Plant Cell Rep DOI 10.1007/s00299-015-1814-9

ORIGINAL PAPER

Silicon improves salt tolerance by increasing root water uptake in Cucumis sativus L. Yong-Xing Zhu1 • Xuan-Bin Xu2 • Yan-Hong Hu1 • Wei-Hua Han1 Jun-Liang Yin3 • Huan-Li Li1 • Hai-Jun Gong1

•

Received: 16 March 2015 / Revised: 10 May 2015 / Accepted: 19 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Key message Silicon enhances root water uptake in salt-stressed cucumber plants through up-regulating aquaporin gene expression. Osmotic adjustment is a genotype-dependent mechanism for silicon-enhanced water uptake in plants. Abstract Silicon can alleviate salt stress in plants. However, the mechanism is still not fully understood, and the possible role of silicon in alleviating salt-induced osmotic stress and the underlying mechanism still remain to be investigated. In this study, the effects of silicon (0.3 mM) on Na accumulation, water uptake, and transport were investigated in two cucumber (Cucumis sativus L.) cultivars (‘JinYou 1’ and ‘JinChun 5’) under salt stress (75 mM NaCl). Salt stress inhibited the plant growth and photosynthesis and decreased leaf transpiration and water content, while added silicon ameliorated these negative effects. Silicon addition only slightly decreased the shoot Na levels per dry weight in ‘JinYou 1’ but not in ‘JinChun 5’ after 10 days of stress. Silicon addition reduced stress-induced decreases in root hydraulic conductivity and/or leaf-

Communicated by L. Tripathi. Y.-X. Zhu and X.-B. Xu contributed equally to this work. & Hai-Jun Gong [email protected] 1

College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China

2

Institute of Soil and Water Conservation, Northwest A&F University, Chinese Academy of Sciences, Yangling 712100, Shaanxi, China

3

College of Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi, China

specific conductivity. Expressions of main plasma membrane aquaporin genes in roots were increased by added silicon, and the involvement of aquaporins in water uptake was supported by application of aquaporin inhibitor and restorative. Besides, silicon application decreased the root xylem osmotic potential and increased root soluble sugar levels in ‘JinYou 1.’ Our results suggest that silicon can improve salt tolerance of cucumber plants through enhancing root water uptake, and silicon-mediated upregulation of aquaporin gene expression may in part contribute to the increase in water uptake. In addition, osmotic adjustment may be a genotype-dependent mechanism for silicon-enhanced water uptake in plants. Keywords Aquaporin  Osmotic potential  Root hydraulic conductance  Salt stress  Silicon  Water uptake and transport Abbreviations Aleaf Supported leaf area DTT Dithiothreitol E Transpiration rate gs Stomatal conductance Lpr Root hydraulic conductance Lsc Leaf-specific conductivity of the stem PIP Plasma membrane intrinsic protein Pn Net CO2 assimilation rate SL Stem length

Introduction Salt stress reduces crop growth and productivity (Zhu and Gong 2014). Among strategies to improve plant salinity tolerance, application of exogenous substances may be a

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promising solution that may save both time and labor. Silicon is the second prevalent element in the soil (Guntzer et al. 2012). Silicon accumulation in plants varies greatly among species (Ma and Yamaji 2008). According to the silicon uptake mode (active, passive, and rejective), plants can be classified as high-, intermediate-, and non-accumulators (Zhu and Gong 2014). Silicon has been demonstrated to be beneficial in mitigating multiple environmental stresses including both biotic stresses (e.g., plant diseases and pest damage) (Dannon and Wydra 2004; Heine et al. 2006; Ranganathan et al. 2006) and abiotic stresses such as salinity (Shi et al. 2013), drought (Liu et al. 2014), freezing (Liang et al. 2008), and heavy metal toxicity (Fuhrs et al. 2009; Dragisic Maksimovic et al. 2012). The alleviative effect of silicon on salt-induced injury has been widely reported in various cultivated crops, such as rice (Gong et al. 2006), wheat (Tuna et al. 2008), sorghum (Yin et al. 2013), barley (Liang et al. 2005), and soybean (Lee et al. 2010). Therefore, application of silicon may be alternative approach to increase agricultural production in salinized soil. Previous researchers have investigated the mechanisms for silicon-mediated salt tolerance. It has been widely reported that silicon addition can reduce Na and Cl accumulation and therefore reduce ion toxicity in plants (Liang 1999; Gong et al. 2006; Shahzad et al. 2013; Zhu and Gong 2014). Gong et al. (2006) suggested that silicon-mediated decreases in Na transport were attributed to decreased apoplastic transport due to silicon deposition in rice, a silicon accumulator. However, in tomato, which is a nonsilicon accumulator (Ghareeb et al. 2011), silicon addition could improve salt tolerance, but Na and Cl accumulations were not decreased (Romero-Aranda et al. 2006), suggesting the involvement of other mechanism(s). Recently, Kurabachew and Wydra (2014) reported that silicon-afforded resistance against bacterial wilt in tomato plants was related to the induction of systemic resistance rather than the formation of mechanical barrier by silicon deposition, and the expressions of genes involved in defense, signal transduction, and transcription were regulated by silicon (Kiirika et al. 2013). Silicon-mediated tolerance has also been attributed to decreased oxidative damage (Gunes et al. 2007a, b; Soylemezoglu et al. 2009). However, whether this is a direct or indirect effect of silicon is still unknown. On the other hand, except ion toxicity, the adverse effects of salinity on plant growth can also be attributed to osmotic stress. However, the possible role of silicon in alleviating salt-induced osmotic stress remains to be investigated. Tolerance to osmotic stress and maintenance of a good water status are important aspects of salt tolerance in plants. Early studies have focused on the effect of silicon on transpirational loss of water, and it was suggested that silicon deposition in rice leaves decreases transpirational rate and

