Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Characterization and developmental expression of a glutamate decarboxylase from maritime pine

  • 273 Accesses

  • 15 Citations

Abstract

Glutamate decarboxylase (GAD, EC 4.1.1.15) is a key enzyme in the synthesis of γ-aminobutyric acid (GABA) in higher plants. A complete cDNA encoding glutamate decarboxylase (GAD, EC 4.1.1.15) was characterized from Pinus pinaster Ait, and its expression pattern was studied to gain insight into the role of GAD in the differentiation of the vascular system. Pine GAD contained a C-terminal region with conserved residues and a predicted secondary structure similar to the calmodulin (CaM)-binding domains of angiosperm GADs. The enzyme was able to bind to a bovine CaM-agarose column and GAD activity was higher at acidic pH, suggesting that the pine GAD can be regulated in vivo by Ca2+/CaM and pH. A polyclonal antiserum was prepared against the pine protein. GAD expression was studied at activity, protein, and mRNA level and was compared with the expression of other genes during the differentiation of the hypocotyl and induction of reaction wood. In seedling organs, GABA levels closely matched GAD expression, with high levels in the root and during lignification of the hypocotyl. GAD expression was also induced in response to the production of compression wood and its expression matched the pattern of other genes involved in ethylene and 2-oxoglutarate synthesis. The results suggest of a role of GAD in hypocotyl and stem development in pine.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Abbreviations

ACO:

1-Aminocyclopropane-1-carboxylic acid oxidase

ACS:

1-Aminocyclopropane-1-carboxylic acid synthase

APX:

Ascorbate peroxidase

AOX:

Ascorbate oxidase

CaM:

Calmodulin

GAD:

Glutamate decarboxylase

LAC:

Laccase

NADP+-IDH:

NADP+-isocitrate dehydrogenase

References

  1. Akama K, Takaiwa F (2007) C-terminal extension of rice glutamate decarboxylase (OsGAD2) functions as an autoinhibitory domain and overexpression of a truncated mutant results in the accumulation of extremely high levels of GABA in plant cells. J Exp Bot 58:2699–2707

  2. Akama K, Akihiro T, Kitagawa M, 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 1522:143–150

  3. Arazi T, Baum G, Snedden WA, Shelp BJ, Fromm H (1995) Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase. Plant Physiol 108:551–561

  4. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (2009) Current protocols in molecular biology. Wiley, New York

  5. Barbosa JM, Singh NK, Cherry JH, Locy RD (2010) Nitrate uptake and utilization is modulated by exogenous gamma-aminobutyric acid in Arabidopsis thaliana seedlings. Plant Physiol Biochem 48:443–450

  6. Barnes JR, Lorenz WW, Dean JFD (2008) Characterization of a 1-aminocyclopropane-1-carboxylate synthase gene from loblolly pine (Pinus taeda L.). Gene 413:18–31

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

  8. Baum G, Lev-Yadun S, Fridmann Y, Arazi T, Katsnelson H, Zik M, Fromm H (1996) Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants. EMBO J 15:2988–2996

  9. Beuve N, Rispail N, Laine P, Cliquet J, Ourry A (2004) Putative role of γ–aminobutyric acid (GABA) as a long-distance signal in up-regulation of nitrate uptake in Brassica napus L. Plant Cell Environ 27:1035–1046

  10. Bormann J (2000) The ‘ABC’ of GABA receptors. Trends Pharmacol Sci 21:16–19

  11. Bouche N, Fromm H (2004) GABA in plants: just a metabolite? Trends Plant Sci 9:110–115

  12. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72:248–254

  13. Cánovas FR (1991) Accumulation of glutamine synthetase during early development of maritime pine (Pinus pinaster) seedlings. Planta 185:372–378

  14. Cánovas F, FR C, García-Gutiérrez A, Gallardo F, Crespillo R (1998) Molecular physiology of glutamine and glutamate biosynthesis in developing seedlings of conifers. Physiol Plant 103:287–294

  15. Cantón FR, Le-Provost G, García V (2004) Transcriptome analysis of wood formation in maritime pine. In: Ritter E, Espinel S, Barredo Y et al (eds) Sustainable forestry, wood products and biotechnology. DFA-AFA Press, Vitoria-Gasteiz, pp 333–348

  16. Carroll AD, Fox GC, Laurie S, Phillips R, Ratcliffe G, Stewart GR (1994) Ammonium assimilation and the role of γ-aminobutyric acid in pH homeostasis in carrot cell suspensions. Plant Physiol 106:513–520

  17. Cercós M, Soler G, Iglesias DJ, Gadea J, Forment J, Talón M (2006) Global analysis of gene expression during development and ripening of citrus fruit flesh. A proposed mechanism for citric acid utilization. Plant Mol Biol 62:513–527

