Plant Cell Reports

, Volume 36, Issue 1, pp 103–116 | Cite as

Activating glutamate decarboxylase activity by removing the autoinhibitory domain leads to hyper γ-aminobutyric acid (GABA) accumulation in tomato fruit

  • Mariko Takayama
  • Chiaki Matsukura
  • Tohru Ariizumi
  • Hiroshi Ezura
Original Article

Abstract

Key message

The C-terminal extension region ofSlGAD3is likely involved in autoinhibition, and removing this domain increases GABA levels in tomato fruits.

Abstract

γ-Aminobutyric acid (GABA) is a ubiquitous non-protein amino acid with several health-promoting benefits. In many plants including tomato, GABA is synthesized via decarboxylation of glutamate in a reaction catalyzed by glutamate decarboxylase (GAD), which generally contains a C-terminal autoinhibitory domain. We previously generated transgenic tomato plants in which tomato GAD3 (SlGAD3) was expressed using the 35S promoter/NOS terminator expression cassette (35S-SlGAD3-NOS), yielding a four- to fivefold increase in GABA levels in red-ripe fruits compared to the control. In this study, to further increase GABA accumulation in tomato fruits, we expressed SlGAD3 with (SlGAD3OX) or without (SlGAD3ΔCOX) a putative autoinhibitory domain in tomato using the fruit ripening-specific E8 promoter and the Arabidopsis heat shock protein 18.2 (HSP) terminator. Although the GABA levels in SlGAD3OX fruits were equivalent to those in 35S-SlGAD3-NOS fruits, GABA levels in SlGAD3ΔCOX fruits increased by 11- to 18-fold compared to control plants, indicating that removing the autoinhibitory domain increases GABA biosynthesis activity. Furthermore, the increased GABA levels were accompanied by a drastic reduction in glutamate and aspartate levels, indicating that enhanced GABA biosynthesis affects amino acid metabolism in ripe-fruits. Moreover, SlGAD3ΔCOX fruits exhibited an orange-ripe phenotype, which was associated with reduced levels of both carotenoid and mRNA transcripts of ethylene-responsive carotenogenic genes, suggesting that over activation of GAD influences ethylene sensitivity. Our strategy utilizing the E8 promoter and HSP terminator expression cassette, together with SlGAD3 C-terminal deletion, would facilitate the production of tomato fruits with increased GABA levels.

Keywords

E8 promoter Fruit ripening GABA HSP terminator Tomato 

Supplementary material

299_2016_2061_MOESM1_ESM.pdf (2.2 mb)
Supplementary material 1 (PDF 2233 kb)

