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Plant Physiol. (1999) 119: 817-828
NADH-Glutamate Synthase in Alfalfa Root Nodules. Genetic
Regulation and Cellular Expression1
Gian B. Trepp,
Martijn van de Mortel,
Hirofumi Yoshioka,
Susan S. Miller,
Deborah A. Samac,
J. Stephen Gantt, and
Carroll P. Vance*
Institut für Pflanzenwissenschaften Eidgenössische Technische
Hochschule-Zürich, 8092 Zürich, Switzerland
(G.B.T.); Department of Agronomy and Plant Genetics (G.B.T., M.v.d.M.,
H.Y., S.S.M., C.P.V.), Department of Plant Pathology (D.A.S.), and
Department of Plant Biology (J.S.G.), University of Minnesota, 411 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, Minnesota 55108; University of Minnesota, 411 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, Minnesota 55108Plant Pathology Laboratory, Nagoya University, Chikusa, Nagoya 464-01,
Japan (H.Y.); and United States Department of Agriculture, Agricultural
Research Service, Plant Science Research Unit, 411 Borlaug Hall, 1991 Upper Buford Circle, University of Minnesota, St. Paul, Minnesota 55108 (D.A.S., C.P.V)
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ABSTRACT |
NADH-dependent glutamate synthase
(NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen
assimilation in alfalfa (Medicago sativa L.) root
nodules. Here we report that in alfalfa, a single gene, probably with
multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were
performed to assess the location of NADH-GOGAT transcript in alfalfa
root nodules. In wild-type cv Saranac nodules the
NADH-GOGAT gene is predominantly expressed in infected
cells. Nodules devoid of bacteroids (empty) induced by
Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript
detectable by in situ hybridization, suggesting that the presence of
the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early
symbiotic zone to low in the late symbiotic zone was observed. In
33-d-old nodules expression was seen in only a few cell layers in the
early symbiotic zone. This pattern of expression was also observed for
the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa
plants carrying chimeric  -glucuronidase promoter fusions. The
results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an
88-bp direct repeat.
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INTRODUCTION |
In most plants primary assimilation of inorganic N results from
the collaborative activity of two enzymes, GS (EC 6.3.1.2) and GOGAT
(EC 1.4.1.14) (Lea et al., 1990 ; Lam et al., 1996). GS catalyzes the
ATP-dependent amination of glutamate, producing Gln. GOGAT catalyzes
the reductive transfer of the amido group of Gln to the  -keto
position of 2-oxoglutarate, yielding two molecules of glutamate (Boland
and Benny, 1977). GOGAT, together with GS, maintains the flow of N from
NH4+ into Gln and glutamate.
These products are then used for several other aminotransferase
reactions in the synthesis of amino acids (Lea et al., 1990). Kinetic
and inhibitory studies suggest that GOGAT is the rate-limiting step
through the GS/GOGAT cycle (Chen and Cullimore, 1989; Baron et al.,
1994).
In higher plants GOGAT occurs as two distinct forms, using Fd or NADH
as a reductant. In addition to reductant specificity, the enzymes
differ in molecular mass, kinetics, and antigenicity (Lea et al., 1990;
Temple et al., 1998). Fd-GOGAT (EC 1.4.7.1) is localized in
chloroplasts (Wallsgrove et al., 1979; Becker et al., 1993). The
monomeric enzyme is suggested to be an Fe-S protein (Hirasawa and
Tamura, 1984; Knauff et al., 1991) with a molecular mass of 140 to 160 kD (Lea et al., 1990; Temple et al., 1998). Fd-GOGAT cDNAs have been
cloned from maize (Sakakibara et al., 1991), tobacco (Zehnacker et al.,
1992), barley (Avila et al., 1993), alfalfa (Vance et al., 1995),
Arabidopsis (Lam et al., 1996), and spinach (Nalbantoglu et al., 1994).
Application of NO3 , which
strongly induces nitrate reductase (EC 1.6.6.1), has only a minor
effect on the expression of Fd-GOGAT in leaves, suggesting that the
major role of Fd-GOGAT is probably the reassimilation of ammonia
derived through photorespiration (Somerville and Ogren, 1980; Kendall
et al., 1986; Sakakibara et al., 1992).
Like Fd-GOGAT, the plant NADH-GOGAT protein (EC 1.4.1.14) is a monomer
with a molecular mass of 200 to 240 kD (Gregerson et al., 1993b; Vance
et al., 1995; Temple et al., 1998). cDNA clones have been reported for
Arabidopsis (Lam et al., 1996) and alfalfa (Gregerson et al., 1993b),
and the enzyme has been purified from pea (Chen and Cullimore, 1988),
lupine (Boland and Benny, 1977), rice (Hayakawa et al., 1992), and
alfalfa (Anderson et al., 1989). Additionally, genes corresponding to a
rice (Goto et al., 1998) and an alfalfa NADH-GOGAT cDNA (Vance et al.,
1995) have been isolated and characterized. In alfalfa the NADH-GOGAT
transcript is 7.2 kb long and encodes a 2194-amino acid protein,
including a 101-amino acid precursor peptide. The NADH-GOGAT
gene is 14 kb long and is composed of 22 exons interrupted by 21 introns. In green tissues of alfalfa NADH-GOGAT enzyme activity and the abundance of its mRNA is low to nondetectable (Gregerson et al., 1993b). However, NADH-GOGAT activity increases severalfold during alfalfa root nodule development (Gregerson et al., 1993b) and is
the major form of GOGAT in this tissue (Vance et al., 1995). This
increase corresponds with an increase in enzyme protein and mRNA
(Gregerson et al., 1993b). Moreover, this increase in NADH-GOGAT expression appears to be restricted to nodules capable of
N2 fixation. In contrast, other nodule-enhanced
enzymes involved in N2 assimilation, such as
aspartate aminotransferase (EC 2.6.1.1), GS, PEP carboxylase (EC
4.1.1.31), and Asn synthetase (EC 6.3.5.4), were expressed in both
effective and ineffective nodules (Groat and Vance, 1981; Vance and
Gantt, 1992; Vance et al., 1994).
Further understanding of the regulation of NADH-GOGAT in
alfalfa nodules necessitates the identification of cellular patterns of
transcript accumulation and the definition of elements in the 5
flanking region of the gene that controls expression in nodules. In
this report we document NADH-GOGAT transcript accumulation in cells of
both effective and ineffective nodules during root nodule development
and relate this expression to that of nitrogenase and leghemoglobin. In
addition, promoter deletion analysis using the GUS reporter gene in
transgenic alfalfa is performed to define elements controlling root
nodule specificity.
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MATERIALS AND METHODS |
Plant Material and Bacterial Strains
Alfalfa (Medicago sativa L.) cv Saranac and a single
gene-recessive genotype "ineffective Saranac"
(in1 Saranac) forming
non-N2-fixing, early-senescing nodules (Peterson
and Barnes, 1981) were used in this study. Seeds were obtained from Dr.
J.F.S. Lamb (U.S. Department of Agriculture, Agricultural Research
Service, St. Paul, MN). Although alfalfa is an outcrossing-tetraploid
species, precluding the formation of isogenic lines, more than 90% of
the in1 Saranac genotype comes from the cv
Saranac background.
