Acta Physiol Plant (2013) 35:3099–3107 DOI 10.1007/s11738-013-1343-5
ORIGINAL PAPER
Application of silicon improves salt tolerance through ameliorating osmotic and ionic stresses in the seedling of Sorghum bicolor Lina Yin • Shiwen Wang • Jianye Li Kiyoshi Tanaka • Mariko Oka
•
Received: 18 November 2012 / Revised: 30 April 2013 / Accepted: 3 July 2013 / Published online: 12 July 2013 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2013
Abstract Silicon has been widely reported to have a beneficial effect on improving plant tolerance to biotic and abiotic stresses. However, the mechanisms of silicon in mediating stress responses are still poorly understood. Sorghum is classified as a silicon accumulator and is relatively sensitive to salt stress. In this study, we investigated the short-term application of silicon on growth, osmotic adjustment and ion accumulation in sorghum (Sorghum bicolor L. Moench) under salt stress. The application of silicon alone had no effects upon sorghum growth, while it partly reversed the salt-induced reduction in plant growth and photosynthesis. Meanwhile, the osmotic potential was lower and the turgor pressure was higher than that without silicon application under salt stress. The osmolytes, the sucrose and fructose levels, but not the proline, were significantly increased, as well as Na? concentration was decreased in silicon-treated plants under salt stress. These
Communicated by S. Renault.
results suggest that the beneficial effects of silicon on improving salt tolerance under short-term treatment are attributed to the alleviating of salt-induced osmotic stress and as well as ionic stress simultaneously. Keywords Ionic stress Osmotic stress Salt tolerance Silicon Sugar Water potential Abbreviations DAT Days after treatment DW Dry weight E Transpiration rate FW Fresh weight gs Stomatal conductance LA Leaf area PN Net photosynthetic rate Wp Osmotic potential Wp Turgor pressure Ww Water potential
Electronic supplementary material The online version of this article (doi:10.1007/s11738-013-1343-5) contains supplementary material, which is available to authorized users. L. Yin S. Wang (&) State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation, Northwest A&F University, Xinong Road No. 26, Yangling 712100, Shaanxi, China e-mail:
[email protected] L. Yin K. Tanaka M. Oka Faculty of Agriculture, Tottori University, Koyama, Minami, 4-101, Tottori 680-8553, Japan S. Wang J. Li College of Natural Resources and Environment, Northwest A&F University, Yangling 712100, Shaanxi, China
Introduction Silicon is the second most abundant mineral element in the soil after oxygen and comprises 31 % of the earth’s crust, and is also a major constituent of many plants (Epstein 1999; Gong et al. 2006). Although silicon is not generally considered to be an essential element for the majority of plants, its uptake has been widely found to be beneficial in improving the biotic and abiotic stress tolerance (Liang et al. 2007; Ma and Yamaji 2008; Epstein 2009); for instance, alleviating heavy metal stress (Neumann and Nieden 2001), increasing tolerance to salt and drought
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(Hattori et al. 2005; Kafi and Rahimi 2011), and improving the resistance to pests and pathogens (Fauteux et al. 2006). The beneficial effects of silicon on plant growth are particularly distinct under stress conditions; however, the mechanisms of silicon-mediated stress responses are still poorly understood (Epstein 2009). Improvement of salt tolerance by silicon supplementation has been reported in various crop species, such as rice (Oryza sativa L.), wheat (Triticum durum), tomato (Lycopersicon esculentum, Mill), and so on (Yeo et al. 1999; Romero-Aranda et al. 2006; Tuna et al. 2008). The role of silicon in enhancing salt tolerance in previous researches include: reduces Na? uptake by inhibiting bypass flow or restricting transpiration in rice (Yeo et al. 1999; Gong et al. 2006), improves the water status in tomato (RomeroAranda et al. 2006), increases antioxidant enzyme activity in cucumber (Cucumis sativus L.) (Zhu et al. 2004), and increases the plasma membrane H?