Utilization of carbon and nitrogen compounds by Frankia in synthetic media and in root nodules of Alnus glutinosa, Hippophae rhamnoides, and Datisca cannabina

ANTOON D. L. AKKERMANS, WIM ROELOFSEN, JAN BLOM, KERSTIN HUSS-DANELL,' AND REINT HARKINK Laboratory of Microbiology, Agricultrrral Utziversiry, Wagetzitzgetz, The Netherlands

Received December 1, 1982

AKKERMANS, A. D. L., W. ROELOFSEN, J. BLOM, K. HUSS-DANELL, and R. HARKINK. 1983. Utilization ofcarbon and nitrogen compounds by Frankia in synthetic media and in root nodules of Alrzus gl~rtinosa, Hippophae rhamnoides, and Datisca cannabina. Can. J . Bot. 61: 2793-2800.

Frankia AvcIl utilizes only Tweens and fatty acids as sole carbon sources but NZ, NH4+, NO3-, and various amino acids as nitrogen sources. The yield in propionate medium was increased by COZ. Cells grown in media with Tween 80 or acetate as C source contain the glyoxylate-cycle enzymes, isocitrate lyase and malate synthase. These enzymes are repressed in cells grown in media with propionate as well as in the symbiotic stage of Frankia in the nodules. The nature of the carbon compounds utilized by Frankia in the nodule symbiosis is discussed. Vesicle clusters derived from root nodules of Alnus respire succinate (however, only at a very low rate, which probably is due to damage of the membrane of the endophyte). Vesicle clusters derived from Al~zus and Hippophae nodules also take up O2 when supplied with a mixture of glutamate, malate, and NAD, indicating that the reduction equivalents could be transported from the host to the endophyte via the malate-aspartate shuttle. Vesicle clusters derived from Datisca respire succinate at a relatively high rate. Electron micrographs of Datisca nodules showed the presence of clusters of mitochondria embedded in the endophyte material, which might be of importance in the energy supply of the endophyte. These mitochondria are partly disintegrated during the preparation of the vesicle-cluster suspensions.

AKKERMANS, A. D. L., W. ROELOFSEN, J . BLOM, K. HUSS-DANELL et R. HARKINK. 1983. Utilization of carbon and nitrogen compounds by Frankia in synthetic media and in root nodules of Alnus glutinosa, Hippophae rhamnoides, and Datisca cannabina. Can. J . Bot. 61: 2793-2800.

La souche AvcI1 de Frankia utilise seulement le Tween et des acides gras comme sources de carbone, mais elle peut utiliser NZr NH4+, NO3- et divers acides aminCs comme sources d'azote. Dans un milieu de propionate, le COZ augmente le rendement. Les cellules cultivCes avec le Tween 80 ou de 1'acCtate comme source de carbone contiennent les enzymes du cycle du glyoxylate, l'isocitrate lyase et la malate synthase. Ces enzymes sont rCpressCes chez les cellules cultivees dans un milieu contenant du propionate, ainsi que dans le stade symbiotique du Frankia dans les nodules. Les auteurs discutent la nature des composes organiques utilisCs par le Frankia dans la symbiose nodulaire. Les groupes de vCsicules provenant des nodules racinaires d' Alnus ne respirent du succinate qu'A un taux tr2s faible, ce qui est probablement dO aux dommages subis par la membrane de l'endophyte. Les groupes de vCsicules provenant d'Alnus et d'Hippophae absorbent aussi de 1'02 lorsqu'on leur fournit un melange de glutamate, de malate et de NAD; cela montre que des Cquivalents rCducteurs peuvent Ctre transportCs de l'h6te vers l'endophyte par la navette malate-aspartate. Les groupes de vCsicules provenant de Datisca respirent du succinate ti un taux relativement ClevC. Des micrographies Clectroniques de nodules de Datisca montrent que des groupes de motochondries sont enfouis dans la substance de l'endophyte; ces groupes de motochondries pourraient avoir un r6le important dans l'apport d'Cnergie A l'endophyte. Ces mitochondries se dCsint2grent partiellement pendant la prCparation des suspensions de groupes de vCsicules.

