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First published online July 9, 2004; 10.1104/pp.103.031534 Plant Physiology 135:1849-1862 (2004) © 2004 American Society of Plant Biologists Novel Expression Pattern of Cytosolic Gln Synthetase in Nitrogen-Fixing Root Nodules of the Actinorhizal Host, Datisca glomerata1,[w]Department of Environmental Horticulture (A.M.B., P.A.O.), and Section of Plant Biology (T.M.M.), University of California, Davis, California 95616; USDA-ARS, Department of Plant Pathology, Washington State University, Pullman, Washington 99164–6430 (P.A.O.); Department of Botany, University of Wisconsin, Madison, Wisconsin 53706 (K.R.J.); Biology Department, Ithaca College, Ithaca, New York 14850 (S.M.S.); Department of Molecular Biology, Agricultural University Wageningen, 6703 HA Wageningen, The Netherlands (K.P.); and Albrecht von Haller Institute for Plant Sciences, Plant Biochemistry, 37077 Göttingen, Germany (K.P.)
Gln synthetase (GS) is the key enzyme of primary ammonia assimilation in nitrogen-fixing root nodules of legumes and actinorhizal (Frankia-nodulated) plants. In root nodules of Datisca glomerata (Datiscaceae), transcripts hybridizing to a conserved coding region of the abundant nodule isoform, DgGS1-1, are abundant in uninfected nodule cortical tissue, but expression was not detectable in the infected zone or in the nodule meristem. Similarly, the GS holoprotein is immunolocalized exclusively to the uninfected nodule tissue. Phylogenetic analysis of the full-length cDNA of DgGS1-1 indicates affinities with cytosolic GS genes from legumes, the actinorhizal species Alnus glutinosa, and nonnodulating species, Vitis vinifera and Hevea brasilensis. The D. glomerata nodule GS expression pattern is a new variant among reported root nodule symbioses and may reflect an unusual nitrogen transfer pathway from the Frankia nodule microsymbiont to the plant infected tissue, coupled to a distinctive nitrogen cycle in the uninfected cortical tissue. Arg, Gln, and Glu are the major amino acids present in D. glomerata nodules, but Arg was not detected at high levels in leaves or roots. Arg as a major nodule nitrogen storage form is not found in other root nodule types except in the phylogenetically related Coriaria. Catabolism of Arg through the urea cycle could generate free ammonium in the uninfected tissue where GS is expressed.
Root nodules are plant organs that are specialized for assimilation of the nitrogen derived from nitrogen fixation by symbiotic bacteria. Cytosolic Gln synthetase (GS1; EC 6.3.1.2) is the key enzyme in primary ammonia assimilation in root nodules of legumes and actinorhizal (Frankia-nodulated) plants (Forde et al., 1989  ; Guan et al., 1996; Baker and Parsons, 1997). Plant GS is typically expressed at high levels in the infected tissue of root nodules, where in most cases the substrate for the enzyme, ammonium, is released directly from the nitrogen-fixing microsymbiont (O'Gara and Shanmugam, 1976). In the actinorhizal plant Alnus glutinosa, GS gene expression is coincident with nif gene expression in Frankia-infected nodule cells (Guan et al., 1996), and GS protein is localized in mature infected tissue (Hirel et al., 1982). In many legumes, nodule GS regulation includes additional tissue-specific and developmental components (Padilla et al., 1987; Carvalho et al., 2000; Morey et al., 2002).
Datisca glomerata is an actinorhizal species in the Datiscaceae, a plant family which together with the Coriariaceae forms one of four phylogenetically separate actinorhizal subclades, within the larger angiosperm nitrogen-fixing clade that encompasses legumes and actinorhizal plant taxa (Swensen, 1996
GS Genes Expressed in Datisca Root Nodules
Two cDNAs with sequence homology to plant cytosolic GS were cloned from D. glomerata root nodules. The first, designated DgGS1-1, was isolated from a Datisca nodule cDNA library and is 1,445 nucleotides in length, encoding a deduced polypeptide of 356 amino acids, with a predicted molecular mass of 39 kD. This polypeptide and molecular mass correspond to the range of predicted structures for cytosolic GS subunits in other plant taxa (Forde and Cullimore, 1989
To examine the relative abundance of DgGS1-1 transcripts in various organs of D. glomerata, gel blots of total RNA were hybridized to probes representing the conserved coding region of the full-length DgGS1-1 cDNA (DgGS1-COD) and the partial cDNA of DgGS1-2. RNA from all organs hybridized to the probe (Fig. 1A). Expression levels were higher in the root and nodules compared to leaves, flowers, and developing fruits. DgGS1-2 hybridization was comparable to that of the DgGS1-COD probe in nodules and roots relative to other organs (Fig. 1B), probably indicating cross-hybridization with the conserved coding region. Hybridization of organ RNA blots with the DgGS1-1 3'-untranslated region (UTR) probe gave results similar to that of DgGS1-2 (data not shown; DgGS1-COD was not compared).
