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Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo-inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologs display phosphohydrolase activity with a high specificity for the 5-β-phosphate of inositol pyrophosphates. We further show that heterologous expression of Arabidopsis PFA-DSP1-5 rescues wortmannin-sensitivity and deranged inositol pyrophosphate homeostasis caused by the deficiency of the PFA-DSP-type inositol pyrophosphate phosphohydrolase Siw14 in yeast. Heterologous expression in Nicotiana benthamiana leaves provided evidence that Arabidopsis PFA-DSP1 also displays 5-β-phosphate specific inositol pyrophosphate phosphohydrolase activity in planta. Our findings lay the biochemical basis and provide the genetic tools to uncover the roles of inositol pyrophosphates in plant physiology and plant development.

In plants, phosphate (Pi) homeostasis is regulated by the interaction of Pi starvation response transcription factors (PHRs) with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates (PP-InsPs). Recently, ITPK1 was shown to generate the PP-InsP InsP7 from InsP6in vitro, but the importance of this activity in Pi signaling remained unknown. Here, we show that uncontrolled Pi accumulation in ITPK1-deficient plants is accompanied by impaired Pi-dependent InsP7 and InsP8 synthesis. Reciprocal grafting demonstrates that Pi starvation responses are mainly controlled by ITPK1 activity in shoots. Nuclear magnetic resonance assays and PAGE analyses with recombinant protein reveal that besides InsP6 phosphorylation, ITPK1 is also able to catalyze ATP synthesis using 5-InsP7 but not any other InsP7 isomer as a P-donor when ATP is low. Additionally, we show that the dynamic changes in InsP7 and InsP8 to cellular Pi are conserved from land plant species to human cells, suggesting that Pi-dependent PP-InsP synthesis is a common component of Pi signaling across kingdoms. Together, our study demonstrates how Pi-dependent changes in nutritional and energetic states modulate ITPK1 activities to fine-tune the synthesis of PP-InsPs.

The combinatorial phosphorylation ofmyo-inositolresults in the generation of different inositol phosphates (InsP), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6is also the precursor of higher phosphorylated forms called inositol pyrophosphates (PP-InsPs), such as InsP7and InsP8, which are characterized by a diphosphate moiety and are also ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in animals and yeast, their biosynthesis and functions in plants has remained largely elusive because plant genomes do not encode canonical InsP6kinases. Recently, it was shown that Arabidopsis ITPK1 catalyzes the phosphorylation of InsP6to the natural 5-InsP7isomerin vitro. Here, we demonstrate that Arabidopsis ITPK1 contributes to the synthesis of InsP7in planta. We further find a critical role of ITPK1 in auxin-related processes including primary root elongation, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity towards exogenously applied auxin. Notably, 5-InsP7binds to recombinant auxin receptor complex, consisting of the F-Box protein TIR1, ASK1 and the transcriptional repressor IAA7, with high affinity. Furthermore, a specific increase in 5-InsP7in a heterologous yeast expression system results in elevated interaction of the TIR1 homologs AFB1 and AFB2 with various AUX/IAA-type transcriptional repressors. We also identified a physical interaction between ITPK1 and TIR1, suggesting a dedicated channeling of an activating factor, such as 5-InsP7, to the auxin receptor complex. Our findings expand the mechanistic understanding of auxin perception and lay the biochemical and genetic basis to uncover physiological processes regulated by 5-InsP7.