A Novel γ-Hydroxybutyrate Dehydrogenase

In plants, γ-aminobutyrate (GABA), a non-protein amino acid, accumulates rapidly in response to a variety of abiotic stresses such as oxygen deficiency. Under normoxia, GABA is catabolized to succinic semialdehyde and then to succinate with the latter reaction being catalyzed by succinic semialdehyde dehydrogenase (SSADH). Complementation of an SSADH-deficient yeast mutant with an Arabidopsis cDNA library enabled the identification of a novel cDNA (designated as AtGH-BDH for Arabidopsis thaliana γ-hydroxybutyrate dehydrogenase), which encodes a 289-amino acid polypeptide containing an NADP-binding domain. Constitutive expression of AtGHBDH in the mutant yeast enabled growth on 20 mm GABA and significantly enhanced the cellular concentrations of γ-hydroxybutyrate, the product of the GHDBH reaction. These data confirm that the cDNA encodes a polypeptide with GHBDH activity. Arabidopsis plants subjected to flooding-induced oxygen deficiency for up to 4 h possessed elevated concentrations of γ-hydroxybutyrate as well as GABA and alanine. RNA expression analysis revealed that GHBDH transcription was not up-regulated by oxygen deficiency. These findings suggest that GHBDH activity is regulated by the supply of succinic semialdehyde or by redox balance. It is proposed that GHBDH and SSADH activities in plants are regulated in a complementary fashion and that GHBDH and γ-hydroxybutyrate function in oxidative stress tolerance. In plants, γ-aminobutyrate (GABA), a non-protein amino acid, accumulates rapidly in response to a variety of abiotic stresses such as oxygen deficiency. Under normoxia, GABA is catabolized to succinic semialdehyde and then to succinate with the latter reaction being catalyzed by succinic semialdehyde dehydrogenase (SSADH). Complementation of an SSADH-deficient yeast mutant with an Arabidopsis cDNA library enabled the identification of a novel cDNA (designated as AtGH-BDH for Arabidopsis thaliana γ-hydroxybutyrate dehydrogenase), which encodes a 289-amino acid polypeptide containing an NADP-binding domain. Constitutive expression of AtGHBDH in the mutant yeast enabled growth on 20 mm GABA and significantly enhanced the cellular concentrations of γ-hydroxybutyrate, the product of the GHDBH reaction. These data confirm that the cDNA encodes a polypeptide with GHBDH activity. Arabidopsis plants subjected to flooding-induced oxygen deficiency for up to 4 h possessed elevated concentrations of γ-hydroxybutyrate as well as GABA and alanine. RNA expression analysis revealed that GHBDH transcription was not up-regulated by oxygen deficiency. These findings suggest that GHBDH activity is regulated by the supply of succinic semialdehyde or by redox balance. It is proposed that GHBDH and SSADH activities in plants are regulated in a complementary fashion and that GHBDH and γ-hydroxybutyrate function in oxidative stress tolerance. γ-Aminobutyrate (GABA) 1The abbreviations used are: GABA, γ-aminobutyrate; SSA, succinic semialdehyde; SSADH, succinic semialdehyde dehydrogenase; GHB, γ-hydroxybutyrate; GHBDH, γ-hydroxybutyrate dehydrogenase; GABA-T, γ-aminobutyrate transaminase; RT, reverse transcriptase. is a four-carbon non-protein amino acid that is present in virtually all of the prokaryotic and eukaryotic organisms as a significant component of the free amino acid pool (1Bown A.W. Shelp B.J. Plant Physiol. (Bethesda). 1997; 115: 1-5Crossref PubMed Scopus (321) Google Scholar, 2Shelp B.J. Bown A.W. McLean M.D. Trends Plant Sci. 1999; 4: 446-452Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). In bacteria, it is involved in carbon and nitrogen metabolism (3Metzer E. Helpern Y.S. J. Bacteriol. 1990; 172: 3250-3256Crossref PubMed Google Scholar), whereas in mammals, it functions as an inhibitory neurotransmitter (4Bormann J. Trends Neurosci. 1988; 11: 112-116Abstract Full Text PDF PubMed Scopus (569) Google Scholar). The role of GABA in plants is uncertain; however, GABA accumulates rapidly in response to a variety of abiotic stresses such as oxygen deficiency or cold temperature (1Bown A.W. Shelp B.J. Plant Physiol. (Bethesda). 1997; 115: 1-5Crossref PubMed Scopus (321) Google Scholar, 2Shelp B.J. Bown A.W. McLean M.D. Trends Plant Sci. 1999; 4: 446-452Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar, 5Bown A.W. Shelp B.J. Biochem. (Life Sci. Adv.). 