Identification of two CiGADs from Caragana intermedia and their transcriptional responses to abiotic stresses and exogenous abscisic acid

Abstract

BackgroundGlutamate decarboxylase (GAD), as a key enzyme in the γ -aminobutyric acid (GABA) shunt, catalyzes the decarboxylation of L-glutamate to form GABA. This pathway has attracted much interest because of its roles in carbon and nitrogen metabolism, stress responses, and signaling in higher plants. The aim of this study was to isolate and characterize genes encoding GADs from Caragana intermedia, an important nitrogen-fixing leguminous shrub.MethodsTwo full-length cDNAs encoding GADs (designated as CiGAD1 and CiGAD2) were isolated and characterized. Multiple alignment and phylogenetic analyses were conducted to evaluate their structures and identities to each other and to homologs in other plants. Tissue expression analyses were conducted to evaluate their transcriptional responses to stress (NaCl, ZnSO4, CdCl2, high/low temperature, and dehydration) and exogenous abscisic acid.ResultsThe CiGADs contained the conserved PLP domain and calmodulin (CaM)-binding domain in the C-terminal region. The phylogenetic analysis showed that they were more closely related to the GADs of soybean, another legume, than to GADs of other model plants. According to Southern blotting analysis, CiGAD1 had one copy and CiGAD2-related genes were present as two copies in C. intermedia. In the tissue expression analyses, there were much higher transcript levels of CiGAD2 than CiGAD1 in bark, suggesting that CiGAD2 might play a role in secondary growth of woody plants. Several stress treatments (NaCl, ZnSO4, CdCl2, high/low temperature, and dehydration) significantly increased the transcript levels of both CiGADs, except for CiGAD2 under Cd stress. The CiGAD1 transcript levels strongly increased in response to Zn stress (74.3-fold increase in roots) and heat stress (218.1-fold increase in leaves). The transcript levels of both CiGADs significantly increased as GABA accumulated during a 24-h salt treatment. Abscisic acid was involved in regulating the expression of these two CiGADs under salt stress.DiscussionThis study showed that two CiGADs cloned from C. intermedia are closely related to homologs in another legume, soybean. CiGAD2 expression was much higher than that of CiGAD1 in bark, indicating that CiGAD2 might participate in the process of secondary growth in woody plants. Multiple stresses, interestingly, showed that Zn and heat stresses had the strongest effects on CiGAD1 expression, suggesting that CiGAD1 plays important roles in the responses to Zn and heat stresses. Additionally, these two genes might be involved in ABA dependent pathway during stress. This result provides important information about the role of GADs in woody plants’ responses to environmental stresses.

Gamma-aminobutyric acid (GABA) alleviates salt damage in tomato by modulating Na+ uptake, the GAD gene, amino acid synthesis and reactive oxygen species metabolism

“…However, there were significant differences in expression sites in different plants under NaCl. CiGAD1 was expressed in the stem, leaf and seed coat of Caragana intermedia, while CiGAD2 was highly expressed in the bark [17]. The increase in expression of CiGAD1 and CiGAD2 induced GABA accumulation within 24 h of salt treatment [17].…”

Identification of the WRKY gene family and functional analysis of two genes in Caragana intermedia

“…The expression patterns of miR2118 and its target genes ( CiDR1 and CiDR2 ) from C. intermedia are altered under drought stress, and constitutive overexpression of cin-miR2118 in tobacco enhances the plant’s tolerance to drought stress [ 46 ]. Abiotic stresses (NaCl, ZnSO 4 , CdCl 2 , high/low temperature, and dehydration) induce the expression of the glutamate decarboxylase-encoding genes CiGAD1 and CiGAD2 , except for CiGAD2 under Cd stress, and ABA has been shown to be involved in regulating the expression of CiGADs in response to salt stress [ 52 ]. However, there is limited knowledge of the function of WRKYs in C. intermedia .…”

Gamma-aminobutyric acid (GABA) alleviates salt damage in tomato by modulating Na+ uptake, the GAD gene, amino acid synthesis and reactive oxygen species metabolism

“…Among these plants, the GABA content was enhanced approximately 20-fold in Arabidopsis seedlings under 150 mmol•L -1 NaCl [13], and GABA levels increased signi cantly in seedlings of lentils treated with 25-100 mmol•L -1 NaCl [15]. Furthermore, it has been demonstrated that the synthesis-related accumulation of endogenous GABA is closely related to exogenous GABA supplementation, which increased by 29% in hulless barley and by 1-fold in Caragana treated with 0.5 mmol•L -1 and 10 mmol•L -1 GABA, respectively, under salt stress [16][17]. Therefore, it is believed that endogenous GABA, which is affected by salt stimulation and exogenous GABA induction, plays a vital role in improving plant resistance to salt stress via regulatory metabolic pathways [18][19][20].…”

“…However, there were signi cant differences in expression sites in different plants under NaCl. CiGAD1 was expressed in the stem, leaf and seed coat of Caragana intermedia, while CiGAD2 was highly expressed in the bark [17]. The increase in expression of CiGAD1 and CiGAD2 induced GABA accumulation within 24 h of salt treatment [17].…”

“…CiGAD1 was expressed in the stem, leaf and seed coat of Caragana intermedia, while CiGAD2 was highly expressed in the bark [17]. The increase in expression of CiGAD1 and CiGAD2 induced GABA accumulation within 24 h of salt treatment [17]. GAD2 expression in all parts of Arabidopsis and tobacco was signi cantly enhanced, accompanied by increased GAD activity and increased GABA content [12,13].…”