From the Laboratory of Molecular Biology and Biotechnology, Research Center of Medicinal Resources, Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan
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ABSTRACT |
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In plants, Ser is synthesized through a couple of
pathways. 3-Phosphoglycerate dehydrogenase (PGDH), the first enzyme
that is involved in the phosphorylated pathway of Ser biosynthesis, is
responsible for the oxidation of 3-phosphoglycerate to
phosphohydroxypyruvate. Here we report the first molecular cloning and
characterization of PGDH from Arabidopsis thaliana.
Sequence analysis of cDNA and a genomic clone revealed that the
PGDH gene is composed of three exons, encoding a 623-amino acid
polypeptide (66,453 Da). The deduced protein, containing three of the
most conserved regions in the NAD-dependent 2-hydroxyacid
dehydrogenase family, has 38-39% identity to its animal and bacterial
counterparts. The presence of an N-terminal signal sequence for
translocation into plastids was confirmed by particle-gun bombardment
experiments using green fluorescence protein as a reporter protein for
subcellular localization. Southern hybridization analysis and
restriction fragment length polymorphism mapping indicated that PGDH is
a single-copy gene that is mapped to the upper arm of chromosome 1. Northern hybridization analysis indicated preferential expression of
PGDH mRNA in root tissues of light-grown plants, suggesting that
the phosphorylated pathway of Ser biosynthesis plays an important role
in supplying Ser to non-photosynthetic tissues. The recombinant enzyme
overproduced in Escherichia coli displayed hyperbolic
kinetics with respect to 3-phosphoglycerate and NAD+.
3-Phosphoglycerate dehydrogenase
(PGDH1; EC 1.1.1.95), the
first enzyme in the Ser biosynthetic pathway from 3-phosphoglycerate (3-PGA), catalyzes the oxidation of 3-PGA to form
phosphohydroxypyruvate by utilizing NAD+ as a cofactor.
Phosphohydroxypyruvate is subsequently transaminased by phosphoserine
aminotransferase to yield phosphoserine. In the final step,
dephosphorylation of phosphoserine to Ser is performed by phosphoserine
phosphatase (Fig. 1). The molecular
cloning and biochemical characterization of PGDH have been reported for
a variety of bacterial (1-3) and animal (4-6) sources. In higher plants, the biochemical characterization of PGDH enzyme preparation has
been carried out in pea (7) and spinach (8); however, no investigation
on molecular cloning and characterization was reported.
INTRODUCTION
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ABSTRACT
INTRODUCTION
REFERENCES
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Fig. 1.
Main route of Ser biosynthesis in higher
plants. Arrow 1, ribulose-1,5-bisphosphate
carboxylase/oxygenase, arrow 2, PGDH; arrow 3,
phosphoserine aminotransferase; arrow 4, phosphoserine
phosphatase; arrow 5, phosphoglycolate phosphatase;
arrow 6, glycolate oxidase; arrow 7,
amino-acid:glyoxylate aminotransferase; arrow 8, glycine
decarboxylase-serine hydroxymethyltransferase complex; arrow
9, serine hydroxymethyltransferase; arrow 10,
glycine-hydroxypyruvate aminotransferase; arrow 11,
Alanine-hydroxypyruvate aminotransferase; arrow 12,
serine:glyoxylate aminotransferase; arrow 13, glycerate
dehydrogenase; arrow 14, hydroxypyruvate reductase;
arrow 15, 3-phosphoglycerate phosphatase; arrow
16, glycerate kinase. C, chloroplast/plastid
localization; M, mitochondrion localization; P,
peroxisome localization.
Ser can be formed by more than two pathways in higher plants (9, 10). The photorespiratory pathway (11) of Ser biosynthesis via glyoxylate and Gly is the major route of Ser formation in photosynthetic tissues under light conditions. The glycine decarboxylase multienzyme complex (GDC), along with the enzyme serine hydroxymethyltransferase, is responsible for the respiratory conversion of Gly to Ser (12, 13). The cDNAs encoding the four component enzymes of GDC (14-17) and serine hydroxymethyltransferase (18, 19) from plants have been cloned and characterized. The other two Ser biosynthetic pathways from 3-PGA via either phosphorylated or non-phosphorylated intermediates are proposed to be important Ser sources in non-photosynthetic tissues or under dark conditions, depending on the type of tissues involved (20) (Fig. 1). For these pathways, 3-PGA is supplied by glycolysis or the pentose phosphate pathway.
