Plant Physiol. (1999) 119: 593-598
Expression of a Soybean Gene Encoding the
Tetrapyrrole-Synthesis Enzyme Glutamyl-tRNA Reductase in
Symbiotic
Root Nodules1
Indu Sangwan and
Mark R. O'Brian*
Department of Biochemistry, State University of New York, Buffalo,
New York 14214
 |
ABSTRACT |
Heme and chlorophyll accumulate to
high levels in legume root nodules and in photosynthetic tissues,
respectively, and they are both derived from the universal tetrapyrrole
precursor 
-aminolevulinic acid (ALA). The first committed step in
ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase
(GTR) in plants. A soybean (Glycine max) root-nodule
cDNA encoding GTR was isolated by complementation of an
Escherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected
roots but accumulated to high levels in root nodules. The induction of
Gtr mRNA in developing nodules was subsequent to that of
the gene Enod2 (early nodule)
and coincided with leghemoglobin mRNA accumulation. Genomic analysis
revealed two Gtr genes, Gtr1 and a 3
portion of Gtr2, which were isolated from the soybean
genome. RNase-protection analysis using probes specific to
Gtr1 and Gtr2 showed that both genes were
expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression of
Gtr1 and Gtr2 were similar to each other
and to total Gtr mRNA in leaves and nodules of mature
plants and etiolated plantlets. The data indicate that
Gtr1 is universal for tetrapyrrole synthesis and that a
Gtr gene specific for a tissue or tetrapyrrole is
unlikely. We suggest that ALA synthesis in specialized root nodules
involves an altered spatial expression of genes that are otherwise
induced strongly only in photosynthetic tissues of uninfected plants.
| |
INTRODUCTION |
Soybean (Glycine max) and numerous other legumes can
establish a symbiosis with rhizobia, resulting in the formation of root nodules comprising specialized plant and bacterial cells (for review,
see Mylona et al., 1995
). Rhizobia reduce atmospheric nitrogen to
ammonia within nodules, which is assimilated by the plant host to
fulfill its nutritional nitrogen requirement. The high energy
requirement for nitrogen fixation necessitates efficient respiration by
the prokaryote within the microaerobic milieu of the nodule. The plant
host synthesizes a nodule-specific hemoglobin (leghemoglobin) that
serves to facilitate oxygen diffusion to the bacterial endosymbiont and
to buffer the free oxygen concentration at a low
tension (for review, see Appleby, 1992). Both of these functions
require that the hemoglobin concentration be high, and, indeed, it
exceeds 1 mM in soybean nodules (Appleby, 1984)
and is the predominant plant protein in that organ. Once thought to be
confined to legume nodules, hemoglobins are found throughout the plant
kingdom, and leghemoglobin likely represents a specialization of a
general plant phenomenon (for review, see Hardison, 1996). A gene
encoding a nonsymbiotic hemoglobin has been identified in soybean and
other legumes (Andersson et al., 1996); therefore, expression in
nodules involves the specific activation of a subset of genes within a
gene family. Leghemoglobin genes may have arisen from gene duplication,
followed by specialization (Andersson et al., 1996).
Hemes and chlorophyll are tetrapyrroles synthesized
from common precursors; chlorophyll is quantitatively the major
tetrapyrrole in plants, with heme and other tetrapyrroles being present
in minor amounts. Legume root nodules represent an exception, in which
heme is synthesized in high quantity in the absence of chlorophyll, thus requiring the activity of enzymes not normally expressed highly in
nonphotosynthetic tissues. Heme is synthesized from the universal
tetrapyrrole precursor ALA by seven successive enzymatic steps;
chlorophyll formation diverges after the synthesis of protoporphyrin, the immediate heme precursor (for review, see O'Brian, 1996). Biochemical and genetic evidence shows that soybean heme biosynthesis genes are strongly induced in root nodules (Sangwan and O'Brian, 1991,
1992, 1993; Madsen et al., 1993; Kaczor et al., 1994; Frustaci et al.,
1995; Santana et al., 1998), and immunohistochemical studies demonstrate that induction is concentrated in infected nodule cells
(Santana et al., 1998).
