Plant Physiology and Biochemistry 78 (2014) 27e36

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Research article

Silicon improves seed germination and alleviates oxidative stress of bud seedlings in tomato under water deficit stress Yu Shi a, Yi Zhang b, Hejin Yao c, Jiawen Wu a, Hao Sun a, Haijun Gong a, * a

College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, PR China College of Horticulture, Shanxi Agricultural University, Taigu 030801, Shanxi, PR China c College of Teacher Education, Quzhou University, Quzhou 324000, Zhejiang, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 October 2013 Accepted 8 February 2014 Available online 22 February 2014

The beneficial effects of silicon on plant growth and development under drought have been widely reported. However, little information is available on the effects of silicon on seed germination under drought. In this work, the effects of exogenous silicon (0.5 mM) on the seed germination and tolerance performance of tomato (Solanum lycopersicum L.) bud seedlings under water deficit stress simulated by 10% (w/v) polyethylene glycol (PEG-6000) were investigated in four cultivars (‘Jinpengchaoguan’, ‘Zhongza No.9’, ‘Houpi L402’ and ‘Oubao318’). The results showed that the seed germination percentage was notably decreased in the four cultivars under water stress, and it was significantly improved by added silicon. Compared with the non-silicon treatment, silicon addition increased the activities of superoxide dismutase (SOD) and catalase (CAT), and decreased the production of superoxide anion (O2
) and hydrogen peroxide (H2O2) in the radicles of bud seedlings under water stress. Addition of silicon decreased the total phenol concentrations in radicles under water stress, which might contribute to the decrease of peroxidase (POD) activity, as observed in the in vivo and in vitro experiments. The decrease of POD activity might contribute to a less accumulation of hydroxyl radical ($OH) under water stress. Silicon addition also decreased the concentrations of malondialdehyde (MDA) in the radicles under stress, indicating decreased lipid peroxidation. These results suggest that exogenous silicon could improve seed germination and alleviate oxidative stress to bud seedling of tomato by enhancing antioxidant defense. The positive effects of silicon observed in a silicon-excluder also suggest the active involvement of silicon in biochemical processes in plants. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Tomato Water deficit Seed germination Silicon Oxidative damage Antioxidant defense

1. Introduction Seed germination is usually the most crucial phase during seedling establishment (Hubbard et al., 2012). However, this process may be delayed or even entirely prevented by various abiotic stresses, such as salinity (Haghighi et al., 2012) and drought (Hubbard et al., 2012). In the arid and semi-arid regions, water deficit is one of the mains factors limiting seed germination and crop production. Application of exogenous substances may be a feasible pathway to increase seed germination under these stress conditions.

Abbreviations: CAT, catalase; DW, dry weight; GI, germination index; GP, germination percentage; MDA, malondialdehyde; PEG-6000, polyethylene glycol6000; POD, peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase. * Corresponding author. Tel.: þ86 29 8708 2613. E-mail address: [email protected] (H. Gong). http://dx.doi.org/10.1016/j.plaphy.2014.02.009 0981-9428/Ó 2014 Elsevier Masson SAS. All rights reserved.

In normal conditions, the production of reactive oxygen species (ROS) and antioxidant defense in plant cells are in a dynamic balance. However, under stress conditions, the balance can be broken, causing an over-production of ROS and inducing oxidative damage (Gong et al., 2005). To minimize the adverse impact, plants have developed the antioxidant defense system against ROS, including the antioxidant enzymes [such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT)] and non-enzymatic compounds (Gong et al., 2005, 2008). SOD converts O2
into H2O2, which is then catalyzed into water and molecular oxygen by CAT (Wang et al., 2009). POD can also detoxify H2O2 using various substrates (such as phenol) as electron donor (Wang et al., 2009). It has been shown that the phenol level can affect the activity of POD in plants (Dragisi c Maksimovi c et al., 2007). Under water deficit conditions, high capability of antioxidant defense is essential for plants to scavenge ROS and therefore avoid oxidative damage (Masoumi et al., 2011; Yang et al., 2010).

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Y. Shi et al. / Plant Physiology and Biochemistry 78 (2014) 27e36

