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Article

Genomic Architecture of AP2/ERF Superfamily Genes in Marigold (Tagetes erecta) and Insights into the Differential Expression Patterns of AP2 Family Genes During Floral Organ Specification

by
Hang Li
,
Guoqing Chen
,
Shirui Hu
,
Cuicui Liu
,
Manzhu Bao
and
Yanhong He
*
National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(5), 1231; https://doi.org/10.3390/agronomy15051231
Submission received: 18 April 2025 / Revised: 15 May 2025 / Accepted: 17 May 2025 / Published: 18 May 2025
(This article belongs to the Section Horticultural and Floricultural Crops)
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Abstract

:
The APETALA2/Ethylene-Responsive Factor (AP2/ERF) superfamily is one of the largest transcription factor families in plants, playing diverse roles in development, stress response, and metabolic regulation. Despite their ecological and economic importance, AP2/ERF genes remain uncharacterized in marigold (Tagetes erecta), a valuable ornamental and medicinal plant in the Asteraceae family known for its unique capitulum-type inflorescence with distinct ray and disc florets. Here, we conducted a comprehensive genome-wide analysis of the AP2/ERF superfamily in marigold and identified 177 AP2/ERF genes distributed across 11 of the 12 chromosomes. Phylogenetic analysis revealed their classification into the AP2 (28 genes), ERF (143 genes), RAV (4 genes), and Soloist (2 genes) families based on domain architecture. Gene structure and motif composition analyses demonstrated group-specific patterns that correlated with their evolutionary relationships. Chromosome mapping and synteny analyses revealed that segmental duplications significantly contributed to AP2/ERF superfamily gene expansion in marigold, with extensive collinearity observed between marigold and other species. Expression profiling across different tissues and developmental stages indicated distinct spatio-temporal expression patterns, with several genes exhibiting tissue-specific expression in Asteraceae-specific structures. In floral organs, TeAP2/ERF145 exhibited significantly higher expression in ray floret corollas compared to disc florets, while TeAP2/ERF103 showed stamen-specific expression in disc florets. Protein interaction network analysis revealed AP2 as a central hub with extensive predicted interactions with MADS-box and TCP family proteins. These findings suggest that AP2 family genes may collaborate with MADS-box and CYC2 genes in regulating the characteristic floral architecture of marigold, establishing a foundation for future functional studies and molecular breeding efforts to enhance ornamental and agricultural traits in this economically important plant.

1. Introduction

Transcription factors (TFs) are key regulators of gene expression that control various biological processes in plants, including growth, development, metabolism, and responses to environmental stimuli [1,2]. Among the numerous plant TF families, the APETALA2/Ethylene-Responsive Factor (AP2/ERF) superfamily represents one of the largest and most functionally diverse groups [3,4]. This superfamily is characterized by the presence of the highly conserved AP2/ERF DNA-binding domain, typically consisting of 60–70 amino acids [5,6]. Based on the number and structural features of the AP2/ERF domains, it is conventionally classified into four major families: AP2 (containing two AP2/ERF domains), ERF (containing a single AP2/ERF domain), RAV (containing one AP2/ERF domain and one B3 domain), and Soloist (a divergent group). The ERF family is further divided into the ERF and DREB (Dehydration-Responsive Element-Binding protein) subfamilies [4,7,8]. This classification system provides a framework for understanding the functional diversity of AP2/ERF proteins.
The AP2/ERF superfamily has been extensively characterized in model plants and several crops, revealing their crucial roles in various biological processes. The AP2 family members primarily regulate floral meristem determinacy, floral organ identity, seed development, and fruit ripening [9,10]. ERF family members are predominantly involved in responses to biotic and abiotic stresses, including pathogen infection, drought, salinity, and extreme temperatures [11,12]. RAV family members function in leaf senescence, flowering time control, and responses to ethylene and brassinosteroid hormones [13]. The Soloist family, though small, has been implicated in various stress responses, with members from different species shown to enhance tolerance to salt stress and pathogen resistance [14,15]. The functional significance of these genes makes them attractive targets for crop improvement through molecular breeding and genetic engineering approaches.
The Asteraceae (Compositae) family is one of the largest and most successful plant families, comprising approximately 10% of all flowering plants with an estimated 32,000–34,000 species distributed worldwide [16]. The remarkable evolutionary success of this family is largely attributed to its unique inflorescence structure, the capitulum, which represents one of the most significant innovations in plant reproductive morphology [17,18]. The capitulum, often perceived as a single flower, is actually a complex inflorescence consisting of numerous individual florets arranged on a common receptacle and surrounded by protective bracts [19]. A distinctive characteristic of many Asteraceae species is the presence of dimorphic florets within a single capitulum: peripheral ray florets with enlarged, zygomorphic corollas that often function primarily for attraction, and central disc florets with actinomorphic corollas that are typically hermaphroditic and ensure reproduction [20]. In Arabidopsis and most model plant species, the outermost floral organs are referred to as sepals and petals, following the classical floral architecture. However, in Asteraceae, these structures have undergone significant evolutionary modifications resulting in distinct terminology. The first whorl organs are referred to as pappus in Asteraceae and often appear as scales, bristles, or hair-like structures that aid in seed dispersal [20,21]. Similarly, the second whorl organs are termed corolla in Asteraceae, which, in ray florets, often fuse to form the characteristic ligule structure [22]. This specialized terminology reflects the unique evolutionary adaptations in Asteraceae floral architecture and highlights the morphological innovations that contribute to the success of this diverse plant family [19].
The molecular mechanisms underlying capitulum development and floret differentiation in Asteraceae have garnered substantial research interest in recent years. Investigations into model Asteraceae species, such as sunflower (Helianthus annuus), gerbera (Gerbera hybrida), and chrysanthemum (Chrysanthemum morifolium), have revealed complex regulatory networks involving multiple transcription factor families [23,24]. Notably, CYCLOIDEA2 (CYC2)-like TCP transcription factors are crucial for determining ray and disc floret identity and establishing flower symmetry in Asteraceae [25,26]. Based on the classic ABCDE model of floral organ development, MADS-box genes, particularly A-, B-, C-, and E-class genes, have been implicated in the differentiation of ray and disc florets in Asteraceae [27,28]. The regulatory networks of TCP proteins, in conjunction with MADS-box transcription factors, are vital for establishing floral dorsoventral asymmetry and regulating cell proliferation and differentiation, thereby collectively shaping floral architecture [29,30].
Within the classic ABCDE model of floral development, AP2 occupies a unique position as the only non-MADS-box transcription factor, fulfilling the A-class function that establishes the foundation for proper floral organ patterning and identity determination [31,32]. In Arabidopsis, mutations in the AP2 gene result in homeotic transformations where sepals are converted to carpel-like structures and petals to stamen-like structures [33]. Despite the well-established role of AP2 genes in flower development in model species, their function in Asteraceae capitulum development and floret differentiation remains largely unexplored. The limited studies available suggest that AP2 genes in Asteraceae may have evolved specialized functions related to the unique architecture of the capitulum and dimorphic florets. In chrysanthemum, the AP2/ERF member WRINKLED1 (WRI1) shows differential expression between contrasting ray floret types, suggesting its involvement in determining ray floret morphology. These AP2/ERF proteins likely mediate auxin and ethylene signaling to regulate the distinctive characteristics of ray florets [28]. This functional divergence of AP2 genes in Asteraceae compared to non-Asteraceae species may represent an evolutionary adaptation contributing to the diversity and success of the capitulum inflorescence across the family.
Marigold (Tagetes erecta), native to South America, is an economically important ornamental plant in the Asteraceae family, valued for its distinctive flowers, medicinal properties, and as a source of carotenoids, particularly lutein [34,35]. The inflorescence of marigold is a capitulum, characterized by peripheral ray florets and central disc florets [36]. Specifically, ray florets possess enhanced corollas but lack stamens, while disc florets contain complete floral structures [37]. This distinctive floral structure makes marigold an excellent model system for studying floral development and organ differentiation in Asteraceae. The recent publication of the chromosome-scale genome assembly of marigold [38] has provided an unprecedented opportunity to investigate the molecular mechanisms underlying its development and adaptive traits. Previous studies in marigold have identified and characterized several MADS-box genes and CYC2 genes involved in floral development [39,40,41,42], providing valuable insights into the genetic control of flower organ formation. However, a systematic analysis of the AP2/ERF superfamily—particularly AP2 genes that function as key regulators of flower organ development alongside MADS-box genes—has not yet been conducted in marigold.
In this study, we conducted a genome-wide identification and comprehensive characterization of AP2/ERF superfamily genes in marigold. We analyzed their phylogenetic relationships, gene structures, protein motifs, chromosomal distribution, and duplication patterns. Additionally, we investigated the expression profiles of AP2 family genes across different tissues and developmental stages, with a particular focus on floral organs in ray and disc florets. To better understand the regulatory networks involving AP2 family genes, we also performed protein interaction network analysis based on homology with Arabidopsis proteins, revealing potential interactions between AP2 family, MADS-box, and TCP family proteins. Our findings provide valuable insights into the evolutionary history and potential functions of AP2/ERF superfamily genes in marigold, establishing a foundation for future functional studies and genetic improvement of this economically important plant.

