Exogenous Putrescine Modulates Nitrate Reductase-Dependent NO Production in Cucumber Seedlings Subjected to Salt Stress

Abstract

Polyamines (PAs) are small aliphatic compounds that participate in the plant response to abiotic stresses. They also participate in nitric oxide (NO) production in plants; however, their role in this process remains unknown. Therefore, the study aimed to investigate the role of putrescine (Put) in NO production in the roots of cucumber seedlings subjected to salt stress (120 mM NaCl) for 1 and 24 h. In salinity, exogenous Put can regulate NO levels by managing NO biosynthesis pathways in a time-dependent manner. In cucumber roots exposed to 1 h of salinity, exogenous Put reduced NO level by decreasing nitrate reductase (NR)-dependent NO production and reduced nitric oxide synthase-like (NOS-like) activity. In contrast, during a 24 h salinity exposure, Put treatment boosted NO levels, counteracting the inhibitory effect of salinity on the NR and plasma membrane nitrate reductase (PM-NR) activity in cucumber roots. The role of endogenous Put in salt-induced NO generation was confirmed using Put biosynthesis inhibitors. Furthermore, the application of Put can modulate the NR activity at the genetic and post-translational levels. After 1 h of salt stress, exogenous Put upregulated CsNR1 and CsNR2 expression and downregulated CsNR3 expression. Put also decreased the NR activation state, indicating a reduction in the level of active dephosphorylated NR (dpNR) in the total enzyme pool. Conversely, in the roots of plants subjected to 24 h of salinity, exogenous Put enhanced the NR activation state, indicating an enhancement of the dpNR form in the total NR pool. These changes were accompanied by a modification of endogenous PA content. Application of exogenous Put led to an increase in the amount of Put in the roots and reduced endogenous spermine (Spm) content in cucumber roots under 24 h salinity. The regulatory role of exogenous Put on NO biosynthesis pathways may link with plant mechanisms of response to salt stress.

Keywords: nitrate reductase (NR); nitric oxide; nitric oxide synthase-like activity; polyamines; putrescine; salt stress.

Figure 1

The level of NO in roots of cucumber treated with 1 mM Put and/or 120 mM NaCl for 1 h and 24 h. (a) Bio-imaging of NO generation in root apical segments of cucumbers using DAF-2 DA dye. Bar, 100 µm. (b) Relative efficiency of Put and/or NaCl for NO release from root apical segments of cucumber seedlings. (c) NO level in cucumber roots measured colorimetrically using the Griess reagent. The average value of untreated plants was 9.40 ± 2.00 nmol NO₂⁻ × gFW⁻¹. Significance was evaluated at: p < 0.1 (*); p < 0.001 (***).

Figure 2

The level of NO in roots of cucumber treated with 0.1 mM DFMA, 0.1 mM DFMO, and/or 120 mM NaCl for 1 h and 24 h. (a) Bio-imaging of NO generation in root apical segments of cucumbers using fluorescent method with DAF-2 DA dye. Bar, 100 µm. (b) Relative efficiency of DFMA, DFMO, and/or NaCl for NO release from root apical segments of cucumber seedlings. (c) NO level in cucumber roots measured using the Griess reagent. The average value of untreated plants was 5.43 ± 0.70 nmol NO₂⁻ × gFW⁻¹. Significance was evaluated at: p < 0.1 (*); p < 0.05 (**); p < 0.001 (***).

Figure 3

NO level in roots of cucumber treated with 0.1 mM WO, 0.1 mM L-NAME, and/or 120 mM NaCl for 1 h and 24 h. (a) Bio-imaging of NO generation in root apical segments of cucumbers using a fluorescent method with DAF-2 DA dye. Bar, 100 µm. (b) Relative efficiency of WO, L-NAME, and/or NaCl for NO release from root apical segments of cucumber seedlings. (c) NO level in cucumber roots measured using the Griess reagent. The average value of untreated plants was 9.08 ± 1.63 nmol NO₂⁻ × gFW⁻¹. Significance was evaluated at: p < 0.1 (*); p < 0.05 (**); p < 0.001 (***).

