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. 2023 Feb 22;21(6):1140–1158. doi: 10.1111/pbi.14024

A novel R2R3‐MYB transcription factor FaMYB5 positively regulates anthocyanin and proanthocyanidin biosynthesis in cultivated strawberries (Fragaria × ananassa)

Leiyu Jiang 1, † , Maolan Yue 1, † , Yongqiang Liu 1, † , Nating Zhang 1, Yuanxiu Lin 2, Yunting Zhang 2, Yan Wang 2, Mengyao Li 1, Ya Luo 1, Yong Zhang 1, Xiaorong Wang 1,2, Qing Chen 1,✉, Haoru Tang 1,2,✉
PMCID: PMC10214752  PMID: 36752420

Summary

Flavonoids have a major contribution to the fruit quality in cultivated strawberries and are regulated by MYB, bHLH and WD40 transcriptional factors. We reported here the identification of the FaMYB5, an R2R3‐MYB transcription factor, which positively regulated the accumulation of anthocyanins and proanthocyanidins through the trans‐activation of the F3'H and LAR. The strawberry FaEGL3 and FaLWD1/FaLWD1‐like interact with the R2R3‐FaMYB5 to form an MYB‐bHLH‐WD40 complex (MBW), enhancing the regulatory efficiency. The R2R3‐FaMYB5 was constitutively expressed in various tissues and in fruits of different developmental stages, which was strikingly contrasting to the fruit‐specific expression patterns of FaMYB10. Meanwhile, R2R3‐FaMYB5 failed to promote a stable accumulation of anthocyanin glycosides in the mature fruits of the myb10 mutant, mainly due to the suppressed expression of TT19. The R2R3‐FaMYB5 was regulated by an antisense long noncoding RNA lncRNA‐myb5. Additionally, the R2R3‐FaMYB5 protein could interact with FaBT2 and was degraded through the ubiquitin/26 S proteasome pathway. Transcriptome and metabolome data showed that R2R3‐FaMYB5 enhanced the gene expression and the metabolite accumulation involved in the flavonoid, phenylpropanoid and lignin biosynthesis pathways. Collectively, we conclude that the FaMYB5 is an R2R3‐MYB activator involved in the composition of MBW, which positively regulates the biosynthesis of anthocyanin and proanthocyanidin. These findings provided new insights into the molecular mechanisms that regulate flavonoids in strawberry fruits.

Keywords: R2R3‐FaMYB5, anthocyanin, proanthocyanidin, regulation network, MBW complex, strawberry

Introduction

Cultivated strawberry (Fragaria × ananassa, 2n = 8x = 56), a worldwide horticultural crop, has considerable commercial importance and healthy benefit due to its sweet flavour, nutritional value and attractive appearance (Sturzeanu et al., 2011). Flavonoids such as anthocyanins and proanthocyanidins (PAs), not only play important roles for plants in response to biotic and abiotic stresses, pollination and seed dispersion but also contribute essentially to strawberry fruit quality (Wen et al., 2020). Currently, the identification and successful application of important flavonoid‐related regulatory genes to strawberry improvement have always been the breeding focus.

The anthocyanin and PA biosynthesis in strawberries are controlled by the structural genes involved in the flavonoid biosynthetic pathway (Pillet et al., 2015; Schaart et al., 2013), which are divided into early biosynthetic genes (EBGs) and late biosynthetic genes (LBGs) according to their positions in the biosynthesis pathway (Wang et al., 2020). Then, they are modified and transported to vacuoles for stable storage by glutathione S‐transferases (GSTs), multidrug and toxic extrusion (MATE), H+‐ATPase and ATP‐binding cassette (ABC) proteins (Ku et al., 2020; Pucker and Selmar, 2022). Importantly, this biosynthetic process is regulated by the MBW ternary complex composed of R2R3‐MYB, bHLH and WD40 transcription factors (TFs), among which R2R3‐MYB TFs play core roles (Lin‐Wang et al., 2014; Schaart et al., 2013). Generally, the MBW complex regulates the LBGs in the flavonoid pathway, as well as some modification‐ and transportation‐related genes, whereas EBGs and phenylpropanoid pathway genes are mediated by independent R2R3‐MYB TFs (Li et al., 2020; Xu et al., 2015; Yan et al., 2021). In strawberries, the FaMYB9/FaMYB11‐FabHLH3‐FaTTG1 MBW complex regulates the PA metabolism in the fruits (Schaart et al., 2013). However, no consensus has been reached about the MBW that regulates anthocyanin metabolism. To date, the existing reports take FaMYB10 as the core factor and speculated that FaMYB10‐FabHLH3/FabHLH33‐FaTTG1 may regulate the anthocyanin metabolism in strawberries (Hossain et al., 2018; Lin‐Wang et al., 2014; Wang et al., 2020). There is no direct evidence that these MBWs have more effective regulation on flavonoid metabolism in strawberry fruits. Unfortunately, among the 111 MYBs (including 105 R2R3‐MYBs) and 61 MYB‐related TFs in strawberries (Zheng et al., 2016), the functions of most MYBs are currently unclear. Whether any other MYBs are involved in strawberry MBW still needs to be confirmed. According to the different types of MYB domains and the number of repeats, the plant MYBs could be divided into 1R‐MYB (MYB‐related), R2R3‐MYB, R1R2R3‐MYB and 4R‐MYB (Dubos et al., 2010). The functions of the reported MYB5 vary among species. In Arabidopsis, AtMYB5 hardly affected the accumulation of PAs in seeds but was closely related to leaf trichome morphology (Gonzalez et al., 2009; Li et al., 2009). The apple MdMYB5 was related to secondary cell wall formation. Ectopic overexpression of MdMYB5 in Arabidopsis increased plant lignin and cellulose content (Chen et al., 2021). Nevertheless, ectopic expression of VvMYB5a and VvMYB5b in tobacco significantly promoted the accumulation of anthocyanin and PA in petals (Deluc et al., 2005, 2008). FhMYB5 was also demonstrated to positively regulate the biosynthesis of anthocyanin and PA (Li et al., 2019). In addition, some MYB5 genes have been shown to promote PA accumulation but inhibit or have no effect on anthocyanin accumulation. For example, ectopic overexpression of CsMYB5a in tobacco increased the PA content in flowers and inhibited anthocyanin accumulation; overexpression of CsMYB5e also increased the PA content but did not affect anthocyanins (Jiang et al., 2018). Moreover, overexpression of PgMYB5‐like in tobacco leaves only promoted the accumulation of the individual intermediate in the flavonoid pathway but could not produce anthocyanins (Arlotta et al., 2020). In strawberries, the FaMYB5 was previously presumed as an R3‐MYB (1R‐MYB) that negatively regulated flavonoid metabolism (Lin‐Wang et al., 2014; Schaart et al., 2013). Recently, the FaMYB5 from ‘Benihoppe’ was shown to act as an R2R3‐MYB, regulating citric acid metabolism in fruits (Liu et al., 2022b). Thus, some puzzling questions arise (1) which type of FaMYB5 is present in cultivated strawberries, R3 or R2R3; (2) Does it regulate flavonoid metabolism, and if so, what is the regulatory mechanism; (3) how does it differ from FaMYB10.

Comparative transcriptomics and metabolomics are widely used in horticultural plants as they provide abundant information for unravelling the regulatory mechanisms of TFs (Bai et al., 2021; Liu et al., 2023). For example, it was used to reveal the principle of PGPR‐mediated salt tolerance in tomatoes (Mellidou et al., 2021). In this study, we confirmed that the FaMYB5 was an R2R3‐ rather than an R3‐MYB TF located in the nucleus, which showed a constitutive expression in various strawberry tissues. Transient overexpression, dual‐luciferase and EMSA experiments demonstrated that the novel R2R3‐FaMYB5 could bind to the promoters of F3'H and LAR genes to promote the biosynthesis of both anthocyanin and PA in strawberry fruits. Protein–protein interaction and co‐expression experiments showed that the FaMYB5 formed an MBW complex with FaEGL3 and FaLWD1/FaLWD1‐like TFs, which positively regulates anthocyanin and PA metabolism. Meanwhile, FaMYB5 was also subject to feedback regulation by the FaBT2 protein and the gene was mediated by an antisense long noncoding RNA lncRNA‐myb5. In addition, the analysis of comparative transcriptome and metabolome showed that FaMYB5 not only affected the expression of the flavonoid biosynthetic structural genes, flavonoid‐related genes such as glycosyltransferase and laccase, as well as the lignin biosynthesis pathway genes, but also enhanced the accumulation of metabolites in lignin metabolism. The results of this study provided us with a new and comprehensive landscape of flavonoid regulation in the strawberry plant and could be further used in ongoing molecular breeding programs.

