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Abstract This study was conducted to comprehensively evaluate the effects of salicylic acid and sodium molybdate on cold tolerance of an ornamental plant Bougainvillea glabra and to provide a theoretical guidance for landscape maintenance. B. glabra plants were treated with 0.5 mmol/L salicylic acid and 2.0 μmol/L alone or in combination， and then exposed to low temperature stress before physiological indices were measured. The results showed that all salicylic acid and sodium molybdate treatments reduced the relative conductivity and malondialdehyde （MDA） content of B. glabra to varying extents under the stress of low temperature， and more significant effect was achieved by using the two agents in combination. Oxygen free radicals production rate increased with decreasing temperature from 20 to 6 ℃， but declined with temperature decreasing from 3 to -3 ℃. The SOD activity of the control （CK） was significantly lower than that of other treatments at 0 and -3 ℃. The treatments with salicylic acid and sodium molybdate alone and in combination increased POD activity of B. glabra plants， especially at 0 ℃， as the POD activity of treatments T1， T2 and T3 was significantly higher than that of CK at 0 ℃. In addition， under low temperature stress， the contents of soluble sugar， starch and proline increased initially and decreased subsequently with temperature decreasing. The soluble sugar content at 3 ℃， starch and proline contents at 0 and -3 ℃ in treatments with salicylic acid and sodium molybdate alone and in combination were significantly higher than those of CK. All above results proved that salicylic acid and sodium molybdate are able to improve cold tolerance of B. glabra， and better effect can be achieved by using them together.
Key words Bougainvillea glabra; Salicylic acid; Sodium molybdate; Low temperature stress; Cold tolerance; Physiological mechanism
Bougainvillea glabra Choisy is a plant in the family Nyctaginaceae， native to Brazil， and is now widely grown in the subtropical region of China. It is a common ornamental plant in southern China. Its flower can be used as a traditional Chinese medicine for promoting blood flow and regulating menstruation[1]. B. glabra has been largely introduced in subtropical region as a garden tree species in recent years for its beautiful， colorful flowers and evergreen leaves. However， the cold winter weather in subtropical regions of China causes huge damage to B. glabra. Therefore， it is an important issue to assay the cold tolerance and cold tolerance mechanism of B. glabra in these regions. Salicylic acid （SA） is a phenolic phytohormone that is synthesized in a wide range of plant species and has been reported to increase plant resistance[2]. Low temperature acclimation can increase the content of free salicylic acid in the leaves of cucumber seedlings by more than 2.5 times， indicating that there is a close relationship between salicylic acid level and cold tolerance of plants[3]. In recent years， studies have reported that salicylic acid improves the seedling cold tolerance of corn[4]， sugar cane[5]， anthurium[6] and other species. Molybdenum is one of the essential trace elements for plants， and its physiological function in plants is mainly achieved through molybdenum enzymes[7]. Appropriate molybdenum level can increase the antioxidant capacity of defense system， maintain the equilibrium between the production and scavenging of reactive oxygen species （ROS）， and thus improve the cold tolerance of winter wheat[8]. But whether salicylic acid and sodium molybdate used in combination can enhance the cold tolerance of B. glabra is still unknown， because they have different properties and mechanism of action. Therefore， in this study， B. glabra plants were treated with salicylic acid and sodium molybdate alone or in combination， and then exposed to low temperature stress， before relative conductivity， malondialdehyde （MDA） content， soluble sugar content， starch content， protein content， proline content， H2O2 content， oxygen free radical production rate， superoxide dismutase （SOD） activity and peroxidase （POD） activity were quantified to evaluate the cold resistance of the plants in each treatment. The data may provide a scientific basis for the introduction and cultivation of tropical tree species in subtropical areas.
Materials and Methods
Description of study area and experimental materials
The field trial was conducted from October to December 2014 in the campus of Kunming University in Kunming， Yunnan Province， characterized by a subtropical climate. The average annual precipitation is 1 031 mm， the average annual temperature is 14.5 ℃， the average temperature in the hottest month （July） is 19.7 ℃， and that in the coldest month （January） is 7.5 ℃. The soil is red clay loam， with a pH value of 5.2， saturated water content of 29.1% and water holding capacity of 38.3%. The contents of total nitrogen， available nitrogen， total phosphorus and available phosphorus in the soil are 31.4， 22.5， 12.4 and 4.21 mg/kg， respectively. The 7year old healthy， pestfree and similarsized B. glabra plants grown in the campus of Kunming University were selected as the experimental materials. They were pruned to a height of about 1.5 m. The crown of the trees was ballshaped， with a diameter of about 100 cm.