Plants of these two genotypes were maintained in greenhouse sand
benches and inoculated with effective Sinorhizobium meliloti 102F51 as described by Egli et al. (1989). For analysis of NADH-GOGAT expression in bacterial conditioned ineffective nodules, cv Saranac seeds were surface-sterilized (70% ethanol for 10 min) and planted in
sterilized sand inoculated with ineffective S. meliloti
F642, dctA (dicarboxylic acid uptake deficient; Yarosh et
al., 1989) or 7154, exoH (acid exopolysaccharide
succinylation deficient; Leigh et al., 1987). These strains were
generous gifts of Turlough Finan (McMaster University, Hamilton,
Ontario, Canada) and Ann Hirsch (University of California, Los
Angeles), respectively. The plants inoculated with mutant
Sinorhizobium strains were grown in growth chambers
(Conviron PGV 36, Controlled Environment, Ltd., Winnipeg,
Manitoba, Canada2).
To exclude contamination with wild-type Sinorhizobium,
nitrogenase activity was assayed as H2 evolution
in an open-flow system adapted from Minchin et al. (1983) using a
nitrogenase-activity analysis system (Morgan Scientific, Haverhill,
MA). For all studies the planting date was designated as d 0.
PCR for NADH-GOGAT
Two degenerate oligonucleotide primers were synthesized for the
amplification of NADH-GOGAT genomic sequences. Each primer is
complementary to conserved sequences in the Fd-GOGAT of tobacco (Zehnacker et al., 1992) and barley (Avila et al., 1993) and the NADH-GOGAT of alfalfa (Gregerson et al., 1993b) and Escherichia coli (Oliver et al., 1987) (see Fig. 1). The primer sequences are as follows: primer 1, GCNAT(A/C/T)AA(A/G)CA (A/G)GTNC;
primer 2, CCNCCNGTCAT(A/G)TA(T/C)TC(A/G)CA. Fifty microliters of
PCR reaction contained 0.1 µg of genomic alfalfa DNA, 2.5 mM MgCl2, 1×
Taq DNA polymerase buffer (50 mM KCl,
10 mM Tris-HCl, 0.1% Triton X-100, pH 9.0)
(Promega), 200 µM of each deoxyribonucleotide triphosphate, 25 pmol of primer, and 5 units of Taq DNA
polymerase (Promega). PCR was performed for 30 cycles and the
amplification conditions were 95°C for 1 min followed by 45°C for
30 s and 72°C for 2 min; final elongation was at 72°C for 5 min. The amplification products were purified and cloned into a pGEM-T
vector (Pharmacia). The PCR product was sequenced by chain terminators
with the T7 primer using Sequenase (Amersham). Sequence analysis was
performed using the Genetics Computer Group (Madison, WI)
sequence-analysis software package.
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| Figure 1.
Diagrammatic representation of the portion of the
alfalfa NADH-GOGAT gene used for PCR. Exons are
indicated by boxed regions, and introns are represented by lines. PCR
was performed over three exons (exon 14 [E14], exon 15 [E15], exon
16 [E16]) and two introns (intron 14 [I14] and intron 15 [I15])
of the reported alfalfa gene using primer 1 (AIKQVA) and primer 2 (CEYMTGG). The putative binding for the (Fe-S) cluster is indicated by
arrows, and the putative flavin mononucleotide binding site is
underlined.
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Preparation of RNA Probes
All probes used in this study were generated from linearized
pBluescript KS+ plasmids (Stratagene), containing
a 1.8-bp (BamHI-NotI) NADH-GOGAT cDNA fragment
(Gregerson et al., 1993b), a 1.1-bp nifH gene
fragment, or a 0.9-bp leghemoglobin cDNA fragment. Each construct was
linearized with the appropriate enzymes and transcribed in the sense
and antisense directions using T3 or T7 RNA polymerases and
[35S]UTP as described by the manufacturer
(Stratagene). The nifH gene fragment and the leghemoglobin
cDNA fragment were generous gifts from Mike Sadowsky (University of
Minnesota, St. Paul) and Ann Hirsch, respectively. The probes were
partially degraded to a length of 150 nucleotides by heating at 60°C
in a 0.06 M
Na2CO3/0.04 M NaHCO3 solution (Cox and
Goldberg, 1988).
In Situ Hybridization
The tissue for all in situ hybridizations was collected as
follows. The planting date is considered to be d 0; at harvest dates,
only the two top root nodules on the main root were used for subsequent
studies. In situ hybridization was carried out essentially as described
by Fleming et al. (1993). Tissues were fixed in 4% paraformaldehyde
and 0.25% glutaraldehyde in 50 mM sodium phosphate buffer,
pH 7.2. The tissues were then rinsed twice in the same buffer and twice
in deionized water, and dehydrated in a graded ethanol series. After
the absolute ethanol was replaced with xylene, tissues were embedded in
Paraplast (Oxford Labware, St. Louis, MO). The embedded tissues were
sectioned at a thickness of 7 µm and affixed to
poly-L-Lys-coated slides. Hybridizations were performed in
mineral oil as described by Heintzen et al. (1994), and the final wash
conditions were 0.1× SSC (1× SSC = 0.15 M NaCl, 15 mM sodium citrate) including 1 mM DTT at 50°C for 20 min. In situ hybridization slides of effective nodules were
exposed to emulsion for 10 to 14 d, and those performed with ineffective nodules were exposed for 26 to 28 d. After
development the sections were stained with 0.05% toluidine blue O,
dehydrated, and mounted with Permount (Fisher Scientific). Sections
were viewed and photographed with a Labophot microscope (Nikon)
equipped with dark- and bright-field optics.
Construction of Chimeric Genes and Plant Transformation
Standard DNA techniques were used for DNA manipulations (Sambrook
et al., 1989). Sequence analyses of these chimeric genes demonstrated
that each insert was correctly cloned. Nine promoter constructs were
generated, one transcriptional and eight translational fusions. The 3
end of the transcriptional fusion (GScript) is 103 bp 5 of the
translational start codon of NADH-GOGAT. Constructs GLate
and G1 through G7 are translational fusions to the GUS gene, and the 3
end of all these constructs is 8 bp past the ATG of the
NADH-GOGAT gene. GLate, G5, and G7 are restriction fragments from the originally isolated 5 upstream region of the
NADH-GOGAT gene. G1, G2, G3, G4, and G6 are PCR products
that have been amplified using a common 3 primer and a specific 5
primer. All promoter sequences were cloned in the vector pBI101.2
(Clontech, Palo Alto, CA).
The promoter-GUS constructions were introduced into Agrobacterium
tumefaciens LB4404 by electroporation. Transgenic alfalfa plants
were obtained essentially as described by Austin et al. (1995) using a
clone selected from the alfalfa cultivar, RegenSY (Bingham, 1991).
Regenerated plants were propagated by cuttings. Verification that
plants were transformed with the respective constructs was obtained by
PCR analysis of DNA extracted from the regenerated plants. Cuttings
were maintained in vermiculite and inoculated with S. meliloti strain 102F51. Nodule tissues were collected 12 to
26 d after inoculation for GUS enzymatic assays and histochemical
detection.