Results

FaMYB5 is an R2R3‐MYB TF and constitutively expressed in different tissues

The previous study postulated that FaMYB5 might exist as an R3‐MYB repressor with a deletion at the N terminal (Schaart et al., 2013). However, the amplified full sequences of FaMYB5 (GeneBank, MW700311) contained the complete R2R3 domains in cultivated strawberry varieties ‘Benihoppe’, ‘Xiaobai’, ‘Snowwhite’ and ‘Toyonoka’ (Figures 1a and S1). Hence, we newly renamed it R2R3‐FaMYB5 to distinguish it from the previous R3‐FaMYB5. Bioinformatics analysis revealed that (Figures S1 and S2), the CDS sequence of the R2R3‐FaMYB5 was 1041 bp in length and encoded a protein with 346 aa. The amino acid sequence contained the highly conserved bHLH interaction motif (DLx2Rx3Lx6Lx3R) in the R3 domain. No anthocyanin characteristic motifs (ANDV and KPRPRS/TF) or repression motifs (LxLxL and TLLLFR) were found (Stracke et al., 2001). Further phylogenetic analysis showed that (Figure 1b), R2R3‐FaMYB5 had a relatively close relationship to the RcMYB5, sharing 86.7% amino acid sequence similarity. The R2R3‐FaMYB5 was distantly related to AtMYB5 with a sequence similarity of 44.5%. In addition, the R2R3‐FaMYB5 fell into a clade that is distant from that of FaMYB9, FaMYB11, FaMYB10 and CsMYB5a. Subcellular localization results reflected that the R2R3‐FaMYB5 was localized in the nucleus, whereas the R3‐FaMYB5 was localized in the nucleus and cytoplasm (Figure 1c). R2R3‐FaMYB5 exhibited the highest transcription levels in strawberry fruits and gradually increased as the fruit turned red (Figure 1d). It was also expressed in other tissues, such as roots, stems, runners, young leaves, functional leaves and flowers, with the transcription levels relatively lower in the runners and stems. By contrast, FaMYB10 was predominantly expressed in fruits, mounted and maintained at very high levels when fruits start to accumulate anthocyanins. These results indicated that the R2R3‐FaMYB5 may have a wider spatial and temporal regulation spectrum than that of FaMYB10.

Figure 1.

Figure 1

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FaMYB5 was an R2R3‐MYB located in the nucleus and constitutively expressed in different tissues. (a) R2R3 domains were compared by ESPript 3.0 online website. (b) Phylogenetic relationships between R2R3‐FaMYB5 (in red) and its closest homologues in different species. Multiple sequence alignments of MYB5 were performed through ClustalW and the phylogenetic tree was constructed using the neighbour‐joining method with 1000 bootstrap replicates. Accession IDs (GenBank): PaMYB5 (Prunus avium), XP_021816704; PpMYB5 (Prunus persica), XM_007205236; PmMYB5 (Prunus mume), XP_008220661; MdMYB5 (Malus domestica), NP_001315731; RcMYB5 (Rosa chinensis), XP_024199204; R2R2‐FaMYB5, MW700311; FvMYB5 (Fragaria vesca), XP_004295005; R3‐FaMYB5, AFL02459; VvMYB5b (Vitis vinifera), AAX51291; VvMYB5a, AAS68190; ClMYB5 (Citrus limon), QBG79825; PhPH4 (Petunia × hybrida), AAY51377; StMYB5 (Solanum tuberosum), XP_006354225; GmMYB5 (Glycine max), XP_006593823; MtMYB5 (Medicago truncatula), XP_003601609; AtMYB5 (Arabidopsis thaliana), NP_187963; FhMYB5 (Freesia hybrida), QAX87835; OsMYB5 (Oryza sativa), BAA23340; ZmMYB5 (Zea mays), XP_008656780; RrMYB5 (Rosa rugosa), AYP10274; PgMYB5‐like (Punica granatum), ADG65150; FaMYB9, OK001453; FaMYB11, OK001454; CsMYB5a (Camellia sinensis), ATC41981; CsMYB5e, ATC41985; FaMYB10, OK001452. (c) Subcellular localization of R2R3‐FaMYB5 and R3‐FaMYB5 (fused with eGFP). The nuclear marker was pCAMBIA‐35 S‐SV40 NLS‐mCherry‐NOS. Scale bars represent 20 μm. (d) Expression profiles of R2R3‐FaMYB5 and FaMYB10 in different tissues and fruit developmental stages. Rt, root; St, stem; Ru, runner; YL, young leave; FL, functional leave; Fw, flower; SG, small green; BG, big green; Wt, white; IR, initially red; PR, partially red; FR, full red. Multiple comparisons were tested using the Turkey's test, and significant differences (P < 0.05) were indicated by different letters; error bars show ± SEs.

R2R3‐FaMYB5 promotes the accumulation of anthocyanins and PAs in cultivated strawberry fruits

As expected, overexpression (OE) of FaMYB10 significantly enhanced the fruit skin and flesh pigment accumulation in strawberry fruits (Figures 2a–c and S3). Interestingly, the OE of the R2R3‐FaMYB5 showed a similar effect, while the OE of the R3‐FaMYB5 almost had no phenotypic changes (Figures 2a–c and S3). HPLC analysis showed that in the R2R3‐FaMYB5 OE fruits, the content of pelargonidin 3‐O‐glucoside (Pg3G) increased from 0 to 975 μg g−1 fresh weight (FW) and cyanidin 3‐O‐glucoside (Cy3G) increased from 0 to 113 μg g−1 FW in the flesh (Figures 2c and S4). Pg3G and Cy3G also increased largely in the skin. In ‘Benihoppe’ fruits, OE of R2R3‐FaMYB5 also promoted the accumulation of Pg3G and Cy3G in the skin and flesh (Figures S5 and S6a, b). Noteworthy, the PA content in the R2R3‐FaMYB5 OE skin and flesh was significantly increased as well (Figures 2d and S6c). The qPCR results demonstrated that the R2R3‐FaMYB5 elevated the expression of most biosynthetic structural genes the same as those regulated by FaMYB10. Among these regulated genes, C4H, F3'H and LAR had the highest fold increment (Figures 2e and S6d). The transportation‐related genes, such as TT12 and AHA10 were also upregulated in expression. The positive regulators of PAs, FaMYB9 and FaMYB11 (Schaart et al., 2013), also elevated in the flesh (Figure 2e). The enhancement of those genes was coordinated with the increasing trend of anthocyanins and PAs. It was worth noting that, OE of the R2R3‐FaMYB5 slightly elevated the transcription levels of FaMYB10 in ‘Xiaobai’ fruits but had no promoting effect in ‘Benihoppe’ and ‘Snowwhite’ fruits (Figures 2e, 5b and S6d). Interestingly, the OE of R3‐FaMYB5 did not change anthocyanin content and only slightly raised the transcription level of C4H and F3'H but far less than that induced by R2R3‐FaMYB5 (Figures 2c and S7). All these results indicated that the R2R3‐FaMYB5 TF, rather than being an R3‐MYB negative mediator, was indeed a new R2R3‐MYB activator that at least positively regulates both anthocyanins and PAs.

Figure 2.

Figure 2

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Overexpression of R2R3‐FaMYB5 in strawberry cv ‘Xiaobai’ led to an increment in anthocyanin and PA content. (a) Transient overexpression of R2R3‐FaMYB5 restored anthocyanin biosynthesis in ‘Xiaobai’ fruits. 35 S:: FaMYB10 was shown as a positive control and 35SN (empty vector) was used as a negative control. Scale bars represent 10 mm. (b) Transcription levels of FaMYB10 and FaMYB5 in different samples. (c) Pelargonidin 3‐O‐glucoside (Pg3G) and cyanidin 3‐O‐glucoside (Cy3G) were detected by HPLC. (d) PAs were quantified with DMACA reagent through a full‐wavelength microplate reader. (e) Relative expression levels of genes involved in overexpression samples of R2R3‐FaMYB5 (in red), FaMYB10 (in blue) and 35SN (in white). Chalcone synthase (CHS), chalcone isomerase (CHI), flavonol 3‐hydroxylase (F3H), flavonol 3′‐hydroxylase (F3′H), flavonoid 3′,5′‐hydroxylase (F3′5′H), dihydroflavonol‐4‐reductase (DFR), anthocyanidin synthase (ANS), UDP‐glucose flavonoid‐3‐O‐glycosyltransferase (F3GT), O‐methyltransferase (OMT), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR), TRANSPARENT TESTA (TT), H+‐ATPase 10 (AHA10), Fra a allergen (Fra a), phenylalanine ammonia lyase (PAL), cinnamate 4‐hydroxylase (C4H), 4‐coumarate:coenzyme A ligase (4CL). Significant differences were compared with 35SN by Student's t‐test (**P < 0.01; *P < 0.05); error bars show ± SEs (b, d, e). Samples were represented by different colors in b, d and e, 35SN in white, 35 S::R2R3‐FaMYB5 in red, 35 S::FaMYB10 in blue and 35 S::R3‐FaMYB5 in grey.

Figure 5.

Figure 5

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R2R3‐FaMYB5 could not restore anthocyanin accumulation in strawberry cv ‘Snowwhite’ (myb10 mutant) due to low expression of FaTT19. (a) Transient overexpression of FaMYB10, R2R3‐FaMYB5 and R3‐FaMYB5 in ‘Snowwhite’ fruits. Scale bars represent 10 mm. (b) Transcription levels of FaMYB10 and FaMYB5 in different samples. (c) Transient overexpression of FaTT19 alone or ‘R2R3‐FaMYB5 + FaTT19’ in ‘Snowwhite’ fruits. Scale bars represent 10 mm. (d) Transcription levels of FaMYB5 and FaTT19 in different samples. (e) Pg3G and Cy3G were detected by HPLC in ‘Snowwhite’ flesh. (f) Relative expression levels of genes involved in overexpression samples of R2R3‐FaMYB5 (in red), FaMYB10 (in blue) and 35SN (in white). (g) Total anthocyanins were detected by the pH differential method in ‘Snowwhite’ fruits. Multiple comparisons were tested using Turkey's test and significant differences (P < 0.05) were indicated by different letters; error bars show ± SEs (b, d, f, g).

