The maintenance of cellular phosphate (Pi) homeostasis is of great importance in living organisms. The SPX domain-containing protein 1 (SPX1) proteins from both Arabidopsis and rice have been proposed to act as sensors of Pi status. The molecular signal indicating the cellular Pi status and regulating Pi homeostasis in plants, however, remains to be identified, as Pi itself does not bind to the SPX domain. Here, we report the identification of the inositol pyrophosphate InsP₈ as a signaling molecule that regulates Pi homeostasis in Arabidopsis. Polyacrylamide gel electrophoresis profiling of InsPs revealed that InsP₈ level positively correlates with cellular Pi concentration. We demonstrated that the homologs of diphosphoinositol pentakisphosphate kinase (PPIP5K), VIH1 and VIH2, function redundantly to synthesize InsP₈, and that the vih1 vih2 double mutant overaccumulates Pi. SPX1 directly interacts with PHR1, the central regulator of Pi starvation responses, to inhibit its function under Pi-replete conditions. However, this interaction is compromised in the vih1 vih2 double mutant, resulting in the constitutive induction of Pi starvation-induced genes, indicating that plant cells cannot sense cellular Pi status without InsP₈. Furthermore, we showed that InsP₈ could directly bind to the SPX domain of SPX1 and is essential for the interaction between SPX1 and PHR1. Collectively, our study suggests that InsP₈ is the intracellular Pi signaling molecule serving as the ligand of SPX1 for controlling Pi homeostasis in plants.

在活生物体中，维持细胞内磷酸盐（Pi）的动态平衡非常重要。已经提出了来自拟南芥和水稻的含SPX域的蛋白1（SPX1）蛋白可作为Pi状态的传感器。但是，由于Pi本身不与SPX域结合，因此指示细胞中Pi的状态和调节植物中Pi稳态的分子信号仍有待确定。在这里，我们报告肌醇焦磷酸盐InsP ₈作为调节拟南芥中Pi动态平衡的信号传导分子的鉴定。InsPs的聚丙烯酰胺凝胶电泳分析显示，InsP ₈水平与细胞Pi浓度成正相关。我们证明了二磷酸肌醇五磷酸激酶（PPIP5K），VIH1和VIH2的同源物多余地合成了InsP ₈，并且vih1 vih2双突变体过度积累了Pi。SPX1与Pi饥饿反应的中央调节剂PHR1直接相互作用，以在Pi充足的条件下抑制其功能。但是，这种相互作用在vih1 vih2双突变体中受到了损害，导致了Pi饥饿诱导基因的组成型诱导，表明如果没有InsP ₈，植物细胞将无法感知细胞Pi的状态。此外，我们证明了InsP ₈可以直接绑定到SPX1的SPX域，并且对于SPX1和PHR1之间的相互作用必不可少。总体而言，我们的研究表明InsP ₈是细胞内Pi信号分子，充当SPX1的配体，用于控制植物体内的Pi稳态。