Plant Science 149 (1999) 85–94

Isolation and characterization of a cDNA clone encodingasparagine synthetase from root nodules of Elaeagnus umbellata

Ho Bang Kim, Sang Ho Lee, Chung Sun An *Department of Biology, Seoul National Uni6ersity, Seoul 151-742, South Korea

Received 15 September 1998; received in revised form 24 June 1999; accepted 28 June 1999

Abstract

A cDNA clone encoding asparagine synthetase (AS) was isolated from a root nodule cDNA library of Elaeagnus umbellata bycompetitive hybridization. The clone, pEuNOD-AS1, coded for 585 amino acid residues with molecular weight of 65.8 kDa andpI value of 6.12. Expression of AS was highly enhanced in the root nodule, and its expression pattern during nodule developmentwas very similar to that of nifH, showing highest level 6–8 weeks after inoculation and decreased thereafter. In situ hybridizationresult showed AS transcripts were strongly detected in the infected cells of fixation zone, where nifH transcripts were also detected.These results suggest its expression may be under metabolic control rather than developmental control of the root nodule.Genomic Southern hybridization revealed the presence of at least two AS genes in the genome of E. umbellata. © 1999 ElsevierScience Ireland Ltd. All rights reserved.

Keywords: Asparagine synthetase; cDNA; Elaeagnus umbellata ; In situ hybridization; Nodule-enhanced expression; Root nodule

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1. Introduction

Actinorhizal root nodules are nitrogen-fixingsymbioses involving the actinomycete Frankia androots of dicotyledonous plants belonging to eightdifferent families and 25 genera. Most are capableof high rates of nitrogen fixation comparable tothose found in legumes [1]. Actinorhizal root nod-ules resemble lateral roots with a central vascularsystem and originate from pericycle, while legumi-nous root nodules resemble shoots with peripheralvascular systems and originate from cortex [2].Due to the presence of apical meristem activity, itsnodule lobes show indeterminate growth patternand thus consist of four different zones, which aremeristem zone, prefixation zone, fixation zone andsenescence zone [3]. For these reasons, the acti-norhizal root nodules may be useful system tostudy many aspects of plant development.

Ammonia fixed by dinitrogenase in root noduleshould be transported to other parts of the plantthrough xylem after assimilation into amino acids.The type of amino acids to be transported throughxylem are different according to plant species,developmental stage or environmental conditions.Generally symbiotic legumes and actinorhizalplants can be divided into amide transporters(temperate legumes and actinorhizal plants such asDatisca, Elaeagnus, Myrica, etc.) and the ureidetransporters (tropical legumes and actinorhizalplants such as Alnus, etc.) [4]. Until now, studiesof molecular aspects of enzymes related with am-monia assimilation in root nodules of actinorhizalplants were concentrated into Alnus, a ureidetransporter. Recently, cDNA clones encoding glu-tamine synthetase, a component of GS/GOGATenzyme complex, and acetylornithine transami-nase, an enzyme related with citrulline biosynthe-sis, from the root nodule of Alnus glutinosa wereisolated and their expression patterns were charac-terized [5].

* Corresponding author. Tel.: +82-2-880-6678; fax: +82-2-872-6881.

E-mail address: ancs@plaza.snu.ac.kr (C.S. An)

0168-9452/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0 1 6 8 -9452 (99 )00124 -7

H.B. Kim et al. / Plant Science 149 (1999) 85–9486

Asparagine (Asn) is a major amino acid formtransporting nitrogen in plants faced with condi-tions of excess ammonia (for example, germina-tion, growth on fertilizers and nitrogen fixation)and limitation of carbon source [6,7]. Asn is anideal amino acid form for transport of reducednitrogen, because of its higher N:C ratio (2:4) thanglutamine (2:5) and its stability [8]. The synthesisof Asn is mediated by asparagine synthetase (AS,EC 6.3.5.4), which catalyzes the ATP-dependenttransamination reaction transferring the amidegroup of glutamine (or ammonia) to aspartate,resulting in the formation of glutamate and Asnwhile hydrolyzing ATP to AMP and PPi;

L-Asp+L-Gln+ATP

�L-Asn+L-Glu+AMP+PPi.

