Oxidative Medicine and Cellular Longevity
Volume 2015 (2015), Article ID 612363, 11 pages
http://dx.doi.org/10.1155/2015/612363

Research Article

Sulfur Dioxide Enhances Endogenous Hydrogen Sulfide Accumulation and Alleviates Oxidative Stress Induced by Aluminum Stress in Germinating Wheat Seeds

Dong-Bo Zhu,¹ Kang-Di Hu,¹ Xi-Kai Guo,¹ Yong Liu,¹ Lan-Ying Hu,¹ Yan-Hong Li,¹ Song-Hua Wang,² and Hua Zhang¹

¹School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, China
²Life Science College, Anhui Science and Technology University, Bengbu 233100, China

Received 28 October 2014; Accepted 21 November 2014

Academic Editor: Guangdong Yang

Copyright © 2015 Dong-Bo Zhu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Aluminum ions are especially toxic to plants in acidic soils. Here we present evidences that SO₂ protects germinating wheat grains against aluminum stress. SO₂ donor (NaHSO₃/Na₂SO₃) pretreatment at 1.2 mM reduced the accumulation of superoxide anion, hydrogen peroxide, and malondialdehyde, enhanced the activities of guaiacol peroxidase, catalase, and ascorbate peroxidase, and decreased the activity of lipoxygenase in germinating wheat grains exposed to Al stress. We also observed higher accumulation of hydrogen sulfide (H₂S) in SO₂-pretreated grain, suggesting the tight relation between sulfite and sulfide. Wheat grains geminated in water for 36 h were pretreated with or without 1 mM SO₂ donor for 12 h prior to exposure to Al stress for 48 h and the ameliorating effects of SO₂ on wheat radicles were studied. SO₂ donor pretreatment reduced the content of reactive oxygen species, protected membrane integrity, and reduced Al accumulation in wheat radicles. Gene expression analysis showed that SO₂ donor pretreatment decreased the expression of Al-responsive genes TaWali1, TaWali2, TaWali3, TaWali5, TaWali6, and TaALMT1 in radicles exposed to Al stress. These results suggested that SO₂ could increase endogenous H₂S accumulation and the antioxidant capability and decrease endogenous Al content in wheat grains to alleviate Al stress.

1. Introduction

Aluminum ions (Al³⁺) together with silicon and iron are the three most abundant mineral elements in soil. Whereas silicon and iron are required for plant growth, Al is toxic. Many different mechanisms have been advanced to explain Al toxicity in plants [1, 2]. One of the primary causes of Al toxicity is oxidative stress due to accumulation of reactive oxygen species (ROS), such as the superoxide anion () and hydrogen peroxide (H₂O₂), bringing about lipid peroxidation in plant cells [3–5]. Plants have developed several strategies to counteract oxidative stress caused by Al, such as activation of antioxidants, and exudation of organic acids as a mechanism for Al exclusion [6]. Recently, a range of signaling molecules, such as inositol 1,4,5-triphosphate (IP₃), salicylic acid, hydrogen peroxide (H₂O₂) and nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H₂S), were found to participate in plant’s resistance to Al-induced oxidative stress [4, 7–10].

Sulfur dioxide (SO₂) is a colorless, nonflammable gas with a penetrating odor. Low concentrations of SO₂ have been found to play a physiological role in vivo in animal models, participating in various biological processes [11]. The physiological processes regulated by SO₂ in animals include cardiac function [11], inhibition of L-calcium channels in cardiomyocytes [12], and improvement in pulmonary vascular structural remodeling [13]. In plants, the toxic effects of SO₂ on growth and development have been extensively studied [14, 15]. Exposure to high concentrations of SO₂ can cause visible foliar damage, a decline in photosynthesis, an inhibition of plant growth, and structural disorganization and cell death [16–19]. On the other hand, many reports show that low levels of atmospheric SO₂ might be beneficial to plants [20]. SO₂ can be metabolized and used as a sulfur source for plant growth, especially when the sulfur supply in soil is insufficient for normal growth [20]. Recently, low concentrations of SO₂ were found to induce transcriptome reprogramming associated with oxidative signaling and biotic defence responses in plants, suggesting a physiological role of SO₂ in plant [21].

In plants, sulfate is taken up from soil by high-affinity transporters. Sulfate is largely transported to shoots where it can be activated by ATP via ATP sulfurylase in the leaves. The product is reduced by 5′-adenylylsulfate (APS) reductase to sulfite which can be reduced to H₂S by sulfite reductase [22]. SO₂ can also be produced endogenously from sulfur-containing amino acids [23]. The endogenous production of SO₂ also suggests that it has a physiological role in plants.

In order to establish the role of SO₂ in alleviating Al stress, we investigated the effects of SO₂ pretreatment on H₂S and ROS accumulation and the antioxidant system in whole wheat grains and in wheat radicles. We also analyzed endogenous H₂S and Al content as a means of understanding the mechanism of the role of SO₂. We speculated that SO₂ might act as an antioxidant molecule to alleviate Al toxicity during wheat grain germination.

