Genome-Wide Analysis of YABBY Gene Family Reveals ZmYABBY8 as a Central Regulator Involved in Drought and Heat Stress Tolerance in Maize

TL;DR

This study identifies ZmYABBY8 as a key gene in maize that helps plants tolerate drought and heat stress.

Contribution

The study reveals ZmYABBY8 as a central regulator of drought and heat stress tolerance in maize through genome-wide and functional analysis.

Findings

ZmYABBY8 is a hub gene significantly induced under drought stress and has stress-responsive promoter elements.

Maize YABBY genes show functional diversification, with distinct expression patterns under abiotic stress.

The YABBY gene family is evolutionarily conserved but functionally diverse in maize.

Abstract

The YABBY transcription factor family plays a critical role in the development of lateral organs and the establishment of polarity in plants. However, its evolutionary dynamics and regulatory functions in response to abiotic stress in maize (Zea mays) remain unclear. In this study, we conducted a genome-wide analysis of the maize YABBY gene family, employing phylogenetic analysis, transcriptomics, co-expression networks, and molecular experiments. A total of 12 ZmYABBY genes were identified from 26 maize inbred lines and classified into five conserved subfamilies. Evolutionary analysis indicated that the family is structurally stable, predominantly shaped by purifying selection, with limited lineage-specific variation among hybrid populations, highlighting its high evolutionary conservation. In contrast, transcriptomic analysis revealed functional diversification: ZmYABBY genes were preferentially expressed in floral organs and exhibited distinct response patterns under drought and heat stress. Notably, co-expression network analysis identified ZmYABBY8 as a hub gene that was significantly induced under drought stress, as validated by RT-qPCR. Furthermore, its promoter region was found to be enriched with conserved stress-responsive cis-elements, including ABRE and DRE. Subcellular localization further confirmed that ZmYABBY8 is localized to the nucleus. In summary, the maize YABBY gene family is evolutionarily conserved yet functionally diversified, with ZmYABBY8 acting as a potential hub linking development and stress responses, making it a promising candidate for improving stress tolerance in maize.

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1. Introduction

YABBY is a family of plant-specific transcription factors characterized by two conserved domains: an N-terminal C2C2-type zinc finger domain that is involved in DNA binding and a C-terminal YABBY domain, which resembles the HMG box and primarily facilitates protein–protein interactions and transcriptional regulation [1]. The first identified YABBY gene, CRABS CLAW (CRC), was discovered in Arabidopsis thaliana [2]. Subsequent studies have demonstrated that YABBY genes play crucial and non-redundant roles in lateral organ development, the establishment of abaxial–adaxial polarity, and the differentiation of floral organs in both dicotyledonous and monocotyledonous plants [3,4,5]. As significant members of the transcription factor superfamily, YABBYs, along with other families such as WRKY, MYB, bHLH, NAC, MADS, and AP2/ERF, contribute to a complex transcriptional regulatory network in plants. They are extensively involved in various biological processes, including growth and development, organ morphogenesis, metabolic regulation, and responses to environmental stimuli [1,6,7,8,9,10,11,12,13].

In addition to their well-established roles in organ development, increasing evidence indicates that the YABBY gene family is also involved in plant responses to abiotic stresses and hormone-mediated signaling pathways. The expression of YABBY genes can be induced or repressed by various environmental factors such as drought, high salinity, low temperature, heat stress, and hormonal signals like abscisic acid (ABA) [14]. For example, AcYABBY4, a member of the YABBY family of transcription factors in pineapple (Ananas comosus), plays a crucial regulatory role in the plant’s response to salt stress [15]; in rice, OsYABBY6 is induced by cold and drought stress [16]; and OsYABBY4 interacts with SLR1 to regulate gibberellin (GA) metabolism, suggesting a potential role in modulating stress adaptation through hormonal networks [17]. In maize, members, including ZmYABBY2, ZmYABBY5, ZmYABBY8, and ZmYABBY9, are significantly upregulated under nutrient stress [18]. YABBY family members in wheat [19] and soybean [20] also exhibit responsiveness to salt and drought stresses. Collectively, these studies indicate that the YABBY gene family is broadly involved in stress responses across both monocotyledonous and dicotyledonous plants. Current research on the functional roles of the YABBY family has primarily focused on developmental regulation. However, its involvement in abiotic stress responses, particularly regarding the evolutionary dynamics and regulatory mechanisms in the important maize crop, remains to be systematically elucidated.

