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.
Highlights
Proanthocyanidins (PAs), called condensed tannins (CTs), result from condensation of flavan-3-ols, and belong to one of the main classes of polyphenolic compounds synthesized via the flavonoid biosynthetic pathway [1]
Ectopic overexpression of SsMYB3 leads to a large reduction in anthocyanin level and enhanced accumulation of CTs in transgenic tobacco flowers. These results suggest that SsMYB3 is a functional PA regulator and may be a useful molecular tool for metabolic engineering of PA biosynthesis in plants
Using the NCBI conserved domain search program, an Nterminal R2R3 repeat corresponding to the DNA-binding (MYB) domain was detected in the deduced amino acid sequence of SsMYB3 (Fig 2A)
Summary
Proanthocyanidins (PAs), called condensed tannins (CTs), result from condensation of flavan-3-ols, and belong to one of the main classes of polyphenolic compounds synthesized via the flavonoid biosynthetic pathway [1]. Few studies have reported on the key structural and regulatory genes involved in the flavonoid pathway (especially in biosynthesis of PAs and anthocyanins) in coleus. The expression of the structural genes of anthocyanin and PA pathways was measured by qPCR using RNA isolated from the SsMYB3-overexpressing tobacco flowers.
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