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• W1995006983 abstract "In plants, Ser is biosynthesized by two different pathways: a photorespiratory pathway via Gly and a plastidic pathway via the phosphorylated metabolites from 3-phosphoglycerate. In contrast to the better characterization of the photorespiratory pathway at a molecular level, the molecular regulation and significance of the plastidic pathway are not yet well understood. An Arabidopsis thaliana cDNA encoding 3-phosphoserine phosphatase, the enzyme that is responsible for the conversion of 3-phosphoserine to Ser in the final step of the plastidic pathway of Ser biosynthesis, was cloned by functional complementation of an Escherichia coli serB − mutant. The 1.1-kilobase pair full-length cDNA, encoding 295 amino acids in its open reading frame, contains a putative organelle targeting presequence. Chloroplastic targeting has been demonstrated by particle gun bombardment using an N-terminal 60-amino acid green fluorescence protein fusion protein. Southern hybridization suggested the existence of a single-copy gene that mapped to chromosome 1. 3-Phosphoserine phosphatase enzyme activity was detected in vitro in the overexpressed protein in E. coli. Northern analysis revealed preferential gene expression in leaf and root tissues of light-grown plants with an ∼1.5-fold abundance in the root compared with the leaf tissues. This indicates the possible role of the plastidic pathway in supplying Ser to non-photosynthetic tissues, in contrast to the function of the photorespiratory pathway in photosynthetic tissues. This work completes the molecular cloning and characterization of the three genes involved in the plastidic pathway of Ser biosynthesis in higher plants. In plants, Ser is biosynthesized by two different pathways: a photorespiratory pathway via Gly and a plastidic pathway via the phosphorylated metabolites from 3-phosphoglycerate. In contrast to the better characterization of the photorespiratory pathway at a molecular level, the molecular regulation and significance of the plastidic pathway are not yet well understood. An Arabidopsis thaliana cDNA encoding 3-phosphoserine phosphatase, the enzyme that is responsible for the conversion of 3-phosphoserine to Ser in the final step of the plastidic pathway of Ser biosynthesis, was cloned by functional complementation of an Escherichia coli serB − mutant. The 1.1-kilobase pair full-length cDNA, encoding 295 amino acids in its open reading frame, contains a putative organelle targeting presequence. Chloroplastic targeting has been demonstrated by particle gun bombardment using an N-terminal 60-amino acid green fluorescence protein fusion protein. Southern hybridization suggested the existence of a single-copy gene that mapped to chromosome 1. 3-Phosphoserine phosphatase enzyme activity was detected in vitro in the overexpressed protein in E. coli. Northern analysis revealed preferential gene expression in leaf and root tissues of light-grown plants with an ∼1.5-fold abundance in the root compared with the leaf tissues. This indicates the possible role of the plastidic pathway in supplying Ser to non-photosynthetic tissues, in contrast to the function of the photorespiratory pathway in photosynthetic tissues. This work completes the molecular cloning and characterization of the three genes involved in the plastidic pathway of Ser biosynthesis in higher plants. Multiple pathways are responsible for Ser biosynthesis in plant cells. The oxidative photosynthetic carbon cycle constitutes the principal biochemical pathway in photorespiration. Gly and Ser are intermediates in the photorespiratory metabolism of glycolate to 3-phosphoglycerate (3-PGA) 1The abbreviations used are: 3-PGA, 3-phosphoglycerate; GDC, glycine decarboxylase multienzyme complex; SHMT, serine hydroxymethyltransferase; PGDH, 3-phosphoglycerate dehydrogenase; PSAT, 3-phosphoserine aminotransferase; PSP, 3-phosphoserine phosphatase; GFP, green fluorescence protein.in leaves (Fig. 1) (1Bryan J.K. Miflin B.J. Lea P.J. The Biochemistry of Plants: A Comprehensive Treatise. 16. Academic Press, New York1988: 175-176Google Scholar). Ser biosynthesis via this pathway has received much attention and is well documented (2Walker J.L. Oliver D.J. Arch. Biochem. Biophys. 