Leaf Metabolomics Reveals Grade-Associated Candidate Metabolites and Physiological Differences in Apple Nursery Plants

Nursery-plant grading relies on morphological traits, but leaf metabolic variation associated with nursery-plant grade remains unclear. In this study, untargeted HPLC-QTOF-MS metabolomics was applied to mature and young leaves of Grade I, II, and III apple nursery plants to explore grade-associated metabolic variation and its relationship with growth performance. After quality filtering, 198 positive-ion features were retained from mature leaves, while 259 positive-ion and 8 negative-ion features were retained from young leaves. Multivariate analysis showed clear grade-associated separation in both leaf types, with stronger discrimination in young leaves. A combination of orthogonal partial least squares (OPLS) modeling and trend analysis identified 20 and 28 differential features in mature and young leaves, respectively. Cross-model prioritization further highlighted key metabolites putatively annotated as methyl nicotinate and 1-palmitoyl-sn-glycero-3-phosphocholine in mature leaves, and methyl nicotinate, nicotinamide riboside, cis-jasmone, and methyl (9Z,14Z)-12,13,16-trihydroxyoctadeca-9,14-dienoate in young leaves. These key metabolites were potentially associated with NAD precursor metabolism, membrane lipid remodeling, and oxylipin-related signaling. Correlation analysis showed that they were associated with both initial grading traits and post-transplant growth performance. These metabolites represent candidate molecular correlates of grade-associated physiological variation and growth performance in apple nursery plants.