Comprehensive Analysis of ZmTBL Genes Reveals Their Roles in Maize Development and Abiotic Stress Responses

Abstract

Trichome birefringence-like proteins function as polysaccharide O-acetyltransferases that catalyze the O-acetylation of cell wall polysaccharides and play widespread roles in regulating plant growth and stress responses. However, no TBL genes have been functionally characterized in maize, and their biological properties remain largely unexplored. Through bioinformatic analysis, we identified 74 maize TBL genes (designated ZmTBL1–ZmTBL74) among the maize genome. Comprehensive analyses of their phylogenetic relationships, basic physicochemical and sequence characteristics, putative upstream regulatory transcription factors and expression patterns were conducted. Expression profiling and qPCR analyses revealed that ZmTBLs respond widely to abiotic stresses, including heat and cold. Association analyses demonstrated that sequence variations in ZmTBL57 and ZmTBL69 correlate with maize agronomic traits. These findings elucidate the molecular characteristics and evolutionary history of maize TBL genes and underscore their roles in abiotic stress responses. In summary, the foundation established by this work will facilitate further functional characterization of TBL genes in maize.

1. Introduction

As a structural scaffold surrounds plant cells, the cell wall plays important roles in several biological processes, such as maintaining the form of the plant cell, allowing intercellular communication, responding to external environmental stimuli and defending against pathogenic microorganisms. Polysaccharides, including pectins, mannan, xyloglucan and xylan, are main components of the cell wall. Acetylation is a common modification of polysaccharide, which is important in regulating cell wall structure and plant growth [1,2]. For example, as a major structural polysaccharide in plants, xylan requires acetylation for its correct folding and proper integration into the cell wall architecture; acetylation of pectin regulates both gel formation capacity and cellular elongation processes [3,4]. As a plant-specific gene family, TBL proteins are recognized as O-acetyltransferases that catalyze the acetylation of cell wall polysaccharides. Based on structural analysis and catalytic activity experiments of Arabidopsis TBL protein XOAT1, the catalytic domain of TBL protein adopts a unique structure, bearing some similarities to the a/b/a topology of members of the GDSL-like lipase/acylhydrolase family. XOAT1 catalyzes xylan acetylation through an acyl-enzyme formation intermediate via a double displacement bi-bi mechanism. This mechanism depends on a Ser-His-Asp triad and likely uses an Arg residue in the “RNQxxS” motif to conduct the role of an oxyanion hole [5].

In recent years, the functions of TBL proteins in plant growth and development have been gradually elucidated. ALTERED XYLOGLUCAN9 (AXY9), a member of the TBL gene family in Arabidopsis, was found to be causative for the modulation of xyloglucan acetylation. An insertional mutant of AXY9 exhibited severe growth defects and a collapsed xylem [6]. AtTBL37 is highly expressed in fast-growing plant tissues, participates in O-Acetylation of polysaccharides and affects the thickening and strength of secondary cell walls. Mutation of AtTBL37 leads to a dwarf plant and significantly reduces plant defense against insects [7]. The loss function of TBL34 and TBL35 can reduce the 3-O-monoacetylation and 2,3-di-O-acetylation of cell wall xylan, which lead to aberrant secondary wall structure; weaken cell wall strength, causing xylem vessel collapse; and subsequently impair plant growth [8,9]. Pectin acetylation is critical for the gelling property of pectins and the elongation of cells; a mutant of TBL44/PMR5 was defective in pectin acetylation and was also shown to have reduced cell expansion [10]. Two cotton TBL genes, GhTBL7 and GhTBL58, were found to regulate cotton fiber elongation; silencing of GhTBL7 and GhTBL58 led to reduced fiber length [11].