Maize (Z. mays L.), recognized as the highest-yielding cereal crop globally, plays a critical role in ensuring global food security and promoting sustainable agriculture (FAO, 2023) [21]. However, with the intensification of climate change, abiotic stresses such as high temperature and drought have emerged as major environmental factors limiting maize yield and quality [22]. Heat stress can disrupt pollination and grain filling, reducing photosynthetic efficiency, while drought leads to leaf wilting, stomatal closure, and impaired carbon assimilation, ultimately causing significant yield losses and posing a severe threat to maize production [23]. Various transcription factor families associated with drought and heat tolerance, including bZIP, NAC, DREB, and WRKY, have been identified in maize in recent years [24]. However, the evolutionary dynamics and regulatory mechanisms of the YABBY gene family under heat and drought stress remain unclear. A comprehensive investigation integrating pan-genome variation, evolutionary constraints, regulatory networks, and functional prioritization under climate-related stresses such as heat and drought is still lacking. Therefore, systematically identifying the ZmYABBY gene family and characterizing its evolutionary features and stress-responsive patterns is of great significance for uncovering its potential novel functions in stress adaptation and advancing breeding strategies for stress tolerance in maize.

In this study, we conducted a comprehensive genome-wide analysis of the YABBY transcription factor family using 26 representative maize inbred lines. By integrating comparative genomics, phylogenetic reconstruction, selection pressure analysis, synteny comparison across major maize heterotic groups, expression profiling under heat and drought stress, and regulatory network analyses, we systematically characterized the evolutionary conservation and functional diversification of maize YABBY genes. Furthermore, we identified ZmYABBY8 as a central regulatory hub that potentially links developmental processes with abiotic stress responses, comprising a promising candidate for future functional validation and stress-resilient maize breeding.

2. Results

2.1. Identification of Members of the Maize YABBY Gene Family

In this study, 12 ZmYABBY genes were identified in the maize genome, each containing the two characteristic YABBY conserved domains. To further investigate the evolutionary relationships of the YABBY gene family in maize, a phylogenetic tree was constructed. Based on previously reported YABBY proteins from A. thaliana , the maize YABBY proteins were classified into five subfamilies: FIL/YAB3, YAB5, YAB2, CRC, and INO (Figure 1a). This classification was further validated through a conserved motif analysis (Figure 1b), which showed that members within the same subfamily shared unique motif patterns. Multiple sequence alignment confirmed that all ZmYABBY proteins contain the conserved N-terminal C2C2 zinc finger domain and the C-terminal YABBY domain (Figure 1c), validating their identity as bona fide YABBY family members. Additionally, a total of 12 YABBY genes were consistently identified across all 26 maize genomes, indicating that the YABBY family represents a conserved core gene set in maize (Supplementary Table S1).

Beyond subfamily classification, the phylogenetic tree (Figure 1a) also elucidates broader evolutionary relationships. Within each of the five subfamilies, YABBY genes from monocot species (Z. mays, O. sativa, B. distachyon, and S. bicolor) tend to cluster together, whereas those from dicot species (A. thaliana, G. max, and S. lycopersicum) form distinct clusters. This consistent separation across all subfamilies highlights the significant evolutionary divergence between monocots and dicots.

2.2. The ZmYABBY Genes Are Subjected to Differential Selective Pressures Among Maize Varieties

To investigate the selective pressures acting on YABBY genes during maize diversification, we calculated the non-synonymous (Ka) to synonymous (Ks) substitution ratios (Ka/Ks) for each ZmYABBY gene across 26 maize varieties. The Ka/Ks ratio serves as an indicator of selective pressure, with values < 1, =1, and >1 indicating purifying selection, neutral evolution, and positive selection, respectively. The majority of ZmYABBY genes exhibited Ka/Ks values concentrated below 1 across all varieties (Figure 2a). As shown in the distribution plot, the peak Ka/Ks values for most genes fell within the 0–0.5 range. Notably, ZmYABBY8 displayed the most constrained distribution, with Ka/Ks values consistently approaching zero across varieties, indicating that it has been subjected to strong purifying selection to maintain its conserved function. Conversely, ZmYABBY6 displayed a broader distribution range, with some Ka/Ks values falling between 1.0 and 1.6, implying that this gene may have undergone positive selection in certain maize varieties.

Additionally, as highlighted in Figure 2b, among them, YABBY9, YABBY10, YABBY7, YABBY5 and YABBY6 have Ka/Ks ratio values of higher than 1 across various maize varieties, indicating that these genes may have experienced varying degrees of positive selection during maize domestication or diversification. In comparison, the remaining YABBY genes predominantly exhibited Ka/Ks values between 0 and 1, with relatively narrow distributions, suggesting that they have mainly been subjected to purifying selection, thereby maintaining functional conservation during maize diversification.