Silicon (Si) is the second most abundant mineral element after oxygen in the earth’s crust (Gottardi et al., 2012). It is considered a non-essential element for the majority of plants. Despite this, its beneficial effects on the growth of plants have been widely reported, especially when they are subjected to biotic and abiotic stresses, such as diseases, pests, lodging, drought, salinity, heavy metal, radiation, high or low temperature and nutrient imbalance (Ma and Yamaji, 2008; Guntzer et al., 2012; Gonzalo et al., 2013; Liu et al., 2013; Mateos-Naranjo et al., 2013; Van Bockhaven et al., 2013). Silicon-mediated drought tolerance has been observed in several plant species, such as rice (Agarie et al., 1998), maize (Gao et al., 2006), wheat (Gong et al., 2005, 2008; Gong and Chen, 2012), sunflower (Gunes et al., 2008) and sorghum (Hattori et al., 2005; Sonobe et al., 2011). The mechanisms for silicon-mediated drought tolerance are still not fully understood, but have been suggested to be associated with the decrease in transpirational loss of water (Agarie et al., 1998), enhancement of water uptake by roots (Hattori et al., 2005), and the involvement in physiological metabolism (Gong et al., 2005; Gunes et al., 2008) in plants. In early years, Agarie et al. (1998) suggested that silicon deposition in the leaves can decrease the transpiration and therefore increase drought tolerance. However, added silicon does not always decrease the plant transpiration under drought, as observed in sorghum (Hattori et al., 2005) and wheat (Gong et al., 2005). Hattori et al. (2005) suggested that silicon-mediated improvement of drought tolerance in sorghum may be associated with the enhancement of water uptake ability, rather than decrease of transpiration. Their later study (Sonobe et al., 2011) confirmed that silicon mediates the active accumulation of soluble sugar and amino acids in sorghum roots, therefore decreasing the root osmotic potential, which induces increased water uptake. Silicon is also suggested to keep the mineral balance of plants under water deficit. For instance, silicon addition could increase the Ca and K levels in drought-stressed maize leaves (Kaya et al., 2006). One possible mechanism for the increased nutrient uptake could be associated with silicon-mediated activation of Hþ-ATPase in the plasma membrane (Kaya et al., 2006). Moreover, silicon can also increase the activities of antioxidant enzymes and reduce oxidative damage in different plant species when subjected to drought stress (Gong et al., 2005; Gunes et al., 2008). These findings suggest that silicon may improve the drought tolerance of plants through promoting the uptake and utilization of water and nutrients, and modulating physiological activities (such as enhancing the activities of antioxidant enzymes). However, most of these studies were conducted at seedling stage or later stages of plant growth and development. Previous research has suggested that silicon has positive effects on seed germination in different plant species under normal conditions. For instance, white oat seeds with higher germination percentage and vigor are obtained with 300 and 450 mg L1 of K2SiO3, respectively (Toledo et al., 2011). It has also been shown that different levels of exogenous silicon (Na2SiO3) had considerable effects on the germination percentage (GP) and germination index (GI) of borage seeds, with the highest germination percentage being obtained at 1.5 mM silicon (Torabi et al., 2012). However, up to now, little information is available on the effects of silicon on seed germination under drought/water deficit stress conditions. Although the beneficial effects of silicon for plants under drought have been reported by many researchers, most of previous studies were conducted on silicon-accumulating plant species, in which physical barrier induced by silicon deposition on plant surface may have contributed to the observed drought tolerance directly or indirectly. It is proposed that, silicon exerts its

beneficial roles mainly through two mechanisms: the protective effects as a mechanical/physical barrier by amorphous silica due to silicon deposition, and the biochemical functions of aqueous silicic acid as a modulator of plant stress tolerance (Ma and Yamaji, 2008; Cooke and Leishman, 2011). The formation of amorphous on leaves can decrease plant transpiration (Agarie et al., 1998) and impede attack by pathogens and pests (Ma and Yamaji, 2008). However, it is still lack of evidence that silicon has a direct biochemical function (i.e. in planta mechanism) in plants. Clarification of the biochemical functions of silicon will help to understand the exact mechanisms of silicon-induced tolerance of plants to environmental stresses. Tomato (Solanum lycopersicum L.) is one of the largest cultivated and consumed vegetables in the world. Compared with the silicon accumulators like rice and wheat, tomato displays passive absorption and much less accumulation of silicon (Liang et al., 2007). Therefore, it has been classified as “silicon excluder” (Nikolic et al., 2007). Hence, using tomato as an experimental material to investigate the effects of silicon on stress tolerance can decrease the effect of mechanical barrier induced by silicon deposition to a greatest extent, and the observed silicon effect is supposed to be mainly due to its biochemical function. The purpose of this study was to investigate whether exogenous silicon could improve the seed germination of tomato e a “silicon excluder” under water deficit stress. If yes, one would expect the possible involvement of silicon’s biochemical function in the improvement, and it would be interesting to investigate whether the plant antioxidant defense was involved, and whether there was a certain relationship among silicon addition, POD activity and phenol level. Polyethylene glycol molecule with a Mr  6000 (PEG6000), which is inert and non-ionic, has frequently been used to induce osmotic stress (and thus simulate drought stress) and maintain uniform water potential throughout the experiment. Besides, PEG-6000 is large enough so that it does not penetrate membranes and thus stays in apoplast fluid (Huang and Song, 2013). Therefore, in this work, the effects of silicon on the germination characteristics, production of reactive oxygen species, lipid peroxidation, antioxidant defense responses, and phenol level during seed germination of tomato under PEG-induced water deficit stress were investigated. A potential use of silicon fertilizer in water-saving irrigation in tomato in arid or semi-arid regions is discussed. 2. Materials and methods 2.1. Plant materials and treatments Four tomato (S. lycopersicum L.) cultivars were used in the study: Jinpengchaoguan (abbreviated as ‘Jinpeng’), Zhongza No.9 (abbreviated as ‘Zhongza’), Houpi L402 (abbreviated as ‘Houpi’) and Oubao 318 (abbreviated as ‘Oubao’). The seeds were sterilized in 55  C water bath for 25 min, immersed in distilled water for 6 h, and then transferred to Petri dishes (80 seeds per dish) lined with two layers of filter paper moistened with four different solutions as follows: (1) Si/PEG ¼ distilled water; (2) þSi/PEG ¼ 0.5 mM sodium silicate solution (Bodi Chemical Co., Ltd., Tianjin, China); (3) Si/þPEG ¼ 10% (w/v) PEG-6000 solution (Purity  99%, Wako Pure Chemical Industries, Ltd., Japan); and (4) þSi/þPEG ¼ 10% (w/ v) PEG-6000 plus 0.5 mM sodium silicate solution. The experiments were conducted in a germination programmed incubator with constant temperature at 28  1  C and relative humidity of 70%, and the culture lasted for 7 days. Every morning, 3 ml of each solution was added into the corresponding Petri dish. Each treatment had 3 replicates. The pH in all solutions was adjusted to 6.2  0.1 with 1 M H2SO4.

