Activating glutamate decarboxylase activity by removing the autoinhibitory domain leads to hyper γ-aminobutyric acid (GABA) accumulation in tomato fruit
- 681 Downloads
The C-terminal extension region of SlGAD3 is likely involved in autoinhibition, and removing this domain increases GABA levels in tomato fruits.
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 (SlGAD3 OX ) or without (SlGAD3ΔC OX ) 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 SlGAD3 OX fruits were equivalent to those in 35S-SlGAD3-NOS fruits, GABA levels in SlGAD3ΔC OX 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, SlGAD3ΔC OX 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.
KeywordsE8 promoter Fruit ripening GABA HSP terminator Tomato
We thank all of our laboratory members for their helpful discussions. This study was supported by the “Japan-France Joint Laboratory Project”, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and the “Research and Development Program for New Bio-industry Initiative”, Bio-oriented Technology Research Advancement Institution (BRAIN). ‘Micro-Tom’ tomato seeds (Accession No. TOMJPF00001) were obtained from the Gene Research Center, University of Tsukuba, through the National Bioresource Project (NBRP) of MEXT, Japan.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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