Silicon Improves Ion Homeostasis and Growth of Liquorice (Glycyrrhiza Uralensis Fisch. and Glycyrrhiza In ata Bat.) Under Salt Stress by Reducing Plant Na+ Uptake


 Silicon effectively alleviates the damage caused by salt stress in plants and can improve plant salt tolerance. However, the details of the mechanism by which silicon improves salt tolerance of liquorice are limited, and the effects of foliar application of silicon on different liquorice species under salt stress are not known. Here, the effects of foliar spray of silicon on the growth, physiological and biochemical characteristics, and ion balance of Glycyrrhiza uralensis Fisch. and Glycyrrhiza inflata Bat. were investigated. High salt stress resulted in the accumulation of a large amount of Na+, decreased photosynthetic pigment content, perturbed ion homeostasis, and eventually inhibited the both liquorice species growth. These effects were more pronounced in G. uralensis, as G. inflata is more salt tolerant than G. uralensis. Foliar spraying of silicon effectively reduced the decomposition of photosynthetic pigments, improved gas exchange parameters, and promoted photosynthesis. It also effectively inhibited lipid peroxidation and electrolyte leakage and enhanced osmotic adjustment of plants. Further, silicon application increased the K+ concentration, reduced Na+ absorption, transport and accumulation in the plants. The protective effects of silicon were more pronounced in G. uralensis than those in G. inflata. In conclusion, silicon reduces Na+ absorption, improves ion balance, and alleviates the negative effects of salt stress in the two liquorice species studied, but the effect is liquorice species-dependent. These findings may inform novel strategies for protecting liquorice plants against salt stress and also provide a theoretical basis for the evaluation of salt tolerance and the scientific cultivation of liquorice.


Introduction
Soil salinisation is the main factor restricting agricultural development worldwide 1 . It has been estimated that 20% of the total cultivated land and 33% of the irrigated agricultural land is affected by high salinity 2 . Ion toxicity and osmotic stress are the main manifestations of salt damage in plants 3 . Ion toxicity causes plant roots and shoots to accumulate many harmful ions, such as Na + , under salinity stress. When the ions enter the plant cell, they destroy the water and ion balance, damage the organelle structure, and inhibit plant growth, even leading to plant death 4 . According to some studies, ion toxicity caused by Na + is more likely to cause irreversible damage to plant than osmotic stress 5 . Further, in plants, an antagonistic relationship exists between Na + and K + , whereby high concentrations of Na + directly interfere with and inhibit the absorption of K + by the plasma membrane 6 . The importance of K + in plant growth is second only to that of nitrogen, as the activation of more than 60 plant enzymes depends on K +7 . Therefore, maintaining a high K + /Na + ratio under salt stress is essential for improving the salt tolerance of plants 1 .
Osmotic stress produces high levels of reactive oxygen species (ROS) under salt stress. Excessive ROS destroy macromolecules, lead to lipid peroxidation, eventually inhibit plant growth 8 . ROS clearance is mainly through enzymes and non-enzymatic antioxidant defence systems. Enzymes mainly include superoxide dismutase (SOD) and catalase (CAT), while non-enzymatic antioxidants mainly include the accumulation of compatible penetrants such as proline, soluble sugar, and soluble protein 9 .
Silicon, which is usually absorbed by higher plants in the form of H 4 SiO 4 10 , is the second most abundant element in the Earth's crust after oxygen 11 . While it is considered a non-essential element for the growth and development of higher plants 12 , many studies have shown that silicon plays an important role in alleviating biotic and abiotic stresses in plants [13][14][15][16] . Compared with other silicon application methods, foliar spray is the most effective [17][18] . Importantly, excessive application of silicon does not cause environmental pollution 10 .