Besides regulating growth and development, TBL proteins also participate in various plant biotic and abiotic stress responses. Two TBL genes in rice, OsTBL1 and OsTBL2, are essential for leaf blight resistance in rice by mediating xylan acetylation. The ostbl1 single mutant and the tbl1 tbl2 double mutant are more susceptible to rice blight disease and displayed a stunted growth phenotype with varying degrees of dwarfism [12]. TBR can regulate plant Zn tolerance by modulating the pectin methylesterification of root cell walls, and this function is conserved across dicot and monocot plant species [13]. In Arabidopsis, TBL27 can modulate the xyloglucan O-acetylation level and influence Al sensitivity by affecting the Al-binding capacity in hemicellulose [14]. TBL10 possibly functions as an acetyltransferase and is required for the O-acetylation of rhamnogalacturonan-I, and the tbl10 mutant displayed enhanced tolerance to drought [15]. GhTBL84 functions as a negative regulator of cold response; compared with wildtype plants, GhTBL84 knock-down plants are more resistant to low-temperature stress [16]. As the cell wall is considered a protective barrier of plant cells, adjustment of the cell wall under stresses is an important phenomenon in plant adaptation. O-acetylation of polysaccharides are important to cell wall assembly and cell wall physicochemical properties [9]. Although it is widely accepted that function of TBL is achieved by O-acetylation of polysaccharides, mechanisms of TBL genes in regulating plant biotic and abiotic stresses remain to be further analyzed. In summary, these studies indicate that TBL genes play a critical role in the environmental adaptation of plants.

As a major food crop around the world, the yield of maize (Zea mays) is seriously affected by multiple abiotic stresses, including low temperature, high temperature, and drought [17,18,19]. Therefore, it is necessary to understand the functional mechanism of maize abiotic stress response to improve maize production performance under abiotic stresses. So far, although the function of hundreds of genes in maize abiotic stress response have been verified, the incomplete understanding of abiotic stress response systems still limits the progress of stress-resistant maize breeding [20,21,22]. In plants, an increasing number of TBL genes have been reported. Besides Arabidopsis, TBL genes were also identified and characterized in cotton, rose, poplar and D. officinale [16,23,24,25]. However, the systematic identification and functional characterization of the TBL gene family in maize remain unreported to date, and the function of ZmTBLs in maize abiotic stress response is also unclear. In this study, we conducted a genome-wide analysis of the maize TBL family, systematically identifying 74 ZmTBL genes. We subsequently performed comprehensive molecular characterization, including phylogenetic reconstruction, gene structure organization, chromosomal localization, duplication event analysis, abiotic stress-responsive expression profiling and genetic variations among maize populations. Our results elucidate the molecular architecture and evolutionary dynamics of ZmTBL genes, demonstrate their functional involvement in abiotic stress adaptation and reveal the potential utility of ZmTBL natural variations for maize improvement.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Seeds of the maize inbred line B73 were surface-sterilized and germinated on moistened filter paper in a growth chamber maintained at 28 °C for 2–3 days. Uniformly germinated seedlings were transplanted into plastic pots (10 cm × 10 cm × 9 cm) filled with potting soil and grown under the same conditions until reaching the three-leaf stage. For stress treatments, three-leaf stage seedlings were subjected to heat (50 °C for 4 h) or cold (4 °C for 24 h) stress in controlled chambers, while control plants remained at 28 °C. Leaf samples were subsequently collected from all groups for total RNA extraction and analysis of ZmTBL gene expression levels [26].

2.2. Identification and Evolutionary Analysis of ZmTBL Genes

All 46 previously characterized Arabidopsis thaliana TBR/TBL genes were obtained [27], and the corresponding protein sequences were retrieved from Ensembl Plants (http://plants.ensembl.org/ (accessed on 8 October 2024)) and aligned to construct a Hidden Markov Model (HMM). Maize protein sequences were similarly sourced from Ensembl Plants. Putative ZmTBL proteins were identified from this dataset using HMMsearch (ver 3.1b2) with default parameters [28]. Candidate proteins lacking both conserved domains (PF13839 and PF14416), as verified by SMART analysis (https://smart.embl.de/ (accessed on 9 October 2024)) [29], were excluded. This process identified 74 genes as ZmTBLs, designated ZmTBL1 through ZmTBL74. Subsequently, multiple sequence alignment and phylogenetic reconstruction were performed using MEGA X (ver X) [30], incorporating the 74 ZmTBL sequences alongside TBL proteins from rice (Oryza sativa) and Arabidopsis thaliana. Phylogenetic trees were generated via the Neighbor-Joining method employing pair-wise deletion and 1000 bootstrap replicates. Finally, synteny analysis of ZmTBL genes was conducted using MCScanX (ver 1.0) with default parameters [31].