Figure 4

Total activity, actual activity, and activation state of NR in roots of cucumber seedlings treated with 1 mM Put and/or 120 mM NaCl for (a–c) 1 h and (d–f) 24 h. The average value of control plants, based on the results of individual experiments, was 0.85 ± 0.16 µmol NO₂⁻ × gFW⁻¹ × h⁻¹ for total NR activity (a,d) and 0.36 ± 0.13 µmol NO₂⁻ × gFW⁻¹ × h⁻¹ for actual NR activity (b,e). Significance was evaluated at: p < 0.05 (**).

Figure 5

PM-NR activity in roots of cucumber seedlings treated with 1 mM Put and/or 120 mM NaCl for (a) 1 h and (b) 24 h. The average value of control plants, based on the results of individual experiments, was (a) 0.65 ± 0.04 µmol NO₂⁻ × mg⁻¹ × h⁻¹ and (b) 0.18 ± 0.04 µmol NO₂⁻ × mg⁻¹ × h⁻¹ for PM-NR activity. Significance was evaluated at: p < 0.05 (**); p < 0.001 (***).

Figure 6

Relative expression of CsNR genes in roots of cucumber seedlings treated with 1 mM Put and/or 120 mM NaCl for (a–c) 1 h and (d–f) 24 h. The expression of CsNR genes was normalized using the TIP41-like gene as reference. Significance was evaluated at: p < 0.1 (*); p < 0.05 (**); p < 0.001 (***).

Figure 7

NOS-like activity in roots of cucumber seedlings treated with 1 mM Put and/or 120 mM NaCl for (a) 1 h and (b) 24 h. The average value of control plants, based on the results of individual experiments, was 11.75 ± 2.10 nmol NADPH × mg⁻¹ × min⁻¹. Significance was evaluated at: p < 0.1 (*); p < 0.05 (**).

Figure 8

Endogenous PA levels in roots of cucumber seedlings treated with 1 mM Put and/or 120 mM NaCl for (a–c) 1 h and (d–f) 24 h. The average value of control plants, based on the results of individual experiments, was 42.24 ± 16.04 µg Put × gFW⁻¹, 79.56 ± 9.59 µg Spd × gFW⁻¹ and 6.03 ± 1.68 µg Spm × gFW⁻¹ for 1 h (a–c) and 24.23 ± 4.25 µg Put × gFW⁻¹, 59.12 ± 10.31 µg Spd × gFW⁻¹, and 3.81 ± 0.27 µg Spm × gFW⁻¹ for 24 h (d–f). Significance was evaluated at: p < 0.05 (**); p < 0.001 (***).

Figure 9

Effect of exogenous Put on NO production and NR activity in roots of cucumber seedlings exposed to salt stress. (a) During short-term (1 h) salt stress exogenous Put diminished NR-dependent NO production in cucumber roots. At the same time, exogenous Put reduced NO generation through NOS-like activity. However, exogenous Put did not modulate the activity of PM-NR, which may not modify NO biosynthesis via the NiNOR enzyme and the non-enzymatic pathway in the apoplast of cucumber roots. It should be noted that the observed changes in the NO level might result from the possible influence of exogenous Put on other pathways of NO production in cucumber roots during salt stress. (b) Under longer (24 h) periods of salt stress, exogenous Put triggered the stimulatory effect of NaCl on NO production in cucumber roots. Exogenous Put enhanced the total NR activity and PM-NR activity. The stimulation of PM-NR activity may increase NO generation in the apoplast of plant roots. Exogenous Put might affect other possible pathways of NO biosynthesis during longer salt stress. (c) Time-dependent regulation of NR activity at the genetic and post-translational level by exogenous Put in cucumber roots subjected to salt stress. Markings: ↑, stimulation; ↑, possible stimulation; ↓, inhibition; −, no changes; −, possible no changes. HA, hydroxylamine; mETC, mitochondrial electron transport chain; NiNOR, plasma membrane-bound nitrite-NO reductase; NOS-like activity, nitric oxide synthase-like activity; NR, nitrate reductase; PM-NR, plasma-membrane nitrate reductase; Put, putrescine.