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 InsP 7 isomers in Arabidopsis and rice, and identify 4/6-InsP 7 and a PP-InsP 4 isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP 7 , 5-InsP 7 , and InsP 8 increase several fold in shoots after P i resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP 6 compartmentalization. Notably, ITPK1 is critical for P i -dependent 5-InsP 7 and InsP 8 synthesis in planta and its activity regulates P i starvation responses in a PHRdependent manner. Furthermore, we demonstrated that ITPK1-mediated conversion of InsP 6 to 5-InsP 7 requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP 7 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.

29The combinatorial phosphorylation of myo-inositol results in the generation of different inositol 30 phosphates (InsP), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6 31 is also the precursor of higher phosphorylated forms called inositol pyrophosphates (PP-InsPs), 32 such as InsP7 and InsP8, which are characterized by a diphosphate moiety and are also 33 ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in 34 animals and yeast, their biosynthesis and functions in plants has remained largely elusive 35 because plant genomes do not encode canonical InsP6 kinases. Recently, it was shown that 36 Arabidopsis ITPK1 catalyzes the phosphorylation of InsP6 to the natural 5-InsP7 isomer in vitro. 37 Here, we demonstrate that Arabidopsis ITPK1 contributes to the synthesis of InsP7 in planta. We 38 further find a critical role of ITPK1 in auxin-related processes including primary root elongation, 39 leaf venation, thermomorphogenic and gravitropic responses, and sensitivity towards 40 exogenously applied auxin. Notably, 5-InsP7 binds to recombinant auxin receptor complex, 41 consisting of the F-Box protein TIR1, ASK1 and the transcriptional repressor IAA7, with high 42 affinity. Furthermore, a specific increase in 5-InsP7 in a heterologous yeast expression system 43 results in elevated interaction of the TIR1 homologs AFB1 and AFB2 with various AUX/IAA-44 type transcriptional repressors. We also identified a physical interaction between ITPK1 and 45 TIR1, suggesting a dedicated channeling of an activating factor, such as 5-InsP7, to the auxin 46 receptor complex. Our findings expand the mechanistic understanding of auxin perception and 47 lay the biochemical and genetic basis to uncover physiological processes regulated by 5-InsP7. 48 49 degradation of Aux/IAA transcriptional repressors to activate AUXIN RESPONSE FACTOR 59 (ARF) transcription factors (Gray et al., 2001; Dharmasiri et al., 2005; Kepinski and Leyser, 60 2005; Prigge et al., 2020). Unexpectedly, in a crystal structure of the auxin receptor complex 61 consisting of insect-purified ASK1-TIR1 and an IAA7 degron peptide, insect-derived InsP6 62 occupied the core of the leucine-rich-repeat (LRR) domain of TIR1 (Tan et al., 2007). While the 63 functional importance of InsP6 in auxin perception remains elusive, this molecule serves as a 64 major phosphate store in seeds and as precursor of InsP7 and InsP8, in which the myo-inositol 65 ring contains one or more energy-rich diphosphate moieties. 66 PP-InsPs regulate a wide range of important biological functions, such as vesicular trafficking, 67 ribosome biogenesis, immune response, DNA repair, telomere length maintenance, phosphate 68 homeostasis, spermiogenesis, insulin signaling and cellular energy homeostasis in yeast and 69

The combinatorial phosphorylation of myo-inositol results in the generation of different inositol phosphates (InsPs), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6 is also an important precursor of the higher phosphorylated inositol pyrophosphates (PP-InsPs), such as InsP7 and 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 InsP6 kinases. Recent work has shown that Arabidopsis (Arabidopsis thaliana) INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1 (ITPK1) and ITPK2 display in vitro InsP6 kinase activity and that, in planta, ITPK1 stimulates 5-InsP7 and InsP8 synthesis and regulates phosphate starvation responses. Here we report a critical role of ITPK1 in auxin-related processes that is independent of the ITPK1-controlled regulation of phosphate starvation responses. Those processes include primary root elongation, root hair development, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity to exogenously applied auxin. We found that the recombinant auxin receptor complex, consisting of the F-Box protein TRANSPORT INHIBITOR RESPONSE1 (TIR1), ARABIDOPSIS SKP1 HOMOLOGUE 1 (ASK1) and the transcriptional repressor INDOLE-3-ACETIC ACID INDUCIBLE 7 (IAA7), binds to anionic inositol polyphosphates with high affinity. We further identified a physical interaction between ITPK1 and TIR1, suggesting a localized production of 5-InsP7, or another ITPK1-dependent InsP/PP-InsP isomer, to activate the auxin receptor complex. Finally, we demonstrate that ITPK1 and ITPK2 function redundantly to control auxin responses, as deduced from the auxin-insensitive phenotypes of itpk1 itpk2 double mutant plants. Our findings expand the mechanistic understanding of auxin perception and suggest that distinct inositol polyphosphates generated near auxin receptors help to fine-tune auxin sensitivity in plants.

