The R2R3-MYB Transcriptional Repressor TgMYB4 Negatively Regulates Anthocyanin Biosynthesis in Tulips (Tulipa gesneriana L.)

Anthocyanins play a paramount role in color variation and significantly contribute to the economic value of ornamental plants. The conserved activation complex MYB-bHLH-WD40 (MBW; MYB: v-myb avian myeloblastosis viral oncogene homolog; bHLH: basic helix–loop–helix protein; WD40:WD-repeat protein) involved in anthocyanin biosynthesis has been thoroughly researched, but there have been limited investigations into the function of repressor factors. In this study, we characterized TgMYB4, an R2R3-MYB transcriptional repressor which is highly expressed during petal coloration in red petal cultivars. TgMYB4-overexpressing tobaccos exhibited white or light pink petals with less anthocyanin accumulation compared to control plants. TgMYB4 was found to inhibit the transcription of ANTHOCYANIDIN SYNTHASE (TfANS1) and DIHYDRO-FLAVONOL-4-REDUCTASE (AtDFR), although it did not bind to their promoters. Moreover, the TgMYB4 protein was able to compete with the MYB activator to bind to the :bHLHprotein, thereby suppressing the function of the activator MBW complex. These findings demonstrate that TgMYB4 plays a suppressive role in the regulation of anthocyanin synthesis during flower pigmentation.


Introduction
Anthocyanins are flavonoid compounds that are commonly present in various parts of plants, such as the flowers, fruits, leaves, roots, and stems, adding to their vivid hue [1].These vibrant colors are crucial for attracting pollinators, facilitating seed dispersion, and providing protection against damage from ultraviolet radiation [2][3][4].Anthocyanin pigments have been the subject of extensive research, leading to notable scientific advancements over the last 150 years [5].
The anthocyanin biosynthesis pathway has been extensively characterized, and the structural genes responsible for encoding the enzymes in the biochemical reactions for anthocyanin synthesis have been identified and isolated from different plant species [6].Transcription factors play a crucial role in regulating the transcriptional expression of these structural genes.Specifically, the highly conservative MBW complex, composed of R2R3-MYB transcription factors, bHLH transcription factors, and WD40 proteins, serves as the core of the anthocyanin regulatory network [5,7,8].MBW is a highly organized complex, wherein each subunit possesses specific functions.MYB proteins directly bind to the promoters of structural genes to regulate gene transcription; bHLH proteins typically interact with MYB proteins to enhance regulation; and WD40 proteins primarily contribute to the stability of the complex [9][10][11][12].
The highly conserved bHLH-binding motif and ERF motif are identified as common characteristics in MYB repressor proteins, which may determine the suppression function.The EAR motif is identified as the most predominant form of the transcriptional repression motif in plants [22].In numerous studies, mutation or deletion of the EAR motif has demonstrated an essential role in the repressive activity of subgroup 4 R2R3-MYBs [13,19,28,29].Meanwhile, the bHLH-binding motif is particularly important for MYB repressors to compete with MYB activators.PrtMYB182 in poplar, lacking a functional bHLH-binding motif, was unable to exert repression, while mutations of the ERF domain did not have any impact on its repressive activity [25].In contrast, the repressive function of MdMYB16 in apples is dependent on the presence of the ERF domain rather than the bHLH-interacting motif [13].The significance of the EAR motif and protein-protein interactions in the repressive activity of subgroup 4 R2R3-MYBs has been documented, and there are notable variations across different species [9,13,19,20,[28][29][30][31].
Anthocyanin constitutes the principal component of floral pigmentation, which plays a crucial role in the ornamental value of flowers.The regulatory mechanisms of anthocyanin biosynthesis though the MYB-bHLH-WD40 complex have been well indicated in many ornamental plants.Additionally, several anthocyanin repressors have been isolated in recent years, including PhMYB27 and PhMYBX in petunias [9], CmMYB21 in chrysanthemums [39], LhR3MYB1/2 in lilies [14], MlROI1 and MlRTO in Mimulus [15,40], FhMYBX in Freesia hybrida [41], and MaMYBx in Grape hyacinths [16].In this study, TgMYB4 was identified as a subgroup 4 R2R3-MYB, which contains a bHLH-binding motif at the N-terminal and an ERF domain at the C-terminal.A functional assay revealed that it negatively regulates anthocyanin synthesis by inhibiting the transcription of structural genes.Additionally, the protein interaction between TgMYB4 and TgbHLH42-1 was characterized, which plays a crucial role in its repressive activity.

