Glyoxalase I activity affects Arabidopsis sensitivity to ammonium nutrition

Abstract

Elevated methylglyoxal levels contribute to ammonium-induced growth disorders in Arabidopsis thaliana. Methylglyoxal detoxification pathway limitation, mainly the glyoxalase I activity, leads to enhanced sensitivity of plants to ammonium nutrition. Ammonium applied to plants as the exclusive source of nitrogen often triggers multiple phenotypic effects, with severe growth inhibition being the most prominent symptom. Glycolytic flux increase, leading to overproduction of its toxic by-product methylglyoxal (MG), is one of the major metabolic consequences of long-term ammonium nutrition. This study aimed to evaluate the influence of MG metabolism on ammonium-dependent growth restriction in Arabidopsis thaliana plants. As the level of MG in plant cells is maintained by the glyoxalase (GLX) system, we analyzed MG-related metabolism in plants with a dysfunctional glyoxalase pathway. We report that MG detoxification, based on glutathione-dependent glyoxalases, is crucial for plants exposed to ammonium nutrition, and its essential role in ammonium sensitivity relays on glyoxalase I (GLXI) activity. Our results indicated that the accumulation of MG-derived advanced glycation end products significantly contributes to the incidence of ammonium toxicity symptoms. Using A. thaliana frostbite1 as a model plant that overcomes growth repression on ammonium, we have shown that its resistance to enhanced MG levels is based on increased GLXI activity and tolerance to elevated MG-derived advanced glycation end-product (MAGE) levels. Furthermore, our results show that glyoxalase pathway activity strongly affects cellular antioxidative systems. Under stress conditions, the disruption of the MG detoxification pathway limits the functioning of antioxidant defense. However, under optimal growth conditions, a defect in the MG detoxification route results in the activation of antioxidative systems.

Keywords: Ammonium nutrition; D-Lactate dehydrogenase; Dicarbonyl stress; Glyoxalase; Methylglyoxal; Mitochondrial Complex I mutant.

Fig. 1

Influence of impairment of methylglyoxal and d-lactate detoxification pathways on the phenotype of Arabidopsis grown on nitrate (NO₃⁻) or ammonium (NH₄⁺) as the sole nitrogen source. Treatment of wild-type (WT_Col-0) seedlings with S-p-bromobenzylglutathione cyclopentyl diester (BBGD) (a), fresh weight (FW) of rosettes of long-term grown d-lactate dehydrogenase insertion lines (dldh) (mean ± SD; n = 8–9) (b) and the length of seedlings roots of glyoxalase I.3 (glxI.3) and II.5 (glxII.5) insertion lines in a vertical-plate agar assay using nitrate- (c) or ammonium-contained (d) medium (mean ± SD; n = 10–11). Phenotype of long-term grown glyoxalase I.3 (glxI.3) and glyoxalase II.5 (glxII.5) insertion lines cultivated on nitrate- (e) or ammonium-supplied (f) medium. Scale bar represents 2 cm. Representative photos are shown. Statistically significant differences by ANOVA (p ≤ 0.05) with Tukey’s post hoc test are indicated by different letters above the bars

Fig. 2

Impact of glyoxalase gene disruption on the corresponding glyoxalase activity in Arabidopsis plants long-term grown on nitrate (NO₃⁻) or ammonium (NH₄⁺) as the sole nitrogen source. Glyoxalase I (GLXI) activity in glyoxalase I.3 (glxI.3) insertion lines (a) and glyoxalase II (GLXII) activity in glyoxalase II.5 (glxII.5) insertion lines (b) as compared to wild-type (WT_Col-0) plants. Data are presented as mean ± SD (n = 3). Statistically significant differences by ANOVA (p ≤ 0.05) with Tukey’s post hoc test are indicated by different letters above the bars

Fig. 3

Role of DJ-1 proteins in Arabidopsis grown on nitrate (NO₃⁻) or ammonium (NH₄⁺) as the sole nitrogen source. Relative transcript level of DJ-1 genes in 9-week-old wild-type (WT_Col-0) plants (mean ± SD; n = 3) (a). Root length of 7-day-old dj-1 seedlings grown on nitrate- (b) or ammonium-contained (c) medium in a Petri dish experiment (mean ± SD; n = 9–10). Statistically significant differences by ANOVA (p ≤ 0.05) with Tukey’s post hoc test are indicated by different letters above the bars

