Mastering ectomycorrhizal symbiosis: the impact of carbohydrates

Mycorrhiza formation is the consequence of a mutualistic interaction between certain soil fungi and plant roots that helps to overcome nutritional limitations faced by the respective partners. In symbiosis, fungi contribute to tree nutrition by means of mineral weathering and mobilization of nutrients from organic matter, and obtain plant-derived carbohydrates as a response. Support with easily degradable carbohydrates seems to be the driving force for fungi to undergo this type of interaction. As a consequence, the fungal hexose uptake capacity is strongly increased in Hartig net hyphae of the model fungi Amanita muscaria and Laccaria bicolor. Next to fast carbohydrate uptake and metabolism, storage carbohydrates are of special interest. In functional A. muscaria ectomycorrhizas, expression and activity of proteins involved in trehalose biosynthesis is mainly localized in hyphae of the Hartig net, indicating an important function of trehalose in generation of a strong carbon sink by fungal hyphae. In symbiosis, fungal partners receive up to approximately 19 times more carbohydrates from their hosts than normal leakage of the root system would cause, resulting in a strong carbohydrate demand of infected roots and, as a consequence, a more efficient plant photosynthesis. To avoid fungal parasitism, the plant seems to have developed mechanisms to control carbohydrate drain towards the fungal partner and link it to the fungus-derived mineral nutrition. In this contribution, current knowledge on fungal strategies to obtain carbohydrates from its host and plant strategies to enable, but also to control and restrict (under certain conditions), carbon transfer are summarized.     

Accepted 17 Jun. 2008Received 16 Mar. 2008

Trehalose is a non-reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose-6-phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In.silico expression profiling of all Arabidopsis trehalose-6-phosphate synthases (TPSs) and trehalose-6-phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

Extensive expression regulation and lack of heterologous enzymatic activity of the Class II trehalose metabolism proteins from Arabidopsis thaliana

Trehalose metabolism has profound effects on plant growth and metabolism, but the mechanisms involved are unclear. In Arabidopsis , 21 putative trehalose biosynthesis genes are classified in three subfamilies based on their similarity with yeast TPS1 (encoding a trehalose-6-phosphate synthase, TPS) or TPS2 (encoding a trehalose-6-phosphate phosphatase, TPP). Although TPS1 (Class I) and TPPA and TPPB (Class III) proteins have established TPS and TPP activity, respectively, the function of the Class II proteins (AtTPS5-AtTPS11) remains elusive. A complete set of promoter- β -glucurinidase/green fluorescent protein reporters demonstrates their remarkably differential tissue-specific expression and responsiveness to carbon availability and hormones. Heterologous expression in yeast furthermore suggests that none of the encoded enzymes displays significant TPS or TPP activity, consistent with a regulatory rather than metabolic function for this remarkable class of proteins.     

Trehalose synthesis inhibitor: A molecular in silico drug design

Infectious diseases are serious public health problems, affecting a large portion of the world's population. A molecule that plays a key role in pathogenic organisms is trehalose and recently has been an interest in the metabolism of this molecule for drug development. The trehalose-6-phosphate synthase (TPS1) is an enzyme responsible for the biosynthesis of trehalose-6-phosphate (T6P) in the TPS1/TPS2 pathway, which results in the formation of trehalose. Studies carried out by our group demonstrated the inhibitory capacity of T6P in the TPS1 enzyme from Saccharomyces cerevisiae, preventing the synthesis of trehalose. By in silico techniques, we compiled sequences and experimentally determined structures of TPS1. Sequence alignments and molecular modeling were performed. The generated structures were submitted in validation of algorithms, aligned structurally and analyzed evolutionarily. Molecular docking methodology was applied to analyze the interaction between T6P and TPS1 and ADMET properties of T6P were analyzed. The results demonstrated the models created presented sequence and structural similarities with experimentally determined structures. With the molecular docking, a cavity in the protein surface was identified and the molecule T6P was interacting with the residues TYR-40, ALA-41, MET-42, and PHE-372, indicating the possible uncompetitive inhibition mechanism provided by this ligand, which can be useful in directing the molecular design of inhibitors. In ADMET analyses, T6P had acceptable risk values compared with other compounds from World Drug Index. Therefore, these results may present a promising strategy to explore to develop a broad-spectrum antibiotic of this specific target with selectivity, potency, and reduced side effects, leading to a new way to treat infectious diseases like tuberculosis and candidiasis.     

