Abstract
Here, we have investigated the possible effect of UV-B light on the folding/unfolding properties and stability of Arabidopsis thaliana MYB4 (AtMYB4) transcription factor in vitro by using biophysical approaches. Urea-induced equilibrium unfolding analyses have shown relatively higher stability of the wild-type recombinant AtMYB4 protein than the N-terminal deletion forms after UV-B exposure. However, as compared to wild-type form, AtMYB4Δ2 protein, lacking both the two N-terminal MYB domains, showed appreciable alteration in the secondary structure following UV-B exposure. UV-B irradiated AtMYB4Δ2 also displayed higher propensity of aggregation in light scattering experiments, indicating importance of the N-terminal modules in regulating the stability of AtMYB4 under UV-B stress. DNA binding assays have indicated specific binding activity of AtMYB4 to a putative MYB4 binding motif located about 212 bp upstream relative to transcription start site of AtMYB4 gene promoter, while relatively weak DNA binding activity was detected for another putative MYB4 motif located at -908 bp in AtMYB4 promoter. Gel shift and fluorescence anisotropy studies have shown increased binding affinity of UV-B exposed AtMYB4 to the promoter proximal MYB4 motif. ChIP assay has revealed binding of AtMYB4 to the promoter proximal (-212 position) MYB4 motif (ACCAAAC) in vivo. Docking experiments further revealed mechanistic detail of AtMYB4 interaction with the putative binding motifs. Overall, our results have indicated that the N-terminal 62–116 amino acid residues constituting the second MYB domain plays an important role in maintaining the stability of the C-terminal region and the overall stability of the protein, while a promoter proximal MYB-motif in AtMYB4 promoter may involve in the regulation of its own expression under UV-B light.
Highlights
The myeloblastosis viral oncogene homolog (MYB) domain proteins represent one of the largest families of transcription factors in plants, playing key roles in various developmental and stress-responsive processes
Previous studies in Arabidopsis have indicated role of MYB4 as negative regulator of cinnamate 4-hydroxylase (C4H) gene, which encodes for cinnamate 4-hydroxylase, the second enzyme in phenylpropanoid pathway involved in the formation of multiple UV-B protective secondary metabolites
The N-terminal MYB domains serve as the site for interactions with DNA, while the Cterminal region has been shown to be involved in mediating protein-protein interaction and transcriptional repression function with the possession of a EAR repression motif [12]
Summary
The MYB domain proteins represent one of the largest families of transcription factors in plants, playing key roles in various developmental and stress-responsive processes. In Arabidopsis, rice, maize, soybean and other plants, several members of MYB transcription factor family have been identified as key regulators of various cellular processes, including cell cycle and cell morphogenesis, biotic and abiotic stress responses [4, 5, 6]. Members of R2R3-MYB subfamily regulate multiple responses in plants, such as biotic and abiotic stresses [7, 8], hormone signaling, phenylpropanoid biosynthesis [9], determination of cell shape and regulation of differentiation [10]. More than over 100 R2R3- type MYB members have been reported in dicots and monocots [11]
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