Introduction Frankia, an actinomycetous, filamentous micro-

organism, produces at least two types of nongrowing cells, viz. spores and vesicles. Spores are formed within sporangia, can germinate after dissemination under proper conditions, and therefore are real resting stages of Frankia. Vesicles, however, are spherical structures formed at the tips of the hyphae and have no function as a resting stage. Their formation is dependent on the culture conditions and may vary among strains. Recent observations on Frankia strain C D I ~ indicated that vesicles, in contrast to spores, are formed only in media

'Present address: Department of Plant Physiology, Univer- sity of UmeH, S-901 87 UmeH, Sweden.

[Traduit par le journal]

free from combined nitrogen (25, 26). Under these conditions Frankia is able to fix small quantities of N2.

Within the nodule symbiosis, Frankia produces large numbers of vesicles under N2-fixing conditions. The shape of the vesicles is in part determined by the host: in Alnus nodules with strain CpIl as microsymbiont, the vesicles are spherical and have many similarities with the vesicles observed in free-living Frankia; however, in root nodules of Myrica and Comptonia spp. the same Frankia strain forms club-shaped vesicles (20).

There are good reasons to believe that vesicles are involved in N2 fixation, like nongrowing heterocysts in filamentous cyanobacteria and bacteroids of Rhizobium in leguminous root nodules (2, 4, 25). In all these nongrowing types of cells more N2 is fixed than needed

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2794 CAN. I. BOT. VOL. 61, 1983

and the surplus is excreted. In the nodules the main part of this nitrogen is utilized by the host.

Very little information is available about the changes in the metabolism during the transition of a growing hyphal cell of Fraizkia into a nongrowing vesicle. To understand this process we investigated the growth requirements of Frankia in pure cultures and measured the activities of the enzymes involved in the carbon and nitrogen metabolism. In addition, a study was made of the cell metabolism of Fraizkia in the root nodules. In the present paper data are presented on the metabolic activity of Frankia strain AvcI1, grown in synthetic media and grown in the root nodules of A. glutinosa. These data are compared with observations on Frankia spp. in root nodules of A. glutinosa, Datisca cannabina, and Hippophae rhamnoides of which pure cultures have not yet been isolated.

Materials and methods Microorganisms

Frnnkia AvcI1, isolated from nodules of A. viridis ssp. crispa, was obtained from Dr. D. Baker and Dr. J . G. Torrey (Harvard University, Petersham, MA, U.S.A. 01366). It was cultivated aerobically at 25°C in triplicate in nonshaken 100-mL erlenmeyer flasks containing 50 mL of medium. The medium contained (grams per litre) CaC12.2H20 (0.1), MgS04.7H20 (0.2), NH4C1 (0.1), K2HP04 (1.0), NaH2P04. H20 (0.067), FeNa-EDTA (0.01), biotine (0.002), and trace elements according to Allen and Arnon (7). The carbon source was sodium propionate (0.5 g/L) unless otherwise men- tioned. For determining the effect of CO? on the growth of Frankia, Erlenmeyer flasks with 50 mL medium were inocu- lated and subsequently placed in desiccators with or without NaOH pellets. After 7 , 14, and 21 days of incubation, cells were harvested. During the time of incubation the oxygen concentration in the dessicators remained 21%. The C 0 2 concentration in the dessicator without NaOH pellets was kept at 0.03%. The pH of the medium increased during the time of the experiment from 6.9 to 7.0 (without C02) and 7.1 (with COz).

