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Plant Physiol. (1998) 117: 1411-1421
Characterization of Two Glutamate Decarboxylase cDNA Clones from
Arabidopsis
Frank J. Turano* and
Tung K. Fang
United States Department of Agriculture, Agricultural Research
Service, Climate Stress Laboratory, Beltsville, Maryland 20705
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ABSTRACT |
Two distinct cDNA clones encoding for
the glutamate decarboxylase (GAD) isoenzymes GAD1 and
GAD2 from Arabidopsis (L.) Heynh. were characterized.
The open reading frames for GAD1 and GAD2 were expressed in
Escherichia coli and the recombinant proteins were
purified by affinity chromatography. Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1 and
GAD2 encode for 58- and 56-kD peptides, respectively.
The enzymatic activities of the pure recombinant GAD1 and GAD2 proteins
were stimulated 35- and 13-fold, respectively, by
Ca2+/calmodulin but not by Ca2+ or calmodulin
alone. Southern-blot analysis of genomic DNA suggests that there is
only one copy of each gene in Arabidopsis. The GAD1 transcript and a corresponding 58-kD peptide were detected in roots
only. Conversely, the GAD2 transcript and a
corresponding 56-kD peptide were detected in all organs tested. The
specific activity, GAD2 transcript, and 56-kD peptide
increased in leaves of plants treated with 10 mM
NH4Cl, 5 mM NH4NO3, 5 mM glutamic acid, or 5 mM glutamine as the sole
nitrogen source compared with samples from plants treated with 10 mM KNO3. The results from these experiments
suggest that in leaves GAD activity is partially controlled by gene
expression or RNA stability. Results from preliminary analyses of
different tissues imply that these tendencies were not the same in
flower stalks and flowers, suggesting that other factors may control
GAD activity in these organs. The results from this investigation
demonstrate that GAD activity in leaves is altered by different
nitrogen treatments, suggesting that GAD2 may play a unique role
in nitrogen metabolism.
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INTRODUCTION |
GAD (EC 4.1.1.15) catalyzes the conversion of Glu to GABA in the
presence of the cofactor PLP. GAD is present in Escherichia coli (Smith et al., 1992 ), mammals (Erlander and Tobin, 1991), and
plants (Satyanarayan and Nair, 1990). In plants the enzyme has a unique
feature, a CaM-binding domain at the carboxy terminus (Baum et al.,
1993; Arazi et al., 1995; Gallego et al., 1995). CaM binding has
been demonstrated in GAD isolated from petunia (Baum et al., 1993) and
fava bean (Ling et al., 1994). In addition, the last 30 amino acids of
the GAD1 gene product from Arabidopsis has been shown to
bind CaM (Arazi et al., 1995). In vitro analyses have shown that
Ca2+ and CaM stimulate GAD activity 1- to 9-fold
(Ling et al., 1994; Snedden et al., 1995; Cholewa et al., 1997; Johnson
et al., 1997) in partially purified protein preparations, and nearly
20-fold in purified preparations (Snedden et al., 1996). These findings suggest that GAD may be stimulated in vivo by
Ca2+ signal pathways. This hypothesis is
consistent with data collected from studies demonstrating the rapid
increase in cytoplasmic Ca2+ concentrations
(Knight et al., 1991, 1992; Price et al., 1994; Cholewa et al.,
1997) and GABA titers (Wallace et al., 1984; Mayer et al., 1990;
Cholewa et al., 1997) in plant cells upon exposure to various
environmental stimuli.
Despite a better understanding of the cellular factors that may
stimulate GAD activity, the physiological roles of the enzyme or the
product, GABA, have not been clearly established in plants. Since
elevated GAD activity is usually seen in tissues with low cytoplasmic
pH (Satyanarayan and Nair, 1990), and the synthesis of GABA consumes a
proton, GABA metabolism has been proposed to regulate cytoplasmic pH in
plant tissues subjected to various stress conditions (Streeter and
Thompson, 1972; Davies, 1980). However, Cholewa et al. (1997)
demonstrated that GABA accumulation may be stimulated by
Ca2+ and not by decreased cytoplasmic pH when
plants are subjected to an abrupt cold-shock treatment. But several
other physiological roles for GABA have been proposed. Selman and
Cooper (1978) suggested that GABA may provide a direct temporary
reserve of carbon and nitrogen for Glu or an indirect reserve for
protein synthesis. Since GABA is an inhibitor of neuron transmission in
animals, Wallace et al. (1984) suggested that increased levels of GABA could alter the eating habits of insects. Recently, Ramputh and Bown
(1996) demonstrated that elevated levels of GABA in the diet of
oblique-banded leaf-roller larvae decreased their growth, development, and survival. In addition, Chen et al. (1994) questioned whether GABA
in plants was involved in the control of ion channels, as in animal
neurons.
Baum et al. (1996) overexpressed a truncated version
of a petunia GAD gene, which lacked the CaM-binding site, in transgenic tobacco plants and demonstrated that the CaM-binding domain was required for normal plant development and for the maintenance of GABA
and Glu levels. These results provide some evidence that GAD is
involved in nitrogen metabolism. Other investigators demonstrated that
GAD may not be solely involved in the maintenance of cytoplasmic pH.
Robinson et al. (1991) and Carroll et al. (1994) showed that GABA was a
major nitrogen source for freshly assimilated ammonium in nonstressed
cell suspensions. Tuin and Shelp (1994) demonstrated that the in situ
synthesis of GABA in developing soybean cotyledons was via GAD, but
that GABA was rapidly metabolized to provide tricarboxylic acid cycle
intermediates for amino acid metabolism, possibly via the GABA shunt.
In addition, Cholewa et al. (1997) reported a
Ca2+/CaM-independent increase in GABA when cells
were treated with Glu or butyrate. Together, these results suggest not
only that GABA synthesis may be a response to stress but that GAD may
perform a unique physiological role in nitrogen and/or carbon
metabolism via the GABA shunt.
