- Email: [email protected]
Cytochemical location of urease in the cell wall of two different lichen phycobionts
Tissue and Cell 36 (2004) 373–377
Cytochemical location of urease in the cell wall of two different lichen phycobionts A.M. Millanesa , B. Fontaniellaa , M.L. Garc´ıab , M.T. Solasc , C. Vicentea,∗ , M.E. Legaza a
Laboratory of Plant Biology, Faculty of Biology, Complutense University, C/Jos´e Antonio Novias s/n, 28040 Madrid, Spain b Center of Electron Microscopy, Complutense University, 28040 Madrid, Spain c Department of Cell Biology, Faculty of Biology, Complutense University, 28040 Madrid, Spain Received 9 December 2003; received in revised form 15 June 2004; accepted 30 June 2004
Abstract The enzyme urease has been located in the cell wall of recently isolated phycobionts from Evernia prunastri and Xanthoria parietina lichens. Cytochemical detection is achieved by producing a black, electron-dense precipitate of cobalt sulfide proceeding from CO2 evolved from urea in the presence of cobalt chloride. Cellular fractionation reveals that about 80% of total urease activity was associated to the cell wall on both phycobionts whereas only 20% was recovered as soluble protein. © 2004 Elsevier Ltd. All rights reserved. Keywords: Cell wall; Evernia prunastri; Phycobiont; Urease; Xanthoria parietina
1. Introduction The plant cell wall is revealed as a dynamic structure to which many proteins are attached to develop different biochemical functions. Proteases in the cell wall are related to the turnover of extensin and some proline-rich proteins (Showalter, 1993) whereas several endoglycanases are related to the enzymatic relaxation of structural cell wall polysaccharides, such as cellulose, callose, xyloglucans and pectins (Cosgrove, 1997). Invertase (-fructofuranosidase) in the cell wall of companion cells is involved in the loading of hydrolyzed sucrose to parenchymatous cells of Saccharum officinarum stalks (Oparka, 1990) whereas ␣-arabinosidase catalyzes the hydrolysis of the arabinoside moiety of extensin (Stolle-Smits et al., 1999). Oxido-reductases such as peroxidases are related to the catabolism of lignin by lignin-degrading fungi (Kim et al., 2002), to the biosynthesis of lignin in response to wounding (Lehner and Cardemil, 2003) and to the generation of active oxygen species related to plant defense mechanisms ∗
Corresponding author. Tel.: +34 91 3944565; fax: +34 91 3945034. E-mail address: [email protected] (C. Vicente).
0040-8166/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2004.06.007
(Kawano, 2003) as well as to the oxidative catabolism of auxin (Boyer et al., 1979). On the other hand, polyamine oxydases catabolize the excess of polyamines produced during active growth and differentiation. Some hydrolases have been also related to cell wall structure. RNase is located in the cell wall of both components of the lichen Evernia prunastri and it is solubilized into the cytoplasm following desiccation or freezing (Fontaniella et al., 2000a). Urease in lichens has been transiently associated to the cell wall of the algal partner during the active secretion of the enzyme to the external medium (P´erez-Urria and Vicente, 1989; P´erez-Urria et al., 1989; Vicente and P´erez-Urria, 1989). However, Legaz and Vicente (1989) find that a substantial amount of urease activity is associated to cell wall fractions of disrupted thalli of the terricolous species Cladina dendroides. Since urease shows to be an induced enzyme regulated through seasonal variations (Legaz et al., 1986a) and, in addition, it has been related to the binding of some fungal lectins on the surface of algal cells (Molina et al., 1993), the true location of this enzyme is a crucial point to support the hydrolysis of exogenous urea as well as the compatibility relationships between a fungus and its potential phycobionts.
374
A.M. Millanes et al. / Tissue and Cell 36 (2004) 373–377
In this paper, location of urease has been achieved through a specific cytochemical reaction using phycobionts recently isolated from two different lichens species, E. prunastri and Xanthoria parietina.
