Characterization and regulation of PiDur3, a permease involved in the acquisition of urea by the ectomycorrhizal fungus Paxillus involutus

Urea, which is known to be a source of nitrogen for the growth of many organisms, represents an important fertilizer in forest soils. Since most trees form symbiotic associations with ectomycorrhizal fungi, the capacities of these symbionts to take up and assimilate urea would determine the efficiency of urea nitrogen salvaging by plants. We showed that Paxillusinvolutus, an ectomycorrhizal basidiomycete, is capable of using urea as sole nitrogen source. We report the molecular characterization of an active urea transporter (PiDur3) isolated from this fungus. We demonstrated that the import of urea is a minor event on ammonium condition, since the expression of PiDUR3 is repressed by the high intracellular glutamine pool. Interestingly, on urea nutritive condition, the uptake of urea is rather mediated by the intracellular urea pool and particularly by urease efficiency.     

Molecular characterization,function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS,NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum

External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structures of the ectomycorrhizal symbiosis. Here, we have cloned and characterized Hebeloma cylindrosporum AMT1, GLNA and GDHA genes, which encode a third ammonium transporter, a glutamine synthetase and an NADP-dependent glutamate dehydrogenase respectively. Amt1 can fully restore the pseudohyphal growth defect of a Saccharomyces cerevisiae mep2 mutant, and this is the first evidence that a heterologous member of the Mep/Amt family complements this dimorphic change defect. Dixon plots of the inhibition of methylamine uptake by ammonium indicate that Amt1 has a much higher affinity than the two previously characterized members (Amt2 and Amt3) of the Amt/Mep family in H. cylindrosporum. We also identified the intracellular nitrogen pool(s) responsible for the modulation of expression of AMT1, AMT2, AMT3, GDHA and GLNA. In response to exogenously supplied ammonium or glutamine, AMT1, AMT2 and GDHA were downregulated and, therefore, these genes are subjected to nitrogen repression in H. cylindrosporum. Exogenously supplied nitrate failed to induce a downregulation of the five mRNAs after transfer of mycelia from a N-starved condition. Our results demonstrate that glutamine is the main effector for AMT1 and AMT2 repression, whereas GDHA repression is controlled by intracellular ammonium, independently of the intracellular glutamine or glutamate concentration. Ammonium transport activity may be controlled by intracellular NH4+. AMT3 and GLNA are highly expressed but not highly regulated. A model for ammonium assimilation in H. cylindrosporum is presented.     

Identification of genes differentially expressed in extraradical mycelium and ectomycorrhizal roots during Paxillus involutus-Betula pendula ectomycorrhizal symbiosis

The development of ectomycorrhizal symbiosis leads to drastic changes in gene expression in both partners. However, little is known about the spatial regulation of symbiosis-regulated genes. Using cDNA array profiling, we compared the levels of expression of fungal genes corresponding to approximately 1,200 expressed sequenced tags in the ectomycorrhizal root tips (ECM) and the connected extraradical mycelium (EM) for the Paxillus involutus-Betula pendula ectomycorrhizal association grown on peat in a microcosm system. Sixty-five unique genes were found to be differentially expressed in these two fungal compartments. In ECM, a gene coding for a putative phosphatidylserine decarboxylase (Psd) was up-regulated by 24-fold, while genes coding for urea (Dur3) and spermine (Tpo3) transporters were up-regulated 4.1- and 6.2-fold in EM. Moreover, urea was the major nitrogen compound found in EM by gas chromatography-mass spectrometry analysis. These results suggest that (i) there is a spatial difference in the patterns of fungal gene expression between ECM and EM, (ii) urea and polyamine transporters could facilitate the translocation of nitrogen compounds within the EM network, and (iii) fungal Psd may contribute to membrane remodeling during ectomycorrhiza formation.     