Silicon accumulates in the cell wall of plants in the form of SiO 2 , which not only enhances the mechanical function of the cell wall, but also acts as a physical barrier to reduce water loss and improve the plant's ability to resist adverse environmental conditions [19][20] . Silicon also regulates polyamine metabolism, enhances H + -ATPase activity in the plasma membrane and the tonoplast, promotes Na + excretion from the cell, and reduces Na + translocation and damage to plants [21][22] . Further, this element improves the activity of antioxidant enzymes (such as SOD and CAT), reduces electrolyte leakage (EL) [23][24] , improves cell membrane stability, increases the water content of plant tissues, and improves photosynthetic function to promote plant growth 25  Xinjiang is a vast territory and the main producer of liquorice in China. However, 31.1% of the existing cultivated land therein is damaged by salinity, so that 18% of that land is severely salinized, and 33% shows medium-level salinisation, which is detrimental to the growth of liquorice 31 . According to recent studies, at salt concentration exceeding 100 mmol L -1 , the germination rate, germination potential, and germination index of G. uralensis seeds signi cantly decrease with a prolonged germination time. The plant height, chlorophyll content, and antioxidant enzyme activity of the seedlings are signi cantly reduced under these conditions, eventually leading to low biomass and low effective component accumulation in G. uralensis [32][33] .
The ameliorating effect of silicon application on plants under salt stress has been reported for many commercial crops, such as rice 34 , wheat 35 , sun ower 36 , sorghum 37 and maize 38 , but the mechanisms involved vary among plant species. Further, to date, the understanding of the mechanism by which silicon improves the salt tolerance of liquorice is limited, and it is not known whether the application of silicon differently affects different liquorice species. In the current study, we focused on G. uralensis and G. in ata in Xinjiang, and explored the effects of foliar spray of silicon on the growth, physiological and biochemical characteristics, and ion balance of liquorice under salt stress, to clarify the mechanisms whereby silicon alleviates salt stress in these two liquorice species. This study provides a theoretical basis for the evaluation of salt tolerance and the scienti c cultivation of liquorice.

Materials And Methods
Plant material and growth conditions. Experimental design.
The treatments were arranged in a completely randomised block design with three replicates. To explore the effects of leaf application of silicon on the two liquorice species under different salt concentrations, the following treatments were chosen for detailed analysis: (1) control (CK); (2) control + 3 mM silicon (CK + Si); (3) 6 g kg -1 NaCl (6S); (4) 6 g kg -1 NaCl + 3 mM silicon (6S + Si); (5) 12 g kg -1 NaCl (12S); and (6) 12 g kg -1 NaCl + 3 mM silicon (12S + Si). Silicon was applied as a foliar spray in the form of potassium silicate (K 2 SiO 3 ). To account for the effect of potassium ions, a corresponding amount of potassium chloride was added to the treatments without silicon.
Ten seeds of liquorice were sown per plastic pot (23.5 ´ 16 ´ 18 cm 3 ) containing 5 kg of sandy soil. After the emergence of 2 or 3 true leaves (15 d after sowing), six liquorice seedlings of the same height and growth, robust and disease-free were retained in each pot and were watered every day. NaCl and Si treatments were conducted simultaneously 4 weeks after sowing. Except for the treatments, other management measures were consistent with local eld management practices. After 100 d of treatment, the growth parameters and physiological and biochemical characteristics of each liquorice species were determined.
Determination of growth parameters of liquorice plants.
Plant height was measured using a tape measure, and stem thickness was measured using a Vernier calliper. The leaves, stems, and roots of liquorice were washed and placed in an oven at 105 °C for 30 min, dried at 75 °C for 46 h, and weighed.
Determination of gas exchange attributes.
Net photosynthesis (P n ), net transpiration rate (T r ), stomatal conductance (g s ), and intercellular CO 2 concentration (C i ) were recorded between 9.00 a.m., and 12.00 a.m. using a Li-6400 photosynthesis instrument (Li-COR, Lincoln, NE, USA). Three similarly sized healthy and fully expanded leaves from the top of the stem from plants under each treatment were analysed at a leaf temperature of 28 °C, irradiance of 1200 mmol m -2 s -1 , and CO 2 concentration of 400 mmol mol -1 .
Determination of chlorophyll content.
The veins of fresh leaves were removed, cut into pieces, and weighed. Then, 10 mL of 80% (v/v) acetone was added to 0.5 g of the plant material and extracted in the dark until the leaves were colourless. Next, the supernatant was obtained after centrifugation at 4000 r min −1 for 10 min. Then we added 4 mL of 80% acetone to 1 ml of supernatant, and the absorbance values of the extracts was determined at 470 nm, 646 nm, and 663 nm using a Shimadzu UV-1900 spectrophotometer (Kyoto, Japan). The contents of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were calculated by Lichtenthaler and Determination of antioxidant enzyme activities.
