EIN3-LIKE1, MYB1, and ETHYLENE RESPONSE FACTOR3 Act in a Regulatory Loop That Synergistically Modulates Ethylene Biosynthesis and Anthocyanin Accumulation

Abstract

Ethylene regulates climacteric fruit ripening, and EIN3-LIKE1 (EIL1) plays an important role in this process. In apple (Malus domestica), fruit coloration is accompanied by ethylene release during fruit ripening, but the molecular mechanism that underlies these two physiological processes is unknown. In this study, we found that ethylene treatment markedly induced fruit coloration as well as the expression of MdMYB1, a positive regulator of anthocyanin biosynthesis and fruit coloration. In addition, we found that MdEIL1 directly bound to the promoter of MdMYB1 and transcriptionally activated its expression, which resulted in anthocyanin biosynthesis and fruit coloration. Furthermore, MdMYB1 interacted with the promoter of ETHYLENE RESPONSE FACTOR3, a key regulator of ethylene biosynthesis, thereby providing a positive feedback for ethylene biosynthesis regulation. Overall, our findings provide insight into a mechanism involving the synergistic interaction of the ethylene signal with the MdMYB1 transcription factor to regulate ethylene biosynthesis and fruit coloration in apple.

Figure 1.

Ethephon induces anthocyanin accumulation in cv Red Delicious apples. A, The cv Red Delicious apples were grown in bags beginning at 30 DAFB. They were debagged at 120 DAFB and treated with the indicated concentrations of ethephon solutions (250, 500, and 1,000 mg L⁻¹) or 1-MCP (1 µL L⁻¹) and then were stored in a phytotron at 24°C with constant high light (70 μmol m⁻² s⁻¹) for 6 d. Untreated fruits were used as the control. Representative images were taken. The assay was performed in three replicates. B and C, Measurements of anthocyanin content (B) and ethylene production (C) in ethephon- or 1-MCP-treated and control apples. The assay was performed in three replicates. The results represent means of these three replicates. Error bars indicate sd.

Figure 2.

Expression analysis of ethylene and anthocyanin synthetic genes in ethephon- or 1-MCP-treated and control cv Red Delicious apples by reverse transcription-quantitative PCR (RT-qPCR). Ethylene biosynthetic genes are MdACS1, MdACS3a, MdACS4, MdACS5a, MdACS5b, and MdACS6, and anthocyanin biosynthetic genes are MdMYB1, MdDFR, and MdUFGT. The value for day 0 was set to 1. RT-qPCR was performed with three technical replicates and three biological replicates. The results represent means of these three replicates. Error bars indicate sd.

Figure 3.

Binding of MdEIL1 to the MdMYB1 promoter. A, Identification of the MdEIL1 protein that binds the cis-element of the MdMYB1 promoter with an electrophoretic mobility shift assay (EMSA). The total proteins were extracted from ethephon- or 1-MCP-treated and untreated apple plants. – and + represented samples without or with the addition of total protein extracted from the untreated apple plants, respectively. Eth and 1-MCP represent samples with the addition of total protein extracted from the ethephon- or 1-MCP-treated apple plants, respectively. B, Expression analysis of MdEIL1 in ethephon- or 1-MCP-treated and control apples by RT-qPCR. The value for day 0 was set to 1. RT-qPCR was performed with three technical replicates and three biological replicates. The results represent means of these three replicates. Error bars indicate sd. C, Top, schematic diagram of the MdMYB1 promoter showing the potential MdEIL1-binding sites (MdMYB1-1, MdMYB1-2, and MdMYB1-3). The predicted ATGTA sequences are indicated by black boxes. Red lines represent the fragments amplified in the ChIP-PCR assay. Bottom, EMSA showing the MdEIL1-HIS fusion protein bound to the MdMYB1-3 site of the MdMYB1 promoter and the free and bound DNAs separated on an acrylamide gel. D, EMSA showing that the MdEIL1-HIS fusion protein bound directly to the MdMYB1-3 site of the MdMYB1 promoter. Unlabeled probes were used as competitors. Mut represents a mutated probe in which the ATGTA motif was replaced by CTTGC. E, ChIP-PCR assay of MdEIL1 binding to the promoter of the MdMYB1 gene. Chromatin from the empty vector control (GFP) and 35S:MdEIL1-GFP apple calli (MdEIL1-GFP) were immunoprecipitated with anti-GFP antibodies or without antibodies. Three regions (MdMYB1-1, MdMYB1-2, and MdMYB1-3) were examined by RT-qPCR. The enrichment of the wild type was set to 1. RT-qPCR was performed with three technical replicates and three biological replicates to examine the enrichment of MdMYB1 fragments. The results represent means of these three replicates. Error bars indicate sd. Asterisks denote Student’s t test significance: **, P < 0.01. F, Y1H assay showing MdEIL1 interaction with the promoter of MdMYB1. The promoter fragment of MdMYB1 (MdMYB1-3) was fused to the pHIS2 vector, and the MdEIL1 gene was fused to the pGAD vector. The columns represent the addition of the pHIS2-MdMYB1-3_pro vector. The rows represent the addition of the pGAD and pGAD-MdEIL1 vectors.