2. Materials and methods 2.1. Plant material E. prunastri (L.) Ach, and X. parietina (L) Th. Fr., growing on Quercus rotundifolia Willd., were collected in La Quinta (El Pardo, Madrid), dry in air flow and stored in the dark no more than 2 weeks. 2.2. Isolation of lichen phycobionts Phycobionts were isolated from recently collected thalli of E. prunastri and X. parietina according to Fontaniella et al. (2000b). 2.3. Isolation of algal cell walls and extraction of protein from phycobiont cell walls Recently isolated phycobionts were mechanically disrupted and dispersed in 3.0 mL 75 mM phosphate buffer, pH 6.9. Homogenates were centrifuged at 3200 × g for 10 min at 2 ◦ C (Brunner and Honegger, 1985). Supernatants were stored and pellets were resuspended in the same volume of buffer containing 0.1% Triton X-100 (w/v). Mixtures were continuously shaken at 4 ◦ C for 2 h, and then dialyzed against 75 mM phosphate buffer, pH 6.9, to remove completely the detergent. After this, mixtures were newly centrifuged at 3200 × g for 15 min at 2 ◦ C, pellets were resuspended in 3.0 mL of 75 mM phosphate buffer, pH 6.9, and used to assay urease activity. Alternatively, pellets were resuspended in 3.0 mL of aqueous 0.5 M NaCl. Mixtures were subjected to 4 × 15 s periods of sonication at 20 K cycles s−1 with ice-cold protection for 2 min and subsequently stored at 4 ◦ C for 12 h. Mixtures were then centrifuged at 12,000 × g for 20 min at 2 ◦ C and supernatants were dialyzed against 75 mM phosphate buffer, pH 6.9 for 4 h at 4 ◦ C (Legaz and Vicente, 1989). Protein was measured in dialysate according to Lowry et al. (1951). 2.4. Assay of urease activity Suspensions of isolated algal cell walls as well as supernatants containing cell-free extracts were assayed for urease activity in reaction mixtures containing a volume of cell wall suspension equivalent to 20 g of cell wall protein (20 g of protein from the supernatant), 40 mol urea and 150 mol potassium phosphate, pH 6.9, in a final volume of 3.0 mL (P´erez-Urria and Vicente, 1989). Incubation was carried out for 30 min at 37 ◦ C and the ammonia produced was estimated according to Conway (1962). A unit of specific activity was
defined as 1.0 mol ammonia produced per mg protein per min. 2.5. Cytochemical detection of urease Isolated phycobionts were collected by centrifugation, washed with distilled water and incubated with 0.1 M Fast Green (from Sigma Chemical Co., St. Louis, MO) in 75 mM phosphate buffer, pH 6.9, to block phenolics in order to avoid cobalt chelation (Lison, 1960). After this incubation, algal cells were incubated in 40 mM urea and 10 mM CoCl2 for 30 min at 30 ◦ C, according to Gomori (1952). Carbon dioxide produced during the enzymatic hydrolysis of urea produced insoluble cobalt carbonate when it was achieved in the presence of cobalt chloride. Cobalt sulfide was revealed as a black, electron-dense precipitate after addition of ammonium sulfate. Then, cells were washed with abundant distilled water, incubated for 30 s at room temperature with (NH4 )2 S, newly washed with distilled water and observed under light microscope. Ultrastructural analysis was performed by softening cells in a phenol:acetic acid (1/1, v/v) mixture for 3 days and then the material was dehydrated and fixed as previously described (Molina et al., 1993), including after dehydration a step during which cell were treated with uranile acetate. Embedding was done in propylene oxide-araldite II resin (stan˚ dard mixture) for 3 days at 70 ◦ C. Ultrathin sections (600 A), obtained with a OmU2 Reichert ultratome, were examined on a Philips E.M. 300 electron microscope.
3. Results Urease activity has been located by using recently isolated phycobionts from E. prunastri and X. parietina thalli. External envelops of algal cells treated for the cytochemical detection of urease appeared superficially stained when they were visualized under light microscope. The cells, used as control, that were not incubated on urea but on cobalt chloride, did not show any deposit on their cell walls (data not shown). To assure the specific location of cobalt sulfide deposits, thin sections of algal cells were observed under transmission electron microscope. Micrographs shown in Figs. 1 and 2, clearly revealed that cobalt appeared mainly associated to the cell wall of both Evernia (Fig. 1E) and Xanthoria (Fig. 2E) phycobionts whereas these deposits were not found in control cells incubated in Fast Green before treatment with ammonium sulfide in absence of urea (Figs. 1C, D and 2C, D). Large storage bodies can be seen inside the cells (Figs. 1E and 2E) as well as a more or less disorganized pyrenoid (Figs. 1E and 2C, D). Some dense, small granules in the chloroplast could be interpreted as soluble, intracellular urease (Figs. 1E and 2E). When isolated cell walls were used as a source of urease to assay this enzyme activity, more than 80% of total activ-
A.M. Millanes et al. / Tissue and Cell 36 (2004) 373–377
375
Fig. 1. Cytochemical detection of urease in phycobiont cells of E. prunastri by transmission electron microscopy. (A) Control cells recently isolated from lichen thalli incubated in 0.1 M Fast Green before treatment with ammonium sulfide. (B) Magnification of the zone in A indicated by a rectangle. (C) Control cells recently isolated from lichen thalli incubated in 0.1 M Fast Green before incubation for 30 min at 30 ◦ C in 10 mM CoCl2 in absence of urea and then treated with ammonium sulfide. (D) Magnification of the zone in C indicated by a rectangle. (E) Algal cells treated with Fast Green and incubated for 30 min at 30 ◦ C in a solution of 40 mM urea containing 10 mM CoCl2 and then treated with ammonium sulfide. Only in this case, black deposits of CoS in the cell wall reveals urease activity (open arrows) and white arrow heads indicate possible intracellular enzyme activity. Black arrow indicates storage bodies. Pyrenoid is symbolized as py. Bar = 2.0 m in A and C, and 1.0 m in B, D, and E.
