanistic insights into carbon\u0026ndash;nitrogen crosstalk.\u003c/p\u003e\u003cp\u003eNitrogen uptake in maize is primarily mediated by two major gene families: nitrate transporters (NRTs) and ammonium transporters (AMTs). Based on substrate affinity, nitrate transport systems are classified into high-affinity transport systems (HATS) and low-affinity transport systems (LATS) [9, 10]. As a low-affinity nitrate transporter, ZmNPF6.6 shows a significant correlation between allelic variation and nitrogen uptake efficiency in maize, rendering it a promising candidate for molecular marker-assisted breeding. As reported by Yuan et al., maize root architecture, including traits such as lateral root density and root hair length, is dynamically regulated by nitrogen availability. Auxin response factors including ZmARF34 and ZmAUX/IAA12 modulate root nitrogen-foraging capacity by integrating nitrogen signals with hormone signaling pathways [1]. At the level of nitrogen assimilation, glutamine synthetase (GS) and glutamate synthase (GOGAT) constitute the GS/GOGAT cycle, which is the central pathway for ammonia incorporation [11]. ZmGS1.3 is specifically expressed in the vascular bundles of leaves, where it assimilates reduced nitrogen products transported from the roots. In contrast, ZmGS1.4 is expressed predominantly in root tips, where it facilitates the direct assimilation of soil ammonium [12, 13]. This tissue-specific expression pattern enables efficient spatial partitioning of nitrogen metabolism. Furthermore, ZmGDH1 (glutamate dehydrogenase) displays markedly enhanced activity under nitrogen-deficient conditions. By catalyzing both the reductive amination and deamination of α-ketoglutarate, it functions as a metabolic safety valve that helps maintain carbon\u0026ndash;nitrogen balance [14].\u003c/p\u003e\u003cp\u003eKernel development in maize depends on the precise coordination between photosynthetic carbon fixation and nitrogen assimilation. From 7 to 49 d after silking, the sucrose content in the kernels exhibits a single-peaked pattern correlated with sucrose synthase (SS) activity. Meanwhile, the peak activity of ADP-glucose pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is strongly influenced by nitrogen supply [15, 16]. Under sufficient nitrogen application (200 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the peak in AGPase activity occurred 7\u0026ndash;10 d earlier, promoting more efficient starch accumulation. In contrast, excessive nitrogen fertilization (\u0026gt;\u0026thinsp;300 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) constrains AGPase activity and reduces the rate of starch synthesis [17]. This non-linear response highlights the complex interactions within the carbon\u0026ndash;nitrogen metabolic network.\u003c/p\u003e\u003cp\u003eRoot\u0026ndash;microbe interactions also play a critical role in maintaining carbon\u0026ndash;nitrogen balance. Mycorrhizal fungi enhance nitrogen acquisition through extensive extraradical mycelial networks, whereas maize roots secrete flavonoids (e.g., coumarins) to attract nitrogen-fixing microbes in the rhizosphere, thereby establishing a mutualistic carbon-for-nitrogen exchange [18, 19]. Yuan Lixing et al. reported that the symbiosis between maize and the beneficial bacterium Bacillus velezensis SQR9 improved NUE by 15\u0026ndash;20%, which was the particularly significant effect under nitrogen-deficient conditions [1].\u003c/p\u003e\u003cp\u003eThe eukaryotic translation initiation factor eIF4E1 functions as a cap-binding protein and plays a central role in the initiation of mRNA translation [20]. By recognizing the 5\u0026prime; cap structure (m\u003csup\u003e7\u003c/sup\u003eGpppX) of mRNAs, it associates with eIF4G and eIF4A to form the eIF4F translation initiation complex, thereby mediating the assembly of the 40S ribosomal subunit [21, 22]. Although eIF4E1 is evolutionarily highly conserved, its biological functions exhibit substantial diversification across species.\u003c/p\u003e\u003cp\u003eIn Arabidopsis, \u003cem\u003eeIF4E1\u003c/em\u003e (\u003cem\u003eAt4g18040\u003c/em\u003e) has been identified as a key regulator of nitrate signaling. The research group led by Wang Yong at Shandong Agricultural University isolated the nitrate response-deficient mutant Mut36 through EMS mutagenesis screening. This mutant carries a G\u0026rarr;A point mutation in the second exon of the \u003cem\u003eeIF4E1\u003c/em\u003e gene, resulting in impaired translation initiation [22, 23]. Notably, the phosphorylation of eIF4E1 serves as a critical switch for its functional regulation. The laboratory of Yu Feng at Hunan University discovered that the Arabidopsis receptor kinase FERONIA, in response to the small peptide signal RALF1, phosphorylates eIF4E1 at Tyr118 and Thr140 [24]. Phosphorylated eIF4E1 exhibited a 3.5-fold increase in binding affinity for mRNAs encoding root hair growth-related genes (such as ROP2 and RSL4), thereby promoting polar root hair development through spatially restricted protein synthesis. This finding establishes a direct molecular link between nitrogen signaling and the regulation of cellular growth patterns [25].\u003c/p\u003e\u003cp\u003eTo address the research gap in translational initiation regulation within maize nitrogen response mechanisms, this study performed the first systematic analysis of the \u003cem\u003eeIF4E1\u003c/em\u003e gene family at the genome-wide level. Using bioinformatics approaches, six maize ZmeIF4E1 family members were identified and comprehensively characterized in terms of their phylogenetic relationships, gene structures, conserved motifs, chromosomal localizations, and promoter cis-acting elements. In addition, by integrating public transcriptomic data with qRT-PCR experiments, the spatiotemporal expression specificity of these family members across multiple tissues and different maize developmental stages was examined. To directly evaluate their involvement in the nitrogen response, the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene expression changes in the roots and leaves of seedlings were analyzed under low-nitrogen (0.2 mM KNO\u003csub\u003e3\u003c/sub\u003e) and high-nitrogen (10 mM KNO\u003csub\u003e3\u003c/sub\u003e) stress conditions. This study aimed to determine whether the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene could be considered a potential target for genetic improvement.