Biochemical and Structural Insights into the Mechanism of DNA Recognition by Arabidopsis ETHYLENE INSENSITIVE3.

Jinghui Song,Xing Zhang,Jinying Peng,Chengqi Yi,Lulu Liu,Chenxu Zhu,Hongwei Guo,Xing Wen,Jin-Song Zhang

doi:10.1371/journal.pone.0137439

Jinghui Song, Xing Zhang + Show 7 more

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https://doi.org/10.1371/journal.pone.0137439

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Journal: PloS one	Publication Date: Sep 9, 2015
Citations: 26	License type: CC BY 4.0

Affiliation: Peking University, Center for Life Sciences

Abstract

Gaseous hormone ethylene regulates numerous stress responses and developmental adaptations in plants by controlling gene expression via transcription factors ETHYLENE INSENSITIVE3 (EIN3) and EIN3-Like1 (EIL1). However, our knowledge regarding to the accurate definition of DNA-binding domains (DBDs) within EIN3 and also the mechanism of specific DNA recognition by EIN3 is limited. Here, we identify EIN3 82–352 and 174–306 as the optimal and core DBDs, respectively. Results from systematic biochemical analyses reveal that both the number of EIN3-binding sites (EBSs) and the spacing length between two EBSs affect the binding affinity of EIN3; accordingly, a new DNA probe which has higher affinity with EIN3 than ERF1 is also designed. Furthermore, we show that palindromic repeat sequences in ERF1 promoter are not necessary for EIN3 binding. Finally, we provide, to our knowledge, the first crystal structure of EIN3 core DBD, which contains amino acid residues essential for DNA binding and signaling. Collectively, these data suggest the detailed mechanism of DNA recognition by EIN3 and provide an in-depth view at molecular level for the transcriptional regulation mediated by EIN3.

Highlights

The plant hormone ethylene regulates numerous growth and developmental processes, including inhibition of cell expansion, regulation of seed germination, promotion of leaf and flower senescence, induction of fruit ripening and abscission, and response to pathogens and stress [1,2,3,4,5]
The results showed that ETHYLENE INSENSITIVE3 (EIN3) 1–352 was capable of binding to the ETHYLENE RESPONSE FACTOR1 (ERF1) probe, consistent with previous experiments using EIN3 1–359 (Fig 1C and 1D; S3A Fig) [24]
Our results reveal that the length of spacing between EIN3-binding sites (EBSs) can significantly constrain EIN3’s affinity towards cognate DNA target and that a 10 bp spacing is optimal for EIN3 binding

Summary

Introduction

The plant hormone ethylene regulates numerous growth and developmental processes, including inhibition of cell expansion, regulation of seed germination, promotion of leaf and flower senescence, induction of fruit ripening and abscission, and response to pathogens and stress [1,2,3,4,5]. A largely putative ethylene signaling pathway from hormone perception at the endoplasmic reticulum membrane to transcriptional regulation in the nucleus was proposed based on genetic and molecular studies [6,7,8]. Ethylene is perceived by a five-member receptor family including ETR1, ETR2, ERS1, ERS2, and EIN4 [9,10,11,12,13,14]. In the absence of ethylene, the receptors constitutively activate a negative regulator CTR1, which acts upstream of the positive regulator EIN2 [15,16,17]. Downstream of EIN2, the signal is transmitted to two necessary transcription factors ETHYLENE INSENSITIVE3 (EIN3) and EIN3-Like (EIL1) by protecting.

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