Metabolomics profiling of flavonoids in the flesh samples of ‘Xiaobai’

In the experiments of detecting Pg3G and Cy3G by HPLC, at least five unknown peaks were found in R2R3‐FaMYB5 OE samples (Figures S4 and S5). To clarify what they were, the flavonoids extracted from the R2R3‐FaMYB5 OE flesh samples were analysed in a targeted metabolomics approach. Collectively, 28 flavonoids (including 20 anthocyanins and five PAs) were identified (Table 1). Sixteen anthocyanins were significantly increased in the OE flesh samples (fold change [FC] > 2 and P value <0.05 as the screening cut) compared with the control. Among them, cyanidin O‐rutinoside was 92 times higher. Cy3G, cyanidin 3‐O‐rutinoside chloride and cyanidin chloride, which could not be detected in the regular fruits, accumulated to a large amount in the R2R3‐FaMYB5 OE fruits. In addition, delphinidin O‐malonylhexoside content also increased significantly, 77 times more than that of the control. It was noteworthy that, the relative content of Cy3G was much lower than cyanidin O‐acetylhexoside, cyanidin O‐rutinoside and cyanidin‐3‐O‐galactoside chloride among the ten cyanidins. This indicated that the modification of cyanidins was not limited to 3‐O‐glucoside in strawberry fruits after overexpressing R2R3‐FaMYB5. Finally, the level of the five free PA oligomers did not change, contrasting with the increase in total PAs.

Table 1.

Flavonoid compounds in strawberry flesh of overexpressing R2R3‐FaMYB5 and 35SN by targeted metabolomics

NO. Compounds Q1 (Da) RT (min) Relative quantification Fold change P value
R2R3‐FaMYB5 35SN
1 Pelargonidin 3‐O‐glucoside 449.1 1.647 (6.3 ± 1.6) × 106 (3.0 ± 0.5) × 105 21.3 0.022
2 Pelargonidin chloride 287.0 3.773 (7.5 ± 0.7) × 105 (4.3 ± 0.4) × 104 17.7 0.003
3 Pelargonidin 3,5‐di‐O‐glucoside 611.1 1.544 (3.8 ± 1.4) × 104 (4.0 ± 1.4) × 103 9.5 0.052
4 Pelargonidin 3‐O‐malonyl‐malonylhexoside 605.1 1.129 (3.4 ± 0.8) × 104 (1.9 ± 0.4) × 104 1.7 0.047
5 Cyanidin O‐rutinoside 595.2 1.525 (4.9 ± 1.3) × 106 (5.3 ± 0.4) × 104 92.1 0.024
6 Cyanidin O‐acetylhexoside 489.1 3.739 (5.1 ± 0.6) × 106 (9.3 ± 1.2) × 105 5.5 0.006
7 Cyanidin O‐syringic acid 465.1 1.555 (2.4 ± 0.5) × 106 (3.5 ± 0.5) × 104 68.3 0.001
8 Cyanidin‐3‐O‐galactoside chloride 465.1 1.560 (2.5 ± 0.6) × 106 (3.7 ± 0.6) × 104 68.1 0.018
9 Cyanidin 3‐O‐malonylhexoside 535.1 3.739 (2.3 ± 0.6) × 106 (3.8 ± 0.3) × 105 6.0 0.004
10 Cyanidin 3‐O‐glucoside 465.1 1.614 (3.7 ± 0.8) × 105 0 NA 0.000
11 Cyanidin 3‐O‐rutinoside chloride 611.1 1.561 (5.3 ± 1.8) × 104 0 NA 0.000
12 Cyanidin chloride 303.1 3.424 (1.0 ± 0.1) × 105 0 NA 0.000
13 Cyanidin 3‐O‐glucosyl‐malonylglucoside 697.1 3.745 (4.6 ± 0.4) × 104 (2.2 ± 0.4) × 104 2.1 0.002
14 Cyanidin‐3,5‐di‐O‐glucoside 627.1 1.370 (6.8 ± 2.1) × 103 (6.4 ± 1.1) × 103 1.1 0.782
15 Delphinidin O‐malonylhexoside 551.1 3.661 (1.4 ± 0.2) × 106 (1.8 ± 0.2) × 104 77.7 0.007
16 Delphinidin 3‐β‐D‐Glucoside 481.1 1.483 (1.8 ± 0.5) × 104 (3.5 ± 0.2) × 103 5.1 0.035
17 Delphinidin chloride 319.0 4.797 (5.8 ± 1.8) × 103 (5.4 ± 0.7) × 103 1.1 0.694
18 Peonidin chloride 317.1 3.812 (1.2 ± 0.2) × 105 (2.2 ± 0.4) × 104 5.2 0.001
19 Peonidin O‐hexoside 463.1 3.646 (7.5 ± 0.8) × 104 (3.3 ± 1.1) × 104 2.3 0.006
20 Malvidin‐3‐O‐glucoside chloride 509.1 1.708 (1.8 ± 0.2) × 104 (6.3 ± 0.6) × 103 2.8 0.000
21 Gentisin 290.9 4.514 (1.7 ± 0.1) × 104 (1.6 ± 0.1) × 104 1.1 0.244
22 Pseudopurpurin 332.9 3.496 (1.4 ± 0.3) × 104 (4.0 ± 2.0) × 103 3.5 0.009
23 Hibiscetin 366.9 1.018 (4.9 ± 0.2) × 103 (5.3 ± 0.7) × 103 0.9 0.410
24 Proanthocyanidin B2 577.1 1.895 (1.7 ± 0.3) × 106 (1.9 ± 0.4) × 106 0.9 0.730
25 Proanthocyanidin B3 577.1 1.908 (1.4 ± 0.2) × 106 (1.6 ± 0.3) × 106 0.9 0.459
26 Proanthocyanidin A3 577.1 1.996 (4.8 ± 1.2) × 105 (6.0 ± 1.7) × 105 0.8 0.368
27 Proanthocyanidin A2 577.0 2.001 (2.6 ± 0.7) × 105 (3.1 ± 1.0) × 105 0.8 0.495
28 Proanthocyanidin A1 577.0 1.900 (2.8 ± 0.7) × 105 (3.2 ± 1.0) × 105 0.9 0.592
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Q1—molecular weight. RT—retention time. NA—not applicable.

Relative quantification, calculated by the area of each individual peak, and the data are expressed in mean ± standard deviation.

To fully explore the changing flavonoids and other metabolites influenced by R2R3‐FaMYB5, the quasitargeted metabolomics technology was adopted. A total of 468 metabolites were initially identified and divided into 51 categories (Table S3). Among them, 51 flavonoid metabolites were included, which were categorized into anthocyanins, flavones, flavonols, catechin derivatives, flavonoids, flavanones, isoflavonoids and PAs. 67 differential metabolites with 57 being increased, and 10 decreased, were screened using the defended criteria (VIP >1.0, FC >1.5 or <0.667 and P value <0.05). Quercetin‐O‐malonylhexoside (flavonoids, FC = 57) had the highest FC value, followed by genistin (flavonoids, FC = 36), p‐Coumaric acid (phenylpropanoids and polyketides, FC = 28), heptamethoxyflavone (flavonoids, FC = 24), prunindihydrochalcone (phenols, FC = 16) and apigenin O‐malonylhexoside (flavonoids, FC = 15). L‐phenylalanine (FC = 4) and p‐Coumaric acid (FC = 28) were important metabolites in the initial synthesis stage of flavonoids. The increase in their content guaranteed the subsequent substantial accumulation of flavonoids (Figure 3). Furthermore, four quercetin glycosides (quercetin‐O‐malonylhexoside, quercetin 3‐D‐galactoside, quercetin 5‐O‐hexoside and quercetin‐O‐glucoside) were significantly increased, which was consistent with the significant upregulation of F3'H (Figure 3). In addition to flavonoids, the accumulation of metabolites such as phenolamides, phenols, polyphenols and alkaloids was also significantly enhanced, suggesting that R2R3‐FaMYB5 could regulate the metabolic pathways of other phenolic substances. In particular, the intermediate metabolites of lignin biosynthesis, such as caffeic acid (FC = 5) and p‐coumaraldehyde (FC = 2), increased obviously.

Figure 3.

Figure 3

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Detailed portion of flavonoid biosynthesis pathway that revealed the various expressions of related genes and different contents of metabolites after overexpressing R2R3‐FaMYB5. Cell color indicated log2 fold change (R2R3‐FaMYB5/35SN) values from 0 (light red) to 12 (dark red). Each cell indicated different transcripts of the genes and the transcript name were listed in Table S5. The blue circle and the number to its right represented that the metabolite was identified by quasitargeted or targeted metabolomics methods and its fold change value in R2R3‐FaMYB5/35SN.

