Dynamic regulation of anthocyanin biosynthesis at different light intensities by the BT2-TCP46-MYB1 module in apple

Abstract

Teosinte branched1/cycloidea/proliferating (TCP) transcription factors play a broad role in plant growth and development, but their involvement in the regulation of anthocyanin biosynthesis is currently unclear. In this study, anthocyanin biosynthesis induced by different light intensities in apple (Malus domestica) was found to be largely dependent on the functions of the MdMYB1 and MdTCP46 transcription factors. The expression of MdTCP46 was responsive to high light intensity, and under these conditions it promoted anthocyanin biosynthesis by direct interactions with MdMYB1 that enhanced the binding of the latter to its target genes. MdTCP46 also interacted with a bric-a-brac/tramtrack/broad (BTB) protein, MdBT2, that is responsive to high light intensity, which ubiquitinated MdTCP46 and mediated its degradation via the 26S proteasome pathway. Our results demonstrate that the dynamic regulatory module MdBT2-MdTCP46-MdMYB1 plays a key role in modulating anthocyanin biosynthesis at different light intensities in apple, and provides new insights into the post-transcriptional regulation of TCP proteins.

Keywords: Malus domestica; Anthocyanin accumulation; TCP transcription factor; apple; high-light intensity; post-transcriptional regulation.

Fig. 1.

Apple MdTCP46 interacts with MdMYB1. (A) Yeast two-hybrid assays. The ORFs of the MdTCP46 and MdMYB1 sequences lacking the autonomously activated fragments were fused to the pGAD424 and pGBT9 vectors, respectively. The pGAD424 and pGBT9 empty vectors were used as negative controls. –T/–L, SD medium lacking Trp and Leu; –T/–L/–H/–A, SD medium lacking Trp, Leu, His, and Ade. (B) Pull-down assays. The ORFs of MdMYB1 and MdTCP46 were fused to the pET32a and pGEX4T-1 vectors, respectively. The MdMYB1-HIS and MdTCP46-GST fusion proteins were obtained from E. coli expression. GST proteins were used as negative controls. The eluted solution was probed using HIS and GST antibodies. (C) Bimolecular fluorescence complementation assays. The ORFs of MdTCP46 and MdMYB1 were fused to the YFP^N and YFP^C vectors, respectively. Fluorescence was examined using a confocal laser-scanning microscope. Scale bars are 10 μm.

Fig. 2.

Overexpression of MdTCP46 promotes anthocyanin biosynthesis in apple. (A) Multiple alignment of the bHLH domain in 10 TCP proteins obtained from the NCBI database. PbTCP, Pyrus × bretschneideri (XP_009360352.1); PmTCP, Prunus mume (XP_008221641.1); RcTCP, Rosa chinensis (XP_024194958.1); FvTCP, Fragaria vesca (XP_004297311.1); PaTCP, Parasponia andersonii (PON70380.1); JrTCP, Juglans regia (XP_018817718.1); SiTCP, Sesamum indicum (XP_011071008.1); TcTCP, Theobroma cacao (XP_017969954.1); MdTCP46, Malus × domestica (MDP0000319941); AtTCP15, Arabidopsis thaliana (AT1G69690.1). The bHLH motif is indicated. (B) Transcription of MdTCP46 under different light intensities using qRT-PCR. Plants were treated for 3 d and expression was normalized to the actin gene. Values are relative to the low-light treatment, which was set as 1. (C) Detection of the MdTCP46-GST fusion protein after dark and moderate light treatments. ‘Red Delicious’ apple fruit were treated with low or high light for 3 d. Total proteins were extracted from the peels and were incubated with purified MdTCP46-GST protein for 0–6 h. For treatment with the proteasome inhibitor MG132, total proteins of fruit subjected to low light were pre-treated with 100 µM MG132 for 0.5 h before the sampling began. ACTIN was used as an internal reference. (D) Apple peel injection assays. Uncolored ‘Red Delicious’ fruit were injected with mixed vectors or A. tumefaciens solutions and stored in a phytotron under the high-light treatment for 3 d. pIR, IL60-1+IL60-2; MdTCP46-pIR, IL60-1+MdTCP46-IL60-2; TRV, TRV1+ TRV2; MdTCP46-TRV, TRV1+MdTCP46-TRV2. pIR are overexpressors (OX); TRV are antisense suppressors (Anti). (E) Anthocyanin contents of the fruit peels shown in (D). Contents are expressed relative to pIR, the value of which was set as 1. (F) Expression of genes related to anthocyanin biosynthesis in the fruit peels shown in (D) as determined by qRT-PCR. Expression was normalized to the actin gene. Values are expressed relative to pIR, which were set as 1. (G) qRT-PCR detection of MdTCP46 expression levels in Arabidopsis Col-0 seedlings and in overexpressing transgenic lines. The lower panels show the ACTIN gene, which was used as the internal control. (H) Phenotypes of Arabidopsis Col-0 and MdTCP46-overexpressing lines and (I) relative expression of MdTCP46. Expression was normalized to the ACTIN gene. Values are expressed relative to Col-0, which was set as 1. All experiments were performed three times with similar results, and representative data from one experiment are shown. Data are means (±SD), n=6. Different letters indicate significant differences as determined by one-way ANOVA and LSD tests (P<0.05).