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of eIF4E1 in the Maize Genome\u003c/h2\u003e\u003cp\u003eThe eIF4E1 sequence was retrieved from the maize genome (Zm-B73-REFERENCE-NAM-5.0.55) using the Basic Local Alignment Search Tool (BLASTP; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Maize gff3, protein, coding sequence, and genome files were downloaded from the Plant Genome Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The Hidden Markov Model (HMM) profile for the eIF4E1 protein domain, IF4E (Pfam: pfam01652), was obtained from the Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The Arabidopsis AT4g18040 protein sequence was downloaded from the Arabidopsis Information Resource (TAIR) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and used for BLASTP alignment with the maize protein database. After integrating the HMMER and BLASTP search results(HMMER search (Pfam: pfam01652) with E-value\u0026thinsp;\u0026le;\u0026thinsp;1e-5; BLASTP search against maize protein database with E-value\u0026thinsp;\u0026le;\u0026thinsp;1e-10 and identity\u0026thinsp;\u0026ge;\u0026thinsp;50%), non-redundant protein sequences were submitted to the NCBI CD-search (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SMART server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://smart.embl-heidelberg.de/\u003c/span\u003e\u003cspan address=\"https://smart.embl-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to confirm the presence of this conserved domain. Proteins containing the IF4E domain were classified as members of the \u003cem\u003eeIF4E1\u003c/em\u003e gene family in maize and designated based on their chromosomal locations.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChromosomal Localization of the\u003c/b\u003e \u003cb\u003eZmeIF4E1\u003c/b\u003e \u003cb\u003eGene\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe chromosomal location of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene was determined using the annotated maize genome. For synteny analysis, pairwise genome comparisons were performed using BLASTP searches (E-value\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e) with the NCBI BLAST suite to identify putative homologous gene pairs. Chromosomal mapping was visualized using TBtools (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePhylogenetic Analysis of eIF4E1 Protein\u003c/h3\u003e\n\u003cp\u003eUsing MEGA 11.0 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.megasoftware.net/\u003c/span\u003e\u003cspan address=\"https://www.megasoftware.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), eIF4E1 protein sequences from Arabidopsis, wheat, maize, rice, and sorghum were aligned and subjected to phylogenetic analysis. The aligned sequences were processed using the NJ method with the Poisson model and pairwise deletion, and a phylogenetic tree was constructed using 1000 bootstrap replicates.\u003c/p\u003e\n\u003ch3\u003eGene Structure and Conserved Motif Analysis of ZmeIF4E1\u003c/h3\u003e\n\u003cp\u003eThe nucleotide sequence structure of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene was analyzed using maize gff3 files. TBtools software was used to generate UTR-CDS distribution diagrams. The upstream 2.0 kb sequence of each \u003cem\u003eZmeIF4E1\u003c/em\u003e gene was selected as the promoter region and extracted from the maize genome. The conserved motifs of the \u003cem\u003eZmeIF4E1\u003c/em\u003e protein were identified using the Motif Elicitation (Multiple Expectation Maximization for Motif Elicitation, MEME) online platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/\u003c/span\u003e\u003cspan address=\"http://meme-suite.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Conserved motifs were visualized using TBtools software.\u003c/p\u003e\n\u003ch3\u003eMaize Seedling Growth and Nitrogen Treatment\u003c/h3\u003e\n\u003cp\u003eSeeds of maize variety B73 were first subjected to flotation in sterile water to remove non-viable or shriveled kernels. One subset of seeds was sown in experimental fields for stage-specific sampling, while another subset was surface sterilized by soaking in a 2.6% sodium hypochlorite solution for 30 min. After five rinses with sterile water, the sterilized seeds were germinated for 48 h in a constant-temperature incubator at 28\u0026deg;C. Germinated seeds were then transferred to PhytoTC seed germination bags containing half-strength MS medium and cultured for two weeks in a plant growth chamber,Plant growth chamber conditions: 16 h light (300 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)/8 h dark photoperiod, day/night temperature 27℃/22℃, relative humidity 60%. Following the removal of endosperm tissue, seedlings were transplanted into PhytoTC bags filled with 2.5 mM ammonium succinate (NH\u003csub\u003e4\u003c/sub\u003eSuc) medium and grown for an additional 2 d. Plants with stable growth were treated with 0.2 mM KCl, 0.2 mM KNO\u003csub\u003e3\u003c/sub\u003e, 10 mM KCl, or 10 mM KNO\u003csub\u003e3\u003c/sub\u003e, respectively, for 2 h.\u003c/p\u003e\n\u003ch3\u003eRNA Extraction, cDNA Synthesis, and qRT-PCR Analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from maize tissues at various growth stages, as well as from the leaves and roots of nitrogen-treated controls, using an RNA Plant Extraction Kit (CWBIO, Cat. CW0581S, China). cDNA was synthesized using the HiFiScript All-in-One RT Master Mix for qPCR Kit (CWBIO, Cat. CW3371). All samples were stored at \u0026minus;\u0026thinsp;20\u0026deg;C until further use. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the SuperStar Universal SYBR Master Mix Kit (CWBIO, Cat. CW3360). Relative gene expression was quantified using a LightCycler\u0026reg; 480 instrument (Roche) with a reference housekeeping gene as the internal control, following the 2\u003csup\u003e\u0026minus;∆Ct\u003c/sup\u003e method [26]. Data analysis and visualization were performed with GraphPad Prism 8.