Experimental design and sample collection
The plants were assigned to each of the four treatments： 0.5 mmol/L salicylic acid （T1）， 2.0 μmol/L sodium molybdate （T2）， 0.5 mmol/L salicylic acid plus 2.0 μmol/L sodium molybdate （T3）， and deionized water （control， CK）. The concentration of the chemicals had been determined in previous experiments.
There were five plants in each treatment， and salicylic acid and/or sodium molybdate solution （300 ml） was sprayed evenly over the surface of all leaves of each plant at 5：00-6：00 pm every day， till leaf surfaces were fully wet and the solution began to drip. The treatments were repeated for five days. Seven days after the last spraying， the mature， robust， pestfree and similarsized branches were selected from each treatment， placed in an incubator at -3， 0， 3， 6 or 25 ℃ （control） for 12 h， before their physiological and biochemical indices were determined.
Measurement items and methods
The relative conductivity of the samples was determined using DDS11A conductivity meter. Soluble sugar content was quantified by phenolsulfuric acid assay. MDA content was determined by dual wavelength spectrophotometry. The content of free proline was quantified using ninhydrin method. The activity of superoxide dismutase （SOD） was measured using nitroblue tetrazolium （NBT） method. The content of H2O2 was determined after extraction with acetone[9]. Peroxidase （POD） activity was assayed using guaiacolH2O2 system. The rate of oxygen free radical （OFR） production was determined using the mixture of paminobenzenesulfonic acid and αnaphthylamine[10]. Every measurement was repeated three times.
Comprehensive evaluation of the effects of salicylic acid and sodium molybdate alone or in combination on cold tolerance of B. glabra
The membership value of each index in the four treatments was calculated， and the sum of the membership values of the indices in each treatment was calculated to reflect the cold resistance of the plants[11-12]. The parameters negatively correlated with cold tolerance include relative conductivity， MDA content， oxygen free radical production rate and H2O2 content， and their membership values were calculated with the formula as follows： X（μ）= 1- （X-Xmin）/（Xmax-Xmin）
The parameters positively correlated with cold tolerance include SOD activity， POD activity， soluble sugar content， starch content， protein content and free proline content. Their membership values were calculated with the formula as follows：
X（μ）=（X-Xmin）/（Xmax-Xmin）
In the formulas， X（μ） is the membership value of each parameter， Xmax is the maximum value of a parameter among all treatments， and Xmin is the minimum value of the parameter among all treatments.
Data processing
The average value of the three repetitions of each measurement was used in the final computation. All data were tested with Duncan’s new complex range （P<0.05） using SPSS 17.0， and histograms were plotted in Excel 2003.
Results and Analysis
Effects of salicylic acid and sodium molybdate on relative conductivity and MDA content in B. glabra
The treatments with salicylic acid and sodium molybdate alone and in combination had an impact on the relative conductivity of B. glabra at 3， 0 and -3 ℃. At 3 ℃， the relative conductivity in treatments T1 and CK was the highest， significantly higher than that in treatments T2 and T3. With temperature decreasing from 3 to 0 ℃， the relative conductivity of all these treatments slightly increased， and that of Treatment CK was the highest， followed by that of Treatment T1， and that of treatments T2 and T3 was the lowest， and differences were all statistically significant. The relative conductivity of all these treatment increased sharply with temperature decreasing from 0 to -3 ℃， and that of Treatment CK was significantly higher than that of Treatment T3 at -3 ℃.
At 25 ℃， the MDA content of Treatment T2 was higher than that of all other treatments， while that of Treatment CK was the lowest， and the difference was statistically significant between them， indicating that treatments with salicylic acid and/or sodium molybdate damaged the leaves of B. glabra. At 6 ℃， the MDA content of Treatment CK was significantly higher than that of all other treatments. At 0 ℃， the MDA content of Treatment T3 was significantly higher than that of T1 and CK， while the MDA content of CK was significantly lower than that of the other three treatments， indicating that mesophyll cells of Treatment CK were seriously injured. At -3 ℃， the MDA content of Treatment T1 was significantly higher than that of the other three treatments.
Effects of salicylic acid and sodium molybdate on oxygen free radical production rate and H2O2 content in B. glabra As shown in Fig. 2， the oxygen free radical production rate of treatments T2 and T3 was significantly higher than that of treatments T1 and CK at 25 ℃， indicating that sodium molybdate had damaged the leaves of B. glabra. The rate of oxygen free radical production of Treatment T1 was significantly lower than that of the other three treatments at 6 and 3 ℃. With temperature dropping from 3 to -3 ℃， the rate of oxygen free radical production in all treatments declined gradually.
