Abstract
NADPH-dependent dihydroflavonol reductase (DFR) plays an important role in both anthocyanin biosynthesis and proanthocyanidin synthesis in plants. A specific and quantitative RT-PCR assay for transcription from the DFR promoter detected high expression with limited variability in rice tissues. A 440 bp minimal promoter region was identified by transfection of β-glucuronidase (GUS) reporter constructs into Jeokjinju variety. Alignment of the region with orthologous promoters revealed three conserved segments containing both bHLH (-386 to -381) and Myb (-368 to -362) binding sites. Transfection of β-glucuronidase constructs with targeted point mutations in the minimal promoter defined two sites important for promoter function to the transcription factor binding consensus sequences. The expression study showed that the bHLH binding domain (-386 to -381) is essential for DFR expression, and that a Myb binding domain (-368 to -362) is also required for full expression of the DFR gene, while the two bHLH binding domains (-104 to -99 and -27 to -22) nearest to the transcriptional start site are not necessary for DFR expression.
Highlights
The accumulation of anthocyanins or proanthocyanidins in plant organs generally requires the presence of at least two families of regulatory proteins: Myc-type proteins containing a basic helix-loop-helix domain, and R2R3 Myb proteins
Studies in maize report that a member of each of these families is necessary to activate anthocyanin biosynthesis in all tissues, and a direct physical interaction between Myc and Myb proteins is required for activation [3]
The petunia basic helix-loop-helix (bHLH) homolog, Jaf13, was unable to activate anthocyanin synthesis when introduced into maize bHLH mutants [7]. These results suggest that interactions between bHLH proteins and their partner Myb proteins are very specific, and the expression of these genes is restricted to certain tissues and developmental stages, which may vary by species [8]
Summary
The accumulation of anthocyanins or proanthocyanidins in plant organs generally requires the presence of at least two families of regulatory proteins: Myc-type proteins containing a basic helix-loop-helix (bHLH) domain, and R2R3 Myb proteins. Genes belonging to these two families are present in numerous copies in plant genomes and encode similar proteins with varying patterns of expression that are associated with the regulation of diverse genes and pathways in different tissues and at different stages of growth [1, 2]. Subsequent research has revealed significant variations on this general theme in different plant species.
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