NUDIX Hydrolases Target Specific Inositol Pyrophosphates and Regulate Phosphate and Iron Homeostasis, and the Expression of Defense Genes in Arabidopsis

Inositol pyrophosphates (PP-InsPs) are important signaling molecules that regulate diverse cellular processes in eukaryotes, including energy homeostasis and phosphate (P _i ) signaling. However, the enzymes responsible for their turnover in plants remain largely unknown. Using a non-hydrolysable PP-InsP analog in a pull-down approach, we identified a family of Arabidopsis Nudix hydrolases (NUDTs) that group into two closely related subclades. Through in vitro assays, heterologous expression systems, and higher-order gene-edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange (SAX)-HPLC, PAGE, and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that their PP-InsP pyrophosphatase activity is enantiomer-selective and Mg ²⁺ -dependent. Specifically, subclade I NUDTs preferentially hydrolyze 4-InsP ₇ , while subclade II NUDTs target 3-InsP ₇ , with minor activity against other PP-InsPs, including 5-InsP ₇ . In higher-order mutants of subclade II NUDTs, we observed defects in both P _i and iron homeostasis, accompanied by increased levels of 1/3-InsP ₇ and 5-InsP ₇ , with a markedly larger increase in 1/3-InsP ₇ . Furthermore, ectopic expression of NUDTs from both subclades induced local P _i starvation responses (PSRs). However, RNA-seq analysis of WT and nudt12/13/16 plants suggests that these NUDTs have PSR-independent roles, potentially involving 1/3-InsP ₇ in the regulation of plant defense. Expanding beyond subclade II NUDTs, we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we found that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to subclade I NUDTs. Collectively, our study provides new insights into the roles of NUDTs in regulating PP-InsP signaling pathways in plants and highlights cross-kingdom conservation of enzymes involved in PP-InsP metabolism.