Characterization of Three Ammonium Transporters of the Glomeromycotan Fungus Geosiphon pyriformis

Members of the Glomeromycota form the arbuscular mycorrhiza (AM) symbiosis. They supply plants with inorganic nutrients, including nitrogen, from the soil. To gain insight into transporters potentially facilitating nitrogen transport processes, ammonium transporters (AMTs) of Geosiphon pyriformis, a glomeromycotan fungus forming a symbiosis with cyanobacteria, were studied. Three AMT genes were identified, and all three were expressed in the symbiotic stage. The localization and functional characterization of the proteins in a heterologous yeast system revealed distinct characteristics for each of them. AMT1 of G. pyriformis (GpAMT1) and GpAMT2 were both plasma membrane localized, but only GpAMT1 transported ammonium. Neither protein transported the ammonium analogue methylammonium. Unexpectedly, GpAMT3 was localized in the vacuolar membrane, and it has as-yet-unknown transport characteristics. An unusual cysteine residue in the AMT signature of GpAMT2 and GpAMT3 was identified, and the corresponding residue was demonstrated to play an important role in ammonium transport. Surprisingly, each of the three AMTs of G. pyriformis had very distinct features. The localization of an AMT in the yeast vacuolar membrane is novel, as is the described amino acid residue that clearly influences ammonium transport. The AMT characteristics might reflect adaptations to the lifestyle of glomeromycotan fungi.     

福建红树林植物丛枝菌根侵染研究

2010年5月和12月,对福建沿海3个红树林生长区(洛阳江、九龙江口、漳江口)的红树林植物丛枝菌根（AM）侵染状况进行研究。结果表明：（1）红树林生长区中6种植物根内均发现AMF侵染结构,其中桐花树、秋茄、鱼藤和芦苇的丛枝为Arum（疆南星）型;（2）6种植物的丛枝菌根侵染率差异较大,老鼠簕的侵染率最高,鱼藤最低;（3）桐花树和秋茄的丛枝菌根侵染率呈显著差异,而其在不同生长区之间无差异;（4）桐花树和秋茄的丛枝菌根侵染率在不同时间呈显著差异,而钝草的丛枝菌根侵染率在不同时间的差异不显著。     

丛枝菌根（AM）与植物的抗逆性

菌根是土壤中的菌根真菌与高等植物的根系形成的一种联合体 ,是自然界中一种普遍的植物共生现象。除十字花科、莎草科、藜科等少数几个科不能或不易形成菌根外 ,大多数植物包括苔藓、蕨类、裸子植物、被子植物都能形成菌根。 AM是其中的一类 ,也是目前研究较多的一类。AM的内外生菌丝不仅增加了根系的吸收面积 ,还能分泌多种有益物质。所以 ,AM对植物养分、水分的吸收与运输 ,对植物抵抗各种胁迫是十分有益的。1　AM与植物的抗旱性随着全球干旱面积的不断增加 ,淡水资源的日益减少 ,干旱研究已受到人们极大的关注。由于菌根的特殊作用和…     

Ethylene Alleviates the Suppressive Effect of Phosphate on Arbuscular Mycorrhiza Formation

Phosphorus is one of the most important macronutrients required for plant growth. Plants have evolved many strategies for inorganic phosphorus (Pi) acquisition, including the symbiotic pathways, involving the formation of mycorrhiza. With regard to arbuscular mycorrhiza (AM), high Pi availability has long been known to negatively affect this association, although the underlying mechanism is unknown. In the present study, the interactive role played by ethylene and Pi in AM regulation was investigated in the tomato-Rhizophagus irregularis symbiosis. AM fungal colonization was analysed in epi, rin and NRO ethylene-responsive mutants under different Pi availability conditions, with a focus on the late stages of the interaction. Although Pi inhibited mycorrhizal parameters in the ethylene-insensitive rin mutant and wild-type cultivars, it did not alter the mycorrhization of the epi tomato mutant, which exhibits a constitutive ethylene-induced response. As with the colonization parameters, root ethylene content and the expression of AM-related and ethylene receptor 6 genes were inhibited by Pi in wild-type cultivars and rin mutants, but were unaffected or slightly activated in epi plants. The application of ethephon offsets the negative impact on the mycorrhizal development caused by the application of Pi. This compensation effect is dose dependent and was ineffective in the NRO mutant, which is more insensitive to the action of ethylene. Our results provide evidence that ethylene signalling negatively affects the suppressive effect of Pi on AM formation and suggests an overlap between this suppressive effect and the regulatory mechanism of Pi-starvation response pathway in plants mediated by ethylene.     

