ITPK1 is an InsP₆/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 InsP₇ isomers in Arabidopsis and rice, and identify 4/6-InsP₇ and a PP-InsP₄ isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP₇, 5-InsP₇, and InsP₈ increase several fold in shoots after P_i resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP₆ compartmentalization. Notably, ITPK1 is critical for P_i-dependent 5-InsP₇ and InsP₈ synthesis in planta and its activity regulates P_i starvation responses in a PHR-dependent manner. Furthermore, we demonstrated that ITPK1-mediated conversion of InsP₆ to 5-InsP₇ requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP₇ 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.