Sugar- and Nitrogen-Dependent Regulation of an Amanita muscaria Phenylalanine Ammonium Lyase Gene

Abstract

The cDNA of a key enzyme of secondary metabolism, phenylalanine ammonium lyase, was identified for an ectomycorrhizal fungus by differential screening of a mycorrhizal library. The gene was highly expressed in hyphae grown at low external monosaccharide concentrations, but its expression was 30-fold reduced at elevated concentrations. Gene repression was regulated by hexokinase.

Together with fine roots of woody plants a wide range of soil fungi form a characteristic symbiotic structure, i.e., the ectomycorrhiza. During mycorrhizal development both fungal hyphae and root cortical cells undergo a number of morphological changes (15). Fully developed ectomycorrhizas are characterized by a fungal mantle ensheathing the infected root and a labyrinthic network of highly branched hyphae between root cortical cells, i.e., the Hartig net. In addition, physiological adaptation which enables the function of symbiosis, including the controlled exchange of metabolites and nutrients between the two types of organisms, takes place (13, 24). Most important in this respect is the exchange of plant-derived carbohydrates for fungus-derived amino acids (10, 17).

In a recent study, we showed that the expression of a fungal monosaccharide transporter gene is highly dependent on both the external concentration of monosaccharides and the period of exposure to monosaccharides (18).

Identification of an A. muscaria PAL.

As a result of application of a procedure for differential screening (19) of a cDNA library obtained from Picea abies/Amanita muscaria mycorrhizas (19a), a clone that codes for a phenylalanine ammonium lyase (PAL) was identified. This cDNA (nucleotide sequence accession no. AJ010143) has a length of 2,311 bp and codes for a protein of 740 amino acids with a molecular mass of 80,167 Da. The best homology for the deduced protein was obtained with phenylalanine ammonium lyases of Rhodosporidium toruloides (3) and Rhodotorula rubra (9), two yeast-like basidiomycetes that both showed identities of 41% and similarities of 64% with the deduced protein. A. muscaria pal (Ampal) is encoded by a single-copy gene in the genome of A. muscaria. Digestion of genomic DNA with different restriction enzymes that do not cut within the cDNA fragment of Ampal used for hybridization revealed only single hybridization bands (data not shown).

Sugar- and nitrogen-dependent expression of Ampal.

Expression of R. rubra and R. toruloides pal is repressed by sugar but induced by external phenylalanine (3, 9). Also, for the ascomycete Neurospora crassa a PAL enzyme which is phenylalanine induced and nitrogen repressed but not sugar repressed was described (23).

To determine the effect of external monosaccharides on Ampal expression, increasing hexose concentrations were added to carbon-starved mycelia. Low monosaccharide concentrations (up to 2 mM) resulted in a strong Ampal expression, while at higher hexose concentrations at least 30-fold reduction of Ampal expression was obtained (Fig. 1A).

FIG. 1.

Ampal expression in mycelia grown in the presence of different monosaccharide concentrations, glucose analogues, or nitrogen sources. Changes in the transcript level of Ampal during fungal growth in submersed culture were investigated. Different monosaccharide concentrations, nitrogen sources, or glucose analogues were added to mycelia depleted of glucose (panels A, B, and C) and nitrogen (panel D). Total RNA was extracted as described by Nehls et al. (18), separated on agarose gels, transferred to nylon membranes, and hybridized with either Ampal cDNA or 5.8S rRNA. All Northern blot experiments were performed in triplicate. (A) Effects of different monosaccharide concentrations. Mycorrhiza-free P. abies roots (lanes 1); A. muscaria/P. abies mycorrhizas (lanes 2); mycelia grown in the absence of glucose (lanes 3) or in 0.5 (lanes 4), 2 (lanes 5), 5 (lanes 6), 26 (lanes 7), and 100 (lanes 8) mM glucose; or mycelia grown in 1 (lanes 9) and 28 (lanes 10) mM fructose. (B) Time course of Ampal expression. (C) Effects of glucose analogues. Mycelia were grown in 40 mM glucose (lanes 1), 40 mM fructose (lanes 2), 40 mM 2-deoxyglucose (lanes 3), and 40 mM 3-O-methyl-glucose (lanes 4). (D) Effects of different nitrogen sources. Lanes: 1, no nitrogen; 2, 2 mM KNO₃; 3, 2 mM NH₄⁺; 4, 2 mM leucine; 5, 2 mM phenylalanine; 6, 2 mM alanine; 7, 2 mM arginine; 8, 2 mM glutamine; 9, 2 mM aspartate.

By 40 min after addition of glucose to carbon-depleted mycelia, a fourfold reduction of the Ampal mRNA content had already occurred (Fig. 1B). After 1 h of exposure, the transcript level was approximately 30-fold reduced and close to the limit of detection, revealing that there was a high turnover rate for the Ampal mRNA. Ampal expression started to rise after 16 days of exposure, by which time glucose in the growth medium had been completely consumed. The expression returned to the initial transcript level after 4 further days of growth (data not shown).