Fresh leaves (0.5 g) were added to phosphate buffer solution (pH 7.0), ground into a homogenate, and centrifuged at 10,000 r min -1 for 10 min. The supernatant was diluted to 25 mL using the same buffer solution. The samples were then stored in an ice-box for the determination of SOD and catalase CAT activities. All operations were performed at 0-4 °C. SOD activity was determined using the nitrogen blue tetrazole method 40 , and CAT activity was determined using the colorimetric method 41 .
Determination of soluble sugar, soluble protein, and proline contents.
For soluble sugar determinations, fresh leaves (0.2 g) were ground into a homogenate in 6 mL of distilled water, and then incubated in a water bath at 100 °C for 20 min. After cooling, the samples were centrifuged at 3000 r min -1 for 10 min. The extract (1.0 mL) was then mixed with 5 mL of anthrone reagent, and absorbance value at 620 nm was measured by a Shimadzu UV-1900 spectrophotometer (Kyoto, Japan). The soluble sugar content was then calculated using a standard curve of sucrose.
Soluble protein was determined using the Coomassie Brilliant Blue G-250 method 42 . Fresh leaves (0.2 g) were added to phosphate buffer solution (pH 7.0), ground into a homogenate, and centrifuged at 5,000 r min -1 for 10 min. Coomassie Brilliant Blue G-250 reagent was added to 1 mL of the supernatant and the absorbance was read at 595 nm. The protein content was calculated using a standard curve of bovine serum albumin.
For proline content determinations, fresh liquorice leaves (0.5 g) were placed in 5 mL of 3% sulfosalicylic acid solution, and centrifuged at 5000 r min -1 for 10 min. The supernatant (2 mL) was added to 2 mL of glacial acetic acid and 2 mL of acidic ninhydrin reagent, and the mixture was heated in a boiling water bath for 30 min. After cooling, 4 mL of toluene was added, the samples were shaken for 30 s, and centrifuged at 5000 r min -1 for 10 min. Using toluene as a blank control, sample absorbance was measured at 520 nm by a Shimadzu UV-1900 spectrophotometer (Kyoto, Japan), and the proline content was calculated using a standard curve 43 .
Determination of lipid peroxidation and EL. To determine EL, fresh liquorice leaves were cleaned, cut into 2-cm pieces, and placed in a test tube containing 10 mL of distilled water. The samples were shaken on an oscillating table at 25 °C for 24 h to determine electrical conductivity 1 (EC 1 ). The test tube was then placed in a water bath at 100 °C for 30 min to determine electrical conductivity 2 (EC 2 ) 45 . EL was calculated using the following formula: EL (%) = EC 1 /EC 2 ´ 100.
Determination of Na + and K + concentrations, transfer, and absorption.
Dry plant samples (0.1 g; various portions, as speci ed) were digested in a mixture of nitric acid and perchloric acid (volume ratio 2:1). The leaf and root concentrations of Na + and K + were measured using a ame photometer (FP640, Shanghai Precision Scienti c Instrument Co., Ltd., Shanghai, China). Sodium uptake at the liquorice root surface, and ion (Na + and K + ) translocation from the root to the shoot were calculated using the methods described by Yan, et al. 46 and Ali, et al. 38 , respectively.
Statistical analysis.
The data were analysed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to test the effect of different treatments on each index in the same liquorice species (P < 0.05), and Duncan's multiple comparisons test was used to determine signi cant differences between different treatments of the same liquorice. When the variances were not uniform, the data were converted to logarithm or square-root values before analysis. All gures were prepared using Origin 9 (OriginLab, Northampton, MA, USA).

Results
Plant growth.
Salt stress signi cantly inhibited the growth of the two liquorice species. The inhibitory effect on plant growth was more pronounced in G. uralensis than that in G. in ate. In particular, under the 12S treatment, the plant height, root dry weight, and shoot dry weight of G. uralensis decreased by 55%, 88%, and 84%, respectively, compared with the corresponding CK values; for G. in ata, these indicators decreased by 41%, 86%, and 83%, respectively, in the same comparison. In addition, under the 12S treatment, the plant height, root dry weight, and shoot dry weight of G. in ata were 57%, 39%, and 42% higher, respectively, than those of G. uralensis (Table 1). This inhibitory effect was alleviated by silicon (Table 1). Under the 12S + Si treatment, the plant height, root dry weight, and shoot dry weight of G. uralensis increased by 74%, 81%, and 74%, respectively, compared with the corresponding 12S values; for G. in ata, these indicators increased by 19%, 48%, and 49%, respectively, in the same comparison. Furthermore, the application of silicon with the CK treatment increased the growth indicators of the two liquorice species.
