J. Pineal Res. 2016; 60:206–216
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12304
Journal of Pineal Research
Glutathione-dependent induction of local and systemic defense against oxidative stress by exogenous melatonin in cucumber (Cucumis sativus L.) Abstract: Melatonin is involved in defending against oxidative stress caused by various environmental stresses in plants. In this study, the roles of exogenous melatonin in regulating local and systemic defense against photooxidative stress in cucumber (Cucumis sativus) and the involvement of redox signaling were examined. Foliar or rhizospheric treatment with melatonin enhanced tolerance to photooxidative stress in both melatonintreated leaves and untreated systemic leaves. Increased melatonin levels are capable of increasing glutathione (reduced glutathione [GSH]) redox status. Application of H2O2 and GSH also induced tolerance to photooxidative stress, while inhibition of H2O2 accumulation and GSH synthesis compromised melatonin-induced local and systemic tolerance to photooxidative stress. H2O2 treatment increased the GSH/oxidized glutathione (GSSG) ratio, while inhibition of H2O2 accumulation prevented a melatonininduced increase in the GSH/GSSG ratio. Additionally, inhibition of GSH synthesis blocked H2O2-induced photooxidative stress tolerance, whereas scavenging or inhibition of H2O2 production attenuated but did not abolish GSH-induced tolerance to photooxidative stress. These results strongly suggest that exogenous melatonin is capable of inducing both local and systemic defense against photooxidative stress and melatonin-enhanced GSH/GSSG ratio in a H2O2-dependent manner is critical in the induction of tolerance.
Introduction As sessile organisms, plants are constantly exposed to a variety of abiotic and biotic stresses throughout their lifecycle. Various environmental stresses inhibit plant growth via different mechanisms, but most directly or indirectly impair multiple physiological processes, such as photosynthesis, photorespiration, and respiration, and result in increased reactive oxygen species (ROS) levels, which disrupts ROS homeostasis [1, 2]. As strong oxidizers, ROS at excessive levels have detrimental effects as a result of their ability to cause lipid peroxidation, DNA damage, protein denaturation, carbohydrate oxidation, pigment breakdown, enzyme activity impairment, and eventually cell death [3]. Hence, the ability to maintain ROS at moderate levels is crucial to the mechanisms by which plants resist various abiotic and biotic stresses. Melatonin (N-acetyl-5-methoxytryptamine), a highly conserved molecule, is ubiquitous throughout animals, plants, and all the other kingdoms [4–6]. Since the first studies showed that melatonin indeed exists in plants [7, 8], numerous subsequent studies have proven melatonin plays important roles in the regulation of plant growth and development [5] and defense against biotic and abiotic stresses such as pathogens, extreme temperature, excess copper, salinity, and drought [9, 10]. The most frequently mentioned function of melatonin in plants is the effective 206
Hao Li1, Jie He1, Xiaozhen Yang1, Xin Li2, Dan Luo1, Chunhua Wei1, Jianxiang Ma1, Yong Zhang1, Jianqiang Yang1 and Xian Zhang1 1
Department of Horticulture, Northwest A&F University, Shaanxi, China; 2Key Laboratory of Tea Biology and Resources Utilization, Tea Research Institute, Chinese Academy of Agricultural Sciences, Ministry of Agriculture, Zhejiang, China
Key words: Cucumis sativus, glutathione, hydrogen peroxide, local/systemic defense, melatonin Address reprint requests to Xian Zhang, College of Horticulture, Northwest A&F University, No. 3, Taicheng Road, Yangling 712100, Shaanxi, China. E-mail: [email protected] Received October 9, 2015; Accepted December 11, 2015.
reduction of ROS accumulation and consequent alleviation of oxidative stress. Solid evidence suggests that melatonin can not only directly scavenge some ROS, but can also modulate antioxidant enzymes and enhance cellular antioxidants such as GSH [11, 12]. However, only a few studies have focused on the signaling pathways involved in melatonin-induced tolerance to abiotic stress in plants [13, 14]. High levels of ROS are harmful to plant tissues; however, extensive studies have shown that moderate ROS levels as second messengers are necessary for plant growth, development, and defending against stresses [15]. Foliar application of H2O2 induced antioxidant enzyme activity and alleviated paraquat- or cold-induced oxidative stress in cucumber leaves [16, 17]. Studies using vitro animal cells have reported that ROS production can be promoted by melatonin at low or high levels [12], although melatonin is a well-documented antioxidant. However, the induction of ROS by the pro-oxidant action of melatonin is not correlated with cytotoxicity, and it is both concentration dependent and cell-type dependent [12]. Accordingly, it will be of interest to examine whether ROS as signal substances are involved in induction of stress tolerance by melatonin in plants. At a whole-organism level, the resistances in various plant tissues across diverse environments are nonindependent. Plants can mount a systemic response to environmental
Signaling in melatonin-induced defense stresses in order to establish long-lasting protection against a broad spectrum of biotic or abiotic stresses in tissues far from the primary site of attack, and these mechanisms are termed ‘systemic acquired resistance’ (SAR) or ‘systemic acquired acclimation’ (SAA) [18, 19]. Both SAR and SAA are dependent on some movably systemic signals, which are transported from the primary site of stimuli to distant tissues in order to induce systemic responses. Salicylic acid is required for SAR, and Park et al. [20] provide evidence that methyl salicylate functions as the critical mobile signal. When plants are exposed to drought or salinity, abscisic acid is produced in roots and transported to leaf tissue in order to induce stomatal closure and antioxidant defense systems [21, 22]. In contrast, some regulators that are not transported, such as brassinosteroids, can induce systemic tolerance to abiotic or biotic stresses via movement of H2O2 acting as a second messenger [23]. Melatonin is amphiphilic, which enables it to cross cell membranes easily [24]. Moreover, melatonin and natural auxin, indole-3-acetic acid (IAA), share tryptophan as a biosynthetic precursor and have similar structural moieties. Therefore, melatonin, akin to IAA, is likely transported over long distances. This is consistent with increased melatonin content in the leaves of water hyacinths after exogenous melatonin application to growth media [25]. Similarly, NaCl stress increases endogenous melatonin content in both roots and cotyledons, thus implicating the transport of melatonin from roots to cotyledons [26]. However, whether melatonin could act as a mobile signal and induce systemic tolerance to stress is unclear. In the present study, we analyzed the photooxidative stress tolerance and antioxidant system changes in both melatonin-treated leaves and untreated leaves of cucumber plants that were treated with melatonin on specific leaves or roots. We also investigated the roles and relationships of H2O2 and melatonin-increased GSH/GSSG ratio in these plants. Our results strongly suggest that melatonin is capable of inducing both local and systemic defense against photooxidative stress. Furthermore, the melatoninenhanced GSH/GSSG ratio is critical in the induction of tolerance.
Materials and methods Plant material Cucumber (Cucumis sativus L. cv. Jinyan No. 4) was used because of its known sensitivity to photooxidative stress. Seeds were germinated in 50-hole plates filled with a mixture of peat, vermiculite, and perlite (2:1:1, v:v:v) and then germinated and grown into plants in a glasshouse. When the first true leaf was fully expanded, seedlings were transplanted into plastic pots (15 cm diameter and 15 cm deep, with one seedling per pot) containing a peat-vermiculite mixture (2:1, v:v). Plants were then maintained in growth chambers at a temperature of 25°C/20°C (day/night), photosynthetic photon flux density (PPFD) of 600 lmol/m2/s, relative humidity of 65%–70%, and photoperiod of 14/10 hr (day/night). Plants were watered daily and fertilized weekly with Hoagland nutrient solution. When
the fourth true leaves were fully expanded, plants were then used in the experiments. Experimental design To determine the effects of melatonin on plant tolerance to photooxidative stress, the four-leaf-stage cucumber plants were first sprayed with melatonin at concentrations of 0, 5, 50, 200, or 1000 lM (the first treatment concentration used distilled water as control). Melatonin (SigmaAldrich, St. Louis, MO, USA) solutions were prepared by dissolving the solute in ethanol followed by dilution with Milli-Q water [ethanol/water (v/v) = 1/10,000]. Each plant was sprayed with 20 mL of solution. Twelve hours later, the plants were sprayed with 50 lM Methyl viologen (MV; Sigma-Aldrich, St. Louis, MO, USA) and then were exposed to light at a strength of 300 lmol/m2/s at 25°C for 12 hr. The level of stress tolerance was accessed by measuring changes in the maximal photochemical efficiency (Fv/Fm), malondialdehyde (MDA) content, and relative electrical conductivity (REC). Accumulations of superoxide anions (O2) and H2O2 were determined in plants with 50 lM melatonin and MV treatments. A 50 lM concentration of melatonin was used in the following experiments. To determine whether melatonin could induce systemic defense against photooxidative stress, either the third leaf from the bottom or the root of a plant was pretreated with 50 lM melatonin, and the two treatments were designated as MTL or MTR respectively. Twelve hours later, the fourth leaf of MTL and all leaves of MTR were sprayed with 50 lM MV. Again, 12 hr later, stress tolerance was measured on the basis of changes in Fv/Fm, MDA content, and REC. To investigate the effects of melatonin on GSH redox status, the plants were sprayed with 50 lM melatonin or distilled water as control. To examine the roles of H2O2 and GSH redox status in melatonin-induced local tolerance against photooxidative stress, the leaves were pretreated with 5 mM dimethylthiourea (DMTU; a H2O2 and O2 scavenger) [16, 23], 100 lM diphenyleneiodonium (DPI; an inhibitor of NADPH oxidases and oxidative burst, which produces H2O2) [16, 27], or 1 mM buthionine sulfoximine (BSO; an inhibitor of GSH biosynthesis) [28]. After 8 hr, the leaves were sprayed with 50 lM melatonin, 5 mM H2O2, or 5 mM GSH. Twelve hours later, the plants were treated with 50 lM MV. To examine the roles of H2O2 and the GSH redox status in melatonin-induced systemic defense against photooxidative stress, the fourth leaves of MTL plants and all leaves of MTR plants were pretreated with 5 mM DMTU, 100 lM DPI, or 1 mM BSO. To investigate the effects of H2O2 on GSH redox status, cucumber plants were sprayed with 5 mM H2O2 or distilled water as control. To investigate the role of H2O2 in melatonin-induced GSH redox status, cucumber plants were pretreated with 5 mM DMTU or 100 lM DPI, and then, 8 hr later, were sprayed with 50 lM melatonin. GSH redox status was determined at 6 hr after the melatonin treatment. To determine the interaction between H2O2 and GSH, leaves were pretreated with 5 mM DMTU, 100 lM DPI, or 1 mM BSO, and then, 8 hr later, were sprayed 207
Li et al. with 5 mM GSH or 5 mM H2O2. An additional 12 hr later, the plants were treated with 50 lM MV. To determine the effects of GSH on MV-induced H2O2 accumulation, the plants were pretreated with 5 mM GSH, and then, 12 hr later, were treated with 50 lM MV.