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therefore keeps water storage under salt and drought conditions (Matoh et al. 1986; Savant et al. 1999). However, silicon-mediated reduction in transpiration is not observed in every case. For example, Gong et al. (2006) found that silicon addition increased the leaf transpirational rate of rice seedlings in saline conditions. These studies suggest a possible role of silicon in regulating water uptake in plants. Recently, Liu et al. (2014) reported that silicon was involved in improving root water uptake in sorghum plants under osmotic stress, implying an important role of silicon in the regulation of water uptake under stress conditions. In plants, the main restraint of water transport is mainly in roots (Rubio-Asensio et al. 2014). Root hydraulic conductance, which reflects the water uptake capacity of roots, is associated with the driving force, root anatomy, root surface, and root water permeability (Liu et al. 2014). Hydrostatic forces and osmotic gradient are important driving force for root water uptake (Javot and Maurel 2002). Root anatomy and surface play important roles in the regulation of water uptake via apoplastic pathway (Liu et al. 2014). Root water permeability can be regulated by aquaporins (Maurel et al. 2008). Under water stress conditions, the aquaporin activity and osmotic gradient play key roles in regulating water uptake by roots (Liu et al. 2014). Although the role of silicon in reducing Na and Cl accumulation (and therefore ion toxicity) has been widely reported in previous studies (see review by Zhu and Gong 2014), there have been very few reports concerning the possible role of silicon in regulating root water uptake and transport in plants under salt stress. Until recently, Liu et al. (2015) reported that silicon pre-treatment could improve root water uptake by up-regulating the expression of plasma membrane aquaporins in one sorghum cultivar at the early stage of salt stress. However, in their study, the role of osmotic adjustment in root water uptake under salt stress was not investigated. The regulative role of silicon on water uptake and aquaporin expression also needs to be confirmed in different plants. Cucumber (Cucumis sativus L.) is an important vegetable and is highly sensitive to salinity (Huang et al. 2009). Compared with the cereal plant rice, a silicon accumulator, cucumber accumulates much lower concentration of silicon due to a lower density of transportermediated transport (Mitani and Ma 2005). Previous study has shown that silicon could mitigate salt toxicity of cucumber seedlings through increasing the activities of major antioxidant enzymes and thus decreasing membrane oxidative damage (Zhu et al. 2004). However, whether this is a direct or indirect effect of silicon is unknown, and the effects of silicon on Na accumulation and water uptake remain to be investigated. The purpose of this study was to investigate the role of silicon on Na accumulation, root water uptake, and the underlying mechanism in two

Plant Cell Rep

cucumber cultivars under salt stress. Our results suggest that silicon improves salt tolerance of cucumber plants mainly through enhancing root water uptake, and siliconmediated up-regulation of aquaporin gene expression can in part contribute to the increase in water uptake. We also found that osmotic adjustment is a genotype-dependent mechanism for silicon-enhanced water uptake in plants under salt stress. The study may help further understand the mechanism for silicon-mediated salt tolerance in plants and provide a theoretical basis for silicon fertilizer application to solve soil salinization in vegetable production.

NE, USA). The recent fully expanded leaf was placed in a chamber at a photon flux density of 800 lmol m-2 s-1. Each treatment included eight replications.

Materials and methods

Leaf water status

Plant material and treatment

The relative water content, bound water content, and free water content of leaves were determined on fresh materials. The relative water content was determined according to Machado and Paulsed (2001). The free water and bound water contents were determined according to Ming et al. (2012). Each treatment included six replications.

Seeds of two cucumber (Cucumis sativus L.) cultivars ‘JinYou 1’ and ‘JinChun 5’ (the latter being more salt tolerant than the former, Zhang and Wu 2009) were rinsed thoroughly with distilled water and germinated on moist gauze incubator at 28 °C for 2 days. The germinated seeds were sown in quartz sands in the university greenhouse in which the temperature was set to 28 °C/18 °C, 12 h/12 h (day/night). The seedlings were transferred to 15 L plastic containers at twoleaf stage; each container had 10 plants with continuously aerated 1/4 strength of Hoagland nutrient solution. Three days after transplanting, the strength of Hoagland solution was increased to 1/2. Silicon and salt treatment were started 7 days after transplanting by adding sodium silicate (Na2SiO39H2O) and sodium chloride (NaCl) to the nutrient solution. Silicon was supplied at a concentration of 0.3 mM. NaCl concentration was 75 mM. The pH of the nutrient solution was adjusted to 6.0 every 2 days using 0.2 M H2SO4 or 1 M KOH. The culture solutions were renewed every 6 days. The plant samples were collected, and measurements were made from 8:00 to 13:00 h in this study. Biomass Plants were harvested after 15 days of treatment. The shoots were quickly washed in double-distilled water and then dried in an oven at 70 °C for 48 h. The roots were washed in double-distilled water quickly and then in cold 10 mM CaCl2 for 10 min, after which they were dried at 70 °C for 48 h (Husain et al. 2004). Each treatment included 10 replications. Photosynthetic parameters The net CO2 assimilation rate (Pn), stomatal conductance (gs), and transpiration rate (E) were assayed with a portable photosynthesis system (Li-6400; LI-COR Inc., Lincoln,

Sodium content The dried shoots or roots were digested in 0.5 M HNO3 (Husain et al. 2004). The digested plant material was then filtered, diluted as required with distilled water, and analyzed for Na? concentrations using an atomic absorption spectrophotometer (Model Z2000, Hitachi Polarized Zeeman, Japan). Each treatment included three replications.

Leaf-specific conductivity of the stem The leaf-specific conductivity of the stem (Lsc) was measured according to Dichio et al. (2013) and was calculated as Lsc = F9SL/DP/Aleaf (kg H2O s-1 m-1 MPa-1), where F is the flow rate (kg s-1), SL is the stem length, DP (expressed in MPa) is the hydrostatic gradient, and Aleaf is the ‘supported leaf area’ of each stem segment. Each treatment included five replications. Water uptake Plant water uptake was measured by a gravimetric method. The seedlings were transferred into 500 mL bottles (one seedling per bottle with the top being sealed with a sealing film) 2 days before the measurement. Each seedling (including the bottle) was weighed every 2 h between 8:00 a.m. and 12:00 a.m. Each treatment included 3–4 replications. Root hydraulic conductance Root hydraulic conductance (Lpr) measurements were carried out as described by Trubat et al. (2012). The root surface area was determined using a root scanner (Model MRS-9600TFU2L, Microtek, Shanghai, China). Root hydraulic conductance was calculated as Lpr = Qv/ (S 9 P) = Jv/P, where Qv (m3 s-1) is water flux density, S (m2) is root surface area, P (MPa) is the pressure put on root, and Jv (m s-1) is the flow rate. Each treatment included three replications.