Analysis of GUS Activity
The detection of GUS activity in nodules was performed by the
procedure described by Jefferson (1987). The GUS-stained tissues were
fixed in 4% paraformaldehyde and 0.25% glutaraldehyde in 50 mM sodium phosphate buffer, pH 7.2, rinsed in deionized
water, and stored in 70% ethanol until they were photographed. GUS
activity was determined as described by Jefferson (1987). Nodules were collected and homogenized in extraction buffer. After centrifugation the supernatant was used to quantitate GUS activity using
4-methylumbelliferyl -D-glucuronic acid as a substrate.
Tissues from regenerated nontransformed plants were used to quantitate
background activity. Enzyme activity was determined with a
minifluorometer (TKO-100; Hoefer Scientific, San Francisco, CA). The
protein concentration was determined using protein assay (Bio-Rad). The
kinetic data obtained were further analyzed using a statistical program
(Statistix, Analytical Software, Tallahassee, FL). Because the
sample population for each promoter-deletion construct did not have a
normal distribution, the data were transformed (log[x + 1]) to achieve homogeneity of variance. The transformed data were then
analyzed using analysis of variance and the corresponding LSD was calculated. The level of significance was
set at P < 0.01. Because many differences in GUS activity for
adjacent promoter constructs were not significant, we further analyzed
the data using linear regression. Therefore, the transformed means were plotted against the corresponding length of the deletion (bp) (1/log[bp + 1]). We found a positive linear relation between deletion length and GUS activity (r2 adjusted = 73.37%; P = 0.0041).
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RESULTS |
Screening for Other NADH-GOGAT Isoforms in the Alfalfa Genome by
PCR
To determine if there are other NADH-GOGAT isoforms in alfalfa, we
designed degenerate primers from the conserved regions of the deduced
amino acid sequence of Fd-GOGAT from tobacco (Zehnacker et al., 1992)
and barley (Avila et al., 1993) and the NADH-GOGAT from alfalfa
(Gregerson et al., 1993b) and E. coli (Oliver et al., 1987)
(Fig. 1). PCR reactions were performed
with alfalfa genomic DNA (cv Saranac), and all PCR products of at least
the size of the corresponding NADH-GOGAT cDNA fragment (1.4 bp) were cloned into pGEM-T (Pharmacia). Fourteen positive clones derived from
four independent PCR reactions were greater than 99% similar to the
originally reported NADH-GOGAT gene (Vance et al., 1995) for
exon as well as intron sequences.
Localization of NADH-GOGAT mRNA in Alfalfa Root Nodules by in Situ
Hybridization
To localize the NADH-GOGAT transcript in root nodules, we
performed in situ hybridization. Nodule ultrastructure was classified based on the nomenclature of Vasse et al. (1990). Five distinct zones
were defined along the axis in a longitudinal section through an
alfalfa nodule: the nodule meristem (zone I), passing to the invasion
zone (zone II), the amyloplast-rich interzone (zones II-III [*]),
the N2-fixing zone and proximal inefficient zone (zone III), and the senescent zone (zone IV) (Figs.
2A and 3G). When sections of a 19-d-old
nodule were hybridized with a NADH-GOGAT antisense RNA probe,
hybridization specific for NADH-GOGAT mRNA, seen in the dark field as a
bright silver grain, was observed in the interzone and the
N2-fixing zone (Fig. 2, A and B). No NADH-GOGAT
transcript was detected in the meristem (Fig. 2, C and F), the invasion
zone (Fig. 2, C and D), or the cortical tissue (Fig. 2, E and F).
Enhanced NADH-GOGAT gene expression began just adjacent to
the invasion zone (Fig. 2, C and D). Little NADH-GOGAT hybridization
signal was found in uninfected cells (Fig. 2, C-H), suggesting that
NADH-GOGAT is predominantly expressed in the infected cells. No
significant hybridization signal was obtained when alfalfa root nodules
were probed with a NADH-GOGAT sense RNA probe (Fig. 2, I-L).
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| Figure 2.
Localization of NADH-GOGAT mRNA in alfalfa root
nodules by in situ hybridization. Bright-field (A, C, E, G, I, and K)
and dark-field (B, D, F, H, J, and L) photographs of a longitudinal
section through 19-d-old effective alfalfa nodules. Nodule
ultrastructure was classified based on the nomenclature of Vasse et al.
(1990): meristem (zone I), invasion zone (zone II), interzone (*),
N2-fixing zone (zone III), and senescent zone (zone IV).
The nodule in A to H was hybridized with NADH-GOGAT
35S-labeled antisense RNA probe. Enlargement of the lower
boxed region in A shown in bright-field (C) and dark-field (D)
photographs includes a portion of the meristem (zone I), invasion zone
(zone II), and interzone (*). Enlargement of the upper box in A shown
in bright-field (E) and dark-field (F) photographs includes the central
zone (III), including parenchymal tissue (PA). In bright-field (G) and
dark-field (H) photographs of infected and uninfected cells, arrows in
C through H point to uninfected cells. Bright-field (I and K) and
dark-field (J and L) photographs of a longitudinal section through a
19-d-old effective alfalfa nodule hybridized with NADH-GOGAT
35S-labeled sense RNA probe. Bars in B and J = 800 µm; bars in D, F, H, and L = 80 µm.
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In Situ Localization of NADH-GOGAT mRNA during the Development of
Effective and Ineffective Nodules
We previously reported an increase in the abundance of
NADH-GOGAT-specific transcripts in RNA isolated from developing
effective nodules, from d 8 after inoculation to d 33. A comparable
increase was not detected in either plant- or bacteria-controlled
ineffective nodules (Gregerson et al., 1993b; Vance et al., 1994). To
study the regulation of the NADH-GOGAT gene during nodule
development in effective cv Saranac and plant-controlled ineffective cv
Saranac nodules, in situ hybridizations were performed comparing the
spatial and temporal expression patterns of NADH-GOGAT using 7-, 9-, 19-, and 33-d-old nodules. Hybridization specific for NADH-GOGAT mRNA was detected in effective 7-d-old nodules 2 d before the onset of
N2 fixation (Fig.
3, A and B) (Gantt et al., 1992; G.B.
Trepp and C.P. Vance, unpublished results). Similarly, ineffective
nodules also had a detectable hybridization signal for the NADH-GOGAT transcript at this time (Fig. 3, I and J). By d 9, at the onset of
N2 fixation (Gantt et al., 1992; G.S. Trepp and
C.P. Vance, unpublished results), we observed a strong signal in the
infected cells of effective nodules throughout the central nodule
tissue (Fig. 3, C and D). In plant-controlled ineffective nodules
hybridization to NADH-GOGAT mRNA was also observed in the infected
cells (Fig. 3, K- N). As a result of early senescence the nodules
generally contained a smaller number of infected cells (Fig. 3, M and
N). In these ineffective nodules the signal representing NADH-GOGAT transcript was observed in a thin layer of cells behind the invasion zone where the infected cells were intact.
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| Figure 3.
