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. 2018 Jan 17;8:909. doi: 10.1038/s41598-018-19206-3

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© The Author(s) 2018

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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HbMYC2/3/4 bind the promoter and regulate the expression of the HbPIP2;1-P gene. (a). The pGADT7-HbMYC2, pGADT7-HbMYC3, pGADT7-HbMYC4 plasmids were transformed into Y187 Gold strains containing pHis2.1-PIP-P, after which the strains were cultured in SD/-His/-Leu/-Trp + 3-AT media at 30 °C for 3–5 days. (b) Schematic diagram of the reporter and effector constructs used in the luciferase assay. The firefly luciferase (LUC) reporter was driven by the HbPIP2;1 promoter, and HbMYC2/3/4 were driven by the CaMV 35 S promoter in each of the effector constructs. c. Luciferase assay of the enhancement of the HbPIP2;1 promoter activity by the overexpression of HbMYC2, HbMYC3, and HbMYC4 in protoplasts. The pPIP2:LUC reporter and respective 35 S:MYC2/3/4 effector constructs as well as empty vector controls were co-transformed into Arabidopsis protoplasts. Luciferase activities were quantified using a dual-luciferase assay kit (Promega, USA) and detected by using a GloMax® 96 microplate luminometer (Promega, USA). The values are the means ± SDs from the results of three replicates.