Combination of nitric Oxide and thiamin regulates oxidative defense machinery and key physiological parameters in salt-stressed plants of two maize cultivars differing in salinity tolerance

Advances in Agricultural Science 06 (2018), 01: 34-44

Combination of nitric Oxide and thiamin regulates oxidative defense machinery and key physiological parameters in salt-stressed plants of two maize cultivars differing in salinity tolerance

Cengiz Kaya 1*, Muhammad Ashraf 2 and Osman Sonmez 3

Harran University, Faculty of Agriculture, Department of Soil Science & Plant Nutrition, Şanlıurfa, Turkey.
Pakistan Science Foundation, Islamabad, Pakistan.  
University of Erciyes, Faculty of Agriculture, Department of Soil Science & Plant Nutrition, Develi, Kayseri, Turkey.



A glasshouse experiment was conducted to appraise  the influence of combined nitric oxide (NO) and thiamin (TA) on oxidative defense system and some key physiological attributes in two maize cultivars (DK 5783 and Apex 836) stressed with 0 (control) or 100 mM NaCl.  Of six NO and TA levels used in the initial germination experiment, 2 levels of combined NO and TA (3 +100 or 6+ 125 mg l-1 respectively) were chosen for subsequent studies as seed soaking or as a spray to seedlings. Salinity resulted in rising leaf free proline content and osmolality, but in a decrease in plant dry biomass and maximum fluorescence yield (Fv/Fm) in cultivars. Both modes of applied NO and TA were found to be effective in alleviating the adverse effects of NaCl on shoot growth. Salt stress resulted in enhancing leaf Na+, but reducing leaf K+ and Ca2+ in plants. Both modes of application of NO and TA resulted in increased Ca2+ and K+ contents, but decreased those of  Na+ in salt stressed maize plants. Salt stress caused the enhanced accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA).  Salinity promoted the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in maize. The growth improvement in maize plants due to exogenously-applied  NO and TA in combination  was found to be due to decreased leaf Na+, H2O2 and MDA levels, and altered  activities of SOD, CAT, and POD as well as improved maximum fluorescence yield under saline stress.

Keywords: Maize, Nitric oxide, Thiamin, Salt tolerance, Oxidative stress

How to Cite: Kaya, C., Ashraf, M., & Sonmez, O. (2018). Combination of nitric Oxide and thiamin regulates oxidative defense machinery and key physiological parameters in salt-stressed plants of two maize cultivars differing in salinity tolerance. Advances in Agricultural Science, 6(1), 34-44.


Salt stress leads to huge losses to crop production worldwide. The research related to the effects of salt stress on plants has been under intensive investigation since a long span of time. These salt-induced effects have been explored using a variety of physiological parameters such as rate of photosynthesis, water and ion uptake, osmotic adjustment, and oxidative metabolism, etc. (Liu et al., 2014). Several studies have shown that accumulations of reactive oxygen species (ROS) are linked primarily to the antioxidant enzyme system, which is one of the premier adaptive responses of plants to saline stress (Dong et al., 2014), although  plants generate a variety of antioxidant compounds both enzymatic and non-enzymatic antioxidants to nullify the ROS. The key enzymatic antioxidants include catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) which can effectively scavenge ROS before they cause injury to cells (Noctor and Foyer, 1998; Perveen et al.,  2013). The modulation of the activities of these enzymes could be important in improving tolerance of plants to environmental stresses (Dong et al., 2014).

During the last few decades, researchers have used a variety of  growth substances  as a shot-gun approach to overcome the adverse effects of environmental stresses on plants (Ashraf and Foolad, 2007; Nawaz and Ashraf, 2010). Thiamin is one of such plant growth substances required for growth and differentiation of some plant species (Proebsting et al., 1990; Rapala-Kozik et al., 2012). Some in vitro studies have shown that thiamin is one of the potential antioxidants known so far (Tunc-Ozdemir et al., 2009), so by virtue of being an antioxidant it can play a key role in plant stress tolerance.

Nitric oxide (NO), being aubiquitous signal in plants,  is believed to play a vital role  in plant responses to environmental stresses (Freschi 2013; Liu et al., 2014). NO has also been reported to mitigate the stress-induced oxidative stress in plants (Chen et al.,. 2010; Mihailovic and Drazic, 2011).

