Study On The Regulatory Effect Of Exogenous EBR On Nicotine Content Of Flue-cured Tobacco In Low Temperature Areas During Mature Stage

In recent years,problems such as delayed tobacco leaf harvest,low nicotine content in the upper part of flue-cured tobacco,and decreased industrial availability were raised in some tobacco growing areas in China due to the continuous low temperature and rainy weather encountered during the late growth period of tobacco.2,4-epibrassinolide(EBR)is a widely used brassinolide plant growth regulator,which promotes root development and improves crops’ photosynthetic efficiency and stress resistance.In this study,to provide the basis for regulating nicotine metabolism and improving nicotine content and quality of tobacco leaves by exogenous EBR,the regulation effect of exogenous EBR application concentration and method on nicotine content in tobacco leaves was systematically studied in Sanmenxia tobacco-growing area with low temperature and rainy weather occurred during the mature period of flue-cured tobacco,and the effects of the EBR application method and EBR compound on growth and development,photosynthesis,low-temperature resistance,maturity and senescence characteristics,nitrogen and nicotine metabolism of tobacco plants during the mature period were explored.The main research results are as follows:The effect of EBR application concentration on the nicotine content of tobacco leaves in the areas where low temperatures frequently occur during the mature period of tobacco was studied.The results showed that compared with spraying water(CK1),the plant height,stem circumference,SPAD value,leaf area coefficient,field virus resistance,and total nitrogen and nicotine contents of cured tobacco leaves of Yunyan 87 could be increased in different degrees by spraying 250 mL/plant EBR solution with different concentrations(0.1 mg/L,0.2 mg/L and 0.4 mg/L)on the day of topping.Among them,the effect of spraying medium concentration of EBR(0.2 mg/L,0.05 mg/plant)was relatively good,and the nicotine content of middle and upper cured tobacco was increased by 11.11%and 19.90%,respectively,compared with CK1 treatment.The differences of photosynthetic characteristics,dry matter accumulation,enzyme activities and gene expressions related to nitrogen metabolism,and chemical composition of flue-cured tobacco were studied under two EBR application methods on the day of topping,including leaf-spraying and root-irrigation.Compared with spraying water(CK2),both leaf-spraying and root-irrigation(0.05 mg/plant)EBR could improve the photosynthetic capacity of tobacco plants during the mature period,increase the dry matter accumulation in each part of tobacco plants,and significantly increase the total nitrogen and nicotine contents of middle and upper cured tobacco leaves,and the effect of leaf-spraying treatment was better than that of root-irrigation treatment.The activity of the key enzymes NR and GS involved in nitrogen assimilation in the upper leaves of both application methods was significantly higher than that of CK2,and the relative expression levels of nitrogen assimilation-related genes NtGS2,NtGDH1,and NtNR in the leaf blades of the leaf spray treatment were significantly higher than those of CK2 and root irrigation treatment.The relative expression levels of NtQPT,NtPMT,NtJAT1,NtJAT2,and NtNUP1,which are involved in nicotine synthesis and transport,were extremely significantly higher in both application methods than in CK2,and the expression levels of nicotine synthesis and transport-related genes in the leaf blades of the foliar spray treatment were extremely significantly higher than those of CK2 and root irrigation treatment.Therefore,exogenous EBR can promote the growth and development of tobacco plants,improve nitrogen and nicotine metabolism levels,increase the nicotine content of middle and upper cured tobacco leaves,and the leaf-spraying treatment is more effective than root-irrigation.To further enhance the regulatory effect of exogenous EBR on nicotine content in flue-cured tobacco,the effects of single EBR spraying(C1 treatment),EBR compound C2 treatment(EBR+stabilizer α-Cyclodextrin and Tween 80+low concentrations of cold resistance agents CaCl2 and ZnSO4·7H2O+low concentrations of ripening agent KH2PO4),and EBR compound C3 treatment(EBR+stabilizerα-Cyclodextrin and Tween 80+high concentrations of cold resistance agents CaCl2 and ZnSO4·7H2O+high concentrations of ripening agent KH2PO4)on nicotine content in mature tobacco leaves were studied with an equal amount of water sprayed on the day of topping(CK3)as the control.Compared with CK3,spraying EBR solution and EBR compound can significantly enhance the photosynthetic capacity and root vitality of mature tobacco plants,increase the dry matter accumulation of various parts of tobacco plants,significantly increase the nicotine and potassium content of the middle and upper cured tobacco leaves,and the effect of spraying EBR compound on the leaf surface is better than that of single EBR spraying.Research on the mechanism of EBR compound regulator on the growth,development,and nicotine content of tobacco plants found that 30 to 50 d after spraying the compound,the expression levels of endogenous BR metabolism-related genes NtDWF4,NtBZR1,NtBZR2,and NtHERK2 in the upper leaves of C2 and C3 treatments were significantly higher than those of the single EBR treatment in C1,and the post-dried tobacco nicotine content was also significantly increased.In C3 treatment,the nicotine content of the middle and upper parts of the tobacco after drying was increased by 9.65%and 12.03%compared with C1 treatment,and the quality of the dried tobacco was significantly improved,indicating that EBR and stabilizers(α-Cyclodextrin and Tween 80)prolonged the efficacy of EBR,and the effect on the growth and development of tobacco plants lasted longer.After 30 days of spraying,the field temperature dropped sharply,and the average daily temperature within four days was 14.8℃.Research on the expression of cold-tolerance-related genes in tobacco leaves after treatment with the EBR compound showed that the expression levels of cold-tolerance-related genes NtCBF1,NtCBF2,NtCOR47,and NtCOR78 in the upper leaves of C2 and C3 treatments were significantly higher than those in C1 treatment,indicating that cold-resistant agents(CaCl2 and ZnSO4·7H2O)can significantly improve the resistance of tobacco plants under low-temperature conditions.A study on the chloroplast pigment content in the upper tobacco leaves during different treatments found that compared with CK3 and C1 treatments,C2 and C3 treatments have the effect of increasing the chloroplast pigment content and delaying leaf maturation and senescence.The chlorophyll content in the upper leaves of C3 treatment after 50 d of spraying was significantly lower than that of C2 treatment,indicating that the high concentration of KH2PO4 in C3 treatment has the effect of promoting leaf maturation and yellowing.In conclusion,the application of exogenous EBR with leaf-spraying on the top day of flue-cured tobacco can promote the growth and development of tobacco plants,improve the nitrogen and nicotine metabolism of tobacco plants,increase the nicotine content of middle and upper cured tobacco leaves,and improve the quality of tobacco leaves in tobacco planting areas where low temperature frequently occurs during the mature period of tobacco.Applying exogenous EBR combined with pesticide stabilizers,antifreeze agents,and high-concentration yellowing agents can further improve the application effect.