Acer paxii is an evergreen tree classified under the genus Acer within the family Sapindaceae, and it is endemic to China. It is primarily distributed in Yunnan Province, including the Kunming region. This species thrives in the natural environment of Kunming, adapting well to the local climate and soil conditions. The evergreen nature of Acer paxii allows it to add greenery to the landscape throughout all four seasons, making it highly valuable for ornamental and ecological purposes [1]. Acer paxii is highly resistant, with dense foliage and branches, and is often planted in gardens as avenue trees and shade trees. After being transplanted to Hefei, Anhui Province, Acer paxii leaves turn red in the fall, which is highly ornamental and very suitable for cities, gardens, and road beautification [2].

Several studies have systematically examined and quantified the pigmentation and physiological indicators of leaves in colorful foliage plants, revealing the mechanisms of leaf color change [3]. Anthocyanins play a direct role in influencing the key mechanisms of leaf color transition to purple or red hues [4]. Anthocyanins, a group of naturally occurring compounds classified under the flavonoid group, are produced via the phenylpropanoid biosynthetic pathway in plants [5]. These naturally occurring pigments, displaying diverse hues spanning crimson, violet, and indigo, are extensively present in multiple plant structures such as blossoms, berries, foliage, and root systems [6]. The production of anthocyanins in plant cells is governed by an intricate regulatory network, in which critical enzymes play a central role in coordinating this process. Within the flavonoid biosynthetic pathway, these enzymes are indispensable for driving precise biochemical transformations. The biosynthesis of anthocyanins begins with the action of chalcone synthase (CHS), which catalyzes the formation of chalcones from phenylpropanoid precursors. These chalcones are then isomerized by chalcone isomerase (CHI) to produce flavanones. Flavanone 3-hydroxylase (F3H) further modifies these intermediates, and dihydroflavonoid 4-reductase (DFR) catalyzes the reduction of dihydroflavonols to leucoanthocyanidins. Finally, anthocyanidin synthase (ANS) plays a pivotal role in converting leucoanthocyanidins into colored anthocyanidins, which are subsequently stabilized into anthocyanins. These enzymatic steps collectively ensure the biosynthesis of anthocyanins. The compounds are responsible for the striking colors of plant tissues and significantly contribute to the regulation of pigment biosynthesis and diversity in plants [7].

Beyond enzymatic catalysis, anthocyanin biosynthesis is precisely orchestrated via the regulatory influence of specific transcriptional activators [8]. Transcription factors not only indirectly influence the accumulation of anthocyanin by modulating the transcriptional activity of genes involved in the anthocyanin biosynthetic pathway (such as CHS, DFR, and ANS), but also directly participate in modulating critical steps within the anthocyanin biosynthetic pathway. For example, in apples (Malus × domestica), MdERF1B regulates anthocyanin and proanthocyanidin biosynthesis [9]. MdLNC499 connects the functions of MdWRKY1 and MdERF109, playing a crucial part in the modulation of anthocyanin accumulation in apple fruit [10]. Besides, MYB can function independently or in a ternary complex with two other Transcription Factors (bHLH and WD40, called MBW) [11]. In poplar, metabolic changes induced by the activation of PdCHS and PdANS via PdMYB113 present a key mechanism for coloration in ornamental species [12]. In the red autumn leaves of toothed oak (Quercus dentata), the transcription factor QdNAC regulates anthocyanin accumulation by interacting directly with QdMYB [13]. A genome-wide study of Pelargonium lanceolatum identified 158 R2R3-MYB transcription factors, with MYB113 specifically linked to anthocyanin biosynthesis [14]. Additionally, transcriptome analysis conducted when the leaves of Populus euramericana ‘Zhonghuahongye’ were changing color, from the initial reddish-purple through an intermediate stage to the final greenish-purple phase, showed that bHLH35 and bHLH63 serve as negative regulators of anthocyanin synthesis [15].

