DNA Binding Mechanism of Arabidopsis ERF96 to GCC boxes

Ethylene response factors (ERFs) belong to a large family of plant-specific transcription factors which participate in biotic and abiotic stresses in plants. ERF96 has been classified as group IX and contains one conserved DNA binding domain named AP2/ERF domain, which specifically binds to GCC box (AGCCGCC). Previous study demonstrated that ERF96 involved in plant defense response. Since functional analysis for ERF proteins is still rare, we aim to study the molecular mechanism of ERF96 protein binding to GCC box or other non-GCC box sequences. Size-exclusive chromatography and dynamic light scattering identified that full-length recombinant ERF96 is monomer and mono-disperse in the solution. Fluorescein-based electrophoretic mobility shift assay (fEMSA) showed the binding ability of ERF96 to different lengths of GCC box, and the results indicated that flanking region of GCC box influence binding affinity of R16A, W23A, R31A and W41A. Site-directed mutagenesis of ERF96 with R16A, R19A, R21A, W23A, R31A, R39A, and W41A indicated that there is no significant change of binding ability in ERF96 single mutation. However, the binding of ERF96 was abolished in double mutation with R19AR21A and R31AR39A. This results showed that Arg19, Arg21, Arg31, and Arg39 are the important residues for DNA binding. Furthermore, we examined binding ability of ERF96 to non-GCC box sequence, such as P box, CS1 box, and DRE box. The results indicated that ERF96 can bind to P box, CS1 box and DRE box. Taken together, recombinant ERF96 showed the monomer form in solution and Arg19, Arg21, Arg31, and Arg39 of ERF96 are the key residues to bind to GCC box DNA. In addition, ERF96 not only bound to GCC box, but also could bind to P box, CS1 box and DRE box. We proposed that ERFs transcription factor might involve in the transcription activation via non-GCC box binding ability.