GLYI4 Plays A Role in Methylglyoxal Detoxification and Jasmonate-Mediated Stress Responses in Arabidopsis thaliana

Abstract

Plant hormones play a central role in various physiological functions and in mediating defense responses against (a)biotic stresses. In response to primary metabolism alteration, plants can produce also small molecules such as methylglyoxal (MG), a cytotoxic aldehyde. MG is mostly detoxified by the combined actions of the enzymes glyoxalase I (GLYI) and glyoxalase II (GLYII) that make up the glyoxalase system. Recently, by a genome-wide association study performed in Arabidopsis, we identified GLYI4 as a novel player in the crosstalk between jasmonate (JA) and salicylic acid (SA) hormone pathways. Here, we investigated the impact of GLYI4 knock-down on MG scavenging and on JA pathway. In glyI4 mutant plants, we observed a general stress phenotype, characterized by compromised MG scavenging, accumulation of reactive oxygen species (ROS), stomatal closure, and reduced fitness. Accumulation of MG in glyI4 plants led to lower efficiency of the JA pathway, as highlighted by the increased susceptibility of the plants to the pathogenic fungus Plectospherella cucumerina. Moreover, MG accumulation brought about a localization of GLYI4 to the plasma membrane, while MeJA stimulus induced a translocation of the protein into the cytoplasmic compartment. Collectively, the results are consistent with the hypothesis that GLYI4 is a hub in the MG and JA pathways.

Keywords: GLYI4; arabidopsis; methyl-jasmonate; methylglyoxal.

Figure 1

Glyoxalase (GLYI4) gene expression analysis in Arabidopsis Col-8 and glyI4 mutant after methylglyoxal (MG) treatment. qRT-PCR analysis of GLYI4 in Col-8 and glyI4 leaves treated with MG and a mock solution. Fold change is relative to the expression in mock-treated plants and normalized to the reference gene PP2AA3. GLYI4 gene expression analyses were performed 24 h after MG treatment of five-week-old plants. Shown data are means of three biological replicates. Error bars represent SEM. Different letters indicate significant statistical differences (Two-ways ANOVA, Tukey’s post-hoc, p < 0.0001).

Figure 2

Analysis of MG-scavenging pathway metabolites in Arabidopsis Col-8 and glyI4 mutant. MG, lactate, and glutathione (GSH) amounts were analyzed in Col-8 and glyI4 leaves. Shown data are means of three biological replicates. Error bars represent SEM. Asterisk indicates the statistically significant difference between Col-8 and glyI4 plants (t-test; * p < 0.05).

Figure 3

MG-protein adducts level in Arabidopsis Col-8 and glyI4 leaves. (A) Western blot performed with anti-methylglyoxal monoclonal antibody using Col-8 and glyI4 total protein extracts (40 g) from five-week-old plant leaves (W). Ponceau S staining (P) was used to normalize protein loading. Protein molecular weight markers are also shown (M). (B) Densitometric analysis of the most intense MG protein-adduct band, as indicated by the arrow in A. Fold-change is relative to the expression in Col-8 plants and normalized to the Ponceau stained band. Shown data are means of three biological replicates. Error bars represent SEM. Asterisks indicate the statistically significant difference between Col-8 and glyI4 (t-test; ** p < 0.005).

Figure 4

Fitness parameters of Arabidopsis Col-8 and glyI4. Leaf area (cm²), dry weight (mg), flowering time (days), and seed production (mg), per plant, were analyzed in Col-8 and glyI4 plants. Error bars represent SEM. Asterisk indicates a statistically significant difference between glyI4 and Col-8 (t-test; * p < 0.05, n = 15). The experiments have been repeated three times with similar results.

Figure 5

Detection of ROS in Arabidopsis Col-8 and glyI4 leaves. Detection on Col-8 and glyI4 leaves was carried out by using 2′,7′-DCFH₂-DA or buffer (negative technical control). Fluorescence was observed under an LSM 710 confocal microscope with Plan Neofluar 20/1.30 objective. Two laser excitations lines were used [i.e., 488 for probe detection (green) and 561 nm for chlorophyll autofluorescence (red)]. Bar corresponds to 50 μm.

Figure 6

Confocal microscope representative images of stomatal guard cells of Arabidopsis Col-8 and glyI4 leaves. Abaxial peeled epidermis of Col-8 and glyI4 leaves was stained with acridine orange (AO) and observed by the confocal microscope. AO is colored in green in not acidified cell compartments while AO red color indicates an acidification of cellular compartments. Magnification of guards cells is also shown (lower panels). Fluorescence images were acquired using a Zeiss LSM 710 confocal microscope with Plan Neofluar 20/1.30 objective (upper panel) and Plan Neofluar 63/1.30 objective (lower panel). Fluorescence emissions of AO in the red and green channels (615 to 660 nm and 530 to 540 nm, respectively) after excitation with a 488-nm laser were obtained. Bar corresponds to 100 μm (upper panels) or 10 μm (lower panels).

Figure 7

Bacterial proliferation and disease rate in Arabidopsis Col-8 and glyI4 plants infected with Pst DC3000 and P. cucumerina, respectively. (A) Plants were challenged with Pst DC3000 by dipping the aerial part in a bacterial suspension (10⁶ colony-forming units (CFU)/mL). The values reported represent means of the log of the proliferation values, recorded at zero and three days post inoculation. Data represent the average from six infected plants. The experiment was repeated two more times with similar results. Error bars represent SEM. Different letters mean significant statistical differences (Two-ways ANOVA, Tukey’s post-hoc, p < 0.0001). (B) Plants were infected with P. cucumerina by inoculating leaves with 6 L-drop of a suspension containing 5 × 10⁶ conidia/mL. Disease symptoms were recorded at seven days post inoculation by measuring the diameter of the necrotic lesions (mm). The figure shows a representative experiment from three independent repetitions. Data represent the average from 45 lesions produced on 12 plants per genotype. Error bars represent SEM. Asterisk indicates significant statistical differences compared with Col-8 (t-test, *** p < 0.0001).

Figure 8

PDF1.2 gene expression analysis in Arabidopsis Col-8 and glyI4 mutant after methyl jasmonate (MeJA) and MeJA+MG treatment. qRT-PCR analysis of PDF1.2 expression in Col-8 and glyI4 leaves treated with a mock solution, MeJA or MeJA+MG. Fold change is relative to the expression in mock-treated plants and normalized to the reference gene PP2AA3. PDF1.2 gene expression analyses were performed 24 h after each treatment using five-week-old plants. Shown data are means of three biological replicates. Error bars represent SEM. Different letters indicate statistically significant difference between treatments (two-way ANOVA; Tukey’s test p < 0.0001).

Figure 9

GLYI4 localization in Arabidopsis protoplasts. glyI4 leaf protoplasts were MeJA- and mock-treated and then transformed with pUC-35S::GLYI4- enhanced yellow fluorescent protein (EYFP). Fluorescence images were acquired using a Zeiss LSM 710 confocal microscope with Plan Neofluar 40/1.30 objective. Two laser excitation wavelengths were used (i.e., λexc = 514 nm for EYFP (green), and λexc = 561 nm for chloroplast autofluorescence (red)). The merged images are also presented. Bars correspond to 50 μm.