The tree peony cultivars ‘Baixueta’, ‘Yaohuang’, ‘Huangguan’, ‘High Noon’, ‘Jinge’, and ‘Roufurong’ were cultivated under adequate conditions of light and moisture in the Tree Peony Garden at Northwest A&F University, Shaanxi Province, China ( Figure 1 ). The middle petal samples from 9 plants of each cultivar were collected at five stages defined according to a previous method (S1, unpigmented compact bud; S2, slightly pigmented soft bud; S3, slightly pigmented flower about to bloom; S4, pigmented blooming flower; S5, pigmented fully blooming flower with exposed anthers) (Zhou et al., 2011), of which spots of ‘Huangguan’, ‘High Noon’, ‘Jinge’, and ‘Roufurong’ were removed. N. tabacum and N. benthamiana plants were cultivated under controlled conditions in a programmable incubator, maintaining a 16 h light/8 h dark photoperiod at 25°C. Color-related values (L*, a*, b*, C*, and h) of fresh petals were quantified using a tristimulus color meter (CR-400, Konica Minolta, Osaka, Japan). The petals for the remaining tests were promptly frozen in liquid nitrogen, and subsequently stored at -80°C.

The frozen petals of four tree peony cultivars were quickly pulverized into a fine powder using liquid nitrogen. Approximately 0.3 g of petal powder was dissolved in a solution of methanol-hydrochloric acid (99: 1, V/V) (for anthocyanins) and a separate solution of methanol (for other flavonoids) (6 ml each), respectively. Next, the samples were extracted at 4°C for 24 h in the dark, and agitated once every 6 h. The supernatant of the samples was collected following centrifugation at 10,000 r/min for 10 minutes. The extraction solution was scanned with the full wavelength of 200~850 nm by Multiskan Spectrum (SP-Max 2300A2, Shanghai, China), and the maximum absorption value at about 520 nm was taken to calculate the total anthocyanin concentration. Each sample was subjected to three biological replicates. The content of other flavonoid components was assessed by HPLC (LC-2030C 3D, Shimadzu, Kyoto, Japan) system equipped with a 4.6 × 250 mm C18 column and a diode array detector (Shimadzu, Kyoto, Japan). Mobile phases comprised of eluent A, a 0.04% aqueous solution of formic acid, and eluent B, acetonitrile. The gradient progressed as follows: 5% B (0 min), 40% B (40 min), 100% B (45 min), 100% B (55 min), 5% B (60 min). The flow rate was maintained at 0.5 ml/min while the column temperature was set to 40°C. The mean values and SDs were determined based on three replicates per sample.

The total RNA from petals at S1-S5 in each tree peony cultivar was extracted using Plant RNA Kit (Tiangen, Beijing, China). First-strand cDNA was synthesized utilizing the PrimeScript™ RT Master Mix reverse transcription kit (Takara, Dalian, China). Subsequently, qPCR was conducted using TB Green TaKaRa Premix Ex Taq™ II (TaKaRa, Dalian, China). The conditions for amplification entailed one cycle at 95°C for 15 s, followed by 45 cycles comprising 95°C for 5 s, 58°C for 30 s, and 72°C for 31 s. Specific primers are detailed in Supplementary Table S1 . Psubiquitin gene was used as an internal reference for the expression level normalization by employing the 2^−ΔΔCT method (Livak and Schmittgen, 2001). Three biological replicates were independently conducted for each qPCR assay.

