Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis

Abstract

Plants produce a wide array of natural products, many of which are likely to be useful bioactive structures. Unfortunately, these complex natural products usually occur at very low abundance and with restricted tissue distribution, thereby hindering their evaluation. Here, we report a novel approach for enhancing the accumulation of natural products based on activation tagging by Agrobacterium-mediated transformation with a T-DNA that carries cauliflower mosaic virus 35S enhancer sequences at its right border. Among approximately 5000 Arabidopsis activation-tagged lines, we found a plant that exhibited intense purple pigmentation in many vegetative organs throughout development. This upregulation of pigmentation reflected a dominant mutation that resulted in massive activation of phenylpropanoid biosynthetic genes and enhanced accumulation of lignin, hydroxycinnamic acid esters, and flavonoids, including various anthocyanins that were responsible for the purple color. These phenotypes, caused by insertion of the viral enhancer sequences adjacent to an MYB transcription factor gene, indicate that activation tagging can overcome the stringent genetic controls regulating the accumulation of specific natural products during plant development. Our findings suggest a functional genomics approach to the biotechnological evaluation of phytochemical biodiversity through the generation of massively enriched tissue sources for drug screening and for isolating underlying regulatory and biosynthetic genes.

Figure 1.

pap1-D Phenotypes. (A) pap1-D (left) and Col-0 (right) flowers. (B) Roots of pap1-D (left) and Col-0 (right) plants. (C) Six-week-old adult pap1-D (front) and Col-0 (back) plants.

Figure 2.

Enhanced Expression of Phenylpropanoid Biosynthetic Genes in pap1-D. RNA gel blot hybridization was conducted with total RNA isolated from 6-week-old pap1-D and Col-0 wild-type plants. DFR, dihydroflavonol reductase; GST, glutathione S-transferase; UBQ, ubiquitin.

Figure 3.

Effect of pap1-D Mutation on Accumulation of Phenylpropanoid Products. (A) to (F) HPLC profiles of phenylpropanoid metabolites in extracts from wild-type Col-0 ([A], [C], and [E]) and pap1-D ([B], [D], [F]) plants. (A) and (B) Soluble phenolics: peak 1, rhamnose (Rha)-Glc-Rha-quercetin; peak 2, quercetin conjugate; peak 3, Rha-Glc-Rha-kaempferol; peak 4, Glu-Rha-quercetin; peak 5, Rha-Rha-quercetin; peak 6, Glc-Rha-kaempferol; peaks 7 and 8, kaempferol conjugates; peak 9, sinapic acid; peak 10, Rha-Rha-kaempferol. (C) and (D) Anthocyanidins; the inset in (D) shows the UV light absorption spectrum of the major anthocyanidin eluting at 21.5 min. (E) and (F) Wall-bound phenolics: peak 1, trans-4-coumaric acid; peak 2, sinapic acid; peak 3, cis-4-coumaric acid.

Figure 4.

Molecular Characterization of PAP1. (A) Genomic context of T-DNA insertion in pap1-D and structure of pMN-PAP1. Basta^r, Basta resistance; Kan^r, Kanamycin resistance; LB, left border; pBS, pBluescript KS+ plasmid; RB, right border; 4 × 35S denotes four copies of 35Se. (B) Sequence homology of R2R3 MYB. Proteins were aligned using the ClustalW software program. Red shading denotes 100% conserved residues, and yellow shading denotes matching residues with PAP1. R2 and R3 MYB domains are shown. GenBank accession numbers PAP1 (AF325123), PAP2 (AF325124), AN2 (AAF66727), C1 (AAA33482), P1 (AAB67720), P (AAC49394), GL1 (P27900), Mixta (CAA55725), and c-myb (AAA52031). (C) Gel blot hybridization of total RNA from 4-week-old pap1-D and Col-0 plants.1.0 denotes transcript size in kilobases. (D) Neighbor-joining phylogenetic tree built by using full-length proteins, showing the anthocyanin MYB family branch. The human gene c-myb is used as an outgroup.

Figure 5.

Overexpression of PAP1 or PAP2 Enhances Pigmentation in Arabidopsis and Tobacco. (A) to (E) Arabidopsis plants transformed with pMN20-2 ([A] and [D]), pMN-PAP1 ([B] and [E]), and pCHF3:PAP2 (C). (A) to (C) show six-week-old plants. (D) and (E) show flowers on 12-week-old plants. (F) to (J) Tobacco plants transformed with pCHF3 ([F] and [I]), pCHF3:PAP1 (G), and pCHF3:PAP2 ([H] and [J]). Plantlets in (F) to (H) were photographed at age 4 weeks, and flowers in (I) and (J) at 10 weeks after transfer to soil. pCHF3-PAP1 plants had brilliant flower pigmentation, identical to that of pCHF3-PAP2 (data not shown).