Storage of P in the fungus

After P uptake from soil solution, fungi incorporate P into the cytosolic pool. The concentration is kept constant to maintain various cell functions, such as energy transfer and biosynthesis of phospholipids, nucleic acids and precursors of carbohydrate polymers such as UDP-glucose (Ezawa et al., 2002). Excess P is transported into the vacuoles, effectively buffering the cytosolic P concentration (Klionsky et al., 1990; Shirahama et al., 1996). Up to 40% of total P is transferred into the vacuoles (Bolan, 1991) and stored there as osmotically inactive polyphosphate granules. Polyphosphates exist as mobile (n_<

100) or immobile, long chained molecules (n > 100) (Gerlitz and Werk, 1994). Due to their rapid depletion under P limiting conditions, polyphosphates have been suggested to be the main source of fungal derived phosphate for host plants (Rasmussen et al., 2000;

Pfeffer et al., 2001; Viereck et al., 2004). ³¹P-nuclear magnetic resonance (NMR) studies have enabled the differentiation and quantification of polyphosphates and various organic P forms (Martin et al., 1983; Martin et al., 1985; Grellier et al., 1989; Rasmussen et al., 2000; Pfeffer et al., 2001; Viereck et al., 2004). Besides vacuoles, long chained, precepitated orthophosphate residues are also stored in fungal tubular cisterns (Bücking and Heyser, 1999) with vacuolar granules containing phosphate and Ca found in both ecto- and endomycorrhizas (White and Brown, 1974; Chilvers and Harley, 1980; Strullu et al., 1981; Strullu et al., 1982).

Knowledge of P storage in OM is limited: there are few studies about the cellular location of stored P or the forms of P which are stored (Richardson et al., 1992), which suggest the presence of polyphosphate bodies. As P storage systems are evolutionarily well conserved within fungi (Beever and Burns, 1981; Terpitz and Kothe, 2012), OM are likely to parallel the syndromes found in ECM and AM .

2.3.5.1 Phosphorus storage in ECM

The transformation of accumulated inorganic phosphate into mobile polyphosphate with a medium chain length and the transformation of mobile into immobile polyphosphate either with long chain lengths or in granules occurs in ECM (Gerlitz and Werk, 1994). However, polyphosphates in ECM occur mainly in a dispersed soluble form in vacuoles of living, biochemically active hyphae (Cole et al., 1998). A high P concentration in the fungus is maintained by the hydrolysis of polyphosphate, which is then catabolised by polyphosphatases or by reversal of polyphosphatekinase (Cox et al., 1980; Capaccio and Callow, 1982). Large granules are infrequent or even absent in the vacuole of living

hyphae (Cole et al., 1998; Ashford et al., 1999) and Orlovich and Ashford (1993) showed these are mainly an artefact of specimen preparation. Very high cellular P contents, mostly balanced by potassium ions, occur in larger, spherical vacuoles, which can contain polyphosphate granules. Cole (1998) stated that those large vacuoles, are in a fixed position located in close association with the plasma membrane. If they move, they move along the membrane and can also function as a place of storage throughout the hyphae.

Most of the vacuole system are tubular (Ashford, 1998; Cole et al., 1998), and have less contact with the plasma membrane and Allaway and Ashford (2001) proposed that the fixed storage vacuoles associated with the plasmalemma, are interconnected by tubules.

The vacuole system in Pisolithus tinctorius (Pers) Coker & Couch (Sclerodermataceae) hyphae has been shown to be both motile and interconnected. The apical cells of its fungal tips and to a lesser extent the basal hyphal cells in more mature regions can increase their motile activity and interconnectedness in response to changing environmental conditons (Hyde and Ashford, 1997). Both tubules and fixed spherical vacuoles contain a number of elements including high levels of P and potassium (Orlovich and Ashford, 1993; Hyde and Ashford, 1997; Ashford, 1998; Cole et al., 1998). The distribution of P is similar in both spherical vacuoles and tubules, suggesting that both might play a role in the longitudinal long distance hyphal movement of P (Hyde and Ashford, 1997). Inorganic phosphate absorbed by hyphae can also be stored as soluble orthophosphate (Harley and Loughman, 1963) or soluble polyphosphate (Martin et al., 1983; Loughman and Ratcliffe, 1984). In vivo transport of P can be observed in intact systems using radioactive tracers (³²P and ³³P)showing translocation throughout the fungal network and towards the roots of the host plants (Lindahl et al., 2001; Lindahl and Olsson, 2004; Cameron et al., 2007; Wu et al., 2012). In addition, it is also possible to generate elemental maps of mycorrhizal roots showing the distribution of P through the use of micro-particle-induced-X-ray emission (Bücking and Heyser, 1999; Bücking and Shachar-Hill, 2005; Orłowska et al., 2008). Ectomycorrhizal basidiomycetes growing in axenic culture can store P as orthophosphate or polyphosphate, depending on species or culture conditions (Martin et al., 1983; Martin et al., 1985; Mousain and Salsac, 1985;

Cairney and Chambers, 1997; Gerlitz and Gerlitz, 1997). Those diverse storage forms the biochemical possibilities and the interconnected tubular vacuole system might be the reason for the high P concentration in the ECM.

2.3.5.2 Phosphorus storage in AM

Of the various cellular functions where fungi supply the plant with P (Kornberg et al., 1999), the most important is the temporary storage of inorganic P in the vacuolar P pool of AM hyphae (Ezawa et al., 2002) (Table 2.3-3). Chain-length of polyphosphate in AM fungi

is variable (Ezawa et al., 1999), longer in extraradical than in intraradical hyphae but both soluble and long-chain granular forms occur in the intra- and extraradical hyphae (Solaiman et al., 1999). Many factors may affect the solubility of polyphosphate in vivo, including pH, chain-length, concentration of polyphosphate and counter ions such as metal cations and polyamines (Harold, 1966; Cramer and Davis, 1984). The efficiency of the AM mycorrhizal association in P nutrition of a host plant is highly geared to the ability of the AM to accumulate P when external supply is high and remobilise this stored pool of P under limiting conditions, thereby maintaining a continuous suppy of P to the plant (Bücking and Heyser, 2000).

2.3.6 Transport of P in the fungus-plant