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Article

Identification and Functional Analysis of the EPF/EPFL Gene Family in Maize (Zea mays L.): Implications for Drought Stress Response

by
Hanchao Xia
1,†,
Qi Wang
2,†,
Ziqi Chen
2,
Xiaopeng Sun
3,
Fangfang Zhao
4,
Di Zhang
1,
Jianbo Fei
2,5,
Rengui Zhao
1,* and
Yuejia Yin
2,*
1
College of Agronomy, Jilin Agricultural University, Changchun 130118, China
2
Institute of Agricultural Biotechnology/Jilin Provincial Key Laboratory of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences (Northeast Innovation Center of Agricultural Science and Technology in China), Changchun 130033, China
3
Hubei Academy of Agricultural Sciences, Wuhan 430064, China
4
Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang Province, College of Life Science and Technology, Harbin Normal University, Harbin 150025, China
5
Agricultural College, Jilin Agricultural Science and Technology College, Jilin 132101, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(8), 1734; https://doi.org/10.3390/agronomy14081734 (registering DOI)
Submission received: 19 June 2024 / Revised: 20 July 2024 / Accepted: 30 July 2024 / Published: 7 August 2024
(This article belongs to the Section Crop Breeding and Genetics)
Browse Figures
Versions Notes

Abstract

:
Maize, a vital cereal in global agriculture, faces significant yield challenges due to drought exacerbated by climate change. This study explores the genetic and molecular bases of drought resilience in maize, focusing on the EPF/EPFL gene family known for its role in stomatal regulation. Through a genome-wide analysis across seven grass species, we identified and characterized 16 ZmEPF/EPFL genes in maize. Focusing on their gene structure, expression patterns, and evolutionary relationships. The study integrated genome-wide searches, phylogenetic analysis, gene expression profiling under drought and other abiotic stresses, and qRT-PCR validation to elucidate the functional roles of these genes in drought response. Our results demonstrate that specific ZmEPF/EPFL genes are differentially expressed under varying drought conditions, suggesting their involvement in the plant’s adaptive response to water scarcity. Furthermore, interaction analyses reveal that these genes are linked to key processes such as stomatal development and oxidative stress management. This study provides a comprehensive overview of the ZmEPF/EPFL gene family’s contribution to stomatal development and drought tolerance, offering insights that could guide future breeding strategies for drought-resistant maize varieties.