1989; 8: 21-25Google Scholar, 6Kinnersley A.M. Turano F.J. Crit. Rev. Plant Sci. 2000; 19: 479-509Crossref Google Scholar). These stresses initiate a signal transduction pathway in which increased cytosolic Ca2+ stimulates Ca2+/calmodulin-dependent activity of the anabolic enzyme, glutamate decarboxylase (Fig. 1). Under normoxia, GABA is catabolized via GABA transaminase (GABA-T, EC 2.6.1.19) to succinic semialdehyde (SSA), which in turn is oxidized via an NAD-dependent succinic semialdehyde dehydrogenase (SSADH, EC 1.2.2.16) to succinate. Under oxygen deficiency, SSADH activity is probably restricted by increases in reducing potential and adenylate energy charge (7Busch K.B. Fromm H. Plant Physiol. (Bethesda). 1999; 121: 589-597Crossref PubMed Scopus (110) Google Scholar, 8Busch K.B. Piehler J. Fromm H. Biochemistry. 2000; 39: 10110-10117Crossref PubMed Scopus (27) Google Scholar), thereby contributing to the accumulation of GABA. Research on bacterial and animal systems indicates the existence of an alternative pathway for SSA catabolism to γ-hydroxybutyrate (GHB) that involves the enzyme γ-hydroxybutyrate dehydrogenase (GHBDH, might also be designated as succinic semialdehyde reductase, EC 1.1.1.61) (GenBank™ accession numbers AJ250267, L21902, and AAC41425) (9Andriamampandry C. Siffert J.-C. Schmitt M. Garnier J.-M. Staub A. Muller C. Gobaille S. Mark J. Maitre M. Biochem. J. 1998; 334: 43-50Crossref PubMed Scopus (26) Google Scholar, 10Schaller M. Schaffhauser M. Sans N. Wermuth B. Eur. J. Biochem. 1999; 265: 1056-1060Crossref PubMed Scopus (41) Google Scholar). Mamelak (11Mamelak M. Neurosci. Biobehav. Rev. 1989; 13: 187-198Crossref PubMed Scopus (163) Google Scholar) reviewed evidence for elevated GHB levels in mammalian tissues in response to anoxia or excessive metabolic demand and suggested that GHB functions as an endogenous protective agent when energy supplies are limited. Recently, Allan et al. (12Allan W.L. Smith R. Shelp B.J. Application Bulletin AB-0015. Agilent Technologies Inc., Mississauga, Ontario, Canada2003: 4Google Scholar, 13Allan, W. L., Peiris, C., Brown, A. W., and Shelp, B. J. (2003) Can. J. Plant Sci., in pressGoogle Scholar) detected GHB in plant tissues and reported that it accumulates in response to oxygen deficiency. The conversion of SSA to GHB is reductive (i. e. fermentative), and like other common fermentation reactions such as lactate and alcohol dehydrogenases (14Drew M.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 223-250Crossref PubMed Scopus (948) Google Scholar), GHBDH may be involved in the stress tolerance of plants. In this report, we identify an Arabidopsis GHBDH cDNA by functional complementation of an SSADH-deficient yeast mutant in conjunction with metabolite analysis. We further demonstrate that GHB accumulation in Arabidopsis subjected to oxygen deficiency does not result from up-regulation of gene expression and propose that GHBDH activity and GHB play a role in oxidative stress tolerance. Isolation of a Putative GHBDH cDNA from Arabidopsis by Complementation of an SSADH-deficient Yeast Mutant—A Saccharomyces cerevisiae mutant of uga2 is unable to use GABA as a nitrogen source and is defective in SSADH activity, suggesting that UGA2 is the structural gene for this enzyme (15Ramos F. El Guezzar M. Grenson M. Wiame J.-M. Eur. J. Biochem. 1985; 149: 401-404Crossref PubMed Scopus (72) Google Scholar). To demonstrate that SSADH activity is attributable to UGA2 and resides at the YBR006w locus, a ura3 uga2 mutant (strain 22641c) was transformed by a centromere-based plasmid library representing the genome of strain Σ1278b (16Marini A.M. Vissers S. Urrestarazu A. Andre B. EMBO J. 1994; l13: 3456-3463Crossref Scopus (246) Google Scholar). Several genomic clones restored normal growth to the uga2 mutant on GABA (0.1%) as the sole nitrogen source. An Arabidopsis thaliana [L.] Heynh (Landsberg erecta ecotype) cDNA library (entire seedlings at two-leaf stage) constructed in a yeast expression vector (pFL61) containing the phosphoglycerate kinase promoter and the yeast URA3 gene (17Minet M. Dufour M.E. Lacoute F. Plant J. 1992; 2: 417-422PubMed Google Scholar) was kindly provided by M. Minet. Wild type yeast Σ1278b and its mutant strain 22641c (15Ramos F. El Guezzar M. Grenson M. Wiame J.-M. Eur. J. Biochem. 