References

  1. Abdou AM, Higashiguchi S, Horie K, Kim M, Hatta H, Yokogoshi H (2006) Relaxation and immunity enhancement effects of gamma-aminobutyric acid (GABA) administration in humans. BioFactors 26:201–208CrossRefPubMedGoogle Scholar
  2. 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. doi:10.1093/jxb/erm120 CrossRefPubMedGoogle Scholar
  3. Akihiro T, Koike S, Tani R, Tominaga T, Watanabe S, Iijima Y, Aoki K, Shibata D, Ashihara H, Matsukura C, Akama K, Fujimura T, Ezura H (2008) Biochemical mechanism on GABA accumulation during fruit development in tomato. Plant Cell Physiol 49:1378–1389. doi:10.1093/pcp/pcn113 CrossRefPubMedGoogle Scholar
  4. Alba R, Payton P, Fei Z, McQuinn R, Debbie P, Martin GB, Tanksley SD, Giovannoni JJ (2005) Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development. Plant Cell 17:2954–2965. doi:10.1105/tpc.105.036053 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 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. doi:10.1104/pp.108.2.551 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 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 268:19610–19617PubMedGoogle Scholar
  7. 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 15:2988–2996PubMedPubMedCentralGoogle Scholar
  8. Bemer M, Karlova R, Ballester AR, Tikunov YM, Bovy AG, Wolters-Arts M, Rossetto Pde B, Angenent GC, de Maagd RA (2012) The tomato FRUITFULL homologs TDR4/FUL1 and MBP7/FUL2 regulate ethylene-independent aspects of fruit ripening. Plant Cell 24:4437–4451. doi:10.1105/tpc.112.103283 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Bouché N, Fromm H (2004) GABA in plants: just a metabolite? Trends Plant Sci 9:110–115. doi:10.1016/j.tplants.2004.01.006 CrossRefPubMedGoogle Scholar
  10. Clark SM, Di Leo R, Van Cauwenberghe OR, Mullen RT, Shelp BJ (2009) Subcellular localization and expression of multiple tomato γ-aminobutyrate transaminases that utilize both pyruvate and glyoxylate. J Exp Bot 60:3255–3267. doi:10.1093/jxb/erp161 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, Van Montagu M, Leemans J (1985) Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acids Res 13:4777–4788CrossRefPubMedPubMedCentralGoogle Scholar
  12. Deikman J, Xu R, Kneissl ML, Ciardi JA, Kim KN, Pelah D (1998) Separation of cis elements responsive to ethylene, fruit development, and ripening in the 5′-flanking region of the ripening-related E8 gene. Plant Mol Biol 37:1001–1011CrossRefPubMedGoogle Scholar
  13. Forde BG, Lea PJ (2007) Glutamate in plants: metabolism, regulation, and signaling. J Exp Bot 58:2339–2358. doi:10.1093/jxb/erm121 CrossRefPubMedGoogle Scholar
  14. Fujisawa M, Nakano T, Ito Y (2011) Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation. BMC Plant Biol 11:26CrossRefPubMedPubMedCentralGoogle Scholar
  15. Fujisawa M, Nakano T, Shima Y, Ito Y (2013) A large-scale identification of direct targets of the tomato MADS Box transcription factor RIPENING INHIBITOR reveals the regulation of fruit ripening. Plant Cell 25:371–386. doi:10.1105/tpc.112.108118 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Fujisawa M, Shima Y, Nakagawa H, Kitagawa M, Kimbara J, Nakano T, Kasumi T, Ito Y (2014) Transcriptional regulation of fruit ripening by tomato FRUITFULL homologs and associated MADS box proteins. Plant Cell 26:89–101. doi:10.1105/tpc.113.119453 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Fukuwatari Y, Sato N, Kawamori R, Watanabe Y, Yoshida K, Ying R, Matsuda K, Fujii A, Uzawa M, Sato R (2001) A study on the antihypertensive action and safety of tablets containing γ-aminobutyric acid (GABA). Eastern Med (in Japanese) 17:1–7Google Scholar
  18. Gut H, Dominici P, Pilati S, Astegno A, Petoukhov MV, Svergun DI, Grütter MG, Capitani G (2009) A common structural basis for pH- and calmodulin-mediated regulation in plant glutamate decarboxylase. J Mol Biol 392:334–351. doi:10.1016/j.jmb.2009.06.080 CrossRefPubMedGoogle Scholar
  19. Hirai T, Kim YW, Kato K, Hiwasa-Tanase K, Ezura H (2011a) Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter. Transgenic Res 20:1285–1292. doi:10.1007/s11248-011-9495-9 CrossRefPubMedGoogle Scholar
  20. Hirai T, Kurokawa N, Duhita N, Hiwasa-Tanase K, Kato K, Kato K, Ezura H (2011b) The HSP terminator of Arabidopsis thaliana induces a high level of miraculin accumulation in transgenic tomatoes. J Agric Food Chem 59:9942–9949. doi:10.1021/jf202501e CrossRefPubMedGoogle Scholar
  21. Inoue K, Shirai T, Ochiai H, Kasao M, Hayakawa K, Kimura M, Sansawa H (2003) Blood-pressure-lowering effect of a novel fermented milk containing gamma-aminobutyric acid (GABA) in mild hypertensives. Eur J Clin Nutr 57:490–495CrossRefPubMedGoogle Scholar