丛枝菌根真菌对连翘幼苗抗旱性的影响

采用集球囊霉（Glomus fasciculatum Gerd.＆ Trappe emend.Walker ＆ Koske）、缩球囊霉（Glomus constrictum Trappe）单独接种和混合接种于连翘（Forsythia suspensa（Thunb.）Vahl）幼苗,研究丛枝菌根真菌对连翘抗旱性的影响.结果表明：在干旱胁迫条件下,随着菌根侵染率的提高,连翘幼苗叶绿素和脯氨酸含量增加,SOD活性增强,丙二醛含量和膜透性降低,苗木枯死率下降.菌根真菌通过促进苗木快速累积游离脯氨酸,提高SOD酶活性,减缓干旱对细胞膜的破坏,延缓了植物受伤害的速度,提高连翘幼苗的抗旱性;在不同接种处理间,混合接种效果最好,其次为缩球囊霉单独接种.     

3种农业措施对柑橘丛枝菌根发育的影响

研究了土施N肥(2L 0.5%尿素)、覆草(5~6cm厚杂草)和翻耕(20cm表层土翻耕2次)三种措施,对田间条件下的国庆1号/枳和国庆4号/枳的菌根侵染率和孢子密度的影响。结果表明,覆草极显著地提高了2种柑橘根系的孢子密度,而对菌根侵染率无显著影响;施N肥极显著降低2种柑橘的孢子密度;翻耕极显著抑制了2种柑橘的菌根侵染率。2种柑橘的根系菌根侵染率和孢子密度间都存在显著或极显著正相关。     

丛枝菌根在退化土壤恢复中的生态学作用

土壤退化(包括土壤侵蚀、贫瘠化、盐碱化、沙化、酸化)不仅为全球所关注,而且是关系到我国农业可持续发展的重大问题.全球1.3×108km2的总土地面积中,因人为原因引起的退化面积为2.0×107km2,这些退化土壤中,耕地近5×108ha,约占总耕地面积的1/3.     

蕨类植物的VA菌根与蕨类植物系统演化关系的研究

通过对云南热带、亚热带生长的２５６种蕨类植物ＶＡ菌根的调查,发现蕨类植物ＶＡ菌根营养者所占的比例低于被子植物;在真蕨类植物中,植物具有由ＶＡ菌根营养经兼性ＶＡ菌根营养向自养方向进化的趋势。     

丛枝菌根在退化土壤恢复中的生态学作用

土壤退化 (包括土壤侵蚀、贫瘠化、盐碱化、沙化、酸化 )不仅为全球所关注 ,而且是关系到我国农业可持续发展的重大问题。全球 1.3× 10 8ｋｍ2 的总土地面积中 ,因人为原因引起的退化面积为 2 0×10 7ｋｍ2 ,这些退化土壤中 ,耕地近 5× 10 8ｈａ ,约占总耕地面积的 1/ 3。我国南方丘陵区土壤退化问题也突出 (水土流失面积 8 0× 10 7ｈａ ,养分贫瘠化 1 9×10 7ｈａ ,污染土壤 3 2× 10 6ｈａ ,酸化土壤 3 2×10 6ｈａ) ,因而探讨恢复和重建退化土壤的途径已成为该地区农业持续发展的重要内容[5] 。菌根 (ｍｙｃｏｒｒｈｉｚａ)是土…     

Identification of Membrane-Associated Proteins Regulated by the Arbuscular Mycorrhizal Symbiosis

