Abstract Background Acer pseudosieboldianum (Pax) Komarov, is a colorful leaf species belonging to the family Aceraceae, mainly distributed in Northeast China, Russia, and northern Korea. The leaves of Acer pseudosieboldianum are green in spring and summer, and turning red in autumn, which is of high ornamental value. In previous study, a mutant maple was selected with alternating red-green leaf color in spring and summer. However, the reason for the color mutation was not clear. Therefore, UPLC /LC-MS and RNA-seq were used to analyze the anthocyanin components and related differentially expressed genes in the spring leaf color changes of A. pseudosieboldianum mutant, which can provide broader insights into the complex coloration process of leaf color. Results The results showed that the mutant leaves contained a total of 50 anthocyanin metabolites. In all differential metabolites of anthocyanins, Cyanidin-3,5-O-diglucoside, Cyanidin-3-O-glucoside, Cyanidin-3-O-sambubioside not only had higher content, but also showed significant changes at different stages. Especially, the consistent high content of anthocyanins in Cyanidin-3-O-glucoside, which are the main pigments for leaf color. In addition, 11,522 genes were found to be significantly differentially with 5,477 genes up-regulated, and 6,045 genes down-regulated. We identified relevant information for differentially expressed genes (DEGs) associated with leaf color, including 20 structural genes involved in anthocyanin biosynthesis, 12 transcription factors, and eight genes related to anthocyanin transport. Conclusions Among all anthocyanins of A. pseudosieboldianum mutant leaf, Cyanidin-3-O-glucoside remained high in all three stages of leaves, which is main substances for the leaf color. Additionally, 20 structure gene, 12 transcription factors and some genes associated with anthocyanin synthesis and transport were screened and there was a complex metabolic network in mutant leaves. This study provided a basis for resource innovation and landscaping applications of Acer plants by analyzing the anthocyanin metabolites and expression of DEGs in the leaf coloring process. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-025-11378-3. Keywords: Acer Pseudosieboldianum, Transcription, Mutant, Anthocyanin, Candidate genes Introduction The leaf color of higher plants depends mainly on the content and ratio of chlorophyll, carotenoids, and anthocyanin [[38]1]. Anthocyanins are an important class of pigments widely present in angiosperms. They form stable water-soluble compounds after glycosylation and are localized within plant vacuoles, imparting rich colors to various plant organs [[39]2]. Due to its direct influence on the coloration of plant organs, the biosynthesis of anthocyanins has become a recent focal point in the field of ornamental crop coloration mechanisms. The biosynthetic pathway of anthocyanins is a branch of the flavonoid biosynthesis pathway, which has been deeply studied in many plants, and it has been clarified that the anthocyanin biosynthesis pathway in plants is generally similar [[40]3]. Phenylalanine is the starting material for the anthocyanin metabolic pathway, and under the catalysis of phenylalanine ammonia-lyase, chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3’-hydroxylase (F3′H), flavonoid 3′5′-hydroxylase (F3′5′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and other related enzymes, various types of anthocyanins are synthesized. Subsequently, they undergo a series of glycosylation and methylation steps under the action of flavonoid glucosyltransferase (UFGT) and methyltransferase (MT), combined with UDP-glucose to form stable anthocyanins [[41]4–[42]6]. Proanthocyanidins (PA) are important secondary metabolites in plants and are involved in the coloring process of plants. In plants, proanthocyanidins can be converted from leucoanthocyanidins and anthocyanidins under the action of specific enzymes. Anthocyanidin reductase (ANR) can convert anthocyanidins into flavan-3-ols and eventually into proanthocyanidins [[43]7–[44]8]. In addition to structural genes in the anthocyanin biosynthetic pathway, many transcription factors have been reported to regulate the formation of plant organ colors by modulating the expression of structural genes [[45]9]. Currently, three main classes of transcription factors involved in color formation are known: MYB, bHLH, and WD40. They regulate the expression of structural genes