Mechanism of ATS-induced flower thinning in Weihai Gold apple revealed by metabolomics analysis

【Objective】The experiment was conducted to explore the secondary differential metabolites of Weihaijin apple after ammonium thiosulfate (ATS) treatment and its mechanism of flower thinning, and to provide scientific basis for efficient and high-quality production and chemical flower thinning. Apple flower and fruit thinning is an important technology to adjust the tree load, control the size of the tree, ensure the high quality and efficient production of apples, and improve the competitiveness on the fruit market. Chemical flower and fruit thinning is a labor- saving and efficient thinning method. By spraying appropriate chemical agents, the side flowers and side fruits in apple clusters are weakened, shriveled, shrunk, and finally fallen off, and only the central flowers and fruits are retained. It is a laborsaving management widely used in developed countries. ATS is a kind of sulfur-based nitrogen fertilizer, which can cause damage to style, reduce pollen germination, effectively reduce crop load and improve fruit quality after spraying. As a common flower thinning agent, it has achieved good application effect in fruit production. The predecessors have clarified the appropriate spraying concentration and period of ATS on apples, and its effect on the physiological changes of side fruits has gradually becomeclear. However, the changes of secondary metabolites in the metabolic pathways of ATS-treated nuts and their removal mechanisms have rarely been reported at home and abroad. Therefore, it is of great significance to study the mechanism of ATS flower thinning from the perspective of metabolomics for the popularization and application of apple chemical flower thinning technology.【Methods】The metabolites were detected by ultra- high performance liquid chromatography- tandem mass spectrometry (UPLC-MS/MS), and the metabolites were qualitatively and quantitatively analyzed by extensive targeted metabolomics to screen and compare the differential metabolites of siliques after thinning.【Results】 A total of 85 differential metabolites were detected, including 34 flavonoids, 16 phenolic acids, 8 lignans and coumarins, 6 tannins and 4 lipids, among which flavonoids accounted for the highest proportion. KEGG pathway enrichment showed that biosynthesis of secondary metabolism and flavonoid biosynthesis with different treatments were the most significantly differential metabolic pathways. Two metabolites related to sugar metabolism were screened out based on secondary metabolic biosynthesis, which were trans-2-hydroxy-cassia bark acid ester and 6-hydroxy feruloyl-CoA; Nine key metabolites were identified based on the flavonoid biosynthetic metabolic pathway. The metabolites with decreased levels included crocetin, wintersweet glycoside, quercetin-3-O-rutinoside, 5, 7-dihydroxy-3', 4', 6-trimethoxyflavone, isorhamnetin-3, 7-O-diglucoside, 3', 5-dihydroxy-4', 6, 7-trimethoxyflavone, and apigenin-6-C-(2''-glucuronidyl)-xyloside. The metabolites with increased levels included alfalfa glycoside-7-Oglucuronide and kaempferol- 3-O- (2''-O- acetyl) glucoside.【Conclusion】A total of 1946 metabolites were detected by extensive targeted metabolomics analysis of apple fruits. Based on the screening of flavonoid biosynthetic metabolic pathways, 11 kinds of flavonols (quercetin-3-O-(4"-O-glucosyl) rhamnoside, quercetin-3-O-apiosyl(1→2) galactoside, isorhamnetin-3, 7-O-diglucoside, quercetin-3-O-neohesperidinoside, kaempferol-7-O-glucuronide, quercetin-3-O-(6"-O-acetyl) glucopyranoside, myricetin-3- O-xyloside, quercetin-3-O-(6"-O-malonyl) glucopyranoside-5-O-glucoside, myricetin-3-O-rhamnopyranoside (myricetin), quercetin-3-O-acacia glycosides, quercetin-3-O-rutinoside (rutin)), and 4 kinds of flavonoids (eupatilin; 3', 5-dihydroxy-4', 6, 7-trimethoxyflavone, Chimonanthus praecoxin, apigenin-6- C-(2"-glucuronic acid) xylopyranoside, eupatilin) were found to be the key metabolites related to ATS elimination. Through KEGG metabolic pathway enrichment analysis, it was found that the most significantly enriched differential metabolite pathways were biosynthesis of secondary metabolism and flavonoid biosynthesis. The results can provide a theoretical basis for the popularization and application of chemical flower and fruit thinning agents and the high-quality development of apple industry.