2.3. Computational Prediction of ZmTBL Proteins’ Physicochemical Characteristics

To obtain the physicochemical characteristics of ZmTBL proteins, all ZmTBL sequences were collected and submitted to ExPASy (https://web.expasy.org/protparam/ (accessed on 10 October 2024)) [32], and the online tool ProtParam was used to predict the number of amino acids, molecular weight, isoelectric point and the average hydrophobicity index of ZmTBLs. Gene structure of ZmTBL genes were characterized with GSDS 2.0 (https://gsds.gao-lab.org/ (accessed on 12 October 2024)) [33]. To predict the subcellular localization of ZmTBL proteins, all ZmTBL protein sequences were submitted to CELLO (http://cello.life.nctu.edu.tw/ (accessed on 12 October 2024)) [34]. Protein–Protein interaction information of ZmTBLs identified based on RLL-Y2H method was collected from the website MaizeNetome (http://minteractome.ncpgr.cn/ (accessed on 15 March 2025)) [35].

2.4. Spatiotemporal Expression Analysis and Upstream Regulator Prediction of ZmTBLs

To analyze the spatiotemporal expression pattern of ZmTBLs, expression data of 11 maize tissues, including seedling, roots, leaves, seeds, shoot apical meristems, internodes, tassel, cob, coleoptite, pericarp and anthers, was collected from maize gene expression altas dataset on website qTeller (https://qteller.maizegdb.org/ (accessed on 15 November 2024)). HEMI software was used to draw the heatmap of ZmTBL expression profile containing 11 maize tissues [36].

For cis-regulatory motif identification, genomic segments 2 kb upstream of each ZmTBL gene were collected from maize genome. These sequences were analyzed by using PlantTFDB (https://planttfdb.gao-lab.org/ (accessed on 18 November 2024)) and 104 maize transcription factor ChIP-seq datasets [37]. Subsequently, expression correlation analyses were performed between ZmTBL genes and their corresponding predicted upstream transcription factors. All candidate transcription factors exhibiting correlation coefficients over 0.5 with ZmTBL genes were designated as putative upstream regulators. Regulatory networks were visualized by Gephi (ver 0.10). Expression data for both upstream regulators and ZmTBL genes were retrieved from the qTeller platform (https://qteller.maizegdb.org (accessed on 13 February 2025)) within MaizeGDB [38].

2.5. Expression Profiling and qPCR Analyze of the ZmTBLs Under Abiotic Stresses

Gene expression profiles of ZmTBL genes under various abiotic stress treatments were obtained from the qTeller database and a previous study [39]. FPKM/TPM values were then used to generate a heat map via HEMI (version 1.0.1) software. For qPCR validation, total RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China), followed by cDNA synthesis with HiScript III qRT SuperMix for qPCR (Vazyme, Nanjing, China). Gene-specific primers were designed for all 74 ZmTBL genes by using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed on 18 November 2024)) and employed in qRT-PCR analysis using SYBR Green master mix (ChamQ SYBR qPCR Master Mix, Vazyme, Nanjing, China). Each treatment included three independent biological replicates. The primers used are listed in Table S5.

2.6. Association Analysis Between ZmTBLs Variations and Maize Agronomic Traits

To analyze the relationship between ZmTBLs and maize agronomic traits, genotypic and phenotypic data of maize association mapping population containing 540 inbred lines were used to perform association analysis [40]. From 1.23 million high-quality SNPs (MAF ≥ 0.05), 2604 SNPs were identified within 74 ZmTBL genes loci. In total, 17 agronomic traits of maize were also collected from previous studies [41]. The MLM was chosen to detect the SNPs significantly associated with maize agronomic traits and drought tolerance by using the TASSEL5.0 program [42].

3. Results

3.1. Identification and Phylogenetic Analysis of ZmTBLs

According to conserved domains in TBL genes reported in a previous study, we identified a total of 74 TBL genes among maize genome (called ZmTBL1-ZmTBL74, Table S1). To analyze the phylogenetic relationships of TBLs among different plant species, a phylogenetic tree was constructed by using 74 ZmTBL proteins, 68 rice OsTBL proteins, and 46 Arabidopsis TBL proteins (Figure 1A; Table S2). The phylogenetic tree topology classified these TBL proteins into six groups (named Group I–VI), which contain 23/6/14/5/17/9 ZmTBL proteins, respectively (Figure 1B, Table S1). Compared with TBL members in Arabidopsis and rice, the numbers of ZmTBLs belonging to group I/IV/VI were obviously expanded. Meanwhile, rice has more TBL members belonging to group V (29 OsTBLs compared with 17 ZmTBLs and 11 AtTBLs), and Arabidopsis has more TBL members belonging to group II (10 AtTBLs compared with 6 OsTBLs and 6 ZmTBLs). These results suggest a potential function divergence of TBL proteins among different groups and their roles in species adaptation.