Two different types of AS have been described inE. coli and yeast; a Gln-dependent form and anammonia-dependent form [9,10]. Although allplant ASs studied up to date appear to be Gln-de-pendent form, AS in maize roots can use ammoniaas a substrate effectively under the condition ofexcess ammonia [11]. Biochemical study of AS hasbeen hampered by its extremely low stability, con-taminating asparaginase activity and specific non-protein inhibitors [12]. Molecular and geneticstudies have been used to circumvent the difficultyof biochemical study. Significant progress has beenmade in understanding expression and regulationof AS through the isolation and characterizationof cDNA and/or genomic clones from non-nodu-lating plants such as asparagus [13], Arabidopsis[14], broccoli [15] and maize [16], and especiallyfrom legume plants such as pea [17], Lotus japoni-cus [18], alfalfa [19], soybean [20] and broad bean[21]. Nothing, however, has been known on theexpression and regulation of AS from any acti-norhizal plants.

In this paper, a cDNA clone encoding AS wasisolated from the root nodule cDNA library of E.umbellata, a amide transporter [4], by competitivehybridization and its molecular biological aspectswere characterized. Expression pattern of AS indifferent tissues was investigated by Northern hy-bridization. Expression pattern of AS during nod-ule development and distribution of AS transcriptsin the root nodule were analyzed by RT-PCR(Reverse Transcriptase-mediated PolymeraseChain Reaction) and in situ hybridization, respec-tively. This is a first report for isolation and

characterization of cDNA clone encoding enzymerelated with ammonia assimilation in the rootnodule of amide transporting actinorhizal plants.

2. Materials and methods

2.1. Bacterial strain and plant material

Frankia strain EuIK1, a symbiont of E. umbel-lata root nodule, was used to nodulate E. umbel-lata seedlings. Culture methods for E. umbellataseedlings and the Frankia strain were describedpreviously [22]. RNAs for construction of nodulecDNA library were isolated from root nodules atvarious developmental stages 6 months after inoc-ulation. Uninoculated seedlings were used to iso-late RNAs of leaf and root. To study geneexpression level during nodule development, nod-ules of 4, 6, 8, and 10 weeks after inoculation wereharvested and stored at −80°C until used. For insitu hybridization, nodules of 8–10 weeks afterinoculation were used.

2.2. Isolation of DNA and RNA

The method of Doyle and Doyle [25] for isola-tion of genomic DNA was modified to isolate totalRNA from leaves, roots and nodules of E. umbel-lata. Plant tissues were ground in liquid nitrogen.Polyvinypolypyrrolidone (PVPP) was added dur-ing grinding with liquid nitrogen to remove pheno-lic compounds. CTAB (cetyltrimethyl ammoniumbromide) extraction buffer (10–12 ml/g tissue) wasadded to the tissue powder. The homogenate wasincubated at 60°C for 10 min and extracted withphenol and chloroform. The supernatant was pre-cipitated with cold IPA and washed with washingbuffer [76% (v/v) EtOH, 10 mM ammonium ac-etate]. Dried pellet was dissolved with nuclease-free water. Total RNA was differentiallyprecipitated with lithium chloride from total nu-cleic acids and treated with RNase-free DNase toremove genomic DNA. Poly(A)+ RNA for nodulecDNA library construction was purified from totalRNA using oligo d(T) cellulose column(Boehringer Mannheim, Mannheim, Germany) ac-cording to the methods of Ausubel et al. [24].Genomic DNA was isolated from E. umbellataleaves according to Doyle and Doyle [25]. PlasmidDNA was purified according to Sambrook et al.[26].

H.B. Kim et al. / Plant Science 149 (1999) 85–94 87

2.3. Construction and screening of a cDNAlibrary

cDNA was synthesized from 5 mg of poly(A)+

nodule RNA using cDNA synthesis Kit (Strata-gene, La Jolla, CA, USA) according to manufac-turer’s guide. The synthesized cDNA was insertedinto ZAP express vector (Stratagene) and recombi-nant phage DNA was in vitro packaged usingGigapack III Packaging Extract (Stratagene) ac-cording to manufacturer’s guide. Phage DNAsfrom about 10 000 plaques per petri dish (r=4.5cm) were transferred to nylon membranes (Amer-sham, Berckinghamshire, UK) and cross-linked byUV-treatment. Ten phage blots were competitivelyhybridized with nodule cDNA probe and an ex-cess of total RNA from roots and leaves accordingto Mangiarotti et al. [27]. The cDNA probe wassynthesized from 2 to 5 mg poly(A)+ nodule RNA.