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Root architecture Roots were sampled and scanned to measure the surface area and average diameter using a root scanner (Model MRS-9600TFU2L, Microtek, Shanghai, China). Each treatment included three replications.

then the roots were rinsed with distilled water and returned to the culture solution without HgCl2, after which the leaf transpiration rate was measured. The reversibility of the effect of HgCl2 on aquaporin activity was tested by treating the roots with 50 lM HgCl2 for 5 min and 5 mM DTT for 15 min in turn before the transpiration rate was measured. Each treatment included 8 replications.

RNA extraction and quantitative qPCR analysis Root xylem osmotic potential After stress treatment, the root samples of cucumber seedlings were harvested, immediately frozen in liquid nitrogen and then stored at -80 °C. Total RNA was extracted from 100 mg of root samples using TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The first-strand cDNA for qPCR analysis was synthesized from 500 ng of total RNA using HiScriptTMReverse Transcriptase (Vazyme, Nanjing, China) according to the manufacturer’s instructions, including a special step for genomic DNA digestion. qPCR experiments were conducted on a CFX 96 Real-Time PCR system (Bio-Rad) using SYBRÒ Green Master Mix (Vazyme, Nanjing, China) with CsPIP gene-specific primers (Table 1). Primer sequences of reference gene and CsPIPs genes for qPCR are referred to Wan et al. (2010) and Qian et al. (2014), respectively, as listed in Table 1. The relative quantity was calculated using the 2-44t method (Pfaffl 2001). Each treatment included three replications, and each replication included two technical replications. Transpiration rate responding to aquaporin inhibitor (HgCl2) and dithiothreitol (DTT) The aquaporin inhibitor and recovery treatments were according to the method of Liu et al. (2014). One set of plants without HgCl2 treatment (-HgCl2) were used to measure the leaf transpiration rate. The other set of plants were treated with aquaporin inhibitor HgCl2 (50 lM) for 5 min,

The osmotic potential of root xylem sap was assayed according to Yin et al. (2013). The shoot was cut-off at the base of the root system, leaving 4 cm of stem. The xylem sap was force exuded by N2 pressurized to 0.4 MPa. The osmotic potential of xylem sap was determined with a vapor pressure osmometer (Model 5520, Wescor, Logan, UT, USA). The readings (C) (mmol kg-1) were used to calculate the osmotic potential according to the equation: ws = -RTC, where R (MPa L mmol-1 K-1) is molar gas constant of 8.314 9 10-6; T is thermodynamic temperature; and C (mmol kg-1) is recorded from dew point microvolt meter. Each treatment included five replications. Soluble sugar content The root soluble sugars were measured by the spectrophotometric method according to Wu et al. (2013). The samples were extracted with 4 ml of 80 % (v/v) ethanol at 80 °C for 40 min and were then centrifuged at 2000g for 15 min. The precipitate was re-extracted with 2 ml of 80 % (v/v) ethanol and re-centrifuged. The supernatants were used for sugar analyses. Each treatment included three replications. Statistical analysis Data were subjected to analysis of variance using SPSS 19.0, where F tests were significant at P \ 0.05, and the

Table 1 The primers of the CsPIP genes and reference gene Gene

Accession no.

Primer sequences (forward/reverse primer)

CsPIP1;2

KF641170

F 50 -CATTATTTACAACCACGACGAAGCA-30 0

Amplicon length (bp) 165 0

R 5 -GGATTGAAGAAGCATCATGGATTTAGA-3 CsPIP2;1

KF641172

F 50 -TTTGGGTTGGACCTTTCATTGGA-30

158

R 50 -ATACTCATGGCACACAATTATTAGGCTT-30 CsPIP2;4

KF641175

F 50 -GCTGCTCTGCTCTCATCTTGCC-30

167

R 50 -GAAAAATACATGAATAACAGGAGCCCC-30 CsPIP2;5

KF641176

F 50 -CAACCGTGAAAAACCCTGGAATGAC-30

161

R 50 -CATCTTCTTCCTCTCAGTTTGTGGGG-30 ACT

DQ115881

F 50 -GTGGTGGTGAATGAGTAGCC-30 R 50 -TTGGATTCTGGTGATGGTGTC-30

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150

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means were separated by Duncan’s Multiple Range test at P \ 0.05.

Results Plant growth and photosynthesis parameters Under non-stress conditions, silicon addition did not change the dry mass accumulation in cv. JinYou 1, but it slightly increased the dry mass in cv. ‘JinChun 5’ (Fig. 1). Salt stress decreased the plant dry weights: the decrease being 47 and 41 % for cvs. ‘JinYou 1’ and ‘JinChun 5,’ respectively. In the presence of added silicon, the dry weights decreased less under salt stress, and they were significantly higher than those without added silicon (Fig. 1a). In ‘JinYou 1,’ the shoot/root ratio was significantly decreased under salt stress, and there was no significantly difference between salt treatment alone and salt treatment with added silicon (Fig. 1b). In ‘JinChun 5,’ neither salt stress nor silicon addition significantly changed the shoot/root ratio (Fig. 1b). Silicon addition did not influence the leaf photosynthetic rate, stomatal conductance, or transpiration rate of both cucumber cultivars under nonsaline conditions (Table 2). Under salt stress, these parameters were significantly decreased, and they were higher in the presence of added silicon than in the absence of silicon in both cultivars (Table 2). Na accumulation Tissue Na? concentrations expressed in both per dry weight and water content were presented (Fig. 2). Under