Figure 4.

Regulation of the MdMYB1 promoter by MdEIL1. A, Schematic representation of the GUS reporter vector containing the MdMYB1 promoter and the effector vector containing MdEIL1. B, GUS staining analysis and GUS activity detection of pMdMYB1-GUS and pMdMYB1-GUS/MdEIL1-OX transgenic apple calli. MdMYB1-Pro, MdMYB1-Promoter-GUS transgenic apple calli; MdMYB1-Pro/MdEIL1-OX, MdMYB1-Promoter-GUS and 35S:MdEIL1-GFP cotransformed apple calli. The GUS activity of MdMYB1-Pro was used as the reference. Transgenic apple calli were stained using GUS buffer. GUS staining analysis and GUS activity detection were performed in three replicates. The results represent means of these three replicates. Error bars indicate sd. The asterisk denotes Student’s t test significance: *, P < 0.05. C, Transient expression assays showing MdEIL1 promotion of MdMYB1 expression. The promoter fragment of MdMYB1 was cloned into the pGreenII 0800-LUC vector to generate the reporter construct. The effector (35S_pro:MdEIL1) was generated by recombining the MdEIL1 gene into the pGreenII 62-SK vector. In MdMYB1_pro(Mut), the ATGTA motif was replaced by CTTGC. D, Quantitative analysis of luminescence intensity. The value for column 2 (MdMYB1_pro:LUC-35S_pro:62-SK) was set to 1. The transient expression assay was performed in three replicates. The results represent means of these three replicates. Error bars indicate sd. Asterisks denote Student’s t test significance: *, P < 0.05 and **, P < 0.01.

Figure 5.

MdEIL1 promotes anthocyanin biosynthesis in an MdMYB1-dependent manner. Overexpression of MdEIL1 promotes anthocyanin biosynthesis. A, Apple peel injection assays. At 120 DAFB, cv Red Delicious apples were debagged, injected with the mixed vectors, and stored in a phytotron at 15°C with constant high light (70 μmol m⁻² s⁻¹) for 4 d. pIR, IL60-1+IL60-2; MdEIL1-pIR, IL60-1+MdEIL1-IL60-2. B and C, Detection of anthocyanin contents (B) and expression analysis of anthocyanin biosynthetic genes (MdMYB1, MdDFR, MdUFGT, MdF3H, MdCHI, and MdCHS; C) in fruit peels around the injection sites. The anthocyanin content of pIR was set as a control. The value for pIR was set to 1. The apple peel injection assay was performed in three replicates. RT-qPCR was performed with three technical replicates and three biological replicates. The results represent means of these three replicates. Error bars indicate sd. Asterisks denote Student’s t test significance: **, P < 0.01. Overexpression of MdEIL1 promotes anthocyanin biosynthesis in an MdMYB1-dependent manner. D, Apple peel injection assays. At 120 DAFB, cv Red Delicious apples were debagged, injected with the mixed vectors or A. tumefaciens solutions, and stored in a phytotron at 15°C with constant high light (70 μmol m⁻² s⁻¹) for 4 d. pIR, IL60-1+IL60-2; MdMYB1-pIR, IL60-1+MdMYB1-IL60-2; MdMYB1-TRV, TRV1+MdMYB1-TRV2; MdEIL1-pIR, IL60-1+MdEIL1-IL60-2; MdEIL1-pRI/MdMYB1-TRV, IL60-1+MdMYB1-IL60-2/TRV1+MdMYB1-TRV2. E and F, Detection of anthocyanin contents (E) and expression analysis of anthocyanin biosynthetic genes (MdMYB1, MdDFR, MdUFGT, MdF3H, MdCHI, and MdCHS; F) in fruit peels around the injection sites. The anthocyanin content of pIR was set as a control. The value for pIR was set to 1. The apple peel injection assay was performed in three replicates. RT-qPCR was performed with three technical replicates and three biological replicates. The results represent means of these three replicates. Error bars indicate sd. Asterisks denote Student’s t test significance: *, P < 0.05 and **, P < 0.01.

Figure 6.

