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

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.