Although several of the factors that stimulate GAD activity have been
identified, there is still a gap in the knowledge of the role(s) of
transcriptional and/or posttranslational events in the control of GAD
activity. Chen et al. (1994) demonstrated that a 58-kD CaM-binding GAD
was expressed in various floral parts, seeds, stems, roots, and leaves
of petunia. In addition, their data suggested that there was a
correlation between in vitro GAD activity, abundance of the 58-kD
peptide, and levels of GAD transcript in developing leaves and
germinating seeds. However, this phenomenon was not observed in
developing flowers. In open flowers the 58-kD peptide was abundant and
in vitro GABA synthesis was high, but the level of GAD transcript was
almost undetectable. The authors suggested that posttranslational
regulation in leaves and seeds may differ from that of flowers, and the
process may play a role in controlling GAD activity in flowers.
Chen et al. (1994) used immunoblot analysis to monitor the accumulation
of GAD in germinating petunia seeds. They identified three peptides of
48, 58, and 66 kD that cross-reacted with the antiserum to a
recombinant petunia GAD (58 kD). There appeared to be a synchronous
increase and decrease of the 48- and 66-kD proteins, which was
inversely proportional to levels of the 58-kD GAD throughout
germination. These data suggest that there may be several GAD-like
peptides or isoenzymes in plants. Molecular mass determinations of GAD
from potato (45.5 kD) (Satyanarayan and Nair, 1985), and fava bean (62 kD) (Ling et al., 1994) also suggest that there may be distinct GAD
isoenzymes. The presence of several GAD isoenzymes could explain the
discrepancies in apparent molecular masses of GAD from different plant
species and could explain the apparent contradictions concerning
physiological role(s) of GAD in plants. The existence of several
isoenzymes could be due to posttranslational modifications of a single
peptide or to the existence of multiple loci or genes encoding GAD. To
gain a greater understanding of the function and role of the GAD
isoenzymes in plant nitrogen metabolism, we initiated a study of the
isoenzymes and genes in Arabidopsis. In this study we combined
molecular biological approaches with biochemical and immunological
techniques to identify the cDNA clones and the protein/peptide
components that they encode, and to demonstrate that the gene products
bind CaM. In addition, we determined the effects of different nitrogen sources on GAD activity in Arabidopsis.
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MATERIALS AND METHODS |
Plant Material
Arabidopsis (L.) Heynh. ecotype Columbia (Col-0) seeds were
obtained from the Arabidopsis Biological Resource Center (Ohio State
University, Columbus). Plants were grown in (20 × 10 × 6 cm
[length × width × depth]) plastic pots, either in a
peat-vermiculite mixture (Jiffy Mix, Jiffy Products of America,
Batavia, IL)1, or
in vermiculite. Plants grown in soil were watered as needed by
subirrigation throughout the experiment. Plants grown in vermiculite were subirrigated with a complete mineral nutrient solution (Cammaerts and Jacob, 1985) with nitrate (10 mM
KNO3) as a sole nitrogen source. After 15 d,
the pots containing vermiculite were flushed daily for 5 d with 25 mL of sterile distilled water, and the plants were subirrigated for
4 d with different nitrogen treatments (10 mM
KNO3 [control], 10 mM
NH4Cl, 5 mM
NH4NO3, 5 mM
Glu, or 5 mM Gln), as described by Turano et al. (1997).
Plants were maintained at 20°C to 21°C and 60% to 70% RH under
cool-white lights (120-140 µmol PPFD m 2
s1) in a 16-h light/8-h dark cycle.
Crude Protein Extractions
Proteins were extracted from frozen or fresh samples. Frozen
samples (approximately 500 mg) were ground to a fine powder with a
mortar and pestle. The powder was transferred to 1 mL of extraction buffer (50 mM Tes, pH 5.8, 5 mM EDTA, 1 mM MgCl2, 0.1 mM PLP, 1 mM DDT, 0.5% [w/v] PVP-40, and 4 mM Cys).
Fresh PMSF was added to a final concentration of 1 mM in
all extracts. The fresh samples (200 mg) were ground in 400 µL of
extraction buffer as described above. The samples were incubated on ice
for 15 to 30 min. Debris were removed from the sample by centrifugation
at 13,000g for 10 min. The supernatant containing the crude
protein extract was used to determine the protein concentration and GAD
activity.
Protein Determinations and Enzymatic Activity Assays
Protein concentrations were determined using a modification of the
Lowry method as described by Markwell et al. (1978) or by NanoOrange as
described by the manufacturer (Molecular Probes, Inc., Eugene, OR). GAD
activity was determined using a method similar to that of Snedden et
al. (1992). Assays were conducted in 17- × 52-mm glass vials plugged
with a soft rubber stopper. An 18-gauge syringe needle was used to
pierce the stopper and a folded piece of Whatman paper no. 3 (7 × 75 mm) held in place by a small piece of rubber tubing. The Whatman
paper was saturated with 180 µL of 1 N KOH and suspended
in the vial when the rubber stopper was put in place to trap the
CO2 that evolved from the GAD reaction. A small
stopper was placed in the end of the syringe needle to prevent the
escape of CO2 from the reaction. The vials were
maintained on ice prior to the initiation of the reaction. Reactions
from crude protein extracts from plant tissues were conducted in 500 µL with 100 mM pyridine-HCl, pH 5.8, 10 mM
NaCl, 0.1 mM PLP, and 20 mM Glu containing 0.02 µCi/µmol L-(1-14C)Glu with 25 to
50 µL of enzyme extract. Crude protein extracts were assayed at pH
5.8 and not 7.3 because there were problems associated with CaM binding
and the effects of proteases on the CaM-binding domain of GAD in these
extracts. Pure rGAD1 and rGAD2 were assayed in 500 µL with 100 mM 1,3-bis-Tris-propane-HCl, pH 7.3, 10 mM
NaCl, 0.1 mM PLP, and 20 mM Glu containing 0.02 µCi/µmol L-(1-14C)Glu with 25 to
50 µL of enzyme extract.
