Plant pyruvate-dependent gamma-aminobutyrate transaminase: identification of an<i>Arabidopsis</i>cDNA and its expression in<i>Escherichia coli</i>

Cauwenberghe, Owen R. Van; Makhmoudova, Amina; McLean, Michael D.; Clark, Shawn M.; Shelp, Barry J.

doi:10.1139/b02-087

Cited by 54 publications

(43 citation statements)

References 36 publications

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“…Thereafter, cells were permeabilized with either 0.3% (v/v) Triton X‐100 for 30 min. Sources of antibodies used for subsequent immunofluorescence staining of cells were as follows: mouse α‐Myc antibodies and goat α‐rabbit or goat α‐mouse Alexa Fluorr 488 IgGs (Cedar Lane Laboratories); rabbit α‐cottonseed catalase (Kunce et al 1988); mouse α‐tobacco catalase hybridoma media (Princeton University Monoclonal Antibody Facility); rabbit α‐chicken and goat α‐rabbit rhodamine red‐X IgGs (Jackson ImmunoResearch Laboratories); custom chicken α‐Arabidopsis GLYR1 antibodies were raised against a synthetic peptide corresponding to the GLYR1 amino acid sequence ‐EGPVSGSKKPA‐ (residues 117–127) and according to the procedures described in Van Cauwenberghe et al (2002). 4′,6‐diamidino‐2‐phenylindole (DAPI) was purchased from Molecular Probes.…”

Section: Methodsmentioning

confidence: 99%

Glyoxylate Reductase Isoform 1 is Localized in the Cytosol and Not Peroxisomes in Plant Cells

Ching

Gidda

Rochon

et al. 2012

JIPB

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Glyoxylate reductase (GLYR) is a key enzyme in plant metabolism which catalyzes the detoxification of both photorespiratory glyoxylate and succinic semialdehdye, an intermediate of the γ-aminobutyrate (GABA) pathway. Two isoforms of GLYR exist in plants, GLYR1 and GLYR2, and while GLYR2 is known to be localized in plastids, GLYR1 has been reported to be localized in either peroxisomes or the cytosol. Here, we reappraised the intracellular localization of GLYR1 in Arabidopsis thaliana L. Heynh (ecotype Lansberg erecta) using both transiently-transformed suspension cells and stably-transformed plants, in combination with fluorescence microscopy. The results indicate that GLYR1 is localized exclusively to the cytosol regardless of the species, tissue and/or cell type, or exposure of plants to environmental stresses that would increase flux through the GABA pathway. Moreover, the C-terminal tripeptide sequence of GLYR1, -SRE, despite its resemblance to a type 1 peroxisomal targeting signal, is not sufficient for targeting to peroxisomes. Collectively, these results define the cytosol as the intracellular location of GLYR1 and provide not only important insight to the metabolic roles of GLYR1 and the compartmentation of the GABA and photorespiratory pathways in plant cells, but also serve as a useful reference for future studies of proteins proposed to be localized to peroxisomes and/or the cytosol.

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Section: Methodsmentioning

confidence: 99%

Glyoxylate Reductase Isoform 1 is Localized in the Cytosol and Not Peroxisomes in Plant Cells

Ching

Gidda

Rochon

et al. 2012

JIPB

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“…GABA can be metabolized along with a molecule of Pyr by GABA transaminase (GABA-T) to produce Ala and succinic semialdehyde (SSA) (Van Cauwenberghe & Shelp 1999;Van Cauwenberghe et al 2002). GABA-T is mitochondrial (Clark et al 2009), indicating the importance of metabolite transport between the organelle and cytosol.…”

Section: Root-specific Gaba Formation Under Hypoxia and Its Degradationmentioning

confidence: 99%

Characterization of distinct root and shoot responses to low‐oxygen stress in Arabidopsis with a focus on primary C‐ and N‐metabolism

