aspartateaminotransferase effective and ineffective ...aat2 (ec 2.6.1.1) catalyzes the reversible...

Plant Physiol. (1992) 98, 868-8780032-0889/92/98/0868/11/$01 .00/0

Received for publication August 13, 1991Accepted October 21, 1991

Aspartate Aminotransferase in Effective and IneffectiveAlfalfa Nodules1

Cloning of a cDNA and Determination of Enzyme Activity, Protein, and mRNA Levels

J. Stephen Gantt, Ruby J. Larson, Mark W. Farnham, Sudam M. Pathirana, Susan S. Miller, andCarroll P. Vance*

Department of Plant Biology (J.S.G., S.M.P.), Department of Agronomy and Plant Genetics (R.J.L., M.W.F., S.S.M.,C.P. V.), and U.S. Department of Agriculture-Agricultural Research Service, Plant Science Research

(M. W.F., C.P. V.), University of Minnesota, St. Paul, Minnesota 55108

ABSTRACT

Aspartate aminotransferase (AAT) is a key plant enzyme af-fecting nitrogen and carbon metabolism, particularly in legumeroot nodules and leaves of C4 species. To ascertain the moleculargenetic characteristics and biochemical regulation of AAT, wehave isolated a cDNA encoding the nodule-enhanced AAT (AAT-2) of alfalfa (Medicago sativa L.) by screening a root nodule cDNAexpression library with antibodies. Complementation of an Esch-erichia coRi AAT mutant with the alfalfa nodule AAT-2 cDNAverified the identity of the clone. The deduced amino acid se-quence of alfalfa AAT-2 is 53 and 47% identical to animal mito-chondrial and cytosolic AATs, respectively. The deduced molec-ular mass of AAT-2 is 50,959 daltons, whereas the mass ofpurified AAT-2 is about 40 kilodaltons as determined by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, and the pro-tein's N-terminal domain (amino acids 1-59) contains many of thecharacteristics of plastid-targeting peptides. We postulate thatAAT-2 is localized to the plastid. Southem blot analysis suggeststhat AAT-2 is encoded by a small, multigene family. The expres-sion of AAT-2 mRNA in nodules is severalfold greater than thatin either leaves or roots. Northem and western blots showed thatexpression of AAT activity during effective nodule developmentis accompanied by a sevenfold increase in AAT-2 mRNA and acomparable increase in enzyme protein. By contrast, plant-con-trolled ineffective nodules express AAT-2 mRNA at much lowerlevels and have little to no AAT-2 enzyme protein. Expression ofroot nodule AAT-2 appears to be regulated by at least two events:the first is independent of nitrogenase activity; the second isassociated with nodule effectiveness.

AAT2 (EC 2.6.1.1) catalyzes the reversible reaction, gluta-mate + oxaloacetate <-+ aspartate + a-ketoglutarate. The

' This research was supported in part by the National ScienceFoundation, grant No. DCB-8905006, and by the U.S. Departmentof Agriculture, grant No. 87-CRCR-1-2588. Joint contribution fromthe Plant Science Research Unit, U.S. Department of Agriculture,Agricultural Research Service, and the Minnesota Agricultural Ex-periment Station (Paper No. 19,547, Scientific Journal Series).

2 Abbreviations: AAT, aspartate aminotransferase; in,Sa,in,Saranac; IPTG, isopropylthiogalactopyranoside; kb, kilobase pair;RACE, rapid amplification ofcDNA ends; pl, isoelectric point.

enzyme is widely distributed in microbes, animals, and plants,being crucial to nitrogen and carbon metabolism (13). Inplants, AAT plays an essential role in (a) the assimilation ofreduced nitrogen into aspartic acid and asparagine, particu-larly in legume root nodules (9, 25); (b) the transfer of fixedcarbon from mesophyll cells to bundle sheath cells in C4plants (20, 31); and (c) the transfer of reducing equivalents tochloroplasts, mitochondria, and peroxisomes through a ma-late-aspartate shuttle (13). Cytosolic, mitochondrial, and plas-tid forms of the plant enzyme have been documented andsuggested to be controlled by different genetic loci (13). Inanimals, cytosolic and mitochondrial forms of AAT havebeen extensively characterized, and the genes encoding theseproteins have been isolated (e.g. 14, 22). Plant AAT geneshave not been isolated.Root nodules of alfalfa, pea, and lupine, which mainly

assimilate and transport symbiotically fixed nitrogen as as-partate and asparagine, are particularly rich sources of AATactivity (4, 9, 25). Multiple forms of the enzyme have beenpurified from both alfalfa (4, 9) and lupine (25). Alfalfa noduleAAT is composed oftwo forms, AAT- 1 and AAT-2. Similarly,two analogous forms, AAT-P, and AAT-P2, were identifiedin lupine. We have shown that alfalfa AAT- 1 has a nativemolecular mass of 84 kD and a subunit molecular mass of 42kD. By comparison, AAT-2 is slightly smaller, having a nativeand subunit molecular mass of 80 and 40 kD, respectively (4,9). Moreover, antibodies produced against alfalfa AAT-1 andAAT-2 are immunologically distinct. Those produced againstAAT-2 do not recognize AAT-1 polypeptides and, likewise,AAT-1 antibodies do not recognize AAT-2 polypeptides (4).Recent studies with C4 species showed that AATs from Pan-icum and Eleusine display physical characteristics and im-munological properties similar to those described for alfalfa(20, 31). Data from both root nodules of legumes and leavesof C4 species indicate that AAT- 1 and AAT-2 are homodi-meric proteins encoded by separate genes. Similar conclusionshave been reached for animal cytosolic and mitochondrialAATs (e.g. 14, 22).We have used immunochemical techniques to demonstrate

that expression ofAAT shows both developmental and organ-ogenic-related variation (3, 4, 9). Egli et al. (3) showed that

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CLONING AND DEVELOPMENTAL EXPRESSION OF ALFALFA NODULE AAT-2

the in vitro specific activity of AAT and relative amounts ofenzyme protein increased in effective nodules during noduledevelopment. Increased nodule AAT activity and relativeAAT content were due primarily to an increase in the AAT-2 isozyme activity and enzyme protein (9). Ineffective nod-ules, by comparison, had reduced amounts ofAAT-2 enzymeprotein. Additionally, immunoprecipitations of total AATactivity with AAT-2 antibodies showed that AAT-2 composed70% or more of the total activity in alfalfa nodules. Bycontrast, AAT-l predominated in roots. Farnham et al. (4)also demonstrated organ-selective expression of AAT-1 andAAT-2 in wheat, pea, soybean, and maize. Differential expres-sion of AAT-1 and AAT-2 has also been demonstrated inleaves of the C4 species Panicum and Eleusine (20, 31).Because no plant nucleic acid probes corresponding to theAAT genes have been isolated, it is not known if the differ-ential expression of plant AATs results from alterations in thelevels ofAAT mRNAs.