Fig. 3.

MdTCP46 promotes light-induced anthocyanin biosynthesis in apple. Transgenic apple calli (15 d old) and leaves of MdTCP46-overexpressing (-OX) and MdTCP46-antisense (-Anti) were subjected to different light intensities for 5 d. (A) Phenotypes of calli of the transgenic lines and the wild-type (WT) and (B) their anthocyanin contents. The content of low light-treated WT was used as a reference and set to 1. (C) The expression of genes related to anthocyanin biosynthesis in apple calli as determined by qRT-PCR. The expression of the WT was used as a reference and set to 1. (D) Phenotypes of transient transgenic leaves the empty-vector control (EV) and (E) their anthocyanin contents. The content of low light-treated EV was used as a reference and set to 1. All experiments were performed three times with similar results, and representative data from one experiment are shown. Data are means (±SD), n=4. Different letters indicate significant differences as determined by one-way ANOVA and LSD tests (P<0.05).

Fig. 4.

MdTCP46 enhances the transcriptional activity of MdMYB1 on MdDFR and MdUF3GT. (A, B) Apple calli (15 d old) of the wild-type (WT), MdTCP46-overexpression (-OX), MdMYB1-antisense (-Anti), and MdTCP46-OX/MdMYB1-Anti (overexpression of MdTCP46 in the background of MdMYB1-Anti) were subjected to high light for 5 d. (A) Phenotypes and (B) anthocyanin levels. The content of the WT was used as a reference and set to 1. (C, D) Electromobility shift assays using MdDFR and MdUF3GT promoter probes. The MBS sequences for MdMYB1 binding and their equivalent mutant forms (-Mut) are underlined. + indicates the presence of corresponding proteins or probes, and − indicates the absence of corresponding of proteins. 2× and 3× indicate increased protein contents. (E) Schematic representation of the LUC reporter vector containing the MdDFR and MdUF3GT promoters and effector vectors expressing MdTCP46 or MdMYB1 under the control of the 35S promoters. The ORFs of MdTCP46 and MdMYB1 were fused to the pGreenII 62-SK vector. The promoter sequences of MdDFR and MdUF3GT were cloned into the pGreenII 0800-LUC vector. (F, G) LUC/REN activities detected by the reporter systems described in (E), which tested the effects of MdMYB1, MdTCP46, and MdMYB1+MdTCP46 on the expression of (F) MdDFR and (G) MdUF3GT. Empty vector, pGreenII 62-SK + pGreenII 0800-LUC; pMdDFR, pGreenII 62-SK + proMdDFR-pGreenII 0800-LUC; pMdUF3GT, pGreenII 62-SK + proMdUF3GT-pGreenII 0800-LUC; MdMYB1+pMdDFR, MdMYB1-pGreenII 62-SK + proMdDFR-pGreenII 0800-LUC; MdMYB1+pMdUF3GT, MdMYB1-pGreenII 62-SK + proMdUF3GT-pGreenII 0800-LUC; MdTCP46+pMdDFR, MdTCP46-pGreenII 62-SK + proMdDFR-pGreenII 0800-LUC; MdTCP46+pMdUF3GT, MdTCP46-pGreenII 62-SK + proMdUF3GT-pGreenII 0800-LUC; MdTCP46+MdMYB1+pMdDFR, MdTCP46-pGreenII 62-SK + MdMYB1-pGreenII 62-SK + proMdDFR-pGreenII 0800-LUC; MdTCP46+MdMYB1+pMdUF3GT, MdTCP46-pGreenII 62-SK + MdMYB1-pGreenII 62-SK + proMdUF3GT-pGreenII 0800-LUC. LUC/REN activities of the empty vector were used as references and set to 1. All experiments were performed three times with similar results, and representative data from one experiment are shown. Data are means (±SD), n=3. Different letters indicate significant differences as determined by one-way ANOVA and LSD tests (P<0.05).

Fig. 5.

