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.

植物胱抑素（PhyCYSs）是半胱氨酸蛋白酶（CPs）的植物特异性蛋白质抑制剂，可调节蛋白质更新和防御反应。在这里，我们表征的成员PhyCYS基因家族在拟南芥，AtCYS1种子发芽过程中，。AtCYS1成绩单水平和AtCYS1分别通过定量逆转录PCR（qRT-PCR）和GUS染色测定法测量，启动子驱动的β-葡萄糖醛酸苷酶（GUS）活性在发芽过程中逐渐增加。分别通过蛋白质印迹和CP抑制活性分析表明，发芽过程中AtCYS1蛋白的水平和活性上调。此外，在AtCYS1启动子中鉴定出一个典型的脱水反应元件（DRE）。电泳迁移率变动分析（EMSA）显示，DRE结合因子2C（DREB2C）直接与AtCYS1启动子结合。使用短暂激活测定拟南芥叶原生质体表明DREB2A和DREB2C充当的转录激活AtCYS1。在DREB2C过表达系，AtCYS1蛋白水平升高，而内源CP活性降低。但是，AtCYS1过表达株系（35S：AtCYS1）和cys1-1 null突变体（其特征在于AtCYS1蛋白水平的定量失衡）在正常生长条件下显示出延迟的发芽。这些结果表明，DREB2蛋白充当AtCYS1基因的转录激活因子，而AtCYS1水平的不平衡会抑制种子发芽。