versão On-line ISSN 0717-6643
Gayana Bot. v.60 n.1 Concepción 2003
NEW PERSPECTIVES ON THE DETERMINATION OF PHOSPHATASE
ACTIVITY IN ECTOMYCORRHIZAE OF NOTHOFAGUS OBLIQUA
FORESTS IN SOUTHERN CHILE
NUEVAS PERSPECTIVAS EN LA DETERMINACION DE LA ACTIVIDAD DE
LA FOSFATASA EN ECTOMICORRIZAS DE NOTHOFAGUS OBLIQUA
EN BOSQUES DEL SUR DE CHILE
We present a method to localise and quantify surface-bound phosphatase activity (SBPA) in mycorrhizal fungi and mycorrhizae of Nothofagus obliqua (Mirb.) Oerst., using image processed confocal fluorescent microscopy. ELF-97 is a hydrophilic substrate which turns into a strongly fluorescent precipitate upon activation by phosphomonoesterases. In fungal mycelium, this technique has recently been approved by comparison with a standard method (p-nitrophenyl-phosphate). The microscopic technique based on ELF-97 revealed that Paxillus involutus (Batsch: Fr.) Sing. and Austropaxillus boletinoides (Sing.) Brsky. & Jarosch provide different adaptive strategies to changing phosphate concentrations and different pH (3-7). We also analysed SBPA in four mycorrhizal associations of N. obliqua and found that the organisation of the mantle played an essential role regarding the SBPA. In general, mycorrhizal roots shifted SBPA from the root to the mantle. In this context, Pisolithus tinctorius (Pers.) Coker & Couch proved to be the most relevant mycorrhiza partner for N. obliqua by increasing significantly the overall SBPA of the mycorrhiza in respect to non-mycorrhizal roots. In conclusion, ELF-97 fluorescence microscopy in combination with image processing routines determined SBPA and revealed phosphorus (P) adaptation strategies of mycorrhizal fungi and mycorrhizae on a structural-physiological level.
Keywords: Confocal microscopy, ectomycorrhizae of Nothofagus obliqua, ELF-97, phosphatase activity, quantitative fluorescence microscopy.
Nosotros presentamos un método basado en la microscopía confocal de fluorescencia y en el análisis de imágenes, para localizar y cuantificar la actividad de la fosfatasa superficial (SBPA) en hongos ectomicorrícicos y en ectomicorrizas de Nothofagus obliqua (Mirb.) Oerst. El sustrato ELF-97 es hidrofílico, el cual se desarrolla en un precipitado altamente fluorescente bajo su activación a través de fosfomonoesterasas. Recientemente esta técnica ha sido aprobada mediante comparaciones con un método estándar (fosfato de p-nitrofenilo) realizadas en micelio fúngico. La técnica microscópica con ELF-97 reveló que Paxillus involutus (Batsch: Fr.) Sing. y Austropaxillus boletinoides (Sing.) Brsky. & Jarosch poseen diferentes estrategias de adaptación frente a distintas concentraciones de fosfatos y pH (3-7). Nosotros analizamos la SBPA en cuatro asociaciones micorrícicas de N. obliqua y encontramos que la organización del manto juega un rol esencial con respecto a la SBPA. En general, las raíces micorrizadas transfieren la SBPA de la raíz hacia el manto. En este contexto, Pisolithus tinctorius (Pers.) Coker & Couch demostró ser el hongo ectomicorrícico más relevante para N. obliqua, al incrementar significativamente el total de la SBPA de la micorriza en relación a la raíz sin micorrizar. En conclusión, a través de la microscopía confocal de fluorescencia basada en el sustrato ELF-97 y su combinación con el procesamiento de imágenes es posible, tanto en hongos micorrícicos como en micorrizas, determinar la SBPA y revelar las estrategias de adaptación con respecto al fósforo (P) a un nivel fisiológico-estructural.
