Ectopic Expression of the Coleus R2R3 MYB-Type Proanthocyanidin Regulator Gene SsMYB3 Alters the Flower Color in Transgenic Tobacco

Abstract

Proanthocyanidins (PAs) play an important role in plant disease defense and have beneficial effects on human health. We isolated and characterized a novel R2R3 MYB-type PA-regulator SsMYB3 from a well-known ornamental plant, coleus (Solenostemon scutellarioides), to study the molecular regulation of PAs and to engineer PAs biosynthesis. The expression level of SsMYB3 was correlated with condensed tannins contents in various coleus tissues and was induced by wounding and light. A complementation test in the Arabidopsis tt2 mutant showed that SsMYB3 could restore the PA-deficient seed coat phenotype and activated expression of the PA-specific gene ANR and two related genes, DFR and ANS. In yeast two-hybrid assays, SsMYB3 interacted with the Arabidopsis AtTT8 and AtTTG1 to reform the ternary transcriptional complex, and also interacted with two tobacco bHLH proteins (NtAn1a and NtJAF13-1) and a WD40 protein, NtAn11-1. Ectopic overexpression of SsMYB3 in transgenic tobacco led to almost-white flowers by greatly reducing anthocyanin levels and enhancing accumulation of condensed tannins. This overexpression of SsMYB3 upregulated the key PA genes (NtLAR and NtANR) and late anthocyanin structural genes (NtDFR and NtANS), but downregulated the expression of the final anthocyanin gene NtUFGT. The formative SsMYB3-complex represses anthocyanin accumulation by directly suppressing the expression of the final anthocyanin structural gene NtUFGT, through competitive inhibition or destabilization of the endogenous NtAn2-complex formation. These results suggested that SsMYB3 may form a transcription activation complex to regulate PA biosynthesis in the Arabidopsis tt2 mutant and transgenic tobacco. Our findings suggest that SsMYB3 is involved in the regulation of PA biosynthesis in coleus and has the potential as a molecular tool for manipulating biosynthesis of PAs in fruits and other crops using metabolic engineering.

Fig 1. Schematic diagram of the flavonoid biosynthesis pathway, including main branches of anthocyanins, PAs, and flavonols.

PAL, phenylalanine ammonia lyase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavonone 3-hydroxylase; DFR, dihydroflavonol reductase; FLS, flavonol synthase; ANS, anthocyanin synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; UFGT, UDP-glucose: flavonoid 3-O-glucosyltransferase. Asterisks indicate key structural enzymes in PA biosynthesis.

Fig 2. Comparison of SsMYB3 with other functional R2R3-MYB TFs.

(A) Full-length protein sequence alignments of the MYBs by Vector NTI 10.0 software. The R2 and R3 domains are shown; the bHLH motif [D/E]Lx₂[R/K]x₃Lx₆Lx₃R and two possible PA specifically conserved motifs, K[I/V]x₂PKPx₁Rx₂S[I/L] and VI[R/P]TKAx₁RC[S/T], are indicated in the green and blue box, respectively. (B) Phylogenetic tree analysis depicts SsMYB3 belonging to PA-clade 1. The tree was constructed from the ClustalX 1.8 alignment using the neighbor-joining method in the MEGA 5.0 program. Scale bar represents 0.1 substitutions per site, and numbers next to nodes are bootstrap values from 1,000 replicates (only values >50% are shown). SsMYB3 and other known functional PA-biosynthesis regulators are divided into two subclades (PA-clades 1 and 2) with different genomic structures indicated in gray background. MYB proteins with known functions and MYB subgroups (G03, G05, G07, G20, N08, and N09) are indicated. GenBank accession numbers of MYBs in the phylogenetic tree are as follows: AmMIXTA (CAA55725), AmROSEA1 (ABB83826), AmROSEA2 (ABB83827), AmVENOSA (ABB83828), Am308 (JQ0960), AtTT2 (Q9FJA2), AtPAP1 (ABB03879), AtPAP2 (NP_176813), AtMYB4 (BAA21619), AtMYB12 (ABB03913), AtWER (AAF18939), AtGL1 (AAC97387), DkMYB2 (AB503699), DkMYB4 (AB503671), FaMYB1 (AAK84064), GMYB10 (CAD87010), LeANT1 (AAQ55181), MdMYB1 (DQ886414), PmMBF1 (AAA82943), PhAN2 (AAF66727), PhPH4 (AAY51377.1), PhODO1 (AAV98200), PtMYB134 (ACR83705), ZmC1 (AAA33482), ZmPl (AAA19821), ZmP (AAC49394), VvMybPA1 (CAJ90831), VvMybPA2 (EU919682), VvMYBPA1 (BAD18977), VvMYBPA2 (BAD18978), VvMYB4 (ABL61515), and VvMYB5a (AAS68190).