1. Introduction

Maize, a pivotal cereal of the family Gramineae, originates from Central America and stands as one of the world’s most crucial food crops. Beyond its agricultural significance, maize is indispensable in various sectors including the food industry, feed production, and biofuel manufacturing, thereby playing a vital role in sustaining human health and economic well-being. In recent years, exacerbated by global warming and a burgeoning global population, drought has emerged as a formidable challenge, significantly curtailing maize yield and inflicting substantial economic losses annually. Consequently, elucidating the genetic and molecular underpinnings of drought tolerance in maize, identifying novel drought-resilient genes, and breeding drought-resistant varieties have become imperative to ensure the sustainability of the agricultural economy and food security.
Drought tolerance in maize is a multifaceted quantitative trait encompassing a myriad of dynamic intrinsic and extrinsic responses. Among these, plant hormones such as abscisic acid (ABA), indole-3-acetic acid (IAA), brassinolactone (BR), and cytokinins (CK) play pivotal roles in mediating plant responses to abiotic stresses [1]. These hormones facilitate crucial adaptations such as maintaining turgor through osmoregulation and enhancing cellular water retention, thereby sustaining photosynthesis, regulating stomatal behavior, and mitigating premature leaf senescence and mortality [2]. The antioxidant system is another critical component linked to drought resilience, where modulation of genes associated with this system can significantly bolster plant endurance under water-deficient conditions [3]. Additionally, reactive oxygen species (ROS) interact with both Ca2+ and ABA to synergistically control stomatal dynamics, playing a key role in the plant’s stress response mechanism [4]. It has been suggested that DPY1 can be phosphorylated and activated in response to osmotic stress and acts upstream of STRESS-ACTIVATED PROTEIN KINASE 6 (SAPK6, class I SnRK2) when grain receives drought stress, thereby permitting the regulation of downstream genes in response to drought stress [5]. Notably, Gao et al. identified a drought-resistance gene, ZmSRO1d, in maize, which regulates ROS levels in stomata by modulating ZmRBOHC activity, thereby facilitating stomatal closure and enhancing drought tolerance [6]. These discoveries underscore the complex interplay of genetic factors that contribute to the adaptive strategies of plants under drought stress.
In the realm of plant biology, a deepening understanding of the molecular mechanisms governing plant development and adaptive responses is emerging. Multicellular plants orchestrate intracellular and intercellular signaling through a variety of cell–cell communication pathways. Among these, small secreted peptides (SSPs) serve as critical messengers. The EPF/EPFL (epidermal patterning factor/EPF-like) gene family, which encodes plant-specific secretory peptides, is instrumental in modulating stomatal density and patterning on the plant epidermis [7,8]. These peptides are pivotal not only in plant growth and development but also in responding to environmental cues through their signaling peptide, maturation peptide, and cysteine sites [9,10], influencing plant morphology and adaptation primarily by regulating stomatal development, cell differentiation, and pattern formation.
Recent advancements have significantly enriched our understanding of the EPF/EPFL gene family. Jangra’s research identified homologs in monocotyledonous plants, broadening our perspective on their roles across grass species [11]. Jiao’s genome-wide study in sorghum underscored the potential of EPF genes in drought response [12], while Lu’s work in rice revealed their critical functions in stomatal regulation, highlighting distinctions between monocots and dicots in these processes [13]. The regulation of stomatal development by the EPF/EPFL gene family is crucial for enhancing plant resilience to drought, heat, and other abiotic stresses by potentially improving photosynthesis and water use efficiency [14,15].
Systematic identification of this gene family in model organisms like Arabidopsis thaliana and Physcomitrium patens, and in cereals such as maize, underscores their evolutionary significance and functional roles. For instance, the maize ZmSTOMAGEN (EPFL9) gene, which positively regulates stomatal development, shows increased expression under elevated light and drought conditions [16,17]. Conversely, heterologous overexpression of genes such as HvEPF1 from barley, and OsEPF1 and OsEPF2 from rice, in Arabidopsis led to reduced stomatal density [13]. Similar regulatory effects have been observed with the rice OsEPFL9 and wheat TaEPF1B genes, where manipulation of expression levels resulted in significant variations in stomatal density and water use efficiency, thereby enhancing drought tolerance [18,19,20]. Similar results have been observed in potato [21] and poplar [22], indicating that overexpression of EPF/EPFL genes can appropriately reduce stomatal density during early plant development, thus reducing transpiration and water loss while maintaining photosynthesis, thereby enhancing plant drought tolerance and water use efficiency.
Despite these discoveries, a comprehensive genomic assessment of EPF/EPFL genes in cereal crops has not been reported, and most of the members in maize still have unknown functions. This study began with a genome-wide characterization of the EPF/EPFL gene family in maize, followed by an examination of the expression patterns of EPF/EPFL family members during maize development and under abiotic stresses. Through this investigation, the researchers delved into evolutionary relationships and gene structure along with comparative genomic analyses [23,24,25,26,27]. This work is intended to provide a bioinformatics basis for subsequent functional analyses and applications of the EPF/EPFL gene family in maize, thereby contributing valuable insights to the fields of plant genetics and molecular biology. This endeavor not only enriches our understanding of plant adaptive mechanisms but also paves the way for innovative strategies to enhance crop resilience in an era of climatic uncertainties.

2. Materials and Methods

2.1. Identification and Evolutionary Analysis of EPF/EPFL Genes

To identify EPF/EPFL proteins with EPF (PF17181) and Stomagen (PF16851) structural domains in Arabidopsis and various grass species, we conducted a systematic search in the Arabidopsis database (TAIR10, https://www.arabidopsis.org/, (accessed on 29 July 2024)). Initially, 11 genes in Arabidopsis were identified based on these structural domains. Subsequently, BlastP and hmmsearch [28] methods were utilized, with an E-value threshold of 1 × 10−10, to search for homologous genes in members of the grass family, including foxtail millet, maize, rice, sorghum, barley, taro, and wheat. The intersection of the results from these two methods was considered for further analysis. The identified genes were then screened using NCBI’s Conserved Domain search tool (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, (accessed on 29 July 2024)) to confirm the presence of the EPF and Stomagen structural domains. Each gene was systematically named based on its chromosomal location within its respective species. Finally, the protein sequences were aligned using MAFFT 7.520 [29], and a phylogenetic tree was constructed using IQ-TREE [30] with 2000 bootstrap replicates [31].

2.2. Analysis of ZmEPF/EPFL Gene Structures and Their Encoded Proteins

The software MEME Suite V1.1 (http://meme-suite.org/tools/meme, (accessed on 29 July 2024)) was utilized to perform motif analysis of ZmEPF/EPFL family members with a motif count set to 10. The results were subsequently visualized using TBtools 2.012 [32].

2.3. Chromosomal Localization and Basic Information of ZmEPF/EPFL

The gene densities on individual maize chromosomes were calculated from the maize gene annotation file at 350 Kb, converted into a heat map, and combined with the results of chromosomal localization of the maize ZmEPF/EPFL genes using TBtools. Finally, the results were visualized and refined using Adobe Illustrator CC 2022. Additionally, the physicochemical properties and subcellular localization of maize ZmEPF/EPFL family proteins were analyzed using the Expasy tool (https://web.expasy.org/protparam/, (accessed on 29 July 2024)) and the subcellular localization prediction tool (https://wolfpsort.hgc.jp/, (accessed on 29 July 2024)).

