AaMYB61-like and AabHLH137 jointly regulate anthocyanin biosynthesis in Actinidia arguta

Abstract

Background: Red Actinidia arguta has recently become highly popular because of its red appearance resulting from anthocyanin accumulation, and has gradually become an important breeding direction. However, regulators involved in anthocyanin biosynthesis have not been fully characterized in A. arguta.

Results: Here, we demonstrated that a key R2R3-MYB transcription factor, AaMYB61-like, plays a crucial role in A. arguta anthocyanin biosynthesis. The RT-qPCR results revealed that transient overexpression of AaMYB61-like in A. arguta fruit at 90-100 DAFB significantly promoted anthocyanin biosynthesis, as did the gene expression levels of AaCHS, AaCHI, AaF3H, AaLDOX, and AaF3GT, whereas the result of VIGS revealed the opposite results in A. arguta fruit at 105-115 DAFB. A transcriptional activation assay indicated that AaMYB61-like exhibited transcriptional activation activity. Y1H and LUC assays revealed that AaMYB61-like activates the promoters of AaCHS, AaLDOX, and AaF3GT. In addition, AabHLH137 was found to be related to fruit color from the transcriptome data. We demonstrated that AaMYB61-like promotes anthocyanin biosynthesis by interacting with AabHLH137 via Y2H, BiFC, and Agrobacterium-mediated co-transformation.

Conclusions: Our study not only reveals the functions of AaMYB61-like and AabHLH137 in anthocyanin regulation, but also broadly enriches color regulation theory, establishing a foundation for clarifying the molecular mechanism of fruit coloration in kiwifruit.

Keywords: Actinidia arguta; AaMYB61-like; AabHLH137; Anthocyanin biosynthesis; Transcription regulation.

Fig. 1

Anthocyanin accumulation and gene expression in the A. arguta fruits at the three stages. A Appearance of the A. arguta fruit at the different stages: S1, S2, and S3. Scale bar. 1 cm. B Anthocyanins extracted from the fruits at different stages. C Anthocyanin content of the fruits at the different stages. D Relative expression of AaMYB61-like at the different stages. E-L Relative expression of anthocyanin biosynthetic genes at the different stages. The values are the means ± SDs of three replicates (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001)

Fig. 2

Phylogenetic tree analysis and multi-sequence alignments of AaMYB61-like. A Phylogenetic tree analysis of AaMYB61-like and other reported MYB proteins from different species. B Multi-sequence alignment of AaMYB61-like with other MYB proteins. Red lines: R2 and R3 domains. These sequences were obtained from the NCBI database. The accession numbers are as follows: AeMYB61-like (XP_057500969.1), OsMYB61 (XP_025881490.1), TaMYB61-like (XP_044387588.1), AtMYB61 (NP_172425.2), MaMYB61-like (XP_064935242.1), MdMYB61-like (XP_008359848.2), PpMYB86 (XP_007207615.1), CaMYB61 (XP_059458726.1), PvMYB61-like (XP_031261472.1), NtMYB86-like (XP_016436036.1), VvMYB61 (XP_002276091.1), CsMYB61-like (XP_028108727.1) and DlMYB61-like (XP_052206166.1). C Structural diagram of the AaMYB61-like protein. R2-MYB domains (amino acids 13–63) and R3-MYB domains (amino acids 64–114) are highlighted with green rectangles

Fig. 3

Functional identification of AaMYB61-like in A. arguta. A Transient overexpression of AaMYB61-like in the A. arguta fruit at 90–100 DAFB. A. tumefaciens suspensions containing 35S::AaMYB61-like or empty vectors were infiltrated into the fruit. These fruits were stored at room temperature for 24 h and then under 16 h of light and 8 h of darkness for one week. Phenotypic changes were observed and photographed one week after infiltration. Scale bar. 1 cm. B Determination of anthocyanin content in the AaMYB61-like overexpressing fruits. C Relative expression of anthocyanin biosynthetic genes and AaMYB61-like in the AaMYB61-like overexpressing fruits. D Transient silencing of AaMYB61-like in the A. arguta fruit. A. tumefaciens suspensions containing pTRV1 + pTRV2-AaMYB61-like or pTRV1 + pTRV2 were infiltrated into the fruit at 105–115 DAFB via a 1 mL needle syringe. The fruits were stored at room temperature for 24 h and then under 16 h of light and 8 h of darkness for one week. The phenotype changes were observed and photographed one week after infiltration. Scale bar. 1 cm. E Determination of the anthocyanin content in theAaMYB61-like silenced fruits. F Relative expression of structural genes and AaMYB61-like in the silenced AaMYB61-like fruits. Values are the means ± SDs of three replicates. Statistical significance: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