At 25 ℃， Treatment T1 had the highest H2O2 level， followed by Treatment CK， and Treatment T2 had the lowest H2O2 level， and the difference in H2O2 level between them was significant. At 6 ℃， Treatment T1 had the highest H2O2 level， followed by Treatment CK， and treatments T2 and T3 had the lowest H2O2 level， with significant difference from each other. With temperature decreasing from 6 to 3 ℃， the H2O2 level of Treatment CK increased greatly to the maximum value， and that in Treatment T1 also increased， while the H2O2 level in treatments T2 and T3 was significantly lower than that of the other two treatments. At 0 ℃， the four treatments were T1>CK>T3>T2 in decreasing order of H2O2 level， with significant different from each other. At -3 ℃， the H2O2 content of Treatment CK dropped significantly， and was lower than that of treatments T1 and T3. The results showed that the leaves of Treatment CK were severely damaged at 0 and -3 ℃， resulting in a dramatic decrease in H2O2 level.
Yongfu ZHANG et al. Role and Mechanism of Action of Exogenous Salicylic Acid and Sodium Molybdate in Improving Cold Tolerance of Bougainvillea glabra
Effects of salicylic acid and sodium molybdate on SOD and POD activity in B. glabra
As can be seen in Fig. 3， the SOD activity of Treatment T3 was significantly higher than that of the other three treatments at 25 ℃， indicating that salicylic acid in combination with molybdic molybdate inhibited SOD activity of B. glabra at normal temperature. At 6 ℃， the SOD activity of Treatment T2 was significantly higher than that of Treatment T3. At 3 ℃， the SOD activity of Treatment T2 was significantly higher than that treatments of T1 and CK， and that of Treatment T1 was significantly lower than that of the other three treatments. At 0 ℃， the SOD activity of treatments T2 and T3 was significantly higher than that of treatments T1 and CK， while the SOD activity of Treatment CK was significantly lower than that of the other three treatments. At -3 ℃， the SOD activity of Treatment CK was significantly lower than that of the other three treatments. At 25 ℃， the POD activity of treatments T1 and CK was significantly higher than that of Treatment T2. At 6 ℃， the POD activity of Treatment T2 was significantly lower than that of Treatment CK， and the POD activity of Treatment T3 was significantly lower than that of Treatment T1. At 3 ℃， the difference in POD activity was not significant between these treatments. With temperature dropping to 0 ℃， the POD activity of Treatment T2 increased rapidly， while that of Treatment CK decreased significantly. With temperature dropping to -3 ℃， the POD activity of all treatments greatly reduced， indicating that the leaves in all these treatments were seriously damaged.
Effects of salicylic acid and sodium molybdate on soluble sugar and starch contents in B. glabra
As shown in Fig. 4， under low temperature stress， the soluble sugar content of all treatments increased to different degrees， indicating soluble sugar as osmatic regulator has certain effect on improving the cold tolerance of B. glabra. At 25 ℃， the soluble sugar content of all treatments was about 20 mg/g， showing no significant difference from each other. At 6 ℃， the soluble sugar content of treatments T1 and T2 increased by nearly100%， and was significantly higher than that of treatments T3 and CK. At 3 ℃， the soluble sugar content of treatments T1 and T2 slightly decreased， while that of treatments T3 and CK increased. At -3 ℃， the soluble sugar content of all treatments decreased， and that of T1 was significantly higher than that of T2 and CK.
Low temperature stress also had certain effect on the starch content of all treatments. At 25 ℃， the starch content of all treatments was about 30 mg/g， showing no significant difference from each other. When the temperature dropped to 6 ℃， the starch content of all treatments increased. At 3 ℃， T2 had the highest starch content and CK had the lowest， and the difference was significant. At 0 ℃， the starch content of CK was significantly lower than that of T1 and T3. At -3 ℃， the starch content of treatment T3 was significantly higher， and CK was significantly lower than other treatments.
Effects of salicylic acid and sodium molybdate on the contents of protein and free proline of B. glabra
As can be seen from Fig. 5， low temperature stress had little effect on protein content of each treatment. At 25 ℃， Treatment T2 had the highest protein content， and Treatment CK had the lowest， and the difference was statistically significant between them. At 6 ℃， the protein content of Treatment CK was significantly lower than that of treatments T2 and T3. There was no significant difference in protein content among these treatments at 3， 0 and -3 ℃. Low temperature stress led to an increase in proline content of B. glabra. At 25 ℃， the proline content of Treatment T3 was significantly lower than that of other treatments， indicating that the combined use of salicylic acid and sodium molybdate reduced proline content. At 6 ℃， the proline content of Treatment T3 was significantly higher than that of treatments T2 and CK. The proline content of Treatment CK was significantly lower than that of treatments T1 and T3 at 3 ℃， and was significantly lower than that of all other treatments at 0 and -3 ℃. The results proved that treatment with salicylic acid and sodium molybdate can significantly increase the proline content of B. glabra under low temperature stress and thus enhance its cold tolerance.