Fig. 1 Effects of silicon on dry weight (a) and shoot/root ratio (b) of cucumber seedlings under salt stress. The dry weights of seedlings were measured after 15 days of exposure to salt stress. Values are

salt stress, when expressed in dry weight, the Na? concentrations in the root were not significantly changed by addition of silicon in both cucumber cultivars (Fig. 2a, e), except cv. ‘JinYou 1,’ in which added silicon slightly decreased the Na? concentration 15 days after salt stress; when expressed on the basic of water content, the root Na? concentration was obviously decreased by added silicon compared with salt stress alone after 10 and 15 days of treatment (Fig. 2b, f). In the shoots of stressed plants of cv. ‘JinYou 1,’ the Na? concentration was lower in siliconapplied plants than non-silicon treatment (Fig. 2c), with the difference being more obvious when expressed in water content (Fig. 2d). In ‘JinChun 5,’ silicon addition only decreased the shoot Na? concentration on the dry weight basis on the 10th day after salt stress (Fig. 2g), while it decreased the concentration when expressed in water content after 10 and 15 days of salt stress (Fig. 2h). Leaf free water content, bound water content, and relative water content Silicon supplementation had no effect on the leaf free water content and relative water content under control conditions (Fig. 3). Salt treatment decreased the leaf free water contents in both ‘JinYou 1’ and ‘JinChun 5,’ and silicon addition inhibited the decrease, especially on the 10th and 15th days after salt stress. The leaf bound water contents were not changed as a result of salt stress or silicon treatment in either cucumber cultivar. The leaf relative water content was decreased under salt stress from 5 days onward after treatment in cv. ‘JinYou 1,’ and it maintained significantly higher in silicon-applied plants. In cv. ‘JinChun 5,’ the relative water content was not changed by salt stress

mean ± SD of ten replicates. Different letters above bars indicate a significant difference (P \ 0.05)

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Plant Cell Rep Table 2 Effects of silicon on net photosynthetic rate (Pn), stomatal conductance (gs), and transpiration rate (Tr) of cucumber seedlings under salt stress

Cultivar

Treatment

Pn (lmol CO2 m-2 s-1)

gs (mol H2O m-2 s-1)

Tr (mmol H2O m-2 s-1)

JinYou 1

CT

10.6 ± 1.8a

0.61 ± 0.18a

4.1 ± 0.8a

10.9 ± 1.2

a

0.56 ± 0.21

a

3.8 ± 1.0a,b

4.9 ± 1.2

c

0.24 ± 0.16

b

2.0 ± 0.8c

9.9 ± 2.3

b

0.48 ± 0.36

a

3.1 ± 0.7b

CT

13.0 ± 3.0

a

0.40 ± 0.21

a,b

2.9 ± 1.2a

Si

14.8 ± 3.3a

0.57 ± 0.26a

4.3 ± 1.3a

6.3 ± 2.5

c

0.27 ± 0.17

b

1.8 ± 0.8b

10.1 ± 2.4

b

0.42 ± 0.26

a,b

2.8 ± 1.0a,b

Si Na Na ? Si JinChun 5

Na Na ? Si

The measurements were conducted 10 days after salt stress treatment. Values are mean ± SD of eight replicates. Different letters indicate a significant difference (P \ 0.05)

or silicon treatment on the 5th day; from the 10th day onward after salt stress, the leaf relative water content was decreased and obviously improved by added silicon (Fig. 3). Leaf-specific conductivity In control conditions, silicon supplementation had no effect on the leaf-specific conductivity in either cucumber cultivars (Fig. 4). Salt treatment significantly decreased the leaf-specific conductivity in both cultivars, except after 5 days of salt stress in ‘JinChun 5.’ Addition of silicon significantly improved the leaf-specific conductivity from 10th day onward after salt treatment in ‘JinYou 1,’ while it only improved the conductivity on the 15th day after treatment in ‘JinChun 5.’

Fig. 2 Effect of silicon on Na? contents in leaves and roots of c cucumber seedlings under salt stress. The analyses were conducted after 5, 10, and 15 days of salt treatment. Root and shoot Na? concentrations in ‘JinYou 1’ (a–d) and ‘JinChun 5’ (e–h) were expressed in both per dry weight (a, c, e, g) and per water content (b, d, f, h). Values are mean ± SD of three replicates. Different letters above bars indicate a significant difference (P \ 0.05)

Root traits The total root surface area was not changed on the 5th day after salt treatment in the two cultivars (Table 3). At later stress period, the root surface area was decreased by salt stress, and it was higher in silicon-applied plants than those without silicon treatment in the two cultivars. There was no significant difference in root diameter between the treatments regardless of salt treatment or silicon application during the experimental period.

Water uptake rate Expression of root aquaporin genes Salt stress significantly decreased the root water uptake rate in both ‘JinYou 1’ and ‘JinChun 5’ (Fig. 5), and silicon application partly inhibited the decrease throughout the experimental period, except on the 5th day after stress start in ‘JinChun 5,’ where there was no difference in root water uptake rate between the silicon and non-silicon treatments. Root hydraulic conductance In normal growth conditions, silicon application did not change the root hydraulic conductance in either cucumber cultivar (Fig. 6). NaCl treatment decreased the root hydraulic conductance in both cultivars, and silicon addition significantly increased it on the 10th day onward. After 5 days of salt stress, silicon addition did not change the root hydraulic conductivity of stressed plants in cv. ‘JinChun 5,’ whereas in cv. ‘JinYou 1,’ t test between ‘Na’ and ‘Na ? Si’ treatments showed that silicon-applied plants had higher root hydraulic conductivity than those without silicon treatment.

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The expression levels of the major plasma membrane aquaporins in roots of cucumber seedlings (Qian et al. 2014) were investigated after salt stress for 24 h to 9 days (Fig. 7). After salt treatment, the expression levels of CsPIP2-4 and CsPIP1-2 tended to be higher in siliconapplied plants than those without applied silicon in the two cucumber cultivars, except 24 h after salt treatment in cv. ‘JinYou 1,’ where the CsPIP2-4 expression was lower in silicon-applied plants. After 24 h of salt stress, the CsPIP21 expression level was not changed as a result of silicon application in cv. ‘JinYou 1’ but it was lower in siliconapplied plants in cv. ‘JinChun 5’; the expression level of CsPIP2-5 was lower in silicon-applied plants in the two cultivars. From the 3rd day after salt treatment, the expression levels of CsPIP2-1 and CsPIP2-5 tended to higher in silicon-applied plants than plants without silicon application in both cultivars, except on the 5th day, when the expression levels of both genes were lower in silicon-applied plants in cv. ‘JinYou 1.’

