An In Vivo Targeted Deletion of the Calmodulin-Binding Domain from Rice Glutamate Decarboxylase 3 (OsGAD3) Increases γ-Aminobutyric Acid Content in Grains

Abstract

Background: Gamma-aminobutyric acid (GABA) is a non-protein amino acid present in all living things. GABA is mainly synthesized from glutamate by glutamate decarboxylase (GAD). 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 plays a central role in not only stress responses but also many aspects of plant growth and development as a signaling molecules. 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. Previous reports indicated that CaMBD found plant GADs possess an autoinhibitory function because truncation of GAD resulted in extreme GABA accumulation in plant cells. Therefore, we attempted a genetic modification of rice GAD via genome editing technology to increase GABA levels in the edible part of rice.Results: 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. We confirmed that GAD3 has an authentic Ca 2+ / CaMBD that functions as an autoinhibitory domain. 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. 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). 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. 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. 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.(Continued on next page)

Genome editing in cereal crops: an overview

“…In contrast, the levels of asparagine and tryptophan fell to 30% of the WT level. The mutant also showed a higher seed weight (12%) and seed protein content (20%) than WT plants (Akama et al 2020).…”

“…This is another important target of metabolic engineering in rice because it plays a key role in plant stress responses, growth, and development, and as a nutritional component of the grain can also reduce the likelihood of hypertension and diabetes. GABAfortified rice was generated by using CRISPR/Cas9 to knock-out the GAD3 gene (Akama et al 2020). The analysis of free amino acids in the seed revealed a seven-fold increase in GABA levels as well as small increases in the levels of serine and glutamate, and twofold-fivefold increases in the levels of alanine, aspartate, methionine, phenylalanine, leucine and isoleucine.…”

Application of CRISPR/Cas9 in Crop Quality Improvement

“…The accumulation of GABA in mutant tomatoes increased seven-fold [ 77 ]. Furthermore, researchers have also created GABA-rich rice by truncating the C-terminal of the OsGAD3 through CRISPR/Cas9 system and the GABA content increased seven-fold [ 78 ]. Undoubtedly, GABA-rich crops have a beneficial effect on human health.…”

MicroRNA Techniques: Valuable Tools for Agronomic Trait Analyses and Breeding in Rice

“…Great progress has been made by using a transgene-free edited system on protein-coding genes. A series of transgene-free edited crops with increased yield ( Li et al, 2016 ), improved quality with low amylose content ( Zhang et al, 2018a ), high amylose content ( Sun et al, 2017 ; Tuncel et al, 2019 ; Li et al, 2021 ), higher grain fragrance ( Usman et al, 2020 ), enriched γ-aminobutyric acid ( Akama et al, 2020 ), and enhanced biotic stress resistance ( Zhou et al, 2020 ) have been generated. Currently, several studies are also trying to convert the accumulating knowledge of miRNA functions into a transgene-free edited system.…”