Hydrogen peroxide pretreatment induces osmotic stress tolerance by influencing osmolyte and abscisic acid levels in maize leaves

Abstract

Hydrogen peroxide (H₂O₂) functions as a signal molecule in plants under abiotic and biotic stresses. Leaves of detached maize (Zea mays L.) seedlings were used to study the function of H₂O₂ pretreatment in osmotic stress resistance. Low H₂O₂ concentration (10 mM) which did not cause a visual symptom of water deficit (leaf rolling) was applied to the seedlings. Exogenous H₂O₂ alone increased leaf water potential, endogenous H₂O₂ content, abscisic acid (ABA) concentration, and metabolite levels including soluble sugars, proline, and polyamines while it decreased lipid peroxidation and stomatal conductance. Osmotic stress induced by polyethylene glycol (PEG 6000) decreased leaf water potential and stomatal conductance but enhanced lipid peroxidation, endogenous H₂O₂ content, the metabolite levels, and ABA content. H₂O₂ pretreatment also induced the metabolite accumulation and improved water status, stomatal conductance, lipid peroxidation, ABA, and H₂O₂ levels under osmotic stress. These results indicated that H₂O₂ pretreatment may alleviate water loss and induce osmotic stress resistance by increasing the levels of soluble sugars, proline, and polyamines thus ABA and H₂O₂ production slightly decrease in maize seedlings under osmotic stress.

Keywords:

• abscisic acid
• H₂O₂ pretreatment
• osmotic stress
• polyamine
• proline
• soluble sugar

Introduction

Drought stress is the primary cause of crop loss across the world, reducing average yields in most major crop plants. Under drought or water-deficit stress condition, plants exhibit a wide range of biochemical and physiological responses at the molecular, cellular, and whole-plant levels, such as changed leaf water potential (ψ _leaf) and stomatal conductance (g _s), increased abscisic acid (ABA) content, and the accumulation of metabolites including soluble sugars, proline, and amines such as glycine betaine and polyamines. These metabolites accumulate under water-deficit stress and function as osmolytes that contribute to the maintaining of cell turgor, antioxidants, or scavengers that help plants to avoid and/or tolerate stresses (Mahajan & Tuteja 2005; Seki et al. 2007; Fariduddin et al. 2013). The changes in these metabolites at the cellular level are associated with protecting cellular function with maintaining the structure of cellular components. ABA is also essential for various stress responses, including stomatal closure, stress-responsive gene expression, and metabolic changes (Seki et al. 2007).

The production of reactive oxygen species (ROS) is a common phenomenon in plants under biotic and abiotic stress conditions. ROS can induce cellular damage by degradation of proteins, inactivation of enzymes, alterations in the gene, and interfere in various pathways of metabolic importance (Mahajan & Tuteja 2005). To scavenge the ROS, plants possess antioxidant enzymes, including superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, catalase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase, and antioxidants such a reduced glutathione and ascorbate. Hydrogen peroxide (H₂O₂), a major kind of ROS plays an important role in signal transduction for abiotic stress tolerance, although H₂O₂ is toxic at high concentrations (Foyer & Noctor 2009). Some authors suggested that the application of H₂O₂ at low concentrations could improve plant tolerance to abiotic stresses such as drought (He et al. 2009), heat (Gao et al. 2010), and heavy metal stresses (Hu et al. 2009). Hydrogen peroxide induced increases in antioxidant enzyme activities of plants, which are subjected to abiotic stress factors, has also been reported earlier. For instance, Liu et al. (2010) reported that pretreatment of two cucumber varieties with H₂O₂ improved osmotic stress resistance by activating antioxidant system. However, to the best of our knowledge, there is little information available on the effects of H₂O₂ pretreatment on metabolite levels including soluble sugars, proline, and polyamines and ABA content in plants under osmotic stress.

Despite a large number of reports detailing plant tolerance mechanisms to osmotic stress, plant responses to H₂O₂ pretreatment under osmotic stress are not still obvious. The aim of this paper was to investigate effects of H₂O₂ pretreatment for improving osmotic stress tolerance of maize seedlings and the role of some metabolites such as proline, soluble sugars, and polyamines and phytohormone ABA in this process.

