Plant Physiol. (1994) 104: 1411-1417

Plant 6-Aminolevulinic Acid Dehydratase’

Expression in Soybean Root Nodules and Evidence for a Bacterial Lineage of the Alad Gene

Christine M. Kaczor, Michael W. Smith, lndu Sangwan, and Mark R. O’Brian*

Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214 (C.M.K., I.S., M.R.O’B.); and Molecular Genetics Laboratory and Human Genome Center,

The Salk Institute, San Diego, California 92186 (M.W.S).

We isolated a soybean (Clycine max) cDNA encoding the heme and chlorophyll synthesis enzyme 6-aminolevulinic acid (ALA) de- hydratase by functional complementation of an Escherichia coli hemB mutant, and we designated the gene Alad. ALA dehydratase was strongly expressed in nodules but not in uninfected roots, although Alad mRNA was only 2- to 3-fold greater in the symbiotic tissue. Light was not essential for expression of Alad in leaves of dark-grown etiolated plantlets as discerned by mRNA, protein, and enzyme activity levels; hence, its expression in subterranean nod- ules was not unique in that regard. The data show that soybean can metabolize the ALA it synthesizes in nodules, which argues in favor of tetrapyrrole formation by the plant host in that organ. Molecular phylogenetic analysis of ALA dehydratases from 11 organisms indicated that plant and bacterial enzymes have a com- mon lineage not shared by animals and yeast. We suggest that plant ALA dehydratase is descended from the bacterial endosym- biont ancestor of chloroplasts and that the Alad gene was trans- ferred to the nucleus during plant evolution.

Plants synthesize a wide variety of tetrapyrroles that in- clude Chl, hemes, siroheme, and bilins, and these compounds participate in many cellular processes (reviewed by Beale and Weinstein, 1991). Synthesis of hemes and Chl from the universal tetrapyrrole precursor ALA share common enzymic steps and diverge after protoporphyrin IX synthesis for for- mation of the respective metal porphyrin derivative. Chl is by far the predominant tetrapyrrole found in most plants, and its abundance in photosynthetic tissues has rendered it amenable to experimental analysis. Accordingly, much less is known about the expression of other tetrapyrroles that are functionally important but are usually found in low amounts.

One exception is plant hemoglobin expressed in legume root nodules, which makes up approximately 25% of the plant protein mass in the cytosol (Appleby, 1984). Nodules are specialized root organs elicited by nitrogen-fixing rhizobia (reviewed by Nap and Bisseling, 1990; Caetano-Anolles and Gresshoff, 1991; Verma, 1992; Long and Staskawicz, 1993), and hemoglobin is one of numerous nodule-specific plant

This work was supported by National Science Foundation grant IBN-9204778 and by the Cooperative State Research Service, U.S. Department of Agriculture, under agreement 91-37305-6750.

* Corresponding author; fax 1-716-829-2725.

proteins essential for symbiosis. Despite the unique context in which plant hemoglobin is expressed, aspects of heme precursor synthesis in soybean (Glycine max) nodules have features in common with tetrapyrrole formation described in leaves. Plants synthesize ALA from glutamate in leaves by the C5 pathway (reviewed by Beale and Weinstein, 1991; Jahn et al., 1992), and the C5 pathway enzyme GSA amino- transferase is expressed in root nodules (Sangwan and O’Brian, 1993). ALA formation activity is activated in nodules (Sangwan and O’Brian, 1991, 1992), and this is at least in part due to induction of the GSA aminotransferase gene (Gsa) (Sangwan and O’Brian, 1993). The soybean Gsa gene is strongly expressed in leaves of etiolated plantlets and is reduced in older leaves; thus Gsa is regulated in leaves as well.