3.2. Chromosomal Localization and Syntenic Analysis of the ZmTBLs

Chromosomal localization analysis revealed that 73 ZmTBL genes were dispersed across all ten maize chromosomes, while ZmTBL74 resided on an unassigned contig (Figure 2). Gene distribution varied substantially among chromosomes, ranging unevenly from 5 to 12 genes per chromosome, with chromosome 6 containing the highest count (n = 12). To elucidate the expansion patterns and duplication relationships of ZmTBL genes, we performed syntenic analysis by using MCscanX. According to the result of the syntenic analysis, forty-three dispersed duplicates, seven tandem duplicates and two segmental duplicates were identified. Additionally, 16 syntenic ZmTBL gene pairs involving 22 genes were detected, distributed as follows: one pair each on chromosomes 1/7, 2/7, 3/10, 4/5, 4/6, 4/9, 4/10, 5/6 and 8; two pairs on chromosomes 5/9 and 6/9; and three pairs on chromosomes 6/8 (Figure 2). These results suggest that dispersed duplicates and WGD/segmental duplicates are the main drivers of ZmTBL expansion.

3.3. Physicochemical Properties of ZmTBL Proteins

To explore the characteristics of maize TBL proteins, the physical and chemical properties of ZmTBL proteins, including protein size, molecular weight (MW), isoelectric point (pI), the grand averages of hydropathicity (GRAVY) and instability index, were characterized from ExPASy (Table S1). The length of ZmTBL proteins varied from 114 to 865 amino acids, corresponding to molecular weights ranging from 12.07 to 90.79 kDa. Predicted isoelectric points (pI) spanned from acidic 4.70 to basic 11.62. Most ZmTBL proteins were hydrophilic (GRAVY < 0). However, two TBL proteins, named ZmTBL11 and ZmTBL73, were hydrophobic (GRAVY > 0). The instability index ranged from 35.05 to 84.18. Subcellular localization predicted through CELLO indicated diverse locations for different ZmTBL proteins: twenty-nine were predicted in the extracellular space, nineteen in the nucleus, nineteen in the plasma membrane, thirteen in the chloroplast, eleven in the mitochondria, six in the lysosome and five in the cytoplasm (Table S1). These results show the wide difference in physicochemical properties among ZmTBLs and suggest the diversity of functions and catalytic activity of ZmTBLs.

3.4. Gene Structure and Protein Conserved Motifs Analysis of ZmTBLs

Gene structure analysis revealed that ZmTBL genes exhibit variation in exon number, ranging from 1 to 16, with ZmTBL53 possessing the highest number (16 exons). Moreover, the lengths of the 5′-UTR or 3′-UTR varied among different ZmTBL members. Compared with the exon sequence, the length of ZmTBL intron sequences show more diversity. Among all ZmTBL genes, eight contain no intron while ZmTBL58 contains the longest intron over 17 kb (Figure 3). These diversity of UTR and intron lengths among ZmTBLs suggest potential regulatory divergence in gene expression and may cause diversity expression patterns of ZmTBLs.

Conserved motif analysis showed that all 46 Arabidopsis TBL proteins contain both the PMR5 N-terminal (PMR5N) domain and the PC-Esterase/DUF231 domain (Figure S1A). However, these two domains are not universally conserved in maize TBL proteins. Nine maize TBL proteins, including ZmTBL1/6/16/28/36/46/65/69/74, lack the PMR5N domain. Similarly, seven maize TBL proteins, including ZmTBL14/15/26/37/40/47/73, lack the PC-Esterase/DUF231 domain. Additionally, ZmTBL6 contains a pyridine nucleotide-disulphide oxidoreductase domain, which is not found in AtTBL proteins (Figure 4). Previous studies indicate that one serine residue from the GDS motif in the PMR5N domain and two residues (one aspartic acid and one histidine) from the DxxH motif in the PC-Esterase/DUF231 domain form a Ser-His-Asp catalytic triad essential for catalyzing polysaccharide O-acetylation. Unlike Arabidopsis, where all 46 AtTBL proteins possess all three catalytic residues, 21 ZmTBL proteins lack at least one catalytic residue (Figures S1B and S2). These differences in conserved protein domains and catalytic sites may underlie catalytic activity and functional diversity within the maize TBL family.