Identification of TgMYB4: A Repressor Transcription Factor in Tulip Petals
An R2R3-MYB transcription factor was isolated from tulip petals, which contained a 627 bp open reading frame (ORF) and encoded 208 amino acids.In a phylogenetic analysis, it showed the closest evolutionary relationship with AmMYB308 in Antirrhinum majus and was classified into the AtMYB4-like group, and was thereby named TgMYB4 (Figure 1A).Amino acid sequence analysis of TgMYB4 revealed that the N-terminal R2R3 domain was highly conserved and included a bHLH-binding domain (Figure 1A).Two conserved motifs, namely the C1 motif (LlsrGIDPxT/SHRxI/L) and C2 motif (pdLNLD/ELxiG/S), were located in the C-terminal region.In addition, the C2 motif also serves as a diagnostic marker for subgroup 4 MYB proteins and contains the EAR-repressor domain (Figure 1B).identified as a subgroup 4 R2R3-MYB, which contains a bHLH-binding motif at the Nterminal and an ERF domain at the C-terminal.A functional assay revealed that it negatively regulates anthocyanin synthesis by inhibiting the transcription of structural genes.Additionally, the protein interaction between TgMYB4 and TgbHLH42-1 was characterized, which plays a crucial role in its repressive activity.

Identification of TgMYB4: A Repressor Transcription Factor in Tulip Petals
An R2R3-MYB transcription factor was isolated from tulip petals, which contained a 627 bp open reading frame (ORF) and encoded 208 amino acids.In a phylogenetic analysis, it showed the closest evolutionary relationship with AmMYB308 in Antirrhinum majus and was classified into the AtMYB4-like group, and was thereby named TgMYB4 (Figure 1A).Amino acid sequence analysis of TgMYB4 revealed that the N-terminal R2R3 domain was highly conserved and included a bHLH-binding domain (Figure 1A).Two conserved motifs, namely the C1 motif (LlsrGIDPxT/SHRxI/L) and C2 motif (pdLNLD/ELxiG/S), were located in the C-terminal region.In addition, the C2 motif also serves as a diagnostic marker for subgroup 4 MYB proteins and contains the EAR-repressor domain (Figure 1B).The TgMYB4-YFP and nuclear marker (VirD2NLS-mCherry) were co-transformed into N. benthamiana leaves for fluorescence observations.The YFP signal was primarily localized in the nucleus and overlapped with the mCherry signal (Figure 2A).The full length of TgMYB4 was cloned into the pGBKT7 vector and subsequently transferred into the yeast strain AH109 for transcriptional activation analysis.The yeast cell carrying the pGBKT7-TgMYB4 vector was able to grow in SD/-Trp/-Leu medium, but it was unable to The TgMYB4-YFP and nuclear marker (VirD2NLS-mCherry) were co-transformed into N. benthamiana leaves for fluorescence observations.The YFP signal was primarily localized in the nucleus and overlapped with the mCherry signal (Figure 2A).The full length of TgMYB4 was cloned into the pGBKT7 vector and subsequently transferred into the yeast strain AH109 for transcriptional activation analysis.The yeast cell carrying the pGBKT7-TgMYB4 vector was able to grow in SD/-Trp/-Leu medium, but it was unable to grow in the selective medium SD/-Trp/-Leu/-Ade/-His with X-α-gal.This result demonstrated that TgMYB4 did not display any transcriptional activation in yeast (Figure 2B).
We investigated the expression patterns of TgMYB4 in different flower developmental stages of the yellow petal cultivar 'Strong Golden' and its budding cultivar 'Strong Fire' with red petals (Figure S1).The content of anthocyanin in 'Strong Fire' was significantly higher than that in 'Strong Golden'.During petal coloration in 'Strong Fire', the expression level of TgMYB4 was the lowest in S1 but showed significant increases in both S2 and S3, We investigated the expression patterns of TgMYB4 in different flower developmental stages of the yellow petal cultivar 'Strong Golden' and its budding cultivar 'Strong Fire' with red petals (Figure S1).The content of anthocyanin in 'Strong Fire' was significantly higher than that in 'Strong Golden'.During petal coloration in 'Strong Fire', the expression level of TgMYB4 was the lowest in S1 but showed significant increases in both S2 and S3, and ultimately peaked in S4.Conversely, in 'Strong Golden', TgMYB4 exhibited a low expression in S1 and a slight increase in S2-S4.In summary, the expression level of TgMYB4 increased following tulip petal coloration (S2-S4) and it was found to be higher in red petal cultivars as compared to yellow ones (Figure 2C).