Synthesis of the leghemoglobin heme prosthetic group has been a topic of controversy, and it underscores the lack of information about tetrapyrrole synthesis by the plant host in nodules. The hypothesis that nodule hemoglobin heme is a bacterial product has been put forth (Cutting and Schulman, 1969), and that idea is based primarily on the lack of heme synthesis enzyme activities in plant fractions of nodules (Cutting and Schulman, 1969; Porra, 1975; Nadler and Av- issar, 1977) and on the leghemoglobin-defective phenotype of nodules induced by some rhizobial heme synthesis mu- tants (Leong et al., 1982; O’Brian et al., 1987). The former argument is undermined somewhat by the discovery of a soybean nodule ALA formation activity (Sangwan and O’Brian, 1991, 1992, 1993), and the analysis of mutants must be viewed in the context of data that show that nodules elicited by a Rhizobium meliloti hemA mutant are arrested in development at a stage that precedes hemoglobin expression (Dickstein et al., 1991). Similarly, Bradyrhizobium japonicum mutants defective in hemB (Chauhan and O’Brian, 1993) and hemH (Frustaci and O’Brian, 1992, 1993) incite soybean nod- ules that are poorly developed; thus, the absence of late- nodule proteins such as hemoglobin is expected regardless of the source of prosthetic group. Thus, the symbiont that cames out hemoglobin heme synthesis remains unknown.

Evidence suggests that Bradyrhizobium japonicum can take up and utilize soybean-derived ALA (Sangwan and O’Brian,

Abbreviations: ALA, 6-aminolevulinic acid; GSA, glutamate 1 - semialdehyde; PBG, porphobilinogen. -

141 1

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1412 Kaczor et al. Plant Physiol. Vol. 104, 1994

1991), thereby rendering the bacterial hemA gene nonessen- tia1 for that symbiosis (Guerinot and Chelm, 1986). Therefore, plant ALA is not committed to plant tetrapyrrole synthesis, and induction of ALA formation activity cannot in itself be interpreted as evidence for plant heme synthesis in nodules. Very recently, we found that, unlike the hemA gene, the B. japonicum hemB gene is essential for symbiosis (Chauhan and O‘Brian, 1993), suggesting that ALA is the only plant tetra- pyrrole precursor that can be utilized by the bacterial sym- biont. It follows then that soybean nodule enzymes involved in ALA metabolism should be committed to plant tetrapyrrole synthesis; so we have initiated a study of the expression of soybean ALA dehydratase, the enzyme that forms PBG from ALA. In addition, we provide molecular phylogenetic evi- dente for a bacterial origin for plant ALA dehydratases, indicating that the Alad gene was derived from the bacterial ancestor of chloroplasts and was transferred to the nucleus during plant evolution.

MATERIALS A N D METHODS

Plants and Bacteria

Escherichia coli strain RP523 is a hemB mutant of strain C600 and is a hemin auxotroph (10 &mL) (Li et al., 1988). E. coli strains were grown in Luria broth, and ampicillin (50 Fg/mL) was added to media for growth of strains harboring pUC18, pSK Bluescript 11, and their recombinant derivatives. Bradyrhizobium japonicum strain I1 10 was the soybean en- dosymbiont used in the present work and was grown in GSY media as previously described (Frustaci et al., 1991). Soy- beans (Glycine max cv Essex), either inoculated with B. japon- icum or uninoculated, were grown in a growth chamber under a 16-h light/8-h dark regimen at 25OC. Nodules, leaves, and roots were harvested from 23-d-old plants for enzyme assays or RNA extraction.

lsolation and Analysis of Soybean Leaf cDNA Encoding ALA Dehydratase

A soybean leaf cDNA expression vector library in pUC18 was a gift from Dr. M.L. Kahn (Washington State University, Pullman, WA) and was constructed as described by Udvardi et al. (1991). Library DNA was used to transform the E. coli hemB mutant strain RP523 en masse by electroporation, and cells were subsequently plated onto Luria broth (Ausubel et al., 1987) containing isopropyl p- A-thiogalactopyranoside (0.4 mM) and ampicillin in the absence of hemin. Comple- mented transformants were scored as ampicillin-resistant hemin prototrophs. Plasmid DNA was isolated from the transformed cells as described previously (Ausubel et al., 1987) and analyzed initially by agarose gel electrophoresis and retransformation of strain RP523 to confirm that the plasmid conferred hemin prototrophy on the mutant. The cDNA insert of one of the complementing clones, pTK1, was cloned into pBluescript IISK+ to construct pRRK1, and dele- tions were made using an Exo-Mung deletion kit (Stratagene). The nucleotide sequence of both strands of the pRRKl insert was determined by the chain termination method (Sanger et al., 1977) using a Sequence kit (United States Biochemical) according to the manufacturer’s instructions.