ALA is synthesized from Glu in plants by a three-step mechanism called
the C5 pathway (Fig.
1); the latter two steps are committed to
ALA synthesis and are catalyzed by GTR and GSAT, respectively (for
review, see Beale and Weinstein, 1990; Jahn et al., 1991). Plant cDNA
or genes encoding GTR (Gtr, also called HemA) and
GSAT (Gsa) have been identified in several plant species
(Grimm, 1990; Sangwan and O'Brian, 1993; Hofgen et al., 1994; Ilag et
al., 1994; Frustaci et al., 1995; Wenzlau and Berry-Lowe, 1995; Bougri
and Grimm, 1996; Kumar et al., 1996; Tanaka et al., 1996). Two genes for each enzyme have been described, and some genes are reported to be
specific to a tissue, tetrapyrrole, or light regimen (Bougri and Grimm,
1996; Kumar et al., 1996; Tanaka et al., 1996). However, soybean
Gsa1 is highly expressed in both leaves and nodules and contains a cis-acting element in its promoter that binds to
a nuclear factor found in both tissues. (Frustaci et al., 1995). In
this study we isolated soybean Gtr1 and characterized the
genetic basis of GTR expression in root nodules.
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| Figure 1.
C5 pathway for ALA synthesis. The
committed steps for ALA synthesis catalyzed by GTR and GSAT are boxed.
Glutamyl-tRNA synthetase (GluRS) and glutamyl-tRNAGlu also
participate in protein synthesis. The gene designations in plants are
shown in parentheses below the arrows.
|
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The accession number for the Gtr1 gene sequence reported
in this paper is AF105221.
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MATERIALS AND METHODS |
Bacteria and Plants
Escherichia coli strain EV149 is an ALA auxotroph
caused by a mutation in the hemA gene encoding GTR (Verkamp
et al., 1993; provided by Dr. D. Söll, Yale University, New
Haven, CT). It was grown in Luria broth or M9 medium (Ausubel et al.,
1994) containing 50 µg mL
1 ALA and also with
50 to 100 µg mL1 ampicillin when harboring
cDNA library clones. Bradyrhizobium japonicum strain I110
was the soybean symbiont used in the present work and was cultured in
glycerol-salts-yeast extract medium (Frustaci et al., 1991). We used
soybean (Glycine max) cv Essex, an inbred isoline (Lorenzen
et al., 1995), in the present work. Plants were either inoculated with
B. japonicum or uninoculated and grown in a growth chamber
under a 16-h light/8-h dark regimen at 25°C. Etiolated soybean plants
were grown in total darkness for 10 d, and either left in the dark
or exposed to direct light to green for the final 24 h before the
leaves were harvested for RNA isolation.
Isolation of cDNA and Genomic DNA Encoding GTR
Soybean nodule and leaf cDNA expression libraries in pUC18 were
gifts from Dr. M.L. Kahn (Washington State University, Pullman) and
were constructed as described previously (Udvardi and Kahn, 1991). Each
library was used to transform E. coli strain EV149, and
cells were plated on M9 medium containing 100 µg
mL1 ampicillin in the absence of ALA.
Prototrophic colonies were cultured, plasmids were isolated, and the
DNA was then used to retransform strain EV149 to confirm that
prototrophy was conferred by the plasmid rather than by a spontaneous
genomic event. The clones were initially compared by analysis of
restriction digests, and the DNA sequences of both strands of selected
clones were determined.
Genomic DNA encoding Gtr1 was obtained by PCR using primers
that delimited the GTR-encoding leaf cDNA clone and
EcoRI-digested genomic DNA as the template. The resulting
3.6-kb DNA was sequenced, and introns were identified by comparing the
genomic and cDNA sequences. A portion of Gtr2 was obtained
by PCR using primers delimiting the 3 end 610 bp from the
unique EcoRV site to the end of the cloned region of
Gtr1. The template was genomic DNA enriched for a 2-kb
fragment that hybridized to Gtr cDNA in Southern analysis.