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.graphpad.com\u003c/span\u003e\u003cspan address=\"https://www.graphpad.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Data are presented as the mean of three technical replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). Gene-specific primers are listed in Supplementary Dataset 1. Statistical significance was assessed using independent-samples t-tests. Error bars represent SE, and p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*) or \u0026lt;\u0026thinsp;0.01 (**) were considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIdentification of the maize\u003c/b\u003e \u003cb\u003eeIF4E1\u003c/b\u003e \u003cb\u003egene and characterization of its protein physicochemical properties\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter integrating search results from HMMER and BLASTP, nine non-redundant proteins were identified and screened for conserved domains. Among these, six proteins contained the IF4E domain (Pfam: pfam01652) and were classified as members of the maize \u003cem\u003eeIF4E1\u003c/em\u003e gene family. Based on their chromosomal locations (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), the corresponding genes were designated \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e to \u003cem\u003eZmeIF4E1.5\u003c/em\u003e. The nucleotide sequences of these genes ranged from 827 bp (ZmeIF4E1.3.2) to 1243 bp (\u003cem\u003eZmeIF4E1.3.1\u003c/em\u003e). Further analysis revealed the physicochemical properties of the eIF4E1 proteins (Table\u0026nbsp;1). \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e and \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e represented the shortest and longest proteins, consisting of 179 and 229 amino acids, respectively. Their molecular weights ranged from 19,936.53 Da (\u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e) to 26,541.70 Da (\u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e), with theoretical isoelectric points (pI) ranging from 5.66 (\u003cem\u003eZmeIF4E1.5\u003c/em\u003e) to 6.32 (\u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e), indicating that all \u003cem\u003eZmeIF4E1\u003c/em\u003e proteins were weakly acidic. Based on the instability index, two proteins were predicted to be unstable (\u0026gt;\u0026thinsp;40), whereas the remaining four were classified as stable (ranging from 29.98 to 38.31). The aliphatic index values ranged from 61.11 (\u003cem\u003eZmeIF4E1.5\u003c/em\u003e) to 74.98 (\u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e), suggesting moderate thermal stability. The grand average of hydropathicity (GRAVY) values ranged from \u0026minus;\u0026thinsp;0.477 (\u003cem\u003eZmeIF4E1.3.2\u003c/em\u003e) to \u0026minus;\u0026thinsp;0.79 (\u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e), indicating that all ZmeIF4E1 proteins are hydrophilic (Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic Analysis of the\u003c/b\u003e \u003cb\u003eeIF4E1\u003c/b\u003e \u003cb\u003eGene\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the evolutionary relationships of the \u003cem\u003eeIF4E1\u003c/em\u003e gene, a phylogenetic tree was constructed using a total of 33 eIF4E1 protein sequences from five species, including 7 from Arabidopsis, 3 from sorghum, 3 from rice, 14 from wheat, and 6 from maize (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S2). Based on this analysis, the six ZmeIF4E1 proteins were grouped into three clusters: Clusters I\u0026ndash;III. Cluster II contained the largest number of members, with 14 protein sequences, including 2 ZmeIF4E1 proteins and 7 wheat protein sequences. Group III included 12 members, consisting of 3 ZmeIF4E1 proteins and 4 wheat sequences. Group I contained the fewest members, with only 7 protein sequences, including 1 ZmeIF4E1 sequence.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eStructure and Motif Composition of the\u003c/b\u003e \u003cb\u003eZmeIF4E1\u003c/b\u003e \u003cb\u003eGene\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo analyze the structural characteristics of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene family, the coding sequences (CDS) of each member were systematically compared with their genomic DNA sequences using bioinformatics methods, thereby precisely determining the arrangement patterns of coding and non-coding regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results indicated that most genes in this family exhibited structural conservation, containing 4 to 5 introns, and demonstrated subfamily specificity. Specifically, \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e, belonging to Group I, contained five introns, whereas other genes in Groups II and III each harbored four introns. Correspondingly, \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e possessed six exons, whereas the remaining genes each contained five exons.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurther structural comparisons revealed that closely related members, such as ZmeIF4E1.1.3 and ZmeIF4E1.1.2, not only shared identical numbers of coding elements but also exhibited highly similar distribution patterns of untranslated regions (UTRs, green boxes in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) and CDSs (yellow boxes). The CDS regions of these subtypes were more compactly arranged, suggesting strong functional constraints during evolution and reflecting branch specificity. This finding provides critical evidence for the classification and functional differentiation of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene family at the structural level, laying the groundwork for further studies on transcriptional regulation and alternative splicing mechanisms.\u003c/p\u003e\u003cp\u003eTo investigate the conserved sequence features of ZmeIF4E1 family proteins, the MEME online tool was used to identify conserved motifs, resulting in the identification of 10 motifs(MEME parameters: minimum motif width\u0026thinsp;=\u0026thinsp;6, maximum motif width\u0026thinsp;=\u0026thinsp;50, maximum number of motifs\u0026thinsp;=\u0026thinsp;10). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, all ZmeIF4E1 proteins contained Motifs 1, 2, and 3, indicating that these motifs represent the core functional elements of the family. The closely related members ZmeIF4E1.3.1 and ZmeIF4E1.3.2 exhibited highly similar motif compositions and arrangements, each containing Motifs 1, 2, 3, 4, 5, 6, 7, and 10. This sequence-level result supported the phylogenetic grouping. Notably, \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e displayed the simplest motif composition, containing only three core motifs and Motif 4, suggesting potential functional divergence from other family members.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDistribution of cis-acting elements in the\u003c/b\u003e \u003cb\u003eZmeIF4E1\u003c/b\u003e \u003cb\u003egene promoter\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo clarify the transcriptional regulatory pathways associated with the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene, cis-acting regulatory elements in the promoter regions were analyzed using PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) within the 2000-bp upstream sequence. In total, 71 cis-acting elements were identified in the promoters of the six \u003cem\u003eZmeIF4E1\u003c/em\u003e genes. Visualization of the 32 explicitly annotated cis-elements revealed five major categories (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), including 8 nitrogen-responsive elements, 13 light-responsive elements, 9 anaerobic-responsive elements, and two maize protein regulatory elements. In addition to these named cis-elements, the predictions indicated that the remaining 39 sequences contained 28 putative functional elements associated with light response, hormone response, and other regulatory functions (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Nitrogen- and anaerobic-response elements were identified in \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e, \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e, \u003cem\u003eZmeIF4E1.1.3\u003c/em\u003e, and \u003cem\u003eZmeIF4E1.3.2\u003c/em\u003e. Light-responsive elements included both G-box and I-box types. \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e contained both types, whereas \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e, \u003cem\u003eZmeIF4E1.1.3\u003c/em\u003e, and \u003cem\u003eZmeIF4E1.3.2\u003c/em\u003e harbored only the G-box type, suggesting the potential functional differentiation among these members within light regulatory networks. Notably, only six putative hormone-responsive elements were detected in the promoter region of \u003cem\u003eZmeIF4E1.5\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of the\u003c/b\u003e \u003cb\u003eZmeIF4E1\u003c/b\u003e \u003cb\u003eGene in Different Maize Tissues\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further elucidate the role of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene in maize development, its expression profiles across six tissues/organs were analyzed using published transcriptome data obtained from the Phytozome database. Gene expression levels were quantified in FPKM. Genes with FPKM values below 1.00 were classified as non-expressed, while those above this threshold were categorized as follows: low expression (1.00\u0026thinsp;\u0026le;\u0026thinsp;FPKM\u0026thinsp;\u0026lt;\u0026thinsp;5.00), medium expression (5.00\u0026thinsp;\u0026le;\u0026thinsp;FPKM\u0026thinsp;\u0026lt;\u0026thinsp;15.00), and high expression (FPKM\u0026thinsp;\u0026ge;\u0026thinsp;15.00) [27].\u003c/p\u003e\u003cp\u003eqRT-PCR analysis was conducted on the roots and leaves of seedlings at the three-leaf stage. The results (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and S3a) demonstrated significant differences in the expression of \u003cem\u003eZmeIF4E1\u003c/em\u003e family members between roots and leaves. Among them, \u003cem\u003eZmeIF4E1.5\u003c/em\u003e exhibited the highest expression, markedly exceeding that of the other homologs in both tissues. \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e ranked the second, whereas \u003cem\u003eZmeIF4E1.1.3\u003c/em\u003e displayed the lowest expression level.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and S3b, qRT-PCR profiling further revealed distinct tissue-specific expression patterns across four tissues (leaves, roots, leaf sheaths, and internodes) at the jointing stage. \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e displayed exceptionally high expression in leaves, significantly surpassing other homologs, while \u003cem\u003eZmeIF4E1.1.3\u003c/em\u003e also presented elevated expression in leaves, second only to \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e. In contrast, \u003cem\u003eZmeIF4E1.3.2\u003c/em\u003e exhibited relatively low expression across all internode-stage tissues examined.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eqRT-PCR expression analysis was performed on four tissues (leaves, roots, leaf sheaths, and internodes) of maize during the big-flare period. The results (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and S3c) indicated that members of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene family displayed distinct tissue-specific expression patterns. In leaves, \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e and \u003cem\u003eZmeIF4E1.5\u003c/em\u003e exhibited the highest expression levels, significantly exceeding those of the other homologs. \u003cem\u003eZmeIF4E1.1.3\u003c/em\u003e and \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e demonstrated the next highest levels. In roots, \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e had the highest expression, followed by \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e and \u003cem\u003eZmeIF4E1.1.3\u003c/em\u003e. In leaf sheaths, \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e exhibited the most pronounced expression, followed by \u003cem\u003eZmeIF4E1.1.3\u003c/em\u003e. \u003cem\u003eZmeIF4E1.3.2\u003c/em\u003e consistently exhibited the lowest expression in all four tissues.