Plant Physiol. (1994) 104: 453-459

Cloning and Sequence Analysis of a cDNA Encoding Ferric Leghemoglobin Reductase from Soybean Nodules’

Lin li, Manuel Becana, Cautam Sarath, and Robert V. KIucas*

Department of Biochemistry and Center for Biotechnology, University of Nebraska, Lincoln, Nebraska 68583 (L.J., G.S., R.V.K.); and EstaciÓn Experimental de Aula Dei, Apdo 202, 50080 Zaragoza, Spain (M.B.)

~

A cDNA encoding soybean (Glycine max [L.] Merr) ferric leg- hemoglobin reductase (FLbR), an enzyme that i s postulated to play an important role in maintaining leghemoglobin in its functional ferrous state, has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full-length clone of FLbR cDNA was isolated by screening a Xgtl l soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragmentas a probe. The cDNA contained about 1.8 kb and had a coding sequence for 523 amino acids with a predicted molecular mas of 55,729 D, which included a putative 30-residue signal peptide and a 493-residue mature protein. Computer-aided analy- sis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20-50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reduc- tase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypano- thione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleo- tide-disulfide oxidoreductases from various organisms. Northern blot analysis using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater leve1 of expression in nodules and leaves than in roots and stems. Southern blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome.

Lb binds O2 reversibly and acts as an O2 camer in legume root nodules. Lb was originally found to be restricted to the N2-fixing tissue of legumes, but accumulating evidence now suggests that similar hemoglobins or hemoglobin genes might exist universally in plants, implying a more general physio- logical role of hemoglobins in plants (Appleby et al., 1988). One role of Lb in N2 fixation is to facilitate the diffusion of O2 through the cytoplasm of bacteria-infected nodule cells, providing O2 to the terminal oxidases in bacteroids and maintaining a concentration that both meets the large energy demand of N2 reduction and avoids O2 damage to nitrogenase (Witty et al., 1986).

To interact with 02, Lb must be maintained in the Lb” state and not bound in highly stable complexes, such as

The work was supported by the National Research Initiative Competitive Grants I‘rogram/U.S. Department of Agriculture (grant 93-0318) to R.V.K., and by the Direccion General de Investigación, Científica y Técnica CPB92-0058 to M.B. l‘ublished as Joumal Series No. 10405, Agricultura1 Research Division, University of Nebraska.

* Corresponding author; fax 1-402-472-7842. 453

Lb+’NO. From the chemical nature of Lb, Lbf3 is expected to be formed in situ by oxidation of Lb+’ and Lb+’02. Lb+3 can be generated by a typical electron oxidation of Lb+‘ with a midpoint potential of approximately 0.22 V at pH 7 (Hen- derson and Appleby, 1972). An abundance of certain metab- olites in the nodules including activated O2 species, phenolic compounds, and nitrite may mediate this oxidation (Becana and Klucas, 1992), and severa1 indirect lines of evidence also support the presence of Lb+3 in nodules (Klucas et al., 1985). However, Lb+’ and Lbf202 are the dominant forms of Lb in nodules from various species (Klucas et al., 1985), suggesting that mechanisms must exist in vivo for maintaining Lb in this functional state.

Nonenzymatic and enzymatic systems may operate in nod- ules to reduce Lb+3 (Becana and Klucas, 1990, 1992). In this laboratory, an enzyme with FLbR activity has previously been purified to homogeneity and partially characterized (Saari and Klucas, 1984; Becana and Klucas, 1990; Ji et al., 1991, 1992). To gain more information about the structure, function, and biogenesis of FLbR, we have cloned, sequenced, and characterized a cDNA encoding the entire FLbR protein and made comparisons with related enzymes available in protein data bases. We have also analyzed the occurrence of the FLbR gene in the soybean (Glycine max [L.] Merr) genome and its expression in soybean tissues.

MATERIALS AND METHODS

Plant Materials

Soybean (Glycine max [L.] Merr. cv Hobbit) plants inocu- lated with Bradyrhizobium japonicum strain USDA 110 were grown as described earlier (Ji et al., 1991). Nodules, roots, leaves, and stems were harvested after 30 to 35 d of plant growth, frozen immediately in liquid N2, and stored at -8OOC for subsequent isolations of RNA and DNA.

Construction of X g t l l cDNA Library

Total RNA was prepared from soybean root nodules using the guanidinium thiocyanate/CsCl method (Ausubel et al.,

Abbreviations: DIG, digoxigenin; DLDH, dihydrolipoamide de- hydrogenase; FAD, flavin adenine dinucleotide; FLbR, femc leghe- moglobin reductase; GSHR, glutathione reductase; Lb, leghemoglo- bin; Lb+2, ferrous Lb, the reactive species that combines reversibly with oxygen to give Lb+202 (oxyferrous Lb); Lb+3, femc Lb; MERA, mercuric reductase; TYTR, trypanothione reductase.