A sub-cellular proteomic approach was carried out to monitor membrane-associated protein modifications in response to the arbuscular mycorrhizal (AM) symbiosis. Membrane proteins were extracted from Medicago truncatula roots either inoculated or not with the AM fungus Glomus intraradices. Comparative two-dimensional electrophoresis revealed that 36 spots were differentially displayed in response to the fungal colonization including 15 proteins induced, 3 up-regulated and 18 down-regulated. Among them, seven proteins were found to be commonly down-regulated in AM-colonized and phosphate-fertilized roots. Twenty-five spots out of the 36 of interest could be identified by matrix assisted laser desorption/ionisation-time of flight and/or tandem mass spectrometry analyses. Excepting an acid phosphatase and a lectin, none of them was previously reported as being regulated during AM symbiosis. In addition, this proteomic approach allowed us for the first time to identify AM fungal proteins in planta.     

Regulation of the Nitrogen Transfer Pathway in the Arbuscular Mycorrhizal Symbiosis: Gene Characterization and the Coordination of Expression with Nitrogen Flux

The arbuscular mycorrhiza (AM) brings together the roots of over 80% of land plant species and fungi of the phylum Glomeromycota and greatly benefits plants through improved uptake of mineral nutrients. AM fungi can take up both nitrate and ammonium from the soil and transfer nitrogen (N) to host roots in nutritionally substantial quantities. The current model of N handling in the AM symbiosis includes the synthesis of arginine in the extraradical mycelium and the transfer of arginine to the intraradical mycelium, where it is broken down to release N for transfer to the host plant. To understand the mechanisms and regulation of N transfer from the fungus to the plant, 11 fungal genes putatively involved in the pathway were identified from Glomus intraradices, and for six of them the full-length coding sequence was functionally characterized by yeast complementation. Two glutamine synthetase isoforms were found to have different substrate affinities and expression patterns, suggesting different roles in N assimilation. The spatial and temporal expression of plant and fungal N metabolism genes were followed after nitrate was added to the extraradical mycelium under N-limited growth conditions using hairy root cultures. In parallel experiments with ¹⁵N, the levels and labeling of free amino acids were measured to follow transport and metabolism. The gene expression pattern and profiling of metabolites involved in the N pathway support the idea that the rapid uptake, translocation, and transfer of N by the fungus successively trigger metabolic gene expression responses in the extraradical mycelium, intraradical mycelium, and host plant.The arbuscular mycorrhizal (AM) symbiosis brings together the roots of the majority of land plant species and fungi of the phylum Glomeromycota to great mutual advantage (Smith and Read, 2008). AM fungi improve the acquisition of phosphate, nitrogen (N), sulfur, and trace elements such as copper and zinc (Clark and Zeto, 2000; Allen and Shachar-Hill, 2008) and increase the biotic and abiotic stress resistance of their host (Smith et al., 2010). In return, the host transfers up to 20% of its photosynthetically fixed carbon to the AM fungus (Jakobsen and Rosendahl, 1990), which depends on its host plant for its carbon supply (Bago et al., 2000).N is the nutrient whose availability most commonly limits plant growth in natural ecosystems. AM fungi can take up NO₃⁻NH₄⁺ and can also increase access to organic N sources from the soil (Ames et al., 1983; Johansen et al., 1993; Bago et al., 1996; Hodge et al., 2001). The translocation by the fungus can represent a significant route for N uptake by the plant (Johansen and Jensen, 1996). For example, Toussaint et al. (2004) showed that in an in vitro mycorrhiza at least 21% of the total N uptake in the roots came from the fungal extraradical mycelium (ERM); for other mycorrhizal systems, even larger proportions have been described (more than 30% and 50%; Govindarajulu et al., 2005; Jin et al., 2005). Tanaka and Yano (2005) reported that 75% of the N in leaves of mycorrhizal maize (Zea mays) was taken up by the ERM of Glomus aggregatum.A mechanism of N transfer from the fungus to the plant has been proposed (Bago et al., 2001) that involves the operation of a novel metabolic route in which N was translocated from the ERM to the intraradical mycelium (IRM) as Arg but transferred to the plant without carbon as inorganic N. This mechanism has been supported by labeling experiments (Johansen et al., 1996; Govindarajulu et al., 2005; Jin et al., 2005), enzyme activity analysis (Cruz et al., 2007), and limited gene expression data (Govindarajulu et al., 2005; Gomez et al., 2009; Guether et al., 2009). Nevertheless, our molecular knowledge of the metabolic and transport pathways involved and how they are regulated is still rudimentary. A better understanding of the mechanism and regulation of N uptake assimilation, translocation, and transfer to the host is important for potential applications of AM fungi as biofertilizers, bioprotectors, and bioregulators in sustainable agriculture and restoration as well as for understanding the role of AM fungi in natural ecosystems (Bruns et al., 2008).In this study, we postulate that the uptake, translocation, and transfer of N by the fungus triggers the metabolic gene expression responses successively in the ERM, IRM, and host plant, which will result in the synthesis and accumulation of Arg in the ERM, the turnover of Arg to release ammonium in the IRM, and the assimilation of ammonium by the host plant via the glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway inside the root (Fig. 1). To test these predictions, 11 genes involved in the N primary assimilation and metabolism were cloned and verified from Glomus intraradices; six enzymes with full-length coding sequences (CDSs) were functionally characterized by yeast knockout mutant complementation. Two GS proteins were found to have different substrate affinities and expression patterns, suggesting that they have different roles in N assimilation. The time courses of gene expression and N movement in fungal and host tissues were analyzed following nitrate supply to the ERM of a mycorrhiza grown under N-limited conditions. The results substantially increase our knowledge of the identity and regulation of most of the metabolic and transport genes involved in N movement through the AM symbiosis.Open in a separate windowFigure 1.Working model of N transport and metabolism in the symbiosis between plant roots and arbuscular mycorrhizal fungi. N moves (black arrows) from the soil into the fungal ERM, through a series of metabolic conversion reactions into Arg, which is transported into the IRM within the root (host) and there is broken down; N is transferred to and assimilated by the host as ammonia. Red circles refer to the sites of action of the genes identified and analyzed in this study. Blue arrows indicate mechanisms hypothesized to regulate gene expression by N metabolites involved in the pathway.     