DHAR activities were measured by a decrease in A290 and an increase in A265 according to the method of Nakano and Asada [36]. CAT activity was measured as a decline in A240 using the method of Patra et al. [37]. Glutathione and ascorbic acid contents assay
Analysis of chlorophyll fluorescence, lipid peroxidation, and relative electric conductivity Fv/Fm was determined with Protable Chlorophyll Flourometer (PAM2500; Heinz Walz, Effeltrich, Germany), after plants were dark-adapted for 30 min. The level of lipid peroxidation in leaves was assessed by measuring the malonyldialdehyde (MDA) content using 2-thiobarbituric acid as described by Hodges et al. [29]. The relative electric conductivity (REC) was measured and calculated as described by Zhou and Leul [30]. Analysis of H2O2 and O2 0.3 g of leaf samples was homogenized with 3 mL of 1 M HClO4 at 4°C, and the homogenate was centrifuged at 6000 g for 5 min at 4°C. Then, the supernatant was collected, adjusted to pH 6.0–7.0 with 4 M KOH and centrifuged at 12,000 g for 5 min at 4°C. The supernatant was placed onto an AG1x8 prepacked column (Bio-Rad, Hercules, CA, USA) and H2O2 was eluted with 4 mL double-distilled H2O. The sample (800 mL) was mixed with 400 mL reaction buffer containing 4 mM 2,20 -azino-di (3-ethylbenzthiazoline-6-sulfonic acid) and 100 mM potassium acetate at pH 4.4, 400 mL deionized water and 0.25 U of horseradish peroxidase (HRP). H2O2 content was measured at OD412 [31]. Superoxide production was quantified according to the method of Elstner and Heupel [32] with a slight modification. Sodium nitrite was used as a standard solution to calculate the production rate of superoxide. The histochemical staining of O2 and H2O2 was performed according the methods of Jabs et al. [33] and Thordal-Christensen et al. [34]. In case of H2O2, leaf discs (1.5 cm in diameter) were placed in a solution containing 1 mg/mL 3,30 -diaminobenzidine (DAB) in 50 mM Trisacetate (pH 3.8) for 6 hr after a light vacuum infiltration. In case of O2, leaf discs (1.5 cm in diameter) were vacuum infiltrated directly with 0.5 mg/mL NBT in 25 mM K-HEPES buffer (pH 7.8) and incubated at 25°C in the dark for 6 hr. In both cases, leaf discs were rinsed in 95% (v/v) ethanol for 10 min at 90°C, mounted in lactic acid/ phenol/water (1:1:1; v/v), and photographed.
For the measurement of reduced glutathione (GSH), oxidized glutathione (GSSG), ascorbic acid (AsA), and dehydroascorbic acid (DHA), 0.2 g of leaf tissue was homogenized in 2 mL of 5% metaphosphoric acid containing 2 mM EDTA and centrifuged at 4°C for 10 min at 12,000 g. Reduced glutathione and GSSG contents were determined by an enzymatic recycling method as described by Rao et al. [38]. For the total glutathione assay, 0.1 mL of the supernatant was added to a reaction mixture containing 0.2 mM NADPH, 100 mM phosphate buffer (pH 7.5), 5 mM EDTA, and 0.6 mM 5,50 -dithio-bis (2-nitrobenzoic acid), after neutralization with 0.5 M phosphate buffer (pH 7.5). The reaction was started by adding 3 U of GR and was monitored by measuring the changes in absorbance at 412 nm for 1 min. For the GSSG assay, GSH was masked by adding 20 lL of 2-vinylpyridine for 1 hr at 25°C. The GSH concentration was obtained by subtracting the GSSG concentration from the total concentration. Ascorbic acid and DHA were measured following Law et al. [39]. For AsA+DHA content assay, the extract was incubated with 150 mM phosphate buffer solution (pH 7.4) and 10 mM DTT for 20 min to reduce all DHA to AsA. After incubation, 100 lL of 0.5% (w/v) N-ethylmaleimide (NEM) was added to remove excess DTT. AsA was analyzed in a similar manner except that 200 lL deionized H2O was substituted for DTT and NEM. Color was developed in both series of reaction mixtures with the addition of 400 lL 10% (w/v) trichloroacetic acid (TCA), 400 lL 44% phosphoric acid (v/v), 400 lL 70% (v/v) a0 -dipyridyl in ethanol and 200 lL 3% (w/v) FeCl3. The reaction mixtures were then incubated at 37°C for 60 min in a water bath and the absorbance was recorded at 525 nm. The DHA concentration was obtained by subtracting the AsA concentration from the total concentration. Statistical analysis The experiment was a completely randomized design with four replications. Each replicate contained at least 12 plants. Analysis of variance (ANOVA) was used to test for significance, and significant differences (P < 0.05) between treatments were determined using Tukey’s test.