To test Ca2+ and/or CaM stimulation,
CaCl2 and CaM were added alone or in combination
to a final concentration of 1 mM and 0.4 µM,
respectively. The CaM antagonist TFP was dissolved in water and added
to a final concentration of 100 µM. Assays were initiated with the addition of the substrate. The vials were incubated in a
gently shaking water bath at 30°C for 1 h. Vials were
immediately placed on ice and 100 µL of 2 N
H2SO4 was added to the
vessels through the 18-gauge syringe to terminate the reaction. The
reactions were incubated on ice for 30 to 45 min to allow complete
absorption of the CO2. The filter paper for each
vial was placed into a scintillation vial containing 4.5 mL of Formula
A-989 high-flash-point cocktail (Packard Instrument Co., Inc.,
Meriden, CT) and counted in a scintillation counter (model LS 2800, Beckman). In each experiment, enzyme determinations were performed in
triplicate.
Gel Electrophoresis and Immunoblot Analysis
Proteins were separated by SDS-PAGE (8.0% or 9.0%
polyacrylamide) using a Mini-Protean II (Bio-Rad) system. Silver
staining and immunoblot analysis were conducted as described by Turano et al. (1990). Rabbit serum raised against petunia rGAD was kindly provided by Dr. Hillel Fromm (Baum et al., 1993). For the
identification of GAD isoenzymes in different organs, an equal amount
of protein (150 µg) from roots, leaves, flower stalks, flowers, and
siliques of 42-d-old plants was added per lane. Similarly, 150 µg of
protein was added per lane to determine the effects of different
nitrogen sources on GAD isoenzymes in leaves of 24-d-old plants. To
determine the effects of different nitrogen sources on GAD isoenzymes
in roots of 24-d-old plants, 75 µg of protein was added per lane.
GAD Clones
Two expressed sequence tag clones encoding GAD1
(35B3T7) and GAD2 (5B2T7P) were obtained from the
Arabidopsis Biological Resource Center. The clones were sequenced by
the dideoxy chain-termination method using modified T7 DNA polymerase
(Sequenase 2.0, United States Biochemical), as described by the
manufacturer. The data were analyzed with the IntelliGenetics Suite
(IntelliGentics, Inc., Mountain View, CA) on a Sun system.
DNA and RNA Isolation and Gel Electrophoresis
Total DNA was isolated, digested with restriction enzymes, and
blotted to nitrocellulose as described by Turano et al. (1992). For the
determination of organ-specific expression, total RNA was isolated from
roots, leaves, flower stalks, flowers, and siliques of 42-d-old
Arabidopsis plants. To determine the effects of nitrogen on GAD, total
RNA was isolated from roots and leaves of 24-d-old plants. Total RNA
was isolated from each organ, separated by gel electrophoresis using
formaldehyde-formamide gels, blotted to nitrocellulose, and hybridized
as described by Turano et al. (1997).
Construction of rGAD1 and rGAD2 for Expression in
Escherichia coli
The open reading frames for GAD1 and GAD2 were
amplified and cloned in the E. coli expression
vector pKK223-3 (Pharmacia). Oligonucleotide primers were synthesized
with HindIII sites at the 5
(5-GCCCAAGCTTAGGAAACAGAAATGGTGCTCTCCCACGCCG-3)
and 3 (5-GCCCAAGCTTAGCAGATACCACTCG-3) for rGAD1 and the 5
(5-GCCCAAGCTTAGGAAACAGAAATG-GTTTTGACAAAAACCG-3) and 3 (5-GCCCAAGC-TTTAGCACACACCATTCA-3) for rGAD2.
Separate amplification reactions were conducted with 5- and
3-specific primers with the appropriate cDNA clone as a template using
a gene-amplification kit (PanVera Corp., Madison, WI). Amplification reactions were conducted as follows: 94°C for 30 s, 55°C for
30 s, and 72°C for 4 min, for 25 cycles. The amplified fragments were digested with HindIII, ligated into the vector, and
transformed into XL1-Blue MRF (Stratagene) competent cells. The
correct orientation was verified by restriction endonuclease analysis.
E. coli cultures containing rGAD1 or rGAD2 were grown
overnight, approximately 16 h, at 37°C in Luria-Bertani medium
with 100 µg/mL ampicillin. Overnight cultures were diluted 1:100 in fresh Luria-Bertani medium with 100 µg/mL ampicillin and incubated at
25°C with shaking at 200 rpm. After 4 h,
isopropylthio- -galactoside was added to a final concentration of 100 µM to induce expression of the recombinant protein, and
incubation was continued for 3 to 4 h for rGAD1 and 20 h for
rGAD2. Bacterial cells were collected by centrifugation at
6000g for 10 min. Pellets were immediately frozen in liquid
nitrogen and stored at  80°C. The E. coli pellets could
be stored at 80°C for 2 months without a significant decrease in
GAD activity. Pellets containing cells were resuspended in 5 to 7 mL of
extraction buffer (50 mM Tris-HCl, pH 7.5, 0.2 mM EDTA, 2.5% [v/v] glycerol, 2 mM DTT, 0.05 mM PLP, 0.05% [v/v] Triton, 10 µM
leupeptin, 20 µg/mL lysozyme, and 1 mM PMSF). The cells
were lysed by sonication with 12 2-s bursts at 150 W. Cellular debris
were removed by centrifugation at 12,000 rpm for 20 min. The
supernatant was filtered through a 0.45-µm filter.
CaCl2, DTT, and PMSF were added to final
concentrations of 1, 2, and 1 mM, respectively.
The supernatant was loaded onto an approximately 1-mL bed volume
CaM-Sepharose column (Pharmacia) and preequilibrated in binding buffer
(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2.5%
[v/v] glycerol, 1 mM CaCl2, 2 mM DTT, 10 µM leupeptin, and 1 mM
PMSF). The column was washed with 20 bed volumes of wash buffer minus calcium (25 mM Tris-HCl, pH 7.5, 150 mM NaCl,
2.5% [v/v] glycerol, 2 mM DTT, 10 µM
leupeptin, and 1 mM PMSF). CaM-binding proteins were eluted
with 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 150 mM NaCl, 2.5% [v/v] glycerol, and 1 mM PMSF.
