Characterization of MYB proteins acting as transcriptional regulatory factors for carrot phenylalanine ammonia-lyase gene (DcPAL3)

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In suspension-cultured carrot cells, a phenylalanine ammonia-lyase gene, DcPAL3, plays an important role in regulation of anthocyanin synthesis. In the DcPAL3 promoter region, a putative cis-element, box-L, which is committed to the upregulation of promoter activity, has been identified. Here, we isolated DcMYB5 cDNA using yeast one-hybrid screening with box-L as a bait from a cDNA library prepared from cells of a variant cultured cell line constitutively synthesizing anthocyanin. Although expression of DcMYB2, 3, and 4 was observed, all of which were previously isolated by plaque hybridization from a subtracted cDNA library of anthocyanin-synthesizing cells of a normal cultured cell line cultured in medium lacking 2,4-dichlorophenoxyacetic acid, DcMYB5 expression was not observed in cells of the normal cultured cell line; high expression of DcMYB5 was observed in cells of the variant cultured cell line. Although not only DcMYB5 but also DcMYB2, 3, and 4 could bind to the box-L sequence in yeast, DcMYB3 and 5 showed strong transcriptional activation activity for DcPAL3 promoter in carrot protoplasts. These results suggest that DcMYB3 and 5 might play an important role in the upregulation of DcPAL3 promoter activity in the different regulatory paths between the normal and variant cultured cell lines.