The serA gene, which encodes PGDH, has been cloned and
characterized in Escherichia coli (1, 21, 22). E. coli PGDH is a tetramer of identical subunits, each consisting of
three domains for nucleotide binding, substrate binding, and regulatory function. PGDHs from pea (7), E. coli (23, 24), and
Bacillus subtilis (25) are reported to be feedback-inhibited
by Ser, whereas the enzymes from spinach (8) and animals (6, 26) do not
exhibit similar feedback regulation. However, PGDH in rat livers is
regulated at the transcriptional level (6). In this paper, we describe,
for the first time, cDNA and genomic cloning, biochemical
characterization, and expression of PGDH from a higher plant,
Arabidopsis thaliana.
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EXPERIMENTAL PROCEDURES |
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Plant Materials-- A. thaliana ecotype Columbia seeds were germinated and grown on germination medium (27) agar plates under 16/8-h light and dark cycles at 22 °C for 3 weeks. For the dark-treated seedlings that were used for Northern analyses, 2-week-old seedlings were wrapped in aluminum foil and subsequently grown for another week before RNA extraction was carried out.
Isolation of cDNA and Genomic Clones--
cDNA library
screening was carried out using a 32P-labeled
synthetic 50-mer oligonucleotide probe
(5'-GTTACTGTGACACCTCATCTTGGAGCTAGCACAAAAGAAGCTCAGGAAGG-3') that
was based on the deposited sequence of the cDNA insert of the
Arabidopsis expressed sequence tag FAFH01
(GenBankTM accession number ATTS3047). Approximately
2.5 × 105 plaques from the gt11 cDNA library
constructed from A. thaliana whole plants were screened. For
genomic cloning, the cDNA clone CPGDH-5 was used to screen
~4 × 105 amplified plaques from the
Arabidopsis genomic
EMBL3 SP6/T7 library
(CLONTECH).
Hybridization of the membranes (Hybond N+, Amersham Pharmacia Biotech) was carried out at 65 °C in 5× SSPE (0.9 M NaCl, 0.05 M sodium phosphate (pH 7.7), and 5 mM EDTA), 0.5% SDS, 5× Denhardt's solution (28), and 25 µg/ml salmon sperm DNA. The final washing of membranes was conducted at 65 °C in 0.1× SSPE and 0.1% SDS for 10 min.
DNA Subcloning and Sequencing--
The cDNA and genomic DNA
inserts of the isolated clones were subcloned into appropriate cloning
sites of pBluescript II(SK) (Stratagene). Sequencing of
full-length DNA was carried out on both strands using a series of
overlapping exonuclease III-digested clones created with the Exo/Mung
deletion kit (Stratagene). Autosequencing was conducted by the dideoxy
chain termination method with Thermo Sequenase (Amersham Pharmacia
Biotech) using a Shimadzu DNA sequencer (Model DSQ1000).
Nucleic Acid Preparation and Blot Analyses-- Genomic DNA was extracted from the leaves of 3-week-old seedlings as described (29). For Southern analysis, ~5 µg of genomic DNA was digested with restriction enzymes, separated by electrophoresis through a 0.8% (w/v) agarose gel, and transferred to a Hybond N+ membrane. Isolation of total RNA was performed by a modified guanidine HCl method as described (28) from the leaves and roots of 3-week-old seedlings. About 10 µg of total RNA was separated under denaturing conditions on a 1.2% (w/v) agarose gel containing formaldehyde and transferred to a Hybond N+ membrane.
