GRF1 fine‐tunes the copper‐responsive transcription module of CPK3 ‐ bHLH107 by manipulating interaction and phosphor

SUMMARY

Previously, the crucial role of calcium‐dependent protein kinase 3 (CPK3) in Cu ²⁺ ‐triggered immunity (CuTI) has been established in Arabidopsis. CPK3 promotes the phosphorylation of basic helix–loop–helix 107 (bHLH107), thereby activating the expression of 1‐aminocyclopropanecarboxylic acid synthesis 8 ( ACS8 ) and downstream defense responses. However, the precise mechanisms underlying the fine‐tuning of CPK3‐mediated phosphorylation of bHLH107 remain unclear. To elucidate the mechanism underlying the inactivation of the CPK3‐bHLH107 module, we identified a 14‐3‐3 protein, GRF1 (GF14χ), among candidate bHLH107 interactors. Notably, the interaction between GRF1 and bHLH107 was weaker following Cu ²⁺ treatment than under control conditions. In grf1 mutant plants, the transcript level of ACS8 was slightly increased, concomitant with a modest increase in resistance to Pst DC3000. GRF1 interacts with both CPK3 and bHLH107 to maintain an ‘OFF’ state. Cu ²⁺ mediates the phosphorylation of CPK3, attenuating its interaction with GRF1 and promoting its dissociation from GRF1. Subsequently, CPK3 phosphorylates bHLH107 and GRF1, thereby switching the immune response to the ‘ON’ state. Furthermore, wild‐type GRF1, the quadruple phospho‐defective mutant GRF1 ^{S72/88/125/156A} and the quadruple phosphomimetic mutant GRF1  ^{S72/88/125/156D} directly interact with unphosphorylated bHLH107, but not with the double phosphomimetic mutant protein bHLH107 ^S62/72D . We also revealed that GRF1 attenuates CPK3 kinase activity. Collectively, our findings indicated that CuTI is precisely orchestrated through GRF1‐mediated modulation of the CPK3‐bHLH107 module in Arabidopsis.