Multiple Copies of a Simple MYB-Binding Site Confers Trans-regulation by Specific Flavonoid-Related R2R3 MYBs in Diverse Species

Abstract

In apple, the MYB transcription factor MYB10 controls the accumulation of anthocyanins. MYB10 is able to auto-activate its expression by binding its own promoter at a specific motif, the R1 motif. In some apple accessions a natural mutation, termed R6, has more copies of this motif within the MYB10 promoter resulting in stronger auto-activation and elevated anthocyanins. Here we show that other anthocyanin-related MYBs selected from apple, pear, strawberry, petunia, kiwifruit and Arabidopsis are able to activate promoters containing the R6 motif. To examine the specificity of this motif, members of the R2R3 MYB family were screened against a promoter harboring the R6 mutation. Only MYBs from subgroups 5 and 6 activate expression by binding the R6 motif, with these MYBs sharing conserved residues in their R2R3 DNA binding domains. Insertion of the apple R6 motif into orthologous promoters of MYB10 in pear (PcMYB10) and Arabidopsis (AtMY75) elevated anthocyanin levels. Introduction of the R6 motif into the promoter region of an anthocyanin biosynthetic enzyme F3'5'H of kiwifruit imparts regulation by MYB10. This results in elevated levels of delphinidin in both tobacco and kiwifruit. Finally, an R6 motif inserted into the promoter the vitamin C biosynthesis gene GDP-L-Gal phosphorylase increases vitamin C content in a MYB10-dependent manner. This motif therefore provides a tool to re-engineer novel MYB-regulated responses in plants.

Keywords: MYB; anthocyanin; biotechnology; flavonoids; promoter; transcription factor.

FIGURE 1

MYB10 orthologs activate the R6-containing MYB10 promoter in a transient assay in Nicotiana benthamiana. (A) Schematic of the R1 and R6 apple MYB10 promoters fused to the LUC reporter gene in pGreen0800LUC representing the R repeat units (open boxes) and the microsatellite structure upstream of unit 1 (black box) and their position relative to the ATG start codon. (B) Leaves of N. benthamiana were infiltrated with the MdMYB10-R1_pro:LUC or MdMYB10-R6_pro:LUC fusions (named R1:LUC and R6:LUC respectively) on their own (no TF) or co-infiltrated with MdMYB10 or MYB10 orthologs and bHLH partner. Md: Malus domestica, Pc: Pyrus communis, Fa: Fragaria ananassa, At: Arabidopsis thaliana, Ph: Petunia hybrida, Ac: Actinidia chinensis. Luminescence was measured 3 days post-infiltration and is expressed as a ratio of the LUC to REN signals. Data represent means (± SE) of four technical replicate reactions.

FIGURE 2

Apple MYBs from subgroups 5, 6 and MdMYB12 can activate the R6-containing MYB10 promoter. (A) Apple MYB transcription factors (TFs) were co-infiltrated with MdbHLH3 and the MdMYB10-R1_pro:LUC or MdMYB10-R6_pro:LUC fusions (named R1:LUC and R6:LUC respectively) in N. benthamiana leaves and the LUC/REN signal was measured. Data represent means (± SE) of four technical replicate reactions. (B) Phylogenetic tree representing relationships between apple and Arabidopsis MYBs. Numbers indicate subgroups previously described for Arabidopsis MYBs (Stracke et al., 2001). Apple MYBs tested in transient assay in (A) (white diamond) and the MYBs tested positive for transactivation of the R6 promoter (black diamond) are indicated. Deduced amino acid sequences were aligned using CLUSTALX. Protein distances were calculated with PROTDIST using the Jones-Taylor-Thornton matrix and the tree was constructed by the neighbor-joining method.

FIGURE 3

Deduced amino acid sequences were aligned using CLUSTALW. The bHLH binding region within the R3 domain is indicated (gray arrow) and conserved amino acids required for the bHLH-dependant activation (Grotewold et al., 2000) are highlighted above it. Box 1, 2, and 3 show previously described conserved residues specific to dicot anthocyanin-promoting MYBs (Lin-Wang et al., 2010). Double black arrow indicates the signature (K/R)₂₅-(X)x46-(K/R/N)₇₂ specific to the R6-activating MYBs.

