Identification and characterization of evolutionarily conserved inositol pyrophosphate phosphohydrolases in plants
Creators: name: Gaugler, Philipp
Publisher: name: Universitäts- und Landesbibliothek Bonn
Year: 2023
Subjects: subject: ddc:570

description: Inositol phosphates (InsPs), phosphate esters of <em>myo</em>-inositol, are cellular regulators with critical roles in a wide range of cellular processes in eukaryotes. Members of the subgroup containing at least one ‘high-energy’ phosphoanhydride group are termed inositol pyrophosphates (PP-InsPs) and have been of special interest in the last decades, especially for plant scientists. The biosynthesis of PP-InsPs has been uncovered in recent years, enabling the discovery of important functions in plant immunity, nutrient sensing and hormone signaling. However, one of the greatest challenges remains the reliable detection and quantification of these enigmatic molecules in plant extracts, primarily due to their low abundance and susceptibility to hydrolytic activities. The extraction of InsPs from plants labeled with [<sup>3</sup>H]-<em>myo</em>-inositol and subsequent separation by strong anion exchange high-performance liquid chromatography (SAX-HPLC) is a method that has been frequently employed for the analysis of InsPs from all types of organisms, due to its high sensitivity and relative simplicity. Still, many parameters need optimization and a detailed description of the set-up and workflow of this method is critical for success. Therefore, the set-up of a suitable SAXHPLC system, as well as the complete workflow covering plant cultivation, radiolabeling, InsP extraction, separation via SAX-HPLC and subsequent data analysis was described in a detailed step-by-step protocol and visualized by video documentation. The protocol allowed the discrimination and quantification of various InsP species, including multiple non-enantiomeric isomers and various PP-InsPs from the established model plant <em>Arabdiopsis thaliana</em>. The versatility of this method was exemplified by the first analysis of InsPs from <em>Lotus japonicus</em>. Multiple differences in InsP levels between <em>Arabdiopsis</em> and <em>Lotus</em> give new hints for future studies of InsPs in plant systems, whereas the described method represents an optimized and standardized tool to help elucidate the biological roles of InsPs <em>in planta</em>. In addition to these methodological challenges, several aspects of PP-InsP biosynthesis in plants remain elusive. For instance, very little is known about the identity of PP-InsP phosphohydrolases, despite the apparent rapid hydrolysis of PP-InsPs in plant extracts. Therefore, the <em>Arabdiopsis</em> members of the Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP), which are homologs of the recently identified yeast PP-InsP phosphohydrolase Siw14, were characterized. All five homologs, similarly to recombinant Siw14, displayed phosphohydrolase activity <em>in vitro</em>, with high specificity for the 5-β-phosphate of PP-InsPs and minor to minimal activity against 4/6-InsP<sub>7</sub> or 1/3-InsP<sub>7</sub>, respectively. Furthermore, heterologous expression of either one of the homologs rescued wortmannin sensitivity of the siw14Δ yeast deletion mutant and restored elevated InsP<sub>7</sub> levels to wild-type levels. Genetic interaction analyses of InsP and PP-InsP kinases with SIW14, revealed that the wortmannin sensitivity of siw14Δ depends on the presence of Kcs1-derived PP-InsPs like 5-InsP<sub>7</sub>. Although the <em>Arabdiopsis</em> PFA-DSPs appear to be at least partially redundant <em>in vitro</em>, as well as <em>in vivo</em>, SAXHPLC analyses of the <em>Arabdiopsis</em> T-DNA insertion line <em>pfa-dsp1-6</em>, in which PFA-DSP1 is overexpressed, showed a clear reduction of InsP<sub>7</sub> levels compared to Col-<em>0</em>. This finding was strengthened by heterologous expression of PFA-DSP1 in Nicotiana bethamiana leaves, which resulted in a specific reduction of 5-InsP<sub>7</sub>. In conclusion, these experiments showed that the <em>Arabdiopsis</em> PFA-DSPs are evolutionarily conserved and are highly specific 5-β-phosphate PP-InsP phosphohydrolases with very similar <em>in vitro</em> and apparently also <em>in vivo</em> activities. These findings not only broaden the knowledge of PP-InsP degradation in yeast and in plants, but also provide additional genetic tools to uncover the roles of PP-InsPs in plant physiology and plant development.