3.5. Tissue-Specific Expression and Upstream Regulator Analysis of ZmTBLs

To investigate the spatial and temporal expression patterns of ZmTBL genes, the expression profiles of ZmTBLs in various maize tissues and organs were collected. As shown in Figure 5, ZmTBL genes show diversified expression patterns across various maize tissues. For example, several ZmTBL genes, including ZmTBL5/8/30/41/69/70/74, are mainly expressed in root and/or meiotic tassel, while ZmTBL21 and ZmTBL22 are mainly expressed in coleoptile and pericarp. ZmTBL32 tends to express in anther, and ZmTBL17 tends to express in whole seedling. These diversified expression patterns of ZmTBL genes across different tissues suggest their differential functions in maize.

Different transcriptional regulation is one of the reasons for expression pattern difference among different ZmTBL genes. To investigate the upstream regulatory factors of ZmTBLs, we integrated the prediction results from PlantTFDB and 104 transcription factors’ ChIP-seq data collected from previous study [37]. Combined with expression correlation analysis between potential upstream regulators and ZmTBLs, totally 182 upstream regulators were predicted (Figure 6A, Table S3). Significantly, expression of four transcription factors (G2, bHLH43, MYC7 and ARFTF4) displayed strong correlations with transcription levels of ZmTBL27 (R² = 0.64, p = 1.0 × 10⁻²¹), ZmTBL27 (R² = 0.62, p = 9.5 × 10⁻²¹), ZmTBL40 (R² = 0.75, p = 4.0 × 10⁻²⁹) and ZmTBL59 (R² = 0.56, p = 7.6 × 10⁻¹⁸), respectively(Figure 6B). Notably, bHLH43 (also named ZmPIF5) plays a critical role in the response to maize light signaling and photomorphogenesis and regulates maize mesocotyl elongation in dark-grown seedlings [43]. MYC7 (also named ZmMYC2a) can regulate benzoxazinoid biosynthesis and modulate defenses in maize responses to herbivory through JA signaling [44]. G2 is a maize GARP transcription factor that regulates several biological processes including ABA signaling transduction, drought response, pathogen resistance and chloroplast development [45]. Maize auxin response factors ARFTF4 confer phosphorus tolerance by promoting root morphological development and regulate plant growth and multiple abiotic stresses’ response [46]. These findings provide valuable insights into the regulatory network involving ZmTBL genes and their upstream regulators in maize and reveal diverse biological processes influenced by these genes.

3.6. Potential Role of ZmTBLs in Regulating Maize Abiotic Stress Responses

To explore the potential role of ZmTBLs in regulating maize abiotic stress responses, we analyzed expression levels of ZmTBLs under cold and heat stress using publicly available B73 RNA-seq data from maizeGDB (https://maizegdb.org/ (accessed on 15 November 2024)). Subsequently, qRT-PCR was performed to validate ZmTBLs expression under both normal growth conditions and heat/cold stress treatments. Integrating the RNA-seq and qRT-PCR results revealed that heat stress significantly suppressed 16 ZmTBL genes while inducing ZmTBL45. Conversely, cold stress significantly induced 7 ZmTBL genes but suppressed ZmTBL27 and ZmTBL44 (Figure 7A–C). Furthermore, analysis of RNA-seq data from a previous study on varying drought stress was performed. Among 74 ZmTBLs, 8 genes were up-regulated under mild drought stress (DT2), 4 were up-regulated under moderate drought stress (DT3) and 7 were up-regulated under severe drought stress (DT4). Additionally, ZmTBL43 was down-regulated under mild drought stress (DT2), and ZmTBL23 was down-regulated under severe drought stress (DT4) (Figure S3). Meanwhile, A total of 69 interacting proteins of 40 ZmTBLs were identified through the protein interaction group data (http://minteractome.ncpgr.cn/ (accessed on 15 March 2025)). Among ZmTBL interaction protein coding genes, 21 (such as mkk2, bzip129 and myb121) belong to the GO term “stress response” (Figures S4 and S5; Table S4). These results indicate potential regulatory roles for ZmTBL genes in multiple abiotic stress responses.

