Induction of γ-aminobutyric acid plays a positive role to Arabidopsis resistance against Pseudomonas syringae

Abstract

Gamma-aminobutyric acid (GABA) is an important metabolite which functions in plant growth, development, and stress responses. However, its role in plant defense and how it is regulated are largely unknown. Here, we report a detailed analysis of GABA induction during the resistance response to Pseudomonas syringae in Arabidopsis thaliana. While searching for the mechanism underlying the pathogen-responsive mitogen-activated protein kinase (MPK)3/MPK6 signaling cascade in plant immunity, we found that activation of MPK3/MPK6 greatly induced GABA biosynthesis, which is dependent on the glutamate decarboxylase genes GAD1 and GAD4. Inoculation with Pseudomonas syringae pv tomato DC3000 (Pst) and Pst-avrRpt2 expressing the avrRpt2 effector gene induced GAD1 and GAD4 gene expression and increased the levels of GABA. Genetic evidence revealed that GAD1, GAD2, and GAD4 play important roles in both GABA biosynthesis and plant resistance in response to Pst-avrRpt2 infection. The gad1/2/4 triple and gad1/2/4/5 quadruple mutants, in which the GABA levels were extremely low, were more susceptible to both Pst and Pst-avrRpt2. Functional loss of MPK3/MPK6, or their upstream MKK4/MKK5, or their downstream substrate WRKY33 suppressed the induction of GAD1 and GAD4 expression after Pst-avrRpt2 treatment. Our findings shed light on both the regulation and role of GABA in the plant immunity to a bacterial pathogen.

Figure 1

Activation of mitogen‐activated protein kinase (MPK)3/MPK6 induces the expression of glutamate decarboxylase (GAD)1 and GAD4 genes and leads to high level of gamma‐aminobutyric acid (GABA) accumulation (A–C) Fourteen‐d‐old seedlings grown in gas chromatography vials supplied with swimming medium were collected at the indicated times after addition of 5 μmol/L dexamethasone (DEX). After reverse transcription, levels of GAD1 (A), GAD2 (B), and GAD4 (C) transcripts were determined by real‐time quantitative polymerase chain reaction (PCR). Relative expression was analyzed by two‐way analysis of variance (ANOVA) (genotype × time point). Different lowercase letters above the groups indicate statistically different groups (P < 0.0001). Error bars indicate SD (n = 3). (D) Fourteen‐d‐old, soil‐grown seedlings were sprayed with 30 μmol/L DEX. Leaves were collected at the indicated times after application of DEX. Gamma‐aminobutyric acid accumulation was determined using a Hitachi Automatic Amino Acid Analyzer, L‐8900. Gamma‐aminobutyric acid concentration was analyzed by two‐way ANOVA (genotype × time point). Different lowercase letters above the groups indicate statistically different groups (P < 0.001). The numbers above each bar at time zero indicate the low initial GABA level (μg/g FW). Error bars indicate SD (n = 3), FW, fresh weight.

Figure 2

Gamma‐aminobutyric acid (GABA) induction after activation of mitogen‐activated protein kinase (MPK)3/MPK6 in glutamate decarboxylase (gad)1, gad2 and gad4 mutants (A) Fourteen‐d‐old DD, DD gad1, DD gad2, and DD gad4 seedlings were sprayed with 30 μmol/L dexamethasone (DEX). Samples were collected 24 h later for GABA analysis. One‐way analysis of variance (ANOVA) was performed to compare different genotypes with DD at the same time point (*P < 0.05; ****P < 0.0001). Error bars indicate SD (n = 3), FW, fresh weight. (B) Levels of the Flag‐tagged NtMEK2^DD protein in the different lines were detected by immunoblot analysis using an anti‐Flag antibody.

Figure 3

Gamma‐aminobutyric acid (GABA) level and glutamate decarboxylase (GAD) gene expression after Pst, Pst‐hrcC^‐, or Pst‐avrRpt2 inoculation in Arabidopsis (A) Fourteen‐d‐old seedlings of Col‐0 were sprayed with Pst, Pst‐hrcC ^‐, or Pst‐AvrRpt2 (final OD₆₀₀ = 0.4). The shoots of the inoculated seedlings were collected at the indicated times. Gamma‐aminobutyric acid levels were determined using a Hitachi Automatic Amino Acid Analyzer. FW, fresh weight. (B–D) Fourteen‐d‐old seedlings were collected at the indicated times after inoculation with Pst, Pst‐hrcC ^‐, or Pst‐AvrRpt2. After reverse transcription, the levels of GAD1 (B), GAD2 (C), and GAD4 (D) transcripts were determined by real‐time quantitative polymerase chain reaction (PCR). Gamma‐aminobutyric acid concentration and GAD relative expression were analyzed by two‐way analysis of variance (treatment × time point). Different lowercase letters above the groups indicate statistically different groups (P < 0.0001). Error bars indicate SD (n = 3).