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Transpiration rate in response to HgCl2 and DTT In cv. ‘JinYou 1,’ after 7 days of salt treatment, siliconapplied plants showed significantly higher transpiration rate than those treated with salt stress alone (Fig. 8). HgCl2, an aquaporin inhibitor (Knipfer et al. 2011), decreased the transpiration rate markedly, and there was no difference between salt stress alone and salt plus silicon treatment. DTT recovery experiment showed that the transpiration rate was significantly higher in silicon-treated plants than in untreated plants, but the values remained lower than those before HgCl2 was added. In ‘JinChun 5,’ there was no significant difference in transpiration rate between silicon-treated and untreated plants. HgCl2 treatment considerably decreased the transpiration rate of both treatments, and there was no difference between them. After DTT recovery, the transpiration rate increased 96.4 % in silicon-treated seedlings compared with 76.9 % in untreated seedlings (Fig. 8). Root xylem osmotic potential and soluble sugar content Under control conditions, silicon supplementation had no effect on the root xylem osmotic potential (Fig. 9). Salt stress significantly decreased the xylem osmotic potential in both cultivars. Under salt stress, silicon-applied plants had lower root xylem osmotic potential compared with salt stress alone in cv. ‘JinYou 1’; however, there was no significant difference between ‘Na’ and ‘Na ? Si’ treatments in cv. ‘JinChun 5.’ The changes of root soluble sugar content under salt stress were different between the two cucumber cultivars (Fig. 10). Under salt stress, the soluble sugar content in roots was increased in cv. ‘JinYou 1,’ and the increase was greater in silicon-applied plants. However, in cv. ‘JinChun 5,’ the root soluble sugar content was increased on the 5th and 15th days after initiation of salt stress, and silicon-applied plants did not show obvious change as compared to the control; after 10 days of salt stress, the root soluble sugar content was not affected by salt treatment or silicon application. There was a negative linear correlation between root xylem osmotic potential and root soluble sugar content in cv. ‘JinYou 1’ (Fig. 11). However, no obvious correlation between these two parameters was observed in cv. ‘JinChun 5’ (data not shown).

Discussion A previous study by Zhu et al. (2004) has shown the positive effect of silicon on cucumber growth under salt stress. Similar results were also observed in the present

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study (Fig. 1). Silicon-mediated growth improvement may be related to the maintenance of higher photosynthetic rate, which allowed a constant supply of assimilates to the growing tissue, as observed in this study (Table 2). Siliconinduced improvement in cucumber growth under salt stress suggests a potential application of silicon fertilizer to solve soil salinization-caused decrease in vegetable production. Silicon-mediated improvement of plant photosynthesis and growth under salt stress has been proposed to be attributed to the decrease in root Na? uptake and/or its rootto-shoot transport in plants. Liang (1999) found that silicon addition decreased the Na concentrations in both root and shoot of barley. Wang and Han (2007) reported that the Na concentrations in the roots of two alfalfa cultivars were significantly decreased by added silicon. Gong et al. (2006) and Shi et al. (2013) observed that silicon addition decreased Na and Cl concentrations in the shoot of rice under salt stress, respectively. These studies suggest that reducing Na accumulation is an important mechanism for siliconmediated salt tolerance of plants. Zhu et al. (2004) have indicated that added silicon could increase salt tolerance of cucumber. However, the effect of silicon on Na accumulation was not reported in their study. In this experiment, after 15 days of salt stress, added Si only slightly reduced Na? concentration in the shoots and roots of ‘JinYou 1’ but not ‘JinChun 5’ (Fig. 2). This suggests that preventing Na uptake by roots or its root-to-shoot transport is not a universal mechanism for silicon-mediated salt tolerance in plants. In the cucumber cv. ‘JinYou 1,’ in the light of the dramatic improvement in plant dry mass (62 %) and relatively small decrease in Na concentration (only 12 % in root and 19 % in shoot) by silicon addition under salt stress, it is less likely that silicon-mediated increase in salt tolerance of cucumber cv. ‘JinYou 1’ was mainly attributed to a decrease in Na accumulation. Control of Na? influx, efflux, and compartmentation contributes to the ability of plants to tolerate salt stress (Zhu 2001). The plasma membrane Na?/ H? antiporter, which is encoded by SOS1, plays a key role in the maintenance of a low Na level by removing Na from the cytosol (Shi et al. 2000). The tonoplast Na?/H? antiporter involves in Na compartmentation into the vacuole, and it is driven by the tonoplast H?-ATPase and H?-PPase (Blumwald et al. 2000). Liang (1999) found that the activity of plasma membrane H?-ATPase was increased by added silicon in the roots of salt-stressed barley. Liang et al. (2005) reported that the activities of root tonoplast H?-ATPase and H?-PPase in barely were considerably stimulated by silicon addition under salt stress. These studies suggest that added silicon may facilitate Na? removal out of cell by plasma membrane Na?/H? antiporter and Na compartmentation into vacuoles through tonoplast Na?/H? antiporter. However, in this study, we observed

Plant Cell Rep

Fig. 3 Effect of silicon on leaf free water content, bound water content, and relative water content of cucumber seedlings under salt stress. All parameters were measured after 5, 10, and 15 days of salt

treatment. Values are mean ± SD of six replicates. Different letters above bars indicate a significant difference (P \ 0.05)

that the expressions of plasma membrane Na?/H? antiporter and tonoplast H?-PPase in the roots of cv. ‘JinYou 1’ were not increased by addition of silicon under salt stress; on the contrary, their expressions were even lower in silicon-applied plants under stress (data not shown). The results suggest that, in contrast to the findings in barley by

Liang (1999) and Liang et al. (2005), silicon may be not actively involved in the regulation of Na export out of cell or compartmentation into the vacuole in cucumber. We do not know whether silicon addition decreased the root-toshoot Na translocation in ‘JinYou 1.’ But our experiment using trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid,

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Fig. 4 Effect of silicon on leaf-specific conductivity of cucumber seedlings without or with salt stress. Leaf-specific conductivity was measured after 5, 10, and 15 days of salt treatment. Values are

mean ± SD of five replicates. Different letters above bars indicate a significant difference (P \ 0.05)