-ATPase activity in barley (Hordeum Vulgare L.) (Liang et al. 2006). The previously described effects of silicon vary among species, strongly suggests that the biological functions of silicon are complex, and its role in plant salt tolerance has not been well established. Salt stress affects plants in two ways: increased osmotic stress and ionic stress. High concentrations of salts in solution result in increased osmotic stress, which limits the water availability for the plant, and in turn affects water status, stomatal conductance (gs), leaf growth and photosynthesis. Ionic stress is a result of salt accumulation to toxic concentrations in old leaves, which accelerates the senescence of old leaves, increases the toxicity in chloroplasts, and leads to leaf death (Munns and Tester 2008). The main mechanism of salt tolerance also has two aspects: osmotic adjustment and alleviation of ionic toxicity. Osmotic adjustment, which is an important salt tolerance mechanism, helps plants to retain water despite a low water potential, and thus to alleviate the osmotic stress. For alleviation of ionic toxicity, plant usually decreases Na? concentration through Na? exclusion, limiting Na? uptake or compartmentalization Na? at cellular and intracellular level, to avoid Na? arrived toxic concentration in cytoplasm, especially in mesophyll cells (Munns and Tester 2008). Several previous researches have focused on the function of silicon in decreasing Na? accumulation in the shoot and increasing the K?/Na? ratio (Gong et al. 2006; Tuna et al. 2008; Ashraf et al. 2010). In contrast, there is less research on the function of silicon in osmotic stress under salt condition. However, osmotic stress has not only an immediate effect on growth, but also a greater effect on growth rate than ionic toxicity in the early stage of salt stress (Munns and Tester 2008). In addition, previous research focused on the function of silicon in long-term
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salt treatment (over 7 days), but less research has been reported on the effect of silicon in short-term salt treatment conditions. Therefore, there are three points in the function of silicon that need to be further clarified. The first is the beneficial effect of silicon on growth under short-term salt stress, the second is the effect of silicon on osmotic stress, and determining the kind of substances that are induced by silicon and that play important roles in osmotic adjustment under salt stress, and the third is the effect of silicon on ion uptake and accumulation in pant tissue under salt stress. Sorghum is an important crop in the world, which is often grown in areas of relatively low rainfall, high temperatures and saline soils, and its growth is always affected by salt stress (Boursier and La¨uchli 1990). Sorghum is a plant that can take up and accumulate a large amount of silicon (2 to 3 % DW), and it is considered a silicon accumulator (Hattori et al. 2005). This research was designed to investigate the integrated role of silicon in alleviating salt stress in sorghum during the early growth stage under short-term salt stress. Including the possible hypotheses explaining the beneficial effect of silicon under saline conditions that were formulated above, we tested the following hypotheses: The first hypothesis is supplementation of silicon improves plant water status by alleviating salt-induced osmotic stress. The second hypothesis is supplementation of silicon reduces sodium uptake and increases the K?/Na? ratio in sorghum, thereby reducing toxic accumulation of Na? in plant tissues. Fresh weight, leaf area, photosynthesis, water potential, osmotic adjustment substances and ion accumulation were studied under short-term salt stress with or without silicon.
Materials and methods Plant material and growth conditions Sorghum seeds (Sorghum bicolor L. Moench Cv. Gadambalia) were surface sterilized with 10 % H2O2 for 10 min and germinated in a Petri dish for 20 h at 28 °C. The seeds were then placed on a wet filter paper for 2 days at 28 °C in an incubator under dark condition (germination in the dark condition causes extension of shoot, which will facilitate the transplanting into the nutrition solution). On the third day after sowing, seedlings were transplanted into plastic containers with 20 L of culture solution and then placed in an environmental controlled growth chamber. Hoagland solution was used as the culture solution, and it was changed every 3 days. The growth chamber was set to a 14/10-h day/night cycle at a day/night temperature of 28/23 °C and 40 % relative humidity. Photosynthetically active radiation was about 800 lmol m-2 s-1.