Phylogeny of Datisca Nodule GS
A 1,071-bp portion of the D. glomerata nodule GS DNA sequence from clone DgGS1-1 was included in a phylogenetic analysis of published plant GS sequences, including DNA sequences encoding both cytosolic and plastidic isoforms (Fig. 2). The analysis included the same stretch of the amino acids in the coding region of the GS cDNA sequences and did not include any 3' or 5' UTR. The plastidic sequences were designated as the outgroup based on our own previous analyses and that of others (Doyle, 1991
In an attempt to identify ortholog groups for GS sequences (and thus possibly assign Datisca sequences to an ortholog group), several Arabidopsis sequences were included in this analysis. All of the Arabidopsis GS sequences appeared as part of a large Brassicaceae clade that included Raphanus sativus and Brassica napus. It appears that Arabidopsis paralogs are confined to the Brassicaceae clade, having duplicated during the divergence of these species. Therefore, at present, Datisca GS appears to have greatest affinity to the  subclass of legume GS; however, the phylogeny does not yet provide a complete picture of angiosperm GS phylogeny. The sampling of GS1 sequences available for this analysis is relatively sparse, representing only a few of the angiosperm species known to be related to Datisca. Once additional GS sequences become available, the current picture of GS phylogeny will likely be modified.
The area of Frankia-infected tissue in nodules of D. glomerata can be divided into the zone of infection (zone II in Fig. 3A), where Frankia hyphae proliferate to fill the plant cells; the zone of nitrogen fixation (zone III in Fig. 3, A and F), where Frankia vesicles have differentiated and nitrogen fixation takes place; and the zone of senescence (zone IV in Fig. 3F), where Frankia material is degraded by the plant, according to terminology established by Ribeiro et al. (1995)
nifH, encoding a subunit of the nitrogenase enzyme system (Rubio and Ludden, 2002 ), served as a marker to delineate the nitrogen-fixing zone III in the Frankia-infected nodule tissue sector (Fig. 3, A and B). nifH was expressed at highest levels in a zone of infected cells at the earliest stage of nitrogen fixation and at lower levels in the rest of the fixation zone. No nifH expression was observed in the infection zone, where Frankia hyphae proliferate in the cortical cells but where vesicles have not differentiated. nifH expression was not observed in the zone of senescence, where Frankia material is degraded by the plant. The GS partial coding region (DgGS1-COD) antisense probe hybridized strongly to the Datisca nodule tissue. However the pattern of hybridization was unusual for nodule GS; high levels of transcript were detected in all the cells of the uninfected cortical tissue flanking the infection zone (Fig. 3C). The pattern of in situ hybridization with the 3' UTR of the DgGS1-1 full-length cDNA (DgGS1-UTR) was essentially the same as that observed for DgGS1-COD (Fig. 3D). Within the infected tissue, however, no hybridization could be detected with either the DgGS1-COD or the DgGS1-UTR probe, except in some nodules where a low level of hybridization could be seen associated with cells where nifH was first expressed (Fig. 3E). GS expression above background could not be detected in the nodule vascular tissues (Fig. 3, C and D). No tissue hybridization was detected using the DgGS1-COD sense probe (data not shown). In older nodules, a zone of apparent senescence of infected cells could be observed at the base of the infected zone (Fig. 3F). DgGS1-COD and DgGS1-UTR hybridized strongly to the senescent infected cells (Fig. 3, G and H). Starch grains in the Frankia-infected tissue served as a marker for metabolic changes associated with senescence, since starch grains are not found in active, Frankia-infected nodule tissue.