2.4. Cloning and sequence analysis

Positive phage clones from primary and sec-ondary competitive screening were changed intophagemid clones according to in vivo excisionprotocol of manufacturer (Stratagene). PhagemidDNA was deleted unidirectionally with exonucle-ase III and S1 nuclease by using double-strandedNested Deletion Kit (Pharmacia Biotech, Uppsala,Sweden) based on the protocol of Henikoff [28].The nucleotide sequences were determined bydideoxynucleotide chain termination method [29]using Taq polymerase (Promega, Madison, WI,USA) and T7 DNA polymerase (USB, Cleveland,OH, USA). The sequences were analyzed usingPC-GENE (Release 6.01; IntelliGenetics, Moun-tain View, CA, USA), DNASIS/PROSIS (V6.01;Hitachi Software Engineering, Tokyo, Japan) andBLAST search program [30,31].

2.5. DNA and RNA blot analysis

For DNA analysis, genomic DNAs from leavesof E. umbellata (10 mg) digested with several re-striction enzymes (EcoRI, HindIII and BglII)were electrophoresed on 0.8% agarose gel, andtransferred to Hybond-N membrane (Amersham)by capillary blotting method [26]. For RNA analy-sis, total RNAs from leaves, roots and nodules ofE. umbellata (10 mg) were electrophoresed on a 1%glyoxal gel and transferred to Hybond-N mem-

brane (Amersham) by capillary blotting method[26]. The blots were hybridized overnight with32P-labelled AS probe under following condition;6×SSC, 5×Denhardt’s solution, 0.5% SDS(Sodium Dodecyl Sulfate) at 65. The hybridizedblots were washed at 65°C with gradually decreas-ing salt concentration to 0.5×SSC, 0.1% SDS,and exposed to X-ray film.

2.6. RT-PCR

RT-PCR method was used to analyze expres-sion pattern of AS gene in the course of noduledevelopment. Two PCR primers [upper primer(position 1715–1737); 5%-TTCTGGAAGGGCTG-CACTAGGAG-3%, lower primer (position 1913–1937); 5%-TCCCCATCAGGCATAGAATCCAT-T-3%] were designed to specifically amplify 3% UTR(untranslated region) of AS cDNA clone. TotalRNAs (1 mg) from each developmental stage wereused as template for reverse transcription afterRNase-free DNase (Promega) treatment. To assessnitrogenase activity during nodule development,618 bp between position 88 and position 705 ofnifH ORF encoding nitrogenase reductase [32]was amplified. Transcripts of PUB (polyubiquitin)were also amplified as an indirect RT-PCR controlusing two primers specific to 3% UTR of PUBcDNA clone [23]. PCR cycling conditions for AS(nifH and PUB) were 95°C for 5 min for initialdenaturation followed by 95°C for 1 min, 62°C(67°C for nifH) for 1 min and 72°C for 1 min (30cycles) with 10 min final extension at 72°C. Am-plified PCR products were electrophoresed onagarose gel, transferred to nylon membrane, andprobed with inserts of AS, PUB and nifH clone.The hybridization signals of the blot werequantified with an image densitometer (Bio-Rad).

2.7. In situ hybridization

Tissue preparation was performed essentially asdescribed by Cox and Goldberg [33]. Noduleswere fixed overnight in FAA [50% (v/v) EtOH, 5%(v/v) glacial acetic acid, 10% (v/v) formaldehyde]under constant vacuum, dehydrated through agraded ethanol series, and embedded in Paraplast(Oxford, St. Louis. Mo. USA). Tissue sections (10mm thick) were applied to precleaned slide glassestreated with Vectabond (Vector Laboratories,

H.B. Kim et al. / Plant Science 149 (1999) 85–9488

Burlingame, CA, USA). Pretreatment, hybridiza-tion and washing of slides were performed essen-tially as described by McKhann and Hirsch [34],except for addition of RNase (30 mg RNase A/NTE buffer 1 ml) in the course of washing. Anti-sense and sense RNA probes for in situhybridization were prepared from linearized plas-mids with digoxigenin (DIG)-11-rUTP(Boehringer Mannheim) according to manufactur-er’s instruction. Hybridization was performed at42°C for 16 h. After posthybridization treatmentand incubation with antidigoxigenin conjugatedwith alkaline phosphatase, color development wasallowed in a dark cabinet for approximately 1–2day(s) with substrate, and stopped by immersingthe slides in TE (pH 8.0). The sections were dehy-drated through a graded ethanol series and thenmounted with Permount (Fisher Scientific, FairLawn, NJ, USA).