DNA and RNA blots were probed with a 32P-labeled probe synthesized from the cDNA clone CPGDH-5. To investigate the mRNA expression levels of H-protein (a subunit of GDC) and serine hydroxymethyltransferase, 32P-labeled probes synthesized from cDNA inserts of Arabidopsis expressed sequence tag clones 200K16T7 and 111M16T7, respectively, were used. To verify equivalent loadings of RNA on blots, membranes were probed with a 32P-labeled rice rDNA (pRR217) (30). Relative values of mRNA were calculated based on the hybridization intensities of specific signals on the blots quantified by a Fuji BAS-2000 image analyzer.
Restriction fragment length polymorphism mapping was carried out with 30 recombinant inbred lines (31). Hybridization and washing were carried out as described above, except that the final washing was performed in 0.5× SSPE and 0.1% SDS for 10 min at 65 °C. The 32P-labeled genomic clone GPGDH-17 was used as a probe for hybridization. The map distance was kindly calculated by Dr. M. Arnold (Nottingham Arabidopsis Stock Center), based on the restriction fragment length polymorphism profiles generated by EcoRV.
Complementation of E. coli serA Mutant--
To
verify the identity of the cDNA clone CPGDH-5, E. coli
serine
536 auxotrophs (HfrOR11 glu
V42
SerA13 T3r) (1)
transformed with plasmids pTV118N (TaKaRa, Kyoto, Japan) and pPGDH-AB14
were tested for their ability to grow on M9 minimal medium without
supplementation of Ser.
Overexpression of Recombinant Enzyme--
A general method of
DNA engineering was followed as described (28). NcoI sites
were created on both ends of the coding region by polymerase
chain reaction engineering using the synthetic primers 5'-CTACACCATGGCATTTTCATCTCG-3' and 5'-CATGACCATGGATAAAACACCTT-3'. The engineered DNA fragment was inserted into the NcoI
site of pET32a(+) (Novagen), in which the cDNA was placed under a
strong 10 promoter in both the sense and antisense orientations. The plasmids were then introduced into E. coli AD494(DE3) pLysS,
in which the gene for lysogenic T7 RNA polymerase under the
lacUV5 promoter is induced by
isopropyl-1-thio-
-D-galactopyranoside. Transformed
E. coli cells were precultured in LB medium (28) supplemented with carbenicillin (100 mg/liter) at 37 °C overnight and then were added to 200 ml of fresh culture medium and further cultured for 4 h. After adding
isopropyl-1-thio-
-D-galactopyranoside (1 mM)
to induce gene expression, incubation was continued for another 4 h.
Enzyme Assay-- Crude extracts were prepared from 50-ml stationary-phase cultures grown in LB liquid medium. Cells were harvested; washed in 20 ml of 200 mM Tris-HCl (pH 7.5), 200 mM KCl, 1 mM EDTA, and 1 mM dithiothreitol; and then resuspended in the same buffer. The cells were disrupted by sonication, and extracts were centrifuged (15,000 rpm for 15 min at 4 °C) to remove debris and membrane-bound proteins (32). PGDH in the supernatant was assayed in the direction of NADH oxidation at 30 °C. The assay mixture contained 10 µl of enzyme extract, 25 mM Hepes (pH 7.1), 100 µM NADH, 400 mM KCl, and 90 µM phosphohydroxypyruvate in a final volume of 1 ml (6). The reaction was started by the addition of phosphohydroxypyruvate. One unit of enzyme activity is defined as the amount that oxidizes 1 µmol of NADH/min under the indicated condition. For the physiological direction regarding enzymatic reaction, PGDH was assayed as described (6) at 30 °C with some modifications. The assay mixture (1 ml) contained 10 µl of extract, 200 mM Tris-HCl (pH 9.0), 25 mM EDTA, 5 mM 3-PGA, 2.5 mM dithiothreitol, and 0.5 mM NAD+. One unit of enzyme activity is defined as the amount that reduces 1 µmol of NAD+/min under the indicated condition.
