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MITIGATION EFFECTS OF SILICON ONTOMATO PLANTS BEARING FRUIT GROWNAT HIGH BORON LEVELSCengiz Kaya a , A. Levent Tuna b , Murat Guneri c & MuhammedAshraf da Soil Science and Plant Nutrition Department, Harran University,Sanliurfa, Turkeyb Biology Department, Mugla University, Mugla, Turkeyc Horticulture Department, Ortaca Vocational School, MuglaUniversity, Mugla, Turkeyd Botany and Microbiology Department, King Saud University, Riyadh,Saudi ArabiaPublished online: 06 Oct 2011.

To cite this article: Cengiz Kaya , A. Levent Tuna , Murat Guneri & Muhammed Ashraf (2011):MITIGATION EFFECTS OF SILICON ON TOMATO PLANTS BEARING FRUIT GROWN AT HIGH BORON LEVELS,Journal of Plant Nutrition, 34:13, 1985-1994

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Journal of Plant Nutrition, 34:1985–1994, 2011Copyright C© Taylor & Francis Group, LLCISSN: 0190-4167 print / 1532-4087 onlineDOI: 10.1080/01904167.2011.610485

MITIGATION EFFECTS OF SILICON ON TOMATO PLANTS

BEARING FRUIT GROWN AT HIGH BORON LEVELS

Cengiz Kaya,1 A. Levent Tuna,2 Murat Guneri,3 and Muhammed Ashraf4

1Soil Science and Plant Nutrition Department, Harran University, Sanliurfa, Turkey2Biology Department, Mugla University, Mugla, Turkey3Horticulture Department, Ortaca Vocational School, Mugla University, Mugla, Turkey4Botany and Microbiology Department, King Saud University, Riyadh, Saudi Arabia

� Interactive effects of silicon (Si) and high boron (B) on growth and yield of tomato (Lycoper-cison esculentum cv. ‘191 F1’) plants were studied. Treatments were: 1) control (B1), normalnutrient solution including 0.5 mg L−1 B (boron), 2) B1 +Si treatment: 0.5 mg L−1 boron plus2 mM Si, 3) B2 treatment: 3.5 mg L−1 B, 4) B2 +Si treatment: 3.5 mg L−1 B plus 2 mM Si, 5)B3 treatment: 6.5 mg L−1 B, and 6) B3 +Si: 6.5 mg L−1 B plus 2 mM Si. High B reduced drymatter, fruit yield and chlorophyll (Chl) in tomato plants compared to the control treatment, butincreased the proline accumulation. Supplementary Si overcame the deleterious effects of high B onplant dry matter, fruit yield and chlorophyll concentrations. High B treatments increased the activi-ties of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC. 1.11.1.7) and polyphenoloxidase (PPO; EC 1.10.3.1). However, supplementary Si in the nutrient solution containing highB reduced SOD and PPO activities in leaves, but POD activity remained unchanged. These datasuggest that excess B-induced oxidative stress and alterations in the antioxidant enzymes. Boron (B)concentrations increased in leaves and roots in the elevated B treatment as compared to the controltreatment. Concentrations of calcium (Ca) and potassium (K) were significantly lower in the leavesof plants grown at high B than those in the control plants. Supplementing the nutrient solutioncontaining high B with 2 mM Si increased both nutrients in the leaves. These results indicate thatsupplementary Si can mitigate the adverse effects of high B on fruit yield and whole plant biomassin tomato plants.

Keywords: boron toxicity, silicon, tomato, calcium, antioxidant enzymes

INTRODUCTION

Boron (B) toxicity is a well-known nutritional disorder that can limitplant growth and productivity particularly in the arid and semiarid regions of

Received 15 September 2009; accepted 30 April 2010.Address correspondence to C. Kaya, Soil Science and Plant Nutrition Department, Harran Univer-

sity, Sanliurfa 63200, Turkey. E-mail: c kaya70@yahoo.com

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the world. Addition of B to the soil is often encountered from fertilizers andmining (Nable et al., 1997). Boron can be often found in high concentrationsin arid regions where saline soils and saline irrigation water are prevalent.Municipal and other wastewater effluents used for irrigation are also potentsources of excess boron in agricultural systems (Tsadilas, 1997). Although Bdeficiency can be overcome by application of B-enriched fertilizers, toxicitycauses some problem to manage. So in recent years, B toxicity has attractedconsiderable interest because of the high demand for desalinated water, inwhich the B concentration may be too high for healthy irrigation (Parks andEdwards, 2005).

