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Nitrogen assimilation enzymes in mycorrhizas of Norway spruce, Douglas fir and beech
65
Agriculture, Ecoaystels and Environment, 28 (1989) 65-70 Elmevier Science Publlshers B.V., Amsterdam Printed in Czechoslovakia
NITROGEN ASSIMILATIONENZYMESIN flYCORRHIZASOF NORWAYSPRUCE, DOUGLAS FIR AND BEECH CHALOT Michel +, DELL Bernard++, BOTTOMBernard+ and MARTINFrancis+++ +
Universit~ de Nancy I, Laboratoire de Biologie V~g~tale et Foresti@Fe, B.P. 239, 54506 Vandoeuvre-les-Nancy Cedex, France
++ Murdoch University, School of Biological and Environmental Sciences, Murdoch, Western Australia, 6150 Australia +++ Centre de Recherches Foresti~res de Nancy, Laboratoire de Hicrobiolologie Foresti~re, Cha~enoux, 54280 Seichamps, France Abstract NAD and NADPdependant glutamate dehydrogenases (GDH) as well as aspartate aminotransferase (AAT) were analyzed on the fungi cultivated in pure culture, on the mycorrhizas and on the non mycorrhizal host roots. Four associations were studied : Norway spruce-Hebeloma sp., Douglas f i r Laccaria laccata, B~ch-I/ebelom~ crustuliniforme and Beech-Paxi~Zus inVo~utus. NADP-GDHpresent in the fungus and NAD-GDHpresent in non-infec-
ted roots were both detected in spruce and douglas mycorrhizas. By contrast, fungal NADP-GDH~as suppressed in mycorrhizas of beech. In the four associations, AAT found in the mycorrhizas corresponded to the host root isoforms whereas the fungal isoform was strongly repressed. Introduction Higher plants use the glutamine synthetase/glutamate synthase (GS/GOGA~ cycle for ammonia assimilation (LEA and MIFLIN, 1974). In fungi, nitrogen assimilation mainly occurs via the glutamate dehydrogenase (GDH) pathway (HARZLUF, 1981), although recently some non-mycorrhizal fungi have been shown to u t i l i z e the GS/GOC~Tcycle (KUS~ANet a l . , 1987). Glutamate is often the amino-donor substrate in transamination reactions. This amino acid is converted to aspartate by the aspartate aminotransferase (MT) and to aianine by the alanine aminotransferase (AIAT). In mycorrhizal associatons, data have been obtained demonstrating that the fungal pathways of nitrogen assimilation in beech mycorrhizas are modified by the establishment of the symbiosis (MARTINet a l . , 1986). Indeed, nitrogen-15 nuclear magnetic resonance spectroscopy as well as use of en-
66
zyme inhibitors suggest that ammoniumassimilation occurs mainly via the 91utamine synthetase/glutamate synthase pathway, and that glutamate dehydrogenase plays l i t t l e , i f any, part in this process. The present work extends these preliminary data to a few other associaciations and also analyzes the aminotransferases. Materials and Methods Norway spruce (Pieea exoelsa) roots and Hebeloma sp. ectomycorrhizas were obtained from 4-year-old plants grown in nursery conditions. Douglas f i r (Pseudotsuga dougZasii) roots either non-mycorrhizal or ectomycorrhizal with Laeoaria laoeata (strain S 238) were collected from lyear-olm seedlings grown in nursery conditions. Beech (Fagus syZvatiea) roots and
PaxiIZus invoZutu~ (NAUDET strain) ectomycorrhizasas well as Hebeloma orustuZiniforme ectomycorrhizas were collected from 4-6-month-old seedlings grown in a pasteurized peat mix in nursery conditions. The fungi were cultivated in pure culture in Pachlewski's medium. Enzyme activities and protein concentration were determined according to methods described elsewhere (KHALIDet a l . , 1988 ; DELL et a l . , 1988). Electrophoresis was carried out on 6 % polyacrylamide slab gels. The bands of NADP-GDHand NAD-GDHactivities were located by using a tetrazolium assay system (BLUMENTHALand SMITH, 1973) and AAT activity was revealed with Fast violet blue (KHALID et a l . , 1988). Antibodies raised against purified NADP-GDHof Cenococcum geophilum were used in anticatalytic immunoprecipitation tests and to localize NADP-GDH by using indirect immunofluorescenceas described previously by PERROT et al. (1981). Results In the ectomycorrhizal fungus Hebeloma sp., high activities of NADP-GDH were present in extracts from mycelium grown in pure culture as well as from the rhizomorphs and the surrounding mycelium associated with spruce roots, while NAD-GDHactivities remained very low (Table I). By contrast, only NAD-GDHactivity was detected in non-mycorrhizal roots. In the mycor~izas both enzyme activities were present although NADP-specific activity was greater than that of NAD. The aminotransferases (AAT and AIAT) were present in each partner and in the mycorrhizas (Table I). A similar distribution of enzymeactivities vJas observed in the association Douglas fir-Laacaria lacaata (not shown).