either individually or by forming MBW complexes [[46]10]. Leaf color variation is a relatively common mutational trait. Leaf color mutants have become increasingly important in basic research and breeding work. At present, the leaf color mutants are widely used in research and production. The leaf color mutants are excellent materials for studying the physiology and functional genomics of plants, which can be used to study photosynthesis and optical morphology, plant hormone synthesis, plant pathology and so on [[47]11]. A color-leaf mutant with purple leaves, stems and petals was isolated from Tradescantia ‘Wandering Jew’, and in mutant, the contents of chlorophyll a, chlorophyll b and carotenoids decreased significantly, while concentration of the anthocyanins was 6.2-fold higher than that of wild type. Furthermore, the purple pigmentation of leaves in mutant was caused by accumulation of petunidin anthocyanin [[48]12]. Wang et al. found that the expression levels of genes such as CHS, CHI, DFR, HY5, and bHLH are relatively high in purple leaves of Zijuan tea [[49]13]. Transcriptome analysis shows that PAL, C4H, CHS, CHI, DFR, ANS, and LAR are all upregulated in red-leaf poplar varieties [[50]14]. MYB transcription factors play an important role in regulating leaf color in red-leaf varieties. In Sapium sebiferum, SsMYB1 activates anthocyanin biosynthesis by directly binding to the promoters of SsDFR1 and SsANS, thereby promoting their transcriptional activity. Additionally, SsbHLH1 can further enhance this transcriptional activity, positively regulating anthocyanin biosynthesis [[51]15]. Overexpression of PtrMYB119 in poplar can simultaneously activate the transcription of PtrCHS1 and PtrANS2. Similarly, overexpression of PdMYB118 can transcriptionally activate most anthocyanin biosynthesis genes, leading to significant anthocyanin accumulation and resulting in red leaves [[52]16]. Acer pseudosieboldianum is a deciduous tree with high ornamental value due to its vibrant red leaf color and elegant leaf shape in autumn [[53]17]. The leaves of the wild plant in A. pseudosieboldianum were green in spring, and gradually turn red in autumn. In our previous study, a mutant with leaves that intersected red and green in spring and summer was identified, greatly improving the ornamental value of A. pseudosieboldianum in spring and summer. Next, we analyzed and compared the mechanisms underlying the differences in leaf color formation between wild-type and mutant leaves of A. pseudosieboldianum, and the results show that at different stages of leaf development, the expression levels of structural genes CHS, DFR, and ANS were significantly higher in the mutate plant than in the wild plant, and the expression pattern was significantly correlated with anthocyanin contents [[54]18]. However, the temporal changes of anthocyanin metabolites and gene expression during leaf development in this mutant are still unclear and requires further research to provide insights into its leaf coloration. Therefore, in this study, we performed metabolomic data analysis and RNA-seq analysis on different developmental stages of A. pseudosieboldianum leaves. From metabolome analysis, we screened for differential metabolites and their KEGG pathways; and from RNA-seq, we screened for differences in expressed genes. We identified genes responsible for anthocyanin synthesis that could be used as target genes for future breeding in this species. In addition, we identified transcription factors that may be involved in anthocyanin synthesis. This study provides broader insights into the complex coloration process of leaf color. Materials and methods Plant materials The leaves of A. pseudosieboldianum mutant were collected from Yanbian University, Yanji City, Yanbian Korean Autonomous Prefecture, Jilin Province (129°49 E, 42°92 N). We chose healthy maple leaves without disease or insect pests at three stages on May 15, May 30, and June 15, 2021. These stages represent early leaves (VE), middle leaves (VM), and late leaves (VA) during leaf discoloration, and each stage was replicated in three biological samples. After collection, all materials were frozen using liquid nitrogen and stored at -80 °C until they were utilized. UPLC (Ultrahigh performance liquid chromatogram) analysis The frozen leaves from each sample, approximately 0.3 g in fresh weight, were pulverized into powder and subsequently subjected to a 48-hour freeze-drying