Carbon Uptake and the Metabolism and Transport of Lipids in an Arbuscular Mycorrhiza

Both the plant and the fungus benefit nutritionally in the arbuscular mycorrhizal symbiosis: The host plant enjoys enhanced mineral uptake and the fungus receives fixed carbon. In this exchange the uptake, metabolism, and translocation of carbon by the fungal partner are poorly understood. We therefore analyzed the fate of isotopically labeled substrates in an arbuscular mycorrhiza (in vitro cultures of Ri T-DNA-transformed carrot [Daucus carota] roots colonized by Glomus intraradices) using nuclear magnetic resonance spectroscopy. Labeling patterns observed in lipids and carbohydrates after substrates were supplied to the mycorrhizal roots or the extraradical mycelium indicated that: (a) ¹³C-labeled glucose and fructose (but not mannitol or succinate) are effectively taken up by the fungus within the root and are metabolized to yield labeled carbohydrates and lipids; (b) the extraradical mycelium does not use exogenous sugars for catabolism, storage, or transfer to the host; (c) the fungus converts sugars taken up in the root compartment into lipids that are then translocated to the extraradical mycelium (there being little or no lipid synthesis in the external mycelium); and (d) hexose in fungal tissue undergoes substantially higher fluxes through an oxidative pentose phosphate pathway than does hexose in the host plant.     

植物菌根共生磷酸盐转运蛋白

大多数植物能和丛枝菌根（arbuscular mycorrhiza, AM）真菌形成菌根共生体。AM能够促进植物对土壤中矿质营养的吸收,尤其是磷的吸收。磷的吸收和转运由磷酸盐转运蛋白介导。总结了植物AM磷酸盐转运蛋白及其结构特征,分析其分类及系统进化,并综述了AM磷酸盐转运蛋白介导的磷的吸收和转运过程及其基因的表达调控。植物AM磷酸盐转运蛋白属于Pht1家族成员,它不仅对磷的吸收和转运是必需的,而且对AM共生也至关重要,为进一步了解菌根形成的分子机理及信号转导途径提供了理论基础。     

