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. 2017 Jun 9;7:3193. doi: 10.1038/s41598-017-03506-1

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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DCMU-inducible AKIN10 represses EIN3-dependent gene expression. (a,b) DIN6 promoter (DIN6p) and EBS element (EBSp) activities were measured with expression of AKIN10 (a) or EIN3 (b) as well as in the presence of DCMU. (c,d) DIN6p (c) and EBSp (d) activities were measured in combination of active or inactive forms of AKIN10 with and without EIN3. (e) EIN3 protein level was measured as EIN3-fLUC activity with gradient expression of AKIN10 in ein3-1 protoplasts. The fLUC activity was used as a cellular response control. (f) EIN3 protein level was measured as EIN3-fLUC activity in the presence of DCMU in ein3-1 protoplasts. The rLUC activity served as control. All protoplast experiments were repeated three times with consistent results. The means of at least three replicates are shown with standard-error bars. ***P < 0.001, **P < 0.01, and *P < 0.05. (g,h) Protein levels of EIN3-GFP (g) and SAP3-GFP (h) were determined in Arabidopsis protoplasts in the absence and presence of DCMU by protein blot analysis using anti-GFP antibody. All experiments were repeated with consistent results.