3.7. Variations in ZmTBLs Were Associated with Maize Agronomic Traits

To elucidate the relationship between ZmTBL genes and maize agronomic traits, we investigated associations between single nucleotide polymorphisms (SNPs) within ZmTBL gene regions and 17 agronomic traits using a Mixed Linear Model (MLM). The analysis identified 374 statistically significant trait-associated SNPs spanning 53 ZmTBL genes. Notably, a specific SNP locus (chr10.S_581500, AA/GG) within ZmTBL69 demonstrated a significant association with kernel number per row (KNR; Figure 8A). Subsequent population expression profiling revealed significantly elevated ZmTBL69 transcript levels in AA-genotype inbreds compared to GG-genotype inbreds. Phenotypic comparisons indicated that the AA genotype correlated with reduced KNR, decreased plant height and shorter ear length relative to the GG genotype (Figure 8B–E). These findings suggest that ZmTBL69 potentially functions as a negative regulator of plant height and ear development.

Furthermore, a SNP locus (chr10.S_581500, CC/TT) within ZmTBL57 exhibited a significant association with maize ear length (Figure 9A). Significant differences in ZmTBL57 expression were detected between CC- and TT-genotype plants, alongside phenotypic variations in flowering time and ear leaf width. Compared with the CC genotype, inbreds carrying the TT allele showed higher ZmTBL57 expression levels, concomitant with reduced ear length, earlier flowering time and narrower ear leaf width (Figure 9B–E). These results collectively suggest that ZmTBL57 and its variants potentially participate in the molecular regulation of ear morphogenesis, flowering time and leaf development in maize.

4. Discussion

As a common modification on plant cell walls, O-acetylation is essential for the stability of the polysaccharide network and plays critical roles in plant growth and environment adaptation. Genetic and biochemical evidence demonstrates that several TBL family members catalyze O-acetylation of plant cell wall polysaccharides [2]. TBL genes have been reported to not only play important roles in plant growth and development but to also participate in various plant biotic and abiotic stress responses. However, no comprehensive analysis of the maize TBL gene family has been performed, and the function of maize TBL genes remains to be discovered. In this study, we identified 74 TBL genes in maize, more than Arabidopsis (46), rice (68), rose (50) and poplar (64), indicating that the TBL protein family has expanded to varying degrees in different plants during evolution. Then, we performed comprehensive analysis of the ZmTBL family, including phylogeny, gene structure, chromosomal location, gene duplication events, expression patterns and genetic variations among maize population. Based on the results of a phylogenetic analysis, ZmTBLs can be divided into six subgroups; compared with TBLs in Arabidopsis and rice, group I, IV and VI are obviously expanded. According to previous studies, group I corresponds to XOAT (xylan O-acetyltransferase), while groups IV and VI correspond to POAT (pectin O-acetyltransferase) [2]. Compared with Arabidopsis thaliana and rice, maize requires higher leaf and stem mechanical strength to maintain plant growth. As modifications of Xylan and pectin are important in regulating the cell wall’s mechanical strength, the expansion of MOAT (group I) and POAT (groups IV and VI) in maize may be necessary for the precise regulation of the cell wall’s mechanical strength. Based on a previous study, all TBL proteins in Arabidopsis contain two conserved domains and a Gly-Asp-Ser (GDS) motif that is required for acetyltransferase activity [5]. However, 16 ZmTBL proteins do not contain both conserved domains, and 21 ZmTBL proteins lack at least one conserved catalytic amino residue. Notably, except for ZmTBL73, all other 20 ZmTBL proteins lack conserved catalytic amino residue belonging to two groups (group I and group VI), which were expanded in maize. These results suggest a potential function differentiation of ZmTBL proteins during family expansion.