process. Anthocyanins were extracted using 10 mg of lyophilized leaf powder mixed with a 1 mL solution of 0.1% acetic acid in methanol. The mixture was then left overnight at 4 °C in the dark, followed by centrifugation the next day at 12,000 rpm for 10 min. The resulting supernatants were collected and dehydrated using a vacuum centrifuge concentrator (CV100-DNA, Aijimu, Beijing, China). An ACQUITY UPLC system (Waters, Milford, MA, USA) coupled with a triple-Quadrupole Mass Spectrometry (XEVO^®-TQ) employing electrospray ionization (ESI) was employed for anthocyanin analysis. Chromatographic column type: ACQUITY BEH C18 1.7 μm, 2.1 mm×100 mm. The dried extracts were reconstituted in MeOH before analysis. The gradient program utilized 0.1% formic acid (A) and methanol (B) as mobile phases: 0 min (5% B), 6 min (50% B), 12 min (95% B), hold for 2 min, 14 min (5% B), hold for 2 min; flow rate, 0.35 mL/min; temperature, 40 °C; injection volume, 2 µL. Compound identification in the sample extracts was achieved by comparing retention times with standards, analyzing UV-VIS spectra characteristics of peaks, and utilizing mass spectrometric information with Mass Hunter qualitative software. Relative quantification of anthocyanin content was determined from peak areas of the samples, normalized to the intensity of the corresponding standard compounds. RNA extraction, library construction, and RNA-seq Total RNA was extracted from the VE, VM, and VA leaf samples using a mirVana mRNA isolation kit (Ambion, TX, USA) according to the manufacturer’s instructions. The integrity of extracted RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Sequencing libraries were prepared using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. Then, these libraries were sequenced on an Illumina HiSeq™ 2500 platform (Beijing Omics Biotech Co., Ltd., Beijing, China). Quality control and mapping of reads Initial processing of the raw reads involved the removal of low-quality reads, adapters, and reads containing poly-A or poly-N sequences. This step was executed using Trimmomatic (version 0.36) [[55]19]. The obtained highly quality data was called “clean reads” and then assembled from scratch in Trinity software to obtain “unigene” [[56]20]. Gene expression analysis The RNA-seq reads were aligned to the unigene library using Bowtie software, and the aligned results were quantified for expression levels using RSEM [[57]21]. Gene normalization was accomplished using FPKM [[58]22]. Genes were identified as differentially expressed genes (DEGs) between samples based on criteria of a p-value < 0.05 and a fold change greater than 2 or less than 0.5. To analyze the expression patterns of these DEGs, hierarchical cluster analysis was conducted. For functional annotation, GO enrichment analysis of the DEGs was carried out using Cytoscape BINGO plugin [[59]23]. Additionally, the involvement of DEGs in various KEGG pathways was examined using KOBAS software [[60]24]. Quantitative real-time PCR (qRT-PCR) analysis Total RNA was extracted from A. pseudosieboldianum samples according to the instructions of a fluorescence quantitative PCR kit (2×SYBR^® Green premix) and a Gene9600 fluorescence quantitative PCR instrument. Primers were designed by Biotechnology Co. Ltd. in Beijing, China. The gene c110191.graph_c0 was used as the reference gene. All primers used for PCR were listed in Supplemental Table [61]S1. The reaction protocol adhered to the manufacturer’s guidelines: 39 cycles consisting of denaturation at 95 °C for 10 s, annealing at 58 °C, and extension for 30 s. Subsequent to the amplification, a dissociation curve analysis was performed to assess the primer specificity. The amplification data were subjected to analysis using the comparative cycle threshold (Ct) method, employing the formula 2^−△△Ct [[62]25]. The quantitative real-time polymerase chain reaction (qRT-PCR) results were computed as the means of three independently replicated treatments. Results Quantitative and qualitative metabolite testing A total of 50 anthocyanin metabolites were detected in the leaves of three periods (Fig. [63]1A) of the A. pseudosieboldianum mutant. The metabolites were classified into six major groups, namely Cyanidin (Cy), Delphinidin (Dp), Peonidin (Pn), Pelargonidin (Pg), Petunidin (Pt) and Malvidin (Mv) (Supplemental Table [64]S2). When quantitatively analyzing the anthocyanin content in the leaves of A. pseudosieboldianum, Cyanidin was found to be the