  18. Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine tree. Plant Mol Biol Rep 11:113–116

  19. Charboneau L, Paweletz CP, Liotta LA (2004) Laser capture microdissection. In: Bonifacino JS, Dasso M, Harford JB, Lippincott-Schwartz J, Yamda KM (eds) Short protocols in cell biology. Wiley, Hoboken, pp 2, 13–16

  20. Chen Y, Baum G, 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 106:1381–1387

  21. Coleman ST, Fang TK, Rovinsky SA, Turano FJ, Moye-Rowley WS (2001) Expression of a glutamate decarboxylase homologue is required for normal oxidative stress tolerance in Saccharomyces cerevisiae. J Biol Chem 276:244–250

  22. Cona A, Rea G, Angelini R, Federico R, Tavladoraki P (2006) Functions of amine oxidases in plant development and defence. Trends Plant Sci 11:80–88

  23. Crawford LA, Bown AW, Breitkreuz KE, Guinel FC (1994) The synthesis of γ-aminobutyric acid in response to treatments reducing cytosolic pH. Plant Physiol 104:865–871

  24. de Pinto M, De Gara L (2004) Changes in the ascorbate metabolism of apoplastic and symplastic spaces are associated with cell differentiation. J Exp Bot 55:2559–2569

  25. Dellaporta SL, Wood J, Hicks JB (1983) Isolation of DNA from higher plants. Plant Mol Biol Rep 4:19–21

  26. Fait A, Fromm H, Walter D, Galili G, Fernie AR (2008) Highway or byway: the metabolic role of the GABA shunt in plants. Trends Plant Sci 13:14–19

  27. Gallardo F, Cánovas F (2002) H+ fluxes in nitrogen assimilation by plants. In: Rengel Z (ed) Handbook of plant growth. pH as the master variable. Marcel Dekker, Inc., New York, pp 211–226

  28. Gallardo F, Cantón F, García-Gutiérrez A, Cánovas F (1993) Changes in photorespiratory enzymes and glutamate synthases in ripening tomatoes. Plant Physiol Biochem 31:189–196

  29. Gallardo F, Fu J, Cantón FR, García-Gutiérrez A, Cánovas FM, Kirby EG (1999) Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210:19–26

  30. Gavnholt B, Larsen K (2002) Molecular biology of plant laccases in relation to lignin formation. Physiol Plant 116:273–280

  31. Groover A, Jones A (1999) Tracheary element differentiation uses a novel mechanism coordinating programmed cell death and secondary cell wall synthesis. Plant Physiol 119:375–384

  32. Hodges M (2002) Enzyme redundancy and the importance of 2-oxoglutarate in plant ammonium assimilation. J Exp Bot 53:905–916

  33. Jing ZP, Gallardo F, Pascual MB, Sampalo R, Romero J, Torres de Navarra A, Cánovas F (2004) Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase. New Phytol 164:137–145

  34. Kathiresan A, Tung P, Chinnappa CC, Reid DM (1997) γ-aminobutyric acid stimulates ethylene biosynthesis in sunflower. Plant Physiol 115:129–135

  35. Kathiresan A, Miranda J, Chinnappa C, Reid D (1998) γ–aminobutyric acid promotes stem elongation in Stellaria longipes: the role of ethylene. Plant Growth Regul 26:131–137

  36. Kinnersley A, Lin F (2000) Receptor modifiers indicate that 4-aminobutyric acid (GABA) is a potential modulator of ion transport in plants. Plant Growth Regul 32:65–76

  37. Klintborg A, Eklund L, Little CHA (2002) Ethylene metabolism in Scots pine (Pinus sylvestris) shoots during the year. Tree Physiol 22:59–66

  38. Kneller DG, Cohen FE, Langridge R (1990) Improvements in protein secondary structure prediction by an enhanced neural network. J Mol Biol 214:171–182

  39. Lancien M, Roberts MR (2006) Regulation of Arabidopsis thaliana 14-3-3 gene expression by gamma-aminobutyric acid. Plant Cell Environ 29:1430–1436

  40. Love J, Björklund S, Vahala J, Hertzberg M (2009) Ethylene is an endogenous stimulator of cell division in the cambial meristem of Populus. Proc Natl Acad Sci USA 14:5984–5989

  41. Masclaux C, Valadier MH, Brugiere N, Morot-Gaudry JF, Hirel B (2000) Characterization of the sink/source transition in tobacco (Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence. Planta 211:510–518

  42. O’Neil KT, DeGrado WF (1990) How calmodulin binds its targets: sequence independent recognition of amphiphilic alpha-helices. Trends Biochem Sci 15:59–64