The cellular structure of the Hartig net: coenocytic and transfer cell-like organization

The organization of the Hartig net in the mature state was studied in mycorrhizas of Amanita muscaria (Pers. ex. Fries) Hook. Picea abies (L.) Karst. grown in vitro. The tips of the fan-like branched hyphae contain dense cytoplasm with a large number of mitochondria and rER frequently stretched in the direction of the hyphal growth, indicating that active transfer of solutes between host and fungus is localized here. Lack of septation of the hyphae and their intimate juxtaposition, leaving no space in between, result in a coenocytic, transfer cell-like structure of the Hartig net. The multinucleate status of the cells is shown. The advantage of this organization for nutrient exchange between host and fungus and for nutrient transport within the hyphal system is discussed.     

Improved drought tolerance without undesired side effects in transgenic plants producing trehalose

Most organisms naturally accumulating trehalose upon stress produce the sugar in a two-step process by the action of the enzymes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Transgenic plants overexpressing TPS have shown enhanced drought tolerance in spite of minute accumulation of trehalose, amounts believed to be too small to provide a protective function. However, overproduction of TPS in plants has also been found combined with pleiotropic growth aberrations. This paper describes three successful strategies to circumvent such growth defects without loosing the improved stress tolerance. First, we introduced into tobacco a double construct carrying the genes TPS1 and TPS2 (encoding TPP) from Saccharomyces cerevisiae. Both genes are regulated by an Arabidopsis RuBisCO promoter from gene AtRbcS1A giving constitutive production of both enzymes. The second strategy involved stress-induced expression by fusing the coding region of ScTPS1 downstream of the drought-inducible Arabidopsis AtRAB18 promoter. In transgenic tobacco plants harbouring genetic constructs with either ScTPS1 alone, or with ScTPS1 and ScTPS2 combined, trehalose biosynthesis was turned on only when the plants experienced stress. The third strategy involved the use of AtRbcS1A promoter together with a transit peptide in front of the coding sequence of ScTPS1, which directed the enzyme to the chloroplasts. This paper confirms that the enhanced drought tolerance depends on unknown ameliorated water retention as the initial water status is the same in control and transgenic plants and demonstrates the influence of expression of heterologous trehalose biosynthesis genes on Arabidopsis root development.     

TREHALOSE SYNTHESIS IN EUGLENA GRACILIS (EUGLENOPHYCEAE) OCCURS THROUGH AN ENZYME COMPLEX1

The aim of this study was to isolate and characterize a trehalose‐synthesizing enzyme from Euglena gracilis Klebs. After purification by anion exchange chromatography, gel filtration, isoelectric focusing, and native electrophoresis, trehalose‐6‐phosphate synthase (TPS, EC 2.4.1.15) and trehalose‐6‐phosphate phosphatase (TPP, EC 3.1.3.12) activities could not be separated. Consequently, a TPS/TPP enzyme complex of about 250 kDa was suggested as responsible for trehalose synthesis in E. gracilis. The TPS activity was shown to be highly specific for glucose‐6‐P, and UDP‐Glc was the preferred glucose donor, but GDP‐Glc and CDP‐Glc could also act as TPS substrates. The TPP activity was highly specific for trehalose‐6‐P. In vitro phosphorylation assays revealed rapid decreases in TPS and TPP activities. These changes corresponded to variations in the elution profile of gel filtration chromatography after the phosphorylation treatment. Taken together, these results suggest that the proposed TPS/TPP complex might be regulated through a protein phosphorylation/dephosphorylation‐mediated mechanism that could affect the association state of the complex. Such a regulatory mechanism might lead to a rapid change in trehalose synthesis in response to variations in environmental conditions.     