The AP2/ERF superfamily, also known as AP2/ethylene-responsive element binding factors, constitutes a major and highly conserved group of transcriptional regulators in plants [16]. Each member of the AP2/ERF family has a conserved AP2 domain [17], which is responsible for DNA binding and typically comprises 60 to 70 amino acids [18]. Based on the number of conserved AP2/ERF domains and variations in their structural organization, the AP2/ERF TFs are classified into five distinct subgroups AP2 (APETALA2), DREB (dehydration-responsive element binding proteins), ERF (ethylene-responsive factors), RAV (related to ABI3/VP1), and Soloist [19]. Structural characterization revealed distinct domain organizations among subfamilies: the AP2 group contains dual AP2/ERF domains, whereas ERF members feature a single AP2 domain. Notably, RAV proteins are characterized by the presence of both an AP2 domain and a unique B3 DNA-binding motif [20]. Phylogenetic analysis revealed that both DREB and ERF subgroups diverged into six distinct clades, with DREB lineages categorized as B1-B6 and ERF lineages grouped into A1-A6 clusters [21].

The AP2/ERF transcription factors regulate multiple aspects of plant biology, including developmental processes, hormonal signaling pathways [22]. The AP2/ERF transcription factors have been reported to be involved in Arabidopsis thaliana flower development [23]. PsERF1 controls the ripening of plum fruit by means of the ethylene pathway [24]. The AP2/ERF family gene MaERF11 in bananas plays a crucial role in ethylene-mediated fruit ripening through its function as a transcriptional repressor of the MaACO1 and MaACS1 promoters, thereby modulating the ripening process [25]. Throughout apple fruit maturation, MdERF2 functions as an inhibitory factor in ethylene production by downregulating MdACS1 expression, consequently modulating ripening progression [26]. Several AP2/ERF TFs have emerged as crucial regulators in enhancing plant adaptation to environmental stresses [27]. The AP2/ERF superfamily has been characterized on a genomic scale among various species. In Arabidopsis thaliana 147 gene members were identified [28]. A total of 163 gene members have been reported in Oryza sativa [29]. In Populus trichocarpa, the literature has documented the identification and characterization of 202 gene homologs in prior research [30]. In Pinus massoniana, 88 AP2/ERF members were identified [31]. In Actinidia eriantha, 158 AP2/ERFs were identified [32]. In Citrus reticulata, 126 AP2/ERFs were identified [33]. Vitis vinifera has 149 AP2/ERF members [34].

Accumulating evidence has demonstrated that AP2/ERF transcription factors play a regulatory role in the biosynthesis of anthocyanins. In Arabidopsis thaliana mutants, mutations in AtERF4 and AtERF8, reduce the rate and extent of anthocyanin production [35]. In ‘Zaosu’ pear, PbERF22 has been shown to positively regulate anthocyanin synthesis [36]. In ‘RedZaosu’ pearfruits, the control of anthocyanin buildup is mediated through the interaction between ethylene-responsive factors Pp4ERF24 and Pp12ERF96 with MYB114 [37]. The transcription factor ZbERF3 enhances anthocyanin biosynthesis in Zanthoxylum bungeanum when induced by ethylene [38]. In Litchi chinensis Sonn, ethylene enhances anthocyanin accumulation by activating positive regulators of anthocyanin biosynthesis, such as LcERF1/22/25/37, or by suppressing the negative regulator LcERF64 [39]. In Lily (Lilium spp.), LhERF4 has been identified as a negative regulator of anthocyanin accumulation, functioning through the suppression of LhMYBSPLATTER expression, a key player in the anthocyanin biosynthesis pathway [40]. These investigations indicate that AP2/ERF transcription factors could significantly influence the regulation of anthocyanin biosynthesis.

Current understanding of AP2/ERF-mediated regulation of anthocyanin accumulation in leaf pigmentation is still incomplete. Through genome-wide analysis of the Acer paxii genome, a number of 123 ApAP2/ERF genes were identified. We systematically investigated their evolutionary relationships and structural organization, and putative regulatory elements. And we measured pigment levels in green and red leaves of Acer paxii, conducting transcriptome sequencing, and performing qRT-PCR. These findings establish the foundation for subsequent explorations concerning the functional responsibilities and underlying mechanisms of AP2/ERF members within Acer paxii.