Fig. 5 Effect of silicon on root water uptake rate of cucumber seedlings without or with salt stress. Root water uptake rate of cucumber seedlings was measured after 5, 10, and 15 days of salt

treatment. Values are mean ± SD of 3–4 replicates. Different letters above bars indicate a significant difference (P \ 0.05)

an apoplastic tracer (Gong et al. 2006), showed that silicon had no effect on transpirational bypass flow in either of the two cucumber cultivars (data not shown). This is in contrast to our earlier results observed in rice, a silicon accumulator, where silicon deposition markedly decreases Na transport through transpirational bypass flow (Gong et al. 2006), implying inter-species differences in silicon effect on Na transport through apoplastic pathway due to silicon accumulation differences. Despite of this, this at least

suggests that silicon did not reduce the sodium uptake through blockage of transpirational bypass flow in cucumber cv. ‘JinYou 1.’ In a word, we did not find any evidence that silicon was actively involved in preventing Na accumulation or enhancing Na compartmentation, suggesting the involvement of other mechanism in siliconmediated salt tolerance in cucumber. Many studies have suggested that the increase in antioxidant defense contributes to silicon-mediated salt

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Fig. 6 Effect of silicon application on root hydraulic conductance (Lp) of cucumber seedlings under salt stress. Values are mean ± SD of three replicates. Different lower case letters above bars indicate a significant difference between treatments (P \ 0.05). Different

Table 3 Effect of silicon on root surface area and mean diameter of cucumber seedlings under salt stress

capital letters above bars of Na and Na ? Si treatments in cv. JinYou 1 at day 5 indicate a significant difference between them according to t test at P \ 0.05

Root surface area (cm2) JinYou 1

Root mean diameter (mm) JinChun 5

JinYou 1

JinChun 5

5 days CT

236.0 ± 86.8a

304.3 ± 32.6a

0.53 ± 0.09a

0.59 ± 0.02a

Si Na

a

259.5 ± 50.9 285.7 ± 40.7a

a

282.4 ± 56.2 269.4 ± 13.0a

a

0.57 ± 0.06 0.57 ± 0.03a

0.60 ± 0.04a 0.52 ± 0.03a

Na ? Si

310.3 ± 53.2a

299.3 ± 41.4a

0.55 ± 0.03a

0.56 ± 0.07a

316.5 ± 27.3a

321.8 ± 59.5a

0.57 ± 0.06a

0.55 ± 0.03a

297.2 ± 68.6

a

303.0 ± 24.4

a

a

0.58 ± 0.03a

277.2 ± 32.0

a

227.1 ± 23.5

b

a

0.53 ± 0.04a

290.5 ± 42.5

a

312.5 ± 39.3

a

a

0.53 ± 0.04

0.54 ± 0.00a

10 days CT Si Na Na ? Si

0.57 ± 0.09 0.52 ± 0.05

15 days CT Si Na Na ? Si

398.3 ± 98.5a

433.5 ± 146.2a

0.55 ± 0.04a

0.56 ± 0.05a

329.3 ± 48.5

a

a

a

0.53 ± 0.05a

276.6 ± 44.4

b

247. 7 ± 105.6

a

0.50 ± 0.05a

385.9 ± 43.1

a

a

a

0.57 ± 0.00a

486.0 ± 196.8

b

448.8 ± 58.8

0.56 ± 0.05 0.51 ± 0.02 0.60 ± 0.07

Values are mean ± SD of three replicates. Different letters indicate a significant difference (P \ 0.05)

tolerance (Zhu and Gong 2014). However, whether this is a direct or indirect effect of silicon is hard to prove. In tomato, a non-silicon accumulator, Kurabachew and Wydra (2014) showed that silicon-mediated resistance against bacterial wilt was related to the induction of systemic resistance rather than the formation of mechanical barrier, and silicon regulated the expressions of genes involved in defense, signal transduction, and transcription (Kiirika et al. 2013). A transcriptome study by Ghareeb et al. (2011) on the silicon-induced resistance against bacterial wilt in

tomato suggested a priming effect. These studies suggest that silicon-mediated stress tolerance is not merely due to mechanical barrier but is related to the modulation of physiological metabolism, especially in non-silicon-accumulating plants. Water uptake and conservation are important aspects of salt tolerance study in plants. The ability of plants to maintain a good water status can mitigate ion toxicity by a dilution effect and therefore improve salt tolerance (Romero-Aranda et al. 2006). In this study, when expressed

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Plant Cell Rep b Fig. 7 Effects of silicon on the expression levels of CsPIP genes

under salt stress. The roots were sampled after 1, 3, 5, 7, and 9 days of salt treatment without or with silicon application. The relative expression was determined by q-PCR. Values are mean ± SD of three replications, and each replication included two technical replications. Different letters indicate a significant difference (P \ 0.05)

on tissue water content basis, the Na concentrations in roots and shoot were significantly decreased by added silicon after 10 days of salt treatment in both cultivars, suggesting that silicon may be involved in improving water status of cucumber plants under salt stress. Investigation on plant water relations showed that silicon addition significantly

Fig. 8 Effect of an aquaporin inhibitor (HgCl2) and an anti-inhibitor (dithiothreitol, DTT) on the transpiration rate of cucumber seedlings under salt stress with or without silicon application. The measurements were conducted on the new fully expanded leaves with a

portable photosynthesis system (Li-6400) after 7 days of salt treatment. Values are mean ± SD of eight replicates. Different letters above bars indicate a significant difference (P \ 0.05)

Fig. 9 Effect of silicon application on the osmotic potential of cucumber root xylem sap under salt stress. The osmotic potential was measured after 5, 10, and 15 days of exposure to salt stress. Values

are mean ± SD of five replicates. Different letters indicate a significant difference (P \ 0.05)

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Plant Cell Rep

Fig. 10 Effect of silicon on the soluble sugar content in the roots of cucumber seedlings under salt stress. The measurements were conducted after 5, 10, and 15 days of salt stress. Values are mean ± SD of three replicates. Different letters indicate a significant difference (P \ 0.05)

