The role of silicon in physiology of the medicinal plant (Lonicera japonica L.) under salt stress

Silicon(Si) is the only element which can enhance the resistance to multiple stresses. However, the role of silicon in medicinal plants under salt stress is not yet understood. This experiment was conducted to study the effects of silicon addition on the growth, osmotic adjustments, photosynthetic characteristics, chloroplast ultrastructure and Chlorogenic acid (CGA) production of Honeysuckle plant (Lonicera japonica L.) under salt-stressed conditions. Salinity exerted an adverse effect on the plant fresh weight and dry weight, whilst 0.5 g L−1 K2SiO3·nH2O addition obviously improved the plant growth. Although Na+ concentration in plant organs was drastically increased with increasing salinity, higher levels of K+/Na+ ratio was obtained after K2SiO3·nH2O addition. Salinity stress induced the destruction of the chloroplast envelope; however, K2SiO3·nH2O addition counteracted the adverse effect by salinity on the structure of the photosynthetic apparatus. K2SiO3·nH2O addition also enhanced the activities of superoxide dismutase and catalase. To sum up, exogenous Si plays a key role in enhancing its resistance to salt stresses in physiological base, thereby improving the growth and CGA production of Honeysuckle plant.

imbalances or more usually, a combination of these factors 14 . Salinity influences photosynthetic capacity and its effects vary with the salt concentration, duration of stress and the cultivar used. Photosynthetic ability of some plant species reduced in the presence of salinity 15,16 , while that of other species reduced only at high stress and long term stress 17 .
Also, salinity is one of the major stresses responsible for changes in metabolic activity of plants. Plants have evolved several adaptive mechanisms to handle with the salinity in their environments, but the understanding of these mechanisms still remains incomplete. It has been shown that reactions to salt and drought stress might be responsible for the increase or decrease in the content of relevant natural products; however, scientific background in this field is still rare 18 . Medicinal plants under salt stress conditions accumulate higher concentrations of secondary compounds than control plants which are cultivated under standard conditions. Chlorogenic acid (CGA) is a type of polyphenol and one kind of secondary metabolites, which has anti-inflammatory, anti-oxidative and anticancer properties [19][20][21] . CGA could inhibit inflammatory cell infiltration, notably neutrophil recruitment into lung 22 and inhibit inflammatory cytokines release through suppressing nuclear factor kappa B (NF-kB) activation 23 . Keeping this in view, accumulation of CGA in the Honeysuckle plant under salt stress conditions was studied in this experiment.
Several investigators have reported that silicon enhanced the salt tolerance of wheat 24 , barley 25,26 , maize 27 and tomato (Solanum lycopersicum L. cv. Hong mei) 28 . However, its role in plant biology has been poorly understood and the attempts to associate Si with metabolic or physiological activities have been inconclusive 29 .
The objective of this study was to elucidate the salt-tolerant mechanisms of the Honeysukle plant on physiological bases, therefore revealing the role of Silicon in the amelioration of salt hazards and enhancement of secondary metabolite in the medicinal plant.

Materials and methods
Experimental layout. The experiment was conducted in a greenhouse at College of Resources and Environmental Sciences, Nanjing Agricultural University, during September 14 to October 25, 2013. Honeysuckle (Lonicera japonica L.) seedlings were obtained from BoZhou city, AnHui Province. Nearly 30-day-old nursery seedlings were transplanted into plastic pots (24 cm in diameter, 18 cm in depth), one seedling per pot. The pots were filled with clean sand and each pot was watered with 1.5 L Hoagland Solution. The experiment was arranged in a completely randomized design (CRD) and each treatment had three replications.
Plants were subjected to salt stress by adding sodium chloride (NaCl) solutions, with the calculated amount of NaCl and K 2 SiO 3 ·nH 2 O dissolved in Hoagland Solution. The experiment consisted of three treatments involving varying salinity levels and three levels of K 2 SiO 3 ·nH 2 O in sand (Table 1).To minimize salt shock, NaCl concentration was raised stepwise in aliquots of 50 mM every other day until the final salinity levels were achieved. The greenhouse temperature and relative humidity varied 25-30 °C and 50-80%, respectively for the entire growth period. Photosynthesis is determined at the 30 th day after salt treatment, respectively. Plants were sampled (the whole plant) at the 40 th day after salt treatment to assess the growth, osmotic adjustments, chloroplast ultrastructrue and chlorogenic acid (CGA) accumulation. After washing and drying, leaves were frozen in liquid N 2 and stored at − 80 °C until biochemical analysis.
