Abstract
γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that has hypotensive effects. Tomato (Solanum lycopersicum L.) is among the most widely cultivated and consumed vegetables in the world and contains higher levels of GABA than other major crops. Increasing these levels can further enhance the blood pressure-lowering function of tomato fruit. Glutamate decarboxylase (GAD) is a key enzyme in GABA biosynthesis; it has a C-terminal autoinhibitory domain that regulates enzymatic function, and deleting this domain increases GAD activity. The tomato genome has five GAD genes (SlGAD1–5), of which two (SlGAD2 and SlGAD3) are expressed during tomato fruit development. To increase GABA content in tomato, we deleted the autoinhibitory domain of SlGAD2 and SlGAD3 using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 technology. Introducing a stop codon immediately before the autoinhibitory domain increased GABA accumulation by 7 to 15 fold while having variable effects on plant and fruit size and yield. This is the first study describing the application of the CRISPR/Cas9 system to increase GABA content in tomato fruits. Our findings provide a basis for the improvement of other types of crop by CRISPR/Cas9-based genetic modification.
Highlights
Satoko Nonaka[1,2], Chikako Arai[3], Mariko Takayama[1], Chiaki Matsukura1,2 & Hiroshi Ezura[1,2] γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that has hypotensive effects
Loss of function analyses revealed that the expression of SlGAD2 and/or SlGAD3, and SlGABA-T1 is important for GABA biosynthesis and catabolism, respectively[14, 18]
We predicted that inducing the stop codon at 30 and 48 amino acids for SlGAD2 and 37 amino acids for SlGAD3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)[9] would lead to increased GABA accumulation in tomato fruits
Summary
Satoko Nonaka[1,2], Chikako Arai[3], Mariko Takayama[1], Chiaki Matsukura1,2 & Hiroshi Ezura[1,2] γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that has hypotensive effects. Tomato (Solanum lycopersicum L.) is among the most widely cultivated and consumed vegetables in the world and contains higher levels of GABA than other major crops Increasing these levels can further enhance the blood pressure-lowering function of tomato fruit. It is noteworthy that the fruit specific overexpression of a mutant SlGAD3, in which 87 C-terminal nucleotides (corresponding to 29 amino acids) were deleted, was even more effective in increasing the GABA level (11- to 18-folds that of WT red-stage tomato)[19]. This was likely the consequence of an increased GAD activity because plant GADs generally have an autoinhibitory domain at the C-terminus. As enhanced GABA productivity by C-terminal truncation of GAD has been reported in several plant species[21,22,23], this trait would be widely applicable for improving GABA accumulation in plant cells
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