2. Materials and Methods

2.1. Materials and Treatments

Wheat (Triticum aestivum L.) grains were supplied by the Anhui Aidi Agricultural Technology Co., Ltd., Anhui Province, China. Sodium bisulfite (NaHSO₃) and anhydrous sodium sulfite (Na₂SO₃) were used as sulfur dioxide (SO₂) donors according to Laisk et al. [24]. Wheat grains were sterilized by 0.1% HgCl₂ for 3 min and washed extensively with H₂O and then dried with filter papers. Wheat grains of similar size were selected and allocated randomly in Petri dish (9 cm diameter × 1.2 cm depth, 50 grains per dish). Wheat grains were germinated in H₂O or aqueous solutions of AlCl₃ at 5, 10, 15, 20, 25, 30, 60, and 90 mM for 48 h at 25°C and the length of coleoptiles and radicles and radicle number were recorded. To test the protective role of SO₂ on germination and seedling growth of wheat grains under Al stress, grains were pretreated with H₂O or 0.4, 0.8, 1.2, 1.6, or 2.0 mM SO₂ donor for 12 h and subsequently subjected to a semi-inhibitory AlCl₃ concentration (15 mM). AlCl₃ solutions were renewed every 12 h and geminating grains were sampled every 12 h for further analysis.

2.2. Determination of MDA, , and H₂O₂

The contents of MDA, , and H₂O₂ were determined by the method of Zhang et al. [25].

2.3. Assays of LOX, CAT, APX, and POD Activities

Activity of lipoxygenase (LOX, EC 1.13.11.12) was determined following the description by Surrey [26] and those of catalase (CAT, EC1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and guaiacol peroxidase (POD, EC 1.11.1.7) were assayed according to Hu et al. [27]. Wheat grains were homogenized in ice-cold 50 mM phosphate buffer (pH 7.8) containing 1.0 mM EDTA. The homogenate was centrifuged at 15,000 g at 4°C for 10 min. The supernatant was used for activity determination.

2.4. Assays of Reducing Sugars and Soluble Protein

Wheat grains (0.5 ± 0.05 g) were ground in 5 mL of phosphate buffer (pH 7.0, 200 mM), the homogenate was centrifuged at 10,000 g for 30 min, and the supernatant was used for detection of reducing sugars and soluble protein content. Reducing sugar content was measured according to Miller [28].

For detection of soluble protein, 0.1 mL supernatant was mixed with 0.9 mL H₂O and 5 mL Coomassie brilliant blue for 5 min and the absorbance recorded at 595 nm using the method described by Bradford [29].

2.5. Preparation of Wheat Radicles

Wheat grains were geminated in H₂O for 36 h in the dark at 25°C and the average of radicle length was approximately 1.0 cm. The geminated wheat grains were pretreated with or without 1 mM SO₂ donor for 12 h and then exposed to 0 or 400 μM AlCl₃ for 48 h.

2.6. Detection of Plasma Membrane Integrity, Al Accumulation, and ROS Production in Radicles

Plasma membrane integrity of wheat radicles was detected following the method of Yamamoto et al. [30]. Radicles were stained with Evans blue solution (0.025% [w/v] Evans blue in 100 μM CaCl₂, pH 5.6) for 10 min, then washed three times with 100 μM CaCl₂ solutions, and photographed. Staining intensity of Evans blue is positively correlated with more damaged plasma membrane.

Al content in radicles was visualized by staining tissues with hematoxylin. Hematoxylin stain was prepared as described by Polle et al. [31]. Wheat radicles were washed with H₂O for 30 min and then stained with solution of 0.2% hematoxylin and 0.02% NaIO₃ for 30 min at room temperature. Radicles were then immersed in H₂O for 30 min to remove excess stain and photographed. Staining intensity of hematoxylin is positively correlated with Al uptake.

ROS distribution in radicle tips was detected by 2′,7′-dichlorofluorescin diacetate (DCFH-DA) following the method of LeBel et al. [32]. Radicle tips were incubated in a solution containing 100 μM CaCl₂ and 10 μM DCFH-DA for 20 min and then washed three times with H₂O. The fluorescence was detected with a Nikon 80i microscope (excitation at 488 nm and emission at 525 nm). For each treatment, ten individual roots from ten seedlings were examined and similar results were obtained.

2.7. Real-Time Quantitative RT-PCR Analysis in Wheat Radicles

Radicle tips were prepared for RNA extraction according to Li et al. [33]. Total RNA was isolated by grinding with liquid nitrogen according to the manufacturer’s instructions (CWBIO, Beijing, China). cDNA was generated from total RNA with a reverse transcription kit (Prime Script RT Master Mix, Takara, Kyoto, Japan). Quantitative PCR was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR Premix Ex Taq (TaKaRa Bio Inc., China) according to the manufacturer’s instructions. cDNA was amplified by PCR using the following primers: Taβ-actin forward (5′-CTATCCTTCGTTTGGACCTT-3′) and reserve (5′-AGCGAGCTTCTCCTTTATGT-3′); TaWali1 forward (5′-CTGATGGAGTCGAGCAAGG-3′) and reserve (5′-CCGAAGTAGCGATTTAGGAGT-3′); TaWali2 forward (5′-AGCCTACTGCTCCGCCTTGT-3′) and reserve (5′-CGTTTCGTCGGCATCTCC-3′); TaWali3 forward (5′-GACGAGCCCTAAGAAGACG-3′) and reserve (5′-CACGGAGCAATGACAACAG-3′); TaWali5 forward (5′-TGGACCCTGCAAGAAGTAC-3′) and reserve (5′-GCTGAACAACAAGCAACACC-3′); TaWali6 forward (5′-TACGGAATAGACAGGACAAGG-3′) and reserve (5′-CAGCATTTCGGGAACTCG-3′); TaALMT1 forward (5′-TGCCACGCTGAGTAAAGG-3′) and reserve (5′-CGCTGACGCTACGAAGAA-3′). Relative gene expression was presented as values relative to the corresponding gene expression in control, after normalization to the control Taβ-actin transcript levels.