Gas exchange attributes.
The P n , g s , T r , and C i parameters of liquorice plants were generally negatively affected (Fig. 1). Under the 6S treatment, the P n , g s , and T r of G. uralensis were reduced by 54%, 64%, and 62%, respectively, compared with the corresponding CK values;for G. in ata, the corresponding parameters were reduced by 26%, 22%, and 21%, respectively, in the same comparison. Under the 12S treatment, the P n , g s , T r , and C i of G. in ata were decreased by 70%, 76%, 77%, and 20%, respectively, compared with the corresponding CK values; for G. uralensis, the corresponding parameters were also signi cantly decreased, except for C i , which was increased by 41% in the same comparison. In addition, the P n , g s , and T r of G. in ata were 148%, 277%, and 267% higher than those of G. uralensis under the 6S treatment, respectively. Nevertheless, compared with the 6S values, the 6S + Si treatment increased the P n , g s , T r , and C i of G.
Compared with the CK values, 12S treatment signi cantly reduced the chlorophyll content of the two liquorice species (Fig. 2). Under the 12S treatment, the chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents of G. uralensis were reduced by 65%, 53%, 63%, and 65%, respectively, compared with the corresponding CK values; for G. in ata, the corresponding parameters were reduced by 51%, 53%, 52%, and 57%, respectively, in the same comparison. Further, under the 12S treatment, the chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents of G. in ata were 44%, 22%, 40%, and 24% higher than those of G. uralensis, respectively. The application of silicon with the 12S treatment resulted in an increase in the chlorophyll a (49% and 30%), chlorophyll b (33% and 5%), total chlorophyll (46% and 26%), and carotenoid (66% and 50%) contents of G. uralensis and G. in ata, accordingly. Further, the application of silicon with the CK treatment also increased the chlorophyll content of the two liquorice species.
Antioxidant enzyme activities.
With the increasing salt concentration, the activities of SOD and CAT in G. in ata were signi cantly increased, while those in G. uralensis increased initially and then decreased (Fig. 3). Compared with the corresponding CK values, the SOD and CAT activity in G. uralensis increased by 28% and 128%, respectively, under the 6S treatment; the corresponding parameters of G. in ata increased by 9% and 62%, respectively, in the same comparison. Compared with the 6S values, the SOD and CAT activities in G. in ata increased by 14% and 64%, respectively, under the 12S treatment; the corresponding parameters in G. uralensis decreased by 12% and 22%, respectively, in the same comparison. In addition, under the 12S treatment, the SOD and CAT activities in G. in ata were 20% and 69% higher than those of G. uralensis, respectively. Compared with the salt treatment, the application of silicon signi cantly increased the SOD and CAT activities in the two liquorice species (P < 0.05).
Soluble sugar, soluble protein, and proline contents.
The soluble sugar, soluble protein, and proline contents in the leaves of G. uralensis rst increased and then decreased with an increasing salt concentration (Fig. 4). Compared with the corresponding CK values, the 6S treatment increased the soluble sugar content of G. uralensis and G. in ata by 101% and 61%, soluble protein content by 97% and 38%, and proline content by 107% and 99%, respectively. However, under the 12S treatment, the soluble sugar, soluble protein, and proline contents of G. uralensis decreased by 30%, 17%, and 20%, respectively, compared with the corresponding 6S values. In addition, under the 12S treatment, the soluble protein and proline contents of G. in ata were 47% and 29% higher than those of G. uralensis, respectively. Compared with the absence of silicon, application of silicon signi cantly reduced the proline content of the two liquorice species (P < 0.05), and increased the soluble sugar and soluble protein contents. Further, the application of silicon with the 12S treatment resulted in a 19% decrease of the proline content of G. uralensis, and a 53% and 20% increase in the soluble sugar and soluble protein contents, respectively, in this species.
