Dual mechanisms regulating glutamate decarboxylases and accumulation of gamma-aminobutyric acid in tea (Camellia sinensis) leaves exposed to multiple stresses

Abstract

γ-Aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in the central nervous system. It has multiple positive effects on mammalian physiology and is an important bioactive component of tea (Camellia sinensis). GABA generally occurs at a very low level in plants but GABA content increases substantially after exposure to a range of stresses, especially oxygen-deficiency. During processing of tea leaves, a combination of anoxic stress and mechanical damage are essential for the high accumulation of GABA. This is believed to be initiated by a change in glutamate decarboxylase activity, but the underlying mechanisms are unclear. In the present study we characterized factors regulating the expression and activity of three tea glutamate decarboxylase genes (CsGAD1, 2, and 3), and their encoded enzymes. The results suggests that, unlike the model plant Arabidopsis thaliana, there are dual mechanisms regulating the accumulation of GABA in tea leaves exposed to multiple stresses, including activation of CsGAD1 enzymatic activity by calmodulin upon the onset of the stress and accumulation of high levels of CsGAD2 mRNA induced by a combination of anoxic stress and mechanical damage.

Figure 1

Contents of GABA (A) and Glu (B) of picked, mechanically damaged, tea (C. sinensis cv. Jinxuan) leaves under aerobic and anoxic treatments. GABA, gamma-aminobutyric acid. Glu, glutamate. Data are expressed as mean ± S.D. (n = 3). Different means with different letters are significantly different from each other (p ≤ 0.05).

Figure 2. Phylogenetic analysis of CsGADs and related functionally characterized GADs.

GAD, glutamate decarboxylase. Sequences were aligned using the ClustalX program. The neighbor-joining phylogenetic tree was generated by MEGA 5 software. The proteins included in the tree are represented by GenBank accession number.

Figure 3

Analysis of recombinant CsGADs enzyme activities in E. coli (A) and overexpressed in N. benthamiana (B). (A) One unit of enzyme activity was defined as the amount (μmol) of GABA produced by the action of one mg protein per min. The white columns indicate the activities of CsGADs recombined in E. coli. The grey columns indicate the activities of the CsGADs in the presence of CaM. *shows the significant difference (p < 0.05) of CsGAD1 activity in the absence and presence of CaM. N.D. indicates that the CsGAD3 had no activity converting glutamate to GABA. (B) Vector, empty vector group as control. CsGAD1, CsGAD2, CsGAD3: overexpression of CsGAD1, CsGAD2, and CsGAD3 respectively. The GABA content in the Vector group was defined as 1. Data represent the mean value ± standard deviation of three independent experiments performed in triplicate. Different means with different letters are significantly different from each other (p ≤ 0.05). GAD, glutamate decarboxylase.

Figure 4

Analysis of CaM binding ability of recombinant CsGADs expressed in E. coli (A) C-terminal truncated regions of the CsGADs (B) enzyme activities of CsGADΔCs (C), and comparison of properties of amino acids in the CaM binding domains of GADs (D). GAD, glutamate decarboxylase. GADΔC, C-terminal truncated glutamate decarboxylase. (A) SDS-PAGE of proteins retained by CaM-affinity chromatography. Only CsGAD1 was visible following PAGE, suggesting that only CsGAD1 had CaM-binding ability. (B) The clusters of Trp (W) and Lys (K) present in the C-proximal regions of GADs, which are important for in vitro binding of CaM, are indicated by asterisks and a thick line, respectively. The positions of two pseudosubstrate residues are highlighted in red. Based on comparison with the PhGAD (L16977), AtGAD1 (At5g17330), OsGAD1 (AB056060), OsGAD2 (AB056061), MdGAD1 (KC812242), MdGAD2 (KC812243), and MdGAD3 (KC812244), these amino acids of CsGADs were truncated to express CsGADΔCs in E. coli. (C) One unit was defined as the amount (μmol) of GABA produced by the action of one mg protein in min. The white columns indicate the activities of recombinant CsGADΔCs in E. coli. The grey columns indicate the activities of the CsGADΔCs in the presence of CaM. (D) The α-helix was presented using the EMBOSS-Lite-Protein Analysis Tools. The Trp (W) -centered (blue arrows) hydrophobic residues are clustered on one side, with the hydrophilic region on the other side. The Trp (W) residue and the Lys (K) (black arrows) cluster, contribute to hydrophobic and electrostatic interactions and are critical for efficient binding of CaM to the PhGAD-CaM binding domain. The Glu (E) (red arrows) residues function as pseudosubstrates.

Figure 5. Effect of anoxic treatment on the transcript levels of the genes involved in formation and metabolism of GABA in the picked tea leaves.

GABA-T, GABA transaminase. GAD, glutamate decarboxylase. GDH, glutamate dehydrogenase. SSADH, succinic semialdehyde dehydrogenase. SSR, succinic semialdehyde reductase. The tea leaves were from C. sinensis cv. Jinxuan. Data are expressed as mean ± S.E. (n = 3). Transcript abundance was calculated based on the difference in cycle threshold (Ct) values between the target gene and beta-actin transcripts normalized by the 2^−△△Ct relative quantification method. The mRNA levels of the genes in the picked tea leaves at 0 h were defined as 1. Different means with different letters are significantly different from each other (p ≤ 0.05).

Figure 6. Hypothetical model of GABA formation in intact and picked tea leaves exposed to anoxic stress.

GABA, gamma-aminobutyric acid. GABA-T, GABA transaminase. GAD, glutamate decarboxylase. GDH, glutamate dehydrogenase. SSADH, succinic semialdehyde dehydrogenase. SSR, succinic semialdehyde reductase.