~ r a n k i a cells used for enzyme studies were cultivated for 14 days in media containing (grams per litre) Tween 80 (1.0), K2HP04 (0.3), NaH2P04.H20 (0.2), MgS04.7H20 (0.2), KC1 (0.2), Fe-citrate (0.01), CaC03 (0. l), biotine (0.0001), nicotinic acid (0.0001), Ca-pantothenate (0.0001), pyridoxine- HC1 (0.0001), riboflavine (0.0001), thiamine-HC1 (0.0001), ZnS04.7H20 (0.0006), H3B03 (0.00 15), MnS04.7H20 (0.0008), CuS04.7H20 (0.0001), (NH4)6M~04.4H20 (0.0002), and CoS04.7H20 (0.00001). The nitrogen source in the medium was either NH4C1 (0.06 g/L), casamino acids (Difco 479744) (1 .O g/L), or one of the following amino acids (0.4 mM): alanine, aspartate, glutamate, leucine, or tyrosine.

Cells were harvested by centrifugation. The pellet was washed three times. The protein content of the cells was determined by adding 2 mL of 1 N NaOH to the pellets and boiling the mixture for 30 min. Samples of 0.5 mL were mixed with 2.5 mL Na2C03 (80 g.L-') and 2.5 mL KNa-tartrate (1.2 g.L-') /CuS04 (0.6 g.L-') mixture. After 30 min,

0.5 mL of Folin reagent was added to the mixture and the absorbance was measured at 660 nm.

Cellfree extracts of the cells were prepared by sonication for 2 min (Branson sonifier B- 12) and centrifugation for 10 min at 10 000 x g. The following enzymes were assayed: glutamine synthetase (GS) using the transferase assay (23); glutamine 2-oxoglutarate aminotransferase (GOGAT) (21); L-glutamate dehydrogenase (GDH) (15); glutamate-oxaloacetate transam- inase (GOT) (18); acetylornithine glutamate transacetylase (AGT) (27); aspartate dehydrogenase (AspDH) (1 9); L-alanine dehydrogenase (AlaDH) (29); tyrosine aminotransferase (TAT) (16); leucine aminotransferase (LAT) (1); L-aspartate ammonia lyase or aspartase (AAL) (28); L-alanine aminotrans- ferase (AAT) (22); L-amino acid oxidase (LAOD) (8).

Plant material Plants of Alnus glutinosa (L.) Vill. and Hippophne

rhamnoides L. spp. rhamnoides were cultivated and nodule homogenates were prepared as described previously (5, 17). Plants of Dntiscn cnnnnbina were raised from seeds and inoculated with nodule homogenates. Nodules used for inoculation were collected in Swatt , Pakistan, and were kindly supplied by Dr. Ashraf Chaudhary (Quaid-i-Azam University, Islamabad, Pakistan).

Transmission electron micrographs (TEM) Nodules of D. cannabinn were fixed in a 2% solution of

glutaraldehyde in 0.05 M phosphate buffer (pH 6.8) and treated for 1 h with 1% Os04 in phosphate buffer (0.05 M, pH 6.8). Material was embedded in epon. Ultrathin sections (ca. 500 A) were stained for 7 rnin with uranyl acetate and 7 min with lead citrate.

Vesicle-cluster suspensions of the Datisca endophyte were prepared by homogenization and filtration through 100-pm and 20-pm filters, respectively (5, 17). The homogenization buffer contained HEPES (2-(N-2-hydroxyethylpiperazin-N1- y1)ethanesulfonic acid) (25 mM, pH 7.4), sucrose (300 mM), MgC12 (2mM), KC1 (1 mM), PVP (polyvinylpyrrolidone) (4%), defatted bovine serum albumin (0.1% w/v), EDTA (2 mM), Na2S204 (20 mM), and dithioerythriol(5 mM). After subsequent removal of the latter two compounds by centrifuga- tion, the vesicle clusters were fixed and embedded for TEM as described above.