67 TABLE 1 Specific activities (nKal/mg protein) of GDH, AAT and AIAT in rnycorrhizal and non-mycerrhizal spruce roots and in the mycorrhizal fungus ~ llp. means of 3 determinations) N/U)P-GDH Spruce- Hebek~ma mycorrhizas (1) Non-mycorrhizal roots (1)
6.2
Fungus (pure culture) (2)
AIAT
1.5
13.1
5.6
• 0
0.8
22.0
2.4
20.0
0.8
18.4
12.2
9.6
6.2
7.6
5.5
Rhizomorphs (1)
4.4
#0
Fluffy mycelium surrounding mycorrhizas (1)
3.0
• 0
(I) (2)
A
AAT
4-year-old spruce seedlings 8-day-old cultures
SPRUCE-H~'SELO~ $p.
B
BEECH-//£BELOMACRUSTU/'INIFOR/,~
r •
M
NM
M
F
f
NM
M
F
NM
M
F
NM
--1
'
i •
1
I
I ! I
I NAD - GDH
NADP- GDH
I
NAD- GDH
NADP- GDH
Fig. i : Electrophoretic patterns of NAD-GDH and NADP-GDH from ectomycorrhizas (Hltfungu s in pure culture (F} and non-mycorrhiza] roots (NM), 25 ug of protein (crude extractsl were added to each wel],
68
These results contrast with those obtained with Beech ectomycorrhizas. Indeed, in the associations Beech-pc~llu~ involutu~, Beech-Hebeloma
o~tulinifo~me, only low )~DP-GI)H a c t i v i t i e s were found (DELL e t a l . , 1988) and even not detected in Beech-~e¢o~ius su1~leis mycorrhizas (F. MARTIN, private comnmication).
A
B
C
D
E
F
F-" I[
Rf
•~
Fi
0.28
0.52 0,58
I I
6.3
8.7
5.0
traces
13.0
11.5
Fi). 2 : Electrophoretic patterns of aspartate aminotransferase (AAT) in spruce ectomycorrhizas, in excised mycorrhizal tissues and in each partner of the association. Figures below the diagram denote specific a c t i v i t i e s of the enzyme. A : whole m~corrhiza (Spruce-Hebelo~a sp.) B C D E F
: : : : :
Vascular cylinder Sheath + Hartig net Peripheral mycelium )~on-mycorrhizal roots Fungus in pure culture (11-day -old culture)
69
In the spruce-HebeZo~ sp. association, gel electrophoresis confirmed the presence of NAD-GDH in the host c e l l s (one band) and the presence of a high level of NADP-GDHa c t i v i t y in the fungus (one major band and one minor band). In the mycorrhizas, NAD and NADP-GDHwere both detected (Fig. 1A). I t can be noticed that NADP-GDHa c t i v i t i e s revealed on gels ~ deamtnation were also detected with NAD. Identical banding patterns were also obtained with the Douglas fir-Laecaria Zaeeata association (not shown). In the Beech-Hebeloma crustuliniforme association, the single band of IIADP-GDH a c t i v i t y found in the fungus was only detected as traces in the mycorrhiza where considerable NAD-GDHa c t i v i t y was revealed. The same band corresponding to NAD-GDHwas present in non-mycorrhizal roots (Fig. 1B). Likewise, ectomycorrhizas of Beech-Paxillus invoZutus gave a single NADGDH band identical to the host plant GDH (not shown). Antibodies raised against NADP-GDHof Cenococcum g e o p h i l ~ were to a large extent recognised by NADP-GDHof HebeZoma Sp. and were then used to confirm the fungal o r i g i n of this enzyme in spruce mycorrhizas. Anticat a l y t i c immunoprecipitation tests suppressed NADP-GDHa c t i v i t i e s in crude extracts of the ectomycorrhiza. Indirect inununofluorescence techniques were also capable of l o c a l i z i n g the enzyme which was restricted to the mycelial layers surrounding the host roots (data not presented). As for aspartate aminot~ansferase, the d i s t i n c t isoforms found in the four ectomycorrhizas, always corresponded to the host root isoforms, whereas the fungal isoform found ifl the fungus cultivated in pure culture, was not detected (Fig. 2A, E, F). Dissection of the mycorrhizal tissues in spruce confirmed these results : the vascular cylinder free of fungus and the cortical region including host c e l l s and fungal hyphae revealed identical isoforms, while no a c t i v i t y was found in the peripheral mycel i a l layer (Fig. 2B, C, D). Conclusion According to the enzyme and the type of association investigated, isoenzymes found in mycorrhizas are variable. In a l l the associations investigated, fungal AAT was strongly repressed whereas fungal NADP-GDHwas only repressed in beech ectomycorrhizas. In this latter symbiosis, the present results suggest that fungal NADP-GDHmay be regulated by the host plant. I t is obvious that the fungal pathways of nitrogen assimilation are modified by the establishment of the symbiosis, however the mechanism of regulation is not understood and requires additional iRvestigation$.