Antioxidant enzyme extraction and activity assay Antioxidant enzyme activities were assayed in leaves by using spectrophotometric methods. 0.3 g of leaf were ground with 3 mL ice-cold 25 mM HEPES buffer (pH 7.8) containing 0.2 mM EDTA, 2 mM AsA, and 2% PVP. The homogenates were centrifuged at 4°C for 20 min at 12,000 g, and the resulting supernatants were used for the determination of enzymatic activity. SOD activity was assayed by following the Stewart and Bewley [35] method based on photochemical reduction of NBT. APX and 208
Results Melatonin is a well-documented antioxidant and plays important roles in alleviating environmental stress-induced oxidative stress in plants. In this study, we analyzed the effects of melatonin at different doses on cucumber tolerance to MV treatment. MV causes photooxidative stress, which can be assessed by assessing reductions in Fv/Fm and increases in both MDA content and REC. As shown in Fig. 1, application of melatonin at appropriate
Signaling in melatonin-induced defense (A)
(B)
Fig. 1. The relationship between melatonin and resistance to methyl viologen (MV)-mediated photooxidative stress. (A) Leaves of cucumber (Cucumis sativus) seedlings at the four-leaf stage were pretreated with 0, 5, 50, 200, or 1000 lM melatonin (MT) and, 12 hr later, were sprayed with 50 lM MV, and were then exposed to 300 20 lmol/m2/s light intensity for 12 hr. The maximum quantum yield of photosystem II (Fv/Fm), malondialdehyde (MDA) content, and relative electric conductivity (REC) were monitored to assess changes in tolerance. (B) Histochemical staining and quantification assay of O2 and H2O2 were performed after plants were pretreated with 50 lM MT and were then sprayed with 50 lM MV or distilled water. Data of Fv/Fm are the means of six replicates (SD). Data of MDA content, REC, O2 and H2O2 are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
concentrations (5–1000 lM) alleviated MV-induced declines in Fv/Fm and increases in MDA content and REC. The most effective melatonin concentration was 50 lM. In the plants pretreated with 50 lM melatonin, Fv/Fm was 57.2% higher, while MDA content and REC were 30.5% and 31.8% lower, respectively, after MV treatment, relative to the control plants. The protective effect of melatonin against photooxidative stress was attenuated, and even disappeared, with melatonin concentrations both higher and lower than 50 lM. Moreover, pretreatment with 50 lM melatonin significantly alleviated MV-induced accumulation of O2 and H2O2. To determine whether melatonin treatments induced stress tolerance in systemic leaves, we treated the third leaves from the bottom of the cucumber plant (called local leaves) with melatonin (in the MTL treatment) and then subjected the fourth leaves (i.e., upper leaves) to MV treatment 12 hr later. As shown in Fig. 2A, treatment of the local leaves with melatonin reduced both the MV-induced decline of Fv/Fm and increases in MDA content and REC in the untreated upper leaves. Thus, melatonin treatment induced photooxidative stress tolerance not only in the treated local leaves, but also in untreated upper systemic leaves. We were interested in extending the analysis to determine whether melatonin treatment on cucumber roots could also induce defense or stress responses in untreated leaves of the same plants. To test this, we investigated the effects of the rhizospheric application of melatonin (in the MTR treatment) on leaf resistance to photooxidative stress. As shown in Fig. 2B,
the MTR treatment also alleviated both the MV-induced decline of Fv/Fm and the increases in MDA content and REC in untreated leaves. Antioxidant systems, including antioxidant enzymes and nonenzymatic oxidants, play critical roles in defending against oxidative stress. Under normal growth conditions, catalase (CAT) activity, GSH content and redox status in both local and upper leaves of plants in the MTL treatments and in leaves of plants in the MTR treatments were significantly elevated, with some exceptions (Table 1). After the MV treatment, the declined activities of enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), and dehydroascorbate reductase (DHAR), and the declines of GSH and AsA redox status of both local and systemic leaves were significantly alleviated by melatonin treatments of both third leaves and roots. Cellular GSH plays important roles in defending against oxidative stress. Thus, we investigated whether melatonininduced local and systemic defense against oxidative stress was associated with the GSH redox status. First, the effects of melatonin on the GSH redox status were examined. GSH levels were increased from 3 hr after melatonin treatment and reached their peaks at 6 hr (Fig. 3). However, GSSG levels in melatonin treated plants were almost unchanged, compared to those in plants as control. Finally, the ratio of GSH/GSSG was significantly induced by melatonin treatment. Then, we determined the effects of H2O2 and GSH on cucumber tolerance to oxidative stress. Application of H2O2 or GSH also significantly alleviated both the 209
Li et al. (A)
(B)
Fig. 2. Enhanced tolerance to photooxidative stress in untreated systemic leaves by melatonin application to local leaves or roots. (A) The third leaf of cucumber (Cucumis sativus) seedlings at the four-leaf stage were pretreated with 50 lM melatonin (MTL); 12 hr later, the fourth leaf from the bottom, corresponding to the upper leaf, was exposed to 50 lM MV. (B) The cucumber seedlings roots were pretreated with 50 lM melatonin (MTR); 12 hr later, the leaves were exposed to 50 lM MV. In both (A and B), the Fv/Fm, MDA content, and REC were monitored to assess changes in tolerance. Data of Fv/Fm are the means of six replicates (SD). Data of MDA and REC are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
Table 1. The response of the antioxidant systems in melatonin-treated leaves and untreated leaves of plants with MTL/MTR and/or MV treatments
Treatment CK-U MTL-U MV-U MTL+ MV-U CK-L MTL-L MV-L MTL+ MV-L CK MTR MV MTR+ MV
SOD activity (unit/g FW)
APX activity (lmol/g FW/min)
DHAR activity (lmol/g FW/min)
CAT activity (lmol/g FW/min)
GSH+GSSG content (lmol/g FW)
GSH/GSSG ratio
ASA+DHA content (lmol/g FW)
ASA/DHA ratio
3.83 3.96 2.82 3.37
0.20a 0.38a 0.08c 0.21b
2.74 3.15 1.06 1.97
0.20a 0.37a 0.04c 0.22b
1.38 1.43 1.14 1.36
0.26a 0.03a 0.06b 0.04a
0.58 0.78 0.70 0.62
0.07b 0.03a 0.03ab 0.10b
0.57 0.77 0.61 0.65
0.03b 0.05a 0.05b 0.07ab
9.73 14.58 2.72 4.83
1.64b 1.12a 0.08d 0.27c
2.93 3.92 1.31 3.17
0.01b 0.42a 0.01c 0.06b
7.77 8.37 1.38 4.95
1.20a 1.83a 0.26c 1.25b
4.60 4.22 2.67 3.50
0.35a 0.38a 0.20c 0.39b
3.19 2.95 1.39 2.74
0.22a 0.16a 0.13b 0.46a
1.32 1.40 0.97 1.18
0.05a 0.06a 0.08c 0.07b
0.70 0.91 0.66 0.74
0.08b 0.10a 0.08b 0.07b
0.48 0.61 0.57 0.62
0.02b 0.01a 0.03a 0.00a
10.15 14.48 4.06 8.27
1.59b 0.44a 0.49c 0.65b
5.70 5.89 2.39 5.26
0.15a 0.22a 0.82c 0.16b
8.16 7.71 3.34 9.37
0.62a 0.42a 0.25b 0.45a
4.16 4.23 2.76 3.72
0.25a 0.24a 0.28c 0.17b
3.11 3.01 1.53 2.24
0.20a 0.43a 0.20c 0.25b
1.40 1.48 1.07 1.34
0.04a 0.09a 0.08b 0.04a
0.65 0.65 0.68 0.67
0.08a 0.03a 0.05a 0.02a
0.56 0.61 0.55 0.58
0.01b 0.01a 0.01b 0.01b
10.89 13.45 4.57 7.22
0.57b 1.27a 0.61c 1.18d
5.81 5.86 2.46 3.99
0.19a 0.48a 0.20c 0.04b
8.24 11.16 3.39 7.75
0.66b 0.19a 0.67c 0.92b
The plants were treated as described in Fig. 2 and the samples were harvested at 12 hr after MV treatment. The activities of the antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and catalase (CAT) as well as the redox status of glutathione and ascorbic acid were analyzed. Data are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
MV-induced decline of Fv/Fm and increases of MDA content and REC (Fig. 4A). However, pretreatment of the cucumber leaves with DMTU (a H2O2 and O2 scavenger), DPI (an inhibitor of NADPH oxidases and oxidative burst), and BSO (an inhibitor of GSH biosynthesis) almost 210
completely abolished the melatonin-induced local and systemic tolerance to oxidative stress (Fig. 4). In addition, DMTU, DPI, and BSO treatments alone had no significant effect on the cucumber tolerance to MV stress (Fig. S1).
Li et al. (A)
(B)
Fig. 2. Enhanced tolerance to photooxidative stress in untreated systemic leaves by melatonin application to local leaves or roots. (A) The third leaf of cucumber (Cucumis sativus) seedlings at the four-leaf stage were pretreated with 50 lM melatonin (MTL); 12 hr later, the fourth leaf from the bottom, corresponding to the upper leaf, was exposed to 50 lM MV. (B) The cucumber seedlings roots were pretreated with 50 lM melatonin (MTR); 12 hr later, the leaves were exposed to 50 lM MV. In both (A and B), the Fv/Fm, MDA content, and REC were monitored to assess changes in tolerance. Data of Fv/Fm are the means of six replicates (SD). Data of MDA and REC are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
Table 1. The response of the antioxidant systems in melatonin-treated leaves and untreated leaves of plants with MTL/MTR and/or MV treatments
Treatment CK-U MTL-U MV-U MTL+ MV-U CK-L MTL-L MV-L MTL+ MV-L CK MTR MV MTR+ MV
SOD activity (unit/g FW)
APX activity (lmol/g FW/min)
DHAR activity (lmol/g FW/min)
CAT activity (lmol/g FW/min)
GSH+GSSG content (lmol/g FW)
GSH/GSSG ratio
ASA+DHA content (lmol/g FW)
ASA/DHA ratio
3.83 3.96 2.82 3.37
0.20a 0.38a 0.08c 0.21b
2.74 3.15 1.06 1.97
0.20a 0.37a 0.04c 0.22b
1.38 1.43 1.14 1.36
0.26a 0.03a 0.06b 0.04a
0.58 0.78 0.70 0.62
0.07b 0.03a 0.03ab 0.10b
0.57 0.77 0.61 0.65
0.03b 0.05a 0.05b 0.07ab
9.73 14.58 2.72 4.83
1.64b 1.12a 0.08d 0.27c
2.93 3.92 1.31 3.17
0.01b 0.42a 0.01c 0.06b
7.77 8.37 1.38 4.95
1.20a 1.83a 0.26c 1.25b
4.60 4.22 2.67 3.50
0.35a 0.38a 0.20c 0.39b
3.19 2.95 1.39 2.74
0.22a 0.16a 0.13b 0.46a
1.32 1.40 0.97 1.18
0.05a 0.06a 0.08c 0.07b
0.70 0.91 0.66 0.74
0.08b 0.10a 0.08b 0.07b
0.48 0.61 0.57 0.62
0.02b 0.01a 0.03a 0.00a
10.15 14.48 4.06 8.27
1.59b 0.44a 0.49c 0.65b
5.70 5.89 2.39 5.26
0.15a 0.22a 0.82c 0.16b
8.16 7.71 3.34 9.37
0.62a 0.42a 0.25b 0.45a
4.16 4.23 2.76 3.72
0.25a 0.24a 0.28c 0.17b
3.11 3.01 1.53 2.24
0.20a 0.43a 0.20c 0.25b
1.40 1.48 1.07 1.34
0.04a 0.09a 0.08b 0.04a
0.65 0.65 0.68 0.67
0.08a 0.03a 0.05a 0.02a
0.56 0.61 0.55 0.58
0.01b 0.01a 0.01b 0.01b
10.89 13.45 4.57 7.22
0.57b 1.27a 0.61c 1.18d
5.81 5.86 2.46 3.99
0.19a 0.48a 0.20c 0.04b
8.24 11.16 3.39 7.75
0.66b 0.19a 0.67c 0.92b
The plants were treated as described in Fig. 2 and the samples were harvested at 12 hr after MV treatment. The activities of the antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and catalase (CAT) as well as the redox status of glutathione and ascorbic acid were analyzed. Data are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
MV-induced decline of Fv/Fm and increases of MDA content and REC (Fig. 4A). However, pretreatment of the cucumber leaves with DMTU (a H2O2 and O2 scavenger), DPI (an inhibitor of NADPH oxidases and oxidative burst), and BSO (an inhibitor of GSH biosynthesis) almost 210
completely abolished the melatonin-induced local and systemic tolerance to oxidative stress (Fig. 4). In addition, DMTU, DPI, and BSO treatments alone had no significant effect on the cucumber tolerance to MV stress (Fig. S1).