Transcriptome sequencing to screen differentially expressed genes in the R2R3‐FaMYB5 OE fruits

The whole transcriptome sequencing method was furtherly used to analyse the mRNAs, lncRNAs, microRNAs and circRNAs alternations in the FaMYB5 OE flesh samples. A total of 108 623 mRNA transcripts, 17 986 lncRNA transcripts, 1218 circRNAs, 173 microRNA mature bodies and 185 microRNA precursors were identified (Table S4). We obtained 6896 differentially expressed (DE) mRNAs, including 6088 being upregulated and 808 being downregulated (Figure S8a). In the flavonoid biosynthesis pathway, most of the structural genes including those of the EBGs, LBGs as well as those taking part in the sequestration and transportation process were elevated. The largest increment occurred in the expression of C4H, F3'H and LAR (Figure 3). This was in line with the qPCR results. Meanwhile, the flavonoid‐related TFs such as the bHLH3, MYB1 and the anthocyanin regulatory C1 were all upregulated. In addition, the transcription levels of genes involved in the lignin synthesis pathway, such as HCT, CCoAoMT, CCR, CAD, COMT and CAD were also augmented (Figure 3), indicating that R2R3‐FaMYB5 also regulate the lignin metabolism. KEGG analysis of the DEGs showed that pathways including the metabolic, biosynthesis of secondary metabolites, plant–pathogen interaction and phenylpropanoid biosynthesis were significantly enriched (Figure S8e).

A total of 370 and 119 lncRNA transcripts were differentially up or downregulated (Figure S8b). Among these DE lncRNAs, the two transcripts, TCONS_00041928 and TCONS_00071989, were dramatically disturbed (FC > 1000). Two of the DE lncRNAs, TCONS_00102355 and TCONS_00090344 were predicted to regulate the transcription levels of R2R3‐FaMYB5 by co‐location prediction methods (Bao et al., 2019). Their regulation roles were further validated (in the following results section). 37 circRNAs were found to be differentially expressed with circ_0000537 and circ_0000141 being the utmost impacted ones (FC > 120) (Figure S8c). Among the 12 DE miRNAs (Figure S8d; Table S4), fve‐miR408 was downregulated and predicted to be able to target the genes F3H and AHA10. RT‐qPCR was employed to validate the observed up‐/down regulation of genes. A strong correlation (R2 = 0.8593) between the qPCR results and the transcriptome expression data was observed (Figure S9), indicating that the RNA‐seq data in this study were highly reliable. All these data implied that R2R3‐FaMYB5 had taken part in the empirical regulation of lignins, anthocyanins and PAs, contrasting to the specific roles of FaMYB10 in anthocyanins and FaMYB9/11 in PAs (Castillejo et al., 2020; Medina‐Puche et al., 2014; Schaart et al., 2013).

R2R3‐FaMYB5 transactivates F3'H and LAR

OE of R2R3‐FaMYB5 significantly increased the transcription of LAR and F3'H in the fruits of three cultivated strawberry varieties (Figures 2e, 5f and S6d), as well as in diploid transgenic strawberry plants (Figure S16). To test whether R2R3‐FaMYB5 directly participated in the activating of target genes in the flavonoid biosynthesis pathway, the deduced promoters of C4H (GeneBank, OK001457), F3'H (OK001460, OK001461), LAR (OK001458) and ANR (OK001459), more than 1500 bp upstream of the transcription start site of the genes were amplified in strawberry. Using the reporter system of dual‐luciferase, it was demonstrated that R2R3‐FaMYB5 could transactivate F3'H and LAR but had no obvious enhanced activity toward C4H and ANR (Figure 4a,b). EMSA results showed that R2R3‐FaMYB5 had a significant binding effect on the MYB recognition site (AACCTAA) in the F3'H promoter (Figure 4c). Similarly, this binding was also present with the MYB recognition site (ACCAACAACCAAA) in the LAR promoter (Figure 4d). Based on these results, the F3'H gene was transiently overexpressed in ‘Benihoppe’. The pigment was significantly accumulated with the recovery of Cy3G in the flesh (Figure 4e,f), which reflected that R2R3‐FaMYB5 could affect anthocyanin metabolism by directly regulating the expression of F3'H.

Figure 4.

Figure 4

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R2R3‐FaMYB5 could regulate the expression of F3'H and LAR. (a) Schematic diagram of the dual‐luciferase transient expression vectors. R2R3‐FaMYB5 was inserted into 35SN as an effector, and target promoters of C4H, F3'H, LAR and ANR were inserted into a pGreenII0800‐LUC vector as the reporters. (b) Validation of the effect of R2R3‐FaMYB5 on C4H, F3'H, LAR and ANR promoters by dual‐luciferase assay. Significant differences were determined by Student's t‐test (**, P < 0.01; *, P < 0.05); each assay measures at least 6 samples; error bars show ± SEs. (c) Detection of the binding ability of R2R3‐FaMYB5 to the FaF3'H promoter by a rapid agarose gel EMSA. Each lane contains a 2.5 ng purified fluorescence probe and 500 ng (+) or 1000 ng (++) GST‐FaMYB5 protein. (d) Detection of the binding ability of R2R3‐FaMYB5 to the FaLAR promoter by a rapid agarose gel EMSA. (e) Transient overexpression of FaF3'H in ‘Benihoppe’ fruits led to pigment accumulation. Scale bars represent 10 mm. (f) Pg3G and Cy3G were detected by HPLC.

R2R3‐FaMYB5 does not upregulate the expression of TT19 in fruits

R2R3‐FaMYB5 was also overexpressed in the cultivated strawberry cv. ‘Snowwhite’, a myb10 mutant whose fruit does not accumulate anthocyanins (Castillejo et al., 2020; Wang et al., 2020), to explore whether it could recover anthocyanin accumulation independent from FaMYB10. Interestingly, anthocyanins were restored in ‘Snowwhite’ by OE of FaMYB10 rather than R2R3‐FaMYB5 (Figure 5a,b,e). The fruit pH, a factor affecting fruit color, did not change significantly neither (Figure S10). Further qPCR analysis suggested that R2R3‐FaMYB5 still significantly promoted the expression of most structural genes involved in anthocyanin metabolism, including PAL, C4H, F3'H and LAR (Figures 5f and S11). However, unlike FaMYB10, R2R3‐FaMYB5 could not promote the expression of TT19 (Figures 2e, 5f and S6d), which encodes a carrier to transport anthocyanin to the tonoplasts (Luo et al., 2018). In addition, TT19 was barely expressed in the flesh of ‘Snowwhite’. Although the OE of TT19 restored its transcription levels to be equivalent to that of regular ‘Benihoppe’ and ‘Xiaobai’ fruits, no anthocyanin accumulation was detected neither (Figure 5c,d). However, the co‐overexpression of R2R3‐FaMYB5 and TT19 recovered the anthocyanins content in the flesh to a certain level, even though it was still much lower than that in ‘Xiaobai’ (Figures 2c and 5c,g).

R2R3‐FaMYB5 forms a new complex with FaEGL3 and FaLWD1/FaLWD1‐like to regulate flavonoid metabolism

Based on the yeast two‐hybrid and BiFC results (Figure 6a–c), R2R3‐FaMYB5, FaEGL3 (bHLH, GeneBank: MW700313) and FaLWD1 (W40, MW700314) / FaLWD1‐like (W40, MW700315) could interact with each other. Co‐overexpression of ‘R2R3‐FaMYB5 + FaEGL3’, ‘R2R3‐FaMYB5 + FaEGL3 + FaLWD1’ and ‘R2R3‐FaMYB5 + FaEGL3 + FaLWD1‐like’ all greatly promoted the accumulation of fruit anthocyanins (Figures 7a,b and S12). No drastic changes were observed between the above co‐overexpression groups and the one where R2R3‐FaMYB5 was expressed alone. However, a much larger extent of enhancement of PAs was achieved in those co‐overexpression groups (Figure 7c). Notably, the increment of FHT, F3'H, ANS, LAR, TT12, AHA10, PAL, C4H and CHS expression was further enhanced by the addition of the ternary components, especially FaEGL3 (Figure 7d). Similarly, genes such as LAR, TT12 and CHS were also augmented in the ‘R2R3‐FaMYB5 + FaEGL3 + FaLWD1’ group. The changing pattern of these genes in the ‘R2R3‐FaMYB5 + FaEGL3’ OE fruits was similar to that was observed in the ‘R2R3‐FaMYB5 + FaEGL3 + FaLWD1‐like’ OE fruits. Taken together, ‘R2R3‐FaMYB5‐FaEGL3‐FaLWD1/like’ functionalized as a new effective MBW to mediate a wide range of flavonoid biosynthesis genes in strawberries.

Figure 6.

Figure 6

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Protein–protein interactions between R2R3‐FaMYB5, FaEGL3 and FaLWD1/FaLWD1‐like. (a) Yeast two‐hybrid experiments. The pGADT7 was an AD empty vector; pGADT7‐T + pGBKT7‐53 was a positive control and pGADT7‐T + pGBKT7‐Lam was a negative control. (b) Schematic diagram of the BiFC vectors. (c) BiFC assays revealed the interactions. Scale bars represent 20 μm.

Figure 7.

