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| Truncation of the Calmodulin Binding Domain in Rice Glutamate Decarboxylase 4 (OsGAD4) Leads to Accumulation of γ-Aminobutyric Acid and Confers Abiotic Stress Tolerance in Rice Seedlings |
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An in vitro Ca2+/CaM assay showed that the C-terminal region of putative GAD4 has the ability to bind to Ca2+/CaM. CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD from the OsGAD4 gene. Rice calli infected with Agrobacterium carrying an all-in-one vector harboring guide RNAs and CRISPR/Cas9 was used to regenerate rice plants. We produced 27 independent transgenic lines (T0) through genome editing. |
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Nadia Akter, Ummey Kulsum, Nobuto Yasuda, Kazuhito Akama Research Square 2023 August 23, https://doi.org/10.21203/rs.3.rs-3267727/v1 BackgroundGamma-aminobutyric acid (GABA) is a non-protein amino acid known as a major inhibitory neurotransmitter. It is mainly synthesized from glutamate via the enzyme glutamate decarboxylase (GAD). GAD is present in all organisms, but only plant GAD has been shown to bind Ca2+/calmodulin (CaM). This binding suppresses the auto-inhibition of Ca2+/CaM binding domain (CaMBD) when the active site of GAD is unfolded, resulting in stimulated GAD activity. OsGAD4 is one of the five GAD genes in the rice genome. Conserved motifs in the C-terminal amino acid sequence suggest the potential ability of this GAD isoform to bind to Ca2+/CaM. Our aim was to produce GABA-fortified rice plants by genome editing of the OsGAD4 gene and comprehensive study of the truncated mutants. ResultsAn in vitro Ca2+/CaM assay showed that the C-terminal region of putative GAD4 has the ability to bind to Ca2+/CaM. CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD from the OsGAD4 gene. Rice calli infected with Agrobacterium carrying an all-in-one vector harboring guide RNAs and CRISPR/Cas9 was used to regenerate rice plants. We produced 27 independent transgenic lines (T0) through genome editing. DNA sequence analysis revealed the truncation of CaMBD (216 bp). Genome-edited line (#14 − 1) produced 12.9 mg GABA/100 g grain, which is almost 9-fold higher than the wild-type. Abiotic stresses such as salinity, flooding, and drought significantly enhanced GABA accumulation in #14 − 1 plants at various time points compared with wild-type controls in the same stress conditions. Moreover, upregulated mRNA expression in vegetative tissues seem to be correlated with the stress responsiveness of OsGAD4 when exposed to the above-mentioned stresses. The stress tolerance of OsGAD4 genome-edited plants was demonstrated by the higher survival rate, suggesting induced tolerance against abiotic stresses in rice. ConclusionsTrimming of the intact Ca2+/CaMBD of the C-terminal region of OsGAD4 induced extreme GABA accumulation in plant tissues. This elevated level of GABA was found to be linked to abiotic stress tolerance in rice plants at the vegetative stage.
See https://www.researchsquare.com/article/rs-3267727/v1
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(98).png "Truncation of the Calmodulin Binding Domain in Rice Glutamate Decarboxylase 4 (OsGAD4) Leads to Accumulation of γ-Aminobutyric Acid and Confers Abiotic Stress Tolerance in Rice Seedlings")