Results and discussion Free-living stage

Frankia strain Avcll can utilize only a few organic compounds as sole carbon source, viz., Tweens and a number of low-molecular fatty acids (9, 13). No growth or only very restricted growth was observed in media with glucose, amino acids, or organic dicarboxylic acids, such as malate and succinate, as sole C source. These results may be explained by the absence of a proper uptake system for these compounds or by the lack of enzymes needed for the degradation of these compounds. It may also be that the compounds can be utilized only when additional carbon sources are present. Previous experiments (10, 13) indicated that a number of amino acids can be taken up, provided that another C compound, e.g., Tween 80, is present. The

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uptake of both types of C source has been demonstrated (1 1, 13). In a particular growth experiment, cells were cultivated in a medium containing Tween 80 (200 mg. L-') and casamino acids ( 1000 mg.L-' ) ( 13). At the end of the growth approximately 92 mg glutamate and aspar- tate were taken up, while the other amino acids were not metabolized. This value is equivalent to ca. 46mg C. The cell yield in this particular experiment was 2 1.8 mg C.L-'. Assuming that Frankia, like other aerobic microorganisms, converts half of the C source into C 0 2 and utilizes the remaining part for biosynthesis, it is possible that the uptake of glutamate and aspartate is sufficient to explain the final yield of the cells. In another experiment with Tween 80 (1 g.L-') and aspartate (50 m g . ~ - I ) , 40 mg aspartate.L-' was consumed and the cell yield was 24.2mg C.L-' (10). There again it is likely that a significant part of the cell material originated from aspartate. Still the growth of Frankia AvcIl was dependent on the presence of Tween as a second C source. Although the utilization of Tween 80 as sole C source has been demonstrated (13), it is also likely that this compound functions as a surface-active compound which enhances the uptake of amino acids. No observations on this subject are available yet.

Fatty acids have been found to be excellent carbon substrates for many Frankia strains, including strain AvcIl. The latter strain prefers propionate as c source and gives significantly higher yield on propionate than on acetate (up to 1.5 g substrate C.L-' in the medium) (Table 1). Growth of the strain on propionate as C source was dependent on the presence of C02 . Nonshaken cultures incubated in the absence of C 0 2 gave signifi- cantly lower yields on propionate (Fig. 1). This suggests that dark fixation of C 0 2 is important in this organism.

Organic dicarboxylic acids, e.g., succinate, are not taken up by strain AvcI 1 and therefore cannot be utilized as C source, either in the presence or in the absence of other C compounds such as acetate (10).

In accordance with the growth requirements described before, cell-free extracts of strain AvcIl contained a number of tricarboxylic acid (TCA) cycle enzymes, viz., isocitrate dehydrogenase, succinate dehydrogen- ase, fumarase, and malate dehydrogenase (12), and showed cyanide-sensitive O2 uptake in the presence of proper electron donors. It is likely that this organism, like other aerobic organisms, can oxidize acetyl-CoA in the TCA cycle and generate ATP in the respiratory chain.

So far, the enzymes catalyzing irreversible steps in glycolysis, viz., hexokinase, pyruvate kinase, and pyruvate dehydrogenase, could not be demonstrated, indicating that acetyl-CoA probably can not be gener- ated via glycolysis (12). This explains why strain AvcI1 does not grow on glucose.

Frankia Avcll , like other fatty acid decomposers,

TABLE 1. Yield of Frn~lkin AvcIl after growth for 19 days in media with acetate

or propionate as carbon source --

Carbon substrate Yield (mg C.L-') (mg protein.L-')

7 14 21 d a y s

FIG. 1. Growth of Frankia AvcIl on propionate-NH4+ medium in the presence (a) and absence (0) of COz (0.03%) in the gas phase. Bars denote extremes of replicates.

contains glyoxylate-cycle enzymes, isocitrate lyase (ICL) and malate synthetase (MS), to bypass the decarboxylation steps in the TCA cycle (10,12). The acetyl-CoA which is formed by degradation of the fatty acids is partly degraded in the TCA cycle and the remaining part is utilized for biosynthesis via the glyoxylate cycle. Succinate which is formed from acetyl-CoA can be converted to pyruvate by succinate dehydrogenase, fumarase, and malic enzymes. Subse- quently, pyruvate can be converted to phosphoenolpy- ruvate by pyruvate orthophosphate dikinase and utilized for biosynthesis. The key enzymes involved in the pathways for gluconeogenesis have been demonstrated in cell-free extracts (12).

