Seedlings of A. commutatum “Red Valentine” that were 2-year-old were potted in the greenhouse of the South China Botanical Garden, Chinese Academy of Sciences (Guangzhou, China) and received natural light with a 60% shade cloth. The temperature and relative humidity of the greenhouse range from 15 to 34°C and 75–99%, respectively. Three stages for leaf development were defined as follows: stage 1, curly and white (S1, 7 days), stage 2, unfolded and light pink (S2, 28 days), and stage 3, mature and dark red (S3, 35 days). The leaves of three developmental stages, roots, stems, and flowers samples were frozen in liquid nitrogen immediately after collection before being stored at −80°C in November 2019 for analysis of anthocyanin content and gene expression levels.

To study the effect of light on anthocyanin biosynthesis and related gene expression, the A. commutatum seedlings at stage S2 were grown under 12 h light/dark cycle or dark conditions for 5 days in a phytotron (DGXM-508, Jiangnan Instrument Factory, Ningbo, China) at 28°C with 8,000 Lux light intensity. Subsequently, leaf samples were collected for analysis of gene expression and anthocyanin content. Following growth for 5 days, samples were immediately stored at −80°C for further study. Each sample contained three biological replicates.

The pH difference method was used to measure the total anthocyanin content in A. commutatum and transgenic Nicotiana tabacum cv. NC89 (tobacco) tissues (Wrolstad et al., 1982). Briefly, fresh samples (0.1 g) were extracted with 2 ml methanol (with 0.05% hydrochloric acid) overnight at 0°C. The absorbance at 510 and 700 nm was measured using a microplate reader (Tecan Infinite, Männedorf, Switzerland). The following equation was used to calculate the total anthocyanin content: Δ_A × DF × M × 1,000/(ε × W). where Δ_A = (A₅₁₀ – A₇₀₀)pH 1.0 -(A₅₁₀ – A₇₀₀)pH 4.5, DF is the dilution factor, M is the molecular weight of cyanidin-3-glucoside (449.2 g mol⁻¹), ε is the molar absorptivity (26,900, molar extinction coefficient (L mol⁻¹· cm⁻¹) for cyanidin-3-glucoside), and W is the sample weight (g).

The leaf transcriptome data of A. commutatum (Accession number: PRJNA793608) were used to screen for R2R3-MYB-like and bHLH-like genes by BLASTx searching from Nr and Swiss-Prot protein databases based on (i) similarity scores with the known anthocyanin-related TF genes in other species and (ii) correlations with anthocyanin structural gene expression. The transcripts were obtained from transcriptome assembly data, and the coding sequences were cloned using the SMARTer RACE cDNA Amplification Kit (Takara Biomedical Technology Co., Ltd.; Beijing, China), and amplified via PCR with 2× Super Pfx DNA Polymerase (Cowin Biotech Co., Ltd.; Taizhou, China) and the pClone007 Versatile Simple Vector Kit (TSINGKE Biotech Co., Ltd.; Beijing, China) for Sanger sequencing and subsequent analyses. DNAMAN (v8.0.8.789) was used to analyze the amino acid sequence alignment and MEGA (v7.0.26) was used to construct phylogenetic trees using the neighbor-joining method with 1,000 bootstrap replicates. The GenBank accession numbers of the MYB and bHLH proteins are listed in Supplementary Table S1.

Total RNA was isolated from tobacco as well as the roots, stems, flowers, and leaves of A. commutatum using a plant RNA kit (Polysaccharides and Polyphenolics-rich, Hua Yueyang, Beijing, China) with RNase-free DNase I (Takara Biomedical Technology Co., Ltd.; Beijing, China) to remove genomic DNA contamination. RNA (1 μg) was used for cDNA synthesis using the TransScript II One-Step gDNA Removal and cDNA Synthesis kit (TransGen Biotech Co., Ltd.; Beijing, China).