TgMYB4 Negatively Regulates the Synthesis of Anthocyanin
The analysis of protein characterization and expression profiles implied that TgMYB4 might play a negative role in anthocyanin synthesis.To confirm this observation, two independent TgMYB4-overexpressing transgenic tobacco lines (OE-1# and OE-2#) were generated.The petals of the control plants displayed a pink color, while the transgenic lines showed white or light pink petals (Figure 3A, B).The quantification assays revealed a significant decrease in total anthocyanin contents in the petals of TgMYB4-transgenic

TgMYB4 Negatively Regulates the Synthesis of Anthocyanin
The analysis of protein characterization and expression profiles implied that TgMYB4 might play a negative role in anthocyanin synthesis.To confirm this observation, two independent TgMYB4-overexpressing transgenic tobacco lines (OE-1# and OE-2#) were generated.The petals of the control plants displayed a pink color, while the transgenic lines showed white or light pink petals (Figure 3A,B).The quantification assays revealed a significant decrease in total anthocyanin contents in the petals of TgMYB4-transgenic tobacco plants (Figure 3C).Furthermore, a qRT-PCR assay showed that genes involving in the anthocyanin synthesis pathway, including NtCHS, NtCHI, NtF3H, NtF3 ′ H, NtDFR, and NtANS, were significantly down-regulated in TgMYB4 transgenic lines (Figure 3D).
To further elucidate the role of TgMYB4 in the synthesis of anthocyanin in tulips, a virus-induced gene silencing (VIGS) assay was carried out.The presence of TRV was confirmed through genomic PCR (Figure 4A).The qRT-PCR assay revealed that the expression level of TgMYB4 was substantially suppressed in the silenced petals (Figure 4B).After 3 days of treatment, the petals of the TRV2 control started to display a red hue, whereas the TgMYB4-silenced petals displayed a more pronounced coloration.Additionally, the total anthocyanin content was significantly reduced in TgMYB4-silenced petals compared to the TRV2 control (Figure 4D).These results indicated that TgMYB4 functioned as a negative regulator of anthocyanin biosynthesis.tobacco plants (Figure 3C).Furthermore, a qRT-PCR assay showed that genes involving in the anthocyanin synthesis pathway, including NtCHS, NtCHI, NtF3H, NtF3′H, NtDFR, and NtANS, were significantly down-regulated in TgMYB4 transgenic lines (Figure 3D).To further elucidate the role of TgMYB4 in the synthesis of anthocyanin in tulips, a virus-induced gene silencing (VIGS) assay was carried out.The presence of TRV was confirmed through genomic PCR (Figure 4A).The qRT-PCR assay revealed that the expression level of TgMYB4 was substantially suppressed in the silenced petals (Figure 4B).After 3 days of treatment, the petals of the TRV2 control started to display a red hue, whereas the TgMYB4-silenced petals displayed a more pronounced coloration.Additionally, the total anthocyanin content was significantly reduced in TgMYB4-silenced petals compared to the TRV2 control (Figure 4D).These results indicated that TgMYB4 functioned as a negative regulator of anthocyanin biosynthesis.

TgMYB4 Suppresses the Activation of Anthocyanin Biosynthesis Genes Indirectly
To further characterize the working mechanisms of TgMYB4, we explored the regulatory relationship between TgMYB4 and structural genes involved in anthocyanin biosynthesis.The promoters of AtDFR from Arabidopsis thaliana and TfANS1 from Tulipa fos-

TgMYB4 Suppresses the Activation of Anthocyanin Biosynthesis Genes Indirectly
To further characterize the working mechanisms of TgMYB4, we explored the regulatory relationship between TgMYB4 and structural genes involved in anthocyanin biosynthesis.The promoters of AtDFR from Arabidopsis thaliana and TfANS1 from Tulipa fosteriana were used as the targeted genes for further analyses [42,43].
For the dual-luciferase reporter assay, the full length of TgMYB4 was combined into the pGreenII 62-SK as an effector, and the promoter fragments of TfANS1 (-732-0 bp) and AtDFR (-520-0 bp) were cloned into the pGreenII 0800-LUC vector as reporters (Figure 5A).The luminescence live images revealed that the fluorescence signal was considerably weaker in the co-transformation of the TgMYB4-SK effector and the pTfANS1 or pAtDFR reporter compared to the control group.When TgMYB4-SK was co-transformed with pGreen0800-pTfANS1 and pGreen0800-pAtDFR, the ratio of LUC (firefly luciferase) to REN (Renilla luciferase) significantly decreased by 42.5% and 75.6%, respectively, compared to the control group.The observations of fluorescence signal and quantitative measurement of the LUC/REN ratio indicated that TgMYB4 was able to suppress the transcription activities of TfANS1 and AtDFR (Figure 5B-E).The Y1H assay was performed to investigate the interaction between TgMYB4 and promoters of TfANS1 and AtDFR.The prey and bait vectors were constructed as shown in Figure 5F.The results showed that only the positive control grew normally on SD/-Trp/-Leu/-His media supplemented with 2.5 mM 3-AT.The yeast cells co-transformed with pGADT7-TgMYB4 prey and the pHIS2-pAtDFR/pTfANS1 bait could not grow on the selected medium.The Y1H assay demonstrated that TgMYB4 could not bind to promoters of TfANS1 and AtDFR.These results suggest that TgMYB4 might inhibit the expression of structural genes through an indirect approach.