FIGURE 4

Insertion of the R6 domain in the promoter of MYB10 orthologs leads to autoregulation. (A) Schematic of the engineered pear MYB10 and AtMYB75 promoters fused to the LUC reporter gene in pGreen0800LUC. (B) Dual luciferase promoter assay of the R6 engineered promoters in N. benthamiana. Promoters, with or without the R6 domain, were co-infiltrated with MdMYB10 and MdbHLH3, PcMYB10 and AtbHLH2, or AtMYB75 and AtbHLH2. Luminescence of LUC and REN was measured 3 days post-infiltration and expressed as a ratio of LUC to REN. Data represent means (± SE) of four technical replicate reactions.

FIGURE 5

Insertion of the R6 motif into the promoter of AtMYB75 induces AtMYB75 overexpression and ectopic anthocyanin accumulation. (A) Schematics of the engineered AtMYB75 constructs transformed into Arabidopsis. (B) Phenotype of representative T2 lines transformed by AtMYB75_pro:AtMYB75 [denoted as native promoter (NP)], AtMYB75-R6_pro:AtMYB75 (denoted as R6) and 35S_pro:AtMYB75 (denoted as 35S). (C) Expression analysis by qPCR of the transcripts of the AtMYB75 gene in each transformed lines. Expression is given relative to actin and error bars represent the standard errors of the means calculated from four technical replicates. (D) Total anthocyanin content in each lines measured by HPLC and expressed as μg of Cy-glu equivalent per gram of fresh weight (FW).

FIGURE 6

Insertion of R6 domain into the pear MYB10 promoter induces PcMYB10 overexpression and ectopic anthocyanin accumulation. (A) Phenotype of representative transgenic pear lines harboring the PcMYB10-R6_pro:PcMYB10 construct. (B) Expression analysis by qPCR of the transcripts of the PcMYB10 gene in fully expanded mature leaves of each transgenic line. Expression is given relative to pear actin and error bars represent the standard errors of the means calculated from four technical replicates. (C) Total anthocyanin content of fully expanded mature leaves of each line measured by HPLC and expressed as μg of Cy-glu equivalent per gram of fresh weight (FW). Error bars represent the standard errors of the means calculated from three replicates.

FIGURE 7

Insertion of the R6 motif into the AcF3′5′H promoter induces accumulation of delphinidin derivatives. (A) Schematic of the engineered F3′5′H promoter fused to the LUC reporter gene in pGreen0800LUC. (B) Actinidia eriantha kiwifruit were infiltrated by Agrobacterium suspension containing AcMYB110 (i), AcMYB110 and F3′5′H_pro: F3′5′H (ii), AcMYB110 and F3′5′H-R6_pro: F3′5′H (iii) constructs and corresponding empty vectors (iv). Photos were taken 4 days post-infiltration. (C) Measurement of cyanidin and delphinidin derivatives in kiwifruits expressing corresponding constructs, expressed as concentration of cyanidin-gal equivalent and (D) presented as a ratio of delphinidin to cyanidin.

FIGURE 8

Effect of inserting the R6 motif in the GGP promoter. (A) Schematic of the engineered GGP promoter fused to the LUC reporter gene in pGreen0800LUC representing the position of two R6 insertions upstream of the 5′ UTR and uORF (open box and dash line respectively). (B) Dual luciferase assay of the GGP promoter harboring the R6 motif fused to the LUC reporter. Promoter constructs were co-infiltrated in N. benthamiana with MdMYB10 and MdbHLH3. Luminescence of LUC and REN was measured 3 days post-infiltration and expressed as a ratio of LUC to REN. Data represent means (± SE) of four replicate reactions. (C) The GGP cDNA, placed under its own promoter engineered with two copies of the R6 motif and harboring or not a single point mutation of the uORF start codon (GGPmut-R12_pro:GGP and GGPwt-R12_pro-GGP, respectively), or placed under the control a 35S promoter (35S_pro:GGP), or the corresponding empty vector (EV) were co-infiltrated with AcMYB110 (or the MYB8 negative control). Ascorbate content was measured at 5 dpi. Data represent means (± SE) of six biological replicates. One-way ANOVA test was applied and resulting grouping information was obtained using the Tukey method, columns sharing the same letters are not significantly different at P < 0.05.