Materials and methods

Plant material, growth, and H₂O₂/osmotic stress treatment

Maize (Zea mays L.) seeds were provided from Black Sea Agricultural Research Institute, Samsun, Turkey, and were surface sterilized with 0.1% HgCl₂ for 3 min followed by repeated washings with sterilized distilled water. Seeds were sown in plastic pots (16 cm height, 18 cm top, and 12 cm bottom diameter) containing peat and sand (5:1). Seedlings were grown in a growth chamber with the following parameters: 16 h light and 8 h darkness at 25 ± 2°C, relative humidity 60 ± 5%, and photon flux density at the surface of the leaves 400 µmol m⁻²s⁻¹. When the fourth leaves were fully expanded, the seedlings were excised 2 cm high from soil and rinsed in distilled water in test tubes wrapped with aluminum foil for 1 h to relieve the effects of wound stress due to excision. After that the seedlings were pretreated with distilled water with or without 10 mM H₂O₂ for 6 h, and then they were submitted to the osmotic stress treatments with 3% polyethylene glycol 6000 (PEG 6000) containing osmotic potential of −0.3 MPa for 12 h or kept in distilled water for 12 h in the growth chamber with an air temperature of 25°C ± 2 and relative humidity 50 ± 2%. Hence, the seedlings were submitted to four treatments: treated with distilled water (Control), pretreated with H₂O₂ and not osmotic stressed (H₂O₂), osmotic-stressed only (PEG), and pretreated with H₂O₂ and osmotic stressed (H₂O₂ + PEG). After the treatments, samples of the second leaves were used for the following analyses.

Measurements of leaf water status

Leaf water potential (ψ _leaf) was measured with a PSYPRO thermocouple psychrometer (Wescor, USA). Discs about 6 mm in diameter were cut from the youngest fully expanded leaves of the plants and sealed in the C-52 psychrometer chamber. Samples were equilibrated for 60 min before the readings were recorded by a water potential data logger in the psychrometric mode.

Degree of leaf rolling (%), a visual indication of internal plant water deficit (Kadioglu & Terzi 2007), was measured as a percentage reduction in leaf width of leaf midportion by the rolling according to Saglam et al. (2008).

Stomatal conductance

Stomatal conductance (g _s) was monitored by an AP4 dynamic diffusion porometer (Delta T Devices, UK) after calibration with a standard calibration plate following manufacturer's instructions.

Lipid peroxidation

Lipid peroxidation was measured in the term of malondialdehyde (MDA) content (ε = 155 mM⁻¹ cm⁻¹), a product of lipid peroxidation, following the method of Heath and Packer (1968). Leaf samples (0.5 g) were homogenized in 10 ml of 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 15,000g for 5 min. To the 1 ml aliquot of the supernatant, 4 ml of 0.5% (w/v) thiobarbituric acid (TBA) in 20% (w/v) TCA was added. The mixture was heated at 95°C for 30 min and then quickly cooled in an ice bath. After centrifugation at 10,000g for 10 min, the absorbance of the supernatant was recorded at 532 and 600 nm. The MDA content was determined using a standard curve and expressed as nmol MDA g⁻¹ dry weight.

H₂O₂ content

Endogenous H₂O₂ content was determined according to modified method of Velikova et al. (2000). Leaves (0.25 g) were ground in 3 ml of 5% TCA with 0.1 g activated charcoal at 0°C. The homogenate was centrifuged at 12,000g for 15 min. To 0.5 ml aliquot of the supernatant, 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0) and 0.75 ml of 1 M KI were added. The absorbance was measured at 390 nm, and H₂O₂ content was expressed as µmol g⁻¹ dry weight.

Determination of proline and total soluble sugars

Determination of proline content was achieved according to the method of Bates et al. (1973). Dry leaf sample (0.2 g) was homogenized in 10 ml of 3% (w/v) aqueous sulfosalicylic acid solution. Homogenate was centrifuged at 10,000g for 10 min. Supernatant was filtered through Whatman No 2 filter paper. Later, 1 ml filtrate was mixed with equal volumes of glacial acetic acid and ninhydrin reagent (1.25 g ninhydrin, 30 ml of glacial acetic acid, 20 ml 6 M H₃PO₄), and incubated for 1 h at 100°C. The reaction was stopped by placing the test tubes in an ice bath. The samples were rigorously mixed with 3 ml toluene. After 50 min, the light absorption of the toluene phase was estimated at 520 nm on a UV-VIS spectrophotometer. The proline concentration was determined using a standard curve. Free proline content was expressed as µg proline g⁻¹ dry wt.