Fig. 2. Cytochemical detection of urease in phycobiont cells of X. parietina by transmission electron microscopy. (A) Control cells recently isolated from lichen thalli incubated in 0.1 M Fast Green before treatment with ammonium sulfide. (B) Magnification of the zone in A indicated by a rectangle. (C) Control cells recently isolated from lichen thalli incubated in 0.1 M Fast Green before incubation for 30 min at 30 ◦ C in 10 mM CoCl2 in absence of urea and then treated with ammonium sulfide. (D) Magnification of the zone in C indicated by a rectangle. (E) Algal cells treated with Fast Green and incubated for 30 min at 30 ◦ C in a solution of 40 mM urea containing 10 mM CoCl2 and then treated with ammonium sulfide. Only in this case, black deposits of CoS in the cell wall reveals urease activity (open arrows). Black arrow indicates storage bodies. Pyrenoid is symbolized as py. Bar = 2.0 m in A and C, and 1.0 m in B, D, and E.
376
A.M. Millanes et al. / Tissue and Cell 36 (2004) 373–377
Table 1 Location of urease activity after separation of a cell wall-enriched fraction from isolated phycobionts of E. prunastri and X. parietina Phycobiont isolated from
E. prunastri X. parietina
Urease activity (milliunits) Cell wall
Supernatant
17.1 ± 2.0 5.8 ± 0.33
3.7 ± 0.41 1.6 ± 0.13
ity detected from E. prunastri phycobiont was associated to these structures whereas particulate cell wall urease from X. parietina phycobionts represented about 78% of total urease activity of algal cells (Table 1).
4. Discussion Several attempts to locate urease in lichens symbionts were achieved in the past. Legaz and Vicente (1981) separated both bionts from E. prunastri thallus and assayed urease activity in cell-free extracts prepared from isolated fungi and algae. They concluded that urease activity was largely restricted to the phycobionts. Later, Legaz et al. (1986) found that algal cell secreted urease when some Antarctic lichen thalli were floated on liquid media containing urea. The ability to secrete the enzyme was extended to many other lichen species (P´erez-Urria and Vicente, 1989; P´erez-Urria et al., 1989) and, consequently, urease activity was transiently located in isolated algal cell walls (Legaz and Vicente, 1989). On this bases, Vicente (1990) hypothesized about the occasional location of urease in the algal cell wall while the enzyme was secreted outside the cells. However, Molina et al. (1993) provided indirect probes about the constancy of the occurrence of urease in algal cell wall by using a ferritinlabelled lectin able to bind to the polygalactoside moiety of urease. Cytochemical reaction described by Gomori has been used in the present work, using phycobionts recently isolated from two epiphytic lichen species, E. prunastri and X. parietina. Since light microscopy does not provide sufficient resolution to analyze the specific location of cobalt deposits, a more precise analysis has been provided by transmission electron microscopy. Micrographs obtained in this way clearly show that electron-dense deposits of cobalt revealing urease activity are mainly located at the cell wall of both phycobionts (Figs. 1 and 2) although the occurrence of some polydisperse, small granules inside the cells indicates intracellular urease activity. This implies that urea is mainly hydrolyzed at the cell wall sites for the enzyme during the uptake of this metabolite from the medium, since the breakdown of algal cells and the assay of urease activity in both cell wall precipitate and cellfree extract indicates that about 80% of total urease activity can be recovered as cell wall-particulate protein whereas only 20% is recovered as soluble enzyme (Table 1). This distribution of urease activity can be seen as surprising. However, it must be considered that a lichen thallus is
composed by about 90% of a fungus (mycobiont) and 10% of algal cells in terms of total volume (Collins and Farrar, 1978). Thus, the bulk of exogenous urea probably is captured by the fungal component from the intercellular spaces whereas the main opportunity for algal cells is a superficial hydrolysis of this urea in contact with the external surface of the cell wall to internalise ammonia later. The high activity value of cell wall urease is in agreement with the conservation of enzyme activity by urease immobilization in inert matrices, such as cellulose (Wang et al., 1998), alginate (Das et al., 1998) or gelatine (El-Shora, 2001), matrices that conserve a hydrophilic environment sufficient to carry out catalysis depending on the immobilized enzyme.
Acknowledgements This work was supported by a grant from the Ministerio de Ciencia y Tecnolog´ıa (Spain), BFI2000-0610. We wish to thank Ms. Raquel Alonso for her excellent technical assistance.