Fig. 4

Influence of disabled methylglyoxal and d-lactate detoxification pathways on advanced glycation end-product formation and protease activity in Arabidopsis plants long-term grown on nitrate (NO₃⁻) or ammonium (NH₄⁺) as the sole nitrogen source. The level of methylglyoxal-derived hydroimidazolone 1 (MG-H1) (a) and the activity of proteolytic enzymes in d-lactate dehydrogenase (dldh) and glyoxalase I.3 (glxI.3) insertion lines grown under nitrate (b) or ammonium (c) conditions (mean ± SD; n = 3–4). The signal intensity for the whole lanes quantified by densitometry is given in the table above the representative protein gel blot (mean ± SD; n = 4). The fold changes were calculated relative to the value for wild-type (WT_Col-0) plants, which is presented as 100% (protease activity) or 1 (blot). Statistically significant differences by ANOVA (p ≤ 0.05) with Tukey’s post hoc test are indicated by different letters above the bars

Fig. 5

Cellular antioxidative defense and oxidative damage to proteins in Arabidopsis plants grown on nitrate (NO₃⁻) or ammonium (NH₄⁺) as the sole nitrogen source. The protein level of ascorbate peroxidase (APX) isoforms and glutathione reductase (GR) (a), the profile of protein carbonylation in leaf tissues (b). The capacity of non-protein antioxidant systems (c) and enzymatic antioxidant systems (d) in d-lactate dehydrogenase (dldh) and glyoxalase I.3 (glxI.3) insertion lines (mean ± SD; n = 3). Two chloroplastic APX isoforms, stromal and thylakoidal APX (s/tAPX) or cytosolic and peroxisomal (c/pAPX), are shown. The signal intensity for band (GR and APX) (mean ± SD; n = 4) or the whole lane (carbonylated proteins) (mean ± SD; n = 3) quantified by densitometry is given in the table above the representative protein gel blot. The fold changes on blots were calculated relative to the value for wild-type (WT_Col-0) plants grown on NO₃⁻, which is presented as 1. Statistically significant differences by ANOVA (p ≤ 0.05) with Tukey’s post hoc test are indicated by different letters above the bars

Fig. 6

Methylglyoxal production in Arabidopsis plants overcoming ammonium syndrome that were long-term grown on nitrate (NO₃⁻) or ammonium (NH₄⁺) as the sole nitrogen source. Phenotype (a), triosephosphate isomerase (TPI) (mean ± SD; n = 4) (b), and non-phosphorylating NADP⁺-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP⁺-GAPDH) (mean ± SD; n = 3–4) (c) activities as well as methylglyoxal (MG) level (mean ± SD; n = 3–5) (d) in frostbite1 (fro1) as compared to wild-type (WT_C24)). Statistically significant differences by ANOVA (p ≤ 0.05) with Tukey’s post hoc test are indicated by different letters above the bars

Fig. 7

Methylglyoxal and d-lactate detoxification routes in Arabidopsis plants overcoming ammonium syndrome that were grown long-term on nitrate (NO₃⁻) or ammonium (NH₄⁺) as the sole nitrogen source. Glyoxalase I (GLXI) (mean ± SD; n = 8–9) (a) and glyoxalase II (GLXII) (mean ± SD; n = 4) (b) activities, d-LDH expression (mean ± SD; n = 3) (c), and mitochondrial d-lactate oxidation (mean ± SD; n = 3) (d) in frostbite1 (fro1) as compared to wild-type (WT_C24). Statistically significant differences by ANOVA (p ≤ 0.05) with Tukey’s post hoc test are indicated by different letters above the bars

Fig. 8

Tolerance to methylglyoxal (MG)-induced cellular damage in Arabidopsis plants overcoming ammonium syndrome that were grown on nitrate (NO₃⁻) or ammonium (NH₄⁺) as the sole nitrogen source. The level of methylglyoxal-derived hydroimidazolone 1 (MG-H1) in frostbite 1 and wild-type (WT_C24) leaf tissues (a). Phenotypic effect of external application of methylglyoxal (MG) to frostbite 1 and WT_C24) seedlings (b). The activity of proteolytic enzymes in frostbite1 (fro1) leaf tissues as compared to WT_C24 (mean ± SD; n = 3) (c). The signal intensity for the whole lanes quantified by densitometry (mean ± SD; n = 3) is given in the table above the representative protein gel blot. Statistically significant differences by ANOVA (p ≤ 0.05) with Tukey’s post hoc test are indicated by different letters above the bars

Fig. 9

The capacity of protein (a) and non-protein (b) antioxidant systems in in frostbite 1 and wild-type (WT_C24) leaf tissues. Data are presented as mean ± SD (n = 3). Statistically significant differences by ANOVA (p ≤ 0.05) with Tukey’s post hoc test are indicated by different letters above the bars

Fig. 10

Scheme showing the role of methylglyoxal (MG) metabolism under conditions of ammonium stress. MG metabolism in nitrate-grown wild-type (WT) plants (a), ammonium-grown wild-type plants (b), and mutants frostbite 1 and glx1.3 grown under conditions of ammonium stress, respectively (c) and (d)