Samples were immediately assayed as described above.
Amplification and Radioactive Labeling of GAD Probes and
Hybridization
One oligoprimer (17-mer) was synthesized (Bio-Synthesis,
Inc., Lewisville, TX) to the 3 regions of each of the GAD genes. The
primers, designated 3primGAD1 (5-GATCGATATAGAGAAAG-3) and 3primGAD2
(5-TAGATACCTTGCCTTCC-3), were used in separate PCRs with the
M13-forward primer and their corresponding cDNA clones as the templates
for use in a gene-amplification kit (Perkin-Elmer). Reactions were
conducted as follows: 94°C for 30 s, 45°C for 30 s, and
72°C for 2 min, for 25 cycles. Each of the amplified DNA fragments,
404 and 332 bp, unique to the 3 regions of GAD1 and GAD2 (Fig. 1), respectively,
were gel purified, amplified a second time, and used as gene-specific
probes in genomic Southern and RNA-blot analyses. Amplification of the
DNA probes, total RNA isolation, blotting, prehybridizations, and
hybridizations were performed as described by Turano et al. (1997). A
probe encoding for a 26S rRNA gene (F.J. Turano, unpublished data) was
used as an internal control on RNA blots to ensure equal loading of RNA per lane. Data were quantified on a beta scanner (Betagen,
IntelliGentics, Inc.).
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| Figure 1.
Nucleotide sequence of Arabidopsis (At) GAD cDNA
clones. The nucleotide sequence of the 3 noncoding region of the cDNA
clone corresponding to GAD1 (A) and the full-length cDNA
clone corresponding to GAD2 (B) are shown in the sense
strand. A, Nucleotides are numbered from the putative stop codon of
GAD1. B, The amino acids are numbered from the putative start codon of
GAD2. C, Schematic representation of the 3 gene-specific probes. Thin
lines represent either the 5 or 3 noncoding regions, the open boxes
represent the coding regions, and the thick bars represent the
gene-specific probe for either GAD1 or
GAD2.
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RESULTS |
Sequence Analysis of GAD Clones
The nucleotide sequence (1509 bp) for the coding region of 35B3T7
(GAD1) was available in GenBank (accession no. U10034), and
an additional 250 bp of sequence was determined for the 5 (data not
shown) and 3 noncoding (Fig. 1A) regions. The full-length GAD1 clone was 1760 bp. A partial nucleotide sequence (465 bp) for 5B2T7P (GAD2) was available in GenBank (accession
no. T04804). The remainder of the cDNA was sequenced; the full-length
clone was 1676 bp long and encoded a 494-amino acid peptide (Fig. 1B). The nucleotide identity between GAD1 and GAD2 was
74% and 28% in the coding and 3 noncoding regions, respectively.
Oligoprimers (17-mer) specific for GAD1 and GAD2,
designated 3primGAD1 and 3primGAD2 (see ``Materials and Methods''),
were synthesized to the 3 regions of each of the cDNA clones. The
primers were used to make 404- and 332-bp DNA probes unique to the 3
regions of GAD1 and GAD2 (Fig. 1C), respectively.
GAD1 and GAD2 have open reading frames that
encode for proteins containing 502- and 494-amino acid residues,
respectively. Theoretically (estimated using PROSITE [version 14.0, Medical Biochemistry Department, Geneva, Switzerland]),
GAD1 encodes for a 57.1-kD peptide and GAD2
encodes for a 56.1-kD peptide. The deduced amino acid sequences of the
two Arabidopsis GAD cDNA clones were aligned with the deduced amino
acid sequences of petunia (Baum et al., 1993) and tomato (Gallego et
al., 1995) GAD cDNA clones (Fig.
2). The GAD1 and
GAD2 gene products had 82% amino acid identity. The gene
product for GAD1 had 85% and 76% amino acid identity with
the petunia and tomato GAD sequences, respectively. The gene product
for GAD2 had 79% and 74% amino acid identity with the
petunia and tomato GAD sequences, respectively. Neither of the
Arabidopsis peptides contained putative organellar target sequences.
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| Figure 2.
Comparison of the deduced amino acid sequences for
Arabidopsis (At) GAD1 and GAD2 with GAD from tomato (tomGAD; Gallego et
al., 1995; accession no. X80840) and petunia (petGAD; Baum et
al., 1993; accession no. L16797). The Lys associated with the putative
PLP-binding motif and the Trp associated with the putative CaM-binding
domains are indicated in bold type.
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The amino acid residues Ser-X-X-Lys are conserved at amino acid
residues 274 to 277 and 273 to 276 in the GAD1 and GAD2 peptide sequences, respectively. This motif is common among
PLP-requiring enzymes (Tanase et al., 1979). The identity and position
of the Ser-X-X-Lys motif in the Arabidopsis GAD peptides are conserved with the motif in the products of the gadA and
gadB genes from E. coli (Smith et al., 1992),
which have been shown to require PLP for GAD activity, and the putative
PLP sites of petunia GAD (Baum et al., 1993) and tomato GAD (Gallego et
al., 1995). A putative CaM-binding region, located at the
carboxy termini of the two peptides, was variable among the sequences.
Both peptides contained Trp residues in this region, Trp-488 (GAD1) and
Trp-480 (GAD2), which has been shown to be essential for CaM
binding in petunia GAD (Arazi et al., 1995). Arazi et al. (1995)
demonstrated that the last 30 amino acids of GAD1 were sufficient for
CaM binding (Arazi et al., 1995).
CaM Binding and Ca2+/CaM Stimulation of rGAD1 and
rGAD2
To demonstrate CaM binding and/or Ca2+/CaM
stimulation, the open reading frame of GAD1 or GAD2 was overexpressed
in E. coli. The recombinant proteins were
purified by affinity chromatography (Fig.