70
References BLUMENTHAL, K.M., SMITH, E . L . J . Biol. Chem. 248: 6002-6008. 1973. DELL, B., BOTTON, B., MARTIN, F., LE TACON, F. New Phytol. (in press). 1988. KHALID, A., BOUKROUTE,A., BOTTON, B., MARTIN, F. Plant Physiol. Biochem. 26: 17-28. 1988. KUSNAN, M.B., BERGER, M.G., FOCK, H . P . J . Gen. rlicrobiol. 133: 1235-1242. 1987. LEA, P.J., MIFLIN, B.B. Nature 251: 614-616. 1974. MARTIN, F., STEWART, G., GENETET, I . , LE TACON, F. New Phytol. 102: 8594. 1986. MARZLUF, G.A. Microbiol. Reviews. 45: 437-461. 1981. PERROT, C., VIDAL, J., BURLET, A., GADAL, P. Planta. 151: 226-231. 1981.
Chalot, M., Dell, B., Botton, B. and Martin, F., 1989: Nitrogen assimilation enzymes in mycorrhizas of Norway spruce, douglas fir and beech. Agric., Ecosystems Environ., 28: 65-70.
Agriculture, Ecoaystels and Environment, 28 (1989) 65-70 Elmevier Science Publlshers B.V., Amsterdam Printed in Czechoslovakia
NITROGEN ASSIMILATIONENZYMESIN flYCORRHIZASOF NORWAYSPRUCE, DOUGLAS FIR AND BEECH CHALOT Michel +, DELL Bernard++, BOTTOMBernard+ and MARTINFrancis+++ +
Universit~ de Nancy I, Laboratoire de Biologie V~g~tale et Foresti@Fe, B.P. 239, 54506 Vandoeuvre-les-Nancy Cedex, France
++ Murdoch University, School of Biological and Environmental Sciences, Murdoch, Western Australia, 6150 Australia +++ Centre de Recherches Foresti~res de Nancy, Laboratoire de Hicrobiolologie Foresti~re, Cha~enoux, 54280 Seichamps, France Abstract NAD and NADPdependant glutamate dehydrogenases (GDH) as well as aspartate aminotransferase (AAT) were analyzed on the fungi cultivated in pure culture, on the mycorrhizas and on the non mycorrhizal host roots. Four associations were studied : Norway spruce-Hebeloma sp., Douglas f i r Laccaria laccata, B~ch-I/ebelom~ crustuliniforme and Beech-Paxi~Zus inVo~utus. NADP-GDHpresent in the fungus and NAD-GDHpresent in non-infec-
ted roots were both detected in spruce and douglas mycorrhizas. By contrast, fungal NADP-GDH~as suppressed in mycorrhizas of beech. In the four associations, AAT found in the mycorrhizas corresponded to the host root isoforms whereas the fungal isoform was strongly repressed. Introduction Higher plants use the glutamine synthetase/glutamate synthase (GS/GOGA~ cycle for ammonia assimilation (LEA and MIFLIN, 1974). In fungi, nitrogen assimilation mainly occurs via the glutamate dehydrogenase (GDH) pathway (HARZLUF, 1981), although recently some non-mycorrhizal fungi have been shown to u t i l i z e the GS/GOC~Tcycle (KUS~ANet a l . , 1987). Glutamate is often the amino-donor substrate in transamination reactions. This amino acid is converted to aspartate by the aspartate aminotransferase (MT) and to aianine by the alanine aminotransferase (AIAT). In mycorrhizal associatons, data have been obtained demonstrating that the fungal pathways of nitrogen assimilation in beech mycorrhizas are modified by the establishment of the symbiosis (MARTINet a l . , 1986). Indeed, nitrogen-15 nuclear magnetic resonance spectroscopy as well as use of en-
66
