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.

在植物中，磷酸盐 (P _i ) 稳态受 PHR 转录因子与独立 SPX 蛋白相互作用的调节，SPX 蛋白充当肌醇焦磷酸盐的传感器。在这项研究中，我们结合了不同的方法来全面了解肌醇（焦）磷酸盐代谢如何受 P _i调节并依赖于肌醇磷酸盐激酶 ITPK1。_{我们发现肌醇焦磷酸比低肌醇磷酸对 P i}的反应更强，这是一种跨界保守的反应。使用毛细管电泳电喷雾电离质谱 (CE-ESI-MS)，我们可以分离拟南芥和水稻中不同的 InsP ₇异构体，并鉴定 4/6-InsP ₇以及植物中迄今未见报道的PP-InsP _{4异构体。}我们发现肌醇焦磷酸盐 1/3-InsP ₇、5-InsP ₇和 InsP _{8在 P i}补给后在芽中增加数倍，并且肌醇焦磷酸盐的组织特异性积累依赖于 ITPK1 活性和 MRP5 依赖性 InsP ₆区室化. 值得注意的是，ITPK1 对于植物中P _i依赖性 5-InsP ₇和 InsP ₈合成至关重要，其活性以 PHR 依赖性方式调节 P _{i饥饿反应。}此外，我们证明了 ITPK1 介导的 InsP _6转化到 5-InsP ₇需要高 ATP 浓度，而拟南芥ITPK1 具有 ADP 磷酸转移酶活性，可在低 ATP 下特异性去磷酸化 5-InsP ₇。总的来说，我们的研究提供了对 P _i依赖性营养和能量状态变化的新见解，以及调节性肌醇焦磷酸盐的合成。