Key genes related to anthocyanin biosynthesis were screened from the A. commutatum “Red Valentine” leaf RNA-Seq database (BioProject ID: PRJNA793608). We identified 10 anthocyanin biosynthetic genes: AcCHS, AcCHS2, AcCHI, AcF3H, AcF3'H, AcDFR1, AcDFR3, AcANS, AcUFGT1, and AcUFGT2. The expression levels of AcMYB1 and AcbHLH1 as well as these 10 genes were analyzed in the root, stem, flower, and three developmental leaf stages of A. commutatum. The expression levels of AcMYB1 in transgenic tobacco leaves and flowers were also determined. The expression levels of nine structural genes and two bHLH TF genes (NtPAL, NtCHS, NtCHI, NtF3H, NtF3'H, NtF3'5'H, NtIDFR, NtANS, NtUFGT, NtAn1a, and NtAn1b, respectively) involved in anthocyanin biosynthesis were compared in the transgenic and control tobacco leaves and corollas. Finally, the mRNA abundance of six anthocyanin structural genes (NbCHS, NbCHI, NbF3H, NbDFR, NbANS, NbUFGT) in Nicotiana benthamiana leaves were examined 8 days after infiltration. Gene expression levels were determined using real-time quantitative PCR (qRT-PCR) with PerfectStart Green qPCR SuperMix (TransGen Biotech Co., Ltd., Beijing, China) with a LightCycler 480 II (Roche, Mannheim, Germany) according to the following conditions: 94°C for 30 s, 45 cycles at 94°C for 5 s, 60°C for 30 s. Elongation factor 1-alpha (AcEF-1α, Accession number: OM688333), NtActin (Accession number: X69885), and NbActin (Accession number: JQ256516) were used as reference genes. The Ct value of each sample was calculated via LightCycler 480 software (Roche, version: 1.5.1.62) and the relative expression was determined using the 2^−ΔΔCT method (Livak and Schmittgen, 2001), where ΔCt = Ct (target gene)–Ct (reference gene) and ΔΔCT = ΔCt (experimental group)–ΔCt (control group). Gene-specific primer pairs are listed in Supplementary Table S2. Three biological replicates and three technical replicates were performed.

The AcMYB1 and AcbHLH1 open reading frames, without the stop codons, were inserted into the BglII and KpnI sites of the pSAT6-EYFP-N1vector which is a yellow fluorescent protein (YFP) driven by the 35S Cauliflower mosaic virus (35S) promoter. The final plasmids, 35S::AcMYB1-YFP, 35S::AcbHLH1-YFP, control 35S::YFP, and nuclear marker 35S::mCherry, were introduced into A. thaliana mesophyll protoplasts by polyethylene glycol (PEG)-mediated transient transformation (Yoo et al., 2007). Co-transformation with the control 35S::YFP and nuclear marker 35S:: mCherry were used as a negative control. After incubation at 20°C for 20 h, the protoplasts were detected using a Zeiss LSM 510 confocal microscope (Zeiss, Jena, Germany).

Yeast two-hybrid (Y2H) analysis was performed using the Matchmaker Two-Hybrid System 3 (Clontech; Takara Bio USA, Inc.; San Jose, CA, United States). The coding region of AcbHLH1 was inserted into the bait vector pGBK-T7 (GAL4 DNA-binding domain), and AcMYB1 was fused to the prey vector pGAD-T7 (GAL4 activation domain). Vector pGADT7-T and pGBKT7-53 were used as the positive controls. All constructs were transformed into the yeast strain AH109 (Clontech, Takara Bio USA, Inc.; San Jose, CA, United States) using the PEG/LiAC method, according to the protocol handbook. All transformed yeast cells were selected on a synthetic drop-out medium without leucine and tryptophan (SD–Leu–Trp) at 30°C for 3 days. Colonies that survived from double selection plates were then screened for growth on a quadruple selection SD medium lacking adenine, histidine, leucine, and tryptophan (SD-Ade-His-Leu-Trp) containing 30 mM 3-amino-1,2,4-triazole (3-AT) solution and 25 mg/L X-α-Gal.

For the biomolecular fluorescence complementation (BiFC) assay, the AcMYB1 and AcbHLH1 ORFs, without stop codons, were inserted into the pSPYNE and pSPYCE vectors containing the N- and C-terminal halves of YFP (Walter et al., 2004). Recombinant plasmids were then introduced into A. thaliana mesophyll protoplasts by PEG-mediated transient transformation (Waadt et al., 2008). After incubation at 20°C for 20 h, the protoplasts were observed using a Zeiss LSM 510 confocal microscope (Zeiss, Jena, Germany).

