Conservation of heat stress acclimation by the IPK2-type kinases that control the synthesis of the inositol pyrophosphate 4/6-InsP7 in land plants

Inositol pyrophosphates (PP-InsPs) are soluble cellular messengers that integrate environmental cues to induce adaptive responses in eukaryotes. In plants, the biological functions of various PP-InsP species are poorly understood, largely due to the absence of canonical enzymes found in other eukaryotes. The recent identification of a new PP-InsP isomer with yet unknown enantiomeric identity, 4/6-InsP7 in the eudicot Arabidopsis thaliana, further highlights the intricate PP-InsP signalling network employed by plants. Yet, the abundance of 4/6-InsP7 in land plants, the enzyme(s) responsible for its synthesis, and the physiological functions of this species are all currently unknown. In this study, we show that 4/6-InsP7 is ubiquitous in the studied land plants. Our findings demonstrate that the Arabidopsis inositol polyphosphate multikinase (IPMK) homologs, AtIPK2α and AtIPK2β phosphorylates InsP6 to generate 4/6-InsP7 as the predominant PP-InsP species in vitro. Consistent with this, AtIPK2α and AtIPK2β act redundantly to control 4/6-InsP7 production in planta. Notably, activity of these IPK2 proteins is critical for heat stress acclimation in Arabidopsis. Our parallel investigations using the liverwort Marchantia polymorpha suggest that the PP-InsP synthase activity of IPK2 and role of IPK2 in regulating the heat stress response are conserved in land plants. Furthermore, we show that the transcription activity of heat shock factor (HSF) is regulated by IPK2 proteins, providing a mechanistic framework of IPK2-controlled heat stress tolerance in land plants. Collectively, our study indicates that IPK2-type kinases have played a critical role in transducing environmental cues for biological processes during land plant evolution.Author summary: Inositol pyrophosphates (PP-InsPs) are eukaryote-specific cellular messengers that control a plethora of critical physiological processes, ranging from cellular metabolism to cellular energetics, and nutrient sensing. The identification of a new inositol pyrophosphate species, 4/6-InsP7, suggests the presence of a more diverse PP-InsP signalling network in plants. To date, the molecular basis of 4/6-InsP7 production and its physiological function in plants remained completely elusive. We report the identification of a non-archetypal function of inositol polyphosphate multikinase (IPMK/IPK2) that catalyzes the synthesis of 4/6-InsP7, and the kinase activity is critical for controlling heat stress acclimation. Furthermore, we show that the role of IPK2 in generating 4/6-InsP7 and regulating heat stress response is conserved in the studied land plants.