Nitric oxide improves thermotolerance in spring maize by inducing varied genotypic defense mechanisms
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The investigation aimed at determining the effect of nitric oxide on antioxidant defense system of spring maize (Zea mays L.) genotypes namely, LM 11 (stress susceptible) and CML 32 (stress tolerant), that showed differential tolerance towards heat stress. Seed priming with a NO donor, sodium nitroprusside (SNP) improved seedling growth and induced varied defense mechanisms, under stress conditions. 75 μM SNP improved seedling lengths and their biomasses. It specifically enhanced catalase (CAT) activity in the roots of stressed seedlings of the two genotypes. However, it could induce CAT activity only in LM 11 shoots, under heat stress. It also enhanced peroxidase (POX) activity in CML 32 roots. However, such induction of POX activity with SNP treatment was not observed in LM 11 roots. This showed that NO increased the H2O2 scavenging efficiency of CML 32 genotype by enhancing the cumulative activation of CAT and POX in its roots. However, it did not induce activation of any of the H2O2 detoxifying enzymes in CML 32 shoots which showed that ascorbate–glutathione cycle remained non-operational in shoots of SNP-treated seedlings of the tolerant genotype, under high temperature stress. With seed priming, superoxide dismutase (SOD) activity increased in both the tissues of LM 11 seedlings. The shoots of SNP primed CML 32 seedlings, however, did not show any effect on SOD activity which illustrated that nitric oxide might act as a direct scavenger of superoxide radicals in CML 32 seedlings. SNP decreased the contents of H2O2 and MDA and increased proline content in seedlings of both the genotypes indicating reduced oxidative damage. The results thus showed that nitric oxide might induce different mechanisms of stress tolerance in these maize genotypes.
KeywordsSpring maize Temperature stress Antioxidant response Sodium nitroprusside
Nitric oxide (NO) is a signal molecule that participates in a range of biochemical and physiological processes of plants. These include photosynthesis, flowering, fruit ripening, and response to various abiotic and biotic stresses (Oz et al. 2015). A cross talk between NO signaling and reactive oxygen species (ROS) has been reported in the literature (Yildiztugay et al. 2014). NO not only activates antioxidant enzymes through signaling, but also acts as an antioxidant itself (Boogar et al. 2014; Parankusam et al. 2017). It acts either through the cGMP-dependent or -independent pathway and modulates the activities of protein kinases (Arasimowicz and Floryszak-Wieczorak 2007). It has been reported to alter the response of plants towards unfavorable environmental conditions such as temperature, salinity, drought, UV radiations, etc. (Singh et al. 2014; Oz et al. 2015).
High temperature is the main abiotic aspect that affects the development and productivity of crops (Hasanuzzaman et al. 2013). It affects physiological, biochemical, and gene regulation pathways of plants (Bita and Gerats 2013). It induces excessive generation of reactive oxygen species (ROS) such as superoxide (O2−·), singlet oxygen (1O), hydroxyl radicals (OH), and hydrogen peroxide (H2O2) that are extremely harmful to all cellular components. ROS are generated at controlled levels under non-stress conditions. However, exposure to stress conditions leads to malfunctioning of the ROS scavenging system, causing an imbalance between utilization and production of ROS thereby leading to their accumulation. This imbalance results in lipid peroxidation, DNA damage, protein denaturation, and pigment bleaching in plants subjected to high temperature stress (Ding et al. 2012; Miller et al. 2010). It has been reported that high temperature stress hastened the phenological stages of plants, thereby reducing yield and quality of grains (Farooq et al. 2011; Tewari and Tripathy 1999).
The detrimental effects of high temperature stress have been reported to be reduced by the application of sodium nitroprusside (SNP), a nitric oxide donor. Its role in alleviating oxidative damage by inducing antioxidant enzymes under high temperature stress has been cited earlier (Hasanuzzaman et al. 2012). With sodium nitroprusside (SNP) application, increased activities of superoxide dismutase (SOD) and catalase (CAT) were observed in heat exposed plants (Song et al. 2006).
Spring maize (Zea mays L.) belongs to Gramineae family and serves as an important food as well as livestock feed. It is a good source of nutrition as it contains carbohydrates, proteins, fats, fiber, and minerals (Enyisi et al. 2014). It has gained vast importance as a commercial crop due to the production of a large number of products from its grain. Moreover, its cultivation in the spring season helps in crop diversification program as it provides new prospects to utilize the vacated fields. Besides, it possesses high yield potential among all the cereals and is, therefore, known as the “queen of cereals”. Due to these reasons, spring maize is attaining more popularity among farmers and is currently being practiced in agriculture (Ranum et al. 2014). However, high temperature stress is a major problem confronted by spring maize. It led to reduced photosynthesis that further resulted in pollen desiccation, delayed silking, wilting, leaf firing, stunted growth and abortion, ultimately leading to poor yield in spring maize (Naveed et al. 2014).