Localization of NADH-GOGAT transcript in effective
and plant-gene-controlled ineffective alfalfa nodules during
development. Longitudinal sections through effective (A-H) and
plant-controlled ineffective (I-T) alfalfa root nodules. The
bright-field and dark-field image pairs are of 7- (A, B, I, and J), 9- (C, D, K, L, M, and N), 19- (E, F, O, P, Q, and R), and 33- (G, H, S,
and T) d-old root nodules. Bars = 400 µm.
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At d 19 effective nodules had a very strong signal for NADH-GOGAT
transcript in the infected cells. Expression was highest in the
interzone and decreased gradually to a low signal in the proximal
regions of the nodule (Fig. 3, E and F). In contrast, every 19-d-old
ineffective cv Saranac nodule examined contained large senescing zones.
Additionally, the infected cells in these nodules were arranged in two
patterns. One formed an enlarged 8- to 15-cell-wide symbiotic zone
followed by a large senescent zone (Fig. 3, O and P), and the second
formed a very short 2- to 4-cell-wide symbiotic zone followed by a
large senescent zone (Fig. 3, Q and R). In both types the signal
appeared to be restricted to the infected cells. In 33-d-old nodules
the in situ hybridization signal for NADH-GOGAT mRNA in cv Saranac
nodules was observed only in the 5- to 15-cell-wide zone including the
interzone and the distal part of the N2-fixing
zone (Fig. 3, G and H). No NADH-GOGAT transcripts were apparent in the
proximal part of the N2-fixing zone or the
senescent zone. The signal specific for NADH-GOGAT mRNA at 33 d in
ineffective nodules had the same distribution pattern that was
described for the effective nodule (Fig. 3, S and T). However,
hybridization was very weak and several attempts had to be made to
obtain a sufficient signal.
Localization of NADH-GOGAT, nifH, and Leghemoglobin
in 33-d-old Alfalfa Root Nodules Using Serial Sections
We performed in situ hybridizations to test whether the expression
pattern of NADH-GOGAT in 33-d-old nodules was unique or whether this is a common pattern for genes involved in
N2 fixation. We made serial sections, which
allowed us to directly compare the expression patterns of
NADH-GOGAT, a subunit of the nitrogenase protein
(nifH), and leghemoglobin (mslbc3) in the same
nodule. The signal for NADH-GOGAT mRNA agrees with the previously
described pattern (Fig. 3, G and H). NADH-GOGAT gene
expression was detected in a 5- to 15-cell-wide zone including the
interzone (zone II-III [*]) and the N2-fixing
zone (zone III) (Fig. 4, A and B). The signal corresponding to leghemoglobin gene expression was detected throughout the nodule interior, including the invasion zone (zone II),
the interzone (zone II-III [*]), and the
N2-fixing zone (zone III). However, expression
appeared strongest in a 5- to 15-cell-wide zone including the interzone
(zone II-III [*]) and the N2-fixing zone (zone
III) (Fig. 4, C and D). The signal specific for nifH gene
expression was in the N2-fixing zone (zone III)
in a 5- to 15-cell-wide area similar to that observed for NADH-GOGAT
(Fig. 4, E and F).
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| Figure 4.
In situ localization of the NADH-GOGAT,
nifH, and leghemoglobin transcripts in 33-d-old alfalfa
root nodules using serial sections. The bright-field and dark-field
image pairs are of longitudinal sections through 33-d-old root nodules
that were hybridized with 35S-labeled NADH-GOGAT (A and B),
leghemoglobin (C and D), or nifH (E and F) antisense RNA
probe. Bars = 400 µm.
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Localization of NADH-GOGAT Transcripts in Ineffective Nodules
Formed on Effective cv Saranac by Ineffective S. meliloti Strains
NADH-GOGAT appears to be expressed predominantly in infected
cells. We addressed the question of whether the presence of S. meliloti was necessary for NADH-GOGAT gene
expression in root nodules. Therefore, we examined ineffective nodules
derived through the infection of two S. meliloti
fix mutants.
Nodules formed by S. meliloti F642, which is deficient in
dicarboxylic acid transport (Yarosh et al., 1989), are generally much
smaller than wild-type nodules. These nodules contain S. meliloti; however, the S. meliloti does not
differentiate into fully developed bacteroids and therefore cannot fix
atmospheric dinitrogen. In 19-d-old nodules induced by S. meliloti F642, NADH-GOGAT transcripts were detected in the
infected cells (Fig. 5, A and B). The
transcript abundance is low, comparable to that of ineffective cv
Saranac.
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| Figure 5.
In situ localization of NADH-GOGAT transcripts in
nodules induced by S. meliloti F642 and 7154. The
bright-field (A and D) and dark-field (B, C, E, and F) images are of
longitudinal sections through 19-d-old root nodules induced by
S. meliloti F642 (A-C) and S. meliloti
7154 (D-F). The sections in A, B, D, and E were hybridized with
35S-labeled NADH-GOGAT antisense RNA probe, whereas those
in C and F were hybridized with sense riboprobes and photographed under
dark-field conditions. Bars = 400 µm.
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S. meliloti strain 7154 (Leigh et al., 1987), which is
deficient in the acid exopolysaccharide succinylation, induces aborted infection threads and thus the S. meliloti is not released
into plant cells. S. meliloti 7154 is able to induce the
formation of root nodules; however, they are tumor-like,
non-N2-fixing nodules. In 19-d-old nodules formed
by S. meliloti 7154, we were unable to detect a significant
hybridization signal to NADH-GOGAT transcripts (Fig. 5, D and E).
Control experiments performed with sense riboprobes showed no
significant hybridization signal (Fig. 5, C and F).
Analysis of the 5 Upstream Region of the NADH-GOGAT
Gene in Transgenic Alfalfa Plants
Our previous data (Gregerson et al., 1993b; Vance et al., 1995)
demonstrated that during the development of effective alfalfa nodules
NADH-GOGAT transcripts increased some 10- to 20-fold, whereas roots and
leaves accumulated little or no transcript. Moreover, a translational
fusion made with the putative promoter region of NADH-GOGAT fused to
the GUS gene directed reporter gene activity to the nodules of alfalfa
and trefoil (Vance et al., 1995). These data, together with the in situ
hybridization results described above, led us to further investigate
the transcriptional regulation of the NADH-GOGAT gene. To
further investigate that the 5 flanking region of
NADH-GOGAT conferred expression in root nodules, GUS enzyme
activity was evaluated in alfalfa plants transformed with either a
translational (GLate) or a transcriptional (GScript) fusion. GUS enzyme
activity was 3- to 20-fold higher in nodules than in leaves, stems, and
roots, irrespective of whether reporter gene activity was controlled by
a translational (GLate) or a transcriptional (GScript)
NADH-GOGAT promoter construct (Table
I). The differences in GUS enzyme
activity between GLate and GScript were not significant. In an attempt
to identify elements in the 5 flanking region of the gene that direct
and limit expression to the root nodule, chimeric reporter genes were
constructed containing sequences of various lengths upstream of the
transcriptional start site fused to GUS (Fig.
6). These constructs were used to
transform alfalfa, and the regenerated plants were assayed for GUS
activity and stained for histochemical localization of GUS (Fig. 6A).
