Overview of primers used in this study.

Inositol pyrophosphates (PP-InsPs) are nutrient messengers whose cellular levels are precisely regulated. Diphosphoinositol pentakisphosphate kinases (PPIP5Ks) generate the active signaling molecule 1,5-InsP8. PPIP5Ks harbor phosphatase domains that hydrolyze PP-InsPs. Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSPs) and NUDIX phosphatases (NUDTs) are also involved in PP-InsP degradation. Here, we analyze the relative contributions of the three different phosphatase families to plant PP-InsP catabolism. We report the biochemical characterization of inositol pyrophosphate phosphatases from Arabidopsis and Marchantia polymorpha. Overexpression of different PFA-DSP and NUDT enzymes affects PP-InsP levels and leads to stunted growth phenotypes in Arabidopsis. nudt17/18/21 knock-out mutants have altered PP-InsP pools and gene expression patterns, but no apparent growth defects. In contrast, Marchantia polymorpha Mppfa-dsp1ge, Mpnudt1ge and Mpvip1ge mutants display severe growth and developmental phenotypes and associated changes in cellular PP-InsP levels. Analysis of Mppfa-dsp1geand Mpvip1ge mutants supports a role for PP-InsPs in Marchantia phosphate signaling, and additional functions in nitrate homeostasis and cell wall biogenesis. Simultaneous elimination of two phosphatase activities enhanced the observed growth phenotypes. Taken together, PPIP5K, PFA-DSP and NUDT inositol pyrophosphate phosphatases regulate growth and development by collectively shaping plant PP-InsP pools.