3. Results and discussion

3.1. Isolation of a cDNA clone encoding AS fromroot nodule of E. umbellata

A cDNA library using 5 mg poly(A)+ RNAfrom root nodule of E. umbellata was constructedto isolate nodule-specific/enhanced cDNA clones.Primary plaque forming units (pfu) of the cDNAlibrary were 5.5×105 with 95% recombinationefficiency and average size of the cDNA insert wasabout 1.5 kb. The 105 phage clones were competi-tively hybridized with 32P-labelled single-strandnodule cDNA probe and an excess of total RNAsfrom roots and leaves, resulting in isolation of 117putative nodule-specific/enhanced clones. A clonewith a 0.85 kb insert showed nucleotide sequencehomology with previously reported AS and cross-hybridized with 37 AS homologues out of 117clones. Full nucleotide sequence of a cDNA clonewith 2 kb insert out of 37 clones, which namedpEuNOD-AS1, was determined and analyzed.

3.2. Nucleotide and deduced amino acid sequenceanalysis of EuNOD-AS1

Asparagine synthetase clone, pEuNOD-AS1,coded for 585 amino acid residues (Fig. 1) withmolecular weight of 65.8 kDa and pI (isoelectricpoint) value of 6.12. The deduced amino acid

sequence of EuNOD-AS1 showed overall sequenceidentities of 74–88, 50, 53 and 55% with those ofother plant species, human, yeast (Asn2), and E.coli (AsnB), respectively. Gln-dependent AS(AsnB) and ammonia-dependent AS (AsnA) wereknown in E. coli [9,35] and the deduced aminoacid sequence of EuNOD-AS1 showed muchhigher sequence identity with the former (55%)than with the latter (14%).

The multiple alignment of deduced amino acidsequence of EuNOD-AS1 with those of previouslyreported nodule-forming leguminous plantsshowed many conserved sequence motifs through-out the overall sequence, except for highly variableregions in C-terminus (Fig. 1). A highly conservedregion among AS and glutamine aminotrans-ferases (E. coli and yeast) is a region of six aminoacid residues, His30, Arg31, Gly32, Pro33, Asp34, andAla35 [36]. The proline residue is not conserved inthe glutamine amidotransferases, whereas the ala-nine residue is not conserved in plant AS. Theglutamine-binding domain of purF-type glutamineamidotransferases is thought to reside in the cata-lytic triad, Cys2, Asp29, and His102 of N-terminus[36]. These residues reside to positions 2, 34, and104, respectively, in all known plant AS.

The N-terminus of EuNOD-AS1 contains theinvariant Cys2 residue, which has been shownthrough site-directed mutagenesis of the humanAS gene to be essential for Gln-dependent ASactivity [37]. However, the variation of histidineconsisting of catalytic triad in yeast Asn2 and E.coli AsnB raises questions about whether thisresidue of the catalytic triad is absolutely requiredfor Gln-dependent AS activity. Further, there areseveral other conserved regions that may playstructural or functional roles for ATP- and/oraspartate-binding sites. Along with the resultshowing higher sequence identity with Gln-depen-dent form than ammonia-dependent form, thepresence of glutamine-binding domain onEuNOD-AS1 strongly supports that EuNOD-AS1uses glutamine as a substrate.

EuNOD-AS1 does not contain hydrophobic sig-nal peptide sequence or transit peptide sequencefor targeting into plastids at N-terminus (data notshown). This result indicates EuNOD-AS1 plays arole as a cytosolic enzyme, although AS enzymeactivities were detected in proplastids fraction aswell as soluble fraction of soybean nodules [38].

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3.3. Expression pattern of EuNOD-AS1

The expression pattern of AS in leaf, root andnodule of E. umbellata was examined by Northernhybridization with full insert of pEuNOD-AS1 as

a probe. As shown in Fig. 2, AS mRNA wasspecifically detected in nodule as a 2.2 kb tran-script size. But, AS mRNAs of leaf and root werealso very weakly detected after longer exposure(data not shown).

Fig. 1. Multiple alignment of amino acid sequence deduced from nucleotide sequence of pEuNOD-AS1 with ASs of nodule-form-ing leguminous plants using Clustal W. Filled circles indicate conserved amino acid residues for purF-type Gln-binding domain.Identical residues are indicated by dots. Dashes indicate gaps introduced to maximize sequence similarity. The sources of ASpolypeptide sequences used in this alignment are: EUAS1, E. umbellata (Genbank Acc. No. AF061740); GMAS1 and GMAS2,Glycine max (U77679 and U77678); LJAS1 and LJAS2, Lotus japonicus (X89409 and X89410); PSAS1 and PSAS2, Pisum sati6um(X52179 and X52180); MSAS, Medicago sati6a (U89923); VFAS, Vicia faba (Z72354).