Glycine-Glomus-Rhizobium Symbiosis: V. Effects of Mycorrhiza on Nodule Activity and Transpiration in Soybeans under Drought Stress

Soybean (Glycine max [L.] Merr.) plants were nodulated (Bradyrhizobium japonicum) and either inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe or left uncolonized. All plants were grown unstressed for 21 days initially. After this period, some VAM and non-VAM plants were exposed to four 8-day drought cycles while others were kept well watered. Drought cycles were terminated by rewatering when soil moisture potentials reached −1.2 megapascal. Nodule development and activity, transpiration, leaf conductance, leaf and root parameters including fresh and dry weight, and N and P nutrition of VAM plants and of non-VAM, P-fed plants grown under the same controlled conditions were compared. All parameters, except N content, were greater in VAM plants than in P-fed, non-VAM plants when under stress. The opposite was generally true in the unstressed comparisons. Transpiration and leaf conductance were significantly greater in stressed VAM than in non-VAM plants during the first half of the final stress cycle. Values for both VAM and non-VAM plants decreased linearly with time during the cycle and converged at a high level of stress (−1.2 megapascal). Effects of VAM fungi on the consequences of drought stress relative to P nutrition and leaf gas exchange are discussed in the light of these findings and those reported in the literature.     

Translocation and Utilization of Fungal Storage Lipid in the Arbuscular Mycorrhizal Symbiosis

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Carbon is transferred from the plant to the fungus as hexose, but the main form of carbon stored by the mycobiont at all stages of its life cycle is triacylglycerol. Previous isotopic labeling experiments showed that the fungus exports this storage lipid from the intraradical mycelium (IRM) to the extraradical mycelium (ERM). Here, in vivo multiphoton microscopy was used to observe the movement of lipid bodies through the fungal colony and to determine their sizes, distribution, and velocities. The distribution of lipid bodies along fungal hyphae suggests that they are progressively consumed as they move toward growing tips. We report the isolation and measurements of expression of an AM fungal expressed sequence tag that encodes a putative acyl-coenzyme A dehydrogenase; its deduced amino acid sequence suggests that it may function in the anabolic flux of carbon from lipid to carbohydrate. Time-lapse image sequences show lipid bodies moving in both directions along hyphae and nuclear magnetic resonance analysis of labeling patterns after supplying 13C-labeled glycerol to either extraradical hyphae or colonized roots shows that there is indeed significant bidirectional translocation between IRM and ERM. We conclude that large amounts of lipid are translocated within the AM fungal colony and that, whereas net movement is from the IRM to the ERM, there is also substantial recirculation throughout the fungus.     

Enzymatic Studies on the Oxidation of Sugar and Sugar Alcohol

During the course of studies on the oxidative metabolism of d-sorbitol by acetic acid bacteria, it was found that d-sorbitol was almost quantitatively converted to 5-keto-d-fructose via l-sorbose by a certain strain of Gluconobacter suboxydans. In addition to 5-keto-d-fructose, three γ-pyrone compounds, kojic acid, 5-oxymaltol, and 3-oxykojic acid, 2-keto-l-gulonate, and several organic acids such as succinic, glycolic, and glyceric acids were confirmed in the culture filtrate of this bacterium.

• The most suitable carbon source for 5-ketofructose fermentation by Gluconobacter suboxydans Strain 1 was confirmed to be d-sorbitol or l-sorbose using growing and resting cells. d-Fructose had little effect on the formation of this dicarbonylhexose.

• The optimal pH for the formation from l-sorbose by intact cells was found to be at 4.2.

• The activity of the pentose phosphate cycle in the resting cells was calculated as 13~17 μatoms/hr/mg of dry cells by the use of the manometric techniques.

• There was no strain tested so far which could accumulate a large amount of 5- keto-d-fructose from d-sorbitol except this bacterium.

• The experimental results shown in this paper makes the prediction that a certain dehydrogenating system of l-sorbose is functional in the organism, and the metabolic pathways of d-sorbitol via l-sorbose and 5-keto-d-fructose is proposed.