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After growing for 17 days (seven leaves), uniform plants were selected for salt and silicon treatment. The silicon solution was prepared according to Sonobe et al. (2009). Plants were exposed to 0 mM (control) or 100 mM NaCl and combined with 0 mM or 0.83 mM silicon. The treatment was continued for 7 days and samples were taken at 1, 3, and 7 days after treatment (DAT). During the treatment, the solution was renewed every 2 days, and the pH was adjusted every day to 6.5 with 0.1 M HCl or KOH according to Sonobe et al. (2009). Harvest and leaf gas exchange Plants were harvested on 1, 3, and 7 DAT, and the fresh weight (FW) and leaf area (LA) were determined. The root, stem, and leaves of the plants were separated, the FW of each part was recorded and total weight was calculated by adding the three parts together. The LA was measured with a leaf area meter (AAL-410, Hayashi Denko Inc., Tokyo, Japan). On the 3 DAT, the net photosynthetic rate (PN), transpiration rate (E), and gs of new fully expanded leaves were measured using a portable photosynthesis system (LI6400, LI-COR., Lincoln, NE, USA). The leaf was placed in 6 cm2 chambers at a photo flux density of 1,000 lmol m-2s-1 PAR, a flow rate through the chamber of 500 mLs-1, and a leaf temperature of 28 °C. This experiment was repeated twice and each repetition of the experiment included six replicates. Measurement of leaf water potential and osmotic potential Leaf water potential (Ww) was measured after treatment for 1, 3, and 7 days. The fully expanded leaves (the third or fourth leaf from the apex) were selected and were measured between 10:00 and 11:00 AM using a pressure chamber (Model 1000, PMS instrument Co., Covallis, OR, USA). After measurement of Ww, leaves were placed in 0.5 mL tubes, and frozen in liquid nitrogen for 30 min. The frozen samples were allowed to thaw at room temperature. After thawing the samples, 0.5 mL tubes were drilled at the bottom, put into 1.5 mL tubes, and centrifuged at 4,000 rpm for 5 min to gather the cell sap. The osmolarity of the collected sap was determined using a dew point microvolt meter (Model 5520, Wescor, Logan, UT, USA), and the osmotic potential (Wp) was calculated. The turgor pressure (Wp) was calculated as the difference between Ww and Wp. Each treatment included six replicates. Osmotic solute contents determination The proline content was determined according to Bates et al. (1973). Proline was extracted using 3 % sulfosalicylic
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acid (5 ml). The 1-mL extraction volume was mixed with 1 mL of a mixture of glacial acetic acid and 6-M orthophosphoric acid (3:2, v/v) and 50-mg ninhydrin. After incubation for 1 h at 100 °C, the tube was cooled down to room temperature and 5-mL toluene was added, and the absorbance of the upper phase was spectrophotometrically determined at 520 nm. The proline concentration was determined using a standard curve. Soluble sugar was measured using the Shimadzu sugar analysis system (HPLC, Shimadzu, Kyoto, Japan). Leaves (0.5 g FW) were homogenized in 8 mL 85 % (v/v) ethanol, and heated at 80 °C for 1 h in a water bath. Then the extracted solution was dried, and the residue was dissolved in 1-mL water and passed through a membrane filter (pore size 0.45 lm). Aliquots (10 lL) were subjected to HPLC in a Shim-pack ISA-07/S2504 column set at 65 °C with a guard column. Buffer A that contained 0.1-M potassium borate (pH 8.0) and buffer B that contained 0.4-M potassium borate (pH 9.0) were used to separate out the sugar, with a linear gradient of 100 % buffer A to 100 % buffer B at 2 %/min from 0 to 50 min, 100 % buffer B from 50 to 65 min, and subsequently to 100 % buffer A from 65 to 90 min at a flow rate of 0.6 mL min-1. The L-arginine reagent (1 % L-arginine containing 3 % borate) was delivered at a flow rate of 0.5 mL min-1, and the reaction oven was set at 150 °C. The fluorescence detector was set at 320-nm excitation and 430-nm emission wavelengths. Each treatment included three replicates. Measurement of ion concentrations Dried leaf material was milled to a powder for ion analysis. The powder was weighed and digested in nitric acid in a glass tube at 200 °C on a hot stove, and H2O2 (20 lL) was added 2–3 times during the digestion. When digestion was finished, only dry white residue was left. The residue was resolved with 10 % nitric acid (v/v). Na?, K?, Ca2?, and Mg2? were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Ciros CCD, Rigaku, Tokyo, Japan). The ion content was expressed as mg g-1 dry weight (DW). Each treatment included three replicates. Statistical analysis This experiment was conducted twice and each treatment of the experiment included three or six replicates. Data were subjected to an analysis of variance (ANOVA) using SAS software version 8.0. Differences between the means were compared by least significant differences (LSD) (P \ 0.05).