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TABLE 2. Activities (nanomoles per milligram protein per minute) of enzymes involved in the nitrogen metabolism of Frankia AvcIl grown for 14 days in media

with Tween 80 as carbon source and various sources of nitrogen

N source in medium

Enzyme NH4+ Cas Alanine Aspartate Glutamate Leucine Tyrosine

GS 258 1180 1105 975 GOGAT 0 0 0 0 GDH(NADH) 0 ND ND ND GOT 147 112 26 65 AGT 0 2 ND 2 AspDH ND ND ND 0 AlaDH ND ND 0 ND TAT 0 0 0 0 L AT 0 0 0 0 A AT 0 0 0 ND AAL 0 0 ND 0 LAOD 0 0 0 0

NOTE: ND, not determined; 0, below detection level; cas, casamino acids

Glyoxylate-cycle enzymes usually are repressed in microorganisms when succinate or precursors of it can be taken up from the medium. When Frankia AvcI1 is grown in media with propionate as organic C source, both ICL and MS are repressed (10). Propionate can be converted to succinate by carboxylation. Evidences for this pathway are the C 0 2 dependence of the growth (Fig. 1) and the presence of propionyl-CoA carboxylase activity in cellfree extracts (J. Blom, unpublished results). No repression takes place when the cells are cultivated in media with acetate and succinate, which is possibly due to the inability to take up succinate in sufficiently high quantities.

The data on the activities of the enzymes involved in the decomposition of carbon substrates and pathways for gluconeogenesis by strain AvcIl are in agreement with the growth experiments. Both types of experiments indicate that this strain can utilize only a restricted number of carbon substrates. However, the strict dependence on fatty acids certainly is not a feature of all Frankia strains. Several authors now have isolated Frankia strains which can grow on various other carbon compounds, including glucose and succinate (24, see also other papers in this symposium). A metabolic study of these strains with different types of growth require- ments will therefore be very fruitful.

Frankia AvcIl can utilize N2, NH4+, NO3-, and various amino acids as N sources. The use of amino acids has been demonstrated by measurement of the uptake of amino acids from the medium during growth of the cells (10, 13). Especially glutamate and aspartate were good N sources. The use of ammonium and nitrate ions as N sources was demonstrated by measuring the uptake of lSN-labeled compounds (3, 9). In both cases

the atom percentage of 15N of the cells at the end of the growth period was much less than the label of the N compound added to the medium. This can be explained in part by the additional uptake of unlabeled N2. Direct evidence for it was derived from the presence of acetylene-reducing activity of the strain (R. Baas and A. D. L. Akkermans, to be published).

Little is known about the pathways of assimilation of nitrogen in Frankia. In cellfree extracts of Frankia Avcll , activity was detected for glutamine synthetase and glutamate-oxaloacetate transaminase but not for various other aminotransferases, amino acid dehydro- genases, L-amino acid oxidase, and GOGAT, irrespec- tive of the N source in the medium (Table 2).

Symbiotic stage To understand which carbon compounds are supplied

by the host to Frankia, a study was made of the cell metabolism of vesicle clusters isolated on a 20-pm filter from nodule homogenates of A. glutinosa and H . rhamnoides. By using this filtration method ( 3 , smaller plant-cell debris, mitochondria, and soluble enzymes in the plant cytosol are separated from the vesicle clusters. In vesicle clusters from root nodules of both A . glutinosa and H . rhamnoides a number of TCA-cycle enzymes and glycolytic enzymes were detected, irrespective of the nature of the Frankia strain (5, 17). No activity was observed of hexokinase, pyruvate kinase, and pyruvate dehydrogenase. The latter enzymes, however, were detectable in the soluble plant fraction of the nodule homogenate. Moreover, the glyoxylate-cycle enzymes ICL and MS were not detectable. The observations made on vesicle clusters from strain AvcIl on A. glutinosa are in agreement with those obtained with

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FIG. 2. Electron micrograph of a section of an infected cell of a root nodule of D. cannabina. Near the electron-dense vesicles ( a ) of the endophyte, conglomerates of plant mitochondria (b) are visible. The centre of the host cell is filled with vacuoles (c).

vesicle clusters of an unidentified Sp+ strain in A. glutinosa and a Sp- strain in H. rhamnoides. The latter two types of Frankia are not yet available in pure culture so their growth requirements are not known.