2. Materials and Methods

2.1. Plant Materials

The marigold inbred line V5, exhibiting typical Asteraceae capitulum structure with peripheral ray florets and central disc florets, was developed through 5 generations of self-crossing of cultivar ‘Vanilla’. Plants were grown in the Floriculture Teaching Experimental Base at Huazhong Agricultural University, Wuhan, Hubei Province, China (30°28′36.5″ N, 114°21′59.4″ E) under natural field conditions during the fall of 2024, with standard management practices.

2.2. Identification, Physicochemical Property Analysis, and Subcellular Localization of AP2/ERF Genes in Marigold

Marigold genome and protein sequences were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_026213115.1/, accessed on 30 December 2024) [37]. The Hidden Markov Model (HMM) profile and seed alignment file for the AP2/ERF domain (PF00847) were retrieved from the Pfam 37.2 database (https://www.ebi.ac.uk/interpro/entry/pfam/PF00847/logo/, accessed on 30 December 2024). HMMER 3.0 software (http://hmmer.org, accessed on 30 December 2024) was used with the HMM profile to identify putative AP2/ERF genes in the marigold genome [43]. All candidate proteins were verified for the presence of the AP2/ERF domain using the Conserved Domain Database (https://www.ncbi.nlm.nih.gov/cdd/, accessed on 30 December 2024).
Physicochemical properties of the identified AP2/ERF proteins, including amino acid length, molecular weight, theoretical isoelectric point (pI), instability index, and aliphatic index, were predicted using the ExPASy ProtParam tool (https://web.expasy.org/protparam/, accessed on 5 January 2025) [44]. Subcellular localization was predicted using the WoLF PSORT platform (https://wolfpsort.hgc.jp/, accessed on 5 January 2025) [45].

2.3. Phylogenetic Analysis of AP2/ERF Genes

Arabidopsis AP2/ERF protein sequences were obtained from The Arabidopsis Information Resource (TAIR) database (http://www.arabidopsis.org/, accessed on 3 January 2025) [46]. Multiple sequence alignment of AP2/ERF proteins from marigold and Arabidopsis was performed using MAFFT 7.487 software (https://mafft.cbrc.jp/alignment/software/, accessed on 3 January 2025) with default parameters. A maximum-likelihood phylogenetic tree was constructed using IQ-TREE 2.3.6 software [47] with the best-fit evolutionary model VT + R8 as determined by ModelFinder [48]. The reliability of the tree topology was assessed using the Ultrafast Bootstrap approximation (1000 replicates) and SH-aLRT test (1000 replicates) [49]. The final phylogenetic tree was visualized and annotated using iTOL 7.0 (https://itol.embl.de/, accessed on 5 January 2025) [50].

2.4. Gene Structure Analysis and Motif Identification

The exon–intron structures of marigold AP2/ERF genes were analyzed and visualized using TBtools V2.225 software based on the genome annotation file [51]. Conserved motifs in AP2/ERF proteins were identified using the MEME Suite v5.5.7 online tool (http://meme-suite.org, accessed on 5 January 2025) [52] with the following parameters: maximum number of motifs, 10; motif width, 6–50; any number of repetitions per sequence.

2.5. Chromosomal Mapping and Synteny Analysis

Based on the marigold genome annotation file, a chromosome localization map of AP2/ERF genes was constructed via TBtools V2.225 software. Core parameters were input, including chromosome assignment, start/end positions of 177 genes, and corresponding chromosome physical lengths. Gene density for each chromosome was calculated using 500 kb genetic intervals. A gradient color scale was employed to characterize gene density distribution. Finally, a standardized chromosome distribution map was generated [53]. According to the linear distribution order of genes on chromosomes, members of this family were systematically named from TeAP2/ERF001 to TeAP2/ERF177.
For comparative genomic analysis, genome annotation files for Arabidopsis (Arabidopsis thaliana) and lettuce (Lactuca sativa) were obtained from The Arabidopsis Information Resource (http://www.arabidopsis.org/, accessed on 5 January 2025) and the NCBI database (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_002870075.3/, accessed on 5 January 2025), respectively. TBtools software was used to analyze gene duplication events within marigold and to identify syntenic relationships between marigold and the other two species through whole-genome alignment.