2.8. Statistical Analysis

Statistical significance was tested by one-way ANOVA, and the results are expressed as the mean values ± SD (standard deviation) of three independent experiments. Each experiment was repeated three times.

3. Results

3.1. Inhibitory Effect of Al on Wheat Grain Germination

The effect of Al stress on wheat seedling growth and development was examined following incubation of grain in AlCl₃ with concentrations ranging from 5 mM to 90 mM (Table 1). At concentrations of 5 mM or below, germination percentage, coleoptile length, and radicle number are almost unaffected, but radicle length was reduced by 13%, suggesting that the radicle is the primary target of Al toxicity. At 15 mM Al, germination percentage was almost halved compared with that of control and this concentration was selected for further experiments. At 90 mM Al, radicle growth was completely inhibited, but very stunted coleoptile growth was still observed.

Table 1: Inhibitory effect of Al stress on the germination of wheat grains. Wheat grains were exposed to 0, 5, 10, 15, 20, 25, 30, 60, or 90 mM AlCl₃ for 48 h.

Al³⁺ concentration
(mM)
Germination percentage (%)
Radicle length
(cm)
Coleoptile length
(cm)
Radicle number
(50 grains)

0
64 ± 1.2a
3.1 ± 0.8a
1.5 ± 0.3ab
178 ± 7.8a

5
66 ± 1.1a
2.7 ± 0.5ab
1.6 ± 0.2a
168 ± 8.9a

10
51 ± 2.3b
1.9 ± 0.3b
1.3 ± 0.3ab
162 ± 7.6a

15
35 ± 3.8c
1.1 ± 0.2c
1.1 ± 0.2bc
142 ± 6.3b

20
28 ± 4.2cd
0.7 ± 0.3cd
0.8 ± 0.2cd
80 ± 5.6c

25
21 ± 5.1de
0.4 ± 0.3d
0.6 ± 0.2de
68 ± 6.1c

30
15 ± 4.7ef
0.2 ± 0.2d
0.3 ± 0.3e
45 ± 5.3d

60
8 ± 5.2f
0.1 ± 0.1d
0.3 ± 0.2e
22 ± 3.5e

90
7 ± 6.3f
0 ± 0e
0.2 ± 0.2e
0 ± 0f

Values are the means ± SD (). Values are the means ± SD (). Different letters mean significance of difference between different treatments ( < 0.05).

3.2. SO₂ Donor Ameliorates Al Stress in Germinating Wheat Grain

To establish whether the SO₂ donor Na₂SO₃/NaHSO₃ had a toxic effect on wheat grain germination, grains were germinated in different SO₂ donor concentrations ranging from 0.4 to 2.0 mM for 36 h (see Table  S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/612363). Table  S1 shows there was no significant change in germination percentage, coleoptile length, radicle length, or radicle number between water control and SO₂ donor treatment, establishing that the concentrations of SO₂ donor used in this work exhibited no visible toxic effects. To test the ability of SO₂ donor to alleviate Al stress, wheat grains were pretreated with SO₂ donor concentrations ranging from 0.4 to 2.0 mM for 12 h prior to incubation with 15 mM Al (Table 2 and Figure 1). At all SO₂ donor concentrations used, SO₂ pretreatment was effective in alleviating the toxic effects of Al in a dose dependent manner. The optimal SO₂ donor concentration for alleviating Al stress was 1.2 mM, a concentration where the germination percentage was increased by 51%, radicle and coleoptile length by 28% and 26%, respectively, compared with those exposed to Al. This result clearly shows that SO₂ alleviates Al-induced inhibition of wheat grain germination and seedling growth.

Table 2: Effects of SO₂ donor pretreatment on wheat grain germination under 15 mM Al³⁺ stress. Wheat grains were pretreated with 0, 0.4, 0.8, 1.2, 1.6, and 2.0 mM SO2 for 12 h and subsequently subjected to 15 mM AlCl₃ for further 48 h, and then germination was investigated.

SO₂ donor concentration
(mM)
0.0
0.4
0.8
1.2
1.6
2.0

Germination percentage (%)
37 ± 3.3a
42 ± 2.7a
44 ± 3.5a
56 ± 3.8a
48 ± 2.7a
47 ± 3.1a

Length of radicle (cm)
1.42 ± 0.6a
1.72 ± 0.4a
1.80 ± 0.7a
1.82 ± 0.7a
1.78 ± 0.8a
1.62 ± 0.4a

Length of coleoptile (cm)
4.64 ± 0.4a
4.70 ± 0.6a
5.20 ± 0.5a
5.83 ± 0.8a
5.40 ± 0.8
5.23 ± 0.6a

Radicle number (50 grains)
127 ± 7.3a
135 ± 8.1a
139 ± 8.1a
148 ± 7.9a
130 ± 6.7a
119 ± 7.1a

Values are the means ± SD (). Different letters mean significance of difference between different treatments ( < 0.05).

