Arg156 in the AP2-Domain Exhibits the Highest Binding Activity among the 20 Individuals to the GCC Box in BnaERF-B3-hy15, a Mutant ERF Transcription Factor from Brassica napus.

Jing Zhuang,Meng-Yao Li,Yan-Jun Liu,Ai-Sheng Xiong,Bei Wu

doi:10.3389/fpls.2016.01603

Abstract

To develop mutants of the ERF factor with more binding activities to the GCC box, we performed in vitro directed evolution by using DNA shuffling and screened mutants through yeast one-hybrid assay. Here, a series of mutants were obtained and used to reveal key amino acids that induce changes in the DNA binding activity of the BnaERF-B3 protein. With the BnaERF-B3-hy15 as the template, we produced 12 mutants which host individual mutation of potential key residues. We found that amino acid 156 is the key site, and the other 18 mutants host the 18 corresponding individual amino acid residues at site 156. Among the 20 individuals comprising WT (Gly156), Mu3 (Arg156), and 18 mutants with other 18 amino acid residues, Arg156 in the AP2-domain is the amino acid residue with the highest binding activity to the GCC box. The structure of the α-helix in the AP2-domain affects the binding activity. Other residues within AP2-domain modulated binding activity of ERF protein, suggesting that these positions are important for binding activity. Comparison of the mutant and wild-type transcription factors revealed the relationship of protein function and sequence modification. Our result provides a potential useful resource for understanding the trans-activation of ERF proteins.

Highlights

Plants are affected by various abiotic stresses during their life cycle
Our results revealed that Arg156 in the AP2-domain is the amino acid residue with the highest binding activity to the GCC box in the mutant and wild type BnaERF-B3
We cloned a member of the ERF family gene, named BnaERF-B3-hy15; the DNA shuffling strategy was used to shuffle the BnaERF-B3-hy15 gene, and three mutants (Mu1, Mu2, and Mu3) of this gene were obtained from variant mutants

Summary

Introduction

Plants are affected by various abiotic stresses during their life cycle. Drought, and extreme temperature are major factors limiting plant growth and development, and considerably affect the efficiency of agricultural production. When plants are exposed to adverse stress, a complex signal transduction network will mediate resistance to the stress (Seki et al, 2001; Shinozaki et al, 2003; Gutterson and Reuber, 2004). The AP2/ERF transcription factor family is one of the families involved in plant development, signal transduction, and stress response; this family can be further divided into four subfamilies: AP2, RAV, DREB, and ERF (Sakuma et al, 2002; Zhuang et al, 2008).

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