GUS activity in transgenic plants was quantified by a fluorometric assay (Jefferson, 1987) (Fig. 6B). Nodules taken from plants containing the longest deletion, GLate ( 2700), had the greatest total GUS activity. Although total GUS activity was reduced in nodules from plants containing deletions G1, G2, G3, and G4, statistical analysis of
adjacent deletions showed no significant differences (P < 0.01). However, when the region between G4 (356) and G5 (241) was deleted, GUS activity decreased significantly (P < 0.01). In contrast, when the region from G5 (241) to G6 (151) was deleted, the GUS activity increased dramatically; the deletion of G6 (151) to G7
(82) reduced all GUS activity from nodules to background levels (P < 0.01).
View this table:
[in this window]
[in a new window]
|
Table I.
Organ-specific GUS activity ±SE in
transgenic alfalfa containing the GUS gene driven by the NADH-GOGAT
promoter
|
|
View larger version (22K):
[in this window]
[in a new window]
| Figure 6.
Diagram of the NADH-GOGAT promoter-GUS
gene constructs. A, Eight sequences containing the putative
NADH-GOGAT promoter were translational fusions to the GUS
gene; the length of these truncations is measured with respect to the
transcriptional start. The boxed region in construct G1 marks the
location of the 88-bp direct repeat. The number of individual
transformants tested for each construct is indicated (#). Mean GUS
activity (pmol min1 mg1 protein) is
indicated with the heading "Mean." LSD values between
adjacent deletions were calculated, and the level of significance was
set at P < 0.01. N.S., Not significant; *, significant at P < 0.01. B, Distribution of GUS activity per construct measured from
individual independently transformed plants.
|
|
The two largest constructs, GLate (2700) and G1 (1064), directed
reporter gene activity to the N2-fixing zone
(zone III) of effective root nodules (Fig.
7, A and B), consistent with in situ
hybridization data. However, it was not unusual to find the root nodule
meristem (zone I), the invasion zone (zone II), and/or the vascular
tissue staining in some plants carrying GLate and G1 promoter
constructs. When the G1 construct was shortened, resulting in construct
G2, significantly different patterns of GUS localization were noted
(Fig. 7C). In root nodules the G2 promoter fragment (655) directed
reporter gene expression to the vascular tissue and/or the root nodule
meristem, but not the central zone of the nodule. These data indicate
that elements within the 5 upstream region between G1 (1064) and G2
(655) control reporter-gene expression in the
N2-fixing zone. Within this region is an 88-bp direct repeat. Plants carrying deletions G3, G4, G5, and G6 had a
staining pattern similar to that of plants carrying deletion G2 (data
not shown). Construct G7 resulted in a loss of all GUS staining in
nodules (Fig. 7D). These data indicate that two regions of the 5
upstream region contribute to nodule activity. The region from 1064
to 655 appears to control reporter gene activity in the interior
N2-fixing zone, whereas the region from 155 to
89 contains elements that affect activity in other zones of the
nodule.
View larger version (40K):
[in this window]
[in a new window]
| Figure 7.
GUS staining of the NADH-GOGAT promoter
deletions. For deletions GLate (A) and G1 (B), individual transformants
with GUS activity close to the mean had a GUS staining pattern similar
to the results obtained for in situ hybridizations. A loss in tissue
specificity was observed in deletion G2 (C), whereas no GUS staining
was observed in deletion G7 (D). E, The 409-bp region between deletion
G1 and G2. The 88-bp direct repeat is overlined, and the location of
the conserved octanucleotide motif is underlined.
|
|
| |
DISCUSSION |
To accurately determine the expression pattern of
NADH-GOGAT in root nodules through in situ hybridization
experiments and promoter deletion analysis studies, we thought it
necessary to assess whether alfalfa contains multiple
NADH-GOGAT genes. Previous results (Gregerson et al., 1993b;
Vance et al., 1995) indicated that NADH-GOGAT occurred as
either a small gene family or a single gene with multiple alleles. The
fact that all PCR products amplified from genomic DNA with degenerate
primers were greater than 99% identical across introns gives strong
support for the latter interpretation. Additionally, the
NADH-GOGAT gene has been mapped to a single locus on the
genetic map of alfalfa (G.B. Kiss, personal communication). However,
multiple alleles of a single gene would be expected in this
outcrossing, tetraploid species. Multiple alleles have also been
reported for the aspartate aminotransferase and PEP carboxylase genes
(Gregerson et al., 1993a; Pathirana et al., 1997). A single form of
NADH-GOGAT is also consistent with biochemical data showing a single form of NADH-GOGAT in alfalfa (Anderson et al., 1989). In
contrast, two isoforms of NADH-GOGAT have been documented in bean root
nodules (Chen and Cullimore, 1988), and a preliminary report suggests
two distinct cDNAs (M. Lara, L. Blanco, and C.P. Vance, unpublished
results).
In situ hybridization experiments give new insights into the regulation
of the NADH-GOGAT gene in developing effective and ineffective alfalfa root nodules. The transcript specific for NADH-GOGAT is triggered in the infected cells of the interzone of
alfalfa root nodules. Toward the proximal end of zone III (the N2-fixing zone), NADH-GOGAT expression
declines as infected cells become older. Similar results have been
reported for leghemoglobin (de Billy et al., 1991), GS (Temple et al.,
1995), and PEP carboxylase (Pathirana et al., 1997). The interzone
seems to be an area where several developmental changes occur. In
addition to the enhanced expression of several genes encoding enzymes
of the N and C metabolism, bacteroid morphology shifts from
nonfixing type 3 to larger, more pleiomorphic
N2-fixing type 4 (Vasse et al., 1990). Also in
this region, plant cells enlarge substantially, amyloplasts accumulate, and in pea and vetch the expression of the early nodulins PsENOD5, PsENOD12, and VsENOD12 declines (Scheres et al., 1990; Franssen et al.,
1992; Vijn et al., 1995).
In previous studies, which relied on RNA-blot analysis, we detected
little to no NADH-GOGAT transcript in plant-controlled ineffective
nodules (Gregerson et al., 1993b; Vance et al., 1995). The enhanced
sensitivity of detection by in situ hybridization coupled to cellular
localization of transcript allowed us to determine that NADH-GOGAT is
expressed in plant-controlled ineffective nodules at d 7, synchronous
with expression occurring in effective nodules. By d 9 and 19 in
ineffective nodules, NADH-GOGAT expression was limited to
only a few layers of infected cells. At these same times in the
effective symbiosis, NADH-GOGAT transcripts were detected throughout
the nodule. Although it is difficult to estimate precise quantitative
differences in the amount of transcripts using in situ hybridization,
it appears that the amount of NADH-GOGAT transcript is substantially
lower in the cells of the ineffective symbiosis than in the cells of
the effective symbiosis. Thus, not only do ineffective nodules have a
reduced number of cells expressing NADH-GOGAT, but they also
have a lower total amount of transcript.
The reduced amount of NADH-GOGAT gene expression probably
results from several factors. Nodules formed on ineffective cv Saranac plants and nodules formed by S. meliloti F642 show early
senescence. Egli et al. (1989) and Pladys and Vance (1993) showed
cellular deterioration and enhanced proteolysis in 9-d-old ineffective cv Saranac nodules, resulting in a substantial reduction of infected cells. Alternatively, the reduced amount of transcript could be attributable to either the lack of
NH4+ (or product derived from
NH4+) or the failure of S. meliloti to become fully differentiated into competent bacteroids.
