Developmental stage- and concentration-specific sodium nitroprusside application results in nitrate reductase regulation and the modification of nitrate metabolism in leaves of Medicago truncatula plants

Abstract

Nitric oxide (NO) is a bioactive molecule involved in numerous biological events that has been reported to display both pro-oxidant and antioxidant properties in plants. Several reports exist which demonstrate the protective action of sodium nitroprusside (SNP), a widely used NO donor, which acts as a signal molecule in plants responsible for the expression regulation of many antioxidant enzymes. This study attempts to provide a novel insight into the effect of application of low (100 μΜ) and high (2.5 mM) concentrations of SNP on the nitrosative status and nitrate metabolism of mature (40 d) and senescing (65 d) Medicago truncatula plants. Higher concentrations of SNP resulted in increased NO content, cellular damage levels and reactive oxygen species (ROS) concentration, further induced in older tissues. Senescing M. truncatula plants demonstrated greater sensitivity to SNP-induced oxidative and nitrosative damage, suggesting a developmental stage-dependent suppression in the plant's capacity to cope with free oxygen and nitrogen radicals. In addition, measurements of the activity of nitrate reductase (NR), a key enzyme involved in the generation of NO in plants, indicated a differential regulation in a dose and time-dependent manner. Furthermore, expression levels of NO-responsive genes (NR, nitrate/nitrite transporters) involved in nitrogen assimilation and NO production revealed significant induction of NR and nitrate transporter during long-term 2.5 mM SNP application in mature plants and overall gene suppression in senescing plants, supporting the differential nitrosative response of M. truncatula plants treated with different concentrations of SNP.

Keywords: Medicago truncatula; antioxidants; hydrogen peroxide; nitrate reductase; nitric oxide; nitrosative stress; oxidative stress; sodium nitroprusside.

Figure 1. Phenotypic response of Medicago truncatula plants five days after application of varying SNP concentrations by vacuum infiltration. Senescing (65d) plants treated with 2.5 mM SNP show increased damage levels indicated by wilted, chlorotic leaves.

Figure 2. Effect of SNP application on photosynthetic pigment content (A) Τotal chlorophyll (α + β) content, (B) Carotenoid content (where upper rows represent measurements made with 40 d plants, while lower rows indicate measurements made with 65 d plants). Asterisks denote statistically different values according to the Tukey pairwise comparison test (p < 0.05). Values are means ± SE (n = 3).

Figure 3. Effect of SNP application on cellular damage as indicated by lipid peroxidation (MDA content). (A) quantitative analysis by spectrophotometry, where upper row represents measurements made with 40 d plants, while lower row indicates measurements made with 65 d plants. (B) qualitative analysis by in situ histochemistry using Schiff’s reagent. Figure presents 40 d old plants; similar trend was observed with 65 d plants (data not shown). Asterisks denote statistically different values according to the Tukey pairwise comparison test (p < 0.05). Values are means ± SE (n = 3).

Figure 4. Effect of SNP application on hydrogen peroxide levels. (A) quantitative analysis by spectrophotometry, where upper row represents measurements made with 40 d plants, while lower row indicates measurements made with 65 d plants. (B) qualitative analysis by in situ histochemistry using DAB reagent. Figure presents 65 d old plants; similar trend was observed with 40 d plants (data not shown). Asterisks denote statistically different values according to the Tukey pairwise comparison test (p < 0.05). Values are means ± SE (n = 3).

Figure 5. Effect of SNP application on nitrite-derived nitric oxide content and NR enzymatic activity. (A) NO measurements made with mature (40 d) and senescing (65 d) plants after lower (100 μΜ) and higher (2.5 mM) SNP application. (B) Measurements of NR enzymatic activity in mature (40 d) and senescing (65 d) plants after lower (100 μΜ) and higher (2.5 mM) SNP application (Upper and lower rows represent measurements made with 40 d and 65 d plants respectively). Asterisks denote statistically different values according to the Tukey pairwise comparison test (p < 0.05). Values are means ± SE (n = 3).

Figure 6. NO effect on gene expression profiles of NO-responsive genes (NR and nitrate/nitrite transporters). Gene expression analysis was determined by qRT-PCR in leaves of Medicago truncatula Jemalong A17 plants vacuum-infiltrated with 100 μΜ and 2.5 mM SNP at 3/24 h in mature (40 d (A) and senescing (65 d (B) plants. Asterisks denote statistically different values according to pairwise fixed reallocation randomization test (p < 0.05) (n = 3).