DREB2 Family Transcription Factors Promote the Expression ofPhytocystatin1inArabidopsis thalianaDuring Seed Germination

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 thePhyCYSgene family inArabidopsis thaliana,AtCYS1, during seed germination.AtCYS1transcript level andAtCYS1promoter-driven beta-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 theAtCYS1promoter. Electrophoretic mobility shift assays (EMSAs) revealed that the DRE-binding factor 2C (DREB2C) binds directly to theAtCYS1promoter. Transient transactivation assays usingArabidopsisleaf protoplasts showed that DREB2A and DREB2C act as transcriptional activators ofAtCYS1. InDREB2Coverexpression lines, AtCYS1 protein levels were elevated, while the endogenous CP activity was reduced. However,AtCYS1overexpression lines (35S:AtCYS1) and thecys1-1null 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 theAtCYS1gene, and an imbalance in the level of AtCYS1 inhibits seed germination.