Growth status analysis. FW is the fresh weight, and DW is the dry weight after oven-drying samples at 70 °C until constant weight. Elemental analysis. Leaves, stems and roots were separated and washed with ultra-pure water to eliminate salts in the surface of these samples. Subsequently they were dried at 70 °C until constant  weight and grinded in a stoneware mortar, from which were taken 100 mg for acid digestion. This procedure was carried out in Teflon reactors using an acid mixture of HNO 3 :HClO 4 (3:1, v/v) during 2 h at 280 °C until the liquid was almost transparent. After cooling overnight, the digestion products were diluted with distilled water to a total volume of 100 ml. Elemental concentrations in the extracts were determined by ICP-AES (Inductively Coupled Plasma Atomic Emission Spectrometry, Optima 2100DV, Perkin Elmer, USA). Si was determined following the method of Nayar et al. 30 .
Transmission electron microscope assay. The fifth fully expanded leaves, numbered basipetally, were sampled for ultramicroscopic observation on day 30 after the onset of the final concentration of salt treatment. For transmission electron microscope, fresh leaves were cut into 2 segments of 1 mm each and placed immediately in a freshly prepared the stationary liquid of 2.5% (w/v) glutaraldehyde in 50 mM sodium phosphate buffer (PH 7.4). The segments were then de-gassed and fixed under vacuum for 4 h at room temperature. After washing in the same buffer, they were post-fixed in 1% (w/v) osmium tetroxide (in the same buffer) for 1 h. After fixation, the samples were rinsed three with the same buffer for 15 min. Rapid dehydration was accomplished by placing the sample in 50% ethanol for 30 min, followed by changes in 70%, 90%, 95%, 100% ethanol (30 min each). The specimens were then placed in low-viscosity epoxy resin. UL-trathin sections (80 nm) were mounted on uncoated nickel grids (300 meshes) and sequentially stained with uranyl acetate and lead citrate before being examined at 80 kV under a transmission electron microscope (H-7650, JEOL, Japan).
The determination of leaf photosynthesis. The tested photosynthetic parameters included photosynthetic rate (A), Transpiration rate (E), stomatal conductance (gs) and Intercellular CO 2 concentration (Ci). They were determined by portable photosynthesis system Li-6400XT (Li-cor Inc., Lincolin, NE, USA). The PAR was provided by a red/blue light-emitting diode (model Li 6400-02B; Li-cor Inc., NE, USA) and was set at 400 or 700 umol photons m-2s-1 to match the PAR used when exposing plants to control or high light conditions, respectively. The atmospheric CO 2 concentration was controlled with a CO 2 injection system controlled by the Li-6400 (Li-cor Inc., Lincolin, NE, USA). The cuvette temperature was maintained at 25 °C with a relative humidity of 65%.
Pre-segmentation of CGA. Leaves washed with ultra-pure water to eliminate salts in the surface of these samples. Subsequently were dried at 70 °C until constant weight and grinded in a stoneware mortar, from which were taken 1.00 g for pre-segmentation. 1.0 g of leaf tissue of both control and stressed plants was homogenized in 14 mL 60% (v/v) ethylalcohol, and the pH of homogenate was 4.0. Homogenate was extracted in an ultrasonic cleaner (Xokeji, NJ, CHN) 15 min, then leached by Buchner funnel using a 0.45 um filter membrane. Estimation of CGA in the Honeysuckle plant (Lonicera japonica L.) was done using HPLC. Chromatography was done in RP 18 columns with a flow rate of 1.0 mL min −1 using 1% phosphate buffer in HPLC acetonitrile (87:13) as carrier solvent. The above step was repeated for three times, and then collected and amalgamated the filter liquor for HPLC analysis. The sample load was 20 uL. Column temperature was maintained at 25 °C and absorbance was measured at 327 nm.