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Results Plant growth and photosynthesis In the absence of salt stress, silicon had no effect on the growth and LA of sorghum seedlings (Table 1). Salt treatment alone significantly reduced sorghum FW by 12, 31, and 40 % after treatment for 1, 3, and 7 days, respectively, compared to controls. However, when plants were grown with 100-mM NaCl plus 0.83-mM silicon, the reduction in the plant FW was only 0, 21, and 22 % after treatment for 1, 3, and 7 days, respectively. Similarly, NaCl alone reduced total plant LA by 11, 28, and 42 %, respectively, but the reduction was only 0, 16, and 25 % after treatment for 1, 3, and 7 days, respectively, when silicon was added to the salt-treated plants. As shown in Fig. 1, the application of silicon did not influence the PN, gs and E in the sorghum seedling under non-saline conditions. However, these parameters were significantly decreased when plants were treated with 100-mM NaCl, and this reduction was reserved by silicon application. Compared with NaCl treatment alone, the PN, gs and E were increased by 30, 42, and 41 %, respectively, by supplementary silicon following the NaCl treatment. Leaf Ww and Wp Silicon supplementation with and without NaCl, decreased the leaf Ww (Fig. 2a). The Wp was
had no effect on leaf Ww both but NaCl treatment significantly after salt treatment for 3 days not affected by silicon in non-
Table 1 Effect of NaCl (0 or 100 mM) and silicon (0 or 0.83 mM) on FW of leaf, stem and root, and LA of sorghum seedlings
salinized plants too, but it was decreased by both NaCl alone and NaCl plus silicon treatments (Fig. 2b). However, there was a larger reduction in the Wp when plants were treated with NaCl plus silicon, which showed a decrease of 73, 58, and 72 % after treatment for 1, 3, and 7 days, respectively, compared with the control. While it showed a decrease of 22, 34, and 35 %, respectively, under treatment with NaCl alone. The leaf Wp, calculated as the difference between Ww and Wp, was markedly increased by silicon under salt stress (Fig. 2c). Proline and water soluble sugar contents The leaf proline content was not affected by supplementary silicon under non-saline conditions. However, NaCl treatment significantly increased the proline content, while silicon application decreased it by 94, 86, and 88 % after treatment for 1, 3, and 7 days, respectively, compared with NaCl treatment alone (Table 2). In contrast to the changes in the proline levels, leaf water soluble sugar levels were increased by application of silicon under NaCl treatment. Compared with control, silicon quickly and significantly increased the sucrose content under salt treatment for 1 day, demonstrating a 4.4-fold increase compared to a 1.8fold increase for salt treatment alone (Table 2). Under nonsaline conditions, silicon increased the leaf fructose content except at 3 DAT. Under salt stress, it increased more at 7 DAT (98 % increase) compared to no silicon supplementation. Glucose content in the leaf was increased by salt stress after 7 days of treatment; however, silicon had no effect on the glucose content both with and without NaCl
Control
Silicon
NaCl
NaCl ? silicon
1 DAT Leaf FW (g) Stem FW (g) Root FW(g) Total FW (g) 2
LA (cm per plant)
3.0 ± 0.0a
2.8 ± 0.1a,b
a
2.3 ± 0.1
a
