WRKY33-PIF4 loop is required for the regulation of H₂O₂ homeostasis

The reactive oxygen species (ROS) are continuously produced and are essential for mediating the growth and development of plants. However too much accumulation of ROS can result in the oxidative damage to cells, especially under the adverse environmental conditions. Plants have evolved sophisticated strategies to regulate the homeostasis of H₂O₂. In this study, we generated transgenic Arabidopsis plants in the Ws ecotype (Ws) background in which WRKY33 is co-suppressed (csWRKY33/Ws). Compared with Ws, csWRKY33/Ws plants accumulate more H₂O₂. RNA-seq analysis indicated that in csWRKY33/Ws plants, expression of oxidative stress related genes such as ascorbate peroxidase 2 (APX2) is affected. Over-expression of APX2 can rescue the phenotype of csWRKY33/Ws, suggesting that the changes in the growth of csWRKY33/Ws is duo to the higher accumulation of H₂O₂. Analysis of the CHIP-seq data suggested that WRKY33 can directly regulate the expression of PIF4, vice versa. qPCR analysis also confirmed that the mutual regulation between WRKY33 and PIF4. Similar to that of csWRKY33/Ws, and the accumulation of H₂O₂ in pif4 also increased. Taken together, our results reveal a WRKY33-PIF4 regulatory loop that appears to play an important role in regulating the growth and development of seedlings by mediating H₂O₂ homeostasis.