EcoRV-digested genomic DNA was size-fractionated using a 1 to 5 M NaCl gradient, as described previously
(O'Brian and Maier, 1987). Fractions of 0.5 mL were analyzed by
Southern blotting to determine those enriched for either the 1- or 2-kb
homologous fragment. Gtr2 was found in the 2-kb fraction,
whereas Gtr1 was found in the 1-kb fraction. Errors in PCR
were ruled out as the basis for differences in DNA sequence between the
respective portions of Gtr1 and Gtr2 by
sequencing DNA from three independent PCR reactions. In addition,
RNase-protection analysis revealed differences in Gtr
transcripts based on sequence variations (see below). Sequence analysis
was carried out using Genetics Computer Group (Madison, WI)
software (Devereaux et al., 1984).
Analysis of RNA
Isolation of RNA from leaves, roots, and nodules and preparation
of poly(A+) RNA were carried out as described
previously (Sangwan and O'Brian, 1993). RNA-blot analysis was carried
out with poly(A)+ RNA under high-stringency
conditions. Gtr1 and Gtr2 mRNAs were analyzed by
RNase-protection analysis using a kit (Hybspeed, Ambion, Austin, TX).
The protocol used took advantage of differences in the RNA sequence
between the two genes by digesting unpaired nucleotides in imperfect
RNA hybrids. Antisense probes of 100 and 98 bp, complementary to
Gtr1 and Gtr2 mRNA, respectively, were prepared
using an in vitro transcription kit (MAXIscript, Ambion) according to
the manufacturer's instructions. The specificity of each antisense probe for the cognate mRNA was established by RNase-protection analysis
using in vitro-synthesized complementary sense-strand RNAs.
Conditions in which each antisense RNA would form an RNase-sensitive
duplex of an imperfect hybrid and a stable duplex with a perfect hybrid
were as follows. Hybridizations were carried out overnight at 47°C in
hybridization buffer described by Ausubel et al. (1994) rather than the
buffer provided in the kit. Hybridized RNA was digested for 45 min at
30°C with 20 units of RNase T1 and 20 units of RNase A. Then, 8 × 104 cpm of probe was used in each reaction,
and products were analyzed as autoradiograms of 7.5% acrylamide gels.
Gtr1 and Gtr2 mRNAs were analyzed using antisense
probes of almost the same size and of the same specific activity and
analyzed on the same gels. Therefore, the relative amounts of each
transcript in tissues could be assessed. Autoradiogram bands were
quantified using an imaging densitometer (model GS-700, Bio-Rad) in the
transmittance mode and the Molecular Analyst software package. Several
exposures were analyzed to quantitations made in the linear region of
the densitometer.
| |
RESULTS |
Isolation of Soybean cDNA Encoding GTR
E. coli strain EV149 is defective in hemA,
the gene encoding GTR, and behaves as an ALA auxotroph (Verkamp et al.,
1993). To isolate soybean-nodule cDNA encoding GTR, strain EV149 was transformed en masse with a soybean-nodule cDNA expression library, and
cells that were functionally complemented were selected as ampicillin-resistant, ALA-prototrophic colonies on agar medium. Eight
complementing plasmids had identical restriction-enzyme patterns, and
partial DNA sequencing of the 3 ends of the clones revealed identical
sequences, with variation only in the length of the polyadenylated
tail. One clone, pGTRN1, was chosen for further analysis. The insert of
pGTRN1 contained a 1629-bp open reading frame that encoded a peptide
542 amino acids in length beginning with a Met codon (Fig.
2). In addition, a termination codon was
identified upstream of the Met codon and in the same reading frame,
showing that the entire coding region was present in the cloned cDNA.
This peptide was highly homologous to GTRs from other plants, with the
highest identity to that from cucumber (83%; Tanaka et al., 1996).
This homology, along with complementation of the E. coli
hemA mutant, provides strong evidence that the cloned cDNA encodes
GTR.
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| Figure 2.
Nucleotide sequence and deduced product of cDNA
encoding soybean GTR. The underlined nucleotides denote the sequence
found in a leaf cDNA clone. The deduced protein shares 83% identity
with GTR from cucumber.
