Fragaria vesca seeds of RG (Ruegen, runnerless, everbearing, and red fruited) were grown in flowerpots and cultivated in photoperiodic conditions of 16 h of light and 8 h of dark at a temperature of 22°C, and a relative humidity of 50%.

All the protein sequences of F. vesca were downloaded from GDR database¹ and the genome database version 4.0.2a was used. An HMM search with the MYB DNA-binding domain HMM profile (PF000249) was used to blast against strawberry protein database and the e-value was set as 1.0 × 10^––3. Because of the different splicing models for many genes in version 4.0.2a, a total of 499 MYB gene transcripts were obtained as a result. For the different splicing of each MYB gene, the longest transcript was preserved and the remaining was removed, resulting in 242 MYB transcripts obtained. To confirm the presence of MYB DNA-binding domain, the protein sequences of the 242 MYB transcripts were used as queries to search against Pfam², SMART³, and HMMER⁴ database, the e-value was set as 1.0 × 10^––5. In the end, a total of 118 MYB protein sequences containing more than two MYB DNA-binding domains were obtained. The isoelectric points and molecular weights of the corresponding MYB proteins were obtained from the online database of ExPASY⁵.

The 125 R2R3-MYB genes identified in Arabidopsis were obtained from a previous study (Dubos et al., 2010). The MYB protein sequences were downloaded from The Arabidopsis Information Resource (TAIR)⁶. The sequences of MYB proteins from Arabidopsis and strawberry were aligned using ClustalW (Thompson et al., 1994), and the BLOSUM matrix was used. The parameters of gap opening and extension penalties were 25 and 1, respectively, and the other parameters were default. The phylogenetic tree was constructed using the Neighbour Joining Tree Method in Mega5 (Tamura et al., 2011). The bootstrap was 1,000 replicates. Evolutionary distances were computed with P-distance method and the positions containing gaps and missing data were eliminated with partial deletion option (95%). For the MYB and bHLH proteins, and other corresponding proteins from different species, the phylogenetic tree was constructed as above.

Full CDS of bHLH and MYB genes were amplified and then inserted into BD vector pLexA/pGBKT7 and AD vector pB42AD/pGADT7, respectively, between EcoR I and Xho I using ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd.). All sequences of the recombinant plasmids were verified by sequencing (Sangon Biotech Co., Ltd. at Shanghai). The LexA yeast two-hybrid was performed as described previously (Xu et al., 2019). For the GAL4 yeast two-hybrid assay, the bait and prey vectors were co-transformed into AH109 yeast cells as described by Lu et al. (2015). The primers used were listed in Supplementary Table 1.

The 2.0 kb upstream sequences before the ATG of FvCHS2 and FvDFR2 genes were amplified from woodland strawberry of RG and inserted into the reporter vector pLacZ using homologous recombination method as above. The recombinant effector plasmids with inserted MYB or bHLH genes and the reporter plasmids were co-transformed into EGY48 yeast cells and grown at 30°C for 3 days. Then the DNA-binding activity were detected as described in the previous study (Li et al., 2020). To investigate whether bHLH can affect the bioactivity of MYB protein, the cDNA encoding the full-length bHLH was inserted into a modified pLexA vector, which lacks the BD domain. The two effector plasmids harboring bHLH and MYB genes, respectively, were co-expressed with the reporter plasmid in EGY48 yeast cells. Transformed colonies were selected on SD-Trp-His-Ura medium plus X-gal as described previously (Li et al., 2020). The sequences of the CHS2 and DFR2 promoters were listed in Supplementary Table 2 and the potential cis-elements of the CHS2 and DFR2 promoters for MBW binding were analyzed by PlantCARE⁷ (Lescot et al., 2002). The primers used were listed in Supplementary Table 1.

