Arabidopsis GLYI4 Reveals Intriguing Insights into the JA Signaling Pathway and Plant Defense

Abstract

Plant hormones play a central role in various physiological functions and mediate defense responses against (a)biotic stresses. Jasmonic acid (JA) has emerged as one of the key phytohormones involved in the response to necrotrophic pathogens. Under stressful conditions, plants can also produce small molecules, such as methylglyoxal (MG), a cytotoxic aldehyde. The enzymes glyoxalase I (GLYI) and glyoxalase II primarily detoxify MG. In Arabidopsis thaliana, GLYI4 has been recently characterized as having a crucial role in MG detoxification and emerging involvement in the JA pathway. Here, we investigated the impact of a GLYI4 loss-of-function on the Arabidopsis JA pathway and how MG affects it. The results showed that the glyI4 mutant plant had stunted growth, a smaller rosette diameter, reduced leaf size, and an altered pigment concentration. A gene expression analysis of the JA marker genes showed significant changes in the JA biosynthetic and signaling pathway genes in the glyI4 mutant. Disease resistance bioassays against the necrotroph Botrytis cinerea revealed altered patterns in the glyI4 mutant, likely due to increased oxidative stress. The MG effect has a further negative impact on plant performance. Collectively, these results contribute to clarifying the intricate interconnections between the GLYI4, MG, and JA pathways, opening up new avenues for further explorations of the intricate molecular mechanisms controlling plant stress responses.

Keywords: Arabidopsis; GLYI4; jasmonic acid; methylglyoxal; oxidative stress; plant defense; plant growth.

Figure 1

Growth index of Col-8 and glyI4 treated with Mock, MeJA, MG, and MeJA + MG. Leaf area and rosette diameter 7 (A) and 14 days (B) after chemical treatments, respectively. Leaf area (LA) (C) was calculated based on two treatment-responsive leaves (the 5th and the 11th) using the following formula: LA = L × W, where LA (leaf area); L (leaf length); and W (leaf width). (D) The rosette diameter (d) was measured with a ruler. Letters above the bars indicate significant differences between Col-8 and glyI4 plants among the different treatments (two-way analysis of variance; Šídák’s test; n = 15; p-value < 0.0001).

Figure 2

Chlorophyll content in Col-8 and glyI4 treated with Mock, MeJA, MG, and MeJA + MG. (A) Chlorophyll a (Chl a) content; (B) chlorophyll b (Chl b) content; (C) chlorophyll a/b ratio. Letters above the bars indicate significant differences between Col-8 and glyI4 among the treatments (two-way analysis of variance; Šídák’s test; n = 3; p-value < 0.0001).

Figure 3

Carotenoid and anthocyanin contents in Col-8 and glyI4 treated with Mock, MeJA, MG, and MeJA + MG. (A) Carotenoid content; (B) anthocyanin content. Letters above the bars indicate significant differences between Col-8 and glyI4 among the different treatments (two-way analysis of variance; Šídák’s test; n = 3; p-value < 0.0001).

Figure 4

Transcript levels of JA marker genes in Col-8 and glyI4 treated with Mock, MeJA, MG, and MeJA + MG. Expression analyses were performed 24 h after the chemical treatment of 5-week-old plants. The expression is relative to the reference gene PP2AA3. Error bars represent standard deviation. Letters above the bars indicate significant differences between Col-8 and glyI4 plants among the different treatments (two-way analysis of variance; Šídák’s test; n = 3; p-value < 0.0001).

Figure 5

Botrytis cinerea resistance bioassay in Col-8 and glyI4 treated with Mock, MeJA, MG, and MeJA + MG. (A) Representative photographs of B. cinerea disease symptoms in Col-8 and glyI4 mutant plants 3 days after inoculation. (a) General overview of the plant after infection; (b) focus on a single leaf affected by disease symptoms. (B) Distribution of disease symptoms in Col-8 and glyI4. The bars indicate the frequency distribution (in %) of disease symptoms. The evaluation of this disease is based on the diameter of the lesion on the leaves and includes 5 classes: I, lesion of 2 mm; II, lesion of 2 mm + chlorosis; III, lesion between 2 and 4 mm + chlorosis; IV, lesion larger than 4 mm + chlorosis; V: widespread leaf necrosis. A black asterisk above the bars indicates that there is a statistically significant difference between Mock + B. cinerea and each treatment (χ² test, n = 9; p-value < 0.05). The asterisk above the bracket indicates significant differences in the response to any treatment between glyI4 and Col-8.

Figure 6

PDF1.2 transcript levels in Col-8 and glyI4 treated with Mock, MeJA, MG, and MeJA + MG and infected with Botrytis cinerea. The analysis was carried out 3 days after inoculation. Error bars represent standard deviation. The expression is relative to PP2AA3. Letters above the bars indicate significant differences between Col-8 and glyI4 among the different treatments (two-way analysis of variance; Šídák’s test; n = 3; p value < 0.0001).

Figure 7

Detection of ROS in Col-8 (A) and glyI4 (B) leaves after Mock, MeJA, MG, and MeJA + MG treatments and Botrytis cinerea infection. The detection of ROS was carried out by using DCFH₂-DA or buffer (negative technical control). Fluorescence was observed under an LSM 710 confocal microscope with a Plan Neofluar 20/1.30 objective. Laser excitation lines were used, i.e., 488 for probe detection (green) and 561 nm for chlorophyll auto-fluorescence (red). The bar corresponds to 50 μm. The merged-, green-, and red-channel images are shown.

Figure 8

Quantification of green fluorescence detected using DCFH₂-DA in Col-8 (A) and glyI4 (B) after Mock, MeJA, MG, MeJA + MG treatments and Botrytis cinerea infection. The integrated density mean is reported. Letters above the brackets indicate a statistically significant difference between samples (one-way analysis of variance; Tukey’s test; n = 3; p-value < 0.0001). Quantification has been performed using ImageJ, version 1.52a.

Figure 9

Summary of the main results obtained. (A) The GlyI enzyme family is involved in MG scavenging. (B) In glyI4 mutant plants, characterized by defective MG scavenging, higher carotenoid, anthocyanin, and chlorophyll b contents; higher ROS production; and an up-regulation of the MeJA-dependent LOX3, JAZ1, JAZ3 genes are observed. On the contrary, plant growth and chlorophyll a content are compromised. Additionally, a set of MeJA-dependent genes such as AOS, AOC1, OPR3, ACX1, MPF2, EIN3, ORA59, and PDF1.2 are downregulated. Finally, glyI4 mutant plants show altered resistance to the necrotrophic fungal pathogen Botrytis cinerea.