Dispersed Polyphosphate in Fungal Vacuoles in Eucalyptus pilularis/Pisolithus tinctorius Ectomycorrhizas

Ashford, A. E.; Vesk, Peter A.; Orlovich, David A.; Markovina, Anne-Laure; Allaway, W. G.

doi:10.1006/fgbi.1999.1140

Cited by 57 publications

(40 citation statements)

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“…Several previous reports have concluded that almost all calcium in fungal cells is in the vacuole, which contains polyphosphate as the predominant polyanion (12). Polyphosphate granules within vacuoles have long been reported (35,40), although some investigators have argued that the particulate nature of the polyphosphate in electron micrographs is an artifact of the staining procedure (2). In living cells, we frequently observed tiny particles in large vacuoles, moving rapidly by Brownian motion.…”

Section: Discussionmentioning

confidence: 70%

Role of Four Calcium Transport Proteins, Encoded bynca-1,nca-2,nca-3, andcax, in Maintaining Intracellular Calcium Levels in Neurospora crassa

et al. 2011

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We have examined the distribution of calcium in Neurospora crassa and investigated the role of four predicted calcium transport proteins. The results of cell fractionation experiments showed 4% of cellular calcium in mitochondria, approximately 11% in a dense vacuolar fraction, 40% in an insoluble form that copurifies with microsomes, and 40% in a high-speed supernatant, presumably from large vacuoles that had broken. Strains lacking NCA-1, a SERCA-type Ca 2؉ -ATPase, or NCA-3, a PMC-type Ca 2؉ -ATPase, had no obvious defects in growth or distribution of calcium. A strain lacking NCA-2, which is also a PMC-type Ca 2؉ -ATPase, grew slowly in normal medium and was unable to grow in high concentrations of calcium tolerated by the wild type. Furthermore, when grown in normal concentrations of calcium (0.68 mM), this strain accumulated 4-to 10-fold more calcium than other strains, elevated in all cell fractions. The data suggest that NCA-2 functions in the plasma membrane to pump calcium out of the cell. In this way, it resembles the PMC-type enzymes of animal cells, not the Pmc1p enzyme in Saccharomyces cerevisiae that resides in the vacuole. Strains lacking the cax gene, which encodes a Ca 2؉ /H ؉ exchange protein in vacuolar membranes, accumulate very little calcium in the dense vacuolar fraction but have normal levels of calcium in other fractions. The cax knockout strain has no other observable phenotypes. These data suggest that "the vacuole" is heterogeneous and that the dense vacuolar fraction contains an organelle that is dependent upon the CAX transporter for accumulation of calcium, while other components of the vacuolar system have multiple calcium transporters.

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Section: Discussionmentioning

confidence: 70%

Role of Four Calcium Transport Proteins, Encoded bynca-1,nca-2,nca-3, andcax, in Maintaining Intracellular Calcium Levels in Neurospora crassa

et al. 2011

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“…Esses resultados demonstram que o P absorvido pode ter outros destinos na célula. Pisolithus tinctorius associado a Eucalyptus pilularis armazena o P absorvido nos vacúolos celulares, na forma de polifosfatos, podendo translocá-lo em seguida para a planta hospedeira (Ashford et al, 1999). Em termos de EUN, os isolados monocarióticos M5 e M11 foram os mais eficazes na utilização do nutriente para produção de biomassa micelial (Quadro 1).…”

Section: Resultsunclassified

“…No que se refere à EUN, o isolado monocariótico M11 foi o que apresentou a maior eficiência de utilização de K para produção de biomassa (Quadro 1). Grandes quantidades de K são encontradas nos vacúolos das células fúngicas e na região de contato de P. tinctorius associado a E. pilularis (Ashford et al, 1999), comprovando o papel de fungos ectomicorrízicos na aquisição desse nutriente. Os fungos ectomicorrízicos podem contribuir em alguns casos com 5 % do total de K absorvido pela planta hospedeira (Jentschke et al, 2000).…”

Section: Concentraçãounclassified

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Formação de ectomicorrizas por monocários e dicários de Pisolithus sp. e interações nutricionais em Eucalyptus grandis

Silva

Costa²,

Rocha³

et al. 2007

Rev. Bras. Ciênc. Solo

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RESUMOA germinação de basidiósporos de Pisolithus spp. dá origem a monocários, caracterizados por possuírem um único núcleo haplóide por célula. No campo, o eucalipto associa-se a micélios dicarióticos de Pisolithus spp., não havendo relatos sobre a capacidade dos monocários em estabelecer a associação ectomicorrízica com a planta hospedeira nessas condições. Embora os monocários de Pisolithus sp. sejam capazes de formar a associação ectomicorrízica in vitro, nada se sabe sobre a capacidade dessas estirpes em promover a absorção de nutrientes e o crescimento do eucalipto. O objetivo deste trabalho foi estudar a formação de ectomicorrizas por isolados monocarióticos e dicarióticos de Pisolithus sp. em Eucalyptus grandis, sob condições de casa de vegetação, bem como investigar as relações entre o estabelecimento da associação e o crescimento e a absorção de P, Ca, Mg, K, Cu e Zn pelas plantas. Caracterizou-se, também, a produção de massa seca micelial e a absorção de nutrientes pelos isolados fúngicos in vitro. Os isolados monocarióticos e dicarióticos testados variaram na produção de massa seca micelial e na capacidade de absorção de nutrientes. Em geral, os monocários apresentaram maiores índices de eficiência de utilização de nutrientes do que os dicários. Todos os isolados monocarióticos e dicarióticos formaram ectomicorrizas típicas quando associados com E. grandis. A presença dos isolados fúngicos monocarióticos associados às raízes laterais resultou em aumento do diâmetro radial das células da epiderme radicular, característico das ectomicorrizas de eucalipto, indicando que os monocários, à semelhança dos dicários, são capazes de produzir reguladores de crescimento. As médias de percentagem de colonização das raízes pelos isolados monocarióticos e dicarióticos variaram de 12 a 76 %. A absorção de Ca, Mg e K foi estimulada de forma expressiva pela presença das ectomicorrizas, com aumentos de até 760 vezes, demonstrando a relevância dessa associação no suprimento desses macronutrientes, especialmente o Ca. Alguns isolados monocarióticos são tão eficientes quanto os dicários na colonização radicular e na absorção de nutrientes. A caracterização dos monocários de Pisolithus sp. é necessária para se estabelecer a seleção e o cruzamento dos isolados com características desejáveis, visando ao

