Abstract
Arbuscules are the most conspicuous structures of arbuscular mycorrhizas. Due to their large surface area, they are regarded as putative sites of nutrient exchange between host and symbiont. A new staining technique for arbuscules is presented in which arbuscules are selectively stained by acidotropic (i.e. based on the ion-trap mechanism) accumulation of neutral red (activity stain). Criteria to distinguish acidotropic staining from staining due to chemical affinity are presented together with approaches to minimize background staining and to check for complete penetration of the dye through the root tissues. The non-toxic staining technique allows detection of arbuscules in living roots within two to six hours. The histochemical properties of neutral red, microscopic techniques, potential pitfalls, the arbuscular life cycle, and aspects of future research are discussed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Agerer R 1991 Characterization of ectomycorrhiza. Methods Microbiol. 23, 25–73.
Alexander T, Meier R, Toth R and Weber H C 1988 Dynamics of arbuscule development and degeneration in mycorrhizas of Triticum aestivum L. and Avena sativa L. with reference to Zea mays L. New Phytol. 110, 363–370.
Alexander T, Toth R, Meier R and Weber H C 1989 Dynamics of arbuscule development and degeneration in onion, bean, and tomato with reference to vesicular-arbuscular mycorrhizae in grasses. Caqn. J. Bot. 67, 2505–2513.
Anderson R G W1989 Postembedding detection of acidic compartments. Methods Cell Biol. 31, 463–472.
Boller T and Wiemken A 1986 Dynamics of vacuolar compartmentation. Ann. Rev. Pl. Physiol. 37, 137–164.
Brundrett M C, Piché Y and Peterson R L 1984 A new method for observing the morphology of vesicular-arbuscular mycorrhizae. Can. J. Bot. 62, 2128–2134.
Carling D E and Brown M F 1982 Anatomy and physiology of vesicular-arbuscular and nonmycorrhizal roots. Phytopathology 72, 1108–1114.
Conn H J 1977 H.J. Conn's biological stains. A handbook on the nature and uses of the dyes employed in the biological laboratory. Waverly Press, Baltimore, MD, USA. 692 p.
Gianinazzi-Pearson V 1996 Plant cell responses to arbuscular mycorrhizal fungi: Getting to the roots of the symbiosis. Plant Cell 8, 1871–1883.
Gianinazzi-Pearson V, Smith S E, Gianinazzi S and Smith F A 1991 Enzymatic studies on the metabolism of vesicular arbuscular mycorrhizas. V. Is HC-ATPase a component of ATP-hydrolysing enzyme activities in plant fungus interfaces? New Phytol. 117, 61–74.
Göke G 1985 Methoden der Interferenzmikroskopie. 4. Differentieller Interferenzkontrast (DIK). Mikrokosmos 74, 274–294.
Gupta H S and De D N 1988 Acridine orange-induced vacuolar uptake of cytoplasmic organelles in plant cells: An ultrastructural study. J. Pl. Physiol. 132, 254–256.
Guttenberger M 2000 Arbuscules of vesicular-arbuscular mycorrhizal fungi inhabit an acidic compartment within plant roots. Planta, in press.
Holley D and Peterson R L 1979 Development of a vesiculararbuscular mycorrhiza in bean roots. Can. J. Bot. 57, 1960–1978.
Ishida M, Aihara M S, Allen R D and Fok A K 1997 Acidification of the young phagosomes of Paramecium is mediated by proton pumps derived from acidosomes. Protoplasma 196, 12–20.
Lulai E C and Morgan W C 1992 Histochemical probing of potato periderm with neutral red: A sensitive cytofluorochrome for the hydrophobic domain of suberin. Biotec. Histochem. 67, 185–195.
Millner P D and Kitt D G 1992 The Beltsville method for soilless production of vesicular-arbuscular mycorrhizal fungi. Mycorrhiza 2, 9–15.
Phillips J M and Hayman D S 1970 Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. br. mycol. Soc. 55, 158–161.
Schaffer G F and Peterson R L 1993 Modifications to clearing methods used in combination with vital staining of roots colonized with vesicular-arbuscular mycorrhizal fungi. Mycorrhiza 4, 29–35.
Schüß ler A, Schnepf E, Mollenhauer D and Kluge M 1995 The fungal bladders of the endocyanosis Geosiphon pyriforme, a Glomus-related fungus: cell wall permeability indicates a limiting pore radius of only 0.5 nm. Protoplasma 185, 131–139.
Sheppard C J R and Török P 1997 Effects of specimen refractive index on confocal imaging. J. Microscopy 185, 366–374.
Smith F A and Smith S E 1996 Mutualism and parasitism: biodiversity in function and structure in the 'arbuscular' (VA) mycorrhizal symbiosis. Adv. bot. Res. 22, 1–43.
Smith F A and Smith S E 1997 Structural diversity in (vesicular)-arbuscular mycorrhizal symbioses. New Phytol. 137, 373–388.
Smith S E and Dickson S 1991 Quantification of active vesiculararbuscular mycorrhizal infection using image analysis and other techniques. Aust. J. Pl. Physiol. 18, 637–648.
Sousa C, Sáe Melo T, Gèze M, Gaullier J M, Mazière J C and Santus R 1996 Solvent polarity and pH effects on the spectroscopic properties of neutral red: Application to lysosomal microenvironment probing in living cells. Photochem. Photobiol. 63, 601–607.
Stadelmann E J and Kinzel H 1972 Vital staining of plant cells. Methods Cell Biol. 5, 325–372.
Stevens T H 1992 The structure and function of the fungal VATPase. J. exp. Biol. 172, 47–55.
Taiz L 1992 The plant vacuole. J. exp. Biol. 172, 113–122.
Toth R and Miller R M 1984 Dynamics of arbuscule development and degeneration in a Zea mays mycorrhiza. Am. J. Bot. 71, 449–460.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Guttenberger, M. A rapid staining procedure for arbuscules of living arbuscular mycorrhizas using neutral red as acidotropic dye. Plant and Soil 226, 211–218 (2000). https://doi.org/10.1023/A:1026568428770
Issue Date:
DOI: https://doi.org/10.1023/A:1026568428770