Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis

Abstract

Many eukaryotic proteins regulating phosphate (Pi) homeostasis contain SPX domains. We have previously shown that these domains act as cellular receptors for inositol pyrophosphate (PP-InsP) signaling molecules, suggesting that PP-InsPs may regulate Pi homeostasis. Here we report that simultaneous deletion of two diphosphoinositol pentakisphosphate kinases VIH1 and 2 in Arabidopsis impairs plant growth and leads to constitutive Pi starvation responses. We demonstrate that VIH1 and VIH2 are bifunctional cytosolic enzymes able to generate and break-down PP-InsPs. Point-mutants targeting the kinase and phosphatase active sites have opposing effects on plant Pi content and Pi starvation responses, while VIH1 and VIH2 protein levels remain constant in different Pi growth conditions. Enzymatic assays reveal that ATP-Mg 2+ substrate levels can shift the relative kinase and phosphatase activities of fulllength diphosphoinositol pentakisphosphate kinases. Deletion of phosphate starvation response transcription factors rescues vih1 vih2 mutant phenotypes, placing diphosphoinositol pentakisphosphate kinases and PP-InsPs in plant phosphate signal transduction cascades. We propose that VIH1 and VIH2 relay changes in cellular ATP concentration to changes in PP-InsP levels, allowing plants to maintain cellular Pi concentrations constant and to trigger Pi starvation responses.

An ATP-responsive metabolic cassette comprised of inositol tris/tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPK1) buffers diphosphosphoinositol phosphate levels

“…The availability of pairs of enantiomeric substrates, not available in the radiolabeled form, beside meso -compounds, allowed analysis of the enantiospecificity of the enzyme. Of particular interest to us were InsP 4 substrates, since Ins(3,4,5,6)P 4 is elevated in itpk1 and ipk1 mutants that share a common misregulation of phosphate homeostasis [ 10 , 11 , 38 ] attributed to deregulated diphosphoinositol phosphate synthesis [ 15 , 16 ]. Of the enantiomeric pairs Ins(1,2,4,6)P 4 /Ins(2,3,4,6)P 4 , Ins(1,3,4,5)P 4 /Ins(1,3,5,6)P 4 and Ins(1,4,5,6)P 4 /Ins(3,4,5,6)P 4 (all shown in Supplementary Figure S1 ), only the latter pair were substrates under ATP-regenerating conditions.…”

“…Others have shown that synthesis of 1,5-InsP 8 (also known as 1,5-bis-PP-InsP 4 ) occurs by 1-phosphorylation of 5-InsP 7 and is mediated by VIH1 and VIH2 [ 14 ] (see Figure 1 ). Deletion of Vih1 and Vih2 recapitulates constitutive Pi starvation response [ 15 , 16 ]. These works confirm the identity of plant InsP 8 previously described [ 14 , 17 ].…”

Phosphate Homeostasis − A Vital Metabolic Equilibrium Maintained Through the INPHORS Signaling Pathway

“…These enzymes contain both a kinase and a histidine acid phosphatase domain, which compete with each other ( Mulugu et al, 2007 ). Their enzymatic and structural properties have mainly been uncovered through studies of the mammalian enzymes ( Wang et al, 2012 ; Weaver et al, 2013 ; Gu et al, 2017 ; Nair et al, 2018 ; Randall et al, 2019 ), but the essential features could be confirmed for the plant and yeast members of the family ( Pöhlmann et al, 2014 ; Dong et al, 2019 ; Zhu et al, 2019 ). The kinase domain phosphorylates InsP 6 and 5-InsP 7 to generate 1-InsP 7 and 1,5-InsP 8 , respectively, whereby 5-InsP 7 appears to be the preferred substrate over InsP 6 ( Wang et al, 2012 ; Weaver et al, 2013 ).…”

“…Importantly, the phosphatase activity of PIPP5Ks is inhibitable by P i in the low millimolar range—a concentration range that is commonly found in the cytosol—and their kinase activity is boosted by an increasing ATP concentration ( Gu et al, 2017 ; Zhu et al, 2019 ). Since cellular ATP concentration diminishes with cellular P i availability ( Boer et al, 2003 ), P i limitation in the cytosol should tip the equilibrium between the kinase and phosphatase activities in favor of dephosphorylation, decreasing the pools of 1,5-InsP 8 and 1-InsP 7 .…”

A genetically validated approach to detect inorganic polyphosphates in plants

“…We have recently uncovered a molecular connection between the Pi starvation response controlled by inositol pyrophosphate (PP-InsPs) signaling molecules and their SPX receptors, and polyP metabolism in yeast (Wild et al, 2016). PP-InsPs and SPX domains also control the Pi starvation response in Arabidopsis (Zhu et al, 2018), and we have characterized the short-chain polyphosphatase TRIPHOSPHATE TUNNEL METALLOENZYME 3 (AtTTM3) (Martinez et al, 2015;Lorenzo-Orts, Witthoeft, et al, 2019) and a specific polyP-binding domain (Lorenzo-Orts, Hohmann, et al, 2019) in plants. We thus speculate that polyPs may exist in plants and that they could form a relevant Pi store.…”