Figure 7

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‘R2R3‐FaMYB5 + FaEGL3 + FaLWD1/FaLWD1‐like’ complex markedly promoted anthocyanin and PA biosynthesis in ‘Xiaobai’ fruits. (a) Regular strawberry fruits compared with overexpressing R2R3‐FaMYB5, ‘R2R3‐FaMYB5 + FaEGL3’, ‘FaEGL3 + FaLWD1’, ‘FaEGL3 + FaLWD1like’, ‘R2R3‐FaMYB5 + FaEGL3 + FaLWD1’ and ‘R2R3‐FaMYB5 + FaEGL3 + FaLWD1‐like’. Scale bars represent 10 mm. (b) Pg3G and Cy3G were detected by HPLC in ‘Xiaobai’ flesh. (c) PAs was quantified with DMACA. Multiple comparisons were tested using Turkey's test and significant differences (P < 0.05) were indicated by different letters; error bars show ± SEs. (d) Relative expression levels of genes involved in anthocyanin and PA biosynthesis. Significant differences were compared with overexpressing R2R3‐FaMYB5 alone by Student's t‐test (**P < 0.01; *P < 0.05); error bars show ± SEs.

LncRNA‐myb5 impacts R2R3‐FaMYB5 at the transcription levels

Based on the transcriptome sequencing data, two homologous lncRNA transcripts (TCONS_00102355 and TCONS_00090344), nominated as LncRNA‐myb5 (GeneBank, OK001456) were found in cultivated strawberries. It was located on the antisense strand of R2R3‐FaMYB5. To explore whether it could affect the transcription levels of R2R3‐FaMYB5, an OE vector of LncRNA‐myb5 was constructed and transiently expressed in the ‘Benihoppe’ strawberry. Although there was no obvious pigmentation in the flesh from the appearance, further HPLC analysis revealed that the content of Pg3G increased and Cy3G was also produced. In addition, PAs increased slightly as well (Figure S14). The subsequent qPCR analysis showed that the transcription levels of R2R3‐FaMYB5 increased by 70%, and PAL, C4H, CHS, LAR, F3'H and TT12 were upregulated (Figure 8a). This indicated that LncRNA‐myb5 may affect the anthocyanin and PA pathways by fine‐tuning the transcription levels of R2R3‐FaMYB5.

Figure 8.

Figure 8

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LncRNA‐myb5 and FaBT2 affected R2R3‐FaMYB5 at the transcription and protein levels, respectively. (a) Expression levels of genes in overexpressing lncRNA‐myb5 samples. (b) Protein–protein interactions between R2R3‐FaMYB5, FaEGL3, FaLWD1, FaLWD1‐like and FaBT2 by yeast two‐hybrid assays. (c) Schematic diagram of the modified dual‐luciferase transient expression vectors. R2R3‐FaMYB5 was linked to renilla luciferase for fusion expression, and pCAMBIA‐35SN‐FaBT2 was used as an effector. (d) FaBT2 caused the degradation of the R2R3‐FaMYB5 protein in vivo. Each assay measures at least six samples. Significant differences were determined by Student's t‐test (**P < 0.01; *P < 0.05); error bars show ± SEs. (e) FaBT2 induced the degradation of R2R3‐FaMYB5 protein in vitro. Total proteins of transgenic strawberry calli (35 S::6 × His‐FaBT2‐6 × HIS) and wild‐type calli (WT) were extracted in protein extraction buffer supplemented with 100 μM MG132 or 0.1% DMSO. Total proteins from 35 S::3 × Flag‐FaMYB5‐3 × FLAG calli were mixed with the above protein extracts in degradation buffer, respectively, and then incubated at 37 °C for 0, 1, 2, 4 and 6 h. These samples were analysed by western blot using anti‐FLAG or anti‐Actin antibodies. (f) Protein ubiquitination experiments in vitro. The FaMYB5‐FLAG and FaBT2‐HIS active proteins were purified from the transgenic calli proteins by using Protein A + G Agarose for Immunoprecipitation. Then, they were incubated with human E1, E2 (UbcH5a) and ubiquitin at 30 °C for 2 h. The mixture was analysed by western blot using anti‐FLAG, anti‐HIS or anti‐Ub antibodies. IB—immunoblotted.

FaBT2 efficiently mediates the R2R3‐FaMYB5 protein level

Transient OE of R2R3‐FaMYB5 was always accompanied by an increase in the transcription levels of the FaMYB1 repressor (Figures 2e, S6d and S11). However, when FaMYB1 was transiently overexpressed alone in strawberries, the pigment accumulation decreased slightly. Co‐overexpression of R2R3‐FaMYB5 and FaMYB1 only caused a slight decrease in pigment (Figure S15), which indicated that FaMYB1 may play a limited role in inhibiting the accumulation of strawberry flavonoids. Interestingly, the BTB‐BACK‐TAZ domain protein FaBT2 (GeneBank, OK001455), a potential factor of the 26 S proteasome system, was upregulated after the OE of R2R3‐FaMYB5 (Table S4). FaBT2 was a homologue of MdBT2 with an amino acid similarity of 76%. The MdBT2 was reported to negatively regulate anthocyanin or PA metabolism in apples (An et al., 2018a,b, 2019; Wang et al., 2018). Yeast two‐hybrid experiments revealed that FaBT2 could interact with R2R3‐FaMYB5, R3‐FaMYB5 and FaEGL3 (Figure 8b). To verify whether FaBT2 could cause the degradation of R2R3‐FaMYB5 protein in vivo, the R2R3‐FaMYB5 was co‐overexpressed with FaBT2 after fusion with REN fluorescent protein (Figure 8c). The results of the modified dual‐luciferase experiments showed that FaBT2 induced the degradation of R2R3‐FaMYB5 protein efficiently (Figure 8d). In vitro protein degradation experiments also revealed that after mixing the total proteins of FaBT2‐HIS and R2R3‐FaMYB5‐FLAG transgenic calli, the R2R3‐FaMYB5 protein level gradually decreased with increasing incubation time in DMSO treatment (Figure 8e). However, this decreasing trend was not obvious after the addition of MG132 (26 S proteasome inhibitor). Further, R2R3‐FaMYB5‐FLAG and FaBT2‐HIS proteins were extracted from the transgenic calli by immunoprecipitation method and then co‐incubated with ubiquitin, human E1 and E2 for 2 h. The results showed that after the addition of FaBT2, more high molecular mass forms of R2R3‐FaMYB5 were detected by anti‐FLAG and anti‐Ubi antibodies in the reaction mixture (Figure 8f). These results suggested that FaBT2 was able to induce the degradation of R2R3‐FaMYB5 protein through the ubiquitin/26 S proteasome pathway.

Discussion

Potential genes related to flavonoid metabolism regulated by R2R3‐FaMYB5

Plant anthocyanins and PAs are synthesized on the endoplasmic reticulum and transported to the vacuole for stable storage after modifications (Ku et al., 2020; Pucker and Selmar, 2022). The flavonoid biosynthesis pathway has been relatively clearly studied in strawberries, but the understanding of modification, transport and storage processes is very limited (Chen et al., 2018; Gao et al., 2020; Luo et al., 2018). Therefore, only the currently known genes related to flavonoid biosynthesis and transport were analysed by qPCR, and whether other genes affected by R2R3‐FaMYB5 were unknown. The content and proportion of some metabolites in the metabolome data increased much, including Pg3G, cyanidin O‐rutinoside, cyanidin O‐syringic acid, cyanidin‐3‐O‐galactoside chloride, Cy3G, cyanidin 3‐O‐rutinoside chloride and delphinidin O‐malonylhexoside. This suggested that R2R3‐FaMYB5 may regulate glycosylation modifications. Transcriptome data proved that the transcription levels of genes such as anthocyanidin 3‐O‐glucosyltransferase5/6, UDP‐glycosyltransferase U74F2/U74E1/U73C3 and UDP‐glucose flavonoid 3‐O‐glucosyltransferase3/6/7 had increased from several to hundreds of times. According to existing reports in other plants (Fukuchi‐Mizutani et al., 2003; Song et al., 2016; Zhao et al., 2012), we speculated that R2R3‐FaMYB5 may regulate these genes involved in the biosynthesis of those modified metabolites. In addition, GST, MATE, H+‐ATPase and ABC proteins played important roles in the transportation and storage of anthocyanins and PAs (Baxter et al., 2005; Behrens et al., 2019; Gomez et al., 2009; Luo et al., 2018). The transcription levels of genes such as Glutathione S‐transferase L2/F11/X1/X6/U9/3, ABC transporter A/B/G, Vacuolar‐sorting receptor 6 and ATPase 10 also increased significantly after overexpressing R2R3‐FaMYB5, which uncovered that R2R3‐FaMYB5 could also regulate these genes. However, the reported GST (TT19) in the strawberry was not its target (Luo et al., 2018). The TT19 was barely expressed in the root, stem, leaf and calli of Xiaobai strawberry, but it increased hundreds of times in red fruit, which may explain why the R2R3‐FaMYB5 transgenic calli did not show more coloration than the fruit (Figures S17c‐d). Besides, laccase‐5 and laccase‐9 resume expression, and the log2FC values of their different transcripts were all around 10. In Arabidopsis, TT10 encodes a laccase‐like enzyme involved in the oxidative polymerization of flavonoids in the seed coat (Pourcel et al., 2005). It's still unexplored whether R2R3‐FaMYB5 could directly regulate laccase‐5 and lactase‐9 in flavonoid metabolism. Moreover, transcription factors such as ERF2, ERF5, MYB39, MYB308, MYB330, WRKY33 and WRKY46 also increased significantly (Table S4). Whether these transcription factors are directly regulated by R2R3‐FaMYB5 and related to flavonoid metabolism was worth exploring.

