Loss of the R2R3 MYB Transcription Factor RsMYB1 Shapes Anthocyanin Biosynthesis and Accumulation in Raphanus sativus.

Da-Hye Kim,Jong-Yeol Lee,Sun-Hyung Lim,Jundae Lee,Juhee Rhee

doi:10.3390/ijms222010927

Abstract

The red or purple color of radish (Raphanus sativus L.) taproots is due to anthocyanins, which have nutritional and aesthetic value, as well as antioxidant properties. Moreover, the varied patterns and levels of anthocyanin accumulation in radish roots make them an interesting system for studying the transcriptional regulation of anthocyanin biosynthesis. The R2R3 MYB transcription factor RsMYB1 is a key positive regulator of anthocyanin biosynthesis in radish. Here, we isolated an allele of RsMYB1, named RsMYB1Short, in radish cultivars with white taproots. The RsMYB1Short allele carried a 4 bp insertion in the first exon causing a frame-shift mutation of RsMYB1, generating a truncated protein with only a partial R2 domain at the N-terminus. Unlike RsMYB1Full, RsMYB1Short was localized to the nucleus and the cytoplasm and failed to interact with their cognate partner RsTT8. Transient expression of genomic or cDNA sequences for RsMYB1Short in radish cotyledons failed to induce anthocyanin accumulation, but that for RsMYB1Full activated it. Additionally, RsMYB1Short showed the lost ability to induce pigment accumulation and to enhance the transcript level of anthocyanin biosynthetic genes, while RsMYB1Full promoted both processes when co-expressed with RsTT8 in tobacco leaves. As the result of the transient assay, co-expressing RsTT8 and RsMYB1Full, but not RsMYB1Short, also enhanced the promoter activity of RsCHS and RsDFR. We designed a molecular marker for RsMYB1 genotyping, and revealed that the RsMYB1Short allele is common in white radish cultivars, underscoring the importance of variation at the RsMYB1 locus in anthocyanin biosynthesis in the radish taproot. Together, these results indicate that the nonsense mutation of RsMYB1 generated the truncated protein, RsMYB1Short, that had the loss of ability to regulate anthocyanin biosynthesis. Our findings highlight that the frame shift mutation of RsMYB1 plays a key role in anthocyanin biosynthesis in the radish taproot.

Highlights

Anthocyanins are flavonoid-derived metabolites with multiple functions, which include attracting pollinators or seed dispersal agents, protecting plants against damage from UV radiation, and contributing to cold and drought stress responses [1,2]
We evaluated the function of RsMYB1Short and RsMYB1Full on anthocyanin biosynthesis via transient assay in radish cotyledons
We demonstrated that anthocyanin biosynthesis is determined in part by allelic variation at the RsMYB1 locus in the form of the RsMYB1Short and RsMYB1Full alleles

Summary

Introduction

Anthocyanins are flavonoid-derived metabolites with multiple functions, which include attracting pollinators or seed dispersal agents, protecting plants against damage from UV radiation, and contributing to cold and drought stress responses [1,2]. The anthocyanin biosynthetic pathway has been well characterized and genes encoding the relevant enzymes and transcriptional regulators have been identified in many plant species [6]. Anthocyanin biosynthesis is transcriptionally regulated by R2R3 MYB transcription factors (TFs), basic helix-loop-helix (bHLH) TFs, and WD40 repeat proteins, which interact to form MYB–bHLH–WD40 (MBW) complexes [7,8]. Among these regulators, the MYB proteins contain two conserved imperfect repeats (named R2 and R3) in their. The bHLH factors form transcriptional complexes at the promoters of anthocyanin biosynthetic genes by interacting with the R3 region of their R2R3 MYB partners [11,12]. The WD40 partners act as a docking platform and stabilize the interaction between the MYB and bHLH TFs, rather than exerting a direct regulatory function [7,13]

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Discussion

Conclusion