The present study was conducted to examine to what extent salinity stress has triggered oxidative stress in maize plants and whether an extra supply of combined nitric oxide and thiamin could mitigate the adverse effects of salt stress on antioxidative defense system.


Materials and methods

Plant growth conditions

The study was carried out during May and June 2013 at the Research Station of the Agriculture Faculty, University of Harran (Turkey) under glasshouse conditions maize (Zea mays L. cvs. DK5783 and Apex 836). Seeds of each of two maize cultivars DK 5783 and Apex 836 were planted at a rate of five seeds per pot. Each  pot contained 10 kg air-dried loamy clay soil. The chemical characteristics of the soil used were as follows. pH (1.2.5 water, v.v)  7.3, electrical conductivity  0.45 dS/m, N  and K 1.25 and 1.40 g/kg, respectively. Nitrogen, phosphorus, and potassium were added to the soil  at the rates of 100, 50 (P2O5)and 120 (K2O) mg/kg, respectively. Salt treatment in the soil was developed before planting the seeds by adding appropriate amount of NaCl to irrigation water to keep salt level to 100 mM. The electrical conductivity of the soil was checked weekly during the whole experimentation.

After germination, thinning of the seedlings was done to maintain  three seedlings in each pot, and then they were further allowed to grow for 35 days under 27±2 C and  mean daytime RH 60-70%. The experiment was arranged in the glasshouse in a randomized complete block design replicated thrice. Every day an aliquot of 50 mL to 500 mL of water depending on plant size and time from planting was applied to each pot.

Two levels (100 and 125 mg/l) of thiamin and two (3 and 6 mg/l) of nitric oxide   in combination were applied as presowing seed treatment or through leaves to 10 days old seedlings. The salt used for NO source was sodium nitroprusside (SNP). All seed samples were sterilized with NaOCl solution (1% v/v) before use. For presowing seed treatment, the seed samples were soaked for 24 h in 3 mg/l NO +100mg/l TA or 6 mg/l NO +125 mg l-1 TA solution. However, for foliar spray, the plants were applied 50 ml per pot of NO and TA solution prepared in 0.01% tween-20.The foliar spray was initiated when the seedlings were 10 days old and continued up to day 35. Data for different growth and physio-biochemical attributes were measured before terminating the experiment.


Chlorophyll fluorescence measurements

Maximal quantum yield (Fv/Fm) was measured using a portable chlorophyll fluorometer (Photosynthesis Yield Analyzer Mini-PAM, Walz, Germany). The data were recorded using previously dark-adapted leaves for 30 min.


Leaf free proline content

The protocol described by Bates et al. (1973) was employed to determine leaf free proline content. The filtrate obtained by grinding 0.5 g fresh leaf sample  in 10 ml of 3% aqueous sulfosalicylic acid, was reacted appropriately with   acid-ninhydrin solution and  glacial acetic acid. The mixture was subjected to 100 oC for one hour. Thereafter the mixture was treated with 4 ml of toluene and its absorbance read at 520 nm.


Leaf osmolality

The leaf samples were placed in liquid nitrogen for 72 h and thereafter they were slightly pressed to extract the sap. The sap so extracted was centrifuged at 5,000g for 5 minutes. The filtrate was fed to a Cryo-osmometer (Osmomat 030, Ganotec).to determine osmomolality.


Soluble protein content

The protocol described by Bradford (1976) was used to determine soluble protein content of fresh leaf samples.


Antioxidant enzyme assays

For determining the activities of different enzymatic antioxidants 0.5 g of fresh leaf material was triturated in 50 mM sodium phosphate buffer (pH 7.0) containing 1% soluble polyvinyl pyrolidine. The mixture was centrifuged at 20,000 g for 15 minutes at 4° C and then used to determine the activities of the key antioxidant enzymes such as POD, CAT and SOD.

The method described by Kraus and Fletcher (1994) was used to determine CAT activity, whereas that of Beauchamp and Fridovich (1971) was used to determine the SOD actvity. The POD activity was determined following the method described by Chance and Maehly (1955).