Fig. 4

Interaction verification between AaMYB61-like and anthocyanin-related genes. A Schematic diagrams showing the MYB binding sites in the promoters of anthocyanin-related genes. B Y1H assay showsing the interaction between AaMYB61–like and the promoters of anthocyanin-related genes. C Schematic diagrams showing the reporter and effector constructs used in the Dual-LUC assay. D-F Luciferase activity and fluorescence intensity of the AaCHS, AaLDOX, and AaF3GT in N. benthamiana leaves. The values are means ± SDs for three replicates. Statistical significance: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. The right area was injected with 35S::AaMYB61-like and promoter::LUC as the experimental treatment, and the left area was injected with the pBI121 empty vector and promoter::LUC as the control. Reded and blue represent high and low luminescence intensities, respectively. Images were captured three days after infiltration

Fig. 5

Interaction verification between the AaMYB61-like and AabHLH137 proteins. A Phylogenetic tree analysis of AabHLH137 and other reported bHLH proteins from different species. These sequences were obtained from the NCBI database. The accession numbers are as follows: AcbHLH137 (PSR95969.1), VvbHLH80 (XP_002284464.1), PpbHLH137 (XP_007209284.1), CsbHLH137 (XP_028125498.1), DlbHLH137 (XP_052176766.1), PvbHLH137 (XP_031277311.1), NtbHLH137 (NP_001312696.1), MdbHLH137 (XP_028959633.1), SlbHLH137 (XP_010322508.1), TabHLH137 (XP_044439635.1), MaBC1 (XP_064966686.1) and OsBC1 (NP_001409737.1). B Relative expression of AabHLH137 at the different stages. C Relative expression of AabHLH137 in the AaMYB61-like overexpressing sample. The values are the means ± SDs of three replicates. Statistical significance: ** P ≤ 0.01, *** P ≤ 0.001. D Y2H assay revealed a physical interaction between AaMYB61-like and AabHLH137. pGADT7-largeT + pGBKT7-53 and pGADT7-largeT + pGBKT7-lamC were used as positive and negative controls, respectively. AD-empty + BD-AaMYB61-like: pGADT7 empty and pGBKT7-AaMYB61-like combination; AD-AabHLH137 + BD-AaMYB61-like: pGADT7-AabHLH137 and pGBKT7-AaMYB61-like combination; DDO: SD/-Trp/-Leu; QDO: SD/-Trp/-Leu/-His/-Ade. E BiFC assay revealed that AaMYB61-like interacts with AabHLH137 in N. benthamiana leaves. The fluorescence was successfully observed in tobacco leaves co-infiltrated with AaMYB61-like-nYFP and AabHLH137-cYFP. Different fluorescence fields, including bright, DAPI, YFP, and merged field, from the same cells are shown. Scale bar: 25 μm

Fig. 6

AaMYB61-like and AabHLH137 jointly promote anthocyanin biosynthesis in A. arguta. A Co-overexpression of AabHLH137 and AaMYB61-like in the A. arguta fruit at 90–100 DAFB. The pBI121 empty vector was used as a control. Scale bar. 1 cm. B Anthocyanins extracted from fruits subjected to the different treatment. C Anthocyanin content of the fruits subjected to the different treatment. D-L Relative expression of AabHLH137 and structural genes in the anthocyanin biosynthesis pathway. Different letters above the top of the bar chart indicate significant differences according to one-way ANOVA with Tukey’s multiple comparisons test (P < 0.05)

Fig. 7

Proposed model by which AaMYB61-like-AabHLH137 regulates fruit color in A. arguta. The AaMYB61-like/AabHLH137 complex regulates fruit color by acting on structural genes (AaCHS, AaLDOX, and AaF3GT)