MdBT2 interacts with MdTCP46. (A) Yeast two-hybrid assays. The ORF of MdTCP46 and the indicated regions of MdBT2 (i.e. MdBT2, MdBT2 N terminal, and MdBT2 C terminal; see schematic diagram to right) were fused to the pGAD and pGBD vectors. The pGAD and pGBD empty vectors were used as negative controls. –T/–L, SD medium lacking Trp and Leu; –T/–L/–H/–A, SD medium lacking Trp, Leu, His, and Ade. (B) Pull-down assays. The ORFs of MdBT2 and MdTCP46 were fused to the pET32a and pGEX4T-1 vectors, respectively. The MdBT2-HIS and MdTCP46-GST fusion proteins were obtained from E. coli expressions. GST proteins were used as negative controls. The eluted solution was probed with HIS and GST antibodies. (C) Bimolecular fluorescence complementation assays. The ORFs of MdBT2 and MdTCP46 were fused to the YFP^N and YFP^C vectors, respectively. Fluorescence was examined using a confocal laser-scanning microscope. Scale bars are 10 μm.

Fig. 6.

MdBT2 negatively regulates light-induced anthocyanin biosynthesis. Apple calli (15 d old) of the wild-type (WT) and transgenic MdBT2-overexpressing (-OX) and MdBT2-antisense (-Anti) lines were subjected to different light intensities for 5 d. (A) Phenotypes and (B) anthocyanin levels. The anthocyanin content the WT under the low-light treatment was used as a reference and set to 1. (C) Expression of MdBT2 in response to the different light intensities as determined using qRT-PCR. Expression was normalized to the ACTIN gene. Values are expressed relative to the low-light treatment, which was set to 1. (D) Detection of the MdBT2-HIS fusion protein after low-and high-light treatments. ‘Red Delicious’ fruit were subjected to the light treatments for 3 d. The total proteins extracted from the peels were incubated with purified MdBT2-HIS protein for 0–6 h. For the treatment with the proteasome inhibitor MG132, the total proteins fruit subjected to low light were pre-treated with 100 µM MG132 for 0.5 h before the sampling began. ACTIN was used as an internal reference. All experiments were performed three times with similar results, and representative data from one experiment are shown. Data are means (±SD), n=3. Different letters indicate significant differences as determined by one-way ANOVA and LSD tests (P<0.05).

Fig. 7.

MdBT2 negatively regulates MdTCP46-promoted anthocyanin biosynthesis. Transgenic apple calli (15 d old) and leaves of MdTCP46-overexpressing (-OX), MdTCP46-OX/MdBT2-OX (overexpression of MdBT2 in the background of MdTCP46-overexpression), and MdTCP46-OX/MdBT2-Anti (antisense suppression of MdBT2 in the background of MdTCP46-overexpression were subjected to high light intensity for 5 d. (A) Phenotypes of calli of the transgenic lines and the wild-type (WT) and (B) their anthocyanin contents. The content of the WT was used as a reference and was set to 1. (C) Phenotypes of transient transgenic leaves and the empty vector (EV) and (D) their anthocyanin contents. The content of the EV was used as a reference and was set to 1. All experiments were performed three times with similar results, and representative data from one experiment are shown. Data are means (±SD), n=4. Different letters indicate significant differences as determined by one-way ANOVA and LSD tests (P<0.05).

Fig. 8.

MdBT2 degrades the MdTCP46 protein. (A) Detection of MdTCP46-GST fusion proteins in stability assays. Protein extracts were taken from 15-d-old calli grown under dark conditions for the wild-type (WT) and transgenic lines of MdBT2-overexpression (-OX) and MdBT2-antisense (-Anti) and were treated for 0.5 h with either 100 µM of the proteasome inhibitor MG132 dissolved in DMSO or in DMSO alone (blank control), before being incubated with MdTCP46-GST protein for 0–6 h. ACTIN was used as an internal reference. (B) MdBT2 promotes the ubiquitination of the MdTCP46 protein in vivo. MdTCP46-GFP was immunoprecipitated using the GFP antibody from the two transgenic calli MdTCP46-GFP and MdTCP46-GFP/MdBT2-OX. The immunoprecipitated proteins were examined using antibodies for ubiquitin (left) and GFP (right).

Fig. 9.

Proposed model of the functioning of MdTCP46 in light-induced anthocyanin biosynthesis. (A) MdTCP46 interacts with MdMYB1 to increase its transcriptional activity and to enhance its binding to target genes, thereby promoting light-mediated anthocyanin biosynthesis. Under low light, MdBT2 interacts with MdTCP46 to ubiquitinate and degrade it, thus negatively regulating MdTCP46-promoted anthocyanin biosynthesis. Under high light, the transcription of MdTCP46 is up-regulated and anthocyanin biosynthesis is stimulated. Light inhibits MdBT2 expression, which in turn releases the MdBT2-enhanced degradation of the MdTCP46 protein, thereby contributing to light-induced anthocyanin biosynthesis. (B) The central role of MdBT2 in anthocyanin biosynthesis mediated by multiple stresses. (1) An et al. (2018d); (2) An et al. (2019c); (3) An et al. (2020); (4) present study.