References Boyer, N., Gaspar, T., Lamond, M., 1979. Changes in isoperoxidases and in the growth rate of Bryonia dioica internodes after mechanical rubbing. Z. Pflanzenphysiol. 93, 459–470. Brunner, U., Honegger, R., 1985. Chemical and ultrastructural studies on the distribution of sporopollenin-like biopolymers in six genera of lichen phycobionts. Can. J. Bot. 63, 2221–2230. Collins, C.A., Farrar, J.F., 1978. Structural resistances to mass transfer in the lichen Xanthoria parietina. New Phytologist 81, 71–83. Conway, E.J., 1962. Microdiffusion Analysis and Volumetric Error. Crosby Loockwood, London. Cosgrove, D.J., 1997. Relaxation in a high-stress environment: the molecular bases of extensible cell walls and cell enlargement. Plant Cell 9, 1031–1041. Das, N., Kayasth, A.M., Malhotra, O.P., 1998. Immobilization of urease from pigeon pea (Cajanus cajan L.) in polyacrylamide gels and calcium alginate beads. Biotechnol. Appl. Biochem. 28, 251. El-Shora, H.M., 2001. Properties and immobilization of urease from leaves of Chenopodium album. Bot. Bull. Acad. Sin. 42, 251–258. Fontaniella, B., Vicente, C., Legaz, M.E., 2000a. The cryoprotective role of polyols in lichens: effects on the redistribution of RNase in Evernia prunastri thallus during freezing. Plant Physiol. Biochem. 33, 621–627. Fontaniella, B., Molina, M.C., Vicente, C., 2000b. An improved method for the separation of lichen symbionts. Phyton (Austria) 40, 323–328. Gomori, G., 1952. Microscopic Histochemistry. The University of Chicago Press, Chicago, 243 pp. Kawano, T., 2003. Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and growth induction. Plant Cell Rep. 21, 829–837. Kim, Y.S., Wi, S.G., Grunwald, C., Schmitt, U., 2002. Immuno electron microscopic localization of peroxidases in the differentiating xylem of Populus spp. Holzforschung 56, 355–359. Legaz, M.E., Vicente, C., 1981. Location of several enzymes of l-arginine catabolism in Evernia pnmastri thallus. Z. Naturforsch. 36c, 692–693. Legaz, M.E., Vicente, C., 1989. Regulation of urease activity Cladina dendroides and its photobiont by lichen phenols. Plant Sci. 63, 15–24.
A.M. Millanes et al. / Tissue and Cell 36 (2004) 373–377 Legaz, M.E., Avalos, A., de Torres, M., Escribano, M.I., Gonz´alez, A., Martin- Falquina, A., P´erez-Urria, E., Vicente, C., 1986a. Annual variations in arginine metabolism and phenolics content of Evernia pnmastri. Environ. Exp. Bot. 26, 385–396. Legaz, M.E., D´ıaz-Santos, E., Vicente, C., 1986b. Lichen substrates and urease production and secretion: a physiological approach using four Antarctic lichens. Biochem. Syst. Ecol. 14, 375–379. Lehner, G., Cardemil, L., 2003. Differences in wound-induced changes in cell-wall peroxidase activities and isoform patterns between seedlings of Prosopis tamarugo and Prosopis chilensis. Tree Physiol. 23, 443–452. Lison, L., 1960. Histochimie et Cytochimie Animales. Gauthier-Villards, Paris. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Molina, M.C., Mu˜niz, E., Vicente, C., 1993. Enzymatic activities of algalbinding protein and its algal cell wall receptor in the lichen Xanthoria parietina. An approach to the parasitic basis of mutualism. Plant Physiol. Biochem. 31, 131–142. Oparka, K.J., 1990. What is phloem unloading? Plant Physiol. 94, 393–396.
377
P´erez-Urria, E., Vicente, C., 1989. Purification and some properties of a secreted urease from Evernia prunastri. J. Plant Physiol. 133, 692–695. P´erez-Urria, E., Vicente, C., Xavier Filho, L., 1989. Screening of urease production and secretion by seven species of Finish lichens. Biochem. Syst. Ecol. 17, 359–363. Showalter, A.M., 1993. Structure and function of plant cell wall proteins. Plant Cell 5, 9–23. Stolle-Smits, T., Beekhuizen, J.G., Pijnenburg, M., Recourt, K., Derksen, J., Voragen, A.G., 1999. Changes in cell wall polysaccharides of green bean pods during development. Plant Physiol. 121, 363– 372. Vicente, C., 1990. The concept of endohabitat in phycobiont-containing lichens. II. The role of secretable enzymes. Endocyt. Cell Res. 7, 61–72. Vicente, G., P´erez-Urria, E., 1989. Production and secretion of urease by Evernia prunastri thallus and its symbionts. Endocyt. Cell Res. 6, 87–97. Wang, J., Song, X.J., Wu, M., Ren, Z.J., Yuan, B., Lei, Z.L., 1998. Studies on immobilization of urease on cellulose acetate membrane. Chem. J. Chin. Univ. 19, 1104–1106.