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454 Ji et al. Plant Physiol. Vol. 104, 1994

1987). Poly(A+) mRNA was isolated from the total RNA preparation using an oligo(dT)-cellulose column (Pharmacia). The double-stranded, blunt-ended cDNA was synthesized from the nodule poly(A+) mRNA (5 pg) using a cDNA synthesis kit (Pharmacia). An EcoRI/NotI adapter (Pharmacia) was ligated to each end of the blunt-ended cDNA. The EcoRI- terminated cDNAs were phosphorylated and ligated to the dephosphorylated Xgtll vector (BRL) at the EcoRI restriction site. The ligated products were packaged into infectious phage particles in vitro using a Packagene Lambda DNA packaging system (Promega) and transformed into a host Escherichin coli strain, Y1088 (BRL). The original cDNA li- brary was amplified and stored at 4OC in 50 m~ Tris-HC1 (pH 7.5), 100 m NaC1, 20 m MgC12, 0.1% gelatin with 2% chloroform or at -8OOC in 7% DMSO. The resulting cDNA library consisted of approximately 3.5 x 10" bacteriophage particles with 90% recombinants.

Amplification of FLbR cDNA Fragment by PCR

One group of degenerate oligonucleotides was designed as primers for the PCR and as an intemal probe for the PCR products according to the N-terminal amino acid sequence of FLbR (Ji et al., 1991) (Fig. 1). The degenerate nucleotides

' were based on the most frequent codon usages among the pyridine nucleotide-disulfide oxidoreductases (Carothers et al., 1989; Williams, 1991). The set of primers spanned the entire coding region of the N-terminal 50 amino acids of soybean FLbR. The sense primers 1 and 2 and antisense primer 3 corresponded to the amino acid residues of the N-terminal sequence 1 to 6,5 to 12, and 43 to 50, respectively. Oligonucleotide 4 was the intemal probe and corresponded to amino acid residues 18 to 27.

The soybean FLbR cDNA was amplified using the GeneAmp RNA/DNA PCR kit and the AmpliWax PCR Gem- mediated Hot Start technique (Perkin-Elmer Cetus, Norwalk, CT). PCR was performed using a Perkin-Elmer DNA thermal cycler 480, using the manufacturer-supplied protocols. PCR products were treated with proteinase K (BRL) to remove any possible attached DNA polymerase (Crowe et al., 1991). The 3' overhang at the end of the PCR products was removed by polishing with a T4 DNA polymerase. The blunt-ended PCR products were size fractionated by agarose gel electrophore- sis. The PCR fragments with the predicted size were purified by a QIAEX gel extraction kit (QIAGEN) and cloned into M13 vectors (BRL) at the PstI sites. Independent clones containing inserts of proper size were isolated. The PCR clones with predicted nucleotide sequences were identified by in situ hybridization (Sambrook et al., 1989) of trans- formed E. coli JMlOl (BRL) colonies as well as by Southem blot analysis using the intemal probe. The DNA sequences of the inserts were determined using the Center for Biotech- nology DNA-sequencing facility (University of Nebraska).

cDNA Cloning and Sequencing

Soybean FLbR cDNA clones were isolated by screening the Xgtl 1 cDNA library constructed from soybean nodule RNA. Approximately 8 X 105 recombinant phages were screened using standard methods (Ausubel et al., 1987; Sambrook et

al., 1989). The sequence-confirmed, PCR-amplified FLbR DNA fragment (150 bp) was used as a probe for filter hybridization. The DNA probe was labeled with DIG-11- dUTP by random priming and hybrids were detected accord- ing to the manufacturer-supplied protocols (Genius nucleic acid labeling system, Boehringer-Mannheim). Positive clones were identified and isolated by rescreening and plaque puri- fication (Ausubel et al., 1987). The FLbR cDNA inserts from positive clones were subcloned into M13 vectors (BRL) at EcoRI sites. The insert DNAs of the clones were partially sequenced. Two clones, FLbRlmll and FLRlm41, were com- pletely sequenced on both strands by the standard dideoxy- nucleotide chain termination method with Sequenase (ver- sion 2.0, United States Biochemical), using [35S]dATP (Amer- sham). DNA sequences and deduced peptide sequences were analyzed using the Genetics Computer Group software pack- age with either the EMBL, SwissProt, or GenBank data base.