Immunoblots of nodule extract-SDS (SDS-polyacrylamide) electropherograms (Fig. 4) displayed two single bands representing GS subunits. The mass of the major polypeptide band (43 kD) was slightly larger than the predicted monomer from the translated amino acid sequence of DgGS1-1 (39 kD). This major nodule polypeptide band corresponded to the single band observed in the root extracts (Fig. 4) and falls within the reported molecular-mass range of cytosolic GS subunits (Forde and Cullimore, 1989
GS Protein Has the Same Nodule Localization Pattern as the Transcript With the same antibodies as those used in the protein blot, GS protein in D. glomerata nodules was localized distinctly to the uninfected cortical tissue, and therefore showed the same spatial pattern as that observed for DgGS1-1 transcripts (Fig. 3I). In uninfected cortical parenchyma cells, the green fluorescing fluorescein isothiocyanate (FITC)-signal of the secondary antibody was restricted to the cytoplasm and appeared also slightly accumulated around amyloplasts (Fig. 3J). GS hybridization signal was not detected in the Frankia-infected tissue, in the nodule periderm, or in the central vascular cylinder (Fig. 3I). No FITC-fluorescent signal was detected in control sections lacking primary antibody (Fig. 3K).
Extracts of leaves, nodule-free roots, and nodules all had substantial amounts of GS activity. As shown in Table I, the specific enzyme activities (units per gram protein) were similar in all three organs.
Arg Is a Major Nodule Amino Acid
Amino acids were analyzed in extracts from nodule, root, leaf, and stem exudates (xylem sap), as well as from Frankia cells fractionated from the D. glomerata nodule tissue (Table II). In the root nodule extract, the three major amino acids were Gln, Arg, and Glu. In root tissue, leaf, and xylem sap, Gln, Glu, and Asp were the major amino acids; Arg was present only at low levels compared with Gln or Glu. Gln was the major transport amino acid in the xylem sap, as reported previously (Wheeler and Bond, 1970
In extracts prepared from a nodule fraction enriched for Frankia, the major amino acids were Asp and Ser, with lesser proportions of Ala, Gly, and Glu (Table II). Ser and Gly were markedly higher in Frankia than in the nodule as a whole, especially as a fraction of the respective amino acid pools. Ornithine was also present at high levels compared with nodule tissue. Glu and Gln, by contrast, were markedly less abundant in the Frankia extracts than in the whole nodule extracts.
Primary assimilation of fixed nitrogen in the root nodules of D. glomerata has a novel spatial organization. Plant GS protein was not detected in the Frankia-infected sector of D. glomerata root nodules by an antibody that recognizes both GS1 and GS2 in a range of plant taxa (Bennett and Cullimore, 1989 ). Transcripts hybridizing either to the conserved coding region (DgGS1-COD) or to the 3' UTR of DgGS1-1, a cDNA with high sequence homology to plant cytosolic GS genes that is expressed abundantly in D. glomerata root nodules, also were not detectable in the infected sector, with the exception of low-level gene expression in a band of early nitrogen-fixing cells and in senescent infected cells. Instead, high levels of both GS mRNA and protein were found in the surrounding uninfected tissue.