Cytochemical location of urease in the cell wall of two different lichen phycobionts A.M. Millanesa , B. Fontaniellaa , M.L. Garc´ıab , M.T. Solasc , C. Vicentea,∗ , M.E. Legaza a
Laboratory of Plant Biology, Faculty of Biology, Complutense University, C/Jos´e Antonio Novias s/n, 28040 Madrid, Spain b Center of Electron Microscopy, Complutense University, 28040 Madrid, Spain c Department of Cell Biology, Faculty of Biology, Complutense University, 28040 Madrid, Spain Received 9 December 2003; received in revised form 15 June 2004; accepted 30 June 2004
Abstract The enzyme urease has been located in the cell wall of recently isolated phycobionts from Evernia prunastri and Xanthoria parietina lichens. Cytochemical detection is achieved by producing a black, electron-dense precipitate of cobalt sulfide proceeding from CO2 evolved from urea in the presence of cobalt chloride. Cellular fractionation reveals that about 80% of total urease activity was associated to the cell wall on both phycobionts whereas only 20% was recovered as soluble protein. © 2004 Elsevier Ltd. All rights reserved. Keywords: Cell wall; Evernia prunastri; Phycobiont; Urease; Xanthoria parietina
1. Introduction The plant cell wall is revealed as a dynamic structure to which many proteins are attached to develop different biochemical functions. Proteases in the cell wall are related to the turnover of extensin and some proline-rich proteins (Showalter, 1993) whereas several endoglycanases are related to the enzymatic relaxation of structural cell wall polysaccharides, such as cellulose, callose, xyloglucans and pectins (Cosgrove, 1997). Invertase (-fructofuranosidase) in the cell wall of companion cells is involved in the loading of hydrolyzed sucrose to parenchymatous cells of Saccharum officinarum stalks (Oparka, 1990) whereas ␣-arabinosidase catalyzes the hydrolysis of the arabinoside moiety of extensin (Stolle-Smits et al., 1999). Oxido-reductases such as peroxidases are related to the catabolism of lignin by lignin-degrading fungi (Kim et al., 2002), to the biosynthesis of lignin in response to wounding (Lehner and Cardemil, 2003) and to the generation of active oxygen species related to plant defense mechanisms ∗
Corresponding author. Tel.: +34 91 3944565; fax: +34 91 3945034. E-mail address: [email protected] (C. Vicente).
0040-8166/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2004.06.007
(Kawano, 2003) as well as to the oxidative catabolism of auxin (Boyer et al., 1979). On the other hand, polyamine oxydases catabolize the excess of polyamines produced during active growth and differentiation. Some hydrolases have been also related to cell wall structure. RNase is located in the cell wall of both components of the lichen Evernia prunastri and it is solubilized into the cytoplasm following desiccation or freezing (Fontaniella et al., 2000a). Urease in lichens has been transiently associated to the cell wall of the algal partner during the active secretion of the enzyme to the external medium (P´erez-Urria and Vicente, 1989; P´erez-Urria et al., 1989; Vicente and P´erez-Urria, 1989). However, Legaz and Vicente (1989) find that a substantial amount of urease activity is associated to cell wall fractions of disrupted thalli of the terricolous species Cladina dendroides. Since urease shows to be an induced enzyme regulated through seasonal variations (Legaz et al., 1986a) and, in addition, it has been related to the binding of some fungal lectins on the surface of algal cells (Molina et al., 1993), the true location of this enzyme is a crucial point to support the hydrolysis of exogenous urea as well as the compatibility relationships between a fungus and its potential phycobionts.
374
A.M. Millanes et al. / Tissue and Cell 36 (2004) 373–377
In this paper, location of urease has been achieved through a specific cytochemical reaction using phycobionts recently isolated from two different lichens species, E. prunastri and Xanthoria parietina.