Lipid peroxidation and EL.
Salt treatment signi cantly increased the MDA and EL of the two liquorice species (P < 0.05; Fig. 5).
Under the 6S and 12S treatments, the MDA of G. uralensis increased by 82% and 161%, respectively, and EL increased by 49% and 141%, respectively, compared with the corresponding CK values. For the same conditions, the MDA of G. in ata increased by 52% and 118%, respectively, and EL increased by 25% and 68%, respectively, compared with the corresponding CK values. Further, under the 12S treatment, the MDA and EL of G. uralensis were 36% and 48% higher than those of G. in ata, respectively. Furthermore, the application of silicon with the 12S treatment resulted in the reduction of the MDA of G. uralensis by 33% and that of EL by 16%; for G. in ata, the MDA was reduced by 19% and EL was reduced by 22%.
Accumulation of Na + and K + in the root and leaf.
The accumulation of Na + in the root and leaf of the two liquorice species increased signi cantly with an increasing salt concentration, with a simultaneous signi cant decrease in the accumulation of K + (Fig. 6).
Under the 12S treatment, Na + levels in the root and leaf of G. uralensis increased by 109% and 265%, respectively, while K + levels decreased by 31% and 58%, respectively, compared with the corresponding CK values. Under the same conditions, Na + levels in G. in ata increased by 181% and 246%, accordingly, and K + levels decreased by 37% and 37%, accordingly, compared with the corresponding CK values. In addition, under the 6S treatment, the Na + and K + levels in the root, and K + levels in the leaf of G. in ata were 44%, 27%, and 50% higher, respectively, than those in G. uralensis. However, without silicon application (the CK, 6S, and 12S treatments), the Na + accumulation in the leaf of G. uralensis was 49%, 35%, and 57% higher than that in G. in ata, respectively. The application of silicon with the 6S treatment signi cantly reduced Na + accumulation in the leaf of G. uralensis (27%), and increased K + accumulation in the leaf of this species (28%).
Ion translocation and uptake.
Salt treatment signi cantly increased Na + transport (root-leaf) and Na + uptake on the root surface and decreased the K + /Na + ratio in both liquorice species (Fig. 7). Under the 6S and 12S treatments, compared with the corresponding CK values, Na + transport in G. uralensis increased by 56% and 73%, respectively; Na + uptake on the root surface increased by 1.81-and 22-fold, respectively; K + transport (root-leaf) decreased by 30% and 40%, respectively; and the K + /Na + ratio decreased by 59% and 81%, respectively. In addition, the K + transport and K + /Na + ratio in G. in ata were 18% and 72% higher than those of G.
uralensis under the 6S treatment, respectively, while the Na + transport and Na + uptake on the root surface in G. uralensis were 95% and 85% higher than those of G. in ata, respectively. While the application of silicon decreased the Na + transport, and increased the K + transport and K + /Na + ratio, it only signi cantly affected the Na + uptake on the root surface under high salt concentration, decreasing the Na + uptake by 52% in G. uralensis and 46% in G. in ata.

Discussion
Salt stress is one of the main adverse environmental conditions faced by plants 1 . It inhibits plant growth and reduces plant yields, mainly because of ion toxicity and osmotic stress, and even leads to plant death 47 . According to many studies, silicon can be used to effectively alleviate the harmful effects of salt stress on plants and promote plant growth in saline environments 48-49 . In the current study, we analysed the growth indicators, gas exchange parameters, chlorophyll content, osmotic regulators, membrane damage indicators, antioxidant enzyme activity, and ion balance in two liquorice species. We showed that silicon application alleviated the negative effects of salt stress to a certain extent with the extent of the mitigating effect depending on the liquorice species. Although some investigations on the effect of silicon on the growth of liquorice under salt stress have been reported [50][51][52] , this is the rst report demonstrating that foliar spray with silicon enhances the salt tolerance of G. uralensis and G. in ata.