To overexpress AcMYB1 in tobacco, the coding sequence of AcMYB1 was inserted into the pGreen-C17 vector, which was triggered under the control of the CaMV 35S promoter. The resulting vector, pGreen-C17-AcMYB1, was transferred into Agrobacterium tumefaciens strain EHA105 and then transformed into tobacco via the leaf disc method (Zhang et al., 2007). Empty vector-infected tobacco plants were used as controls. The T1-generation leaves and corollas from the three transgenic lines and control plants were collected for the anthocyanin concentration (Section Anthocyanins Content Analysis) and gene expression (Section RNA Extraction, cDNA Synthesis, and Gene Expression Analysis) analyses.

The transient over-expression experiments in N. benthamiana leaves were performed according to the method described by Palapol et al. (2009). Briefly, 35S-promoter-driven AcMYB1 and AcbHLH1 constructs were inserted into Agrobacterium tumefaciens strain GV3101 via electroporation. N. benthamiana plants with 6–8 leaves were solely or simultaneously infiltrated with a needleless 1-ml syringe into the abaxial sides of the fourth or fifth leaves, and Agrobacteria with an empty vector was used as a control. Anthocyanin content was measured and digital photographs were taken 8 days after infiltration. Anthocyanin content was measured with the pH difference method as described in Section Anthocyanins Content Analysis.

All experiments were performed on at least three independently grown biological replicates. All values represent the mean ± SE. Differences between the treatment groups were examined using SPSS software (one-way ANOVA, Duncan test), and the significant differences (p < 0.05) are indicated with different letters, respectively.

Leaves of A. commutatum “Red Valentine” has white color and stay curly at leaf-S1. At leaf-S2, leaf color changes from white to light pink and appears deep red at leaf-S3 (Figure 1A). Anthocyanin content in the leaves of A. commutatum significantly increased with developmental age. Additionally, trace amounts or no anthocyanins were detected in the roots, stems, and flowers (Figure 1B).

In this study, R2R3-MYB and bHLH TFs were isolated from the leaf transcriptome database of A. commutatum “Red Valentine.” The candidate genes were cloned from the cDNA of A. commutatum leaves by RACE PCR, sequenced, and named AcMYB1 and AcbHLH1. The results showed that AcMYB1 and AcbHLH1 contained 969 and 2,115 bp ORFs, encoding proteins of 322 and 712 amino acids, respectively. The neighbor-joining phylogenetic tree showed that AcMYB1 clustered with monocot-positive anthocyanin regulators, including AaMYB2, LhMYB6, LhMYB12, and MaAN2 (Figure 2A). Similarly, AcbHLH1 formed a cluster with AabHLH1 and other bHLH TFs associated with anthocyanin biosynthesis in several plant species (Figure 2B). Alignment analysis showed that a highly conserved R2R3 domain is contained in the AcMYB1 protein for DNA binding at the N-terminus, which contains a crucial bHLH-interacting motif in the R3 domain for interactions with bHLH TFs (Figure 2C) (Zimmermann et al., 2004). We also found an N-terminal MYB-interacting region and conserved bHLH domain in the AcbHLH1 protein (Supplementary Figure S1) (Pattanaik et al., 2008). These results suggested that AcMYB1 and AcbHLH1 may interact and play roles in anthocyanin biosynthesis.

The gene expression analyses showed that all the candidate structural genes, including AcMYB1 and AcbHLH1, had significantly higher expression levels in the three developmental leaf stages compared to those in the other tissues and reached a peak at leaf stage two except AcF3H (Figure 3). Furthermore, the correlation analysis revealed that the gene expression correlation coefficient between AcMYB1 or AcbHLH1 and structural genes was in the range of 0.73–0.99 and 0.75–0.98. This result implies that transcript abundance of AcMYB1 and AcbHLH1 are correlated with those of the most anthocyanin structural genes and they are likely involved in the regulation of anthocyanin biosynthesis in A. commutatum.

