Abstract
GABA (Gamma-aminobutyric acid) is a non-protein amino acid widely known as major inhibitory neurotransmitter. It is synthesized from glutamate via the enzyme glutamate decarboxylase (GAD). GAD is ubiquitous in all organisms, but only plant GAD has ability to bind Ca2+/calmodulin (CaM). This kind of binding suppresses the auto-inhibition of Ca2+/calmodulin 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 rice genome. It was confirmed that OsGAD4 has ability to bind to Ca2+/CaM. Moreover, it exhibits strongest expression against several stress conditions among the five OsGAD genes. In this study, CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD from the OsGAD4 gene, to remove its autoinhibitory function. DNA sequence analysis of the genome edited rice plants revealed the truncation of CaMBD (216 bp). Genome edited line (#14–1) produced 11.26 mg GABA/100 g grain, which is almost nine-fold in comparison to wild type. Short deletion in the coding region for CaMBD yielded in mutant (#14–6) with lower GABA content than wild type counterpart. Abiotic stresses like salinity, flooding and drought significantly enhanced GABA accumulation in #14–1 at various time points compared to wild-type and #14–6 under the same stress conditions. Moreover, upregulated mRNA expression in vegetative tissues seems correlated with the stress-responsiveness of OsGAD4 when exposed to the above-mentioned stresses. Stress tolerance of OsGAD4 genome edited lines was evidenced by the higher survival rate indicating the gene may induce tolerance against abiotic stresses in rice. This is the first report on abiotic stress tolerance in rice modulated by endogenous GABA.
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Data availability
The vectors used in this study will be available upon request.
Abbreviations
- bp:
-
Base pairs
- CaM:
-
Calmodulin
- CaMBD:
-
Calmodulin binding domain
- CRISPR/Cas:
-
Clustered regularly interspaced palindromic repeats/CRISPR-associated protein
- GABA:
-
γ-Aminobutyric acid
- GAD:
-
Glutamate decarboxylase
- gRNA:
-
Guide RNA
- Ni:
-
Nipponbare
- PAM:
-
Protospacer adjacent motif
- RT-qPCR:
-
Reverse transcription quantitative polymerase chain reaction
- TBP2 :
-
TATA-binding protein 2
- UTR:
-
Untranslated region
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Acknowledgements
Our sincere gratitude goes to Dr. Masaki Endo (NARO, Tsukuba, Japan) and Dr. Seiichi Toki (Ryukoku University, Kyoto, Japan) for providing the plasmids (pU6gRNA and pZH_gYSA_MMCas9).
Funding
This work was supported by JSPS KAKENHI (Grant No. 23K05166), Cabinet Office, Government of Japan, Moonshot Research and Development Program for Agriculture, Forestry and Fisheries (funding agency: Bio-oriented Technology Research Advancement Institution) grant no. JPJ009237, JST A-STEP TRYOUT (Grant No. JPMJTM20FU), and the Shimane Industrial Promotion Foundation.
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KA conceptualized the study and designed the experiments. Experimentation, data collection, and analysis were performed by NA. UK, MM and NY performed additional experiments. The draft of the manuscript was written by NA. All the authors read and approved the manuscript.
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Akter, N., Kulsum, U., Moniruzzaman, M. et al. 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. Mol Breeding 44, 21 (2024). https://doi.org/10.1007/s11032-024-01460-1
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DOI: https://doi.org/10.1007/s11032-024-01460-1