Y. Shi et al. / Plant Physiology and Biochemistry 78 (2014) 27e36

2.2. Measurements 2.2.1. Germination characteristics Seeds were considered to be germinated when the radicle emerged through the seed coat and reached more than 2 mm in length. The number of germinated seeds for each cultivar and treatment was recorded every day. Germination percentages (GP) and germination index (GI) were calculated using the following equations: GP ¼ n/N  100%, where n is the number of germination, and N represents the total number of tested seeds; P GI ¼ (Gt/Dt), where Gt is the number of seed germinated at t day, and Dt represents the corresponding day of germination; 2.2.2. Determination of antioxidant enzyme activity Antioxidant enzymes were extracted by using the methods of Gong et al. (2005) with minor modifications. Fresh radicles (1.0 g each) were homogenized in 8 ml ice-cold 50 mM sodium phosphate buffer (pH 7.0) using a pre-cooled mortar and pestle. The homogenate was centrifuged at 9661  g for 20 min at 4  C, and the supernatant was used to determine SOD, POD and CAT activities. SOD activity was assayed according to the nitrobluetetrazolium method (Gong et al., 2005). The reaction mixture (3 ml) contained 50 mM sodium phosphate buffer (pH 7.3), 13 mM methionine, 75 mM nitrobluetetrazolium, 0.1 mM ethylene diaminetetra acetic acid, 4 mM riboflavin and 0.2 ml of extract. The SOD activity was measured colorimetrically at 560 nm by UVeVis spectrophotometer (Shimadzu UV-2450, Japan). One unit of SOD was defined as the amount of enzyme required to cause 50% inhibition of nitrobluetetrazolium reduction. Enzyme activity was expressed as U$mg1 protein. POD and CAT activities were measured with the methods described by Yang et al. (2010) with slight modifications. The POD reaction mixture (3 ml) consisted of 10 mM guaiacol, 50 mM H2O2, 0.2 M phosphate buffer (pH 6.0) and 0.1 ml of enzyme extract. The POD activity was measured by spectrophotometry at 470 nm. The CAT assay mixture (3 ml) contained 15 mM sodium phosphate buffer (pH 7.0), 10 mM H2O2, and 0.1 ml of enzyme extract. The CAT activity was measured by spectrophotometry at 240 nm. One unit of POD or CAT activity was defined as 1.0 change in absorbance per min. The enzyme activities of POD and CAT were expressed as U$mg1 protein. Protein content was determined according to the method of Bradford (1976). 2.2.3. Determination of silicon effect on POD activity in vitro The composition of reaction mixture for determination of in vitro POD activity was the same as described above. The effect of silicon on POD activities in vitro were determined by adding 0.5 mM Si(OH)4 to the reaction medium. Si(OH)4 was prepared by passing potassium silicate through cation-exchange resin (001  7, Cangzhou Bon Adsorber Technology Co. Ltd., Cangzhou, Hebei, China). The enzyme solutions were extracted from the fresh radicles (control group) of each tomato cultivar. 2.2.4. Detection of reactive oxygen species level The levels of O2
and H2O2 in the radicles were examined by histochemical stain according the method of Xu et al. (2012) with some modifications. For localizing the O2
, the radicles were immersed and infiltrated under vacuum with 0.5 mg ml1 of 3,30 -diaminobenzidine (Sigma) in 50 mM TriseHCl (pH 3.8) under dark place for 8 h and washed in alcohol: lactic acid: glycerin (3:1:1, v/v) for 10 min and then stored in 95% ethanol until photographed under a Epi-Fluorescence microscope (BX51, Olympus, Japan).

