Screening and identification of several metabolites associated with cold resistance in peach buds based on widely targeted metabolomics

Abstract: 【Objective】Peach has a long history of cultivation and wide distribution in China. Temperature is the main factor affecting the growth and development of peach trees as well as determining their natural distribution, so it is of great theoretical and practical significance to clarify its physiological and biochemical mechanisms in response to low temperature for breeding and screening peach cold- resistant varieties. Metabolomics, as important components of systems biology, can reveal differences in metabolite profiles among different species, between different tissues of the same species, and between the same tissues of the same species under different conditions of adversity stress. The aim of this study was to investigate the metabolites that would affect the cold resistance of peach buds and to facilitate a deeper understanding of the metabolic pathways of cold resistance in peach.【Methods】The material used for the cold treatment to identify the differential metabolites was 6- year- old Zhongnong Jinhui, and the annual branches were taken from the upper and middle periphery of the tree, cut into 30 cm segments, wrapped in plastic wrap and placed in the refrigerator for stress treatment. All the samples need-ed for the experiment were put in at once, and the time of starting the treatment was recorded. The branches were removed at 0 h, 24 h and 72 h, and the leaves on the branches were removed before the branches were thawed, and the peach buds of the same size and without mechanical damage on the branches were selected and peeled out with forceps and dissecting needles and then were quickly put into liquid nitrogen. metabolite assay. Four significantly different metabolites, aminopurine, adenosine, xanthine nucleoside and DL-homocysteine were selected from the metabolome assay, and the standards were prepared in distilled water at different concentrations. Fifteen uniformly growing Zhongtao Kangzhen 1 rootstock plants were treated at -4 ℃ in a light incubator for 16 h of light and 8 h of darkness. The seedlings were sprayed with different solutions of the standards at a dosage of about 15 mL per plant every 24 h, using tap water as control. Finally, the proline content, the expression of the key gene PpP5CS for proline synthesis and the cold resistance related gene PpCBF2 in the leaves were measured on 1, 3 and 7 days after the treatment. It is known that the increase of proline content in the plant tissues under low temperature conditions can improve the cold resistance of plants, therefore, proline content was used as a physiological indicator for cold resistance breeding. Similarly, the relative expression of PpCBF2 gene could indicate the response of seedlings to the low temperature stress.【Results】A total of 172 functionally annotated metabolites were detected in the peach buds by comparing information from LC- MS/MS detection platform and database. The secondary classification of these metabolites could be classified into 10 categories, among them the amino acids and their derivatives accounted for the highest percentage, followed by the phenolic acids, flavonoids, organic acids and lipids. The content of these 172 metabolite components was analyzed, and 32 major metabolites were found in the peach buds, including 7 amino acid derivatives, 5 flavonoids, 4 phenolic acids, 4 lipids, 3 organic acids, 2 alkaloids, 2 nucleotides and their derivatives, 1 tannin, and 4 others. According to the Fold change (FC ≥ 1.5) combined with p-value＜0.05 analysis, 32 metabolites with significant differences were screened, among them 8 were down-regulated and 24 were up-regulated, and their secondary classifications were mainly amino acids and their derivatives, nucleotides and their derivatives, flavonoids, alkaloids, lipids, etc. We randomly selected 4 metabolites from the up-regulated metabolites to perform validation experiments on seedlings. The standards of these four significantly different metabolites, xanthine nucleoside, adenosine, DL-homocysteine and aminopurine were selected to prepare solutions for in vitro validation experiments. The results of the validation experiment showed that the proline content showed an overall increasing trend followed by a decreasing trend and reached a peak on the 3th day of the -4 ℃ treatment. However, by comparing the increase of the proline in the control and treated groups, we found that the increase of the proline content in the control group which sprayed with tap water was not obvious by comparing with the previous and the subsequent treatments. The peak of the proline content on the 3th day was only twice as much as that in the control group, while the increase of the proline content in the plants caused by spraying other substances was strengthened obviously, especially by the spray of xanthine, reaching 6 times as much as that of the control. Similar to the changes in the proline content, the expression of the PpP5CS, a key gene for proline synthesis, in the leaves of seedlings treated with xanthine nucleoside likewise peaked on day 3 of treatment and was four times as much as that of the control. The expression of the PpCBF2 in the control group sprayed with tap water gradually increased with time and reached the highest value on day 7, which was 1246 times as high as that on day 0. The expression of the PpCBF2 in the leaves of seedlings treated with DL-homocysteine and xanthine nucleoside gradually increased with the time of stress treatment and reached the highest value on day 3, which was 13 810 and 3650 times as high as that on day 0, respectively; The expression of the PpCBF2in the leaves of seedlings treated with aminopurine and adenosine increased gradually with the prolongation of stress, reaching the highest value on the first day, 1464 and 1169 times as high as that on day 0, respectively, and then gradually decreased on the third day.【Conclusion】The results of the validation experiment showed that the proline content of seedlings treated with four substances, namely xanthine nucleoside, adenosine, DL-homocysteine, and aminopurine, increased during the low temperature stress compared with the control group sprayed with tap water, indicating that all four substances have the effect of regulating the cold resistance of peach buds.