Therefore, this investigation was carried out to study the role of nitric oxide in inducing thermotolerance in spring maize.
Materials and methods
Determination of antioxidant enzymes
Superoxide dismutase, peroxidase, and glutathione reductase were extracted with 0.1 M sodium phosphate buffer (pH 7.5) containing 1 mM EDTA, 1% polyvinylpyrrolidone (PVP), and 10 mM β-mercaptoethanol. Pyrogallol was used as substrate for determining superoxide dismutase (SOD) activity (Marklund and Marklund 1974). It was determined using 0.5 ml of 6 mM pyrogallol solution, 1.5 ml of 100 mM Tris HCl buffer (pH 8.2), and 0.1 ml enzyme extract. The absorbance was determined at 420 nm for 3 min with 30 s interval. The substrates, guaiacol and H2O2 were used for determining peroxidase activity (Shannon et al. 1966). The assay system had 3 ml of 50 mM guaiacol (in 100 mM phosphate buffer, pH 6.5) and 0.1 ml enzyme extract to which 0.1 ml of 800 mM H2O2 was added to initiate the reaction. The absorbance was recorded at 470 nm for 3 min with 30 s interval. The activity of glutathione reductase was determined using glutathione (oxidized) as substrate, following the methodology of Esterbaur and Grill (1978). For this, 0.1 ml of 1.5 mM MgCl2, 0.1 ml of 0.2 mM EDTA, 0.2 ml of 0.025 mM NADPH, 0.2 ml of 200 mM sodium phosphate buffer (pH 7.5), and 0.2 ml of enzyme extract were added to the spectrophotometric cuvette followed by the addition of 0.2 ml of 0.25 mM glutathione (oxidized). The absorbance was recorded at 340 nm for 3 min with 30 s interval. The molar extinction coefficient was 6.22 mM−1 cm−1 for NADPH.
Catalase activity was determined after homogenizing the tissue with sodium phosphate buffer (50 mM, pH 7.5). It was assayed by taking 1.8 ml sodium phosphate buffer (50 mM, pH 7.5), 0.2 ml enzyme extract and 0.1 ml of 39 mM H2O2 as substrate. The absorbance was recorded at 240 nm for 3 min with 30 s interval (Chance and Maehly 1955). Ascorbate peroxidase activity was determined after homogenizing the tissue with 50 mM sodium phosphate buffer (pH 7.5) having 1% PVP and 1 mM ascorbic acid. It was assayed by taking 0.8 ml of 0.5 mM ascorbate, 1 ml of 39 mM H2O2, 1 ml of 50 mM sodium phosphate buffer (pH 7.0) and 0.2 ml enzyme extract. Absorbance was measured spectrophotometrically at 290 nm for 3 min with 30 s interval (Nakano and Asada 1987). The content of proteins was also determined (Lowry et al. 1951).
Determination of H2O2 and MDA contents
H2O2 was extracted with sodium phosphate buffer (10 mM, pH 7.0) and estimated by the method described by Sinha (1971). 2 ml supernatant was mixed with 2 ml of solution containing glacial acetic acid and 5% potassium dichromate (1:3 v/v), and filtered using Whatman filter paper no. 1; and then, absorbance was read against the reagent blank at 570 nm. H2O2 content was determined from standard curve prepared by taking different concentration of H2O2 ranging from 20 to 100 µmol. MDA was extracted using 5% trichloroacetic acid and assayed by mixing the supernatant with an equal volume of 20% (w/v) TCA having 0.5% thiobarbituric acid (TBA). Absorbance was read at 532 nm and corrected by subtracting the absorbance at 600 nm for non-specific turbidity (Heath and Packer 1968). Extinction coefficient of 155 mM−1 cm−1 was used for calculating malondialdehyde content.
Determination of nitric oxide and proline contents
For extraction of nitric oxide, the tissue was homogenized with sodium acetate buffer (50 mM, pH 3.6) having 4% zinc acetate. It was then centrifuged for 15 min at 10,000 g. The supernatants were collected and pooled after repeated washings of the pellet. To the pooled extract, 0.1 g charcoal was added and the mixture was again filtered. 1 ml of filtrate was then mixed with 1 ml of Greiss reagent and the solution was kept at room temperature for 30 min. Thereafter, it was spectrophotometrically measured at 540 nm. The content of nitric oxide was determined by taking NaNO2 (10–50 nmol) as standard (Zhou et al. 2005). Methodology of Bates et al. (1973) was followed for determining proline content. For extracting proline content, the tissue was homogenized with 3% sulfosalicylic acid. The extract was then filtered. Estimation was carried out by adding 2 ml acidic ninhydrin solution and 2 ml glacial acetic acid to 2 ml of filtrate. The mixture was kept at 100 °C for 60 min. The tubes were then kept in an ice bath and 4 ml toluene was added to them. Absorbance of the upper toluene layer was read at 520 nm using toluene as blank.
