Activating glutamate decarboxylase activity by removing the autoinhibitory domain leads to hyper γ-aminobutyric acid (GABA) accumulation in tomato fruit
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- Takayama, M., Matsukura, C., Ariizumi, T. et al. Plant Cell Rep (2017) 36: 103. doi:10.1007/s00299-016-2061-4
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The C-terminal extension region ofSlGAD3is 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 (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.
KeywordsE8 promoter Fruit ripening GABA HSP terminator Tomato
|Funder Name||Grant Number||Funding Note|
|“Japan-France Joint Laboratory Project”, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan|
|“Research and Development Program for New Bio-industry Initiative”, Bio-oriented Technology Research Advancement Institution (BRAIN)|