2.3. Collinearity Analysis of YABBY Family Genes in Maize and Other Plants

Synteny analysis serves as a robust methodology for examining the functional and evolutionary relationships among genes across various species. In this study, we investigate the evolutionary relationships of YABBY genes within major maize heterotic groups by selecting inbred lines that represent six distinct heterotic groups subjected to collinearity analysis with the temperate reference line B73. As shown in the collinearity plot (Figure 3a), most lines exhibited extensive syntenic blocks with B73 across the majority of chromosomes. The results indicate that the YABBY gene family exhibits an evolutionary pattern characterized by overall conservation coupled with localized divergence. At a genome-wide level, most accessions demonstrate a high degree of consistency with B73 in the chromosomal distribution of YABBY genes. Specifically, complete one-to-one gene correspondences were observed among the tropical lines CML52, CML247, and Ki3, as well as between the specialty maize group (HP301 and P39) and B73, as indicated by continuous collinear lines connecting each YABBY gene to its B73 ortholog (Figure 3a, highlighted in red). This suggests that the core genomic architecture of this gene family has been subjected to strong evolutionary constraint. However, a lineage-specific variation was detected on chromosome 10, where the collinear counterpart corresponding to B73 chromosome 10 (ZmYABBY12) lacked a syntenic counterpart in Oh7B (temperate non-stiff stalk), Mo18W (mixed background), and Ki3 (tropical). This lineage-specific absence suggests that, while the YABBY gene family maintains overall syntenic conservation, certain members, particularly ZmYABBY12, may have undergone gene loss or rapid divergence in specific heterotic groups. Such variation could reflect local adaptation or selection pressure during the diversification of maize lineages, highlighting candidate genes that may contribute to heterotic group differentiation.

To investigate the evolutionary conservation of YABBY genes across Poaceae, we performed a comparative synteny analysis between maize and eight other plant species. As shown in the synteny dot plot (Figure 3b), extensive collinear blocks were observed between Z. mays and four representative grass species. Specifically, after homologous gene counting, we identified 26, 28, 24, and 26 syntenic YABBY gene pairs between Z. mays and B. distachyon, O. sativa, S. bicolor, and S. italica, respectively. Given that these grass species are all diploid, the YABBY gene family exhibits a relatively high degree of conservation among members of the Poaceae family. To assess the deeper evolutionary conservation of YABBY genes beyond that of the monocots, we extended our syntenic analysis to four dicot species: A. thaliana (Brassicaceae), G. max (Fabaceae), S. lycopersicum, and Solanum tuberosum (both Solanaceae). The resulting synteny maps (Figure 3b) revealed markedly fewer conserved orthologs compared to the results for the grass species. The number of syntenic YABBY orthologs between maize and each dicot species was substantially lower: one with Arabidopsis, four with soybean, and five each with tomato and potato. Despite the low numbers, these syntenic relationships were non-randomly distributed; the maize orthologs were consistently located on specific chromosomes. Visual inspection of Figure 3b reveals that Zm-2 was the most frequently shared chromosome, participating in syntenic pairs with all four dicot species. Additionally, Zm-5, Zm-7, and Zm-9 contributed to syntenic pairs with the Solanaceae species (S. lycopersicum and S. tuberosum), while Zm-9 also shared synteny with G. max.

2.4. Three-Dimensional Structure and Interaction Analysis of Maize YABBY Proteins

To gain functional insights, we predicted the three-dimensional structures of all 12 ZmYABBY proteins (Figure 4a) and constructed a protein–protein interaction (PPI) network using the STRING database, visualized with Cytoscape (Figure 4b). All 12 YABBY proteins in maize were subjected to three-dimensional structural modeling. The models demonstrate that each maize YABBY protein consists of a single polypeptide chain, with a tertiary structure primarily composed of α-helices, extended strands, and random coils, lacking β-turns. Each protein contains a YABBY domain module (colored blue) and a C2C2 zinc finger domain module (colored pink), indicating a high degree of conservation (Figure 4a).

To explore putative functional associates of maize YABBY proteins, we constructed a protein–protein interaction network using the STRING database (Figure 4b). The network shows that ZmYABBY proteins interact with multiple protein families involved in transcription regulation (MADS8, VP1), development (MWP1, DL1, RS2), and plant architecture (BL1-like, ZAG6, LOP1). Interaction degree analysis revealed that ZmYABBY3 occupies a central position, with more connecting edges than any other ZmYABBY protein, suggesting that it may play a broader regulatory role. Notably, members from different subfamilies exhibit distinct interaction patterns with specific functional partners. These subfamily-specific profiles, together with the structural variations observed in Figure 4a, indicate that the YABBY family in maize has undergone functional diversification, with different members potentially specializing in distinct developmental processes.