This partitioning of plant GS in the uninfected cortical tissue, at a distance from the locus of nitrogen fixation, contrasts markedly with the high levels of GS expression observed at sites of assimilation of recently fixed N, located in the central infected zone in legume nodules (Miao et al., 1991
In protein gels of D. glomerata nodule extracts, the finding of a single major band at 43 kD corresponding to the root GS band corroborates the likelihood that DgGS1-1 encodes a root cytosolic GS isoform (Forde and Cullimore, 1989
Based on the GS phylogenetic analysis, DgGS1-1 clearly belongs among the cytosolic sequences, where it is most closely allied with GS from nonnodulating H. brasilensis (rubber) and the actinorhizal species A. glutinosa but is part of a larger clade that includes the
There is not a close correlation between the phylogenetic grouping of the cytosolic GS sequences from the two actinorhizal species, D. glomerata and A. glutinosa, and their tissue localization patterns in the nodule; in Datisca, DgGS1-COD expression was confined to the uninfected nodule cortical cells, whereas in A. glutinosa nodules, GS was expressed highly in the infected cells and in the vascular tissue (Guan et al., 1996
What is the basis for the novel expression pattern of cytosolic GS in D. glomerata root nodules, and how is GS tissue localization related to the organization of nodule nitrogen assimilation? The protein localization pattern in the nodule shows that no plant GS is unaccounted for in the infected tissue of the nodule, since the antibodies used for GS localization cross-react with the root (cytosolic) and leaf (plastidic) isoforms in D. glomerata, as in a wide range of other plant taxa (Bennett and Cullimore, 1989
The absence of detectable GS in D. glomerata infected tissue may be due to metabolic regulation of gene expression by the particular nitrogen compounds released by Frankia into the host cytoplasm. The band of cells in the infected tissue expressing GS coincides with the earliest expression of nifH. In nodules of A. glutinosa, where the spatial and temporal expression of plant GS coincides with nifH expression, Guan et al. (1996)
If ammonia is not the major N compound transferred by Frankia into mature infected host tissue, then N must be transferred primarily by amino acid efflux. In soybeans (Glycine max), a substantial fraction of fixed nitrogen can be assimilated in the Bradyrhizobium bacteroids via AlaDH and exported as Ala (Waters et al., 1998
Root nodule nitrogen metabolism is a result of the interplay among primary assimilation, nitrogen storage, and nitrogen export from the nodule. To construct a unified model of nitrogen assimilation in D. glomerata nodules requires an understanding of how these functions are integrated among all the cellular and tissue compartments. In ureide-exporting legume nodules for example, there is a complex partitioning of metabolic functions between infected and uninfected nodule cortical tissues and among different organelles (Schubert, 1986
The one exception to this general pattern is to be found in nodules of Coriaria myrtifolia (Wheeler and Bond, 1970
The amino acid profile of symbiotic Frankia in D. glomerata nodules differs from that of the host tissue. Ser and Gly were particularly elevated in the Frankia extracts compared to total nodule amino acid pools, as was Orn. Gln and Glu pools on the other hand were unusually low in the symbiotic Frankia when compared either with the endogenous amino acid profile of free-living Frankia cultures that had been induced for nitrogen fixation (Berry et al., 1990 ) or with total nodule pools of D. glomerata (Table II). Ala similarly did not occur at notably high levels within the total Frankia amino acid pool. Thus if ammonia is assimilated in the symbiotic Frankia as Gln, Glu, or Ala, the activity of endogenous transferases and/or the amino acid export mechanism matches the rate of synthesis, even at low concentrations of these amino acids. In the only study to date of amino acid export from Frankia, Ser and lesser amounts of Gln were exported from an Alnus-infective Frankia strain induced in vitro for nitrogen fixation and exposed to a pulse of exogenous 13N-ammonium (Berry et al., 1990).
The distinctive spatial localization pattern of GS protein and transcripts in D. glomerata root nodules suggests that the organization of metabolic and regulatory networks governing nitrogen assimilation represents a new variant type, compared with legumes or with A. glutinosa. Because of the complexity of nitrogen and carbon metabolism in the Datisca-Frankia nodule symbiotic system, we expect that isotopic labeling combined with NMR spectroscopy will be needed to clarify the successive steps in this unique organization of nitrogen assimilation. Such information will provide a basis for further exploration of the gene regulatory interactions of Frankia and the host in D. glomerata nodule nitrogen assimilation.