Li et al. (A)
(B)
Fig. 5. The role of H2O2 in melatonininduced increases in the GSH redox status. (A) Kinetics of changes in GSH redox status in plants treated with distilled water or 5 mM H2O2. (B) Plants were pretreated with 5 mM DMTU or 100 lM DPI, and then, 8 hr later, were treated with 50 lM melatonin. After 6 hr, the GSH redox status was analyzed. Data are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
respectively. Pretreatment with BSO completely abolished alleviation of both the MV-induced decline of Fv/Fm and increases of MDA and REC by H2O2 (Fig. 6A). In contrast, pretreatment with DMTU or DPI attenuated but did not completely abolish GSH-induced oxidative stress tolerance. In addition, application of GSH effectively inhibited MV-induced H2O2 accumulation (Fig. 6B,C).
lower untreated leaves [23]. As was shown for EBR, melatonin led to a systemic induction of oxidative stress tolerance in upper untreated leaves in the present study (Fig. 2A). Furthermore, rhizospheric treatment of melatonin also alleviated MV-induced oxidative stress in untreated leaves (Fig. 2B). Taken together, these results confirm that melatonin is capable of inducing both local and systemic defense against oxidative stress.
Discussion
Melatonin-induced local and systemic defense against oxidative stress is dependent on GSH
Exogenous melatonin induces local and systemic defense against photooxidative stress in cucumber Melatonin is a well-documented antioxidant and plays important roles in alleviating environmental stress-induced oxidation. In this study, application of MV caused severe photooxidative stress by generating O2, which was then converted into H2O2 (Fig. 1). However, pretreatment with melatonin alleviated MV-induced photooxidative stress and this role of melatonin was dose dependent. With melatonin concentrations both higher and lower than the most effective concentration (around 50 lM), the protective effect of melatonin against photooxidative stress was attenuated, and even disappeared. A previous study indicated that 24-epibrassinolide (EBR) treatments enhanced tolerance to paraquat-activated photooxidative stress not only in treated leaves, but also in upper and 212
The antioxidant potential of melatonin results from its ability to directly scavenge free radicals and induce antioxidant systems including antioxidant enzymes and nonenzymatic antioxidants. However, strong evidence has demonstrated melatonin is unable to directly scavenge O2–, and the earlier conclusion of melatonin directly scavenging H2O2 has been dismissed [40, 41]. Thus, removal of MV-induced O2– and H2O2 by melatonin was likely dependent on the induction of antioxidant systems via some signaling pathways. O2– is easily converted to H2O2 by SOD, while H2O2 can be scavenged by an AsA and/or a GSH regenerating cycle and CAT [42]. It is reported that exogenous melatonin increased contents and reduction/oxidation ratio of AsA and GSH via upreglating activities of some key enzymes and alleviated dark-inducecd H2O2 accumulation and senescence [11]. Consistently, we found
Signaling in melatonin-induced defense (A)
(B)
Fig. 6. Interaction between H2O2 and GSH in enhanced tolerance to photooxidative stress. (A) Plants were pretreated with DMTU (5 mM) and DPI (100 lM) or BSO (1 mM), 8 hr before GSH or H2O2 treatments. The Fv/Fm, MDA content, and REC were monitored to assess changes in tolerance. (B) DAB staining and (C) a quantification assay of H2O2 were performed after plants were pretreated with GSH (5 mM), and then, 12 hr later, were sprayed with 50 lM MV or distilled water. Data of MDA and REC are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
Fig. 7. Various ways to alleviate ROS stress in view of H2O2, GSH, and melatonin.
AsA and GSH levels and redox status and the activities of some key enzymes (APX and DHAR) involved in AsAGSH cycle were significantly increased by melatonin in both local and systemic leaves under oxidative stress (Table 1), but CAT activity was almost unchanged. Therefore, AsA and/or GSH cycle but not CAT is responsible for the reduction of H2O2 by melatonin. As an essential co-substrate and reductant, GSH can not only directly remove H2O2 via peroxidase metabolism, but also is required for regeneration of AsA [43]. It has been indicated that melatonin can increase GSH synthesis
under environmental stresses [11, 44]. Our experiments revealed that melatonin application induced GSH synthesis and increased the GSH/GSSG ratio under natural growth conditions (Fig. 3, Table 1). Furthermore, GSH application induced tolerance to MV-activated oxidative stress by inhibiting accumulation of ROS, while pretreatment with BSO, an inhibitor of GSH biosynthesis, completely blocked melatonin-induced local and systemic resistance against oxidative stress (Fig. 4). As such, induction of local and systemic defense against oxidative stress by melatonin was dependent on induction of the GSH 213
Li et al. (A)
(B)
Fig. 5. The role of H2O2 in melatonininduced increases in the GSH redox status. (A) Kinetics of changes in GSH redox status in plants treated with distilled water or 5 mM H2O2. (B) Plants were pretreated with 5 mM DMTU or 100 lM DPI, and then, 8 hr later, were treated with 50 lM melatonin. After 6 hr, the GSH redox status was analyzed. Data are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
respectively. Pretreatment with BSO completely abolished alleviation of both the MV-induced decline of Fv/Fm and increases of MDA and REC by H2O2 (Fig. 6A). In contrast, pretreatment with DMTU or DPI attenuated but did not completely abolish GSH-induced oxidative stress tolerance. In addition, application of GSH effectively inhibited MV-induced H2O2 accumulation (Fig. 6B,C).
lower untreated leaves [23]. As was shown for EBR, melatonin led to a systemic induction of oxidative stress tolerance in upper untreated leaves in the present study (Fig. 2A). Furthermore, rhizospheric treatment of melatonin also alleviated MV-induced oxidative stress in untreated leaves (Fig. 2B). Taken together, these results confirm that melatonin is capable of inducing both local and systemic defense against oxidative stress.
Discussion
Melatonin-induced local and systemic defense against oxidative stress is dependent on GSH
Exogenous melatonin induces local and systemic defense against photooxidative stress in cucumber Melatonin is a well-documented antioxidant and plays important roles in alleviating environmental stress-induced oxidation. In this study, application of MV caused severe photooxidative stress by generating O2, which was then converted into H2O2 (Fig. 1). However, pretreatment with melatonin alleviated MV-induced photooxidative stress and this role of melatonin was dose dependent. With melatonin concentrations both higher and lower than the most effective concentration (around 50 lM), the protective effect of melatonin against photooxidative stress was attenuated, and even disappeared. A previous study indicated that 24-epibrassinolide (EBR) treatments enhanced tolerance to paraquat-activated photooxidative stress not only in treated leaves, but also in upper and 212
The antioxidant potential of melatonin results from its ability to directly scavenge free radicals and induce antioxidant systems including antioxidant enzymes and nonenzymatic antioxidants. However, strong evidence has demonstrated melatonin is unable to directly scavenge O2–, and the earlier conclusion of melatonin directly scavenging H2O2 has been dismissed [40, 41]. Thus, removal of MV-induced O2– and H2O2 by melatonin was likely dependent on the induction of antioxidant systems via some signaling pathways. O2– is easily converted to H2O2 by SOD, while H2O2 can be scavenged by an AsA and/or a GSH regenerating cycle and CAT [42]. It is reported that exogenous melatonin increased contents and reduction/oxidation ratio of AsA and GSH via upreglating activities of some key enzymes and alleviated dark-inducecd H2O2 accumulation and senescence [11]. Consistently, we found
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Effects of DMTU, DPI, and BSO on tolerance to photooxidative stress in cucumber plants.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12304
Journal of Pineal Research
Glutathione-dependent induction of local and systemic defense against oxidative stress by exogenous melatonin in cucumber (Cucumis sativus L.) Abstract: Melatonin is involved in defending against oxidative stress caused by various environmental stresses in plants. In this study, the roles of exogenous melatonin in regulating local and systemic defense against photooxidative stress in cucumber (Cucumis sativus) and the involvement of redox signaling were examined. Foliar or rhizospheric treatment with melatonin enhanced tolerance to photooxidative stress in both melatonintreated leaves and untreated systemic leaves. Increased melatonin levels are capable of increasing glutathione (reduced glutathione [GSH]) redox status. Application of H2O2 and GSH also induced tolerance to photooxidative stress, while inhibition of H2O2 accumulation and GSH synthesis compromised melatonin-induced local and systemic tolerance to photooxidative stress. H2O2 treatment increased the GSH/oxidized glutathione (GSSG) ratio, while inhibition of H2O2 accumulation prevented a melatonininduced increase in the GSH/GSSG ratio. Additionally, inhibition of GSH synthesis blocked H2O2-induced photooxidative stress tolerance, whereas scavenging or inhibition of H2O2 production attenuated but did not abolish GSH-induced tolerance to photooxidative stress. These results strongly suggest that exogenous melatonin is capable of inducing both local and systemic defense against photooxidative stress and melatonin-enhanced GSH/GSSG ratio in a H2O2-dependent manner is critical in the induction of tolerance.