2.3 ± 0.0
a
7.5 ± 0.1
b
174.2 ± 6.2
2.3 ± 0.1
a
2.1 ± 0.1
a
7.2 ± 0.1
a,b
178.8 ± 7.5
a,b
2.6 ± 0.2b
3.1 ± 0.1a
2.0 ± 0.2
a
2.3 ± 0.1a
2.0 ± 0.3
a
2.2 ± 0.2a
6.6 ± 0.1
b
7.6 ± 0.5a
154.1 ± 4.3
c
195.0 ± 3.6a
3 DAT Leaf FW (g) Stem FW (g) Root FW(g) Total FW (g) 2
LA (cm per plant) All parameters were measured 1, 3, and 7 days after treatment (DAT) Values are mean ± SE from six replicates. Different letters in the same line indicate statistical significance difference (P \ 0.05)
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4.9 ± 0.5a
4.9 ± 0.4a
3.4 ± 0.2b
3.8 ± 0.5a,b
a
3.3 ± 0.3
a
2.3 ± 0.1
b
2.5 ± 0.3a,b
3.9 ± 0.2
a
2.9 ± 0.0
a
3.3 ± 0.7a
8.5 ± 0.3
b
9.7 ± 1.5a,b
197.2 ± 7.7
b
3.3 ± 0.4
a
4.1 ± 0.6
a
12.3 ± 1.4
12.1 ± 0.9 a
a,b a
238.1 ± 26.4a,b
274.9 ± 21.0
282.6 ± 28.7
10.6 ± 0.9a
10.3 ± 0.3a
5.8 ± 0.3c
7.6 ± 0.2b
Stem FW (g) Root FW(g)
a
7.5 ± 0.5 6.7 ± 0.3a
a
7.6 ± 0.3 6.6 ± 0.1a
c
3.9 ± 0.2 5.1 ± 0.2b
5.1 ± 0.2b 6.8 ± 0.4a
Total FW (g)
24.8 ± 1.6a
24.5 ± 0.4a
14.7 ± 0.5c
19.5 ± 0.9b
570.2 ± 34.2a
572.3 ± 2.5a
330.1 ± 16.7c
433.9 ± 29.5b
7 DAT Leaf FW (g)
LA (cm2 per plant)
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3103
30
0.0
a
25 20
10
5 0 Si
NaCl
NaCl+Si
b -0.6
a a a a
b a a b
-0.9
b a
-1.2
a
-1.5
0.20
Control
a
a
Si
NaCl
NaCl+Si
-1.8
0.15
a
0.0
b 0.10
0.05
0.00 Control
Si
NaCl
NaCl+Si
T (mmol H2 O m-2 s-1)
4
3
a
a
a
Osmotic potential (MPa)
-0.3
-0.6
-0.9
b
-1.2
2
c c
c c
c c
b
b a
-1.5
a
a
b -1.8 1.8
1
1.5 0 Control
Si
NaCl
NaCl+Si
Fig. 1 Effects of NaCl (100 mM) and silicon (Si, 0.83 mM) on net photosynthetic rate (PN), stomatal conductance (gs) and transpiration rate (E) of sorghum leaves. All parameters were measured after being treated for 3 days. Mean ± SE, n = 6. Different letters in one measure indicate statistically significant differences at P \ 0.05
Turgor pressure (MPa)
Cond (mol H2 O m-2 s-1)
7 DAT
3 DAT
-0.3
15
Control
1 DAT
a b
Water potential (MPa)
P (µmol CO2 m-2 s-1)
a
1.2
a
0.9
a b
0.6
b b b
0.3
treatment. The silicon-induced increase of water soluble sugar contents was mainly attributed to sucrose changes under salt stress. Ion accumulation in leaves In the absence of NaCl, silicon application had no effect on Na?, K?, and Ca2? concentration, except a slight decrease in Mg2? concentration in the leaves (Table 3). Salt stress dramatically increased the Na? concentration, while silicon supplementation dramatically decreased it. As shown in Table 3, NaCl alone increased Na? concentration by 807, 771, and 754 % after treatment for 1, 3, and 7 days, respectively, but the increase was only 575, 385, and 283 % when silicon was applied. The K? concentration
a
c c b b b
0.0
1 DAT
3 DAT
7 DAT
Fig. 2 Effects of NaCl (100 mM) and silicon (Si, 0.83 mM) on water potential (a), osmotic potential (b) and turgor pressure (c) of sorghum leaves. All parameters were measured 1, 3, and 7 days after treatment (DAT). Mean ± SE, n = 6. Different letters in one measure indicate statistically significant differences at P \ 0.05
was not affected by silicon application under salt stress conditions during all treatment periods, but the K?/Na? ratio was increased 1.4-, 1.8-, and 2.3-fold compared with NaCl treatment alone. Under salt stress, the Ca2? concentration was not affected by silicon application, while the Mg2? concentration was slightly decreased.