2.4. Collinearity Analysis of EPF/EPFL Genes across Genomes

Collinearity across the genome and between genomes was assessed using the McscanX [33] module within TBtools, complemented by CGSuite for comprehensive analysis. Results were visually represented, with collinear gene pairs manually annotated for clarity.

2.5. ZmEPF/EPFL Protein-Protein Interaction

To elucidate the functional protein interaction network, we utilized the online tool STRING [34] (https://string-db.org/, (accessed on 29 July 2024)) to construct the protein interaction network for maize ZmEPF/EPFL genes. The analysis was performed with parameters set to a required score of 0.4 and an FDR stringency of 5%.

2.6. Expression Profiling of the ZmEPF/EPFL Gene Family

RNA-seq datasets for the maize inbred line B73 across various developmental stages and tissue types [35], as well as under heat and salt stress conditions [36], were obtained from MaizeGDB. Following Z-Score normalization of expression data, the expression patterns of ZmEPF/EPFL gene family members were visualized as heatmaps using the TBtools Heatmap functionality.

2.7. Drought-Induced Expression Patterns of ZmEPF/EPFL Genes

These data were obtained from the RNA-seq data of B73 in response to drought published by Prof. Mingqiu Dai from Huazhong Agricultural University. Meanwhile, we compared different RNA-seq data with the same differential expression trend. Heatmaps were drawn using the Heatmap tool in TBtools to analyze the expression patterns of the ZmEPF gene family under different drought conditions.

2.8. Drought Stress in Maize and qRT-PCR Analysis

Seeds of the maize inbred line B73 were surface sterilized and subsequently planted in pots containing 200 g of vermiculite. Following germination, the seedlings were cultivated under controlled environmental conditions: 14 h of light and 10 h of darkness, with a light intensity of 70 µmol/m2/s, a daytime temperature of 28 °C, a nighttime temperature of 22 °C, and a relative humidity of 60 ± 5%. Upon reaching the five-leaf stage, the seedlings were subjected to drought stress simulated by applying 20% PEG6000. Leaves (specifically the third leaf) were harvested at 12, 24, and 36 h post-treatment and immediately frozen at −80 °C for subsequent analysis.
Total RNA was extracted from the plant tissue using the Ultrapure RNA Kit (CW Bio, Taizhou, China). The integrity of the RNA was assessed via electrophoresis, and its purity and concentration were quantified using a NanoDrop ND-2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The RNA was then reverse transcribed into cDNA using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China).
To examine the expression patterns of maize ZmEPF/EPFL genes under drought stress, 12 representative genes were selected for quantitative real-time PCR (qRT-PCR) analysis. Specific primers for these genes were designed using the Primer-BLAST tool from NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi, (accessed on 29 July 2024)). The qRT-PCR was performed using SYBR™ Green PCR Universal Master Mix on a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Carlsbad, CA, USA). Each cDNA sample was analyzed in triplicate to ensure reproducibility. The expression data were processed using the Relative Quantification (RQ) method to assess relative changes in gene expression.

3. Results

3.1. Comprehensive Identification and Phylogenetic Delineation of EPF/EPFL Gene Family in Seven Poaceae Species

The EPF/EPFL gene family has been widely studied in Poaceae such as rice and wheat. In order to gain a clearer understanding of its evolutionary relationship in different Poaceae and explore the potential function of ZmEPF/EPFL in maize. We have studied the genomes of seven Poaceae species—Setaria italica, Hordeum vulgare, Oryza sativa, Sorghum bicolor, Panicum miliaceum, Triticum aestivum, 14, 12, 12, 12, 14, 35, and 16 EPF/EPFL family genes were identified. According to the chromosomal localization and established naming conventions in Arabidopsis, these genes have been systematically named. To elucidate the evolutionary trajectories of these gene families, we constructed a phylogenetic tree containing newly identified genes and Arabidopsis reference genes, with a total of 126 encoded proteins (Figure 1). Subsequently, it was divided into four main subfamilies: EPFL1/2/3, EPFL4/5/6/8, EPFL9, and EPF1/2&EPFL7, with the first two subfamilies showing significant expansion. Not only that, but we also found that different ZmEPF/EPFL genes exist in all subfamilies, among which ZmEPF1, ZmEPF2-1 and the reporter genes OsEPF1 and OsEPF2 have a closer genetic distance, indicating that their functions may be similar. In order to understand the specific functions of the EPF/EPFL genes in maize, we analyzed the information of the ZmEPF/EPFL genes in detail.