Identification of Genes Differentially Expressed in Extraradical Mycelium and Ectomycorrhizal Roots during Paxillus involutus-Betula pendula Ectomycorrhizal Symbiosis

The development of ectomycorrhizal symbiosis leads to drastic changes in gene expression in both partners. However, little is known about the spatial regulation of symbiosis-regulated genes. Using cDNA array profiling, we compared the levels of expression of fungal genes corresponding to approximately 1,200 expressed sequenced tags in the ectomycorrhizal root tips (ECM) and the connected extraradical mycelium (EM) for the Paxillus involutus-Betula pendula ectomycorrhizal association grown on peat in a microcosm system. Sixty-five unique genes were found to be differentially expressed in these two fungal compartments. In ECM, a gene coding for a putative phosphatidylserine decarboxylase (Psd) was up-regulated by 24-fold, while genes coding for urea (Dur3) and spermine (Tpo3) transporters were up-regulated 4.1- and 6.2-fold in EM. Moreover, urea was the major nitrogen compound found in EM by gas chromatography-mass spectrometry analysis. These results suggest that (i) there is a spatial difference in the patterns of fungal gene expression between ECM and EM, (ii) urea and polyamine transporters could facilitate the translocation of nitrogen compounds within the EM network, and (iii) fungal Psd may contribute to membrane remodeling during ectomycorrhiza formation.     

AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient Arabidopsis roots

Despite the fact that urea is a ubiquitous nitrogen source in soils and the most widespread form of nitrogen fertilizer used in agricultural plant production, membrane transporters that might contribute to the uptake of urea in plant roots have so far been characterized only in heterologous systems. Two T-DNA insertion lines, atdur3-1 and atdur3-3, that showed impaired growth on urea as a sole nitrogen source were used to investigate a role of the H+/urea co-transporter AtDUR3 in nitrogen nutrition in Arabidopsis. In transgenic lines expressing AtDUR3-promoter:GFP constructs, promoter activity was upregulated under nitrogen deficiency and localized to the rhizodermis, including root hairs, as well as to the cortex in more basal root zones. Protein gel blot analysis of two-phase partitioned root membrane fractions and whole-mount immunolocalization in root hairs revealed the plasma membrane to be enriched in AtDUR3 protein. Expression of the AtDUR3 gene in nitrogen-deficient roots was repressed by ammonium and nitrate but induced after supply of urea. Higher accumulation of urea in roots of wild-type plants relative to atdur3-1 and atdur3-3 confirmed that urea was the substrate transported by AtDUR3. Influx of 15N-labeled urea in atdur3-1 and atdur3-3 showed a linear concentration dependency up to 200 microM external urea, whereas influx in wild-type roots followed saturation kinetics with an apparent Km of 4 microM. The results indicate that AtDUR3 is the major transporter for high-affinity urea uptake in Arabidopsis roots and suggest that the high substrate affinity of AtDUR3 reflects an adaptation to the low urea levels usually found in unfertilized soils.     

Degradation and compartmentalization of urea in Chlamydomonas reinhardii

Urea is accumulated against a concentration gradient in Chlamydomonas reinhardii. Only energy generated from photosynthesis is used for this accumulation, while degradation of urea utilizes other energy sources. Exogenous supplied urea is distributed between two pools, one large nonmetabolic and one metabolic pool. Ammonia inhibits the transport from the nonmetabolic to the metabolic pool.The nonmetabolic pool is probably located in the chloroplast, and the accumulation of urea is due to an active transport into the chloroplast.Non-Standard Abbreviations N medium medium without nitrogen source - TCA trichloroacetic acid - TCAI trichloroacetic acid insoluble fraction     

Species-specific measurements of ectomycorrhizal turnover under N-fertilization: combining isotopic and genetic approaches

Ectomycorrhizal fungi play a significant role in the transfer of nutrients between plant and soil pools. Here we combine natural abundance ¹⁴C measurements with restriction fragment length polymorphism (RFLP) to study the effects of nitrogen fertilization on the residence time of carbon within ectomycorrhizal species. We show that the carbon in ectomycorrhizal fungi turns over every 4–5 years, indicating that these fungi are relatively long-lived. Moreover, ectomycorrhizal fungi responded in a species-specific way to fertilization. Cenococcum geophilum contained younger carbon on average in nitrogen-fertilized plots than in control plots, even though turnover rates of the community as a whole did not shift significantly. Our results suggest that the response of ectomycorrhizal fungi to N availability is complex, and alterations in tissue turnover within this microbial pool may vary depending on community structure.     