R2R3‐FaMYB5 participates in the composition of MBW complex, independent from that of FaMYB10

Flavonoid metabolism is regulated by the MBW complex composed of MYB, bHLH and WD40 TFs (Xu et al., 2015). In Arabidopsis, PAP1/PAP2‐TT8/GL3/EGL3‐TTG1 regulates anthocyanin metabolism (Gonzalez et al., 2008; Xu et al., 2014), TT2‐TT8‐TTG1 regulates PA metabolism in seeds (Baudry et al., 2004). In Petunia hybrida, PH4‐AN1‐AN11 regulates anthocyanin metabolism in flowers (Quattrocchio et al., 2006; Spelt et al., 2002). In strawberries, FaMYB9/FaMYB11‐FabHLH3‐FaTTG1 regulates the PA metabolism pathway in fruits (Schaart et al., 2013), and FaMYB10‐FabHLH3/FabHLH33‐FaTTG1 may regulate the anthocyanin metabolism (Hossain et al., 2018; Lin‐Wang et al., 2014; Wang et al., 2020). However, it should be noted that FaMYB10 was expressed exclusively in fruit tissues of mature stages. Given these patterns, the observed phenotype that fruit does not accumulate anthocyanins in the wild strawberry ‘Yellow Wonder YW5AF7’ (myb10 mutant), while the petiole remains red (Slovin et al., 2009). Similarly, in the myb10 mutant cultivated strawberry ‘Snowwhite’, fruits do not accumulate anthocyanins (Wang et al., 2020). However, we found that some flowers of ‘Snowwhite’ are pink in the field, and under the conditions of low temperature, salt stress and disease, anthocyanin accumulation can be restored near achenes (Figure S13a). This evidence furtherly indicated the existence of regulators other than FaMYB10. In this study, we found an R2R3‐FaMYB5 TF could regulate the metabolism of anthocyanin and PA in strawberry fruits, as well as in transgenic calli (Figure S17a,b). However, no interaction was observed between R2R3‐FaMYB5 and FabHLH3 (Figure S13b,c), which was different from previous reports (Schaart et al., 2013). R2R3‐FaMYB5 and FaTTG1 only interacted in the yeast two‐hybrid experiment but not in the BiFC experiment (Figure S13), which was different from FaMYB9, FaMYB10 and FaMYB11. We noticed that in addition to the FaTTG1 (homologue of AtTTG1), the WD40 TFs FaLWD1 and FaLWD1‐like also existed in cultivated strawberries, which were homologous to AN11 in Petunia hybrid (Spelt et al., 2002). Yeast two‐hybrid, BiFC, and co‐overexpression experiments demonstrated that R2R3‐FaMYB5, FaEGL3, FaLWD1/FaLWD1‐like could form a new MBW complex to regulate flavonoid metabolism in strawberries, which was different from the MBW documented previously (Hossain et al., 2018; Lin‐Wang et al., 2014; Schaart et al., 2013; Wang et al., 2020). Moreover, R2R3‐FaMYB5 and FaMYB10 all promoted the expression of most structural genes in the flavonoid biosynthesis pathway, including PAL, C4H, 4CL‐2 and F3'H. Different from FaMYB10, R2R3‐FaMYB5 could specifically regulate the PA‐related genes, such as LAR, MYB9 and MYB11, while FaMYB10 had a greater influence on TT19, CHI, DFR2, ANS and F3GT genes. Despite the findings that OE of R2R3‐FaMYB5 could slightly trigger the expression of FaMYB10 in cv. Xiaobai (Figure 2e), it was unlikely that R2R3‐FaMYB5 exerted its function upstream of FaMYB10 since it had no influence on FaMYB10 in the other two varieties. In considering the different expression patterns of R2R3‐FaMYB5 and FaMYB10, we speculated that the MBW with R2R3‐FaMYB5 as the core factor regulated the metabolism of anthocyanin and PA during the entire developmental stage of the strawberry fruits, while FaMYB10 mainly regulated the anthocyanin metabolism at the fruit maturation stage.

Regulation of R2R3‐FaMYB5 via lncRNAs and FaBT2 at the transcription and protein levels

Studies had confirmed that lncRNAs could regulate the expression of target genes through cis‐ or trans‐activation at the transcription, post‐transcriptional and epigenetic levels (Kopp and Mendell, 2018; Sun et al., 2018). Diverse mechanisms were involved, including genomic imprinting (Wutz et al., 2002), chromatin remodelling (Rinn et al., 2007), cell cycle regulation (Hung et al., 2011), splicing regulation (Tripathi et al., 2010), mRNA degradation (Gong and Maquat, 2011), translation regulation (Yoon et al., 2012) and so on. Since we were not able to determine whether lncRNA‐myb5 was located in the nucleus or cytoplasm, the way it regulated the R2R3‐FaMYB5 was still unclear. Considering that lncRNA‐myb5 was on the antisense strand of the R2R3‐FaMYB5, we inferred that it was more likely to affect the transcription of R2R3‐FaMYB5 rather than translation. Moreover, after overexpression of R2R3‐FaMYB5, the transcription levels of the two lncRNA‐myb5 transcripts, TCONS_00102355 and TCONS_00090344, increased 8‐fold (Table S4). We speculated that this change formed a self‐activation route for R2R3‐FaMYB5. According to the co‐location prediction method (Bao et al., 2019), another lncRNA TCONS_00084252 was also assumed to target and regulate the expression of R2R3‐FaMYB5, which was located about 70 kb downstream of R2R3‐FaMYB5 in the genome. TCONS_00084252 was similar to lncRNA‐myb5 in transcription levels, with an increasement of more than 30 times in the R2R3‐FaMYB5 OE fruits. It might have a feedback effect on R2R3‐FaMYB5. The transcription levels of lncRNAs TCONS_00057627, TCONS_00009175, TCONS_00035610, TCONS_00033620 and TCONS_00009172 were also found to have a positive correlation with R2R3‐FaMYB5 significantly in the co‐expression analysis (Kopp and Mendell, 2018). They were greatly upregulated in samples overexpressing R2R3‐FaMYB5, while its relationship with R2R3‐FaMYB5 was still unknown.

Besides, we found that the FaMYB1 and FaBT2 could inhibit the excessive R2R3‐FaMYB5 production in strawberries. FaMYB1 may affect R2R3‐FaMYB5 by competitively binding to FaEGL3 (Aharoni et al., 2001; Paolocci et al., 2011). However, we proved that FaMYB1 was not so effective in preventing the promoting role of the R2R3‐FaMYB5. LaFountain and Yuan (2021) speculated that the role of the R2R3‐MYB repressors, such as FaMYB1 and AtMYB4, was more in modulating the absolute anthocyanin intensity by serving as a ‘break’ rather than an ‘off‐switch’. It seems more simple and more effective to directly affect the protein levels of the MBW members, such as MYBs and bHLHs. In apples, MdBT2 inhibits anthocyanin and PA biosynthesis by triggering MdMYB9 and MdMYB1 degradation (An et al., 2018a; Wang et al., 2018). MdBT2 also directly interacts with and degrades MdMYB73 through the ubiquitin/26 S proteasome pathway to regulate malate accumulation and vacuolar acidification (Zhang et al., 2020). We discovered that FaBT2 could interact with R2R3‐FaMYB5 or FaEGL3 and induce the degradation of R2R3‐FaMYB5 protein efficiently through the ubiquitin/26 S proteasome pathway. However, the detailed process of ubiquitination still needs to be further explored.

Other potential functions of R2R3‐FaMYB5 beyond flavonoid metabolism

Reports show that the regulatory effects on flavonoids metabolism by MYB5 TFs are inconsistent across plants, with some MYB5 TFs promoting both PA and anthocyanin accumulation (Deluc et al., 2008; Li et al., 2019), others promoting PA but inhibiting or not regulating anthocyanins (Jiang et al., 2018) and still others regulating only individual intermediate in the flavonoid pathway (Arlotta et al., 2020). In addition, MYB5 TFs also regulate leaf trichome morphology and secondary cell wall formation (Chen et al., 2021; Deluc et al., 2008; Li et al., 2009). Recently, FaMYB5 was shown to regulate citric acid metabolism in fruits (Liu et al., 2022b). Strangely, the citric acid content of R2R3‐FaMYB5 OE fruits did not change significantly in our metabolomics, while the same was true for the fruit pH (Table S3; Figure S10). It is not clear whether this is caused by differences in strawberry varieties or different developmental stages of the fruit. Finally, overexpression of R2R3‐FaMYB5 also promoted the accumulation of phenolamines, phenols, polyphenols and alkaloids (Table S3), the regulatory mechanisms of which also need to be further explored.

