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22 Nitrogen Transport and Metabolism in Mycorrhizal Fungi and Mycorrhizas 405
gene under conditions of N starvation, suggests that the transporter can also
be involved in the prevention of amino acid loss by hyphal leakage in the
absence of a suitable N source (Nehls et al. 1999).
A gene named HcBap1 has recently been isolated from H. cylindrosporum
by functional complementation of a yeast strain deficient in amino acid transporters
(Wipf et al. 2002).
5 Reduction of Nitrate to Nitrite and Ammonium
5.1 Reduction of Nitrate to Nitrite
Nitrate assimilation in fungi follows the same pathway as that described for
yeasts and plants. After transport into the cells, nitrate is converted to ammonium
by two successive reductions catalysed respectively by nitrate reductase
and nitrite reductase. Although nitrate is one of the most abundant nitrogen
sources in nature, numerous fungi more readily use ammonium, especially
ectomycorrhizal fungi which live predominantly in forest soils where a high
organic material content maintains an acidic pH. Under these circumstances,
nitrification is inhibited and ammonium is usually the main form of mineral
nitrogen (Vitousek and Matson 1985). However, it has been shown that ectomycorrhizal
fungi are also able to utilize NO 3 – which, for a few species, is capable
of promoting better growth than ammonium (Scheromm et al. 1990;
Anderson et al. 1999).
The enzyme complex nitrate reductase which is a molybdoflavoprotein catalyzes
the reduction of NO 3 – to NO2 – by reduced pyridine nucleotides. The
enzyme of higher plants has a high molecular weight, varying from 220 to
600 kDa, depending on the organisms in which it occurs (Notton and Hewitt
1978). In fungi, nitrate reductase has been extensively studied in Neurospora
crassa where it is found as a 228-kDa homodimer (Garrett and Nason 1969)
and in Aspergillus nidulans where the enzyme has a molecular mass of
180 kDa (Minagawa and Yoshimoto 1982). In plants and fungi, the polypeptide
is located in the cytosolic soluble fraction, but is weakly bound to the
plasmalemma and tonoplast in Neurospora crassa (Roldan et al. 1982).
Nitrate reductase generally appears to be unstable and,due to the difficulties
experienced in purifying the enzyme, information on its properties in mycorrhizal
fungi is very scarce. However, nitrate reduction by partially purified
enzyme preparations has been investigated in Hebeloma cylindrosporum by
Plassard et al. (1984a). The Michaelis constants for nitrate, NADPH and FAD
were found to be 152, 0.185, and 22.7 mM,respectively.In Pisolithus tinctorius,
nitrate reductase exhibited less affinity for nitrate (Km: 328 mM) and for
NADPH (Km: 49.6mM; Aouadj et al. 2000), but the enzyme was similar to those
found in nonmycorrhizal fungi. Such values are in the same range as those
found in higher plant tissues and suggest that ectomycorrhizal fungi have