ALA Dehydratase Protein and Enzyme Activity

The enzyme activity of ALA dehydratase was measured as PBG formation from ALA as described previously (Sangwan and O’Brian, 1991). Extracts of E. coli and of soybean leaves, roots, and nodule cytosol were prepared as demibed previ- ously (Sangwan and O’Brian, 1993). The absence of bacterial contamination in plant nodule fractions is observed as the lack of ALA synthase activity in the plant fractions (Sangwan and O’Brian, 1991). Reactions were camed out for 1 h at 37OC im Tricine buffer (pH 8), 8 m ALA, ancl 20 mM DTT using 0.5 mg of protein per reaction. Control reactions were incubated on ice. PBG was quantitated spectrophotometri- cally after reaction with the Ehrlich reagent as described by Sangwan and O‘Brian (1992). ALA dehydratase was detected in plant tissue extracts immunologically using antibodies raised against the spinach enzyme (Schaumburg et al., 1992) in sloi blots. Cross-reactive protein on nitrocellulose filters was cliscemed visually using peroxidase-conjugated goat anti-rabbit IgG as described previously (Ausubel et al., 1987).

RNA lsolation and Analysis

Nodules, leaves, and roots were excised, frozen in liquid nitrogen, and homogenized in a blender with homogeniza- tion buffer and phenol (223 , w/v/v). The homogenization buffer contained 500 mM Tris (pH 8), 10 mM MgC12, 1 m EDTA, 100 m NaC1, 0.5% (w/v) deoxycholate, and 1 m P-mercaptoethanol. Total RNA was isolated from the ho- mogenate as described previously (Ausubel et iil., 1987), and poly(A)+ RNA was isolated using oligo(dT)-cellulose col- umns. Northem blot analysis of poly(A)+ RNA was camed out as previously described under high-stringency conditions (Ausubel et al., 1987). Ubiquitin cDNA was provided by Dr. D.P.S. Verma (Fortin et al., 1988). Relative amounts of RNA detected in northem blots were estimated by ijcanning den- sitometry of the autoradiograms.

Analysis of ALA Dehydratase Sequences and Construction of Phylogenetic Trees

Deduced amino acid sequences of 11 ALA dehydratases were trimmed to the conserved regions and progressively aligned with the TREE program of Feng and Doolittle (1990), which uses the Dayhoff Mutation Matrix (D‘iyhoff, 1978). Matrix distances are calculated from the number of shared residues in pairwise comparisons, their raxtdomized se- quences, and self-comparisons of each (Feng and Doolittle, 1990). The ALA dehydratase sequences analyzed in the pres- ent work are from soybean, pea (Boese et al., l991), spinach and Selaginella martensii (Schaumburg et al., 1992), human (Weknur et al., 1986), mouse (Bishop et al., 15189), Saccharo- myces cerevisiae (yeast, Myers et al., 1987), E. coli (Li et al., 1989), Bacillus subtilis (Hansson et al., 1991). B. japonicum (Chauhan and O’Brian, 1993), and Methanothtrmus sociabilis (Brocld et al., 1992). The rat ALA dehydratase (Bishop et al., 1986) was not included in the analysis because of its near identity to the mouse sequence.