Figure 1: Effects of SO₂ pretreatment on wheat grain germination under 15 mM Al stress. Wheat grains were pretreated with 0, 0.4, 0.8, 1.2, 1.6, and 2.0 mM SO₂ for 12 h, subsequently subjected to 15 mM Al for further 48 h, and then photographed.

3.3. Effect of SO₂ Donor on the Contents of Reducing Sugars and Soluble Protein in Al-Stressed Wheat Grain

Figure 2(a) shows the changes in reducing sugars in germinating wheat grains preincubated in SO₂ donor or H₂O for 12 h followed by incubation in Al for 48 h. Within 12 h pretreatment in H₂O and 24 h of Al treatment, the content of reducing sugar decreased gradually, whereas reducing sugar in the SO₂ donor pretreatment remained stable and slightly increased at 24 h. Thereafter reducing sugar content increased steadily in both treatments followed by a slight decrease at 48 h. The content of reducing sugars in SO₂ donor pretreated grain was always significantly higher than the counterpart of only Al treatment.

(a)

(b)

Figure 2: Effect of SO₂ pretreatment on the contents of reducing sugar and soluble protein in Al-treated grain as shown in (a) and (b), respectively. Wheat grains were pretreated with water (Al) or 1.2 mM SO₂ donor (S + Al) for 12 h (shown from 0′ to 12′/0 h of pretreatment time) and then exposed to 15 mM Al for further 48 h (shown as 12′/0, 12, 24, 36, and 48 h). The symbols * and ** in this figure and following ones stand for significant difference between Al-treated grains with and without SO₂ pretreatment at and , respectively.

The content of soluble protein increased gradually and peaked on 24 h of Al stress followed by a slight decrease (Figure 2(b)). Though the mean values of soluble protein in SO₂ donor pretreatment were higher than those pretreated in H₂O, they are not significantly different.

3.4. Effect of SO₂ Donor Pretreatment on Contents of Endogenous H₂S, , H₂O₂, and MDA

H₂S, which can be produced from sulfite, is involved in plant growth regulation including various abiotic stresses [8, 22]. To investigate whether exogenous SO₂ application can induce endogenous H₂S production, we measured the concentration of H₂S in Al-stressed wheat grain. Generally, H₂S accumulated during wheat grain germination following pretreatment with water or SO₂, but SO₂ donor pretreatment significantly enhanced H₂S concentration at 12 h of pretreatment and 12 h, 36 h of Al stress (Figure 3(a)).

(a)

(b)

(c)

(d)

Figure 3: Effects of SO₂ pretreatment on the accumulation of endogenous H₂S (a), superoxide anion () (b), hydrogen peroxide (H₂O₂) (c), and malondialdehyde (MDA) (d) in germinating wheat grains under Al stress. The numbers (0′, 12′/0, 12, 24, 36, and 48) or letters (CK or SO₂) presented are the same as mentioned in Figure 2. Al: Al stress without SO₂ pretreatment; S + Al: Al stress with SO₂ pretreatment.

To study the protective role of SO₂ in the Al-stressed wheat grain, reactive oxygen species , H₂O₂, and malondialdehyde (MDA) were determined with time. As shown in Figure 3(b), a rapid accumulation of was observed when H₂O-pretreated grains were exposed to Al. During the first 12 h of Al exposure, the increase in content was very rapid, but this was followed by a slow decrease. In contrast, the content of in SO₂ pretreatment increased slowly till 36 h of Al stress followed by a decrease. SO₂ pretreatment maintained significantly lower level of in Al-stressed wheat grains compared with grains incubated in H₂O and exposed to Al.

H₂O₂ in both treatments increased gradually during pretreatment time and 36 h of Al stress and decreased at 48 h (Figure 3(c)). However, H₂O₂ content in SO₂ pretreatment was significantly lower than that in water pretreatment when exposed to Al stress.

During the 12 h pretreatment time, no significant difference was observed in MDA content in wheat grains whether pretreated with SO₂ donor or H₂O (Figure 3(d)). After exposure to Al, the content of MDA in water pretreated grains increased rapidly till 48 h of Al stress. An increase of MDA content was also observed in SO₂ pretreatment at 12 h of Al stress, but thereafter MDA content remained stable until 36 h. SO₂ pretreatment dramatically reduced the amount of MDA from 24 h to 48 h of Al stress in comparison with grains pretreated in water.

3.5. Effects of SO₂ Donor Pretreatment on POD, CAT, APX, and LOX Activities

Activities of POD, CAT, APX, and LOX were determined with time in SO₂ donor and H₂O-pretreated grains exposed to Al (Figure 4). Figure 4(a) shows the time course of POD activity following pretreatment in SO₂ donor or H₂O for 12 h when POD activity showed almost a twofold increase. During Al stress, POD activity exhibited a gradual increase in both treatments, but SO₂ pretreatment maintained significantly higher level of POD activity during Al stress.

