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

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
Affiliations

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

Leiyu Jiang et al. Plant Biotechnol J. 2023 Jun.

Abstract

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: MBW complex; R2R3-FaMYB5; anthocyanin; proanthocyanidin; regulation network; strawberry.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
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 3
Figure 3
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.
Figure 4
Figure 4
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.
Figure 5
Figure 5
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).
Figure 6
Figure 6
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
‘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.
Figure 8
Figure 8
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.
Figure 9
Figure 9
Putative regulatory network of anthocyanin and PA biosynthesis in strawberry fruits by R2R3‐FaMYB5. Putative activations are indicated in a dotted line. Ubi—ubiquitin.
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