3.2. The Gene Structure, Encoded Protein Motifs, and Conserved Domains of the EPF/EPFL Gene Family in Maize

Investigation into the gene architecture and protein motifs of the ZmEPF/EPFL gene family revealed distinct patterns of conservation and diversification. The ZmEPF/EPFL gene family is divided into three branches in maize containing a total of eight unique protein motifs (Figure 2A), emphasizing the functional heterogeneity within this gene family. Notably, motif 6 was prevalent in all branches, suggesting that they play an important role in the ZmEPF/EPFL family. Conserved structural domains are key in determining the phylogenetic relationships of protein families, further confirming the accuracy of the classification. Analysis showed that ZmEPFL9-2 contains a unique stomatal-like structural domain, while ZmEPF9-1 and ZmEPF9-3 contain a unique PLNO03207 structural domain, while all other family members mainly contain EPF structural domains (Figure 2B). In addition, an examination of the gene structures identified nine genes in the family that contain two introns, six genes that contain one intron, and ZmEPFL1-1, which does not contain an intron. In addition, all members of the family contain two to three exons, and notably, ZmEPFL4-1, ZmEPFL5, ZmEPFL6, and ZmEPFL8 lack untranslated regions (UTRs), (Figure 2C), suggesting that they may have undergone significant evolution.

3.3. Chromosome Localization and Synteny Relationship of ZmEPF/EPFL Genes

We investigated in detail the basic information of 16 members of ZmEPF/EPFL (Table S1) and studied their localization on chromosomes (Figure 3) to reveal variation among family members. Specifically, the number of amino acids of these members ranged from 113 to 216, with ZmEPFL4-2 having the highest number of amino acids at 216 and ZmEPFL5 having the lowest number of amino acids at 113. whereas the predicted isoelectric point (pI) ranged from 6.81 to 10.7. In addition, the Instability Index had a wide range, ranging from 46.2 to 75.99, and the Aliphatic Index leaves showed significant variation, ranging from 56.43 to 86.42. And all members showed hydrophilic (GRAVY < 0). Notably, our prediction of subcellular localization of all members showed that ZmEPF1, ZmEPFL2-1, ZmEPFL3, and ZmEPFL4-1 localized to the cell wall. Whereas 11 members, including ZmEPFL1-1 and ZmEPFL1-2, localized to chloroplasts, ZmEPFL4-2 localized to mitochondria alone.
Subsequently, in a study of gene localization, we found that among the 10 chromosomes of maize, the distribution of ZmEPF/EPFL genes varied in each of the 9 chromosomes, except for chromosome VII. Specifically, chromosomes 1, 3, and 4 contain three family members each, and two family members in chromosome 10, while the remaining five chromosomes are all distributed with only one family member.

3.4. Analysis of Covariance within the ZmEPF/EPFL Family and Evolutionary Relationships among Species

Gene duplication is the driving force behind the generation of genes within the same family, with the main duplication modes being tandem duplicates and segmental duplicates. In this study, we identified four homologous gene pairs among 16 ZmEPF/EPFL genes and did not find the presence of multiple genes interrelated, implying the existence of a complex evolutionary relationship between ZmEPF/EPFL (Figure 4).
The collinear genes among species are generated during the process of species evolution. A species tree was constructed with the dicot plant Arabidopsis thaliana as the outgroup to compare its evolutionary relationship with Poaceae plants. The evolutionary relationship showed that Oryza sativa, Triticum aestivum, and Setaria italica form one branch in the Poaceae family, while Panicum miliaceum, Sorghum bicolor, and Zea mays form another branch, indicating their evolutionary closeness or distance. The collinearity analysis was conducted among these species, and their EPF/EPFL genes were annotated (Figure 5). Starting from Arabidopsis, three genes showed collinearity, as do Oryza sativa, Hordeum vulgare, and Setaria italica. In the other branch, starting from Panicum miliaceum, it decreased to two genes, then increased to three in Sorghum bicolor, and reached four in Zea mays. Therefore, the number of collinear genes varies among different evolutionary branches, and the species tree and collinearity relationships complement each other, indicating the flow of EPF/EPFL genes among different species.