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The H2O2 level in radicles was detected by using a fluorescent dye 2,7-dichlorofluorescin diacetate (D6883, Sigma) as described by Xu et al. (2012). Radicles were immersed in 10 mM 2,7dichlorofluorescin diacetate in 10 mM MES-Tris buffer (pH 6.1) containing 10 mM NaN3 for 30 min at 37  C, and were washed three times in MES-Tris buffer (10 min each) to remove excess dye and then viewed under a Leica laser scanning confocal microscope (A1R/A1, Nikon, Japan) at excitation wavelength of 488 nm and emission wavelength of 530 nm. 2.2.5. Determination of phenol content Phenol was extracted according to the method of Schaller et al. (2012) with some modifications. Radicles (0.5 g each) were homogenized with 95% (v/v) ethanol. The extract was well mixed with equal volume of Folin-Ciocalteu-reagent for 3 min, and then half its volume of 10% Na2CO3 was added. After 1 hour’s standing, the absorbance of the reaction solution was read at 760 nm by spectrophotometry. The phenol content was expressed in mg g1 DW or mg g1 H2O. 2.2.6. Determination of malondialdehyde content MDA content was determined by thiobarbituric acid reaction (Bailly et al., 1996) with slight modifications. Fresh radicles (0.5 g each) were homogenized in 8 ml of 0.1% (w/v) trichloroacetic acid, and the homogenates were centrifuged at 4830  g for 10 min at 4  C. To measure MDA, 1.5 ml of the supernatant was added into 1.5 ml of 0.5% (w/v) trichloroacetic acid. The mixture was reacted at 100  C for 20 min before being cooled in an ice bath. After centrifugation at 7888  g for 10 min, the absorbance of the supernatant was measured at 450 nm, 532 nm and 600 nm in spectrophotometer. The MDA content was calculated using the formula: MDA content (mmol g1 DW) ¼ [6.452 (OD532  OD600)  0.559 *OD450] *8/(DW*1.5) 2.2.7. Determination of silicon content Silicon content in radicles was measured according to the method of Iwasaki et al. (2002) with some modifications. The radicles were washed thoroughly and quickly with distilled water, and were then further washed in cold CaCl2 solution (0.5 mM) for 15 min, followed by a quick rinse in distilled water (Liang et al., 2005). After the wash, the radicles were then dried in an oven (Fuma, Shanghai, China) at 65  C for 2 days and ground with mortar and pestle. The concentration of silicon in radicles was determined by digesting 20 mg of ground plant materials in 1 ml of the mixture of 1 M HCl and 2.3 M HF (v:v, 1:2) and shaken overnight. The samples were centrifuged at 10,000 g for 10 min, and 0.5 ml of the supernatant was added in plastic tubes. Then 3 ml of 20% glacial acetic acid and 1 ml of 54 g/l (NH4)6Mo7.4H2O were added, and incubated for 5 min, followed by addition of 0.5 ml of 20 g/l tartaric acid and 0.1 ml reductant. Thirty minutes after addition of the reductant, the sample absorbance was measured at 650 nm with a spectrophotometer (Shimadzu UV-2450, Japan). To prepare the reductant, firstly, solution A and B were prepared: solution A, 2 g Na2SO3 and 0.4 g 1-amino-2-naphthol- 4-sulfoacid were dissolved in 25 ml of ultrapure water; solution B: 25 g NaHSO3 was dissolved in 200 ml of ultrapure water. Then the solution A and B were mixed together and quantified to 250 ml. 2.3. Statistical analysis Data were statistically analyzed with SAS software (SAS Institute, Cary, NC) using Duncan’s multiple range test at the 0.05 level.

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3. Results 3.1. Germination responses The effects of water deficit stress and silicon addition on the germination percentages (GP) of four tomato cultivars are shown in Fig. 1. Water stress significantly reduced the GP of each cultivar, as compared with the control. No significant differences, across all cultivars, were observed in GP by added silicon under non-stress conditions. However, silicon addition caused significant increases in GP in each cultivar (P < 0.05) under water stress, and on the 7th day, the GP rised by 34.7%, 27.6%, 21.9% and 38.2% in Jinpeng, Zhongza, Houpi and Oubao, respectively. The germination index of four tomato cultivars was significantly reduced after 7 days of water deficit stress (Fig. 2A), and added silicon significantly alleviated the stress-induced reduction. However, under non-stress conditions, silicon application exerted no significant effect on the germination index in each cultivar. The length and fresh weight of bud seedlings were significantly decreased under water stress (Fig. 2B and C). Addition of silicon could increase the seedling length in Zhongza and Houpi and fresh weight in zhongza and Oubao, but it did not affect the growth of Jinpeng under water stress (Fig. 2B and C). 3.2. Activities of SOD and CAT As shown in Table 1, as compared with the control, the activities of SOD and CAT in the radicles of each cultivar were increased