Protein Interaction between TgMYB4 and TgbHLH42-1
TgMYB4 contains a bHLH-interacting motif (Figure 1B), which was identified as a conserved domain of subgroup 4 R2R3-MYBs and was shown to interact with the bHLH The Y1H assay was performed to investigate the interaction between TgMYB4 and promoters of TfANS1 and AtDFR.The prey and bait vectors were constructed as shown in Fig- ure 5F.The results showed that only the positive control grew normally on SD/-Trp/-Leu/-His media supplemented with 2.5 mM 3-AT.The yeast cells co-transformed with pGADT7-TgMYB4 prey and the pHIS2-pAtDFR/pTfANS1 bait could not grow on the selected medium.The Y1H assay demonstrated that TgMYB4 could not bind to promoters of TfANS1 and AtDFR.These results suggest that TgMYB4 might inhibit the expression of structural genes through an indirect approach.

Protein Interaction between TgMYB4 and TgbHLH42-1
TgMYB4 contains a bHLH-interacting motif (Figure 1B), which was identified as a conserved domain of subgroup 4 R2R3-MYBs and was shown to interact with the bHLH component of the MBW complex.TgbHLH42-1 plays a positive role in the regulation of anthocyanin in tulips, and has a high homology to AtTT8 [44].In order to dissect the working mechanism of TgMYB4, we explored the relationship between TgMYB4 and TgbHLH42-1.
Yeast two-hybrid assays were performed to investigate whether TgMYB4 could interact with TgbHLH42-1.The CDSs of TgMYB4 and TgbHLH42-1 were cloned and inserted into the pGBKT7 and pGADT7 vector as bait and prey plasmids, respectively (Figure 6A).All transformed yeast grew well on SD/-Trp/-Leu media, whereas only positive control and yeast cells harboring TgMYB4 bait and TgbHLH42-1 prey conducts survived and exhibited a blue color on SD/-Trp/-Leu/-Ade/-His media supplemented with X-α-gal (Figure 6B).Moreover, a firefly luciferase complementation imaging assay (LCI) was conducted in tobacco leaves through transient overexpression of TgbHLH42-1 fused with the N-terminus of luciferase (nLUC) and TgMYB4 fused with the C-terminus of luciferase (cLUC) (Figure 6C).Through live luminescence imaging, significant LUC fluorescence signals were exclusively observed in the presence of TgbHLH42-1_nLUC and TgMYB4_cLUC, while marginal signals were detected in the other groups (Figure 6D).This result provided further evidence showing the interaction between TgMYB4 and TgbHLH42-1.

The Relationship between TgMYB4 and the MYB-bHLH-WD40 Complex
We previously demonstrated that the AtPAP1-TgbHLH42-1 complex could activate the transcription of AtDFR through dual luciferase assays [44].In this study, we investigated whether TgMYB4 could attenuate the ability of the MYB-bHLH complex to activate AtDFR.The ratio of LUC to REN demonstrated that the expression of AtDFR was acti- Moreover, a firefly luciferase complementation imaging assay (LCI) was conducted in tobacco leaves through transient overexpression of TgbHLH42-1 fused with the Nterminus of luciferase (nLUC) and TgMYB4 fused with the C-terminus of luciferase (cLUC) (Figure 6C).Through live luminescence imaging, significant LUC fluorescence signals were exclusively observed in the presence of TgbHLH42-1_nLUC and TgMYB4_cLUC, while marginal signals were detected in the other groups (Figure 6D).This result provided further evidence showing the interaction between TgMYB4 and TgbHLH42-1.

The Relationship between TgMYB4 and the MYB-bHLH-WD40 Complex
We previously demonstrated that the AtPAP1-TgbHLH42-1 complex could activate the transcription of AtDFR through dual luciferase assays [44].In this study, we investigated whether TgMYB4 could attenuate the ability of the MYB-bHLH complex to activate AtDFR.The ratio of LUC to REN demonstrated that the expression of AtDFR was activated by AtPAP1, whereas it was repressed by AtMYB4 and unchanged in the presence of TgbHLH42-1.The highest ratio was observed when both AtPAP1 and TgbHLH42-1 were present.However, the addition of TgMYB4 led to a slight reduction in the LUC to REN ratio, with no clear dose-dependent effect observed.The evidence indicated that TgMYB4 could inhibit the activation effects of the MBW complex on the expression of biosynthesis-related genes (Figure 7A).Moreover, we investigated whether the function of TgMYB4 was influenced after interaction with TgbHLH42-1.As depicted in Figure 7B, with the increase in the TgbHLH42-1protein, the inhibitory impact of TgMYB4 on AtDFR diminished gradually and eventually ceased.The results presented above suggested that TgMYB4 might play an inhibitory role through competing with bHLH proteins in the MBW complex.