Total soluble sugar content was determined by phenol sulfuric acid method (Dubois et al. 1956). Dry leaves (0.1 g) were extracted with 5 ml of 80% ethanol by boiling the samples in glass tubes in a 95°C water bath for 10 min. After extraction, the tubes were centrifuged at 580g for 5 min, and the supernatants of the extractions were used for sugar analysis. To 900 µl of distilled water, 100 µl of the sample was added, and the mixture was shaken vigorously. To 1 ml of sample, 1 ml of 5% phenol, and 5 ml of H₂SO₄ were added, and the mixture was stirred. After cooling at room temperature for 15 min, the absorbance of the sample was recorded at 490 nm.

Determination of polyamines

Extraction of leaf samples and High Performance Liquid Chromatography (HPLC) determination of biogenic amines (putrescine (Put), spermine (Spm), and spermidine (Spd)) were carried out according to the procedures developed by Ben-Gigirey et al. (1998). To extract biogenic amines, 10 ml of 0.4 M perchloric acid containing a known amount of 1,7-diaminoheptane as an internal standard was added to 5 g of leaf samples, and the mixture was homogenized for 3 min. The homogenate was centrifuged at 3000g at 4°C for 10 min. The supernatant was collected, and the residue was extracted twice with the same volume of 0.4 M perchloric acid solution. All supernatants were combined, and the final volume was adjusted to 25 ml with 0.4 M perchloric acid. The extract was filtered through Whatman paper No. 1. One milliliter of each of the extract was used for the HPLC analysis followed by derivatization with dansyl chloride.

Derivatization of biogenic amines was carried out according to developed method of Ben-Gigirey et al. (1998). One milliliter of each extracted sample or standard amine solution was mixed with 200 µl of 2 M sodium hydroxide and 300 µl of sodium hydrogen carbonate. Two milliliters of a dansyl chloride solution (10 mg/ml in acetone) was added to the mixture and then incubated in water bath at 40°C for 45 min. One hundred microliters of 25% ammonium hydroxide were added to stop the reaction and to remove residual dansyl chloride. After 30 min incubation at room temperature, the final volume was adjusted to 5 ml by adding acetonitrile. Finally, the mixture was centrifuged at 2500g for 5 min, and the supernatant was filtered through 0.2 µm-pore-size filter (Pall Co., Acrodisc Syringe Filters, 25 mm, 0.2 µm, Woongki Science Co., Seoul, Korea). The filtered supernatant was kept at −25°C until assayed by HPLC. All samples were subjected to HPLC injection.

Quantitative analyses of polyamines were carried out using a HPLC unit (Shimadzu, LC 20 AT/Prominence, Japan), consisted of two pumps and a UV–VIS detector. Separation was achieved using a C₁₈ Supelco column 5 µm (250 mm × 4.6 mm). The mobile phase was ammonium acetate (0.1 M; solvent A) and acetonitrile (solvent B) at the flow rate 1 ml min⁻¹ with gradient elution program for 35 min. The sample volumes injected were 10 µl. The samples were monitored at 254 nm. Each HPLC run took about 35 min, and afterward the column was conditioned again with the mixture of 65% solvent A and 35% solvent B.

Determination of ABA

Leaves were grounded in liquid nitrogen, homogenized in distilled water at a ratio of 1:7 (w/v), and placed overnight in the dark at 4°C. The extracts were centrifuged at 10,000g for 10 min at 4°C, and the resulting supernatant was diluted four times in standard TBS buffer. ABA in these extracts was quantified using the Phytodetek ABA enzyme linked immunosorbent assay (ELISA) kit (Agdia/Linaris) according to the manufacturer's instructions.

Statistical analysis

All experiments were repeated three times with five biological replicates. Variance analysis of mean values was performed with Duncan Multiple Comparison test (one-way analysis of variance [ANOVA]) using SPSS software for Microsoft Windows (Ver. 13.0, SPSS Inc., USA). Statistical significance of the means between control and treatments were evaluated at the 5% (P < 0.05) probability level.