Mustroph

Barding

Kaiser

et al. 2014

Plant Cell & Environment

100

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Oxygen deficiency, caused by flooding of all or a portion of a plant, leads to significant gene regulatory and metabolic responses associated with survival. When oxygen-deprived in light, aerial organs and root systems respond in distinct manners because of their respective autotrophy and heterotrophy, as well as intrinsic differences in cell biology and organ function. To better understand organ-specific responses to oxygen deficiency, we monitored changes in the metabolome of roots and shoots of Arabidopsis thaliana seedlings using gas chromatography-mass spectrometry and (1) H-nuclear magnetic resonance spectroscopy. Only roots accumulated high amounts of γ-aminobutyrate (GABA) and lactate, whereas both organs accumulated alanine (Ala) upon hypoxia. Meta-analysis of gene regulation data revealed higher induction of mRNAs coding for fermentative enzymes in roots as compared with shoots. However, the elevation in GABA level was not correlated with changes in transcript abundance, supporting the proposal that post-translational mechanisms are important in metabolic acclimation to hypoxia. The biosynthesis, degradation and function of GABA and Ala during oxygen deprivation and re-aeration is discussed. Finally, a systematic survey of low-oxygen mediated regulation of genes associated with primary metabolism across organs and cell types reveals exciting new avenues for future studies.

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“…GABA is then transported into mitochondria by an, as yet, unidentified pathway, where it is catabolized by enzymes of the GABA shunt. In mitochondria, GABA is converted into succinic‐semialdehyde (SSA) by GABA‐transaminase (GABA‐T) (Van Cauwenberghe et al. , 2002; Palanivelu et al.…”

Section: Introductionmentioning

confidence: 99%

A mitochondrial GABA permease connects the GABA shunt and the TCA cycle, and is essential for normal carbon metabolism

et al. 2011

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SUMMARYIn plants, c-aminobutyric acid (GABA) accumulates in the cytosol in response to a variety of stresses. GABA is transported into mitochondria, where it is catabolized into TCA cycle or other intermediates. Although there is circumstantial evidence for mitochondrial GABA transporters in eukaryotes, none have yet been identified. Described here is an Arabidopsis protein similar in sequence and topology to unicellular GABA transporters. The expression of this protein complements a GABA-transport-deficient yeast mutant. Thus the protein was termed AtGABP to indicate GABA-permease activity. In vivo localization of GABP fused to GFP and immunobloting of subcellular fractions demonstrate its mitochondrial localization. Direct [ 3 H]GABA uptake measurements into isolated mitochondria revealed impaired uptake into mitochondria of a gabp mutant compared with wild-type (WT) mitochondria, implicating AtGABP as a major mitochondrial GABA carrier. Measurements of CO 2 release, derived from radiolabeled substrates in whole seedlings and in isolated mitochondria, demonstrate impaired GABA-derived input into the TCA cycle, and a compensatory increase in TCA cycle activity in gabp mutants. Finally, growth abnormalities of gabp mutants under limited carbon availability on artificial media, and in soil under low light intensity, combined with their metabolite profiles, suggest an important role for AtGABP in primary carbon metabolism and plant growth. Thus, AtGABPmediated transport of GABA from the cytosol into mitochondria is important to ensure proper GABA-mediated respiration and carbon metabolism. This function is particularly essential for plant growth under conditions of limited carbon.

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Plant pyruvate-dependent gamma-aminobutyrate transaminase: identification of anArabidopsiscDNA and its expression inEscherichia coli

Cited by 54 publications

References 36 publications

Glyoxylate Reductase Isoform 1 is Localized in the Cytosol and Not Peroxisomes in Plant Cells

Glyoxylate Reductase Isoform 1 is Localized in the Cytosol and Not Peroxisomes in Plant Cells

Characterization of distinct root and shoot responses to low‐oxygen stress in Arabidopsis with a focus on primary C‐ and N‐metabolism

A mitochondrial GABA permease connects the GABA shunt and the TCA cycle, and is essential for normal carbon metabolism

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