3) and tested for
Ca2+/CaM stimulation (Table
I). E. coli
extracts overexpressing the open reading frames for GAD1 or GAD2 were
loaded onto CaM-Sepharose columns. The columns were washed extensively
to remove nonspecific proteins (see ``Materials and Methods'' for
details). CaM-binding proteins were eluted with 2 mM EGTA.
Purified CaM-binding proteins were analyzed by silver staining
SDS-polyacrylamide gels and by immunoblot analysis (Fig. 3). The
estimated molecular masses for rGAD1 and rGAD2 were 58 and 56 kD,
respectively. These findings are similar to the estimated molecular
masses derived from the deduced amino acid sequences of the two cDNA
clones described above. In crude and purified extracts, the 58 and
56-kD peptides cross-reacted with rabbit serum raised against petunia
rGAD. Furthermore, neither the 58- nor the 56-kD peptides was observed
in crude E. coli extracts containing only the vector
pKK223-3. These results confirm that the recombinant proteins were
expressed in E. coli, were purified to homogeneity, and were
CaM-binding proteins. The purified recombinant proteins were assayed
for GAD activity in the presence of Ca2+, CaM,
Ca2+/CaM, or Ca2+/CaM/TFP
(Table I). Neither rGAD1 nor rGAD2 was stimulated by Ca2+ or CaM alone. However, both rGAD1 and rGAD2
were stimulated 35- and 13- fold, respectively, by
Ca2+/CaM. In both cases, the
Ca2+/CaM stimulation was significantly reduced by
the CaM antagonist TFP. These data confirm that both rGAD1 and rGAD2
encode for CaM-binding proteins and that both are enzymatically
stimulated by Ca2+/CaM.
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| Figure 3.
Characterization of rGAD1 and rGAD2 by SDS-PAGE
and immunoblot analysis. Crude protein extracts (20 µg/lane) from
E. coli containing the expression vector pKK223-3 (lanes
1 and 7) or the open reading frame for GAD1 (lanes 2 and
8) or GAD2 (lanes 4 and 10) cloned into pKK223-3 were
resolved in a SDS 8.0% polyacrylamide gel. Protein extracts from
E. coli expressing the open reading frame for
GAD1 or GAD2 were purified by affinity
chromatography using CaM-Sepharose. The eluted peptides (0.075 µg/lane) rGAD1 (lanes 3 and 9) and rGAD2 (lanes 5 and 11) were
resolved by SDS-PAGE. Lane 6 contains a molecular mass marker (10-kD
ladder, Life Technologies). The proteins in lanes 1 through 6 were
stained with silver; the proteins in lanes 7 through 11 were blotted
onto nitrocellulose and incubated with antiserum raised against petunia
rGAD. The positions of the rGAD1 (58 kD) and rGAD2 (56 kD) peptides are
indicated.
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Table I.
Stimulation of pure rGAD1 or rGAD2 at pH 7.3
rGAD1 and rGAD2 were assayed in 100 mM
1,3-bis-Tris-propane-HCl, pH 7.3, 10 mM NaCl, 100 µM PLP, and 20 mM glutamate
([1-C14]L-glutamate 0.02 µCi/mmol). When
indicated, CaCl2 and CaM were added to final concentrations
of 1 mM and 0.4 µM, respectively. The values
are the averages ± SE of two separate experiments.
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Southern-Blot Analysis
Southern-blot analysis was used to determine the number of copies
of GAD1 and GAD2 genes in Arabidopsis (Fig.
4). Specific 3 probes for the cDNA
corresponding to GAD1 and GAD2 were hybridized under stringent conditions to Southern blots of Arabidopsis genomic DNA
digested with BamHI, EcoRI, or
HindIII. It was evident that under the hybridization
conditions used in these experiments, the probes were gene specific,
since there were different hybridization patterns for each probe. The
hybridization patterns for both GAD1 and GAD2
were simple; one band was apparent in each digest. Based on the
simplicity of the hybridization patterns, comparisons of the
intensities of these Southern blots with those of GDH1 (data not shown), a single-copy gene (Melo-Oliveira et al., 1996), and the
initial results from the identification of genomic clones (data not
shown), it appears that both GAD1 and GAD2
represent single-copy genes.
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| Figure 4.
Southern-blot analysis of Arabidopsis genomic DNA
digested with BamHI (B), EcoRI (E), or
HindIII (H), separated by electrophoresis, transferred
to nitrocellulose membranes, and hybridized under stringent conditions
with gene-specific probes for either GAD1 or
GAD2 (see ``Materials and Methods'' for details).
Molecular masses are indicated.
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Organ Specificity
Specific activity, immuno-, and RNA-blot analyses were used to
determine the level of activity, peptide, and/or transcript(s) for each
GAD isoenzyme in different plant organs (Fig.
5). The specific activity of GAD was
highest in flower stalks and lowest in siliques of 42-d-old plants.
Protein extracts from different organs were separated by SDS-PAGE
(9.0% polyacrylamide), and immunoblot analysis was used to identify
the GAD peptides (Fig. 5A). A 58-kD peptide was identified only in root
samples, and based on the migration of the peptide in the gel, this
subunit was designated GAD1. A 56-kD peptide, designated GAD2, was
identified in protein samples from all organs. The transcript
corresponding to GAD1 was detected only in root samples. The
transcript corresponding to GAD2 was readily detected in all
tissues tested, except for siliques, but was visible in samples from
siliques upon longer exposure (data not shown). The GAD2
transcript was abundant in samples from roots, young leaves, and
flowers, but the level of transcript was relatively lower in flower
stalks and old leaves. The results from this experiment suggest that
GAD1 encodes for a 58-kD peptide and that GAD2
encodes for a 56-kD peptide. These findings are consistent with the
estimated sizes of the peptides deduced from the amino acid sequences
and the results obtained from SDS-PAGE and immunoblot analysis of the
recombinant proteins described above.