2.6. RNA Extraction from Various Tissues and Organs

During the flowering period, we collected samples for tissue-specific expression analysis, including roots, stems, leaves, receptacles, bracts, seeds, floral organs from disc florets (pappus, corollas, stamens, pistils, and ovaries) and ray florets (pappus, corollas, pistils, and ovaries). Additionally, we also collected flowers buds at different developmental stages: flower buds with diameter of 0–1 mm (ray florets and outermost disc florets primordia formation), flower buds with diameter of 4–5 mm (stamen development in outermost disc florets), flower buds with diameter of 9–10 mm (floral organ morphological completion) [40]. All plant materials were rapidly frozen in liquid nitrogen and stored at −80 °C for RNA extraction.
Total RNA was isolated using the SteadyPure RNA Extraction Kit (Accurate Biotechnology, Changsha, China). RNA concentration was determined using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was assessed by 1% agarose gel electrophoresis. First-strand cDNA was synthesized using the Evo M-MLV RT Mix Kit (Accurate Biotechnology, Changsha, China) according to the manufacturer’s instructions.

2.7. Expression Analysis of AP2 Family Genes

Quantitative real-time PCR (qRT-PCR) was performed to analyze the expression patterns of AP2 family genes across different tissues and developmental stages. PCR reactions were conducted using the SYBR Green Premix Pro Taq HS qPCR Kit III (Low Rox Plus) (Accurate Biotechnology, Changsha, China) on the QuantStudio™ 6 Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The marigold β-actin gene was used as an internal reference for expression normalization [36]. Gene-specific primers were designed using Primer Premier 5.0 software (https://www.premierbiosoft.com/primerdesign/, accessed on 15 January 2025) (Supplementary Table S1). Each sample was analyzed with three technical replicates. Relative expression levels were calculated using the 2−ΔΔCt method [54]. The expression data were normalized and visualized as heatmaps to reveal tissue-specific expression patterns of AP2 family genes.

2.8. Protein–Protein Interaction Network Analysis

To gain insights into the potential regulatory relationships of AP2 family proteins in marigold, we analyzed their putative protein–protein interactions using the STRING database v12.0 (https://string-db.org/, accessed on 4 March 2025). The amino acid sequences of 28 identified AP2 family proteins from marigold were used as queries against Arabidopsis proteins to predict functional associations, as Arabidopsis has the most well-characterized interactome among plant species. The analysis was performed with the following parameters: medium confidence score (0.400), evidence from experiments, databases, co-expression, neighborhood, gene fusion, and co-occurrence were all included. Arabidopsis was used as the reference organism for homology-based predictions. For network visualization, homologous proteins with interaction scores above the threshold were imported into Cytoscape v3.10.2. The final network representation integrated AP2 family proteins with their interaction partners from MADS-box and TCP (a homolog of CYC2-like genes) proteins. The network layout was organized using the edge-weighted spring embedded algorithm, with node size and color intensity proportional to the number of interaction partners (degree of connectivity). Edge thickness was set to represent the confidence score of the predicted interactions.

3. Results

3.1. Identification and Classification of AP2/ERF Superfamily Genes in Marigold

To identify AP2/ERF superfamily genes in the marigold genome, we performed a comprehensive analysis using the Hidden Markov Model (HMM) profile of the AP2/ERF domain (PF00847). After validation through conserved domain analysis, we identified a total of 177 AP2/ERF genes in the marigold genome, which were subsequently named TeAP2/ERF001 to TeAP2/ERF177 based on their chromosomal positions (Supplementary Table S2).
The identified AP2/ERF proteins varied considerably in their physicochemical properties. The length of these proteins ranged from 52 to 624 amino acids, with molecular weights ranging from 6.00 to 69.10 kDa. The theoretical isoelectric points (pI) varied from 4.23 to 9.87, with 107 proteins exhibiting acidic properties (pI < 7) and 70 displaying basic properties (pI > 7). The instability index analysis revealed that approximately 52% of the AP2/ERF proteins were unstable (instability index > 50), while the remaining 48% were stable. Subcellular localization prediction indicated that most of TeAP2/ERF proteins were localized in the nucleus, consistent with their function as transcription factors (Supplementary Table S3).
Based on the number and configuration of AP2/ERF domains, the 177 marigold TeAP2/ERF genes were classified into four families: AP2 (28 genes), ERF (143 genes), RAV (4 genes), and Soloist (2 genes) (Table 1). Members of the AP2 family contain two AP2 domains, while ERF family members possess a single AP2 domain, and RAV family members have one AP2 domain and one B3 domain.

3.2. Phylogenetic Analysis of AP2/ERF Superfamily Members in Marigold

To understand the evolutionary relationships of AP2/ERF genes in marigold, we constructed a maximum-likelihood phylogenetic tree using the full-length protein sequences of AP2/ERF superfamily members from marigold and Arabidopsis (Figure 1). The resulting tree clearly separated the AP2/ERF proteins into distinct clades corresponding to the AP2, ERF, RAV, and Soloist families, confirming our initial classification.
Within the AP2 family, 28 marigold AP2 proteins formed three distinct groups, consistent with the classification in Arabidopsis. Based on their sequence similarities and phylogenetic positions, these groups likely represent functional equivalents of the AP2, ANT (AINTEGUMENTA), and AIL (AINTEGUMENTA-LIKE) groups described in other plant species [55]. In the ERF family, containing the ERF and DREB subfamilies, were further divided into 12 groups (ERF-B1 to B6 and DREB-A1 to A6), following the nomenclature proposed by Nakano et al. [3] (Table 1). The clustering pattern of marigold AP2/ERF proteins with their Arabidopsis counterparts suggests functional conservation across these evolutionarily distant species. Additionally, the uneven distribution of marigold AP2/ERF proteins across different groups, with notable expansions in the ERF-B3 and DREB-A4 groups, suggests differential selection pressures on specific groups during the evolutionary history of marigold.

3.3. Gene Structure and Conserved Motif Analysis

To gain insights into the structural diversity and functional evolution of AP2/ERF genes in marigold, we analyzed their exon–intron structures and conserved motifs (Supplementary Figure S1). The analysis revealed distinct structural patterns among different families and groups, which generally corresponded with their phylogenetic relationships.
The AP2 family genes exhibited the most complex gene structures, containing four to eight exons. In contrast, most ERF subfamily and DREB subfamily genes generally had one to two exons, RAV family genes consistently contained one exon, and Soloist family genes had six exons. These structural patterns are consistent with those observed in other plant species [56], suggesting evolutionary conservation of gene structures within each family.
The distribution of introns within the AP2/ERF domain was also family-specific. AP2 family genes typically contained introns within their AP2/ERF domains, while the majority of ERF subfamily and DREB subfamily genes lacked introns in this region. This differential intron pattern likely reflects the ancient divergence between the AP2 family and ERF family and may have functional implications for the regulation of gene expression and alternative splicing.
Conserved motif analysis using the MEME program identified 10 distinct motifs (designated as Motifs 1–10) across the marigold AP2/ERF proteins (Supplementary Figures S1 and S4). Motifs 1, 2, 3, and 4 were identified as the core components of the AP2/ERF domain and were present in all AP2/ERF proteins, confirming their conserved nature across the superfamily. The distribution of the remaining motifs showed family- and group-specific patterns. For instance, Motifs 5 and 8 were specifically present in the AP2 family. Motifs 9 and 10 were exclusively found in B3 of ERF subfamily proteins. Motif 6 was only present in the DREB subfamily and Motif 7 was only present in A1 and A4 of the DREB subfamily.