Palabas claves: Actividad fosfatasa, ectomicorrizas, ELF-97, microscopía confocal, microscopía cuantitiva de fluorescencia
Surface-bound phosphatase activity (SBPA) of fungi, roots, and mycorrhizae is an important parameter which describes the ability of plants to catalyse phosphorous (P) mineralisation. SBPA of mycorrhizal associations is of great interest in areas of limited P supply, because it permit access to sources of phosphate which are inaccessible for plants under isolated conditions (Smith & Read 1997). This holds especially in soils of volcanic origin, which represent a natural sink for P due to their highly active soil components (Scheffer & Schachtschabel 1998). In the humid temperate forests of southern Chile, developed on volcanic soils, ectomycorrhizal Nothofagus species are among the most widespread overstorey dominants. However, little is known so far about the importance of mycorrhizal associations for the P-supply of Nothofagus under different soil conditions (Veblen et al. 1996, Donoso et al. 1999).
The present work evaluates the relevance of several ectomycorrhizae for the P-supply of Nothofagus obliqua (Mirb.) Oerst., introducing a new method for the determination of SBPA of fungal hyphae of Paxillus involutus (Batsch: Fr.) Sing. and Austropaxillus boletinoides (Sing.) Brsky. & Jarosch. The method developed is based on enzyme activated fluorescence of ELF-97 substrate (van Aarle et al. 2001) and image processed confocal Laser-Scanning-Microscopy. In addition to the determinations in fungal hyphae, we applied this technique on non-mycorrhizal root systems of N. obliqua and compared the results to the mycorrhizae of P. involutus, Cenococcum geophilum Fr., Pisolithus tinctorius (Pers.) Coker & Couch and Descolea antartica Sing.
CULTIVATION OF FUNGI AND MYCORRHIZAE OF N. OBLIQUA
Ectomycorrhizal (EM) fungi Paxillus involutus and A. boletinoides were grown in liquid Modified Melin-Norkrans media (Molina & Palmer 1982). Fungi were cultivated in the dark at 25°C and pH = 5 with 0, 30, 60, and 100 % of dissolved phosphate (100 % = 0.5 g KH2PO4 l-1 + 0.25 g (NH4)2HPO4 l-1). Fungi were cultivated for a period of two weeks in accordance with Straker & Mitchell (1986). Roots and mycorrhizal associations of N. obliqua with P. involutus, C. geophilum, P. tinctorius and D. antartica were cultivated in mini-rhizotrons.
LOCALISATION AND QUANTIFICATION OF SURFACE BOUND PHOSPHATASE ACTIVITY
EM fungi, non-mycorrhizal roots, and EM of N. obliqua were harvested and incubated with ELF-97 substrate solution for 15 min (Alvarez et al. 2002). After washing, fungal hyphae were observed directly with confocal Laser-Scanning Microscopy (LSM) (LSM 510, ZEISS, Göttingen, Germany) in specially designed microscopic object slides. Roots and mycorrhizae were embedded in agar at 40°C. After cooling, fractions were cut with a vibratom (model 1000, Lancer Sherwood Medical Company) into sections of 30 µm and observed with LSM, equipped with a 364 nm wavelength UV-argon-laser (model Enterprise II-653, Coherent, Santa Clara, CA, USA). Three-channel picture series were recorded (see Fig. 1e-h). Channel 1: bright field pictures. Channel 2: 385-470 nm band pass filter for auto fluorescence of fungal mycelium. Channel 3: 560-615 nm band pass filter for enzymatically activated ELF?-97 fluorescence intensities. SBPA was determined in EM fungi at different pH = 3-7, roots and EM of N. obliqua at pH = 5. Image processing routines for segmentation and quantification of SBPA and mycorrhizal morphology were written in IDL5.4 (Interactive Data Language, Research Systems, Co, USA) (Härtel et al. 2003).