The AP2/ERFs represent a major class of regulatory proteins in plant systems, extensively present across multiple biological processes. These molecular regulators are involved in developmental regulation and environmental adaptation mechanisms in plants [52,53]. Through comprehensive genomic analysis, a total of 123 ApAP2/ERFs were identified in Acer paxii, showing a comparatively reduced quantity when contrasted with certain plant species such as Erianthus fulvus (145) [54], Actinidia eriantha (158) [32] and Solanum tuberosum L. (181) [55]. Through phylogenetic and structural domain analysis, the 123 ApAP2/ERF genes were categorized into five groups, and the ERF subgroup emerged as the most predominant cluster, comprising 63 sequences that represent approximately 51.2% of the complete set. This classification pattern demonstrates consistency with the genomic organization observed across various plant species, including Myrica rubra [56], Rhododendron [57], and Coptis chinensis Franch [58]. Subfamilies such as DREB, ERF, and AP2 evolved distinct roles in stress responses, hormone signaling, and development, influenced by lineage-specific selective pressures [59]. Structural conservation of the AP2 domain contrasts with functional divergence in flanking regions, enabling varied cis-element recognition [60].

Gene duplication plays a critical role in driving the adaptive expansion and functional specialization of gene lineages [61]. Gene replication, including whole-genome duplication, tandem duplication, and segment duplication, etc., significantly impacts the functional diversity of gene families by providing raw genetic material for evolutionary innovation. For example, in the ERF subfamily, whole-genome duplication events in Brassicaceae and Poaceae led to the retention of duplicated genes, enabling functional divergence in stress responses and development. Tandem duplications, particularly in Subgroup IX, contributed to over half of its gene copies, with ancient events giving rise to functionally distinct clades like IXb from IXa [62].

The AP2/ERF transcription factor family has undergone significant functional differentiation and evolutionary expansion, driven by gene duplication. We analyzed duplicated genes based on their cis-regulatory elements and transcriptome data. Conserved gene pairs share more than 80% motif similarity in light-responsive, MYB binding, and ABA-responsive elements. This high conservation suggests these pairs maintain ancestral functions through gene dosage effects, with purifying selection acting to preserve their regulatory architecture [63]. Divergent gene pairs like ApERF019 and ApERF110 exhibit striking differences in their regulatory elements. While ApERF110 contains 20 light-responsive elements, ApERF019 possesses 78, along with distinct ABA-responsive motifs. This regulatory divergence suggests potential subfunctionalization, with ApERF019 showing enhanced light-responsive regulatory capacity compared to its duplicate ApERF110, which retains other ancestral functions.

Tandem duplicates show significantly greater motif conservation than segmental pairs. RNA-seq analysis revealed that among the 19 tandem-duplicated AP2/ERF gene pairs, the 16 pairs (84.2%) exhibited similar expression patterns in red and green leaves, suggesting potential functional redundancy. However, three pairs (15.8%) showed significant divergence, including ApERF0121 (highly expressed in red leaves) and its tandem duplicate ApERF120 (lowly expressed), indicating possible subfunctionalization or neofunctionalization after duplication. Similarly, among 27 segmentally duplicated pairs, 20 (74.1%) maintained similar expression patterns whereas 7 (25.9%) showed differential expression, exemplified by ApERF048 (red-leaf enriched) and its duplicate ApERF056 (red-leaf suppressed). This pattern of expression divergence aligns closely with the findings from studies on the AP2/ERF family in Solanaceae [64]. These observations together indicate that changes in expression patterns following gene duplication events are a significant driver of gene functional divergence.

Traditionally, conserved motifs in transcription factors have been associated with roles like protein interactions, DNA binding, and transcriptional regulation [65]. Motif analysis in the present research demonstrated that motifs 1, 2, 5, and 6 are predominantly linked to the AP2 domain within the ApAP2/ERF gene family. Specifically, motif 2 shows a connection with the subfamilies of ERF and DREB. These findings suggest that while the motifs in the AP2/ERF gene family exhibit significant evolutionary stability, the newly identified motifs may be linked with unique functional roles in plants, warranting further investigation. Introns boost gene expression by enhancing transcription, mRNA stability, and translation efficiency. They enable alternative splicing, allowing single genes to produce multiple protein variants. Evolutionarily, introns provide recombination sites that drive genetic innovation [66]. In our study, we observed that Acer paxii AP2/ERF members, particularly those in the AP2 subfamily, predominantly retain introns, which is a striking contrast to other species where AP2/ERF genes often exhibit fewer introns [43,56]. This divergence may reflect lineage-specific evolutionary dynamics.