Strawberry fruits at green stage (about 15 days post anthesis, DPA), white stage (about 22 DPA), pre-turning stage (white receptacles with red achenes), turning stage (2 or 3 days after the pre-turning stage), and ripen stage (3 or 4 days after the turning stage) were harvested. The achenes were removed from all the fruits. and then the total RNA was extracted following the manufacturer’s instructions of RNA Plant Plus Reagent (Tiangen, Beijing, China, Cat.DP437). DNase I was used to remove the genomic DNA contamination. First-strand cDNA was synthesized using EasyScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, China). Real-time qPCR was performed using the CFX96 Touch^TM Real-Time PCR System (Bio-Rad, United States) with 2 × M5 HiPer SYBR Premix EsTaq (with Tli RNaseH) (Meibio, China, Cat. MF787-01). The qPCR reaction was performed by pre-denaturing at 95°C for 1 min, followed by 40 cycles of denaturing at 95°C for 5 s, annealing at 60°C for 15 s, and extension at 72°C for 15 s. Melting curve detection from 65 to 95°C was done after the cyclic reactions. Transcript abundance of target genes were calculated using 2^–ΔCt method in comparison with the internal control gene, Fv26S or FvActin. Three biological replicates were used in each assay. The experiments were repeated at least three times. The primer sequences were listed in Supplementary Table 3.

The vectors used to produce constructs for the BiFC assays were pXY104 and pXY106, which carry fragments encoding the C- and N-terminal halves of YFP (cYFP and nYFP), respectively. The full-length cDNAs of MYB and bHLH genes were cloned into pXY104 and/or pXY106 vector to generate corresponding recombinant plasmids. All constructs were transformed into Agrobacterium tumefaciens strain GV3101 and infiltrated into tobacco (Nicotiana benthamiana) epidermal leaves in the given combinations as described previously (Lu et al., 2015; Xu et al., 2016). After incubation in the dark for 36–40 h, the tobacco leaf samples were examined by confocal microscopy (Leica TCS SP5II) to detect the expression of various fluorescent proteins. The intensity of the laser was set at 30%, the wavelength at 514 nm, and the voltage at 700 v. All the primers used were listed in Supplementary Table 1.

The pGreen0800-Pro_DFR2:Luc vector was described previously (Li et al., 2020). The full length CDS of MYB and bHLH genes including FvMYB22, FvMYB64, FvMYB105, and FvbHLH33 were cloned into pGreen62SK vector and served as effectors. The reporter strain GV3101 harboring Pro_DFR2:Luc was mixed with each of the effector strain harboring pGreen62SK, pGreen62SK-bHLH33 or one of the pGreen62SK-FvMYB plasmids (pGreen62SK-FvMYB22, pGreen62SK-FvMYB64, or pGreen62SK-FvMYB105) individually, as well as combinations of plasmids indicated. The mixed culture of each combination was infiltrated into tobacco leaves as described in the results of this article. After incubation in the dark for 36–40 h, the N. benthamiana leaves were treated with 1 mM l-luciferin sodium salt (Yeasen, Shanghai, China, Cat. 40903ES02) and kept in the dark for an additional 10 min before the bioluminescence signal were imaged and the intensity measured by a luminescent imaging workstation (5200; Tanon, Shanghai, China). The primers used for luciferase reporter assay are listed in Supplementary Table 1.

The method of anthocyanin detection was described in previous studies (Xu et al., 2018; Li et al., 2020). The method of proanthocyanidins detection was described in previous study (Li et al., 2018). DMACA, (−)-epicatechin and 4-hydroxy-3-methoxybenzaldehyde used in the experiments were from Bide Pharmatech. Ltd. (Shanghai, China).

The construct of pGreen62SK, pGreen62SK-bHLH33 or one of the pGreen62SK-FvMYB plasmids (pGreen62SK-FvMYB22, pGreen62SK-FvMYB64, or pGreen62SK-FvMYB105) was used for transient overexpression. The method of injection was described in previous study (Zhang et al., 2020). The octoploid strawberry of “HongYan” was used for transient overexpression. The fruit were collected at 7 days after transfection and the measurement of proanthocyanidins was described in the same previous study (Xu et al., 2018). The expression of FvbHLH33, FvMYB22, FvMYB64, FvMYB105, FvCHS2, and FvDFR2 was confirmed by qRT-PCR assay.

