Comparative metabolic and transcriptomic analysis of the pericarp of Sucui 1, Cuiguan and Huasu pears

Abstract:【Objective】In China, pear is a popular fruit approved by the consumers. Appearance is an important index to measure the economic value of fruits. For pears, the evaluation of appearance quality is mainly focused on the pericarp color, and the occurrence of fruit russet will greatly reduce its com- modity value. Sucui 1 has an emerald green color, smooth pericarp, rare brown russet and excellent ap- pearance. Therefore, the object of this study was to reveal the biochemical and molecular mechanisms that establish the characteristics of the pericarp in Sucui 1, so as to provide a theoretical basis for the large-scale popularization of Sucui 1.【Methods】Sucui 1 (with less or no russet) and its parents Cui- guan (with russet) and Huasu (russet-free) were collected as experimental materials. Detection of metab-olites was carried out by using Ultra Performance Liquid Chromatography tandem mass spectrometry (UPLC-MS). The area peak of all metabolites was integrated and corrected by MultiaQuant. Data was normalized by prcomp of R software, and analyzed by using Principal Component Analysis, Cluster Analysis, and Orthogonal Partial Least Squares- Discriminant Analysis. Differential metabolites be- tween two cultivars were further screened based on VIP ≥ 1 and Fold change ≥ 2 and Fold change ≤ 0.5. RNA-seq was carried out by using Illumina and clean reads were obtained. Transcripts were ana-lyzed and mapped to pear genome database using Tophat2/Hisat2/STAR. Fragments Per Kilobase of transcript sequence per million base pairs sequenced were set as expression level of transcripts or genes. Differentially expressed genes (DEGs) were screened using edgeR based on the value of padj＜0.05 and |log2FoldChange| ＞1. Joint analysis of the results of metabolism and RNA- seq was performed. The DEGs and their metabolites of the same group were mapped to the KEGG pathway map, and the path-ways with both differential metabolites and DEGs were displayed. Differentially expressed genes were verified by qRT-PCR.【Results】A total of 586 substances were detected in the pericarp of three culti-vars. 210 different metabolites in the pericarp of Cuiguan and Huasu mature fruits (HP vs CP) were ob-tained. Among these, 55% was secondary metabolites and 45% was primary metabolites including lip-ids (12%), the soluble sugars and organic acids (28%). 156 different metabolites were found between Cuiguan and Suicui 1 (SP vs CP), of which 64% was secondary metabolites and 36% was primary me-tabolites with 17% lipids and 20% soluble sugars and organic acids. Secondary metabolites mainly in-cluded flavonoids (31% in the HP vs CP and 34% in the SP vs CP), phenolic acids (13% in the HP vs CP and 17% in the SP vs CP), lignin and coumarins (4% in the HP vs CP and 5% in the SP vs CP). Further analysis of differential metabolites showed that in comparison of Sucui 1 and Huasu, a total of 10 sub- stances were specifically up-regulated and 29 substances were specifically down-regulated in Cuiguan pericarp. Among them, lipids and phenolic acids were the most main metabolites. For transcriptome da-ta, a total of 10.97 Gb clean bases were obtained and above 85% transcripts were mapped to pear ge-nome. 12 841 DEGs were obtained in the HP vs CP with 5866 up-regulated and 6975 down-regulated. 16 165 DEGs were obtained in the SP vs CP with 7973 up-regulated and 8632 down-regulated. KEGG analysis of DEGs showed that compared with Sucui 1 and Huasu, cutin, suberin and wax biosynthesis process, terpenoid skeleton biosynthesis process, flavonoid synthesis process, phenylalanine metabo- lism process, phenylpropanoid synthesis process, phenylalanine acid process, tyrosine and tryptophan biosynthesis process, and chlorophyll metabolism process were significantly enriched in Cuiguan. Tak-ing together with the results of differential metabolite analysis, three metabolic pathways including cu-tin, suberin and wax biosynthesis process, phenylpropanoid biosynthesis process and flavonoid biosyn-thesis process were selected for further analysis. There were 4 DEGs in cutin, suberin and wax biosyn-thesis process. Among them, CYP86A1 and HHT1 were significantly up-regulated in CP. CYP86B1 and CER1 were significantly down-regulated in CP. It was worth noting that the expression of HHT1 and CYP86A1 in SP was slightly higher than that in HP. There was no differential expression of the other two DEGs in SP vs HP. Four POD genes and one 4CL gene of phenylpropanoid biosynthesis pathway were up-regulated in CP, but not in SP vs HP. CAD of phenylpropanoid biosynthesis pathway was down-regulated in CP. All the six DEGs (two CHS, one DFR, one CYP75B1, one F3H and one ANS) in flavo- noid biosynthesis were down-regulated in SP.【Conclusion】The lower transcript accumulation of POD and 4CL in phenylpropanoid biosynthesis pathway and HHT1, CYP86A1 and CER1 in cutin, suberin and wax biosynthesis pathway in Sucui 1 might contribute to reduce the synthesis of lignin polymer, in-hibit the synthesis of thrombus, and decrease the mechanical strength of the cuticular layer of pericarp by inhibiting the synthesis of wax in pericarp cells, and consequently, the occurrence of fruit russet of Sucui 1 was greatly reduced. Vice versa, the higher transcript abundance of these genes in Cuiguan peri-carp might be correlated to the formation of lignin, thrombus and wax, enhancing the mechanical strength of the pericarp, so that it was not easy to break in the period of rapid expansion of fruit, result-ing in stress-resistant embolization of cells and the formation of russet in Cuiguan pericarp. DEGs relat-ed to anthocyanin in the synthetic branch of flavonoids were down-related in Sucui 1, saving more dihy-droflavonol as a substrate for the synthesis of flavonols, improving the storage resistance of Sucui 1.The causes of the excellent russet-free pericarp were revealed, which provided a theoretical basis for the large-scale popularization of Sucui 1. This finding is also of great significance of the development of molecular assisted breeding in pears.