Based on the data summarized before it has been assumed that organic dicarboxylic acids, probably succinate, are utilized as carbon and energy sources by the symbiotic form of Frankia. Direct evidence for this hypothesis should have been derived from respiration studies with purified vesicle clusters. Vesicle clusters derived from 1 g (fresh weight) nodules of A. glutinosa showed a NADH-dependent O2 uptake with a K, of 0.4 mM NADH and a V,,, of 182 nmol g-' min-I (6). Addition of ADP had no enhancing effect on the O2 consumption. Carbon compounds were respired only very slowly, if at all. Addition of succinate resulted in only very little enhancement of the endogenous O2 uptake, usually not more than 2 nmol 02.g-'min-'. This rate could not be enhanced by addition of ADP. This may indicate that membranes were damaged.

Significant O2 uptake by vesicle clusters has been observed when a mixture of malate (5 mM), glutamate (5 mM), and NAD (1 mM) was added (3, 17). Under these incubation conditions the rate of O2 uptake was ca. 20% of the rate in the presence of NADH (3). This O2 uptake has been explained by a combined action of MDH and GOT in the vesicle clusters. Both enzymes have been detected in cellfree extracts of vesicle clusters and function in a constant generation of NADH from NAD. In this way excess of oxaloacetate produced by the action of MDH is removed by GOT. Since both enzymes are also present in the plant cytosol, it is possible that excess of reduction equivalents generated in the plant cytosol can be transported to the vesicle clusters via the malate-aspartate shuttle (5).

Similar results have been obtained with vesicle clusters derived from root nodules of H. rhamnoides and D. cannabina (3), with the exception that in the latter case significant respiration of succinate was observed. Addition of succinate (final concentration 10 mM)

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FIG. 3. Electron micrograph of a section of a vesicle cluster in a 20-km residue of D. canrlabirla nodule homogenate. The plant vacuoles and the cytoplasm in the centre ( c ) of the cluster are absent. The mitochondria ( b ) located in the spaces between the hyphae (4 of the endophyte are largely disintegrated, leaving empty holes in the vesicle clusters. The vesicles ( a ) are seen as electron-dense, swollen cells orientated to the inner side of the cluster. 'The vesicle clusters were incubated in nitro blue tetrazolium and NADH for 10 min. Note the formazan crystals inside the vesicles.

resulted in an enhancement of respiration rate to 13 nmol O2-g-'.min-'. This was ca. 20% of the O2 uptake in the presence of NADH. Subsequent addition of ADP resulted in an increase of the rate to 20 nmo1.g-Ismin-'. These results indicate that succinate can be respired and that the membranes are at least partly intact. Evidence for the hypothesis that succinate is consumed inside the vesicle clusters has been derived from observations on the localization of tetrazolium reduction. Light microscopic observations have demonstrated formazan crystals in the vesicle clusters after incubation of vesicle-cluster suspen- sions with tetrazolium, succinate, and phenazine metho- sulphate. These observations are in agreement with pre- vious observations on vesicle clusters from A. glutinosa nodules (5 ) .

A principal question is still whether the succinate respiration of the isolated vesicle clusters from Datisca indeed is localized inside the Frankia cells and not in plant material, associated with the clusters. Electron micrographs of root nodules of D. cannabina clearly show the presence of islands of mitochondria packed together in between the peripheral hyphae of the vesicle clusters (Fig. 2). Since the plant cytoplasm in this symbiosis is so firmly intermingled with the Frankia cells, it is unlikely that plant and endophyte material can be separated simply by isolation of vesicle clusters on a 20-pm filter. To get an idea of the structure of the isolated vesicle clusters in a 20-pm residue, electron microscopic observations were made on vesicle cluster suspensions. As shown in Fig. 3, the structure of the

