CPK3-mediated cytosolic-nuclear translocation of bHLH107 recruits HY5 to regulate the Cu ²⁺ -triggered upregulation of ACS8

Copper serves as a micronutrient for plant growth and development and has been a key component of copper-based antimicrobial compounds (CBACs) for protection against plant diseases for more than 130 years. We previously revealed that nanomolar- to-micromolar concentrations of Cu ²⁺ elicit plant immune responses by activating the expression of the ethylene synthesis rate-limiting enzyme ACS8 , which is dependent on the promoter copper response element (CuRE) cis -element. Here, we genetically confirmed that Cu ²⁺ -induced resistance to Pseudomonas syringae pv. tomato ( Pst ) DC3000 is dependent on the CuRE in ACS8 . Upon screening for CuRE-binding transcription factors, bHLH107, which is required for Cu ²⁺ -triggered activation of ACS8 expression and resistance to Pst DC3000, was identified via DNA-pull-down and mass spectrometry (MS) assays. Calcium-dependent protein kinase 3 (CPK3) interacts with and phosphorylates bHLH107 at Ser62 and Ser72 to mediate bHLH07 translocation from the cytoplasm into the nucleus, where it interacts with Arabidopsis ELONGATED HYPOCOTYL5 (HY5). HY5 directly binds to the G-box and acts as a coactivator to promote bHLH107 binding to the CuRE cis -element and to increase transcription of ACS8 upon Cu ²⁺ treatment. Overall, we revealed a CPK3-bHLH107-HY5 module that regulates the Cu ²⁺ -responsive regulatory network upstream of ACS8 that is involved in the cytosolic-nuclear translocation of bHLH107.