Phylogenetic trees of PsMYB4 and PsEGL3 were constructed from evolutionary distance data with Neighbor-joining method by MEGA 6.0. The gene names and login numbers used in phylogenetic trees were shown in Supplementary Tables S2, S3 . The open reading frames (ORFs) of PsMYB4 and PsEGL3, lacking the termination codon, were individually inserted into pCAMBIA1302-GFP vectors. Next, the resulting constructs were transformed into the competent Agrobacterium strain GV3101 (Yan et al., 2012). Agrobacterium cultures transformed by each construct were resuspended in infiltration buffer containing 10 mM MES, 100 mM AS and 10 mM MgCl₂, after which they were injected into leaves of N. benthamiana. Three days after injection, the GFP fluorescence signal was observed using a Nikon C2-ER confocal laser scanning microscope (Nikon, Tokyo, Japan). The experiment was conducted in triplicate. The primers are shown in Supplementary Table S4 .

For gene functional analysis, the recombinant pCAMBIA1302 vectors carrying PsMYB4 and PsEGL3 were transferred into Agrobacterium GV3101, respectively. The leaf discs of N. tabacum were utilized as for transgenic transformation, following a previously described methodology (Horsch et al., 1985). Subsequently, the plantlets were transplanted into soil, subsequently cultivated in a controlled greenhouse environment until blooming. The transgenic lines were validated through qRT-PCR using specific downstream primers targeting PsMYB4 and PsEGL3, along with a specific upstream primer derived from the CaMV35S promoter. The petals of T2 transgenic plants were collected for the quantification of color-related values, including L*, a*, C*, and h. Meanwhile, the flavonoid contents were determined as described in the section HPLC analysis, while the structural genes were detected using qRT-PCR. All specific primers are listed in Supplementary Table S5 .

The TRV1 and TRV2 vectors were employed to perform VIGS experiments in P. suffruticosa ‘High Noon’ for the purpose of characterizing the functions of PsMYB4 and PsEGL3. PsMYB4 and PsEGL3 fragments measuring 203 and 318 bp were inserted into the TRV2 vectors using PstI and Xhol, respectively. Target fragments and linear TRV2 vectors were treated with T4 DNA ligase to generate TRV2-PsMYB4 and TRV2-PsEGL3. Recombinant vectors and empty vectors of TRV1 and TRV2 were introduced into Agrobacterium strain GV3101, resuspended in an infiltration buffer with 10 mM MES, 100 mM AS and 10 mM MgCl₂, and incubated at room temperature for 2 h. Agrobacterium strain GV3101 cells containing TRV1 mixed with TRV2 (CK), TRV1 mixed with TRV2-PsMYB4, TRV1 mixed with TRV2-PsEGL3 at a 1:1 ratio were injected into the buds of ‘High Noon’ at S2 stage. The injection volume of each combination was 1 ml, and the injection should be gentle and slow to ensure that the suspension evenly penetrates into the flower buds. The injection was repeated two days later. The petal samples (spots removed) were collected after blooming to detect the flavonoid contents as described in the section HPLC analysis. In addition, qRT-PCR was performed to determine gene expression levels. Specific primers utilized in this study are documented in Supplementary Table S6 .

To test the yeast two-hybrid between PsMYB4 and PsEGL3, the ORF of PsMYB4 was fused into pGADT7 AD vector by homologous recombination to generate pGADT7-PsMYB4 recombinant vector. Simultaneously, the ORF of PsEGL3 was fused into pGBKT7 BD vector to generate pGBKT7-PsEGL3 recombinant vector. Subsequently, pGADT7-PsMYB4 and pGBKT7-PsEGL3, pGADT7 empty vector and pGBKT7-PsEGL3 (negative control), as well as pGBKT7–53 and pGADT7-T (positive control) were co-transformed into AH109 yeast competent cells, respectively. After confirming the absence of any self-activation activity, yeast cells successfully co-transformed were grown on SD/-Trp/-Leu, SD/-Trp/-Leu/-His, SD/-Trp/-Leu/-His/-Ade adding X-α-Gal screening medium respectively. The specific primers utilized are documented in Supplementary Table S7 .