3.5. Expansion and Contraction Analysis of Poaceae Plants and Their EPF/EPFL Gene Families

To gain insight into the role of the ZmEPF/EFL gene family in growth and development, we predicted the interaction networks of all ZmEPF/EFL proteins and showed all results containing support from previous experimental results. Our analysis shows that most of the ZmEPF/EFL proteins, such as ZmEPF1, ZmEPF2-1, and ZmEPF2-2, have been shown to be involved in the morphogenesis of the stomatal complex and cell growth regulation. Some of these ZmEPF/EPFL proteins interact with ribosomal proteins and mitochondrial structures, suggesting that they are important coding proteins in plants. In addition, we predict that some of the ZmEPF/EPFL proteins interact with proteins related to stomatal complex development, such as the Subtilisin-like protease SBT1.2 and the BHLH transcription factor. Interestingly, we found that six EPF/EPFL proteins in the reciprocal network, ZmEPFL4-1, ZmEPF2-1, and ZmEPFL4-2, were confirmed to interact indirectly with phosphorylation-associated proteins (A0A1D6HFF1), whereas ZmEPFL9-1, ZmEPFL9-2, and ZmEPFL9-3 were confirmed to interact with proteins associated with oxidoreductase activity (A0A1D6MCX1, A0A1D6NGD1) indirectly. In addition, the protein families of several other proteins, including Zm00001eb159500, have not been identified. Our functional predictions suggest that these proteins also play crucial roles in stomatal development and cell physiology (Figure 6).

3.6. Expression Analysis of ZmEPF/EPFL Genes

In our investigation into the expression patterns of ZmEPF/EPFL genes across diverse tissues and developmental stages, we delved into the dynamics of the ZmEPF/EPFL gene family. ZmEPF/EPFL genes exhibit tissue-specific expression profiles. Predominantly, members of the ZmEPF/EPFL family are mainly expressed in leaves and embryos, but not detected in seeds and endosperm. It is worth noting that the expression levels of ZmEPFL1-1, ZmEPFL1-2, ZmEPFL2-2, ZmEPFL6, and ZmEPFL8 are increased in leaves, suggesting their potential importance in plant growth and development. On the other hand, the upregulation of ZmEPFL9-1 and ZmEPFL4-2 in embryos suggests their involvement in seed germination. Furthermore, the high expression of ZmEPF1 and ZmEPFL5 in the spericarp at their possible role in regulating seed dehydration processes (Figure 7A).
To investigate the expression dynamics of ZmEPF/EPFL genes under abiotic stress conditions, we retrieved the selfed line B73 RNA-seq data from the GDB database (https://www.maizegdb.org, (accessed on 29 July 2024)). Subsequently, we analyzed ZmEPF/EPFL family members’ expression patterns in response to drought, salinity, and concurrent stressors, as recorded by. Our examination focused on seedlings subjected to drought, salinity, and their combined stresses. Our findings revealed that ZmEPFL2-2, ZmEPFL6, ZmEPFL8, ZmEPFL1-2, and ZmEPFL9-3 exhibited lower expression under drought conditions, on the contrary, the expression of ZmEPF1 and ZmEPFL4-2 was significantly higher Additionally, the expression of ZmEPF2-1, ZmEPFL9-3, ZmEPFL3, and ZmEPF2-2 was significantly increased under salt stress, and it is noteworthy that ZmEPFL1-1, ZmEPFL2-1, and ZmEPFL5 showed higher expression levels under both salt and drought stresses (Figure 7B).

3.7. Expression Pattern Analysis of ZmEPF/EPFL Genes under Varied Drought Conditions and qRT-PCR Validation

Understanding gene function often hinges on deciphering their expression dynamics. In our quest to unravel the role of ZmEPF/EPFL family genes under drought stress, we scrutinized their expression profiles amidst drought treatments. Leveraging data from prior studies and conducting genome-wide RNA-seq analysis on B73 selfing unaffiliated lines, we explored the responses of ZmEPF/EPFL genes to drought stress conditions. The drought stress levels were delineated as WW (well-watered), DT2 (soil moisture content of 30~35%), DT3 (soil moisture content of 20~25%), and DT4 (soil moisture content of 10~15%), as outlined in a previously published work (Zhang et al., 2019) [37]. Heat map analysis unveiled that a majority of ZmEPF/EPFL genes exhibited responses to drought stress in the B73 genotype, with diverse degrees of up- and down-regulation observed across most gene members under drought conditions (Figure 8).
Subsequently, we examined the expression profiles of 16 ZmEPF/EPFL genes under drought stress. The 16 genes analyzed showed different expression levels under WW and DT4 conditions. Particularly interesting was the observation that seven genes (ZmEPF2-1, ZmEPF2-2, ZmEPFL1-1, ZmEPFL1-2, ZmEPFL2-1, ZmEPFL9-1, ZmEPFL9-3, and ZmEPF1) showed a pattern of increasing and then decreasing expression levels from WW to DT4, and that expression levels of four genes (ZmEPFL4-2) increased and then decreased with soil conditions. ZmEPFL8, ZmEPFL3, and ZmEPFL6 showed a decreasing pattern of expression levels with decreasing water content in the soil, while the remaining five genes (ZmEPFL5, ZmEPFL2-2, ZmEPFL4-1, ZmEPFL9-2, and ZmEPFL1-1) showed the opposite pattern. Intriguingly, most of the genes had the lowest overall expression levels in the WW condition and the highest overall expression levels in the DT3 condition blind.

