DREB2 Family Transcription Factors Promote the Expression of Phytocystatin1 in Arabidopsis thaliana During Seed Germination
2021
Kim, T.Y. | Nguyen, T.T. | Baek, J.W. | Song, Y.H. | Hong, J.C. | Lim, C.O.
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.
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