DcMYB1 Acts as a Transcriptional Activator of the Carrot Phenylalanine Ammonia-lyase Gene (DcPAL1) in Response to Elicitor Treatment, UV-B Irradiation and the Dilution Effect

Abstract

Expression of a carrot phenylalanine ammonia-lyase (PAL) gene (DcPAL1) in suspension-cultured carrot cells is induced by treatment with a fungal elicitor, ultraviolet B (UV-B) irradiation, and by transferring and diluting cells with fresh medium (the dilution effect). Box-L-like sequences are known as important cis-elements of genes for enzymes involved in the phenylpropanoid biosynthetic pathway. Six sequences, box-L0 to box-L5, exist in the DcPAL1 gene promoter region. In this study, we isolated cDNA encoding the R2R3 type of MYB transcription factor, DcMYB1, using yeast one-hybrid screening with box-L1 or box-L5 as target elements. DcMYB1 bound to boxes-L0, L1, L3/4, and L5 sequences (ACC(A/T)(A/T)CC) in vitro, and in yeast cells and carrot protoplasts. Transient expression of DcMYB1 could up-regulate DcPAL1 promoter activity in carrot protoplasts. Results of the transient expression experiment for the deletion-mutated promoters of boxes-L0, L1, L3, and L5 suggest that these box-L-like sequences were required for the complete activation of the DcPAL1 promoter by DcMYB1. Expression of DcMYB1 transcripts was induced 0.5 h after elicitor treatment or UV-B irradiation, and 2 h after the dilution effect. Induction of DcPAL1 expression occurred 1 h after DcMYB1 expression in all stress treatments, and repression of DcMYB1 expression by RNA interference caused cessation of the up-regulation of DcPAL1 expression in the elicitor treatment or with UV-B irradiation. These results suggest that DcMYB1 is the main regulatory factor acting on box-L sequences in the DcPAL1 gene that respond to environmental cues.