significantly under water deficit stress, and silicon addition further enhanced the activities of SOD and CAT. However, under non-stress conditions, added silicon did not have any significant effect on their activities. 3.3. ROS levels The radicles of each cultivar showed higher O2
and H2O2 accumulation under water deficit stress as compared to the corresponding control. Under non-stress conditions, added silicon had no significant effects on the O2
(Fig. 3 a, e; b, f; c, g; d, h) and H2O2 (Fig. 4 a, e; b, f; c, g; d, h) accumulation in the radicles of each cultivar. Silicon addition significantly decreased the O2
(Fig. 3 i, m; j, n; k, o; l, p) and H2O2 (Fig. 4 i, m; j, n; k, o; l, p) accumulation in radicles of all stressed seedlings. 3.4. POD activity Under non-stress conditions, added silicon did not have any significant effect on the POD activities (Table 1). Under water deficit stress, the activities of POD in the radicles of each cultivar were obviously increased, whereas addition of silicon inhibited the increase (Table 1). In order to investigate whether silicon itself affected the activity of POD, we measured the POD activity in the presence of 0.5 mM Si(OH)4 in the reaction mixture. The results showed that inclusion of silicon in the reaction mixture significantly decreased the POD activity in the radicles of each cultivar in vitro (Fig. 5).

Fig. 1. Effect of silicon on the seed germination percentage of four tomato cultivars under polyethylene glycol-simulated water deficit stress. The stress lasted for 7 days. Data are shown as means  SE of three replicates. CT, control; Si, silicon; PEG, polyethylene glycol; P þ Si, polyethylene glycol plus silicon. Data with the same letters at the same tomato cultivar are not significantly different between treatments at P  0.05 at each time point.

Y. Shi et al. / Plant Physiology and Biochemistry 78 (2014) 27e36

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3.5. Phenol contents The total phenol contents in the radicles were expressed in both per dry weight and water content in order to reflect its metabolism and substrate concentration of POD, respectively. In non-stress conditions, when expressed on the base of per dry weight (Fig. 6A), the phenol contents of radicles were increased in Jinpeng and Zhongza, but decreased in Houpi and not changed in Oubao by addition of silicon. Water stress decreased the phenol contents in three of the four cultivars investigated, with the exception of Oubao. Silicon addition significantly decreased the phenol contents in the radicles of stressed seedlings in most of the cultivars, except for Houpi, where the decrease was not statistically significant. When expressed on the base of water content (Fig. 6B), the phenol contents of radicles were increased notably in most of the cultivars (except for Zhongza) under water deficit stress as compared to the control (Fig. 6B). Silicon addition significantly decreased the phenol contents in the radicles of stressed seedlings. However, under non-stress conditions, added silicon had no significant effects on the phenol contents in the radicles of each cultivar. 3.6. MDA contents Under non-stress conditions, added silicon caused significant decreases in MDA levels in Zhongza and Houpi radicles, but it did not affect the MDA levels in Jinpeng and Oubao (Table 2). After 7 days of water deficit stress, the MDA contents in radicles of four tomato cultivars were significantly increased as compared to the corresponding controls, and silicon addition significantly inhibited the increase of MDA levels in all four cultivars (Table 2). 3.7. Silicon contents Fig. 2. Effect of silicon on the seed germination index, length and fresh weight of bud seedlings of four tomato cultivars under polyethylene glycol-simulated water deficit stress. Data are shown as means  SE of 3 replicates for germination index, 24 replicates for seedling length, and 8 replicates for seedling fresh weight. Bars with the same letters at the same tomato cultivar are not significantly different between treatments at P  0.05. CT, control; Si, silicon; PEG, polyethylene glycol; PEG þ Si, polyethylene glycol plus silicon.

To remove free silicon in apoplastic space, the radicles were firstly washed in distilled water and then further washed in cold CaCl2 solution. The results showed that silicon addition did not significantly increase the silicon concentrations of radicles (Table 3). 4. Discussion

Table 1 Effects of silicon on the activities of superoxide dismutase, catalase and peroxidase in tomato radicles under polyethylene glycol-simulated water deficit stress. Data are shown as means of three replicates. Means  SE followed by the same letters at the same tomato cultivar are not significantly different between treatments at P  0.05.CAT, catalase; PEG, polyethylene glycol; POD, peroxidase; SOD, superoxide dismutase. Treatments

Activities of enzymes (U mg1 protein) Jinpeng

SOD

CAT

POD

Si/PEG þ Si/PEG Si/þPEG þSi/þPEG Si/PEG þSi/PEG Si/þPEG þSi/þPEG Si/PEG þ Si/PEG Si/þPEG þSi/þPEG