Introduction As sessile organisms, plants are constantly exposed to a variety of abiotic and biotic stresses throughout their lifecycle. Various environmental stresses inhibit plant growth via different mechanisms, but most directly or indirectly impair multiple physiological processes, such as photosynthesis, photorespiration, and respiration, and result in increased reactive oxygen species (ROS) levels, which disrupts ROS homeostasis [1, 2]. As strong oxidizers, ROS at excessive levels have detrimental effects as a result of their ability to cause lipid peroxidation, DNA damage, protein denaturation, carbohydrate oxidation, pigment breakdown, enzyme activity impairment, and eventually cell death [3]. Hence, the ability to maintain ROS at moderate levels is crucial to the mechanisms by which plants resist various abiotic and biotic stresses. Melatonin (N-acetyl-5-methoxytryptamine), a highly conserved molecule, is ubiquitous throughout animals, plants, and all the other kingdoms [4–6]. Since the first studies showed that melatonin indeed exists in plants [7, 8], numerous subsequent studies have proven melatonin plays important roles in the regulation of plant growth and development [5] and defense against biotic and abiotic stresses such as pathogens, extreme temperature, excess copper, salinity, and drought [9, 10]. The most frequently mentioned function of melatonin in plants is the effective 206
Hao Li1, Jie He1, Xiaozhen Yang1, Xin Li2, Dan Luo1, Chunhua Wei1, Jianxiang Ma1, Yong Zhang1, Jianqiang Yang1 and Xian Zhang1 1
Department of Horticulture, Northwest A&F University, Shaanxi, China; 2Key Laboratory of Tea Biology and Resources Utilization, Tea Research Institute, Chinese Academy of Agricultural Sciences, Ministry of Agriculture, Zhejiang, China
Key words: Cucumis sativus, glutathione, hydrogen peroxide, local/systemic defense, melatonin Address reprint requests to Xian Zhang, College of Horticulture, Northwest A&F University, No. 3, Taicheng Road, Yangling 712100, Shaanxi, China. E-mail: [email protected] Received October 9, 2015; Accepted December 11, 2015.
reduction of ROS accumulation and consequent alleviation of oxidative stress. Solid evidence suggests that melatonin can not only directly scavenge some ROS, but can also modulate antioxidant enzymes and enhance cellular antioxidants such as GSH [11, 12]. However, only a few studies have focused on the signaling pathways involved in melatonin-induced tolerance to abiotic stress in plants [13, 14]. High levels of ROS are harmful to plant tissues; however, extensive studies have shown that moderate ROS levels as second messengers are necessary for plant growth, development, and defending against stresses [15]. Foliar application of H2O2 induced antioxidant enzyme activity and alleviated paraquat- or cold-induced oxidative stress in cucumber leaves [16, 17]. Studies using vitro animal cells have reported that ROS production can be promoted by melatonin at low or high levels [12], although melatonin is a well-documented antioxidant. However, the induction of ROS by the pro-oxidant action of melatonin is not correlated with cytotoxicity, and it is both concentration dependent and cell-type dependent [12]. Accordingly, it will be of interest to examine whether ROS as signal substances are involved in induction of stress tolerance by melatonin in plants. At a whole-organism level, the resistances in various plant tissues across diverse environments are nonindependent. Plants can mount a systemic response to environmental
Signaling in melatonin-induced defense stresses in order to establish long-lasting protection against a broad spectrum of biotic or abiotic stresses in tissues far from the primary site of attack, and these mechanisms are termed ‘systemic acquired resistance’ (SAR) or ‘systemic acquired acclimation’ (SAA) [18, 19]. Both SAR and SAA are dependent on some movably systemic signals, which are transported from the primary site of stimuli to distant tissues in order to induce systemic responses. Salicylic acid is required for SAR, and Park et al. [20] provide evidence that methyl salicylate functions as the critical mobile signal. When plants are exposed to drought or salinity, abscisic acid is produced in roots and transported to leaf tissue in order to induce stomatal closure and antioxidant defense systems [21, 22]. In contrast, some regulators that are not transported, such as brassinosteroids, can induce systemic tolerance to abiotic or biotic stresses via movement of H2O2 acting as a second messenger [23]. Melatonin is amphiphilic, which enables it to cross cell membranes easily [24]. Moreover, melatonin and natural auxin, indole-3-acetic acid (IAA), share tryptophan as a biosynthetic precursor and have similar structural moieties. Therefore, melatonin, akin to IAA, is likely transported over long distances. This is consistent with increased melatonin content in the leaves of water hyacinths after exogenous melatonin application to growth media [25]. Similarly, NaCl stress increases endogenous melatonin content in both roots and cotyledons, thus implicating the transport of melatonin from roots to cotyledons [26]. However, whether melatonin could act as a mobile signal and induce systemic tolerance to stress is unclear. In the present study, we analyzed the photooxidative stress tolerance and antioxidant system changes in both melatonin-treated leaves and untreated leaves of cucumber plants that were treated with melatonin on specific leaves or roots. We also investigated the roles and relationships of H2O2 and melatonin-increased GSH/GSSG ratio in these plants. Our results strongly suggest that melatonin is capable of inducing both local and systemic defense against photooxidative stress. Furthermore, the melatoninenhanced GSH/GSSG ratio is critical in the induction of tolerance.
Materials and methods Plant material Cucumber (Cucumis sativus L. cv. Jinyan No. 4) was used because of its known sensitivity to photooxidative stress. Seeds were germinated in 50-hole plates filled with a mixture of peat, vermiculite, and perlite (2:1:1, v:v:v) and then germinated and grown into plants in a glasshouse. When the first true leaf was fully expanded, seedlings were transplanted into plastic pots (15 cm diameter and 15 cm deep, with one seedling per pot) containing a peat-vermiculite mixture (2:1, v:v). Plants were then maintained in growth chambers at a temperature of 25°C/20°C (day/night), photosynthetic photon flux density (PPFD) of 600 lmol/m2/s, relative humidity of 65%–70%, and photoperiod of 14/10 hr (day/night). Plants were watered daily and fertilized weekly with Hoagland nutrient solution. When
the fourth true leaves were fully expanded, plants were then used in the experiments. Experimental design To determine the effects of melatonin on plant tolerance to photooxidative stress, the four-leaf-stage cucumber plants were first sprayed with melatonin at concentrations of 0, 5, 50, 200, or 1000 lM (the first treatment concentration used distilled water as control). Melatonin (SigmaAldrich, St. Louis, MO, USA) solutions were prepared by dissolving the solute in ethanol followed by dilution with Milli-Q water [ethanol/water (v/v) = 1/10,000]. Each plant was sprayed with 20 mL of solution. Twelve hours later, the plants were sprayed with 50 lM Methyl viologen (MV; Sigma-Aldrich, St. Louis, MO, USA) and then were exposed to light at a strength of 300 lmol/m2/s at 25°C for 12 hr. The level of stress tolerance was accessed by measuring changes in the maximal photochemical efficiency (Fv/Fm), malondialdehyde (MDA) content, and relative electrical conductivity (REC). Accumulations of superoxide anions (O2) and H2O2 were determined in plants with 50 lM melatonin and MV treatments. A 50 lM concentration of melatonin was used in the following experiments. To determine whether melatonin could induce systemic defense against photooxidative stress, either the third leaf from the bottom or the root of a plant was pretreated with 50 lM melatonin, and the two treatments were designated as MTL or MTR respectively. Twelve hours later, the fourth leaf of MTL and all leaves of MTR were sprayed with 50 lM MV. Again, 12 hr later, stress tolerance was measured on the basis of changes in Fv/Fm, MDA content, and REC. To investigate the effects of melatonin on GSH redox status, the plants were sprayed with 50 lM melatonin or distilled water as control. To examine the roles of H2O2 and GSH redox status in melatonin-induced local tolerance against photooxidative stress, the leaves were pretreated with 5 mM dimethylthiourea (DMTU; a H2O2 and O2 scavenger) [16, 23], 100 lM diphenyleneiodonium (DPI; an inhibitor of NADPH oxidases and oxidative burst, which produces H2O2) [16, 27], or 1 mM buthionine sulfoximine (BSO; an inhibitor of GSH biosynthesis) [28]. After 8 hr, the leaves were sprayed with 50 lM melatonin, 5 mM H2O2, or 5 mM GSH. Twelve hours later, the plants were treated with 50 lM MV. To examine the roles of H2O2 and the GSH redox status in melatonin-induced systemic defense against photooxidative stress, the fourth leaves of MTL plants and all leaves of MTR plants were pretreated with 5 mM DMTU, 100 lM DPI, or 1 mM BSO. To investigate the effects of H2O2 on GSH redox status, cucumber plants were sprayed with 5 mM H2O2 or distilled water as control. To investigate the role of H2O2 in melatonin-induced GSH redox status, cucumber plants were pretreated with 5 mM DMTU or 100 lM DPI, and then, 8 hr later, were sprayed with 50 lM melatonin. GSH redox status was determined at 6 hr after the melatonin treatment. To determine the interaction between H2O2 and GSH, leaves were pretreated with 5 mM DMTU, 100 lM DPI, or 1 mM BSO, and then, 8 hr later, were sprayed 207
Li et al. with 5 mM GSH or 5 mM H2O2. An additional 12 hr later, the plants were treated with 50 lM MV. To determine the effects of GSH on MV-induced H2O2 accumulation, the plants were pretreated with 5 mM GSH, and then, 12 hr later, were treated with 50 lM MV.