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Fig. 2. Expression of AS in different organs of E. umbellata.Blot containing 10 mg total RNA per lane were hybridizedwith the 32P-labeled insert of pEuNOD-AS1 (A). Gel wasstained with ethidium bromide before blotting to ensure equalloading of RNA (B). Arrows indicate the positions of rRNAbands. L, leaf; R, root; N, nodule.

GOGAT [43]. Expression of nifH and AS wasdecreased 10 WAI, when PUB was highly ex-pressed (Fig. 3). Polyubiquitin is closely relatedwith senescence of cells, tissues, organs, and entireorganisms [44].

The spatial expression of AS in E. umbellataroot nodule was determined by in situ hybridiza-tion of longitudinal sections of nodules with DIG-labeled antisense and sense RNA probes,respectively (Fig. 4A, B, C and F). In the course ofin situ hybridization, antisense RNA probe ofnifH was used as a marker gene to confirm in-

Fig. 3. RT-PCR analysis of the expression of AS and PUBduring nodule development. nifH transcripts encoding nitro-genase reductase were amplified as a marker gene to assessnitrogenase activity during nodule development. All totalRNAs used in this experiment were treated with RNase-freeDNase. RT-PCR products separated on agarose gel weretransferred on nylon membrane, and probed with 32P-labeledDNAs containing nifH of Frankia EuIK1 [32] and inserts ofpEuNOD-AS1 and pEuNOD-PUB1 [23]. The hybridizationsignals were quantified using an image densitometer (BIO-RAD). Data are the mean values of two independent experi-ments. Error bars indicate standard deviation. R, root; N4, 4weeks after inoculation (WAI); N6, 6 WAI; N8, 8 WAI; N10,10 WAI.

Asn is a major nitrogen-containing compoundin the xylem sap of temperate legumes such asalfalfa and pea. An increased AS activity has beencorrelated with the accumulation of Asn in alfalfaroot nodules [39]. AS activity has been known tobe increased in the root nodules of plant speciestransporting fixed nitrogen product into amideforms [19,40,41].

To examine AS expression pattern during nod-ule development, RT-PCR method was used.First, nifH transcripts encoding nitrogenase reduc-tase were amplified as a positive control and toolto assess nitrogenase activity during nodule devel-opment. nifH showed highest expression level 6–8weeks after inoculation (WAI), and decreasedthereafter (Fig. 3). AS showed highest expressionlevel 6–8 WAI, and decreased thereafter (Fig. 3),like nifH expression pattern. PUB, however,showed different expression pattern; it was firstlyexpressed highly 4 WAI, decreased thereafter dur-ing 6–8 WAI and reaccumulated 10 WAI (Fig. 3).

Klucas [42] suggested that the initial decline innitrogenase activity indicates the beginning ofnodule senescence. Decrease of nitrogen fixationcapacity in alfalfa nodule were accompanied bydecreases in nodule soluble proteins, GS and

H.B. Kim et al. / Plant Science 149 (1999) 85–94 91

Fig. 4. In situ localization of AS transcript in longitudinal sections of E. umbellata root nodules. Antisense probe of nifH was alsoused as a marker gene to identify infected cells in fixation zone. Antisense and sense RNA probes for in situ hybridization wereprepared from linearized plasmids with digoxigenin (DIG)-11-rUTP (Boehringer Mannheim) according to manufacturer’sinstruction. Boxed regions of panel a and d were detailed on panel b and e, respectively. Arrows indicate hybridization signalswith purple color. A, longitudinal section probed with antisense probe of AS; B, detailed view of fixation zone of panel A; C, senseprobe of AS; D, antisense probe of nifH; E, detailed view of fixation zone of panel D; F, sense probe of nifH. MZ, meristem zone;FZ, fixation zone; VS, vascular system; PD, periderm; IC, infected cell; UC, uninfected cell. Bars of panel A, C, D and F=250mm, bars of panel B and E=50 mm.

fected and nitrogen-fixing cells in the root nodule[45,3] (Fig. 4D and E). In situ hybridization usingAS antisense RNA probe showed AS transcriptswere detected only at the infected cells completelyfilled with Frankia hyphae in fixation zone (Fig.4A and B), where nifH transcripts were also de-tected (Fig. 4D and E). No hybridization signalwas detected with AS and nifH sense RNA probe(Fig. 4C and F).