1986; 248: 626-628Crossref PubMed Scopus (46) Google Scholar). The glycine decarboxylase multienzyme complex (GDC) (3Srinivasan R. Oliver D.J. Plant Physiol. (Bethesda). 1995; 109: 161-168Crossref PubMed Scopus (44) Google Scholar) and serine hydroxymethyltransferase (SHMT) (4Turner S.R. Hellens R. Ireland R. Ellis N. Rawsthorne S. Mol. Gen. Genet. 1993; 236: 402-408Crossref PubMed Scopus (41) Google Scholar) are responsible for the photorespiratory conversion of Gly to Ser in plant mitochondria. Servaites and Orgen (5Servaites J.C. Orgen W.L. Plant Physiol. (Bethesda). 1977; 60: 461-466Crossref PubMed Google Scholar) demonstrated that 50% of the initial Ser synthesis remained after chemical inhibition of the photorespiratory pathway, suggesting that a second pathway is activated for Ser formation. In the phosphorylated pathway, an alternative pathway of Ser biosynthesis that takes place in the plastids, Ser is synthesized from 3-PGA via a series of reactions catalyzed by 3-phosphoglycerate dehydrogenase (PGDH), 3- phosphoserine aminotransferase (PSAT), and 3-phosphoserine phosphatase (PSP) (Fig. 1). This pathway is proposed to be important in supplying Ser to non-photosynthetic tissues (6Reynolds P.H. Blevins D.G. Plant Physiol. (Bethesda). 1986; 81: 283-293Crossref Google Scholar) and fast proliferating tissues (7Stolz M. Dörnemann D. Z. Naturforsch. Sect. C Biosci. 1994; 49: 63-69Crossref PubMed Scopus (9) Google Scholar) and in the dark (8Saito K. Takagi Y. Ho C.-L. Takahashi H. Noji M. Plant Mol. Biol. 1997; 33: 359-366Crossref PubMed Scopus (19) Google Scholar, 9Ho C.-L. Noji M. Saito M. Yamazaki M. Saito K. Plant J. 1998; 16: 443-452Crossref PubMed Google Scholar, 10Ho C.-L. Noji M. Saito M. Saito K. J. Biol. Chem. 1999; 274: 397-402Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) when the photorespiratory rate is low. A non-phosphorylated pathway of Ser biosynthesis via glycerate and hydroxypyruvate (11Keys A.J. Stumpf P.K. Conn E.E The Biochemistry of Plants. 5. Academic Press, New York1980: 359-374Google Scholar) was also proposed to take place in plant tissues in the dark. In animals, however, this pathway is assumed to be involved in Ser catabolism (12Snell K. Trends Biochem. Sci. 1986; 11: 241-243Abstract Full Text PDF Scopus (61) Google Scholar). This could also be the case in plant cells because glycerate dehydrogenase, which catalyzes glycerate to hydroxypyruvate, was found to be tissue-specific and strongly expressed in light-grown leaves (13Greenler J.M. Becker W.M. Plant Physiol. (Bethesda). 1990; 94: 1484-1487Crossref PubMed Scopus (14) Google Scholar). Actually, it is unknown to what extent glycerate can be converted to Ser in plant cells. The molecular studies on the phosphorylated Ser biosynthetic pathway in plastids started with the cDNA cloning of spinach PSAT (8Saito K. Takagi Y. Ho C.-L. Takahashi H. Noji M. Plant Mol. Biol. 1997; 33: 359-366Crossref PubMed Scopus (19) Google Scholar). The first molecular cloning and characterization of ArabidopsisPGDH (9Ho C.-L. Noji M. Saito M. Yamazaki M. Saito K. Plant J. 1998; 16: 443-452Crossref PubMed Google Scholar) and PSAT (10Ho C.-L. Noji M. Saito M. Saito K. J. Biol. Chem. 1999; 274: 397-402Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), the first two enzymes in this pathway, have recently been carried out in our laboratory. BothArabidopsis PGDH and PSAT are plastidic proteins, and their catalytic activities are not sensitive to Ser inhibition. The genes encoding Arabidopsis PGDH and PSAT have a common feature in their mRNA expression patterns, in which they demonstrated root-preferential expression (9Ho C.-L. Noji M. Saito M. Yamazaki M. Saito K. Plant J. 1998; 16: 443-452Crossref PubMed Google Scholar, 10Ho C.