Subcellular Organelle Localization of PGDH by Green Fluorescent
Protein (GFP)--
To verify the subcellular localization of PGDH in
plant cells, a 246-base pair (bp) polymerase chain reaction-amplified
fragment encoding the N-terminal region of
Arabidopsis PGDH of the cDNA clone CPGDH-5, using
primers 5-AGTGTGGTCGACATGGCATTTTCATCTTCGT-3' and
5'-CCTGGCCATGGCCTTTGGAAGAGTTGGATT-3', was subcloned into
plasmid CaMV35S-sGFP(S65T)-NOS3' (33), with a CaMV35S promoter at its 5'-end, GFP as the reporter gene, and nopaline synthetase terminator as
the transcription terminator, to yield plasmid CPGDH-GFP. Plasmid constructs 35S-TP-sGFP(S65T) (33), carrying transit peptide sequence
obtained from the ribulose-1,5-bisphosphate carboxylase/oxygenase small
subunit polypeptide of Arabidopsis (34), and pCS-C-GFP, carrying mitochondrial cysteine synthase isoform C (35), were used as
positive controls for plastid and mitochondrion localization, respectively. Plasmid CaMV35S-sGFP(S65T)-NOS3', carrying GFP alone without a transit peptide insert, was used as a positive control for
cytosolic protein.
These plasmids were used for subsequent particle-gun bombardment. Particle-gun bombardment was carried out using the Helios Gene-Gun system (Bio-Rad) following the standard protocol provided by the supplier. A microcarrier loading quantity of 0.5 mg of gold/target and a DNA loading ratio of 2 µg of DNA/mg of gold were chosen, and 3-week-old Arabidopsis seedlings were bombarded at a pressure of 100 p.s.i. Plates were incubated for 20 h under illumination conditions at 22 °C after bombardment. Signals from individual leaves were viewed with an Olympus fluorescent microscope (BX50-FLA) using a Chroma dual band filter, FITC, and TRITC (Olympus Corp.), which provide excitations at 475-490 and 545-565 nm and emissions at 510-530 and 585-620 nm.
Miscellaneous Techniques--
SDS-polyacrylamide gel
electrophoresis, protein quantitation, and primer extension were
carried out as described (28).
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RESULTS |
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Arabidopsis PGDH Is Closely Related to Its Counterparts from B. subtilis, Synechocystis sp., and Mammals-- Phage plaques produced from an Arabidopsis whole plant cDNA library were screened with a synthetic 50-mer oligonucleotide probe based on the sequence of A. thaliana expressed sequence tag clone FAFH01, which shows high homology to PGDH from B. subtilis. Among the four positive clones selected for further studies, CPGDH-5, which contains the largest cDNA insert (2.2 kilobase pairs), was subcloned and sequenced. Sequence analysis revealed an open reading frame of 1881 nucleotides, encoding for 623 amino acid residues. The first ATG triplet, which is 14 nucleotides away from the 5'-end of CPGDH-5, is designated as the translational start point because the sequence around the Met codon (AGTCATGGC) matches well with the consensus sequence for plant gene initiation codons (AACAATGGC) (36). This is further supported by the primer extension result that mapped the transcriptional start point at 38 bp before the translational start site. A 3'-untranslated region of 265 nucleotides downstream of the translational stop codon (TAA) is present in the cDNA sequence. The AATAAA polyadenylation signal is located 129 nucleotides upstream of the poly(A) tail.
The deduced amino acid sequence of Arabidopsis PGDH has been aligned with PGDHs from other organisms (Fig. 2). A phylogenetic tree (Fig. 3) indicates that Arabidopsis PGDH is closely related to the enzymes from B. subtilis, Synechocystis sp., and mammals. These proteins form a family distinct from other bacterial and yeast PGDHs. The deduced 66,453-Da protein, containing three of the most conserved regions in the NAD-dependent 2-hydroxyacid dehydrogenase family, has 38-39% similarity to the amino acid sequences of PGDHs from other organisms (Fig. 2). The first pattern is based on a Gly-rich region that probably corresponds to the NAD-binding domain, Gly-X-Gly-X2-Gly-X17-Asp (37). Two other patterns contain a number of conserved charged residues, some of which may play a role in the catalytic mechanism. The 623 amino acid residues of Arabidopsis PGDH, sharing the three-dimensional structure of E. coli PGDH (22), is the longest sequence among all. It differs from the rest mainly due to its C-terminal domain and N-terminal extension.
