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The root-specific glutamate decarboxylase (GAD1) is essential for sustaining GABA levels in Arabidopsis

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Abstract

In plants, as in most eukaryotes, glutamate decarboxylase catalyses the synthesis of GABA. The Arabidopsis genome contains five glutamate decarboxylase genes and one of these genes (glutamate decarboxylase1; i.e.GAD1) is expressed specifically in roots. By isolating and analyzing three gad1 T-DNA insertion alleles, derived from two ecotypes, we investigated the potential role of GAD1 in GABA production. We also analyzed a promoter region of the GAD1 gene and show that it confers root-specific expression when fused to reporter genes. Phenotypic analysis of the gad1 insertion mutants revealed that GABA levels in roots were drastically reduced compared with those in the wild type. The roots of the wild type contained about sevenfold more GABA than roots of the mutants. Disruption of the GAD1 gene also prevented the accumulation of GABA in roots in response to heat stress. Our results show that the root-specific calcium/calmodulin-regulated GAD1 plays a major role in GABA synthesis in plants under normal growth conditions and in response to stress.

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References

  • Akama, K., Akihiro, T., Kitagawa, M. and Takaiwa, F. 2001. Rice (Oryza sativa) contains a novel isoform of glutamate decarboxylase that lacks an authentic calmodulin-binding domain at the C-terminus. Biochim. Biophys. Acta 1522: 143.

    PubMed  Google Scholar 

  • The Arabidopsis Genome Initiative. 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796.

    Google Scholar 

  • Arazi, T., Baum, G., Snedden, W. A., Shelp, B. J. and Fromm, H. 1995. Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase. Plant Physiol. 108: 551.

    PubMed  Google Scholar 

  • Aurisano, N., Bertani, A. and Reggiani, R. 1995. Involvement of calcium and calmodulin in protein and amino-acid metabolism in rice roots under anoxia. Plant Cell Physiol. 36: 15259.

    Google Scholar 

  • Baum, G., Chen, Y., Arazi, T., Takatsuji, H. and Fromm, H. 1993. A plant glutamate decarboxylase containing a calmodulin binding domain. Cloning, sequence, and functional analysis. J. Biol. Chem. 268: 19610–19617.

    PubMed  Google Scholar 

  • Baum, G., Lev-Yadun, S., Fridmann, Y., Arazi, T., Katsnelson, H., Zik, M. and Fromm, H. 1996. Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants. EMBO J. 15: 29886.

    Google Scholar 

  • Bechtold, N., Ellis, J. and Pelletier, G. 1993. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris, Ser. III 316: 11949.

    Google Scholar 

  • Beeckman, T. and Engler, G. 1994. An easy technique for the clearing of histochemically stained plant tissue. Plant Mol. Biol. Rep. 12: 37–42.

    Google Scholar 

  • Bieleski, R. L. and Turner, N. A. 1966. Separation and estimation of amino acids in crude plant extracts by thin-layer electrophoresis and chromatography. Anal. Biochem. 17: 278.

    PubMed  Google Scholar 

  • Blanc, G., Barakat, A., Guyot, R., Cooke, R. and Delseny, M. 2000. Extensive duplication and reshuffling in the Arabidopsis genome. Plant Cell 12: 10932.

    Google Scholar 

  • Bouchè, N. and Bouchez, D. 2001. Arabidopsis gene knockout: phenotypes wanted. Curr. Opin. Plant Biol. 4: 111.

    PubMed  Google Scholar 

  • Bouchè, N. and Fromm, H. 2004. GABA in plants: just a metabolite? Trends Plant Sci. 9: 110.

    PubMed  Google Scholar 

  • Bouchè, N., Fait, A., Bouchez, D., Møller, S. G. and Fromm, H. 2003a. Mitochondrial succinic-semialdehyde dehydrogenase of the c-aminobutyrate shunt is required to restrict levels of reactive oxygen intermediates in plants. Proc. Natl. Acad. Sci. USA. 100: 68438.

    Google Scholar 

  • Bouchè, N., Lacombe, B. and Fromm, H. 2003b. GABA signalling: a conserved and ubiquitous mechanism. Tr. Cell Biol. 13: 607.

    Google Scholar 

  • Bouchez, D., Camilleri, C. and Caboche, M. 1993. A binary vector based on Basta resistance for in planta transformation of Arabidopsis thaliana. C. R. Acad. Sci. Paris, Ser. III 316: 11883.

    Google Scholar 

  • Breitkreuz, K. E., Allard, M. W., van Cauwenberghe, O. R., Jakobs, C., Talibi, D., Andrè, B. and Shelp, B. J. 2003. A novel gamma-hydroxybutyrate dehydrogenase: identification and expression of an Arabidopsis cDNA and potential role under oxygen deficiency. J. Biol. Chem. 278: 41552–41556.

    PubMed  Google Scholar 

  • Chen, Y., Baum, G. and Fromm, H. 1994. The 58-kilodalton calmodulin-binding glutamate decarboxylase is a ubiquitous protein in petunia organs and its expression is developmentally regulated. Plant Physiol. 106: 13817.

    Google Scholar 

  • Chiu, W., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H. and Sheen, J. 1996. Engineered GFP as a vital reporter in plants. Curr. Biol. 6: 325.