Ineffective root nodules have been used to evaluate the roleof NH4+ availability in nodule gene expression (1, 2, 12, 37).In most studies, ineffective nodules were induced by mutantstrains of rhizobia. In efforts to ascertain how changes in plantgenes affect symbiosis and nodule gene expression, severalplant gene-controlled ineffective alfalfa genotypes have beendeveloped (3, 24). Comparative studies of nodule physiologyand biochemistry with plant gene-controlled ineffective andeffective genotypes have been invaluable. In alfalfa, fourgenetic systems, designated in,, in2, in3, and in4in5, conditionineffective symbiosis (24). All are simply inherited, requirethe nulliplex condition, and are recessive to effective sym-biosis. These genotypes are ineffective with all strains ofRhizobium meliloti. Nodule size and bacteroid developmentof the in, genotype most closely approximates the effectivewild-type nodules. In the "Saranac" background, in, nodules(in,Sa) are comparable to effective nodules in size and initialdevelopment (3), but bacteroids quickly deteriorate, leghe-moglobin is reduced, and nodules senesce early. Further com-parisons have shown that nitrogenase activity and the activityof enzymes involved in nodule nitrogen and carbon metabo-lism are strikingly reduced in in,Sa nodules as compared witheffective parental Saranac nodules (3). The primary lesionresulting in ineffectiveness of in, nodules is unknown.To ascertain further how AAT affects plant nitrogen and

carbon metabolism and to understand molecular events in-volved in regulating AAT activity, we have cloned an alfalfaAAT-2 cDNA. Using this cDNA, we have investigated thechanging levels of AAT-2 mRNA and compared these levelsto AAT activity and AAT-2 protein during effective andineffective nodule development.

MATERIALS AND METHODS

Plant Material

Alfalfa (Medicago sativa L.) cv Saranac and the single gene,recessive ineffective plant genotype in,Sa (24), seeds wereobtained from Dr. D. K. Barnes, USDA-ARS (St. Paul, MN).Over 90% of the in,Sa genotype is from cv Saranac back-ground. The fact that alfalfa is an outcrossing tetraploidspecies precludes formation of isogenic lines. Plants were

grown in glasshouse sandbenches inoculated with effectiveRhizobium meliloti as previously described (3). The date thatseeds were planted in the sandbench was designated day 0.For developmental analyses, plant material was collected ondays 5, 7, 8, 9, 12, 19, and 33. Plants were harvested at 8:00AM and roots (day 5), nodules on 2 mm root sections (days 7and 8), or nodules (day 9 and older) were hand-collected ontoice and used immediately for measurement of AAT activityand enzyme protein and for isolation of RNA.

Protein Extraction and Enzyme Assays

Triplicate samples of roots or nodules were ground inextraction buffer (100 mM Mes, pH 6.8, 100 mm sucrose, 2%2-mercaptoethanol, 15% ethylene glycol, 2 mM PMSF, and0.2 mM antipain) and centrifuged 15 min at 1 5,500g to obtainthe soluble protein fraction (3). AAT activity was assayed invitro by spectrophotometrically monitoring the disappearanceofNADH at 340 nm as described by Griffith and Vance (9).Protein content ofextracts andAAT and nitrogenase activitieswere determined as previously described (3).

SDS-PAGE and Westem Blotting

Soluble proteins in cell-free extracts were electrophoresedin 10% SDS-polyacrylamide gels and electrophoreticallytransferred to nitrocellulose as described by Farnham et al.(4). Each lane was loaded with 50 ,ug protein. Rabbit poly-clonal antibodies to alfalfa nodule AAT-2 (9) were mademonospecific by affinity purification (28) and used to detectAAT-2 enzyme protein on western blots (3).

RNA Isolation and Northem Blots

Total RNA was isolated from freshly collected root andnodule samples of effective Saranac and ineffective in,Saaccording to procedures detailed in Silflow et al. (27). Tissue(1 g/10 mL buffer) was homogenized in 10 mM Tris(pH 8.8) containing 1% triisopropylnaphthalenesulfonic acid(Kodak), 6% 4-aminosalicylic acid, 50 mm NaCl, and 6% n-butanol with a Polytron (Brinkmann Instruments) on highspeed for 10 to 20 s. After several extractions withphenol:chloroform:isoamyl alcohol (25:24:1, v/v), total RNAwas precipitated in ethanol. Poly(A)+ RNA was obtained byone cycle of oligo(dT)-cellulose chromatography.

Total RNA (10 4g/lane) and poly(A)+ RNA (0.5 ug/lane)were electrophoretically separated on formaldehyde-1.5%agarose gels and transferred to Zetaprobe (Bio-Rad) in 10 xSSC (1 x SSC: 150 mm NaCl, 15 mM trisodium citrate)overnight. Northern blots were probed with denatured 32P-labeled pAAT37 cDNA prepared by the random primermethod (5). Prehybridization and hybridization of northernblots were as described by the manufacturer. After hybridi-zation, blots were washed sequentially with: 2 x SSC/.1%SDS at room temperature for 15 min; 0.5 x SSC/. 1% SDSat room temperature for 15 min; and 0.1 x SSC/0.1% SDSat 65°C for 15 min. Radioactivity on blots was quantifiedwith an AMBIS Radioanalytic Imaging System (San Diego,CA). The amount of radioactivity that hybridized to AAT-2mRNA was determined, with similar results, from three sep-

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Plant Physiol. Vol. 98, 1992

arate blots. After quantitation of radioactivity, filters wereexposed to x-ray film. The radioactivity from the blotswas normalized for poly(A)+ RNA by 32P-labeled poly(U)hybridization.