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt the tasseling stage, qRT-PCR analysis of six tissues (leaves, roots, leaf sheaths, internodes, male flowers, and female flowers) revealed further spatial variation (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and S3d). \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e exhibited the highest expression in internodes, followed by \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e. In leaves and leaf sheaths, \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e exhibited relatively higher expression than other homologs. \u003cem\u003eZmeIF4E1.5\u003c/em\u003e displayed the highest expression in pistils, whereas \u003cem\u003eZmeIF4E1.3.2\u003c/em\u003e exhibited the lowest expression levels across all six tissues.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDuring the grain-filling stage, qRT-PCR analysis of four tissues (leaves, roots, leaf sheaths, and internodes) confirmed distinct expression profiles (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and S3e). In leaf sheaths, \u003cem\u003eZmeIF4E1.1.3\u003c/em\u003e exhibited the highest expression, followed by \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e. In roots, \u003cem\u003eZmeIF4E1.5\u003c/em\u003e and \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e showed elevated expression, whereas \u003cem\u003eZmeIF4E1.3.2\u003c/em\u003e had the lowest expression across all four tissues.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of\u003c/b\u003e \u003cb\u003eZmeIF4E1\u003c/b\u003e \u003cb\u003ein Response to Nitrogen\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further explore the potential role of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene in nitrogen response, we analyzed the expression patterns of the six \u003cem\u003eZmeIF4E1\u003c/em\u003e genes in maize leaves and roots subjected to low-nitrogen (0.2 mM KNO\u003csub\u003e3\u003c/sub\u003e) and high-nitrogen (10 mM KNO\u003csub\u003e3\u003c/sub\u003e) treatments using qRT-PCR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the expression level of \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e in leaves under low-nitrogen treatment (0.2 mM KNO\u003csub\u003e3\u003c/sub\u003e) was significantly higher than that in the corresponding KCl control (0.2 mM). Similarly, \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e expression in roots under low-nitrogen treatment was markedly elevated compared with the KCl control. In contrast, the expression of all six \u003cem\u003eZmeIF4E1\u003c/em\u003e genes in leaves was significantly repressed under high-nitrogen treatment (10 mM KNO\u003csub\u003e3\u003c/sub\u003e) relative to the 10 mM KCl control, indicating that high nitrogen availability negatively affected leaf development. Notably, in roots treated with 10 mM KNO\u003csub\u003e3\u003c/sub\u003e, the expression levels of \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e, \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e, \u003cem\u003eZmeIF4E1.1.3\u003c/em\u003e, and \u003cem\u003eZmeIF4E1.3.2\u003c/em\u003e were significantly higher than the 0.2 mM KCl control.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEfficient nitrogen utilization is a central goal of the genetic improvement of maize. Previous studies have primarily focused on transcriptional regulators, such as NLP and BZIP transcription factors, and nitrogen transporters, including NRT and AMT families [28, 29]. However, the role of translational regulation, which is a crucial step in gene expression in plant nitrogen response, has only recently begun to be elucidated in Arabidopsis [22, 23] and remains largely unexplored in maize. This study presents the first systematic identification of the \u003cem\u003eeIF4E1\u003c/em\u003e gene family in the maize genome and a comprehensive analysis of the expression patterns of its members under nitrogen stress, thereby offering important insights into the translational regulatory mechanisms that may affect NUE in maize.\u003c/p\u003e\u003cp\u003eThis study successfully identified six members of the maize \u003cem\u003eeIF4E1\u003c/em\u003e gene family, each containing the characteristic IF4E (Pfam: pfam01652) cap-binding domain, thereby confirming the evolutionary conservation of their molecular function. Phylogenetic analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S2) demonstrated that maize eIF4E1 proteins formed a well-supported clade with homologs from Arabidopsis and rice, reflecting the high degree of conservation of eukaryotic translation initiation mechanisms [30, 31]. Notably, substantial variation was observed among eIF4E1 family members in physicochemical properties, such as isoelectric point and instability index, which suggests potential functional divergence within the family that may enable adaptation to diverse cellular environments in maize (Table\u0026nbsp;1). Most \u003cem\u003eZmeIF4E1\u003c/em\u003e genes lacked introns within their coding regions, which served as structural features shared with the Arabidopsis \u003cem\u003eAt4g18040\u003c/em\u003e (\u003cem\u003eeIF4E1\u003c/em\u003e) homolog. This intronless architecture may facilitate rapid and constitutive expression, supporting the sustained demand for core translation initiation factors under basal conditions and enabling prompt responses to environmental changes.\u003c/p\u003e\u003cp\u003eTo thoroughly analyze the transcriptional regulatory mechanisms by which the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene family responds to external environmental signals, this study adopted a systematic analysis of cis-acting elements in the promoter regions of the six family members (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The diverse composition of these elements suggests that the ZmeIF4E1 family may integrate multiple environmental and endogenous signals through complex transcriptional regulatory networks, thereby playing a crucial role in maize growth, development, and stress responses. Notably, both nitrogen response elements and anaerobic response elements co-occurred in the promoter regions of ZmeIF4E1.1.1, ZmeIF4E1.1.2, ZmeIF4E1.1.3, and ZmeIF4E1.3.2. This co-occurrence suggests the potential collaborative participation of these genes in nitrogen signal perception and hypoxic environment responses, reflecting the cross-talk between signaling pathways. This finding is consistent with mechanisms previously reported in Arabidopsis [10]. Previous research has indicated that At4g18040 directly participates in nitrogen response regulation via the nitrate signaling pathway, with its functional knockout mutant exhibiting significant nitrate utilization defects [22]. These findings suggest that maize may achieve the functional specificity of eIF4E1 in nitrogen signaling pathways through conserved transcriptional regulatory mechanisms, offering new insights into nitrogen signal transduction in monocotyledons.