The ORFs of PsMYB4 and PsEGL3 lacking the termination codon were PCR-amplified. PsMYB4 fragment was fused into pUC-SPYNE vector to generate PsMYB4-nYFP, and PsEGL3 fragment was fused into pUC-SPYCE vector to generate PsEGL3-cYFP using BamHI and Xhol restriction sites. The recombinant vectors were co-transformed transiently into N. benthamiana leaves as described by previous study (Hellens et al., 2005). BZIP63-nYFP co-transformed with BZIP63-cYFP was the positive control. The negative controls consisted of PsMYB4-nYFP co-transformed with 35S-cYFP and PsEGL3-cYFP co-transformed with 35S-nYFP. The YFP fluorescence signal was visualized 72 h after injection. The experiment was conducted in triplicate. All primers utilized in this study are shown in Supplementary Table S8 .

The promoters of PsCHS, PsCHI and PsDFR were cloned from genomic DNA of ‘High Noon’ petals based on previous genome DNA sequences (China National Gene Bank, CNP0003098) and transcriptome cDNA sequences (Genome Sequence Archive, CRA005005) of tree peony. The analysis and prediction of cis-elements in promoters were conducted using online website PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html). The PsMYB4 and PsEGL3 ORFs were cloned into pCAMBIA1302 vectors as effectors, while the promoters of PsCHS, PsCHI, and PsDFR were cloned into pGreenII0800-LUC vectors as reporters. All effectors and reporters were transformed into Agrobacterium strain GV3101 and subsequently co-transformed into N. benthamiana leaves according to the method of previous study (Hellens et al., 2005). The empty pCAMBIA1302 vectors co-transformed with different LUC recombination vectors were regarded as negative controls. At 48 h post-infiltration, the enzyme activities of LUC and REN were assessed using a dual-luciferase assay system on GloMax^® Discover (Promega, Madison, USA). The LUC/REN ratio determined the activity, and three biological replicates were conducted per assay. All primers utilized in the present study are documented in Supplementary Table S9 .

The flavonoid content and composition of the tree peony petals of yellow cultivar ‘High Noon’ and purple red cultivar ‘Roufurong’ were characterized in a previous study (Luo et al., 2021). Thus, the present study employed high-performance liquid chromatography (HPLC) experiments in the other four cultivars ‘Baixueta’ (white), ‘Yaohuang’ (yellowish), ‘Huangguan’ (light yellow), ‘Jinge’ (reddish yellow) at five blooming stages. As a result, no anthocyanins were detected in ‘Baixueta’, ‘Yaohuang’, ‘Huangguan’ and ‘High Noon’ except for ‘Jinge’ and ‘Roufurong’, which was consistent with their floral color phenotype ( Figure 2 ). Our previous research has shown that ‘High Noon’ has the highest tetrahydroxychalcone (THC) content and co-dominates the formation of its yellow flowers with rich flavones and flavonols such as kaempferol (Km), apigenin (Ap), chrysoeriol (Ch) and isorhamnetin (Is) (Luo et al., 2021). However, the contents of flavones and flavonols in ‘Baixueta’, ‘Yaohuang’, ‘Huangguan’ and ‘Jinge’ were much higher than that of THC, and the contents of various flavonoids in ‘Baixueta’ and ‘Yaohuang’ were obviously lower than those in ‘Huangguan’ and ‘Jinge’ ( Figure 2 ), which may be the reason for their lighter flower color. Among them, the content of Km, one of the flavonols in ‘Baixueta’, ‘Huangguan’ and ‘Jinge’ was obviously higher than that of other flavonoids, and its change trend was consistent in ‘Baixueta’ and ‘Huangguan’. In regard to ‘Jinge’, the content of Km exhibited an initial upward trend followed by a subsequent decline. It is noteworthy that anthocyanin was detected in ‘Jinge’, and the total anthocyanin content increased significantly at S3 and then decreased gradually at S4 and S5. This is consistent with the simultaneous red and yellow phenotype of ‘Jinge’ petals from S3 to S5, during which the red color gradually decreases.

