DREB2 Family Transcription Factors Promote the Expression of Phytocystatin1 in Arabidopsis thaliana During Seed Germinat
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RESEARCH ARTICLE
DREB2 Family Transcription Factors Promote the Expression of Phytocystatin1 in Arabidopsis thaliana During Seed Germination Taeyoon Kim1 · Trang Thi Nguyen1 · Juwan Baek1 · Young Hun Song2 · Jong Chan Hong1 · Chae Oh Lim1 Received: 20 July 2020 / Revised: 24 August 2020 / Accepted: 25 August 2020 © Korean Society of Plant Biologist 2020
Abstract Phytocystatins (PhyCYSs) are plant-specific proteinaceous inhibitors of cysteine proteases (CPs) that regulate protein turnover and defense responses. Here, we characterized a member of the PhyCYS gene family in Arabidopsis thaliana, AtCYS1, during seed germination. AtCYS1 transcript level and AtCYS1 promoter-driven β-glucuronidase (GUS) activity gradually increased during germination, as measured by quantitative reverse transcription PCR (qRT-PCR) and GUS staining assays, respectively. The level and activity of the AtCYS1 protein were up-regulated during germination, as shown by western blot and CP inhibitory activity analyses, respectively. In addition, a canonical dehydration-responsive element (DRE) was identified in the AtCYS1 promoter. Electrophoretic mobility shift assays (EMSAs) revealed that the DRE-binding factor 2C (DREB2C) binds directly to the AtCYS1 promoter. Transient transactivation assays using Arabidopsis leaf protoplasts showed that DREB2A and DREB2C act as transcriptional activators of AtCYS1. In DREB2C overexpression lines, AtCYS1 protein levels were elevated, while the endogenous CP activity was reduced. However, AtCYS1 overexpression lines (35S:AtCYS1) and the cys1-1 null mutant, characterized by a quantitative imbalance in the AtCYS1 protein level, showed delayed germination under normal growth conditions. These results suggest that DREB2 proteins act as transcriptional activators of the AtCYS1 gene, and an imbalance in the level of AtCYS1 inhibits seed germination. Keywords Phytocystatin · Dehydration-responsive element-binding factor · Seed germination · Transgenic plants Abbreviations ABA Abscisic acid AtCYS Arabidopsis Phytocystatin CP Cysteine protease DREB2 Dehydration-responsive element-binding factor 2 EMSA Electrophoretic mobility shift assay GA Gibberellin GST Glutathione S-transferase GUS β-Glucuronidase PhyCYS Phytocystatin Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12374-020-09278-y) contains supplementary material, which is available to authorized users. * Chae Oh Lim [email protected] 1
Division of Life Science, Systems and Synthetic Agrobiotech Center, PMBBRC, Gyeongsang National University, Jinju 52828, Korea
Department of Applied Biological Chemistry, Seoul National University, Seoul 08826, Korea
2
qRT-PCR Quantitative reverse transcription PCR X-Gluc 5-Bromo-4-chloro-3-indolyl-ß-D-glucuronide
Introduction Phytocystatins (PhyCYSs) are a family of plant-specific proteinase inhibitors implicated in the turnover of storage proteins and response to environmental stresses. PhyCYSs inhibit the activity of legumain-like (C13 family) and papain-
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