Analysis of flavor metabolite changes in fruits of Jiping 5 apple grafted onto Yanxing crabapple under salt stress based on untargeted metabolomics

【Objective】Soil salinization is a major environmental constraint affecting apple cultivation in coastal regions of northern China, including the Bohai Rim area, where the high soil salt content limits tree growth and reduces fruit quality. Although moderate salt stress has been reported to enhance fruit flavor quality in certain horticultural crops such as cherry tomato and cucumber, the metabolic responses of apple fruits to salt stress and their associations with flavor changes remain largely unclear. Using the salt-tolerant rootstock Yanxing crabapple grafted with Jiping 5 apple, this study systematically investigates the changes in fruit metabolite profiles under five different concentrations of salt stress (0, 0.2%, 0.4%, 0.6%, and 0.8% soil salt contents). The objective is to clarify the activation or suppression effects of salt stress on specific metabolic pathways, to identify key differential metabolites associated with flavor quality, and to characterize how salt stress concentration affects the metabolic features of fruit flavor quality, thereby providing a theoretical basis for high-quality apple cultivation in saline-al-kali soils.【Methods】Jiping 5 apple was grafted onto Yanxing crabapple rootstock. Five salt stress treatments were applied: 0 (CK), 0.2%, 0.4%, 0.6%, and 0.8% soil salt contents. Fruit samples were collected at maturity. Non-targeted metabolomics analysis was performed using UPLC-MS/MS. Data processing included peak detection, alignment, normalization, and filtering (RSD ＞30% in QC samples removed). PCA and OPLS- DA were conducted using the ropls package in R. Differential metabolites were screened based on VIP＞1.0 and P＜0.05. Metabolite annotation and KEGG pathway enrichment analysis were performed using HMDB, Metlin, KEGG databases, and the scipy. stats package in Python.【Results】A total of 1854 metabolites were identified across all samples. The major categories included organooxygen compounds (17.91% ), carboxylic acids and derivatives (13.05% ), fatty acyls (8.68% ), prenol lipids (8.63% ), and flavonoids (8.20% ). PCA showed gradual separation of samples along PC1 (36.20% variance explained) with increasing salt concentration, indicating significant metabolic reprogramming. PLS-DA further confirmed distinct clustering among treatment groups. The number of differential metabolites increased progressively with salt concentration: 297 (0.2% vs CK), 422 (0.4% vs CK), 588 (0.6% vs CK), and 764 (0.8% vs CK). Upregulated metabolites dominated all comparisons, accounting for 75.8% , 84.6% , 86.2% , and 91.4% , respectively. Venn diagram analysis revealed 223 common differential metabolites across all four treatment groups, representing a core salt-responsive metabolome. Unique differential metabolites increased with salt concentration, reaching 250 in the 0.8% vs CK comparison. VIP analysis identified Multinoside A, cyclomorusin, ursolic acid, and myricetin as highly responsive metabolites across multiple comparisons. Under low to moderate salt stress (0.2%-0.4%), flavonoids and terpenoids were prominent, while under high salt stress (0.8%), carbohydrate derivatives and terpenoids became more important. KEGG pathway analysis showed that the citrate cycle (TCA cycle), pentose and glucuronate interconversions, fructose and mannose metabolism, and galactose metabolism were consistently enriched across all treatment groups. Four pathways were constitutively activated under all salt concentrations: ABC transporters, biosynthesis of plant secondary metabolites, phenylpropanoid biosynthesis, and amino acid metabolism (especially arginine and proline metabolism). Quantitative analysis of representative metabolites revealed the following trends. TCA cycle intermediates (citrate, isocitrate, cis-aconitate, and 2-oxoglutarate) increased progressively with salt concentration, reaching peak levels at 0.8%. Amino acids and derivatives (L-glutamine, L-citrulline, Nacetyl-L-glutamate, and acetyl-β-alanine) generally increased under salt stress, with L-citrulline and Nacetyl-L-glutamate showing the highest levels at 0.6%-0.8%. Sugar derivatives (maltotriose, neohesperose, and phosphorylated sorbitol) decreased with increasing salt concentration, most notably at 0.8%. Terpenoid aroma compounds (β- ionone, rhododendrol, and caryophyllene oxide) showed overall decreasing trends, with the most pronounced reductions at 0.4% and 0.6%.【Conclusion】Salt stress significantly reprograms the fruit metabolome of Jiping 5 apple grafted on Yanxing crabapple. Four core pathways constitute the conserved metabolic features in response to salt stress: (1) The TCA cycle, which enhances energy metabolism and carbon skeleton supply; (2) Amino acid metabolism, leading to accumulation of compatible solutes such as glutamine and citrulline for osmotic adjustment; (3) Phenylpropanoid biosynthesis, which supports flavonoid-mediated antioxidant defense; (4) ABC transporters, involved in ion compartmentation and secondary metabolite transport. These pathways are consistently activated across all tested salt concentrations, indicating a robust and conserved metabolic response. The effect of salt stress on fruit flavor quality is concentration-dependent. Under low to moderate salt stress (0.2%-0.4%), the TCA cycle and amino acid metabolism are moderately activated. Sugar accumulation remains largely unaffected, while organic acids (citrate, and isocitrate) and certain aroma-related com-pounds show moderate increases. This metabolic profile suggests enhanced flavor intensity without significant loss of sweetness. Under high salt stress (0.8% ), however, sugar derivatives decrease, organic acids accumulate to higher levels, and terpenoid aroma compounds decline. These changes collectively lead to a reduced sugar-to- acid ratio and diminished aroma, indicating a deterioration of fruit flavor quality. This study provides a systematic metabolomic characterization of salt stress responses in fruits of apple grafted on a salt-tolerant rootstock. The findings establish a theoretical basis for high-quality apple cultivation in saline-alkali soils of the Bohai Rim region.