Both the medium-salt (6S) and high-salt (12S) treatments signi cantly reduced the biomass of the two liquorice species, with the lowest values obtained at high salt concentrations (Table 1). Under the same salt treatment, the growth of G. in ata was better than that of G. uralensis, indicating that G. in ata is more salt tolerant than G. uralensis. The increase in Na + under salt treatment hindered the photosynthesis by reducing photosynthetic pigment levels and further reducing the photosynthetic rate 17 , thereby inhibiting plant growth and reducing the biomass (Table 1). Consistent with the ndings for mung bean 8 and cucumber 53 , salt stress reduced chlorophyll concentration and carotenoid levels in the leaves of the two liquorice species (Fig. 2). In addition, under high salt stress, the P n , T r , and g s of the two liquorice species were also signi cantly reduced. However, the C i of G. uralensis was signi cantly increased under high salt stress, indicating that when the salt concentration exceeds the tolerance limit of a plant, the stomatal protection mechanism is damaged, and non-stomatal factors play a major role in photosynthesis 54 . Further, foliar spray with silicon signi cantly increased chlorophyll and carotenoid levels in plant leaves under salt stress 17,55 . These observations suggest that silicon promotes the biosynthesis of photosynthetic pigments under salt stress. This may be attributed to the notion that silicon alleviates the damage to chloroplast under salt stress 51 , enhances RuBisCo protein expression, and promotes the synthesis of chlorophyll and carotenoids 48 . Similarly, foliar spraying with silicon alleviated the negative effects of salt stress on gas exchange parameters in the two liquorice species (Fig. 1). The promoting effect was different for the different liquorice species. In the current study, the effect of silicon was more pronounced for G. uralensis (Fig. 2).
Under salt stress, the damage to plant cell and the decrease in photosynthesis are mainly caused by excessive absorption and accumulation of Na +17, 35 . Under high salt stress, the ability of the root system of G. uralensis to intercept Na + was signi cantly decreased, and the accumulation of Na + in the root and leaf increased, which led to disturbance in the synthesis of photosynthetic pigments 56 , a decrease in the net photosynthetic rate, and a signi cant reduction of plant biomass (Figs 1a, 2, and Table 1). This indicates that the ability of the root to intercept Na + was limited under these conditions. In high-salt environments, the accumulation of Na + was increased in roots and the transport ability of Na + was enhanced toward the shoot, thus increasing Na + accumulation in the leaf and damaging mesophyll cells 57 . When the Na + concentration in the leaf exceeds 1.3 mg g -1 , the chloroplast structure is destroyed, chlorophyll degradation accelerates, and photosynthesis is inhibited 14 . In addition, the transportation and absorption of Na + on the root surface of G. uralensis were 1.95 and 1.84 times higher than those in G.
in ata under medium-salinity conditions. This indicates that the ability of G. in ata roots to intercept Na + is greater than that of G. uralensis, which might account for the relatively high salt tolerance of G. in ata.
K + is the key regulator of cell homeostasis 7 and plays an important role in inducing cell elongation, maintaining osmotic regulation in plants, and promoting photosynthesis 58-59 . Therefore, excessive Na + levels often lead to K + de ciency. Al-Huqail, et al. 48 reported that high salt concentrations signi cantly increase the Na + content in maize and greatly reduce the K + content, resulting in an increased Na + /K + ratio and plant growth inhibition. In G. uralensis, compared with the application of silicon in the soil, foliar spray alone effectively increased K + levels and reduced Na + levels 50 . This may be associated with the upregulation of genes involved in potassium uptake (OsAKT1 and OsSHAK1) and xylem load (OsSKOR) by silicon 34 , which promote the increase of K + transport and increase the H + -ATPase activity, forming a mechanical barrier to reduce Na + transport 60 . Therefore, we believe that the capacity of silicon to enhance K + -selective transport and increase the K + /Na + ratio might be one of the main mechanisms to improve plant growth and productivity under salt stress, which is in agreement with the ndings of previous reports 61 . In addition, silicon not only reduced the Na + transfer and damage to the shoot under salt stress, but also decreased the Na + absorption by the root in the two liquorice species, which is consistent with the previous ndings in rice 62 and chickpea 63 . There are at least two possible explanations: (1) silicon deposition on the root cell wall enhances the mechanical strength of the cell wall, thus reducing Na + absorption 11 ; and (2) silicon decreases Na + accumulation in the root apex and cortex by upregulating the expression of ZmSOS1 and ZmSOS2 transporters 64 . Taken together, the reduction of Na + uptake and transport may be a potential mechanism of silicon-mediated enhancement of salt tolerance of liquorice.