Southern Blot Analysis

Genomic DNA was isolated from both soybean leaves and root nodules using an improved procedure based on methods described by Sambrook et al. (1989) andAusube1 et al. (1987). Five grams of frozen plant tissue were ground to a fine powder in liquid N2 with a mortar and pestle. The frozen tissue powders were added to 25 mL of extraction solution containing 100 m Tris-HC1 (pH €9, 100 m~ EDTA, 250 m~ NaCl, 100 pg mL-' of proteinase K (United States Biochemi- cal), and 20 pg mL-' of RNase (United States Biochemical) and incubated at 55OC for 1 h with gentle shaking. The lysate was centrifuged at 8000g at 4OC for 10 min to pellet debris, and the supematant was slowly added to 25 mL of isopro- pano1 at room temperature. The DNA was recovered by spooling onto a sterile, twisted glass rod. The adhered DNA on the rod was washed twice in 80% ethanol and then resuspended in 1 mL of 10 m Tris-HC1 (pH 8.0), 1 m~ EDTA. This crude DNA solution was extracted with phenoll chloroform and precipitated with 0.1 volume of 3 M sodium acetate (pH 4.5) and 2.5 volumes of ethanol. The purified DNAs were digested with EcoRI, HindIII, and PstI, and a parallel set of samples was separated by electrophoresis on a 0.7% agarose gel (Sambrook et al., 1989). After a sample was transferred to a nylon membrane, the membrane was cut into two, each piece containing one set of samples. One filter was hybridized at 65OC with the PCR DNA fragment (150 bp) probe and the other with the FLbR-coding DNA (1800 bp) probe. Both probes were labeled with DIG-11-dUTP by ran- dom priming, and hybrids were detected as described earlier. The Southem blot analysis of the cloned FLbR cDNA was camed out using EcoRI-digested A-DNA that was obtained from the positive Xgtll clones by zinc chloride precipitation (Santos et al., 1991) and the DIG-11-dUTP-labeled PCR fragment (150 bp) as a probe.

Northern Blot Analysis

Total RNA was isolated from soybean roots, stems, leaves, and nodules using guanidinium isothiocyanate/phenol ex- traction and LiCl/isopropanol precipitation (Ausubel et al., 1987; Chomczynski and Sacchi, 1987; Puissant and Houde-

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Cloning of Soybean Ferric Leghemoglobin Reductase 455

N-terminal Amino Acid Sequence

1 25 50ASGSDENDVWIGGGPGGYVAAIKAAOLGLKTTCIEKRGTLGGTCINVGC

1 2 4 3

Sense Primers

1. 5' GC(AG) TC(AG) GG(AG) TC(AG) GA(CT) GA(AG) AA 3'

2. 5' GA(CT) GA(AG) AA(CT) GA(CT) GT(AGCT) GT(AGCT)

GT(AGCT) AT 3'

Antisense Primer

3. 5' CA (AG)CC (AG)AC (AG)TT (CT)A(AG) (AG)CA

(AGCT)GT (AGCT)CC 3'

Internal Probe

4. 5' (CT)TG (AG)GC (AG)GC (CT)TT AAT AGC AGC AAC

(AG)TA TCC 3'

Figure 1. Sequence of the FLbR N terminus and correspondingdegenerate oligonucleotides used as primers for the PCR and as aninternal probe for identifying the PCR products. The sequencestranslated into oligonucleotides are underlined and numbered cor-respondingly. The nucleotides in parentheses represent the degen-erate nucleotides.

bine, 1990). RNA samples (about 20 jig) were separated onan agarose-formaldehyde gel, transferred to a nylon mem-brane (Boehringer-Mannheim), and hybridized with 20 to 30ng mlT1 of the DIG-11-dUTP-labeled FLbR-coding DNA(1800 bp) (Sambrook et al., 1989). The filter was washed andhybrids were detected as described for the DNA Southernblots.

RESULTS

Amplification of cDNA by PCR

The PCR technique was used to generate the cDNA probefor facilitating cloning and analysis of the soybean FLbR geneusing a group of highly degenerate oligonucleotide primers,based on the FLbR N terminus and the most frequent codonusages of the DLDHs in the available data base (Fig. 1). Theseprimers were used to amplify single-copy sequences from thecomplex soybean nodule RNA or cDNA mixtures by PCR togenerate the cDNA probe for hybridization studies and forscreening the soybean nodule cDNA library for a full-lengthFLbR clone. After 30 to 40 cycles of amplification, the dom-inant PCR products of the expected size of 150 and 140 bpwere amplified from soybean nodule cDNA mixtures usingprimer 1/primer 3 and primer 2/primer 3, respectively.

Six clones containing the 150-bp insert and four clonescontaining the 140-bp insert were identified by in situ hy-bridization of transformed bacterial colonies with the internalprobe. The sequences of the inserted DNA, four from theformer and two from the latter, were determined by anautomated DNA sequencer. One of the 150-bp inserts re-vealed a nucleotide sequence the deduced amino acid se-quence of which matched with the experimentally deter-mined N-terminal amino acid sequence of soybean FLbR.This cDNA fragment was used for the subsequent cDNA

library screening and Southern blot analysis of the genomicDNA.