Fig. 11 Correlation between root xylem osmotic potential and root soluble sugar content in cv. ‘JinYou 1.’ Pooled data from Figs. 9a, 10a

increased the leaf relative water content after 5 days of salt stress in ‘JinYou 1’ and 10 days of salt stress in ‘JinChun 5’ (Fig. 3), which confirmed that silicon addition improved the water status of cucumber plants under salt conditions. Reduction in plant transpiration has long been considered an important mechanism for silicon-mediated drought tolerance. It was believed that silicon deposition on the leaf surface may decrease cuticular transpiration (Savant et al. 1999). Gao et al. (2006) found that silicon could decrease stomatal transpiration in maize leaves. However, in this study, the leaf transpirational rate was increased by silicon application in cucumber under salt stress (Table 2). Similar

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results have also been reported in sorghum (Hattori et al. 2005) and rice (Chen et al. 2011) under drought stress and tomato (Romero-Aranda et al. 2006) and rice (Shi et al. 2013) under salt stress. Hattori et al. (2008) found that added silicon had no effect on the leaf transpirational rate of water-stressed cucumber seedlings. Therefore, the effect of silicon on leaf transpirational water loss may be related to plant species and stress conditions. Our results suggest that other factor(s), rather than reduction in transpirational loss of water, were attributed to silicon-mediated improvement in water status of cucumber plants. Water transport affects the water status of leaves. Leafspecific conductivity is a measure of the hydraulic capacity of stem to supply leaves with water (Dichio et al. 2013). In this study, silicon did not change the leaf-specific conductivity on the 5th day after salt treatment, and it increased the conductivity from 10 days after salt stress in cv. ‘JinYou1’; in cv. ‘JinChun 5,’ silicon application increased the leaf-specific conductivity on the 15th day under salt stress, but it did not change the conductivity on the 5th and 10th day. The increases in leaf-specific conductivity in the two cucumber cultivars might have contributed to the improvement in leaf water status at the late period under salt stress (Fig. 3). Leaf-specific conductivity is controlled by the structure and size of vessels and the formation of embolisms (Liu et al. 2014). Gao et al. (2004) suggested that the deposited silicon in cell walls interlaced with organic macromolecules to form amorphous colloidal complexes, which affected the wetting properties of xylem vessels and therefore water transport in maize. Diogo and Wydra (2007) suggested that silicon-enhanced resistance to

Plant Cell Rep

bacterial wilt in tomato was related to its induced changes in polysaccharide structure of xylem vessel walls: silicon inhibited the non-blockwise de-esterification of homogalacturonan and induced increased branching or new deposition of rhamnogalacturonan, which might strengthen the pit membranes of vessels. These studies suggest that silicon may affect cell wall properties of xylem vessels and therefore regulate water transport in plants. The possible effects of silicon on structure and size of vessels and formation of embolisms in relation to water transport in cucumber remain to be investigated. Although addition of silicon could increase the leafspecific conductivity, it suggested that leaf-specific conductivity is not a main limiting factor for water transport under water stress (Liu et al. 2014). Therefore, siliconmediated improvement in water status in cucumber might be mainly attributed to the increased water uptake. In this study, silicon-mediated increase in cucumber transpiration (Table 2) also suggests a higher root water uptake rate, as was the case (Fig. 5). Root water uptake rate is related to root water uptake ability, which can be assessed by determination of root hydraulic conductance (Lp, Liu et al. 2014). In the liquid component of soil–plant–air continuum, the Lp is usually the lowest (Vandeleur et al. 2009); therefore, its improvement may markedly increase root water uptake. In this work, silicon-mediated increase in root water uptake rate corresponded with an increase in Lp (Figs. 5, 6). These results suggest that silicon-mediated improvement in cucumber water status might partly be attributed to the increase of Lp under salt stress. Lp is dependent on the root anatomy, the dynamics of root permeability to water and driving force (Sonobe et al. 2010; Liu et al. 2014). In this study, we did not investigate the root anatomic characteristics of cucumber seedlings, but we observed no change in the root mean diameter regardless of salt stress or silicon treatment (Table 3). In a previous study, Liu et al. (2014) did not find any changes in vessel diameter and vessel number as a result of water deficit stress or silicon addition in sorghum. It seems less likely that the increase of Lp by silicon addition was mainly due to the change of root anatomy in cucumber plants, although further investigations are still needed. In the radial move of water from the external growth medium to the root xylem, there are three pathways: the apoplastic, symplastic, and transcellular routes, with the latter two being collectively considered as ‘cell-to-cell’ pathway (Chaumont and Tyerman 2014). Under water stress, the cell-to-cell pathway plays a more important role in root water transport (Steudle 2000; Javot and Maurel 2002). In the cell-to-cell pathway, the hydraulic conductance can be largely determined by the amount and activity of aquaporins, especially under water stress (Steudle 2000; Vandeleur et al. 2009). In this study, the transcript levels of

four major PIPs genes in cucumber (Qian et al. 2014) were investigated (Fig. 7). Silicon addition basically increased the transcript levels of four PIPs genes (CsPIP2-4, CsPIP12, CsPIP2-1, and CsPIP2-5) in both cultivars under salt stress (Fig. 7). However, there were some cases where the expression levels of PIPs genes were lower in silicon-added plants than the non-silicon-treated plants under stress: the transcript levels of CsPIP2-1 and CsPIP2-5 on the 5th day in ‘JinYou 1’ (Fig. 7c, d) and on the 1st day in ‘JinChun 5’ (Fig. 7g, h). It is noted that the transcript levels of CsPIP24 and CsPIP1-2 contributed to about 80 % of the total CsPIPs expression in cucumber roots (Qian et al. 2014). Therefore, the silicon-mediated increases in expression levels of CsPIP2-4 and CsPIP1-2 could counteract the lower expression of CsPIP2-1 and CsPIP2-5. In ‘JinYou 1,’ on the 1st day after salt stress treatment, silicon addition did not induce a higher expression of CsPIP2-4 as seen in ‘JinChun 5,’ which may suggest a cultivar difference. Although the genetic backgrounds of these two cucumber cultivars are not clear, previous research showed that ‘JinChun 5’ is more salt-tolerant than ‘JinYou 1’ (Zhang and Wu 2009). The cultivar difference in reaction to silicon has also been observed in bacterial wilt resistance in tomato. Diogo and Wydra (2007) found that silicon-induced resistance to bacterial wilt is more effective in the resistant genotype than in the moderately resistant genotype. By and large, in this study, silicon addition increased the CsPIPs expression levels under salt stress, which is consistent with the results of Liu et al. (2015). Siliconmediated increase in CsPIPs expression might have contributed to the increase of root hydraulic conductance in cucumber under salt stress. However, in this study, we also observed that, with compared to the control, salt stress alone increased the CsPIPs expression, while the root Lp was decreased. This suggests that silicon-mediated improvement in root water uptake and Lp under salt stress might also be attributed to other mechanism. The involvement of aquaporins in water uptake can be determined by measuring the changes of transpiration rate in response to HgCl2—an inhibitor of aquaporins (Knipfer et al. 2011). In this study, the transpiration rate was markedly decreased by HgCl2 treatment in both cultivars, regardless of silicon application, with the decreases being 60–73 % (Fig. 8), suggesting the participation of aquaporins in water uptake in cucumber plants. In cv. ‘JinYou 1,’ silicon addition significantly promoted the transpiration rate of salt-stressed plants. The transpiration rate was depressed by HgCl2, and the difference between plants without or with added silicon disappeared (Fig. 8), while DTT could partly recover the transpiration, especially in silicon-added plants. In cv. ‘JinChun 5,’ DTT treatment recovered the transpiration to the level of control in siliconadded plants, while the transpiration in non-silicon-treated