Isolation and Characterization of AAT-2 cDNA

A cDNA synthesis kit (Pharmacia) was used to constructan oligo-dT-primed alfalfa nodule cDNA library in the vectorXgtl 1. Insert size of the library ranged from 0.5 to 5.5 kb.The library was screened with affinity-purified monospecificantisera prepared against alfalfa AAT-2 (9) with horseradishperoxidase-conjugated goat anti-rabbit antibodies as a detec-tion system. Approximately 2 x 105 recombinants werescreened. From this screening, six recombinant antigen-pro-ducing bacteriophages were purified, and the sizes of thecDNA inserts were determined. The recombinant containingthe largest cDNA was subcloned into pBluescript KS- (Stra-tagene), and its nucleotide sequence was determined fromnested deletion (11) fragments by the dideoxy terminationmethod with Sequenase (U.S. Biochemical).The RACE procedure (7), as modified below, was used to

obtain cDNA clones that encode the N-terminal amino acidsof AAT-2. One microgram of nodule poly(A)+ RNA wasreverse-transcribed in Taq polymerase buffer (50 mM KCI, 10mM Tris, pH 8.8 at 42C, 0.1% Triton X-100, 1.5 mM MgC12)supplemented with MgCl2 to a final concentration of 3.75mM and containing 1.5 units/gL RNasin (Promega Biotec), 1mm of each dNTP, 10 units of avian myeloblastosis virusreverse transcriptase (Promega Biotec), and 10 pmol of re-verse transcriptase primer (5'-AACTCCAAGATTGAGCTT-3') that is complementary to nucleotides 346 to 329. Afterincubation at 42°C for 1 h and at 52°C for 30 min, unextendedprimers were removed with a Centricon 100 spin filter (Ami-con) and the cDNA was tailed with dA and then diluted to0.5 mL in preparation for amplification. Amplification of thesingle-stranded, tailed cDNA was carried out in a 50 '4Lreaction mixture that contained Taq polymerase buffer, 200,M of each dNTP, 0.1 g/gL nuclease-free BSA, 25 pmolAAT2-5AMP primer (5'-ACATTAGTCGCAACAGCCAT-3'), complementary to nucleotides 243 to 224, 10 pmol dT-adaptor primer (5'-GACTCGAGGATCCAAGCTTTTTTT-TTTTTTTTTT-3'), 25 pmol adaptor primer (5'-GACT-CGAGGATCCAAGCTTT-3'), 10 gL of the cDNA, and 2.5units Taq polymerase (Promega Biotec). The mixture wascycled 25 times in a Perkin-Elmer Cetus DNA Thermal Cycleras follows: 94°C for 30 s; 50°C for 30 s; and 72°C for 30 s.Amplification products were subcloned into the vectorpCR1000 (Invitrogen) and sequenced as described above.

DNA Extraction and Southem Blots

Genomic DNA was isolated from leaf tissue of a single,clonally propagated Saranac alfalfa plant by a modificationof the procedure of Saghai-Maroof et al. (26). Lyophilizedleaf tissue (0.5 g) was ground to a fine powder with a mortarand pestle and then incubated in 10 mL of fresh extractionbuffer (1% Cetrimide, 0.1 M Tris, pH 7.5, 0.7 M NaCl, 40 mmEDTA, 1% 2-mercaptoethanol) at 60°C for 1 to 2 h withgentle shaking. Chloroform:isoamyl alcohol (24:1, v/v; 10

mL) was added and the phases were mixed gently for 10 min.The resulting emulsion was centrifuged at 10OOg for 45 minat 20°C. The aqueous phase was collected and reextractedwith an equal volume of chloroform-isoamyl alcohol as de-scribed above. The aqueous phase was collected, and DNAwas precipitated with 2.5 volumes of cold ethanol at -20°Covernight. Precipitated DNA was spooled out and rinsed threetimes with cold 70% ethanol containing 0.3 M ammoniumacetate. DNA pellets were thoroughly dried and then dissolvedin 0.4 to 0.8 mL TE (10 mm Tris, pH 7.5, 1 mM EDTA).For Southern blot analysis, approximately equal molar

amounts of genomic alfalfa (10 ,ug) and R. meliloti (0.01 g)DNA were digested to completion with various restrictionenzymes and electrophoresed in a 0.8% agarose gel. DNA wasdenatured and transferred to Immobilon-N (Millipore). Filterswere hybridized with denatured 32P-labeled pAAT37 cDNAin a solution containing: 6 x SSC, 0.5% SDS, 5 x Denhardt'ssolution, 100 ,g/mL salmon sperm DNA, and 10 mm EDTA,at 65°C for 16 h. Southern blots were washed three times in2 x SSC, 0.5% SDS for 5 min each at 25°C, followed by threemore washes in 0.1 x SSC, 0.1% SDS, twice for 10 min at25°C, and once for 30 min at 65°C.

Complementation of an Escherichia coli Mutant LackingAAT Activity

The AAT-deficient Escherichia coli mutant DL39 (8, 15)was obtained from the E. coli Genetics Stock Center, YaleUniversity, courtesy of B. J. Bachmann. Strain DL39 carriesthe aspC13 marker, requires aspartate for growth, and haslow to nondetectable levels ofAAT activity. The mutant wasmaintained on selection medium (8, 15) supplemented withaspartate (50 ug/mL). Competent DL39 cells were trans-formed with pAAT37 DNA. After transformation, cells weresuspended in selection medium supplemented with aspartate(50 ,g/mL) and containing 1 mm IPTG and incubated in aroller drum at 37°C for 3 h. Cells were collected by centrifu-gation at 4°C, washed twice by gentle resuspension in selectionmedium that lacked aspartate, and then spread on a Petridish containing selection medium, 50 sg/mL ampicillin, and0.1 mM IPTG. Plates were incubated at 37°C and coloniesarising from transformed cells appeared after 60 to 72 h.Individual colonies were picked and streaked onto fresh selec-tion medium containing 50 gg/mL ampicillin. Individualcolonies arising on these plates were used for enzyme analysisand western blot evaluation.

Liquid cultures of E. coli strains DL39, DL39/pKS-, andDH5-a (which is AAT+) were grown in supplemented mini-mal medium containing 50 g/mL aspartate, whereas DL39/pAAT37 was grown in minimal medium lacking aspartate.Cultures of DL39/pKS- and DL39/pAAT37 also contained50 Ag/mL ampicillin with or without 1 mm IPTG. After 48 hof growth, cells were collected by centrifugation and eitherresuspended in SDS-sample buffer and disrupted by boilingfor 15 min or resuspended in enzyme extraction buffer (9)and disrupted by three 15-s cycles of sonication. Cell lysatesfrom both disruption schemes were clarified by centrifugation,and the supernatants were utilized for SDS-PAGE and west-ern blotting or enzyme activity, native-PAGE isozyme stain-ing, and immunotitration of activity.

870 GANTT ET AL.




Western blots were performed as described previously (3,4) and AAT was detected with affinity-purified AAT-2 mon-ospecific antibodies. Enzyme activity, native PAGE isozymestaining, and immunotitration of AAT activity were per-formed according to Griffith and Vance (9) and Farnhamet al. (4).