3.8. Validation of Drought Stress on Maize EPF/EPFL Gene Family

To corroborate the expression changes of the EPF/EPFL gene family under drought stress, we subjected maize B73 to drought stress conditions and selected 12 genes for quantification of their expression levels using qPCR (Figure 9). The results, depicted in Figure 8, unveiled distinct expression patterns within the EPF and EPFL gene families following exposure to drought stress. Notably, ZmEPF5 and ZmEPFL2-2 exhibited relatively stable expression levels at 12 h of drought treatment, followed by an increase after 24 h. On the contrary, the expression levels of ZmEPFL3, ZmEPFL6, and ZmEPFL8 gradually decreased with the drought stress time. Additionally, we observed that some ZmEPF/EPFL genes exhibited irregular expression changes when subjected to drought stress, with most showing a trend of initially increasing and then decreasing. For example, ZmEPFL2-2 and ZmEPFL9-3 showed up-regulation at 12 h and down-regulation at 24 h. The expression of ZmEPFL2-2 and ZmEPFL9-3 was also up-regulated at 12 h and down-regulated at 24 h. These findings suggest that the pattern of expression changes in the EPF/EPFL gene family under drought stress is inconsistent, with different dynamics observed at the 12 h time point. Thus, temporal variations in expression levels may occur at different durations of stress exposure.

4. Discussion

The EPF/EPFL gene family is a unique gene family in plants that plays a crucial role in plant growth, development, and adaptation to adversity. Although some members of the EPF/EPFL family have been identified in other plant species, a comprehensive genomic analysis of EPF/EPFL genes in maize has not yet been performed, and their regulatory functions remain unclear. In this study, we conducted a comprehensive survey and expression analysis of the EPF/EPFL family using genomic data, providing fundamental insights for further exploration of developmental and stress responses in maize.
This study identified the EPF/EPFL gene family members in seven Poaceae plants, with wheat having the highest number of genes. This may be due to the fact that common wheat is an allohexaploid derived from the natural hybridization of three diploid ancestors, undergoing whole-genome duplication events during evolution, leading to the presence of homologous genes in the A, B, and D genomes.
Exploring protein motifs in ZmEPF/EPFL will deepen our understanding of their unique functions in development and stress adaptation. In the study of motif composition, all members of ZmEPF/EPFL were found to have motif 6, indicating its potential conservation. However, there is no similar phenomenon in Secale cereale L. [38], suggesting that the EPF/EPFL family may have undergone a complex evolutionary process. In addition, the analysis of the ZmEPF/EPFL gene structure will help us to further explore its specific function in the evolutionary process [39]. Amino acid sequence analysis showed that most of the ZmEPF/EPFL genes have highly conserved EPF structural domains, indicating that the evolutionary trajectory of the EPF/EPFL family is relatively conserved. Most of the ZmEPF/EPFL genes belonging to the same subgroup exhibited similar intron-exon structures, but a few genes showed different and specific structures. In addition, the number and length of introns and exons varied among genes in different branches, which may be due to prolonged evolutionary processes.
Protein–protein interactions showed that ZmEPF/EPFL proteins interacted with proteins related to cell growth and stomatal development, as shown in Figure 6, and the experimentally verified protein interactions can be broadly categorized into three models, each containing three ZmEPF/EPFL members, which, interestingly, is in line with our results of using motif for ZmEPF/EPFL gene family analysis, indicating the deterministic role of different motifs on gene function. Meanwhile, we found that most of the ZmEPF/EPFL proteins are associated with stomatal complex morphogenesis. Weng et al. utilized CO stress in Arabidopsis and found that CO positively regulates stomatal initiation and distribution by regulating the expression of EPF2 and STOMAGEN (EPF9) [40]. Lu et al. also demonstrated that elevated expression of OsEPF1 or OsEPF2 greatly reduced stomatal density in rice by different transgenic means, and on the contrary, knockdown of OsEPFL9 resulted in transgenic plants with fewer stomata in rice than in WT [13]. Caspar et al. identify potential orthologs of the key toolbox genes in a hornwort, finding that n bHLH and EPF components are required for moss stomatal development and patterning, further supporting a single ancient genetic origin of stomata in the ancestor to all stomatous land [41]. Furthermore, the EPF/EPFL family has been shown to regulate spike and seed development in rice [42]. However, we did not find that ZmEPF/EPFL proteins have direct interactions with proteins related to ABA and Ca2+ signaling, etc., but six EPF/EPFL proteins, ZmEPFL4-1, ZmEPF2-1, and ZmEPFL4-2, were shown to interact indirectly with proteins related to phosphorylation (A0A1D6HFF1), and thus we speculate that these gene family members may have ABA-like ability to precisely regulate stomatal opening and closing by passing information through phosphorylation/dephosphorylation reactions [43]. ZmEPFL9-1, ZmEPFL9-2, and ZmEPFL9-3 were shown to interact indirectly with oxidoreductase-activated proteins (A0A1D6MCX1 and A0A1D6NGD1), which may imply that these three members also utilize ROS-mediated signaling to enhance drought tolerance in plants [44] or interact with Ca2+ and ABA to synergize with the regulation of stomatal opening and closure in plant leaves [45].
The EPF/EPFL family has been shown to be associated with stomatal development in a variety of crops, and factors such as stomatal density, stomatal distribution, and other factors play a huge role in plant response to drought stress. Dunn et al. found that down-regulation of the expression level of some EPF genes in wheat improved water utilization [18]; Nerva et al. found that in four grape varieties, different expression levels of the VvEPFL9, VvEPF1, and VvEPF2 genes were differentially expressed in four grape species, causing changes in stomatal density, size, and number, and ultimately leading to intraspecific variability in response to drought stress in grape [46]. Similarly, ZmEPF8 in maize has shown high expression levels under GA, ABA, MeJA, IAA, and SA treatments, suggesting that this gene may respond to drought stress through multiple pathways [47]. These results explain our variation in ZmEPF/EPFL gene expression under drought stress. Therefore, we suggest that ZmEPF/EPFL similarly responds to the maize response to drought stress by regulating the development of stomata in the leaves, altering the overall traits of stomata, and influencing the plant’s water utilization. In this study, we found that the expression of ZmEPF1-1 increased significantly with the extension of drought stress, and the expression of ZmEPFL3 and ZmEPFL8 decreased significantly. Therefore, we speculate that gene editing (Crispr-Cas9) may be able to accelerate the breeding of new drought-tolerant maize varieties by regulating the expression of the above genes.