Vasse et al. (1990) noted that bacteroids in ineffective cv Saranac do
not develop beyond the type 3 stage. Bacteroids in this stage in
normal N2-fixing nodules are smaller, have a
less pleiomorphic shape, and do not fix N2
compared with type 4 bacteroids.
The intense hybridization of NADH-GOGAT transcript seen in infected
cells and the absence of a hybridization signal in other developmental
zones and uninfected cells suggest that the presence of
bacteria/bacteroid may be a prerequisite for NADH-GOGAT expression. This point is further supported by the absence of NADH-GOGAT transcript in empty nodules induced by S. meliloti 7154. Although the
presence of bacteria/bacteroid appears to be a prerequisite to trigger NADH-GOGAT transcription, expression does not require active
nitrogenase. Two lines of evidence support this thinking. First, we
found that NADH-GOGAT transcripts can be detected in 7-d-old
nodules, significantly earlier than nitrogenase activity
can be measured (Gantt et al., 1992). Second, transcripts of
NADH-GOGAT can be detected, albeit at reduced levels,
formed in response to fix bacteria such as
S. meliloti F642 or from the plant-controlled ineffective
genotype in1 Saranac.
Although the presence of bacteria/bacteroid may be a prerequisite to
trigger NADH-GOGAT gene expression, a different mechanism may be involved in the down-regulation of the gene. In 33-d-old nodules
NADH-GOGAT transcripts are restricted to a 5- to
15-cell-wide zone, including the interzone and the proximal part of the
N2-fixing zone. Although bacteroids are present
in the distal part of 33-d-old nodules, no signal for the
NADH-GOGAT transcript could be detected. By comparing
NADH-GOGAT to nifH and leghemoglobin expression, it appears that the NADH-GOGAT expression pattern is similar
to that of nifH, but not to that of leghemoglobin.
Therefore, similar or common factors may be involved in the regulation
of NADH-GOGAT and nifH. Vasse et al. (1990)
reported that in the proximal part of the
N2-fixing zone, the bacteroid morphology changes
from type 4 to type 5. Moreover, they suggested that the type 5 bacteroid is inefficient in N2 fixation. It would
be tempting to suggest that N2 fixation is
restricted to the areas where both nifH and NADH-GOGAT are expressed. However, additional data are
needed to link the changes in bacteroid morphology with the
disappearance of nifH and NADH-GOGAT
transcripts.
The analysis of the 5 upstream region of the NADH-GOGAT
gene led to the identification of four possible regulatory regions, one
responsible for directing expression in the
N2-fixing zone, two positive elements, and one
negative element. Earlier we reported that the 5 upstream region of
the NADH-GOGAT gene directs expression of the GUS reporter
gene in the alfalfa root nodule meristem (zone I), invasion (zone II),
interzone (zone II-III [*]), and N2-fixing (zone III) zones (Vance et al., 1995). Detection of the NADH-GOGAT message by in situ hybridization showed that the transcript is absent
in the meristem (zone I) and invasion (zone II) zones of the nodule.
This discrepancy could be the result of two factors: (a) the
integration site of the promoter reporter gene construct into the plant
genome influences transcription and thus distorts localization; or (b)
the GUS protein and/or reaction substrate could diffuse during the
infiltration and incubation procedure and therefore be another source
of distortion.
However, for constructs GLate and G1, plants with GUS enzyme activity
closest to the mean had a histochemical staining pattern similar to the
results obtained from in situ hybridization. These data indicate that
the two constructs contain elements necessary for expression of the
NADH-GOGAT gene in the N2-fixing zone
of a root nodule. By contrast, a consistent loss of
N2-fixing-zone specificity was observed in
nodules of G2 plants, suggesting that an element for nodule-infected,
cell-specific expression lies between G1 (1064) and G2 (655). It is
interesting that this region contains an 88-bp direct repeat (Fig. 7E).
Of the 408 bp found in the region controlling
N2-fixing-zone specificity, 176 bp or 43% of the
sequence occur in this direct repeat. However, the conserved elements
5-AAAGAT and 5-CTCTT, which have been associated with nodule
specificity of late-nodulin genes (Sandal et al., 1987; de Bruijn and
Schell 1993), were found neither in the 88-bp direct repeat nor in the
G1 (1064) to G2 (655) region. By comparing the 88-bp direct repeat
against 5 upstream regions of other nodulin and nodule-enhanced genes,
we found a 14-bp AT-rich sequence also found in the alfalfa
leghemoglobin promoter (5-TTAATTAATAAATA-3). This region contains a
sequence similar to the proposed octanucleotide motif on the opposite
strand (5-AATAAATA-3), which has been associated with positive
regulatory elements in several other late-nodulin gene promoters (Forde
et al., 1990). Using gel-mobility-shift analysis, it was demonstrated
that this motif is part of the NAT2 binding site in the soybean
leghemoglobin (lbc3) (Jensen et al., 1988) and the soybean
N23 promoter (Jacobsen et al., 1990). In French bean, the
promoter of the nodule-enhanced GS (gln ) contains this motif within
two 21-bp AT-rich repeats that are binding sites for PNF-1
(Phaseolus nodule
factor-1) (Forde et al., 1990). When the region
of the gln promoter that contains these repeats was inserted in the
90 position of the cauliflower mosaic virus 35S promoter, the
construct was able to direct expression of the GUS-reporter gene in
birdsfoot trefoil root nodules (Shen et al., 1992). However, the
function of AT-rich elements, which serve as binding sites for nuclear
proteins, is still unclear. Although such elements are often closely
associated with positive regulatory elements in a wide variety of plant
genes (Forde, 1994), it has also been found that these AT-rich binding
sites can be deleted without affecting gene expression (Forde, 1994).
Although the region between G1 (1064) and G2 (655) seems to be
important for reporter-gene expression in the
N2-fixing zone of a root nodule, other than the
AT-rich binding site, the deleted region does not contain regions with
sequence homology to other reported sequences that have been associated
with nodule specificity of late-nodulin genes (Sandal et al., 1987;
Forde et al., 1990; de Bruijn and Schell 1993). These observations led
us to two conclusions: (a) the deleted region may contain unknown
elements responsible for nodule-specific expression; and (b) the
deleted region contains elements involved in a physiological response
of the plant cell, e.g. low oxygen partial pressure. Further analysis
of this important region of the NADH-GOGAT promoter using
finer deletions and gel-mobility-shift analysis may resolve this
question.
Although construct G5 (240) has the same histochemical staining as
construct G4 (343), we measured a significant decrease in GUS
activity (P < 0.01), suggesting the presence of positive element(s) associated with the deleted region. A significant increase in enzyme activity (P < 0.01) was measured between deletion G5 (240) and G6 (151), suggesting the presence of negative element(s) associated with this region. However, sequence analysis of the deleted
regions did not reveal similarities to previously reported positive and
negative elements. Between deletion G6 (151) and G7 (82) we
measured a significant decrease in GUS activity (P < 0.01) to
background levels, indicating the presence of positive element(s)
associated with this region. Although deletion G7 (82) still
contained the putative TATA box (21), the region between G6 (151)
and G7 (82) lies within the area generally associated with elements
responsible for regulating the frequency of transcription initiation
(Kuhlemeier et al., 1987). Such elements have been identified in
animals as the conserved CAAT and GC boxes. However, in plants such
boxes are often substituted (Kreis et al., 1986; Kuhlemeier et al.,
1987) or absent (Kuhlemeier et al., 1987).
