Arabidopsis Type-III Phosphatidylinositol 4-Kinases β1 and β2 are Upstream of the Phospholipase C Pathway Triggered by Cold Exposure

Phosphatidylinositol-4-phosphate (PtdIns4P) is the most abundant phosphoinositide in plants and the precursor of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P-2]. This lipid is the substrate of phosphoinositide-dependent phospholipase C (PI-PLC) that produces diacylglycerol (DAG) which can be phosphorylated to phosphatidic acid (PtdOH). In plants, it has been suggested that PtdIns4P may also be a direct substrate of PI-PLC. Whether PtdIns4P is the precursor of PtdIns(4,5)P-2 or a substrate of PI-PLC, its production by phosphatidylinositol-4-kinases (PI4Ks) is the first step in generating the phosphoinositides hydrolyzed by PI-PLC. PI4Ks can be divided into type-II and type-III. In plants, the identity of the PI4K upstream of PI-PLC is unknown. In Arabidopsis, cold triggers PI-PLC activation, resulting in PtdOH production which is paralleled by decreases in PtdIns4P and PtdIns(4,5)P-2. In suspension cells, both the PtdIns4P decrease and the PtdOH increase in response to cold were impaired by 30 mu M wortmannin, a type-III PI4K inhibitor. Type-III PI4Ks include AtPI4KIII alpha 1, beta 1 and beta 2 isoforms. In this work we show that PtdOH resulting from the PI-PLC pathway is significantly lowered in a pi4kIII beta 1 beta 2 double mutant exposed to cold stress. Such a decrease was not detected in single pi4kIII beta 1 and pi4kIII beta 2 mutants, indicating that AtPI4KIII beta 1 and AtPI4KIII beta 2 can both act upstream of the PI-PLC. Although several short-term to long-term responses to cold were unchanged in pi4kIII beta 1 beta 2, cold induction of several genes was impaired in the double mutant and its germination was hypersensitive to chilling. We also provide evidence that de novo synthesis of PtdIns4P by PI4Ks occurs in parallel to PI-PLC activation.