  22. Itkin M, Seybold H, Breitel D, Rogachev I, Meir S, Aharoni A (2009) TOMATO AGAMOUS-LIKE 1 is a component of the fruit ripening regulatory network. Plant J 60:1081–1095. doi:10.1111/j.1365-313X.2009.04064 CrossRefPubMedGoogle Scholar
  23. Jakoby WD (1962) Enzyme of γ-aminobutyrate metabolism (bacterial). Methods Enzymol 5:765–778CrossRefGoogle Scholar
  24. Kazami D, Ogura N, Fukuchi T, Tsuji K, Anazawa M, Maeda H (2002) Antihypertensive effect of Japanese taste seasoning containing γ-amino butyric acid on mildly hypertensive and high-normal blood pressure subjects and normal subjects. Nippon Shokuhin Kagaku Kogaku Kaishi (in Japanese) 49:409–415CrossRefGoogle Scholar
  25. Kitagawa M, Ito H, Shiina T, Nakamura N, Inakuma T, Kasumi T, Ishiguro Y, Yabe K, Ito Y (2005) Characterization of tomato fruit ripening and analysis of gene expression in F1 hybrids of the ripening inhibitor (rin) mutant. Physiol Plant 123:331–338. doi:10.1111/j.1399-3054.2005.00460.x CrossRefGoogle Scholar
  26. Klee HJ, Hayford MB, Kretzmer KA, Barry GF, Kishore GM (1991) Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants. Plant Cell 3:1187–1193. doi:10.1105/tpc.3.11.1187 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Koike S, Matsukura C, Takayama M, Asamizu E, Ezura H (2013) Suppression of γ-aminobutyric acid (GABA) transaminases induces prominent GABA accumulation, dwarfism and infertility in the tomato (Solanum lycopersicum L.). Plant Cell Physiol 54:793–807. doi:10.1093/pcp/pct035 CrossRefPubMedGoogle Scholar
  28. Kurokawa N, Hirai T, Takayama M, Hiwasa-Tanase K, Ezura H (2013) An E8 promoter-HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin. Plant Cell Rep 32:529–536. doi:10.1007/s00299-013-1384-7 CrossRefPubMedGoogle Scholar
  29. Lee JM, Joung JG, McQuinn R, Chung MY, Fei Z, Tieman D, Klee H, Giovannoni J (2012) Combined transcriptone, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation. Plant J 70:191–204. doi:10.1111/j.1365-313X.2011.04863.x CrossRefPubMedGoogle Scholar
  30. Liu L, Shao Z, Zhang M, Wang Q (2015) Regulation of carotenoid metabolism in tomato. Mol Plant 8:28–39. doi:10.1016/j.molp.2014.11.006 CrossRefPubMedGoogle Scholar
  31. Matsumoto Y, Ohno K, Hiraoka Y (1997) Studies on the utilization of functional food materials containing high levels of gamma-aminobutyric acid (Part 1). Ehime Kougi Kenkyu Houkoku (in Japanese) 35:97–100Google Scholar
  32. Mubarok S, Okabe Y, Fukuda N, Ariizumi T, Ezura H (2015) Potential use of a weak ethylene receptor mutant, Sletr1-2, as breeding material to extend fruit shelf life of tomato. J Agric Food Chem 63:7995–8007. doi:10.1021/acs.jafc.5b02742 CrossRefPubMedGoogle Scholar
  33. Nagaya S, Kawamura K, Shinmyo A, Kato K (2010) The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells. Plant Cell Physiol 51:328–332. doi:10.1093/pcp/pcp188 CrossRefPubMedGoogle Scholar
  34. Nambeesan S, Datsenka T, Ferruzzi MG, Malladi A, Mattoo AK, Handa AK (2010) Overexpression of yeast spermidine synthase impacts ripening, senescence and decay symptoms in tomato. Plant J 63:836–847. doi:10.1111/j.1365-313X.2010.04286.x CrossRefPubMedGoogle Scholar
  35. Oeller PW, Min-Wong L, Taylor LP, Pike DA, Theologis A (1991) Reversible inhibition of tomato fruit senescence by antisense RNA. Science 254:437–439CrossRefPubMedGoogle Scholar
  36. Okabe Y, Asamizu E, Saito T, Matsukura C, Ariizumi T, Brès C, Rothan C, Mizoguchi T, Ezura H (2011) Tomato TILLING technology: development of a reverse genetics tool for the efficient isolation of mutants from Micro-Tom mutant libraries. Plant Cell Physiol 52:1994–2005. doi:10.1093/pcp/pcr134 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Okada T, Sugishita T, Murakami T, Murai H, Saikusa T, Horino T, Onoda A, Kajimoto O, Takahashi R, Takahashi T (2000) Effect of the defatted rice germ enriched with GABA for sleeplessness, depression, autonomic disorder by oral administration. Nippon Shokuhin Kagaku Kogaku Kaishi (in Japanese) 47:596–603CrossRefGoogle Scholar
  38. Owens DF, Kriegstein AR (2002) Is there more to GABA than synaptic inhibition? Nat Rev Neurosci 3:715–727. doi:10.1038/nrn919 CrossRefPubMedGoogle Scholar
  39. Park KB, Oh SH (2007) Production of yogurt with enhanced levels of gamma-aminobutyric acid and valuable nutrients using lactic acid bacteria and germinated soybean extract. Bioresour Technol 98:1675–1679. doi:10.1016/j.biortech.2006.06.006 CrossRefPubMedGoogle Scholar