It is now evident that B toxicity is manifested primarily at three differentsites within a cell: 1) disruption of cell wall development; 2) metabolic dis-ruption by binding of B to the ribose moieties of ATP, NADH or NADPHand 3) disruption of cell division and development by binding to ribose,either as the free sugar or within RNA (Stangoulis and Reid, 2002). Addi-tionally, accumulation of high concentrations of B in leaf tissues might leadto osmotic imbalances (Reid et al., 2004).

Under stress conditions such as B toxicity, higher activities of antioxidantenzymes and higher contents of non-enzymatic biomolecules are importantfor plants to tolerate the stress (Gunes et al., 2007). Antioxidant-inducedreduction in B-toxicity damage has also been shown in some plants (Guneset al., 2006). For protection against oxidative stress, plant cells contain somekey antioxidant enzymes such as superoxide dismutase (SOD; EC 1.15.1.1),and peroxidase (POD; EC 1.11.1.7) (Molassiotis et al., 2006). Altered activ-ities of these antioxidant enzymes and antioxidants commonly have beenreported, and are used as indicators of oxidative stress in crops (Mittler,2002; Ashraf, 2009).

Silicon (Si) is one of the potential beneficial elements for the growthof higher plants (Marschner, 1995; Epstein, 1999). It also plays a vital rolein the maintenance of plant growth under various stresses. For example,metal toxicities in several species have been shown to be ameliorated bySi. Silicon also prevents the local accumulation of manganese (Mn) on theleaf blades by using a more homogeneous distribution of Mn (Horst, 1988).Silicon was found to lower the apoplastic Mn concentration in cowpea (Horstet al., 1999). The authors suggested that Si can modify the cation bindingproperties of the cell walls as earlier proposed by Mera and Beveridge (1993).The effect of Si application on aluminum (Al) toxicity has also been wellstudied. For example, in maize, the concentration of Al3+ in culture solutionis strongly reduced with the addition of Si (Ma et al., 1997). In Sorghum bicolorsilicon inhibited Al penetration into the root cortex, which suggests that Simakes a complex with Al in the medium and/or roots and contributes to thedetoxification of Al (Hodson and Sangster, 1993). Furthermore, formationof B-Si (boron-silicate) complexes in the soil leads to lower B availability toplants and consequently lowers tissue B (Gunes et al., 2007; Inal et al., 2009).

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Besides these two reports on different species, there is hardly any informationavailable in the literature on the effect of supplementary Si on B toxicity.So, the objectives of the present work were to test the effectiveness of Si inameliorating B toxicity in tomato plants. It was also aimed to investigate theeffects of high B on antioxidant systems and the distribution of B, Si, calcium(Ca) and potassium (K) in leaves and roots of tomato plants.

MATERIALS AND METHODS

Plant Culture and Treatments

An experiment was conducted under glasshouse conditions from Febru-ary to June 2005 with tomato (Lycopersicon esculentum Mill.) cv. ‘191 F1’.Environmental conditions were typical of those for a small-scale tomatocrop grown under glasshouse conditions. Temperature was controlled usingheater during the growing season with the aim of keeping daytime temper-ature in the 20–30◦C ranges and night time temperature above 10◦C. Threeseeds of tomato were sown directly in to each plastic pot containing 8 kgof peat, perlite and sand mixture in equal ratios. After germination, theseedlings were thinned to one plant per pot.

The containers were covered with black plastic sheets to reduce evapo-ration. The basic nutrient solution used in this experiment was a modifiedHoagland and Arnon (1940) formulation. All chemicals used were of ana-lytical grade, and composition of the nutrient solution was (mg L−1): 270nitrogen [N as nitrate (NO3) form], 31 phosphorus (P), 234 potassium (K),200 calcium (Ca), 64 sulfur (S), 48 magnesium (Mg), 2.8 iron (Fe), 0.5manganese (Mn), 0.5 boron (B), 0.02 copper (Cu), 0.05 zinc (Zn), and 0.01molybdenum (Mo). The pH of the nutrient solution was adjusted to 6.0daily using 0.01 mol L−1 potassium hydroxide (KOH) and/or sulfuric acid(H2SO4).

Twenty days after germination different treatments of boron (B) andsilicon (Si) were initiated. The treatments were: 1) control (B1), normalnutrient solution including 0.5 mg L−1 B (boron), 2) B1 + Si treatment:0.5 mg L−1 boron plus 2 mM Si, 3) B2 treatment: 3.5 mg L−1 B, 4) B2 + Sitreatment: 3.5 mg L−1 B plus 2 mM Si, 5) B3 treatment: 6.5 mg L−1 B, and6) B3 + Si: 6.5 mg L−1 B plus 2 mM Si. Boron and silicon treatments werestarted by adding boric acid (H3BO3) and sodium silicate (Na2SiO3) to thenutrient solution immediately after 10-day-old seedlings were transplantedinto the pots. Control and high boron-stressed plants were treated with anequivalent amount of sodium chloride. Double-distilled water was used forthe preparation of nutrient solution.