Genetic Transformation of Tobacco with the Trehalose Synthase Gene from Grifola frondosa Fr. Enhances the Resistance to Drought and Salt in Tobacco

Trehalose is a non-reducing disaccharide of glucose that functions as a protectant in the stabilization of biological structures and enhances the tolerance of organisms to abiotic stress. In the present study, we report on the expression of the Grifolafrondosa Fr. trehalose synthase (TSase) gene for manipulating abiotic stress tolerance in tobacco (Nicotiana tabaccum L.). The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and was transferred into tobacco by Agrobacterium tumefaciens EHA105. Compared with non-transgenic plants, transgenic plants were able to accumulate high levels of products of trehalose, which were increased up to 2.126-2.556 mg/g FW, although levels were undetectable in non-transgenic plants. This level of trehalose in transgenic plants was 400-fold higher than that of transgenic tobacco plants cotransformed with Escherichia coli TPS and TPP on independent expression cassettes, twofold higher than that of transgenic rice plants transformed with a bifunctional fusion gene (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of E. coli, and 12-fold higher than that of transgenic tobacco plants transformed the yeast TPS1 gene.It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and had morphological alterations of their roots. Interestingly, our transgenic plants have obvious morphological changes, including thick and deep-coloured leaves, but show no growth inhibition; moreover, these morphological changes can restore to normal type in T2 progenies. Trehalose accumulation in 35S-35S:TSase plants resulted in increased tolerance to drought and salt, as shown by the results of tests on drought, salt tolerance, and drought physiological indices, such as water content in excised leaves, malondialdehyde content, chlorophyll a and b contents, and the activity of superoxide dismutase and peroxidase in excised leaves. These results suggest that transgenic plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought and salt.     

Trehalose Biosynthesis in Response to Abiotic Stresses

Trehalose is a non‐reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose‐6‐phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In‐silico expression profiling of all Arabidopsis trehalose‐6‐phosphate synthases (TPSs) and trehalose‐6‐phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

Accepted 17 Jun. 2008Received 16 Mar. 2008

Trehalose is a non-reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose-6-phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In.silico expression profiling of all Arabidopsis trehalose-6-phosphate synthases (TPSs) and trehalose-6-phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

红色亚栖热菌TPS/TPP海藻糖合成途径中相关基因的克隆、表达及功能鉴定

通过构建红色亚栖热菌(Meiothermus ruberCBS-01)的基因组DNA文库,克隆得到该嗜热菌海藻糖合成途径中的磷酸海藻糖合成酶(TPS)和磷酸海藻糖磷酸酯酶(TPP)基因。以pET21a为表达载体,将磷酸海藻糖合成酶和磷酸海藻糖磷酸酯酶在大肠杆菌中进行表达并纯化,利用薄层层析的方法验证了这两个酶的活性。同时,本研究检测了红色亚栖热菌在各种环境压力下细胞内含物成分的变化情况,发现在高渗环境压力的诱导下,该菌会在胞内积累大量的6-磷酸海藻糖,而并非海藻糖,这为进一步研究TPS/TPP和TreS途径在细胞体内的作用奠定了基础。     

Trehalose metabolism in Arabidopsis: occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues

Axenically grown Arabidopsis thaliana plants were analysed for the occurrence of trehalose. Using gas chromatography-mass spectrometry (GC-MS) analysis, trehalose was unambiguously identified in extracts from Arabidopsis inflorescences. In a variety of organisms, the synthesis of trehalose is catalysed by trehalose-6-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-6-phosphate phosphatase (TPP; EC 3.1.3.12). Based on EST (expressed sequence tag) sequences, three full-length Arabidopsis cDNAs whose predicted protein sequences show extensive homologies to known TPS and TPP proteins were amplified by RACE-PCR. The expression of the corresponding genes, AtTPSA, AtTPSB and AtTPSC, and of the previously described TPS gene, AtTPS1, was analysed by quantitative RT-PCR. All of the genes were expressed in the rosette leaves, stems and flowers of Arabidopsis plants and, to a lower extent, in the roots. To study the role of the Arabidopsis genes, the AtTPSA and AtTPSC cDNAs were expressed in Saccharomyces cerevisiae mutants deficient in trehalose synthesis. In contrast to AtTPS1, expression of AtTPSA and AtTPSC in the tps1 mutant lacking TPS activity did not complement trehalose formation after heat shock or growth on glucose. In addition, no TPP function could be identified for AtTPSA and AtTPSC in complementation studies with the S. cerevisiae tps2 mutant lacking TPP activity. The results indicate that while AtTPS1 is involved in the formation of trehalose in Arabidopsis, some of the Arabidopsis genes with homologies to known TPS/TPP genes encode proteins lacking catalytic activity in trehalose synthesis.     

Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase of Escherichia coli

To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in E. coli and purified to near homogeneity; its molecular weight was 88,300, as expected. The K(m) values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, the k(cat) values of TPSP were similar to those of TPS/TPP, resulting in a 3.5- to 4.0-fold increase in the catalytic efficiency (k(cat)/K(m)). The K(m) and k(cat) values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-beta-D-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.     

Trehalose-6-phosphate synthase/phosphatase complex from bakers' yeast: purification of a proteolytically activated form.

A protein of about 800 kDa with trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) activity was purified from bakers' yeast. This TPS/P complex contained 57, 86 and 93 kDa polypeptides. The 86 and 93 kDa polypeptides both appeared to be derived from a polypeptide of at least 115 kDa in the native enzyme. A TPS-activator (a dimer of 58 kDa subunits) was also purified. It decreased the Michaelis constants for both UDP-glucose (three-fold) and glucose 6-phosphate (G6P) (4.5-fold), and increased TPS activity at 5 mM-UDP-glucose/10 mM-G6P about three-fold. It did not affect TPP activity. The purification of TPS/P included an endogenous proteolytic step that increased TPS activity about three-fold and abolished its requirement for TPS-activator, but did not change TPP activity. This activation was accompanied by a decrease of some 20 kDa in the molecular mass of a cluster of SDS-PAGE bands at about 115 kDa recognized by antiserum to pure TPS/P, but by no change in the 57 kDa band. Phosphate inhibited TPS activity (Ki about 5 mM), but increased TPP activity about six-fold (Ka about 4 mM). Phosphate (6 mM) stimulated the synthesis of trehalose from G6P and UDP-glucose and decreased the accumulation of trehalose 6-phosphate.     

Contribution of trehalose biosynthetic pathway to drought stress tolerance of Capparis ovata Desf

Trehalose and the trehalose biosynthetic pathway are important contributors and regulators of stress responses in plants. Among recent findings for trehalose and its metabolism, the role of signalling in the regulation of growth and development and its potential for use as a storage energy source can be listed. The xerophytic plant Capparis ovata (caper) is well adapted to drought and high temperature stress in arid and semi‐arid regions of the Mediterranean. The contribution of trehalose and the trehalose biosynthetic pathway to drought stress responses and tolerance in C. ovata are not known. We investigated the effects of PEG‐mediated drought stress in caper plants and analysed physiological parameters and trehalose biosynthetic pathway components, trehalose‐6‐phosphate synthase (TPS), trehalose‐6‐phosphate phosphatase (TPP), trehalase activity, trehalose and proline content in drought stress‐treated and untreated plants. Our results indicated that trehalose and the trehalose biosynthetic pathway contributed to drought stress tolerance of C. ovata. Overall growth and leaf water status were not dramatically affected by drought, as both high relative growth rate and relative water content were recorded even after 14 days of drought stress. Trehalose accumulation increased in parallel to induced TPS and TPP activities and decreased trehalase activity in caper plants on day 14. Constitutive trehalose levels were 28.75 to 74.75 μg·g·FW^?1, and drought stress significantly induced trehalose accumulation (385.25 μg·g·FW^?1 on day 14) in leaves of caper. On day 14 of drought, proline levels were lower than on day 7. Under drought stress the discrepancy between trehalose and proline accumulation trends might result from the mode of action of these osmoprotectant molecules in C. ovata.     

Characterization and identification of field ectomycorrhizae of Boletus edulis and Cistus ladanifer

Field ectomycorrhizae sampled under Boletus edulis and Cistus ladanifer have been characterized and described in detail based on standard morphological and anatomical characters. The described ectomycorrhiza has traits typical of Boletales: whitish with three differentiated plectenchymatous layers in the mantle in plan view forming ring-like structures and rhizomorphs with highly differentiated hyphae. The inflated, smooth cystidia-like clavate end cells on the surface of the rhizomorphs and their slightly twisted external hyphae are additional characterizing features. The Hartig net occupies 1 1/2 rows of cortical cells, partly reaching the endodermis. Not all hyphae have clamps. The identification of the fungal symbiont as B. edulis was confirmed by ITS rDNA sequence comparison between mycorrhizas and sporocarps. The singularity of this symbiotic association, as well as its ecological and practical implications, are discussed.