2.5. Expression Analysis of YABBY Family Genes in Different Maize Tissues and Under High-Temperature and Drought Stress Conditions

To investigate potential functions of ZmYABBY genes, we analyzed their expression patterns across 19 maize tissues using RNA-Seq data, including data for the auricle, blade, coleoptile tip, ear, embryo, flag leaf, floret, husk, internode, kernel, radicle root, root, seedling leaf, seedling meristem, seedling root, sheath, silk, spikelets, and tassel (Figure 5a). Expression clustering analysis revealed organ-specific expression patterns for several genes. Several genes showed preferential expression in reproductive tissues: ZmYABBY12, ZmYABBY4, and ZmYABBY10 demonstrated high expression in the female inflorescence, while ZmYABBY8 and ZmYABBY6 exhibited high expression in both female and male inflorescences. ZmYABBY7 and ZmYABBY3 were highly expressed in spikelets, whereas ZmYABBY9 was specifically expressed in the male inflorescence. ZmYABBY5, ZmYABBY11, and ZmYABBY1 showed high expression in the silk. Few genes showed prominent expression in the vegetative tissues, indicating that the YABBY family primarily plays specialized roles in floral organ morphogenesis in maize.

Under drought and heat stress (Figure 5b), most responsive genes were affected by heat stress rather than drought. ZmYABBY1, ZmYABBY8, ZmYABBY5, and ZmYABBY9 were downregulated exclusively under heat stress, while ZmYABBY6 was upregulated specifically under heat stress. ZmYABBY2 was the only gene responsive to both stresses, upregulated under drought but downregulated under heat. These results suggest that the maize YABBY gene family may participate in responses to abiotic stresses such as heat and drought. Notably, the expression of most YABBY genes is more strongly influenced by heat stress, likely because the female and male inflorescences are the most sensitive to high temperatures during differentiation and flowering, and these genes are highly expressed in the respective inflorescences.

2.6. Co-Expression Network and Enrichment Analysis of Maize YABBY Family Genes

To explore functional relationships among ZmYABBY genes, in this study, we performed a co-expression analysis of maize YABBY genes utilizing the online platform Maize Netome, developed by Huazhong Agricultural University. The co-expression network was calculated and visualized using Cytoscape version 3.9.1 (Figure 6a). As shown by the network topology in Figure 6a, ZmYABBY8 occupied the most central position (largest node with most connecting edges), followed by ZmYABBY12, ZmYABBY4, ZmYABBY3, and ZmYABBY7. This centrality suggests that these genes may serve as hub regulators within the YABBY co-expression network. The enrichment results show that, for the cellular component, YABBY genes are primarily localized in the nucleus and intracellular membrane-bounded organelles, supporting their role as transcriptional regulators. In the biological process category, significant enrichment was observed for flower development, reproductive structure development, embryo development, and other developmental processes, as well as regulation of gene expression and cellular/metabolic processes. For molecular function, YABBY genes were enriched in DNA binding, nucleic acid binding, and sequence-specific transcription factor activity. Taken together, the central network positions of specific ZmYABBY genes (Figure 6a) and their enriched GO terms (Figure 6b) indicate that maize YABBY genes function as nuclear transcription factors that play critical roles in regulating gene expression associated with plant growth, development, and reproduction.

2.7. RT-qPCR Was Used to Quantify the Expression of Maize YABBY Genes Under Heat and Drought Stress Conditions

This study analyzes the expression of ZmYABBY genes under heat and drought stress. To investigate the responses of ZmYABBY genes to abiotic stress, the expression levels of five ZmYABBY genes were examined under heat and drought treatments at different time points (6, 12, 24, and 36 h) (Figure 7b–f). Under heat stress, most ZmYABBY genes exhibited significantly lower expression levels than those of the control at multiple time points. In particular, ZmYABBY2 (Figure 7b), ZmYABBY5 (Figure 7c), and ZmYABBY8 (Figure 7e) were markedly downregulated, especially at early time points (6–12 h), indicating a negative response to heat. ZmYABBY6 demonstrated a transient increase in expression at 24 h (Figure 7d), suggesting a time-dependent response. Under drought stress, ZmYABBY genes displayed more variable expression patterns. ZmYABBY8 was significantly upregulated at 12, 24, and 36 h (Figure 7e), indicating a positive response to drought stress. ZmYABBY9 showed increased expression at 36 h (Figure 7f), while ZmYABBY2 and ZmYABBY5 were moderately downregulated at early time points. ZmYABBY6 maintained relatively stable expression throughout the treatment.

Co-expression analysis under stress conditions identified ZmYABBY8 as a central node-responsive node (Figure 7g,h). Further stress-specific analyses revealed significant network reconfiguration. Under heat stress, the co-expression between ZmYABBY8 and ZmYABBY2 was markedly enhanced (Figure 7g). In contrast, under drought stress, ZmYABBY8 formed a newly emerged strong co-expression module with ZmYABBY6 (Figure 7h). Notably, ZmYABBY8 displayed distinct expression plasticity, being significantly downregulated under high-temperature stress while being strongly induced under drought conditions. Collectively, these results indicate that ZmYABBY genes respond differentially to heat and drought stress, reflecting functional divergence within the YABBY family, with ZmYABBY8 serving as a key regulatory hub integrating multiple stress-responsive pathways.