1985; 149: 401-404Crossref PubMed Scopus (72) Google Scholar) were maintained on yeast minimal medium (18Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar). Competent yeast cells were made and transformed with the Arabidopsis cDNA expression library (19Dohlmen R.J. Strasser A.W. Höner C.B. Hollenberg C.P. Yeast. 1991; 7: 691-692Crossref PubMed Scopus (319) Google Scholar). URA + transformants were selected on solid SD medium (0.67% bactoyeast nitrogen base without amino acids, 2% glucose, 2% bacto-agar) supplemented with 0.5% ammonium sulfate and 0.35 mm uracil (18Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar), washed from each plate with liquid SD medium, and re-selected on SD medium supplemented with 20 mm GABA as the sole nitrogen source. Plasmids were isolated from selected colonies, amplified in Escherichia coli strain dH5α cells on LB medium supplemented with ampicillin (50 μg/ml) according to standard protocols (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), and re-introduced into the yeast mutant for re-selection on 20 mm GABA. Plasmid DNA was isolated from a 2-ml overnight culture derived from a single colony (21Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Short Protocols in Molecular Biology. 2nd ed. John Wiley & Sons, Inc., New York1992Google Scholar). Plasmids bearing one of four independent cDNA clones isolated by complementation were sent to Genologics (Agricultural and Food Laboratory Services Branch, Guelph, Ontario, Canada) for sequencing (ABI PRISM Dye Terminator Cycle Sequencing Ready reaction kit on the ABI PRISM Sequencer Model 377, PerkinElmer Life Sciences). Vector primers used for sequencing were PGK5′ (5′-TCA AGA TCA TCA AGG AAG TAA TTA T-3′) and PGK3′ (5′-TAT TTT AGC GTA AAG GAT GAG GAS A-3′). All four of the cDNAs encoded the gene product. Growth and Metabolite Analyses of an SSADH-deficient Yeast Mutant Expressing the Putative Arabidopsis GHBDH cDNA—The transformed yeast strains were grown in 50 ml of liquid SD medium to an A 600 of 0.5. Cells were washed twice and resuspended in nitrogen-free SD medium. Approximately equal volumes (full inoculating loop) were streaked onto nitrogen-free SD plates supplemented with GABA, proline, or (NH4)2SO4 (20 mm N). Growth on the plates was checked after 4 days at 28 °C. Single colonies from wild type yeast (Σ1278b) and 22641c yeast transformed with the empty yeast vector (pFL61) or Arabidopsis complement (GHBDH) were used to inoculate 1-ml aliquots of liquid SD medium and grown overnight in a rotary shaker (150 rpm, 28 °C). The overnight culture was divided into three aliquots and used to inoculate 50 ml of nitrogen-free liquid SD medium supplemented with GABA, proline, or (NH4)2SO4 (20 mm N). The cells were grown to mid-log phase (A 600 of 1.0 ± 0.1) at 28 °C for 12–96 h depending on the strain. Approximately 1.2 × 109 cells were harvested by centrifugation (5000 × g, 4 °C, 5 min), and the pellet was washed twice with 50 ml of cold 0.3 m sorbitol and suspended in 1 ml of 95% methanol and1gof silica sand. This mixture was vigorously vortexed (3 × 20 s) and centrifuged (20,500 × g, 5 min). The supernatant then was transferred to a second Microfuge tube, and the pellet was extracted with an additional 1 ml of 70% methanol by vortexing. After centrifugation (20,500 × g, 5 min), the second supernatant was combined with the first. These methanol-derived extracts were analyzed for GHB and GABA levels by gas chromatography-mass spectrometry (22Gibson K.M. Aramaki S. Sweetman L. Nyhan W.L. DeVivo D.C. Hodson A.K. Jakobs C. Biomed. Environ. Mass Spectrom. 1990; 9: 89-93Crossref Scopus (92) Google Scholar, 23Kok R.M. Howells D.W. Van DenHeuvel C.C.M. Guérand W.S. Thompson G.N. Jakobs C. J. Inherit. Metab. Dis. 1993; 16: 508-512Crossref PubMed Scopus (40) Google Scholar). Metabolite and Expression Analyses of Arabidopsis—Arabidopsis seeds were stratified at 4 °C for 48 h and then grown in Fox sandy loam (pH 6.5) at 22/18 °C day/night temperature and a 11/13-h day/night photoperiod. Plants were grown individually and with sufficient spacing in seedling trays to preclude shading between adjacent plants. They were fertilized twice weekly with a half-strength modified nutrient solution (24Shelp B.J. Penner R. Zhu Z. Can. J. Plant Sci. 1992; 72: 883-888Crossref Google Scholar) and subirrigated as needed with water. To investigate gene expression as a function of development and organ, rosette leaves 1–3, rosette