\u003c/p\u003e\u003cp\u003eAmong the light-responsive elements, two distinct types were identified: G-box and I-box. Notably, significant differences were identified among family members. ZmeIF4E1.1.1 contained both light-responsive elements, whereas ZmeIF4E1.1.2, ZmeIF4E1.1.3, and ZmeIF4E1.3.2 contained only the G-box element. This differential distribution suggests potential functional specialization among members of the photoperiod regulation network. ZmeIF4E1.1.1 may integrate more complex light signals through multiple light-responsive elements, whereas other members primarily rely on G-box-mediated photoregulatory pathways [23]. This finding was consistent with studies in higher plants, where light signals frequently regulated gene expression through G-box elements, providing clues for investigating the coordinated regulation between maize photosynthetic products and nitrogen utilization.\u003c/p\u003e\u003cp\u003eThe promoter region of ZmeIF4E1.5 contained only six putative hormone-responsive elements, exhibiting a regulatory pattern markedly different from that of other family members. We hypothesized that this gene could not directly respond to nitrogen, light, or hypoxia signals, but could achieve transcriptional regulation through rare cis-acting modules or hormone-related pathways. Furthermore, its expression may be strongly influenced by distant enhancers and three-dimensional chromatin architecture. This unique property suggests that ZmeIF4E1.5 may perform specialized functions within the family, warranting further experimental investigation, such as promoter deletion analysis, EMSA, or ChIP-seq, to elucidate its precise regulatory mechanisms. In summary, this study revealed significant structural differences in the promoters of \u003cem\u003eZmeIF4E1\u003c/em\u003e gene family members at the cis-acting element level, providing theoretical support for their functional differentiation in response to nitrogen, light, and hypoxic stress. These findings not only offer new evidence for refining the molecular regulatory network of nitrogen efficiency in maize but also identify potential cis-regulatory targets for improving crop nitrogen use efficiency through molecular design breeding.\u003c/p\u003e\u003cp\u003eThis study revealed that the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene was constitutively expressed across multiple maize tissues, including roots, stems, leaves, leaf sheaths, internodes, stamens, and pistils, although its expression levels varied considerably, demonstrating clear spatiotemporal specificity (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and S3). These findings indicate that members of this gene family could play extensive and finely tuned roles in the post-transcriptional regulation of diverse growth and developmental processes in maize. By integrating public transcriptomic data from the Phytozome database with qRT-PCR validation results, we further elucidated the dynamic expression patterns of this gene family across different developmental stages. At the three-leaf stage (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and S3a), ZmeIF4E1.5 emerged as the predominant subtype expressed in both roots and leaves, followed by ZmeIF4E1.1.1, whereas ZmeIF4E1.1.3 exhibited the lowest expression levels. This suggests that ZmeIF4E1.5 may play a dominant role in establishing foundational translational mechanisms during early seedling development.\u003c/p\u003e\u003cp\u003eBy the jointing stage (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and S3b), the expression profiles shifted markedly, where ZmeIF4E1.1.2 exhibited strong leaf-specific accumulation, ZmeIF4E1.1.3 was also elevated in leaves, and ZmeIF4E1.3.2 remained low across all tissues. This suggests a strong reliance on specific eIF4E1 subtypes to support leaf development during this phase.\u003c/p\u003e\u003cp\u003eAt the big-flare stage (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and S3c), the expression profiles displayed a pronounced tissue-specific divergence. ZmeIF4E1.1.1 and ZmeIF4E1.5 were strongly expressed in leaves, with ZmeIF4E1.1.1 also demonstrating preferential accumulation in roots. In contrast, ZmeIF4E1.1.2 was highly abundant in leaf sheaths, indicating that different family members achieve complementary functions in processes such as photosynthesis, nutrient uptake, and structural maintenance.\u003c/p\u003e\u003cp\u003eDuring reproductive growth, ZmeIF4E1.1.1 reached peak expression in internodes at the tasseling stage (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and S3d), whereas ZmeIF4E1.5 was specifically enriched in pistils, suggesting critical roles in floral organ development and post-pollination translational control.\u003c/p\u003e\u003cp\u003eAt the grain-filling stage (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and S3e), ZmeIF4E1.1.3 and ZmeIF4E1.1.2 presented the elevated transcript levels in leaf sheaths, while both ZmeIF4E1.5 and ZmeIF4E1.1.1 were upregulated in roots. These expression patterns imply enhanced capacities for resource transport and nitrogen assimilation to meet the high metabolic demands of grain filling.