Comprehensive evaluation of the cold tolerance of B. glabra in the four treatments using membership functions
The cold tolerancerelated physiological and biochemical indices of B. glabra in these treatments responded differently to low temperature stress. A single index is not enough to completely reflect the cold tolerance of plants. Comprehensive analysis on all the indices revealed that the four treatments were T3>T1>T2>CK in decreasing order of cold tolerance （Table 1）. In addition， we also found that conductivity and proline content are closely related to cold tolerance， and thus they can be used as the indices to evaluate the cold tolerance of B. glabra plants treated by salicylic acid and sodium molybdate. Our results also showed that salicylic acid and sodium molybdate can improve the cold tolerance of B. glabra under low temperature stress， and better effect can be achieved by using them together.
Discussion
Under normal circumstances， the generation and scavenging of reactive oxygen species or free radicals in cells are in a dynamical equilibrium， so that they have no harm to the organisms. However， this equilibrium is destroyed under low temperature stress， and peroxidation produces new free radicals， leading to further membrane lipid peroxidation， which produces a large amount of MDA， and the integrity of the membrane is destroyed， resulting in tissue damage or death eventually[13]. These changes occur significantly earlier than those in external morphological structure， so membrane permeability can be used as an index to evaluate chilling injury[14]. Zhang et al.[15] found that the increase in relative conductivity of rice seedlings under low temperature stress was reduced after treatment with salicylic acid. Our results also indicated that salicylic acid and sodium molybdate can reduce the relative conductivity of B. glabra under low temperature stress， and better effect can be achieved by using them together. In addition， treatment with salicylic acid and sodium molybdate can also effectively reduce the MDA content of B. glabra under low temperature stress， and alleviate the injury caused by low temperature. Low temperature has different effects on the enzymes in plants[16]. POD synergize with SOD and catalase （CAT） to scavenge free radicals， and thus improve cold tolerance of plants[17]. However， when the temperature further decreases to a certain point， the cell membrane structure will be impaired， resulting in reduced enzyme activity. Zhang et al.[15] reported that exogenous salicylic acid increased POD activity under low temperature stress， which was contrary to the results of Ruffer et al.[18] and Durner et al.[19]， but similar to our results. Under low temperature stress， application of molybdenum can significantly increase the activity of antioxidant enzymes such as SOD， POD and CAT， and significantly reduce the rate of superoxide anion production in winter wheat leaves[8]. A suitable amount of molybdenum can improve the SOD activity in Turfgrass seashore[20]， broccoli[21] and tobacco[22]， and alleviate the damage caused by oxygen free radicals. The study of Wang et al.[23] showed that spraying salicylic acid reduced membrane lipid peroxidation and membrane structure damage in Trichosanthes kirilowii leaves. Our data showed that the oxygen radical production rate of all treatments increased at first with temperature decreasing from 25 to 6 ℃， and then declined with temperature decreasing from 3 to -3 ℃， indicating that the leaves of B. glabra were severely damaged when the temperature was below 3 ℃. Under low temperature stress， the H2O2 content of B. glabra plants treated with salicylic acid and sodium molybdate changed less than that of untreated ones， indicating that salicylic acid and sodium molybdate can be effective alleviate the damage caused by low temperature. All the treatments with salicylic acid and/or sodium molybdate had certain effects on the activity of SOD and POD of B. glabra under low temperature stress， and POD activity increased firstly and then decreased， which may be due to that the chemicals activate PODencoding gene at early stage， leading to an increase in its activity， and then with increasing damage to the membrane system over time， POD activity begins to decrease.
The accumulation of osmotic regulators， which is closely associated with chilling injury， is an adaptive response of plants to low temperature stress， and it can enhance the stability of cell membrane and the ability of cells to resist dehydration[24-25]. Therefore， it is generally considered that plants with higher level of osmotic regulators have higher cold tolerance. Zhang et al.[15] found that under low temperature stress， pretreatment with salicylic acid increased the contents of proline and soluble sugar in rice seedlings. The study of Yu et al.[26] showed that the combined use of boron and molybdenum promoted the accumulation of soluble sugar and free proline in seashore paspalum under low temperature stress， improved the stability and integrity of cell membrane structure， increasing its cold tolerance. Our results indicated that with temperature decreasing， the contents of soluble sugar， starch and free proline in all treatments increased at first and then decreased， while the content of protein changed little. The B. glabra plants treated with salicylic acid and sodium molybdate produced more osmotic regulators to protect themselves from the damage caused by low temperature. Salicylic acid can reduce osmotic potential by inducing the synthesis and accumulation of proline and soluble sugar， to improve the cold tolerance of B. glabra. The cold tolerance of woody plants is a quantitative trait that are controlled by multiple genes. Each trait related to cold tolerance has certain but small effect on plant cold tolerance[27]. It is necessary to evaluate the combined effect of the physiological indices in each treatment on cold tolerance. In the present study， we calculated the average membership value of the 10 physiological indices in each treatment， according to which the four treatments were T3>T1>T2>CK. B. glabra plants can be severely injured or even die at 0 to -3 ℃， however， salicylic acid in combination with sodium molybdate significantly improves the cold tolerance of B. glabra. The results may provide a scientific basis for the introduction and cultivation of tropical tree species in subtropical areas.
