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Microorganisms have evolved different strategies to cope with environmental stresses. Stress induces a cellular stage that usually leads to the synthesis of specific proteins and metabolites in order to adapt and survive until better conditions return. Heat-shock proteins or hydrophilins are a good example, conserved in evolution, of common cell response against hostile environmental conditions (Garay-Arroyo et al., 2000; Hohmann, 2002). A common response of organisms to drought, salinity, and low-temperature stresses is the accumulation of sugars and other compatible solutes as osmoprotectants that act by stabilizing biomolecules (Voit, 2003). One such compound is trehalose (α-D-glucopyranosyl, α-D-glucopyranoside), a nonreducing disaccharide, consisting of two molecules of glucose bound by an α,α-1,1-glycosidic linkage. Trehalose is also present in bacteria, eukaryotic microorganisms, plants, insects and invertebrates, but up to now has not been found in vertebrates (Benaroudj et al., 2001). In all organisms where trehalose is present, it has been found to play an important physiological role as protectant against abiotic stress (Crowe et al., 1992). Trehalose accumulation to protect the cell is likely to be an evolutionarily old mechanism, since even Archaebacteria accumulate trehalose in response to stress (Nicolaus et al., 1988). Trehalose protects the cells by stabilizing cell structures and enables proteins to maintain their native conformation under stress conditions (Singer & Lindquist, 1998; Eroglu et al., 2000). Interestingly, the presence of trehalose interferes with protein refolding, and that explains why trehalose is quickly degraded after stress has ceased (Singer & Lindquist, 1998). Trehalose is commonly found in fungi and at particularly high concentrations in resting cells and survival forms, such as spores and sclerotia. In addition to its major function as carbohydrate reserve, and similarly to other organisms, in fungi trehalose also protects against several adverse conditions such as heat, desiccation, freezing (van Laere, 1989), hydrostatic pressure (Iwahashi et al., 2000), nutrient starvation, and several abiotic stresses such as osmotic, oxidative or chemical stresses (Zähringer et al., 2000). In arbuscular mycorrhizal (AM) fungi, trehalose and glycogen are the main storage carbohydrates (Bécard et al., 1991). However, these fungi preferentially accumulate carbon (C) in the form of triacylglycerols (TAGs) (up to 95%) during their life cycle. AM fungi obtain the C through the symbiosis with their partner, the plant. Carbon is drawn from the root cortex, by the so-called intraradical mycelium in the form of hexoses and transformed quickly to trehalose and glycogen, and further to lipids (Shachar-Hill et al., 1995; Pfeffer et al., 1999). Translocation of C to the distal part of the fungal colony, the extraradical mycelium, has been shown to take place mainly in the form of glycogen and TAGs (Bago et al., 2003). This extraradical mycelium spreading into the soil is responsible for the mineral nutrient uptake that benefits its plant host, and ultimately produces the asexual propagules, the chlamydospores, that complete the life cycle. Trehalose has been shown to be present in extraradical mycelium as well as in spores of AM fungi, and in all cases it has been attributed a mere role as intermediate C storage (Bécard et al., 1991; Bago et al., 2002, 2003). Although there are reports showing the accumulation of trehalose in mycorrhizal plants in response to drought stress (Schellenbaum et al., 1998, 1999), the possible role of trehalose as cellular stress protectant in extraradical mycelium has not been investigated.
In yeast and other fungi, trehalose synthesis takes place in a two-step process catalyzed by the trehalose synthase enzyme complex comprising four subunits (Bell et al., 1992; de Virgilio et al., 1993; Vuorio et al., 1993; Reinders et al., 1997). In the first step, a glucosyl residue is transferred from uridine-5-diphosphoglucose (UDPG) to glucose-6-phosphate to form trehalose-6-phosphate. This reaction is catalyzed by the trehalose-6-phosphate synthase subunit (TPS1). In a second step, the phosphate group is removed from trehalose-6-phosphate to render trehalose and orthophosphate. This step is catalyzed by a second enzyme, the trehalose-6-phosphate phosphatase (TPS2). Two other subunits without catalytic activity have been shown to form part of the complex and to participate in the regulation of the trehalose synthesis, TPS3 and TSL1. Investigations in other fungi, including the filamentous fungus A. nidulans and A. niger, have shown that they synthesize trehalose using a an enzymatic complex that includes at least TPS1 and TPS2 (Borgia et al., 1996; Wolschek & Kubicek, 1997). Expression of TPS1, TPS2, and TSL1 in yeast is strongly stimulated by all stress conditions, while TPS3 expression is only weakly stimulated by heat shock (Winderickx et al., 1996; Gasch et al., 2000; Causton et al., 2001). Mutants for TPS1 and TPS2 are unable to accumulate trehalose and display a low thermotolerance phenotype. None of the genes responsible for trehalose synthesis and their encoding enzymes has been isolated so far in AM fungi.