112.9 113.2 161.3 188.6 18.4 18.9 37.2 42.1 232.4 231.9 281.0 249.1

           

Zhongza 0.9c 0.3c 1.1b 1.2a 0.1c 0.3c 0.3b 0.5a 2.8c 5.0c 2.6a 1.6b

97.3 95.9 155.2 182.9 34.7 35.4 73.0 86.3 206.8 210.3 279.0 233.2

           

Houpi 0.7c 3.3c 3.4b 1.6a 0.5c 0.7c 0.7b 0.6a 4.3c 2.9c 4.5a 2.9b

105.1 105.8 142.3 164.8 24.4 25.6 53.0 64.4 266.6 265.4 347.2 306.9

Oubao            

1.6c 2.4c 1.6b 3.3a 0.8c 0.8c 1.3b 0.7a 3.0c 2.8c 2.6a 1.8b

119.5 118.5 192.5 237.1 21.5 22.7 46.7 57.0 132.9 130.9 194.9 159.7

           

0.6c 1.6c 1.0b 1.5a 0.3c 0.4c 0.7b 2.0a 3.2c 1.8c 3.3a 2.6b

The positive effects of silicon on seed germination have been observed in white oat (Toledo et al., 2011) and borage (Torabi et al., 2012) under normal conditions. However, in this study, we did not observe any positive effect of silicon on the seed germination in the investigated tomato cultivars in normal water conditions (Fig. 1). This suggests that the effect of silicon on seed germination under normal environmental conditions may be associated with plant species. Although there has been some work on the effect of silicon on seed germination in normal conditions, to our knowledge, little work has been done under water stress. In this study, the results demonstrated that water deficit stress resulted in a delayed and decreased germination, which could be improved by addition of silicon, as can be seen from the increased GP and GI, especially for cv. Oubao and Zhongza (Figs. 1 and 2). Silicon-mediated improvement of seed germination in tomato under water deficit stress suggests a potential application of silicon fertilizer in arid and semiarid regions in the world. In Xinjiang, China, which belongs to an arid region, there are large areas of tomato, most of which has been directly seeded. Therefore, application of silicon may be an available pathway to improve tomato seed germination in this region and reduce water irrigation in the mean time. There were

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differences in the effects of silicon on the growth of bud seedlings among cultivars (Fig. 2), suggesting cultivar-dependant silicon effect. Plants have an antioxidant system for scavenging toxic ROS, and efficient running of this defense system is essential for plants for tolerate various adverse environmental stresses (Gong et al., 2005). In the present study, the results showed that the activities of SOD and CAT in the radicles of all cultivars were increased under water deficit condition (Table 1). The increases in activities of SOD and CAT might be an adaptive response, which facilitated the

scavenging of reactive oxygen species. Similar results were also reported in soybean (Masoumi et al., 2011). The effects of exogenous silicon on the SOD and CAT activities of plants under water deficit stress have been previously reported. In drought stressed wheat (Gong et al., 2005) and maize (Li et al., 2007), it was found that application of silicon increased the SOD and CAT activities in the leaves. In drought stressed sunflower, Gunes et al. (2008) found that the SOD activities were decreased by applying silicon, while the CAT activities were increased by supplemental silicon. In drought stressed Elaeagnus angustifolia L. seedlings, it was found

Fig. 3. Effect of silicon on superoxide (O2
) levels in the radicles of four tomato cultivars under polyethylene glycol-simulated water deficit stress. The stress lasted for 6 days. The O2
accumulation was detected the radicles of Jinpeng (a, e, i, m), Zhongza (b, f, j, n), Houpi (c, g, k, o) and Oubao (d, h, l, p) using 3,30 diaminobenzidine staining method. CT, control; Si, silicon; PEG, polyethylene glycol; PEG þ Si, polyethylene glycol plus silicon.

Y. Shi et al. / Plant Physiology and Biochemistry 78 (2014) 27e36

that the SOD activities were increased by exogenous silicon, while the CAT activities were decreased by silicon addition (Zari et al., 2010). These studies were conducted at seedling stage or later plant growth stages. In the seed germination period, however, the research on silicon effect is still lacking under water stress. In this work, compared with the PEG treatment alone, þSi/þPEG treatment could further increase the SOD and CAT activities in the radicles of the four cultivars (Table 1). The differences in siliconmediated activity changes of antioxidant enzymes might be associated with differences in culture conditions and growth stages. High activities of SOD and CAT induced by added silicon might help to scavenge the excessive ROS (Figs. 3 and 4) and protect plant cell from oxidative damage under water deficit stress (Gong et al., 2005). The decrease in oxidative stress mediated by silicon might have contributed to the improvement of germination of tomato seeds exposed to water deficit stress (Figs. 1 and 2). In the present work, the POD activities were significantly increased in the radicles of each cultivar under water deficit stress, while application of silicon decreased the activities (Table 1). The increase of POD activity under water stress might be an adaptive response and contribute to stress tolerance. However, the siliconmediated decrease in POD activity suggests its decreased H2O2

