Comparative metabolomics analysis of thorn and branch of　‘Goutou’ sour orange based on UPLC-MS/MS technique

Abstract：【Objective】Most of the thorns and branches of citrus initially formed in the same position of leaf axil, and finally developed into the thorns and branches separately, indicating that there were differ- ent regulation mechanisms of development between them. However, little is known about the molecular mechanism of the thorn development of citrus. In a previous study, we found that the differentially ex- pressed genes between the thorn and branch of citrus mainly focused on the pathways related to the syn- thesis of secondary metabolites. In order to further elucidate the regulatory factors of the differential de- velopment of the thorn and branch, the differences of the internal structure and metabolites between the thorn and branch were investigated, which would provide the clues and basis for further study of the regulation mechanism of the thorn development.【Methods】The top parts of the young thorns and branches (1 cm in length) were collected from the leaf axils of the new shoots from 5-year-old‘Goutou’Sour Orange seedlings. The differences of the internal structure of the thorn and branch were ob- served and analyzed using the paraffin section method. The differential metabolites between the thorn and branch were detected and screened by UPLC-MS/MS and widely targeted metabolome technology. The metabolome data were analyzed based on MWDB (metware database) and OPLS-DA (orthogonal signal correction and partial least squares-discriminant analysis) model, and the VIP (variable import in project) and fold change values were used to screen the differential metabolites between the thorn and branch. Statistical pathway enrichment analysis of the differential metabolites was performed based on the KEGG database.【Results】The results of cross sections of the thorn and branch showed that there were obvious differences between the internal structure of the thorn and branch. There were secretory canal, leaf or lateral branch primordium in the epidermal cells of the branch, but not in the thorn. How- ever, the other internal structures of the thorn were similar to the branch. The results of paraffin section also showed that citrus thorn had the same vascular tissue as branch or stem, which was different from that of some other plants, such as rose prickles or tomato trichomes. The comparative metabolomics analysis of the thorn and branch was performed based on widely targeted metabolome technology. The result of PCA (principal component analysis) of the two groups showed that the scores of principal com- ponents PC1 and PC2 were 51.20% and 18.70% respectively, indicating that the trend of separation of the thorn and branch samples was obvious. The assessment of OPLS-DA model established for metabo- lome data analysis showed that the difference between the two groups of samples was obvious, and the difference among the samples in the group was not obvious, indicating that the OPLS-DA model estab- lished in this analysis had meeted the excellent model standard. The results of widely targeted metabo- lome showed that a total of 895 metabolites were detected, of which 138 differentially accumulated me- tabolites were identified between the thorn and branch, including 81 up-regulated metabolites and 57 down-regulated metabolites in the branch. These differential metabolites were mainly related to the secondary metabolites, such as flavonoids, phenylpropanoids, terpenes, alkaloids, polyphenols, organic ac- ids and derivatives, vitamins and derivatives. Cluster analysis of differential metabolites also showed that there were significant differences between the screened metabolites of the thorn and branch, and most of the differential secondary metabolites were accumulated more in the branch than in the thorn. KEGG enrichment analysis showed that 91 of the differential metabolites were mainly concentrated in 74 KEGG pathways, and the enriched pathways mainly included metabolic pathway (48 metabolites), secondary metabolite synthesis (41 metabolites), flavone and flavonol biosynthesis (16 metabolites), biosynthesis of amino acids (15 metabolites), flavonoid biosynthesis (14 metabolites), aminoacyl-tRNA biosynthesis (13 metabolites), ABC transporters (11 metabolites), glutathione metabolism (11 metabolites), tryptophan metabolism (9 metabolites) and phenylpropanoid biosynthesis (8 metabolites) pathways, etc.【Conclusion】Differences in the internal structure of the thorn and branch of citrus were found, and there were leaf primordia and lateral branch primordia in the epidermal cells of the branch, but not in the thorn. A total of 138 differentially accumulated metabolites were identified between the thorn and branch, including 81 up-regulated metabolites metabolites and 57 down-regulated metabo- lites, which were mainly related to the secondary metabolites. The differential metabolites were mainly concentrated on metabolic pathway and secondary metabolite synthesis pathway. These results indicat- ed that the differential accumulation of the secondary metabolites played an important regulatory role in the differential development of the thorn and branch. All the above results would lay the foundation for the research on the regulation mechanism of the citrus thorn development. The key genes will be screened according to the results of the comparative metabolome and transcriptome in the future.