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PGDH Gene of Arabidopsis Is Mapped to Chromosome 1--
The
cDNA clone CPGDH-5 was used to screen the
Arabidopsis genomic DNA library. Among various clones,
GPGDH-17 was selected for further studies. The 7.1-kilobase pair
fragment that covers the PGDH structural gene and the 5'- and
3'-flanking regions are presented in Fig.
4A. Analysis of genomic and
cDNA sequences revealed the presence of two introns. All of the
exon/intron junctions had the consensus GT/AG splice donor and acceptor
sites. The sequences for both the coding regions and the 3'- and
5'-untranslated regions of the cDNA clone are identical to those
for the genomic clone, demonstrating that this gene is actively
transcribed. The transcriptional start site of the genomic clone was
determined using primer extension analysis. A single major
transcriptional start point was confirmed to be located 38 bp before
the translational start site. An AT-rich sequence is located at 27 to
34 bp with respect to the transcriptional start site (+1), and a
potential CAAT sequence is located at
79 to
82 bp (Fig.
4B).
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Using the coding sequence of PGDH as a probe, the Southern blot results (Fig. 5) suggest that Arabidopsis PGDH is a single-copy gene. Upon digestion with BglII, EcoRI, EcoRV, SacI, and XbaI, several bands were observed due to the presence of several restriction sites for these endonucleases in the genomic sequence. The Arabidopsis PGDH gene was mapped to the upper arm of chromosome 1 between the markers g3786 and g3829.
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Arabidopsis PGDH Is a Plastidic Protein-- The 60-amino acid leader sequence exhibits the general features of a transit peptide for transportation of protein to plastid. It starts with Met-Ala; is rich in hydroxylated amino acids, Ser and Thr (19/60); is rich in small hydrophobic amino acids, Ala and Val (13/60); is essentially deficient in acidic amino acids, Asp and Glu (1/60); and has a net positive charge (pI 11.5). Prediction by the PSORT program2 also suggested its localization in chloroplasts. The recombinant fusion protein CPGDH-GFP, containing the N-terminal 82-amino acids fused with GFP, could be detected in intact tissues after delivering the constructs into Arabidopsis leaves by particle-gun bombardment. The observed signals in the construct containing the predicted N-terminal transit peptide of PGDH from Arabidopsis were observed as green fluorescence that lighted up the chloroplasts (data not shown). They were similar to those exhibited by GFP fused with the transit peptide of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit polypeptide of Arabidopsis (34), which was already known to be sufficient for translocation of a passenger protein to chloroplasts (33). Despite a fainter degree of signals exhibited by CPGDH-GFP compared with the positive control, the fluorescent pattern observed for CPGDH-GFP was clearly distinct from those exhibited by mitochondrial and cytosolic proteins. These results confirmed that the N-terminal sequence of Arabidopsis PGDH is sufficient for translocation of passenger protein into chloroplasts, and thus, PGDH is a plastidic protein.
Arabidopsis PGDH Can Functionally Complement E. coli
serA Mutant--
The identity of the isolated cDNA
clone CPGDH-5 was confirmed by successful complementation of the
E. coli serine auxotroph
536 (HfrOR11 glu
V42
SerA13 T3rr)
(1) (Fig. 6). Mutant E. coli
cells were transformed with the expression plasmid pPGDH-AB14, in which
the expression of PGDH is regulated by the lacZ promoter.
Transformants could grow on M9 minimal medium in the absence of Ser,
whereas pTV118N-transformed E. coli
536 cells were not
able to grow without supplementation of Ser, indicating the
authenticity of CPGDH-5 encoding the functional PGDH. The shortened
cDNA with 95 amino acids truncated at the N terminus was not able
to complement the E. coli
536 mutant. This failure of
complementation could be due to deletion of amino acid residues
conserved among PGDHs from different organisms and thus necessary for
functional expression.