2. Materials and methods 2.1. Plant material E. prunastri (L.) Ach, and X. parietina (L) Th. Fr., growing on Quercus rotundifolia Willd., were collected in La Quinta (El Pardo, Madrid), dry in air flow and stored in the dark no more than 2 weeks. 2.2. Isolation of lichen phycobionts Phycobionts were isolated from recently collected thalli of E. prunastri and X. parietina according to Fontaniella et al. (2000b). 2.3. Isolation of algal cell walls and extraction of protein from phycobiont cell walls Recently isolated phycobionts were mechanically disrupted and dispersed in 3.0 mL 75 mM phosphate buffer, pH 6.9. Homogenates were centrifuged at 3200 × g for 10 min at 2 ◦ C (Brunner and Honegger, 1985). Supernatants were stored and pellets were resuspended in the same volume of buffer containing 0.1% Triton X-100 (w/v). Mixtures were continuously shaken at 4 ◦ C for 2 h, and then dialyzed against 75 mM phosphate buffer, pH 6.9, to remove completely the detergent. After this, mixtures were newly centrifuged at 3200 × g for 15 min at 2 ◦ C, pellets were resuspended in 3.0 mL of 75 mM phosphate buffer, pH 6.9, and used to assay urease activity. Alternatively, pellets were resuspended in 3.0 mL of aqueous 0.5 M NaCl. Mixtures were subjected to 4 × 15 s periods of sonication at 20 K cycles s−1 with ice-cold protection for 2 min and subsequently stored at 4 ◦ C for 12 h. Mixtures were then centrifuged at 12,000 × g for 20 min at 2 ◦ C and supernatants were dialyzed against 75 mM phosphate buffer, pH 6.9 for 4 h at 4 ◦ C (Legaz and Vicente, 1989). Protein was measured in dialysate according to Lowry et al. (1951). 2.4. Assay of urease activity Suspensions of isolated algal cell walls as well as supernatants containing cell-free extracts were assayed for urease activity in reaction mixtures containing a volume of cell wall suspension equivalent to 20 g of cell wall protein (20 g of protein from the supernatant), 40 mol urea and 150 mol potassium phosphate, pH 6.9, in a final volume of 3.0 mL (P´erez-Urria and Vicente, 1989). Incubation was carried out for 30 min at 37 ◦ C and the ammonia produced was estimated according to Conway (1962). A unit of specific activity was
defined as 1.0 mol ammonia produced per mg protein per min. 2.5. Cytochemical detection of urease Isolated phycobionts were collected by centrifugation, washed with distilled water and incubated with 0.1 M Fast Green (from Sigma Chemical Co., St. Louis, MO) in 75 mM phosphate buffer, pH 6.9, to block phenolics in order to avoid cobalt chelation (Lison, 1960). After this incubation, algal cells were incubated in 40 mM urea and 10 mM CoCl2 for 30 min at 30 ◦ C, according to Gomori (1952). Carbon dioxide produced during the enzymatic hydrolysis of urea produced insoluble cobalt carbonate when it was achieved in the presence of cobalt chloride. Cobalt sulfide was revealed as a black, electron-dense precipitate after addition of ammonium sulfate. Then, cells were washed with abundant distilled water, incubated for 30 s at room temperature with (NH4 )2 S, newly washed with distilled water and observed under light microscope. Ultrastructural analysis was performed by softening cells in a phenol:acetic acid (1/1, v/v) mixture for 3 days and then the material was dehydrated and fixed as previously described (Molina et al., 1993), including after dehydration a step during which cell were treated with uranile acetate. Embedding was done in propylene oxide-araldite II resin (stan˚ dard mixture) for 3 days at 70 ◦ C. Ultrathin sections (600 A), obtained with a OmU2 Reichert ultratome, were examined on a Philips E.M. 300 electron microscope.
3. Results Urease activity has been located by using recently isolated phycobionts from E. prunastri and X. parietina thalli. External envelops of algal cells treated for the cytochemical detection of urease appeared superficially stained when they were visualized under light microscope. The cells, used as control, that were not incubated on urea but on cobalt chloride, did not show any deposit on their cell walls (data not shown). To assure the specific location of cobalt sulfide deposits, thin sections of algal cells were observed under transmission electron microscope. Micrographs shown in Figs. 1 and 2, clearly revealed that cobalt appeared mainly associated to the cell wall of both Evernia (Fig. 1E) and Xanthoria (Fig. 2E) phycobionts whereas these deposits were not found in control cells incubated in Fast Green before treatment with ammonium sulfide in absence of urea (Figs. 1C, D and 2C, D). Large storage bodies can be seen inside the cells (Figs. 1E and 2E) as well as a more or less disorganized pyrenoid (Figs. 1E and 2C, D). Some dense, small granules in the chloroplast could be interpreted as soluble, intracellular urease (Figs. 1E and 2E). When isolated cell walls were used as a source of urease to assay this enzyme activity, more than 80% of total activ-
A.M. Millanes et al. / Tissue and Cell 36 (2004) 373–377
375
Fig. 1. Cytochemical detection of urease in phycobiont cells of E. prunastri by transmission electron microscopy. (A) Control cells recently isolated from lichen thalli incubated in 0.1 M Fast Green before treatment with ammonium sulfide. (B) Magnification of the zone in A indicated by a rectangle. (C) Control cells recently isolated from lichen thalli incubated in 0.1 M Fast Green before incubation for 30 min at 30 ◦ C in 10 mM CoCl2 in absence of urea and then treated with ammonium sulfide. (D) Magnification of the zone in C indicated by a rectangle. (E) Algal cells treated with Fast Green and incubated for 30 min at 30 ◦ C in a solution of 40 mM urea containing 10 mM CoCl2 and then treated with ammonium sulfide. Only in this case, black deposits of CoS in the cell wall reveals urease activity (open arrows) and white arrow heads indicate possible intracellular enzyme activity. Black arrow indicates storage bodies. Pyrenoid is symbolized as py. Bar = 2.0 m in A and C, and 1.0 m in B, D, and E.