DHAR activities were measured by a decrease in A290 and an increase in A265 according to the method of Nakano and Asada [36]. CAT activity was measured as a decline in A240 using the method of Patra et al. [37]. Glutathione and ascorbic acid contents assay
Analysis of chlorophyll fluorescence, lipid peroxidation, and relative electric conductivity Fv/Fm was determined with Protable Chlorophyll Flourometer (PAM2500; Heinz Walz, Effeltrich, Germany), after plants were dark-adapted for 30 min. The level of lipid peroxidation in leaves was assessed by measuring the malonyldialdehyde (MDA) content using 2-thiobarbituric acid as described by Hodges et al. [29]. The relative electric conductivity (REC) was measured and calculated as described by Zhou and Leul [30]. Analysis of H2O2 and O2 0.3 g of leaf samples was homogenized with 3 mL of 1 M HClO4 at 4°C, and the homogenate was centrifuged at 6000 g for 5 min at 4°C. Then, the supernatant was collected, adjusted to pH 6.0–7.0 with 4 M KOH and centrifuged at 12,000 g for 5 min at 4°C. The supernatant was placed onto an AG1x8 prepacked column (Bio-Rad, Hercules, CA, USA) and H2O2 was eluted with 4 mL double-distilled H2O. The sample (800 mL) was mixed with 400 mL reaction buffer containing 4 mM 2,20 -azino-di (3-ethylbenzthiazoline-6-sulfonic acid) and 100 mM potassium acetate at pH 4.4, 400 mL deionized water and 0.25 U of horseradish peroxidase (HRP). H2O2 content was measured at OD412 [31]. Superoxide production was quantified according to the method of Elstner and Heupel [32] with a slight modification. Sodium nitrite was used as a standard solution to calculate the production rate of superoxide. The histochemical staining of O2 and H2O2 was performed according the methods of Jabs et al. [33] and Thordal-Christensen et al. [34]. In case of H2O2, leaf discs (1.5 cm in diameter) were placed in a solution containing 1 mg/mL 3,30 -diaminobenzidine (DAB) in 50 mM Trisacetate (pH 3.8) for 6 hr after a light vacuum infiltration. In case of O2, leaf discs (1.5 cm in diameter) were vacuum infiltrated directly with 0.5 mg/mL NBT in 25 mM K-HEPES buffer (pH 7.8) and incubated at 25°C in the dark for 6 hr. In both cases, leaf discs were rinsed in 95% (v/v) ethanol for 10 min at 90°C, mounted in lactic acid/ phenol/water (1:1:1; v/v), and photographed.
For the measurement of reduced glutathione (GSH), oxidized glutathione (GSSG), ascorbic acid (AsA), and dehydroascorbic acid (DHA), 0.2 g of leaf tissue was homogenized in 2 mL of 5% metaphosphoric acid containing 2 mM EDTA and centrifuged at 4°C for 10 min at 12,000 g. Reduced glutathione and GSSG contents were determined by an enzymatic recycling method as described by Rao et al. [38]. For the total glutathione assay, 0.1 mL of the supernatant was added to a reaction mixture containing 0.2 mM NADPH, 100 mM phosphate buffer (pH 7.5), 5 mM EDTA, and 0.6 mM 5,50 -dithio-bis (2-nitrobenzoic acid), after neutralization with 0.5 M phosphate buffer (pH 7.5). The reaction was started by adding 3 U of GR and was monitored by measuring the changes in absorbance at 412 nm for 1 min. For the GSSG assay, GSH was masked by adding 20 lL of 2-vinylpyridine for 1 hr at 25°C. The GSH concentration was obtained by subtracting the GSSG concentration from the total concentration. Ascorbic acid and DHA were measured following Law et al. [39]. For AsA+DHA content assay, the extract was incubated with 150 mM phosphate buffer solution (pH 7.4) and 10 mM DTT for 20 min to reduce all DHA to AsA. After incubation, 100 lL of 0.5% (w/v) N-ethylmaleimide (NEM) was added to remove excess DTT. AsA was analyzed in a similar manner except that 200 lL deionized H2O was substituted for DTT and NEM. Color was developed in both series of reaction mixtures with the addition of 400 lL 10% (w/v) trichloroacetic acid (TCA), 400 lL 44% phosphoric acid (v/v), 400 lL 70% (v/v) a0 -dipyridyl in ethanol and 200 lL 3% (w/v) FeCl3. The reaction mixtures were then incubated at 37°C for 60 min in a water bath and the absorbance was recorded at 525 nm. The DHA concentration was obtained by subtracting the AsA concentration from the total concentration. Statistical analysis The experiment was a completely randomized design with four replications. Each replicate contained at least 12 plants. Analysis of variance (ANOVA) was used to test for significance, and significant differences (P < 0.05) between treatments were determined using Tukey’s test.