Measurement of Antioxidant enzyme activities. The activity of SOD was determined following
Giannopolitis and Ries 31 by measuring its ability to inhibit the photoreduction of nitroblue tetrazolium (NBT). Activities of catalase (CAT) and peroxidase (POD) were appraised using the method of Chance and Maehly 32 with some modification. The CAT reaction solution (3 ml) contained 50 mM phosphate buffer (PH 7.0), 5.9 mM H2O2, and 50 ul enzyme extract. Then the reaction was initiated by adding the enzyme extract. Changes in absorbance of the reaction solution at 240 nm were read every 40 s. One unit CAT activity was defined as an absorbance change of 0.01 units per min. The POD reaction solution (3 ml) contained 50 mM phosphate buffer (PH 6.0), 20 mM guaiacol, 40 mM H2O2, and 30 ul enzyme extract. Changes in absorbance of the reaction solution at 470 nm were determined after every 15 s. The activity of each enzyme was expressed on protein basis. Statistical analysis. Data were statistically analyzed using analysis of variance (ANOVA) by SPSS v.20 for Windows, and presented as treatment mean ± SE of three measurements. Tukey's multiple range tests were calculated for the significant data at P < 0.05.

Results
Plant growth status. The effect of K 2 SiO 3 ·nH 2 O addition on the fresh weight and dry weight of Honeysuckle plant (Lonicera japonica L.) was shown in Table 2. The highest value of the fresh weight and dry weight for no salt treatments was obtained in 0.5 g L −1 K 2 SiO 3 ·nH 2 O addition treatments, implying that the optimum dose of K 2 SiO 3 ·nH 2 O addition (0.5 g L −1 K 2 SiO 3 ·nH 2 O) did enhance the plant growth. The same trends, however, were observed in 0.5 g L −1 K 2 SiO 3 ·nH 2 O × 100 mM NaCl treatment and only for plant fresh weight. Additionally, higher concentration of K 2 SiO 3 ·nH 2 O addition (1.0 g L −1 ) exerted no effect on the plant fresh weight and dry weight. As a whole, NaCl concentration of < 200 mM did not affect the plant growth, suggesting the tested variety of Honeysuckle itself was a more salt-tolerant Scientific RepoRts | 5:12696 | DOi: 10.1038/srep12696 one. These results implied that K 2 SiO 3 ·nH 2 O addition with 0.5 mg L −1 promoted the growth of the Honeysuckle, therefore increasing salt tolerance of the plant to a certain extent.
Osmotic adjustments. Salt stress significantly affected the ionic uptake and distribution in the plant organs (Table 3). Na + concentration in leaves, stems and roots was significantly increased under salt-stressed conditions as compared with the control. However, this increment in Na + concentration was partially counteracted in 100 mM NaCl treatment by 0.5 mg L −1 K 2 SiO 3 ·nH 2 O application. Additionally, an interesting phenomenon was investigated that Na + concentration in plant organs was much lower than K + concentration, indicating that selective absorption of K + over Na + (high S K+/Na+ ) could be one of the mechanism for this plant adapting to salt stress. Salt stress also caused the declined Ca 2+ uptake by plant; whereas 1 g L −1 K 2 SiO 3 ·nH 2 O application increased the Ca 2+ concentration to a high level of approximately equal to that in the control treatment. Thus, it was concluded that K 2 SiO 3 ·nH 2 O application played an important role in osmotic adjustments, subsequently enhancing the salt tolerance of the Honeysuckle plant.
Chloroplast ultrastructure. There were no significant differences in ultrastructure of chloroplasts between the control and control plus remission of K 2 SiO 3 ·nH 2 O (Fig. 1). Salt stress induced alterations in the structure of chloroplasts as compared to the control. The chloroplasts in salt-stressed plants were separated from the plasmamembrane, where chloroplasts were in closely contact with the membrane. The envelope membrane in the NaCl treated samples appeared to be ruptured. However, exogenous remission of K 2 SiO 3 ·nH 2 O alleviated the structural changes of chloroplasts induced by salt stress. Remission of K 2 SiO 3 ·nH 2 O maintained a well-preserved internal lamellar system in the chloroplasts of salt-stressed leaves and the chloroplasts contained less osmiophilic plastoglobuli. These results suggested that K 2 SiO 3 ·nH 2 O application helped to maintain the integrity of chloroplast ultrastructrue, thus executed a normal physiological functions of the plant exposed to salt stress.

