Metabolomics analysis of differential metabolites in the fruit pulp of‘Tubtim Siam Red Ruby Pummelo’ （Citrus grandis）

【Objective】Tubtim Siam Red Ruby Pummelo originated from a Thailand's southern city, and fruit flesh was ruby-like, so it got the name for granted. It is currently one of the major cultivars of excellent pummelo in southern China. It is mainly eaten fresh, and little has been reported on the analysis of its fruit characteristic components, drug and health care research and development. The systematic comparisons of pulp metabolites and screened characteristic metabolites between Tubtim Siam Red Ruby Pummelo (TSRRP) and Yuenan Qingyou pummelo (YNQY), Hongyu Xiangyou pummelo (HYX), Star Ruby grapefruit (XLBPTY), Sanhong Miyou pummelo (SHMY), and Xingluo Hongyou pummelo (XLHY) are considerable value for the utilization of pummelo germplasm resources and the promotion of medical and health care fields.【Methods】Liquid chromatography- mass spectrometry was used to analyze the non-targeted metabolomics of pulp components of six pummelo cultivars. Thedetected data unit variance scaling (UV) was normalized and the samples were analyzed for holistic properties using principal component analysis (PCA). Meanwhile, the log transformation and mean-centered scaling of the collected data were performed, and subsequently the orthogonal partial least squaresdiscriminant analysis (OPLS-DA) was conducted using the R package Meta boAnalyst. Meanwhile, the values of the variable importance in projection (VIP), fold change (FC), and p-value of student's t-test were screened for differential metabolites. In addition, TBtools-Ⅱ and the R package ClusterProfiler software were performed for clustering heatmap and kyoto encyclopedia of genes and genomes (KEGG) enrichment of the screened metabolites.【Results】UPLC-MS/MS detected a total of 1019 metabolites. The PCA score plot showed that the contributions of principal components 1 and 2 were 23.33% and 19.02%, respectively. QC samples were densely scattered, indicating stable and high quality metabolic data. TSRRP pummelo was clearly separated from the other cultivars. The six cultivars were grouped using OPLS-DA analysis. Metabolites were screened by VIP≥1, FC＞2 or FC＜0.5 and P＜0.05. In the TSRRP vs YNQY comparison, 213 metabolites were identified as differentially expressed, with 138 upregulated and 75 downregulated. In the TSRRP vs HYX comparison, 237 metabolites were identified, with 165 upregulated and 72 downregulated. In the TSRRP vs SHMY comparison, 264 metabolites were identified, with 196 upregulated and 68 downregulated. In the TSRRP vs XLBPTY comparison, 386 metabolites were identified, with 193 upregulated and 193 downregulated. In the TSRRP vs XLHY comparison, 390 metabolites were identified, with 259 upregulated and 131 downregulated. An upset plot was conducted for 606 differential metabolites. There were 55 metabolites with simultaneous differences in the five combinations. They included 12 phenylpropanoids and polyketides, 12 lipids and lipid-like molecules, 10 organic acids and derivatives, 8 organoheterocyclic compounds, 5 benzenoids, 2 amino acids and derivatives, 2 nucleosides and nucleotides analogues, 2 organic oxygen compounds, 1 lignan, neolignan and related compound, and 1 coumarin and lignan. Phenylpropanoids and polyketides, lipids and lipid-like molecules accounted for the highest proportion of 21.82%, while organic acids and derivatives, organoheterocyclic compounds, and benzenoids also occupied large proportions of 18.18%, 14.55%, and 9.09%, respectively. Metabolite differences between cultivars were demonstrated, and 55 significantly different metabolite expressions were analyzed by Euclidean clustering. Significant accumulation took place in metabolites of 11- hydroxydrim-7- en-6- one, 8- hydroxy-7 (11)-eremophilen-12, 8-olide, 4, 4'-cyclohexylidenebisphenol, α-cyperone, L-valine, 3-amino-4-methylpentanoic acid, DL-isoleucine, D-tyrosine, L-tyrosine, DL-tyrosine, and DL-m-tyrosine, while tilmisartan and scopolamine lactone contents in TSRRP pummelo were low. In addition, the samples cluster analysis based on the Euclidean distance showed that TSRRP was clearly distinguished from the other cultivars, with YNQY clustered into one unit with XLBPTY and one branch clustered with XLHY, SHMY, and HYX pummelo. KEGG pathway enrichment analysis showed that phenylpropanoid, glucosinolate, aminoacyl-tRNA, and cyanoamino acid biosynthesis were the most significant metabolic pathways. Up-regulation of sinapinate and 1-O-sinapoyl-β-D-glucose was found in the phenylpropanoid biosynthetic pathway. L-tyrosine, L-valine, and DL-isoleucine were up-regulated in the glucosinolate, aminoacyl-tRNA, cyanoamino acid biosynthetic pathways. 1-Pentadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine, indole-3-acetaldehyde, scoparone, and xanthotoxol were up- regulated and scopoletin was down-regulated in the other secondary metabolite biosynthetic pathways.【Conclusion】Differential metabolites in the pulp of TSRRP compared to five pummelos by metabolomics. 55 differential metabolites were screened by UPLC-MS/MS. The significantly enriched differential metabolite pathways were phenylpropanoid and glucosinolate metabolism. 10 major metabolites were screened, including sinapi-nate, 1-O-sinapoyl-β-D-glucose, L-tyrosine, L-valine, DL-isoleucine, 1-pentadecanoyl-2-hydroxy-snglycero-3-phosphocholine, indole-3-acetaldehyde, scoparone, xanthotoxol, and scopoletin, respectively. The active ingredients content of TSRRP was significantly higher than other cultivars, indicating that it may have higher medicinal and edible value, which can provide a certain theoretical basis for the development of functional products and medicines.