RESULTS

Characterization of an AAT-2 cDNA Clone

A Xgtl 1 cDNA library was constructed from poly(A)+ RNAextracted from 20-d-old root nodules. This library wasscreened with antibodies prepared against purified AAT-2protein. A recombinant bacteriophage containing a 1624 basepair insert was subcloned into a plasmid vector and se-quenced. The cDNA in the recombinant plasmid (pAAT37)lacks a 3' poly(A) tail, as do most of the clones in the library,and does not contain the entire AAT-2 protein coding region.The cDNA does, however, contain a single long open readingframe of 458 codons. The 5' end of this cDNA correspondsto position 80 (Fig. 1).To obtain the entire protein coding sequence of the AAT-

2 gene, we amplified the 5' region of the AAT-2 mRNA bythe RACE procedure (7). Three cDNA clones, each derivedfrom independent RACE reactions, were isolated and se-quenced. The sequence of the cDNA contained in pAAT37exactly matched the sequences of the 3' portion of the poly-merase chain reaction-derived clones from its 5' end to theend of the AAT2-5AMP primer (position 224). This dem-onstrates that the RACE procedure amplified the 5' end ofAAT-2 mRNA. The three RACE-derived cDNA clones areidentical in length and extend the pAAT37 cDNA sequenceby 79 nucleotides. The first ATG triplet is found at position59 and is followed by a 464 codon open reading frame thatencodes a 50,959 D protein. The nucleotides flanking thismethionine codon, AAATATGGC, fit the plant consensustranslation initiation sequence, AACAATGGC, well (10).These data suggest that we have determined the completeAAT-2 coding sequence.When the deduced amino acid sequence was used to search

a protein sequence data base, significant similarities werefound with animal and bacterial AATs. Alignment of thealfalfa AAT-2 amino acid sequence with mouse cytosolicAATshows similarity throughout the entire mouse protein (Fig. 2).Comparison ofthe alfalfa AAT-2 sequence with that ofmousemitochondrial AAT shows similarity throughout the entireprocessed form (N-terminus corresponding to position 61) ofthe mitochondrial protein (Fig. 2). A comparison ofthe aminoacid sequences of alfalfa AAT-2 with mouse cytosolic andmitochondrial AATs in the region corresponding to the ma-ture mouse mitochondrial protein shows that alfalfa AAT-2is slightly more similar to mouse mitochondrial AAT (53%identical) than it is to mouse cytosolic AAT (47%).The N-terminal end ofAAT-2 is blocked, precluding deter-

mination of its sequence (M.W. Farmham, C.P. Vance, un-published). However, as deduced from the cDNA sequence,the amino acid sequence of AAT-2 from position 1 to about60, where it can be aligned with animal cytosolic and mito-chondrial AATs, is very similar to a plastid or mitochondrial

targeting sequence (35). It contains just two acidic aminoacids, is positively charged, and contains a high proportion(23%) of serine or threonine.

Confirmation that pAAT37 does encode an AAT was dem-onstrated by using this plasmid to rescue an aspartate-requir-ing AAT- mutant of E. coli phenotypically (8). WhenpAAT37, but not the vector alone, was transformed into theDL39 strain ofE. coli carrying the aspC13 mutation, virtuallyall cells that gained the ampicillin-resistance marker of thevector were also able to grow on medium lacking aspartate.The pAAT37-transformed mutant showed AAT activity com-parable to AAT+ E. coli (Table I). In vitro staining for AATisozymes on native polyacrylamide gels showed that thepAAT37 complemented mutant had two or three activitybands that migrated slightly more slowly than alfalfa AAT-2and were strikingly different than E. coli AAT (data notshown). Moreover, western blots of proteins from DL39 cellsseparated by SDS-PAGE and probed with monospecific AAT-2 antibodies showed no cross-reactive AAT polypeptides,whereas comparable blots of the pAAT37-transformed lineshowed three bands near 40 kD in size that were highlyreactive with AAT-2 antibodies (Fig. 3). These data not onlyconfirm that our clone encodes AAT protein, but also showthat an enzymatically active protein can be synthesized. Res-cue of the aspC13 mutation by pAAT37 was somewhatsurprising, because the lacZ open reading frame, into whichthe cDNA was inserted, does not coincide with the AAT-2open reading frame. The fact that the pAAT37 plasmid cancomplement the aspC13 mutation and that multiple AATbands on both western blots and on native gels stained forenzyme activity suggest that pAAT37 has multiple transla-tional start sites that are functional in E. coli or that theprotein is proteolytically cleaved.AAT-2 mRNA was initially characterized by northern blot

analysis of poly(A)+ RNA extracted from uninfected roots,nodules, and leaves (Fig. 4). The size of the mRNA is between1700 and 1800 nucleotides and is independent of the tissuefrom which it was extracted. Comparing the relative amountof the mRNA in the different tissues by direct countingthrough image analysis suggests that nodules contain about15- to 20-fold more AAT-2 mRNA than do uninfected rootsand leaves.To estimate the number of alfalfa AAT-2 genes, we hybrid-

ized 32P-labeled AAT-2 cDNA to alfalfa DNA digested witha variety of restriction enzymes (Fig. 5). All restriction digestsshow between two and four hybridizing fragments, suggestingthat AAT-2 is encoded by a small multigene family or thatthe AAT-2 gene contains several introns. Alternatively, thehybridization pattern could reflect allelic variation for a singlegene in this tetraploid species.

Nitrogenase Activity, AAT Enzyme Activity, and AAT-2Protein

Although we have previously published a developmentalsequence study for alfalfa nodules, AAT activity, and AATenzyme protein (3), we thought it important to repeat thatexperiment sampling nodules at early time points more fre-quently for this current study. Nodule initiation and visibleemergence of nodules from roots occurs between days 7 and