Fig. 2. Cytochemical detection of urease in phycobiont cells of X. parietina by transmission electron microscopy. (A) Control cells recently isolated from lichen thalli incubated in 0.1 M Fast Green before treatment with ammonium sulfide. (B) Magnification of the zone in A indicated by a rectangle. (C) Control cells recently isolated from lichen thalli incubated in 0.1 M Fast Green before incubation for 30 min at 30 ◦ C in 10 mM CoCl2 in absence of urea and then treated with ammonium sulfide. (D) Magnification of the zone in C indicated by a rectangle. (E) Algal cells treated with Fast Green and incubated for 30 min at 30 ◦ C in a solution of 40 mM urea containing 10 mM CoCl2 and then treated with ammonium sulfide. Only in this case, black deposits of CoS in the cell wall reveals urease activity (open arrows). Black arrow indicates storage bodies. Pyrenoid is symbolized as py. Bar = 2.0 m in A and C, and 1.0 m in B, D, and E.
376
A.M. Millanes et al. / Tissue and Cell 36 (2004) 373–377
Table 1 Location of urease activity after separation of a cell wall-enriched fraction from isolated phycobionts of E. prunastri and X. parietina Phycobiont isolated from
E. prunastri X. parietina
Urease activity (milliunits) Cell wall
Supernatant
17.1 ± 2.0 5.8 ± 0.33
3.7 ± 0.41 1.6 ± 0.13
ity detected from E. prunastri phycobiont was associated to these structures whereas particulate cell wall urease from X. parietina phycobionts represented about 78% of total urease activity of algal cells (Table 1).
4. Discussion Several attempts to locate urease in lichens symbionts were achieved in the past. Legaz and Vicente (1981) separated both bionts from E. prunastri thallus and assayed urease activity in cell-free extracts prepared from isolated fungi and algae. They concluded that urease activity was largely restricted to the phycobionts. Later, Legaz et al. (1986) found that algal cell secreted urease when some Antarctic lichen thalli were floated on liquid media containing urea. The ability to secrete the enzyme was extended to many other lichen species (P´erez-Urria and Vicente, 1989; P´erez-Urria et al., 1989) and, consequently, urease activity was transiently located in isolated algal cell walls (Legaz and Vicente, 1989). On this bases, Vicente (1990) hypothesized about the occasional location of urease in the algal cell wall while the enzyme was secreted outside the cells. However, Molina et al. (1993) provided indirect probes about the constancy of the occurrence of urease in algal cell wall by using a ferritinlabelled lectin able to bind to the polygalactoside moiety of urease. Cytochemical reaction described by Gomori has been used in the present work, using phycobionts recently isolated from two epiphytic lichen species, E. prunastri and X. parietina. Since light microscopy does not provide sufficient resolution to analyze the specific location of cobalt deposits, a more precise analysis has been provided by transmission electron microscopy. Micrographs obtained in this way clearly show that electron-dense deposits of cobalt revealing urease activity are mainly located at the cell wall of both phycobionts (Figs. 1 and 2) although the occurrence of some polydisperse, small granules inside the cells indicates intracellular urease activity. This implies that urea is mainly hydrolyzed at the cell wall sites for the enzyme during the uptake of this metabolite from the medium, since the breakdown of algal cells and the assay of urease activity in both cell wall precipitate and cellfree extract indicates that about 80% of total urease activity can be recovered as cell wall-particulate protein whereas only 20% is recovered as soluble enzyme (Table 1). This distribution of urease activity can be seen as surprising. However, it must be considered that a lichen thallus is
composed by about 90% of a fungus (mycobiont) and 10% of algal cells in terms of total volume (Collins and Farrar, 1978). Thus, the bulk of exogenous urea probably is captured by the fungal component from the intercellular spaces whereas the main opportunity for algal cells is a superficial hydrolysis of this urea in contact with the external surface of the cell wall to internalise ammonia later. The high activity value of cell wall urease is in agreement with the conservation of enzyme activity by urease immobilization in inert matrices, such as cellulose (Wang et al., 1998), alginate (Das et al., 1998) or gelatine (El-Shora, 2001), matrices that conserve a hydrophilic environment sufficient to carry out catalysis depending on the immobilized enzyme.
Acknowledgements This work was supported by a grant from the Ministerio de Ciencia y Tecnolog´ıa (Spain), BFI2000-0610. We wish to thank Ms. Raquel Alonso for her excellent technical assistance.