Discussion
Anthocyanins are a class of flavonoids that exhibit various functions such as attracting pollinators, defending against phytopathogens, protecting against UV light damage, and mitigating oxidative stress [5,45,46].In ornamental plants, anthocyanins play a substantial role in red, purple, pink, and blue coloration, greatly contributing to their high value [14,47,48].Investigations into anthocyanin regulation began over 30 years ago with the cloning of the first transcription factor, Colorless1 (C1), from maize [49,50].Subsequently, investigations into other species, particularly P. hybrida, Antirrhinum majus, and A. thaliana, have significantly contributed to the advancement of the MBW model in understanding the regulation of anthocyanin [51][52][53][54][55].In the past 20 years, the discovery of the anthocyanin repressor has improved our understanding of the regulatory mechanism of anthocyanin synthesis.
MYB repressors play important roles in regulating anthocyanin accumulation by providing feedback to the regulatory network [9,41,56].In petunias, PhMYB27 and PhMYBX serve to inhibit the production of anthocyanin, while their transcription was activated by the MBW complex.This illustrated a complex hierarchical and feedback regulation between the repressors and activating factors involved in anthocyanin regulation

Discussion
Anthocyanins are a class of flavonoids that exhibit various functions such as attracting pollinators, defending against phytopathogens, protecting against UV light damage, and mitigating oxidative stress [5,45,46].In ornamental plants, anthocyanins play a substantial role in red, purple, pink, and blue coloration, greatly contributing to their high commercial value [14,47,48].Investigations into anthocyanin regulation began over 30 years ago with the cloning of the first transcription factor, Colorless1 (C1), from maize [49,50].Subsequently, investigations into other species, particularly P. hybrida, Antirrhinum majus, and A. thaliana, have significantly contributed to the advancement of the MBW model in understanding the regulation of anthocyanin [51][52][53][54][55].In the past 20 years, the discovery of the anthocyanin repressor has improved our understanding of the regulatory mechanism of anthocyanin synthesis.
MYB repressors play important roles in regulating anthocyanin accumulation by providing feedback to the regulatory network [9,41,56].In petunias, PhMYB27 and PhMYBX serve to inhibit the production of anthocyanin, while their transcription was activated by the MBW complex.This illustrated a complex hierarchical and feedback regulation between the repressors and activating factors involved in anthocyanin regulation [9,38,[57][58][59].R2R3-MYB repressors, such MtMYB2 in Medicago truncatula; PpMYB18 in Prunus persica; and CsMYB3 in Citrus sinensis, typically exhibit high expressions during the anthocyaninpigmented stage, and are activated by the MBW complex.It was suggested that the roles of these R2R3-MYB repressors might not be attributed to completely preventing anthocyanins synthesis, but to modulating the absolute anthocyanin intensity [9,19,20,29].In this study, TgMYB4 was characterized as an S4 R2R3-MYB anthocyanin repressor with a high expression level in anthocyanin-pigmented stages in tulip petals (Figures 1-3).The LUC assay demonstrated that TgMYB4 could suppress the expression of anthocyaninbiosynthesis-related genes and attenuate the activating effects of AtPAP1 and TgbHLH42-1 (Figure 5).However, in the presence of both the TgMYB4 repressor and MYB activator, the activating factors play a prominent role, despite an abundance of TgMYB4 (Figure 7A).In summary, TgMYB4 might suppress the transcription of anthocyanin-related genes to protect plants from excessive anthocyanin accumulation.
The R2R3 MYBs repressors are classified into two types according to the phylogenetic relationships: 'FaMYB1-like' and 'AtMYB4-like'.In this study, TgMYB4 was identified as an 'AtMYB4-like' repressor, which primarily regulates the general phenylpropanoid steps to reduce the accumulation of phenolic acids and lignans (Figure 1B).However, TgMYB4 overexpression in tobacco plants reduced anthocyanin accumulation in flower petals, resulting in a white or light pink color.Moreover, the expression of the structural genes NtCHS, NtCHI, NtF3H, NtF3 ′ H, NtDFR, and NtANS was consistently down-regulated in TgMYB4 transgenic lines (Figure 3).In addition, the luciferase assay revealed the inhibition of TgMYB4 on the expression of TfANS1 and AtDFR (Figure 5).Incidentally, narcissus (Narcissus tazetta) NtMYB2 and apple (Malus domestica) MdMYB16 were categorized in the 'AtMYB4-like' group; however, they inhibit the production of anthocyanin by suppressing the transcription of multiple genes in the anthocyanin pathway [13,60].PtMYB165 and PtMYB194 in poplars fell into the 'FaMYB1-like' clade phylogenetically, and could greatly decrease the accumulation of various phenylpropanoids rather than flavonoids [61].In general, the R2R3-MYB repressors typically play a key role in suppressing the biosynthesis of diverse metabolites in the phenylalanine pathway.Although these repressors exhibit a certain degree of division of labor, it is not strictly exclusive.
The highly conserved bHLH-binding motif and ERF motif are important for the suppression function of R2R3-MYB repressor proteins.For AtMYB4 and PpMYB18 (P.persica), mutations in either the bHLH-binding motif or the ERF motif significantly affected their inhibitory function.In poplars, PrtMYB182 without a functional bHLHbinding motif lost its repressor function, whereas mutations of the ERF motif did not lead to a noticeable decrease in repressor function [25].On the contrary, the repressive function of MdMYB16 was unaffected by the removal of the bHLH-interacting motif.However, it was completely inactivated when the EAR motif was deleted [13].Here, TgMYB4 contained a bHLH-binding motif, a C1 motif, and an ERF motif (Figure 1A).This protein demonstrated the capacity to interact with TgbHLH42-1 (Figure 6), and the inhibition of AtDFR expression was diminished in the presence of an excess amount of TgbHLH42-1 (Figure 7B).These results suggest that the physical interaction between TgMYB4 and TgbHLH42-1 plays a significant role in the repressive function of TgMYB4.

