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.

GABA（γ-氨基丁酸）是一种非蛋白质氨基酸，被广泛认为是主要的抑制性神经递质。它是通过谷氨酸脱羧酶（GAD）从谷氨酸合成的。GAD普遍存在于所有生物体中，但只有植物GAD具有结合Ca ²⁺ /钙调蛋白(CaM)的能力。当 GAD 活性位点展开时，这种结合会抑制 Ca ²⁺ /钙调蛋白结合域 (CaMBD) 的自动抑制，从而刺激 GAD 活性。OsGAD4是水稻基因组中的五个GAD基因之一。确认OsGAD4具有与Ca ²⁺ /CaM结合的能力。此外，它在五个OsGAD基因中针对几种应激条件表现出最强的表达。在这项研究中，通过CRISPR/Cas9介导的基因组编辑来修剪OsGAD4基因中CaMBD的编码区，以消除其自抑制功能。基因组编辑水稻植株的 DNA 序列分析揭示了 CaMBD (216 bp) 的截短。基因组编辑品系 (#14–1) 产生 11.26 毫克 GABA/100 克谷物，几乎是野生型的九倍。CaMBD 编码区的短缺失导致突变体 (#14-6) 的 GABA 含量低于野生型对应物。与相同胁迫条件下的野生型和 #14-6 相比，盐度、洪水和干旱等非生物胁迫显着增强了 #14-1 在不同时间点的 GABA 积累。此外，当暴露于上述应激时，营养组织中 mRNA 表达的上调似乎与OsGAD4的应激反应性相关。OsGAD4基因组编辑品系的胁迫耐受性通过较高的存活率得到证明，表明该基因可能诱导水稻对非生物胁迫的耐受性。这是关于内源 GABA 调节水稻非生物胁迫耐受性的第一份报告。