-L. Noji M. Saito M. Saito K. J. Biol. Chem. 1999; 274: 397-402Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). These results suggested that the phosphorylated pathway in plastids is regulated at the mRNA expression level, so as to ensure Ser formation in non-photosynthetic tissues, in which photorespiratory Ser biosynthesis is suppressed. To confirm this hypothesis, it is necessary to isolate the cDNA and gene encoding PSP, the terminal enzyme in the plastidic pathway, and to examine its biochemical properties and mRNA expression pattern. In this study, we describe the successful molecular cloning and characterization of PSP, the enzyme that is responsible for the dephosphorylation of 3-phosphoserine to Ser in the phosphorylated pathway of Ser biosynthesis. Together with our previous work onArabidopsis PGDH and PSAT, the first molecular cloning and characterization of the entire plastidic pathway of Ser biosynthesis in the plant kingdom were completed. Arabidopsis thaliana ecotype Columbia seeds were germinated and grown on germination medium (14Valvekens R.D. Van Montagu M. Van Lijsebettens M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5536-5540Crossref PubMed Scopus (1058) Google Scholar) 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 1 week before RNA extraction was carried out. cDNA cloning of PSP was carried out by functional complementation of E. coli SK472 (CGSC 6102) (λ − , relA1, spoT1, thi-1. zji-202::Tn10, serB22), which was obtained from theE. coli Genetic Stock Center (Department of Biology, Yale University, New Haven, CT). Approximately 1 × 105cells were transformed with plasmid DNA recovered from the λYESA. thaliana cDNA expression library (15Elledge S.J. Mulligan J.T. Ramer S.W. Spottswood M. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1731-1735Crossref PubMed Scopus (322) Google Scholar) by both heat shock and the electroporation transformation method (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), plated on M9 minimal medium with 50 μg/ml ampicillin, and then incubated at 37 °C for 1 week. To isolate a longer cDNA clone, FPSP4, the cDNA clone that was isolated by functional complementation of E. coli serB −mutant SK472, was used as probe to screen 5 × 105amplified plaques. Hybridization of the membranes (Hybond N+, Amersham Pharmacia Biotech) was carried out at 65 °C in 5× SSPE (0.9m NaCl, 0.05 m sodium phosphate (pH 7.7), and 5 mm EDTA), 0.5% SDS, 5× Denhardt's solution, and 25 μg/ml salmon sperm DNA. Membranes were washed at maximum stringency in 1× SSPE and 0.1% SDS for 10 min twice with final washing conducted at 65 °C in 0.1× SSPE and 0.1% SDS for 10 min and were then exposed to Fuji x-ray film. Genomic cloning of PSP was carried out by polymerase chain reaction amplification using primers 5′-CTCCAGTCGACATGGAAGCATTAACTACTT-3′ and 5′-CGCGGATCCTATAGAGGCTTTTAGTCCAAT-3′. Autosequencing was conducted by dideoxy chain termination methods with Thermo Sequenase (Amersham Pharmacia Biotech) using a Shimadzu Model DSQ1000 DNA sequencer. Genomic DNA was extracted from the leaves of 3-week-old seedlings as described (16Dellaporta S.L. Wood J. Hicks J.B. Plant Mol. Biol. Rep. 1983; 1: 19-23Crossref Scopus (6378) Google Scholar). Total RNA was isolated from the leaves and roots of 3-week-old seedlings by a modified guanidine HCl method as described (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). RNA blots were probed with 32P-labeled probe synthesized from the cDNA clones CPSP-1 and CPGDH-5 (10Ho C.-L. Noji M. Saito M. Saito K. J. Biol. Chem. 1999; 274: 397-402Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and CPSAT-5 (9Ho C.-L. Noji M. Saito M. Yamazaki M. Saito K. Plant J. 1998; 16: 443-452Crossref PubMed Google Scholar). To investigate the mRNA expression levels of GDC and SHMT,32P-labeled probes synthesized from cDNA inserts ofArabidopsis expressed sequence tag clones 200K16T7 and 111M16T7, respectively, were used. To verify equivalent loading of RNA on blots, membranes were probed with a 32P-labeled rice rDNA (pRR217) (18Takaiwa F. Oono K. Sugiura M. Plant Mol. Biol. 1985; 4: 355-364Crossref PubMed Scopus (145) Google Scholar). Hybridization and washing conditions were the same as those described above. Relative values of mRNA transcripts 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 (19Lister C. Dean C. Plant J. 1993; 4: 745-750Crossref Scopus (518) Google Scholar). Hybridization and washing were carried out as described above, except the final washing condition was reduced to 0.5× SSPE and 0.1% SDS for 10 min at 65 °C. The map distances were