| In this study,the physiological and biochemical changes of tobacco leaves at 25℃（T0,normal temperature）,16℃（T1,mild low temperature）,10℃（T2,severe low temperature）and 4℃（T3,extremely severe low temperature）were studied with the Hong Da（with strong low temperature resistance）and K326（with weak low temperature resistance）which are in the prosperous long-term as test materials.In addition,tobacco grown at 4℃which was treated with exogenous glycine betaine（0,0.0125,0.05,0.1,0.15,0.2mol/L）and exogenous salicylic acid（0,10,25,50,100,150mg/L）at different concentrations.Next,we explored the stress mitigation mechanism of exogenous salicylic acid and glycine betaine on tobacco under low temperature stress at 4℃,and selected the optimal application concentration,so as to provide technical reference for the use of exogenous glycine betaine and salicylic acid to alleviate low temperature stress of tobacco.The main results are as follows:1.Physiological response of tobacco leaves under low temperature stress1)under low temperature stress,chlorophyll a,chlorophyll b,and chlorophyll a+b in tobacco leaves all decreased,and the degree of decrease increased with the increase of cold stress days and low temperature stress intensity.Among them,the decrease of chlorophyll in K326 cultivars with weak cold resistance was generally greater than that in Hong Da cultivars with strong cold resistance.Under low temperature stress,the chlorophyll a/b ratio of Hong Da was not significantly changed compared with CK in the group which was treated at 16℃.The chlorophyll a/b ratio in other treatments showed an upward trend,and the chlorophyll a/b ratio increased faster with the increase of stress days and the decrease of stress temperature.2)At 16℃and 10℃the relative electrical conductivity（REC）and malondialdehyde（MDA）of tobacco leaves were first increased and then decreased along with the increase of stress days,but continued to increase at 4℃.The higher the low temperature stress was,the higher the increase of leaf REC and MDA content was.Among them,the increase of REC and MDA content of K326 cultivar with weak cold resistance was generally higher than that of Hong Da cultivar with strong cold resistance.3)Low temperature stress effectively activated the protective enzyme system of tobacco leaves,and the activities of superoxide dismutase（SOD）,peroxidase（POD）and catalase（CAT）varied with the increase of stress days under different treatments.However,under the temperature stress of different gradients,the changes of the three protective enzyme activities were consistent,that is,the enzyme activity under the low temperature treatment at 10℃was generally higher than that under the low temperature treatment at 16℃,and the enzyme activity under the low temperature treatment at 4℃was the lowest.Enzyme activity was inhibited under 4℃low temperature treatment compared with 10℃low temperature treatment.4)At low temperature,the contents of soluble sugar（SS）and soluble protein（SP）in tobacco leaves were first increased and then decreased along with the increase of stress days.The accumulation rate of SS and SP contents in tobacco leaves under 10℃low temperature treatment was faster and increased more than that under 16℃low temperature treatment.When the low temperature intensity increased to 4℃,the response rate of SS and SP to the low temperature in the initial stage of stress was slower,and the maximum increase rate was smaller than that under 10℃low temperature treatment.Under the same low temperature treatment,the increase of osmotic substance content of low-temperature tolerant varieties was generally greater than that of low-temperature intolerant varieties.5)The activity of polyphenol oxidase（PPO）and phenylalanine ammonia-lyase（PAL）in tobacco leaves increased continuously with the increase of treatment days under the induction of low temperature.When the treatment temperature dropped from 16℃to 10℃,the peak value of PPO and PAL activity increased along with the decrease of temperature.When the temperature continued to drop to the treatment level of 4℃,the peak value of PPO and PAL activity in leaves was lower than that in the low-temperature treatment at 10℃,and the maximum activity of PPO and PAL was inhibited.At 10℃and 16℃,the lower the temperature was,the stronger the ability of the leaves to accumulate phenolic substances.However,the stress continued to increase to 4℃,and the ability of phenolic substances to accumulate was inhibited.6)The cold resistance of plants under each treatment was comprehensively analyzed by using the