Subcellular localization analysis in A. thaliana leaf protoplasts revealed that both AcMYB1 and AcbHLH1 were specifically localized in the cell nucleus (Figure 4). In A. commutatum, AcMYB1 and AcbHLH1 showed conserved interacting motifs, similar mRNA expression patterns in different tissues, and co-localization in the nucleus, suggesting a possible interaction. To test this hypothesis, we used Y2H and BiFC assays. For the Y2H assay, all the transformed colonies grew well on SD/-Leu/-Trp, indicating their successful transformation. The co-transformed colonies of pGADT7-AcMYB1 + pGBKT7-AcbHLH1 displayed distinct blue coloration on SD/-Leu/-Trp/-His/-Ade, indicating that AcMYB1 and AcbHLH1 physically interacted (Figure 5A). In the BiFC assays, YFP fluorescence signals were observed only when pSPYNE/AcMYB1 and pSPYCE/AcHLH1 were co-expressed and no fluorescence was detected in cells that contained control vectors (Figure 5B). These results further confirm that AcbHLH1 and AcbMYB1 may interact in A. commutatum cells.

To investigate the role of AcMYB1 in regulating the anthocyanin biosynthetic pathway, overexpression (OE) under the control of the CaMV-35S promoter was carried out in tobacco. More than 20 independent transgenic lines were generated using genomic PCR. The results of phenotypic changes showed that pigment levels were significantly increased in both vegetative and reproductive tissues relative to that in the control plants. In particular, the OE-AcMYB1 line showed a remarkable change in leaf color, and the mature leaves of the transgenic plants displayed an observably darker red color than that of the control plants. In addition, the corolla color of OE-AcMYB1 tobacco changed from light pink to deep red, and increased anthocyanin accumulation was markedly visible in the anthers, filaments, calyxes, ovary walls, and seed coats (Figures 6A–L). Anthocyanin content determination confirmed that the anthocyanin extracted from the three lines of OE-AcMYB1 tobacco leaves and corollas were markedly higher than those in the controls (Figures 6M,N).

To reveal the potential target genes in OE-AcMYB1 transgenic plants, the mRNA expression level of structural genes and two bHLH TF genes involved in anthocyanin biosynthesis were verified by RT-PCR assay in tobacco leaves and corollas. A high expression level of AcMYB1 was first confirmed in three lines by qRT-PCR (Figures 7A,B). Expression analysis showed that the 11 anthocyanin regulatory genes were significantly upregulated in the leaves of all three transgenic lines compared to those of the control plants (Figure 7C). However, in the corollas of the three transgenic tobacco plants, only NtCHI, NtANS, NtUFGT, and NtAn1b showed significantly higher expression levels than that of the control (Figure 7D). These results indicated that AcMYB1 could upregulate or activate the expression of key anthocyanin structural genes and bHLH TFs, ultimately promoting anthocyanin accumulation in transgenic tobacco.

An Agrobacterium-mediated transient assay was further performed to investigate the regulation of AcMYB1 and AcbHLH1 on anthocyanin biosynthesis. The results showed that a slight accumulation of anthocyanin was detected in the leaves inoculated with the control or AcbHLH1 construct, while patches of anthocyanin were observed in AcMYB1 and AcMYB1 + AcbHLH1 inoculated leaves (Figures 8A–E). The anthocyanin content in AcMYB1 + AcbHLH1 leaves is 1.36 times that of the AcMYB1 leaves (Figure 8E). Moreover, the expression of anthocyanin biosynthetic genes were strongly up-regulated in the leaves infiltrated with AcMYB1 and AcMYB1 + AcbHLH1 constructs (Figure 8F). Interestingly, NbCHI, NbANS, NbDFR, and NbUFGT displayed significantly higher expression levels in simultaneous inoculation of 35S::AcMYB1 and 35S::AcbHLH1 constructs than solely infiltrating with 35S::AcMYB1. Taken together, we predicted that the ability of AcMYB1 in anthocyanin regulation could enhance by the interaction with AcbHLH1.

Comparative analysis was conducted between 2-year-old A. commutatum “Red Valentine” seedlings after growing in the dark or light for 5 days. As shown in Figure 9A, the leaf color was bright red in light treatment plants and pale pink in dark-grown plants. The leaf anthocyanin content in light treatment seedlings is 11.2-fold higher than that of the seedlings growing in dark (Figure 9B). Furthermore, the transcriptional level of AcMYB1, AcbHLH1, and 10 anthocyanin structural genes in light-grown conditions were all noticeably higher in comparison with the seedlings growing in dark (Figure 9C). The results reveal that light can strongly induce the expression of anthocyanin-related genes and promote the anthocyanin accumulation in A. commutatum “Red Valentine” leaf.