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Biochemical Properties of Recombinant Arabidopsis PGDH Produced in E. coli-- Recombinant PGDH was overproduced in E. coli AD494 cells using a pET32a(+) vector system with a strong promoter. The recombinant protein was visualized on SDS-polyacrylamide gel as the expected 90-kDa protein in the insoluble fraction of crude extract as an inactive form. However, production of PGDH in the soluble fraction was too low to be visualized by SDS-polyacrylamide gel electrophoresis. Nevertheless, the soluble form of the protein exhibited PGDH activity of 0.14 ± 0.01 units/mg of protein in the physiological direction, catalyzing the oxidation of 3-PGA to phosphohydroxypyruvate, and of 10.95 ± 1.36 units/mg of protein in the opposite direction, reducing phosphohydroxypyruvate to 3-PGA. Enzyme activity was not detected in the cells transformed by the cDNA in the antisense orientation relative to the promoter. The construct of an insert with a 95-amino acid truncation at the N terminus could not be overexpressed in E. coli AD494 cells.
Double-reciprocal plots of the data for the initial rates demonstrated Km values of 0.35 and 0.12 mM for phosphohydroxypyruvate and NADH, respectively, at pH 7.1. The activity was inhibited by phosphohydroxypyruvate (~90 µM), as reported for the rat enzyme (6). This inhibition could be released by 100-400 mM KCl. Km values for 3-PGA and NAD+ were 1.19 and 0.01 mM, respectively, at pH 9.0. Ser, Thr, Val, Gly, Trp, O-acetyl-L-Ser, and Cys (in the range of 5-50 mM) had no effect on the reaction rates in both orientations.
Preferential PGDH Expression in Root Tissues-- The mRNA abundance of PGDH was examined in leaf and root tissues from both light-grown and dark-treated plants. The highest level of PGDH mRNA expression was observed in light-grown root tissues (Fig. 7A). It was ~2-3-fold higher than the mRNA expression in dark-grown root and leaf tissues. A minor amount of mRNA expression (~1:15 of roots in light) was detected in the light-grown leaf tissues. The preferential expression of PGDH in root tissues of light-grown plants was in contrast with the expression pattern exhibited by H-protein (a component protein of GDC) (13) and serine hydroxymethyltransferase (18, 19), which accumulated primarily in the light-grown leaf tissues (Fig. 7, B and C). These RNA blot analyses suggested that the regulation of the PGDH gene is mainly exerted at the level of transcription or by stability of mRNA.
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DISCUSSION |
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This is the first investigation on the molecular characterization of plant PGDH, a key enzyme committed to the entry step of the phosphorylated pathway of Ser biosynthesis. The isolated cDNA contains an open reading frame encoding the entire PGDH polypeptide of A. thaliana. The deduced protein with a molecular mass of 66,453 Da, sharing the three-dimensional structure of the E. coli enzyme (22), is composed of three distinct domains: a nucleotide-binding domain, a substrate-binding domain, and a regulatory domain or a Ser-binding domain in each subunit of the tetrameric protein of E. coli PGDH. The main contact points between the subunits are at the level of the coenzyme-binding domains and the regulatory domains, indicating the importance of these zones for tetramerization. The deduced amino acid sequence has high similarity to eukaryotes (human and rat), but not yeast. Surprisingly, the nucleotide- and substrate-binding domains of B. subtilis PGDH exhibit more similarity to the eukaryotic enzymes than to other bacterial PGDH enzymes (E. coli and Haemophilus influenza), whereas the yeast enzyme is closer to the latter. This suggests that there are two different types of PGDH that may have evolved at its origin before diverging to eukaryotes and prokaryotes. Three of the most common regions in the NAD-dependent 2-hydroxyacid dehydrogenase family are conserved in Arabidopsis PGDH.
Alignment of the Arabidopsis PGDH sequence with bacterial, yeast, and mammal sequences reveals the presence of a presequence, presumably targeting the nuclear encoded protein to the chloroplasts/plastids. The essential common features of the chloroplast presequence are exhibited by the first 60 deduced amino acid residues at the N terminus. The exact cleavage site of the transit peptide was difficult to determine. Based on the multiple alignment results (Fig. 2) showing a low homology between various organisms at its first 80 amino acids, together with our complementation experiment and attempts to overexpress PGDH protein with its 95 amino acids truncated at the N terminus, the cleavage site is most probably located between 80 and 90 amino acids away from the N terminus. Even if the protein is actually processed after entering the chloroplasts, the kinetic properties may not change much. From our recent results with phosphoserine aminotransferase (38), the full-length proteins and the proteins with the transit peptide truncated exhibited essentially the same properties.
