Targeted Metabolic Profiling Indicates Apple Rootstock Genotype-Specific Differences in Primary and Secondary Metabolite Production and Validate Quantitative Contribution From Vegetative Growth

Abstract

Previous reports regarding rhizodeposits from apple roots are limited, and complicated by microbes, which readily colonize root systems and contribute to modify rhizodeposit metabolite composition. This study delineates methods for collection of apple rhizodeposits under axenic conditions, indicates rootstock genotype-specific differences and validates the contributions of vegetative activity to rhizodeposit quantity. Primary and phenolic rhizodeposit metabolites collected from two apple rootstock genotypes, G935 and M26, were delineated 2 months after root initiation by utilizing gas chromatography/liquid chromatography-mass spectrometry (GC/LC-MS), respectively. Twenty-one identified phenolic compounds and 29 sugars, organic acids, and amino acids, as well as compounds tentatively identified as triterpenoids were present in the rhizodeposits. When adjusted for whole plant mass, hexose, erythrose, galactose, phloridzin, kaempferol-3-glucoside, as well as glycerol, and glyceric acid differed between the genotypes. Phloridzin, phloretin, epicatechin, 4-hydroxybenzoic acid, and chlorogenic acid were among the phenolic compounds found in higher relative concentration in rhizodeposits, as assessed by LC-MS. Among primary metabolites assessed by GC-MS, amino acids, organic acids, and sugar alcohols found in relatively higher concentration in the rhizodeposits included L-asparagine, L-cysteine, malic acid, succinic acid, and sorbitol. In addition, putative ursane triterprenoids, identified based on accurate mass comparison to previously reported triterpenoids from apple peel, were present in rhizodeposits in high abundance relative to phenolic compounds assessed via the same extraction/instrumental method. Validation of metabolite production to tree vegetative activity was conducted using a separate set of micropropagated trees (genotype MM106) which were treated with a toxic volatile compound (butyrolactone) to inhibit activity/kill leaves and vegetative growth. This treatment resulted in a reduction of total collected rhizodeposits relative to an untreated control, indicating active vegetative growth contributes to rhizodeposit metabolites. Culture-based assays indicated an absence of bacterial or fungal endophytes in roots of micropropagated G935 and M26 plants. However, the use of fungi-specific primers in qPCR indicated the presence of fungal DNA in 30% of the samples, thus the contribution of endophytes to rhizodeposits cannot be fully eliminated. This study provides fundamental information for continued research and application of rhizosphere ecology driven by apple rootstock genotype specific rhizodeposition.