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The gene corresponding to the complementing clone was designated
Gtr1. A single complementing clone was isolated from a leaf cDNA library using the same selection procedure described for the
nodule library. The cDNA sequence was identical to pGTRN1 except that
an additional 70 bp was found immediately prior to the
poly(A+) tail (underlined sequence in Fig. 2).
The sequence variation likely arose from differential processing of
Gtr1 mRNA rather than from transcription of two genes (see
below). RNase-protection analysis of leaf and nodule mRNA using an
antisense probe corresponding to the 70-bp region showed that the
additional sequence was not leaf specific and was present as a minor
species (data not shown). The basis for this variation was not
studied further.
Gtr Is Induced in Root Nodules
Hemoglobin synthesis is highly induced in root nodules, as is
Glu-dependent ALA-synthesis activity and Gsa expression
(Sangwan and O'Brian, 1991, 1992, 1993; Frustaci et al., 1995). To
determine the expression pattern of Gtr, RNA-blot analysis
was performed on poly(A+) RNA from various
tissues from soybean plants using a portion of the Gtr cDNA
as a probe. Gtr mRNA accumulated to very low levels in
uninfected roots but was strongly expressed in root nodules to a level
somewhat lower than was observed in leaves from the same plants (Fig.
3A). These observations indicate that,
like Gsa (Sangwan and O'Brian, 1993; Frustaci et al., 1995;
also see Fig. 3A), induction of ALA synthesis is correlated with the
activation of Gtr.
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| Figure 3.
Northern analysis of Gtr mRNA from
soybean tissues. A, Poly(A)+ RNA (approximately 5 µg) was
analyzed from leaves (L), roots (R), and nodules (N) of 24-d-old
plants. A single filter was probed separately with radiolabeled cDNA
from Gtr, Gsa, and ubiquitin
(Ubi), and the filter was stripped after each
hybridization and exposure. Ubiquitin was used as a
control for a constitutively expressed gene. B, Leaves from illuminated
(I) or dark-treated (D) etiolated plantlets were analyzed for
Gtr, Cab, and Ubi mRNA.
Cab was used as a control for a light-regulated gene.
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Evidence indicates that ALA synthesis is induced by light in
photosynthetic tissues of some plants, at least in part because of
induction of the Gtr (HemA) gene (Ilag et al.,
1994; Bougri and Grimm, 1996; Tanaka et al., 1996). To assess the light
requirement for soybean Gtr mRNA expression in leaves,
RNA-blot analysis was carried out with poly(A+)
from leaves of etiolated plants grown completely in the dark or those
exposed to light for 24 h prior to harvesting (Fig. 3B). Cab (chlorophyll
a/b-binding protein) was
used as a control for a light-regulated gene (Chang and Walling, 1992). Gtr transcripts accumulated to high levels in etiolated
leaves. Exposure to light resulted in an approximately 3-fold increase in expression (see also Fig. 6). Thus, although Gtr mRNA is
modestly light induced, light is not required for expression.
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| Figure 6.
Analysis of Gtr1 and
Gtr2 mRNA. A, Sequence comparison of a 3 portion of
Gtr1 and Gtr2 DNA. The sequence of
Gtr1 is shown, with differences in Gtr2
shown below it. "X" denotes a gap in one sequence where a
nucleotide is present in the other. The underlined sequence denotes the
antisense riboprobe used to analyze Gtr1 (probe 1) in B
and C. The same region was used as a probe for Gtr2
(probe 2), except that it contained the nucleotide additions,
deletions, and substitutions noted in the figure. B, Antisense probe 1 and probe 2 are specific for Gtr1 and
Gtr2 mRNA, respectively. RNA sense strand 1 (S1) and
sense strand 2 (S2) are identical to portions of Gtr1
and Gtr2 mRNA, respectively, and were synthesized in
vitro and used in RNase-protection assays with radiolabeled antisense
probes 1 and 2. Each probe formed an RNase-resistant duplex with the
perfectly complementary hybrid only. C, RNase-protection analysis of
RNA from leaves (L) and nodules (N) of 24-d-old plants and from leaves
of illuminated (I) and dark-treated (D) etiolated plantlets using
riboprobes specific to Gtr1 or Gtr2
(probes 1 and 2, respectively). The intensities of bands in the first
two rows can be directly compared. Gtr2 (long) is a
longer exposure of the autoradiogram above it, which allows a better
comparison of Gtr2 between tissues.