Energy requirement for the mobilization of vacuolar arginine in Neurospora crassa

The bulk of the intracellular arginine pool in exponentially growing mycelia of Neurospora crassa is sequestered in the vacuoles. Vacuolar arginine effluxes from the vacuoles into the cytosol and is catabolized to ornithine and urea upon nitrogen starvation. The energy requirement for mobilization has been studied by treating nitrogen-starved mycelia with inhibitors or respiration or glycolysis or an uncoupler of respiration. Mobilization was inhibited by the inhibitors or the uncoupler of respiration, but not by the inhibitors of glycolysis. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge of the treated mycelia. The inhibitors of glycolysis reduced the ATP pool but had no effect on the energy charge. The results indicate that mobilization of arginine from the vacuoles requires metabolic energy. The forms of this energy and the mode of its association with the mobilization process are discussed.     

连续三年夜间增温和施氮对云杉外生菌根及菌根真菌多样性的影响

以西南亚高山针叶林建群种粗枝云杉(Picea asperata)为研究对象,采用红外加热模拟增温结合外施氮肥(NH₄NO₃ 25 g N m^-2 a^-1)的方法,研究连续3a夜间增温和施肥对云杉幼苗外生菌根侵染率、土壤外生菌根真菌生物量及其群落多样性的影响。结果表明:夜间增温对云杉外生菌根侵染率的影响具有季节性及根级差异。夜间增温对春季(2011年5月)云杉1级根,夏季(2011年7月)和秋季(2010年10月)云杉2级根侵染率影响显著。除2011年7月1级根外,施氮对云杉1、2级根侵染率无显著影响。夜间增温对土壤中外生菌根真菌的生物量和群落多样性无显著影响,施氮及增温与施氮联合处理使土壤中外生菌根真菌生物量显著降低,但却提高了外生菌根真菌群落的多样性。这说明云杉幼苗外生菌根侵染率对温度较敏感,土壤外生菌根真菌生物量及其群落多样性对施氮较敏感。这为进一步研究该区域亚高山针叶林地下过程对全球气候变化的响应机制提供了科学依据。     

Effect of cultural conditions on the concentrations of metabolic intermediates during growth and sporulation of Bacillus licheniformis.

Intracellular concentrations of adenine nucleotides and intermediates of the Embden-Meyerhof pathway and the tricarboxylic acid cycle have been determined during growth and sporulation of Bacillus licheniformis in a variety of different media. The ATP pool was independent of growth rate and nitrogen source, but the use of glucose as a carbon source resulted in a twofold elevation in the ATP pool during exponential growth. The intracellular phosphoenolpyruvate pool was at least twofold higher during gluconeogenesis than during glycolysis. The finding that the use of glutamate as the sole nitrogen source resulted in at least a fivefold elevation of the alpha-ketoglutarate pool suggests a role for alpha-ketoglutarate in the repression of the enzymes of the tricarboxylic acid cycle responsible for alpha-ketoglutarate synthesis. Not one of the metabolites assayed appears to function as a signal of the nutrient deprivation which accompanies the initiation of sporulation.     

用混合氮源在室外培养产油链带藻的可行性研究

为探索两株链带藻（Desmodesmus sp.T28-1和Desmodesmus sp.NMX451）在室外培养的最优氮源,首先在室内就不同氮源（尿素、硝酸钠、碳酸铵以及尿素和硝酸钠混合氮源）下微藻的生长和油脂积累做了研究,筛选出最优的混合氮源在室外进行了培养的可行性研究。室内研究结果表明两株链带藻在尿素下培养油脂含量最低,在铵氮下培养生物量最低。且NMX451在混合态氮下的油脂产率显著性的高于其他氮源下的油脂产率。对两株链带藻在混合氮源下的脂肪酸组分做进一步分析,结果表明油脂组分适合生物柴油生产要求,估算的生物柴油品质达到国际和国内生产标准。将两株链带藻置于室外140 L柱式反应器中用混合氮源进一步扩大培养,结果表明NMX451比T28-1的油脂含量和油脂产率高,生产成本更低,且脂肪酸组分更适宜生物柴油生产。研究表明用混合氮源在室外培养微藻是非常可行的培养方法,也说明NMX451比T28-1在生物柴油生产方面具有更好的潜力。     

Intraspecific variation in use of different organic nitrogen sources by the ectomycorrhizal fungus Hebeloma cylindrosporum

