Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms, such as membrane trafficking, telomere maintenance, ribosome biogenesis, and apoptosis. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate biosynthesis of inositol pyrophosphates: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Here, we report that PPIP5K homologs are widely distributed in plants and that Arabidopsis thaliana VIH1 and VIH2 are functional PPIP5K enzymes. We show a specific induction of inositol pyrophosphate InsP₈ by jasmonate and demonstrate that steady state and jasmonate-induced pools of InsP₈ in Arabidopsis seedlings depend on VIH2. We identify a role of VIH2 in regulating jasmonate perception and plant defenses against herbivorous insects and necrotrophic fungi. In silico docking experiments and radioligand binding-based reconstitution assays show high-affinity binding of inositol pyrophosphates to the F-box protein COI1-JAZ jasmonate coreceptor complex and suggest that coincidence detection of jasmonate and InsP₈ by COI1-JAZ is a critical component in jasmonate-regulated defenses.

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.

Inositol phosphates (IPs) comprise a network of phosphorylated molecules that play multiple signaling roles in eukaryotes. IPs synthesis is believed to originate with IP ₃ generated from PIP ₂ by phospholipase C (PLC). Here, we report that in mammalian cells PLC-generated IPs are rapidly recycled to inositol, and uncover the enzymology behind an alternative “soluble” route to synthesis of IPs. Inositol tetrakisphosphate 1-kinase 1 (ITPK1)—found in Asgard archaea, social amoeba, plants, and animals—phosphorylates I(3)P ₁ originating from glucose-6-phosphate, and I(1)P ₁ generated from sphingolipids, to enable synthesis of IP ₆ . We also found using PAGE mass assay that metabolic blockage by phosphate starvation surprisingly increased IP ₆ levels in a ITPK1-dependent manner, establishing a route to IP ₆ controlled by cellular metabolic status, that is not detectable by traditional [ ³ H]-inositol labeling. The presence of ITPK1 in archaeal clades thought to define eukaryogenesis indicates that IPs had functional roles before the appearance of the eukaryote.

Diphospho-myo-inositol polyphosphates, also termed inositol pyrophosphates, are molecular messengers containing at least one high-energy phosphoanhydride bond and regulate a wide range of cellular processes in eukaryotes. While inositol pyrophosphates InsP₇ and InsP₈ are present in different plant species, both the identity of enzymes responsible for InsP₇ synthesis and the isomer identity of plant InsP₇ remain unknown. This study demonstrates that Arabidopsis ITPK1 and ITPK2 catalyze the phosphorylation of phytic acid (InsP₆) to the symmetric InsP₇ isomer 5-InsP₇ and that InsP₆ kinase activity of ITPK enzymes is evolutionarily conserved from humans to plants. We also show by ³¹P NMR that plant InsP₇ is structurally identical to the in vitro InsP₆ kinase products of ITPK1 and ITPK2. Our findings lay the biochemical and genetic basis to uncover physiological processes regulated by 5-InsP₇ in plants.

Most Gram-negative phytopathogenic bacteria inject type III effector (T3E) proteins into plant cells to manipulate signaling pathways to the pathogen’s benefit. In resistant plants, specialized immune receptors recognize single T3Es or their biochemical activities, thus halting pathogen ingress. However, molecular function and mode of recognition for most T3Es remains elusive. Here, we show that the Xanthomonas T3E XopH possesses phytase activity, i.e., dephosphorylates phytate (myo-inositol-hexakisphosphate, InsP₆), the major phosphate storage compound in plants, which is also involved in pathogen defense. A combination of biochemical approaches, including a new NMR-based method to discriminate inositol polyphosphate enantiomers, identifies XopH as a naturally occurring 1-phytase that dephosphorylates InsP₆ at C1. Infection of Nicotiana benthamiana and pepper by Xanthomonas results in a XopH-dependent conversion of InsP₆ to InsP_5. 1-phytase activity is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene, and for induction of jasmonate- and ethylene-responsive genes in N. benthamiana.

Inositol polyphosphate binding specificity of the jasmonate receptor is largely determined by the F-box protein COI1.