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Biochemical and physiological studies on polyphosphate metabolism in plants

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Defense Thèse de doctorat : Univ. Genève, 2019 - Sc. 5359 - 2019/06/25
Abstract As other living organisms, plants rely on phosphate (P i ) as an essential macronutrient required for growth. One way to store P i in both prokaryotic and eukaryotic cells is in form of inorganic polyphosphates (polyP). PolyP are linear polymers formed by P i subunits linked by high energy phosphoanhydride bonds. PolyP are versatile molecules which can adopt different chain lengths, bind to cations and other positively charged molecules and accumulate in various subcellular compartments. Although polyP are very ancient polymers, they have evolved to have many different functions. In bacteria, polyP are mainly involved in stress responses, as for instance nutrient deprivation or metal toxicity. In yeast cells, polyP represent the main P i pool and maintain both P i and ion homeostasis. In humans, polyP regulate blood clotting, bone calcification, rRNA biogenesis and the cell cycle. While polyP have been found in bacterial, fungal and human cells, it is not yet known whether plants can accumulate polyP. The enzymes involved in polyP synthesis and breakdown are known in bacteria and lower eukaryotes, including yeast, amoeba, protozoans and algae. Although plants do not have homologs of polyP biosynthetic enzymes, they contain structurally-related proteins. Here I report two plant proteins with features similar to polyP metabolizing enzymes. One of them, the TRIPHOSPHATE TUNNEL METALLOENZYME 3 (TTM3) harbors a domain which is similar in structure to the yeast polyP kinase Vtc4 (Hothorn et al., 2009). In vitro characterization of TTM3 by our lab and others (Moeder et al., 2012; Martinez et al., 2015) demonstrated that Arabidopsis TTM3 is not producing polyP, as previously thought, but rather cleaves tripolyphosphate into pyrophosphate and P i . My work shows that TTM3 is expressed together with the CELL DIVISION CYCLE PROTEIN 26 (CDC26) from a plant polycistronic transcript. Polycistronic transcripts are rare in eukaryotes and had not been reported in plants thus far. I characterize CDC26 as a member of the plant Anaphase Promoting Complex/Cyclosome, an E3 ubiquitin ligase involved in cell cycle progression. CDC26 has an essential role in embryo development and can regulate plant growth. The CDC26-TTM3 polycistronic transcript has been conserved in the entire plant lineage for over 700 million years (from algae to dicotyledon plants), suggesting an important role of this transcript in cell cycle regulation, as well as a putative connection between the cell cycle and polyP metabolism in plants. A protein from the plant Ricinus communis L. (castorbean) was found to harbor a domain of unknown function named CHAD (conserved histidine α-helical domain). In bacteria, CHADs are often fused to TTM domains, or expressed as stand-alone domains from operons encoding polyP metabolizing enzymes. I obtained a crystal structure of the CHAD-containing protein from castorbean, which revealed a basic surface patch common in polyP-metabolizing enzymes. Next I found that CHAD-containing proteins from all kingdoms of life specifically bind polyP with μM to nM affinity in grating-coupled interferometry assays. A complex structure of a bacterial CHAD and polyP showed that CHAD interacts with polyP through its basic surface patch. A fluorescent-tagged version of CHAD localized in the nucleus and nucleolus of plant cells. Co-expression of CHAD with a bacterial polyP kinase demonstrated that CHAD can bind polyP in vivo, as previously observed in bacteria. My work defines CHAD as a novel polyP-binding module which may serve as a tool to further investigate the presence of polyP in plants.
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URN: urn:nbn:ch:unige-1213533
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LORENZO-ORTS, Laura. Biochemical and physiological studies on polyphosphate metabolism in plants. Université de Genève. Thèse, 2019. doi: 10.13097/archive-ouverte/unige:121353 https://archive-ouverte.unige.ch/unige:121353

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Deposited on : 2019-07-26

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