The ectomycorrhizal (ECM) fungus Hebeloma cylindrosporum is an appropriate model to study the intraspecific functional diversity of ECM fungi in forest ecosystems. Numerous metabolic genes, specifically genes related to nitrogen assimilation, have been characterised for this species and the spatial and temporal structures of its natural populations have been extensively worked out. In this paper, we reveal the extent to which intraspecific variation exists within this fungus for the ability to use organic nitrogen, an important functional characteristic of ECM fungi. In addition to ammonium and nitrate, H. cylindrosporum can use at least 13 different amino acids out of 21 tested as sole nitrogen source, as well as urea and proteins. By screening 22 genetically different wild type haploid strains we identified obvious differences in use of six nitrogen sources: alanine, glycine, phenylalanine, serine, bovine serum albumin and gelatine. Of the 22 haploid strains, 11 could not use at least one of these six nitrogen sources. The inability of some haploid strains to use a nitrogen source was found to be a recessive character. Nevertheless, obvious differences in use of the four amino acids tested were also measured between wild type dikaryons colonising a common Pinus pinaster root system. This study constitutes the basis for future experiments that will address the consequences of the functional diversity of an ECM fungus on the functioning of the ECM symbiosis under natural conditions.     

Regulation of genes involved in nitrogen utilization on different C/N ratios and nitrogen sources in the model ectomycorrhizal fungus Hebeloma cylindrosporum

Nitrogen (N) utilization by ectomycorrhizal fungi is an essential aspect of their ecosystem function. N deposition changes both the N pools and the carbon/nitrogen (C/N) ratio of the substrates where ectomycorrhizal fungi are found, and it is important to understand how these changes affect the N forms used by ectomycorrhizal fungi. To overcome the difficulties of studying ectomycorrhizal fungi in situ, we investigated all known N genes in the model fungus, Hebeloma cylindrosporum in a culture study. In addition to studying the regulation of all known N utilization genes, we aimed to understand whether there are gene clusters that undergo similar regulation. Lastly we studied how C/N ratio, N transporter type, and N source affected relative gene expression levels. We grew the D2 strain of H. cylindrosporum on a range of inorganic and organic N sources under low, medium, and high C/N ratios. We found three gene clusters that were regulated in a similar pattern. Lastly, we found C/N ratio, N source and N transporter type all affected gene expression levels. Relative expression levels were highest on the high C/N ratio, BSA and diLeucine N sources, and inorganic N transporters were always expressed at higher levels than organic N transporters. These results suggest that inorganic N sources may always the default preference for H. cylindrosporum, regardless of both the N sources and the C/N ratio of the substrate.     

Urea is a dynamic pool of bioavailable nitrogen in coral reefs

Urea may be an important source of nitrogen in low nutrient coral reef environments because corals and other organisms can assimilate it easily and it is found throughout ocean waters. We measured the distribution and concentrations of urea in seagrass beds, areas of schooling fish, coral formations and bottom sediments in the Upper Florida Keys Reef Tract. The flux of urea from bottom sediments was also measured. Ambient concentrations of urea in the offshore reefs were similar to the concentrations of nitrate and ammonium. Seagrass beds, areas of schooling fish and coral formations had elevated concentrations of urea that were up to eight times higher than nitrate in the system. Numerous ephemeral hotspots of urea that were 8–20 times the ambient urea concentration existed in seagrass beds, areas of schooling fish, and above sediments. Coastal areas and inland canals had high urea concentrations where urban runoff and septic effluents were prevalent, but there was no anthropogenic influence in the offshore habitats. Urea concentrations above bottom sediments were not different from ambient concentrations and benthic flux chamber incubations showed biological activity in carbonaceous sediments but no net urea production. The decrease in urea concentrations from coasts and inland waterways to a consistent ambient concentration in the offshore reef system and ephemeral hotspots of high urea concentration suggest that urea is a dynamic pool of bioavailable nitrogen in the reefs of the Upper Florida Keys.     