Antioxidant enzyme extraction and activity assay Antioxidant enzyme activities were assayed in leaves by using spectrophotometric methods. 0.3 g of leaf were ground with 3 mL ice-cold 25 mM HEPES buffer (pH 7.8) containing 0.2 mM EDTA, 2 mM AsA, and 2% PVP. The homogenates were centrifuged at 4°C for 20 min at 12,000 g, and the resulting supernatants were used for the determination of enzymatic activity. SOD activity was assayed by following the Stewart and Bewley [35] method based on photochemical reduction of NBT. APX and 208
Results Melatonin is a well-documented antioxidant and plays important roles in alleviating environmental stress-induced oxidative stress in plants. In this study, we analyzed the effects of melatonin at different doses on cucumber tolerance to MV treatment. MV causes photooxidative stress, which can be assessed by assessing reductions in Fv/Fm and increases in both MDA content and REC. As shown in Fig. 1, application of melatonin at appropriate
Signaling in melatonin-induced defense (A)
(B)
Fig. 1. The relationship between melatonin and resistance to methyl viologen (MV)-mediated photooxidative stress. (A) Leaves of cucumber (Cucumis sativus) seedlings at the four-leaf stage were pretreated with 0, 5, 50, 200, or 1000 lM melatonin (MT) and, 12 hr later, were sprayed with 50 lM MV, and were then exposed to 300 20 lmol/m2/s light intensity for 12 hr. The maximum quantum yield of photosystem II (Fv/Fm), malondialdehyde (MDA) content, and relative electric conductivity (REC) were monitored to assess changes in tolerance. (B) Histochemical staining and quantification assay of O2 and H2O2 were performed after plants were pretreated with 50 lM MT and were then sprayed with 50 lM MV or distilled water. Data of Fv/Fm are the means of six replicates (SD). Data of MDA content, REC, O2 and H2O2 are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
concentrations (5–1000 lM) alleviated MV-induced declines in Fv/Fm and increases in MDA content and REC. The most effective melatonin concentration was 50 lM. In the plants pretreated with 50 lM melatonin, Fv/Fm was 57.2% higher, while MDA content and REC were 30.5% and 31.8% lower, respectively, after MV treatment, relative to the control plants. The protective effect of melatonin against photooxidative stress was attenuated, and even disappeared, with melatonin concentrations both higher and lower than 50 lM. Moreover, pretreatment with 50 lM melatonin significantly alleviated MV-induced accumulation of O2 and H2O2. To determine whether melatonin treatments induced stress tolerance in systemic leaves, we treated the third leaves from the bottom of the cucumber plant (called local leaves) with melatonin (in the MTL treatment) and then subjected the fourth leaves (i.e., upper leaves) to MV treatment 12 hr later. As shown in Fig. 2A, treatment of the local leaves with melatonin reduced both the MV-induced decline of Fv/Fm and increases in MDA content and REC in the untreated upper leaves. Thus, melatonin treatment induced photooxidative stress tolerance not only in the treated local leaves, but also in untreated upper systemic leaves. We were interested in extending the analysis to determine whether melatonin treatment on cucumber roots could also induce defense or stress responses in untreated leaves of the same plants. To test this, we investigated the effects of the rhizospheric application of melatonin (in the MTR treatment) on leaf resistance to photooxidative stress. As shown in Fig. 2B,
the MTR treatment also alleviated both the MV-induced decline of Fv/Fm and the increases in MDA content and REC in untreated leaves. Antioxidant systems, including antioxidant enzymes and nonenzymatic oxidants, play critical roles in defending against oxidative stress. Under normal growth conditions, catalase (CAT) activity, GSH content and redox status in both local and upper leaves of plants in the MTL treatments and in leaves of plants in the MTR treatments were significantly elevated, with some exceptions (Table 1). After the MV treatment, the declined activities of enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), and dehydroascorbate reductase (DHAR), and the declines of GSH and AsA redox status of both local and systemic leaves were significantly alleviated by melatonin treatments of both third leaves and roots. Cellular GSH plays important roles in defending against oxidative stress. Thus, we investigated whether melatonininduced local and systemic defense against oxidative stress was associated with the GSH redox status. First, the effects of melatonin on the GSH redox status were examined. GSH levels were increased from 3 hr after melatonin treatment and reached their peaks at 6 hr (Fig. 3). However, GSSG levels in melatonin treated plants were almost unchanged, compared to those in plants as control. Finally, the ratio of GSH/GSSG was significantly induced by melatonin treatment. Then, we determined the effects of H2O2 and GSH on cucumber tolerance to oxidative stress. Application of H2O2 or GSH also significantly alleviated both the 209
Li et al. (A)
(B)
Fig. 2. Enhanced tolerance to photooxidative stress in untreated systemic leaves by melatonin application to local leaves or roots. (A) The third leaf of cucumber (Cucumis sativus) seedlings at the four-leaf stage were pretreated with 50 lM melatonin (MTL); 12 hr later, the fourth leaf from the bottom, corresponding to the upper leaf, was exposed to 50 lM MV. (B) The cucumber seedlings roots were pretreated with 50 lM melatonin (MTR); 12 hr later, the leaves were exposed to 50 lM MV. In both (A and B), the Fv/Fm, MDA content, and REC were monitored to assess changes in tolerance. Data of Fv/Fm are the means of six replicates (SD). Data of MDA and REC are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
Table 1. The response of the antioxidant systems in melatonin-treated leaves and untreated leaves of plants with MTL/MTR and/or MV treatments
Treatment CK-U MTL-U MV-U MTL+ MV-U CK-L MTL-L MV-L MTL+ MV-L CK MTR MV MTR+ MV
SOD activity (unit/g FW)
APX activity (lmol/g FW/min)
DHAR activity (lmol/g FW/min)
CAT activity (lmol/g FW/min)
GSH+GSSG content (lmol/g FW)
GSH/GSSG ratio
ASA+DHA content (lmol/g FW)
ASA/DHA ratio
3.83 3.96 2.82 3.37
0.20a 0.38a 0.08c 0.21b
2.74 3.15 1.06 1.97
0.20a 0.37a 0.04c 0.22b
1.38 1.43 1.14 1.36
0.26a 0.03a 0.06b 0.04a
0.58 0.78 0.70 0.62
0.07b 0.03a 0.03ab 0.10b
0.57 0.77 0.61 0.65
0.03b 0.05a 0.05b 0.07ab
9.73 14.58 2.72 4.83
1.64b 1.12a 0.08d 0.27c
2.93 3.92 1.31 3.17
0.01b 0.42a 0.01c 0.06b
7.77 8.37 1.38 4.95
1.20a 1.83a 0.26c 1.25b
4.60 4.22 2.67 3.50
0.35a 0.38a 0.20c 0.39b
3.19 2.95 1.39 2.74
0.22a 0.16a 0.13b 0.46a
1.32 1.40 0.97 1.18
0.05a 0.06a 0.08c 0.07b
0.70 0.91 0.66 0.74
0.08b 0.10a 0.08b 0.07b
0.48 0.61 0.57 0.62
0.02b 0.01a 0.03a 0.00a
10.15 14.48 4.06 8.27
1.59b 0.44a 0.49c 0.65b
5.70 5.89 2.39 5.26
0.15a 0.22a 0.82c 0.16b
8.16 7.71 3.34 9.37
0.62a 0.42a 0.25b 0.45a
4.16 4.23 2.76 3.72
0.25a 0.24a 0.28c 0.17b
3.11 3.01 1.53 2.24
0.20a 0.43a 0.20c 0.25b
1.40 1.48 1.07 1.34
0.04a 0.09a 0.08b 0.04a
0.65 0.65 0.68 0.67
0.08a 0.03a 0.05a 0.02a
0.56 0.61 0.55 0.58
0.01b 0.01a 0.01b 0.01b
10.89 13.45 4.57 7.22
0.57b 1.27a 0.61c 1.18d
5.81 5.86 2.46 3.99
0.19a 0.48a 0.20c 0.04b
8.24 11.16 3.39 7.75
0.66b 0.19a 0.67c 0.92b
The plants were treated as described in Fig. 2 and the samples were harvested at 12 hr after MV treatment. The activities of the antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and catalase (CAT) as well as the redox status of glutathione and ascorbic acid were analyzed. Data are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
MV-induced decline of Fv/Fm and increases of MDA content and REC (Fig. 4A). However, pretreatment of the cucumber leaves with DMTU (a H2O2 and O2 scavenger), DPI (an inhibitor of NADPH oxidases and oxidative burst), and BSO (an inhibitor of GSH biosynthesis) almost 210
completely abolished the melatonin-induced local and systemic tolerance to oxidative stress (Fig. 4). In addition, DMTU, DPI, and BSO treatments alone had no significant effect on the cucumber tolerance to MV stress (Fig. S1).
Li et al. (A)
(B)
Fig. 2. Enhanced tolerance to photooxidative stress in untreated systemic leaves by melatonin application to local leaves or roots. (A) The third leaf of cucumber (Cucumis sativus) seedlings at the four-leaf stage were pretreated with 50 lM melatonin (MTL); 12 hr later, the fourth leaf from the bottom, corresponding to the upper leaf, was exposed to 50 lM MV. (B) The cucumber seedlings roots were pretreated with 50 lM melatonin (MTR); 12 hr later, the leaves were exposed to 50 lM MV. In both (A and B), the Fv/Fm, MDA content, and REC were monitored to assess changes in tolerance. Data of Fv/Fm are the means of six replicates (SD). Data of MDA and REC are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
Table 1. The response of the antioxidant systems in melatonin-treated leaves and untreated leaves of plants with MTL/MTR and/or MV treatments
Treatment CK-U MTL-U MV-U MTL+ MV-U CK-L MTL-L MV-L MTL+ MV-L CK MTR MV MTR+ MV
SOD activity (unit/g FW)
APX activity (lmol/g FW/min)
DHAR activity (lmol/g FW/min)
CAT activity (lmol/g FW/min)
GSH+GSSG content (lmol/g FW)
GSH/GSSG ratio
ASA+DHA content (lmol/g FW)
ASA/DHA ratio
3.83 3.96 2.82 3.37
0.20a 0.38a 0.08c 0.21b
2.74 3.15 1.06 1.97
0.20a 0.37a 0.04c 0.22b
1.38 1.43 1.14 1.36
0.26a 0.03a 0.06b 0.04a
0.58 0.78 0.70 0.62
0.07b 0.03a 0.03ab 0.10b
0.57 0.77 0.61 0.65
0.03b 0.05a 0.05b 0.07ab
9.73 14.58 2.72 4.83
1.64b 1.12a 0.08d 0.27c
2.93 3.92 1.31 3.17
0.01b 0.42a 0.01c 0.06b
7.77 8.37 1.38 4.95
1.20a 1.83a 0.26c 1.25b
4.60 4.22 2.67 3.50
0.35a 0.38a 0.20c 0.39b
3.19 2.95 1.39 2.74
0.22a 0.16a 0.13b 0.46a
1.32 1.40 0.97 1.18
0.05a 0.06a 0.08c 0.07b
0.70 0.91 0.66 0.74
0.08b 0.10a 0.08b 0.07b
0.48 0.61 0.57 0.62
0.02b 0.01a 0.03a 0.00a
10.15 14.48 4.06 8.27
1.59b 0.44a 0.49c 0.65b
5.70 5.89 2.39 5.26
0.15a 0.22a 0.82c 0.16b
8.16 7.71 3.34 9.37
0.62a 0.42a 0.25b 0.45a
4.16 4.23 2.76 3.72
0.25a 0.24a 0.28c 0.17b
3.11 3.01 1.53 2.24
0.20a 0.43a 0.20c 0.25b
1.40 1.48 1.07 1.34
0.04a 0.09a 0.08b 0.04a
0.65 0.65 0.68 0.67
0.08a 0.03a 0.05a 0.02a
0.56 0.61 0.55 0.58
0.01b 0.01a 0.01b 0.01b
10.89 13.45 4.57 7.22
0.57b 1.27a 0.61c 1.18d
5.81 5.86 2.46 3.99
0.19a 0.48a 0.20c 0.04b
8.24 11.16 3.39 7.75
0.66b 0.19a 0.67c 0.92b
The plants were treated as described in Fig. 2 and the samples were harvested at 12 hr after MV treatment. The activities of the antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and catalase (CAT) as well as the redox status of glutathione and ascorbic acid were analyzed. Data are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
MV-induced decline of Fv/Fm and increases of MDA content and REC (Fig. 4A). However, pretreatment of the cucumber leaves with DMTU (a H2O2 and O2 scavenger), DPI (an inhibitor of NADPH oxidases and oxidative burst), and BSO (an inhibitor of GSH biosynthesis) almost 210
completely abolished the melatonin-induced local and systemic tolerance to oxidative stress (Fig. 4). In addition, DMTU, DPI, and BSO treatments alone had no significant effect on the cucumber tolerance to MV stress (Fig. S1).