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Expression and Lineage of Plant 6-Aminolevulinic Acid Dehydratase 1413

Table 1. Functional complementation of E . coli hemB mutant strain RP523 with sovbean cDNA clone oTKl

Doubling Time of ALAD Activity Crowth Strain

Plus hemin No hemin

min nmol PBC h-' mg-' protein

C6OO[pUC18] 11.2 41 39 RP523[pUC18] O 44 No growth RP523 [ pTK1 ] 6.6 43 62

RESULTS

lsolation of a ALA Dehydratase-Encoding cDNA

E. coli strain RP523 is defective in hemB, the gene encoding ALA dehydratase, and behaves as a hemin auxotroph (Li et al., 1988). Strain RP523 was transformed en masse with a soybean leaf cDNA expression vector library, and cells that were functionally complemented were screened as ampicillin- resistant hemin prototroph colonies on agar media. Fifteen prototrophic transformants were isolated that bore identical plasmids as judged from restriction digest pattems, and one of them, pTK1, was chosen for subsequent experiments. pTKl conferred on strain RP523 hemin prototrophy and ALA dehydratase activity, which was approximately one-half of that found in parent strain C600 (Table I). The insert of pTKl contained an open reading frame of 412 amino acids begin- ning with a Met codon (Fig. l), and the deduced protein shared 88% identity with the ALA dehydratase from pea (Boese et al., 1991). This identity, along with the complemen- tation of the E. coli hemB mutant, provides very strong evidence that the cloned cDNA encodes soybean ALA de- hydratase, and the corresponding gene is designated Alad. The cDNA sequence shows that Alad mRNA is polyadenyl- ated at the 3' end (Fig. I), indicating that the RNA was processed in the nucleus. Because ALA dehydratases are plastid-localized proteins (Smith, 1988), it must be targeted to that organelle from the cytoplasm, and the amino-terminal region of the soybean Alad product is predicted to contain a chloroplast transit peptide (Fig. 1). The predicted transit peptide is based on the known sequence of the mature spinach ALA dehydratase (Schaumburg et al., 1992) and on features that typify such domains (Gavel and von Heijne, 1990).

Expression of the AIad Cene in Soybean Tissues

Poly(A)+ RNA from leaves, nodules, and uninfected (asym- biotic) roots of 23-d-old plants was probed with ALA dehy- dratase cDNA in northem blots to discern Alad transcripts in those tissues (Fig. 2) . Alad mRNA was expressed in root nodules as discerned by a single band, and the steady-state leve1 was 2- to 3-fold greater than was found in uninfected roots (Fig. 2). The modest increase of Alad message in nodules compared to roots differs from that of Gsa, which is much greater in the symbiotic tissue than in uninfected roots (Sang- wan and O'Brian, 1993; Fig. 2). The single band at 1.6 kb in a11 tissues examined suggested that the message represents a single gene or gene family. Antibodies raised against spinach

ALA dehydratase (Schaumburg et al., 1992) were used to detect the enzyme in soybean tissues; cross-reactive protein was found in leaves and nodules but not in uninfected roots (Fig. 3). ALA dehydratase enzyme activities in the respective tissues were consistent with the antibody data, showing activity in leaves and nodules but no discemible activity in uninfected roots (Fig. 3). It is unlikely that the ALA dehydra- tase found in nodule cytosol was due to B. japonicum contam- ination during fractionation because bacterial extracts ex- pressed only one-fourth of the activity of that found in the plant cytosol(4 versus 16 nmol h-' mg-' protein). In addition, ALA synthase activity, a bacterial marker enzyme, is absent in the plant fraction of nodules. These observations indicate that Alad gene expression in nodules is controlled primarily at the synthesis or tumover of the enzyme and not of the mRNA; Boese et al. (1991) reached a similar conclusion for control of ALA dehydratase in pea leaves. Although we