Inorganic nutrient analysis and dry weight measurement

Plant samples were dried well in an oven at 65 oC and their dry weights measured. For determining inorganic nutrients, the well ground plant samples were placed in a muffle furnace at 550 °C for 6 h. The ash was dissolved in 5 mL of 2 M hot HCl and the final volume was raised to 50 mL by adding distilled water (Chapman and Pratt 1982).  An ICP was used to quantify sodium (Na+), Ca2+, and K+.


Determination of lipid peroxidation and hydrogen peroxide

The leaf malondialdehyde (MDA) content, a product of lipid peroxidation, was determined using the method described by Cakmak and Horst (1991) with some modifications as suggested by Weisany et al. (2012).

The quantification of H2O2 in leaves was carried out following Loreto and Velikova (2001). Half gram of fresh leaves was triturated in 3 mL of 1% (w/v) tri-chloro-acetic acid (TCA). The mixture was  centrifuged at 10,000 rpm  for 10 minutes. 0.75 mL of 10 mM K-phosphate buffer (pH 7.0) and 1.5 mL of 1M KI were added to an aliquot of 0.75 ml of the supernatant and then its OD read at 390 nm.

Statistical analysis

A statistical package SAS version 9.1 was employed to work out analysis of variance of data for each attribute. The Least Significance Difference (LSD) test at P = 0.05 was employed to determine the differences among data means within each parameter.



While screening nine maize cultivars at the germination stage (unpublished data) cv. DK 5783 was found to be salt tolerant and cv. Apex 836 as salt sensitive. Also while optimizing the levels of NO and TA, out of six two levels (3 and 6 mg l-1) of NO and two (100 and 125 mg l-1) TA in combination were found more effective in promoting seed germination of the two maize cultivars under saline conditions and thus they were used for further glasshouse studies.


Some key growth parameters

Salt stress caused a significant reduction in plant dry biomass and maximum fluorescence yield (Fv/Fm) in both maize cultivars (Tables 1 and 2). However, these salt-induced reductions were less in cv. DK 5783 than those in cv. Apex 836.Combined application of varying levels of NO and TA as presowing seed treatment or foliar spray improved both attributes in the salt stressed plants of both maize cultivars, however, the effect of the former mode of application was slightly better than that of the latter in improving plant dry biomass  in the salt-stressed maize plants.

Leaf osmolality (LO) and free proline content increased in both maize cultivars subjected to saline stress Cv. Apex 836 had slightly greater leaf osmomolality than that in DK 5783, whereas the reverse was true for proline content (Table 2). Both modes of application of NO and TA considerably decreased proline content and leaf osmolality in salt stressed plants of both maize cultivars exposed to saline regimes.


Inorganic elements

Saline stress caused a marked increase in leaf Na+ in the plants of both maize cultivars, however, it was significantly higher in the salt sensitive cultivar Apex 836 than that  in the salt tolerant cultivar DK5783 (Table 3). Both presowing seed treatment and foliar spray of both growth substances  in combination  significantly decreased leaf  Na+. Salinity stress also lowered down leaf Ca2+ and K+ content in both maize cultivars, but these reductions being greater  in the salt sensitive cultivar Apex 836. Overall, pre-sowing seed treatment  with combined NO and TA was more efficient in decreasing leaf Na+, and  foliar application in enhancing leaf Ca2+ and K+ contents.


Antioxidant enzymes and ROS 

Salt stress caused a significant improvement in  the activities of SOD, CAT  and POX in both maize cultivars and these enhancement being greater  in  cv. DK 5783 as compared to  those in cv. Apex 836. Both modes of combined NO and TA applications decreased the activities of all antioxidant enzymes examined (Table 4).

Leaf H2O2 and MDA contents were found to increase in both cultivars exposed to salt and they being higher in the salt sensitive cultivar (Table 5). Both modes of applications of the two growth regulators, i.e. NO and TA in combination, caused a marked reduction in the levels of leaf H2O2 and MDA contents in both maize cultivars. However, presowing seed treatment of the two growth regulators was found to be more efficient in decreasing the ROS than foliar application.



