A Metabolic Profiling Analysis Revealed a Primary Metabolism Reprogramming in Arabidopsis glyI4 Loss-of-Function Mutant

Abstract

Methylglyoxal (MG) is a cytotoxic compound often produced as a side product of metabolic processes such as glycolysis, lipid peroxidation, and photosynthesis. MG is mainly scavenged by the glyoxalase system, a two-step pathway, in which the coordinate activity of GLYI and GLYII transforms it into D-lactate, releasing GSH. In Arabidopsis thaliana, a member of the GLYI family named GLYI4 has been recently characterized. In glyI4 mutant plants, a general stress phenotype characterized by compromised MG scavenging, accumulation of reactive oxygen species (ROS), stomatal closure, and reduced fitness was observed. In order to shed some light on the impact of gly4 loss-of-function on plant metabolism, we applied a high resolution mass spectrometry-based metabolomic approach to Arabidopsis Col-8 wild type and glyI4 mutant plants. A compound library containing a total of 70 metabolites, differentially synthesized in glyI4 compared to Col-8, was obtained. Pathway analysis of the identified compounds showed that the upregulated pathways are mainly involved in redox reactions and cellular energy maintenance, and those downregulated in plant defense and growth. These results improved our understanding of the impacts of glyI4 loss-of-function on the general reprogramming of the plant's metabolic landscape as a strategy for surviving under adverse physiological conditions.

Keywords: glyoxalase I; metabolite profiling; methylglyoxal; oxidative stress; plant defense; plant growth.

Figure 1

Metabolic pathway analysis plot. Color intensity (white to red) reflects increasing statistical significance, and circle diameter varies with the pathway impact. The graph was made by plotting on the y-axis the −log₁₀ transforms of p-values from the pathway enrichment analysis and on the x-axis the pathway impact values derived from the pathway topology analysis. The top three most significant pathways (p-value < 0.05; pathway impact (pi) > 0) are depicted: arginine biosynthesis (p-value = 0.0022868, pi = 0.08641); pyrimidine metabolism (p-value = 0.0022904, pi = 0.09611); cysteine and methionine metabolism (p-value = 0.032033, pi = 0.04138). The metabolomic analysis was repeated three times using different biological samples giving the same results.

Figure 2

Metabolic pathway analysis plot. Color intensity (white to red) reflects increasing statistical significance, and circle diameter varies with the pathway impact. The graph was obtained by plotting on the y-axis the −log₁₀ transforms of p-values from the pathway enrichment analysis and on the x-axis the pathway impact values derived from the pathway topology analysis. The top three most significant pathways (p-value < 0.05; pathway impact (pi) > 0) are depicted: purine metabolism (p-value = 1.3987 × 10⁻⁶, pi = 0.18529); phenylalanine, tyrosine, and tryptophan biosynthesis (p-value = 0.0039386, pi = 0.09009); zeatin biosynthesis (p-value = 0.025118; pi = 0.0271). The metabolomic analysis was repeated three times using different biological samples giving the same results.

Figure 3

PAL2 (a), CAD4 (b), and EPSPS (c) transcript levels relative to the reference gene UBI10 in Col-8 and the glyI4 mutant. An asterisk indicates a statistically significant difference between genotypes (Mann–Whitney test p-value < 0.05). Error bars represent means ± SDs (n = 3).

Figure 4

Antioxidant enzyme activities and lipid peroxidation in the glyI4 mutant. (a) superoxide dismutase (SOD); (b) catalase (CAT); (c) TBARS. Asterisks indicate statistically significant difference between genotypes. (Mann–Whitney test p-value < 0.05). Error bars represent means ± SDs (n = 3).