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Key words Bougainvillea glabra; Salicylic acid; Sodium molybdate; Low temperature stress; Cold tolerance; Physiological mechanism
Bougainvillea glabra Choisy is a plant in the family Nyctaginaceae， native to Brazil， and is now widely grown in the subtropical region of China. It is a common ornamental plant in southern China. Its flower can be used as a traditional Chinese medicine for promoting blood flow and regulating menstruation[1]. B. glabra has been largely introduced in subtropical region as a garden tree species in recent years for its beautiful， colorful flowers and evergreen leaves. However， the cold winter weather in subtropical regions of China causes huge damage to B. glabra. Therefore， it is an important issue to assay the cold tolerance and cold tolerance mechanism of B. glabra in these regions. Salicylic acid （SA） is a phenolic phytohormone that is synthesized in a wide range of plant species and has been reported to increase plant resistance[2]. Low temperature acclimation can increase the content of free salicylic acid in the leaves of cucumber seedlings by more than 2.5 times， indicating that there is a close relationship between salicylic acid level and cold tolerance of plants[3]. In recent years， studies have reported that salicylic acid improves the seedling cold tolerance of corn[4]， sugar cane[5]， anthurium[6] and other species. Molybdenum is one of the essential trace elements for plants， and its physiological function in plants is mainly achieved through molybdenum enzymes[7]. Appropriate molybdenum level can increase the antioxidant capacity of defense system， maintain the equilibrium between the production and scavenging of reactive oxygen species （ROS）， and thus improve the cold tolerance of winter wheat[8]. But whether salicylic acid and sodium molybdate used in combination can enhance the cold tolerance of B. glabra is still unknown， because they have different properties and mechanism of action. Therefore， in this study， B. glabra plants were treated with salicylic acid and sodium molybdate alone or in combination， and then exposed to low temperature stress， before relative conductivity， malondialdehyde （MDA） content， soluble sugar content， starch content， protein content， proline content， H2O2 content， oxygen free radical production rate， superoxide dismutase （SOD） activity and peroxidase （POD） activity were quantified to evaluate the cold resistance of the plants in each treatment. The data may provide a scientific basis for the introduction and cultivation of tropical tree species in subtropical areas.
Materials and Methods
Description of study area and experimental materials
The field trial was conducted from October to December 2014 in the campus of Kunming University in Kunming， Yunnan Province， characterized by a subtropical climate. The average annual precipitation is 1 031 mm， the average annual temperature is 14.5 ℃， the average temperature in the hottest month （July） is 19.7 ℃， and that in the coldest month （January） is 7.5 ℃. The soil is red clay loam， with a pH value of 5.2， saturated water content of 29.1% and water holding capacity of 38.3%. The contents of total nitrogen， available nitrogen， total phosphorus and available phosphorus in the soil are 31.4， 22.5， 12.4 and 4.21 mg/kg， respectively. The 7year old healthy， pestfree and similarsized B. glabra plants grown in the campus of Kunming University were selected as the experimental materials. They were pruned to a height of about 1.5 m. The crown of the trees was ballshaped， with a diameter of about 100 cm.
Experimental design and sample collection
The plants were assigned to each of the four treatments： 0.5 mmol/L salicylic acid （T1）， 2.0 μmol/L sodium molybdate （T2）， 0.5 mmol/L salicylic acid plus 2.0 μmol/L sodium molybdate （T3）， and deionized water （control， CK）. The concentration of the chemicals had been determined in previous experiments.
There were five plants in each treatment， and salicylic acid and/or sodium molybdate solution （300 ml） was sprayed evenly over the surface of all leaves of each plant at 5：00-6：00 pm every day， till leaf surfaces were fully wet and the solution began to drip. The treatments were repeated for five days. Seven days after the last spraying， the mature， robust， pestfree and similarsized branches were selected from each treatment， placed in an incubator at -3， 0， 3， 6 or 25 ℃ （control） for 12 h， before their physiological and biochemical indices were determined.
