The core metabolome and root exudation dynamics of three phylogenetically distinct plant species

Abstract

Root exudates are plant-derived, exported metabolites likely shaping root-associated microbiomes by acting as nutrients and signals. However, root exudation dynamics are unclear and thus also, if changes in exudation are reflected in changes in microbiome structure. Here, we assess commonalities and differences between exudates of different plant species, diurnal exudation dynamics, as well as the accompanying methodological aspects of exudate sampling. We find that exudates should be collected for hours rather than days as many metabolite abundances saturate over time. Plant growth in sterile, nonsterile, or sugar-supplemented environments significantly alters exudate profiles. A comparison of Arabidopsis thaliana, Brachypodium distachyon, and Medicago truncatula shoot, root, and root exudate metabolite profiles reveals clear differences between these species, but also a core metabolome for tissues and exudates. Exudate profiles also exhibit a diurnal signature. These findings add to the methodological and conceptual groundwork for future exudate studies to improve understanding of plant-microbe interactions.

Fig. 1. Root exudation dynamics in three species.

Principal component plots of root exudates collected after 0.5 h, 2 h, 4 h, 1 d, 4 d of B. distachyon a, A. thaliana b, and M. truncatula c. Details of this dataset are given in Supplementary Figs. 2 and 3. Total number of metabolites: 63. Number of jars with 3–5 plants: 3–4 for each timepoint. One representative experiment out of three total is displayed.

Fig. 2. B. distachyon root exudation dynamics in different growth conditions.

B. distachyon was grown for 3 weeks in sterile, nonsterile, and sucrose-supplemented conditions followed by exudate collection in the same 0.5 strength MS medium. Fresh weight of root and shoot a and root/shoot ratio b at 3 weeks. Data are averages ± SEM, n = 15 individual plants, *=p < 0.05, ***=p < 0.0005 (two-sided t-test). Root morphology of plants is described in Supplementary Fig. 4. c Principal component analysis of exudate profiles. d Metabolite classes detected in the entire dataset, and classes enriched (bold, blue) or depleted (italic, red) in sucrose-supplemented or nonsterile conditions compared to sterile conditions (Fishers exact test). Values are in percent of total number of metabolites (n = 109 metabolites of 4–5 jars with 3 plants per growth condition). One representative experiment out of three is displayed.

Fig. 3. Metabolites distinct in B. distachyon exudates from different growth conditions.

Normalized peak height of metabolites significantly different between growth conditions (light blue: sterile, red: sucrose supplemented, dark blue: nonsterile)(Anova/Tukey test, p < 0.05, p-values are given in Supplementary Data 1). Sucrose-supplemented vs sterile conditions a, nonsterile vs sterile conditions b. Displayed are all conditions as reference, black boxes and labeling to the left indicate in which conditions a metabolite is distinct, and if it its abundance is increased or decreased compared to control. S: Sterile, NS: nonsterile, 4-hydroxy-2: 4-hydroxy-2-quinolinecarboxylic acid, *: and/or isomers. Data are averages ± SEM, n = 4–5 jars per growth condition with 3 plants each. Metabolites are colored by class: orange: Amino acids, peptides and derivatives, purple: carbohydrates and conjugates, green: lipids and lipid-like, blue: nucleosides, nucleotides and derivatives, red: organic acids, black: other.

Fig. 4. Species-specific patterns of root metabolites.

Heatmap of root metabolites. Each cell represents an averaged value of normalized peak height of metabolites significantly different between plant species (Anova, p < 0.05 and post-hoc Duncan’s multiple range test). Hierarchical clustering identified six clusters of metabolites based on their distribution across analyzed species. Heatmap of shoot metabolites is found in Supplementary Fig. 6, and PCA plots of tissues in Supplementary Fig. 5.

Fig. 5. Diurnal changes in exudate profiles.

a Principal component analysis of A. thaliana (yellows), B. distachyon (reds) and M. truncatula (blues) exudate metabolites at the end of day (EOD) and end of night (EON). Percent of metabolites that are significantly different between species at end of day b, end of night c, and for each species between end of day and night d. Colored by high (dark green) to low (light green) intensity. e Fold difference of metabolites significantly increased in end of day vs. end of night A. thaliana (yellow, top graph), in B. distachyon (red), M. truncatula (blue), or in two species (bottom graph, indicated in subtitles). One representative experiment out of three total is displayed. Compounds are grouped by decreasing fold change within one group and are colored by class: orange: Amino acids, peptides and derivatives, purple: carbohydrates and conjugates, green: lipids and lipid-like, blue: nucleosides, nucleotides and derivatives, red: organic acids, black: other. #: EOD and/or EON value below detection limit. *: and/or isomers. Data are averages ± S.E. N = 4–8 jars per timepoint with 3–5 plants each for a–e, n = 74 metabolites in total for a–d, Anova/Tukey test, p < 0.05 for significant differences in e. Details of this dataset are given in Supplementary Fig. 5.

Fig. 6. Core metabolites in roots and exudates.

Metabolites detected in A. thaliana, B. distachyon, and M. truncatula roots or in roots as well as exudates. Metabolites are colored by class: orange: Amino acids, peptides and derivatives, purple: carbohydrates and conjugates, green: lipids and lipid-like, blue: nucleosides, nucleotides and derivatives, red: organic acids, black: other. *: and/or isomers. # changing significantly diurnally in exudates of at least one species (Anova/Tukey test, p < 0.05, p-values are given in Supplementary Data 1). Specific metabolites of this dataset are presented in Fig. 7.

Fig. 7. Specific metabolites in roots and exudates.

Metabolites detected in roots and/or exudates of one or several species. A specific metabolite is present in roots (orange box), roots and exudates (purple box), or exudates (green box). Its presence in A. thaliana, B. distachyon, M. truncatula is indicated by the connecting colored lines. Number of jars with 3–5 plants: 3–8 for each timepoint. One representative experiment out of three total is displayed. Metabolites are colored by class: orange: Amino acids, peptides and derivatives, purple: carbohydrates and conjugates, green: lipids and lipid-like, blue: nucleosides, nucleotides and derivatives, red: organic acids, black: other. *: and/or isomers. # changing significantly diurnally (Anova/Tukey test, p < 0.05, p-values are given in Supplementary Data 1). Common metabolites of this dataset are presented in Fig. 6.