Metabolic basis of fruit quality difference between Liuyangjindan and Suichuanjindan

【Objective】The cultivation history of Suichuanjindan (Fortunella) in Suichuan County and Jiangxi Province spans over a millennium, while Liuyangjindan represents a reintroduced cultivar derived from Suichuanjindan through regional introduction, also possessing a long cultivation history. Divergent growth environments have led to variations in fruit quality and phenological periods between the two cultivars. Fruit quality differences are primarily manifested in indicators such as soluble sugars and vitamin C content. Liuyangjindan exhibits delayed initial bud appearance and shorter flowering periods compared to Suichuanjindan, though their fruit maturation times are similar. This study employs metabolomics approaches to conduct differential analysis of metabolites in both kumquat fruits, investigating the impacts of differential metabolites and metabolic pathways on fruit development. It specifically elucidates the varying accumulation patterns of flavonoids between the two cultivars, providing theoretical foundations for the production applications of kumquat fruits in food and medicine from a metabolomics perspective. The findings also offer references for cross-regional introduction, breeding, and industrial promotion of high-quality kumquat germplasm resources.【Methods】In this study, fruits from Liuyangjindan and Suichuanjindan were utilized as experimental materials and divided into four tissue groups: whole fruit, peel, seed, and pulp. Non-targeted metabolomics analysis was performed using Ultra Performance Liquid Chromatography coupled with Triple Quadrupole Mass Spectrometry (UPLC- QqQ- MS/MS) technology. Metabolite data obtained were subjected to principal component analysis (PCA), followed by t- test- based screening to identify differential metabolites in each tissue group. The screened differential metabolites underwent KEGG pathway enrichment analysis to investi-gate significant metabolic pathways in different tissue groups. For targeted metabolomics analysis, the accumulation and distribution of 24 flavonoid compounds (including 13 flavonols, 8 flavones, 2 flavanones, and 1 isoflavone) were quantified in both kumquat cultivars. Statistical significance analysis was conducted on the detected flavonoid contents to evaluate inter-cultivar differences.【Results】The study found that each group of the two kumquats showed obvious discrete distribution in PCA analysis, indicating that there were differences in metabolic pathways between the two kumquats. More than one thousand differential metabolites were screened out in each group of the two kumquats by t test. The study revealed significant metabolic pathway differences between the two kumquat fruit cultivars. Within the Porphyrin metabolism pathway, differential metabolites primarily concentrated in the ALA (5- aminolevulinic acid) biosynthesis and metabolic pathways, showing upregulated expression trends in Liuyangjindan, indicating enhanced activity in this pathway. The Amino sugar and nucleotide sugar metabolic pathways were the most significant metabolic pathways in the two kumquats. D-glucosaminidase, UDP-D-galactose and D-fructose 6-phosphate within the pathway were important intermediates in the biosynthesis of plant polysaccharides. Their upward trend indicatedmore active polysaccharide synthesis and greater polysaccharide accumulation in the pulp of Liuyangjindan. Suichuanjindan exhibited more active pyruvate metabolism, resulting in higher organic acid accumulation than Liuyangjindan. Divergences in the nicotinate and nicotinamide metabolism pathway indicated variations in energy metabolism processes between the two cultivars. Among the 24 flavonoids examined, Myricetin-3-O-glucoside, Naringenin, and Puerarin exhibited similar distribution patterns between the two types of kumquat fruits, showing no significant differences. Vicenin-2 and Rutin showed differential distributions, while the remaining flavonoids displayed significant distribution differences between the two fruit varieties. Vitexin-2-O-rhamnoside, Myricetin-3-O-galactoside, Hesperidin, and Isoquercitrin were the four most abundant flavonoids detected. Among these, Vitexin-2-O-rhamnoside, the most abundant flavones, primarily distributed in peel and pulp tissues, showing comparable levels in Liuyangjindan's peel and pulp but slightly higher peel content in Suichuanjindan. Distribution patterns of eight flavones varied across tissues: Vicenin-2 and Apigenin exhibited tissue-specific localization. Suichuanjindan showed higher Vicenin-2 content in peel tissues, while Liuyangjindan displayed the opposite trend. Apigenin content was higher in Liuyangjindan's pulp but more abundant in Suichuanjindan's pericarp. Except for myricetin and its glycosides (Myricetin-3-O-galactoside and Myricetin-3-O-glucoside), flavonols primarily accumulated in peel and seeds of both cultivars, with relatively lower concentrations in pulp tissues. For flavanones, Naringin showed higher pulp tissue content in both cultivars, while Naringenin predominantly dsitributed in seeds. Naringin is one of the primary bitter compounds in citrus fruits. The slightly higher naringin content detected in Liuyangjindan may negatively affect its fruit quality. However, the naringin levels in the peels of both kumquat varieties were only around 10 μg · g-1 , significantly lower than those found in the peels of pomelos and sweet oranges. This may partially explain the high edibility of kumquats. In contrast, the Isoflavone Puerarin was detected at very low concentrations in both varieties, at approximately 0.005 μg·g-1 .【Conclusion】The study suggests that the more active biosynthesis and metabolism of ALA in the fruits of the Liuyangjindan variety, which might be responsible for its shorter maturation period compared to the Suichuanjindan. This accelerated maturation could potentially lead to inferior fruit quality in the Liuyangjindan variety. Conversely, the pulp of Liuyangjindan exhibits higher polysaccharide accumulation, making it a more suitable raw material for polysaccharide extraction. Although the Suichuanjindan accumulates higher levels of organic acids, its balanced soluble sugar content and sugar-to-acid ratio ultimately make it a more desirable variety for fresh consumption. The two varieties of kumquat fruits exhibit abundant flavonoid contents with significantly different distribution patterns between cultivars. Among the relatively abundant flavonoids such as Vitexin-2-O-rhamnoside, Hyperoside, Isoquercitrin, myricetin-3-O-galactoside, and vicenin-2, all except vicenin-2 demonstrate similar primary accumulation tissues in both kumquat varieties, though quantitative differences exist. Specifically, the peel of Suichuanjindan is more suitable for extracting Hyperoside, Quercetin, and Isoquercitrin, while its seeds can be utilized for kaempferol-3-O-rutinoside extraction. Conversely, the peel and pulp of Liuyangjindan serve as ideal materials for vitexin-2-O-rhamnoside extraction. Further analysis revealed that certain significantly differential metabolic pathways (e.g., Terpenoid backbone biosynthesis in peel tissues and α-Linolenic acid metabolism in pulp tissues) may result from environmental differences between the two cultivation regions. The specific mechanisms underlying these environmental impacts require joint analysis with ecological factors for clarification.