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ATTTTTCGCA ACTCTTTCTG TTGAGTTGCT TTGGTTTTTT

GANTT ET AL. Plant Physiol. Vol. 98, 1992

CCTCTTCACA ACAAAAAT ATG GCA TCA TCT TCA TTA CTC 79M A S S S L L

TCC TCT GTA CCT TCA CAC TCT GCT TCA CTT TCG ATC CTC GAC ACC MC ATC MG GGA MG CTT MG CTTS S V P S

GGA ACC MC GGT TTGG T N G L

ATC TGC ATG GCT GTTI C M A V

CTT GGA GTT TCT GML G V S E

AGA ACG GAA GAA CTAR T EE L

GGG GM MC AAA GAGG E N K E

CTC GGA GCA GAC MTL G A D N

TCT CTG CGA CTA GGTS L R L G

ACG TGG GGT MT CACT W G N H

CCC MG ACA GTT GGCP K T V G

GTG CTA CTT CAT GGAV L L H G

GCT GAT GTA ATT CMA D V I Q

AGC CTT GAT GM GATS L D E D

TCA TAC AGT AAA MCS Y S K N

GM TCT GCA ACA AGGE S A T R

CAC GGG GCT AGG ATTH G A R I

GM ATG ATG GCT GGAE M M A G

AGT GGA MG GAT TGGS G K D W

CAG AGT GAC MT ATGQ S D N M

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AAA MG GCA GM M-K K A E N

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GCC ACT GTC CM GGA T V Q G

CCT GGA GCA AAA GT-P G A K V

GTA CCA TGG TCT GAIV P W S E

GAT ATA MG TCG GC-D I K S A

GAT CCA ACA CCA GA)D P T P E

GAT GTT GCT TAC CA(D V A Y Q

TCA CGT GGC ATG GA)S R G M E

GGA GCT ATT AAT GTIG A I N V

GCT CGA CCA ATG TAIA R P M Y

GCT CTC TTT GAT GA)A L F D E

CTG TAT GAT AGT AT'L Y D S I

ATG TTC TCA TTC ACUM

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ACA MG GAT GGA AGG ATT TCC CTG GCA GGAT K D G R I S L A G

TTG TCG CTG GCC AM TGT GM TAC CTT GCA GAT GCT ATT ATC GAT TCA TAT CAT AAT GTC AGC TGA AACL S L A K C E Y L A D A I I D S Y H N V S *

GCAGTGA MCATGCTTT TGMGCMGC ATATATTTTT GGTGAGTATT ATACCAAATC ATAGTTATTG ACACATTACA ATMTT

TTAT TATGTATGCA TTGTTTGTCA TACATATGTA CCCAAAGTCC CTTTGGAAAT CATGTTGTM CCTTGAATM GTTGMTCA

A AGTGTTGATG TMCAAACGA GTCTCTTTTG CAGACTTGM GCMGTTGM CCTAATTATT GATTTAGAGT ATGTTT

1321

1390

1459

1542

1625

1702




1 20 40 60Ms AAT-2 MASSSLLSSVPSHSASLSI LDTNI KGKLKLGTNGLRFNNEGINNFSNLRSSGR ICM4AVATNVSRFEGIPMAPPDP ILMmM AAT MA.LHSSRILSGMAAAFHP.LAAA.S.RAS.WWTHVE.GMmC AAT MAPP.V.AQV.Q ... VLVF

80 100 120 140Ms AAT-2 GVSEAFKADTSDVKLNLGVGAYRTEELQPYVLNWKKAE-NLMLERGENKEYLPIEGLAAFNKATAELLLGADNPAIKQQMmMAAT ..T....R..NSK.1.. DDNGK PS.R...-AQIAAKNLD G...E.C..S...A..EN.EVL.SG

MmCAAT KLTAD.RD.PDPR.V. D.S..W..P..R.V.QKIANDNSL.H L E.RSCASR.V..DNS...REN

160 180 200 220Ms AAT-2 RVATVQGLSGTGSLRLGAALIERYFP-GAK-- VL-I SNPTWGNHRNI FNDARVP-WSEYRYYDPKTVGLDFEGMI ED IMmMAAT .FV...TI A..V..SFLQ.F.KFSRD-----.FLPK.S....TP..R..GMQ-LOG C.F..S.ALMmCAAT ..GG..S.G A..I..DFLG.WYN-.TDNKNTPIYV.S...E..NAV.SA.GFKDIRP.C.W.AEKR LQ.FLN.L

240 260 280 300KSAPEGTFVLLHGCAHNPTGIDPTPEQWEKIADVIQQKNHFPFFDVAYQGFASGSLDEDAASVRLFVSRGMEVLVAQSYSSKI ..QSVL ..A. V.R....KE..S.VKK..L.A.M DG.K..WA..H.IEQ.IN.CLC ... A

EN FSIFV..A T. KQ..A.M.RRFL.. S D.EK..WAI.Y E.F.LFC...F.

320 340 360 380KNLGLYAERVGAI NVI SSSPESATRVKSQLKRLARPMYSNPPVHGARIVANIVGTPALFDEWKAEMEMMAGR IKTVRQAL.. M G FT.VCKDA.E.K..E....I.I..L..... LN....A.T.LTS.D.RKQ.LQ.VKG..D..ISN.TQ.

.N.... NLT.VGKESD.VL L.MEKIV.ITWU .... AQ. ATLSD.E. .K GNVKT .D. .L.M.SE.

Figure 2. Comparison of the deduced alfalfaAAT-2 amino acid sequence (Ms AAT-2) withthose of mouse mitochondrial (MmM AAT; 21)and mouse cytosolic (MmC AAT; 21) AATs.Identical residues are identified by dots(.... ). Dashed lines (- - -) indicate gaps intro-duced in the sequences to maximize identity.Residue numibering is from the N-terminal aminoacid of alfalfa AAT-2.

400 420 440 460Ms AAT-2 YDSI SSKDKSGKDUSF I LKQIGMFSFTGLNKSQSDNMTNKWH IYMTKDGRI SLAGLSLAKCEYLADAI IDSYHNVSMmMAAT VSNL-K.EG.SHN.QH.TD C....KPE.VERL.KEFSV ..........VTSGNVG...H..-----.Q.TK

MmCAAT RARLEALKTP.T-..H.TE PK.VEYLV.EK...LLPS...NMC..TTKNLD.V.TS.HEAVTKIQ

10, a period during which nitrogenase activity increases fromzero to its maximum specific activity. Because we obtainedsamples on days 7 and 10 in our previously published study,we did not know the sequence of events in the importantintervening days. Additionally, our western blots for AATprotein in the previous study were developed with serum thatreacted with a 38 kD protein that was not AAT, making theinterpretation of data open to question. Thus, we wanted torepeat the experiment with monospecific AAT-2 serum. Also,we wanted to extend our understanding of the relationshipsbetween nitrogenase and AAT-2 activity by evaluating thesynthesis of AAT-2 enzyme protein and mRNA during thisperiod.Even though small white nodule outgrowths began to

emerge from roots of both effective Saranac and ineffectivein,Sa by 7 and 8 d after planting and inoculation, nitrogenaseactivity, estimated by acetylene reduction, was not detecteduntil day 9 in Saranac nodules (Fig. 6), about 24 h earlierthan our previous report (3). Nitrogenase specific activity ofeffective Saranac nodules continued to increase through day12, remained constant to day 19, and then decreased slightlyby day 33. By comparison, nitrogenase specific activity ofin,Sa nodules was not detectable through day 12 and was only5% that ofeffective nodules on days 19 and 33. The differencein nitrogenase activity between Saranac and injSa reconfirmthe ineffective nature of in,Sa nodules (3, 24). This differencein phenotype was also reflected in dry matter accumulation.By day 33, shoot dry weights of Saranac and injSa averaged0.41 and 0.22 g, respectively.With the exception of not having evaluated early nodule

development and nitrogenase on day 8 and 9 in previous

studies, these current data are similar to those analyses ofalfalfa nodule development (3). The present study extends thecomparison of Saranac and in,Sa by showing that differencesin nitrogenase activity between the two are first detectable by9 d after inoculation and planting. Moreover, sequentialanalysis of nodules on days 7, 8, and 9 were required tofacilitate comparisons ofnodule development and nitrogenasewith AAT activity, enzyme protein, and mRNAs as describedbelow.