1 CAATTCGGCACGAGATGGCTTCTTCAATCCCTAATGCGCCTTCTGCGTTCAATTCTCAGA 60 i r h e M A S S l P N A P S A F N S O S

61 GCTACGTTGGTCTCAGAGCGCCACTGAGGACCTTCAACTTTTCTTCTCCTCAAGCTGCCA 120 Y V G L R A P L R T F N F S S P Q A A K

121 AAATTCCTCGCTCCCAACGCCTTTTCGTCGTCAGAGCCTCCGATTCGGAGTTCGAAGCCG 180 I P R S O R L F V V R A S D S E F E A A

181 CCGTTGTCGCCGGTAAGGTTCCGCCGGCGCCTCCCGTGCGGCCCAGACCGGCGGCTCCGG 240 V V A G K V P P A P P V R P R P A A P V

241 TTGGAACACCGGTGGTTCCTTCACTTCCACTTCACCGGCGTCCTCGTCGGAACCGGAAGT 300 G T P V V P S L P L H R R P R R N R K S

301 CCCCGGCGCTTCCGTCGGCTTTTCAGGAAACGAGCATTTCGCCGGCGAATTTCGTGTATC 360 P A L R S A F O E T S I S P A N F V Y P

361 CGCTTTTCATTCACGAACGTGAAGAGGATACTCCAATTGGGGCTATGCCTGGATGCTACA 420 L F l H E G E E D T P l G A M P G C Y R

421 GGCTTGGGTGGACGCATGGACTTGTAGAAGAGGTTGCAAAAGCACGGGATGTTGGTGTTA 480 L G U R H G L V E E V A K A R D V G V N

481 ATAGTGTTGTGCTCTTCCCCAAAATTCCAGATGCTTTGAAGTCTCCCACAGGAGATGAAG 540 S V V L F P K I P D A L K S P T G D E A

541 CATACAATGAAAATGGTTTAGTGCCTCGAACAATACGTTTGCTCAAGGATAAGTACCCAG 600 Y N E N G L V P R T I R L L K D K Y P D

601 ACCTTCTTATCTATACAGATGTTGCATTAGATCCTTATTCATCAGATGGGCATGATGGCA 660 L V I Y T D V A L D P Y S S D G H D G I

661 TAGTTAGAGAAGATGGAGTTATTATGAATGATGAGACAGTTCATCAGCTATGTAAACAAG 720 V R E D G V I M N D E T V H O L C K O A

721 CTGTAGCCCAGGCCCAAGCTGGAGCAGATGTTGTCAGTCCTAGTGATATGATGGATGGTC 780 V A Q A Q A G A D V V S P S D M M D G R

781 GGGTAGGAGCACTGCGTGCAGCTCTGGATGCTGAAGGCTTTCAGCATGTTTCTATAATGT 840 V G A L R A A L D A E G F O H V S I M S

841 CCTATACAGCAAAGTATGCAAGTTCTTTTTATGGTCCATTTAGAGAGGCACTAGACTCAA 900 Y T A K Y A S S F Y G P F R E A L D S N

901 ACCCCCGGTTTGGAGACAAGAAAACTTATCAGATGAACCCAGCTAATTACAGAGAGGCTC 960 P R F C D K K T Y Q M N P A N Y R E A L

961 TGACTGAGATGCGGGAGGATGAATCTGAAGGAGCTGACATTCTCTTGGTGAAGCCTGGTC 1020 T E M R E D E S E G A D I L L V K P G L

1020 TTCCTTACTTGGATATCATAAGGCTGCTCAGGGATAATTCTCCTTTGCCAATTGCAGCAT 1080 P Y L D I I R L L R D N S P L P I A A Y

1081 ACCAGGTTTCTGGTCAATATGCAATGATAAAGGCTGCCGGTGCTCTCAAAATGATAGACG 1140 Q V S G E Y A M I K A A G A L K M I D E

1141 AAGAAAAGGTTATGATGGAGTCACTGATGTGCCTCCGAAGGGCCGGTGCTGATATCATCC 1200 E K V M H E S L M C L R R A G A D I I L

1201 TCACATATTCTGCTCTCCAAGCTGCCAGATGTTTGTGTGGAGAGAAGAGGTGAAGTTCTC 1260 T Y S A L O A A R C L C G E K R "

1261 TGATTATGTAGGGCGTTGTTCATGTAGAAGGTTGAAGAGTTTATAATACCAGTATCTGCT 1320 1321 GGATTTTGGTTATTGTAAATTGTTTAAGAGGGACATGGAGGTTTGTGTATAGAGAGACAT 1380 1381 TCATAATAAAATATTATGGCCTCGTTTGATTTAATATATTTAAGGACATAATAGAAAAAG 1440 1441 ACTTGGCCCTTGTTTTTGGTGTTAATTGTAACTTTACGTGTGGATTGTCAACCCATTGTA 1500 1501 TGAATGAAAGTGTGCACTCACCTGCAGAATATTTCGTACTGTAAAAAAAAAAAAAAAAAA 1560 1561 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 1595

Figure 1. Nucleotide sequence and deduced product of the cloned cDNA insert of pTK1 encoding soybean ALA dehydratase. The underlined region represents the putative leader peptide.

