ATP-Dependent but Proton Gradient-Independent Polyphosphate-Synthesizing Activity in Extraradical Hyphae of an Arbuscular Mycorrhizal Fungus

Arbuscular mycorrhizal (AM) fungi benefit their host plants by supplying phosphate obtained from the soil. Polyphosphate is thought to act as the key intermediate in this process, but little is currently understood about how polyphosphate is synthesized or translocated within arbuscular mycorrhizas. Glomus sp. strain HR1 was grown with marigold in a mesh bag compartment system, and extraradical hyphae were harvested and fractionated by density gradient centrifugation. Using this approach, three distinct layers were obtained: layers 1 and 2 were composed of amorphous and membranous materials, together with mitochondria, lipid bodies, and electron-opaque bodies, and layer 3 was composed mainly of partially broken hyphae and fragmented cell walls. The polyphosphate kinase/luciferase system, a highly sensitive polyphosphate detection method, enabled the detection of polyphosphate-synthesizing activity in layer 2 in the presence of ATP. This activity was inhibited by vanadate but not by bafilomycin A₁ or a protonophore, suggesting that ATP may not energize the reaction through H⁺-ATPase but may act as a direct substrate in the reaction. This report represents the first demonstration that AM fungi possess polyphosphate-synthesizing activity that is localized in the organelle fraction and not in the cytosol or at the plasma membrane.Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that form symbiotic associations with most land plants (29). These fungi promote the growth of host plants via enhanced uptake of phosphate (P_i) and thus play important roles in the terrestrial phosphorus cycle. In the symbiotic phase, AM fungi take up P_i from soil through an extensive network of extraradical hyphae and rapidly accumulate inorganic polyphosphate (polyP). This accumulation was as rapid as that for a polyP-hyperaccumulating bacterium found in activated sludge (6). PolyP is a linear polymer of three to hundreds of molecules of P_i linked by high-energy phosphoanhydride bonds and has been found across all classes of organisms (19). Although polyP is considered to play a central role in long-distance translocation of P_i in AM fungal associations (4, 10, 30, 31), the translocation mechanism, metabolism, and dynamics in the fungi have not been elucidated due to the difficulty in obtaining sufficient fungal material for analysis.Many enzymes/genes involved in polyP synthesis/metabolism have been identified and characterized in prokaryotes (19). For instance, exopolyphosphatase hydrolyzes the terminal high-energy bonds of polyP, and polyphosphate glucokinase (PPGK) transfers the terminal P_i residue to glucose. Polyphosphate kinase 1 (PPK1) is responsible both for polyP synthesis, using ATP as a phosphoryl donor, and for the reverse ATP-generating reaction. This enzyme is bound to the plasma membrane (18) and has been found in a wide range of bacteria (17). Unlike the case for prokaryotes, knowledge of polyP synthesis/metabolism in eukaryotes remains limited. The first eukaryotic PPK genes, DdPPK1 (32) and DdPPK2 (14), were identified from the social slime mold Dictyostelium discoideum. The products of these genes, as known for bacterial PPK1s, are responsible both for polyP synthesis and for the ATP-generating reaction and have been suggested to be associated with vacuoles or small vesicles (14, 32). Although several homologues of bacterial PPK1 genes have now been found in the genomes of eukaryotic microorganisms (17), yeast Candida humicola is the only organism apart from D. discoideum for which PPK-like activity has been confirmed (22). The model organism Saccharomyces cerevisiae is known to accumulate polyP, to up to 10% of its dry weight (19). A unique polyP synthetic pathway different from those of PPK1 has been proposed for S. cerevisiae based on the observation that vacuolar-type H⁺-ATPase (V-ATPase)-defective mutants could not accumulate polyP (23). In this hypothetical pathway, P_i would be polymerized by an analogous system (enzyme) of mitochondrial F₁-ATPase on the vacuolar membrane, using the proton motive force created by V-ATPase (23). On the other hand, Hothorn et al. (16) demonstrated very recently that vacuolar transporter chaperone 4 (VTC4), a small transmembrane protein associated with the membrane, polymerizes P_i by using the γ-P_i residue of ATP as a phosphoryl donor in S. cerevisiae.More than 2 decades ago, Capaccio and Callow (3) reported the presence of polyP-hydrolyzing, -metabolizing (PPGK), and -synthesizing (PPK-like) activities in the soluble (cytosolic) fractions of the hyphae of the AM fungus Glomus mosseae. Recently, polyP-hydrolyzing activity was found in both the cytosolic and insoluble (membrane) fractions and then characterized (8). PPGK activity has also been confirmed in the cytosolic fraction, although the activity was quite low and hexokinase (ATP-hexose phosphotransferase) activity appeared to dominate in the glucose phosphorylation process (9). PPK-like activity, however, could not be detected in the same fraction (10), and this seems likely because all other prokaryotic (reviewed in reference 17) and eukaryotic (14, 16, 22, 32) polyP-synthesizing enzymes, so far, are associated with membranes. These observations suggest that AM fungi possess a polyP-synthesizing enzyme that is probably associated with membranes and that ATP may be essential in the synthesis as a phosphoryl donor or via H⁺-ATPase, as suggested by Ogawa et al. (23). In this study, a cell fractionation technique was applied to demonstrate polyP-synthesizing activity in an AM fungus, and then the role of ATP in the synthesis was investigated.