Measurement items and methods
The relative conductivity of the samples was determined using DDS11A conductivity meter. Soluble sugar content was quantified by phenolsulfuric acid assay. MDA content was determined by dual wavelength spectrophotometry. The content of free proline was quantified using ninhydrin method. The activity of superoxide dismutase （SOD） was measured using nitroblue tetrazolium （NBT） method. The content of H2O2 was determined after extraction with acetone[9]. Peroxidase （POD） activity was assayed using guaiacolH2O2 system. The rate of oxygen free radical （OFR） production was determined using the mixture of paminobenzenesulfonic acid and αnaphthylamine[10]. Every measurement was repeated three times.
Comprehensive evaluation of the effects of salicylic acid and sodium molybdate alone or in combination on cold tolerance of B. glabra
The membership value of each index in the four treatments was calculated， and the sum of the membership values of the indices in each treatment was calculated to reflect the cold resistance of the plants[11-12]. The parameters negatively correlated with cold tolerance include relative conductivity， MDA content， oxygen free radical production rate and H2O2 content， and their membership values were calculated with the formula as follows： X（μ）= 1- （X-Xmin）/（Xmax-Xmin）
The parameters positively correlated with cold tolerance include SOD activity， POD activity， soluble sugar content， starch content， protein content and free proline content. Their membership values were calculated with the formula as follows：
X（μ）=（X-Xmin）/（Xmax-Xmin）
In the formulas， X（μ） is the membership value of each parameter， Xmax is the maximum value of a parameter among all treatments， and Xmin is the minimum value of the parameter among all treatments.
Data processing
The average value of the three repetitions of each measurement was used in the final computation. All data were tested with Duncan’s new complex range （P<0.05） using SPSS 17.0， and histograms were plotted in Excel 2003.
Results and Analysis
Effects of salicylic acid and sodium molybdate on relative conductivity and MDA content in B. glabra
The treatments with salicylic acid and sodium molybdate alone and in combination had an impact on the relative conductivity of B. glabra at 3， 0 and -3 ℃. At 3 ℃， the relative conductivity in treatments T1 and CK was the highest， significantly higher than that in treatments T2 and T3. With temperature decreasing from 3 to 0 ℃， the relative conductivity of all these treatments slightly increased， and that of Treatment CK was the highest， followed by that of Treatment T1， and that of treatments T2 and T3 was the lowest， and differences were all statistically significant. The relative conductivity of all these treatment increased sharply with temperature decreasing from 0 to -3 ℃， and that of Treatment CK was significantly higher than that of Treatment T3 at -3 ℃.
At 25 ℃， the MDA content of Treatment T2 was higher than that of all other treatments， while that of Treatment CK was the lowest， and the difference was statistically significant between them， indicating that treatments with salicylic acid and/or sodium molybdate damaged the leaves of B. glabra. At 6 ℃， the MDA content of Treatment CK was significantly higher than that of all other treatments. At 0 ℃， the MDA content of Treatment T3 was significantly higher than that of T1 and CK， while the MDA content of CK was significantly lower than that of the other three treatments， indicating that mesophyll cells of Treatment CK were seriously injured. At -3 ℃， the MDA content of Treatment T1 was significantly higher than that of the other three treatments.
Effects of salicylic acid and sodium molybdate on oxygen free radical production rate and H2O2 content in B. glabra As shown in Fig. 2， the oxygen free radical production rate of treatments T2 and T3 was significantly higher than that of treatments T1 and CK at 25 ℃， indicating that sodium molybdate had damaged the leaves of B. glabra. The rate of oxygen free radical production of Treatment T1 was significantly lower than that of the other three treatments at 6 and 3 ℃. With temperature dropping from 3 to -3 ℃， the rate of oxygen free radical production in all treatments declined gradually.
At 25 ℃， Treatment T1 had the highest H2O2 level， followed by Treatment CK， and Treatment T2 had the lowest H2O2 level， and the difference in H2O2 level between them was significant. At 6 ℃， Treatment T1 had the highest H2O2 level， followed by Treatment CK， and treatments T2 and T3 had the lowest H2O2 level， with significant difference from each other. With temperature decreasing from 6 to 3 ℃， the H2O2 level of Treatment CK increased greatly to the maximum value， and that in Treatment T1 also increased， while the H2O2 level in treatments T2 and T3 was significantly lower than that of the other two treatments. At 0 ℃， the four treatments were T1>CK>T3>T2 in decreasing order of H2O2 level， with significant different from each other. At -3 ℃， the H2O2 content of Treatment CK dropped significantly， and was lower than that of treatments T1 and T3. The results showed that the leaves of Treatment CK were severely damaged at 0 and -3 ℃， resulting in a dramatic decrease in H2O2 level.