Each treatment was replicated three times and each replicate includedfive plants (i.e., 15 plants per treatment). The pH of the nutrient solutionwas adjusted each time to 5.5 with a minimum amount of 0.1 mM potassium

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hydroxide (KOH). The volume of the nutrient solution applied to the rootzone of plants ranged from 200 to 1000 mL from February to June dependingon plant age. Plants were harvested after fruit set to assess biomass andafter fruit ripening (four weeks after fruit set) to determine some otherparameters. At fruit-set stage, two plants from each replicate were harvestedand separated into shoots and roots for dry weight determination after dryingat 70◦C for 48 h. At fruit-harvest stage, fruits of the remaining three plantsfrom each replicate were harvested and both individual and total fruit weightper plant were recorded.

Relative Water Content (RWC) and Proline Content

Leaf relative water content (RWC) was calculated based on the methodsof Yamasaki and Dillenburg (1999). Free proline was extracted from 500 mgof the leaves in 3% (w:v) aqueous sulfosalicylic acid and its concentration wasestimated by ninhydrin reagent (Bates et al., 1973). The absorbance of thefraction with toluene aspired from the liquid phase was read at 520 nm. Pro-line concentration was determined from a calibration curve and expressedas µmol proline g−1 fresh weight.

Protein Content and Enzyme Determination

Protein content in the plant extracts for enzymes was determined ac-cording to Bradford (1976) using bovine serum albumin V as a standard.

Leaves (0.5 g) were homogenized in 50 mM sodium phosphate buffer(pH 7.0) containing 1% soluble polyvinyl pyrolidine (PVP). The ho-mogenate was centrifuged at 20,000 g for 15 min at 4◦ C and the super-natant used for assays of the activities of peroxidase (POD) and superoxidedismutase (SOD).

The activity of SOD was assayed by monitoring its ability to inhibit thephotochemical reduction of nitoblue tetrazolium (NBT) (Beauchamp andFridovich, 1971). One unit of SOD was defined as the amount of enzymenecessary to inhibit the reduction of cytochrome C by 50%.

The activity of POD was assayed by adding an aliquot of the tissue extract(100 µL) to 3 mL of assay solution, consisting of 3 mL of reaction mixturecontaining 13 mM guaiacol, 5 mM hydrogen peroxide (H2O2) and 50 mMNa-phosphate buffer (pH 6.5) (Chance and Maehly, 1955). An increaseof the optical density at 470 nm for 1 min at 25◦C was recorded using aspectrophotometer. The POD activity was expressed as change in absorbancemin−1 mg−1 protein. The increase in A470 was measured for 3 min and activityexpressed as �A470/mg protein/min.

Polyphenol oxidase (PPO) activity was assayed with 4-methylcatechol asa substrate according to the method of Zauberman et al. (1991). A halfgram of fresh leaf was ground with 10 mL of 0.1 mol L−1 sodium phosphate

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buffer (pH 6.8) and 0.2 g of polyvinylpyrrolidone (PVP, insoluble). Aftercentrifugation at 19,000 g for 20 min, the supernatant was collected as thecrude enzyme extract. The assay of the enzyme activity was performed using1 mL of 0.1 mol L−1 sodium phosphate buffer (pH 6.8), 0.5 mL of 100mmol/l 4-methylcatechol, and 0.5 mL enzyme solution. The increase inabsorbance at 410 nm at 25◦C was recorded automatically for 5 min. Oneunit of enzyme activity was defined as an increase of 0.01 in absorbance permin per mg protein.

Chemical Analysis

Chemical analyses were carried out using plant dry weight. Ground sam-ples were dry-ashed at 550◦C for four h mixed with 2 M hot hydro cholericacid (HCl), filtered, and then brought to a final volume of 50 mL withdistilled water. Calcium (Ca) and potassium (K) were determined in thesesample solutions. The calcium (Ca) and potassium (K) were determined inthe sample solution using an inductively coupled plasma (ICP) (Chapmanand Pratt, 1982). For determining B concentration, samples were dry ashedin a muffle furnace at 500◦C for 6 h. The carbon free residue was then dis-solved in 0.1 M HCl and B was determined by the azomethine-H method(Wolf, 1971). For silicon determination, dried leaves and roots were groundto powder using a pestle and mortar and stored in polyethylene bottles. Thesamples were then microwave digested in a mixture of 3 mL of 62% (w/w)nitric acid (HNO3), 3 mL of 30% (w/w) H2O2, and 2 mL of 46% (w/w) hy-drogen fluoride (HF). The samples so digested were diluted to 100 mL with4% (w/v) boric acid (H3BO3). The Si concentration in the digest solutionwas determined by the colorimetric molybdenum blue method at 600 nm(Ma et al., 2002).