(a)

(b)

(c)

(d)

Figure 4: Effect of SO₂ donor pretreatment on the activities of POD (a), CAT (b), APX (c), and LOX (d) in germinating wheat grains under 15 mM Al stress. Grains were treated and the number or letters presented are the same as mentioned in Figure 2. Al: Al stress without SO₂ pretreatment; S + Al: Al stress with SO₂ pretreatment.

The activity of CAT increased almost twofold during 12 h pretreatment with H₂O or SO₂ donor (Figure 4(b)). After exposure to Al, CAT activity in water pretreatment decreased gradually till 48 h of Al stress, suggesting that CAT activity is very sensitive to Al stress. In contrast, CAT activity in SO₂ pretreatment increased steadily and decreased only slightly at 48 h of Al stress.

As shown in Figure 4(c), SO₂ pretreatment enhanced APX activity in Al-stressed wheat grain. A rapid increase in APX activity occurred during the pretreatment time in H₂O and SO₂. Within the first 12 h of Al stress, APX activity in H₂O-pretreated grains decreased sharply, whereas SO₂ donor pretreatment enhanced APX activity slightly. Thereafter APX activity increased steadily in water pretreated grain, whereas its activity in SO₂ donor pretreatment fluctuated slightly. The APX activity in SO₂ donor pretreated grains was always significantly higher than the counterpart of water pretreatment.

An increase in LOX activity was observed during the first 24 h of Al stress in SO₂ and H₂O-pretreated grains (Figure 4(d)). However, the increase of LOX activity in water pretreatment was more rapid than after SO₂ pretreatment. Thereafter LOX activity in water pretreatment showed a sharp decrease at 36 h of Al stress, while its activity in SO₂ pretreatment decreased at 48 h. At 12 and 24 h of Al stress, SO₂ pretreatment maintained significantly lower level of LOX, while at 36 h LOX activity in SO₂ pretreatment was higher than that of water pretreatment.

3.6. Effects of SO₂ Donor Pretreatment on Localization of Al, Lipid Peroxidation, and ROS Production

To detect ROS production in the radicle tips, we used DCFH-DA fluorescence to indicate ROS accumulation. As shown in Figure 5(a), Al treatment induced higher level of ROS in radicle as intense DCFH-DA fluorescence, while SO₂ donor pretreatment for 12 h followed by Al stress significantly reduced fluorescence. Figure 5(b) shows DCFH-DA fluorescence in maturation zone in radicles. Similarly, intense fluorescence in SO₂ donor pretreatment followed by Al stress was much weaker than that in water pretreated plus Al-stressed radicles, suggesting that SO₂ donor was effective in alleviating oxidative stress in radicles. SO₂ donor treatment alone showed comparable fluorescence intensity as observed in water control.

(a)

(b)

(c)

(d)

Figure 5: ROS staining ((a) on radicle tips; (b) on maturation zone; bar: 200 μm), Evans blue staining (c), and hematoxylin staining (d) in wheat radicles. Initially, wheat grains were geminated in water for 36 h. Then four treatment groups were done as follows, control, 60 h in H₂O; Pre-S, pretreatment with 1 mM SO₂ donor for 12 h, and then exposed to H₂O for 48 h; Al, 12 h in H₂O prior to exposure to 400 μM AlCl₃ for 48 h; Pre-S + Al, 12 h in 1 mM SO₂ donor pretreatment followed by 400 μM AlCl₃ for 48 h.

The radicles were stained with Evans blue to show membrane integrity. The radicles treated with Al alone were stained extensively with Evans blue, while Al-stressed radicles pretreated with SO₂ donor for 12 h were less stained (Figure 5(c)), suggesting SO₂ donor serves to protect cell membrane from Al-induced damage. SO₂ donor treatment alone showed similar Evans blue staining to water control, suggesting no visible damaging effect of SO₂ on radicles.

The hematoxylin staining was used to detect Al accumulation in radicles. As shown in Figure 5(d), the radicles of water control and SO₂ treatment incubated with hematoxylin showed no dark staining but wheat radicles treated with Al alone were stained intensively. In contrast, radicles pretreated with SO₂ donor for 12 h and then exposed to Al for 48 h showed much weaker staining compared with Al stress, especially in the elongation zone.

3.7. Effect of SO₂ Donor Pretreatment on the Relative Expressions of Aluminum Stress Related Genes

We determined the changes in gene expression of aluminum stress related genes in wheat radicles. Radicles were pretreated with or without 1 mM SO₂ donors for 12 h and then exposed to Al for 48 h. As shown in Figure 6, Al stress induced higher expression of TaWali1, TaWali2, TaWali3, TaWali5, and TaWali6 (wheat aluminum induced) in radicles, while pretreatment with SO₂ donor for 12 h followed by Al stress alleviated such expression increase. Besides, the gene expression of TaALMT1 (Al-activated malate transporter) was also attenuated by SO₂ pretreatment.