3.4. Chromosomal Distribution and Duplication Analysis

To investigate the genomic organization and evolutionary expansion of the AP2/ERF superfamily genes in marigold, we mapped their chromosomal positions and analyzed duplication events (Figure 2). The 177 AP2/ERF genes were unevenly distributed across 11 of the 12 marigold chromosomes. Chromosome 1 harbored the highest number of AP2/ERF genes (38 genes), while chromosome 12 contained no AP2/ERF genes.
Analysis of gene duplication events identified 56 pairs of segmentally duplicated TeAP2/ERF genes (Figure 3), predominantly involving ERF subfamily and DREB subfamily members. The prevalence of segmental duplications suggests that whole-genome or segmental duplication events have contributed significantly to the expansion of the AP2/ERF superfamily members in marigold. Calculation of non-synonymous (Ka) to synonymous (Ks) substitution ratios for the duplicated gene pairs revealed that all pairs had Ka/Ks ratios less than 1 (Supplementary Table S5), such as TeAP2/ERF011-TeAP2/ERF043 (Ka/Ks = 0.101) and TeAP2/ERF052-TeAP2/ERF107 (Ka/Ks = 0.093). This indicates that the AP2/ERF superfamily genes are subject to strong purifying selection within the species, with highly conserved functions where mutations likely disrupt their biological functions. Some gene pairs show Ka/Ks ratios approaching 0.5, such as TeAP2/ERF059-TeAP2/ERF119 (Ka/Ks = 0.463) and TeAP2/ERF033-TeAP2/ERF064 (Ka/Ks = 0.366). While still less than 1, these values suggest that certain families may be experiencing weaker purifying selection or contain local sites with positive selection signals that require further validation.
To understand the evolutionary relationships of AP2/ERF genes between marigold and other plant species, we performed synteny analysis with Arabidopsis (a model dicot) and lettuce (a related Asteraceae species) (Figure 4). We identified 49 syntenic AP2/ERF gene pairs between marigold and Arabidopsis and 93 pairs between marigold and lettuce. The higher number of syntenic pairs between marigold and lettuce reflects their closer evolutionary relationship as members of the Asteraceae family. The syntenic gene pairs were predominantly distributed in the AP2 and ERF family members, suggesting that these families may have experienced stronger selection pressure for functional conservation during the divergence of these species.

3.5. Expression Patterns of AP2 Family Genes Reveal Their Roles in Tissue-Specific and Floral Organ Development

AP2 family members function as A-class genes in the classical ABCDE model of floral organ development, playing essential roles in the specification of sepals and petals [32,57,58]. Additionally, given our research focus on understanding the molecular mechanisms controlling the distinctive capitulum architecture in marigold, we prioritized the 28 AP2 family genes and analyzed their expression patterns across different tissues and developmental stages using qRT-PCR.
We analyzed the temporal expression patterns of AP2 family genes during flower bud development, examining flower buds at different developmental stages (0–1 mm, 4–5 mm, and 9–10 mm diameter) (Figure 5 and Figure S2). Several genes showed stage-specific expression patterns. For instance, TeAP2/ERF014, TeAP2/ERF030, TeAP2/ERF038, TeAP2/ERF050, and TeAP2/ERF145 were highly expressed in early-stage flower buds, suggesting their involvement in floral organ initiation, while TeAP2/ERF009, TeAP2/ERF026, TeAP2/ERF036, and TeAP2/ERF042 showed increasing expression as flower development progressed, indicating their potential roles in later stages of floral development.
Based on their predominant expression patterns of the 28 AP2 family genes across different tissues (Figure 6 and Figure S3), these genes could be classified into four groups: (1) those with high expression in vegetative tissues (roots, stems, and leaves), (2) those predominantly expressed in reproductive tissues (receptacle and flowers), (3) those specifically expressed in seeds, and (4) those with broad expression across multiple tissues.
Several AP2 family genes, including TeAP2/ERF030, TeAP2/ERF036, TeAP2/ERF048, TeAP2/ERF138, and TeAP2/ERF154, showed high expression in roots. TeAP2/ERF028, TeAP2/ERF050, and TeAP2/ERF137, were highly expressed in the stems, while TeAP2/ERF015 and TeAP2/ERF114 exhibited high expression in leaves. Twelve genes, including TeAP2/ERF002, TeAP2/ERF009, TeAP2/ERF014, and TeAP2/ERF035 etc., were highly expressed in receptacles. In bracts, TeAP2/ERF026, TeAP2/ERF042, and TeAP2/ERF111 showed notably high expression levels. Interestingly, TeAP2/ERF038, TeAP2/ERF103, and TeAP2/ERF137 showed particularly high expression in seeds, suggesting potential roles in seed development or maturation.
Given the distinctive floral architecture of marigold, characterized by peripheral ray florets and central disc florets, we further investigated the expression patterns of AP2 family genes in different floral organs of these two floret types (Figure 6, Figure 7 and Figure S3). In the corollas, several genes showed differential expression between the two floret types. Notably, TeAP2/ERF114 and TeAP2/ERF145 exhibited significantly higher expression in ray floret corollas compared to disc floret corollas. This elevated expression suggests their potential role in determining the enlarged, showy corolla morphology characteristic of ray florets. In disc florets, which uniquely possess stamens, several genes showed high stamen-specific expression. TeAP2/ERF036 showed the highest expression in stamens among all floral organs, while TeAP2/ERF103 also exhibited strong expression in this male reproductive organ. The stamen-specific expression of these genes suggests their involvement in stamen development or function, which may contribute to the fertility of disc florets.
When comparing the same organs between ray and disc florets, we observed both consistent and differential expression patterns. In pappus, TeAP2/ERF015, TeAP2/ERF137, and TeAP2/ERF151 were highly expressed in both floret types, while TeAP2/ERF042 and TeAP2/ERF111 showed notably higher expression in ray floret pappus. In pistils, TeAP2/ERF035 and TeAP2/ERF154 exhibited high expression in both floret types, indicating conserved functions in pistil development. For ovaries, TeAP2/ERF103 and TeAP2/ERF151 showed consistently high expression in both floret types, suggesting their roles in ovule or seed development. Several genes showed broader expression patterns across multiple floral organs. TeAP2/ERF114 exhibited high expression in both pappus and corollas of ray florets, consistent with the A-class function in the floral organ identity model. Similarly, TeAP2/ERF100, TeAP2/ERF103, and TeAP2/ERF111 were expressed across multiple organs in both floret types, indicating their potential involvement in general floral development processes.

