TY  - GEN
T1  - Quantitative insight into the combinatorial interactions within MYB-BHLH-WD complex in Arabidopsis thaliana
AU  - Umeyama, Mikiya
AU  - Morohashi, Kengo
DO  - 10.17912/micropub.biology.000293
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000293/
AB  - Plants protect themselves from different environmental stresses, and the responses are governed by multiple transcription factors (TFs) via complex forms. Plants evolutionally conserve a heteromeric complex, comprised of MYB, basic helix-loop-helix (bHLH), and WD-repeat proteins (MBW) (Albert et al. 2014; Xu et al. 2015; Lloyd et al. 2017). The combination of MYB and bHLH in MBW is responsible for a wide range of biological events, while the variation of WD-repeat protein is low. In Arabidopsis thaliana, GL3, bHLH, forms a complex with GL1, MYB, and the GL3-GL1 complex generates a trichome in epidermal cells, which is believed to protect the plant body from insects. GL3 also forms a complex with PAP1, another MYB, and the GL3-PAP1 complex regulates the synthesis of anthocyanin which protects against UV and strong light (Fig 1A). Because MBW consists of various MYBs, a competition between MYB to bHLH impacts the activity and the regulation of gene expression (Zhang and Hülskamp, 2019). Thus, the affinity between MYB and bHLH proteins are essential for understanding the mechanism underlying the combinatorial regulation of MBW complex (Lloyd et al. 2017). However, little is known about the quantitative evaluation of affinities between MYBs and bHLH. To acquire affinity information of MBW in a quantitative manner, we focused on GL3, GL1, and PAP1. Because these proteins are expressed in an epidermal cell at early leaf development, GL3, GL1, and PAP1 would integrally impact the trichome formation and anthocyanin biosynthesis via competitive interactions. We quantified the dissociation constants of these three proteins using a quartz crystal microbalance (QCM). QCM is a biosensor to monitor in real time an interaction that occurred on a sensor chip by a frequency of a quartz crystal resonator (Fig 1B) (Fee, 2013; Johannsmann, 2015). We purified the recombinant proteins of GST-tagged GL3, His-tagged GL1, and GST-tagged PAP1, which were expressed in bacteria and confirmed using SDS electrophoresis (Fig 1C). Purified GL1 and GL3 were separately immobilized on the surface of two sensor chips. The immobilization was observed as a difference of frequency. Following the confirmation of stable immobilization, we proportionally added the other proteins, GL3 or PAP1, in a cuvette, because the difference of frequency represents the amount of correlated proteins. Dissociation constants were measured by applying nonlinear fit on Langmuir formula (Fig 1D), resulting in the dissociation constants (KD) of GL3 and GL1, GL3 and PAP1, and PAP1 and GL1, which were 9.53 ± 7.52 µM, 0.29 ± 0.20 µM, and 0.049 ± 0.04 µM, respectively (average of three independent experiments) (Fig 1E). Previously, the KD values between EGL3, GL3, and PAP2 were described using a different system from ours, wherein the range of KD between bHLH and MYBs was 3.5–7.5 µM (Nemie-Feyissa et al. 2015). Since we can not rule out that the GL3-PAP1 interaction is due to GST dimerization, our conclusion should be considered as preliminary. Nevertheless, these KD values are in a similar range with the GL3-GL1 interaction in our study. It is notable that our result shows that the affinity between GL1 and PAP1 was 100-fold higher than that between GL3 and GL1 (Fig. 1F). These results propose that the competitive regulation in the MBW complex should be considered not only between bHLH and MYB but also between MYBs.
PY  - 2020
JO  - microPublication Biology
ER  -