MYB46 and MYB83 bind to the SMRE sites and directly activate a suite of transcription factors and secondary wall biosynthetic genes.

MYB46 and MYB83 are two functionally redundant Arabidopsis thaliana MYB transcription factors that act as master switches regulating secondary wall biosynthesis. Here, we report the identification of the transcriptional responsive elements and global analysis of the direct targets of MYB46 and MYB83. Using the estrogen-inducible direct activation system, we found that a number of previously identified MYB46 downstream transcription factors, including MYB43, MYB52, MYB54, MYB58, MYB63 and KNAT7, are direct targets of MYB46. Promoter deletion coupled with transactivation analysis of the MYB63 promoter led to the identification of a 7 bp sequence that is sufficient to be responsive to MYB46 activation, and therefore this sequence is designated as the secondary wall MYB-responsive element (SMRE). Further single nucleotide mutation together with electrophoretic mobility shift assay mapped the SMRE consensus sequence as ACC(A/T)A(A/C)(T/C). Genome-wide analysis of direct targets of MYB46 demonstrated that it directly regulates the expression of not only a number of downstream transcription factors, but also a suite of secondary wall biosynthetic genes, some of which are also directly activated by secondary wall NAC (SWN) master switches or by MYB46 direct targets. Furthermore, MYB83 was found to bind to the same SMRE consensus sequence and activate the same set of direct targets as MYB46. Our study has revealed that the transcription program regulating secondary wall biosynthesis involves a multileveled feed-forward loop regulatory structure in which MYB46/MYB83 together with their regulators SWNs and their direct targets regulate an array of downstream genes thereby activating the secondary wall biosynthetic program.