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.
Highlights
Gene transcription is partly controlled by transcription factors (TFs), often in combination(s) and within hierarchical networks (Hobert, 2008)
Examination of many sources of apple germplasm (Espley et al, 2009) identified only R1 and R6 variants, suggesting that this mutation has happened only once. To test whether this activation is restricted to the apple MYB10, we over-expressed the anthocyanin-activating MYBs from pear, strawberry, Arabidopsis, petunia, and kiwifruit in Nicotiana benthamiana together with the R1 or R6 versions of the apple MYB10 promoter fused to a luciferase reporter (Hellens et al, 2005)
MdMYB8, an R2R3 MYB from apple, which is not implicated in anthocyanin regulation (Espley et al, 2007), failed to activate the R6 motif, even in the presence of a bHLH partner
Summary
Gene transcription is partly controlled by transcription factors (TFs), often in combination(s) and within hierarchical networks (Hobert, 2008). In these cultivars there are two alleles; the MYB1 allele, which is present in all whitefleshed, green-foliaged apples and has one copy of a 23-base pair sequence (R1) in the proximal promoter sequence of the coding region of the MYB, and secondly the MYB10 allele, which is only found in red-fleshed, red-foliage apple accessions and contains a further five copies of the 23-base pair sequence in a minisatellite-like structure (Espley et al, 2009) This multiple repeat rearrangement (R6) generates a positive auto-regulatory allele, as MYB10 can bind the 23 bp sequence as shown by in vitro binding assays (Espley et al, 2009). We show that the introduction of an R6 motif into promoters of MYB10 orthologs can initiate auto-regulation in species as diverse as pear and Arabidopsis Engineering of this cis-element can change anthocyanin composition, harnessing the activity of different biosynthetic genes to produce a different metabolic profile. This versatility could suit a range of biotechnological applications for promoter engineering in order to produce a temporal or spatial elevation of target pathways
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