kindly calculated by Dr. M. Anderson (NottinghamArabidopsis Stock Center) based on the restriction fragment length polymorphism profiles generated by HhaI. We followed a general method of DNA engineering according to Sambrook et al. (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Primers AB5 (5′-CAGTTCCATGGAAGCATTAACTACTCAAGG-3′) and AB6 (5′-CGCGGATCCTATAGAGGCTTTTAGTCCAAT-3′) were used to create aNcoI site at the 5′-end and a BamHI site at the 3′-end of the open reading frame of PSP. The engineered DNA fragments were inserted into the appropriate sites of pET3d (Novagen), in which the cDNA was placed under a strong φ10 promoter in both sense and antisense orientations. The plasmids were then introduced into E. coli BL21(DE3) pLysS, in which the gene for lysogenic T7 RNA polymerase, under the lacUV5 promoter, is induced by isopropyl-1-thio-β-d-galactopyranoside. PSP activity was determined by measuring the phosphate release from 3-phosphoserine at 30 °C in a volume of 0.5 ml of reaction mixture containing 80 mm Tris-HCl (pH 7.5), 10 mm 3-phosphoserine, 3 mm MgCl2, and 20 μl of extract to start the reaction (20Larsson C. Albertsson E. Physiol. Plant. 1979; 45: 7-10Crossref Scopus (34) Google Scholar). The rate was linear for at least 15 min. After 10 min, 0.25 ml of 15% trichloroacetic acid was added to stop the reaction. Free phosphate was determined (21Fiske C.H. SubbaRow Y.P. J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar), and corrections were made for zero time blank “rates.” Blanks without enzyme were also run. One unit of enzyme is the amount that catalyzes the conversion of 1 μmol of substrate/min under the stated condition. To verify the subcellular localization of PSP in plant cells, primers 5′-CTCCAGTCGACATGGAAGCATTAACTACTT-3′ and 5′-CCTGGCCATGGCTCGTGAGGCTGCACAGAA-3′ were used to amplify a 180-base pair fragment of the 5′-end of PSP for subcloning into plasmid CaMV35S-sGFP(S65T)-NOS3′ (22Chiu W. Niwa Y. Zeng W. Hirano T. Kobayashi H. Sheen J. Curr. Biol. 1996; 6: 325-330Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar) with a cauliflower mosaic virus35S-labeled promoter at its 5′-end, GFP as reporter protein, and nopaline synthetase terminator as transcription terminator. The plasmid construct 35SΩ-TP-sGFP(S65T) (22Chiu W. Niwa Y. Zeng W. Hirano T. Kobayashi H. Sheen J. Curr. Biol. 1996; 6: 325-330Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar), carrying transit peptide sequence obtained from the ribulose-1,5-bisphosphate carboxylase small subunit polypeptide of Arabidopsis (23Krebbers E. Seurinck J. Herdies L. Cashmore A.R. Timko M.P. Plant Mol. Biol. 1988; 11: 745-759Crossref PubMed Scopus (150) Google Scholar), was used as a positive control. 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. Individual leaves were viewed with a fluorescent microscope (BX50-FLA, Olympus) using a Chroma dual band filter, FITC, and TRITC (Olympus). Photographs were taken using Fujichrome ISO 100 film. DNA subcloning and sequencing, SDS-polyacrylamide gel electrophoresis, and protein quantitation were carried out as described (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Plasmid recovery of DNA from the λYESA. thaliana cDNA expression library was carried out as described (15Elledge S.J. Mulligan J.T. Ramer S.W. Spottswood M. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1731-1735Crossref PubMed Scopus (322) Google Scholar). The E. coli serB −mutant strain SK472 is deficient in PSP activity and is unable to grow on M9 minimal medium plates without Ser supplementation. Complemented cells that were ampicillin-resistant were obtained at a frequency of 50 from 1 × 105 cells transformed with a total of 16 μg of plasmid DNA recovered from the λYES ArabidopsiscDNA expression library (15Elledge S.J. Mulligan J.T. Ramer S.W. Spottswood M. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1731-1735Crossref PubMed Scopus (322) Google Scholar). The cDNA clone obtained (FPSP4) was ∼1 kilobase pairs in length, and it starts immediately from the ATG translation codon. To obtain a longer cDNA clone, 4.5 × 105 phage plaques produced from an Arabidopsis whole plant cDNA library were screened with the cDNA clone (FPSP4) isolated by functional complementation, and only two positive clones were isolated. Of the two positive clones selected for further studies, CPSP-1, which contains the largest cDNA insert (1.1 kilobase pairs), was subcloned and sequenced. Sequence analysis revealed that it is 82 base pairs longer than FPSP4, containing an open reading frame of 885 nucleotides that encodes 295 amino acid residues. The poly(A) tail is absent from the cDNA clone. The deduced amino acid sequence of Arabidopsis PSP is aligned with PSP sequences from other organisms in Fig.2 A. The phylogenetic tree (Fig. 2 B) indicates that Arabidopsis PSP is closely related to human PSP (24Collet J.-F. Gerin I. Rider M.H. Veiga-da-Cunha M. Van Schaftingan E. FEBS Lett. 1997; 408: 281-284Crossref PubMed Scopus (57) Google Scholar). Arabidopsis PSP, containing two of the most conserved regions of PSP (DXDST and GDGXXD), has high similarity to amino acid sequences of PSP from other organisms (Fig. 2 A). It differs from the rest mainly due to its N-terminal region. The genomic clone GPSP covering the coding region of PSP was isolated by polymerase chain reaction amplification and has been fully sequenced. Analysis of genomic and cDNA sequences revealed the presence of six short introns, splitting the open reading frame into seven exons. The Southern blot results (Fig. 3) demonstrate a single major fragment being hybridized upon digestion with BamHI, BglII, EcoRI,SacI, and XbaI. Two bands were observed upon digestion with EcoRV due to the presence of restriction sites for this endonuclease in the genomic structure. The results suggested that Arabidopsis PSP is a single-copy gene, and it was mapped to chromosome 1, between the markers g3786 and g3829. The recombinant fusion protein CPSP-GFP, containing the 60 amino acids at the N terminus of CPSP-1 fused to jellyfish GFP, could be detected in intact tissues after delivering the constructs into Arabidopsisleaves by particle gun bombardment. The observed signals in the construct containing the predicted transit peptide of PSP fromArabidopsis (Fig.4 b) were similar to those exhibited by the transit peptide of the ribulose-1,5-bisphosphate carboxylase small subunit polypeptide of Arabidopsis (23Krebbers E. Seurinck J. Herdies L. Cashmore A.R. Timko M.P. Plant Mol. Biol. 1988; 11: 745-759Crossref PubMed Scopus (150) Google Scholar) (Fig. 4 a), which was already known to be sufficient for translocation to chloroplasts (22Chiu W. Niwa Y. Zeng W. Hirano T. Kobayashi H. Sheen J. Curr. Biol. 1996; 6: 325-330Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar). The expression pattern of the plastidic proteins was distinct from the signals observed in the cytosol and nucleus of the empty vector, sGFP(S65T), without a transit peptide (25Noji M. Inoue K. Kimura N. Gouda A. Saito K. J. Biol. Chem. 1998; 273: 32739-32745Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). These results confirmed that the N-terminal sequence ofArabidopsis PSP is sufficient for the translocation of a passenger protein into chloroplasts. Thus,Arabidopsis PSP is a plastidic protein. Recombinant PSP was overproduced in E. coli BL21 cells using a pET3d vector system. The recombinant protein was visualized on SDS-polyacrylamide gel electrophoresis gel as a 37-kDa protein (Fig.5), exhibiting a PSP activity of 3.0 ± 0.6 units/mg of protein. Double-reciprocal plots of the initial rate data demonstrated aK m value of 3.5 mm for 3-phosphoserine. Enzyme activity was reduced rather than completely inhibited in the presence of Ser. Approximately 60% inhibition of PSP activity by 10 mm Ser was observed. Gly did not reduce or inhibit the PSP activity. The mRNA expression level of PSP was examined in leaf and root tissues of both light-grown and dark-treated plants. Northern analyses were also carried out with two other enzymes, PGDH and PSAT, in the plastidic pathway and with two enzymes, H-protein (a subunit of GDC) and SHMT, in the photorespiratory pathway for comparison with PSP expression. The highest level of PSP mRNA expression was observed in light-grown root tissues (Fig.6 C). It is ∼1.5-fold higher than the mRNA expression in light-grown leaf tissues. Lower mRNA expression levels were