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| Figure 5.
Specific GAD activity, immunoblot analysis, and
expression of GAD1 and GAD2 in different
organs of Arabidopsis. Plants were grown in soil and maintained at
20°C to 21°C and 60% to 70% RH under cool-white lights (120-140
µmol PPFD m2 s1) in a 16-h light/8-h dark
cycle until tissues were harvested. Protein and total RNA extractions
were performed on roots (R), flower stalks (FS), flowers (F), siliques
(S), leaves (OL) of 42-d old plants, and leaves of 24-d-old plants
(YL). The specific activity (Spec. Act.) of each organ is
indicated (nanomoles of CO2 per minute per milligram of
protein). The values are the averages of three separate experiments
(±SE). A, For immunoblot analysis, proteins (150 µg/lane) were resolved in an SDS-9.0% polyacrylamide gel, blotted
onto nitrocellulose, and incubated with antiserum raised against
recombinant petunia GAD. The position of GAD1 (58 kD) and GAD2 (56 kD)
peptides are indicated. Total RNA (10 µg) was separated by gel
electrophoresis, blotted onto nitrocellulose, and hybridized with DNA
probes specific for GAD1 (B) or GAD2 (C).
D, A portion of the 26S rRNA probe was used as a control to ensure
equal loading of total RNA into the lanes.
|
|
Effect of Different Nitrogen Sources on GAD
Plants were maintained in soil or vermiculite with complete
nutrient solution and 10 mM KNO3 as a
sole nitrogen source. After 20 d the pots containing vermiculite
were treated with complete mineral nutrient solution containing
10 mM KNO3, 10 mM
NH4Cl, 5 mM
NH4NO3, 5 mM
Glu, or 5 mM Gln as a sole nitrogen source. Determination
of specific GAD activity and immunoblot and northern-blot analyses were
performed on protein and total RNA extracts from leaves (Fig.
6) and roots (Fig.
7) 4 d after treatment with
different nitrogen sources.
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| Figure 6.
Effect of various nitrogen treatments on GAD
in leaves of Arabidopsis. Plants were grown in vermiculite and
maintained in the environmental conditions described in Figure 5.
Plants were watered with a mineral nutrient solution containing nitrate
(10 mM KNO3) for 20 d. The plants were
treated with 10 mM KNO3, 10 mM
NH4Cl, 5 mM NH4NO3, 5 mM Glu, or 5 mM Gln for 4 d prior to the
harvest of the leaves on d 24. One set of plants was grown in soil and
maintained as described in Figure 5. Protein and RNA extractions were
collected and data were analyzed as described in Figure 5. The specific
activity (Spec. Act.; nanomoles of CO2 per minute
per milligram of protein), GAD1 (58 kD) and GAD2 (56 kD) peptides (A),
GAD1 (B), GAD2 (C), and 26S (D)
transcripts are indicated. The results presented above are
representative of three separate experiments. The change in specific
activity and the abundance of the 56-kD peptide and GAD2
transcript are presented as a relative change (percentage) in the
samples from various nitrogen treatments compared with the levels of
GAD activity and abundance of peptide and transcript in controls, which
received a 10 mM KNO3 treatment (E). The
results are a summary of three separate experiments, and the error bars
represent the SEs.
|
|
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| Figure 7.
Effect of various nitrogen treatments on GAD in
roots of Arabidopsis. Plants were treated as described in Figure 6.
Protein and RNA extractions were collected and analyzed as described in
Figure 5. The specific activity (Spec. Act.; nanomoles of
CO2 per minute per milligram of protein), GAD1 (58 kD) and
GAD2 (56 kD) peptides (A), and GAD1 (B),
GAD2 (C), and 26S (D) transcripts are indicated. The
results are representative of three separate experiments.
|
|
The specific activity of GAD in leaves of plants treated with ammonium,
either 10 mM NH4Cl or 5 mM NH4NO3, was
approximately 85% higher than that of the 10 mM
KNO3 (control) treatments (Fig. 6). There were
smaller increases, 58% or 67%, respectively, in the specific activity
from extracts of plants treated with either Glu or Gln when compared
with the control treatment. The 56-kD (GAD2) peptide was observed in
all of the treatments (Fig. 6A), but the abundance of the peptide
varied among nitrogen treatments. The 56-kD peptide (GAD2) was more
abundant in 10 mM NH4Cl- or 5 mM NH4NO3-
treated samples than in samples from plants treated with 5 mM Glu or 5 mM Gln, which were more abundant
than the control (10 mM KNO3).
Neither the 58-kD peptide (GAD1) (Fig. 7A) nor the GAD1
transcript (Fig. 6B) was detected in the leaf samples. The GAD2 transcript was detected in all of the samples (Fig.
6C). The relative change in specific activity and the abundance of 56-kD peptide and GAD2 transcript detected in the various
nitrogen treatments were compared with levels in controls, which
received a 10 mM KNO3 treatment (Fig.
6E). There was a proportional increase in the amount of 56-kD peptide
and GAD2 transcript detected in each treatment compared with
controls. However, the relative change in the level of 56-kD peptide
and GAD2 transcript were approximately 4 times higher than
the increase in the specific activity; i.e. the specific activity
increased 83%, whereas the amount of the 56-kD peptide and
GAD2 transcript increased 314% and 432%, respectively, in
the 5 mM NH4NO3
treatment compared with the control. In summary, these data suggest
that GAD activity is altered by different nitrogen sources, and the
activity is regulated in part by gene expression or RNA stability.
The specific activity of GAD in extracts from roots (Fig. 7) of plants
treated with different nitrogen sources was 3 to 6 times higher than
that of extracts from leaves (Fig. 6) of the same plants. There was one
exception: the specific activity in root extracts from plants
maintained in soil was similar to that of leaves from the same plants.
The specific activity in roots of plants treated with 10 mM
NH4Cl, 5 mM
NH4NO3, 5 mM
Glu, or 5 mM Gln were 3% to 21% lower than that in the
controls (10 mM KNO3 treatments).