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scavenging ability. The decreases in POD activities might be associated with the increases in CAT activities by added silicon (Table 1), which led to a lower H2O2 content (Fig. 4), and therefore there was less demand to activate the H2O2-scavenging enzymes. This is in agreement with a previous report in silicon-applied wheat plants exposed to drought stress (Gong et al., 2008). This may also suggest that CAT plays more important role than POD in scavenging excessive hydrogen peroxide in water stress conditions. In addition, it has been demonstrated that POD can mediate the transformation of H2O2 into much more toxic
OH (Chen and Schopfer, 1999). Therefore, it is possible that silicon-mediated decrease in the POD activity might have been contributed to decreased production of
OH and therefore less oxidative stress to plants. POD uses various substrates as electron donors to reduce H2O2 to water and phenol is one of the substrates (Wang et al., 2009). In the present study, as compared to the control, the increases in phenol concentrations (in mg g1H2O, Fig. 6B) under water stress basically corresponded to the increases in the POD activities (Table 1). The relationship was also observed in the radicles of silicon-added seedlings, as can be seen from the decreases of phenol level and POD activity as compared to the stressed seedlings without added silicon (Fig. 6B; Table 1). Therefore, the

Fig. 4. Effect of silicon on hydrogen peroxide (H2O2) levels in the radicles of four tomato cultivars under polyethylene glycol-simulated water deficit stress. The stress lasted for 6 days. The H2O2 accumulation was detected in the radicles of Jinpeng (a, e, i, m), Zhongza (b, f, j, n), Houpi (c, g, k, o) and Oubao (d, h, l, p) using 2,7-dichlorofluorescin diacetate as a fluorescent probe. CT, control; Si, silicon; PEG, polyethylene glycol; PEG þ Si, polyethylene glycol plus silicon.

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Y. Shi et al. / Plant Physiology and Biochemistry 78 (2014) 27e36 Table 2 Effects of silicon on the concentrations of malondialdehyde in tomato radicles under polyethylene glycol-simulated water deficit stress. Treatments

MDA concentration (nmol g1 DW) Jinpeng

Si/PEG þ Si/PEG Si/þPEG þSi/þPEG

6.28 5.72 11.82 6.18

   

Zhongza 0.04b 0.06b 1.67a 0.27b

12.23 8.91 14.15 7.94

   

Houpi 0.04b 0.05c 0.33a 0.01d

4.69 3.94 6.77 4.26

   

Oubao 0.07b 0.01c 0.24a 0.02c

8.60 8.71 15.35 10.98

   

0.15c 0.04c 0.21a 0.04b

Data are shown as means of three replicates. Means  SE followed by the same letters at the same tomato cultivar are not significantly different between treatments at P  0.05. DW, dry weight; MDA, malondialdehyde; PEG, polyethylene glycol.

Table 3 Effects of exogenous silicon on the silicon concentrations in tomato radicles under polyethylene glycol-simulated water deficit stress. Silicon concentration in radicles (mmol g1 DW)

Treatment

a

Si/PEG þSi/PEG Si/þPEG þSi/þPEG

17.72 18.30 16.63 16.55

Jinpeng Fig. 5. Effect of silicon (0.5 mM) on the activities of guaiacol-peroxidase in vitro. The enzyme was extracted from the radicles of the control plants of each tomato cultivar. Data are shown as means  SE of three replicates. Bars with different letters at the same tomato cultivar are significantly different between CT and Si treatments at P  0.05. CT, control; Si, silicon; POD, peroxidase.

silicon-mediated decrease of POD activities in radicles might be due to silicon-mediated decrease of the phenol level. Another possible reason for silicon-mediated decrease in POD activities may be due to the silicon effect in preventing contact between the enzyme and phenolic substrate, as suggested by Iwasaki et al. (2002). Previous studies have suggested that silicon can influence phenol metabolism and the effect is dependent on plant species and tissues (Dragisi c Maksimovi c et al., 2007; Schaller et al., 2012). In the present work, we observed that the effect of silicon on the phenol content was cultivar-dependent in non-stress conditions (as mg g1 DW, Fig. 6A). Under water deficit, the phenol contents in the radicles were notably decreased by addition of silicon (Fig. 6A), suggesting that silicon could regulate the synthesis or catalysis of phenols. Further investigations are needed to clarify how silicon regulates the phenol metabolism in the radicles. Except for the possible regulative role of silicon on phenol metabolism, the decrease of phenol level under water stress (Fig. 6A) may also be

   

Houpi 0.87a 1.37a 1.33a 0.21a

19.62 20.07 15.76 17.36

Oubao    

1.51a 1.60a 1.07a 0.74a

17.76 20.79 14.06 15.92

   