predominant compound, constituting 95.7% of the total anthocyanin content (Fig. [65]1B). Furthermore, it appears that Cyanidin and Delphinidin are the two most common anthocyanins found in A. pseudosieboldianum, each accounting for 26% of the total type of anthocyanin content (Fig. [66]1C). Furthermore, the anthocyanin metabolite cyanidin-3-O-glucoside was consistently detected as the highest content among anthocyanin metabolites during the three periods of analysis. Fig. 1. [67]Fig. 1 [68]Open in a new tab The leaf morphology during development (A), anthocyanin content (B) and classification (C) of A. pseudosieboldianum mutant. VE (Early stages), VM (Middle stages), and VA (Late stages) represent three stages during leaf discoloration Differential metabolite screening In this study, fold change ≥ 2 or fold change ≤ 0.5 was used to screen differential metabolites. Especially in VE vs. VA, a total of 47 differential metabolites were identified, of which 38 were up-regulated and nine were down-regulated (Table [69]1; Fig. [70]2A). These findings indicate significant changes in the composition of metabolites during the development of leaves in A. pseudosieboldianum mutant, providing valuable insights into the metabolic differences between these stages. Table 1. The statistics of the number of different metabolites in each contrast combination Group name Differential metabolites Up regulated Down regulated VE_vs_VM 28 27 1 VM_vs_VA 44 34 10 VE_vs_VA 47 38 9 [71]Open in a new tab Fig. 2. [72]Fig. 2 [73]Open in a new tab Venn diagram of different metabolites in each contrast combination (A) and important differential metabolites in VE vs. VM (B), VE vs. VA (C), VM vs. VA (D). VE (Early stages), VM (Middle stages), and VA (late stages) represent three stages during leaf discoloration Further analysis of differential metabolites at different stages based on criteria of log[2]FC greater than 1.5 (Fig. [74]2B, C, D) revealed that significant differences in the content of anthocyanin such as Cyanidin-3,5-O-diglucoside, Cyanidin-3-O-sambubioside-5-O-glucoside, Cyanidin-3-O-sambubioside, Delphinidin-3-O-glucoside, Delphinidin-3-O-sophoroside at different stages. These results indicated that these specific anthocyanin metabolites play a prominent role in the leaves of A. pseudosieboldianum mutants throughout different developmental periods. It showed the changes in the content of important anthocyanins in Fig. [75]3. The results showed that the content of most anthocyanins and proanthocyanins gradually increased with leaf development, and Cyanidin-3,5-O-diglucoside, Cyanidin-3-O-glucoside, Cyanidin-3-O-sambubioside not only had higher content, but also showed significant changes at different stages. Especially, the consistent high content of anthocyanins in Cyanidin-3-O-glucoside, which are the main pigments for leaf color. Proanthocyanins are one of the pathways for flavonoid synthesis. In this study, two types of proanthocyanins contents significantly increased indicating that some anthocyanins are ultimately converted into proanthocyanins (Fig. [76]3). Fig. 3. [77]Fig. 3 [78]Open in a new tab The contents of differential metabolite at leaf development stage. VE (Early stages), VM (Middle stages), and VA (late stages) represent three stages during leaf discoloration Functional annotation and enrichment analysis of differential metabolites KEGG Three contrasting combinations of A. pseudosieboldianum leaves at different times were enriched by the KEGG database and differential metabolites were enriched into four pathways. Among them, the number of differential metabolites was most enriched in the pathway of anthocyanin biosynthesis. In the anthocyanin biosynthesis pathway, 50 differential metabolites were enriched, accounting for 71.62% of all differential metabolites; the differential metabolite KEGG pathway was enriched in different periods of the comparison combination. These results indicate that the anthocyanin biosynthesis pathway had the highest number of differential metabolites, suggesting its significant involvement in the metabolic changes observed in A. pseudosieboldianum leaves during different periods of comparison. Statistical analysis of transcriptomic Leaves with various colorations were collected from three developmental stages, the early (VE), middle (VM) and late stages (VA) (Fig. [79]1), with three biological replicates. After filtering the obtained 190,110,067 raw reads, a total of 130,224,215 