The hydrolysis of trehalose into two molecules of glucose is performed by trehalases (Jorge et al., 1997). On the basis of catalytic properties, subcellular localization and mechanisms of regulation, trehalases have been grouped into two classes: acid trehalases, extracellular and vacuolar glycoproteins, permanently active, with a high thermal stability and an optimum pH of approx. 4.5 (Wiemken & Schellenberg, 1982; Londesborough & Varimo, 1984); and neutral trehalases, cytosolic proteins with an optimum pH of approx. 7.0, a much lower thermal stability and a lower substrate affinity (Thevelein, 1984). Acid and neutral trehalases have specific and independent roles. They are distinct types of enzymes, which only share a strict specificity for trehalose. Neutral trehalases are considered key enzymes responsible for the internal trehalose breakdown and they are tightly regulated enzymes. Interestingly, it was shown that exposure to different types of stresses also induced neutral trehalase concomitant to the induction of enzymes of trehalose biosynthesis in different fungi (Zähringer et al., 1997, 2000; d’Enfert et al., 1999). Although in principle paradoxical with the concept of trehalose accumulation during stress, the current hypothesis suggests that neutral trehalase activation might be important for recovery after stress by inducing trehalose mobilization. Consistent with this hypothesis, neutral trehalase mutation produces in yeast the so-called ‘poor heat shock recovery phenotype’. These mutants, in contrast to the wild-type, are impaired in growth at normal temperature after exposure for some hours to sublethal heat shock (Nwaka et al., 1995). In AM fungi, trehalase activity with an optimum pH of c. 7 was reported in germinated sporocarps and extraradical mycelium (Schubert et al., 1992; Schubert & Wyss, 1995). However, no further experiments have been carried out.
To investigate in more detail the role of trehalose in AM fungi, we have studied the gene expression and the activity of two key enzymes in the trehalose metabolism, neutral trehalase (trehalose mobilization) and trehalose-6-phosphate phosphatase (trehalose synthesis), and correlated this with trehalose concentrations in the hyphae. This study is aimed, in particular, at ascertaining whether trehalose turnover plays a role during abiotic stress in AM fungi.
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The best-studied trehalose-inducing condition in fungi is heat stress. In response to mild and severe heat shock, fungal cells respond by activating transcriptionally and/or post-transcriptionally enzymes of the trehalose metabolism with an overall effect of trehalose accumulation (de Virgilio et al., 1991; Cansado et al., 1998a,b; Fillinger et al., 2001; Van Dijck et al., 2002; Gancedo & Flores, 2004). Interestingly, not only enzymes from the trehalose synthase complex are induced upon heat and other stresses but also neutral trehalase, responsible for the internal trehalose breakdown. This phenomenon of trehalose futile cycling with activation of anabolic and catabolic enzymes during heat stress was first observed in the yeast S. cerevisiae (Hottiger et al., 1987). In order to gain an insight into how trehalose metabolism was affected by heat stress and other stresses in AM fungi, we assessed trehalose content as well as enzyme activity of neutral trehalase and trehalose-6-P phosphatase in G. intraradices. To complement this data we also isolated the full-length cDNA of a neutral trehalase and partial cDNA of a trehalose-6-P phosphatase and studied their RNA accumulation during abiotic stress. Interestingly, we found that sublethal heat shock consisting of an increase of 10°C above the normal growth temperature induces trehalose accumulation in G. intraradices and this is possibly the result of a combined action of both synthesis and degradation activities. Neutral trehalase appears to be an important enzyme for the recovery from heat shock in AM fungi. Increase of neutral trehalase activity during heat shock has been extensively studied in S. cerevisiae and S. pombe and found to be mediated by both transcriptional and post-translational regulation by cAMP-dependent protein kinase A (PKA)-mediated phosphorylation (de Virgilio et al., 1991; Nwaka et al., 1995; Ribeiro et al., 1997; Cansado et al., 1998a; Zähringer et al., 1998). In contrast to yeast, we did not observe significant transcriptional activation of GiNTH1 upon heat shock, despite the increase of activity at 2 h, suggesting post-translational regulation of the AM fungal enzyme as the main activation mechanism under these conditions. However, we cannot exclude the possibility that other neutral trehalase genes exist and are regulated in a different manner. Similarly to GiNTH1, neutral trehalases from the filamentous fungi A. nidulans and N. crassa were not transcriptionally activated by heat shock (d’Enfert et al., 1999), although recovery from heat shock in A. nidulans produced a modest but significant RNA accumulation of the corresponding genes that, together with a post-translational activation, could explain the trehalose mobilization observed during heat shock recovery in these fungi (d’Enfert et al., 1999). The presence of a putative cAMP-dependent protein kinase phosphorylation site in the neutral trehalase deduced protein in both AM fungi studied is consistent with a post-transcriptional regulation via cAMP-dependent protein kinase (PKA) by phosphorylation, as has been shown for other fungi (Amaral et al., 1997). It is also in agreement with our results, where maximum neutral trehalase activity was achieved after addition of cAMP to the reaction mix (data not shown). The EF-like Ca2+-binding site localized at the N-terminus of both AM fungal proteins has also been identified in all fungal neutral trehalases. This motif could contribute to a further regulation of enzyme activity, as has been proposed for other fungi (Amaral et al., 1997; Franco et al., 2003).