2.8. Cis-Acting Element Analysis of the ZmYABBY8 Promoter

To investigate the transcriptional regulatory basis of stress responsiveness, we analyzed cis-acting elements within the approximately 2 kb promoter regions of ZmYABBY8 across 26 maize inbred lines (Figure 8). As shown in the cis-element annotation (Figure 8a), the ZmYABBY8 promoter was consistently enriched with stress- and hormone-responsive elements, including ABA-responsive elements (ABRE, ABRE3a, ABRE4), the dehydration-responsive element core (DRE-core), and drought-inducible MYB-binding sites (MBS). Additional elements related to MeJA, ethylene, auxin, gibberellin, light responsiveness, and meristem expression were also detected, indicating a conserved regulatory framework for integrating multiple environmental and developmental signals. However, comparative analysis across 26 inbred lines (Figure 8b) revealed considerable variation in the number and arrangement of these elements among maize subgroups. Tropical and subtropical lines, particularly CML accessions, generally exhibited more complex stress-related promoter architectures, whereas some temperate lines displayed relatively compact patterns. This cis-regulatory diversity suggests that genotype-dependent variation in the ZmYABBY8 promoter structure may contribute to differential transcriptional activation under drought stress across maize varieties.

2.9. Subcellular Localization Analysis of ZmYABBY8 Proteins

To experimentally validate the subcellular localization of maize YABBY proteins, we selected ZmYABBY8 as a representative candidate. Its coding sequence was cloned into a GFP-tagged expression vector and transiently expressed in maize protoplasts (Figure 9). As shown in Figure 9, fluorescence microscopy revealed that the GFP signal derived from the ZmYABBY8–GFP fusion protein was exclusively detected in the nucleus, colocalizing with the nuclear marker mCherry (merged image). In contrast, the free GFP control was distributed throughout the cytoplasm and nucleus. This colocalization confirms the nuclear localization of ZmYABBY8, consistent with the computational prediction using CELLO v2.5. These findings provide experimental evidence that ZmYABBY8 proteins primarily function in the nucleus, consistent with their roles as transcriptional regulators.

3. Discussion

YABBY transcription factors are plant-specific regulatory proteins that play essential roles in lateral organ development and the establishment of abaxial–adaxial polarity, with their core functions being highly conserved across angiosperms [25,26]. Despite this well-established developmental role, the involvement of YABBY genes in abiotic stress responses remains poorly understood, particularly in major gramineous crops such as maize. In this study, leveraging maize pan-genome resources, we conducted a comprehensive identification and evolutionary analysis of the ZmYABBY gene family. By integrating transcriptomic profiling with RT-qPCR validation, we demonstrate that while ZmYABBY genes are evolutionarily conserved and likely retain fundamental developmental functions, specific members exhibit stress-responsive expression patterns, suggesting functional diversification associated with high-temperature and drought adaptation.

Phylogenetic and synteny analyses indicated that ZmYABBY genes are highly conserved among monocotyledonous species, consistent with their indispensable roles in plant development. Ka/Ks analysis further supported this conclusion, with the majority of ZmYABBY genes exhibiting values lower than 1 across 26 maize accessions [27], a hallmark of purifying selection acting to preserve core developmental functions throughout maize evolution. Similar evolutionary patterns have been reported for YABBY families in other crops, highlighting their essential roles in maintaining fundamental aspects of plant morphogenesis [27,28]. Notably, ZmYABBY5, ZmYABBY6, and ZmYABBY9 displayed Ka/Ks values greater than 1 in certain accessions, suggesting that these genes may have experienced episodic positive selection during maize domestication or environmental adaptation. This observation aligns with reports for soybean and wheat, where some YABBY members exhibit diversified selective pressures associated with stress-related functions [19], implying that relaxed constraint or positive selection may have facilitated the acquisition of new functional roles in specific lineages.

Tissue-specific expression analysis revealed that ZmYABBY genes are predominantly expressed in reproductive organs, including female and male inflorescence, spikelets, and silks. This finding further supports their conserved roles in floral organ development and the formation of reproductive structures [26]. Importantly, several genes that are highly expressed in reproductive tissues, such as ZmYABBY8, ZmYABBY6, and ZmYABBY9, also exhibited significant transcriptional responses to heat and drought stresses. Given that the reproductive stage of maize is particularly sensitive to environmental stresses, this overlap suggests that ZmYABBY genes may serve as molecular bridges integrating developmental regulation with stress adaptation [5,29]. The phenomena of ‘development–stress crosstalk’ have been observed in various plant species, where heat stress can trigger the transcriptional reprogramming of developmental regulators, thereby impacting growth and reproductive processes [30]. The dual involvement of YABBY genes in reproductive development and stress responses may therefore represent an evolutionarily conserved strategy for coordinating fitness under adverse conditions.