In high-salt environments, plants accumulate high levels of Na + . This leads to excessive accumulation of ROS, perturbing the balance between their production and elimination, and resulting in cellular oxidative damage 8 . MDA and EL are the main indexes used to evaluate the severity of oxidative cell damage 62 . MDA is an oxidation product of membrane lipids and accumulates when plants are subjected to oxidative stress. Na + accumulation results in the production of high levels of ROS, which destroy the cell membrane structure, leading to an increased MDA content and EL. For instance, salt-induced oxidative damage leads to the rupturing of the plasma membrane in maize, leading to lipid peroxidation 38 . We here showed that both medium and high-salt treatments increased the MDA content and EL in the two liquorice species, especially in G. uralensis (Fig. 5), indicating major damage of the cell membrane in these liquorice species. Of the two species, the damage was more pronounced in G. uralensis. However, the MDA content and EL in the two liquorice species decreased after foliar spraying with silicon. These observations suggest that silicon may counteract the membrane damage caused by salt stress in liquorice. In addition, silicon can alleviate reactive oxygen damage by maintaining membrane integrity and activating the antioxidant defence system 65 . In the current study, silicon enhanced SOD and CAT activities in the two liquorice species under salt stress (Fig. 3), which was consistent with the observations in mung bean 8 and mustard 66 . This effect may be related to the upregulation of genes encoding antioxidant enzymes 67 . We also observed that silicon application enhanced the SOD and CAT activities in G. uralensis to a greater extent than those in G. in ata. Therefore, the regulation of the plant antioxidant system by silicon upon salt stress is different for the two different liquorice species.
The alleviating effect of silicon described above was not only associated with the increased antioxidant enzyme activity, but also with the accumulation of osmotic regulators in plants 48 . Accordingly, upon salt stress, plants produce and accumulate compatible organic solutes as a means of osmotic adjustment, to maintain the normal physiological and biochemical characteristics of intracellular water, and to impede the damage to the cell membrane 54,68 . By contrast, under high-salt stress, the soluble sugar, soluble protein, and proline contents in the leaf of G. uralensis were decreased, indicating that high-salt stress greatly affected the ability of G. uralensis to respond to the adverse conditions, and inhibited plant growth 50 . Proline is a key osmoprotectant and can reduce the damage caused by ROS, reduce lipid peroxidation, and protect protein and membrane structures 54 . In the current study, the application of silicon reduced the proline content in liquorice, which was consistent with the ndings in sun ower 36 and pelargonium 24 . In the presence of silicon, proline was degraded, and used as a source of carbon and nitrogen in plants recovering from stress, as well as a membrane stabiliser and a free-radical scavenger, to reduce lipid peroxidation and EL 15,18 . Therefore, reduction of membrane damage may be another mechanism whereby silicon improves the salt tolerance of plants. However, the mechanism for obtaining silicon to improve plant salt tolerance is a complex process. In addition to many physiological and biochemical aspects, further research is required on protein and gene expression analysis.

Conclusion
High salinity greatly inhibited the growth and development of the two liquorice species studied herein.
Under salt stress, the morphological characteristics (plant height, stem diameter, and biomass), photosynthetic characteristics (gas exchange parameters and photosynthetic pigments), antioxidant enzyme activities (SOD and CAT), ion homeostasis (K and Na transport, and K /Na ratio), and osmotic adjustment (e.g. proline) in G. in ata were better than those in G. uralensis. These observations indicate that G. in ata is more salt tolerant than G. uralensis. Foliar application of silicon effectively reduced the absorption of Na + , improved ion balance, alleviated membrane damage, and promoted the growth of the two liquorice species. Further, the response of G. uralensis to silicon was more pronounced than that of G. in ata, indicating that the protective effect of silicon is different for different liquorice species. This study provides a theoretical basis for the evaluation of salt tolerance in and scienti c arti cial cultivation of liquorice in the future. Effects of salinity and silicon on Na+ concentration in the root (a) and leaf (b), and K+ concentration in the root (c) and leaf (d) in the two liquorice species. Data are presented as the mean ± SE (n = 3). Different letters indicate signi cant differences between the values for one index for the same liquorice species under different treatments (P < 0.05)