In order to identify R2R3-MYB genes in strawberry, the HMM profile (Pfam:00249) of MYB DNA-binding domain was used to blast against the woodland strawberry genome database (F. vesca Whole Genome v4.0.a2) from GDR (see Text Footnote 1). The blast search resulted in a total of 242 MYB genes after removing repetitive redundant sequences. The protein products of 118 MYB genes out of the 242 were verified to contain conserved MYB DNA-binding domains through searching Pfam, SMART and HMMER databases. All 118 MYB gene products contain more than two MYB repeat units. Among them, one contains four MYB repeat units, five contain three MYB repeat units. The remaining 112 contain two MYB repeat units, which are R2R3-MYBs (Supplementary Table 4). Through gene sequence alignment analysis, we found that FvH4_1G22020 shares the same sequence with FvMYB10, thus annotated as FvMYB10. Blast results showed that the sequences of FvH4_2g31100 and FvH4_6g34650 were almost consistent with FaMYB9 and FaMYB11, respectively. We therefore named FvH4_2g31100 and FvH4_6g34650 as FvMYB9 and FvMYB11. The remaining R2R3-MYB genes were named according to the order of their chromosomal locations in the strawberry genome browser (Supplementary Table 4). The physical and chemical property prediction analysis was performed for the 112 R2R3-MYB proteins using ExPASy database. The results showed that the sizes of these MYB proteins are significantly different. For example, the protein product of FvH4_7g19850 is composed of 1703 amino acids, while other MYB proteins are much smaller, containing amino acids of a number ranging from 127 to 562. The isoelectric points of MYB proteins have been predicted to range from 4.82 to 10.10, and the presumed molecular weights are in a range of 15–62.7 kD, except for the protein product of FvH4_7g19850, the molecular weight of which is 185 kD (Supplementary Table 4).

To identify which R2R3-MYB genes in strawberry are possibly involved in flavonoids biosynthesis, the sequences of the 112 strawberry R2R3-MYB genes identified by us and the 125 R2R3-MYB genes from Arabidopsis were phylogenetically analyzed. The results showed that a total of 17 strawberry MYB genes clustered with AtMYBs that are involved in flavonoids biosynthesis (highlighted in black solid circles in Figure 1). We also included R2R3-MYB genes from other species with known functions in flavonoids synthesis for comparison, including those from grape, apple, cotton, tomato, maize and freesia. These R2R3-MYB genes were also phylogenetically analyzed together with the 17 strawberry MYB genes. Taken together,12 strawberry R2R3-MYB genes were identified as potentially involved in flavonoids biosynthesis (highlighted in black solid circles, Supplementary Figure 1). Specifically, FvMYB10 and FvMYB75 clustered with MdMYB10 and MdMYB1, which promote anthocyanin biosynthesis (Espley et al., 2007). FvMYB3, FvMYB9, and FvMYB11 clustered with FaMYB9 and FaMYB11, which were found to complement the phenotype of transparent testa in Arabidopsis tt2-1 mutant (Schaart et al., 2013), suggesting a function of regulating proanthocyanidins biosynthesis. FvMYB64 and FvMYB105 were homologous to VvMYBPA1, which is specific to the regulation of proanthocyanidins biosynthesis in seeds (Bogs et al., 2007). FvMYB45 and FvMYB77 were similar to maize P (Grotewold et al., 1994) and AtMYB111 (Stracke et al., 2007), respectively, suggesting that both of them might be related to flavonols synthesis. FvMYB41 clustered with VvMYB5b, which contributes to the regulation of anthocyanin and proanthocyanidins biosynthesis in the developing grape fruits (Deluc et al., 2008). AtMYB123 did not explicitly cluster with any known genes, but when AtMYB123 was used as a query to blast against strawberry genome, FvMYB21 and FvMYB22 was also among the results, displaying high similarity to AtMYB123, suggesting that FvMYB21 and FvMYB22 are potentially related to proanthocyanidins biosynthesis.

To screen bHLH components of MBW complexes, we used three Arabidopsis bHLH proteins as queries to perform BLASTP searches against F. vesca protein database. The three Arabidopsis bHLH proteins, including AtTT8, AtEGL3, and AtGL3, have been shown to have partially overlapping expression patterns and redundant functions in flavonoids biosynthesis. By the searches, we found that no matter which one of AtTT8, AtEGL3, and AtGL3 was used for blasting, the matching proteins with higher similarity were always the products of FvH4_2g23700, FvH4_7g14230, and FvH4_5g02520 (Supplementary Table 5). A phylogenetic analysis of these three strawberry bHLH proteins and those from other 16 plants was performed, and we found that FvH4_2g23700, FvH4_7g14230, and FvH4_5g02520 clustered with FabHLH3, FabHLH33 and FaMYC1 of Fragaria × ananassa, respectively (Supplementary Figure 2). Consequently, the bHLH proteins from woodland strawberry were named as FvbHLH3, FvbHLH33, and FvMYC1, and considered potential components of MBW complexes for flavonoids biosynthesis.

