Preliminary Study On Identification And Mechanism Of Tea Plant Cold Resistant Agents

As a healthy drink,tea contains many beneficial ingredients.At the same time,tea plants are also an important economic crop,and tea industry plays an important role in the development of agricultural economy in China’s tea area.Tea plants originated in Southwest China,and its optimum growth temperature is 20-25℃.Therefore,the low temperature in winter and spring will affect the growth and development of tea plants,causing freezing damage,reducing the yield and quality of tea plants,and even causing the death of tea plants.The development of green and safe cold-resistant agents based on the metabolites of tea plants is a better way to solve the problem of cold resistance of tea plants.In this study,‘Longjing 43’potted seedlings were treated with low temperature for different time lengths,and the differential metabolites of tea shoots in response to low temperature were identified and screened by metabolite determination and analysis.Then,based on the results of our previous studies on the high content of ascorbic acid in tea plants during cold acclimation,the germinated branches of‘Longjing 43’were used as experimental materials.By spraying exogenous ascorbic acid,the physiological indexes of tea plants were measured after low temperature treatment,and the effect of exogenous ascorbic acid on cold resistance of tea plants was analyzed.The mechanism of ascorbic acid enhancing the cold stress response of tea plants was further analyzed by RNA-seq sequencing technology.In addition,in the analysis of differential metabolites of tea plant young shoots in response to low temperature,it was found that the synthesis precursor glutamic acid of theanine was significantly up-regulated after low temperature treatment.Therefore,theanine was selected to spray the germinated branches of‘Longjing 43’.Moreover,the mixture of ascorbic acid and theanine was sprayed on tea plants,and compared with the single spraying of ascorbic acid and theanine.The main results are as follows:（1）By measuring the metabolome changes of‘Longjing 43’potted tea seedlings after low temperature treatment at different times,it was found that there were 258 differential metabolites at 12 h,208 differential metabolites at 24 h,296 differential metabolites at 48 h,and 210differential metabolites at 72 h.KEGG pathway and classification annotation were performed on the identified differential metabolites.The results showed that most of the screened metabolites belonged to the synthesis pathways of amino acid metabolism,carbohydrate metabolism,nucleotide metabolism and other secondary metabolites in the metabolic pathway.After classifying the differential metabolites,it was found that100 of them belonged to amino acids and their derivatives,most of which showed an up-regulation trend,and 16 amino acids and their derivatives were continuously up-regulated after low temperature treatment,including the synthetic precursor glutamic acid of theanine.（2）After spraying exogenous ascorbic acid on the branches of‘Longjing 43’tea plants,the low temperature treatment was carried out.After sampling and measuring various physiological indexes,the results showed that low temperature treatment could induce the increase of relative conductivity,malondialdehyde（MDA）content,Fv/Fm value and hydrogen peroxide（H₂O₂）content of tea plant young shoots.Exogenous spraying 5 mmol L^-1ascorbic acid could significantly reduce the relative conductivity of tea plant young shoots,reduce MDA content,increase Fv/Fm and reduce H₂O₂content,and enhance the cold resistance of tea plants.Through transcriptome sequencing analysis,it was found that exogenous ascorbic acid could inhibit the expression of genes related to ROS scavenging in tea plant young shoots under cold stress,reduce the activity of catalase（CAT）,inhibit the expression of genes related to ascorbic acid synthesis and metabolism,and induce a large number of differentially expressed genes related to cell wall.Therefore,it is speculated that ascorbic acid may enhance the cold resistance of tea plants by increasing cell wall thickness or cell wall remodeling.（3）After spraying exogenous theanine on the branches of‘Longjing 43’tea plants,the low temperature treatment was carried out.Then the physiological indexes were measured.The results showed that exogenous spraying 1 mmol L^-1theanine could reduce the relative conductivity of tea plant young shoots under cold stress,reduce MDA content,increase Fv/Fm value,and improve the cold resistance of tea plants.（4）After spraying ascorbic acid,theanine and the mixed solution of ascorbic acid and theanine on the branches of‘Longjing 43’tea plants,low temperature treatment was carried out.After analyzing and comparing the determination results of physiological indexes,it was found that exogenous spraying of 5 mmol L^-1ascorbic acid,1 mmol L^-1theanine and mixed spraying of 5 mmol L^-1ascorbic acid and 1 mmol L^-1theanine could reduce the relative conductivity of tea plant young shoots under cold stress,reduce MDA content and H₂O₂content,and increase Fv/Fm.Among them,the effect of mixed spraying 5 mmol L^-1ascorbic acid and 1 mmol L^-1theanine was more obvious.It shows that the mixed spraying of exogenous ascorbic acid and theanine has the best effect on improving the cold resistance of tea plants.