3.6. Protein Interaction Network Analysis of AP2 Family, MADS-Box, and TCP Family Transcription Factors

Based on our expression profile analysis results, which revealed distinct expression patterns and significant differential expression of the 28 AP2 family genes across tissues and between ray and disc florets, we specifically focused on these genes for protein–protein interaction network based on homology with Arabidopsis proteins (Figure 8). The resulting network revealed extensive predicted interactions between AP2 family proteins and members of the MADS-box and TCP transcription factors. AP2 emerged as a central hub in this network, showing strong interactions with multiple MADS-box proteins. The TeAP2/ERF026, TeAP2/ERF035, TeAP2/ERF114, and TeAP2/ERF154 identified in our study correspond to AP2 in Arabidopsis, highlighting their potential conservation of function.
These interactions suggest potential cooperative functions between AP2 family and MADS-box proteins in regulating flower development in marigold. Notably, TCP1, a homolog of CYC2-like genes which are known to regulate floral symmetry in Asteraceae, was also integrated into this network. The predicted interactions between AP2 family, MADS-box, and TCP family proteins provide molecular evidence for the potential cross-talk between these three transcription factor families in controlling floral organ specification and floret identity in marigold. The strength of these predicted interactions, indicated by the thickness of connecting lines in the network, suggests varying degrees of functional association, with particularly strong connections observed between AP2 protein and several MADS-box proteins (including AP3, AGL8, and SOC1).

4. Discussion

4.1. Evolutionary Relationships and Structural Features of AP2/ERF Genes in Marigold

In this study, we conducted a comprehensive genome-wide analysis of the AP2/ERF superfamily members in marigold, an important ornamental plant with distinctive heterogamous capitulum inflorescence, and investigated their potential roles in floral development, particularly in the differentiation of ray and disc florets.
We identified 177 AP2/ERF genes in the marigold genome, which is comparable to the number found in other plant species such as Arabidopsis (145) [7], rice (170) [59], sunflower (288) [60], lettuce (228), and tomato (134) [61]. The classification of TeAP2/ERF genes into different families based on domain architecture and phylogenetic analysis is consistent with previous studies in other plants [7,8]. Notably, the total number of ERF subfamily and DREB subfamily genes (143) far exceeds the number of AP2 family genes (28), and the predominance of ERF family genes (including both ERF and DREB subfamilies) aligns with findings in other species [3,62,63]. This pattern highlights the evolutionary conservation and functional significance of the ERF family members across plant lineages and reflects the central role of ERF and DREB genes in diverse stress response pathways [11]. Comparison between marigold and other Asteraceae species like lettuce reveals interesting patterns in the composition of AP2/ERF superfamily genes (Table 1). While the total number of AP2/ERF superfamily genes in marigold (177) is somewhat lower than in lettuce (228), the proportional distribution across families shows similarities, particularly in the expansion of the ERF family relative to the AP2 family. These observations suggest conserved evolutionary patterns within the Asteraceae family, possibly reflecting shared adaptive pressures.
The gene structure and motif composition analysis revealed distinct patterns among different subfamilies, reflecting their evolutionary divergence and functional specialization. The AP2 family genes typically contained four to eight exons, while most ERF and DREB subfamily genes had only one to two exons. This structural difference has been observed across multiple plant species [56,60] and likely represents an ancient divergence in the evolution of the AP2/ERF superfamily members. The presence of multiple introns in AP2 family genes could provide additional opportunities for alternative splicing and post-transcriptional regulation, potentially contributing to their functional complexity in developmental processes [64]. These family-specific motifs likely contribute to the functional specificity of different AP2/ERF proteins and may serve as transcriptional activation domains, nuclear localization signals, or protein–protein interaction sites [65,66,67]. A comparative analysis of gene structure between marigold and lettuce revealed striking conservation patterns within the AP2/ERF superfamily members [43]. In both Asteraceae species, AP2 family members consistently displayed more complex gene architecture with multiple introns (ranging from four to eight in marigold and four to thirteen in lettuce). By contrast, the vast majority of ERF family members exhibited a markedly simpler structure, predominantly lacking introns entirely. This pronounced structural dichotomy, preserved across these evolutionarily related species, provides compelling evidence for an ancient divergence event that separated the AP2 family members from the ERF family members long before the diversification of the Asteraceae family.
Our analysis of chromosomal distribution and duplication events revealed that segmental duplications have played a major role in the expansion of the AP2/ERF superfamily genes in marigold [68]. The prevalence of segmental duplications suggests that whole-genome or large-scale chromosomal duplication events have been particularly important in the evolutionary history of marigold’s AP2/ERF superfamily genes. The collinearity relationships in the marigold AP2/ERF superfamily genes are primarily driven by purifying selection, while elevated Ka/Ks ratios in specific gene pairs may reflect functional specialization or adaptive evolutionary events that require further verification through functional experiments.
The synteny analysis between marigold and other plant species provided evolutionary evidence for the conservation and divergence of AP2/ERF genes. The higher number of syntenic pairs between marigold and lettuce (93 pairs), compared to marigold and Arabidopsis (49 pairs), reflects their closer evolutionary relationship as members of the Asteraceae family. This observation aligns with previous studies showing higher synteny among closely related species [69]. The preferential conservation of syntenic relationships in certain families, particularly the AP2 and ERF families, suggests stronger functional constraints on these families during evolution, possibly due to their roles in essential developmental and adaptive processes.

4.2. Diverse Expression Patterns Reveal Functional Specialization of AP2 Family Genes in Marigold

The expression analysis of AP2 family genes revealed diverse spatial and temporal expression patterns, suggesting their involvement in various developmental processes in marigold. The tissue-specific expression patterns observed for many AP2 family genes provide valuable clues about their potential functions. The high expression of TeAP2/ERF030, TeAP2/ERF036, TeAP2/ERF048, TeAP2/ERF138, and TeAP2/ERF154 in roots suggests potential roles in root development or root-specific responses, similar to functions reported for certain AP2 genes in Arabidopsis [70] and rice [71]. Similarly, the seed-specific expression of TeAP2/ERF038, TeAP2/ERF130, and TeAP2/ERF137 indicates their potential roles in seed development or maturation, consistent with the functions of AP2 genes in seed development in other plants [72,73,74].
Several genes showed specific expression patterns in Asteraceae-specific structures such as the receptacle and bracts, which are crucial components of the capitulum [75]. TeAP2/ERF002, TeAP2/ERF009, TeAP2/ERF014, TeAP2/ERF035, etc., were highly expressed in receptacles, suggesting their potential roles in the development of this specialized structure that supports the florets. Similarly, TeAP2/ERF026, TeAP2/ERF042, and TeAP2/ERF111 showed high expression in bracts, indicating their possible involvement in the development of these protective structures surrounding the capitulum.
One of the most intriguing findings of our study is the differential expression of several AP2 family genes between ray and disc florets, suggesting their potential roles in determining floret identity or organ development in the complex inflorescence of marigold. The expression of TeAP2/ERF036 and TeAP2/ERF103 specifically in stamens of disc florets, while they are absent in sterile ray florets, suggests their potential roles in stamen development or function. This pattern could be relevant to understanding the evolutionary origin of ray florets, which are thought to have evolved from disc florets through the suppression of stamen development and corolla fusion [19]. Further functional studies of these genes could provide valuable insights into the molecular mechanisms underlying the evolution of the complex capitulum structure in Asteraceae. The preferential expression of TeAP2/ERF111, TeAP2/ERF114, and TeAP2/ERF145 in both pappus and corollas of disc and ray florets aligns with the canonical A-class function in the ABCDE model of floral organ development [76]. However, the additional expression of TeAP2/ERF111 in pistils of ray florets suggests an expanded functional role beyond the classical A-function typically limited to pappus and corollas [77]. The significantly higher expression of TeAP2/ERF145 in ray floret corollas compared to disc floret corollas is particularly interesting, as it may contribute to the development of the enlarged, showy corollas characteristic of ray florets in marigold. Similarly, the differential expression of TeAP2/ERF103, TeAP2/ERF114, and TeAP2/ERF13 between ray and disc florets suggests their potential involvement in establishing the distinct morphological and functional identities of these floret types [78].