Analysis of anthocyanin content and ethylene production in red-flesh and non-red-flesh apples. A to C, Representative red-flesh and non-red-flesh apples (A) and measurements of anthocyanin content (B) and ethylene production (C). Apple cross-breeding groups (red-flesh and non-red-flesh apples) were harvested at 140 DAFB for anthocyanin measurements. They were treated or untreated with 1-MCP (1 µL L⁻¹) and stored at room temperature for 6 d for ethylene measurements. The anthocyanin content of non-red-flesh apples was used as the control. The assay was performed in three replicates. The results represent means of these three replicates. Error bars indicate sd. Asterisks denote Student’s t test significance: **, P < 0.01. D and E, Expression of anthocyanin biosynthetic genes (MdMYB1, MdDFR, MdUFGT, MdF3H, MdCHI, and MdCHS; D) and ethylene biosynthetic genes (MdERF3, MdACS1, MdACS3a, MdACS4, MdACS5a, MdACS5b, and MdACS6; E) in untreated red-flesh and non-red-flesh apples. The value for non-red-flesh apple was set to 1. RT-qPCR was performed with three technical replicates and three biological replicates. The results represent means of these three replicates. Error bars indicate sd. Asterisks denote Student’s t test significance: **, P < 0.01.

Figure 7.

Binding of MdMYB1 to the MdERF3 promoter. A, Schematic diagram of the MdERF3 promoter showing the potential MdMYB1 binding sites. The predicted CCGTTG sequences are indicated by the black line. In the mutated probe (Mut), the CCGTTG motif was replaced by GGAGGT. B, ChIP-PCR assay of MdMYB1 binding to the promoter of the MdERF3 gene. Chromatin from the empty vector control (GFP) and 35S:MdMYB1-GFP apple calli (MdMYB1-GFP) were immunoprecipitated with and without anti-GFP antibodies. Three regions (P1, P2, and P3) were examined by RT-qPCR. The enrichment of GFP was set to 1. RT-qPCR was performed with three technical replicates and three biological replicates to examine the enrichment of MdERF3 fragments. The results represent means of these three replicates. Error bars indicate sd. Asterisks denote Student’s t test significance: **, P < 0.01. C, EMSA results showing that the MdMYB1-HIS fusion protein bound directly to the MdMYB1 promoter. Unlabeled probes were used as competitors. In the mutated probe (Mut), the CCGTTG motif was replaced by GGAGGT. D, Y1H assay showing MdMYB1 interaction with the MdERF3 promoter. The promoter of MdERF3 was fused to the pHIS2 vector, and the MdMYB1 gene was fused to the pGAD vector. The columns represent the addition of the pHIS2-MdERF3_pro vector. The rows represent the addition of the pGAD and pGAD-MdMYB1 vectors.

Figure 8.

MdMYB1 positively regulates the expression of MdERF3. A, Transient expression assays showing that MdMYB1 promoted the expression of MdERF3. The promoter fragment of MdERF3 was cloned into the pGreenII 0800-LUC vector to generate the reporter construct. The effector (35S_pro:MdMYB1) was generated by recombining MdMYB1 into the pGreenII 62-SK vector. In MdERF3_pro(Mut), the CCGTTG motif was replaced by GGAGGT. B, Quantitative analysis of luminescence intensity. The value for column b (MdERF3_pro:LUC-35S_pro:62-SK) was set to 1. The transient expression assay was performed in three replicates. The results represent means of these three replicates. Error bars indicate sd. Asterisks denote Student’s t test significance: **, P < 0.01. C, Ethylene production of apple calli. Wild-type apple calli (WT), MdERF3 overexpression calli (MdERF3-OX), MdMYB1 overexpression calli (MdERF3-OX), MdERF3-TRV transient single-transgenic calli (MdERF3-TRV), and MdERF3-TRV transient calli in the background of MdMYB1-OX (MdMYB1-OX/MdERF3-TRV) were used for measurements of ethylene production. The ethylene production of the wild type was used as the reference. The assay was performed in three replicates. The results represent means of these three replicates. Error bars indicate sd. Asterisks denote Student’s t test significance: *, P < 0.05 and **, P < 0.01.

Figure 9.

Proposed model of the mechanism regulating anthocyanin accumulation and ethylene release during fruit ripening. MdEIL1 binds directly to the promoter of MdMYB1 and enhances its action, leading to increased anthocyanin accumulation. In addition, MdMYB1 binds to the promoter of MdERF3 and activates its expression, resulting in enhanced ethylene production. C₂H₄, Ethylene; ATGTA, MdEIL1-binding sequence; CCGTTG, MdMYB1-binding sequence. Solid arrows show positive regulation.