Figure 6: Effect of SO₂ donor pretreatment on relative gene expression of TaWali1, TaWali2, TaWali3, TaWali5, TaWali6, and TaALMT1 in wheat radicals exposed to Al stress. Initially, wheat grains were geminated in water for 36 h. Then four treatment groups were done as follows, control, 60 h in H₂O; Pre-S, pretreatment with 1 mM SO₂ donor for 12 h, and then exposed to H₂O for 48 h; Al, 12 h in H₂O prior to exposure to 400 μM AlCl₃ for 48 h; Pre-S + Al, 12 h in 1 mM SO₂ donor pretreatment followed by 400 μM AlCl₃ for 48 h.

4. Discussion

In solution, SO₂ is dissociated from its sulfite derivatives (NaHSO₃/Na₂SO₃ 1 : 3 M/M) [34]. Thus NaHSO₃/Na₂SO₃ (1 : 3 M/M) was chosen as an SO₂ donor in our study. Similar to the observation that H₂S could promote wheat grain germination and alleviate oxidative damage against Al stress [8], our results show that SO₂ donor pretreatment alleviates Al stress in germinating wheat seedlings. Wheat grains pretreated for 12 h with the SO₂ donor show an increase in germination percentage, coleoptile length, radicle length, and radicle numbers of wheat. The increase in the contents of reducing sugars and soluble protein suggests that nutrients in wheat grains pretreated with SO₂ donor are rapidly mobilized to provide energy to grain germination. SO₂ donor maintained lower level of H₂O₂, , and MDA probably by activation of the antioxidant system. These results suggest that SO₂ acts as an antioxidant and may function in a way that is similar to what the effects of H₂S, CO, and NO do in plants exposed to heavy metal stress [10, 35].

Sulfite can be reduced by sulfite reductase to H₂S, which is incorporated into O-acetylserine via O-acetyl(thiol)lyase to form cysteine [22]. In RNA interfered mutant of sulfite reductase (SiR), sulfide synthesis in younger leaves was decreased by the impaired SiR activity [36]. In the present study, exogenous SO₂ application can induce endogenous H₂S production in Al-stressed wheat grains (Figure 3(a)), suggesting the interplay between sulfite and the formation of H₂S.

Consistent with previous observations [7], our results show that Al stress caused overproduction of ROS in wheat. To mitigate and repair oxidative damage, plants have evolved an efficient antioxidant system that includes enzymes such as SOD, CAT, and APX that function to scavenge ROS [37]. SOD catalyzes the dismutation of the superoxide radical and H⁺ into H₂O₂. CAT, APX, and POD are responsible for the elimination of H₂O₂ generated by SOD. Al stress brings about a dramatic increase in H₂O₂ and . The elevated levels of H₂O₂ and suggest that antioxidant enzymes in Al-stressed wheat do not efficiently scavenge the overproduction of ROS, and this can result in lipid peroxidation or plasma membrane inhibiting grain germination and seedling growth [8]. Our data show that pretreatment of wheat with SO₂ donor activates antioxidant enzymes including POD, CAT, and APX.

LOX, which catalyzes oxygenation of polyunsaturated fatty acids into lipid hydroperoxides, is considered an indicator of oxidative stress during responses to various environmental stresses [9]. Pretreatment with SO₂ donor lowers LOX activity in Al-stressed wheat radicles compared to seedlings pretreated with H₂O and exposed to Al. The lowering of LOX by SO₂ pretreatment also helps to explain the lower MDA content of Al-stressed grain. Taken together, these data suggest that SO₂ donor reduced oxidative stress by modulation of the antioxidant system.

Our data indicate that the radicle is the primary target for Al toxicity. DCFH-DA fluorescence assay shows that Al incubation induces higher accumulation of ROS in radicle tips and maturation zone. SO₂ donor pretreatment effectively reduces ROS content in subsequent Al stress, suggesting the role of SO₂ in alleviating oxidative stress. Correspondently, Al stress causes membrane injury to radicles, while SO₂ donor effectively alleviates such injury. To understand whether SO₂ donor helps to reduce Al accumulation in radicles, hematoxylin staining was used to indicate Al and the results show that SO₂ donor obviously reduces Al content in radicles, implying a potential role of SO₂ donor treatment as a strategy to reduce Al uptake.

In response to Al stress, many gene expressions are activated, for instance, TaWali (wheat aluminum induced), aluminum-activated malate transporter (TaALMT1) [38–41]. Relative gene expression analysis shows that Al treatment induces higher expression of TaWali, while these gene expression levels are reduced by SO₂ donor pretreatment, suggesting the response to Al stress is attenuated in SO₂ donor pretreatment.

5. Conclusion

In the present study, SO₂ acts as an antioxidant signal to reduce ROS damage in wheat grains and radicles caused by Al stress. Besides, SO₂ also decreases Al uptake. The induced higher level of H₂S suggests an intricate interplay of SO₂ and H₂S in plants. Exogenous application of SO₂ may be reduced to H₂S by sulfite reductase, thus contributing to H₂S production. H₂S in itself acts as an antioxidant signaling molecule in plants’ response to abiotic stress. Thus the nature of SO₂/sulfite functions in alleviating Al stress still needs further research.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contribution

Dong-Bo Zhu, Kang-Di Hu, and Xi-Kai Guo contributed equally to this work.