Isolation of FLbR cDNA Clones

For isolating a full-length FLbR cDNA, a soybean nodulecDNA Xgtll library was screened with the sequence-con-firmed PCR fragment (150 bp) of FLbR. Five positive cloneswere obtained by screening about 8 X 105 primary recombi-nant phage plaques. The cDNA insert sizes of these fiveclones were approximately 1.8 kb as indicated by electropho-retic analysis of the EcoRI digests on an agarose gel (Fig. 2A).The cDNA inserts were hybridized to the PCR fragment ofFLbR as shown by Southern blot analysis (Fig. 2B) and alsocross-hybridized with each other. Partial sequences of fivecDNA clones were determined and found to be identicalexcept for the differences of starting positions at the 5' ends;therefore, all of the cDNAs isolated belonged to a single class.

Two clones, FLRlmll and FLRlm41, were completely se-quenced on both strands. Comparison of the two sequencesshowed that the two cDNA clones were identical except theDNA sequence of FLRlmll was eight nucleotides shorter atits 5' end. The complete sequence of FLbR cDNA consists of1740 bp (Fig. 3). The primary amino acid sequence of FLbRwas deduced from the cDNA sequence, and the predictedN-terminal sequence is identical with the experimentallydetermined 50-residue N-terminal sequence (underlined inFig. 3) of purified protein. An open reading frame of 1572bp is located in the sequence between nucleotide positions22 and 1593 (Fig. 3), which corresponds to a protein of 523amino acids with a calculated molecular mass of 55,729 D.This predicted molecular mass is close to 54,000 D measuredfor purified FLbR (Ji et al., 1991). The N terminus alsocontains a putative 30-residue signal sequence that is rich in

bp bp

-1800

B

Figure 2. Analysis of FLbR cDNA clones by restriction digestionand Southern blotting. A, Agarose gel electrophoresis (1.2% gel) ofthe EcoRI-digested Agtl 1 DNAs from the positive clones of FLbRcDNA. Lane 1, One-kilobase DNA markers (BRL); lanes 2 to 6,agarose gel-purified EcoRI digests of five positive clones. B, South-ern blots of the cloned FLbR cDNAs using the 150-bp PCR-ampli-fied FLbR cDNA fragment as a probe. Lanes 2 to 6 correspond tothe FLbR fragments in A.

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456 J i et al. Plant Physiol. Vol. 104, 1994

CGGCCGCAAAAAAGAAAGATCATGGCGATGGCGATGGCAAACTTGGCTCGACGGAAGGGTTACGCC M A M A N L A R R K G Y A

GTCGTTTTGTCGTCGAGGTCGTCGCTTTGTCTGACAAGGTGGAGGGGGTTCGCGTCCGGA V V L S S R S S L C L T R W R G F A S G TCTGACGAAAACGACGTCGTGGTCATCGGCGGCGGTCCCGGCGGCTACGTGGCGGCCATT S D E N D V V V I G G G P G G Y V A A I

AAAGCGGCGCAGCTGGGTCTGAAAACCACTTGCACTTGCATCG~CGTGGCACTCTCGGCGGT K A A O L G L K T T C I E K R G T L G G

ACCTGCCTCAACGTCGGATGCATCCCTTCCAAGGCACTTTTGCATTCTTCCCACATGTAT T C * L N V G C + I P S K A L L H S S H M Y CATGAGGCTAAACATGCATTTGCCAACCATGGGGTCAAGTTTTCATCTGTCGAGGTTGCT H E A K H A F A N H G V K F S S V E V A TTGCCAGCCATGATGGGCCAGATAAAGCAGTTTTCTAATCTTACCCAAGGTATTGAT L P A M M G Q K D K A V S N L T Q G I D

GGTCTATTTCAGAAGAATAAGGTAACCTATGTCAAAGGATATGGCAAATTAGTTTCGCCG G L F Q K N K V T Y V K G Y G K L V S P TCTGAAATCTCTGTAGACACCACTGAAGGTG-TACTGTGGTTAAGGGCAAGCATATT S E I S V D T T E G E N T V V K G K H I

ATTATTGCCACTGGTTCGGATGTGAAATCTCTGCCCGGTGTCACTATTGATG-GAAA I I A T G S D V K S L P G V T I D E K K

ATTGTTTCATCAACTGGGGCTCTTGCTTTGTCTGAAATCCCCAAGAAGCTCGTAGTCATT I V S S T G A L A L S E I P K K L V V I

GGGGCAGGCTACATTGGGCTGGAAATGGGCTCAGTATGGGGCCGAATTGGCTCCGAGGTA G A G Y I G L E M G S V W G R I G S E V ACAGTTGTTGAGTTTGCATCGGAGATTGTTCCAACCATGGATGCAGACATCCGGAAGCAG T V V E F A S E I V P T M D A D I R K Q