5. Conclusions

This study mapped the EPF/EPFL gene family in maize, identifying 16 members and detailing their structural features and roles in stress response. We analyzed their gene structure, subcellular localization, and evolutionary dynamics using advanced genomic tools. Our results highlight these genes’ involvement in stomatal regulation and adaptive signaling pathways, particularly under drought conditions. This work lays a foundation for further exploration of EPF/EPFL genes in enhancing crop resilience to environmental stresses.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14081734/s1, Table S1: Gene and protein characteristics of the ZmEPF/EPFL members.; Table S2: The list of Qpcr primers.

Author Contributions

R.Z. and Y.Y. designed the experiments and methods. H.X. and Q.W. performed the experiments and data analysis. Z.C., J.F., F.Z., D.Z. and X.S. assisted with the experiments. F.Z. and X.S. participated in the discussion for the experimental direction. R.Z. and Y.Y. supervised and guided the experiments. H.X. and Q.W. drafted the manuscript. H.X. and Q.W. proofread the manuscript. R.Z. and Y.Y. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by National Natural Science Foundation of China (Key Program) (U23A20186); National Natural Science Foundation of China (No.32171928); Jilin Scientific and Technological Development Program 20220101330JC.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Acknowledgments

We thank our contributors for their dedication and compliance through the many stages of this research as well as the editors and anonymous reviewers whose comments helped to greatly improve this paper.

Conflicts of Interest

The authors declare that they have no conflicts of interest or competing interests.