4.3. Integration of AP2 Family, MADS-Box, and CYC2 Protein Regulatory Networks in Marigold Floral Development

The complex floral architecture of the Asteraceae family, characterized by the capitulum inflorescence with distinct ray and disc florets, is likely regulated by intricate genetic networks involving multiple transcription factor families. Our findings on the expression patterns of AP2 family genes, together with previous studies on MADS-box [39] and CYC2 [37] genes in marigold, provide an opportunity to explore the potential integration of these transcription factor networks in controlling floral development (Figure 9). Previous studies in marigold have identified and characterized several MADS-box genes involved in floral organ development, including A-class genes (TeAP1/FUL-like), B-class genes (TeAP3/PI-like), C/D-class genes (TeAG-like), and E-class genes (TeSEP-like). These genes showed distinct expression patterns in ray and disc florets, with some genes exhibiting floret type-specific expression. For instance, TeMADS49, TeMADS45, and TeMADS54 (A-class genes) were highly expressed in pappus of ray and disc florets, similar to the expression patterns we observed for TeAP2/ERF151. This suggests potential co-regulation or interaction between A-class MADS-box genes and A-class AP2 genes in specifying pappus identities in both floret types.
Interestingly, some MADS-box genes, such as TeMADS10 and TeMADS33 (A-class), were specifically expressed in the stamens of disc florets, similar to the expression of TeAP2/ERF103 in our study. This parallel expression pattern suggests a potential cooperative role of these genes in regulating stamen development specifically in disc florets.
Similarly, CYC2 genes in marigold have been shown to play crucial roles in regulating floral symmetry and organ development, particularly in the differentiation of ray and disc florets. Seven CYC2 genes were identified in marigold (TeCYC2a, TeCYC2b, TeCYC2c, TeCYC2d, TeCYC2e1, TeCYC2e2, and TeCYC2g). Among these, TeCYC2b, TeCYC2c, and TeCYC2d were specifically expressed in ray floret corollas, exhibiting an expression pattern similar to the ray floret-specific expression of TeAP2/ERF114 and TeAP2/ERF145 observed in our study. Notably, functional analysis revealed that TeCYC2c inhibits stamen development when overexpressed in Arabidopsis, providing a molecular explanation for the characteristic absence of stamens in marigold ray florets. The expression patterns of TeAP2 genes and TeCYC2 genes in marigold florets suggest potential interactions or regulatory relationships between these transcription factor families. For instance, the ray floret-specific expression of TeCYC2b, TeCYC2c, and TeCYC2d, together with the higher expression of TeAP2/ERF145 in ray floret corollas, suggests a possible genetic interaction in specifying ray floret identity and corolla development. Given the distinctive expression pattern of TeAP2/ERF145 and its potential role in determining ray floret morphology, this gene deserves particular attention in future functional studies to elucidate its precise regulatory mechanisms. Similarly, the expression of TeCYC2a, TeCYC2e1, and TeCYC2e2 in disc floret corollas, contrasted with the notably low expression of all TeAP2 family genes in this tissue, suggests a distinctive regulatory mechanism governing floret differentiation. The absence of TeAP2 family genes expression in disc floret corollas likely contributes to the development of their characteristic tubular morphology. This expression pattern indicates that while CYC family members are active in disc florets, the lack of AP2 family member activity may be a critical factor in determining disc floret identity. Conversely, the potential expression of TeAP2 family genes in ray florets could be instrumental in establishing their distinctive asymmetric structure and elongated ligule formation, highlighting how differential transcription factor activity may drive the morphological diversification of floret types within the composite inflorescence of Asteraceae.
Our protein–protein interaction network analysis further supports this integrated regulatory model, revealing extensive predicted interactions between AP2 family, MADS-box, and TCP family proteins (Figure 8). The emergence of AP2 as a central hub in this network, with strong connections to multiple MADS-box proteins (including AP3, AGL8, and SOC1) and TCP1 (a homolog of CYC2-like genes), provides molecular evidence for the coordinated function of these transcription factor families in floral development. The varying strengths of these predicted interactions suggest differential regulatory relationships, which may contribute to the distinctive development of ray and disc florets in marigold.
In other Asteraceae species, interactions between MADS-box, CYC2, and AP2 family members have been reported to regulate floral development. In chrysanthemum, the A-class gene ClAP2 was found to particularly accumulate in ray floret corollas and form heterodimers with CYC2-like proteins, suggesting their cooperative role in ray floret identity and development [29,79]. Similarly, in gerbera, a regulatory network involving CINCINNATA-like TCP proteins functions upstream of GhCYC to specify ray flower identity. The E-class MADS-box transcription factor GRCD5 was shown to activate GhCYC3 expression during ligule development, while the C-class MADS-box transcription factor GAGA1 appears to contribute to stamen development upstream of GhCYC3 [30]. These findings, together with our results on differential expression of AP2 family genes in marigold florets, demonstrate how intricate genetic networks involving AP2 family, MADS-box, and CYC2 family members collectively regulate the complex floral architecture of the Asteraceae family, with particular importance in controlling heteromorphic flower type identity and organ development.
In the context of the ABCDE model of floral organ development, our results suggest both conservation and divergence of AP2 family member functions in marigold compared to model plants. The expression patterns of several AP2 family genes in sepals and petals are consistent with the A-class function in the classical model. However, their expression in additional organs suggests potential neofunctionalization or subfunctionalization in the complex floral architecture of marigold. This functional divergence might be related to the evolution of the unique capitulum structure in Asteraceae, which represents one of the most successful adaptations in flowering plants [80].