Enhanced salinity tolerance in different plants has been reported to be associated with enhanced growth and yield as well as a variety of physio-biochemical adaptations/mechanisms (Batool et al., 2014; Gupta and Huang, 2014) including low uptake of toxic ions (Kader and Lindberg, 2005; Sabir and Ashraf 2007), low accumulation of ROS (Fayez and Bazaid 2014), upregulation of oxidative defense system (Ashraf 2009; Akram et al., 2012), high accumulation of key osmoprotectants (Ashraf and Foolad, 2007, Kaya et al., 2013a), efficient photo-respiratory machinery




(Muranaka et al., 2002), etc. However, different plant species/cultivars respond differently with regard to these processes. In the present study, although plant dry weight of both maize cultivars reduced significantly under saline conditions, more reduction was observed in the salt sensitive cv. Apex 836 as compared to that in the salt tolerant cv. DK 5783. Exogenous combined application of nitric oxide (NO) and thiamin (TA) improved dry weights of both maize cultivars under saline conditions. Although the growth regulatory effects of NO and TA have been already assessed individually and both have been shown effective in approving growth and yield under saline conditions, little information exists in the literature on the effects of these two bioregulators applied in combination. For example, NO-induced growth improvement has already been observed in different plant species such as rice (Habib and Ashraf, 2014), tomato (Wu et al., 2011), wheat (Xie et al., 2008), Kandelia obovata (Chen et al., 2014), and cucumber (Fan et al., 2012). Exogenously applied thiamin has also been reported to be effective in regulating plant growth and some key processes such as decreased membrane damage, low uptake of Na+, enhanced uptake of K+, and increased leaf relative water contents and total amino acids in sunflower plants under saline stress (Sayed and Gadallah, 2002).

In the present study, maximum fluorescence yield (Fv/Fm) of both maize cultivars decreased significantly, and the maximum reduction was observed in the salt sensitive maize cv. Apex 836. Exogenously-applied varying levels of TA and NO significantly improved Fv/Fm in both cultivars under saline conditions. Salinity-induced reduction in Fv/Fm could be due to the inactivation and destruction of PSII reaction center (Santos et al., 2001; Yan et al., 2012; Ashraf and Harris, 2013; Dong et al., 2014). However, combined TA and NO application ameliorated the adverse effects of salt on this fluorescence parameter. Analogous to this in another study on cotton plants, Dong et al. (2014) inferred that exogenously applied NO improved the activity of PSII under saline conditions. Furthermore, they observed that the salt-induced reduction in Fv/Fm was due to the inhibitory effects of salinity on photosynthetic machinery of cotton plants. It has also been reported that thiamin plays an important role in protecting PSI by minimizing over-accumulation of H2O2 contents as well as up-regulation of oxidative pentose phosphate pathway in plants (Goyer, 2010). Thus, the TA-induced increase in Fv/Fm in both maize cultivars under saline conditions may have been due to the protective effect of TA on D1 protein of PSII (Ashraf and Harris, 2013). In another study, grain soaking in thiamin at the rate of 0.3 mM alleviated the adverse effects of salt stress by reducing NaCl-induced accumulation of proline in wheat seedlings (Al-Hakimi and Hamada, 2001).

Salinity stress increased proline content in both maize cultivars which is parallel to some earlier reports in which salt-induced increase in proline contents has been observed in different plants under saline conditions, e.g. sunflower (Akram et al. 2012), maize (Cha-um and Kirdmanee, 2009), Jerusalem artichoke (Huang et al., 2013), okra (Saleem et al., 2011), cauliflower (Batool et al., 2013), eggplant (Shaheen et al., 2013), etc. Proline is one of the essential osmoprotectants that play a critical role in osmoregulation in different plants under saline stress (Ashraf and Foolad, 2007; Sabir et al., 2011; Akram et al.,. 2012). High accumulation of proline in plant tissues is generally associated with enhanced salt tolerance (Ashraf and Foolad 2007; Banu et al., 2012). In the present study, proline contents were higher in the relatively salt tolerant maize cv. DK 5783 and both seed and foliar applications of NO + TA reduced proline contents in both maize cultivars. A number of reports available in the literature show that both NO and TA have multifunctional roles when applied individually as an external application. However, not a single report could be deciphered from the literature on the effects of combined application of NO and TA on proline accumulation, therefore, there is a need to explore the effects of NO and TA alone or in combination in different plant species. Recently, Fan et al. (2013) suggested that exogenously applied NO alleviated the salt-induced damages to cucumber seedlings and they attributed this alleviation due to the regulation of the content and proportions of different polyamines (Fan et al. 2013).