\u003c/p\u003e\u003cp\u003eOverall, the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene family was constitutively expressed across a broad range of maize tissues, including roots, stems, leaves, and grains, although the expression intensity varied among members, reflecting their diverse contributions to growth and development. Notably, \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e and \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e exhibited tissue-specific responses to nitrogen stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Under low-nitrogen conditions (0.2 mM KNO\u003csub\u003e3\u003c/sub\u003e), \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e was significantly induced in roots, whereas \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e was strongly upregulated in leaves. These patterns were consistent with previous findings in Arabidopsis. For instance, research from Yong Wang\u0026rsquo;s group at Shandong Agricultural University demonstrated that a loss-of-function mutant of Arabidopsis \u003cem\u003eeIF4E1\u003c/em\u003e (\u003cem\u003eAt4g18040\u003c/em\u003e; \u003cem\u003eMut36\u003c/em\u003e) caused severe nitrate uptake defects [22]. It is plausible that \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e in maize functions in sensing low-nitrogen signals in roots through a conserved pathway, thereby enhancing the translational efficiency of specific mRNAs encoding nitrate transporters (NRTs) or key enzymes in nitrogen assimilation. This mechanism could contribute to improved nitrogen uptake and NUE in maize.\u003c/p\u003e\u003cp\u003eWe observed a pronounced tissue-specific response to high-nitrogen treatment (10 mM KNO\u003csub\u003e3\u003c/sub\u003e). The expression of nearly all \u003cem\u003eZmeIF4E1\u003c/em\u003e genes was strongly repressed in leaves, implying the existence of a feedback mechanism to prevent nitrogen overload. In contrast, the expressions of \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e, \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e, \u003cem\u003eZmeIF4E1.1.3\u003c/em\u003e, and \u003cem\u003eZmeIF4E1.3.2\u003c/em\u003e were markedly induced in roots. This sharp divergence between roots and leaves suggests a spatial functional division of labor among eIF4E1 family members in regulating nitrogen responses. As the primary site for nitrogen uptake and initial assimilation, roots may require translational reprogramming under high-nitrogen conditions to efficiently process incoming nitrogen sources. Conversely, leaves, as the central organs for assimilation, may downregulate global translational activity to avoid excessive carbon skeleton consumption, thereby maintaining carbon-nitrogen balance [32\u0026ndash;34]. Such organ-specific regulation can represent an adaptive strategy that enables maize to sustain high-yield traits by optimizing NUE while preserving overall metabolic homeostasis.\u003c/p\u003e\u003cp\u003eAlthough this study revealed the potential roles of the \u003cem\u003eZmeIF4E1\u003c/em\u003e gene family in nitrogen signaling and highlighted its spatiotemporal expression patterns through bioinformatics and expression profiling, certain limitations remain. These findings require direct functional validation through genetic experiments. Generating targeted knockout mutants of \u003cem\u003eZmeIF4E1\u003c/em\u003e genes using CRISPR/Cas9 or creating overexpression lines via transgenic approaches would provide more definitive evidence of their roles in nitrogen uptake, assimilation, and plant growth. Such genetic evidence is critical for establishing precise gene functions. Similar to how the function of eIF4E1 in Arabidopsis was conclusively demonstrated through the T-DNA insertion mutant mut36 [22], functional analyses of homologous genes in maize should require analogous loss-of-function and gain-of-function studies to validate their contributions to nitrogen response and utilization.\u003c/p\u003e\u003cp\u003eMore critically, as a canonical cap-binding protein, the essential function of eIF4E1 lies in regulating translation initiation. Hence, it is crucial to employ ribosome profiling (Ribo-seq) to comprehensively identify the mRNA populations preferentially bound and translationally regulated by each ZmeIF4E1 protein, thereby delineating their downstream targets with precision. For instance, studies in Arabidopsis have indicated that eIF4E1 not only governs global translational activity but also selectively enhances the translation of key genes, such as nitrate transporters (e.g., NRT1.1) and auxin response factors (e.g., ARFs) [22, 24]. Whether maize homologs regulate specific targets, including auxin-related transcription factors (e.g., ZmARF34) or nitrogen metabolic enzymes, through analogous mechanisms remains to be determined [24]. Such validation will require integrated multi-omics strategies that combine Ribo-seq with RNA-seq, RNA immunoprecipitation (RIP), and related molecular techniques[35].\u003c/p\u003e\u003cp\u003eOf particular importance is the translational regulation of Arabidopsis eIF4E1 by phosphorylation. Determining whether maize homologs undergo comparable post-translational modifications is a promising direction for future research. The focused development of specialized genetic resources and in-depth mechanistic analyses of these molecular interactions are essential. Such efforts will advance a more comprehensive understanding of ZmeIF4E1-mediated regulation at the translational level during nitrogen signaling. This knowledge will not only identify potential molecular targets for enhancing NUE in crops via molecular breeding but also broaden comparative insights into the evolutionary conservation and divergence of eIF4E1 function across species.\u003c/p\u003e\u003cp\u003eFurthermore, nitrate plays a pivotal role not only as a nutrient but also as an environmental signal that extends beyond plant physiology to influence broader ecosystems. Recent studies on sludge composting have suggested that nitrate availability can reshape microbial communities, enriching aromatic-ring-degrading microbes that drive complex organic matter breakdown [34, 36]. This parallel highlights the fundamental importance of nitrate as both a metabolic substrate and a signaling molecule, orchestrating biological processes from microbial decomposition in the environment to translational control within plants. The discovery that ZmeIF4E1 responds to nitrate stress underscores this conserved principle across kingdoms, where nitrogen serves not only as a nutrient but also as a signaling hub governing metabolic and developmental outcomes.