Arabidopsis inositol polyphosphate kinase AtIpk2β is phosphorylated by CPK4 and positively modulates ABA signaling

Arabidopsis inositol polyphosphate kinase 2β (AtIpk2β) has multiple functions in plant development and in responding to abiotic stress. Although some related clues suggested a potential role of AtIpk2β in ABA signaling, the defined evidence was still lack. Here we discovered that a key ABA signaling component calcium-dependent protein kinase 4 (CPK4) can interact with AtIpk2β under ABA treated conditions through affinity purification and mass spectrometry detection. The interaction between CPK4 and AtIpk2β were further confirmed by yeast two hybrid and bimolecular fluorescence complementation assays. Expression of AtIpk2β also can be rapidly induced by ABA. In addition, we found that CPK4 can phosphorylate AtIpk2β in vitro and identified five novel phosphorylation sites of AtIpk2β by CPK4 kinase, including Tyr46, Ser48, Ser51, Thr128, Ser147. Overexpression of AtIpk2β in Arabidopsis was more sensitive to ABA in seed germination, primary root inhibition, ABA-responsive gene expression than wild type plants, whereas knockout mutant atipk2β exhibited no significant difference. The AtIpk2β variants containing Tyr46, Thr128, Ser147 mutated to Ala cannot complement the yeast mutant ipk2 growth in high temperature, suggesting that those three amino acid residues are critical for AtIpk2β. These findings provide insight into the modulation of ABA signaling by AtIpk2β.