Heat shock also induces expression of TPS2 and its ortholog tpp1+, coding for the phosphatase of the trehalose synthase complex, in S. cerevisiae and S. pombe, respectively. This runs parallel with an increase in trehalose-6-P phosphatase activity (de Virgilio et al., 1993; Franco et al., 2000). Similarly, we observed a moderate transient increase in GiTPS2 RNA accumulation after 1 h of heat shock, parallel to a higher trehalose-6-P phosphatase activity with a maximum at 2 h. This, together with an increased rate of activity of neutral trehalase indicates an increased turnover of trehalose, because its pool size is only slightly elevated (Fig. 4a). However, after 5 h of exposure to heat stress, both enzymes exhibit reduced activities, compared with the 2 h values. Trehalose concentrations after 5 h of heat stress undergo a 10-fold increase with respect to the control values under normal growth conditions. This increase is of a similar magnitude to that observed in C. albicans, where heat shock produces a trehalose increment from 5 to 50 nmol trehalose mg−1 fresh weight (Zaragoza et al., 1998). We thus assume that in the period between 2 and 5 h of stress treatment, down-regulation of the G. intraradices neutral trehalase precedes that of the trehalose phosphatase, in order to achieve the required amount of trehalose able to protect from the stress. This could be achieved by a faster degradation of the neutral trehalase enzyme.
In yeast and other fungi, disruption of the trehalose-6-P phosphatase gene leads, in parallel to the abolition of trehalose-6-P phosphatase activity, to a thermosensitive phenotype of different magnitudes (Piper & Lockheart, 1988; de Virgilio et al., 1993; Borgia et al., 1996; Franco et al., 2000; Van Dijck et al., 2002; Zaragoza et al., 2002). Interestingly, in S. cerevisiae the thermosensitive phenotype of TPS2 mutants was found to be the result of an accumulation of the intermediate trehalose-6-phosphate rather than a decrease in trehalose concentration (Elliot et al., 1996). Experiments in A. nidulans and C. albicans using trehalose-6-P phosphatase mutants indicate a possible correlation between the accumulation of trehalose-6-phosphate and defects in the cell wall biosynthesis/assembly (Borgia et al., 1996; Zaragoza et al., 2002). How do increases in neutral trehalase activity observed during the time-course of heat shock in G. intraradices fit into this model? A possible hypothesis is a two step-protection mechanism vs heat shock, where the cell would first react to maintain low trehalose-6-phosphate concentrations. A decrease in the amount of trehalose-6-phosphate would be achieved by pushing the reaction to trehalose formation (increased GiTPS2 activity) and further to glucose (increased GiNTH1 activity). Both activities would have to be coordinated to produce a net increase of trehalose that would serve as a protectant for, for example, soluble proteins or cell structures. In a second step, net trehalase activity would have to prevail in order to allow trehalose mobilization for recovery after heat shock. Similar distinct roles for the neutral trehalase activity during heat shock and heat shock recovery have been postulated for A. nidulans, N. crassa and S. cerevisiae (d’Enfert et al., 1999; Wera et al., 1999).