5. Conclusions

In this study, we conducted a comprehensive genome-wide analysis of the AP2/ERF superfamily members in marigold, identifying 177 members classified into four families: AP2 (28), ERF (143), RAV (4), and Soloist (2). These genes exhibited family-specific structural features, conserved motifs, and chromosomal distributions, with significant expansion through segmental duplications under strong purifying selection. Expression profiling revealed tissue-specific and developmental stage-specific patterns, with several TeAP2 family genes showing differential expression between ray and disc florets that may contribute to the distinctive floral architecture of marigold. Protein interaction network analysis suggested cross-talk among AP2 family, MADS-box, and TCP family proteins, providing insights into how these regulatory networks collectively shape the complex capitulum structure. Our findings establish a foundation for future functional studies of TeAP2/ERF superfamily members in marigold and contribute to understanding the molecular mechanisms underlying floral development in Asteraceae.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15051231/s1, Figure S1: Gene structure and conserved motif analysis of AP2/ERF superfamily members in marigold; Figure S2: Relative expression levels of 28 AP2 family genes across sequential stages of flower bud development in marigold; Figure S3: Relative expression levels of 28 AP2 family genes across different marigold tissues; Table S1: Primer information of qRT-PCR; Table S2: Sequences of TeAP2/ERF proteins; Table S3: Analysis of physicochemical properties and subcellular localization of AP2/ERF proteins; Table S4: Conserved motifs identified in marigold AP2/ERF proteins using the MEME motif search tool; Table S5: Ka/Ks analysis for the paralogous gene pairs.

Author Contributions

Conceptualization, H.L., G.C., M.B. and Y.H.; methodology, H.L.; software, G.C. and C.L.; validation, H.L. and G.C.; formal analysis, H.L., G.C. and S.H.; investigation, H.L., G.C. and S.H.; resources, H.L. and G.C.; data curation, H.L., G.C. and C.L.; writing—original draft preparation, H.L. and G.C.; writing—review and editing, M.B. and Y.H.; visualization, G.C.; supervision, M.B. and Y.H.; project administration, M.B. and Y.H.; funding acquisition, Y.H. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Natural Science Foundation of China (No. 32172616).