Shi et al. [19] showed that AS mRNA wasdetected in ineffective nodule of alfalfa, althoughthe expression level was much lower than that ofeffective nodule. AS transcripts were also detectedin both infected and uninfected cells of the symbi-otic zone and in the nodule parenchyma of alfalfa

root nodule. From these data, Shi et al. [19]suggested that initial signal for AS expression inalfalfa nodule is unrelated to the presence of func-tional nitrogenase. On the other hand, our datashowed that the expression pattern of AS duringnodule development was very similar with that ofnifH (Fig. 3). In situ hybridization data alsoshowed AS and nifH transcripts were stronglydetected in infected cells of root nodule (Fig. 4),unlike AS expression pattern in alfalfa root nod-ule. Thus, our data suggests that AS expressionmay be closely related with nitrogen fixation activ-ity during nodule development, that is, AS expres-sion may be under metabolic control rather thandevelopmental control. Similarly to our in situ

H.B. Kim et al. / Plant Science 149 (1999) 85–9492

hybridization result, GS, key enzyme involved inprimary ammonia assimilation in root nodule,were strongly expressed in the infected cells offixation zone of Alnus glutinosa nodule, indicatingthat GS is under metabolic control [5].

3.4. AS genes in the genome of E. umbellata

A genomic Southern hybridization was per-formed using full length cDNA insert of pE-uNOD-AS1 as a probe under high stringencycondition (Fig. 5). Total genomic DNA was di-gested with restriction enzymes, EcoRI, HindIIIand BglII. As shown in Fig. 5, several strong orweak hybridization signals were detected in eachlane; 2.0, 2.5, and 3.7 kb EcoRI fragments, 1.5,3.0, 3.5, 5.5, and 9.6 kb HindIII fragments and8.0, and 15.0 kb BglII fragments. An EcoRI re-striction site is located on the nucleotide sequenceof pEuNOD-AS1, but HindIII and BglII site are

not located on the sequence (data not shown).Along with result that another cDNA clone (pE-uNOD-AS2) showing different digestion patternwas isolated, this hybridization pattern indicatesthat AS may be encoded by at least two genes.Two different AS cDNA clones have been alsoisolated from pea [17], trefoil [18] and soybean[20].

In conclusion, we showed that a high expressionlevel of AS in the root nodule of E. umbellata isclosely related with nitrogen-fixation. Accordingly,we suggest its role as to further assimilate excessammonia made by nitrogen fixation in infectedcells, and its expression may be under metaboliccontrol rather than developmental control of theroot nodule.

Acknowledgements

This work was supported by Non Directed Re-search Fund (D 0189), Korea Research Founda-tion (1996). The authors would like to thank DrAnn M. Hirsch for her kind help in in situhybridization.

References

[1] D.D. Baker, W.B.C.R. Schwintzer, Introduction, in: J.D.Tjepkema (Ed.), The Biology of Frankia and Acti-norhizal Plants, Academic Press, New York, 1990, pp.1–13.

[2] W. Newcomb, S.M. Wood, Morphogenesis and finestructure of Frankia (Actinomycetes): The microsym-biont of nitrogen-fixing actinorhizal root nodules, Int.Rev. Cytol 109 (1987) 1–88.

[3] A. Ribeiro, A.D.L. Akkermans, A. Van Kammen, T.Bisseling, K. Pawlowski, A nodule-specific gene encodinga subtilisin-like protease is expressed in early stages ofactinorhizal nodule development, Plant Cell 7 (1995)785–794.

[4] K.R. Schubert, Products of biological nitrogen fixationin higher plants: synthesis, transport, and metabolism,Ann. Rev. Plant Physiol. 37 (1986) 539–574.

[5] C. Guan, A. Ribeiro, A.D.L. Akkermans, Y. Jing, A.Van Kammen, T. Bisseling, K. Pawlowski, Nitrogenmetabolism in actinorhizal nodules of Alnus glutinosa :expression of glutamine synthetase and acetylornithinetransaminase, Plant Mol. Biol. 32 (1996) 1177–1184.

[6] K.A. Sieciechowicz, K.W. Joy, R.J. Ireland, Themetabolism of asparagine in plants, Phytochemistry 27(1988) 663–671.

[7] A.A. Urquhart, K.W. Joy, Use of phloem exudate tech-nique in the study of amino acid transport in pea plants,Plant Physiol. 68 (1981) 750–754.

Fig. 5. Genomic Southern hybridization of AS. GenomicDNAs (10 mg) digested with restriction enzymes were sepa-rated on 1% agarose gel and corresponding nylon membranewas probed with 32P-labeled insert of pEuNOD-AS1 underhigh stringency. E, EcoRI; H, HindIII; Bg, BglII.