RT-qPCR analysis further revealed a marked expression divergence of ZmYABBY genes under abiotic stress. Under heat treatment, most ZmYABBY genes showed a general trend of downregulation, a response likely reflecting global transcriptional reprogramming that prioritizes stress survival over growth-related processes, a common adaptive strategy in plants under elevated temperatures [31]. In contrast, ZmYABBY8 was significantly upregulated under drought stress, particularly at later stages of treatment, pointing to a potential positive regulatory role in drought tolerance. This stress-dependent expression divergence is not without precedent; similar patterns have been reported for YABBY genes in rice [32] and pineapple [15]. Mechanistically, some YABBY members are believed to participate in stress responses through hormone signaling pathways, particularly abscisic acid (ABA) signaling [33,34]. Given the central role of ABA in coordinating drought responses and developmental regulation, it is plausible that ZmYABBY genes function as regulatory nodes that integrate developmental cues with stress signaling pathways, a hypothesis supported by the enrichment of ABA-responsive elements (ABREs) in their promoter regions.

Among all ZmYABBY members, ZmYABBY8 emerges as a prominent candidate linking developmental regulation with abiotic stress responses. Across 26 maize genomes, ZmYABBY8 exhibits a relatively low Ka/Ks ratio, indicating strong evolutionary conservation and functional constraint. Consistent with this finding, transcriptomic and RT-qPCR analyses reveal a pronounced induction of ZmYABBY8 under drought stress, particularly at later stages of treatment. This apparent contradiction, i.e., strong purifying selection coupled with stress-inducible expression, suggests that ZmYABBY8 occupies a unique regulatory position where its core function is essential under normal conditions, but its modulation is critical for stress adaptation. Co-expression network analysis further positions ZmYABBY8 as a central regulatory hub, implying that it coordinates not only developmental programs but also stress-responsive transcriptional networks. In addition, promoter analysis identifies an enrichment of stress-responsive cis-acting elements, including ABRE and DRE motifs, which likely confer its drought-inducible expression pattern. The significance of ZmYABBY8 as a stress–development integrator is further supported by evidence from other species: PvYABBY14 in switchgrass participates in stress-related pathways [35], and stress-responsive YABBY members have been documented in rice [32] and pineapple [15]. Collectively, these findings point to an evolutionarily conserved role for specific YABBY family members in stress adaptation across angiosperms.

ZmYABBY8, as a key regulatory hub, provides new insights into breeding strategies aimed at balancing growth, development, and stress adaptation. Its dual characteristics of evolutionary conservation and stress-inducible expression suggest that its expression, rather than its protein sequence, can be precisely modulated through approaches such as promoter engineering, gene editing, and marker-assisted selection. This gene represents a mechanism-based breeding target for developing climate-resilient maize. As summarized in the working model (Figure 10), ZmYABBY8 integrates stress signals via its promoter cis-elements and coordinates downstream responses through stress-specific interactions with ZmYABBY2 (heat) and ZmYABBY6 (drought). Future efforts should focus on functional validation and network dissection to facilitate its application in breeding programs.

4. Materials and Methods

4.1. Cis-Acting Element and Population Variation Analysis

The genomic sequences and annotation data for 26 maize genomes were obtained from the study by Hufford et al. [27]. These genomes are classified into six groups based on their characteristics and distribution areas: stiff-stalk heterotic (B73); non-stiff-stalk heterotic (B97, Ky21, M162W, Ms71, Oh43, Oh7B); mixed tropical–temperate ancestry (M37W, Mo18W, Tx303); popcorn (HP301); sweet corn (P39, II14H); and tropical (CML52, CML69, CML103, CML228, CML247, CML277, CML322, CML333, Ki3, Ki11, NC350, NC358, Tzi8). These varieties are primarily sourced from Africa, Asia, and the Americas. The Hidden Markov Model (HMM) configuration file for the YABBY domain (PF04690 and PF24868) was retrieved from the Pfam database (http://pfam.xfam.org, accessed on 4 November 2025). The conserved YABBY domain was identified using HMMER v3.3.2 (http://hmmer.org/) [39] based on the Pfam HMM profile, with an e-value threshold of e < 1 × 10^−5^. Candidate YABBY genes were submitted to SMART (http://smart.embl-heidelberg.de/, accessed on 5 November 2025) [40] and the CD-search module of NCBI (http://hmmer.org/, accessed on 5 November 2025) to confirm the presence of the YABBY domain. Finally, YABBY genes displaying collinearity across the 26 maize germplasms were classified as members of the YABBY family.