汞对外生菌根真菌氮素利用酶活性的影响

在含有0、5、50和150μmol/L Hg2+的液体培养基中培养土生空团菌Cenococcum geophilum Fr.菌株Cg SIV、彩色豆马勃Pisolithus tinctorius(Pers.)Coker et Couch菌株Pt715、松乳菇Lactarius sp.菌株Ld-1和Ld-3,研究汞对外生菌根真菌生长和氮素利用酶活性的影响。结果表明,汞对外生菌根真菌生长有不同程度的抑制作用,其中Cg SIV生物量降幅最小,在高汞浓度的培养基中生物量仅比对照减少9.7%,具有较高的耐汞性。供试菌株均能合成蛋白酶、几丁质酶、脲酶和硝酸还原酶,但不同菌株之间酶活性差异显著。说明外生菌根真菌既有益于寄主植物利用氮源的多样性,又具有对不同氮源的偏嗜性。汞对外生菌根真菌氮素利用酶活性的影响因菌株、酶类和汞浓度的不同而异,原因可能是不同菌株遗传特性的差异,使其在汞胁迫条件下产酶量不同,并表达对汞敏感性不同的等位酶。此外,低浓度(5μmol Hg2+/L)~中浓度(50μmol Hg2+/L)的汞提高或不影响氮素利用酶的活性,对外生菌根真菌氮素利用能力应无抑制作用。在正常和汞胁迫条件下,Pt715和Ld-3的蛋白酶、脲酶、硝酸还原酶和几丁质酶的活性均最高,表现出较强的氮素利用能力。推断在汞污染的土壤上种植桉树和松树,接种Pt715和Ld-3可能改善寄主植物的氮素营养。     

Can ectomycorrhizal colonization of Pinus resinosa roots affect their decomposition?

In many forest ecosystems, fine root litter comprises a large pool of organic carbon and nutrients. In temperate climates ectomycorrhizal fungi colonize the roots of many forest plant species. If ectomycorrhizal colonization influenced root decomposition, it could significantly influence carbon sequestration and nutrient cycling. Fungal tissues and fine roots may decompose at different rates and, therefore, ectomycorrhizal colonization may either hasten or retard root decomposition. Unfortunately, no comparisons of the decomposition of roots and ectomycorrhizal fungi have yet been made. Therefore, we compared decomposition of Pinus resinosa fine roots and ectomycorrhizal fungi from a Pinus resinosa plantation. We also compared the decomposition rates of nonmycorrhizal Pinus resinosa fine roots with roots colonized by nine species of ectomycorrhizal fungi. We found that the several tested isolates of ectomycorrhizal fungi decomposed far more rapidly than the fine roots and that ectomycorrhizal colonization either had no significant effect on root decomposition or significantly increased root decomposition depending on the isolate of fungus. We conclude that the composition of an ectomycorrhizal fungal community may affect carbon and nutrient cycling through its influence on root decomposition.     

Sediment-water fluxes of nutrients in an Antarctic coastal environment: influence of bioturbation

Rates of exchanges of nitrate and ammonium across the sediment-water interface were measured in an inshore marine environment at Signy Island, South Orkney Islands, Antarctica, over 6 months from August 1991 to February 1992. The sediment was a source of ammonium to the water column but a sink of nitrate, although nitrate exchange rates were very variable. Concentration profiles of nitrate and ammonium in the sediment porewater corroborated the measured vertical exchanges. Bioturbation, by a largely amphipod benthic infauna which was confined to the top 2 cm of sediment, was investigated experimentally. Removal of bioturbation depressed sedimentary O₂ uptake by 33% and sedimentary release of NH₄ ⁺ by 50%. In contrast, in the absence of bioturbation, the removal of NO₃ ^– from the water column by the sediment increased in rate. The measured fluxes of ammonium and nitrate from the sediment did not match with the amount of nitrogen mineralised within the sediment, and urea may account for the difference. It is suggested that the export of nitrogen from the bottom sediment may be significant in sustaining primary production in the Antarctic inshore environment. Ammonium and urea are preferred to nitrate as a nitrogen source by phytoplankton. The nitrate concentrations in the sediment porewater were low, but a large pool of nitrate was identified in the top 0–2 cm layer, which was released by KCl extraction or by freezing of the sediment. This extractable pool of nitrate did not equilibrate with the soluble nitrate pool in the sediment, but seemed to be released from components of the benthic infauna, which were also largely confined to the top 0–2 cm. The physiological role of this nitrate is unknown.