Although AAT activity in both genotypes increased duringthe early stages of nodule development (days 7 and 8), therewere differences between in,Sa and Saranac (Fig. 7C). By day12, total AAT activity of effective Saranac nodules was ap-proximately twofold higher than that of ineffective in,Sanodules. Total AAT activity of Saranac continued to increasefrom day 12 to day 33, whereas that of in,Sa declined gradu-ally throughout that same time period. On day 33, Saranactotal nodule AAT was fivefold greater than that of injSanodules.Western blots probed with affinity-purified, monospecific

AAT-2 antibodies confirmed that increased total AAT activityof effective nodules results, in great part, from an increase inAAT-2 enzyme protein (Fig. 7, A and B). During the develop-ment of effective Saranac nodules, little to no staining forAAT-2 enzyme protein can be detected in proteins extractedfrom day 5 roots, which have no visible nodules. Then, asnodules emerge from the roots, a slight increase in AAT-2enzyme protein is noticeable. A prominent increase in AAT-2 enzyme protein occurs by day 12, with a further increaseevident by day 19. With ineffective in,Sa, the intensity ofstaining for AAT-2 enzyme protein is very similar to that of

Ms AAT-2MmM AATMmC AAT

Ms AAT-2MmM AATMmC AAT

Figure 1. Nucleotide sequence of alfalfa AAT-2 cDNA and deduced amino acid sequence of AAT-2. The nucleotide sequence of the cDNA clonepAAT37 extends from position 80 to 1702. Nucleotides 1 to 224 were obtained from cDNA clones derived from three independent RACEreactions, each of which terminated at nucleotide 1.

873




Table I. AAT Activity and Immunotitration of AAT Activity from TotalProtein Extracts of E. coli Strains DH5a (an AAT+ Strain) and DL39(an AAT- Strain) and from DL39 Cells Containing PlasmidspBluescript KS-(DL39/KS-) or pAAT37 (DL39/pAAT37).

E. co/i Proteina Specific MT ActivityCell Line Activitya Immunotitratedb

mg mU-' nm-min`' mgprotein-'

DH5a 11.63 97 0DL39/pKS- 8.80 3 0DL39 10.10 4 0DL39/pAAT37 6.83 60 60DL39/pAAT37C 7.00 67 60

a Values are means of three separate experiments. b Valuesare representative of two separate experiments. MT activity wasimmunotitrated with monospecific AAT-2 antibodies from al-falfa. c Grown in medium containing 1 mm IPTG.

Saranac through day 9. However, on days 12, 19, and 33,AAT-2 enzyme protein was essentially not detectable when50 ,ug of protein was applied on the gel. Egli et al. (3)previously reported similar trends for AAT-2 enzyme proteinand AAT activity, but monospecific antibodies were not usedto stain their western blots, and their results were subject toother interpretations. The data presented here conclusivelyshow that increased total AAT enzyme activity is due primar-

ily to an increase in AAT-2 enzyme protein. Additionally, thelack of AAT enzyme activity in in1Sa nodules is reflected bya lack of AAT-2 enzyme protein.

kD

Figure 4. Northern blot analysis of MT-2 mRNA in poly(A)+ RNAisolated from nodules, roots, and leaves of effective Saranac alfalfa.A total of 0.5 ,g poly(A)+ RNA was applied in each lane. Size markersare indicated in kb. Radioactivity in each lane was quantified byAMBIS (San Diego, CA) radioanalytic image analysis. Data are rep-resentative of four separate blots.

Rh Alfalfa

0 a

60>

9.4

- 2.3-2:0

40>

- 0.5

1 2 3 4 5 6 7

Figure 3. AAT enzyme protein in cells of an MT- mutant of E. coli(DL39) transformed with pMT37. MT-2 polypeptide was detectedby western blots of total protein extracts that were separated bySDS-PAGE. Lane 1, DHa (MT+ strain); lane 2, DL39 (MT- strain);lane 3, DL39/pKS- (DL39 cells containing pBluescript KS-); lane 4,DL39/pMT37 (DL39 cells containing pMT37); lane 5, DL39/pMT37(DL39 cells containing pAAT37 and grown in minimal medium with 1mM IPTG). Lanes 1 to 5 were loaded with 100 jig protein. For sizeand specificity comparisons, pure MT-2 (0.3 gg) and pure MT-1(0.3 Ag) were loaded in lanes 6 and 7, respectively. Blots were probedwith monospecific MT-2 antibodies. All bacterial lanes contained 100Mg protein. The blot is representative of three separate experiments.Molecular mass is indicated in kD.

Figure 5. Southern blot analysis of alfalfa genomic DNA. Alfalfagenomic DNA was digested to completion with EcoRI, Pstl, EcoRV,and Hindlll, size fractionated on an 0.8% agarose gel, transferred toImmobilon (Millipore Corp.), and hybridized with 32P-labeled MT-37cDNA. DNA size markers are indicated in kb. The lane designatedRh is R. meliloti total DNA digested to completion with EcoRI. Theblot is representative of at least four separate blots.

0 -

an-

Z it -j

- 7.5- 4.4- 2.4

- 1.4

- 0.2

874 GANTT ET AL.




0

0

I-

E

0

0

2.

'

o 1 0 20 30 40DAYS AFTER PLANTING (INOCULATION)

Figure 6. Nitrogenase activity of effective Saranac and ineffectivein1Saranac plants throughout nodule development. On day 5, nonodules were present on roots. Small nodules were emerging fromroots on days 7 and 8. By day 9, nodules had emerged from roots.Nitrogenase activity was assayed by the acetylene reduction as-

say. Each data point represents the average of three separatedeterminations.