4.2. Phylogenetic Analysis of the ZmYABBY Gene Family

To classify the maize YABBY proteins, we constructed a phylogenetic tree incorporating both A. thaliana and Z. may YABBY proteins. The phylogenetic analysis utilized YABBY protein sequences from monocotyledonous plants (O. sativa, S. bicolor, B. distachyon, and S. italica) and dicotyledonous plants (A. thaliana, G. max, and S. lycopersicum), in addition to maize YABBY protein sequences. The A. thaliana YABBY protein sequences were obtained from the TAIR10 database (https://www.arabidopsis.org/, accessed on 5 November 2025). YABBY protein sequences from the other species were retrieved from the Plant TFDB database (https://planttfdb.gao-lab.org/, accessed on 6 November 2025). Multiple sequence alignment was conducted using the MUSCLE algorithm, as implemented in MEGA11(State College, PA, USA) [41], with default parameters. The aligned sequences were then used to construct a phylogenetic tree using the Maximum Likelihood (ML) method based on the Jones–Taylor–Thornton (JTT) model. Node support was assessed by 1000 bootstrap replicates. The resulting tree was visualized and annotated using iTOL v6(Heidelberg, Germany) [42].

4.3. Conserved Motif and Domain Analysis of the Maize YABBY Gene Family Members

The amino acid sequences of the maize YABBY gene family members were downloaded from the Phytozome database (https://phytozome-next.jgi.doe.gov/, accessed on 8 November 2025). Conserved domain annotations were obtained using the NCBI Batch CD-Search tool (Bethesda, MD, USA, https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 8 November 2025), and motif information was identified using the MEME Suite (https://meme-suite.org/meme/tools/meme, accessed on 8 November 2025). Based on the phylogenetic tree, conserved motif analysis results, and conserved domain annotations retrieved from NCBI, an integrated schematic diagram was constructed using TBtools-II (Guangzhou, China) [43]. This comprehensive visualization facilitated a comparative analysis of conserved motif distribution and domain conservation among maize YABBY genes.

4.4. Ka/Ks Calculation

The YABBY protein sequences, whole-genome sequences, and annotation files for 26 maize genomes were obtained from the study conducted by Hufford et al. [27]. Batch sequence alignment was performed using ParaAT v2.0(Beijing, China) [44], and Ka/Ks ratios were calculated utilizing the KaKs Calculator [45]. The distribution of Ka/Ks values was visualized as ridge plots using the ChiPlot online tool (ChiPlot LLC, Beijing, China, https://www.chiplot.online/, accessed on 19 November 2025), and a heatmap illustrating the proportion of ZmYABBY genes with Ka/Ks values exceeding 1 was generated.

4.5. Collinearity Analysis of YABBY Family Genes

The target proteins were aligned against the v5 B73 proteome dataset (https://maizegdb.org/, accessed on 11 November 2025) through BLAST (BLAST+ v2.16.0, NCBI, Bethesda, MD, USA) analysis, resulting in the creation of an m8-format file with a significance cutoff of e < 1 × 10^−5^. Gene chromosome collinearity was analyzed using both the m8-format file and the General Feature Format (GFF) file of the v5 B73 genome. Synteny analysis and visualization were conducted using TBtools-II [43].

4.6. Construction of Co-Expression Network of YABBY Genes in Maize, Analysis of Three-Dimensional Structure of Proteins, and Analysis of Protein Interactions

The three-dimensional structures of maize YABBY proteins were predicted using the online tool SWISS-MODEL (https://swissmodel.expasy.org, Basel, Switzerland, accessed on 20 November 2025). The resulting PDB files were analyzed, and the YABBY domain was annotated using PyMOL v2.0(Schrödinger, Inc., New York, NY, USA). Protein–protein interactions of maize YABBY proteins were predicted using the STRING database (Version 12.0, https://string-db.org/, accessed on 22 November 2025), and the results were calculated and visualized using Cytoscape version 3.9.1(San Diego, CA, USA). Furthermore, based on the co-expression network data released on the MaizeNetome website (http://minteractome.ncpgr.cn/searchelement.php, accessed on 25 November 2025), a co-expression network of maize YABBY genes was constructed using the B73 v4 reference genome.

4.7. Expression Profile Analysis of Maize YABBY Genes

The development of maize encompasses both nutritional and reproductive growth. The primary organs involved in nutritional growth are the roots, stems, and leaves, while the main organs of reproductive growth include the flowers, ears, and seeds. Expression data for maize B73 across various tissues and stress conditions (heat and drought) were obtained from the public database PPRD (Plant Public RNA-Seq Database) (http://ipf.sustech.edu.cn/pub/plantrna/, accessed on 15 November 2025). The datasets utilized were PRJNA482146 (tissue expression) and PRJNA368967 (stress expression). Heatmaps were generated using the R package ComplexHeatmap (v2.14.0).

4.8. Cis-Acting Element Analysis

Genome sequences and annotation files for 26 maize lines representing diverse heterotic groups (temperate stiff stalk, temperate non-stiff stalk, tropical, subtropical, and mixed) were obtained from Hufford et al. Promoter sequences, defined as 2000 bp upstream of the coding region, were analyzed for cis-acting elements using TBtools-II [43]. Putative cis-acting regulatory elements were identified by searching the promoter sequences against the PlantCARE database [46] (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 5 January 2026), with default parameters. The number and type of cis-elements were quantified for each promoter. Visualization of element distributions was performed using the TBtools-II Graphic View module.