We studied the transcript levels of MYBs and bHLHs, and their effects on the accumulation of flavonoids in strawberry fruits at five stages of fruit development, including green stage, white stage, pre-turning stage, turning stage and ripe stage. qRT-PCR results showed that the transcript level of a bHLH gene (FvMYC1) and nine MYB genes (FvMYB3, FvMYB9, FvMYB11, FvMYB21, FvMYB22, FvMYB45, FvMYB64, FvMYB77, and FvMYB105) was higher at green stage, but significantly decreased at the other four stages (Figure 2 and Supplementary Figure 3), matching proanthocyanidins biosynthesis pattern, which accumulated mainly at the early stages, especially at the green stage (Figure 3A). Specifically, the expression of FvMYB11, FvMYB21, FvMYB77, and FvMYB105 were not detected during turning stage and ripe stage. By contrast, the expression of FvMYB10 was extremely low in green and white stages, but significantly increased during the late development stages, especially the turning stage, resembling anthocyanin biosynthesis pattern, which accumulated in a large amount during turning and maturation stages (Figure 3B). The expression of FvMYB41 and FvbHLH3 gradually increased accompanying the development of fruit and peaked at ripe stage (Figure 2 and Supplementary Figure 3), also aligned with anthocyanins accumulation (Figure 3B). The expression of FvMYB75 and FvbHLH33, however, was detected at all five stages, with higher expression level at early stages from green to pre-turning than turning and ripe stages (Figure 2 and Supplementary Figure 3). Taken together, the bHLH gene FvMYC1, and the nine MYB genes including FvMYB3, FvMYB9, FvMYB11, FvMYB21, FvMYB22, FvMYB45, FvMYB64, FvMYB77, and FvMYB105, might be involved in proanthocyanidins biosynthesis at early stages of fruit development. FvMYB10, FvMYB41, and FvbHLH3 might be associated with the accumulation of anthocyanins at later stages. The proposed functions of woodland strawberry MYB and bHLH genes are consistent with the phylogenetical analysis (Supplementary Figure 1).

To study whether these potential MYB and bHLH proteins can form MBW complexes, we first investigated the interaction among them in vitro using LexA yeast two-hybrid system. When FvbHLH3 or FvMYC1 were used as a bait (fused with DNA-binding domain, BD), they both could interact with every MYB protein tested (fused with Activation domain, AD) except for FvMYB45 or FvMYB77 (Figure 4). FvbHLH33, however, displayed strong self-activation when fused to LexA BD as a bait. This is also true when only the N-terminal fragment of FvbHLH33 was used (Supplementary Figure 4). Considering that 3-AT can be used to inhibit self-activation in GAL4 yeast two-hybrid system, the interaction between FvbHLH33 and MYB proteins were studied in GAL4 system instead. 20 mM 3-AT was introduced to inhibit self-activation using FvbHLH33 as a bait, and we were able to detect interactions between FvbHLH33 and 10 individual MYB proteins, including FvMYB3, FvMYB9, FvMYB10, FvMYB11, FvMYB21, FvMYB22, FvMYB41, FvMYB75, FvMYB77, and FvMYB105, but not FvMYB45 or FvMYB64 (Supplementary Figure 5).

We also performed BiFC experiments to furtherly investigate the interactions between bHLH and MYB proteins in vivo. As shown in Figure 5, when MYBs or FvbHLH3 were expressed individually, no over-lapping fluorescent signal of YFP was seen. When FvbHLH3 and any of the 10 MYB proteins, including FvMYB3, FvMYB9, FvMYB10, FvMYB11, FvMYB21, FvMYB22, FvMYB41, FvMYB64, FvMYB75, and FvMYB105, were co-expressed in tobacco cells, strong fluorescent signal of YFP was observed. However, when FvbHLH3 and FvMYB45 or FvMYB77 were co-expressed, no YFP signal was detected. Similarly, FvMYC1 or FvbHLH33 was observed to interact with the same 10 MYB proteins, but not FvMYB45 or FvMYB77 (Supplementary Figure 6). The interaction between FvMYB64 and FvbHLH33 was not detected in yeast two-hybrid, but the interaction was detected in BiFC assay, likely due to the difference between the in vitro and in vivo experimental systems. Taken together, the results in vitro and in vivo suggested that the 10 MYB proteins, except for FvMYB45 and FvMYB77, could possibly form functional MBW complexes with FvbHLH3, FvbHLH33, or FvMYC1.