SBPA IN MYCELIUM OF P. INVOLUTUS AND A. BOLETINOIDES
Fig. 1(a-d) shows colour-coded bivariate plots containing structural information ([number of phosphatase activated centres] / [length of the fungal hypha, µm]), and the total enzymatic activity ([Intensity of ELF-97] / [hyphal length, µm]) of SBPA. For P. involutus, two distinct maxima were observed for the SBPA at a P concentration of 30 %, and pH values of 3.5 and 6 (Fig. 1a). The bivariate distribution of the total enzymatic activity differed from the number of phosphatase activated centres per µm fungal hyphae (Fig. 1c). For A. boletinoides instead, Fig. 1(b) shows that only one optimum condition evolved for the SBPA at pH = 5 and at P concentration of 30 % during the growth period. In contrast to the results obtained with P. involutus, the same distribution was observed when the number of phosphatase activated centres was considered (Fig. 1d).
FIGURE 1. Surface bound mono-phosphatase activity (SBPA) of fungal hyphae of P. involutus and A. boletinoides as determined by image processed fluorescence microscopy based on fluorescence activation of ELF-97 substrate.
(a-b): Bivariate colour-coded presentation of SBPA per µm hyphal length of P. involutus (a) and of A. boletinoides (b). (c-d): Bivariate colour-coded presentation of the number of fluorescent centres of precipitated ELF-97 substrate per µm hyphal length of P. involutus (c) and of A. boletinoides (d). For a-d, mean values were derived from three independent experiments.
(e-h): Representative 3-channel fluorescence images of hyphae of P. involutus and A. boletinoides. Channel 1: bright field pictures (blue). Channel 2: 385-470 nm band pass filter for auto fluorescence of fungal mycelium (green). Channel 3: 560-615 nm band pass filter for enzymatically activated ELF-97 fluorescence intensities (red). (e, g): P. involutus at pH = 5 and P = 100 % (e), and at pH = 4 and P = 30 % (g). (f, h): A. boletinoides at pH = 7 and P = 0 % (f), and at pH = 5 and P = 30 % (h). Compare to black and white circles in a-d.
Representative three-channel fluorescence images of the hyphae of P. involutus and A. boletinoides (Fig. 1e-h) outline selected incubation conditions shown in Fig. 1(a-d). For P. involutus, a high number of individual fluorescent ELF-97 centres (with relatively small fluorescent intensities) were formed on the fungal hyphae (Fig. 1e) at P concentration of 100 % and at pH = 5. At P concentrations of 30 % and a pH value of 4 instead, a relatively small number of individual fluorescent ELF-97 centres could be observed (Fig. 1g). However, these centres yielded high fluorescence intensities. For A. boletinoides, only the number of phosphatase activated centres per µm fungal hyphae was changed, while the individual fluorescence intensities of the centres remained constant (Fig. 1f-h).
SBPA OF MYCORRHIZAL AND NON-MYCORRHIZAL ROOT SYSTEMS OF N. OBLIQUIA
Three out of four mycorrhizal root systems of N. obliqua shifted the majority of the SBPA from the root cells to the fungal hyphae of the mantle (Fig. 2a). Only for D. antartica, ~80 % of the SBPA remained with the root cells and did not shift to the mantle. As far as the relative fluorescence intensity of ELF-97 per µm² area of the mycorrhizal / non-mycorrhizal root of N. obliqua is concerned (Fig. 2b), only the mycorrhizae formed with P. tinctorius increased the total enzymatic activity in respect of the non-mycorrhizal root. P. involutus, C. geophilum, and D. antartica instead lowered the enzymatic activity of the root system. While Fig. 2(b) marks essential differences between the total enzymatic activities of the mycorrhizal and non-mycorrhizal roots of N. obliqua, Fig. 2(c) shows that the enzymatic activities of the individual phosphatase centres did not change to a great extent.
