Glutamate receptor-like channel3.3 is involved in mediating glutathione-triggered cytosolic calcium transients, transcriptional changes, and innate immunity responses in Arabidopsis

Abstract

The tripeptide reduced glutathione (GSH; γ-glutamate [Glu]-cysteine [Cys]-glycine) is a major endogenous antioxidant in both animal and plant cells. It also functions as a neurotransmitter mediating communication among neurons in the central nervous system of animals through modulating specific ionotropic Glu receptors (GLRs) in the membrane. Little is known about such signaling roles in plant cells. Here, we report that transient rises in cytosolic calcium triggered by exogenous GSH in Arabidopsis (Arabidopsis thaliana) leaves were sensitive to GLR antagonists and abolished in loss-of-function atglr3.3 mutants. Like the GSH biosynthesis-defective mutant PHYTOALEXIN DEFICIENT2, atglr3.3 showed enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Pathogen-induced defense marker gene expression was also decreased in atglr3.3 mutants. Twenty-seven percent of genes that were rapidly responsive to GSH treatment of seedlings were defense genes, most of which were dependent on functional AtGLR3.3, while GSH suppressed pathogen propagation through the AtGLR3.3-dependent pathway. Eight previously identified putative AtGLR3.3 ligands, GSH, oxidized glutathione, alanine, asparagine, Cys, Glu, glycine, and serine, all elicited the AtGLR3.3-dependent cytosolic calcium transients, but only GSH and Cys induced the defense response, with the Glu-induced AtGLR3.3-dependent transcription response being much less apparent than that triggered by GSH. Together, these observations suggest that AtGLR3.3 is required for several signaling effects mediated by extracellular GSH, even though these effects may not be causally related.

Figure 1.

[GSH]_ext-induced concentration-dependent [Ca²⁺]_cyt rise in the leaf. A, Representative recording curve of 10, 100, and 1,000 µm GSH-induced [Ca²⁺]_cyt transient rise in a detached leaf. B, Averaged peak values with se of the responses (n = 5), which are significantly different to each other at P < 0.01. The dash line indicates the background [Ca²⁺]_cyt.

Figure 2.

Pharmacological study of the [GSH]_ext-induced [Ca²⁺]_cyt transient response in the leaf. A, Representative recording curve of 100 µm [GSH]_ext-induced [Ca²⁺]_cyt rise in the CK and pretreated with 1 mm various Ca²⁺ mobility pathway blockers as indicated. B, Averaged peak values of these responses with se (n = 4 for EGTA and n = 5 for the rest). The asterisk stands for the sd to the CK at P < 0.01. The average background [Ca²⁺]_cyt was around 0.08 µm.

Figure 3.

[GSH]_ext-induced [Ca²⁺]_cyt response was absent in the atglr3.3 mutant. Averaged recording curves with se of 100 µm [GSH]_ext-induced [Ca²⁺]_cyt rise in the leaf cells of the ecotype Columbia (Col-0) and atglr3.3-1 and atglr3.3-2 mutants expressing aequorin (n = 10 for each).

Figure 4.

Overview of the GSH- and Glu-mediated AtGLR3.3-dependent gene expression. A, Number of overlapping genes modulated by GSH and Glu in the ecotype Columbia (Col-0) and atglr3.3 mutant. B, Functional category of the genes modulated by GSH with AtGLR3.3 dependence. C, Functional category of the genes modulated by Glu with AtGLR3.3 dependence. The numbers in and the percentage symbol outside the pie chart indicate the number and percentage of the genes in the category, respectively. U, Unknown; TF, Transcription Factor; S, Signaling; O, Others; D, Defense.

Figure 5.

Role of AtGLR3.3 in the innate immunity and GSH-triggered defense response in the leaf. A, Leaves of ecotype Columbia (Col-0) and atglr3.3-1 and atglr3.3-2 mutants were infiltrated with either CK solution or the solution containing 100 µm GSH 1 d before the inoculation of Pst DC3000 at 5 × 10⁵ cfu mL^–1. The pathogen proliferation was monitored at 0, 1, and 2 DPI. Values presented are average with se from at least four leaves from different plants. At 1, 2, and 3 DPI, there is sd between the Col-0 and two mutants at P < 0.01. Two asterisks indicated significance at P < 0.01. Five independent experiments were conducted and showed comparable results. B, Leaf symptoms of the Col-0, atglr3.3-1, and atglr3.3-2 plants preinfiltrated with CK solution or the solution containing 100 µm GSH 3 DPI with Pst DC3000. C, Same leaves in B were kept 1 d longer, e.g. 4 DPI, in a sealed agar plate.

Figure 6.

Pst DC3000-induced defense market gene expression in ecotype Columbia (Col-0) and atglr3.3 mutants. Pst DC3000 at 5 × 10⁵ cfu mL^–1 was infiltrated into the leaves of Col-0, atglr3.3-1, and atglr3.3-2. The leaves were harvested at 0, 12, 24, and 48 h post infection (HPI) for PR1 (A) and WRKY33 (B) gene expression analysis with qRT-PCR. The transcript abundance was normalized with reference gene Tubulin2 in Col-0 at 0 h post infection. Values presented are average with se from three independent experiments. The asterisk indicated the sd between the mutants and Col-0 at same time point at P < 0.05.

Figure 7.

Pst DC3000 infection assay of ecotype Columbia (Col-0) and atglr3.3 with putative ligands and Asp. The experiment procedure and analysis methods are same as those in Figure 5. Pst DC3000 proliferation in these leaves was monitored at 0 (A) and 2 DPI (B) preinfiltrated with either CK solution or the solution containing 100 µm GSSG, Glu, Asp, Ala, Cys, and Asn. C, The same assay was conducted separately for Ser and Gly. At 2 DPI in B and C, except for Asp, the values of atglr3.3-1 and atglr3.3-2 were significantly higher than that of Col-0 at P < 0.01. The asterisk in B indicated there was sd between the treatment and the CK for the corresponding genotype at P < 0.01.

Figure 8.

Effects of the seven putative ligands of AtGLR3.3 and Asp on the [Ca²⁺]_cyt in the leaf cells of ecotype Columbia (Col-0) and atglr3.3 expressing aequorin. Averaged recording curves with se of 100 µm Glu (A) and Asp (B) induced [Ca²⁺]_cyt rise in the leaf cells of Col-0 and atglr3.3-1 and atglr3.3-2 mutants expressing aequorin (n = 8 for Glu and n = 5 for Asp). C, the average peak values with se of the response to the indicated ligands (n = 8 for GSSG and Glu, n = 5 for Asp, Cys, Ala, Asn, and Ser, and n = 7 for Gly). The asterisk indicated sd at P < 0.01 between the mutants and Col-0 for the same treatment. The dash line indicated the background [Ca²⁺]_cyt.