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TABLE 3. Activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) (nanomoles per gram nodule fresh weight per minute) in dif- ferent fractions of root nodules of Alnus glutinosa

and Hippophae rhamnoidesa

AKKERMANS ET AL. 2799

Nodule fraction GS GDH

A. glutinosa 100-p,m filtrate pellet 520 1396 100-p,m filtrate supernatant 10300 1222 20-p,m residue 550 103 20-p,m filtrate pellet 400 963 20-p,m filtrate supernatant 11450 1365

H. rhamnoides 20-p,m residue 0 8" 20-p,m filtrate pellet 0 35' 20-p,m filtrate supernatant 6800" 671'

"Each value is the average of two independent measurements of different nodule homogenates. Activity of GDH was NADH dependent; no activity was obtained with NADPH.

b16 nmol.mg protein-'min-I. '56 nmolmg protein-'min-'. "774 nrnol.rng protein-'.rnin-'. '79 nmol,mg protein-'.rnin-'.

Frankia cells remained almost unchanged after isola- tion. However, the mitochondria of the plant were largely destroyed. The cytoplasmic spaces between the Frankia cells contained only damaged mitochondria. Because of the close contact between the plant cytoplasm and the endophyte it will be difficult to draw conclusions from in vitro experiments with vesicle- cluster suspensions. There will always be plant material inside these clusters, although the contribution of damaged mitochondria to the respiration of the clusters probably will be small.

The close contact between mitochondria and the microsymbiont in the infected nodule cells doubtless has important consequences for the exchange of metabolites between the symbionts. A further study of the interac- tions between cvtosol. mitochondria. and Frankia possibly can contiibute to the question of which carbon and energy sources are supplied by the host to the microsymbiont.

In the state of symbiosis, Frankia fixes N2 and transports the main part of the fixed nitrogen to the plant. It has been demonstrated that vesicle-cluster suspen- sions derived from root nodules of A. alutinosa " contained almost no ammonia-assimilating enzymes; most of the activities of GS and GDH (NADH) were found in the plant fractions (1 1, 14). Similar observa- tions have been obtained with other kinds of actinorhizal plants, e.g., H. rhamnoides (Table 3) . The low GS and GDH activities that were measured in the 20-km residue could have been associated in part with plant material. Significant differences were observed in the distribution of GS and GDH over the different nodule fractions.

Almost all GS activity was present in the soluble part of the homogenate, while at least part of the GDH was localized in the particulate fraction, which contained mitochondria. It is likely that GS plays an important role in the assimilation of ammonia. Until now no GOGAT activity has been found in alder nodules (14). This might be due to the presence of inhibitory compounds in the nodule extracts which mask the GOGAT activity, since alder nodule extracts inhibited the GOGAT activity of bacteroids from lupin nodules (11). The function of GDH in the nodules is still unclear. Possibly it is localized in the mitochondria and functions in the production of NH4+ needed for the formation of amino acids and amines. This subject needs further investiga- tion.

Acknowledgements Electron micrographs were prepared by M. van

Brake1 and E. Bouw, Technical and Physical Engineer- ing Research Service (TFDL), Wageningen. K. Huss- Dane11 was supported by the Swedish Natural Science Research Council and a fellowship from the Agricultural University of Wageningen. J. Blom was supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). We thank P. Meijer for technical assistance and Dr. A. Choudhany and F. Hateez for providing Datisca material and for stimulative discussions.

1. AKI, K. , and A. ICHIHARA. 1970. Branched-chain amino acid aminotransferase (pig heart mitochondria). In Methods in enzymology. Vol. XVLIA. Edited by H. Tabor and C. White Tabor. Academic Press, London. pp. 807-8 11.

2. AKKERMANS, A. D. L. 1982. Biology of actinorhizal plants and their application in forestry. In Proceedings of the second national symposium on biological nihogen fixation, Helsinki, Finland. Sitra Report 1. ISBN 951-563-069-X. pp. 261-272.

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