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1414 Kaczor et al. Plant Physiol. Vol. 104, 1994

L R N

A lad

Gsa

Ubi

Figure 2. Northern blot analysis poly(A)+ RNA from leaves (L), roots(R), and nodules (N) of 23-d-old plants. RNA (5 Mg) was loaded ontoeach lane, and the figure represents a composite of a single filterprobed separately with cDNA encoding ALA dehydratase (A/ad),CSA aminotransferase (Gsa), or ubiquitin (Ubi). Ubiquitin was usedas a constitutively expressed gene.

cannot strictly rule out a nodule-specific dehydratase as analternative explanation, such a protein would need to behomologous enough to the ubiquitous enzyme to be detectedby the anti-ALA dehydratase antibodies, but the mRNAwould have to be sufficiently disparate to be undetected innorthern blots. Regardless, expression of soybean ALA de-hydratase in root nodules indicates that the plant can utilizethe ALA it synthesizes there, thus inferring tetrapyrrole for-mation by the host in the symbiotic organ. In addition, theinduction of the Alad gene in nodules suggests an increasedcapacity for plant tetrapyrrole formation compared with un-infected roots.

Effect of Light on Alad Expression

Full expression of Chl in soybean and in other angiospermsrequires light, and evidence for light regulation of tetrapyrroleenzymes, including ALA dehydratase, in photosynthetic tis-sues has been put forth (Kasemir and Masoner, 1975). Directlight exposure is an unlikely positive effector of gene expres-sion in subterranean root nodules; hence, we examined

23 days

N

Protein

Activity 16

Etiolated

in*

42 26

Figure 3. Immunoblot and enzyme activities of ALA dehydratasefrom soybean tissue extracts. Protein (25 ^g Per slot) was loadedand probed with antibodies raised against spinach ALA dehydra-tase. The enzyme activity of ALA dehydratase was carried out asdescribed in the text, and the data are expressed as nmol PBGformed h~' mg~' protein and are averages of three trials. Tissueextracts were prepared from leaves (L), roots (R), and nodules (N)of 23-d-old plants and from the leaves of etiolated plantlets thatwere either subjected to complete darkness (D) or illuminated for24 h prior to harvesting (I), in which time they greened.

whether light was necessary for expression of the Alad genein leaves. Poly(A)+ RNA was isolated from leaves of etiolatedsoybean plants grown completely in darkness and fromgreening etiolated plant leaves made so by exposure to lightfor 24 h prior to harvesting. The soybean cab gene was usedas a control for a light-induced gene (Chang and Walling,1992), and it can be seen in northern blots that expression ofthat gene is strongly dependent on light (Fig. 4). However,Alad mRNA levels did not require light for high expression,as seen by the pronounced hybridization signal in RNA fromleaves of dark-grown plantlets. The Alad message was en-hanced only 2-fold by light, which is similar to what wepreviously found for the Gsa gene (Sangwan and O'Brian,1993; Fig. 4). Because leaves of etiolated plants expand con-siderably during the 24-h greening period, it is plausible thatthe modest differences in Alad and Gsa messages expressedin the respective tissues reflect developmental control of thosegenes. The protein and enzyme activity of ALA dehydratasewere expressed in leaves of dark-grown plants at levels atleast as high as was found in light-exposed leaves (Fig. 3).From this, we conclude that full expression of ALA dehydra-tase in leaves does not require light.