Results

ψ_leaf, g_s, and leaf rolling

Low H₂O₂ concentration (10 mM) was preferred for exogenous H₂O₂ pretreatment because leaf rolling used as a visual indication of water deficit was observed in concentrations higher than 10 mM H₂O₂ (data not presented). Exogenous H₂O₂ alone significantly increased ψ_leaf but decreased g_s value in the seedlings compared to the control. Osmotic stress supplied by application of 3% PEG 6000 decreased ψ_leaf and g_s (Figure 1), and thus induced leaf rolling (Table 1). Leaf rolling was observed about 120 min after PEG treatment. In course of time, leaf rolling degree (%) increased (Table 1). Pretreatment of H₂O₂ (H₂O₂ + PEG) improved ψ_leaf and g_s values (Figure 1). Hence, leaf rolling delayed in pretreated seedlings (H₂O₂ + PEG) compared to osmotic-stressed only ones (PEG) (Table 1). The degree of leaf rolling (%) was also low in pretreated seedlings (Table 1).

Figure 1. Effect of exogenous H₂O₂ on leaf water status (Ψ_leaf) (a) and stomatal conductance (g_s) (b) in the leaves of detached maize seedlings under osmotic stress conditions.

Note: The seedlings were submitted to four treatments: treated with distilled water (control), pretreated with H₂O₂ and not osmotic stressed (H₂O₂), osmotic-stressed only (PEG), pretreated with H₂O₂ and osmotic stressed (H₂O₂ + PEG). Vertical bars represent standard deviation of the means of six replicates. Different letters denote significant differences among different treatments at p < 0.05.

Table 1. Effect of exogenous H₂O₂ on leaf rolling in detached maize seedlings under osmotic stress conditions.

Treatments	Beginning time of leaf rolling (min.)	Degree of leaf rolling (%) 18 hour after the treatments
Control	–^a	0
H2O2	–^a	0
PEG	120 ± 15	34 ± 4
H2O2 + PEG	180 ± 15	22 ± 5

Note: The seedlings were submitted to four treatments: treated with distilled water (control); pretreated with H₂O₂, and not osmotic stressed (H₂O₂); osmotic-stressed only (PEG); pretreated with H₂O₂ and osmotic stressed (H₂O₂ + PEG). Data are mean ± SD (n = 6).

^a No rolling.

Lipid peroxidation and H₂O₂ content

As shown in Figure 2, exogenous H₂O₂ alone decreased MDA content. The exposure of osmotic stress greatly increased MDA content in maize seedlings. H₂O₂ pretreatment also alleviated lipid peroxidation under osmotic stress.

Figure 2. Effect of exogenous H₂O₂ on lipid peroxidation (a) and endogenous H₂O₂ content (b) in the leaves of detached maize seedlings under osmotic stress conditions.

Foliar H₂O₂ content after exogenous application was slightly elevated in comparison to control seedlings. Osmotic stress caused a sharp H₂O₂ accumulation in the seedlings. The H₂O₂ pretreatment brought a substantial decline of endogenous H₂O₂ level under osmotic stress (Figure 2).

Proline and total soluble sugars

The proline contents of H₂O₂-treated only seedlings (H₂O₂) interestingly increased as compared to the control seedlings. PEG exposure with H₂O₂ pretreatment also increased proline content. There is no significant increase in pretreated seedlings (H₂O₂ + PEG) compared to osmotic-stressed only ones (Figure 3).

Figure 3. Effect of exogenous H₂O₂ on proline (a) and total soluble sugar contents (b) in the leaves of detached maize seedlings under osmotic stress conditions.

The total soluble sugar content of the control seedlings was measured as 118.9 ± 0.2 mg 100 g⁻¹ dry weight. Exogenous H₂O₂ alone (H₂O₂) significantly increased total soluble sugar content. Osmotic stress enhanced the soluble sugar content more than H₂O₂-treated only group. H₂O₂ pretreatment also apparently induced the soluble sugar production in maize seedlings under osmotic stress (Figure 3).

Polyamines

H₂O₂ treatment alone (H₂O₂) induced polyamine accumulation as compared to the control group. Osmotic stress treatment alone (PEG) also caused a significant increase in polyamine contents in maize leaves. For instance, the increases in Put, Spd, and Spm contents in osmotic-stressed only seedlings were approximately 57, 81 and 29% of the control, respectively. Furthermore, H₂O₂ pretreatment increased overproduction of polyamines under osmotic stress. Thus, H₂O₂ pretreatment increased leaf Put, Spd, and Spm contents by 72, 106, and 68% of the control under the stress, respectively (Figure 4).

Figure 4. Effect of exogenous H₂O₂ on polyamine content in the leaves of detached maize seedlings under osmotic stress conditions.