AAT-2 mRNA Levels

We next examined the amount of AAT-2 mRNA in effec-tive and ineffective nodules. Due to the small amount oftissue from which RNA was extracted, northern blots of totalRNA were used to estimate AAT-2 mRNA levels. RNAsamples from tissue collected after day 9 have significantamounts of rhizobial RNA in addition to plant RNA (Fig. 8).Thus, estimates of the -fold increase in AAT-2 mRNA duringnodule development probably underestimate the actual in-crease. AAT-2 mRNA in effective nodules appears by auto-radiography to increase from a relatively low, basal level thatis maintained through day 7 to a relatively high level on day8. The amount of AAT-2 mRNA continues to increase fromday 8 through day 33. Direct assessment of radioactivityhybridized to effective Saranac AAT-2 mRNAs shows a two-fold increase between days 7 and 8, with a further twofoldincrease by day 9, followed by a further twofold increase today 19, leading to a total increase of about sevenfold betweenday 7 and 19. Similar to AAT activity and AAT-2 enzymeprotein, AAT-2 mRNA increased as nodules formed andfurther increased as nitrogenase activity increased.The overall developmental pattern of AAT-2 mRNAs in

inSa was similar to that of effective Saranac with threeexceptions: (a) the total amount of radioactive pAAT37cDNA probe hybridized to RNA extracted from inSa was

generally about one-third to one-fourth that bound by Sar-anac; (b) the increase in AAT-2 mRNA that occurred afternitrogenase activity is detected and coincident with maximumAAT enzyme activity in effective Saranac did not occur ininSa nodules; and (c) on day 33, the amount of radioactivitybound by injSa decreased about 40% as compared with day19, which did not occur in the effective nodules.

DISCUSSION

We report here the isolation, cloning, and characterizationof plant AAT-2 cDNA sequences. Because pAAT37 did not

contain the complete AAT-2 protein coding region, we usedthe RACE protocol (7) to obtain a nearly complete AAT-2cDNA sequence. The RACE procedure extended the 5' endof the cDNA sequence found in pAAT37 by 79 nucleotides(positions 1-79, Fig. 1) and completed the AAT-2 codingregion. Direct confirmation that pAAT37 does, in fact, encodea plant AAT was conclusively demonstrated by its comple-mentation of an E. coli AAT- mutant (Table I, Fig. 3) withsubsequent expression of AAT activity. Moreover, the ap-

proach of selecting the AAT clone with monospecific anti-bodies to the nodule-enhanced form ofAAT (AAT-2) coupledto western and northern blot analyses indicates that the AATcDNA isolated corresponds to the nodule-enhanced form ofthe gene. Because we can study the effects of alterations inalfalfa AAT-2's primary structure in E. coli and we can alsotransform and regenerate alfalfa, we believe that we can now

directly modify this key plant enzyme.

Animals contain two forms of AAT, one localized in thecytosol and the other in the mitochondrion. The molecular

co10 I0

aI*-

E

12

10

8

6

4

2

0

0 1 0 20 30 40DAYS AFTER PLANTING (INOCULATION)

Figure 7. AAT activity and AAT-2 enzyme protein over the de-velopment of effective Saranac and ineffective inSaranac nodules.A, AAT-2 enzyme protein in 50 zg soluble protein from effectiveSaranac. B, AAT-2 enzyme protein in 50 jsg soluble protein fromineffective inSaranac. C, Total AAT enzyme activity for effectiveSaranac and ineffective in1Saranac. On day 5, no nodules were

present on roots. Small root sections having emerging noduleswere harvested on days 7 and 8. Nodules, free of root tissue,were harvested on the remaining days. Data reflect three separateexperiments.

Saranac A

La1Saranac B

5 7 8 9 12 19 33

TOTAL AAT ACTIVITY C

- - SARANAC) - inl SARANAC

I. I ----..I

875




-4.4

-1.4

B. Ineffective in, Saranac

-4.4

U,.,r

-2.4

-1.4

5 7 8 9 12 19 33

Figure 8. Northern blots of AAT mRNA expression throughout nod-ule development of effective Saranac and ineffective iniSaranacalfalfa. Total RNA was isolated at various days after planting. Rhizo-bium inoculum was applied at planting (day 0). On day 5, no noduleswere present on roots. Small root sections having emerging noduleswere harvested on days 7 and 8. Nodules, free of root tissue, wereharvested on the remaining days. Each lane contains 10 j1g totalRNA. Size markers are indicated in kb. Data are representative offour separate blots.

mass of alfalfa AAT-2 deduced from the cDNA sequence is51 kD, whereas the mass of purified AAT-2 is substantiallysmaller, 40 ± 2 kD. Also, the calculated pI of the 51 kDprotein is 8.5, whereas the measured pI ofthe purified proteinis about 6.5. However, the portion of the alfalfa protein thatis similar to the mature mitochondrial and cytosolic AATs ofmouse (amino acids 60-465) has a molecular mass of about44 kD and a pl of 6.45, very similar to the values measuredfor purified alfalfa AAT-2. Further indirect support that AAT-2 is localized to an organelle is demonstrated by cell fraction-ation studies showing that AAT-2 antibodies are highly cross-reactive with a 40 kD polypeptide found in alfalfa and beannodule mitochondria and amyloplasts (D. Robinson, C.P.Vance, unpublished data). Therefore, we hypothesize thatAAT-2 is localized to the plastid. Currently, we are attemptingto determine the localization of AAT-2 by importing the invitro synthesized protein into isolated organelles. WhetherAAT-2 is localized to the plastid or to the mitochondrion isnot known. However, von Heijne et al. (36) have comparedthe targeting peptides of mitochondrial and chloroplast pro-teins and found that they can be distinguished, with about90% accuracy, by comparing the relative numbers of serines,which typically compose 19% ofthe plastid targeting peptide,and arginines, which are more abundant in mitochondrialtargeting sequences than they are in plastid targeting peptides.When their criteria are applied to the first 59 amino acids ofthe AAT-2 sequence, it is clear that this peptide more closelyresembles a plastid transit peptide than it does a mitochondrialtargeting peptide. This would be consistent with preliminaryin situ immunolocalization data that suggest AAT-2 is foundin nodule amyloplasts and not in mitochondria (D. Robinson,M. Kahn, C.P. Vance, unpublished data).

Because AAT-1 and AAT-2 are both present in alfalfanodules (9), it is important to understand their relative con-tributions to total AAT activity. Because antibodies to AAT-1 and AAT-2 are immunologically distinct (4, 9), specificantibodies can be used to ascertain the contribution of eachform of AAT to total activity. Using immunological tech-niques in conjunction with isozyme staining on native PAGE,Griffith and Vance (9) and Farnham et al. (4) have shownthat the prominent increase in total AAT activity duringeffective nodule development is attributable to increasedAAT-2 enzyme activity. Not only does activity staining forAAT-2 enzyme become more prominent, but the amount ofAAT-2 protein increases in approximately the same propor-tion as does total AAT activity. In comparison, ineffectiveinSa nodules have much less prominent staining for AAT-2enzyme activity and the amount of AAT-2 enzyme protein ismuch lower, both characteristics more resembling roots thannodules. Western blots of AAT- 1 show only a slight increaseduring development of effective nodules (data not shown).