Transcription factors serve as fundamental regulatory components orchestrating transcriptional programs during plant developmental processes. Promoter analysis of ZmTBL genes identified diverse cis-acting transcription factors (TFs) potentially governing their expression. Among 182 candidate upstream regulators, several are functionally characterized mediators of maize growth and stress adaptation. For example, bHLH43 (also named ZmPIF5), predicted to bind the ZmTBL27 promoter, regulates photomorphogenesis and etiolated mesocotyl elongation through light signaling pathways; G2 (GARP family), targeting ZmTBL27, coordinates ABA-dependent signaling, drought response, chloroplast development and pathogen resistance; MYC7/ZmMYC2a, predicted to interact with the ZmTBL40 promoter, modulates benzoxazinoid biosynthesis and herbivore defense via jasmonate signaling; ARFTF4, binding the ZmTBL59 promoter, enhances phosphorus tolerance through root architecture modification while integrating growth with abiotic stress responses. These regulatory associations position ZmTBL genes within established signaling networks controlling maize development and environmental adaptation, warranting further functional validation.

As one of the world’s major food crops, maize yield is seriously threatened by abiotic stresses such as heat, cold and drought. For example, for every 1 °C increase in global temperature, yields of maize reduce by 3.1% on average [47,48]. While significant progress has been made in identifying numerous genes responsive to abiotic stresses, the limited understanding of plants’ abiotic stress response systems has restricted their successful application in crop improvement [49]. Consequently, further elucidation of maize’s abiotic stress response mechanisms remains imperative. The cell wall plays an important role in plant abiotic stress response. As a common cell wall polysaccharides modification, acetylation is an important factor in regulating cell wall assembly and physicochemical activity. In Arabidopsis, mutants defective in polysaccharides O-acetylation also displayed various stress response phenotypes [9]. Leveraging existing transcriptomic data [39,50], we analyzed the expression profiles of ZmTBL genes under temperature (cold, heat) and varying intensities of drought stress. Candidate stress-responsive ZmTBL genes identified by transcriptomics were subsequently validated using qRT-PCR (Figure 7C). Our analysis revealed that 9 ZmTBL genes responded to cold stress and 16 to heat stress. Under drought conditions, nine, four and eight ZmTBL genes exhibited up-regulation in response to mild (DT2), moderate (DT3) and severe (DT4) stress, respectively. Meanwhile, protein–protein interaction showed that ZmTBL proteins interacted with proteins related to plant stress response. These findings indicate that ZmTBL genes, similarly to their homologs in other plant species, are likely involved in regulating maize abiotic stress responses. However, further functional characterization is required to definitively establish the roles of ZmTBL genes in these processes, while gene editing and transformation may be able to accelerate drought-tolerant maize breeding by regulating the expression of the ZmTBL genes.

Natural variations are the basis of crop breeding and improvement and significantly affect maize agronomic traits. Numerous elite alleles have been identified through genetic approaches, which significantly accelerated the breeding process [51]. To explore the potential application value of ZmTBL genes and their genetic variations in maize breeding, 17 agronomic traits data of maize association panels published in previous studies were collected, and high-density SNP markers were used to perform association analyses on the ZmTBL gene and its flanking regions. Through the association analysis, we found 53 ZmTBL genes correlated with 17 agronomic traits, including ZmTBL57 and ZmTBL69, among others. Variation associated with maize ear length in ZmTBL57 is also associated with the expression of ZmTBL57; meanwhile, variation associated with kernel number per row in ZmTBL69 is also associated with the expression of ZmTBL69. However, it is worth noting that in order to have a better insight of ZmTBLs’ functions in regulating maize agronomic traits, further experimental validations are needed. In summary, we revealed the variations in ZmTBL gene expression among maize populations, which were associated with multiple important maize traits. These results reveal that genetic variations in ZmTBLs may have potential applications in maize breeding.

5. Conclusions

This study identified and characterized 74 ZmTBL genes in maize. By using advanced genomic tools, we analyzed gene structure, subcellular localization and evolutionary dynamics of ZmTBLs. Through expression profiling and qPCR validation, we also confirmed the response of ZmTBLs under multiple abiotic stresses. Furthermore, association analysis revealed the potential relationship between ZmTBLs’ variations and maize agronomic traits. Our findings elucidate the molecular characteristics and evolutionary history of maize TBL genes and underscore their roles in abiotic stress responses. This study also suggests that, in the future, expression regulation of ZmTBL genes through genetic editing and transgenic technology can improve the resistance and agronomic traits of maize. Collectively, this work establishes a foundation for further exploration of TBL genes in enhancing crop performance to environmental stresses.