ITPK1 is an InsP(6)/ADP phosphotransferase that controls phosphate signaling in Arabidopsis

In plants, phosphate (P(i)) homeostasis is regulated by the interaction of PHR transcription factors with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates. In this study, we combined different methods to obtain a comprehensive picture of how inositol (pyro)phosphate metabolism is regulated by P(i) and dependent on the inositol phosphate kinase ITPK1. We found that inositol pyrophosphates are more responsive to P(i) than lower inositol phosphates, a response conserved across kingdoms. Using the capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) we could separate different InsP(7) isomers in Arabidopsis and rice, and identify 4/6-InsP(7) and a PP-InsP(4) isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP(7), 5-InsP(7), and InsP(8) increase several fold in shoots after P(i) resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP(6) compartmentalization. Notably, ITPK1 is critical for P(i)-dependent 5-InsP(7) and InsP(8) synthesis in planta and its activity regulates P(i) starvation responses in a PHR-dependent manner. Furthermore, we demonstrated that ITPK1-mediated conversion of InsP(6) to 5-InsP(7) requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP(7) under low ATP. Collectively, our study provides new insights into P(i)-dependent changes in nutritional and energetic states with the synthesis of regulatory inositol pyrophosphates.