Results were expressed as mean ± SD of three replicates. The data on seedling growth were analyzed by one-way analysis of variance with post hoc analysis, the Fisher’s LSD. Data for the other biochemical parameters were analyzed by applying factorial experiment in the CRD (SAS software 9.3).
Results and discussion
Effect of nitric oxide on seedling growth
Effect of nitric oxide on antioxidant defense system
Peroxidases (POX) catalyze the cleavage of H2O2 with the oxidation of a range of substrates (Yali and Bingru 2010). Our results showed that CML 32 seedlings had comparatively higher POX activity than LM 11 seedlings under high temperature stress (Fig. 3c). This showed that CML 32 seedlings had an efficient mechanism of scavenging reactive oxygen species as compared to the susceptible genotype, LM 11. Priming of CML 32 seeds with SNP improved peroxidase activity in their roots during seedling growth under stress conditions (Fig. 3c). An increase in POX activity with nitric oxide treatment has been cited earlier in response to other abiotic stresses (Kausar et al. 2013). This showed that NO enhanced the H2O2 scavenging efficiency of CML 32 seedlings by the cumulative activation of CAT and POX activities in their roots, under high temperature stress. However, such induction of POX activity with SNP treatment was not observed in the roots of LM 11 as well as shoots of both CML 32 and LM 11 genotypes (Fig. 3c, d).
Superoxide dismutase (SOD) regulates the concentrations of O 2 −· and H2O2 molecules and thus plays a vital role in antioxidant defense (Bowler et al. 1992). SOD activity was higher in the roots of the tolerant genotype (CML 32) than those of the susceptible (LM 11) ones at 5 DSG, under high temperature conditions. However, at 9 DSG, the roots of heat-stressed LM 11 seedlings achieved the same levels as those acquired by CML 32 at 5 DSG (Fig. 3e). The high SOD activity in shoots of CML 32 seedlings at 5 DSG might be responsible for scavenging super oxide radicals, thereby assisting the genotype in tolerating heat stress to a higher extent than that of LM 11 seedlings. SNP treatment induced an increase in SOD activity of LM 11 shoots under high temperature (Fig. 3f), thus improving the superoxide scavenging efficiency of the susceptible genotype. Induction of SOD activity with SNP treatment has also been reported in the literature under various abiotic stresses (Kausar et al. 2013). The increase in SOD activity thus provided a defensive pathway in shoots of LM 11 seedlings against superoxide radicals as was suggested by Ostrovskaya et al. (2009).
SNP treatment resulted in reduced SOD activity in CML 32 roots, under high temperature stress (Fig. 3e). The shoots of SNP primed CML 32 seedlings, however, did not affect SOD activity (Fig. 3f). The reduced or unaffected SOD activity in SNP-treated heat-stressed CML 32 seedlings showed that nitric oxide might act as a direct scavenger of superoxide radicals as was suggested by Zhou et al. (2005). As a result, the activation of SOD might not be required by the system for scavenging superoxide radicals. The results thus showed that there are genotypic differences in the mechanism of action of nitric oxide in LM 11 and CML 32 seedlings.
The overall results of H2O2 scavenging enzymes thus showed that the role of H2O2 detoxification was primarily played by the nitric oxide induced CAT and POX activities in CML 32 roots under stress conditions (Fig. 3a, c). However, in the roots of LM 11 genotype, CAT is the principal enzyme responsible for H2O2 detoxification (Fig. 3a). Since APX activity was not required for scavenging H2O2 molecules, it was reduced in the SNP-treated seedlings of the tolerant and susceptible genotypes, under heat stress (Fig. 4a, b). Contrary to our results, there are reports in the literature that showed increased APX activity with SNP treatment under various abiotic stresses (Hasanuzzaman et al. 2012). An important observation of the results showed that nitric oxide did not induce activation of any of the H2O2 detoxifying enzymes in the CML 32 shoots but caused induction of CAT activity in those of LM 11 seedlings. This showed that nitric oxide might induce different mechanisms of heat stress tolerance in the two genotypes. Nitric oxide, being an antioxidant, either acts as a ROS scavenger (H2O2 and O 2 −· ) or as an antioxidant system inducer (Singh et al. 2009). As a direct scavenger of H2O2 molecules, nitric oxide might take over the functions of the H2O2 scavenging enzymes, thereby preventing their activation in CML 32 shoots under high temperature stress (Fig. 6). However, in LM 11 shoots, nitric oxide might act as an inducer of antioxidant system, causing enhanced CAT activity under stress conditions (Fig. 3b).