1.07ab 1.44a 2.11b 0.63b

Data are shown as means of three replicates. Means  SE followed by the same letters at the same tomato cultivar are not significantly different between treatments at P  0.05. DW, dry weight; PEG, polyethylene glycol. a The radicles were washed in both distilled water and 0.5 mM CaCl2 when sampling.

due to the formation of silicon-phenol complexes (Dragisi c Maksimovi c et al., 2012). In this study, we did not observe any obvious accumulation of silicon as a result of silicon addition in the tomato radicles that were washed with both distilled water and CaCl2 (Table 3), suggesting that silicon addition did not increase the level of silicon in the radicle cells or covalently-bound silicon on the cell walls (Table 3). These results suggest that the beneficial effect of exogenous silicon may be mainly due to the biochemical function of the extracellular silicon, which existed in the apoplast. Thus, there is a possibility that the formation of silicon-phenol occurred in the apoplastic space, resulting in decreased POD activities in apoplast. In addition, a direct inhibitory effect of silicon on the POD activities can not be excluded, as has been confirmed in our in vitro experiment (Fig. 5). No significant amount of silicon entering the radicle

Fig. 6. Effect of silicon on the phenol contents in radicles of four tomato cultivars under polyethylene glycol-simulated water deficit stress. The phenol contents were expressed in mg g1 DW (A) or mg g1 H2O (B). Data are shown as means  SE of three replicates. Means followed by the same letters at the same tomato cultivar are not significantly different between treatments at P  0.05. CT, control; Si, silicon; PEG, polyethylene glycol; PEG þ Si, polyethylene glycol plus silicon.

Y. Shi et al. / Plant Physiology and Biochemistry 78 (2014) 27e36

cells may be related to low density of silicon transporters responsible for silicon transport from external solution to cortical cells in tomato (Mitani and Ma, 2005). Under abiotic stress, lipid peroxidation may be one of the most important factors that result in the inhibition of seed germination (Yang et al., 2010). Malondialdehyde (MDA) is a stable metabolite of peroxidation, and its content can reflect oxidative damage of plants (Gong et al., 2008). In this study, the MDA contents in the radicles of the four tomato cultivars were significantly increased under water stress (Table 2), indicating that lipid peroxidation was aggravated by water deficit stress, which might have caused seed deterioration (Yang et al., 2010). Besides, the stress-induced rises of MDA levels were in good correlation with the seed germination inhibition caused by water stress (Figs. 1 and 2; Table 2). Added silicon could decrease the MDA contents and improve the ability of seeds germination, suggesting that silicon could decrease lipid peroxidation in tomato seedlings under water stress. Silicon-mediated decrease in lipid peroxidation was attributed to the increased antioxidant defense and decreased production of ROS, as discussed above. In the present study, we found that addition of silicon could modulate the activities of antioxidant enzymes (Table 1) and alleviate oxidative damage (Table 2) of bud seedlings in tomato under water deficit conditions. However, up to now, little is understood about the in-depth mechanism for silicon-mediated physiological changes. How silicon initiates the physiological reactions of plants to deal with environmental stress under stress still remains to be investigated. Several studies have suggested that silicon application may induce stress tolerance by affecting endogenous plant hormone balance and silicon may be associated with hormone signaling (Fauteux et al., 2006; Brunings et al., 2009; Lee et al., 2010; Kim et al., 2013; Van Bockhaven et al., 2013). However, as Van Bockhaven et al. (2013) pointed out, verifying whether silicon-mediated stress tolerance is really resulted from these signaling pathways will be a challenge in future. In addition, the positive effect of Si on seed germination of tomato under water deficit might partly be due to Simediated improvement in Fe nutrition, as has been shown in Fe deficiency conditions (Gonzalo et al., 2013; Pavlovic et al., 2013). Under Fe deficiency, phenolic compounds are one of the main components of root exudates in many Strategy 1 species (all dicots and monocots with the exception of grasses) (Pavlovic et al., 2013; and references therein). If added silicon could improve the Fe nutrition, one may expect reduced phenol levels and POD activities in radicles. The effect of exogenous silicon on the Fe nutrition in tomato under water deficit still remains to be investigated. In summary, the results show that silicon application could improve the seed germination ability and alleviate oxidative stress of bud seedlings in tomato under water deficit stress by increasing antioxidant defense. These positive effects of silicon observed in a silicon-excluder may suggest its active involvement in biochemical processes in plants. The results also suggest a potential use of silicon fertilizer in water-saving irrigation in tomato, and provide a theoretical basis for application of silicon fertilizer in arid or semiarid regions. Author contributions Conceived and designed the experiments: HG. Performed the experiments: YS. Analyzed the data: YS. Contributed reagents/ materials/analysis tools: YZ HY JW HS. Wrote the paper: YS. Acknowledgments This study is supported by the National Natural Science Foundation of China (31272152), Program for New Century Excellent

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