Evidence for a Bacterial Lineage for PlantALA Dehydratases

The putative metal-binding domain of the B. japonicumALA dehydratase contains some features characteristic of thethe corresponding domain in the plant enzymes (Chauhanand O'Brian, 1993). In the present work, we wanted todetermine whether the anomalous B. japonicum protein couldbe the result of an evolutionary horizontal gene transfer fromthe plant host to the bacterium, in which case the B. japonicum

Alad

Cab

Gsa

Ubi

Figure 4. Effect of light on Alad mRNA levels in leaves of etiolatedplantlets. Poly(A)+ RNA was isolated from leaves of plantlets growncompletely in darkness (D) or illuminated for 24 h prior to harvesting(I), in which time they greened. RNA (5 Mg) was loaded onto eachlane, and the figure represents a composite of a single filter hybrid-ized separately with radiolabeled DNA probes. The cDNA probesused encode ALA dehydratase (Alad), Chl a/b-binding protein(Cab), CSA aminotransferase (Csa), and ubiquitin (Ubi). Cab andUbi were used as controls for light-induced and constitutive genes,respectively.

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Expression and Lineage of Plant 6-Aminolevulinic Acid Dehydratase 1415

ALA dehydratase would be unique in its similarity to the plant enzymes over its entire length. Conserved regions of 11 ALA dehydratases from plants, bacteria, yeast, and mam- mals were progressively aligned as described by Feng and Doolittle (1990), and phylogenetic trees were constructed (Fig. 5 ) . The analysis as depicted in the unrooted matrix tree did not indicate a plant source for the B. japonicum ALA dehydratase, but rather it infers a bacterial origin of plant ALA dehydratases. The sequence similarities between each of the plant-bacteria comparisons (50-58%) were higher than any of those between plants and other eukaryotes (39-47%). Grouping of the archaebacterium M. sociabilis with the eu- bacteria is an unexplained anomaly of the phylogenetic analysis (Fig. 5 ) . The endosymbiotic theory of organelle orig- ination proposes that chloroplasts (and mitochondria) are derived from once free-living bacteria (Gray, 1989). Thus, the deduced phylogeny of ALA dehydratases, along with the known plastid location of the nuclear-encoded enzyme, leads us to postulate that the plant Alad gene is descended from the bacterial ancestor of chloroplasts and was transferred to the nucleus during plant evolution.

DISCUSSION In the present work, we isolated a cDNA encoding soybean

ALA dehydratase and examined the expression of the Alad

1 O matrix

units s o y b e a n Aea L s p i n a c h

S. martensii

6. japonicum

B. subtilis

1L__ M. sociabilis

human

mouse

yeast

Figure 5. Molecular phylogenetic analysis of ALA dehydratase from various organisms. Deduced amino acid sequences of 11 ALA dehydratases were trimmed to the conserved regions and progres- sively aligned with the TREE program of Feng and Doolittle (1990), which uses the Dayhoff Mutation Matrix (Dayhoff, 1978). The ALA dehydratase sequences analyzed in the present work are from soybean (Fig. Z), pea (Boese et al., 1991), spinach and S. martensii (Schaumburg et al., 1992), human (Wetmur et al., 1986), mouse (Bishop et al., 1989), 5. cerevisiae (yeast, Myers et al., 1987), E. coli (Li et al., 1989), 5. subtdis (Hansson et ai., 19911, 6. japonicum (Chauhan and O’Brian, 1993), and M. sociabilis (Brockl et al., 1992). The rat ALA dehydratase (Bishop et al., 1986) was not included in the analysis because of its near identity to the mouse sequence.

gene in root nodules as a step toward assessing whether the plant host can synthesize significant quantities of heme in that organ. Soybean ALA dehydratase was highly expressed in nodules but not in uninfected roots, and thus, the Alad gene was induced by symbiosis. Alad mRNA was only mod- estly higher in nodules than in uninfected roots; hence, the large differences in enzyme expression between the two tissues are likely to be due primarily to protein synthesis or stability. This level of control is unlike that of the GSA aminotransferase gene in nodules, in which the transcript level contributes significantly to differential expression (Sang- wan and O’Brian, 1993). Although Chl synthesis requires light, expression of Alad did not require light in leaves of etiolated plantlets; thus, there is no need to invoke a regula- tory factor to compensate for light to account for the expres- sion of ALA dehydratase in subterranean root nodules. Our studies with etiolated plants do not rule out light regulation of the Alad gene in leaves of plants grown under a normal diumal light/dark cycle.