Cis-acting elements are crucial for controlling gene expression [67]. AP2/ERF transcription factors are known to broadly modulate gene expression and are potentially involved in multiple signaling pathways, such as those regulated by SA, JA/ET, and ABA [68]. Numerous phytohormone-responsive and light-responsive elements, along with elements associated with stress responses, are present within the promoter zone of the ApAP2/ERF genes. Our study identified several key AP2/ERF genes, including ApERF006, ApERF014, ApERF048, ApERF097, and ApERF107, these genes show significantly elevated expression in red leaves which contain various light-responsive and phytohormone-responsive cis-elements and suggest that these genes may integrate environmental light signals with hormonal pathways to regulate leaf discoloration. Additionally, cis-acting elements within the ApAP2/ERF gene family were identified to contain numerous MYB binding sites, which often through formation of MBW complexes with bHLH and WD40 partners. Given the involvement of MYB transcription factors in anthocyanin biosynthesis [11]. For the high-expression genes during red leaf development, our data shows that ApERF048 and ApERF097 contain both MYB binding sites, suggesting that they may bind to MYB sites and be involved in anthocyanin-related pathways, potentially influencing color variation in plants.

Homologous lineage analysis with other species showed a certain number of homologous gene pairs between Acer paxii and Citrus reticulata, Populus trichocarpa, Vitis vinifera, and Arabidopsis thaliana. In this analysis, the ApAP2/ERF genes exhibited strong sequence similarity with Populus trichocarpa but showed relatively lower conservation when compared to Arabidopsis thaliana. These analyses shed light on the genetic divergence and duplication events of the Acer paxii AP2/ERF family, offering valuable insights into its evolutionary history and functional diversification.

Changes in anthocyanin content are an important cause of color change in Acer paxii leaf blades. Due to constraints in experimental resources, we initially focused on the two most representative stages (green and red) to identify genes potentially associated with the initiation and completion of discoloration. The experimental data showed an 8-fold enhancement in anthocyanin accumulation in red leaves relative to green leaves, reflecting pronounced seasonal differences in pigment production. Transcriptomic profiling combined with qRT-PCR verification revealed increased expression levels of certain ApAP2/ERF genes in the red foliage of autumn leaves, such as ApERF006, ApERF014, ApERF048, ApERF97, and ApERF107, and it is worth noting that ApERF048 is homologous to AtERF8, AtERF4, and AtERF8 participated in the anthocyanin production process [35], thus, it is hypothesized that ApERF048 likely exhibits analogous functional roles. Research across various species has demonstrated that AP2/ERF family members regulate anthocyanin biosynthesis [9,69–71]. Based on transcriptome analysis and previous studies, it is speculated that the ApAP2/ERF genes may play a role in regulating anthocyanin biosynthesis in Acer paxii leaves, contributing to their color variation. In future studies, we plan to conduct functional validations including ethylene and ABA treatments, light quality experiments, tobacco overexpression assays, and virus-induced gene silencing (VIGS). These experiments will systematically examine whether the candidate genes regulate leaf color transition through light signaling and hormone response pathways, thereby providing deeper mechanistic insights into this biological process.

In this study, through a genome-wide screening approach, 123 ApAP2/ERF genes were detected in Acer paxii. A series of in-depth bioinformatics analyses were performed to investigate their molecular characteristics, evolutionary connections, structural organization of genes, genomic locations, duplication events, regulatory elements, and transcriptional profiles. The results demonstrate that tandem duplication serves as a major mechanism driving the expansion of ApAP2/ERF genes, likely contributing significantly to organismal development and environmental adaptation. Additionally, differential expression of certain ApAP2/ERF genes was observed during leaf discoloration, accompanied by a significant rise in anthocyanin levels in red leaves during autumn. The study proposed that genes such as ApERF006, ApERF014, ApERF048, ApERF097, and ApERF107 could be crucial in regulating anthocyanin synthesis and leaf coloration in Acer paxii. However, the functional analysis of these transcription factors in Acer paxii remains unexplored, and the molecular mechanisms underlying their control of anthocyanin biosynthesis during color development are yet to be elucidated. Future experiments are planned to validate these gene functions. This research establishes a foundational framework for elucidating the molecular regulatory networks mediated by AP2/ERF transcription factors in Acer paxii, while offering novel perspectives on the genetic mechanisms underlying color variation in ornamental species.