Targeted metabolic profiling indicates apple rootstock genotype-specific differences in primary and secondary metabolite production and validate quantitative contribution from vegetative growth

Authors: Leisso, Rachel; Rudell, David; Mazzola, Mark

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/24/2018
Publication Date: 9/21/2018
Citation: Leisso, R.S., Rudell Jr, D.R., Mazzola, M. 2018. Targeted metabolic profiling indicates apple rootstock genotype-specific differences in primary and secondary metabolite production and validate quantitative contribution from vegetative growth. Frontiers in Plant Science. 9:1336. https://doi.org/10.3389/fpls.2018.01336.
DOI: https://doi.org/10.3389/fpls.2018.01336

Interpretive Summary: Understanding the interaction of the apple root system with the surrounding soil is a topic of much interest due to the potential for research findings to impact fruit production issues including root disease, nutrient acquisition, and drought tolerance. The present study 1) establishes a method for assessment of root-released metabolites in the absence of microbes, which can potentially confound results, and 2) results establish genotype (cultivar) specific differences of two apple rootstock cultivars for a set of pre-determined metabolites, including sugars, sugar alcohols, amino acids, phenolic compounds, and compound tentatively identified as triterpenoids. This research has impact for the fruit industry by increasing fundamental knowledge regarding apple root interaction with the soil, specifically by enabling future research involving functional microbiome assessment with rhizodeposit profiles. Major findings: 1. This study reports methods for axenic collection of apple rhizodeposits. 2. This study delineates the relative levels of a number of compounds in collected rhizodeposits from two apple rootstock genotypes (G935 and M.26), including phenolic compounds, sugars, sugar alcohols, amino acids, organic acids, and several putative triterpenoids. 3. Vegetative activity was confirmed to contribute to rhizodeposit levels. 4. Endophytes were assessed per molecular methods but results were inconclusive, so the contribution of endophytes to rhizodeposits cannot be eliminated.

Technical Abstract: Previous reports regarding rhizodeposits from apple roots are limited, and complicated by microbes, which readily colonize root systems and contribute to or modify rhizodeposit metabolite composition. This study reports a method and results for the quantitative assessment of apple rhizodeposits under axenic conditions. Primary and phenolic rhizodeposit metabolites collected from two apple rootstock cultivars (genotypes), G935 and M26, were delineated two months after root initiation utilizing gas chromatography/liquid chromatography – mass spectrometry, 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 levels in rhizodeposits, and L-asparagine, L-cysteine, malic acid, succinic acid, and sorbitol were among the amino acids, organic acids, and sugar alcohols found in relatively higher abundance in the rhizodeposits. 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 fungal or bacterial-specific primers in qPCR were inconclusive in determining the presence of these microbial groups in root tissues; 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.