GABA is unique in plants, because various biotic and abiotic stresses rapidly induce GABA accumulation in cells (Kinnersley and Turano 2000). Glutamate decarboxylase (GAD) is responsible for the conversion of L-glutamate to GABA, which is the major pathway for the production of GABA in spite of an alternative polyamine pathway associated with GABA synthesis (Shelp et al. Although GABA is a non-protein amino acid, it is ubiquitously present both in prokaryotes and eukaryotes (Shelp et al. Today GABA is well known as a major inhibitory neurotransmitter in animals (Roberts and Eidelberg 1960). Gamma-aminobutyric acid (GABA) was first discovered in potato tuber tissue (Steward et al. This is the first report of the production of GABA-enriched rice via a genome editing. Modified rice contained seven-fold higher GABA content and furthermore displayed significantly higher grain weight and protein content than wild-type brown rice. We have successfully established GABA-fortified rice by using CRISPR/Cas9 genome editing technology. In addition to high level of GABA in #8_8, the average seed weight per grain and protein content were superior to wild-type and #8_1. Free amino acid analysis of the seeds (T 2) indicated that the former line contained seven-fold higher levels of GABA than wild-type, whereas the latter line had similar levels to the wild-type, although in vitro enzyme activities of recombinant GAD proteins based on the GAD3 amino acid sequence elucidated from these two lines in the absence of Ca 2+/bovine CaM were both higher than wild-type counterpart. A very similar rice line (#8_1) carrying AKNRSSRRSGR in OsGAD3 was obtained from one base pair deletion in the N-terminal coding region of the CaMBD. Out of 24 transformed rice (T 1), a genome-edited rice line (#8_8) derived from two independent cleavages and ligations in the N-terminal position encoding OsGAD3-CaMBD and 40 bp downstream of the termination codon, respectively, displayed a AKNQDAAD peptide in the C-terminal region of the putative OsGAD3 in place of its intact CaMBD (bold indicates the trace of the N-terminal dipeptides of the authentic CaMBD). CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD off from the OsGAD3 gene, then introducing this transgene into rice scutellum-derived calli using an all-in-one vector harboring guide RNAs and CRISPR/Cas9 via Agrobacterium to regenerate rice plants. We confirmed that GAD3 has an authentic Ca 2+/CaMBD that functions as an autoinhibitory domain. In this study, we focused on GAD3, one of five GAD genes present in the rice genome, because GAD3 is the predominantly expressed in seeds, as reported previously. Therefore, we attempted a genetic modification of rice GAD via genome editing technology to increase GABA levels in the edible part of rice. Previous reports indicated that CaMBD found plant GADs possess an autoinhibitory function because truncation of GAD resulted in extreme GABA accumulation in plant cells. Furthermore, it is known to be a health-promoting functional substance that exerts improvements in life-style related diseases such as hypertension, diabetes, hyperlipidemia, and so on. GABA plays a central role in not only stress responses but also many aspects of plant growth and development as a signaling molecules. In plants the enzymatic activity of GAD is activated by Ca 2+/calmodulin binding (CaMBD) at the C-terminus in response to various stresses, allowing rapid GABA accumulation in cells. ![]() GABA is mainly synthesized from glutamate by glutamate decarboxylase (GAD). Gamma-aminobutyric acid (GABA) is a non-protein amino acid present in all living things.
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