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Root-nodule ontogeny is broadly divided into early and late
development, with the latter stage commencing with the onset of nitrogen fixation. We compared the temporal expression of
Gtr with those of the nodule-specific genes Enod2
and Lb, which are well-described markers of early and late
development, respectively (for review, see Mylona et al., 1995).
RNA-blot analysis showed that Enod2 mRNA was not
detected in uninfected roots but was found by 10 d postinfection
(Fig. 4). Gtr mRNA was weakly
expressed in 13-d-old nodules and easily discerned by 16 d and,
therefore, does not correspond well with early development. However,
the temporal pattern of Gtr expression correlated well with
that of Lb, which encodes nodule hemoglobin, and therefore
Gtr is likely to be activated later in nodule development
when needed for high levels of heme synthesis.
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| Figure 4.
Temporal expression of Gtr mRNA in
developing nodules and comparison with Enod2 and
Lb. Approximately 5 µg of poly(A+) RNA
from uninfected roots (U) and from nodules 10, 13, 16, 19, and 25 d postinfection were loaded onto each lane. A single filter was
hybridized with each radiolabeled cDNA separately, and the filter was
stripped after each hybridization and exposure.
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Isolation of Genomic DNA Encoding Gtr1 and Evidence for
a Second Gtr Gene
Genomic DNA was isolated by PCR using primers that delimited the
cDNA sequence, and the DNA sequence was determined. The cloned gene
contained two introns 1007 and 513 bp in size (Fig.
5A), which is much larger than the
introns found in the Gtr1 (HemA1) gene of
Arabidopsis (Ilag et al., 1994). The exon sequences were identical to
the corresponding sequence in the cDNA, indicating that the mRNA from
which the cDNA was synthesized is a transcript of the identified gene.
To determine whether more than one Gtr gene was present in
the soybean genome, Southern analysis of genomic DNA was carried out
using restriction enzymes and a radiolabeled probe that would yield
only one fragment if only Gtr were present (Fig. 5B). Each
digested DNA sample yielded two bands of approximately equal intensity,
indicating the presence of two Gtr genes that are very
homologous, at least in the region corresponding to the probe.
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| Figure 5.
Gene structure of Gtr1 and evidence
for two Gtr genes. A, Representation of
Gtr1 showing three exons (white bars) and two introns
(black bars). Restriction sites are shown for HindIII
(H), SphI (S), and EcoRV (RV). The open
and closed arrowheads denote the translation start and termination
sites, respectively. The probe used for the Southern analysis in B is
shown. B, Southern analysis of soybean DNA cut with
EcoRI (RI), SphI (S),
HindIII (H), or EcoRV (RV).
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To further investigate whether there were two Gtr genes, DNA
corresponding to the 3 end of Gtr was amplified from
size-fractionated EcoRV-digested genomic DNA enriched for
either the 1- or 2-kb fragment that hybridized to the probe (Fig. 5B).
DNA sequencing revealed that the 1-kb EcoRV fragment
corresponded to Gtr1, whereas the 2-kb fragment contained a
sequence highly related but not identical to Gtr1 (Fig.
6A), indicating the existence of a second gene tentatively named Gtr2. The isolated 3 portion of
Gtr2 was different from Gtr1 in 5.3% of the
nucleotides and comprised substitutions, deletions, and additions. The
high degree of relatedness of the two genes suggests a gene-duplication
event.