  43. Page RD (1996) TreeView: an application to display phylogenetic trees on personal computers. Comp App Bios 12:357–358

  44. Palanivelu R, Brass L, Edlund A, Preuss D (2003) Pollen tube growth and guidance is regulated by pop2, an arabidopsis gene that controls gaba levels. Cell 114:47–59

  45. Palomo J, Gallardo F, Suárez MF, Cánovas F (1998) Purification and characterization of NADP+-linked isocitrate dehydrogenase from scots pine. Plant Physiol 118:617–626

  46. Pascual MB, Molina-Rueda JJ, Cánovas F, Gallardo F (2008) Spatial distribution of cytosolic NADP+-isocitrate dehydrogenase in pine embryo and seedlings. Tree Physiol 28:1773–1782

  47. Passardi F, Penel C, Dunand C (2004) Performing the paradoxical: how plant peroxidases modify the cell wall. Trends Plant Sci 9:534–540

  48. Pignocchi C, Foyer C (2003) Apoplastic ascorbate metabolism and its role in the regulation of cell signalling. Curr Opin Plant Biol 6:379–389

  49. Plomion C, Pionneau C, Brach J, Costa P, Baillères H (2000) Compression wood-responsive proteins in developing xylem of maritime pine (Pinus pinaster Ait.). Plant Physiol 123:959–969

  50. Plomion C, Leprovost G, Stokes A (2001) Wood formation in trees. Plant Physiol 127:1513–1523

  51. Regan S, Bourquin V, Tuominen H, Sundberg B (1999) Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues. Plant J 19:363–369

  52. Snedden WA, Arazi T, Fromm H, Shelp BJ (1995) Calcium/calmodulin activation of soybean glutamate decarboxylase. Plant Physiol 108:543–549

  53. Snedden WA, Koutsia N, Baum G, 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 271:4148–4153

  54. Sterky F, Bhalerao RR, Unneberg P, Segerman B, Nilsson P, Brunner AM, Charbonnel-Campaa L, Lindvall JJ, Tandre K, Strauss SH, Sundberg B, Gustafsson P, Uhlen M, Bhalerao RP, Nilsson O, Sandberg G, Karlsson J, Lundeberg J, Jansson S (2004) A Populus EST resource for plant functional genomics. Proc Natl Acad Sci USA 101:13951–13956

  55. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res 25:4876–4882

  56. Timell TE (1986) Compression wood in gymnosperms. Springer, Heidelberg

  57. Turano FJ, Fang TK (1998) Characterization of two glutamate decarboxylase cDNA clones from Arabidopsis. Plant Physiol 117:1411–1421

  58. Willis KJ, McElwain JC (2002) The evolution of plants. Oxford University Press, NY, USA

  59. Yevtushenko DP, McLean MD, Peiris S, Van Cauwenberghe OR, Shelp BJ (2003) Calcium/calmodulin activation of two divergent glutamate decarboxylases from tobacco. J Exp Bot 54:2001–2002

  60. Yu G, Liang J, He Z, Sun M (2006) Quantum dot-mediated detection of γ-aminobutyric acid binding sites on the surface of living pollen protoplasts in tobacco. Chem Biol 13:723–731

  61. Zhang G, Bown AW (1997) The rapid determination of γ-aminobutyric acid. Phytochemistry 44:1007–1009

  62. Zik M, Fridmann-Sirkis Y, Fromm H (2006) C-terminal residues of plant glutamate decarboxylase are required for oligomerization of a high-molecular weight complex and for activation by calcium/calmodulin. Biochim Biophys Acta 1764:872–876

Download references

Acknowledgments

We want to thank Fernando Nicolás de la Torre Fazio for his help in the production of polyclonal antibodies. This work was supported by the grants BIO2003-04590, and AGL2009-11404 to F.G, and BIO2006-06216 to F.M.C. from the Spanish Ministry of Science and Innovation, which were partially financed by FEDER funds from the European Union. J.J.M.-R. and M.B.P. were recipients of a FPU and a UAs-CSIC fellowship from the Spanish Ministry of Education and Science, respectively.

Author information

Correspondence to Fernando Gallardo.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 159 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Molina-Rueda, J.J., Pascual, M.B., Cánovas, F.M. et al. Characterization and developmental expression of a glutamate decarboxylase from maritime pine. Planta 232, 1471–1483 (2010). https://doi.org/10.1007/s00425-010-1268-9

Download citation

Keywords

  • Glutamate decarboxylase
  • GABA
  • Isocitrate dehydrogenase
  • Pine
  • Reaction wood
  • Vascular development