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3104 Table 2 Effect of NaCl (0 or 100 mM) and silicon (0 or 0.83 mM) on the sucrose, fructose, glucose and proline contents in sorghum leaves
Acta Physiol Plant (2013) 35:3099–3107 (lmol g-1 FW)
Control
Silicon
NaCl
NaCl ? silicon
1 DAT Sucrose Fructose Glucose Proline
2.78 ± 0.07d
3.26 ± 0.03c
7.83 ± 0.22b
15.14 ± 0.06a
b
a
0.81 ± 0.05
b
0.77 ± 0.11b
20.81 ± 1.65
a
16.46 ± 0.37b
a
0.25 ± 0.04b
0.66 ± 0.03
a
19.15 ± 0.46
b
1.00 ± 0.07
a
20.58 ± 0.65
b
0.14 ± 0.04
0.18 ± 0.02
4.14 ± 0.24
3.42 ± 0.01b
2.90 ± 0.11b
11.27 ± 0.24a
10.58 ± 0.38a
b
b
1.03 ± 0.05
a
0.99 ± 0.02a
17.90 ± 0.02
b
18.99 ± 0.49a
a
0.30 ± 0.05b
3 DAT Sucrose Fructose Glucose All parameters were measured 1, 3, and 7 days after treatment (DAT) Values are mean ± SE from three replicates. Different letters in the same line indicate statistical significance difference (P \ 0.05) Table 3 Effect of NaCl (0 or 100 mM) and silicon (0 or 0.83 mM) on Na?, K?, Ca2?, Mg2? contents and K/Na ratio in sorghum leaves
Proline
0.71 ± 0.05
a,b
18.21 ± 0.23
b
0.68 ± 0.03
c
15.11 ± 0.42
b
0.24 ± 0.02
0.31 ± 0.04
2.25 ± 0.38
4.24 ± 0.08c
3.64 ± 0.06c
7.38 ± 0.38b
20.01 ± 0.82a
c
b
0.80 ± 0.01
c
1.52 ± 0.07a
23.55 ± 0.61
a
22.99 ± 0.12a
5.40 ± 0.35
a
0.64 ± 0.04b
7 DAT Sucrose Fructose Glucose Proline
(mg g-1 DW)
0.79 ± 0.02
c
14.07 ± 0.07
c
0.24 ± 0.02
Control
0.96 ± 0.06
b
15.12 ± 0.20
c
0.28 ± 0.02
Silicon
NaCl
NaCl ? silicon
1 DAT Na? K
?
2.39 ± 0.21c
18.32 ± 0.83a
12.36 ± 0.25b
a
a
a
21.30 ± 0.73a
4.69 ± 0.15a
4.39 ± 0.01b
b
2.01 ± 0.02c
b
1.24 ± 0.18
1.72 ± 0.35b
22.50 ± 0.83
Ca2? Mg
2.34 ± 0.17c 4.57 ± 0.06a,b
2?
?
K /Na
22.23 ± 1.39
?
2.55 ± 0.08
a
9.62 ± 1.15
a
4.48 ± 0.06a,b 2.24 ± 0.03
b
9.30 ± 1.79
a
22.70 ± 1.07
2.32 ± 0.03
3 DAT Na? K
?
2?
?
K /Na All parameters were measured 1, 3, and 7 days after treatment (DAT) Values are mean ± SE from three replicates. Different letters in the same line indicate statistical significance difference (P \ 0.05)
2.43 ± 0.37c
17.25 ± 0.83a
8.86 ± 0.65b
a
a
b
20.10 ± 0.69b
24.73 ± 1.02
Ca2? Mg
2.51 ± 0.30c
?
24.06 ± 0.72
21.30 ± 0.58
5.22 ± 0.11a
4.98 ± 0.11a
4.46 ± 0.06b
4.57 ± 0.04b
2.78 ± 0.06
a
2.66 ± 0.02
b
c
1.84 ± 0.02d
9.85 ± 0.63
a
9.90 ± 0.41
a
c
1.23 ± 0.15
2.27 ± 0.36b
2.20 ± 0.01
7 DAT Na?
2.12 ± 0.11c
2.18 ± 0.07c
15.48 ± 0.69a
6.89 ± 0.21b
K? Ca2?
22.51 ± 0.47a 4.93 ± 0.06a
22.10 ± 1.01a 5.03 ± 0.34a
16.91 ± 0.40b 4.15 ± 0.12b
17.30 ± 0.32b 4.20 ± 0.10b
3.01 ± 0.08a
2.04 ± 0.02c
2.44 ± 0.03b
2.38 ± 0.02b
a
a
c
2. 51 ± 0.49b
Mg2? ?
K /Na
?
10.62 ± 0.59
Discussion
10.14 ± 0.64
1.09 ± 0.17
related to maintaining a high PN, which allowed a constant supply of assimilate to the growing tissue.
