Transposon Insertion At The Promoter Of A Myb Transcription Factor Results In Ectopic Anthocyanins Accumulation In Purple Cauliflower (Brassica Oleracea L Var. Botrytis)

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Flavonoids such as anthocyanins possess significant health benefits to humans and play important physiological roles in plants. An interesting purple (pr) gene mutation in cauliflower confers an abnormal pattern of anthocyanin accumulation, giving rise to intense purple color in very young tissues, curds, and seeds. Through a combination of candidate analysis and fine-mapping, we have isolated the pr gene and verified it via functional complementation in wild type Arabidopsis and cauliflower. The pr gene was found to encode a R2R3-MYB transcription factor that exhibited tissue-specific expression, consistent with anthocyanin accumulation pattern in the mutant. Comparison of the DNA sequences between the WT and mutant alleles revealed that the mutation was caused by an autonomous DNA transposon insertion in the promoter region of the pr allele. The sequence rearrangement generated several cis-acting R response elements (RRE), which may provide new binding sites for regulatory proteins in conferring enhanced expression of the pr gene. GUS activity assay demonstrated that the promoter activity was correlated with the numbers of RREs in the promoter region. The upregulation of the pr gene activated the expression of a bHLH transcription factor (BoTT8), which in turn activated a subset of anthocyanin structural genes BoF3’H, BoDFR, and BoLDOX to produce the striking mutant phenotype. Yeast two-hybrid assays showed that the PURPLE (PR) protein directly interacted with BoTT8 and exhibited stronger binding activity than other BoMYBs isolated. The successful isolation of the pr gene has provided important insights for understanding the regulation of anthocyanin accumulation for breeding Brassica vegetables with enhanced health-promoting properties and visual appeal. Our discovery also has also demonstrated the involvement of transposable elements in gene regulation for phenotypic change in plants.

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