In comparing AAT activity in inSa and Saranac nodules,it is interesting to note that the increase in total enzymeactivity appears to occur in two phases. The first phase occurson days 7 and 8, coincident with nodule emergence from theroot. The second phase, commencing after day 9, occurs afternitrogenase activity is readily detectable (Fig. 6). Only the firstphase increase appears to occur in ineffective inSa nodules,and this increase is temporarily delayed about 24 h (Fig. 7, Aand B). Thus, inSa nodules apparently lack the factor(s)

876 GANTT ET AL.




required for maximum AAT expression. We have seen similarpatterns of development for several other nodule enzymes,including glutamine synthetase and phosphoenolpyruvatecarboxylase (3).The striking increase in AAT-2 mRNA abundance during

nodule development (Fig. 8) suggests that an increase in therate of AAT-2 synthesis accounts for the increase in AAT-2protein and AAT enzyme activity (Fig. 7). The increase inaccumulation of AAT-2 mRNA in effective Saranac nodulesmirrors the apparent biphasic increase in AAT-2 enzymeactivity and enzyme protein. We propose that the increase inAAT-2 mRNA occurs in two independent stages. The initialincrease in expression of AAT-2 is due to an event (signal)involved with organ (nodule) development, because the in-creased abundance of AAT-2 mRNA is coincident with nod-ule initiation and emergence from the root in both Saranacand in,Sa nodules. In the next stage, an additional increase inthe amount of AAT-2 mRNA apparently requires a signalthat is associated with effective nodules for maximal expres-sion ofAAT-2. This second signal, absent from in,Sa nodules,is probably unrelated to bacterial release from infectionthreads into host plant cytosol ofinfected cells, because releaseoccurs in both Saranac and in1Sa (3).

Regulation of AAT activity in ineffective in,Sa nodulesmust also involve posttranslational events, because AAT-2mRNAs are relatively abundant 12 d after inoculation,whereas total AAT activity and AAT-2 enzyme protein re-main low. These observations suggest that AAT-2 mRNAseither are not translated efficiently or, alternatively, AAT-2enzyme protein is rapidly degraded. Early senescence thatoccurs in ineffective injSa nodules and is apparent with lightmicroscopy by day 13 (3) may play a role in such events. Itis currently unknown how senescence affects either the trans-lation of AAT mRNAs or degradation of AAT-2 enzymeprotein.

Although events that occur very early in nodule differentia-tion seem to be controlled by rhizobial signals that functionat very low concentrations (16), additional signals are requiredto yield functionally effective nodules (18). These additionalsignals may be the result of metabolic alterations resultingfrom bacteroid metabolism and/or N2 fixation. For example,because effective bacteroids release free NH4' into the plantcytosol for assimilation (23), and because the interior ofeffective root nodules has a very low 02 concentration (33),both NH4' and 02 have been implicated as signals involvedin the regulation of plant genes during the latter stages ofsymbiosis. However, it has been difficult to demonstrateunequivocally that NH4' and/or anaerobiosis are primarysignals for the regulation of expression of the late nodulegenes. Experiments with alfalfa (2, 19), common bean (1),and pea (37) have shown that NH4+ production by nodules isnot required for maximum expression ofglutamine synthetaseand leghemoglobin. By contrast, glutamine synthetase andleghemoglobin expression in soybean nodules was directlyrelated to the availability of NH4' (17). Likewise, low 02concentrations in nodules have been implicated in regulationof leghemoglobin (35) and sucrose synthase (32). However,direct evidence linking low 02 concentration and the expres-sion of leghemoglobin and sucrose synthase genes is lacking.Irrespective of the nature of signals involved in regulating

nodule gene expression, some appear to be conserved betweenspecies. Regulation ofthe soybean Lbc3 and N-23 gene expres-sion (29), as well as glutamine synthetase from common bean(6) and leghemoglobin glb3 from Sesbania (30), have beenevaluated in transgenic Lotus corniculatus carrying chimericgene fusions containing the 5'-upstream region of these genesfused to reporter genes. In all instances, organ-specific expres-sion was displayed in a heterologous host, indicating thatdiverse legumes contain functionally similar nodule transcrip-tional activation factors and cis-acting elements. With theisolation of the alfalfa nodule enhanced AAT, we have initi-ated experiments aimed at understanding the expression ofgenes encoding enzymes important in the assimilation ofcarbon and nitrogen with the objective of defining regulatorymechanisms common to these genes. Udvardi and Kahn (34)have recently isolated an AAT- 1 cDNA clone from alfalfaand found that its amino acid sequence is about 50% identicalto that of AAT-2. Thus, the genetic character of both AATscan soon be fully compared.

ACKNOWLEDGMENTS

The authors acknowledge the technical support of L.J. Howardand the numerous individuals involved in collecting alfalfa rootnodules from the laboratories of J.S. Gantt and C.P. Vance.

LITERATURE CITED

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7. Frohman MA (1990) RACE: Rapid amplification ofcDNA ends.In MA Innis, DH Gelfand, JJ Sninsky, TJ White, eds, PCRProtocols: A Guide to Methods and Applications. AcademicPress, San Diego, pp 28-38

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13. Ireland RJ, Joy KW (1985) Plant transaminases. In P Christen,DE Metzler, eds, Transaminases. John Wiley, New York, pp376-383

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29. Stougaard J, Joergensen JE, Christensen T, Kuhle A, MarckerKA (1990) Interdependence and specificity of cis-acting regu-latory elements in soybean leghemoglobin lbc3 and N23 genepromoters. Mol Gen Genet 220: 353-360

30. Szabados L, Ratet P, Grunenberg B, de Bruijn F (1990) Func-tional analysis of Sesbania rostrata leghemaglobin glb3 gene5'-upstream in transgenic Lotus corniculatus and Nicotianatabacum plants. Plant Cell 2: 973-986

31. Taniguchi M, Sugiyama T (1990) Aspartate aminotransferasefrom Eleusine coracana, a C4 plant: purification, characteriza-tion and preparation of antibody. Arch Biochem Biophys 282:427-432

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aspartateaminotransferase effective and ineffective ...aat2 (ec 2.6.1.1) catalyzes the reversible...

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