Plant Material
Seeds of Datisca glomerata were collected in Gates Canyon, Vacaville, CA. D. glomerata plants were grown from seed in the greenhouse and inoculated with a slurry containing 0.5 g/plant of crushed nodules from Ceanothus griseus stock plants grown in aeroponics culture. Root nodules for mRNA isolation were harvested into liquid nitrogen at 4, 5, 7, and 11 weeks postinoculation (PI). Nodules for in situ hybridization were harvested at 6 to 10 weeks PI. For immunolocalization, nodules were harvested at 6 weeks PI. For protein analysis, leaves, nodules (6 weeks and 12 weeks PI), and roots (from uninoculated plants) were harvested. Harvested materials were in all cases used immediately or immediately frozen in liquid nitrogen and stored at –80°C. Nodule DNA, organ RNA, and mRNA isolation have been described in Okubara et al. (1999)
For PCR amplification of GS sequences, primers were designed from two stretches of 100% amino acid conservation within the common GS coding regions of eight plant species (
To obtain the second GS species (DgGS1-2), hand slices of Frankia-infected tissue from nodules 11 weeks PI were excised under a dissecting microscope and frozen in liquid nitrogen for RNA isolation. Poly(A) mRNA was isolated from the excised tissue using oligo(dT)25 Dynabeads as recommended by the manufacturer (Dynal Biotech, Lake Success, NY). First-strand cDNA synthesis was carried out using Superscript II MMLV reverse transcriptase (Gibco BRL, Gaithersburg, MD), followed by PCR with primers GS51 and GS32. The PCR product was cloned in pCRII (Invitrogen, Carlsbad, CA), mobilized into Escherichia coli strain INV
A Datisca nodule cDNA library was constructed from equal amounts of mRNA from nodules harvested at 4-, 7-, and 11-weeks PI, as described in Okubara et al. (1999)
For in situ hybridization, 35S-labeled antisense and sense RNA probes were prepared from the internal cDNA fragment of DgGS1-1, designated DgGS1-COD, in pBluescript KS+. For production of antisense RNA, pDgGS1-COD was linearized with XbaI and transcribed with T3 RNA polymerase (LifeTechnologies, Gaithersburg, MD) in the presence of 35S-UTP (Amersham Pharmacia Biotech, Uppsala). For the production of sense RNA, pDgGS1-COD was linearized with EcoRI and transcribed with T7 RNA polymerase (Lifetech). For production of the gene-specific probe DgGS1-UTR, the 307-bp fragment representing the entire UTR of the cDNA was amplified by PCR and cloned in pGEM-T (Promega), yielding pDgGS1-UTR. For production of antisense RNA, pDgGS1-UTR was linearized with SalI and transcribed with T7 RNA polymerase in the presence of 35S-UTP. The preparation of a Frankia nifH antisense probe has been described (Ribeiro et al., 1995
Root nodules were harvested at 6 to 9 weeks PI (3–6 weeks post nodule initiation) and fixed intact in 4% paraformaldehyde, 0.25% glutaraldehyde in sodium phosphate buffer, pH 6.9 for 2 h under vacuum and embedded in paraffin as described by van de Wiel et al. (1990). Seven- to ten-micrometer thick sections were cut and placed on polylysine coated slides. Slides containing adjacent sections were used for the three different probes. In situ hybridization was performed as described by van de Wiel et al. (1990) with modifications according to Ribeiro et al. (1995)
DgGS1-COD (the DgGS1-1 internal fragment) and the DgGS1-2 partial cDNA were labeled with 32P and used as probes of total mRNA isolated from leaf, (green) flowers, and (green) developing fruits, as well as total mRNA from nodules harvested at 6 weeks after inoculation. Total RNA from clone pTa71 (Gerlach and Bedbrook, 1979
The D. glomerata DNA sequences from clone DgGS1-1 and DgGS1-2 were compared to other plant GS genes through phylogenetic analysis. The DgGS1-1 sequence used for comparison was a 1,071 bp portion of the GS cDNA in the coding region and did not include any 3' or 5' UTRs. For DgGS1-2, the entire partial cDNA (880 bp) was used. A total of 47 different DNA sequences encoding both cytosolic and chloroplast GS were obtained from GenBank (Fig. 2) and aligned to each other and to the D. glomerata sequences. The alignment was accomplished using ClustalW (Thompson et al., 1994
Samples of leaves, root nodules (6 weeks or 12 weeks old), and nodule-free roots were frozen at –80°C then ground in a mortar, first with liquid nitrogen, then extracted with a buffer that contained 10 mM Tris-Cl, pH 7.5, 5 mM Glu, 10 mM MgCl2, 10% glycerol, 1 mM dithiothreitol (DTT), 0.1% mercaptoethanol, 0.05% Triton X-100, and 0.02 mM phenylmethylsulfonylfluoride, adapted from Bennett and Cullimore (1989)
