Nitric Oxide Alleviates Salt Stress Inhibited Photosynthetic Performance by Interacting with Sulfur Assimilation in Mustard

Abstract

The role of nitric oxide (NO) and sulfur (S) on stomatal responses and photosynthetic performance was studied in mustard (Brassica juncea L.) in presence or absence of salt stress. The combined application of 100 μM NO (as sodium nitroprusside) and 200 mg S kg(-1) soil (S) more prominently influenced stomatal behavior, photosynthetic and growth performance both in the absence and presence of salt stress. The chloroplasts from salt-stressed plants had disorganized chloroplast thylakoids, but combined application of NO and S resulted in well-developed chloroplast thylakoids and properly stacked grana. The leaves from plants receiving NO plus S exhibited lower superoxide ion accumulation under salt stress than the plants receiving NO or S. These plants also exhibited increased activity of ATP-sulfurylase (ATPS), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) and optimized NO generation that helped in minimizing oxidative stress. The enhanced S-assimilation of these plants receiving NO plus S resulted in increased production of cysteine (Cys) and reduced glutathione (GSH). These findings indicated that NO influenced photosynthesis under salt stress by regulating oxidative stress and its effects on S-assimilation, an antioxidant system and NO generation. The results suggest that NO improves photosynthetic performance of plants grown under salt stress more effectively when plants received S.

Keywords: antioxidant; nitric oxide; photosynthesis; salt stress; sulfur.

Figure 1

SDS-PAGE protein profile of Rubisco of mustard (Brassica juncea L.) leaves treated with 100 μM nitric oxide (NO) and grown with S (200 mg S kg⁻¹ soil; S) in presence or absence of 100 mM NaCl at 30 DAS. Equal amount of protein (40 μl) were loaded on to each lane. The proteins expressed in the range of 14.3–97.4 kDa. LSU, large subunits of Rubisco (native soil S content: 100 mg S kg⁻¹ soil).

Figure 2

In situ accumulation of superoxide ion (${\text{O}}_2^{-}$) by nitro blue tetrazolium (NBT) staining of mustard (Brassica juncea L.) leaves after dehydration. The leaves originated from plants treated with/without 100 mM NaCl, S (200 mg S kg⁻¹ soil; S) or 100 μM nitric oxide (NO) individually or in combinations at 30 DAS. Arrow (→) shows ${\text{O}}_2^{·-}$ accumulation.

Figure 3

Activity of CAT (A), APX (B), GR (C), and NO generation (D) in mustard (Brassica juncea L.) leaves treated with 100 μM nitric oxide (NO) and/or grown with S (200 mg S kg⁻¹ soil; S) in presence or absence of 100 mM NaCl at 30 DAS. Data are presented as treatments mean ± SE (n = 4). Data followed by same letter are not significantly different by LSD test at P < 0.05. APX, ascorbate peroxidase; CAT, catalase; GR, glutathione reductase.

Figure 4

Content of ABA in mustard (Brassica juncea L.) leaves treated with 100 μM nitric oxide (NO) and/or grown with S (200 mg S kg⁻¹ soil; S) in presence or absence of 100 mM NaCl at 30 DAS. Data are presented as treatments mean ± SE (n = 4). Data followed by same letter are not significantly different by LSD test at P < 0.05.

Figure 5

Leaf stomatal response of mustard (Brassica juncea L.) under the effect of various treatments. The stomatal opening and closing response was studied using confocal microscopy in control (A), 100 mM NaCl (B), and S (200 mg S kg⁻¹ soil; S) and 100 μM nitric oxide (NO) with 100 mM NaCl treated plants (C) at 30 DAS. Bar represents 1 μm in the panels (A–C).

Figure 6

Leaf stomatal behavior of mustard (Brassica juncea L.) performed under control (A,B), 100 mM NaCl (C,D), and S (200 mg S kg⁻¹ soil; S) and 100 μM nitric oxide (NO) with 100 mM NaCl (E,F). The effect of stomatal opening and closing was observed under the scanning electron microscopes at 1.50 K X (A,C,E) and 5.0 K X magnifications (B,D,F) in the leaf surface of mustard (Brassica juncea L.) grown under 100 mM NaCl treated plants at 30 DAS.

Figure 7

Ultrastructure of chloroplasts from leaves of mustard (Brassica juncea L.). Transmission electron microscopy micrographs on the representative chloroplasts from the leaves of mustard performed on the control (A,D); 100 mM NaCl (B,E) and S (200 mg S kg⁻¹ soil; S) and 100 μM nitric oxide (NO) with 100 mM NaCl treated plants (C,F) at 30 DAS. Ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and examined by transmission electron microscopy operated at voltage of 120 kV and magnification of 6000 X and 1200 X. Bar represents 100 nm in the (A–C) and 500 nm in the (D–F).(Thy; thylakoid membranes).

Figure 8

Leaf area (A) and plant dry mass (B) of mustard (Brassica juncea L.) treated with 100 μM nitric oxide (NO) and/or grown with S (200 mg S kg⁻¹ soil; S) in presence or absence of 100 mM NaCl at 30 DAS. Data are presented as treatments mean ± SE (n = 4). Data followed by same letter are not significantly different by LSD test at P < 0.05.

Figure 9

Schematic representation of major mechanisms underlying excess-S and NO-mediated alleviation of salt stress in mustard (Brassica juncea L.). The figure shows that NO generated by the supplementation of SNP (NO donor) can react with GSH and yield S-nitrosoglutathione (GSNO). This metabolite can be converted by the enzyme GSNO reductase (GSNOR) into GSSG and NH₃. Additionally, supplementation of excess-S and NO reduced NaCl-induced oxidative stress and influenced photosynthesis by increasing GR-mediated conversion of GSSG into GSH and regulating NO generation in plants. Arrow () indicates signaling between NO and GSH for salt tolerance. Arrow () indicates interaction between NO and ABA. NO induces ABA accumulation which in turn up regulates S-assimilation and controls GSH formation.