  40. Pecker I, Gabbay R, Cunningham FX, Hirschberg J (1996) Cloning and characterization of the cDNA for β-cyclase from tomato reveals decrease in its expression during fruit ripening. Plant Mol Biol 30:807–819. doi:10.1007/BF00019013 CrossRefPubMedGoogle Scholar
  41. Ronen G, Carmel-Goren L, Zamir D, Hirschberg J (2000) An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato. Proc Natl Acad Sci USA 97:11102–11107. doi:10.1073/pnas.190177497 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Saito T, Matsukura C, Sugiyama M, Watahiki A, Ohshima I, Iijima Y, Konishi C, Fujii T, Inai S, Fukuda N, Nishimura S, Ezura H (2008) Screening for γ-aminobutyric acid (GABA)-rich tomato varieties. J Japan Soc Hort Sci 77:242–250CrossRefGoogle Scholar
  43. Saito T, Ariizumi T, Okabe Y, Asamizu E, Hiwasa-Tanase K, Fukuda N, Mizoguchi T, Yamazaki Y, Aoki K, Ezura H (2011) TOMATOMA: a novel tomato mutant database distributing Micro-Tom mutant collections. Plant Cell Physiol 52:283–296. doi:10.1093/pcp/pcr004 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Schauer N, Zamir D, Fernie A (2005) Metabolic profiling of leaves and fruit of wild species tomato: a survey of the Solanum lycopersicum complex. J Exp Bot 56:297–307. doi:10.1093/jxb/eri057 CrossRefPubMedGoogle Scholar
  45. Shelp BJ, Bown AW, McLean MD (1999) Metabolism and functions of gamma-aminobutyric acid. Trends Plant Sci 4:446–452. doi:10.1016/s1360-1385(99)01486-7 CrossRefPubMedGoogle Scholar
  46. Shima Y, Kitagawa M, Fujisawa M, Nakano T, Kato H, Kimbara J, Kasumi T, Ito Y (2013) Tomato FRUITFULL homologues act in fruit ripening via forming MADS-box transcription factor complexes with RIN. Plant Mol Biol 82:427–438. doi:10.1007/s11103-013-0071-y CrossRefPubMedGoogle Scholar
  47. Snedden WA, Arazi T, Fromm H, Shelp BJ (1995) Calcium/calmodulin activation of soybean glutamate decarboxylase. Plant Physiol 108:543–549. doi:10.1104/pp.108.2.543 CrossRefPubMedPubMedCentralGoogle Scholar
  48. 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 271:4148–4153CrossRefPubMedGoogle Scholar
  49. Sorrequieta A, Abriata LA, Boggio SB, Valle EM (2013) Off-the-vine ripening of tomato fruit causes alteration in the primary metabolite composition. Metabolites 3:967–978. doi:10.3390/metabo3040967
  50. Sun HJ, Uchii S, Watanabe S, Ezura H (2006) A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol 47:426–431. doi:10.1093/pcp/pci251 CrossRefPubMedGoogle Scholar
  51. Takayama M, Koike S, Kusano M, Matsukura C, Saito K, Ariizumi T, Ezura H (2015) Tomato glutamate decarboxylase genes SlGAD2 and SlGAD3 play key roles in regulating γ-aminobutyric acid levels in tomato (Solanum lycopersicum). Plant Cell Physiol 56:1533–1545. doi:10.1093/pcp/pcv075 CrossRefPubMedGoogle Scholar
  52. Trobacher CP, Zarei A, Liu J, Clark SM, Bozzo GG, Shelp BJ (2013) Calmodulin-dependent and calmodulin-independent glutamate decarboxylases in apple fruit. BMC Plant Biol 13:144. doi:10.1186/1471-2229-13-144 CrossRefPubMedPubMedCentralGoogle Scholar
  53. Tsushida T, Murai T, Omori M, Okamoto J (1987) Production of a new type tea containing a high level of γ-aminobutyric acid. Nippon Nogeikagaku Kaishi (in Japanese) 7:817–822CrossRefGoogle Scholar
  54. Van de Poel B, Bulens I, Markoula A, Hertog ML, Dreesen R, Wirtz M, Vandoninck S, Oppermann Y, Keulemans J, Hell R, Waelkens E, De Proft MP, Sauter M, Nicolai BM, Geeraerd AH (2012) Targeted systems biology profiling of tomato fruit reveals coordination of the Yang cycle and a distinct regulation of ethylene biosynthesis during postclimacteric ripening. Plant Physiol 160:1498–1514. doi:10.1104/pp.112.206086 CrossRefPubMedPubMedCentralGoogle Scholar
  55. Wilkinson JQ, Lanahan MB, Clark DG, Bleecker AB, Chang C, Meyerowitz EM, Klee HJ (1997) A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants. Nat Biotechnol 15:444–447CrossRefPubMedGoogle Scholar
  56. Yap KL, Yuan T, Mal TK, Vogel HJ, Ikura M (2003) Structural basis for simultaneous binding of two carboxyl-terminal peptides of plant glutamate decarboxylase to calmodulin. J Mol Biol 328:193–204. doi:10.1016/s0022-2836(03)00271-7 CrossRefPubMedGoogle Scholar
  57. Yin YG, Tominaga T, Iijima Y, Aoki K, Shibata D, Ashihara H, Nishimura S, Ezura H, Matsukura C (2010) Metabolic alterations in organic acids and γ-aminobutyric acid in developing tomato (Solanum lycopersicum L.) fruits. Plant Cell Physiol 51:1300–1314. doi:10.1093/pcp/pcq090 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Faculty of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan

Personalised recommendations