Li et al. (A)
(B)
Fig. 5. The role of H2O2 in melatonininduced increases in the GSH redox status. (A) Kinetics of changes in GSH redox status in plants treated with distilled water or 5 mM H2O2. (B) Plants were pretreated with 5 mM DMTU or 100 lM DPI, and then, 8 hr later, were treated with 50 lM melatonin. After 6 hr, the GSH redox status was analyzed. Data are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
respectively. Pretreatment with BSO completely abolished alleviation of both the MV-induced decline of Fv/Fm and increases of MDA and REC by H2O2 (Fig. 6A). In contrast, pretreatment with DMTU or DPI attenuated but did not completely abolish GSH-induced oxidative stress tolerance. In addition, application of GSH effectively inhibited MV-induced H2O2 accumulation (Fig. 6B,C).
lower untreated leaves [23]. As was shown for EBR, melatonin led to a systemic induction of oxidative stress tolerance in upper untreated leaves in the present study (Fig. 2A). Furthermore, rhizospheric treatment of melatonin also alleviated MV-induced oxidative stress in untreated leaves (Fig. 2B). Taken together, these results confirm that melatonin is capable of inducing both local and systemic defense against oxidative stress.
Discussion
Melatonin-induced local and systemic defense against oxidative stress is dependent on GSH
Exogenous melatonin induces local and systemic defense against photooxidative stress in cucumber Melatonin is a well-documented antioxidant and plays important roles in alleviating environmental stress-induced oxidation. In this study, application of MV caused severe photooxidative stress by generating O2, which was then converted into H2O2 (Fig. 1). However, pretreatment with melatonin alleviated MV-induced photooxidative stress and this role of melatonin was dose dependent. With melatonin concentrations both higher and lower than the most effective concentration (around 50 lM), the protective effect of melatonin against photooxidative stress was attenuated, and even disappeared. A previous study indicated that 24-epibrassinolide (EBR) treatments enhanced tolerance to paraquat-activated photooxidative stress not only in treated leaves, but also in upper and 212
The antioxidant potential of melatonin results from its ability to directly scavenge free radicals and induce antioxidant systems including antioxidant enzymes and nonenzymatic antioxidants. However, strong evidence has demonstrated melatonin is unable to directly scavenge O2–, and the earlier conclusion of melatonin directly scavenging H2O2 has been dismissed [40, 41]. Thus, removal of MV-induced O2– and H2O2 by melatonin was likely dependent on the induction of antioxidant systems via some signaling pathways. O2– is easily converted to H2O2 by SOD, while H2O2 can be scavenged by an AsA and/or a GSH regenerating cycle and CAT [42]. It is reported that exogenous melatonin increased contents and reduction/oxidation ratio of AsA and GSH via upreglating activities of some key enzymes and alleviated dark-inducecd H2O2 accumulation and senescence [11]. Consistently, we found
Signaling in melatonin-induced defense (A)
(B)
Fig. 6. Interaction between H2O2 and GSH in enhanced tolerance to photooxidative stress. (A) Plants were pretreated with DMTU (5 mM) and DPI (100 lM) or BSO (1 mM), 8 hr before GSH or H2O2 treatments. The Fv/Fm, MDA content, and REC were monitored to assess changes in tolerance. (B) DAB staining and (C) a quantification assay of H2O2 were performed after plants were pretreated with GSH (5 mM), and then, 12 hr later, were sprayed with 50 lM MV or distilled water. Data of MDA and REC are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
Fig. 7. Various ways to alleviate ROS stress in view of H2O2, GSH, and melatonin.
AsA and GSH levels and redox status and the activities of some key enzymes (APX and DHAR) involved in AsAGSH cycle were significantly increased by melatonin in both local and systemic leaves under oxidative stress (Table 1), but CAT activity was almost unchanged. Therefore, AsA and/or GSH cycle but not CAT is responsible for the reduction of H2O2 by melatonin. As an essential co-substrate and reductant, GSH can not only directly remove H2O2 via peroxidase metabolism, but also is required for regeneration of AsA [43]. It has been indicated that melatonin can increase GSH synthesis
under environmental stresses [11, 44]. Our experiments revealed that melatonin application induced GSH synthesis and increased the GSH/GSSG ratio under natural growth conditions (Fig. 3, Table 1). Furthermore, GSH application induced tolerance to MV-activated oxidative stress by inhibiting accumulation of ROS, while pretreatment with BSO, an inhibitor of GSH biosynthesis, completely blocked melatonin-induced local and systemic resistance against oxidative stress (Fig. 4). As such, induction of local and systemic defense against oxidative stress by melatonin was dependent on induction of the GSH 213
Li et al. (A)
(B)
Fig. 5. The role of H2O2 in melatonininduced increases in the GSH redox status. (A) Kinetics of changes in GSH redox status in plants treated with distilled water or 5 mM H2O2. (B) Plants were pretreated with 5 mM DMTU or 100 lM DPI, and then, 8 hr later, were treated with 50 lM melatonin. After 6 hr, the GSH redox status was analyzed. Data are the means of four replicates (SD). Means denoted by the same letter did not significantly differ at P < 0.05 according to Tukey’s test.
respectively. Pretreatment with BSO completely abolished alleviation of both the MV-induced decline of Fv/Fm and increases of MDA and REC by H2O2 (Fig. 6A). In contrast, pretreatment with DMTU or DPI attenuated but did not completely abolish GSH-induced oxidative stress tolerance. In addition, application of GSH effectively inhibited MV-induced H2O2 accumulation (Fig. 6B,C).
lower untreated leaves [23]. As was shown for EBR, melatonin led to a systemic induction of oxidative stress tolerance in upper untreated leaves in the present study (Fig. 2A). Furthermore, rhizospheric treatment of melatonin also alleviated MV-induced oxidative stress in untreated leaves (Fig. 2B). Taken together, these results confirm that melatonin is capable of inducing both local and systemic defense against oxidative stress.
Discussion
Melatonin-induced local and systemic defense against oxidative stress is dependent on GSH
Exogenous melatonin induces local and systemic defense against photooxidative stress in cucumber Melatonin is a well-documented antioxidant and plays important roles in alleviating environmental stress-induced oxidation. In this study, application of MV caused severe photooxidative stress by generating O2, which was then converted into H2O2 (Fig. 1). However, pretreatment with melatonin alleviated MV-induced photooxidative stress and this role of melatonin was dose dependent. With melatonin concentrations both higher and lower than the most effective concentration (around 50 lM), the protective effect of melatonin against photooxidative stress was attenuated, and even disappeared. A previous study indicated that 24-epibrassinolide (EBR) treatments enhanced tolerance to paraquat-activated photooxidative stress not only in treated leaves, but also in upper and 212
The antioxidant potential of melatonin results from its ability to directly scavenge free radicals and induce antioxidant systems including antioxidant enzymes and nonenzymatic antioxidants. However, strong evidence has demonstrated melatonin is unable to directly scavenge O2–, and the earlier conclusion of melatonin directly scavenging H2O2 has been dismissed [40, 41]. Thus, removal of MV-induced O2– and H2O2 by melatonin was likely dependent on the induction of antioxidant systems via some signaling pathways. O2– is easily converted to H2O2 by SOD, while H2O2 can be scavenged by an AsA and/or a GSH regenerating cycle and CAT [42]. It is reported that exogenous melatonin increased contents and reduction/oxidation ratio of AsA and GSH via upreglating activities of some key enzymes and alleviated dark-inducecd H2O2 accumulation and senescence [11]. Consistently, we found
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Effects of DMTU, DPI, and BSO on tolerance to photooxidative stress in cucumber plants.