    PubMed  Google Scholar 

  • Cholewa, E., Cholewinski, A. J., Sheen, J., Snedden, W. A. and Bown, A. W. 1997. Cold shock-stimulated c-aminobutyrate synthesis is mediated by an increase in cytosolic Ca2+ not by an increase cytosolic H+. Can. J. Bot. 75: 375.

    Google Scholar 

  • Clough, S. J. and Bent, A. F. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735.

    PubMed  Google Scholar 

  • Fiehn, O., Kopka, J., Dormann, P., Altmann, T., Trethewey, R. N. and Willmitzer, L. 2000. Metabolite profiling for plant functional genomics. Nat. Biotechnol. 18: 11571.

    Google Scholar 

  • Forde, B. G. 2002. Local and long-range signaling pathways regulating plant responses to nitrate. Annu. Rev. Plant Biol. 53: 203.

    PubMed  Google Scholar 

  • Ling, V., Snedden, W. A., Shelp, B. J. and Assmann, S. M. 1994. Analysis of a soluble calmodulin binding protein from fava bean roots: identification of glutamate decarboxylase as a calmodulin-activated enzyme. Plant Cell 6: 11353.

    Google Scholar 

  • Palanivelu, R., Brass, L., Edlund, A. F. and Preuss, D. 2003. Pollen tube growth and guidance is regulated by POP2, an Arabidopsis gene that controls GABA levels. Cell 114: 47–59.

    PubMed  Google Scholar 

  • Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  • Schales, O., Mims, V. and Schales, S. 1946. Glutamic acid decarboxylase of higher plants. Arch. Biochem. Biophys. 10: 455.

    Google Scholar 

  • Schmidt, H. 1996. HPLC-determination of c-aminobutyric acid (GABA) in psychotropic medicinal plants. Chromatographie 1: 12–15.

    Google Scholar 

  • Shelp, B. J., Bown, A. W. and McLean, M. D. 1999. Metabolism and functions of c-aminobutyric acid. Trends Plant Sci. 4: 446.

    PubMed  Google Scholar 

  • Snedden, W. A. and Fromm, H. 1999. Regulation of the c-aminobutyrate-synthesizing enzyme, glutamate decarboxylase, by calcium-calmodulin: a mechanism for rapid activation in response to stress. In: H. R. Lerner (Ed) Plant Responses to Environmental Stresses: From Phytohormones to Genome Reorganization, Marcel Dekker, Inc., New York pp. 549.

    Google Scholar 

  • Snedden, W. A. and Fromm, H. 2001. Calmodulin as a versatile calcium signal transducer in plants. New Phytol. 151: 35–66.

    Google Scholar 

  • Snedden, W. A., Arazi, T., Fromm, H. and Shelp, B. J. 1995. Calcium/calmodulin activation of soybean glutamate decarboxylase. Plant Physiol. 108: 543.

    PubMed  Google Scholar 

  • Snedden, W. A., Koutsia, N., Baum, G. and Fromm, H. 1996. Activation of a recombinant petunia glutamate decarboxylase by calcium/calmodulin or by a monoclonal antibody which recognizes the calmodulin binding domain. J. Biol. Chem. 271: 41483.

    Google Scholar 

  • Sokal, R. R. and Rohlf, F. J. 1995. Biometry: The Principles and Practice of Statistics in Biological Research. WH Freeman and Company, NY.

    Google Scholar 

  • Stitt, M., Muller, C., Matt, P., Gibon, Y., Carillo, P., Morcuende, R., Scheible, W. R. and Krapp, A. 2002. Steps towards an integrated view of nitrogen metabolism. J. Exp. Bot. 53: 959.

    PubMed  Google Scholar 

  • Sunkar, R., Kaplan, K., Bouchè, N., Arazi, T., Dolev, D., Talke, I. N., Maathuis, F. J. M., Sanders, D., Bouchez, D. and Fromm, H. 2000. Expression of a truncated tobacco NtCBP4 channel in transgenic plants, and disruption of the homologous Arabidopsis CNGC1 gene confer Pb++ tolerance. Plant J. 24: 533.

    PubMed  Google Scholar 

  • Turano, F. J. and Fang, T. K. 1998. Characterization of two glutamate decarboxylase cDNA clones from Arabidopsis. Plant Physiol. 117: 14111.

    Google Scholar 

  • Yun, S. J. and Oh, S. H. 1998. Cloning and characterization of a tobacco cDNA encoding calcium/calmodulin-dependent glutamate decarboxylase. Mol. Cells 8: 125.

    PubMed  Google Scholar 

  • Zik, M., Arazi, T., Snedden, W. A. and Fromm, H. 1998. Two isoforms of glutamate decarboxylase in Arabidopsis are regulated by calcium/calmodulin and differ in organ distribution. Plant Mol. Biol. 37: 967.

    PubMed  Google Scholar 

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Bouché, N., Fait, A., Zik, M. et al. The root-specific glutamate decarboxylase (GAD1) is essential for sustaining GABA levels in Arabidopsis . Plant Mol Biol 55, 315–325 (2004). https://doi.org/10.1007/s11103-004-0650-z

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  • DOI: https://doi.org/10.1007/s11103-004-0650-z

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