high-quality clean reads were produced. The percentage of the Q30 base was 91.55% or above. The average GC% was 43.81%. After assembly with Trinity software, the sequences produced 41,006 unigenes with an N50 of 88 bp (Supplemental Table [80]S3). Identification and functional analysis of differentially expressed genes (DEGs) A total of 11,522 unigenes were identified as differentially expressed genes (DEGs) between the three discoloration stages of A. pseudosieboldianum, with 5,477 genes up-regulated, and 6,045 down-regulated. There were 5229, 7944, and 8564 DEGs in the three comparative groups, including VE vs. VM, VM vs. VA, and VE vs. VA, respectively. In the VE vs. VM group, 2280 DEGs were up-regulated, whereas 2949 DEGs were down-regulated; In the VM vs. VA group, 3374 DEGs were up-regulated and 4570 DEGs were down-regulated; In the VE vs. VA group, 3774 DEGs were up-regulated and 4790 DEGs were down-regulated (Supplemental Fig. [81]S1). This data is also represented in the Venn diagram of DEGs in different stages, as shown in Fig. [82]4A. These DEGs likely play critical roles in the developmental and color-changing processes in A. pseudosieboldianum leaves across these stages. Fig. 4. [83]Fig. 4 [84]Open in a new tab Venn diagram (A), KEGG annotation (B) and GO annotation (C) of DEGs (VE vs. VA). VE (Early stages), VM (Middle stages), and VA (late stages) represent three stages during leaf discoloration Functional annotation and classification of DEGs To accurately identify and classify the functions of each DEG, classification and enrichment GO analyses were conducted in the early, middle, and late stages. To elucidate specific processes, gene functions, and gene interactions at the transcriptomic level, a KEGG pathway enrichment analysis of DEGs in the leaves of A. pseudosieboldianum was conducted to further reveal the functions of DEGs. We found that a total of 326 pathways were affected by 3,591 DEGs for VE vs. VA (Fig. [85]4B). The GO annotation results of DEGs for VE vs. VM and VM vs. VA are same as VE vs. VA that shown in Supplemental Fig. [86]S2 and Fig. [87]S3 respectively. We identified 4,772, 2,630, and 4,235 DEGs that were assigned to 52 significantly enriched GO terms for VE vs. VA, VE vs. VM, and VM vs. VA, respectively. The most abundant GO terms for VE vs. VA were “metabolic process”, “membrane” and “catalytic activity” in the biological process, cellular component, and molecular function categories, respectively (Fig. [88]4C). Notably, the greatest number of DEGs were mapped to “Plant-pathogen interaction” (382, 10.64%), and the second most significantly enriched pathway was the “RAS signaling pathway” (284, 7.91%). At the same time, 304 and 323 pathways were affected by 3,117 and 3,268 DEGs for VE vs. VM, and VM vs. VA, respectively (Supplemental Fig. [89]S4 and Fig. [90]S5). Candidate genes involved in anthocyanin biosynthesis and qRT-PCR analysis This study identified 20 candidate unigenes encoding seven enzymes related to the flavonoid biosynthesis pathway (ko00942) in the A. pseudosieboldianum transcriptome. The 20 candidate genes were identified as follows: 4 CHS genes, 1 CHI gene, 1 F3H gene, 1 F3’H gene, 1 DFR gene, 3 ANS genes, 3 ANR genes and 6 UFGT genes. The candidate genes in the anthocyanin biosynthesis pathways are shown in Fig. [91]5. Fig. 5. [92]Fig. 5 [93]Open in a new tab DEGs involved in the anthocyanin biosynthesis pathway of early middle and after three periods (Upregulated genes are marked by red arrows, downregulated genes by green arrows, and genes that are not expressed are marked with black bars.) VE (Early stages), VM (Middle stages), and VA (Late stages) represent three stages during leaf discoloration In this study, a total of 4938 unigenes were identified as putative TFs, with 70 subclasses. The unigenes encoding WRKY (384), MYB (321), and bHLH (179) are the most abundant in the A. pseudosieboldianum. Among them, 6 MYB genes, 4 bHLH genes, and 2 WRKY genes were identified to be related to leaf color synthesis according to the KEGG and GO analysis (Supplemental Table [94]S4). In this study, the DEGs encoding the most abundant transporters were ABC transporters (33), glutathione S-transferase (GST) (36), multidrug and toxic compound excretion-associated proteins (MATEs) (21), and H+-ATPases (20). Among these genes, four ABC genes (TRINITY_DN2943_c3_g1, TRINITY_DN8751_c0_g1, TRINITY_DN21795_c0_g1 and TRINITY_DN97_c0_g2 ), two GST gens (TRINITY_DN5896_c0_g1 