In support of a role in trehalose mobilization after heat stress of AM neutral trehalases is the successful complementation of the yeast mutant defective in neutral trehalase with GmNTH1 (see earlier discussion). This mutant, similar to other neutral trehalase yeast mutants, displays the so-called ‘poor heat shock recovery phenotype’ (Nwaka et al., 1995). This is because the lack of neutral trehalase activity prevents the strain from growing properly after 6 h at 50°C. Following transformation and heterologous expression of GmNTH1 under the control of the PMA1 constitutive promoter, the mutant strain was able to recover after heat shock similar to the wild-type strain. It is interesting that despite the fact that GmNTH1 presents higher similarity to the basidiomycetes than to yeast neutral trehalases, the AM fungal protein is able to complement the mutant phenotype. This is not surprising considering that yeast has been proven to be a good model organism for heterologous complementation even across kingdoms (Piotrowski et al., 1998), and has been successfully used for several AM fungal genes (Harrison & van Buuren, 1995; Lanfranco et al., 2005). The functional complementation analysis in yeast using the neutral trehalase gene of G. mosseae confirms the role of neutral trehalase in the recovery after heat shock and proves the functionality of an AM neutral trehalase in this process. Additionally, it suggests that neutral trehalase could promote the recovery after heat shock by activating trehalose hydrolysis. This was confirmed in the recovery experiment, in which concentrations of trehalose quickly returned to basal values after the 24 h recovery period, indicating an active role of the AM neutral trehalase in mobilizing accumulated trehalose after the stress period.
In contrast to the well-established protective role of trehalose during heat stress, it appears that only extreme forms of osmotic stress, such as desiccation and freezing, clearly involve trehalose as an osmoprotectant in yeast (reviewed in Hohmann, 2002). However, in different fungi, moderate osmotic stress induces different trehalose phenotypes. Thus, in S. pombe, there was increased neutral trehalase expression and activity as well as trehalose-6-P phosphatase activity concomitant with increased concentrations of trehalose after exposure to 0.75 m NaCl (Cansado et al., 1998a; Franco et al., 2000). Candida albicans is in general much less effective than S. cerevisiae in accumulating compatible solutes during stress. However, in contrast to S. cerevisiae, osmotic stress (0.3 m NaCl) induces trehalose accumulation and transcription of trehalose-related enzymes to a greater degree than heat or oxidative stress (Enjalbert et al., 2003). In S. cerevisiae 0.5 m NaCl does not induce significant changes in the trehalose content of cells despite increased enzyme activities of TPS1 and NTH1 (Zähringer et al., 2000). The phytopathogenic fungus Botrytis cinerea, which undergoes activation of trehalose metabolism in response to heat stress, is also not responsive to osmotic stress induced by NaCl (0.5–1.5 m) or by sorbitol (0.5–1.0 m) (Döhlemann et al., 2006). Similarly to these two latter fungi, we observed that G. intraradices did not alter its trehalose content in response to 0.5 m NaCl and only underwent moderate transient activations of TPS2 and NTH1 not associated to any transcriptional change.
It has been suggested that chemical stressors (such as arsenite) may modify protein activity or structure by reacting with thiol groups and thus transiently affect cell growth (Chang et al., 1989). Since toxic chemicals did not lead to an increased trehalose concentration in S. cerevisiae cells, it was suggested that an increase in neutral trehalase activity could indicate a direct participation of the enzyme in the defense or detoxification mechanism against this chemical (Zähringer et al., 1997). However, in our study we observed both an increase in neutral trehalase activity and a small but significant increase in trehalose content in response to arsenate. These results could suggest that, in contrast to S. cerevisiae, not only the neutral trehalase enzyme, but also trehalose, is involved in the protection of the fungus against arsenate. It has been shown that mycorrhizal plants accumulate less arsenate than nonmycorrhizal plants when growing on contaminated soils (Liu et al., 2004). As arsenate is transported by the same transporters as orthophosphate, the increased phosphate amount in mycorrhizal plants could account for a lower arsenate accumulation ratio. Since trehalose formation yields inorganic phosphate, this metabolic step has been suggested as a control point for orthophosphate liberation with possible implications in the glycolysis control in yeast (Thevelein & Hohmann, 1995). The trehalose synthase complex is activated allosterically by fructose-6-phosphate and inhibited by free phosphate, implying it would have maximum activity when sugar phosphates accumulate and phosphate drops. NMR studies of mycorrhizal roots show that import of hexoses by intraradical hyphae is quickly channelled to trehalose formation (Shachar-Hill et al., 1995; Pfeffer et al., 1999). In addition, intraradical hyphae are responsible for the transfer of phosphate to the root. In this scenario, one could speculate that synthesis of trehalose/liberation of inorganic phosphate within the fungus stimulated by the hexose import from the plant could favor phosphate liberation/translocation to the plant. Under arsenate stress, increases in trehalose synthesis would fuel this phosphate translocation and decrease the relative accumulation of the toxic arsenate in the plant. At high enough rates of trehalose turnover and sugar phosphate/trehalose shuttling, even the observed low concentrations of trehalose would be sufficient.