| ||FIGURE 2. Surface bound phosphatase activity (SBPA) in root systems as determined by image processed fluorescence microscopy and ELF-97. (a): SBPA (%) on the mantle of the ectomycorrhiza of N. obliqua with P. tinctorius, P. involutus, C. geophilum, and D. antartica (pH=5). (b): Relative Intensity of ELF-97 fluorescence per µm2 area of the mycorrhizal / non mycorrhizal roots of N. obliqua with P. tinctorius, P. involutus, C. geophilum, and D. antartica (pH=5). (c): Density of fluorescence of ELF-97 [I /µm2] inside of the fluorescence centres in the mycorrhizal / non-mycorrhizal root of N. obliqua with P. tinctorius, P. involutus, C. geophilum, and D. antartica (pH=5). For all plots, standard deviations and mean values were derived from three independent experiments.|
For membrane-bound PA in fungal mycelium, the microscopic method based on ELF-97 has recently been shown to reproduce results obtained with a conventional colorimetric method based on p-nitrophenyl-phosphate (Alvarez et al. 2002). Additionally, the microscopic method contributed valuable information about the structure and distribution of phosphatase-active spots on the fungal hyphae (Fig. 1). The different adaptive strategies by which P. involutus and A. boletinoides regulate their phosphatase activity in response to the variation of the nutrient supply with P during its respective growth periods, and in response to the pH-condition during the incubation periods, are reflected in the representative fluorescent image examples (Fig. 1e-h). While A. boletinoides primarily adapts to different P-supply and pH-condition by changing the number of phosphatase active centres on its hyphae (Fig. 1b, d, f, h), P. involutus primarily changes the activity of the individual centres (Fig. 1a, c, e, g).
P. involutus and A. boletinoides adapted to different growth conditions (P-concentrations) in the same way as they respond to a different pH environment (Fig. 1). As reported, a growth period of 14 days is sufficient for the adaptation of cultured fungi to different phosphorus concentrations (Straker & Mitchell 1986). In contrast to the time scale of adaptation to different growth conditions, it seems very unlikely that the fungi can actively respond to the changing pH during the short incubation period applied here (15 min. for ELF-97). A possible explication would be that the isolates respond to different growth conditions by varying the number (A. boletinoides) or the individual intensity (P. involutus) of phosphatase active centres, but that the exposed phosphatases are characterised by optimal activities at narrow pH ranges.
The comparison of SBPA in mycorrhizal and non-mycorrhizal roots of N. obliqua (Fig. 2) shows that mycorrhization does not necessarily favour the overall capacity of the root to cleave P. In fact, three out of four mycorrhizal root systems of N. obliqua provide less integral SBPA than non mycorrhizal roots (Fig. 2b). Out of the tested mycorrhizal associations, only P. involutus increases the integral SBPA significantly and concentrates nearly the entire activity on the hyphal mantle (Fig. 2b). Interestingly, C. geophilum and P. involutus still concentrate the majority of the SBPA on the hyphal mantles, although they lower the integral SBPA (Fig. 2a-b). As can be derived from Fig. 2c, overall variations of the SBPA in the tested mycorrhizal associations cannot be explained by a variation of the phosphatase activity of the existing centres (fluorescence intensity of ELF-97 per centre surface). As might be expected, the majority of the observed differences instead results from changes of the number (total surface) of the active centres.
In conclusion, the total phosphatase activity and physio-anatomical parameters derived with image processed confocal LSM in isolated fungal hyphae as well as in mycorrhizal associations were found to vary intensively between fungi. The external variation of the pH conditions and different phosphorus concentrations during the growth periods of the cultured fungi had different effects on the phosphatase activity of isolated fungal hyphae. Our newly developed method leads to a precise anatomic-physiologic description of SBPA in transversal cuts of roots or mycorrhizae in vivo. None of the existing methods for the determination of SBPA (Antibus et al. 1992, Tibbett et al. 1998, Tisserant et al. 1993) can offer comparable features. Through interactive segmentation of root, mantle, and PA, our method distinguished between SBPA expressed by the root and by the fungi. In the future, this method will be applied to root material that is directly extracted from its natural habitat in order to gain further insight into the strategies of enzyme driven P solubilisation in situ.