In this study we have extended the understanding of
NADH-GOGAT in N2 fixation and N
assimilation by showing that the enzyme is encoded by a single gene and
that NADH-GOGAT transcripts predominantly accumulate in the
infected cell of the N2-fixing zone, but do not
require active N2 fixation for initial
expression. In addition, we have delimited several segments in the 5
upstream region of NADH-GOGAT that affect reporter-gene
expression. One of them between G1 (1064) and G2 (655) contains an
88-bp repeat that may affect expression in the
N2-fixing zone. Although these findings
strengthen our original hypothesis that NADH-GOGAT represents a
rate-limiting step in the assimilation of symbiotically fixed N in
alfalfa nodules, further studies using antisense approaches are
required to verify the strategic role of this important enzyme.
| |
FOOTNOTES |
1
This work was supported in part by National
Science Foundation grant no. IBN-9206890 and ETH-Zurich
fellowship no. 0-28-001-91. This paper is a joint contribution from the
Plant Science Research Unit, U.S. Department of Agriculture,
Agricultural Research Service, and the Minnesota Agricultural
Experiment Station (paper no. 98-1-13-0100, Scientific Journal Series).
*
Corresponding author; e-mail vance004{at}maroon.tc.umn.edu; fax
1-651-649-5058.
Received September 14, 1998;
accepted December 9, 1998.
2
Mention of a trademark, proprietary product, or
vendor does not constitute a guarantee or warranty of the product by
the U.S. Department of Agriculture and does not imply its approval to
the exclusion of other products or vendors that might also be
suitable.
| |
ABBREVIATIONS |
Abbreviations:
GOGAT, Glu synthase.
GS, Gln synthetase.
| |
ACKNOWLEDGMENTS |
We thank Ann Hirsch and Turlough Finan for generously
sharing ineffective S. meliloti strains, Ann
Hirsch and Mike Sadowsky for sharing the leghemoglobin cDNA and the
nifH gene, and Gyorgy B. Kiss and Allen Smith for critically
reading the manuscript. We give special thanks to Nikolaus Amrhein for
his insightful remarks.
| |
LITERATURE CITED |
Anderson MP,
Vance CP,
Heichel GH,
Miller SS
(1989)
Purification and characterization of NADH-glutamate synthase from alfalfa root nodules.
Plant Physiol
90:
351-358
[Abstract/Free Full Text]
Austin S,
Bingham ET,
Mathews DE,
Shahan MN,
Will J,
Burgess RR
(1995)
Production and field performance of transgenic alfalfa (Medicago sativa L.) expressing alpha-amylase and manganese-dependent lignin peroxidase.
Euphytica
85:
381-383
[CrossRef]
Avila C,
Marquez AJ,
Pajuelo P,
Cannell ME,
Wallsgrove RM,
Forde BG
(1993)
Cloning and sequence analysis of a cDNA for barley ferredoxin-dependent glutamate synthase and molecular analysis of photorespiratory mutants deficient in the enzyme.
Planta
189:
475-483
[ISI][Medline]
Baron AC,
Tobin TH,
Wallsgrove RM,
Tobin AK
(1994)
A metabolic control analysis of the glutamine synthetase/glutamate synthase cycle in isolated barley (Hordeum vulgare L.) chloroplasts.
Plant Physiol
105:
415-424
[Abstract]
Becker TW,
Perrot-Rechenmann C,
Suzuki A,
Hirel B
(1993)
Subcellular and immunocytochemical localization of the enzymes involved in ammonia assimilation in mesophyll and bundle-sheath cells of maize leaves.
Planta
191:
129-136
[ISI]
Bingham ET
(1991)
Registration of alfalfa hybrid RegenSY germplasm for tissue culture and transformation research.
Crop Sci
31:
1098
[Free Full Text]
Boland MJ,
Benny AG
(1977)
Enzymes of nitrogen metabolism in legume nodules: purification and properties of NADH-dependent glutamate synthase from lupin nodules.
Eur J Biochem
79:
355-362
[Medline]
Chen F-L,
Cullimore JV
(1988)
Two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.
Plant Physiol
88:
1411-1417
[Abstract/Free Full Text]
Chen F-L,
Cullimore JV
(1989)
Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.
Planta
179:
441-447
Cox KH,
Goldberg RB
(1988)
Analysis of plant gene expression.
In
CH Shaw,
eds, Plant Molecular Biology: A Practical Approach.
IRL Press, Oxford, UK, pp 1-34
de Billy F,
Barker DG,
Gallusci P,
Truchet G
(1991)
Leghemoglobin gene transcription is triggered in a single cell layer in the indeterminate nitrogen-fixing root nodule of alfalfa.
Plant J
1:
27-35
de Bruijn FJ,
Schell J
(1993)
Regulation of plant genes specifically induced in developing and mature nitrogen-fixing nodules: cis-acting elements and trans-acting factors.
In
DPS Verma,
eds, Control of Plant Gene Expression.
CRC Press, Boca Raton, FL, pp 241-258
Egli MA,
Griffith SM,
Miller SS,
Anderson MA,
Vance CP
(1989)
Nitrogen assimilatory enzymes and enzyme protein during development and senescence of effective and plant gene-controlled ineffective alfalfa nodules.
Plant Physiol
91:
898-904
[Abstract/Free Full Text]
Fleming AJ,
Mandel T,
Roth I,
Kuhlemeier C
(1993)
The patterns of gene expression in the tomato apical meristem.
Plant Cell
5:
297-309
[Abstract]
Forde BG
(1994)
AT-rich elements (ATREs) in the promoter regions of nodulin and other higher plant genes: a novel class of cis-acting regulatory element?
In
L Nover,
eds, Plant Promoters and Transcription Factors.
Springer-Verlag, Heidelberg, Germany, pp 87-101
Forde BG,
Freeman J,
Oliver JE,
Pineda M
(1990)
Nuclear factors interact with conserved A/T-rich elements upstream of nodule-enhanced glutamine synthetase from French bean.
Plant Cell
2:
925-939
[Abstract/Free Full Text]
Franssen HJ,
Vijn I,
Yang WC,
Bisseling T
(1992)
Developmental aspects of the Rhizobium-legume symbiosis.
Plant Mol Biol
19:
89-107
[CrossRef][ISI][Medline]
Gantt SJ,
Larson RJ,
Farnham MW,
Pathirana SM,
Miller SS,
Vance CP
(1992)
Aspartate aminotransferase in effective and ineffective alfalfa nodules. Cloning of a cDNA and determination of enzyme activity, protein, and mRNA levels.
Plant Physiol
98:
868-878
[Abstract/Free Full Text]
Goto S,
Akagawa T,
Kojima S,
Hayakawa T,
Yamaya T
(1998)
Organization and structure of NADH-dependent glutamate synthase gene from rice.