To investigate the potential functions of PsMYB4 and PsEGL3 in yellow tree peony flowers, we conducted qRT-PCR analysis to examine the expression patterns of PsMYB4 and PsEGL3 at five different blooming stages across six tree peony cultivars. The results are presented in Figure 3 , and the expression patterns of PsMYB4 and PsEGL3 exhibited a high degree of consistency across all six tree peony cultivars. Overall, their expression levels were higher in ‘High Noon’, ‘Huangguan’ and ‘Jinge’, while lower in ‘Baixueta’, ‘Yaohuang’ and ‘Roufurong’. It indicated that the expression levels of PsMYB4 and PsEGL3 were higher in the darker yellow cultivars, while lower in the purple red, white and light yellow cultivars. Moreover, the expression levels of PsMYB4 and PsEGL3 in ‘High Noon’ and ‘Huangguan’ showed an accordant trend of first increasing and then decreasing, reaching their maximum values at S4. Nevertheless, the expression of PsMYB4 in ‘Jinge’ exhibited a pattern of initial increase, followed by decrease, and subsequent re-increase, reaching its peak at S5, while the expression pattern of PsEGL3 in ‘Jinge’ showed a continuous upward trend, reaching a peak rapidly at S5, which may be related to the presence of anthocyanins in the petals of ‘Jinge’. These results suggested that PsMYB4 and PsEGL3 may inhibit the synthesis of anthocyanins in tree peony yellow flowers.

In the preliminary research, the full-length sequences of PsMYB4 and PsEGL3 genes were isolated, and a homologous alignment revealed that the N-terminus of PsMYB4 contained a conserved R2R3 domain and a bHLH-interacting motif, while its C-terminus also contained C1 and C2 inhibitory motifs (Luo et al., 2021). Similarly, the amino acid sequence of PsEGL3 consisted of a bHLH domain and an N-terminal MYB-interacting region (MIR) (Luo et al., 2021). The MIR of subgroup IIIf bHLH proteins is pivotal for binding to MYB TFs. We further performed cluster analysis on PsMYB4 and other homologous proteins involved in flavonoid biosynthesis, and found that PsMYB4 belonged to the R2R3-MYB repressor and had the closest relationship with SG4 member AtMYB4 in Arabidopsis ( Figure 4A ). Another phylogenetic tree, constructed using bHLH TFs derived from other species that involved in the regulation of anthocyanin synthesis, showed that PsEGL3 clustered into the GL3 clade of subgroup IIIf with high similarity to AtGL3, AtEGL3, CpbHLH1, VvMYCA1, CsMYC2, MdbHLH33 ( Figure 4B ).

To investigate the subcellular localization of PsMYB4 and PsEGL3, the fusion proteins 35S:PsMYB4-GFP and 35S:PsEGL3-GFP were transiently expressed in six-week-old Nicotiana benthamiana leaves, while the protein 35S:GFP served as a negative control. The subcellular localization of proteins was observed after 72h. The 35S:GFP protein distributed extensively in cytoplasm, especially in the cell membrane and nucleus ( Figure 4C ). Whereas the protein 35S:PsMYB4-GFP was exclusively localized in the nucleus, and the protein 35S:PsEGL3-GFP was primarily detected in the nucleus, suggesting that PsMYB4 and PsEGL3 are nuclear proteins ( Figure 4C ).