To summarize, we have identified a novel R2R3‐FaMYB5 that could positively regulate strawberry flavonoids including anthocyanins and PAs by forming a ternary complex with FaEGL3, FaLWD1/FaLWD1‐like in fruits (Figure 9). The transcription of this R2R3‐FaMYB5 itself could be fine‐tuned by a lncRNA‐myb5. Also, it could interact with the FaBT2 protein, subjecting to efficient protein regulation by the proteasomal machinery. These results provided us a new and comprehensive landscape of flavonoid regulation in strawberries and could be further used in ongoing molecular breeding programs.

Figure 9.

Figure 9

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Putative regulatory network of anthocyanin and PA biosynthesis in strawberry fruits by R2R3‐FaMYB5. Putative activations are indicated in a dotted line. Ubi—ubiquitin.

Methods

Plant materials and growth conditions

Four cultivated strawberry (Fragaria × ananassa) varieties ‘Benihoppe’, ‘Xiaobai’, ‘Snowwhite’, ‘Toyonoka’ and tobacco (Nicotiana benthamiana) plants were grown in a greenhouse located at Sichuan Agricultural University. Environmental conditions were controlled at 22 ± 2 °C, 80%–90% relative humidity and 16/8 h light–dark cycle with 220 μmol m−2 s−1. Organs (roots, stems, runners, young leaves, functional leaves, flowers) and fruits at different developmental stages: small green (7 days after fruit setting [DFS]), big green (14 DFS), white (20 DFS), initially red (23 DFS and red surface area less than 1/2), partially red (25 DFS and red surface area more than 1/2), full red (28 DFS and surface area are all red), were harvested from ‘Benihoppe’. All collected samples were immediately snap‐frozen with liquid nitrogen and stored at −80 °C for further use.

Gene cloning and bioinformatics analysis

Total RNA was extracted by a CTAB‐based method and transcribed into cDNAs (Chen et al., 2012). Genes were amplified and cloned into the pBlunt vector (TransGen, Beijing, China) from the four varieties using the primers listed in Table S1. MYB domains were analysed through the Pfam (pfam.xfam.org) and ESPript 3.0 online websites (Robert and Gouet, 2014). Multiple sequence alignments were performed through the ClustalW program in the MEGA 6.06 software package and a phylogenetic tree was constructed using the neighbour‐joining method with 1000 bootstrap replicates.

Subcellular localization and BiFC assays

R2R3‐FaMYB5 and R3‐FaMYB5 were fused to the N‐terminus of the eGFP in the pCAMBIA‐35 S‐eGFP vector through homologous recombination, separately. For BiFC, target genes were cloned into the vectors modified from pSAT1‐nEYFP‐C1 and pSAT1‐cEYFP‐N1 to produce fused YFP‐target proteins (Citovsky et al., 2006). Vectors were transformed into the agrobacterium strain GV3101. Cells were grown overnight at 28 °C in a YEP medium supplemented with appropriate antibiotics. After centrifugation, the collected cells were resuspended in the MMA solution (10 mM MES, 10 mM MgCl2, 500 μM acetosyringone) and incubated at room temperature for additional 3 h on a shaker. The bacterium was suspended in the fresh buffer and adjusted to a final density of OD600 = 1.5. A needless syringe was used to inject the suspensions into the abaxial side mesophyll cells of tobacco leaves. The treated plants were maintained in the greenhouse for 2–4 days before observation under a confocal microscope (Olympus FV1000). All primers used to construct vectors were listed in Table S1.

Anthocyanin and PA detections

Pg3G and Cy3G were detected by HPLC based on the previously described method (Donno et al., 2013; Lin et al., 2018). Total anthocyanins content was carried out according to the pH differential method (Giusti and Wrolstad, 2001). Total PAs was stained by DMACA solution (0.2% [w/v] DMACA, 50% [v/v] methanol and 9% [w/v] HCl) for 1 h, and its content was determined using DMACA assay through full‐wavelength microplate reader as described by Prior et al. (2010).

RT‐qPCR assays

SYBR‐Green (TaKaRa, Dalian, China) was used for detecting the PCR products on a CFX96 Real‐time reaction system (Bio‐Rad). 26 S‐18 S interspacer RNA was applied as a housekeeping gene and the relative quantitative data were analysed using the 2−ΔΔCt method (Ring et al., 2013). All primers used for qPCR were listed in Table S2.

Transient overexpression and transgenic calli assays

The complete CDS sequences of the target genes (R2R3‐FaMYB5, R3‐FaMYB5, FaMYB10, FaBT2, FaEGL3, FaLWD1, FaLWD1‐like, FaF3′H, FaTT19 and FaMYB1) were cloned into the expression vector pCAMBIA‐35SN. Transient overexpression in the strawberry fruits was conducted following the protocols as described (Lin et al., 2018; Spolaore et al., 2001), and at least 15 fruits (white stage) were selected for each replicate (three biological replicates in total). The injected fruits were harvested 6 days later and manually separated into the skin (outer red layer including achenes) and flesh (inner layer without pith) parts and then stored at −80 °C before use. For transgenic strawberry calli, 3 × FLAG‐FaMYB5‐3 × FLAG and 6 × HIS‐FaBT2‐6 × HIS were cloned into a pCAMBIA‐35SN vector and transformed into Agrobacterium GV3101. Then, the transgenic strawberry calli were generated by the Agrobacterium‐mediated leaf disk transformation method, as we described previously (Liu et al., 2022a). For the induction of flavonoid synthesis, the calli were first cultured in dark for 2 weeks and then transferred to a hormone‐free MS medium and continuously treated under light for 96 h.

Total RNA sequencing and analysis

Total RNA was qualitatively controlled and libraries were constructed and sequenced by Novogene (Beijing, China) (Jiang et al., 2022). Three biological replicates were included for each sample. Bioinformatics analysis of the sequencing reads followed the procedures we described previously (Jiang et al., 2022).

Metabolomics detection

Quasitargeted metabolomics and targeted metabolomics methods (Novogene, China) were used to construct metabolomics libraries. The detailed procedure for the detection and analysis of metabolites was described before (Jiang et al., 2022).

Yeast Two‐Hybrid analysis

According to Clontech's Matchmaker™ Gold Yeast Two‐Hybrid Screening Systems user manual, two target genes were cloned to the pGADT7 and pGBKT7 vector, respectively, and the positive interaction was detected on SD/−Trp/−Leu and SD/−Trp/−Leu/−Ade/‐His/+X‐gal/+AbA medium.

Dual‐luciferase assay

To construct the reporter, the deduced promoter sequences of C4H (1789 bp), F3′H (2139 bp), LAR (1968 bp) and ANR (2217 bp) were cloned from ‘Benihoppe’ and inserted into the pGreenII0800‐LUC vector. The pCAMBIA‐35SN‐FaMYB5 was used as an effector. The empty pCAMBIA‐35SN was used as a negative control. Vectors were transformed into the Agrobacterium GV3101 (pSoup‐p19). Dual‐luciferase transient expression assay was performed according to the method described by Liu et al. (2008). Fire luciferase (LUC) and renilla luciferase (REN) were detected using the Dual‐Luciferase Reporter Assay System kit (Promega, USA). Six replicates were measured in each assay. All primers used to construct the vectors were listed in Table S1.

EMSA assay

R2R3‐FaMYB5 was inserted into pGEX‐4T‐1 and the vector was transformed into Rosetta (DE3) expression strain. The GST‐FaMYB5 fusion protein was induced at 18 °C for 12 h by adding IPTG to a final concentration of 0.1 mM. The target protein was purified by GST Sefinose(TM) Resin Kit (Sangon Biotech, Shanghai, China) and then detected by 12% SDS‐PAGE electrophoresis before storage at −80 °C. The MYB binding sites on F3′H and LAR promoter sequences were identified through PlantCARE online website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). For EMSA probes, the nucleotide sequences consisted of the M13‐20 (5′‐GTAAAACGACGGCCAGT‐3′), followed by NNNNNNNN‐NNNNNNNNNNNNNNNNN (25 bp) and by the pBABE‐5 (5′‐CTTTATCCAGCCCTCAC‐3′) were synthesized as PCR templates. The underlined nucleotides represented the putative MYB binding sites ([C/T]AACNG or ACC[A/T]A[A/C]). The 5′‐Cy5 labelled M13‐20 and 5′‐Cy5 pBABE‐5 were used as PCR primers to generate the EMSA fluorescence probes. Binding reactions were carried out at room temperature for 30 min in a 10 μL buffer, in the presence of 10 mM Tris–HCl (pH 7.5), 100 mM KCl, 1 mM EDTA, 5% (v/v) glycerol, 1 mM DTT, 0.01 mg mL−1 BSA, 2.5 ng purified fluorescence probe and 500 ng (+) GST‐FaMYB5 protein. The reactions were analysed by a rapid agarose gel electrophoretic mobility shift assay (Ream et al., 2016), and Cy5‐labelled probes were detected by the GelView 6000Plus Smart Gel Imaging System (BLT PHOTON TECHNOLOGY, Guangzhou, China).