and TRINITY_DN47677_c0_g1 ) and two MATE gens (TRINITY_DN12742_c0_g1 and TRINITY_DN4147_c1_g1 ) were found to be related to the transport of anthocyanin according to the KEGG annotation and GO annotation (Supplemental Table [95]S5). To validate the data from our digital expression analysis, quantitative real-time PCR (qRT-PCR) assays were performed on eight DEGs involved in the anthocyanin biosynthesis (Fig. [96]6). The qPCR expression levels detected in the mutant of A. pseudosieboldianum at three periods were basically consistent with the transcriptome sequencing results. These results indicate that the transcriptomic analysis was reproducible and reliable for additional investigation into key genes involved in anthocyanin accumulation in A. pseudosieboldianum. Fig. 6. [97]Fig. 6 [98]Open in a new tab Quantitative RT-PCR validation of RNA-Seq data of eight candidate genes related to leaf color in A. pseudosieboldianum Discussion Chlorophyll, carotenoids, flavonoids, and alkaloids are the primary plant pigments in nature, imparting different colors to plants. Among them, flavonoids are widely distributed in plants, giving plant organs various colors ranging from light yellow to blue-purple. Anthocyanin is a type of water-soluble natural pigment in the natural world, belonging to the flavonoid compound category. Up to now, more than 600 types of anthocyanins have been discovered in plants, mainly classified into six categories: Cyanidin (Cy), Delphinidin (Dp), Peonidin (Pn), Pelargonidin (Pg), Malvidin (Mv), and Petunidin (Pt) [[99]26–[100]27]. The fractions of the A. pseudosieboldianum mutant leaves were analyzed by UPLC and LC-MS, and the results showed that the mutant leaves contained a total of 50 anthocyanin metabolites, among which Cyanidin-3-O-glucoside had always maintained a high content and is the main pigment for leaf color. This finding is similar to the findings of Rothenberg et al. [[101]28]. The anthocyanin biosynthetic pathway is one of the most well-understood secondary metabolic pathways in plants. Genes involved in anthocyanin biosynthesis are classified into two main categories: structural genes and regulatory genes. Among them, structural genes directly encode key enzymes in anthocyanin biosynthesis, such as CHI, CHS, DFR, ANS, UFGT [[102]29]. In this study, A total of 20 genes encoding enzymes involved in anthocyanin biosynthesis were screened from the transcriptome data of A. pseudosieboldianum. The leaf color of common maple will turn red in the later stage of discoloration, so the gene expression at the VA stage is generally the highest, which is no exception in our experiment. It is worth mentioning that the leaf color of our mutant shows alternating red and green in spring, so some enzyme genes will show high expression content in VE. As a result of this, the expression of most coding enzymes decreased in VE vs. VM stage and then increased in VM vs. VA stage. For example, CHS (TRINITY_DN452_c0_g1, TRINITY_DN759_c6_g1), DFR(TRINITY_DN3960_c0_g1) and ANS(TRINITY_DN18024_c0_g1), these unigenes were down-regulated in the VE vs. VM stage and up-regulated in the VM vs. VA stage. Previously, many studies have confirmed that the coding enzyme, including CHS, CHI, F3H, DFR, ANS and UFGT, are indeed involved in the biosynthesis of flavonoids [[103]30–[104]34]. The results also confirmed that these genes encoding enzymes indeed participate in the early synthesis of anthocyanins in the spring leaves of A. pseudosieboldianum mutant, playing a crucial role in the process. Transcriptional regulation is one of the crucial aspects in the regulation of structural gene expression in the anthocyanin biosynthetic pathway in plants. Its mechanism is quite complex, and currently, three major classes of regulatory factors are well-studied: bHLH, MYB genes, and WD40. For most species, the biosynthesis of anthocyanins is regulated by the formation of protein complexes involving these three classes of transcription factors (TFs), binding to the promoter of structural genes [[105]35]. R2R3-MYB play crucial roles as transcriptional regulators in the production of anthocyanins. MYB transcription factors have been widely reported in a variety of plants, such as Arabidopsis and horticultural plants. The identified transcription factors AtMYB113, AtMYB114, AtMYB75, AtMYB90 can promote the synthesis of anthocyanin. Wang et al. cloned the MdMYB12 and MdMYB22 genes in apples and validated their roles in