Arabidopsis PFA-DSP-Type Phosphohydrolases Target Specific Inositol Pyrophosphate Messengers

Abstract

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 homologues display phosphohydrolase activity with a high specificity for the 5-β-phosphate of inositol pyrophosphates and only minor activity against the β-phosphates of 4-InsP 7 and 6-InsP 7 . 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-DSPtype 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.

Crystal Structure and Enzymology of Solanum tuberosum Inositol Tris/Tetrakisphosphate Kinase 1 (StITPK1)

“…Recently, noncanonical PP-InsP 5 species have been identified in plants. 27,34 CE-MS peaks with chromatographic mobility and parent/daughter ion relationships identical with synthetic D-and/or L-4-PP-InsP 5 [4-PP-InsP 5 /6-PP-InsP 5 ] and the meso-compound 2-PP-InsP 5 have been detected. 27,34 Similar conclusions can be drawn from Both compounds eluted substantially earlier than 3-PP-InsP 5 (or its enantiomer, 1-PP-InsP 5 ) (Figure S3).…”

“…The pronounced inhibition of phosphokinase activity by Ins(3,4,5,6)P 4 says as much, while the reversibility of IPK1's phosphokinase activity (previously described for InsP 6 substrate, 21,27 here tested against 1-PP-InsP 5 and 3-PP-InsP 5 ) extends ITPK1 influence to whole cohorts of inositol pyrophosphate substrates that are widely reported in plants. [1][2][3][6][7][8][9][10]15,23,24,27,34,42 34 By corollary, it seems plausible that VIH1/2 also shows reversible phosphokinase (i.e., ATP-synthesizing) activity: not only does VIH1/2 possess the ATP-grasp fold but also the mammalian homologue PPIP5K2 shows ADP-driven 1,5-[PP] 2 -InsP 4 1-dephosphorylation (ATP-synthesizing) activity. 28,43 It is, perhaps, instructive, therefore, to compare the relative activities of different ATP-grasp kinase proteins of plants.…”

“…23,24 Even so, the activities of ITPK1/2 and VIH1/2 20,21,27 do not account for the full spectrum of inositol pyrophosphates detected in plants. 21,27,34 The possible isomers and enantiomers comprise 1-InsP 7 , 1-PP-Ins (2,3,4,5,6) The ITPK family, including members that synthesize inositol pyrophosphates 20,21,27 or do not, 20,22 is represented in early land plants 29 and in early aquatic vascular plants, the duckweeds in which a lipid-independent pathway of InsP 6 synthesis that uses the favored InsP 4 substrate of plant ITPK1 21 was described. 30−32 Herein, we have solved a crystal structure for a potato enzyme, StITPK1, in complex with ADP.…”

“… These activities are, however, a very small fraction of the Ins­(3,4,5,6)­P 4 1-kinase activity of the enzyme . To date, the only enzymes capable of synthesizing InsP 8 , 1,5-InsP 8 , 1,5-[PP] 2 -Ins­(2,3,4,6)­P 4 , hereafter 1,5-[PP] 2 -InsP 4 , the presumed endogenous [PP] 2 -InsP 4 in plants, are the VIH1/2 enzymes. , Even so, the activities of ITPK1/2 and VIH1/2 ,, do not account for the full spectrum of inositol pyrophosphates detected in plants. ,, The possible isomers and enantiomers comprise 1-InsP 7 , 1-PP-Ins­(2,3,4,5,6)­P 5 , hereafter 1-PP-InsP 5 ; 3-InsP 7 , 3-PP-Ins­(1,2,4,5,6)­P 5 , hereafter 3-PP-InsP 5 ; 4-InsP 7 , 4-PP-Ins­(1,2,3,5,6)­P 5 , hereafter 4-PP-InsP 5 ; and 6-InsP 7 , 6-PP-Ins­(1,2,3,4,5)­P 5 , hereafter 6-PP-InsP 5 . The meso -isomers include 5-PP-InsP 5 and 2-InsP 7 , 2-PP-Ins­(1,3,4,5,6)­P 5 , and hereafter 2-PP-InsP 5 .…”

Intracellular phosphate recycling systems for survival during phosphate starvation in plants

“…Since it is known that Pi starvation enhances accumulation of reactive oxygen species (ROS), which leads to programmed cell death ( Van Camp et al., 1998 ; Overmyer et al., 2000 ; Tyburski et al., 2009 ; Yoshitake et al., 2022 ), that proteins may contribute to plant growth not only through the resupply of Pi from intracellular components but also because increase ROS degradation under Pi starvation. It is known that a phosphohydrolase for inositol pyrophosphates (PP-InsPs), one of the phosphoesters, has a role in Pi starvation response, but it is unclear whether this enzyme is involved in Pi recycling ( Gaugler et al., 2022 ).…”