zyme inhibitors suggest that ammoniumassimilation occurs mainly via the 91utamine synthetase/glutamate synthase pathway, and that glutamate dehydrogenase plays l i t t l e , i f any, part in this process. The present work extends these preliminary data to a few other associaciations and also analyzes the aminotransferases. Materials and Methods Norway spruce (Pieea exoelsa) roots and Hebeloma sp. ectomycorrhizas were obtained from 4-year-old plants grown in nursery conditions. Douglas f i r (Pseudotsuga dougZasii) roots either non-mycorrhizal or ectomycorrhizal with Laeoaria laoeata (strain S 238) were collected from lyear-olm seedlings grown in nursery conditions. Beech (Fagus syZvatiea) roots and
PaxiIZus invoZutu~ (NAUDET strain) ectomycorrhizasas well as Hebeloma orustuZiniforme ectomycorrhizas were collected from 4-6-month-old seedlings grown in a pasteurized peat mix in nursery conditions. The fungi were cultivated in pure culture in Pachlewski's medium. Enzyme activities and protein concentration were determined according to methods described elsewhere (KHALIDet a l . , 1988 ; DELL et a l . , 1988). Electrophoresis was carried out on 6 % polyacrylamide slab gels. The bands of NADP-GDHand NAD-GDHactivities were located by using a tetrazolium assay system (BLUMENTHALand SMITH, 1973) and AAT activity was revealed with Fast violet blue (KHALID et a l . , 1988). Antibodies raised against purified NADP-GDHof Cenococcum geophilum were used in anticatalytic immunoprecipitation tests and to localize NADP-GDH by using indirect immunofluorescenceas described previously by PERROT et al. (1981). Results In the ectomycorrhizal fungus Hebeloma sp., high activities of NADP-GDH were present in extracts from mycelium grown in pure culture as well as from the rhizomorphs and the surrounding mycelium associated with spruce roots, while NAD-GDHactivities remained very low (Table I). By contrast, only NAD-GDHactivity was detected in non-mycorrhizal roots. In the mycor~izas both enzyme activities were present although NADP-specific activity was greater than that of NAD. The aminotransferases (AAT and AIAT) were present in each partner and in the mycorrhizas (Table I). A similar distribution of enzymeactivities vJas observed in the association Douglas fir-Laacaria lacaata (not shown).
67 TABLE 1 Specific activities (nKal/mg protein) of GDH, AAT and AIAT in rnycorrhizal and non-mycerrhizal spruce roots and in the mycorrhizal fungus ~ llp. means of 3 determinations) N/U)P-GDH Spruce- Hebek~ma mycorrhizas (1) Non-mycorrhizal roots (1)
6.2
Fungus (pure culture) (2)
AIAT
1.5
13.1
5.6
• 0
0.8
22.0
2.4
20.0
0.8
18.4
12.2
9.6
6.2
7.6
5.5
Rhizomorphs (1)
4.4
#0
Fluffy mycelium surrounding mycorrhizas (1)
3.0
• 0
(I) (2)
A
AAT
4-year-old spruce seedlings 8-day-old cultures
SPRUCE-H~'SELO~ $p.
B
BEECH-//£BELOMACRUSTU/'INIFOR/,~
r •
M
NM
M
F
f
NM
M
F
NM
M
F
NM
--1
'
i •
1
I
I ! I
I NAD - GDH
NADP- GDH
I
NAD- GDH
NADP- GDH
Fig. i : Electrophoretic patterns of NAD-GDH and NADP-GDH from ectomycorrhizas (Hltfungu s in pure culture (F} and non-mycorrhiza] roots (NM), 25 ug of protein (crude extractsl were added to each wel],
68
These results contrast with those obtained with Beech ectomycorrhizas. Indeed, in the associations Beech-pc~llu~ involutu~, Beech-Hebeloma
o~tulinifo~me, only low )~DP-GI)H a c t i v i t i e s were found (DELL e t a l . , 1988) and even not detected in Beech-~e¢o~ius su1~leis mycorrhizas (F. MARTIN, private comnmication).
