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- Materials and Methods
Arbuscular mycorrhizal fungi (AMF) are obligate plant symbionts that cannot reproduce themselves without colonizing roots. Various functions have been demonstrated for AMF, such as enhancing host nutrient uptake (especially inorganic phosphate, Pi); increasing host resistance to environmental stresses (e.g. drought) or to plant pathogens; forming nutritional connections between different kinds of host in natural grassland ecosystems; and contributing to the maintenance of diversity (Smith & Read, 1997). Therefore exploitation of effective inocula of the fungi has been widely expected (Saito & Marumoto, 2002).
Many pot experiments have shown that colonization by an AMF enhanced the host's Pi uptake, but quantitative evaluation of the AMF's contribution is difficult, especially in natural ecosystems. In the case of nitrogen fixation by Rhizobium spp., the contribution of the bacterium to the host's N acquisition can be evaluated by 15N labelling because the plant does not fix N by itself. However, because AMF and roots both obtain Pi from the same source, specific labelling of Pi cannot distinguish between Pi obtained directly from the soil and that obtained from the AMF. The separation of roots and hyphae into different compartments by using, for example, nylon mesh sheets, or the use of transformed root colonized by AMF, provides an excellent system for assaying AMF activity (Joner & Leyval, 1997; Smith et al., 2003), but even in this system the root compartment contains many hyphae, and quantitative evaluation of the AMF's contribution requires precise determination of the distribution of active hyphae among the compartments.
It has been suggested that AMF convert Pi taken up from the soil into inorganic polyphosphate (poly P) (Callow et al., 1978), a linear polymer of three Pi to thousands of Pi residues connected by high-energy phospho-anhydrate bonds. Poly P is a ubiquitous molecule, and all organisms investigated so far have been found to contain it. AMF have been shown to accumulate poly P in their hyphae (Cox et al., 1980), and to synthesize poly P very rapidly (Ezawa et al., 2003). Poly P has various biological functions (Kornberg et al., 1999), and the biological function of poly P in AMF remains to be clarified. Ezawa et al. (2001) showed that the activity of poly P-glucokinase in AMF was very low, and thus proposed that poly P did not function as a major Pi donor in glucose metabolism in AMF cells. It has been hypothesized that AMF translocate poly P from extraradical to intraradical mycelium (Cox et al., 1980), and depolymerize poly P before supplying Pi to the host plant cells (Solaiman & Saito, 2001). Alkaline phosphatase (ALP) has been considered a key enzyme in determining an AMF's ability to supply Pi, and the ALP activity was induced under high levels of environmental Pi (van Aarle et al., 2002; Olsson et al., 2002). However, transcription of the ALP gene was not induced by environmental Pi (Aono et al., 2004). The ALP expressed specifically in mycorrhizal roots was found to be of fungal origin (Kojima et al., 1998) and has been partially purified (Kojima et al., 2001). Although the involvement of ALP in the metabolism of poly P was questioned because Ba2+-sensitive fungal ALP did not hydrolyse poly P (Ezawa et al., 1999), the enzyme was shown to be at least partially involved in Pi efflux from isolated intraradical hyphae of AMF (Kojima & Saito, 2004).
Until recently, microquantification of poly P was difficult. Commonly used quantification methods, including staining with Toluidine blue O, DAPI or Fura-2, and determination of Pi after acid hydrolysis of poly P, are not highly specific to poly P, nor are they sensitive enough to quantify very small amounts of poly P. An enzymatic assay, in which poly P is converted into ATP by Escherichia coli polyphosphate kinase (PPK) followed by ATP quantification using the luciferin–luciferase system (Ault-Richéet al., 1998), has overcome this difficulty. Application of this technique to AMF has been successful (Ezawa et al., 2003), and it is now possible to measure poly P in small fungal samples (mg levels). Thorough investigation of the poly P dynamics in AMF would help us understand the metabolism and function of poly P in these fungi.
In the present study, we monitored changes in the poly P content of mycorrhizal roots during the colonization process. Because we first aimed to determine the poly P levels in various colonization stages, we periodically sampled mycorrhizal plants to obtain roots with various levels of colonization. We analysed the relationships between poly P content and colonization, host P status, and growth of both host and fungus.