Yongfu ZHANG et al. Role and Mechanism of Action of Exogenous Salicylic Acid and Sodium Molybdate in Improving Cold Tolerance of Bougainvillea glabra
Effects of salicylic acid and sodium molybdate on SOD and POD activity in B. glabra
As can be seen in Fig. 3， the SOD activity of Treatment T3 was significantly higher than that of the other three treatments at 25 ℃， indicating that salicylic acid in combination with molybdic molybdate inhibited SOD activity of B. glabra at normal temperature. At 6 ℃， the SOD activity of Treatment T2 was significantly higher than that of Treatment T3. At 3 ℃， the SOD activity of Treatment T2 was significantly higher than that treatments of T1 and CK， and that of Treatment T1 was significantly lower than that of the other three treatments. At 0 ℃， the SOD activity of treatments T2 and T3 was significantly higher than that of treatments T1 and CK， while the SOD activity of Treatment CK was significantly lower than that of the other three treatments. At -3 ℃， the SOD activity of Treatment CK was significantly lower than that of the other three treatments. At 25 ℃， the POD activity of treatments T1 and CK was significantly higher than that of Treatment T2. At 6 ℃， the POD activity of Treatment T2 was significantly lower than that of Treatment CK， and the POD activity of Treatment T3 was significantly lower than that of Treatment T1. At 3 ℃， the difference in POD activity was not significant between these treatments. With temperature dropping to 0 ℃， the POD activity of Treatment T2 increased rapidly， while that of Treatment CK decreased significantly. With temperature dropping to -3 ℃， the POD activity of all treatments greatly reduced， indicating that the leaves in all these treatments were seriously damaged.
Effects of salicylic acid and sodium molybdate on soluble sugar and starch contents in B. glabra
As shown in Fig. 4， under low temperature stress， the soluble sugar content of all treatments increased to different degrees， indicating soluble sugar as osmatic regulator has certain effect on improving the cold tolerance of B. glabra. At 25 ℃， the soluble sugar content of all treatments was about 20 mg/g， showing no significant difference from each other. At 6 ℃， the soluble sugar content of treatments T1 and T2 increased by nearly100%， and was significantly higher than that of treatments T3 and CK. At 3 ℃， the soluble sugar content of treatments T1 and T2 slightly decreased， while that of treatments T3 and CK increased. At -3 ℃， the soluble sugar content of all treatments decreased， and that of T1 was significantly higher than that of T2 and CK.
Low temperature stress also had certain effect on the starch content of all treatments. At 25 ℃， the starch content of all treatments was about 30 mg/g， showing no significant difference from each other. When the temperature dropped to 6 ℃， the starch content of all treatments increased. At 3 ℃， T2 had the highest starch content and CK had the lowest， and the difference was significant. At 0 ℃， the starch content of CK was significantly lower than that of T1 and T3. At -3 ℃， the starch content of treatment T3 was significantly higher， and CK was significantly lower than other treatments.
Effects of salicylic acid and sodium molybdate on the contents of protein and free proline of B. glabra
As can be seen from Fig. 5， low temperature stress had little effect on protein content of each treatment. At 25 ℃， Treatment T2 had the highest protein content， and Treatment CK had the lowest， and the difference was statistically significant between them. At 6 ℃， the protein content of Treatment CK was significantly lower than that of treatments T2 and T3. There was no significant difference in protein content among these treatments at 3， 0 and -3 ℃. Low temperature stress led to an increase in proline content of B. glabra. At 25 ℃， the proline content of Treatment T3 was significantly lower than that of other treatments， indicating that the combined use of salicylic acid and sodium molybdate reduced proline content. At 6 ℃， the proline content of Treatment T3 was significantly higher than that of treatments T2 and CK. The proline content of Treatment CK was significantly lower than that of treatments T1 and T3 at 3 ℃， and was significantly lower than that of all other treatments at 0 and -3 ℃. The results proved that treatment with salicylic acid and sodium molybdate can significantly increase the proline content of B. glabra under low temperature stress and thus enhance its cold tolerance.
Comprehensive evaluation of the cold tolerance of B. glabra in the four treatments using membership functions
The cold tolerancerelated physiological and biochemical indices of B. glabra in these treatments responded differently to low temperature stress. A single index is not enough to completely reflect the cold tolerance of plants. Comprehensive analysis on all the indices revealed that the four treatments were T3>T1>T2>CK in decreasing order of cold tolerance （Table 1）. In addition， we also found that conductivity and proline content are closely related to cold tolerance， and thus they can be used as the indices to evaluate the cold tolerance of B. glabra plants treated by salicylic acid and sodium molybdate. Our results also showed that salicylic acid and sodium molybdate can improve the cold tolerance of B. glabra under low temperature stress， and better effect can be achieved by using them together.