Statistical Analysis

A two-way analysis of variance (ANOVA) was performed using SAS (SASInstitute, Cary, NC, USA) and the data were considered significant if valueswere higher than F values at P < 0.05.

RESULTS AND DISCUSSION

Key Growth Parameters

High boron concentrations (3.5 and 6.5 mg L−1) reduced both dryweights and relative water contents (RWC) in the tomato plants comparedwith those in control (Table 1). A reduction in growth and increase ofboron (B) concentration in the plant tissues as a consequence of B toxicityhas previously been observed in tomato (Gunes et al., 1999), sunflower

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TABLE 1 Leaf relative water content (RWC), proline, total, shoot, and root dry weights of tomatoplants grown under high boron conditions in the presence of silicon

Total DM Shoot DM Root DM

Treatment∗ RWC (%) Proline (µmolg−1 FW) g/plant

B1 81.1a 3.15c 168.1a 144.2a 23.9aB1+Si 81.4a 3.05c 165.5ab 142.8a 22.7abB2 74.4b 9.30a 158.5bc 138.0b 20.5cB2+Si 74.2b 4.72d 163.6ab 141.9a 21.7bcB3 70.7b 9.08a 153.8c 133.3c 20.5cB3+Si 71.5b 5.57c 162.2ab 140.3b 21.9abc

∗B1, B2 and B3: 0.5, 3.5 and 6.5 mg/l B, respectively; Si: 2 mM.Values followed by different letters, in the same column, are significantly different at P ≤ 0.05.

(Ruiz et al., 2003), barley (Inal et al., 2009; Karabal et al., 2003) and wheat(Sonmez et al., 2009). Supplementary silicon (Si) resulted in an increase indry weights restoring to the levels comparable with the control, but it didnot significantly change RWC. Boron toxicity alleviated by Si supplementhas not been studied widely. Si supplement improves plant growth of barleyunder B toxicity (Gunes et al., 2007; Inal et al., 2009). In the literature thereis a lack of information on the effect of high B on RWC.

High B treatments (3.5 mg L−1 and 6.5 mg L−1) caused an increase inproline content in tomato leaves, but supply of Si reduced proline accumu-lation in the plants as compared with B toxicity conditions (Table 1). Theseresults are in agreement with those of Gunes et al. (2007) in which boronand silicon interaction was found in spinach. Contradictory results havealso been reported on the effect of excess B on proline accumulation inplants. For example, it has been suggested that excess B did not significantlychange proline concentrations of B-tolerant and B-sensitive barley cultivars(Karabal et al. 2003). However, a decrease in proline levels as the result ofB toxicity was reported in mandarin and apple rootstock (Papadakis et al.,2004; Molassiotis et al., 2006), and barley (Inal et al., 2009). Higher boronconcentrations reduced fruit yield and average fruit weight of tomato plants(Table 2). It has been reported that high boron reduces yields of tomato aswell as other plant species (Francois, 1984). However, Si supplementationimproved the yield parameters of tomato plants grown under higher B con-ditions. Interactions between B and Si were significant for fruit yield andaverage fruit weight (P ≤ 0.05), but not for number of fruits.

Enzyme Activities

Both high levels of B resulted in a significant increase in superoxidedismutase (SOD), peroxidise (POD), and polyphenol oxidase (PPO) activi-ties in the tomato plants (Table 3). The enhanced enzyme activities of SOD,POD and PPO in the tomato plants grown at high B suggest that antioxidative

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TABLE 2 Fruit yield, numbers of fruit per plant and average fruit weight of tomato grown under highboron conditions in the presence of silicon

Fruit yield No. of Average fruitTreatment∗ g/plant fruit/plant weight (g/fruit)

B1 4752a 47.0a 101.1bB1+Si 4766a 47.3a 100.8bB2 4300c 44.0ab 97.7cB2+Si 4560b 43.6ab 104.6aB3 3833e 43.3ab 88.5dB3+Si 4166d 40.6b 102.6bF Test ∗ ∗ ∗Interaction BXSi ∗ ns ∗