Protein degradation and ubiquitination assays

Protein degradation experiments were carried out both in vivo and in vitro. For protein degradation in vivo, a modified dual‐luciferase system was used (Sun et al., 2021). A modified pGreenII0800‐miRNA vector was constructed by moving the KpnI and HindIII restriction sites to the 3' end of the REN. FaMYB5 was fused to the REN protein serving as a reporter. The construct pCAMBIA‐35SN‐FaBT2 was used as an effector. Six replicates were measured in each dual‐luciferase assay. For protein degradation in vitro, total proteins of transgenic strawberry calli (35 S::3 × FLAG‐FaMYB5‐3 × FLAG, 35 S::6 × HIS‐FaBT2‐6 × HIS) and wild‐type (WT) were extracted in protein extraction buffer (5 mM EDTA, 250 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1% Triton X‐100, 2 mM PMSF, 1 × Protease inhibitor cocktail for plant cell and tissue extracts [Beyotime, Shanghai, China]) supplemented with 100 μM MG132 (dissolved in DMSO) or 0.1% DMSO. Proteins from 35 S::3 × FLAG‐FaMYB5‐3 × FLAG calli (without MG132) were mixed with the above protein extracts in degradation buffer (25 mM Tris–HCl [pH 8.0], 5 mM DTT, 10 mM NaCl, 10 mM MgCl2, 4 mM PMSF and 10 mM ATP), respectively, and then incubated at 37 °C for 0, 1, 2, 4, 6 h (Zhang et al., 2020). These samples were analysed by western blot using anti‐FLAG (ABT2010, Abbkine) or anti‐Actin (ABL1050, Abbkine) antibodies.

Protein ubiquitination experiments were performed in vitro according to the Ubiquitylation Assay Kit (Abcam, Cambridge, UK). The FaMYB5‐FLAG and FaBT2‐HIS active proteins were purified from the above calli proteins by using Protein A + G Agarose for IP (Beyotime). The reactions (50 μL) contained 1 × Ubiquitinylation Buffer, 1 mM DTT, 5 mM Mg‐ATP, 10 nM human E1, 100 nM human E2 (UbcH5a), 2.5 μM ubiquitin, 500 ng FaMYB5‐FLAG, with or without 500 ng FaBT2‐HIS active proteins. After incubation at 30 °C for 2 h, the reaction mixture was analysed by western blot using anti‐FLAG, anti‐HIS (ABT2050, Abbkine) and anti‐Ub (ABP52661, Abbkine) antibodies.

Statistical analysis

If not specified, all data were analysed by using the IBM SPSS Statistics 23 software, and the statistically significant differences between samples were determined by a Student's t‐test (P < 0.05). Multiple comparisons were conducted using Tukey's test and significant differences (P < 0.05) were indicated by different letters.

Accession numbers

The data that support the findings of this study have been deposited into the CNGB Sequence Archive (CNSA) of China National GeneBank DataBase (CNGBdb, https://db.cngb.org/cnsa) with accession number CNP0002209.

Conflict of interest statement

The authors declare that they have no competing interests.

Author contributions

L.J., M.Y., Yo.L. and H.T. conceived and designed the research; L.J., M.Y. and Yo.L. performed experiments; N.Z., Ya.L. and Yo.Z. collected the samples and conducted fieldwork; Q.C., M.L. and Yu.L. analysed the transcriptome data; Yu.Z., Ya.W. and X.W. carried out metabolomics analyses; L.J., M.Y., Q.C. and H.T. revised the manuscript; L.J. wrote this paper. L.J., M.Y. and Yo.L. contributed equally. All authors had approved the final version and declared that they had no competing interests.

Supporting information

Figure S1. Comparison of the CDS and protein sequences between FvMYB5, R2R3‐FaMYB5 and R3‐FaMYB5.

Figure S2. Alignment of the complete amino acid sequences between R2R3‐FaMYB5 and its homologous genes by ESPript 3.0 online website.

Figure S3. Anthocyanins extraction solutions.

Figure S4. HPLC analysed the anthocyanins content of ‘Xiaobai’ skin and flesh in overexpression samples of R2R3‐FaMYB5, R3‐FaMYB5, FaMYB10 and 35SN.

Figure S5. HPLC analysed the anthocyanins content of ‘Benihoppe’ skin and flesh in overexpression samples of R2R3‐FaMYB5, R3‐FaMYB5, FaMYB10 and 35SN.

Figure S6. Anthocyanins and PAs content in overexpression samples of R2R3‐FaMYB5 in ‘Benihoppe’.

Figure S7. Relative expression levels of genes involved in overexpression samples of R3‐FaMYB5.

Figure S8. Differentially expressed genes between R2R3‐FaMYB5 (XB_5) and 35SN (XB_CK) overexpressing samples in ‘Xiaobai’ by whole transcriptome sequencing.

Figure S9. Correlation between qPCR and RNA‐seq data.

Figure S10. pH value of the flesh in overexpression samples of R2R3‐FaMYB5, FaMYB10 and 35SN.

Figure S11. Relative expression levels of other genes involved in ‘Snowwhite’ after overexpressing R2R3‐FaMYB5, FaMYB10 and 35SN.

Figure S12. Relative expression levels of FaMYB5, FaLWD1, FaEGL3 and FaLWD1‐like in different overexpression samples.

Figure S13. Special ‘Snowwhite’ and interaction between R2R3‐FaMYB5, FaTTG1 and FabHLH3.

Figure S14. Anthocyanins and PAs content in overexpression samples of lncRNA‐myb5 in ‘Benihoppe’.

Figure S15. Overexpression of FaMYB1 had a limited role in inhibiting the accumulation of strawberry anthocyanins.

Figure S16. Transgenic lines overexpressing R2R3‐FaMYB5 in wild strawberry (Ruegen) and relative quantification of F3′H, LAR and C4H in plants.

Figure S17. Detection of anthocyanins and PAs in R2R3‐FaMYB5 transgenic calli after 96 h of light treatment and the expression level of TT19 in different tissues of Xiaobai strawberry.

PBI-21-1140-s001.docx (7.1MB, docx)

Table S1. Primers used in this study.

Table S2. Primers used for qPCR in this study.

Table S3. Metabolites detected by quasitargeted metabolomics in strawberry flesh of overexpressing R2R3‐FaMYB5 and 35SN.

Table S4. Description and expression values of lncRNAs, mRNAs, circRNAs and microRNAs.

Table S5. Transcripts names for color scale mapping.

PBI-21-1140-s002.rar (9.6MB, rar)

Acknowledgements

Yeast two‐hybrid vectors were a gift from Professor Wanchen Li in China. This work was supported by the National Natural Science Foundation of China (No. 31872083).

Contributor Information

Qing Chen, Email: supnovel@sicau.edu.cn.

Haoru Tang, Email: htang@sicau.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Comparison of the CDS and protein sequences between FvMYB5, R2R3‐FaMYB5 and R3‐FaMYB5.

Figure S2. Alignment of the complete amino acid sequences between R2R3‐FaMYB5 and its homologous genes by ESPript 3.0 online website.

Figure S3. Anthocyanins extraction solutions.

Figure S4. HPLC analysed the anthocyanins content of ‘Xiaobai’ skin and flesh in overexpression samples of R2R3‐FaMYB5, R3‐FaMYB5, FaMYB10 and 35SN.

Figure S5. HPLC analysed the anthocyanins content of ‘Benihoppe’ skin and flesh in overexpression samples of R2R3‐FaMYB5, R3‐FaMYB5, FaMYB10 and 35SN.

Figure S6. Anthocyanins and PAs content in overexpression samples of R2R3‐FaMYB5 in ‘Benihoppe’.

Figure S7. Relative expression levels of genes involved in overexpression samples of R3‐FaMYB5.

Figure S8. Differentially expressed genes between R2R3‐FaMYB5 (XB_5) and 35SN (XB_CK) overexpressing samples in ‘Xiaobai’ by whole transcriptome sequencing.

Figure S9. Correlation between qPCR and RNA‐seq data.

Figure S10. pH value of the flesh in overexpression samples of R2R3‐FaMYB5, FaMYB10 and 35SN.

Figure S11. Relative expression levels of other genes involved in ‘Snowwhite’ after overexpressing R2R3‐FaMYB5, FaMYB10 and 35SN.

Figure S12. Relative expression levels of FaMYB5, FaLWD1, FaEGL3 and FaLWD1‐like in different overexpression samples.

Figure S13. Special ‘Snowwhite’ and interaction between R2R3‐FaMYB5, FaTTG1 and FabHLH3.

Figure S14. Anthocyanins and PAs content in overexpression samples of lncRNA‐myb5 in ‘Benihoppe’.

Figure S15. Overexpression of FaMYB1 had a limited role in inhibiting the accumulation of strawberry anthocyanins.

Figure S16. Transgenic lines overexpressing R2R3‐FaMYB5 in wild strawberry (Ruegen) and relative quantification of F3′H, LAR and C4H in plants.

Figure S17. Detection of anthocyanins and PAs in R2R3‐FaMYB5 transgenic calli after 96 h of light treatment and the expression level of TT19 in different tissues of Xiaobai strawberry.

PBI-21-1140-s001.docx (7.1MB, docx)

Table S1. Primers used in this study.

Table S2. Primers used for qPCR in this study.

Table S3. Metabolites detected by quasitargeted metabolomics in strawberry flesh of overexpressing R2R3‐FaMYB5 and 35SN.

Table S4. Description and expression values of lncRNAs, mRNAs, circRNAs and microRNAs.

Table S5. Transcripts names for color scale mapping.

PBI-21-1140-s002.rar (9.6MB, rar)

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