H.B. Kim et al. / Plant Science 149 (1999) 85–94 93

[8] P.J. Lea, B.J. Miflin, Transport and metabolism of as-paragine and other nitrogen compounds within the plant,in: P.K. Stumpf, E.E. Conn (Eds.), The Biochemistry ofPlants, Academic press, New York, 1980, pp. 569–607.

[9] J. Felton, S. Michaelis, A. Wright, Mutations in twounlinked genes are required to produce asparagine aux-otrophy in Escherichia coli, J. Bacteriol. 142 (1980) 221–228.

[10] F. Ramos, J.M. Wiame, Two asparagine synthetases inSaccharomyces cere6isiae, Eur. J. Biochem. 108 (1980)373–377.

[11] I. Stulen, G.F. Israelstam, A. Oaks, Enzymes of as-paragine synthesis in maize roots, Planta 146 (1979)237–241.

[12] G. Tjaden, G.M. Coruzzi, Glutamine and asparaginebiosynthesis: the regulation of genes for enzymes along acommon nitrogen-metabolic pathway, in: D.P.S. Verma(Ed.), Control of Plant Gene Expression, CRC, BocaRaton, 1993, pp. 459–470.

[13] K.M. Davies, G.A. King, Isolation and characterizationof a cDNA for harvest-induced asparagine synthetasefrom Asparagus officinalis L, Plant Physiol. 102 (1993)1337–1340.

[14] H.M. Lam, S.S.Y. Peng, G.M. Coruzzi, Metabolic regu-lation of the gene encoding glutamine-dependent as-paragine synthetase in Arabidopsis thaliana, PlantPhysiol. 106 (1994) 1347–1357.

[15] C.G. Downs, B.J. Pogson, K.M. Davies, E.C. Amira, Anasparagine synthetase cDNA clone (GenBank X84448)from broccoli (PGR95-016), Plant Physiol. 108 (1995)1342.

[16] C. Chevalier, E. Bourgeois, D. Just, P. Raymond,Metabolic regulation of asparagine synthetase gene ex-pression in maize (Zea mays L.) root tips, Plant J. 9(1996) 1–11.

[17] F. Tsai, G.M. Coruzzi, Dark-induced and organ-specificexpression of two asparagine synthetase genes in Pisumsati6um, EMBO J. 9 (1990) 323–332.

[18] R.N. Waterhouse, A.J. Smyth, A. Massonneau, I.M.Prosser, D.T. Clarkson, Molecular cloning and charac-terisation of asparagine synthetase from Lotus japonicus :dynamics of asparagine synthesis in N-sufficient condi-tions, Plant Mol. Biol. 30 (1996) 883–897.

[19] L. Shi, S.N. Twary, H. Yoshioka, R.G. Gregerson, S.S.Miller, D.A. Samac, J.S. Gantt, P.J. Unkefer, C.P.Vance, Nitrogen assimilation in alfalfa: Isolation andcharacterization of an asparagine synthetase gene show-ing enhanced expression in root nodules and dark-adapted leaves, Plant Cell 9 (1997) 1339–1356.

[20] C.A. Hughes, H.S. Beard, B.F. Matthews, Molecularcloning and expression of two cDNAs encoding as-paragine synthetase in soybean, Plant Mol. Biol. 33(1997) 301–311.

[21] H. Kuster, U. Albus, M. Fruhling, S.A. Tchetkova, L.A.Tikhonovitch, A. Puhler, A.M. Perlick, The asparaginesynthetase gene VfAS1 is strongly expressed in the nitro-gen-fixing zone of broad bean (Vicia faba L.) root nod-ules, Plant Sci. 124 (1997) 89–95.

[22] S.C. Kim, C.D. Ku, M.C. Park, C.H. Kim, S.D. Song,C.S. An, Isolation of symbiotic Frankia EuIK1 strainfrom root nodule of Elaeagnus umbellata, Korean J. Bot.36 (1993) 177–182.

[23] H.B. Kim, Structures and Expression Patterns of cDNAClones Encoding Asparagine Synthetase, Chitinase, andPolyubiquitin from the Root Nodule of Elaeagnus um-bellata, Ph.D. dissertation, Seoul National University,Seoul, 1998.

[24] F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore,J.G. Seidman, J.A. Smith, K. Struhl, Current Protocolsin Molecular Biology, John Wiley and Sons, New York,1987, pp. 4.5.1–4.5.3.

[25] J.J. Doyle, J.I. Doyle, Isolation of plant DNA from freshtissue, Focus 12 (1990) 13–15.