cold resistance membership function,and the results showed that:cold resistance T2>T1>T3 of two tobacco varieties.When the temperature dropped from 16℃to 10℃,the plants could reduce the external environment temperature and improve the cold resistance of the plants through self-metabolism regulation.However,under the low temperature stress of4℃,the self-regulation ability of the plants was limited compared with the treatment at 10℃.2.The mitigation effect of exogenous glycine betaine on tobacco under low temperature stress1)The appropriate concentration of exogenous glycine betaine（GB）can effectively improve the chlorophyll content,protective enzymes（SOD/POD/CAT）,osmotic regulators（SS/SP）and the activities of PPO and PAL,the key enzymes of polyphenol metabolism,in tobacco leaves under low temperature stress at 4℃,and the accumulation of phenolic substances is also improved.Hong Da and K326 were in 0.15 mol/L and 0.1 mol/L exogenous GB processing,the chlorophyll content reached the highest level;the protective enzyme activities of the two tobacco varieties were significantly improved when treated with0.0125mol/L～0.1mol/L exogenous GB solution;among the osmotic regulators,the optimal concentration of exogenous GB solution to improve SS content was 0.2mol/L for Hong Da and 0.1mol/L for K326,and the SP content of the two varieties reached the optimal level under the exogenous GB treatment of 0.0125mol/L;when the activity of PPO and PAL was maximized,the applied concentration of exogenous GB of Hong Da was 0.1mol/L,and that of K326 was 0.05mol/L and 0.2mol/L,respectively.In addition,the phenolic substance accumulation of Hong Da and K326 reached the maximum value under the exogenous GB treatment of 0.15mol/L and 0.1mol/L.2)The treatment of exogenous GB solution with appropriate concentration effectively reduced the relative conductivity（REC）and malondialdehyde（MDA）of tobacco leaves under low temperature stress at 4℃,and the application of 0.15mol/L exogenous GB solution maximally inhibited the rise of REC and MDA under low temperature stress.3)By using the cold resistance membership function,the cold resistance of the measured indexes was comprehensively analyzed,and the results showed that the optimal concentration of Hong Da and K326 for low temperature relief was 0.15mol/L and 0.1mol/L.3.Mitigation effect of exogenous salicylic acid on tobacco under low temperature stress1)the application of exogenous salicylic acid（SA）at an appropriate concentration to the root can positively promote the chlorophyll content,the activity of protective enzymes（SOD/POD/CAT）,the content of osmotic regulators（SS/SP）,and the contents of key polyphenol metabolism enzymes（PPO/PAL）and phenolic substances in tobacco under low temperature stress at 4℃.Under the exogenous SA treatment of 50mg/L,the content of chlorophyll a and chlorophyll a+b reached the highest level,and the optimal concentration of chlorophyll b was 10mg/L.Under the exogenous SA treatment,the chlorophyll a/b ratio was significantly lower than that of CK0.Under the action of 50mg/L exogenous SA solution,SOD activity of Hong Da and K326 was maximized.For the two tobacco varieties of Hong Da and K326,the optimal concentration of exogenous SA solution to improve POD activity was100mg/L and 150mg/L,respectively,and the optimal concentration of exogenous SA solution to improve CAT activity was 25mg/L and 50mg/L,respectively.The SS and SP contents in the leaves of the two tobacco varieties could be maximized under the action of exogenous SA of 50mg/L.For the two tobacco varieties,the use concentration of exogenous SA to enhance the activity of PPO and PAL was 100mg/L,and the polyphenol content reached the maximum value under the treatment of exogenous SA of 50mg/L.2)the appropriate concentration of exogenous SA solution effectively reduced the relative conductivity（REC）and malondialdehyde（MDA）content of tobacco leaves under low temperature stress at 4℃.Under the low temperature stress of 4℃,the REC and MDA of Hong Da could be reduced to the maximum extent under the action of exogenous SA with the concentration of 100mg/L and 25mg/L,respectively.However,for K326 variety,50mg/L exogenous SA can simultaneously minimize the content of REC and MDA under 4℃low temperature stress.3)the cold resistance was comprehensively analyzed by using the cold resistance membership function,and the results showed that the optimal concentration of exogenous SA solution of Hong Da and K326 to alleviate low temperature stress was 50mg/L. |
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