Effect of silicon on growth and photosynthesis Effect of silicon on osmotic adjustment Our results showed that salt-induced growth inhibition in sorghum seedlings was partly reversed by silicon supplementation in the culture solution (Table 1). These results are in agreement with previous reports regarding the beneficial effects of silicon on the growth and yield of various cultivated plant species under salt stress conditions (Zhu et al. 2004; Romero-Aranda et al. 2006; Ashraf et al. 2010). This growth improvement in the presence of silicon was
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A deleterious effect of salt is an osmotic stress that causes increased external osmotic pressure, and an osmotic adjustment is an important mechanism in salt tolerance, which is a more rapid response than decreasing the Na? accumulation in the leaf (Munns and Tester 2008). An osmotic adjustment can help the plant to withstand osmotic stress and water deficit (Volkmar et al. 1997). In this study,
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supplementary silicon had no effect on Ww under salt stress, but it decreased the Wp more than no silicon treatment under salt stress (Fig. 2). Lower values of Wp indicate a higher osmotic adjustment capacity, and also a higher capacity for the tissues to take up and retain water. Meanwhile, supplementary silicon helped the plants to maintain a high Wp, which is what drives cell wall expansion and prevents the cell from wilting. Decreasing the Wp and increasing the Wp by addition of silicon under salt stress has also been found in tomato (Romero-Aranda et al. 2006). In addition to salt stress, similar results have also been reported in drought. Ming et al. (2012) showed that silicon increased the Wp in rice, and Sonobe et al. (2009, 2011) reported that silicon decreased both the Ww and the Wp in sorghum under drought stress conditions. These results suggest that improving the osmotic adjustment by supplementary silicon is a universal phenomenon under osmotic stress. Synthesis of organic osmolytes and accumulation in cells are general ways to decrease the cell Wp, help retain water, and improve the salt tolerance (Yang et al. 2007; Lee et al. 2008). Soluble sugar is considered to be one of the key osmolytes in the osmotic adjustment. In this study, silicon supplementation increased the soluble sugar content compared with no silicon treatment under salt conditions. A similar observation that silicon increased the total amount of soluble sugar was also found in wheat and sorghum under drought stress (Pei et al. 2010; Sonobe et al. 2011). We found that silicon improved the sucrose and fructose contents, but not glucose in sorghum under shortterm treatment (Table 2). Proline is another important osmolyte that contributes to osmotic adjustment, and its accumulation in response to osmotic stress has been widely reported (Ashraf and Foolad 2007). In the current study, an increased proline concentration in the sorghum leaf was observed under salt stress, but this increase was reversed by the application of silicon in the culture solution. The actual role of proline in osmotic tolerance remains controversial. In some studies, accumulation of proline under stress has been shown to be generally higher in stress-tolerant plants than in stresssensitive ones (Madan et al. 1995; Nayyar and Wali 2003). However, other research suggested that the accumulation of proline was a symptom of stress injury rather than an indication of stress tolerance (Lutts et al. 1999; De-Lacerda et al. 2003). Since proline biosynthesis is a highly energydemanding process, the reduced production of proline could benefit the plant by saving more energy for coping with stresses. Supplementary silicon alleviated the stress tolerance always accompanied by a decrease in proline levels (Tuna et al. 2008; Pei et al. 2010; Lee et al. 2010). Our results seem to support the view that proline accumulation under stress is a response to injury rather than an
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osmotic adjustment. Thus, supplementary silicon decreased the Wp through accumulation of soluble sugar, but not through accumulation of proline in sorghum under salt stress. Effect of silicon on ion accumulation in the leaf Under saline growth conditions, it has been reported that the beneficial effect of silicon is due to reduction of Na? content in the shoot of wheat, rice, alfalfa (Medicago sativa. L), sugarcane (Saccharum officinarum L.) and purslane (Portulaca oleracea L) (Gong et al. 2006; Wang and Han 2007; Tuna et al. 2008; Ashraf et al. 2010; Kafi and Rahimi 2011). If Na? uptake is reduced by silicon, it could act directly by reducing the bypass flow. Indirect effects are also possible, and any reduction in E could also reduce Na? uptake. In rice, Yeo et al. (1999) and Gong et al. (2006) demonstrated that silicon deposition in the root could reduce Na? uptake by reducing bypass flow. However, Matoh et al. (1986) suggested that the reduction of Na? uptake imposed by silicon in rice results from the restriction