4.9. Plant Growth and Treatment

The experimental materials were cultivated in the greenhouse of the Yazhou Bay National Laboratory in Sanya, China. In this study, the maize inbred line B73 was employed. Seeds of the maize inbred line B73 were obtained from the Maize Genomics and Breeding Team, Yazhouwan National Laboratory, Hainan, China. Seeds were sown in a mixed substrate (soil:vermiculite = 7:3) under a photoperiod of 16 h light and 8 h dark. When the seedlings reached the three-leaf stage (V3), heat and drought stress treatments were administered using 42 °C and 20% PEG 6000 (Sigma-Aldrich, St. Louis, MO, USA), respectively, to simulate high-temperature and drought conditions. Uniform seedlings exhibiting similar phenotypes were selected, and samples were collected from the fully expanded third leaf (counting from the base), which represents metabolically active tissue at this developmental stage. Samples were collected at 6 h, 12 h, 24 h, and 36 h post stress treatment. For each time point, six healthy seedlings were pooled as one biological replicate, with three biological replicates per treatment. All samples were immediately frozen in liquid nitrogen and stored at −80 °C for subsequent RNA extraction.

4.10. Quantitative RT-PCR Validation

Real-time quantitative PCR was performed using the LightCycler^®^ 480 II System (Roche, Basel, Switzerland). First-strand cDNA synthesis was performed with the Hifair^®^III 1st Strand cDNA Synthesis Super Mix kit (YEASEN, Shanghai, China). Using 18S rRNA as the reference gene, RT-qPCR was executed using the Hieff UNICON^®^ ColorGPS qPCR SYBR Green Master Mix (YEASEN, Beijing, China) in a total reaction volume of 20 µL. The amplification protocol included an initial denaturation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. All experiments were replicated in triplicate, and relative gene expression was calculated using the 2^−∆∆Ct^ method [47]. The primer sequences for the genes are provided in Table S2.

4.11. Subcellular Localization of ZmYABB8 Proteins

The subcellular localization of ZmYABBY proteins was initially predicted using CELLO v2.5 (Hsinchu, Taiwan, China, http://cello.life.nctu.edu.tw/, accessed on 17 October 2025). The coding sequences (CDS) of ZmYABBY genes, excluding the stop codon, were amplified employing a high-fidelity DNA polymerase with 2× Phanta Flash Master Mix (Vazyme, Nanjing, China). The amplified fragments were subsequently cloned into the pAN580 vector(Yazhou Bay National Laboratory in Sanya, China) containing a green fluorescent protein (GFP) tag using LightNing^®^ DNA Assembly Mix Plus (Yugong Biotech, Lianyungang, China). Prior to cloning, the pAN580-GFP vector was linearized utilizing XbaI and BamHI restriction enzymes(New England Biolabs, Ipswich, MA, USA). The ZmYABBY–GFP fusion constructs were transiently expressed in maize protoplasts isolated from etiolated seedlings of the B73 inbred line via a PEG-mediated transformation method [48]. Transformed protoplasts were incubated at 23 °C for 14 h in a growth chamber. GFP fluorescence signals were visualized using a laser scanning confocal microscope (LSM900, ZEISS, Oberkochen, Germany), with mCherry serving as a nuclear marker. The primer sequences utilized for vector construction are listed in Supplementary Table S2.

5. Conclusions

In this study, we conducted a comprehensive genome-wide analysis of the YABBY transcription factor family across 26 maize genomes, extending previous investigations that were limited to single reference genomes. Our results reveal that maize YABBY genes constitute a highly conserved core family under strong purifying selection; however, specific members (ZmYABBY5, ZmYABBY6, ZmYABBY9) exhibit signatures of positive selection and stress-responsive expression, indicating functional diversification. By integrating comparative genomics, expression profiling, network analysis, and experimental validation, we identified ZmYABBY8 as a key regulatory hub that coordinates development with heat and drought responses. Its dual characteristics—strong purifying selection coupled with stress-inducible expression—exemplify how a developmentally essential gene can be co-opted for stress adaptation through cis-regulatory evolution. From an applied perspective, ZmYABBY8 represents a promising breeding target: its conserved protein sequence suggests that modulating expression, rather than altering protein function, could enhance stress tolerance without pleiotropic effects. The natural promoter variation among maize lines provides a roadmap for such manipulation through marker-assisted selection or gene editing. Future work should focus on identifying downstream targets, validating promoter haplotypes across germplasm, and exploring whether other YABBY members play complementary roles related to stress. This study offers both a conceptual framework and practical resources for harnessing genes at the intersection of development and stress adaptation, a class of targets that is increasingly vital for climate-resilient crop improvement.