TTTCAGCGTTCTCTTGAGAAGCAAGGCATGAAATTCAAGCTGAAGACCAAGGTGGTTGGA F Q R S L E K Q G M K F K L K T K V V G

GTTGATACTTCTGGGGATGGTGTGAAGCTAACTGTTGAACCATCCGCTGGCGGTGAACAG V D T S G D G V K L T V E P S A G G E Q

ACCATAATTGAAGCAGATGTTGTCCTTGTTTCGGCTGGTAGAACTCCATTCACTTCTGGA T I I E A D V V L V S A G R T P F T S G CTTAATTTGGATAAGATAGGAGTTGAGACTGACAAGTTAGGACGGATTTTGGTAAATGAA L N L D K I G V E T D K L G R I L V N E

AGATTTTCAACAAATGTCTCTGGTGTCTATGCAATCGGAGATGTGATTCCAGGTCCAATG R F S T N V S G V Y A I G D V I P G P M TTGGCACACAAGGCAGAAGAAGATGGTGTTGCTTGTGTTGAGTACTT~CTGGTAAGGTT L A H K A E E D G V A C V E Y L T G K V

GGCCATGTGGATTATGACAAAGTCCCTGGCGTTGTCTATACAAACCCTGAAGTTGCATCT G H V D Y D K V P G V V Y T N P E V A S

G T T G G G A A G A C A G A G G A G C A G G T G A A G G A G T T G G T T C V G K T E E Q V K E T G V E Y R V G K F CCATTCCTGGCTAATAGCAGAGCTAAGGCAATTGACAATGCTGAAGGACTGGTGAAGATA P F L A N S R A K A I D N A E G L V K I

ATTGCTGAAAAGGAGACAGACAAGATATTGGGAGTGCACATTATGGCGCCTAATGCAGGA I A E K E T D K I L G V H I M A P N A G

GAACTCATTCATGAAGCTGCCATAGCACTACAGTATGATGCATCAAGTGAGGACATTGCA E L I H E A A I A L Q Y D A S S E D I A CGTGTGTGCCATGCACATCCAACAATGAGCGAGGCTGTG~GAAGCCGCAATGGCCACT R V C H A H P T M S E A V K E A A M A T TATGACAAGCCGCATTCACATCTAAAGAGCTGGTTGCTACTTTCTTCTCTTGTTTTCATT Y D K P H S H L K S W L L L S S L V F I

TTTGTCCAAGAATTTACAATGACTTGGAGATGATACCACCACCTCTGTTAAATTTAGATTCAA F V Q E F T M T W R E N D

ATTTTTAAATGCAAATTAGCCCCTTGTTTACTTGTTTGCATAATAAGAGGAATGGTTGTG TTGTTCTTATCCTTGTTGAATAAATTATATATATCTCCCCTTCTCTCTGGGACAATATTGTA

60 13 120 33 180 53 240 73 300

93 360 113 420 133 480 153 540 173 600 193 660 213 720 233 780 253 840 273 900 293 960 313 1020 333 1080 353 1140 373 1200 393 1260 413 1320 433 1380 453 1440 473 1500 493 1560 513 1620 523

1680 1740

Figure 3. Nucleotide sequence and deduced amino acid sequence of soybean FLbR. T h e experimentally determined N-terminal amino acid sequence of the purified enzyme is underlined. A putative poly(A+) signal is indicated by t h e overbar. The Cys residues in the presumed active disulfide center are marked with asterisks. The complete DNA sequence was determined for both strands.

basic and hydroxylated amino acids and lacks acidic residues. The putative poly(A+) signal (AATAAA) (overlined in Fig. 3) (Proudfoot and Brownlee, 1976) is found 105 nucleotides downstream from the stop codon (TGA).

Sequence Comparisons of FLbR with Pyridine Nucleotide-Disulfide Oxidoreductases

The FLbR cDNA sequence and deduced peptide sequence were compared to known sequences in the GenBank data base using the FASTA program (Pearson and Lipman, 1988). The FLbR was found to be highly related to the family of flavin disulfide oxidoreductases (Williams, 199 l), especially the DLDH (EC 1.8.1.4) from various sources. The nucleic acid sequence of FLbR showed identity values ranging from 28 to 56% for the DLDHs from E. coli (Stephens et al., 1983), yeast (Browning et al., 1988), pig (Otulakowski and Robinson, 1987), human (Otulakowski and Robinson, 1987; Pons et al., 1988), and pea (Bourguignon et al., 1992); 18 to 25% for GSHRs (EC 1.6.4.2) from pea (Creissen et al., 1991), human

(Krauth-Siegel et al., 1982), and E. coli (Greer and Perham, 1986); 22 to 31% for MERAs (EC 1.16.1.1) from Shigella flexneri (Misra et al., 1985) and Staphylococcus aureus (Lad- daga et al., 1987); and 18% for TYTR (EC 1.6.4.8) from Trypanosoma congolense (Shames et al., 1988). The amino acid percentage identity values ranged from 35 to 50% for the DLDHs, 20 to 25% for GSHRs, 25 to 34% for the MERAs, and 22% for TYTR.