Discussion
Under normal circumstances， the generation and scavenging of reactive oxygen species or free radicals in cells are in a dynamical equilibrium， so that they have no harm to the organisms. However， this equilibrium is destroyed under low temperature stress， and peroxidation produces new free radicals， leading to further membrane lipid peroxidation， which produces a large amount of MDA， and the integrity of the membrane is destroyed， resulting in tissue damage or death eventually[13]. These changes occur significantly earlier than those in external morphological structure， so membrane permeability can be used as an index to evaluate chilling injury[14]. Zhang et al.[15] found that the increase in relative conductivity of rice seedlings under low temperature stress was reduced after treatment with salicylic acid. Our results also indicated that salicylic acid and sodium molybdate can reduce the relative conductivity of B. glabra under low temperature stress， and better effect can be achieved by using them together. In addition， treatment with salicylic acid and sodium molybdate can also effectively reduce the MDA content of B. glabra under low temperature stress， and alleviate the injury caused by low temperature. Low temperature has different effects on the enzymes in plants[16]. POD synergize with SOD and catalase （CAT） to scavenge free radicals， and thus improve cold tolerance of plants[17]. However， when the temperature further decreases to a certain point， the cell membrane structure will be impaired， resulting in reduced enzyme activity. Zhang et al.[15] reported that exogenous salicylic acid increased POD activity under low temperature stress， which was contrary to the results of Ruffer et al.[18] and Durner et al.[19]， but similar to our results. Under low temperature stress， application of molybdenum can significantly increase the activity of antioxidant enzymes such as SOD， POD and CAT， and significantly reduce the rate of superoxide anion production in winter wheat leaves[8]. A suitable amount of molybdenum can improve the SOD activity in Turfgrass seashore[20]， broccoli[21] and tobacco[22]， and alleviate the damage caused by oxygen free radicals. The study of Wang et al.[23] showed that spraying salicylic acid reduced membrane lipid peroxidation and membrane structure damage in Trichosanthes kirilowii leaves. Our data showed that the oxygen radical production rate of all treatments increased at first with temperature decreasing from 25 to 6 ℃， and then declined with temperature decreasing from 3 to -3 ℃， indicating that the leaves of B. glabra were severely damaged when the temperature was below 3 ℃. Under low temperature stress， the H2O2 content of B. glabra plants treated with salicylic acid and sodium molybdate changed less than that of untreated ones， indicating that salicylic acid and sodium molybdate can be effective alleviate the damage caused by low temperature. All the treatments with salicylic acid and/or sodium molybdate had certain effects on the activity of SOD and POD of B. glabra under low temperature stress， and POD activity increased firstly and then decreased， which may be due to that the chemicals activate PODencoding gene at early stage， leading to an increase in its activity， and then with increasing damage to the membrane system over time， POD activity begins to decrease.
The accumulation of osmotic regulators， which is closely associated with chilling injury， is an adaptive response of plants to low temperature stress， and it can enhance the stability of cell membrane and the ability of cells to resist dehydration[24-25]. Therefore， it is generally considered that plants with higher level of osmotic regulators have higher cold tolerance. Zhang et al.[15] found that under low temperature stress， pretreatment with salicylic acid increased the contents of proline and soluble sugar in rice seedlings. The study of Yu et al.[26] showed that the combined use of boron and molybdenum promoted the accumulation of soluble sugar and free proline in seashore paspalum under low temperature stress， improved the stability and integrity of cell membrane structure， increasing its cold tolerance. Our results indicated that with temperature decreasing， the contents of soluble sugar， starch and free proline in all treatments increased at first and then decreased， while the content of protein changed little. The B. glabra plants treated with salicylic acid and sodium molybdate produced more osmotic regulators to protect themselves from the damage caused by low temperature. Salicylic acid can reduce osmotic potential by inducing the synthesis and accumulation of proline and soluble sugar， to improve the cold tolerance of B. glabra. The cold tolerance of woody plants is a quantitative trait that are controlled by multiple genes. Each trait related to cold tolerance has certain but small effect on plant cold tolerance[27]. It is necessary to evaluate the combined effect of the physiological indices in each treatment on cold tolerance. In the present study， we calculated the average membership value of the 10 physiological indices in each treatment， according to which the four treatments were T3>T1>T2>CK. B. glabra plants can be severely injured or even die at 0 to -3 ℃， however， salicylic acid in combination with sodium molybdate significantly improves the cold tolerance of B. glabra. The results may provide a scientific basis for the introduction and cultivation of tropical tree species in subtropical areas.
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