Plant Materials and Growth Conditions
The tulip cultivars 'Strong Fire' and 'Strong Golden' (Figure S1), Nicotiana benthamiana, and early flowering tobacco [62] were used in this study.The plant materials were cultivated in a plant growth room with a 16/8 h light/dark cycle and a temperature range of 18-22 • C. 4.2.Total RNA Extraction, Gene Cloning and Real-Time Quantitative PCR Analysis Total RNA isolation, first-strand cDNA synthesis, gene cloning and qRT-PCR assays were performed using the same methods as described previously [63,64].The specific primers were designed by the web tool Primer-BLAST on the NCBI (National Center for Biotechnology Information) website (https://www.ncbi.nlm.nih.gov/tools/primerblast/index.cgi?LINK_LOC=BlastHome (accessed on 11 December 2023)).All the primer sequences are listed in Table S1.

Multiple Sequence Alignment and Phylogenetic Analysis
Multiple protein sequence alignments were performed using the ClustalW1.81and GenDoc 2.7 programs.The conserved domains were identified using the Conserved Domain Search Service (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?(accessed on 11 December 2023)).The phylogenetic tree was generated using the maximum-likelihood method with 1000 bootstrap replicates in MEGA11.

Vector Construction and Plant Transformation
The coding sequence (CDS) of TgMYB4 was recombined into the pCAMBIA2300s vector under the control of the CaMV 35S promoter.TgMYB4 transgenic tobacco lines were generated through leaf disc transformation [62].All the primer sequences are listed in Table S1.

VIGS Assay in Tulip Petals
The fragment located 313-624 bp downstream of the start codon (ATG) of TgMYB4 was amplified and cloned into the pTRV2 vector.The recombinant pTRV2-TgMYB4 vector, empty pTRV2, and pTRV1 vectors were transformed into A. tumefaciens GV3101.The petals of the red cultivar 'SF' were used for the assay before coloration.The specific steps of the assay were conducted following a previously described protocol [44,65].Firstly, the petals were immersed in a bacterial suspension containing pTRV1 and pTRV2 or pTRV2-TgMYB4 (OD600 = 1.5, Ratio = 1:1 v/v) and vacuum-infiltrated at 0.7 Mpa for 5 min.After infiltration, the petals were washed with distilled water and incubated in darkness at 22 • C for 24 h.All petals were then transferred to a growth chamber with a 16/8 h light/dark cycle for the specified time points.The primer sequences used in VIGS assay are listed in Table S1.

Subcellular Localization
A subcellular localization assay was performed following the established protocol outlined in our previous work [44,65].The ORF of TgMYB4 without a stop codon was amplified and inserted into the L101YFP vector to generate the 35S::TgMYB4-YFP construct.The construct was transformed into A. tumefaciens strain GV3101 for a transient assay in N. benthamiana leaves.After 2-3 days of culture, the leaves were observed thought a confocal laser scanning microscope (Leica TCSSP8).The primer sequences are listed in Table S1.

