Diversification of NRT2 and the origin of its fungal homolog

We investigated the origin and diversification of the high-affinity nitrate transporter NRT2 in fungi and other eukaryotes using Bayesian and maximum parsimony methods. To assess the higher-level relationships and origins of NRT2 in eukaryotes, we analyzed 200 amino acid sequences from the Nitrate/Nitrite Porter (NNP) Family (to which NRT2 belongs), including 55 fungal, 41 viridiplantae (green plants), 11 heterokonts (stramenopiles), and 87 bacterial sequences. To assess evolution of NRT2 within fungi and other eukaryotes, we analyzed 116 amino acid sequences of NRT2 from 58 fungi, 40 viridiplantae (green plants), 1 rhodophyte, and 5 heterokonts, rooted with 12 bacterial sequences. Our results support a single origin of eukaryotic NRT2 from 1 of several clades of mostly proteobacterial NNP transporters. The phylogeny of bacterial NNP transporters does not directly correspond with bacterial taxonomy, apparently due to ancient duplications and/or horizontal gene transfer events. The distribution of NRT2 in the eukaryotes is patchy, but the NRT2 phylogeny nonetheless supports the monophyly of major groups such as viridiplantae, flowering plants, monocots, and eudicots, as well as fungi, ascomycetes, basidiomycetes, and agaric mushrooms. At least 1 secondary origin of eukaryotic NRT2 via horizontal transfer to the fungi is suggested, possibly from a heterokont donor. Our analyses also suggest that there has been a horizontal transfer of nrt2 from a basidiomycete fungus to an ascomycete fungus and reveal a duplication of nrt2 in the ectomycorrhizal mushroom genus, Hebeloma.     

An update on nutrient transport processes in ectomycorrhizas

Nutrient transport, namely absorption from the soil solution as well as nutrient transfer from fungus to plant and carbon movement from plant to fungus are key features of mycorrhizal symbiosis. This review summarizes our current understanding of nutrient transport processes in ectomycorrhizal fungi and ectomycorrhizas. The identification of nutrient uptake mechanisms is a key issue in understanding nutrition of ectomycorrhizal plants. With the ongoing functional analysis of nutrient transporters, identified during sequencing of fungal and tree genomes, a picture of individual transport systems should be soon available, with their molecular functions assessed by functional characterization in, e.g., yeast mutant strains or Xenopus oocytes. Beyond the molecular function, systematic searches for knockout mutants will allow us to obtain a full understanding of the role of the individual transporter genes in the physiology of the symbionts. The mechanisms by which fungal and plant cells obtain, process and integrate information regarding nutrient levels in the external environment and the plant demand will be analyzed.     

Microsatellite markers for Hebeloma species developed from expressed sequence tags in the ectomycorrhizal fungus Hebeloma cylindrosporum

The increasing number of expressed sequence tag (EST) projects dedicated to ectomycorrhizal fungi is translating into the release of large sets of ESTs. The aim of this study was to develop and test simple sequence repeat (SSR) markers from EST databases of the model ectomycorrhizal fungus Hebeloma cylindrosporum. Six SSR markers were found to be both unambiguously scorable and polymorphic among 12 H. cylindrosporum isolates. Two SSR markers were transferable to other Hebeloma species and one marker was interestingly found to be polymorphic among seven H. crustuliniforme isolates.     

Sucrose Utilization of the Ectomycorrhizal Fungi Amanita muscaria and Hebeloma crustuliniforme Depends on the Cell Wall-bound Invertase Activity of their Host Picea abies