To investigate whether Ampal expression is regulated by glucose or by products of glucose metabolism, either glucose analogues or monosaccharides were added to sugar-depleted fungal mycelia (Fig. 1C). Exposure of A. muscaria to 3-O-methyl-glucose, which is imported into cells but does not serve as a substrate for hexokinase (2, 6, 14), like its cultivation in the absence of glucose, led to a strong Ampal gene expression. In contrast, exposure to 2-deoxyglucose (which is phosphorylated by hexokinase but is not further metabolized) resulted in a 30-fold repression of Ampal expression, comparable to that found in the presence of high hexose concentrations. Thus, it could be concluded that monosaccharide-dependent gene repression in A. muscaria occurred in a hexokinase-dependent manner.

Together with recent data about sugar-enhanced expression of an A. muscaria monosaccharide transporter gene (18), these results reveal that in ectomycorrhizal basidiomycetes, as in saprophytic ascomycetes (8, 20), two independent mechanisms of sugar-controlled regulation of gene expression are active.

To examine the effect of nitrogen on Ampal expression, different nitrogen sources were added to nitrogen- and glucose-depleted fungal mycelia (Fig. 1D). In the presence of glucose, Ampal expression was 30-fold decreased, independently of the nitrogen source. In the absence of glucose, addition of amino acids that are not or are only poorly utilized by A. muscaria, e.g., phenylalanine (1), as well as nitrate or ammonium, resulted in maximal Ampal expression. In contrast, amino acids that could be used as a nutrient source (e.g., alanine [1]) led to a sevenfold reduction of Ampal expression. Since ammonium, which is easily used as a nitrogen source, had no effect, the reduction of Ampal expression must have been due to the catabolic degradation of the carbon skeletons of amino acids. Thus, control elements in later stages of glycolysis in addition to hexokinase can be assumed.

Ampal expression in complex fungal tissues.

The Ampal expression level was high in fruiting bodies (Fig. 2). This was surprising because fruiting bodies contain high internal glucose concentrations (26). Since glucose flux through hexokinase is thought to be the signal for sugar-dependent gene repression (22), either the phosphorylation of internal glucose must be low or sugar-dependent gene repression is suppressed by developmental signals in fruiting bodies.

FIG. 2.

Development-dependent expression of Ampal. Lanes: 1, fruiting bodies; 2, P. tremula × tremuloides/A. muscaria mycorrhizas; 3, A. muscaria/P. abies mycorrhizas; 4, A. muscaria mycelium grown in 40 mM glucose.

The symbiotic interaction (12, 15) with the gymnosperm P. abies as well as with the angiosperm Populus tremula × tremuloides led to a strong Ampal gene expression (Fig. 1A and 2). No cross-hybridization with plant mRNA occurred. Due to the fungal access to monosaccharides, increased hexose concentrations can be expected in mycorrhizas. This is supported by an increased expression of an A. muscaria monosaccharide transporter gene in mycorrhizas that occurs only if the hexose concentration exceeds 5 mM (18). As a consequence, Ampal expression should be repressed here. A possible explanation for the strong Ampal expression in mycorrhizas is their structural and functional heterogeneity; mycorrhizas consist of a fungal mantle, which covers the host fine root and hyphae at the fungus/plant interface (the Hartig net) (4, 5, 16, 24). Most probably, only hyphae of the Hartig net and the innermost part of the fungal mantle are exposed to high external monosaccharide concentrations and thus would manifest Ampal repression. Since this fraction of mycorrhizal hyphae makes up only 30 to 50% of the fungal biomass (estimated from microscopical analysis of cross sections) a reduction of Ampal expression of less than a factor of two could be expected in mycorrhizas. This is in agreement with the observed data.

PAL activity is important for cells in two aspects: catabolic degradation of phenylalanine (23) and synthesis of phenolic compounds (21). Since A. muscaria cannot make use of phenylalanine as a nutrient source (1), catabolic degradation of phenylalanine by AmPAL could be excluded. Therefore, it is rather likely that AmPAL is involved in the synthesis of phenolic compounds. This assumption is confirmed by the presence of high contents of phenolic compounds in A. muscaria fruiting bodies (7), where Ampal expression was high.

Secondary metabolites, e.g., phenolic compounds, are involved in the protection against invasion of pathogenic microorganisms as well as in competitor defense (11, 24). Due to sugar-dependent regulation of Ampal expression, high contents of phenolic compounds could thus be assumed to be present in all hyphae that are in contact with other microorganisms, including fungal hyphae in soil as well as in the fungal mantle of ectomycorrhizas. In these areas the external hexose concentration is low (25). This synthesis of antimicrobial phenolic compounds, which occurs preferentially in the fungal mantle, would thus explain the observed host root protection in mycorrhizal symbiosis (11, 24), which is of great ecological importance.