Transcriptional Activation ACTIVITY Assay
For transcriptional activation analyses, the full length of TgMYB4 was amplified and cloned into the pGBKT7 vector containing the GAL4 DNA-binding domain.The recombinant plasmid, pGBKT7-p53, and empty vector pGBKT7 were transformed into yeast strain AH109.All the positive clones were plated onto SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade medium with or without X-α-gal and incubated at 30 • C for 48-72h.The primer sequences are listed in Table S1.

Yeast One-Hybrid Assay
The promoter sequences of TfANS1 and AtDFR were amplified and inserted into pHIS2 as the bait vectors, and the CDS of TgMYB4 was cloned into pGADT7 as the prey vector.The bait and prey plasmids were transformed into the Y187 yeast strain and cultivated on SD/-Trp/-Leu selection plates at 30 • C.Then, the positive clones were transferred to SD/-Trp/-Leu and SD/-Leu/-Trp/-His/-Ade media with 2.5 mmol/L 3-AT and X-α-Gal.The primer sequences are listed in Table S1.

Yeast Two-Hybrid Assay
For yeast two-hybrid assay, the CDSs of TgbHLH42-1 and TgMYB4 were cloned into pGADT7 and pGBKT7 vectors to generate the prey and bait plasmids.The recombinant plasmids were co-transformed into the AH109 strain and grown on SD/-Leu/-Trp medium at 30 • C for 3-4 days.Then, several dilutions of transformants were transferred to SD/-Leu/-Trp/-His/-Ade with 3-AT and X-α-Gal.The primer sequences are listed in Table S1.

Dual Luciferase Assay
The CDSs of TgMYB4, TgbHLH42-1, and AtPAP1 were amplified and ligated into the pGreenII 62-SK effector vector under the control of CaMV 35S.The TfANS1 (GenBank: KC261507) and AtDFR promoter fragments were ligated into the pGreen II 0800-LUC vector as reporters.The vectors were introduced into A. tumefaciens strain GV3101 and transiently expressed in 4-week-old tobacco leaves as previously described by Hu et al. [44].
Luminescence live images were acquired using the plant living imaging system as described by Meng et al. [65].The ratio of LUC to REN, determined using the Dual-Luciferase ® Reporter Assay System (Promega), was calculated to detect the transcriptional activity.The primer sequences are listed in Table S1.

Firefly Luciferase Complementation Imaging Assay (LCI)
A firefly luciferase complementation imaging (LCI) assay was performed following the description of Chen et al. [66] and Li et al. [67].The full lengths of TgbHLH42-1 and TgMYB4 were inserted into pCAMBIA1300-nLUC and pCAMBIA1300-cLUC vectors to create TgbHLH42-1-nLUC and TgMYB4-cLUC constructs.The constructs or empty plasmids were transformed into A. tumefaciens GV3101 and transiently expressed in N. benthamiana leaves.The LUC fluorescence signal was observed thought a live plant imaging system.The primer sequences are listed in Table S1.

Anthocyanin Extraction and Determination
The method of total anthocyanin extraction and determination was performed following to the description of Luo et al. [68] and Shen et al. [69].The lyophilized powder of the petals (0.1 g) was extracted with 1 mL of extraction solution (0.1% HCl in methanol) at 4 • C in the dark for 24 h.After extraction, the samples were centrifuged at 12,000× g for 10 min, and the supernatant was collected for analysis.Finally, the absorbance values were determined via a multifunctional microporous plate detector (TECAN, Beijing, China) at wavelengths of 530 and 657.The content of anthocyanin was calculated as Q Anthocyanins = (A 530 − 0.25 × A 657 ) × M −1 , where A 530 and A 657 correspond to the absorption at specified wavelengths and M represents the weight of the plant material used for extraction.

Statistical Analysis
All experiments in this study were conducted a minimum of three times.The data were expressed as means ± standard error (SE).Statistical analysis was performed using a Student's t-test, with significance levels of p < 0.05 (*) and p < 0.01 (**).Duncan's test was employed to assess significant differences among multiple comparisons via PASS 26.0 software.

Conclusions
In conclusion, TgMYB4 is an R2R3-MYB transcriptional repressor that acts to inhibit anthocyanin synthesis in tulip petals.The bHLH-binding motif in the R3 domain of TgMYB4 is crucial for its repression function, but further investigation is needed to understand its regulatory mechanisms.