The role of apoplastic invertase (β-d -fructofuranoside — fructohydrolase, EC 3.2.1.26) of the host Picea abies for carbohydrate uptake and growth of two of its natural ectomycorrhiza partners was studied. For that purpose, hyphae of Amanita muscaria (Pers. ex Fries) Hock. and Hebeloma crustuliniforme (Bull. ex Fries) Quell., as well as roots and suspension cultured cells of Picea abies (L.) Karst. were used. Apoplastic invertase activity was demonstrated on roots and suspension cultured cells of spruce (in the latter case with 21.7 nkat (g fresh weight)^?1). Inhibition of the root cell wall invertase activity (pH optimum 4.5) by increasing the apoplastic pH allowed determination of the permanent release of sucrose from the root. However, under in vivo conditions at a lower cell wall pH the hydrolysation products glucose and fructose were predominantly found. In contrast to spruce cells and certain fungi, such as Saccharomyces (Novick et al., 1981) or Phycomyces (Ruiz-Herrera et al., 1989) invertase activity of the mycorrhizal fungi Hebeloma and Amanita was negligibly low. Furthermore, sucrose could not be consumed by Amanita and Hebeloma. As a consequence, cultures of these mycorrhizal fungi starved when kept on media with sucrose as sole carbohydrate source. But addition of invertase initiated hyphal growth immediately. Studies on carbohydrate uptake of host and fungal cells confirmed that the monosaccharides glucose and fructose were readily incorporated by spruce and fungal cells, with a clear preference for glucose. From these results it is suggested that apoplastic invertase activity of the host Picea abies is a precondition for the utilization of sucrose by the studied mycorrhizal fungi during the nutritional interaction of the symbiotic partners.     

Ammonium-assimilating enzymes and their regulation in wild and NADP-glutamate dehydrogenase-deficient strains of the ectomycorrhizal fungus Hebeloma cylindrosporum

Hebeloma cylindrosporum strain h 17 was grown on media containing either glutamate or ammonium as nitrogen source. Growth tests and in vitro activity measurements revealed that both glutamine synthetase (GS. EC 6.3.1.2) and NADP-specific glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) are fully functional in wild type mycelia grown on glutamate or ammonium as sole nitrogen source. However, NADP-GDH appeared to be more active than GS in stationary growing mycelia. NADP-GDH is also able to sustain adequate ammonium assimilation in methionine sulfoximine (MSX)-treated mycelia since they grew as well as mycelia fed with ammonium alone. The NADP-GDH also appeared to be L-glutamate inducible whereas GS was repressed by ammonium. The NADP-GDH deficient strain, when transferred from a glutamate containing medium to an ammonium containing medium, exhibited a derepressed GS, although this enzyme did not fully substitute for the deficiency of NADP-GDH in ammonium assimilation. The low NADP-GDH activity of the mutant strain exhibited a reduced mobility on a 6% constant polyacrylamide gel. By contrast, the two enzymes had identical molecular weights, estimated to be ca 295 kDa on gradient polyacrylamide gel. The involvement of NADP-GDH and GS enzymes in nitrogen assimilation is discussed.     

Nitrate reductase activity of nonmycorrhizal Douglas-fir rootlets and of some associated mycorrhizal fungi

Douglas-fir root tips reduced nitrate at much higher rates than seven mycorrhizal fungi, which also differed between species in rate of nitrate reduction. Results are related to nitrogen nutrition of Douglas-fir.Excised, nonmycorrhizal root tips of Douglas-fir reduced nitrate at much higher rates than seven mycorrhizal fungi commonly associated with Douglas-fir. The fungi differed significantly in degree of activity:Cenococcum geophilum, Piloderma bicolor, andRhizopogon vinicolor were highest;Amanita muscaria was intermediate; andHebeloma crustuliniforme, Thelephora terrestris, andLaccaria laccata were lowest.     

Wood mechanics, allometry, and life-history variation in a tropical rain forest tree community

Wood density plays a central role in the life-history variation of trees, and has important consequences for mechanical properties of wood, stem and branches, and tree architecture. Wood density, modulus of rupture, modulus of elasticity, and safety factors for buckling and bending were determined for saplings of 30 Bolivian rain forest tree species, and related to two important life-history axes: juvenile light demand and maximum adult stature. Wood density was strongly positively related to wood strength and stiffness. Species safety factor for buckling was positively related to wood density and stiffness, but tree architecture (height : diameter ratio) was the strongest determinant of mechanical safety. Shade-tolerant species had dense and tough wood to enhance survival in the understorey, whereas pioneer species had low-density wood and low safety margins to enhance growth in gaps. Pioneer and shade-tolerant species showed opposite relationships between species traits and adult stature. Light demand and adult stature affect wood properties, tree architecture and plant performance in different ways, contributing to the coexistence of rain forest species.