In a MBW complex, MYB protein is the decisive factor for the regulation of specific flavonoids synthesis genes. To confirm whether the identified MYB proteins have a potential role in regulating genes involved in flavonoids biosynthesis, we first analyzed whether the promoters of CHS2 and DFR2 contained cis-elements bound by MBW complexes. As shown in Supplementary Figure 7, the promoter regions of CHS2 and DFR2 contain binding elements of MYB proteins. Then, we tested in yeast one-hybrid system to see whether these MYB proteins are able to bind to the promoters of CHS2 and DFR2 genes, which encode key enzymes related to the flavonoids synthesis pathways. As shown in Figure 6, all tested MYB proteins, except for FvMYB75, displayed binding activity to the promoter of CHS2 gene. However, only FvMYB11 and FvMYB21 bound to the DFR2 promoter.

We observed that most of the MYB proteins (except for FvMYB11 and FvMYB21) in strawberry could not bind to the promotor of DFR2 in yeast one-hybrid assay (Figure 6), nor the three bHLH proteins (FvbHLH3, FvbHLH33, or FvMYC1) (Supplementary Figure 8). To explore whether bHLH proteins are in need for those MYBs to bind to the promotor of DFR2, we test the DNA-binding activity of heterodimers formed between five FvMYB proteins (FvMYB10, FvMYB22, FvMYB64, FvMYB75, and FvMYB105) and three FvbHLH proteins (FvbHLH3, FvbHLH33, or FvMYC1) in yeast one-hybrid assay. As shown in Figure 7, transformation of yeast cells with either one of the five MYB proteins fused to AD did not show positive DFR2 promotor binding activity. However, when either one of the three bHLH proteins, FvbHLH3, FvbHLH33 or FvMYC1, was co-expressed with AD-FvMYB22, AD-FvMYB64, or AD-FvMYB105, the report gene of LacZ was activated and the yeast cells displayed blue on media containing X-gal, demonstrating the binding of MYB proteins to the promotor of DFR2 in the presence of bHLH proteins. For FvMYB75 and FvMYB10, the reporter gene LacZ can only be activated in the presence of FvMYC1, but not FvbHLH3 and FvbHLH33 (Supplementary Figure 9), suggesting a requirement of specific bHLH protein for the DNA-binding activity of FvMYB75 or FvMYB10.

To further confirm the requirement of FvbHLH proteins for the DNA-Binding activity of FvMYB proteins in vivo, we transiently expressed FvbHLH33 and three FvMYB proteins (FvMYB22, FvMYB64, and FvMYB105) separately, or as a combination of FvbHLH33 with one of the three FvMYB proteins in N. benthamiana leaves, along with a reporter luciferase gene under the control of DFR2 promoter. As shown in Figures 8A,B, when FvMYB22, FvMYB64, or FvMYB105 was expressed alone, the reporter gene was not significantly induced, whereas these MYB proteins induced the reporter gene in the presence of FvbHLH33, leading to a strong luminescence signal when the substrate of luciferin was added. These results, together with results from yeast one-hybrid suggested that the heterodimers between FvMYBs and FvbHLHs were necessary for activating some flavonoids synthesis genes.

To confirm the biofunction of strawberry MBW complexes in flavonoids synthesis, we co-expressed FvMYB and FvbHLH proteins in the fruits and measured the flavonoids accumulation. FvMYB22, FvMYB64, and FvMYB105 are homologs of MYB proteins in other plants that regulate proanthocyanidin synthesis (Supplementary Figure 1) and mainly expressed at green stage (Figure 2 and Supplementary Figure 3). FvbHLH33 has been shown to interact with all three FvMYB proteins in yeast and tobacco cells. We transiently expressed FvbHLH33 and the three FvMYB proteins individually, or a combination of FvbHLH33 and one of the three FvMYB proteins in green stage strawberry fruit. The accumulation of proanthocyanidins was measured following the expression. As shown in Figures 9A,B and Supplementary Figure 10, the accumulation of procyanidins did not significantly increase following the expression of either FvbHLH33 or any of the three FvMYB proteins alone. However, the expression levels of flavonoid biosynthesis related genes CHS2 and DFR2 was significantly enhanced and the accumulation of proanthocyanidins was significantly increased in strawberry fruits when FvbHLH33 and one of the three FvMYB proteins were co-expressed (Figures 9A,B and Supplementary Figure 10). These results confirmed the function of the identified MBW components in strawberry flavonoids biosynthesis.