leaves 4–6, rosette leaves 7–8, roots, flowers, and siliques were collected from three 6-week-old plants. To investigate the response to oxygen deficiency, all of the rosette leaves were harvested from six 4-week-old plants at time zero with the 24 plants remaining divided equally and randomly between two washbasins covered with tin foil. One contained sufficient water to cover the entire plants (i.e. flooded or treated plants), whereas the other did not contain water (i.e. control plants). At 2 or 4 h, leaves from six plants in each basin were quickly harvested and frozen in liquid nitrogen. For each harvest, the leaves were divided into three replicates of two plants each (mean fresh weight ± S.E. of 0.27 ± 0.002 g) and stored at –80 °C until analysis. For metabolite analysis, the frozen leaf tissue was extracted with 80% ethanol containing 5 nmol of GHB-d6 as an internal control and the water-soluble fraction after washing with chloroform was dried in a Speedvac concentrator. The dried residue was suspended in 500 μl of de-ionized water, and then the extract was filter-sterilized through a 45-μm membrane. Analysis and quantification of GHB content were performed using a Hewlett-Packard 1100 series liquid chromatography/mass spectrometer (Agilent Technologies Inc.) as described previously (12Allan W.L. Smith R. Shelp B.J. Application Bulletin AB-0015. Agilent Technologies Inc., Mississauga, Ontario, Canada2003: 4Google Scholar). For GABA and alanine analysis, a 100-μl of each extract was with and by liquid using a × (Agilent Technologies Inc.) and was detected at as described by et al. C. Agilent Technologies Inc., Mississauga, Ontario, Scholar). For expression analysis by quantification reverse RNA was extracted from leaf tissue using in the plant kit The RNA was treated with 1 of in the of reaction mm (pH mm 1 mm and of for at °C to genomic cDNA was using a as described in the kit using 1 of the RNA and of the of cDNA was used for each reaction. The was as described in the kit To quantification of gene cDNA was a with the gene of using an mixture of as provided in the of two was by the primers and mixture to the reaction at a of The primers used to GHBDH and is described A. McLean M.D. Shelp B.J. Can. J. Scopus Google were as GTA TTT GTA and AGA TTA These primers gene of and for GHBDH and GABA-T, The were °C for 5 at °C for °C for °C for and a at °C for 5 was performed with as described previously (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). control reactions did not contain reverse whereas control reactions contained the cDNA of in a of amplified were performed using of an Arabidopsis GHBDH cDNA by Yeast complementation of an SSADH-deficient yeast mutant 22641c was used to an Arabidopsis GHBDH This mutant of the SSADH activity of the strain when grown on GABA as the sole nitrogen source and and was to the not that is the yeast SSADH Turano F.J. W.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The mutant was transformed with a cDNA expression library from entire seedlings of and containing a cDNA were These when into mutant growth on GABA. The cDNA in the was and to a which amino (Fig. and a of an of and a charge of at The gene the polypeptide designated AtGHBDH for A. thaliana A of the data base does not identify significant with SSA from (9Andriamampandry C. Siffert J.-C. Schmitt M. Garnier J.-M. Staub A. Muller C. Gobaille S. Mark J. Maitre M. Biochem. J. 1998; 334: 43-50Crossref PubMed Scopus (26) Google Scholar) or M. Schaffhauser M. Sans N. Wermuth B. Eur. J. Biochem. 1999; 265: 1056-1060Crossref PubMed Scopus (41) Google Scholar) and NAD-dependent from (GenBank™ accession (GenBank™ accession or (GenBank™ accession the amino of AtGHBDH does to and and dehydrogenase from not The of between AtGHBDH and dehydrogenases is at the (Fig. a containing a that is of the of dehydrogenases D.W. PubMed Scopus Google Scholar). this by amino is an residue that is in and all of the This residue is in the of In all of the NAD-dependent dehydrogenases an residue at this Several other that in in and dehydrogenases to be These and according to the AtGHBDH polypeptide to an NADP-binding and to to a of dehydrogenases that not previously The yeast and the mutant 22641c AtGHBDH well on 20 mm GABA as well as 20 mm or whereas mutant cells the empty did not on GABA (Fig. Mutant cells AtGHBDH or significantly