We have provided evidence for visualization of the targeting of the fusion protein of the N-terminal transit peptide and GFP to leaf chloroplasts. The weaker signals shown by CPGDH-GFP compared with the positive control of the ribulose-1,5-bisphosphate carboxylase/oxygenase transit peptide may be due to its intrinsic nature. PGDH has been detected in the proplastids of soybean (39, 40), and there may be an extrachloroplastic form, too (8). However, Southern blot analysis and restriction fragment length polymorphism results confirmed that Arabidopsis PGDH is a single-copy gene that is mapped to the upper arm of chromosome 1.
We have provided molecular evidence that the mRNA of PGDH is preferentially expressed in roots, similar to that of phosphoserine aminotransferase, being the subsequent enzyme of the phosphorylated pathway (41). Conversely, the mRNA abundance of the H-protein (a subunit of GDC) and serine hydroxymethyltransferase, which both are responsible for Ser biosynthesis in the photorespiratory pathway, in leaves of light-grown plants far exceeded expression in roots or dark-grown leaves. Therefore, we suggest that the phosphorylated pathway may take over as the major route of the Ser biosynthetic pathway in both non-green tissues and green tissues in the dark when the photorespiration rate is low.
Lower enzyme activities of PGDH in the physiological direction compared
with the non-physiological direction were also reported by Slaughter
and Davies (7) under the given conditions. They reported that pea PGDH
was inhibited by Ser, but became progressively less sensitive as the
purification progressed. However, Larsson and Albertsson (8) could not
find Ser or phosphoserine inhibition of the enzyme in spinach
chloroplast extract. Our results concur with those of Larsson and
Albertsson, i.e. Ser does not show any inhibition effect on
the enzyme activities. Hence, Ser levels are unlikely modulated by the
metabolite flux of the pathway from 3-PGA to Ser through enzyme
activities. Although the recombinant protein produced in E. coli has been used effectively to verify gene product function, we
have to be careful when making conclusions regarding regulation since
few potentially post-translational events may occur in E. coli. Nevertheless, our present data indicated that the regulation
of PGDH is mainly exerted at the level of transcription or by stability
of PGDH mRNA.
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ACKNOWLEDGEMENTS |
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We thank the Arabidopsis Biological Resource
Center of Ohio State University for Arabidopsis expressed
sequence tag clones 200K16T7 and 111M16T7. We also thank Dr. M. Arnold
for kindly mapping the gene. We are grateful to Dr. R. Curtiss III for
the gift of E. coli serine auxotroph 536 and Dr. Y. Niwa
(Shizuoka Prefecture University, Shizuoka, Japan) for the gift of
plasmids CaMV35S-sGFP(S65T)-NOS3' and 35S
-TP-sGFP(S65T). The
excellent technical assistance of H. Hoshi and A. Watanabe is greatly
appreciated. The three GFP positive control photographs were taken and
kindly provided by K. Inoue.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan and by Research for the Future Program Grant 96I00302 from the Japan Society of Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequences reported in this paper have been submitted to the DDBJ/GenBankTM/EMBL Data Bank with accession numbers AB003280 (cDNA) and AB010407 (genomic).
To whom correspondence should be addressed. Tel.: 81-43-290-2904;
Fax: 81-43-290-2905; E-mail: ksaito{at}p.chiba-u.ac.jp.
2 GenomeNET Service, Osaka University, Osaka, Japan.
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ABBREVIATIONS |
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The abbreviations used are: PGDH, 3-phosphoglycerate dehydrogenase; 3-PGA, 3-phosphoglycerate; GDC, glycine decarboxylase multienzyme complex; GFP, green fluorescence protein; bp, base pair(s)..
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