Soybean Gtr1 Is Universal for Tetrapyrrole
Synthesis
The identification of two Gtr genes raises the
possibility that high expression in root nodules involves activation of
a gene specific for that tissue or for heme synthesis in general. To examine the expression of the two genes, RNase-protection analysis of
RNA from various tissues was carried out using probes specific to
Gtr1 or Gtr2 (Fig. 6, B and C). In addition, the
experiments were carried out so that the relative quantity
Gtr1 and Gtr2 mRNA in each tissue could be
determined by the intensity of the bands on autoradiograms (see
``Materials and Methods''). Gtr1 mRNA was 15- to 20-fold
more abundant than the Gtr2 message in leaves and nodules of
24-d-old plants and etiolated plantlets (Fig. 6C). Thus, if
Gtr2 is a functional gene, it can account for only a minor
portion of total Gtr expression. In addition, the
qualitative patterns of expression of the two genes were similar, with
the highest levels found in leaves from mature plants and light-exposed
etiolated plantlets and lesser but significant levels found in nodules
and leaves of dark-treated etiolated plants. Therefore, strong
expression of Gtr in different tissues or light conditions
cannot be explained by differential expression of Gtr1 and
Gtr2. Finally, the expression pattern of each gene was
similar to that for total Gtr expression, as discerned by
northern analysis (Fig. 3). The data indicate that Gtr1 is expressed both in nodules for heme synthesis and in leaves, where chlorophyll is the predominant tetrapyrrole, and the findings argue
against significant expression of a Gtr gene specific for a
tissue, tetrapyrrole, or light condition.
| |
DISCUSSION |
We isolated a soybean Gtr gene that is strongly induced
in symbiotic root nodules. Gsa1 is induced similarly
(Sangwan and O'Brian, 1993; Frustaci et al., 1995), and thus ALA
synthesis correlates with the activation of both committed steps of the C5 pathway at the mRNA level. Heterogeneity in
Gtr mRNA could be attributed to the presence of two
transcribed genes, Gtr1 and Gtr2, and the likely
differential processing of Gtr1 transcripts at the 3
end. The temporal expression of Gtr in developing nodules was correlated with that of the soybean hemoglobin gene Lb,
and thus regulation of Gtr is likely to be coordinated with
nodule function rather than with the early stages of nodule
development.
ALA formation in symbiotic root nodules is unique among plants in that
synthesis is high but none is incorporated into chlorophyll. In
addition, synthesis is induced in response to parameters associated with symbiosis rather than photosynthesis, suggesting that there should
be fundamental differences in regulation in these two contexts. In
cucumber a Gtr (HemA) gene specific for
chlorophyll and one for nonchlorophyll tetrapyrroles was proposed based
on the light-dependent regulation of the former (Tanaka et al., 1996).
However, soybean Gtr1 mRNA was strongly expressed in both
leaves and nodules, and Gtr2 was expressed to a lesser
extent in a qualitatively similar manner. In addition, Gtr1
mRNA accumulation did not require light in leaves; therefore,
expression in subterranean nodules did not require a compensatory
regulatory mechanism.
The data suggest that the soybean Gtr1 gene is activated in
tissues where high levels of ALA are necessary for the synthesis of
heme or chlorophyll, and the data argue against a Gtr gene specific for a tissue or tetrapyrrole. Analysis of the Gsa1
gene yielded essentially the same conclusion (Frustaci et al., 1995). Therefore, activation of the two committed steps of the
C5 pathway during nodule development likely
requires an altered spatial pattern of expression of genes normally
induced strongly only in photosynthetic tissue. Evidence suggests that
chlorophyll synthesis is coordinated with chloroplast development
(Beator and Kloppstech, 1993). If so, then the high expression of
Gtr1 in nodules and etiolated leaves indicates that ALA
synthesis can be uncoupled from chloroplast development, and thus
Gtr1 is likely to be affected by separate and independent
signal transduction pathways.
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FOOTNOTES |
1
This work was supported by the Cooperative State
Research Service, U.S. Department of Agriculture, under agreement no.
95-37305-2253.
*
Corresponding author; e-mail mrobrian{at}buffalo.edu; fax
1-716-829-2725.
Received July 9, 1998;
accepted October 23, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ALA, -aminolevulinic acid.
GSA, glutamate
1-semialdehyde.
GSAT, glutamate 1-semialdehyde aminotransferase.
GTR, glutamyl-tRNA reductase.
| |
ACKNOWLEDGMENTS |
We thank Dr. M.L. Kahn for soybean nodule and leaf cDNA
libraries, Dr. D. Söll for E. coli strain EV149, and
Drs. T. Bisseling, K. Marcker, D.P.S. Verma, and L. Walling for soybean
cDNA clones.
| |
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Abstract
Introduction
