Plastidic Pathway of Serine Biosynthesis

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 that catalyzes the conversion of 1 of under the To verify the of PSP in plant primers 5′-CTCCAGTCGACATGGAAGCATTAACTACTT-3′ and were used to a pair fragment of the 5′-end of PSP for into plasmid Y. T. H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) with a promoter at its as and as The plasmid Y. T. H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), sequence obtained from the of Arabidopsis E. J. Plant Mol. Biol. 1988; 11: PubMed Scopus Google Scholar), was used as a These plasmids were used for particle gun gun bombardment was carried out using the the by the leaves were with a using a and were using film. DNA and and protein 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). 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 SK472 is in PSP activity and is to on M9 minimal medium plates without Ser cells that were were obtained at a of 50 from 1 × cells transformed with a of of plasmid DNA recovered from the 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 was in and it from the To a longer cDNA clone, × from an Arabidopsis plant cDNA library were with the cDNA clone isolated by functional and two clones were the two clones for which contains the cDNA was and analysis revealed that it is longer FPSP4, containing an open reading frame of that 295 amino acid The is from the cDNA The amino acid sequence of Arabidopsis PSP is with PSP from in A. The (Fig. indicates that Arabidopsis PSP is to PSP M. Van E. 1997; PubMed Scopus Google Scholar). Arabidopsis PSP, containing two of the of PSP and has to amino acid of PSP from (Fig. from the to its N-terminal The clone the of PSP was isolated by polymerase chain reaction amplification and has been of and cDNA revealed the of the open reading frame into The Southern results (Fig. a fragment with and were with to the of restriction sites for this in the The results suggested that Arabidopsis PSP is a single-copy and it was mapped to chromosome the and The recombinant fusion protein containing the amino acids at the of CPSP-1 to GFP, could be detected in tissues after the into by particle gun The signals in the containing the of PSP were to those by the of the of Arabidopsis E. J. Plant Mol. Biol. 1988; 11: PubMed Scopus Google Scholar) (Fig. which was to be for to Y. T. H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The expression of the plastidic was from the signals in the and of the without a M. K. N. A. Saito K. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). These results that the N-terminal sequence ofArabidopsis PSP is for the of a protein into PSP is a plastidic protein. PSP was in E. coli cells using a pET3d The recombinant protein was on as a protein a PSP activity of of protein. of the initial rate demonstrated m of mm for activity was reduced in the of Approximately inhibition of PSP activity by 10 mm Ser was Gly not the PSP The mRNA expression of PSP was in leaf and root tissues of both light-grown and dark-treated plants. Northern were also carried out with two PGDH and PSAT, in the plastidic pathway and with two of and SHMT, in the photorespiratory pathway for with PSP The of PSP mRNA expression was in light-grown root tissues is ∼1.5-fold higher the mRNA expression in light-grown leaf tissues. mRNA expression levels were detected in the leaf and root tissues. The in roots was higher in These of expression were also for PGDH (Fig. and PSAT (Fig. in the of (Fig. and in the light-grown In this study, the first molecular cloning of PSP from the plant kingdom was from Arabidopsis by functional complementation of E. coli serB In the amino acid two containing are found in and have to the site of of the suggesting that be functional PSP and M. Van E. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of the first of the by in of the suggesting that the first of this is the phosphorylated in PSP M. Van E. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). PSP to a with an that as an M. Van E. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Arabidopsis PSP is a single-copy gene at chromosome and the gene for PGDH, which catalyzes the first step of the phosphorylated pathway of Ser biosynthesis. The significance of their to the regulation of Ser biosynthesis a that The m for PSP in the extract of spinach was to be mm for 3-phosphoserine, and the activity was at (20Larsson C. Albertsson E. Physiol. Plant. 1979; 45: 7-10Crossref Scopus (34) Google Scholar). Arabidopsis recombinant PSP a m of mm for In E. PSP was by Ser J. Biol. Chem. Full Text PDF PubMed Google Scholar). The inhibition is of the the with to The of PSAT and PGDH 3-PGA when PSP is that the pathway as a is The phosphorylated pathway is by the for Ser the of the 3-PGA J. Biol. Chem. Full Text PDF PubMed Google Scholar). Approximately of Arabidopsis PSP activity is at 10 mm This is with the of and Albertsson (20Larsson C. Albertsson E. Physiol. Plant. 1979; 45: 7-10Crossref Scopus (34) Google Scholar) that the spinach PSP activity was reduced at Ser of the of Ser biosynthesis also revealed inhibition of PSP the final step of the pathway K. Biochem. J. 1988; PubMed Scopus Google Scholar). In and animals, Ser is synthesized via the phosphorylated pathway A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), using 3-PGA that was from from the oxidative phosphate The is much in photosynthetic plant tissues Ser biosynthesis can via the photorespiratory pathway The Biochemistry of Plants. Academic Press, New York1980: Scholar). are two of Ser biosynthesized in photosynthetic cells (Fig. in the Ser is synthesized via the photorespiratory pathway, and the in the the phosphorylated pathway is and Albertsson (20Larsson C. Albertsson E. Physiol. Plant. 1979; 45: 7-10Crossref Scopus (34) Google Scholar) suggested a of plant PSP based on their In the study, particle gun bombardment using the recombinant protein that Arabidopsis PSP is a plastidic is a pathway of Ser formation in the Ser in the catalyzed by the series of reactions of GDC and SHMT, is to be the of Ser in light-grown plants. the gene expression of enzymes in the phosphorylated pathway in the leaf tissues of light-grown plants (Fig. indicates the of a of Ser in the is not 3-PGA, the of this pathway, is generated in a in via the of The preferential mRNA expression of PSP is with that of PGDH and PSAT, the initial two enzymes in the phosphorylated pathway of Ser biosynthesis. These three genes were expressed in the root tissues of light-grown plants (Fig. 3-PGA generated the oxidative phosphate pathway as the 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 in the dark when the photorespiratory rate is low. The is by the that both GDC and were detected in both leaf and root tissues of the dark-treated (Fig. and the of PGDH and PSP detected in the root tissues was higher in the leaf tissues from dark-treated plants, a root-preferential expression of of their light The expression of PGDH, PSAT, and PSP in the root tissues of light-grown plants could be to the of Ser the of 3-PGA enzyme activities of PSAT in fast proliferating tissues, root and tissues, is a Ser was (6Reynolds P.H. Blevins D.G. Plant Physiol. (Bethesda). 1986; 81: 283-293Crossref Google Scholar). the expression of genes in the root tissues of light-grown plants is for of suggesting that genes are In are two Ser biosynthesis pathways in is and to and the takes place in the In root tissues, the plastidic pathway is the of Ser biosynthesis, in leaf tissues, are two Ser biosynthetic the plastidic pathway the of Ser in non-photosynthetic tissues in the dark. We the Center of expressed sequence tag clones 200K16T7 and We also Dr. M. Anderson for kindly mapping the We are to Dr. M. K. Stock Center) for the of E. coli serine SK472, Dr. T. University, New and J. of for E. coli Dr. Y. for the Arabidopsis cDNA expression and Dr. Y. University, for the of plasmids and by A. is The was and kindly by K. We Dr. for and reading of this