Peptide sequence comparisons of FLbR with those of DLDH, GSHR, MERA, and TYTR show that the functional domains of these enzymes appear to be highly conserved in FLbR with higher divergence in the surrounding regions (Fig. 4). For example, the amino acids around the disulfide active site are considerably similar among a11 the pyridine nucleo- tide-disulfide oxidoreductases (Williams, 1991) (Fig. 4). The very high sequence identities are also seen for the FAD- (Fig. 4) and NAD(P)H-binding sites (Fig. 4), and the cen- tral and interface domains of the flavin-disulfide oxidoreduc- tases are also observed in the FLbR sequence. High sequence similarity suggests a common origin for the FLbR and these oxidoreductases.

Genomic Blot Analysis of the FLbR Genes

Southern blot analysis of the genomic DNA was performed to estimate the number of FLbR genes in the soybean genome. Total soybean genomic DNA was isolated from leaf and root nodules. The genomic DNAs were digested with EcoRI, HindIII, and PstI, respectively. Based on the restriction map analysis using the Genetics Computer Group software, none of the three restriction enzymes has cleavage sites within the FLbR cDNA. Digests of genomic DNAs from leaf and nodule were separated on an agarose gel by electrophoresis and transferred to a nylon membrane. The blots were hybridized with either the 5’ end coding region-specific probe (150 bp FLbR PCR cDNA fragment) (Fig. 5A) or the entire FLbR cDNA probe (1.74 kb) including the coding and 5’ and 3’ untranslated regions (Fig. 58). The number of restriction fragments hybridized by both probes was consistent with size ranges from 2.3 to 7.0 kb (Fig. 5). Identical results were also observed from blots washed at a low-stringency temper- ature (25OC). These results indicated that the cDNA probes were specific to FLbR genomic sequences and that the soy- bean genome contains two copies of the FLbR gene. The same hybridization pattems were obtained on the blot of genomic DNA from leaves (Fig. 5, lanes 1-3).

Expression of FLbR Gene in Soybean Tissues

To examine the abundance and tissue distribution of FLbR mRNA in soybean plants, northem blot analysis of the total RNA from different tissues was performed using a DIG-11- dUTP-labeled cDNA of the cloned FLbR as a probe. The results of northern blot analysis, performed under stringent conditions, are shown in Fig. 6. In a11 soybean tissues, one major RNA-hybridizing band of approximately 1.8 kb was observed, but the abundance varied considerably. The leaf and nodule had far greater levels of FLbR mRNA than the root and stem.

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Cloning of Soybean Ferric Leghemoglobin Reductase 457

Enzyme-species Positions Amino Acid Sequence

FLbR-Soybean DLDH-Human DLDH-Pea DLDH-Yeast DLDH-EC GSHR-Human GSHR-Ec MERA-Sf MERA-Sa TYTR-TC

FLbR-Soybean DLDH-Human DLDH-Pea DLDH-Yeast

GSHR-Human DLDH-EC

GSHR-Ec MERA-Sf MERA-Sa TYTR-TC

FLbR-Soybean DLDH-Human DLDH-Pea DLDH-Yeast DLDH-Ec GSHR-Human GSHR-Ec MERA-Sf MERA-S.3 TYTR-Tc

Conserved Sequences in FAD-Binding Domain

37-66 N D V V V I G G G P G G Y V A A I K A A Q L G L K T T C I E 42-71 A . . T . . . S . . . . . . . . . . . . . . . F . . V . . . 38-67 . . . . I . . . . . . . . . . . . . . . . . . F . . . . . . 28-56 - . . . I . . . . . A . . . . . . . . . . . . F N . A . V . 6-35 T Q . . . L . . . . A . . S . . F R C . D . . . E . V I V . 21-50 Y . Y L . . . . . S . . L A S . R R . . E . . A R A A V V . 5-34 Y . Y I A . . . . S . . I A S I N R . . M Y . Q . C A L . . 98-127 L H I A . . . S . . A A M A . . L . . V E Q . A R V . L . . 86-115 Y . L L I . . S . G A A F S . . . . . N E N . A . V A M V . 5-33 F . L . I . . A . S . . L E . G W N . . T . Y K . R V A V -