∗B1, B2 and B3: 0.5, 3.5 and 6.5 mg/l B, respectively; Si: 2 mM.Ns: not significant; ∗: P ≤ 0.05.Values followed by different letters, in the same column, are significantly different at P ≤ 0.05.

defence system could be one of the effective components of mechanism oftolerance of tomato plants to B toxicity. In an earlier study on tomato, it hasbeen suggested that high B concentration in the growth medium stimulatesoxidative damage in tomato leaves and induces an increase in antioxidantenzyme activity (Cervilla et al., 2007). Some other studies also show an evi-dence of oxidative damage in plants under excess B. For example, it has beenreported that high boron concentration in the growth medium stimulatedSOD level in apple rootstock (Molassiotis et al., 2006), in barley (Karabalet al., 2003), and tobacco leaves (Garcia et al., 2001).

There seem to be no reports available in the literature on the relationshipbetween excess B and PPO activity. The induction of PPO activity can lead toan increase in the levels of quinones, produced by the oxidation of phenolics(Lopez-Gomez et al., 2007). Quinones, in fact, are considered as importantplant growth regulators (Ranade and David, 1985).

Supplementary Si reduced the activities of all three antioxidant en-zymes (SOD, POD and PPO), but their levels were still higher than those in

TABLE 3 Superoxide dismutase (SOD; unit mg protein−1), polyphenol oxidase (PPO; Unit x100/mgprotein) and peroxidase (POD; �A470/min/mg protein) levels in tomato grown under high boronconditions in the presence of silicon

Treatment∗ SOD PPO POD

B1 134d 2.25c 11.58cB1+Si 126d 2.48c 12.10cB2 274b 2.50c 31.90bB2+Si 192c 3.21d 30.48bB3 409a 5.60a 47.10aB3+Si 294b 3.28c 49.50a

∗ B1, B2 and B3: 0.5, 3.5 and 6.5 mg/l B, respectively; Si: 2 mM.Values followed by different letters, in the same column, are significantly different at P ≤ 0.05.

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unstressed tomato plants. Gunes et al. (2007) also reported that Si reducedthe SOD activity in spinach plants under B toxicity.

Macronutrient Contents

Boron concentration in plant tissues increased with increasing B con-centration in the nutrient solution, but was reduced by additional Si supply.This may have been due to the formation of B-Si (boron-silicate) complexesin the soil and within the plant, resulting to lower B availability (Inal et al.,2009). It has been earlier reported that Si improved tolerance to cadmium(Cd) in maize, and this was attributed to Cd immobilization in the rootsand soil (Liang et al., 2005). Similar mechanism could be possible for theprotective role of Si against B toxicity in the present investigation.

Leaf Ca2+and K+ decreased in the tomato plants grown at high boron.Concentrations of K+ and Ca2+ in the roots were lower in plants grown athigh B than those in normal conditions. Concentrations of K+ and Ca2+

were significantly increased by supplementary Si in most cases (Table 4).Singaram and Prabha (1997), while investigating the boron and calciuminteraction in tomato plants, reported that the translocation of absorbed Cato the aerial parts of the plant was a problem due to the relatively high pHof the translocating plant sap. But this was overcome by the addition of Bwhich helped translocation of absorbed Ca.

Based on the present work, it can be concluded that Si could be used toimprove plant growth under B stress. It seems that Si may play an importantphysiological role in detoxification of B within plants. High B enhances theactivities of some key antioxidant enzymes such as SOD, POD and PPO.Since the data presented here suggest the direct involvement of oxidativestress enzymes in plant tolerance to excess B, so these data will provide abasis for further detailed studies.

TABLE 4 Boron, Ca and K in the leaves of tomato grown under high boron conditions in the presenceof silicon

Treatment∗ B mg/kg Si (%) Ca (%) K (%)

B1 46c 0.08c 3.52b 1.51bB1+Si 32d 1.32a 3.45c 1.44cB2 96b 0.07c 3.69a 1.37dB2+Si 91b 0.69b 3.12d 1.87aB3 139a 0.04c 3.11d 0.78fB3+Si 101b 0.67b 3.50bc 1.03e

∗ B1, B2 and B3: 0.5, 3.5 and 6.5 mg/l B, respectively; Si: 2 mM.Values followed by different letters, in the same column, are significantly different at P ≤ 0.05.

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ACKNOWLEDGMENTS

The authors wish to thank the University of Harran (Turkey) and Uni-versity of Mugla for supporting the present study.

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