concentrations of GABA cells This was by concentrations of however, the concentrations in cells AtGHBDH were in GHB was by all of the strains when grown on or as the sole nitrogen source. These data confirm that the isolated Arabidopsis cDNA encodes a polypeptide with GHBDH of GABA and GHB pool in wild type yeast (Σ1278b) and the SSADH-deficient mutant transformed with the empty vector (pFL61) or ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± in a of in investigate the role of GHBDH in we using that the expression of GHBDH or was plant 1–3, leaves 4–6, leaves 7–8, roots, flowers, and from Arabidopsis plants not and independent of and We then subjected Arabidopsis plants to for up to 4 h to oxygen deficiency and the expression of in leaves as well as the concentrations of GHB, the product of the GHBDH and (Fig. 1). The levels of gene at 2 and 4 h were in flooded plants in control plants (Fig. whereas the concentrations of GABA and alanine as A.W. Shelp B.J. Biochem. (Life Sci. Adv.). 1989; 8: 21-25Google Scholar) were significantly in leaves of flooded plants (Fig. This was by a accumulation of GHB in flooded plants. and a in the pool of GHB to oxygen deficiency W. L., Peiris, C., Brown, A. W., and Shelp, B. J. (2003) Can. J. Plant Sci., in pressGoogle pool in Arabidopsis plants subjected to for up to 4 All of the plants were in the the time The and control and flooded plants, are the ± S.E. of three These metabolic be a of glutamate decarboxylase activity by two cytosolic or in conjunction with and increases in GABA and alanine (Fig. (1Bown A.W. Shelp B.J. Plant Physiol. (Bethesda). 1997; 115: 1-5Crossref PubMed Scopus (321) Google Scholar, 2Shelp B.J. Bown A.W. McLean M.D. Trends Plant Sci. 1999; 4: 446-452Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar, 6Kinnersley A.M. Turano F.J. Crit. Rev. Plant Sci. 2000; 19: 479-509Crossref Google restricted SSADH activity to (7Busch K.B. Fromm H. Plant Physiol. (Bethesda). 1999; 121: 589-597Crossref PubMed Scopus (110) Google Scholar, 8Busch K.B. Piehler J. Fromm H. Biochemistry. 2000; 39: 10110-10117Crossref PubMed Scopus (27) Google Scholar), thereby the accumulation of SSA and the of activity Shelp B.J. 1999; Scopus (40) Google and the of alanine an enzyme that the of alanine from and glutamate Plant Physiol. (Bethesda). 1992; PubMed Scopus Google Scholar). the to pool of it that a significant of the SSA derived GABA flooding-induced oxygen deficiency was to GHB and that this result not be to up-regulation of gene using SSADH in yeast Turano F.J. W.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) and plants N. A. Fromm H. Sci. S. A. PubMed Scopus Google Scholar) that the GABA a role in the accumulation of oxygen probably by a source of reducing for the of or by on suggested that GHB also functions in of oxygen probably by (11Mamelak M. Neurosci. Biobehav. Rev. 1989; 13: 187-198Crossref PubMed Scopus (163) Google Scholar). oxygen deficiency increases the of EMBO J. 1997; 16: PubMed Scopus Google Scholar, W.S. M.C. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and oxygen such as and in plants Annu. Rev. Plant Physiol. Plant Mol. Biol. PubMed Scopus Google however, the of as well as via the is Annu. Rev. Plant Physiol. Plant Mol. Biol. PubMed Scopus Google Scholar). findings suggest that GHBDH and SSADH activities in plants are regulated in a complementary fashion by redox and that GHB functions in oxidative stress tolerance. using such as and R. that GHB is of a fermentation pathway from SSA to and J. Biol. Full Text PDF PubMed Google Scholar, Environ. 1993; PubMed Google Scholar, A. A. Eur. J. Biochem. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). is also a for acid PubMed Scopus Google Scholar, S. 2000; Scopus (27) Google Scholar). In GHB as product and of the neurotransmitter GABA Maitre M. C. P. PubMed Scopus Google Scholar, C. Maitre M. J. 1997; Google Scholar, M. 1997; PubMed Scopus Google Scholar). It may also function as a neurotransmitter Neurosci. Biobehav. Rev. 1994; PubMed Scopus Google Scholar, J. 1998; Google Scholar). its and to its use as a M. Smith 1997; PubMed Scopus Google Scholar, C. J. 1997; PubMed Scopus Google Scholar). A of with GHB in plants may a function or for We M. McLean and A. for and M. for the Arabidopsis cDNA library constructed in a yeast expression

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