detected in the dark-grown leaf and root tissues. The level in dark-grown roots was still higher than in dark-grown shoots. These trends of expression were also similar for PGDH (Fig. 6 A) and PSAT (Fig. 6 B); in contrast, the mRNAs of H-protein (Fig. 6 D) and SHMT (Fig.6 E) accumulated almost absolutely in the light-grown leaves. In this study, the first molecular cloning of PSP from the plant kingdom was successfully achieved from Arabidopsis by functional complementation of E. coli serB mutant SK472. In the deduced amino acid sequence, two highly conserved motifs containing aspartate residues are found in PSP: DXDST and GDGXXD. Both these domains have high similarity to the consensus phosphorylation site of ATPases of the Na+/K+-ATPase family, suggesting that there may be functional homology between PSP and ATPases (26Collet J.-F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Replacement of the first aspartate of the DXDST motif by asparagine or glutamate resulted in complete inactivation of the enzyme, suggesting that the first aspartate of this motif is the phosphorylated residue in human PSP (26Collet J.-F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). PSP belongs to a new phosphotransferase family with an amino-terminal DXDX(T/V) motif that serves as an intermediate phosphoryl acceptor (26Collet J.-F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Arabidopsis PSP is a single-copy gene located at chromosome 1, ∼2.2 centimorgans below marker g3786 and 0.1 centimorgan above the gene for PGDH, which catalyzes the first step of the phosphorylated pathway of Ser biosynthesis. The actual significance of their proximity to the regulation of Ser biosynthesis remains a question that needs further investigation. The K m value for PSP in the chloroplast extract of spinach was reported to be 1.1 mm for 3-phosphoserine, and the activity was Mg2+-dependent at optimum pH 7 (20Larsson C. Albertsson E. Physiol. Plant. 1979; 45: 7-10Crossref Scopus (34) Google Scholar). Arabidopsis recombinant PSP exhibited a higherK m value of 3.2 mm for 3-phosphoserine. In E. coli, PSP was inhibited by Ser (27Pizer L.I. J. Biol. Chem. 1964; 239: 4219-4226Abstract Full Text PDF PubMed Google Scholar). The inhibition is of neither the competitive nor the non-competitive type with respect to Ser. The equilibrium positions of PSAT and PGDH will favor 3-PGA when PSP is inhibited, thereby ensuring that the pathway as a whole is effectively controlled. The phosphorylated pathway is controlled primarily by the demand for Ser rather than the supply of the precursor, 3-PGA (27Pizer L.I. J. Biol. Chem. 1964; 239: 4219-4226Abstract Full Text PDF PubMed Google Scholar). Approximately 60% of Arabidopsis PSP activity is inhibited at 10 mm Ser. This finding is consistent with the report of Larsson and Albertsson (20Larsson C. Albertsson E. Physiol. Plant. 1979; 45: 7-10Crossref Scopus (34) Google Scholar) that the spinach PSP activity was reduced rather than eliminated completely at high Ser concentration. Analysis of the control of mammalian Ser biosynthesis also revealed product inhibition of PSP activity, the final step of the pathway (28Fell D.A. Snell K. Biochem. J. 1988; 256: 97-101Crossref PubMed Scopus (70) Google Scholar). In bacteria and animals, Ser is mainly synthesized via the phosphorylated pathway (29Ichihara A. Greenberg D.M. J. Biol. Chem. 1957; 224: 331-340Abstract Full Text PDF PubMed Google Scholar), using 3-PGA that was derived from glycolysis or from the oxidative or reductive pentose phosphate pathway. The situation is much more complicated in photosynthetic plant tissues since Ser biosynthesis can proceed via the photorespiratory pathway (30Tolbert N.E. Davies D.D. The Biochemistry of Plants. 2. Academic Press, New York1980: 488-525Google Scholar). There are two pools of Ser biosynthesized de novo in photosynthetic cells (Fig. 1): one in the mitochondria, where Ser is synthesized via the photorespiratory pathway, and the other in the chloroplasts, where the phosphorylated pathway is present. Larsson and Albertsson (20Larsson C. Albertsson E. Physiol. Plant. 