Acknowledgments

This work was supported by the Natural Science Foundation of China (31271803, 31301820, 31300133, and 31470013), the Scientific Research Foundation for Returned Overseas Chinese Scholars (SRF for ROCS, MOE), the Natural Science Foundations of Anhui Province (11040606M85), and the Anhui Provincial Education Department (2012AJZR0028, ZD200910).

References

P. R. Ryan, S. D. Tyerman, T. Sasaki et al., “The identification of aluminium-resistance genes provides opportunities for enhancing crop production on acid soils,” Journal of Experimental Botany, vol. 62, no. 1, pp. 9–20, 2011.

Z. Q. Wang, X. Y. Xu, Q. Q. Gong et al., “Root proteome of rice studied by iTRAQ provides integrated insight into aluminum stress tolerance mechanisms in plants,” Journal of Proteomics, vol. 98, no. 26, pp. 189–205, 2014.

W. Huang, X. Yang, S. Yao et al., “Reactive oxygen species burst induced by aluminum stress triggers mitochondria-dependent programmed cell death in peanut root tip cells,” Plant Physiology and Biochemistry, vol. 82, no. 12, pp. 76–84, 2014.

Y.-S. Wang and Z.-M. Yang, “Nitric oxide reduces aluminum toxicity by preventing oxidative stress in the roots of Cassia tora L,” Plant and Cell Physiology, vol. 46, no. 12, pp. 1915–1923, 2005.

K. Tahara, T. Yamanoshita, M. Norisada et al., “Aluminum distribution and reactive oxygen species accumulation in root tips of two Melaleuca trees differing in aluminum resistance,” Plant and Soil, vol. 307, no. 1-2, pp. 167–178, 2008.

Z. J. Ding, J. Y. Yan, X. Y. Xu, G. X. Li, and S. J. Zheng, “WRKY46 functions as a transcriptional repressor of ALMT1, regulating aluminum-induced malate secretion in Arabidopsis,” The Plant Journal, vol. 76, no. 5, pp. 825–835, 2013.

S. J. Zheng and J. L. Yang, “Target sites of aluminum phytotoxicity,” Biologia Plantarum, vol. 49, no. 3, pp. 321–331, 2005.

H. Zhang, Y. H. Li, L. Y. Hu, S. H. Wang, F. Q. Zhang, and K. D. Hu, “Effects of exogenous nitric oxide donor on antioxidant metabolism in wheat leaves under aluminum stress,” Russian Journal of Plant Physiology, vol. 55, no. 4, pp. 469–474, 2008.

H. Zhang, Z.-Q. Tan, L.-Y. Hu, S.-H. Wang, J.-P. Luo, and R. L. Jones, “Hydrogen sulfide alleviates aluminum toxicity in germinating wheat seedlings,” Journal of Integrative Plant Biology, vol. 52, no. 6, pp. 556–567, 2010.

J. Xu, W. Xuan, B. Huang et al., “Carbon monoxide-induced adventitious rooting of hypocotyl cuttings from mung bean seedling,” Chinese Science Bulletin, vol. 51, no. 6, pp. 668–674, 2006.

S. Q. Zhang, J. B. Du, H. F. Jin et al., “Endogenous sulfur dioxide aggravates myocardial injury in isolated rat heart with ischemia and reperfusion,” Transplantation, vol. 87, no. 4, pp. 517–524, 2009.

R. Y. Zhang, J. B. Du, Y. Sun et al., “Sulfur dioxide derivatives depress L-type calcium channel in rat cardiomyocytle,” Clinical and Experimental Pharmacology and Physiology, vol. 38, no. 7, pp. 416–422, 2011.

H.-F. Jin, S.-X. Du, X. Zhao et al., “Effects of endogenous sulfur dioxide on monocrotaline-induced pulmonary hypertension in rats,” Acta Pharmacologica Sinica, vol. 29, no. 10, pp. 1157–1166, 2008.

I. Ziegler, “The effect of SO₂ pollution on plant metabolism,” Residue Reviews, vol. 56, no. 1, pp. 79–105, 1975.

R. Sandhu, Y. Li, and G. Gupta, “Sulphur dioxide and carbon dioxide induced changes in soybean physiology,” Plant Science, vol. 83, no. 1, pp. 31–34, 1992.

D. Yarmolinsky, G. Brychkova, R. Fluhr, and M. Sagi, “Sulfite reductase protects plants against sulfite toxicity,” Plant Physiology, vol. 161, no. 2, pp. 725–743, 2013.

M. Noji, M. Saito, M. Nakamura, M. Aono, H. Saji, and K. Saito, “Cysteine synthase overexpression in tobacco confers tolerance to sulfur-containing environmental pollutants,” Plant Physiology, vol. 126, no. 3, pp. 973–980, 2001.

R. Rakwal, G. K. Agrawal, A. Kubo et al., “Defense/stress responses elicited in rice seedlings exposed to the gaseous air pollutant sulfur dioxide,” Environmental and Experimental Botany, vol. 49, no. 3, pp. 223–235, 2003.

J. J. Yin, X. Liu, H. L. Yi, and M. L. Yang, “Sulfur dioxide induces guard cell death in Vicia faba,” Acta Scientiae Circumstantiae, vol. 30, no. 12, pp. 2512–2517, 2010.

H. Rennenberg, “The fate of excess sulfur in higher plants,” Annual Review of Plant Physiology, vol. 35, no. 4, pp. 121–153, 1984.