To validate the function of PsMYB4 and PsEGL3 in flavonoid biosynthesis, we generated transgenic tobacco lines overexpressing PsMYB4 and PsEGL3. Two PsMYB4 transgenic tobacco lines and three PsEGL3 transgenic tobacco lines were ultimately obtained, named OE-PsMYB4-1/2, and OE-PsEGL3-1/2/3, respectively. Compared to the deep pink color of wild type tobacco, the corolla color of OE-PsMYB4–1 and OE-PsMYB4–2 lines was significantly lighter, while the corolla color of OE-PsEGL3-1, OE-PsEGL3–2 and OE-PsEGL3–3 lines was slightly lighter ( Figure 5A ). Next, the floral color values of transgenic plants were determined, and the results indicated a significant decrease in the a* and C* values of OE-PsMYB4–1 and OE-PsMYB4–2 compared to the wild-type, while the L* values of OE-PsMYB4–1 and OE-PsMYB4–2 were notably higher than that of the wild-type ( Figure 5B ). The color values of three PsEGL3 transgenic lines were not consistent, and the L* values increased significantly in OE-PsEGL3–1 and OE-PsEGL3-2. The L* value in OE-PsEGL3–3 had no significant change, but the a* value and C* value decreased significantly. In contrast, the a* and C* values of OE-PsEGL3–1 and OE-PsEGL3–2 changed inapparently ( Figure 5B ). In accordance with floral color phenotype, the anthocyanin content in the petals of two PsMYB4 transgenic strains was significantly lower than that of the wild-type at p<0.01, whereas the anthocyanin content of three PsEGL3 transgenic strains exhibited a significant decrease compared to the wild-type at p<0.05 ( Figure 5C ).

Furthermore, the gene expression analysis confirmed that the overexpression of PsMYB4 in tobacco resulted in varying degrees of down-regulation of several structural genes, including NtC4H, NtCHS, NtCHI, NtF3H, NtF3’H, NtFLS and NtDFR. Notably, significant down-regulation was observed for NtC4H, NtCHS, NtCHI and NtDFR in the OE-PsMYB4–2 transgenic lines ( Figure 5D ). The results are consistent with the analysis of anthocyanin content and floral color phenotype, suggesting that PsMYB4 may exert inhibitory effects on anthocyanin biosynthesis by down-regulating the expression of structural genes involved in flavonoid biosynthesis, particularly NtC4H, NtCHS, NtCHI, and NtDFR. Compared to the wild-type, the expression levels of NtCHS, NtCHI, NtF3H, NtF3’H, NtFLS and NtDFR exhibited inconsistency in three PsEGL3 transgenic tobacco lines, however, down-regulation of NtC4H was observed across all three transgenic lines ( Figure 5E ). These results indicates that PsEGL3 may not play a major inhibitory role on anthocyanin biosynthesis, but assists PsMYB4 in regulation.

To further investigate the function of PsMYB4 and PsEGL3 in tree peony yellow flowers, the transcription of PsMYB4 and PsEGL3 was disturbed by VIGS system. Finally, we obtained TRV-PsMYB4 and TRV-PsEGL3 tree peony lines, with TRV1 and TRV2 empty vectors injected lines as a control. Next, yellow petals with their spots removed were collected for further analysis. As shown in Figure 6A , the yellow petals of TRV-PsMYB4 and TRV-PsEGL3 lines were lightened, notably in TRV-PsMYB4 line. The contents of flavonoids such as THC, Ap, Ch, Km, Qu and Is in TRV-PsMYB4 line decreased significantly, while the contents of THC, Ap, Lu, Ch and Qu in TRV-PsEGL3 line decreased significantly ( Figure 6B ), consistent with their floral color phenotypes.

The expression of flavonoid biosynthesis-related genes in silenced petals was determined using qRT-PCR methods. As a result, the expression of PsMYB4 in TRV-PsMYB4 petals was down-regulated by 68% compared to the control, while PsEGL3 in TRV-PsEGL3 petals showed a 42% decrease, confirming successful gene silencing in tree peony petals ( Figures 6C, D ). When PsMYB4 was silenced, the expression level of PsEGL3 was largely inhibited by 74%. Similarly, in TRV-PsEGL3 petals, the expression level of PsMYB4 was also decreased by 53%. It indicates that there may be an interaction relationship between PsMYB4 and PsEGL3. The expression of four key flavonoid biosynthesis-related genes (PsCHS, PsCHI, PsF3H and PsDFR) was remarkably increased in TRV-PsMYB4 petals compared to the control line ( Figure 6C ). The expression of PsCHS, PsCHI, PsF3H and PsDFR was also increased in TRV-PsEGL3 petals ( Figure 6D ). Therefore, we speculate that PsMYB4 and PsEGL3 may synergistically regulate the expression of PsCHS, PsCHI, PsF3H and PsDFR. Meanwhile, above up-regulated structural genes may affect the metabolic balance of the branches controlled by DFR and FLS genes, thus inhibiting the biosynthesis of flavonoid components related to yellow flower formation.