the anthocyanin biosynthesis pathway. Xu et al. isolated and validated DcMYB7 from purple carrot varieties, confirming it as a key gene determining anthocyanin synthesis in carrots [[106]36–[107]38]. Based on our results, six MYB genes and four bHLH genes were identified to participate in the biosynthesis of flavonoids and anthocyanins as flavonoid 3’-monooxygenase or detected from the biological process of flavonoid biosynthetic process (GO:0009813). Although the number of WD40 TFs is small in our transcriptome, it is still possible that MBW complexs regulate anthocyanin synthesis. It is noteworthy that MYB and bHLH TFs in this experiment is indeed involved in the biosynthesis of leaf color discoloration. Moreover, there are other TFs also control the regulation of anthocyanin transport. WRKY TFs have been found during flavonoid biosynthesis in purple tea [[108]39]. In A. pseudosieboldianum transcriptome, two WRKY TFs were detected from the biological process of anthocyanin metabolic process (GO:0031538) and regulation of leaf development (GO:2000024). This suggests that they may be involved in regulating the formation of leaf color in A. pseudosieboldianum. The transport and accumulation of anthocyanins significantly affect the color phenotype of plants, but the process of anthocyanin transport from the cytoplasm to the vacuole remains unclear. Currently, four models of anthocyanin transport have been proposed, and four types of proteins, namely GST, MRP, MATE, and BTL-homologue, have been identified as potential participants in the transport of anthocyanins to the vacuole [[109]40, [110]41]. Many studies indicate that GST is involved in the transport of anthocyanins [[111]42]. In this study, 36 genes encoding GST were identified, and some of these GSTs may participate in the transport of anthocyanins. The Multidrug resistance-associated protein (MRP/ABCC) subfamily belongs to the ATP-binding cassette (ABC) superfamily. The ABC superfamily is a large and functionally diverse group of proteins, and different subfamilies play distinct but crucial roles in the transmembrane transport of secondary metabolites in plants [[112]43–[113]45]. Goodman et al. proposed that maize ZmMRP3 is associated with the transport of anthocyanins, and inhibiting the expression of ZmMRP3 leads to a decrease in the accumulation of anthocyanins [[114]46]. In the A. pseudosieboldianum transcriptome, 33 genes encoding ABC transporters were identified. Through Gene Ontology (GO) analysis, two ABC genes in A. pseudosieboldianum were identified to be related to leaf color. MATE transporters are a class of transmembrane transport proteins that perform relatively conservative and fundamental transport functions in most prokaryotes and eukaryotes. H+-ATPase plays a crucial role in the transport of anthocyanins [[115]47]. In the A. pseudosieboldianum transcriptome, some genes encoding MATE and H+-ATPases were identified, suggesting their potential involvement in the transport of anthocyanins. Currently, research on the transport mechanism of anthocyanins is still in its early stages [[116]48]. It is not yet clear which transport mechanism primarily facilitates the transport of anthocyanins in A. pseudosieboldianum leaves. The results of this study may provide useful information for understanding the regulation of anthocyanin transport in Acer pseudosieboldianum and lay the groundwork for future research. Conclusion The results showed that the A. pseudosieboldianum mutant leaves contained a total of 50 anthocyanin metabolites, among which Cyanidin-3,5-O-diglucoside, Cyanidin-3-O-glucoside, Cyanidin-3-O-sambubioside not only had higher content, but also showed significant changes at different stages. Especially, the consistent high content of anthocyanins in Cyanidin-3-O-glucoside, which are the main pigments for leaf color. Additionally, our results indicate the identification of 20 differentially expressed genes (DEGs) related to leaf color synthesis and involved in the anthocyanin pathway. Furthermore, 12 transcription factors and some genes associated with anthocyanin transport were screened. These findings suggest that these genes play crucial roles in regulating anthocyanin biosynthesis and transport in A. pseudosieboldianum mutant leaves, contributing to variations in leaf color. Electronic supplementary material Below is the link to the electronic supplementary material. [117]Supplementary Material 1^ (2MB, docx) Acknowledgements