Biochim Biophys Acta
1387:
298-308
[CrossRef][Medline]
Gregerson RG,
Miller SS,
Petrowski M,
Gantt JS,
Vance CP
(1993a)
Molecular analysis of allelic polymorphism at the AAT2 locus of alfalfa.
Mol Gen Genet
241:
124-128
[Medline]
Gregerson RG,
Miller SS,
Twary SN,
Gantt JS,
Vance CP
(1993b)
Molecular characterization of NADH-dependent glutamate synthase from alfalfa nodules.
Plant Cell
5:
215-226
[Abstract]
Groat GR,
Vance CP
(1981)
Root and nodule enzymes of ammonia assimilation in alfalfa (Medicago sativa L.).
Plant Physiol
67:
1198-1205
[Abstract/Free Full Text]
Hayakawa T,
Yamaya T,
Kamachi K,
Ojima K
(1992)
Purification, characterization, and immunological properties of NADH-dependent glutamate synthase from rice cell cultures.
Plant Physiol
98:
1317-1322
[Abstract/Free Full Text]
Heintzen C,
Melzer S,
Fischer R,
Kappeler S,
Apel K,
Staiger D
(1994)
A light- and temperature-entrained circadian clock controls expression of transcripts encoding nuclear proteins with homology to RNA-binding proteins in meristematic tissue.
Plant J
5:
799-813
[CrossRef][ISI][Medline]
Hirasawa M,
Tamura G
(1984)
Flavin and iron-sulphur containing ferredoxin-linked glutamate synthase from spinach leaves.
J Biochem
95:
983-994
[Abstract/Free Full Text]
Jacobsen K,
Lauren NB,
Jensen EO,
Marcker A,
Poulsen C,
Marcker KA
(1990)
HMG I-like proteins from leaf and nodule nuclei interact with different AT motifs in soybean nodulin promoters.
Plant Cell
2:
85-95
[Abstract/Free Full Text]
Jefferson RA
(1987)
Assaying chimeric genes in plants: the GUS gene fusion system.
Plant Mol Biol Rep
5:
387-405
[CrossRef]
Jensen EO,
Marcker KA,
Schell J,
de Bruijn FJ
(1988)
Interaction of a nodule specific, trans-acting factor with distinct DNA elements in the soybean leghemoglobin lbc3 5 upstream region.
EMBO J
7:
1265-1271
[ISI][Medline]
Kendall AC,
Wallsgrove RM,
Hall NP,
Turner JC,
Lea PJ
(1986)
Carbon and nitrogen assimilation in barley mutants lacking ferredoxin-dependent glutamate synthase.
Planta
168:
316-323
[CrossRef][ISI]
Knauff DB,
Hirasawa M,
Ameyibor E,
Fu W,
Johnson MK
(1991)
Spectroscopic evidence for a [3Fe-4S] cluster in spinach glutamate synthase.
J Biol Chem
266:
15080-15084
[Abstract/Free Full Text]
Kreis M,
Williamson MS,
Forde J,
Schmutz D,
Clark J,
Buxton B,
Pywell J,
Marris C,
Henderson J,
Harris N,
and others
(1986)
Differential gene expression in the developing barley endosperm.
Philos Trans R Soc Lond B Biol Sci
314:
355-366
Kuhlemeier C,
Green PJ,
Chua N-H
(1987)
Regulation of gene expression in higher plants.
Annu Rev Plant Physiol Plant Mol Biol
38:
221-257
[CrossRef][ISI]
Lam H-M,
Coschigano KT,
Oliveira IC,
Melo-Oliveira R,
Coruzzi GM
(1996)
The molecular-genetics of nitrogen assimilation into amino acid in higher plants.
Annu Rev Plant Physiol Plant Mol Biol
47:
569-593
[CrossRef][ISI]
Lea PJ,
Robinson SA,
Stewart GR
(1990)
The enzymology and metabolism of glutamine, glutamate and asparagine.
In
BJ Miflin,
PJ Lea,
eds, Biochemistry of Plants: Intermediary Nitrogen Metabolism, Vol 16.
Academic Press, San Diego, CA, pp 121-159
Leigh VA,
Reed JW,
Hanks JF,
Hirsch AM,
Walker GC
(1987)
Rhizobium meliloti mutants that fail to succinylate their calcofluor-binding exopolysaccharide are defective in nodule invasion.
Cell
70:
579-587
Minchin FR,
Witty JF,
Sheely JF,
Mueller M
(1983)
A major error in the acetylene reduction assay: decreases in nodular nitrogenase activity under assay conditions.
J Exp Bot
34:
641-649
[Abstract/Free Full Text]
Nalbantoglu B,
Hirasowa M,
Moomav C,
Nguyen H,
Knaff DB,
Allen R
(1994)
Cloning and sequencing of the gene encoding spinach ferredoxin-dependent glutamate synthase.
Biochim Biophys Acta
1183:
557-561
[Medline]
Oliver G,
Gosset G,
Sanchez-Pescador R,
Lozoya E,
Ku LM,
Flores N,
Becerril B,
Valle F,
Bolivar F
(1987)
Determination of the nucleotide sequence for the glutamate synthase structural genes in Escherichia coli K12.
Gene
60:
1-11
[CrossRef][Medline]
Pathirana MS,
Samac DA,
Roeven R,
Yoshioka H,
Vance CP,
Gantt JS
(1997)
Analyses of phosphoenolpyruvate carboxylase gene structure and expression in alfalfa nodules.
Plant J
12:
293-304
[Medline]
Peterson MA,
Barnes DK
(1981)
Inheritance of ineffective nodulation and non-nodulation traits in alfalfa.
Crop Sci
21:
611-616
[Abstract/Free Full Text]
Pladys D,
Vance CP
(1993)
Proteolysis during development and senescence of effective and plant gene-controlled ineffective alfalfa nodules.
Plant Physiol
103:
379-384
[Abstract]
Sakakibara H,
Kawabata S,
Hase T,
Sugiyama T
(1992)
Differential effects of nitrate and light on the expression of glutamine synthetases and ferredoxin-dependent glutamate synthase in maize.
Plant Cell Physiol
33:
1193-1198
[Abstract/Free Full Text]
Sakakibara H,
Watanabe M,
Hase T,
Sugiyama T
(1991)
Molecular cloning and characterization of complementary DNA encoding for ferredoxin-dependent glutamate synthase in maize leaf.
J Biol Chem
266:
2028-2035
[Abstract/Free Full Text]
Sambrook J,
Fritsch EF,
Maniatis T
(1989)
Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Sandal NN,
Bojsen K,
Marcker KA
(1987)
A family of nodule specific genes from soybean.
Nucleic Acids Res
15:
1507-1519
[Abstract/Free Full Text]
Scheres B,
van Engelen F,
van der Knaap E,
van der Wiel C,
van Kammen A,
Bisseling T
(1990)
Sequential induction of nodulin gene expression in the developing pea nodule.
Plant Cell
2:
687-700
[Abstract/Free Full Text]
Shen W-J,
Williamson MS,
Forde BG
(1992)
Functional analysis of the promoter region of a nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L.
Plant Mol Biol
19:
837
[Medline]-846
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Abstract
Introduction