Figure 2 .
Figure 2. Characterization of TgMYB4 during flower coloring in T. gesneriana.(A) Subcellular location of TgMYB4 in tobacco leaves.Confocal microscopic images show fluorescence signals of bright field, YFP (yellow), mCherry (red) in epidermal cells.. Merged: merged images of fluorescence signals in bright field.Scale bars = 50 μm.(B) Transcription activity assay.The positive and negative controls contain pGBKT7-p53 and pGBKT7 vectors, respectively.(C) The expression levels of TgMYB4 during flower development in a red petal cultivar (SF: 'Strong Fire') and yellow petal cultivar (SG, 'Strong Gol den').

Figure 2 .
Figure 2. Characterization of TgMYB4 during flower coloring in T. gesneriana.(A) Subcellular location of TgMYB4 in tobacco leaves.Confocal microscopic images show fluorescence signals of bright field, YFP (yellow), mCherry (red) in epidermal cells.. Merged: merged images of fluorescence signals in bright field.Scale bars = 50 µm.(B) Transcription activity assay.The positive and negative controls contain pGBKT7-p53 and pGBKT7 vectors, respectively.(C) The expression levels of TgMYB4 during flower development in a red petal cultivar (SF: 'Strong Fire') and yellow petal cultivar (SG, 'Strong Gol den').

Figure 3 .
Figure 3.The phenotypes of TgMYB4 transgenic tobaccos.(A) Pigmentation of flower petals of the control and TgMYB4 transgenic tobacco plants.The scale bar=1cm.(B) Total anthocyanin content of the control and transgenic lines.(C) The expression level of TgMYB4 in the control and transgenic tobaccos.(D) Expression levels of anthocyanin-related genes in the control and TgMYB4 transgenic tobaccos.Error bars indicate SE (n = 3), ** p < 0.01.

Figure 3 .
Figure 3.The phenotypes of TgMYB4 transgenic tobaccos.(A) Pigmentation of flower petals of the control and TgMYB4 transgenic tobacco plants.The scale bar=1cm.(B) Total anthocyanin content of the control and transgenic lines.(C) The expression level of TgMYB4 in the control and transgenic tobaccos.(D) Expression levels of anthocyanin-related genes in the control and TgMYB4 transgenic tobaccos.Error bars indicate SE (n = 3), ** p < 0.01.Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 6 of 15

Figure 6 .
Figure 6.Protein interactions between TgMYB4 and TgbHLH42-1.(A) Schematic diagram for bait and prey in a yeast two-hybrid assay.(B) TgMYB4 interacts with TgbHLH42-1 in yeast two-hybrid assays.The positive and negative control contain pGADT7-T with pGBKT7-p53 or pGBKT7-lam vectors, respectively.(C) Schematic diagram for c-LUC and n-LUC plasmids.(D) Bimolecular luminescence complementation assay demonstrating that TgMYB4 interacts with TgbHLH42-1 in N.benthamiana leaves.

Figure 6 .
Figure 6.Protein interactions between TgMYB4 and TgbHLH42-1.(A) Schematic diagram for bait and prey in a yeast two-hybrid assay.(B) TgMYB4 interacts with TgbHLH42-1 in yeast two-hybrid assays.The positive and negative control contain pGADT7-T with pGBKT7-p53 or pGBKT7-lam vectors, respectively.(C) Schematic diagram for c-LUC and n-LUC plasmids.(D) Bimolecular luminescence complementation assay demonstrating that TgMYB4 interacts with TgbHLH42-1 in N. benthamiana leaves.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 9 of 15TgMYB4 might play an inhibitory role through competing with bHLH proteins in the MBW complex.

Figure 7 .
Figure 7. Dual luciferase assay to test the activation and repression of the AtDFR promoter by re-MYB, activator MYB and bHLH factors.(A) TgMYB4 inhibited the activation of the MYB-bHLH complex on the AtDFR promoter.(B) Activation effect of co-infiltration of TgMYB4 and TgbHLH42-1 at different ratios on the AtDFR promoter.LUC, firefly luciferase; REN, Renilla luciferase.SK empty vector + pGreen0800.pAtDFR was used as the negative control.Error bars indicate SE (n = 3).Significant differences were analyzed using Duncan's multiple comparison test.

Figure 7 .
Figure 7. Dual luciferase assay to test the activation and repression of the AtDFR promoter by repressor MYB, activator MYB and bHLH factors.(A) TgMYB4 inhibited the activation of the MYB-bHLH complex on the AtDFR promoter.(B) Activation effect of co-infiltration of TgMYB4 and TgbHLH42-1 at different ratios on the AtDFR promoter.LUC, firefly luciferase; REN, Renilla luciferase.SK empty vector + pGreen0800.pAtDFR was used as the negative control.Error bars indicate SE (n = 3).Different letters indicate significant differences analyzed using Duncan's multiple comparison tests.