ABA

ABA content clearly increased in the H₂O₂-treated only and osmotic-stressed only groups compared to the control. When the H₂O₂-osmotic-stressed seedlings (H₂O₂ + PEG) was compared with the osmotic-stressed only (PEG) seedlings, ABA content in H₂O₂ + PEG was slightly lower than those of osmotic-stressed seedlings (Figure 5).

Figure 5. Effect of exogenous H₂O₂ on ABA content in the leaves of detached maize seedlings under osmotic stress conditions.

Discussion

The present study explores the ameliorative effect of H₂O₂ on maize seedlings subjected to osmotic stress and the role of some metabolites in this process. Results of the present study showed that H₂O₂ treatment only caused increased ψ _leaf thanks to stomatal closure partially, and osmolytes accumulation, maintaining turgor potential and osmotic adjustment. Indeed, it is known that one way to maintain the turgor potential is by osmotic adjustment. Osmotic adjustment is the net accumulation of solutes in a cell of a plant in response to a fall in the water potential of the cell's environment. As a consequence of this net accumulation, the osmotic potential of the cell is lowered, which in turn attracts water into the cell, and this tends to maintain turgor pressure (Kirkham 2011). Also we determined that the leaves pretreated with 10 mM H₂O₂ (H₂O₂ + PEG) exhibited improved ψ _leaf and g _s as compared to osmotic-stressed only leaves (PEG). As a result of decreased water loss with H₂O₂ pretreatment, leaf rolling, a visual indication of water loss in plants, retarded and the rolled leaves slightly opened under osmotic stress (Table 1 and Figure 1). Similar results were previously observed in osmotic-stressed wheat seedlings (He et al. 2009) and drought-stressed soybean plants (Ishibashi et al. 2011), but in those studies, parameter of leaf rolling was not used to evaluate water status in the plants. Here, we can express that the ameliorative effect of H₂O₂ on maize plants under osmotic stress can be visually observed by leaf rolling. H₂O₂ pretreatment may limit water loss by increasing the levels of metabolite involved in osmotic adjustment such as soluble sugar, proline, and polyamines under osmotic stress and thus contribute to the stress acclimation of maize plants.

We observed that exogenous applications of H₂O₂ at low concentration significantly decreased lipid peroxidation of maize plants under both osmotic-stressed and nonstressed conditions. Similar results in membrane damage were reported in wheat seedlings under drought by He et al. (2009) and in cucumber plants under osmotic stress by Liu et al. (2010). Compared with the untreated seedlings, decrease in MDA level with 10 mM H₂O₂ pretreatment under osmotic stress might be an induction that exogenous H₂O₂ at low concentrations alleviated membrane damages by inducing the metabolite levels involved in osmotic adjustment.

The role H₂O₂ in stomatal closure was described previously (e.g. Pei et al. 2000; Zhang et al. 2001). Accordance with g _s values, we also determined that endogenous H₂O₂ content slightly increased in H₂O₂-treated only seedlings compared to control. The increase might be a result from penetration of exogenous H₂O₂ to leaf tissues. Also, we observed that endogenous H₂O₂ level increased under osmotic stress. However, H₂O₂ content was significantly low in plants subjected to a combined treatment with H₂O₂ and osmotic stress in comparison with the PEG treatment. He et al. (2009) found the same results in drought-stressed wheat seedlings had arisen from H₂O₂-treated seeds. H₂O₂ accumulation was also alleviated in cucumber leaves under osmotic stress in the case of foliar spray of H₂O₂ (Liu et al. 2010). It was known that the endogenous H₂O₂ concentration depended on the balance between the rates of its production and its enzymatic and nonenzymatic utilization. Thus, our results indicated the induction of ROS scavenging compounds such as proline and polyamines in seedlings pretreated with H₂O₂ under osmotic stress.