Glutathione reductase (GR) activity enhanced from 5 to 9 DSG stages in LM 11 roots, whereas an opposite trend was observed in CML 32 roots, under high temperature stress (Fig. 4c). SNP treatment improved its activity in the roots of both the genotypes under stress conditions (Fig. 4c). SNP-induced GR activity, under high temperature stress, has been reported earlier (Hasanuzzaman et al. 2012). SNP treatment also increased its activity in LM 11 shoots at 5 DSG stage (Fig. 4d). With further seedling growth, GR activity maintained itself at the same levels leading to its reduced activity as compared to stress conditions (Fig. 4d). In the shoots of CML 32 seedlings, GR activity decreased significantly at 9 DSG as compared to 5 DSG under stress conditions. In other words, SNP treatment did not improve GR activity in CML 32 shoots, under stress conditions. It, in fact, decreased GR activity under stress conditions (Fig. 4d). The results thus showed that, with SNP treatment, the Halliwell–Asada pathway becomes inactive in CML 32 shoots, under heat stress. In other words, nitric oxide resulted in non-operational Halliwell–Asada pathway in CML 32 shoots under heat stress.
Hydrogen peroxide, malondialdehyde, nitric oxide, and proline contents
SNP treatment led to a reduction in H2O2 content of LM 11 and CML 32 seedlings than those of untreated ones, under heat stress (Fig. 5a, b). This reduction in H2O2 content with SNP treatment might be due to its direct quenching by nitric oxide or due to changes in the activities of CAT/POX (Groß et al. 2013) that in turn led to reduced cell injury and protection against cell death. The decreased accumulation of H2O2 in SNP-treated plants, under high temperature, has been reported in the literature (El-Beltagi et al. 2016). A similar decline in H2O2 accumulation by nitric oxide, under other stresses, has been reported (Liu et al. 2013).
Malondialdehyde (MDA) content, an indicator for the determination of the degree of oxidative stress (Davey et al. 2005), reflects the extent of membrane injury (Wang et al. 2011). Our results reflected that heat stress led to enhanced MDA content from 5 to 9 DSG stages in the roots of the susceptible and tolerant genotypes (Fig. 5c, d). However, the content was significantly higher in LM 11 seedlings than those of CML 32. This showed that membrane injury was higher in LM 11 as compared to CML 32 seedlings. An increase in lipid peroxidation, under high temperature, has been observed earlier (James et al. 2015). SNP treatment reduced membrane injury in CML 32 and LM 11 genotypes, under stress conditions. This was inferred from the reduced MDA levels in SNP-treated seedlings of the two maize genotypes (Fig. 5c, d). However, MDA content remained comparatively higher in the SNP-treated LM 11 seedlings than those of the other genotype, under heat stress (Fig. 5c, d). Cechin et al. (2015) also showed a similar reduction in MDA content in SNP-treated seedlings, under high temperature stress. The results thus showed that nitric oxide treatment reduced the levels of ROS either by its direct quenching or through activation of antioxidant defense system which in turn led to reduced membrane injury.
Proline acts as an osmolyte as well as an antioxidant in providing defense against abiotic stresses (Gosavi et al. 2014). The content of proline was comparatively higher in LM 11 seedlings than those of CML 32 under high temperature stress (Fig. 6c, d). Accumulation of proline in plants is reported to be a modulation against stress conditions (Sadeghipour 2016). SNP treatment further increased its content in seedlings of the two genotypes under heat stress (Fig. 6c, d). Similar results were observed by Fan and Du (2012) and Liu et al. (2013). The results thus showed that nitric oxide improved heat tolerance in the two genotypes by increasing proline accumulation that might be due to the increased activities of proline synthesizing enzymes or decreased activities of those involved in proline oxidation as suggested by Khan et al. (2013).
It may thus be concluded that nitric oxide improves thermotolerance in spring maize genotypes by inducing varied genotypic mechanisms of antioxidant system.
Author contribution statement
Khushdeep Kaur performed all the experiments discussed in the manuscript entitled, “Nitric oxide improves thermotolerance in spring maize by inducing varied genotypic defense mechanisms”. Kamaljit Kaur designed the overall project, formulated the objectives, and analyzed the results critically for the manuscript entitled, “Nitric oxide improves thermotolerance in spring maize by inducing varied genotypic defense mechanisms”.
We are grateful to Dr. Gurjit Kaur Gill, Sr. Maize Breeder, Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, for providing maize genotypes for this investigation.
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