Root NRT, NiR, AMT, GS, GOGAT and GDH expression levels reveal NO and ABA mediated drought tolerance in Brassica juncea L

Abstract

Little is known about the interactive effects of exogenous nitric oxide (NO) and abscisic acid (ABA) on nitrogen (N) metabolism and related changes at molecular and biochemical levels under drought stress. The present study highlights the independent and combined effect of NO and ABA (grouped as "nitrate agonists") on expression profiles of representative key genes known to be involved in N-uptake and assimilation, together with proline metabolism, N-NO metabolism enzyme's activity and nutrient content in polyethylene glycol (PEG) treated roots of Indian mustard (B. juncea cv. Varuna). Here we report that PEG mediated drought stress negatively inhibited growth performance, as manifested by reduced biomass (fresh and dry weight) production. Total N content and other nitrogenous compounds (NO₃^-, NO₂^-) were decreased; however, NH₄⁺, NH₄⁺/ NO₃^- ratio and total free amino acids content were increased. These results were positively correlated with the PEG induced changes in expression of genes and enzymes involved in N-uptake and assimilation. Also, PEG supply lowered the content of macro- and micro-nutrients but proline level and the activity of ∆¹-pyrroline-5-carboxylate synthetase increased indicating increased oxidative stress. However, all these responses were reversed upon the exogenous application of nitrate agonists (PEG + NO, PEG + NO + ABA, and PEG + ABA) where NO containing nitrate agonist treatment i.e. PEG + NO was significantly more effective than PEG + ABA in alleviating drought stress. Further, increases in activities of L-arginine dependent NOS-like enzyme and S-nitrosoglutathione reductase were observed under nitrate agonist treatments. This indicates that the balanced endogenous change in NO and ABA levels together during synthesis and degradation of NO mitigated the oxidative stress in Indian mustard seedlings. Overall, our results reveal that NO independently or together with ABA may contribute to improved crop growth and productivity under drought stress.

Figure 1

Nitrogen uptake and assimilation, and its interconnection with nitric oxide signaling process in plants.

Figure 2

Phenotypic changes, oxidative stress, and NO-metabolism of 7-d-old B. juncea L. after treatment with PEG (10%) and its combination with NO (100 µM) and ABA (10 µM) (nitrate agonists) for 96 h. (A) Shoot and root phenotypic changes (Scale bar = 2 cm), (B) Evan’s blue uptake, (C, D) endogenous accumulation of NO (Scale bar = 50 µm) (E) S-nitrosothiol (SNO) and S-nitrosoglutathione (GSNO) content, (F, G) activity of L-Agr-dependent nitric oxide synthase-like enzyme (NOSLE) and S-nitrosoglutathione reductase (GSNOR). Values are mean ± S.E. of three independent experiments, each including four biological replicates. Asterisk (*) indicates significant difference compared with control (*p < 0.05).

Figure 3

Content of nitrate (NO₃⁻), nitrite (NO₂⁻), ammonium (NH₄⁺), ammonium- nitrate ratio (NH₄⁺/ NO₃⁻), and total nitrogen (N) of 7-day-old B. juncea L. roots after treatment with PEG (10%) and its combination with NO (100 µM) and ABA (10 µM) (nitrate agonists) for 96 h. Values are mean ± S.E. of three independent experiments, each involving four biological replicates. Asterisk (*) indicates significant difference compared with control (*p < 0.05).

Figure 4

Activity of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (NADPH-GDH and NADH-GDH) of 7-day-old B. juncea L. roots after treatment with PEG (10%) and its combination with NO (100 µM) and ABA (10 µM) (nitrate agonists) for 96 h. Values are mean ± S.E. of three independent experiments, each including four biological replicates. Asterisk (*) indicates significant difference compared with control (*p < 0.05).

Figure 5

Heat map represents ICP-MS content of macro (P, K, Ca, Mg, Na, and S) and micronutrients (Fe, Cu, Mn, and Zn) of 7-d-old B. juncea L. roots after treatment with PEG (10%) and its combination with NO (100 µM) and ABA (10 µM) (nitrate agonists) for 96 h. Values are mean ± S.E. of three independent experiments, each including four biological replicates.

Figure 6

Relative expression levels of key genes related to nitrogen absorption and assimilation including nitrate transporter (BjNRT), ammonium transporter (BjAMT), nitrate reductase (BjNR), nitrite reductase (BjNiR), glutamine synthetase (BjGS), and glutamate synthase (BjGOGAT), and glutamate dehydrogenase (BjGDH) in 7-days-old B. juncea L. roots after treatment with PEG (10%) and its combination with NO (100 µM) and ABA (10 µM) (nitrate agonists) for 96 h. Expression values were calculated relative to the expression of plants grown in control condition under normalization with actin gene (2^−ΔΔCT). Values are mean with CIs of three independent experiments, each including four biological replicates. *p < 0.05 compared with control indicates significant difference.

Figure 7

A schematic comparison of (A) PEG-water stress and (B) nitrate agonists regulated N–NO coordinated underlying physiological phenomenon that shows the relationship of NO generation and scavenging to molecular mechanism of N-assimilation in B. juncea L. roots. Red boxes and narrow arrows represent decrease or down-regulation, while green boxed and wide arrows indicate increase or up-regulation in N–NO pathway. NO₃⁻ nitrate, NO₂⁻ nitrite, NH₄⁺ ammonium, SNO S-nitrosylated glutathione, GSNO S-nitrosoglutathione, NO nitric oxide, Glu glutathione, AA amino acids, GSSG oxidised glutathione, P5CS ∆¹-pyrroline-5-carboxylate synthetase, ProDH proline dehydrogenase, NR nitrate reductase, NiR nitrite reductase, NOSLE l-arginine dependent NOS-like enzyme activity, GSNOR S-nitrosoglutathione reductase, GS glutamine synthetase, GOGAT glutamate synthase, GDH glutamate dehydrogenase.