[26] J. Sambrook, E.F. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, 1989 (Chapter 1,2, 7, 8, 9 and 10).

[27] G. Mangiarotti, S. Chung, C. Zucker, H.F. Lodish,Selection and analysis of cloned developmentally-regu-lated Dictyostelium discoideum genes by hybridizationcompetition, Nucl. Acids Res. 9 (1981) 947–963.

[28] S. Henikoff, Unidirectional digestion with exonucleaseIII creats targetted breakpoint for DNA sequencing,Gene 28 (1984) 351–359.

[29] F. Sanger, S. Nicklen, A.R. Coulson, DNA sequencingwith chain terminating inhibitors, Proc. Natl. Acad. Sci.USA. 74 (1977) 5463–5467.

[30] W.R. Pearson, D.J. Lipman, Improved tools for biologi-cal sequence comparison, Proc. Natl. Acad. Sci. USA. 85(1988) 2444–2448.

[31] S.F. Altschul, W. Gish, W. Miller, E.W. Meyers, D.Lipman, Basic local alignment search tool, J. Mol. Biol.215 (1990) 403–410.

[32] H.B. Kim, C.S. An, Nucleotide sequence and expressionof nifH,D from Frankia EuIK1 strain, a symbiont ofElaeagnus umbellata, Physiol. Plant. 99 (1997) 690–695.

[33] K.H. Cox, R.B. Goldberg, Analysis of plant gene expres-sion, in: C.H. Shaw (Ed.), Plant Molecular Biology: APractical Approach, IRL press, Oxford, 1988, pp. 1–35.

[34] H.I. Mackhann, A.M. Hirsch, In situ localization ofspecific mRNAs in plant tissues, in: B.R. Glick, J.E.Thompson (Eds.), Methods in Plant Molecular Biologyand Biotechnology, CRC press, Boca Raton, 1993, pp.179–205.

[35] R. Humbert, R.D. Simoni, Genetic and biochemicalstudies demonstrating a second gene coding for as-paragine synthetase in Escherichia coli, J. Bacteriol. 142(1980) 212–220.

[36] B. Mei, H. Zalkin, A cysteine-histidine-aspartate cata-lytic triad is involved in glutamine amide transfer func-tion in purF-type glutamine amidotransferases, J. Biol.Chem. 264 (1989) 16613–16619.

[37] G. Van Heeke, S.M. Schuster, The N-terminal cysteineresidue of human asparagine synthetase is essential ofglutamine-dependent activity, J. Biol. Chem. 264 (1989)19475–19478.

[38] M.J. Boland, J.F. Hanks, P.H.S. Reynolds, D.G.Blevins, N.E. Tolbert, K.R. Schubert, Subcellular orga-nization of ureide biogenesis from glycolytic intermedi-ates and ammonium in nitrogen-fixing soybean nodules,Planta 155 (1982) 45–51.

[39] T.C. Ta, F.D.H. Macdowell, M.A. Faris, Metabolism ofnitrogen fixed by nodules of alfalfa (Medicago sati6a L.).

H.B. Kim et al. / Plant Science 149 (1999) 85–9494

II. Asparagine synthesis, Biochem. Cell Biol. 66 (1988)1349–1354.

[40] B.J. Miflin, J.V. Cullimore, Nitrogen assimilation in thelegume Rhizobium symbiosis: a joint endeavour, in:D.P.S. Verma, T. Hohn (Eds.), Genes Involved in Mi-crobe-Plant Interactions, Springer-Verlag, Vienna, 1984,pp. 129–178.

[41] P.H. Reynolds, D.G. Blevins, M.J. Boland, K.R. Schu-bert, D.D. Randall, Enzymes of ammonia assimilation inlegume nodules: a comparison between ureide- andamide-transporting plants, Physiol. Plant. 55 (1982) 255–260.

[42] R.V. Klucas, Studies on soybean nodule senescence,Plant Physiol. 54 (1994) 612–616.

[43] R.G. Groat, C.P. Vance, Root nodule enzymes of am-monia assimilation in alfalfa (Medicago sati6a L.): devel-opmental patterns and response to applied nitrogen,Plant Physiol. 67 (1981) 1198–1203.

[44] W.R. Belknap, J.E. Garbarino, The role of ubiquitin inplant senescence and stress responses, Trends Plant Sci. 1(1996) 331–335.

[45] K. Pawlowski, A.D.L. Akkermans, A. Van Kammen, T.Bisseling, Expression of Frankia nif genes in nodules ofAlnus glutinosa, Plant Soil 170 (1995) 371–376.

.