Immunoblot analysis showed (Fig. 7A) that all samples contained both
the 58- and 56-kD peptides, GAD1 and GAD2, respectively. In most cases,
the level of each peptide detected on immunoblots was approximately the
same. However, there was slight variability in the ratio of the level
of the 58-kD transcript to that of the 56-kD peptide within each
treatment (data not shown). The transcripts for GAD1 and
GAD2 were detected on RNA blots (Fig. 7, B and C,
respectively) in all of the root samples. After normalization of the
data to the 26S probe (data not shown), the amount of GAD1 and GAD2 transcript in each sample remained unchanged.
However, there were small decreases (10%-15%) in the level of
GAD2 transcript in the roots of Gln-treated plants
compared with the control.
| |
DISCUSSION |
Two full-length cDNAs encoding GAD in Arabidopsis have been
characterized. The two clones appear to encode distinct single-copy genes, GAD1 and GAD2, in Arabidopsis. The small
GAD family joins the growing list of small multigene families encoding
enzymes involved in Glu metabolism and/or catabolism, i.e. glutamine
synthetase (Peterman and Goodman, 1991), glutamate synthase
(Coschigano et al., 1998), aspartate aminotransferase (Schultz
and Coruzzi, 1995; Wilkie et al., 1995), and glutamate
dehydrogenase (Melo-Oliveira et al., 1996; Turano et al., 1997)
in Arabidopsis.
The deduced amino acid sequences of both cDNA clones lack target
sequences, suggesting that they are located in the cytosol, which is
consistent with the cellular location reported for numerous plant GAD
isoenzymes (Wallace et al., 1984; Satyanarayan and Nair, 1985;
Breitkreuz and Shelp, 1995). The peptides appear to be divided into three distinct regions: (a) a small, variable region at the amino
termini (from approximately residues 1-35), (b) a large, highly
conserved region (from approximately residues 36-245), and (c) a
small, highly variable region at the carboxy termini (from
approximately residues 246-493). To date, there does not appear to be
a specific function associated with the small, variable region at the
amino termini. The large, highly conserved region contains the GAD
enzyme domain. In this region, GAD1 and GAD2 contain a putative
PLP-binding motif, Ser-X-X-Lys. This motif is conserved in the 58-kD
GAD from petunia (Baum et al., 1993) and the putative GAD cDNA from
tomato (Gallego et al., 1995), and it aligns with the same motif
in the products of the gadA and gadB genes from
E. coli (Smith et al., 1992). Furthermore, the sequence
analysis is consistent with our biochemical analysis of GAD2, which
showed that PLP was required for activity (F.J. Turano and T.K. Fang,
unpublished data). The highly variable region at the carboxy termini
contains the CaM-binding domain. Both peptides contain a Trp residue in
the highly variable carboxy termini, at positions 488 (GAD1) and 480 (GAD2). Similarly, there is a Trp in this region in petunia GAD, which
was shown to be essential for CaM binding (Arazi et al., 1995). In
addition, Arazi et al. (1995) demonstrated that CaM bound to the
carboxy terminus of GAD1. In this report both rGAD1 and rGAD2 were
purified by affinity chromatography using CaM-Sepharose. Enzymatic
activity for pure rGAD1 and rGAD2 was stimulated 35- and 13-fold,
respectively. The stimulation of rGAD1 and rGAD2 by
Ca2+/CaM was inhibited 65% and 81%,
respectively, by the CaM antagonist TFP. Furthermore, GAD activity was
not stimulated by Ca2+ or CaM alone. Together,
these data demonstrate that both Arabidopsis isoenzymes, although
variable in their carboxy termini, bind Ca2+/CaM.
The physiological significance for the variability in the CaM region is
not known, but since there are six CaM and CaM-like genes
differentially expressed in Arabidopsis (Braam and Davis, 1990;
Sistrunk et al., 1994; Ito et al., 1995), it is possible that this
region may be involved in specific binding of different CaM and/or
CaM-like proteins. It is worth noting that the stimulation of rGAD1 and
rGAD by Ca2+/CaM is significantly different, although they
were assayed under similar conditions. It is plausible that the
distinct CaM-binding domains may play a role in the differential
stimulation of the two recombinant proteins. However, it is premature
to overlook the possible function of the distinct amino termini and/or
variations in the enzymatic domains of the two peptides in the
stimulatory process.
Both cDNA clones were expressed in E. coli and the estimated
molecular masses for the recombinant proteins were 58 and 56 kD for
rGAD1 and rGAD2, respectively (Fig. 3). The migration of the
recombinant proteins was similar to those of peptides identified by
immunoblot analyses in crude protein extracts from plants. The data
suggest that GAD1 encodes a 58-kD peptide (GAD1) and GAD2 encodes a 56-kD peptide (GAD2). This gene/peptide
assignment was consistently observed on immunoblots and RNA blots from
different organs (Fig. 5) and from similar analyses comparing extracts
from leaves (Fig. 6) and roots (Fig. 7) of plants subjected to
different nitrogen treatments. Protein and RNA extracts from roots
(Figs. 5, lane R, and 7) had detectable levels of both peptides, 58 and 56 kD, and both transcripts, GAD1 and GAD2,
whereas all other organs (Figs. 5 and 6) had detectable levels of only
the 56-kD peptide (GAD2) and the GAD2 transcript.