1979; 45: 7-10Crossref Scopus (34) Google Scholar) suggested a chloroplast localization of plant PSP based on their physiological investigation. In the present study, particle gun bombardment experiments using the recombinant protein CPSP-GFP confirmed that Arabidopsis PSP is a plastidic protein, indicating there is a pathway of Ser formation in the chloroplasts/plastids. Ser production in the mitochondria, catalyzed by the series of reactions of GDC and SHMT, is conceived to be the major source of Ser supply in light-grown plants. However, the significant gene expression level of Ser-biosynthesizing enzymes in the phosphorylated pathway in the leaf tissues of light-grown plants (Fig. 6, A-C) indicates the coexistence of a source of Ser in the chloroplasts. It is not surprising since 3-PGA, the precursor of this pathway, is generated in a large amount in chloroplasts via the carboxylation of ribulose-1,5-bisphosphate during photosynthesis. The preferential mRNA expression pattern of PSP is consistent with that of PGDH and PSAT, the initial two enzymes in the phosphorylated pathway of Ser biosynthesis. These three genes were preferentially expressed in the root tissues of light-grown plants (Fig. 6,A-C). 3-PGA generated through the oxidative pentose phosphate pathway may serve as the precursor of Ser biosynthesis in the plastids of root tissues (Fig. 1). The phosphorylated pathway of Ser biosynthesis is also proposed to be an important Ser supply in the dark when the photorespiratory rate is low. The above assumption is supported by the fact that both GDC and SHMT were hardly detected in both leaf and root tissues of the dark-treated seedling (Fig. 6, D and E). Moreover, the amount of PGDH and PSP mRNAs detected in the root tissues was significantly higher than in the leaf tissues from dark-treated plants, revealing a root-preferential expression of these genes, irrespective of their light dependence. The high expression level of PGDH, PSAT, and PSP in the root tissues of light-grown plants could be due to the high demand of Ser or the availability of 3-PGA during photosynthesis. High enzyme activities of PSAT in fast proliferating tissues, e.g. root meristems and apical tissues, etc., where there is a high Ser requirement was reported (6Reynolds P.H. Blevins D.G. Plant Physiol. (Bethesda). 1986; 81: 283-293Crossref Google Scholar). Therefore, the highest expression level of these genes in the root tissues of light-grown plants is needed for high demand of Ser, suggesting that these genes are light-regulated. In conclusion, there are two Ser biosynthesis pathways in plants: one is mitochondrially located and related to photorespiration, and the other takes place in the plastids. In root tissues, the plastidic pathway most probably is the major route of Ser biosynthesis, whereas in leaf tissues, where there are two Ser biosynthetic pathways, the plastidic pathway may become the main supply of Ser in non-photosynthetic tissues or in the dark. We thank the ArabidopsisBiological Resource Center of Ohio State University forArabidopsis expressed sequence tag clones 200K16T7 and 111M16T7. We also thank Dr. M. Anderson for kindly mapping the gene. We are grateful to Dr. M. K. B. Berlyn (E. coliGenetic Stock Center) for the gift of E. coli serine auxotroph SK472, Dr. T. Leustek (Rutgers University, New Brunswick, NJ) and Prof. J. Wray (University of St. Andrews, St. Andrews, United Kingdom) for E. coli BNN132, Dr. Y. Komeda (Tokyo University) for the Arabidopsis cDNA λYES expression library, and Dr. Y. Niwa (Shizuoka Prefecture University, Shizuoka, Japan) for the gift of plasmids CaMV35S-sGFP(S65T)-NOS3′ and 35SΩ-TP-sGFP(S65T). Excellent technical assistance by A. Watanabe is greatly appreciated. The GFP-positive control photograph was taken and kindly provided by K. Inoue. We thank Dr. Yves Hatzfeld for useful suggestions and critical reading of this manuscript." @default.
• W1995006983 created "2016-06-24" @default.
• W1995006983 creator A5004402448 @default.
• W1995006983 creator A5052696406 @default.
• W1995006983 creator A5064219257 @default.
• W1995006983 date "1999-04-01" @default.
• W1995006983 modified "2023-09-27" @default.
• W1995006983 title "Plastidic Pathway of Serine Biosynthesis" @default.
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