Plant photosynthesis.
The changes of photosynthetic characteristics in leaf Honeysuckle were shown in Table 4. Salt stress markedly reduced the photosynthetic rate in Honeysuckle leaves, whilst K 2 SiO 3 ·nH 2 O application with the concentration of 1.0 g L −1 unexpectedly increased the photosynthetic rate. Similarly, the transpiration rate and stomatal conductance were significantly decreased with increasing NaCl concentrations no matter what K 2 SiO 3 ·nH 2 O was applied or not. However, 0.5 g L −1 K 2 SiO 3 ·nH 2 O addition appeared no effect on the plant photosynthesis, transpiration rate and stomatal conductance, but even inhibited them in a great deal.
Secondary metabolite of CGA. CGA content in leaf Honeysuckle increased significantly with increasing salinities, as compared with the control (Fig. 2). With exogenous application of K 2 SiO 3 ·nH 2 O, CGA content was gradually decreased, suggesting that CGA production in the plant leaves was hindered although K 2 SiO 3 ·nH 2 O application alleviated plant injury induced by salt stress. Moreover, no significant difference was observed in CGA content of leaf Honeysuckle among all salt-stressed treatments when 1.0 g L −1 K 2 SiO 3 ·nH 2 O was applied. In this experiment, the representative chromatogram with retention time for CGA was 7 min (Fig. 3).

Discussion
Silicon has been extensively shown to increase crop yield and stress tolerance 33 . In this study, we found that K 2 SiO 3 ·nH 2 O addition resulted in an improvement in the growth of salt-stressed Honeysuckle (Lonicera japonica L.) seedlings. These findings were comparable with observations in other crops, for example sorghum studied by Yin 34 , in which the application of silicon alone had no effects upon sorghum growth, while it partly reversed the salt-induced reduction in plant growth. In fact, silicon application resulted in the improvement of growth in many plants exposed to salt stresses that is often associated with many aspects, such as salt concentration, plant salt tolerance in addition to the plant species or genotypes. For rice plant, added Si can increase rigidity of the mature leaves, which have a rougher texture and are held more horizontally, delays leaf senescence and increases chlorophyll content and ribulose-bisphosphate carboxylase activity 35 . In this experiment, it was evident that Si uptake by the plant increased with increasing concentration of exogenous K 2 SiO 3 ·nH 2 O, while inhibited by increasing salinity of hydroponic solution, suggesting Si may be involved in the metabolic or physiological activity in Honeysuckle exposed to salt stress (Table 5).
Maintaining structural integrity and orderliness of chloroplast is necessary in the conversion of light energy for photosynthesis. It was reported that many stressors led to the decrease in the photochemical efficiency and electron transport activity that might be associated with the changes of the structure of photosynthetic apparatus 36 . In the present study, we observed that exogenous Si decreased the separation of plasma membrane from plasmolysis. Moreover, it commendably maintains the integrity of the Na + Concentration ((mg g −1 dwt) Salt levels (mM of NaCl)  Table 3. Effect of K 2 SiO 3 ·nH 2 O addition on the ion distribution within Honeysuckle (Lonicera japonica L.) plant under NaCl-stressed conditions. The values represent the means of three replicates. The means followed by the same letters (lower-case letters within lines and upper-case letters within columns, respectively) did not differ significantly at 5% probability by Tukey's test.