Crude extracts were subjected to SDS-polyacrylamide electrophoresis according to Laemmli (1970)
The transferase activity of Gln synthetase was measured following the procedure of Cullimore and Sims (1980)
Datisca root nodules were fixed at 6 weeks PI in 4% paraformaldehyde and 0.l% glutaraldehyde in 50 mM potassium phosphate buffer, pH 6.9, overnight at 4°C. Fixed tissue was rinsed in buffer and stored in buffer with sodium azide at 4°C before use. Nodules were embedded in HistoPrep (Fisher, Loughborough, UK), cooled in liquid nitrogen, and stored after embedment at –80°C. Frozen sections were cut with a Cryocut 1800 (Leica Microsystems, Vienna) at (–)19°C, knife angle 0° to 3°. Twenty- to thirty-micrometer thick sections were transferred to slides at room temperature, and dried at least 5 s. Polyclonal antibodies against plant GS were obtained courtesy of J.V. Cullimore, as described above for protein blots. Preparatory blots were done prior to incubation to determine the optimal range of concentration of the primary antibody. Tissue sections were incubated in blocking buffer (1% nonfat dry milk in 1x PBS) for 1 h prior to incubation in primary antibody for 15 s. After rinsing in PBS, the sections were incubated with secondary antibody (goat anti-rabbit IgG conjugated with Alexa TM 488 [Molecular Probes, Eugene, OR] 2 g/mL) at 1:100 and 1:200 for 15 s and rinsed in PBS. As controls, the primary antibody was replaced by buffer followed by Alexa-conjugated secondary antibody in buffer (Fig. 3K) or by a different primary antibody also generated in rabbits, which hybridized to the infected tissue (data not shown). In a concentration series of the anti-GS antibody (1:100, 1:50, 1:25, 1:10), the localization pattern was consistent (i.e. hybridization to the infected tissue was not observed) even at the 1:10 dilution. Sections were mounted in 90% glycerol, 10% 1x PBS containing 0.2% p-phenylenediamine (DeWitt and Sussman, 1995
To quantify endogenous amino acid pools, extracts were prepared from several tissues of D. glomerata including whole nodule, leaf, stem exudates, and nodule-Frankia cells. Tissues were either extracted fresh or frozen in liquid nitrogen at the time of harvest and stored at –80°C until use. Leaf, root and whole-nodule extracts for amino acid analysis were prepared by homogenization on ice in 50 mM Tris buffer, 200 mM Suc, 2 mM EDTA, 5 mM DTT, pH 8. Stem exudates from well-watered plants were collected early in the morning by microcentrifugation of small-diameter cut stems at 3,000 rpm for 5 min followed by 4,000 rpm for 5 min, using a swinging-bucket rotor. To obtain an extract enriched in Frankia cell amino acids, a differential filtration method was modified from Lundquist and Huss-Danell (1992). Nodules harvested 6 weeks PI were homogenized on ice in 50 mM Tris buffer, pH 8 (1 g nodule/30 mL buffer) or in 50 mM phosphate buffered saline, pH 7.4. The addition of Suc, DTT, or EDTA to the buffer made no difference in the amino acid profile. The resulting brei containing clusters of Frankia cells was filtered through 167-µm nylon mesh. The filtrate suspension was then filtered through 20-µm nylon mesh. The 20-µm retentate was resuspended in 1 to 3 mL buffer and sonicated on ice for 30 s, using a sonifier (Branson Ultrasonics, Danbury, CT) at 50% duty cycle, output setting 6. The sonicate was microcentrifuged at 15,000 rpm for 20 min at 4°C and the supernatant retained for analysis. Extracts from all the tissues were analyzed for amino acids on a Beckman 6300 Amino Acid Analyzer using Li citrate at the Molecular Structure Facility, University of California, Davis.
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY422224 and AY422225.
The technical assistance of Melinda Klein, Carolyn Woo, and Rochelle Jones is gratefully acknowledged. We also thank Judy Jernstedt for the kind use of laboratory facilities. Received August 8, 2003; returned for revision April 14, 2004; accepted May 2, 2004.
1 This work was supported by the California Agricultural Experiment Station Project (grant no. 6258–H to A.M.B.), by a Katherine Esau Fellowship, University of California, Davis (to K.P.), and by the NSF (grant no. DEB–9815816 to S.M.S.). 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.031534. * Corresponding author; e-mail amberry{at}ucdavis.edu; fax 530–752–1819.
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, and 
subclasses identified previously (Gebhardt et al., 1986
5% of total amino acids) in the nodules of several actinorhizal genera, including Alnus, Myrica, Ceanothus, Hippophae, and Elaeagnus (Leaf et al., 1958