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“…However, polyP granules have been shown to be artifacts caused by ethanol dehydration following chemical fixation and by staining with cationic TBO (33). According to the results of EDXS analysis of freezesubstituted fungal hyphae, phosphorus in the vacuoles is not precipitated but is evenly dispersed, indicating that polyP is distributed in a soluble form in the vacuoles of living hyphae (7,20,33). However, polyP granules have been observed in nonfixed and air-dried algal cells (9) and in cryofixed and freeze-dried hyphae of ectomycorrhizal fungi (17).…”

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confidence: 99%

Direct Labeling of Polyphosphate at the Ultrastructural Level inSaccharomyces cerevisiaeby Using the Affinity of the Polyphosphate Binding Domain ofEscherichia coliExopolyphosphatase

Saito

Ohtomo

Kuga-Uetake

et al. 2005

Appl Environ Microbiol

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Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate and has many biological functions in prokaryotic and eukaryotic organisms. To investigate polyP localization, we developed a novel technique using the affinity of the recombinant polyphosphate binding domain (PPBD) of Escherichia coli exopolyphosphatase to polyP. An epitope-tagged PPBD was expressed and purified from E. coli. Equilibrium binding assay of PPBD revealed its high affinity for long-chain polyP and its weak affinity for short-chain polyP and nucleic acids. To directly demonstrate polyP localization in Saccharomyces cerevisiae on resin sections prepared by rapid freezing and freeze-substitution, specimens were labeled with PPBD containing an epitope tag and then the epitope tag was detected by an indirect immunocytochemical method. A goat anti-mouse immunoglobulin G antibody conjugated with Alexa 488 for laser confocal microscopy or with colloidal gold for transmission electron microscopy was used. When the S. cerevisiae was cultured in yeast extract-peptone-dextrose medium (10 mM phosphate) for 10 h, polyP was distributed in a dispersed fashion in vacuoles in successfully cryofixed cells. A few polyP signals of the labeling were sometimes observed in cytosol around vacuoles with electron microscopy. Under our experimental conditions, polyP granules were not observed. Therefore, it remains unclear whether the method can detect the granule form. The method directly demonstrated the localization of polyP at the electron microscopic level for the first time and enabled the visualization of polyP localization with much higher specificity and resolution than with other conventional methods.Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate (P i ) connected by high-energy bonds. PolyP occurs in a wide range of organisms, including prokaryotes and eukaryotes. PolyP has various biological functions; for example, it acts as a P i reservoir, as an alternative source of highenergy bonds, and as a buffer against alkaline conditions and metals (25). Furthermore, polyP also has regulatory functions such as competence for transformation (18), motility (36, 37), gene expression under stressed conditions (24,35,46), and protein degradation in amino acid starvation (28, 29) in prokaryotic organisms. The regulatory functions of polyP in eukaryotes are less clear, but some important facts are known. Recently, involvement of polyP in apoptosis (43) and enhancement of the mitogenic activities of acidic and basic fibroblast growth factors by polyP (45) have been suggested to occur in mammalian cells.The presence of polyP in cells can be visualized by staining with toluidine blue O (TBO) or 4Ј,6-diamidino-2-phenylindole (DAPI). DAPI is usually used for DNA detection, because blue fluorescence is apparent when the stained tissues are viewed under UV light. However, DAPI-polyP fluoresces yellow at high concentrations when viewed under UV (48). These staining methods have often been used for detecting polyPaccumulating bacteria in activated...

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Dispersed Polyphosphate in Fungal Vacuoles in Eucalyptus pilularis/Pisolithus tinctorius Ectomycorrhizas

Cited by 57 publications

References 53 publications

Role of Four Calcium Transport Proteins, Encoded bynca-1,nca-2,nca-3, andcax, in Maintaining Intracellular Calcium Levels in Neurospora crassa

Role of Four Calcium Transport Proteins, Encoded bynca-1,nca-2,nca-3, andcax, in Maintaining Intracellular Calcium Levels in Neurospora crassa

Formação de ectomicorrizas por monocários e dicários de Pisolithus sp. e interações nutricionais em Eucalyptus grandis

Direct Labeling of Polyphosphate at the Ultrastructural Level inSaccharomyces cerevisiaeby Using the Affinity of the Polyphosphate Binding Domain ofEscherichia coliExopolyphosphatase

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