As known, plants exhibit the accumulation of metabolites including proline, soluble sugars, and amines such as glycine betaine and polyamines under the various stress conditions (Seki et al. 2007). Proline can protect plants from the stress through different mechanisms, including osmotic adjustment, detoxification of ROS, protection of membrane integrity, and stabilization of proteins (Seki et al. 2007; Patade et al. 2011). On the other hand, to the best of our knowledge, only single study investigated the relationship among H₂O₂-pretreatment, the metabolites, ABA, and drought tolerance in plants (Abass & Mohamed 2011). As different from our study, these researches were used in the seeds of common bean for H₂O₂-pretreatment, and they were similarly recorded that H₂O₂-pretreatment of the seeds enhanced the level of some metabolites such as proline, soluble sugars, and polyamines in the shoots under drought condition. Our present data showed that proline content of H₂O₂-treated only seedlings was higher than the other treatments (Figure 3). Based on known properties of the proline, the increases might be ascribed in detoxification of exogenous H₂O₂. The H₂O₂ pretreatment under osmotic stress also induced proline accumulation compared to control seedlings. Thus, we suggested that exogenous H₂O₂ enhanced proline content to alleviate deleterious effects of the osmotic stress.

It is well known that sugar accumulation contributes to regulation of internal osmolarity and protection of the biomolecules and membranes (Seki et al. 2007). In the current study, osmotic stress increased the total soluble sugar content in maize leaves (Figure 3). Furthermore, we found that the highest soluble sugar content was in H₂O₂-pretreated plants under osmotic stress conditions. As mentioned above, Abass and Mohamed (2011) recorded that H₂O₂-pretreatment of common bean seeds led to a significant accumulation of soluble sugars in the shoots under drought stress. In line with these data, we could express that exogenous H₂O₂ applications might protect the water status of the tissues by increasing soluble sugars involved in osmotic regulation.

The results showed that the main polyamines, Put, Spd, and Spm significantly increased under the stress alone or in combination with pretreatment of H₂O₂. Generally, plant species are endowed with a great capacity to enhance polyamine biosynthesis in response to abiotic stresses including drought (Erdei et al. 1996). For the first time, in the present study, the obtained results showed that H₂O₂ pretreatment increased the overproduction of polyamines including Put, Spd, and Spm in osmotic-stressed maize seedlings treated with H₂O₂ at the vegetative stage. Similar to our results, Abass and Mohamed (2011) recorded the same results in common bean plants arising from H₂O₂-treated seeds grown under drought stress. So, we concluded here that as a result of H₂O₂ applications, increased polyamine content might protect the membranes by scavenging of ROS including H₂O₂. Indeed, we determined that exogenous H₂O₂ at low concentrations lessened membrane damages.

We also found that ABA content increased in H₂O₂-treated only seedlings compared to the control group. Accordingly, increased ABA slightly decreased g_s value. ABA was known as a factor responsible for stomatal closure (Seki et al. 2007). On the other hand, ROS-induced ABA accumulation in root tips of wheat seedlings was recorded by Zhao et al. (2001). As similar to results of Hassine et al. (2009), ABA accumulation in H₂O₂-treated only seedlings also stimulated synthesis of the osmolytes. Again, ABA, the key phytohormone involved in response to abiotic stress mainly triggers the drought protection (Mahajan & Tuteja 2005). Here, we determined that PEG treatment increased the content of ABA in maize leaves, while H₂O₂ pretreatment under osmotic stress conditions relieved the production of ABA. Accordingly, g_s values were affected by PEG and/or H₂O₂ applications. Also, our findings showed that H₂O₂ application improved water status under osmotic stress thus ABA content decreased compared to osmotic-stressed only plants due to alleviating of the stress. As similar to our findings, Zhang et al. (2007) recorded that ABA was gradually degraded upon removal of drought stress. On the other hand, high osmolyte level in H₂O₂ + PEG seedlings compared to PEG in spite of decreased ABA showed that other mechanisms induced by H₂O₂ might also induce osmolyte synthesis under osmotic stress. Therefore, the interactions among H₂O₂, ABA, and osmolytes should be studied further.

In summary, the results of this study indicate that delay in leaf rolling may be used for a visual symptom for early detection of stress relief. Furthermore, exogenous H₂O₂ treatment can enhance tolerance of maize seedlings to osmotic stress by increasing some metabolite and phytohormone levels and by decreasing lipid peroxidation and endogenous H₂O₂ content. The protection mechanism of H₂O₂ pretreatment against osmotic stress should be investigated in molecular level further.

Acknowledgments

This work is supported by Turkish National Science Foundation (Project No. 111T511). We thank Prof. Dr. Sevim Köse (Faculty of Marine Science, Karadeniz Technical University) and Assist. Prof. Dr. Serkan KORAL (Faculty of Fisheries, Rize University) for their assistance with analysis of HPLC.