Considering above results, we performed Y2H and BiFC assays to confirm whether PsMYB4 interacts with PsEGL3. In the Y2H assay, the yeast cells co-transformed with pGADT7-T and pGBKT7–53 were employed as positive controls, whereas yeast cells co-transformed with pGADT7 empty vector and pGBKT7-PsEGL3 were designated as negative controls. We observed that yeast cells co-transformed with pGADT7-PsMYB4 and pGBKT7-PsEGL3 grew normally on SD-Leu-Trp-His-Ade screening medium, consistent with the positive control ( Figure 7A ), indicating an interaction between PsMYB4 and PsEGL3 in the yeast system.

The BiFC assay was performed to further validate the interaction between PsMYB4 and PsEGL3. The leaves co-transformed with BZIP63-nYFP and BZIP63-cYFP were positive controls, while leaves co-transformed with PsMYB4-nYFP/35S-cYFP or 35S-nYFP/PsEGL3-cYFP were negative controls. The fluorescence signal of YFP was observed in N. benthamiana leaves co-transformed with recombinant vectors PsMYB4-nYFP and PsEGL3-cYFP, as well as the positive control ( Figure 7B ). However, there was no signal in the negative control leaves ( Figure 7B ). The results obtained from BiFC and Y2H experiments provide evidence for an in vivo interaction between PsMYB4 and PsEGL3.

Overexpression in tobacco and VIGS in tree peony indicate that PsMYB4 and PsEGL3 may synergistically regulate flavonoid biosynthesis-related structural genes, especially PsCHS, PsCHI and PsDFR. To verify this hypothesis, we isolated the promoters of PsCHS, PsCHI and PsDFR from ‘High Noon’ petals. The identification of numerous cis-elements associated with MYB and bHLH TFs, such as the MYB-binding site (5’-CAACNG-3’), bHLH-binding site (5’-CATGTG-3’), and G-box (5’-CACGTG-3’) ( Figure 8A ), provides support for their potential regulatory relationships with PsMYB4 and PsEGL3.

To further clarify whether PsMYB4 and PsEGL3 directly regulate the expression of PsCHS, PsCHI, and PsDFR genes, a LUC transient expression assay was conducted in N. benthamiana. The pCAMBIA 1302 vector harboring PsMYB4 and PsEGL3 functioned as effectors, while the pGreenII0800 LUC vector carrying the promoters of PsCHS, PsCHI, and PsDFR served as reporters ( Figure 8B ). Consequently, both the PsMYB4/Luc-PsCHS and PsMYB4/Luc-PsDFR constructs exhibited significantly lower LUC/REN ratios compared to PsMYB4/Luc-PsCHI, indicating the inhibitory effect of PsMYB4 on the promoters of PsCHS and PsDFR ( Figure 8C ). Nevertheless, PsEGL3 had no significant inhibitory effect on PsCHS, PsCHI, and PsDFR. It is worth noting that the LUC/REN ratios significantly decreased when co-transformed with PsMYB4/PsEGL3/Luc-PsCHS, PsMYB4/PsEGL3/Luc-PsCHI, and PsMYB4/PsEGL3/Luc-PsDFR ( Figure 8C ), indicating that PsMYB4 and PsEGL3 synergistically inhibit the transcription of PsCHS, PsCHI, and PsDFR.