of transpiration because of silica deposition in the cell wall of the leaves. The present study showed that, in salt-stressed plants, supplementary silicon did not decrease the transpiration (Fig. 1), which indicates that the silicon-induced reduction in Na? uptake was not simply through a reduction in volume flow from root to shoot in sorghum. In addition, the concentration of Na? in the leaves did not increase with time when plants were exposed to salt, especially after 7 days, this may ascribe to the severe damage of salt treatment that already caused leaf death after 7 days (supplemental Fig. 1). With supplementation of silicon, the beneficial effects of silicon become obvious with time, and plant exhibited less Na? accumulation in their leaves. The effect of silicon on the K concentration varies in different studies and in different species under salt stress. Wang and Han (2007) showed that under salt stress, silicon increased the K? content in the shoot and leaves in salt tolerant alfalfa, but not in salt sensitive alfalfa. In wheat, K? content was increased with an increasing silicon concentration under salt stress (Tuna et al. 2008). In sugarcane, silicon alone enhanced the K? content with increasing silicon concentration, and while under salt stress, silicon enhanced the K? concentration just under high silicon concentration treatment (Ashraf et al. 2010). Gong et al. (2006) reported that silicon did not enhance the K? content in rice under salt stress. This is in agreement with our results that silicon had no effect on the K? concentration (Table 3). Silicon can also alleviate a K deficiency and enhance K? uptake under K deficient conditions (Miao et al. 2010). Therefore, we assumed that silicon-enhanced leaf K? concentrations are related to the K? and silicon
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concentration in the culture solution, it showed notably under low K? and high silicon conditions.
of ion contents and osmotic potential. Kiyoshi Tanaka helped in drafting the manuscript and interpretation of the results.
The integrated effects of silicon on the osmotic and ionic tolerance
Acknowledgments This study was supported by the National Natural Science Foundation of China (31101597), West Light Foundation of the Chinese Academy of Sciences, Chinese Universities Scientific Fund (Z109021202) and 111 project of Chinese Education Ministry (B12007).
Under salt stress, silicon increased both shoot and root growth, which had a positive effect on the whole plant. The gas exchange measurement revealed that the siliconinduced increase in total plant biomass under salt stress conditions was due to enhanced net CO2 assimilation rates, and similar results have been widely reported (Yeo et al. 1999; Savvas et al. 2009). Salinity may restrict net assimilation either through reduction of the CO2 supply arising from a partial closure of the stomata aperture, or through impairment of the biochemical mechanism of CO2 fixation, independent of altered CO2 diffusion rates, or by both procedures (Greenway and Munns 1980). Photosynthetic gas exchange is known to be highly sensitive to salt accumulation in the leaves (Yeo et al. 1985). Previous researches identified that silicon improved photosynthetic gas exchange through decreasing Na? accumulation in the leaves (Yeo et al. 1999; Savvas et al. 2009). In this study, silicon decreased leaf Na? concentration, and the net CO2 assimilation rate was increased by silicon. Although silicon did not affect the leaf Ww, it substantially decreased the Wp, and maintained a higher turgor potential, which is beneficial in keeping the gs high (Figs. 1, 2). Hence, it can be concluded that the increased PN by supplementation of silicon was attributed to both the decrease of Na? accumulation and the alleviation of osmotic stress. In the present study, different functions are involved in silicon-induced salt tolerance under short-term salt treatment, including enhancing osmotic adjustment and inhibiting Na? accumulation. Some of the responses are more rapid than expected, such as silicon significantly decreasing the osmotic potential after 24 h and inducing more soluble sugar accumulation. This study suggests that silicon induced the stress tolerance not only through structural activity of a physical barrier, but also through the involvement of a metabolic or physiological regulator. Therefore, although direct evidence is still lacking that silicon is a part of essential molecular constituent or metabolite in plant, further research should be conducted to determine the roles of silicon in plant metabolism. Furthermore, since this research is conducted in short time, more researches are needed to investigate the effect of silicon on improving the stress tolerance in sorghum under long-term growth. Author contribution Lina Yin conducted the experiment, collected and analyzed the data, and prepared the draft. Shiwen Wang planned the experiment and revised the manuscript. Jianye Li and Mariko Oka helped measurements
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