A
B
C
D
E
F
F-" I[
Rf
•~
Fi
0.28
0.52 0,58
I I
6.3
8.7
5.0
traces
13.0
11.5
Fi). 2 : Electrophoretic patterns of aspartate aminotransferase (AAT) in spruce ectomycorrhizas, in excised mycorrhizal tissues and in each partner of the association. Figures below the diagram denote specific a c t i v i t i e s of the enzyme. A : whole m~corrhiza (Spruce-Hebelo~a sp.) B C D E F
: : : : :
Vascular cylinder Sheath + Hartig net Peripheral mycelium )~on-mycorrhizal roots Fungus in pure culture (11-day -old culture)
69
In the spruce-HebeZo~ sp. association, gel electrophoresis confirmed the presence of NAD-GDH in the host c e l l s (one band) and the presence of a high level of NADP-GDHa c t i v i t y in the fungus (one major band and one minor band). In the mycorrhizas, NAD and NADP-GDHwere both detected (Fig. 1A). I t can be noticed that NADP-GDHa c t i v i t i e s revealed on gels ~ deamtnation were also detected with NAD. Identical banding patterns were also obtained with the Douglas fir-Laecaria Zaeeata association (not shown). In the Beech-Hebeloma crustuliniforme association, the single band of IIADP-GDH a c t i v i t y found in the fungus was only detected as traces in the mycorrhiza where considerable NAD-GDHa c t i v i t y was revealed. The same band corresponding to NAD-GDHwas present in non-mycorrhizal roots (Fig. 1B). Likewise, ectomycorrhizas of Beech-Paxillus invoZutus gave a single NADGDH band identical to the host plant GDH (not shown). Antibodies raised against NADP-GDHof Cenococcum g e o p h i l ~ were to a large extent recognised by NADP-GDHof HebeZoma Sp. and were then used to confirm the fungal o r i g i n of this enzyme in spruce mycorrhizas. Anticat a l y t i c immunoprecipitation tests suppressed NADP-GDHa c t i v i t i e s in crude extracts of the ectomycorrhiza. Indirect inununofluorescence techniques were also capable of l o c a l i z i n g the enzyme which was restricted to the mycelial layers surrounding the host roots (data not presented). As for aspartate aminot~ansferase, the d i s t i n c t isoforms found in the four ectomycorrhizas, always corresponded to the host root isoforms, whereas the fungal isoform found ifl the fungus cultivated in pure culture, was not detected (Fig. 2A, E, F). Dissection of the mycorrhizal tissues in spruce confirmed these results : the vascular cylinder free of fungus and the cortical region including host c e l l s and fungal hyphae revealed identical isoforms, while no a c t i v i t y was found in the peripheral mycel i a l layer (Fig. 2B, C, D). Conclusion According to the enzyme and the type of association investigated, isoenzymes found in mycorrhizas are variable. In a l l the associations investigated, fungal AAT was strongly repressed whereas fungal NADP-GDHwas only repressed in beech ectomycorrhizas. In this latter symbiosis, the present results suggest that fungal NADP-GDHmay be regulated by the host plant. I t is obvious that the fungal pathways of nitrogen assimilation are modified by the establishment of the symbiosis, however the mechanism of regulation is not understood and requires additional iRvestigation$.
70
References BLUMENTHAL, K.M., SMITH, E . L . J . Biol. Chem. 248: 6002-6008. 1973. DELL, B., BOTTON, B., MARTIN, F., LE TACON, F. New Phytol. (in press). 1988. KHALID, A., BOUKROUTE,A., BOTTON, B., MARTIN, F. Plant Physiol. Biochem. 26: 17-28. 1988. KUSNAN, M.B., BERGER, M.G., FOCK, H . P . J . Gen. rlicrobiol. 133: 1235-1242. 1987. LEA, P.J., MIFLIN, B.B. Nature 251: 614-616. 1974. MARTIN, F., STEWART, G., GENETET, I . , LE TACON, F. New Phytol. 102: 8594. 1986. MARZLUF, G.A. Microbiol. Reviews. 45: 437-461. 1981. PERROT, C., VIDAL, J., BURLET, A., GADAL, P. Planta. 151: 226-231. 1981.
Chalot, M., Dell, B., Botton, B. and Martin, F., 1989: Nitrogen assimilation enzymes in mycorrhizas of Norway spruce, douglas fir and beech. Agric., Ecosystems Environ., 28: 65-70.