plasma membrane. These results demonstrated that silicon was involved in protecting the photosynthetic apparatus. Our results also showed that photosynthetic activity in Honeysuckle (Lonicera japonica L.) was affected by salinity, and the extent of the reduction was dependent on the salt strength. Zollinger 37 found a similar reduction in photosynthetic rate and stomatal conductance in E. purpurea irrigated with increasing concentrations of salinity up to 5 gL −1 . Reduction in photosynthetic rate under salinity can be attributed to both stomatal and non stomatal limitations 38 . In this study, we found a strong negative correlation between stomatal conductance and Na + concentrations both in the aboveground parts and roots, which indicates that plants tend to close their stomatal conductance as a result of specific-ion accumulations. This reduction in stomatal conductance led subsequently to a reduction in photosynthetic rate as indicated by the high correlation between stomatal conductance and photosynthetic rate. Gong et al. 39 reported that silicon did not enhance the K + content of rice under salt stress. This is in agreement with our results that silicon had no effect on the K + concentration. It is well known that high concentrations of Na + can inhibit K + and Ca 2+ uptake through antagonism between these ions 40 . This fact can be attributed to their physicochemical similarity, which promotes ionic competition for binding sites on membrane transporters 41 . Further studies on antioxidant systems showed that the activities of superoxide dismutase, and catalase were continuously enhanced with increasing salinity, thereby protecting the Honeysuckle plants from oxidative injury (Table 6). Moreover, 0.5 g L −1 K 2 SiO 3 ·nH 2 O addition did increase the activities of superoxide dismutase and catalase, demonstrating exogenous Si played an important role in these two enzymes. The data recorded elevated CGA content under both non-silicon kinds of salinity when compared to the with-silicon saline conditions plants suggesting their probable role in the secondary metabolite of CGA. Many hypotheses suggest that the synthesis of secondary metabolites, including saponins, a large diverse group of secondary metabolites defined as amphipathic glycosides in which glycosyl residues are attached to tritrtpenoid (triterpeneor or teroidal) aglycon 42 , is a plant response to environmental factors and part of an adaptative strategy leading to tolerance to abiotic stresses. Just showed as our results, the content of secondary metabolite of CGA is highest at 200 Mm NaCl level without any remission of silicon, indicating that fluctuations in the content of CGA resulting from salinity changes in environmental circumstances might be expected. In fact, the level of CGA like other plant secondary metabolites, can be significantly influenced by the physiological status of the plant as well as the external factors, both abiotic and biotic, including temperature, availability of water and interactions with pathogens and parasites. However, CGA biosynthesis and accumulation by plant tissues in response to saline conditions in plant habitat are still not clear, so the study on Honeysuckle plant (Lonicera japonica L.) might have considerable merit in it.
In this paper, the salt-tolerant mechanisms of the Honeysukle plant on physiological and biochemical bases have been elucidated, which is well consistent with the study aims. The role of silicon in genomics and proteome of the medicinal plant (Lonicera japonica L.) under salt stress will be studied further in future.
A (umol CO 2 m −2 s −1 ) photosynthetic rate Salt levels (mM of NaCl)  Table 4. Photosynthetic rate (A), transpiration rate (E) and stomatal conductance (Gs) of Honeysuckle (Lonicera japonica L.) exposed to nine treatments. The values represent the means of three replicates. The means followed by the same letters (lower-case letters within lines and upper-case letters within columns, respectively) did not differ significantly at 5% probability by Tukey's test.

Conclusions
Plant salt tolerance is a multifaceted physiological trait. In line with physiological observations, medicinal Honeysuckle plant (Lonicera japonica L.) adapted to salt stress under K 2 SiO 3 ·nH 2 O addition is mainly  relied on ionic osmotic adjustment, improvement of superoxide dismutase and catalase activities and stability of chloroplasts ultrastructures. These results clearly highlighted the role of Si in protecting the plant against the hazardous effect of salinity. Also, this study provided the evidence that high salt stress affected the entire quantity of secondary metabolite of CGA produced. Thus, regulation of salinity through K 2 SiO 3 ·nH 2 O addition could be a promising way to obtain better growth and considerable secondary metabolite of the medicinal plant.  Table 6. Effect of K 2 SiO 3 ·nH 2 O addition on superoxide dismutase (SOD), peroxide (POD), and catalase (CAT) contents of Honeysuckle (Lonicera japonica L.) plant under NaCl-stressed conditions. The values represent the means of three replicates. The means followed by the same letters (lower-case letters within lines and upper-case letters within columns, respectively) did not differ significantly at 5% probability by Tukey's test.