Data Availability Statement

The original data underlying this study are available within the article and its Supplementary Materials. For additional inquiries, please contact the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic analysis and classification of AP2/ERF superfamily members in marigold and Arabidopsis. The maximum-likelihood phylogenetic tree was constructed using full-length protein sequences of AP2/ERF superfamily members from marigold (Tagetes erecta) and Arabidopsis (Arabidopsis thaliana). The different colored arcs represent different families: AP2 family (AP2, ANT, and AIL, purple shades), RAV family (brown), Soloist family (orange), DREB subfamily (A1–A6, blue shades), and ERF subfamily (B1–B6, green shades). Marigold proteins are labeled as TeAP2/ERF followed by numbers, while Arabidopsis proteins are shown with their original gene names. Red and green spots indicate AP2/ERF superfamily members from marigold and Arabidopsis, respectively.
Figure 1. Phylogenetic analysis and classification of AP2/ERF superfamily members in marigold and Arabidopsis. The maximum-likelihood phylogenetic tree was constructed using full-length protein sequences of AP2/ERF superfamily members from marigold (Tagetes erecta) and Arabidopsis (Arabidopsis thaliana). The different colored arcs represent different families: AP2 family (AP2, ANT, and AIL, purple shades), RAV family (brown), Soloist family (orange), DREB subfamily (A1–A6, blue shades), and ERF subfamily (B1–B6, green shades). Marigold proteins are labeled as TeAP2/ERF followed by numbers, while Arabidopsis proteins are shown with their original gene names. Red and green spots indicate AP2/ERF superfamily members from marigold and Arabidopsis, respectively.
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Figure 2. Chromosomal distribution of AP2/ERF superfamily genes in marigold. The 177 AP2/ERF genes identified in this study are distributed across 11 of the 12 marigold chromosomes (TeORChr1-TeORChr11). Chromosome numbers are labeled on the left side of each chromosome, with their physical lengths presented in megabases (Mb) using the scale bar on the left. Gene IDs are labeled at their corresponding chromosome positions according to their actual locations. Gene density for each chromosome was calculated using 500 kb genetic intervals, and a gradient color scale was used to characterize gene density distribution, where the red to blue gradient corresponds to high-density to low-density regions (red: gene-rich regions; blue: gene-sparse regions). Blank areas represent genomic regions lacking gene distribution information.
Figure 2. Chromosomal distribution of AP2/ERF superfamily genes in marigold. The 177 AP2/ERF genes identified in this study are distributed across 11 of the 12 marigold chromosomes (TeORChr1-TeORChr11). Chromosome numbers are labeled on the left side of each chromosome, with their physical lengths presented in megabases (Mb) using the scale bar on the left. Gene IDs are labeled at their corresponding chromosome positions according to their actual locations. Gene density for each chromosome was calculated using 500 kb genetic intervals, and a gradient color scale was used to characterize gene density distribution, where the red to blue gradient corresponds to high-density to low-density regions (red: gene-rich regions; blue: gene-sparse regions). Blank areas represent genomic regions lacking gene distribution information.
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Figure 3. Segmental duplications of TeAP2/ERF genes in marigold. Circular representation of the 56 segmental duplication pairs of TeAP2/ERF genes mapped across the 11 marigold chromosomes (Chr1–Chr11). Chromosomes are depicted as colored arcs with gene density represented by the red histogram track along each chromosome. TeAP2/ERF genes are labeled at their respective chromosome positions. Red curved lines connect segmentally duplicated AP2/ERF gene pairs, revealing extensive inter-chromosomal duplication events, while gray lines represent other duplication gene pairs of non-AP2/ERF genes across the genome. The inner heat map bands on each chromosome illustrate gene density with a blue to red gradient (representing low to high density).
Figure 3. Segmental duplications of TeAP2/ERF genes in marigold. Circular representation of the 56 segmental duplication pairs of TeAP2/ERF genes mapped across the 11 marigold chromosomes (Chr1–Chr11). Chromosomes are depicted as colored arcs with gene density represented by the red histogram track along each chromosome. TeAP2/ERF genes are labeled at their respective chromosome positions. Red curved lines connect segmentally duplicated AP2/ERF gene pairs, revealing extensive inter-chromosomal duplication events, while gray lines represent other duplication gene pairs of non-AP2/ERF genes across the genome. The inner heat map bands on each chromosome illustrate gene density with a blue to red gradient (representing low to high density).
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Figure 4. Synteny analysis of AP2/ERF genes between marigold and related species. Syntenic relationships of AP2/ERF genes between (A) marigold and Arabidopsis and (B) marigold and lettuce. Chromosomes of Arabidopsis (Chr1–Chr5) and lettuce (Chr1–Chr9) are represented by orange bars, while green bars represent the corresponding marigold (TeChr1–TeChr12) AP2/ERF genes. Red curves connect syntenic gene pairs between the species, with the intensity of red indicating the strength of syntenic relationships. The grey lines represent other collinear gene pairs of non-AP2/ERF genes across genomes.
Figure 4. Synteny analysis of AP2/ERF genes between marigold and related species. Syntenic relationships of AP2/ERF genes between (A) marigold and Arabidopsis and (B) marigold and lettuce. Chromosomes of Arabidopsis (Chr1–Chr5) and lettuce (Chr1–Chr9) are represented by orange bars, while green bars represent the corresponding marigold (TeChr1–TeChr12) AP2/ERF genes. Red curves connect syntenic gene pairs between the species, with the intensity of red indicating the strength of syntenic relationships. The grey lines represent other collinear gene pairs of non-AP2/ERF genes across genomes.
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Figure 5. Expression profiles of AP2 family genes during flower bud development in marigold. Expression intensity is represented by circle size and color depth, with larger, darker circles indicating higher expression levels. The genes are clustered based on their expression patterns.
Figure 5. Expression profiles of AP2 family genes during flower bud development in marigold. Expression intensity is represented by circle size and color depth, with larger, darker circles indicating higher expression levels. The genes are clustered based on their expression patterns.
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Figure 6. Expression profiles of AP2 family genes in different marigold tissues. The color scale represents normalized expression levels from low (light pink) to high (dark burgundy). Hierarchical clustering of genes (left) groups those with similar expression patterns.
Figure 6. Expression profiles of AP2 family genes in different marigold tissues. The color scale represents normalized expression levels from low (light pink) to high (dark burgundy). Hierarchical clustering of genes (left) groups those with similar expression patterns.
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Figure 7. Differential expression patterns of AP2 family genes in floral organs of ray and disc florets in marigold. To clarify the expression characteristics of AP2 family genes in marigold inflorescences, this study selected 12 candidate genes with normalized expression values > 0.3 in at least one tissue region of either ray florets or disc florets for visualization analysis according to Figure 6. Heatmap showing the expression profiles of 12 TeAP2 family genes across different floral organs of ray florets (pappus, corolla, pistil, ovary) and disc florets (pappus, corolla, stamen, pistil, ovary). Expression levels are represented by a color gradient from light pink (low expression) to dark burgundy (high expression).
Figure 7. Differential expression patterns of AP2 family genes in floral organs of ray and disc florets in marigold. To clarify the expression characteristics of AP2 family genes in marigold inflorescences, this study selected 12 candidate genes with normalized expression values > 0.3 in at least one tissue region of either ray florets or disc florets for visualization analysis according to Figure 6. Heatmap showing the expression profiles of 12 TeAP2 family genes across different floral organs of ray florets (pappus, corolla, pistil, ovary) and disc florets (pappus, corolla, stamen, pistil, ovary). Expression levels are represented by a color gradient from light pink (low expression) to dark burgundy (high expression).
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Figure 8. Protein–protein interaction network of AP2 family, MADS-box, and TCP family transcription factors based on homology with Arabidopsis. Nodes represent transcription factors, with node size and color intensity (from yellow to deep red) indicating the degree of connectivity. Line thickness represents the confidence level of predicted interactions, with thicker lines indicating stronger interaction scores. The TeAP2 labels (TeAP2XXX) represent marigold AP2 family transcription factors corresponding to their Arabidopsis homologs in the network.
Figure 8. Protein–protein interaction network of AP2 family, MADS-box, and TCP family transcription factors based on homology with Arabidopsis. Nodes represent transcription factors, with node size and color intensity (from yellow to deep red) indicating the degree of connectivity. Line thickness represents the confidence level of predicted interactions, with thicker lines indicating stronger interaction scores. The TeAP2 labels (TeAP2XXX) represent marigold AP2 family transcription factors corresponding to their Arabidopsis homologs in the network.
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Figure 9. Expression patterns of AP2 family members alongside MADS-box and CYC2 transcription factor family members in different organs of marigold. The schematic shows tissue-specific expression of TeAP2 family genes (labeled as AP2XXX) in comparison with MADS-box (AP1, FUL, AP3/PI, AG, SEP, AGL6, AGL15, AGL17, BS, MIKC*) and CYC2 (CYC2a, CYC2b, CYC2c, CYC2d, CYC2e1, CYC2e2, CYC2g) family genes across different marigold organs. Genes highly expressed in each tissue are grouped in colored ovals, with the size of the ovals representing the number of genes (1–6) as indicated in the legend.
Figure 9. Expression patterns of AP2 family members alongside MADS-box and CYC2 transcription factor family members in different organs of marigold. The schematic shows tissue-specific expression of TeAP2 family genes (labeled as AP2XXX) in comparison with MADS-box (AP1, FUL, AP3/PI, AG, SEP, AGL6, AGL15, AGL17, BS, MIKC*) and CYC2 (CYC2a, CYC2b, CYC2c, CYC2d, CYC2e1, CYC2e2, CYC2g) family genes across different marigold organs. Genes highly expressed in each tissue are grouped in colored ovals, with the size of the ovals representing the number of genes (1–6) as indicated in the legend.
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Table 1. Comparative analysis of AP2/ERF superfamily member distribution in marigold, lettuce, and Arabidopsis.
Table 1. Comparative analysis of AP2/ERF superfamily member distribution in marigold, lettuce, and Arabidopsis.
FamilySubfamilyGroupTagetes erectaLactuca sativaArabidopsis
thaliana
RAV 446
Soloist 221
AP2AP2 182010
ANT 431
AIL 667
ERFDREBA18146
A23128
A3111
A4242916
A5161415
A661110
ERFB1191915
B2665
B3374517
B4397
B52108
B6182312
Total 177228 *145
* The set of 228 genes analyzed in the comparative section was identified from the lettuce (Lactuca sativa) genome, specifically from the “Genome assembly Lsat_Salinas_v8” (GCA_002870075.3), which is available at the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_002870075.3/, accessed on 5 January 2025).
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Li, H.; Chen, G.; Hu, S.; Liu, C.; Bao, M.; He, Y. Genomic Architecture of AP2/ERF Superfamily Genes in Marigold (Tagetes erecta) and Insights into the Differential Expression Patterns of AP2 Family Genes During Floral Organ Specification. Agronomy 2025, 15, 1231. https://doi.org/10.3390/agronomy15051231

AMA Style

Li H, Chen G, Hu S, Liu C, Bao M, He Y. Genomic Architecture of AP2/ERF Superfamily Genes in Marigold (Tagetes erecta) and Insights into the Differential Expression Patterns of AP2 Family Genes During Floral Organ Specification. Agronomy. 2025; 15(5):1231. https://doi.org/10.3390/agronomy15051231

Chicago/Turabian Style

Li, Hang, Guoqing Chen, Shirui Hu, Cuicui Liu, Manzhu Bao, and Yanhong He. 2025. "Genomic Architecture of AP2/ERF Superfamily Genes in Marigold (Tagetes erecta) and Insights into the Differential Expression Patterns of AP2 Family Genes During Floral Organ Specification" Agronomy 15, no. 5: 1231. https://doi.org/10.3390/agronomy15051231

APA Style

Li, H., Chen, G., Hu, S., Liu, C., Bao, M., & He, Y. (2025). Genomic Architecture of AP2/ERF Superfamily Genes in Marigold (Tagetes erecta) and Insights into the Differential Expression Patterns of AP2 Family Genes During Floral Organ Specification. Agronomy, 15(5), 1231. https://doi.org/10.3390/agronomy15051231

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