E. Giraud, A. Ivanova, C. S. Gordon, J. Whelan, and M. J. Considine, “Sulphur dioxide evokes a large scale reprogramming of the grape berry transcriptome associated with oxidative signalling and biotic defence responses,” Plant, Cell and Environment, vol. 35, no. 2, pp. 405–417, 2012.

T. Rausch and A. Wachter, “Sulfur metabolism: a versatile platform for launching defence operations,” Trends in Plant Science, vol. 10, no. 10, pp. 503–509, 2005.

M. H. Stipanuk, J. E. Dominy Jr., J.-I. Lee, and R. M. Coloso, “Mammalian cysteine metabolism: new insights into regulation of cysteine metabolism,” Journal of Nutrition, vol. 136, no. 6, pp. 1652S–1659S, 2006.

A. Laisk, H. Pfanz, and U. Heber, “Sulfur-dioxide fluxes into different cellular compartments of leaves photosynthesizing in a polluted atmosphere: II. Consequences of SO₂ uptake as revealed by computer analysis,” Planta, vol. 173, no. 2, pp. 241–252, 1988.

H. Zhang, S.-L. Hu, Z.-J. Zhang et al., “Hydrogen sulfide acts as a regulator of flower senescence in plants,” Postharvest Biology and Technology, vol. 60, no. 3, pp. 251–257, 2011.

K. Surrey, “Spectrophotometric method for determination of lipoxidase activity,” Plant Physiology, vol. 39, no. 1, pp. 65–70, 1964.

L.-Y. Hu, S.-L. Hu, J. Wu et al., “Hydrogen sulfide prolongs postharvest shelf life of strawberry and plays an antioxidative role in fruits,” Journal of Agricultural and Food Chemistry, vol. 60, no. 35, pp. 8684–8693, 2012.

G. L. Miller, “Use of dinitrosalicylic acid reagent for determination of reducing sugar,” Analytical Chemistry, vol. 31, no. 3, pp. 426–428, 1959.

M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding,” Analytical Biochemistry, vol. 72, no. 1-2, pp. 248–254, 1976.

Y. Yamamoto, Y. Kobayashi, S. R. Devi, S. Rikiishi, and H. Matsumoto, “Aluminum toxicity is associated with mitochondrial dysfunction and the production of reactive oxygen species in plant cells,” Plant Physiology, vol. 128, no. 1, pp. 63–72, 2002.

E. Polle, C. F. Konzak, and J. A. Kittrick, “Visual detection of aluminum tolerance levels in wheat by hematoxylin staining of seeding roots,” Crop Science, vol. 18, no. 5, pp. 823–827, 1978.

C. P. LeBel, H. Ischiropoulos, and S. C. Bondy, “Evaluation of the probe 2′,7′-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress,” Chemical Research in Toxicology, vol. 5, no. 2, pp. 227–231, 1992.

S.-P. Li, K.-D. Hu, L.-Y. Hu et al., “Hydrogen sulfide alleviates postharvest senescence of broccoli by modulating antioxidant defense and senescence-related gene expression,” Journal of Agricultural and Food Chemistry, vol. 62, no. 5, pp. 1119–1129, 2014.

R. Shapiro, “Genetic effects of bisulfite (sulfur dioxide),” Mutation Research, vol. 39, no. 2, pp. 149–175, 1977.

K.-D. Hu, L.-Y. Hu, Y.-H. Li, F.-Q. Zhang, and H. Zhang, “Protective roles of nitric oxide on germination and antioxidant metabolism in wheat seeds under copper stress,” Plant Growth Regulation, vol. 53, no. 3, pp. 173–183, 2007.

D. Yarmolinsky, G. Brychkova, A. Kurmanbayeva et al., “Impairment in sulfite reductase leads to early leaf senescence in tomato plants,” Plant Physiology, vol. 165, no. 4, pp. 1505–1520, 2014.

P. Sharma and R. S. Dubey, “Involvement of oxidative stress and role of antioxidative defense system in growing rice seedlings exposed to toxic concentrations of aluminum,” Plant Cell Reports, vol. 26, no. 11, pp. 2027–2038, 2007.

K. C. Snowden and R. C. Gardner, “Five genes induced by aluminum in wheat (Triticum aestivum L.) roots,” Plant Physiology, vol. 103, no. 3, pp. 855–861, 1993.

A. Ligaba, I. Dreyer, A. Margaryan, D. J. Schneider, L. Kochian, and M. Piñeros, “Functional, structural and phylogenetic analysis of domains underlying the Al sensitivity of the aluminum-activated malate/anion transporter, TaALMT1,” The Plant Journal, vol. 76, no. 5, pp. 766–780, 2013.

N. Yoshimoto, H. Takahashi, F. W. Smith, T. Yamaya, and K. Saito, “Two distinct high-affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots,” The Plant Journal, vol. 29, no. 4, pp. 465–473, 2002.

T. Kataoka, A. Watanabe-Takahashi, N. Hayashi et al., “Vacuolar sulfate transporters are essential determinants controlling internal distribution of sulfate in Arabidopsis,” The Plant Cell, vol. 16, no. 10, pp. 2693–2704, 2004.