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Figure 1. Evolutionary analysis of the EPF/EPFL gene family in 7 grass species and the model plant Arabidopsis thaliana. Different colors in the rootless tree represent different clades.
Figure 1. Evolutionary analysis of the EPF/EPFL gene family in 7 grass species and the model plant Arabidopsis thaliana. Different colors in the rootless tree represent different clades.
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Figure 2. Analysis of the gene structure, conserved motifs, and conserved domains encoding proteins of the EPF/EPFL family in maize: (A) Composition and distribution of conserved motifs in the ZmEPF/EPFL proteins different colored rectangles represent different motifs. (B) Conserved domain of EPF/EPFL family genes. (C) Structure of exons and introns in EPF/EPFL genes. The areas of UTR, and CDS are depicted using blue boxes, and pink boxes, in that order.
Figure 2. Analysis of the gene structure, conserved motifs, and conserved domains encoding proteins of the EPF/EPFL family in maize: (A) Composition and distribution of conserved motifs in the ZmEPF/EPFL proteins different colored rectangles represent different motifs. (B) Conserved domain of EPF/EPFL family genes. (C) Structure of exons and introns in EPF/EPFL genes. The areas of UTR, and CDS are depicted using blue boxes, and pink boxes, in that order.
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Figure 3. Chromosome distribution of ZmEPF/EPFL family. Each gene’s genomic position is accurately charted on the chromosome, determined by its distinct physical coordinates. The left side displays the chromosome count (Chr01–Chr10), while the right side lists all the genes.
Figure 3. Chromosome distribution of ZmEPF/EPFL family. Each gene’s genomic position is accurately charted on the chromosome, determined by its distinct physical coordinates. The left side displays the chromosome count (Chr01–Chr10), while the right side lists all the genes.
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Figure 4. Genomic covariance analysis of the ZmEPF/EPFL gene family. Syntenic segments in the ZmEPF/EPFL genome are denoted by gray lines, while blue lines represent pairs of ZmEPF/EPFL genes that are collinear.
Figure 4. Genomic covariance analysis of the ZmEPF/EPFL gene family. Syntenic segments in the ZmEPF/EPFL genome are denoted by gray lines, while blue lines represent pairs of ZmEPF/EPFL genes that are collinear.
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Figure 5. EPF/EPFL genes are analyzed for collinearity in the genomes of monocot plants. The curves represent covariance within the species’ genomes.
Figure 5. EPF/EPFL genes are analyzed for collinearity in the genomes of monocot plants. The curves represent covariance within the species’ genomes.
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Figure 6. Depicts the protein-protein interactions (PPI) among ZmEPF/EPFL proteins, with each node representing a protein of different color and each edge indicating an interaction. The red nodes signify the query ZmEPF/EPFL protein. Edge colors correspond to different evidence types: cyan and violet lines denote known interactions from curated databases and experimentally determined interactions, while green, red, and blue lines represent predicted interactions from gene neighborhoods, gene fusions, and gene co-occurrence, respectively. Dark yellow, black, and light blue lines indicate other evidence types derived from text mining, co-expression, and protein homology.
Figure 6. Depicts the protein-protein interactions (PPI) among ZmEPF/EPFL proteins, with each node representing a protein of different color and each edge indicating an interaction. The red nodes signify the query ZmEPF/EPFL protein. Edge colors correspond to different evidence types: cyan and violet lines denote known interactions from curated databases and experimentally determined interactions, while green, red, and blue lines represent predicted interactions from gene neighborhoods, gene fusions, and gene co-occurrence, respectively. Dark yellow, black, and light blue lines indicate other evidence types derived from text mining, co-expression, and protein homology.
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Figure 7. Expression analysis of the ZmEPF/EPFL gene: (A) Embryo, endosperm, whole root, whole seed, Anthers, seed coat, and immature leaves. (B) Samples under stress included control seeds, drought-treated seeds, salt-treated seeds, and drought-and salt-co-treated seeds.
Figure 7. Expression analysis of the ZmEPF/EPFL gene: (A) Embryo, endosperm, whole root, whole seed, Anthers, seed coat, and immature leaves. (B) Samples under stress included control seeds, drought-treated seeds, salt-treated seeds, and drought-and salt-co-treated seeds.
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Figure 8. Expression analysis of ZmEPF/EPFL genes under three different drought degree stresses (WW (well-watered), DT2 (soil moisture content of 30~35%), DT3 (soil moisture content of 20~25%), DT4 (soil moisture content of 10~15%)).
Figure 8. Expression analysis of ZmEPF/EPFL genes under three different drought degree stresses (WW (well-watered), DT2 (soil moisture content of 30~35%), DT3 (soil moisture content of 20~25%), DT4 (soil moisture content of 10~15%)).
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Figure 9. Differential expression plots of ZmEPF/EPFL genes under three different drought levels of stress. ANOVA was used to compare the differences between groups. * denotes significant (0.01 < p < 0.05) ** denotes highly significant (p < 0.01).
Figure 9. Differential expression plots of ZmEPF/EPFL genes under three different drought levels of stress. ANOVA was used to compare the differences between groups. * denotes significant (0.01 < p < 0.05) ** denotes highly significant (p < 0.01).
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MDPI and ACS Style

Xia, H.; Wang, Q.; Chen, Z.; Sun, X.; Zhao, F.; Zhang, D.; Fei, J.; Zhao, R.; Yin, Y. Identification and Functional Analysis of the EPF/EPFL Gene Family in Maize (Zea mays L.): Implications for Drought Stress Response. Agronomy 2024, 14, 1734. https://doi.org/10.3390/agronomy14081734

AMA Style

Xia H, Wang Q, Chen Z, Sun X, Zhao F, Zhang D, Fei J, Zhao R, Yin Y. Identification and Functional Analysis of the EPF/EPFL Gene Family in Maize (Zea mays L.): Implications for Drought Stress Response. Agronomy. 2024; 14(8):1734. https://doi.org/10.3390/agronomy14081734

Chicago/Turabian Style

Xia, Hanchao, Qi Wang, Ziqi Chen, Xiaopeng Sun, Fangfang Zhao, Di Zhang, Jianbo Fei, Rengui Zhao, and Yuejia Yin. 2024. "Identification and Functional Analysis of the EPF/EPFL Gene Family in Maize (Zea mays L.): Implications for Drought Stress Response" Agronomy 14, no. 8: 1734. https://doi.org/10.3390/agronomy14081734

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