Figure 4

Pst‐avrRpt2‐induced transcript accumulation of glutamate decarboxylase (GAD)1 and GAD4 were compromised in mpk3, mpk6, mkk4 mkk5, and wrky33 mutants (A, B) Fourteen‐d‐old seedlings were collected at indicated times after spraying with Pst‐avrRpt2 (OD₆₀₀ = 0.4). Gene expression was quantified by quantitative reverse transcription polymerase chain reaction (RT‐qPCR). One‐way analysis of variance (ANOVA) was performed when three genotypes were compared, and Student's t‐test was performed when two genotypes were compared. Asterisks above the columns indicate statistical difference compared to Col‐0 (**P < 0.01; ***P < 0.001; ****P < 0.0001). Error bars indicate SD (n = 3).

Figure 5

Glutamate decarboxylase (GAD)1, GAD2, and GAD4 genes are important for both γ‐aminobutyric acid (GABA) induction and resistance to Pst and Pst‐avrRpt2 (A, B) Fourteen‐d‐old seedlings of Col‐0, various gad mutants, and rps2 were sprayed with Pst‐AvrRpt2 (final OD₆₀₀ = 0.4). The shoots of the inoculated seedlings were collected at the indicated times. Gamma‐aminobutyric acid levels were determined using a Hitachi Automatic Amino Acid Analyzer. One‐way analysis of variance (ANOVA) (A) and Student's t‐test (B) were performed to compare different genotypes with the wild type at each time point. Asterisks above the columns indicate statistical difference (*P < 0.05; ***P < 0.001; ****P < 0.0001). Error bars indicate SD (n = 3). FW, fresh weight. (C) The leaves of 3‐week‐old plants were infiltrated with Pst and Pst‐avrRpt2 (OD₆₀₀ = 0.001). Bacteria levels were quantified 0 and 3 days post‐inoculation (dpi). Differences in bacterial growth between Pst and Pst‐avrRpt2 were analyzed by two‐way ANOVA, with different lowercase letters above the groups indicating statistically significant differences (P < 0.001). One‐way ANOVA was also performed to compare mutants with the wild type at the same time point (*P < 0.05; **P < 0.01). Error bars indicate SD (n = 3). CFU, colony‐forming units.

Figure 6

Over‐accumulation of γ‐aminobutyric acid (GABA) in plants leads to a dwarf and yellowish phenotype (A) Amino acid sequence alignment of the CaM‐binding domains of glutamate decarboxylase (GAD) proteins in Arabidopsis. Conserved acidic (red), basic (blue) and hydrophobic (green) residues are colored, key anchors are boxed, and potential pseudo‐substrate glutamate/aspartate residues are indicated with asterisks. Deleted residues in transgenic plants are shown with pink backgrounds. (B) Expression of GAD1ΔC, GAD2ΔC, and GAD4ΔC in T2 transgenic plants was detected by immunoblot analysis using anti‐Flag antibody. (C) Expression of GAD1ΔC, GAD2ΔC, and GAD4ΔC in T2 transgenic plants leads to dwarf and yellowish morphological phenotypes. Images were taken at 3 weeks. Bar = 1 cm. (D) Expression of GAD2ΔC in G (green) and Y (yellow) leaves of transgenic line #33 in the T3 generation was detected by immunoblot analysis using anti‐Flag antibody. The Coomassie‐stained gel is shown below as a loading control. The three pairs of G and Y leaves were from three different plants, respectively. Bar = 1 cm. (E) Gamma‐aminobutyric acid concentration in G (green) and Y (yellow) leaves of T3 transgenic line #33. Error bars indicate SD (n = 3). Gamma‐aminobutyric acid levels were analyzed by one‐way analysis of variance (ANOVA). Asterisks above the columns indicate statistical difference (***P < 0.001; ****P < 0.0001). Error bars indicate SD (n = 3).

Figure 7

Over‐accumulation of γ‐aminobutyric acid (GABA) is associated with decreased resistance to Pst‐avrRpt2 in glutamate decarboxylase (GAD)2ΔC transgenic plants (A) Three‐week‐old plant leaves were infiltrated with Pst‐avrRpt2 (OD₆₀₀ = 0.001). The bacteria were quantified 0 and 3 days post‐inoculation (dpi). Leaves used for bacterial counting in Col‐0 and GAD2ΔC #33 were from the same position. Student's t‐test was used to compare different genotypes with the same treatment at any certain time point (**P < 0.01). Error bars indicate SD (n = 3). CFU, colony‐forming units. (B–C) Pst‐avrRpt2 (final concentration OD₆₀₀ = 0.001) and different concentrations of GABA (0, 0.5, 2, or 10 mmol/L GABA) were added to plant culture medium (swimming medium, SW) (B) or Luria‐Bertani medium (C), respectively. Bacterial growth was measured at indicated time points and was analyzed by one‐way analysis of variance. Asterisks indicate statistical difference (**P < 0.01; ***P < 0.001; ****P < 0.0001). Error bars indicate SD (n = 3).