7In plants, phosphate (P i ) homeostasis is regulated by the interaction of P i starvation response 2 8 transcription factors (PHRs) with stand-alone SPX proteins, which act as sensors for inositol 2 9 pyrophosphates (PP-InsPs). Recently, ITPK1 was shown to generate the PP-InsP InsP 7 from 3 0 InsP 6 in vitro, but the importance of this activity in P i signaling remained unknown. Here, we 3 1show that uncontrolled P i accumulation in ITPK1-deficient plants is accompanied by impaired 3 2 P i -dependent InsP 7 and InsP 8 synthesis. Reciprocal grafting demonstrates that P i starvation 3 3 responses are mainly controlled by ITPK1 activity in shoots. Nuclear magnetic resonance 3 4 assays and PAGE analyses with recombinant protein reveal that besides InsP 6 3 5 phosphorylation, ITPK1 is also able to catalyze ATP synthesis using 5-InsP 7 but not any other 3 6InsP 7 isomer as a P-donor when ATP is low. Additionally, we show that the dynamic changes 3 7 in InsP 7 and InsP 8 to cellular P i are conserved from land plant species to human cells, 3 8 suggesting that P i -dependent PP-InsP synthesis is a common component of P i signaling across 3 9 kingdoms. Together, our study demonstrates how P i -dependent changes in nutritional and 4 0 energetic states modulate ITPK1 activities to fine-tune the synthesis of PP-InsPs.4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8The in vivo interaction of PHR1 and SPX1 is influenced by P i 3, 4, 5 , suggesting that this 6 7 mechanism could represent a direct link between P i perception and downstream signaling 6 8 events. However, the dissociation constants for P i itself in a SPX-PHR complex ranged from 6 9 10 mM to 20 mM 3, 4, 5 , while P i levels of only 60 µM to 80 µM have been recorded in the 7 0 cytosol of plant cells 9 . A later study, demonstrated that SPX domains can actually act as 7 1 receptors for inositol pyrophosphates (PP-InsPs) and isothermal titration calorimetry 7 2 experiments revealed that 5PP-InsP 5 (hereafter referred to as 5-InsP 7 ) interacted more strongly 7 3 4 with SPX domains than P i 10 . In these assays, interaction of rice OsPHR2 and OsSPX4 was 7 4 promoted at 5-InsP 7 concentrations as low as 20 µM 10 . Although the isomeric nature of plant 7 5 InsP 7 in vegetative tissues remains unknown, it has been proposed that the activity of PHRs is 7 6 regulated by direct interaction with PP-InsPs. In support of this hypothesis, an Arabidopsis 7 7 mutant for INOSITOL PENTAKISPHOSPHATE 2-KINASE (IPK1), which is compromised in 7 8 the synthesis of the PP-InsP precursor inositol hexakisphosphate (InsP 6 ), exhibits constitutive 7 9PSR and increased P i accumulation when grown on sufficient P i availability 11, 12 . More 8 0 recently, other mutants with compromised synthesis of PP-InsPs have been shown to exhibit 8 1 disturbed PSR and P i over-accumulation phenotypes 13, 14, 15 , and it was observed that the 8 2 levels of different inositol polyphosphates (InsPs) are significantly altered in P i -deficient 8 3 plants 13, 14 . Interestingly, polyacrylamide gel electrophoresis (PAGE) ...