Osmoregulation is important for plant growth, development and response to environmental changes. SNF1-related protein kinase 2s (SnRK2s) are quickly activated by osmotic stress and are central components in osmotic stress and abscisic acid (ABA) signaling pathways; however, the upstream components required for SnRK2 activation and early osmotic stress signaling are still unknown. Here, we report a critical role for B2, B3 and B4 subfamilies of Raflike kinases (RAFs) in early osmotic stress as well as ABA signaling in Arabidopsis thaliana. B2, B3 and B4 RAFs are quickly activated by osmotic stress and are required for phosphorylation and activation of SnRK2s. Analyses of high-order mutants of RAFs reveal critical roles of the RAFs in osmotic stress tolerance and ABA responses as well as in growth and development. Our findings uncover a kinase cascade mediating osmoregulation in higher plants.

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 InsP 8 as a signaling molecule that regulates Pi homeostasis in Arabidopsis. Polyacrylamide gel electrophoresis profiling of InsPs revealed that InsP 8 level positively correlates with cellular Pi concentration. We demonstrated that the homologs of diphosphoinositol pentakisphosphate kinase (PPIP5K), VIH1 and VIH2, function redundantly to synthesize InsP 8 , 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 InsP 8 . Furthermore, we showed that InsP 8 could directly bind to the SPX domain of SPX1 and is essential for the interaction between SPX1 and PHR1. Collectively, our study suggests that InsP 8 is the intracellular Pi signaling molecule serving as the ligand of SPX1 for controlling Pi homeostasis in plants.

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