\u003c/p\u003e\u003cp\u003eIn conclusion, this study systematically identified and characterized the maize \u003cem\u003eeIF4E1\u003c/em\u003e gene family, revealing its distinct expression patterns and member-specific responses to nitrogen signaling. These findings extend the current knowledge of NUE beyond transcriptional and transporter-level regulation into the domain of translational control. Our study establishes a new framework for understanding the molecular mechanisms underlying nitrogen efficiency in maize and provides promising genetic targets, such as \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e and \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e, for crop improvement. Overall, this study offers valuable genetic resources and strategic insights for breeding nitrogen-efficient maize varieties using molecular design approaches.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Horizontal Research Project \u0026ldquo;Key Engineering Technologies for Smart Agriculture\u0026rdquo; (K23LD90), \u0026ldquo;Technical Services for the Development of Modern Agricultural Industries\u0026rdquo; (K24LD179) and the Open Project Program of State Key Laboratory of Crop Biology (2021KF04).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQ. W. and L. Z. designed the project. Q. W. conducted the experiments. The data were analyzed by L. Z., Q. W., and H. Z. The manuscript was written by Q. W., H. Z., L. Z., and P. C. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary information can be found in the online version of this article. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHorizontal Research Project \u0026ldquo;Key Engineering Technologies for Smart Agriculture\u0026rdquo; (K23LD90), \u0026ldquo;Technical Services for the Development of Modern Agricultural Industries\u0026rdquo; (K24LD179) and the Open Project Program of State Key Laboratory of Crop Biology (2021KF04).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQi Wang, [email protected], Liaocheng University CN and Hang Zhou, [email protected], Shandong Shennong Zhiyi Intelligent Technology Co., Ltd CN.\u003c/p\u003e\n\u003cp\u003eCorresponding author: Pengfei Chu, [email protected], Liaocheng University CN, and Lufei Zhao, [email protected], Liaocheng University CN.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaize (\u003cem\u003eZea mays L.\u003c/em\u003e) inbred line B73 is a widely used public genetic resource with no endangered or protected status. No specific ethical approval or permits were required for the collection, cultivation, and experimental use of this material in this study, which complies with the national agricultural research policies of the People\u0026rsquo;s Republic of China.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll raw gene sequences of \u003cem\u003eZmeIF4E1\u003c/em\u003e family members, and promoter cis-acting element analysis results are included in the Supplementary Information (Datasets S1-S4, Figures S1-S3).\u003c/p\u003e\n\u003cp\u003ePublic transcriptome data used in this study were retrieved from Phytozome (https://phytozome-next.jgi.doe.gov/) under accession number: PRJNA10769 (Maize), PRJNA13876 (Sorghum), and EnsemblPlants (https://plants.ensembl.org/) under accession number: GCA_900519105 (Wheat), GCA_001433935 (Rice), and the data of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e is from TAIR (https://www.arabidopsis.org/) .\u003c/p\u003e\n\u003cp\u003eAll other data generated or analyzed during this study are available from the corresponding author (Pengfei Chu, [email protected] and Lufei Zhao, [email protected]) upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Li J, Cao H, Li S, Dong X, Zhao Z, Jia Z, et al. 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Bioresour Technol. 2021;342:125907. https://doi.org/10.1016/j.biortech.2021.125907.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Maize, eIF4E1, Nitrogen use efficiency, Gene expression, Translational regulation","lastPublishedDoi":"10.21203/rs.3.rs-7863718/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7863718/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNitrogen is an essential nutrient for maize growth, and nitrogen use efficiency (NUE) remains below 40%, causing considerable resource waste and environmental pollution. Although previous studies have primarily focused on transcriptional regulation and nitrogen transporters, the contribution of translational control mechanisms to nitrogen responses remains unknown. In this study, the \u003cem\u003eeIF4E1\u003c/em\u003e gene family was systematically identified in maize, and its expression patterns under nitrogen stress were investigated. Bioinformatic analysis revealed six \u003cem\u003eZmeIF4E1\u003c/em\u003e genes, and qRT-PCR assays demonstrated their constitutive expression across diverse tissues, together with member-specific responses to both low (0.2 mM KNO\u003csub\u003e3\u003c/sub\u003e) and high (10 mM KNO\u003csub\u003e3\u003c/sub\u003e) nitrogen treatments. \u003cem\u003eZmeIF4E1.1.1\u003c/em\u003e was specifically induced in roots under low nitrogen, whereas \u003cem\u003eZmeIF4E1.1.2\u003c/em\u003e exhibited strong upregulation in leaves. These findings highlight the potential involvement of eIF4E1-mediated translational regulation in maize nitrogen adaptation and identify promising candidate targets for improving NUE through molecular breeding.\u003c/p\u003e","manuscriptTitle":"Identification and Expression Analysis of the eIF4E1 Gene Family in Maize","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-20 06:58:04","doi":"10.21203/rs.3.rs-7863718/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c4ecf477-2dfe-47ea-ba7e-948ebbeb990f","owner":[],"postedDate":"November 20th, 2025","published":true,"recentEditorialEvents":[{"type":"decision","content":"Withdrawn","date":"2026-05-06T12:00:02+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T12:12:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-20 06:58:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7863718","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7863718","identity":"rs-7863718","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}