The WRKY46–MYC2 module plays a critical role in E-2-hexenal-induced anti-herbivore responses by promoting flavonoid accumulation

Abstract

Volatile organic compounds (VOCs) play key roles in plant–plant communication, especially in response to pest attack. E-2-hexenal is an important component of VOCs, but it is unclear whether it can induce endogenous plant resistance to insects. Here, we show that E-2-hexenal activates early signaling events in Arabidopsis (Arabidopsis thaliana) mesophyll cells, including an H2O2 burst at the plasma membrane, the directed flow of calcium ions, and an increase in cytosolic calcium concentration. Treatment of wild-type Arabidopsis plants with E-2-hexenal increases their resistance when challenged with the diamondback moth Plutella xylostella L., and this phenomenon is largely lost in the wrky46 mutant. Mechanistically, E-2-hexenal induces the expression of WRKY46 and MYC2, and the physical interaction of their encoded proteins was verified by yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays. The WRKY46–MYC2 complex directly binds to the promoter of RBOHD to promote its expression, as demonstrated by luciferase reporter, yeast one-hybrid, chromatin immunoprecipitation, and electrophoretic mobility shift assays. This module also positively regulates the expression of E-2-hexenal-induced naringenin biosynthesis genes (TT4 and CHIL) and the accumulation of total flavonoids, thereby modulating plant tolerance to insects. Together, our results highlight an important role for the WRKY46–MYC2 module in the E-2-hexenal-induced defense response of Arabidopsis, providing new insights into the mechanisms by which VOCs trigger plant defense responses.