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plants was still significantly lower than the control. These results suggest that under salt stress, silicon-enhanced water uptake was mediated by aquaporins in the roots, which further supports our results. Osmotic gradient between soil/growth medium and root xylem sap can facilitate water uptake (Javot and Maurel 2002). In cv. ‘JinYou 1,’ silicon supplementation further decreased the root xylem osmotic potential in stressed plants under salt stress (Fig. 9a). Since the osmotic potential of root xylem sap in silicon-added plants was lower than that of the culture solution (-0.38 MPa for NaCl solution and -0.41 MPa for NaCl ? Si solution), silicon-mediated decrease in root xylem osmotic potential was beneficial for water uptake under salt stress. However, in ‘JinChun 5,’ silicon supplementation did not change the root xylem osmotic potential of salt stress plants (Fig. 9b). Recently, Liu et al. (2014) did not find any change of root xylem sap as a result of silicon addition in sorghum seedlings under water deficit stress. Therefore, the role of osmotic driving force in silicon-mediated enhancement of water uptake under stress conditions is genotype dependent and may also be related to stress types. The decrease of root xylem osmotic potential, i.e., osmotic adjustment, results from the accumulation of compatible organic solutes (Parida and Das 2005; Ming et al. 2012; Misˇic´ et al. 2012). Soluble sugar is one of the most important compatible organic solutes in the osmotic adjustment (Zhu and Gong 2014). In this study, silicon application increased the root soluble sugar content in cv. ‘JinYou 1,’ but not in ‘JinChun 5’ under salt stress (Fig. 10). There is a negative correlation between the root xylem osmotic potential and root soluble sugar content in ‘JinYou 1’ (Fig. 11), suggesting a possible participation of soluble sugar in osmotic adjustment in this cultivar. These results suggest that the regulation of silicon on soluble sugar accumulation in plants under stress is genotype dependent. Root water uptake is also related to root surface area. In ‘JinYou 1,’ before 10 days of salt stress, since silicon addition did not change the root surface area (Table 3), silicon-mediated increase in root water uptake should be attributed to the increase in Lp (Fig. 6). On the 15th day in ‘JinYou 1’ and from the 10th day in ‘JinChun 5’ under salt stress, added silicon increased the root surface area (Table 3), which might have facilitated the water uptake. However, addition of silicon did not change the shoot/root ratio in the two cultivars under salt stress (Fig. 1b). This suggests that the higher water uptake in silicon-added plants has been attributed to the increase in Lp per se, which was associated with the increased expressions of plasma membrane aquaporin genes as discussed above. Compared with silicon-accumulating plants, cucumber, and tomato accumulate much lower concentrations of silicon due to a lower density of transporter-mediated transport and a defect of putative transporter (SIT2)

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(Mitani and Ma 2005). In tomato, silicon mainly accumulates in the root, while it can mediate bacterial wilt resistance (Diogo and Wydra 2007), suggesting an induced resistance. Ghareeb et al. (2011) proposed that silicon primed tomato plants, therefore improving the resistance against bacterial wilt. The study also suggested that the priming effects were mediated via ethylene, jasmonic acid, and/or reactive oxygen species. In cucumber, an involvement of silicon-mediated priming effects in salt tolerance is also possible. Further studies are needed to explore the molecular mechanism (such as signaling pathway) for silicon-mediated salt tolerance in plants. Overall, under stress condition, silicon-mediated upregulation of aquaporin gene expression partly contributed to the increased root hydraulic conductivity and water uptake in both cucumber cultivars. But the up-regulation of aquaporin gene expression is not the only mechanism of silicon-facilitated root water uptake in cucumber, and the genotype-dependent osmotic adjustment may also have contributed to the root water uptake in cucumber. In the stem, silicon addition increased leaf-specific conductivity, which was useful for the stem to supply leaves with water. The improvement of root water uptake and leaf-specific conductivity was beneficial to maintain leaf water statues and contributed to sodium dilution within plants and therefore reduced ion toxicity. Our results suggest that silicon improves salt tolerance of cucumber plants mainly through enhancing root water uptake. This report may enhance our understanding of the mechanism for siliconmediated plant salt tolerance. Author contribution statement Hai-Jun Gong conceived and designed the experiments and revised the manuscript. Yong-Xing Zhu and Xuan-Bin Xu conducted the experiments and drafted the manuscript. Yan-Hong Hu, Wei-Hua Han, Jun-Liang Yin, and Huan-Li Li helped conduct parts of the experiments and analyses. Acknowledgments This study is supported by the National Natural Science Foundation of China (31272152, 31471866), Program for New Century Excellent Talents in University of China (NCET-110441), and Research Fund for the Doctoral Program of Higher Education of China (20120204110020). Conflict of interest This work has not been accepted or published in any other journal. It is not being considered for publication in any other journal. The authors declare that they have no conflict of interest.

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