Conserved Sequences around the Disulfide Active Site * * 67-96 K R G T L G G T C L N V G C I P S K A L L H S S H M Y H E A 73-102 . N E . . . . . . . . . . . . . . . . . . N N . . Y . . M . 69-98 . . . A . . . . . . . . . . . . . . . . . . . . . M Y H E A 58-87 . . . K . . . . . . . . . . . . . . . . . N N . . L F . Q M 37-66 R Y N . . . . V . . . . . . . . . . . . . . V A K V I E . . 52-80 S H K - . . . . . V . . . . V . . . T M W N T A V H S E F M 36-64 . N E - . . . . . V . . . . V . K . V M W . A A Q I R E A 1 129-157 - . . . I . . . . V . . . . V . . . I M l R A A . I A . L R 117-145 - . . . V . . . . V . . . . V . . . T M L R A G E I N G L . 49-74 - - - A . . . . . V . . . . V . K . L M V T G A Q Y M D Q L

Conserved sequences in NAD(P)H-Binding Domain

196-225 203-232 198-227 194-223 163-192 173-202 161-190 259-288

S S T G A L A L S E I P K K L V V I G A G Y I G L E M G S V . . . . . . S . K K V . E . M . . . . . . V . . V . L . . .

. . . . . . S . K . . . . R . T I . . G . I . . . . . . . . D . . S . . E . K . V . E R . L . M . G . I . . . . . . T . D . D . F F . . P A L . E R V A . V . . . . . A V . L A G . T . D . F F Q . E . L . G R S . 1 V . S . . . A V . . A G I T . . E . . V S E T . . . R . A . . . S S V V A . . L A Q A

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

240-269 T . . S . . E . K . V . Q R . A . . . S . . . A A . L . Q M 178-207 S . N E . F Y . E . P . R R V L T V . G . F . S V . F A G 1

Figure 4. Comparison of amino acid sequences of the soybean FLbR with sequences around the disulfide active sites and the FAD-binding and NAD(P)H-binding domains of the flavin-disulfide oxidoreductases. Alignment of amino acid sequences of the soybean FLbR and the DLDH, GSHR, MERA, and TYTR from various sources was determined using the PILEUP program of the University of Wisconsin Genetics Computer Group software package. The gaps between amino acids are presented by dashes. The conserved residues are presented as dots. The disulfide Cys residues of the putative active site are marked with asterisks. The positions of sequences are numbered on the left. The sequences of active site and the FAD- and NAD(P)H-binding domain were analyzed based on the sequence and the x-ray crystal structure of human erythrocyte CSHR (Thieme et al., 1981; reviewed by Carothers et al., 1989, and by Williams, 1991). EC, E. coli; Sf, S. flexneri; Sa, S. aureus; Tc, T. congolense.

DlSCUSSlON

FLbR has been hypothesized to play an important phys- iological role in maintaining Lb in its functional ferrous state during N2 fixation (Becana and Klucas, 1990, 1992; Ji et al., 1991, 1992). Here we have described the cloning of a cDNA encoding soybean FLbR. The specific cDNA probes of FLbR are rapidly generated by the PCR using as primers a set of highly degenerate oligonucleotides derived from the experi- mentally determined N-terminal amino acid sequence (Lee et al., 1988) and by automated DNA sequencing for confirma- tion of the PCR products. The full-length cDNA sequence (1.74 kb) of the cloned FLbR corresponded well with the observed size of the FLbR transcript of about 1.8 kb. The identity of the clone was confirmed by a perfect match of the predicted amino acid sequence with the experimentally de- termined N-terminal partia1 sequence of the purified FLbR from soybean nodules. The calculated molecular mass (55,729 D) from the derived amino acid sequence of the cloned FLbR is very close to the experimentally determined molecular mass (about 54 kD) of the purified FLbR from soybean nodules based on its mobility in SDS-PAGE. These

results suggest that the complete coding region of the mRNA sequence was isolated in cDNA form.

The analysis of the putative FLbR leader sequence (the first 30 amino acids at the N terminus, Fig. 4) showed some features shared by signal sequences of mitochondrial matrix proteins. The FLbR signal sequence is rich in basic and hydroxylated amino acid residues but devoid of acidic and of long stretches of uncharged amino acids and appears to have a "positive hydrophobic polar design" (von Heijne, 1989). These structural properties were suggested to allow signal sequences to fold independently into an amphiphilic a-helix, hence enabling them to interact with the mitochon- drial membrane (Hurt and Schatz, 1987; von Heijne, 1989). The secondary structure prediction and the calculated hydro- phobic nature of the FLbR peptide indicated a possible am- phiphilic a-helix structure at the FLbR leader sequence. Any physiological function related to these observations, however, cannot be suggested until the cellular location of FLbR and its secondary structure are accurately determined.