In leaves (Figs. 5, lanes YL and OL, and 6) there appeared to be a
correlation between the specific activity and the amount of the 56-kD
peptide and GAD2 transcript detected on immunoblots and RNA
blots, respectively. These data suggest that GAD activity may be
controlled by transcriptional events or by RNA stability in leaves. A
similar correlation among the accumulation of GAD transcript, GAD peptide, and in vitro GABA synthesis was observed in
petunia (Chen et al., 1994). Combined, these data suggest that the
transcriptional and posttranscriptional processes that control GAD
activity in the leaves of Arabidopsis and petunia may be similar; however, it is too early to state whether these processes occur in the
leaves of all plant species. Although there appeared to be a
correlation among the in vitro specific activity, the amount of the
56-kD peptide, and the level of GAD2 transcript in leaves, this phenomenon did not appear to be conserved in all plant organs. In
flower stalks and flowers (Fig. 5, lanes FS and F), there appeared to
be little or no correlation among the level of GAD2
transcript, 56-kD peptide, and specific activity. In flower stalks, the
specific activity was higher than that in flowers; likewise, the level of 56-kD peptide was higher in flower stalks than in flowers. However,
the level of GAD2 transcript was lower in flower stalks than
in flowers. The data suggest that GAD 56-kD protein is more stable in
flower stalks than in flowers or leaves of Arabidopsis. These results
suggest that other factors such as posttranslational events may
regulate GAD activity in these organs. Chen et al. (1994) made similar
conclusions when comparing their results concerning developmental
studies of petunia flowers with developmental studies of leaves; in
their study a 58-kD peptide appeared to be more stable in flowers than
in leaves. Also, in the siliques there was little or no GAD activity or
transcript detected, but the 56-kD peptide was readily detected,
suggesting that there could be other factors, such as posttranslational
events, controlling GAD in these organs. However, there could be
factors that affect the in vitro determination of GAD activity in
samples from siliques. First, the amount of GAD2 detected in samples
from siliques by immunoblot analysis was very low compared with other
organs and we may be approaching the limits of our assay method.
Second, because of the possible release of inhibitory compounds during protein isolations, the level of in vitro GAD activity may not be a
true measure of in vivo activity.
In roots (Fig. 7) there was little or no change in the amount of
GAD1 or GAD2 transcripts detected in each
treatment, but there were small differences in the abundance of the 58- or 56-kD peptides. In addition, there was small variability among the
ratio of 58-kD to 56-kD peptides in replica samples from the same
treatment. This may be due to differential stability of the two
peptides during extraction or the different location of the peptides in the root. To our knowledge, there are no data to support the former hypothesis, but results from Ling et al. (1994) suggest that the GAD
isoenzymes may be localized in different regions of the root. They
identified a 62-kD peptide in a region 2 to 17 cm from the root tip,
but never within it. The small variability in the ratio of 58-kD to
56-kD peptides in our experiments could be attributed to the different
proportions of distinct root regions in our samples.
The GAD2 transcript, which encodes a 56-kD peptide, was
detected in all tissues tested, suggesting that it is constitutively expressed. Similarly, the transcript corresponding to a 58-kD peptide
was identified in all of the tissues of petunia (Chen et al., 1994).
Together, these data suggest that these peptides may play a crucial
role in plant development. This hypothesis has been supported by
results from studies of transgenic plants overexpressing a truncated
version of the petunia GAD in tobacco (Baum et al., 1996). Conversely,
the GAD1 transcript, which encodes a 58-kD peptide, was
detected only in roots. Similarly, a 62-kD peptide was identified in
the roots of fava bean (Ling et al., 1994). Together, these data
suggest that there are root-specific GAD isoenzymes for which the
physiological function is unknown.
One goal of this study was to gain a greater understanding of the
function and role of the GAD isoenzymes in nitrogen metabolism. The
GAD2 transcript appears to be constitutively expressed;
however, the level of transcript detected by RNA-blot analyses was
altered by different nitrogen sources. The level of GAD2
transcript increased with 10 mM
NH4Cl, 5 mM
NH4NO3, 5 mM
Glu, or 5 mM Gln treatments. Our data suggest that ammonia,
Glu, and Gln affect GAD activity in leaves by increasing gene
expression or RNA stability. Other investigators have reported
increases in GAD activity or GABA accumulation in plant cells after
different nitrogen treatments. Kishinami and Ojima (1980) demonstrated
that GABA titers increased significantly in rice cultures after
treatment with ammonium or Gln; however, no increase was observed in
cell cultures after Glu treatments. They used inhibitors of glutamine
synthetase and glutamate synthase to demonstrate that ammonium
increased Gln levels, but they did not determine the effects of these
compounds on GAD activity. It appeared from their data that GABA
biosynthesis was via GAD. Tuin and Shelp (1994) suggested that GAD was
involved in nitrogen and carbon metabolism via the GABA shunt. Cholewa et al. (1997) reported a Ca2+/CaM-independent
increase in GABA when asparagus mesophyll cells were treated with Glu.
Furthermore, Baum et al. (1996) demonstrated that overexpression of a
truncated version of a petunia GAD gene lacking the CaM-binding domain
in transgenic tobacco had increased titers of free GABA and decreased
titers of free Glu. The combined findings from these studies suggest
that GAD2 or GAD2 homologs may play a unique physiological role in
nitrogen metabolism.
| |
FOOTNOTES |
*
Corresponding author; e-mail fturano{at}asrr.arsusda.gov; fax
1-301-504-7521.
Received February 11, 1998;
accepted May 14, 1998.
The nucleotide sequence data for the 3 noncoding region of
GAD1 and full-length sequence for GAD2
are available in GenBank under accession nos. AF060094 and
U46665, respectively.
1
Mention of 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 may be suitable.
| |
ABBREVIATIONS |
Abbreviations:
CaM, calmodulin.
GABA,  -aminobutyric acid.
GAD, glutamate decarboxylase.
PLP, pyridoxal 5-phosphate.
rGAD, recombinant GAD.
TFP, trifluoperazine.
| |
ACKNOWLEDGMENTS |
The authors would like to thank Drs. Benjamin F. Matthews, Mark
Tucker, Jane Weisemann, and Kenneth G. Wilson for critical review of
this manuscript. We appreciate the technical assistance of Ms. Sona S. Thakkar, who performed all of the molecular biology experiments, and
Ms. Geraldine G. Glover, who cloned and sequenced rGAD1.
| |
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[CrossRef][Web of Science][Medline]
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Abstract
Introduction