In the current study, the salt sensitive cv. Apex 836 had higher tissue Na+ concentration than that in the salt tolerant cv. DK5783. Both modes of combined application of NO and TA in different doses reduced tissue Na+ content. Furthermore, Ca2+ and K+ concentrations in the leaves of both maize cultivars decreased under saline conditions. Seed application of NO and TA in combination was more effective than foliar application in reducing Na+ contents. While working with wheat plants, Kausar et al. (2013) observed that varying levels of NO caused a slight reduction in Na+ accumulation in root and shoot tissues of wheat plants, however, exogenously applied NO enhanced the activities of SOD, POD and CAT enzymes, and soluble protein and proline contents in wheat plants under saline and non-saline conditions which were pointed out to be major factors of improving growth in wheat plants under saline stress. Similarly, Dong et al. (2014) have observed NO-induced improvement in K+ content, antioxidant enzyme activities, while decrease in Na+/K+ ratio, TBARS and MDA contents under saline regimes in cotton plants.

Hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents increased in both maize cultivars subjected to saline conditions. Of both maize cultivars, salt sensitive cv. Apex 836 accumulated more H2O2 and MDA contents than did the salt tolerant maize cultivar under saline conditions. Foliar and seed combined applications of NO + TA reduced H2O2 and MDA contents in the leaves of both maize cultivars. It has been speculated that efficient scavenging of ROS produced under saline conditions requires the action of several enzymatic and non-enzymatic antioxidants (Ashraf 2009; Akram et al., 2012; Sharma et al., et al. 2012; Kaya et al.,. 2013b). In the present study, seed application of combined NO and TA at varying doses was more effective in reducing the H2O2 and MDA contents than the foliar supplementation. During a study with Arabidopsis thaliana, it has been observed that under adverse environmental conditions, accumulation of thiamin increased which induced tolerance against salinity-induced oxidative stress by decreasing the over-accumulation of ROS particularly that of H2O2 (Tunc-Ozdemir et al., 2009). In the present study, activities of CAT, SOD and POX were found to be accelerated in both maize cultivars on their exposure to saline stress. However, seed and foliar applications of combined NO and TA suppressed the activities of all these antioxidant enzymes. In contrast, the supplementation of SNP, the source of NO (1.0 mM) suppressed the superoxide anions synthesis but enhanced the activities of antioxidants such as CAT and SOD which suggested that the application of SNP at moderate concentration protected plants from oxidative damage under salt stress (Dong et al., 2014).



Overall, salinity stress caused high reductions in plant dry matter and Fv/Fm, but increased proline content and leaf osmolality in both maize cultivars. These suppressions were greater in the salt sensitive Apex 836 than those in the salt tolerant DK 5783. Application of NO and TA in combination as presowing seed treatment or foliar application were effective in mitigating salinity-induced suppression in shoot growth with the exception of foliar application of NO an TA (6+125 mg/l) being not effective for cv. DK 5783. Seed treatment with both levels of combined NO and TA was more effective in improving dry weights than that with foliar treatments. Salt stress increased leaf Na+ contents, but reduced those of  K+ and Ca2+ in both cultivars. Both modes and combined application of NO and TA increased tissue Ca2+ and K+ contents, whereas reduced Na+ in the salt stressed maize plants. Salt stress caused oxidative stress in plants by generating the accumulation of ROS such as MDA and H2O2. In both cultivars increased activities of POD, CAT and SOD were observed under saline regime. Externally-applied NO and TA in combination induced growth improvement in maize was ascribed  to reduced leaf Na+, osmolality, MDA and H2O2 levels, and modulated activities of  CAT, SOD and POD as well as increased Fv/Fm under saline conditions.



This research was supported by The Scientific and Technical Research Council of Turkey (TOVAG-112O375).



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