M. A. is a Postdoctoral Fellow of Mecesup AUS 02-14 (Chile). S. H. is a Feodor-Lynen-Fellow of the Alexander von Humboldt Foundation (Germany) and a Postdoctoral Fellow of CONICET (Argentina). This study is a contribution to Fondecyt 11020989 and Millenium P01-057-F.
Alvarez, M., S. Härtel, R. Godoy, A. Gieseke & W. Heyser. 2002. Phosphatase activity in ectomycorrhizal fungi: a comparative study between a colorimetric and a microscope-based method. Submitted to Mycological Research (Ref. Number: 02-990). [ Links ]
Antibus, R. K., R. L. Sinsabaugh & A. E. Linkins. 1992. Phosphatase activities and phosphorus uptake from inositol phosphate by ectomycorrhizal fungi. Canadian Journal of Botany 70: 794-801. [ Links ]
Donoso, P., M. González, B. Escobar, I. Basso & L. Otero. 1999. Viverización y plantación de raulí, roble y coigüe en Chile. En: Silvicultura de los Bosques Nativos de Chile (eds C. Donoso & A. Lara), pp. 177-244. Editorial Universitaria, Santiago de Chile. [ Links ]
Härtel, S., Zorn-Kruppa, M., Tikhanova, S., Heino, P., Engelke, M. & H. Diehl. 2003. Staurosporine-induced apoptosis in human cornea epithelial cells in vitro. In press. Cytometry. [ Links ]
Molina, R. & J. G. Palmer. 1982. Isolation, maintenance, and pure culture manipulation of ectomycorrhizal fungi. In: Methods and principles of mycorrhizal research. (ed N.C. Schenck), pp. 115-129. The American Phytopathological Society, St. Paul. [ Links ]
Scheffer, F. & P. Schachtschabel. 1998. Lehrbuch der Bodenkunde. 14. Auflage. Ferdinand Enke Verlag, Suttgart, Germany. 491 pp. [ Links ]
Smith, S. E. & D.J. Read. 1997. Mycorrhizal symbiosis. 2nd ed. Academic Press, San Diego, California. 605 pp. [ Links ]
Straker, C. J. & D. T. Mitchell. 1986. The activity and characterization of acid phosphatases in endomycorrhizal fungi of the Ericaceae. New Phytologist 104: 243-256. [ Links ]
Tibbett, M., S. M. Chambers & J. W. G. Cairney. 1998. Methods for determining extracellular and surface-bound phosphatase activities in ectomycorrhizal fungi. In: Mycorrhiza manual. (ed A. Varma), Chapter 14, Springer Verlag, Berlin, Heidelberg, Germany. [ Links ]
Tisserant, B., V. Gianinazzi-Pearson, S. Gianinazzi & A. Gollotte. 1993. In planta histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections. Mycological Research 97: 245-250. [ Links ]
Van Aarle, I., P. A. Olsson & B. Söderström. 2001. Microscopic detection of phosphatase activity of saprophytic and arbuscular mycorrhizal fungi using a fluorogenic substrate. Mycologia 93: 17-24. [ Links ]
Veblen, T. T., R. S. Hill & J. Read. 1996. The ecology and biography of Nothofagus forest. Yale University Press, New Haven, London. UK. 403 pp. [ Links ]
1University of Bremen, Institute of Environmental Science and Technology (UFT), Plant Physiology and Plant Anatomy, Leobenerstrasse, D-28359 Bremen, Germany. E-mail: email@example.com
2Centro de Estudios Científicos del Sur (CECS), Arturo Prat 514, Valdivia, Chile. And Facultad de Ciencias Químicas, Departamento de Química Biológica, Pabellón Argentina, Universidad Nacional de Córdoba, Ciudad Universitaria. 5000 Córdoba, Argentina.