References Boyer, N., Gaspar, T., Lamond, M., 1979. Changes in isoperoxidases and in the growth rate of Bryonia dioica internodes after mechanical rubbing. Z. Pflanzenphysiol. 93, 459–470. Brunner, U., Honegger, R., 1985. Chemical and ultrastructural studies on the distribution of sporopollenin-like biopolymers in six genera of lichen phycobionts. Can. J. Bot. 63, 2221–2230. Collins, C.A., Farrar, J.F., 1978. Structural resistances to mass transfer in the lichen Xanthoria parietina. New Phytologist 81, 71–83. Conway, E.J., 1962. Microdiffusion Analysis and Volumetric Error. Crosby Loockwood, London. Cosgrove, D.J., 1997. Relaxation in a high-stress environment: the molecular bases of extensible cell walls and cell enlargement. Plant Cell 9, 1031–1041. Das, N., Kayasth, A.M., Malhotra, O.P., 1998. Immobilization of urease from pigeon pea (Cajanus cajan L.) in polyacrylamide gels and calcium alginate beads. Biotechnol. Appl. Biochem. 28, 251. El-Shora, H.M., 2001. Properties and immobilization of urease from leaves of Chenopodium album. Bot. Bull. Acad. Sin. 42, 251–258. Fontaniella, B., Vicente, C., Legaz, M.E., 2000a. The cryoprotective role of polyols in lichens: effects on the redistribution of RNase in Evernia prunastri thallus during freezing. Plant Physiol. Biochem. 33, 621–627. Fontaniella, B., Molina, M.C., Vicente, C., 2000b. An improved method for the separation of lichen symbionts. Phyton (Austria) 40, 323–328. Gomori, G., 1952. Microscopic Histochemistry. The University of Chicago Press, Chicago, 243 pp. Kawano, T., 2003. Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and growth induction. Plant Cell Rep. 21, 829–837. Kim, Y.S., Wi, S.G., Grunwald, C., Schmitt, U., 2002. Immuno electron microscopic localization of peroxidases in the differentiating xylem of Populus spp. Holzforschung 56, 355–359. Legaz, M.E., Vicente, C., 1981. Location of several enzymes of l-arginine catabolism in Evernia pnmastri thallus. Z. Naturforsch. 36c, 692–693. Legaz, M.E., Vicente, C., 1989. Regulation of urease activity Cladina dendroides and its photobiont by lichen phenols. Plant Sci. 63, 15–24.
A.M. Millanes et al. / Tissue and Cell 36 (2004) 373–377 Legaz, M.E., Avalos, A., de Torres, M., Escribano, M.I., Gonz´alez, A., Martin- Falquina, A., P´erez-Urria, E., Vicente, C., 1986a. Annual variations in arginine metabolism and phenolics content of Evernia pnmastri. Environ. Exp. Bot. 26, 385–396. Legaz, M.E., D´ıaz-Santos, E., Vicente, C., 1986b. Lichen substrates and urease production and secretion: a physiological approach using four Antarctic lichens. Biochem. Syst. Ecol. 14, 375–379. Lehner, G., Cardemil, L., 2003. Differences in wound-induced changes in cell-wall peroxidase activities and isoform patterns between seedlings of Prosopis tamarugo and Prosopis chilensis. Tree Physiol. 23, 443–452. Lison, L., 1960. Histochimie et Cytochimie Animales. Gauthier-Villards, Paris. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Molina, M.C., Mu˜niz, E., Vicente, C., 1993. Enzymatic activities of algalbinding protein and its algal cell wall receptor in the lichen Xanthoria parietina. An approach to the parasitic basis of mutualism. Plant Physiol. Biochem. 31, 131–142. Oparka, K.J., 1990. What is phloem unloading? Plant Physiol. 94, 393–396.
377
P´erez-Urria, E., Vicente, C., 1989. Purification and some properties of a secreted urease from Evernia prunastri. J. Plant Physiol. 133, 692–695. P´erez-Urria, E., Vicente, C., Xavier Filho, L., 1989. Screening of urease production and secretion by seven species of Finish lichens. Biochem. Syst. Ecol. 17, 359–363. Showalter, A.M., 1993. Structure and function of plant cell wall proteins. Plant Cell 5, 9–23. Stolle-Smits, T., Beekhuizen, J.G., Pijnenburg, M., Recourt, K., Derksen, J., Voragen, A.G., 1999. Changes in cell wall polysaccharides of green bean pods during development. Plant Physiol. 121, 363– 372. Vicente, C., 1990. The concept of endohabitat in phycobiont-containing lichens. II. The role of secretable enzymes. Endocyt. Cell Res. 7, 61–72. Vicente, G., P´erez-Urria, E., 1989. Production and secretion of urease by Evernia prunastri thallus and its symbionts. Endocyt. Cell Res. 6, 87–97. Wang, J., Song, X.J., Wu, M., Ren, Z.J., Yuan, B., Lei, Z.L., 1998. Studies on immobilization of urease on cellulose acetate membrane. Chem. J. Chin. Univ. 19, 1104–1106.