The expression of ALA dehydratase by soybean in nodules strongly suggests that plant-synthesized ALA can be metab- olized by the host as well as by the bacterial symbiont. This conclusion is significant because plant PBG cannot support B. japonicum heme formation in nodules (Chauhan and O’Brian, 1993); hence, the soybean ALA dehydratase is com- mitted to plant tetrapyrrole synthesis. Expressions of soybean Alad and Gsa are comparable to those found in leaves in which copious amounts of Chl are synthesized (Figs. 2 and 3; Sangwan and O’Brian, 1993); thus, it is clear that the activities of those enzymes are sufficient to support heme formation in high quantities. A diminution of the induced ALA formation and ALA dehydratase activities as a function of nodule age (Nadler and Avissar, 1977; Sangwan and O’Brian, 1992) infers that rates of synthesis are highest in young nitrogen-fixing nodules. The data do not prove that hemoglobin heme is provided, in part or in toto, by the eukaryotic symbiont, but it alters the premises on which past arguments have been made (Cutting and Schulman, 1969). In addition, we reiterate that soybean nodules elicited by B. japonicum hemB or hemH mutants are underdeveloped (Frus- taci and O’Brian, 1992; Chauhan and OBrian, 1993); thus, the lack of leghemoglobin, a late-nodule protein, is expected regardless of the source of the prosthetic group.

Molecular phylogenetic analysis suggests that plant and bacterial ALA dehydratases have a common lineage not shared by animals or yeast; we interpret this as evidence that plant ALA dehydratase is derived from the bacterial chloro- plast ancestor and was transferred to the nucleus during plant evolution. Paleobotanical and molecular evidence iden- tifies the lycopsids, the group of primitive tracheophytes to which Selaginella belongs, as the basal lineage in vascular land plants (Raubeson and Jansen, 1992, and refs. therein). Thus, if one assumes that the nuclear location of the bacteria- type Alad genes analyzed herein is the result of a single transfer, the event would have occurred no less than 400 million years ago, and the nuclear-encoded gene should be prevalent in vascular land plants. Transfer of the Alad gene from the chloroplast to the nucleus may be considerably more ancient, and the identification and localization of the

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1416 Kaczor et al. Plant Physiol. Vol. 104, 1994

gene from nonvascular plants (bryophytes) and algae should clarify this.

Separate origins for ALA dehydratase between plants and animals indicates that the Chl pathway in plants is not likely the result of a n evolutionary addition of a unique branch to the heme pathway enzymes of the ancestor from which plants and animals diverged approximately 1 billion years ago. Also, the absence of a cytosolic ALA dehydratase in plants (Smith, 1988) suggests that the eukaryotic type Alad gene predicted to be inherited from that ancestor has been lost or inactivated. Evidence suggests that Chl-specific plant enzymes are descended from the chloroplast ancestor (Fujita e t al., 1992; Bauer et al., 1993; Burke et al., 1993), and the present work points to the same origin for a universal tetra- pyrrole synthesis enzyme.

NOTE ADDED IN PROOF A soybean gene encoding coproporphyrinogen oxidase,

which catalyzes the antepenultimate step in heme synthesis, is highly expressed in root nodules (Madsen et al., 1993). This observation supports the conclusion that the plant host can form heme in nodules.

ACKNOWLEDCMENTS

The authors thank Sharon Cosloy for E. coli strain RP523, Michael Kahn for the soybean leaf cDNA library, H.A.W. Schneider-Poetsch for anti-spinach ALA dehydratase antibodies, Desh Pal Verma for the ubiquitin cDNA, and Linda Walling for cab3 cDNA.

Received October 8, 1993; accepted December 13, 1993. Copyright Clearance Center: 0032-0889/94/104/1411/07. The GenBank accession number for the sequence reported in this

article is U04525.

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