Comparisons of the derived FLbR amino acid sequence with known peptide sequences in protein data bases showed considerable homologies (varied from 20-50% with enzymes

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458 Ji et al. Plant Physiol. Vol. 104, 1994

Kb

12.2-8.16.1-

4.1-3.1-

2.0-

1.0-

Figure 5. Hybridization analysis of soybean genomic DMA for theFLbR genes. Cenomic DNA (about 5 jig) isolated from soybeanleaves (lanes 1-3) and from symbiotic nodules of soybean/rhizobia(lanes 4-6) were digested with either EcoRI, H/ndlll, or Pstl, sepa-rated by electrophoresis on a 0.8% agarose gel (2 \i% of digests persample), transferred to a nylon membrane, and hybridized with a150-bp PCR-amplified FLbR DNA probe (A) or a 1.8-kb full-lengthFLbR cDNA probe (B). Sizes based on the migration of 1-kb DNAladders (BRL) are indicated on the left.

served with genomic DNA either from soybean leaves orfrom root nodules (Fig. 5), suggesting either that the FLbRgenes originated in the soybean plant only or that they arehomologous in both the soybean plant and Bradyrhizobium.We previously reported that the highly purified FLbR fromsoybean root nodules showed three distinct bands on an IEFgel and suggested that they might represent isoforms ormodifications of the protein (Ji et al., 1991). The two copiesof the FLbR gene might be the genetic reflections of theseisoforms or modifications. These suggestions are enforced bythe evidence that only one kind of FLbR transcript was foundamong all of the soybean tissues (Fig. 6), which indicatedthat the two genes were not encoding two different FLbRproteins but rather some isoforms or modifications of theFLbR. Whether the two copies of FLbR gene detected insoybean encode isozymes or represent genetic mutations, orare simply a gene repeat that might regulate the synthesisand distribution of the enzyme in different enzyme complexesin higher plants, is worthy of further investigation.

FLbR is a multifunctional flavoenzyme (Saari and Klucas,1984; Ji et al., 1991, 1992). The enzyme exhibited highactivities for NAD(P)H-dependent diaphorase and Lb+3 re-ductions and, in the absence of any other exogenous electroncarriers, catalyzed NADH oxidation, functioning as anNADH oxidase. If the diversity of enzymatic activities of thesoybean FLbR is to meet a number of physiological demandsin vivo, then expression of the gene throughout the plantand during development is expected. Northern blot analysisshowed that the FLbR gene is expressed in various tissues insoybean plants (Fig. 6). Levels of expression of the FLbR geneare greater in nodules and in leaves than in roots and stems.This finding is consistent with an expected role of FLbR inthe reduction of Lb+3 in nodules. Expression of the FLbRgene in various tissues of the soybean plant suggests that

and species) to the pyridine nucleotide-disulfide oxidored-uctases, especially the DLDHs. This family of pyridine nu-cleotide-disulfide oxidoreductases includes well-character-ized enzymes such as DLDH, GSHR, thioredoxin reductase(EC 1.6.4.5), TYTR, and MERA (Williams, 1991). All enzymesin this family are homodimers containing FAD and redox-active disulfides and catalyze the electron transfer fromNAD(P)H to flavin and then to the disulfide group and tothe substrates (Williams, 1991) with the exceptions of MERAand dithiol oxidase, which require four thiol groups forcatalytic activities (Massey et al., 1988). Human erythrocyteGSHR has been the most extensively investigated at thestructural level. Peptide sequences among human GSHR andother flavoproteins such as DLDH, MERA, TYTR, and thio-redoxin reductase from various species possessed a highdegree of homology throughout the amino acid sequence anda remarkable sequence identity around the disulfide-activesite and the FAD- and NAD(P)H-binding regions (Carotherset al., 1989; Williams, 1991).

Analysis of genomic DNA indicated that two copies ofFLbR gene were present in the soybean genome. The twogenomic DNA sequences were hybridized to the same FLbRcDNA probe, and identical hybridization patterns were ob-

kb

-1.85

0.24

Figure 6. Northern blot analysis of the level and distribution ofFLbR mRNA in soybean plants. Total RNA (20 jig) isolated fromsoybean nodules, leaves, roots, and stems was separated ona formaldehyde-agarose gel, transferred to a nylon membrane,and hybridized with a 1.8-kb full-length FLbR cDNA probe. TheBRL RNA markers were used to estimate sizes of FLbR mRNA.

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Cloning of Soybean Ferric Leghemoglobin Reductase 459

FLbR could serve other functions, as has been observed for DLDH (Williams, 1991). A definitive role of FLbR in root nodule metabolism will be revealed by further cloning the individual FLbR genes and studying their regulations under various environmental conditions and in different develop- ment stages.

Received June 18, 1993; accepted September 29, 1993. Copyright Clearance Center: 0032-0889/94/104/0453/07.

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