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

Abstract

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 A 1 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 polyPhyperaccumulating 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 highenergy 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 ATPgenerating 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), we...

Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity

“…Interestingly, the polyP synthesis in Dictyostelium discoideum vacuoles, possibly acidocalcisomes, is inhibited by carbonyl cyanide m-chlorophenyl hydrazone (CCCP), suggesting that it might also depend on the proton gradient (Gómez-García and Kornberg, 2004). However, an opposite example also exists: polyP synthesis by the organelle fraction from arbuscular fungi was reported to be insensitive to CCCP (Tani et al, 2009). Therefore, it remains to be determined how universal is the requirement for the energized membrane environment for polyP synthesis in eukaryotes.…”

Enzymes of Inorganic Polyphosphate Metabolism

“…The catalytic domain faces the cytoplasm and the polymer must pass the membrane (Hothorn et al 2009). It is very likely that a similar protein provides polyphosphate kinase activity in mycorrhizal fungi where the polyphosphate-synthesizing activity using ATP is localized in the organelle fraction but not in the cytosol or at the plasma membrane (Tani et al 2009). …”

Genomics of Arbuscular Mycorrhizal Fungi

“…To achieve the long-distance translocation from ERM to IRM, Pi is rapidly converted to polyphosphate (polyP) (Ezawa, Cavagnaro, Smith, Smith, & Ohtomo, 2004;Ezawa, Smith, & Smith, 2002;Viereck, Hansen, & Jakobsen, 2004), a phosphate chain composed of three to thousands of molecules linked by phosphoanhydride bonds (Kornberg, Rao, & Ault-Riché, 1999). PolyP synthesis activity has been detected in vacuolar membrane after fractionation of the cellular compartment of IRM (Tani, Ohtomo, Osaki, Kuga, & Ezawa, 2009), and genes coding for protein involved in the synthesis of polyP and a putative vacuolar transporter chaperone complex Vtc4p were found in R. irregularis (Tisserant et al, 2012). In IRM, polyP has to be hydrolyzed to free Pi that will be delivered in the apoplast where a specialized host plant transporter takes care of importation (Pumplin, Zhang, Noar, & Harrison, 2012).…”