Functional characterization of inositol pyrophosphate pyrophosphatases in plants

The consequences of the climate and biodiversity crisis present an increasingly challenge for the agricultural sector to ensure food security for a growing world population. The optimal nutrient supply of plants is a critical factor in maintaining or increasing yields, especially of essential macronutrients such as phosphorus (P). It is therefore important to gain a deeper understanding of the processes involved in P uptake, in form of inorganic phosphate (P_i), and the mechanisms regulating P_i homeostasis in plants. Recent research has identified the involvement of the signaling molecules inositol pyrophosphates (PP-InsPs) in the process of P_i signaling and homeostasis in plants. The biosynthesis of PP-InsPs relies on balanced phosphorylation and dephosphorylation of inositol phosphates (InsPs) and PP-InsPs. However, the current state of knowledge regarding PP-InsP dephosphorylation is notably limited in comparison to InsP/PP-InsP phosphorylation. Therefore, the objective of this thesis was to identify and characterize putative PP-InsP pyrophosphatases in plants. We found that recombinant proteins of all five Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) display pyrophosphatase activity with a high specificity for the 5 β-phosphate of PP-InsPs. Heterologous expression in <em>Nicotiana benthamiana</em> leaves provided evidence that Arabidopsis PFA-DSP1 also displays a 5-β-phosphate-specific PP-InsP pyrophosphatase activity <em>in planta</em>. Moreover, we identified a family of Arabidopsis NUDIX hydrolases (NUDTs) that group into two closely related subclades. Through <em>in vitro</em> assays, heterologous expression systems, and higher-order gene-edited mutants, we found that subclade I NUDTs preferentially hydrolyze 4-InsP₇, while subclade II NUDTs target 3-InsP₇. 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-InsP₇ and 5-InsP₇. Ectopic expression of NUDTs from both subclades induced local P_i starvation responses (PSRs), while RNA-seq analyses comparing WT and <em>nudt12/13/16</em> loss-of-function plants indicate additional PSR-independent roles and a potential involvement of 1/3-InsP₇ in the regulation of plant defense. <br> Collectively, the results presented in thesis show not only new insights into the roles of PFA DSPs and NUDTs in regulating PP-InsP signaling pathways in plants, but also provide the genetic tools to uncover more general roles of PP-InsPs in plant physiology and plant development.