Fig 3. Accumulation of PAs and the gene expression profiles of SsMYB3.

(A) The expression pattern of SsMYB3 and accumulation of PAs in various tissues of coleus: R (root), S (stem), YL (young leaf), ML (mature leaf), OL (old leaf), and F (flower). Transcription pattern of SsMYB3 in leaves under different treatments: (B) light, (C) dark, and (D) wounding. All expression data were normalized to the coleus SsActin gene, and values represent averages of three technical replicates.

Fig 4. Complementation of Arabidopsis PA-deficient tt2 mutant seed phenotype by overexpression of SsMYB3.

(A) Seed coat pigmentation and DMACA staining of wild-type (WT), tt2 mutant, and tt2 35S::SsMYB3 seeds. Seeds of WT and tt2 35S::SsMYB3 show brown pigmentation due to the accumulation of PAs, and their seed coats display dark coloration from staining by DMACA. The PA-deficient tt2 mutant seed coat is yellow and cannot be stained by DMACA. (B) RT-PCR analysis of key structural genes related to PA biosynthesis in immature siliques of WT, tt2 mutant, and tt2 35S::SsMYB3 lines. (C) Protein–protein interactions between SsMYB3 and Arabidopsis PA-related regulator AtTT8 or AtTTG1. In a Y2H assay, SsMYB3 fused to the GAL4-activation domain (pAD-SsMYB3) and GAL4-DNA-binding domain (pBD-SsMYB3); pAD-SsMYB3 was co-transformed with fusion constructs of the GAL4 DNA-binding domain with the WD40 protein AtTTG1 (pBD-AtTTG1) and the MYB-interaction regions from AtTT8 (pBD-AtTT8^aa1-204).

Fig 5. Functional characterization of SsMYB3 by its ectopic expression in tobacco.

(A) Overexpression of SsMYB3 resulted in visibly decreased color in the corolla of transgenic tobacco flowers. (B) Relative anthocyanin contents quantified as (A530–0.25 × A650)/fresh weight (g). (C) The measured relative condensed tannin contents at 500-nm absorbance. (D) RT-PCR expression analysis of SsMYB3 and flavonoid-related regulators in transgenic tobacco flowers. Asterisks indicate a statistically significant difference between wild-type and transgenic plants (P≤ 0.05 by Student’s t-test).

Fig 6. Transcript expression profiles of anthocyanin-related structural genes in flowers of WT and SsMYB3-overexpressing transgenic tobacco lines.

All transcripts expressed in transgenic flowers were quantified relative to those expressed in WT tobacco flowers (2^-ΔΔCt).

Fig 7. Protein–protein interactions between SsMYB3 and tobacco flavonoid-related regulators.

In a yeast two-hybrid assay, the SsMYB3/GAL4-activation domain fusion (pAD-SsMYB3) was co-transformed with fusion constructs of the GAL4-DNA-binding domain with the WD40 protein NtAn11-1 (pBD-NtAn11), the MYB-interaction regions from NtAn1a (pBD-NtAn1a^aa1–195), or NtJAF13-1 (pBD-NtJAF13-1^aa1-203).

Fig 8. A model for regulation of PAs and anthocyanins biosynthesis by SsMYB3 in transgenic tobacco.

In wild tobacco (A), the NtAn2-complex controls anthocyanins biosynthesis by activating expression of late anthocyanin structural genes, such as F3H, DFR, ANS, and UFGT. In SsMYB3-overexpressing tobacco (B), high levels of SsMYB3 compete with NtAn2 to form SsMYB3-complex promoting PAs biosynthesis by specially activating expression of LAR and ANR, and repressing anthocyanins accumulation by inactivating expression of UFGT, through inhibition or destabilization of the formation of NtAn2-complex.