Root-Secreted Coumarins and the Microbiota Interact to Improve Iron Nutrition in Arabidopsis

Abstract

Plants benefit from associations with a diverse community of root-colonizing microbes. Deciphering the mechanisms underpinning these beneficial services are of interest for improving plant productivity. We report a plant-beneficial interaction between Arabidopsis thaliana and the root microbiota under iron deprivation that is dependent on the secretion of plant-derived coumarins. Disrupting this pathway alters the microbiota and impairs plant growth in iron-limiting soil. Furthermore, the microbiota improves iron-limiting plant performance via a mechanism dependent on plant iron import and secretion of the coumarin fraxetin. This beneficial trait is strain specific yet functionally redundant across phylogenetic lineages of the microbiota. Transcriptomic and elemental analyses revealed that this interaction between commensals and coumarins promotes growth by relieving iron starvation. These results show that coumarins improve plant performance by eliciting microbe-assisted iron nutrition. We propose that the bacterial root microbiota, stimulated by secreted coumarins, is an integral mediator of plant adaptation to iron-limiting soils.

Keywords: coumarins; edaphic adaptation; immune regulation; iron nutrition; microbiota; plant growth promotion; root microbiota; secondary metabolites.

Graphical abstract

Figure 1

Coumarin Biosynthesis Is Important for Plant Growth and Root Microbiota Composition in a Naturally Calcareous Soil (A) Diagram of pathways for coumarin biosynthesis and export, and reductive uptake of iron in Arabidopsis. (B and C) (B) SFW and (C) total chlorophyll content of coumarin pathway mutants grown in a non-calcareous (CAS) and a calcareous (IS) soil. Statistical significance was determined by Kruskal-Wallis; each mutant was compared with Col-0 by Wilcoxon Ranked Sum post-hoc. Significance is indicated by red asterisks (^∗, ^∗∗, ^∗∗∗, indicate p < 0.05, 0.01, and 0.001, respectively). For shoot fresh weight measurements, Col-0 n = 171, 204; f6’h1 n = 168, 272; s8h n = 93, 113; cyp82c4 n = 164, 209; and pdr9 n = 172, 169 in CAS and IS, respectively. Chlorophyll content was measured from pooled leaf samples, (Col-0 n = 35, 29; f6’h1 n = 34, 36; s8h n = 19, 14; cyp82c4 n = 34, 30; and pdr9 n = 35, 30 in CAS and IS, respectively). (D) Constrained ordination of root bacterial community composition of coumarin pathway mutants, constrained for the interaction between soil and genotype. Ellipses delineate multivariate normal distribution at 95% confidence. Data are from one representative experiment of three (Col-0 n = 17, 15; f6’h1 n = 18, 14; s8h n = 15, 14; cyp82c4 n = 18, 15; and pdr9 n = 17, 14 in CAS and IS, respectively). p values represent significance of separations between genotypes within each soil determined by pairwise PERMANOVA. Only f6’h1 (orange) and s8h (purple) were significantly separated from Col-0 in IS. See also: Figures S1 and S2.

Figure 2

Coumarin Biosynthesis Restructures the Root Microbiota at the ASV Level (A) Number of deASVs detected in indicated mutants compared with Col-0 in each soil. Data are pooled from three experiments (except s8h, which was included in only one), and filtered for ASVs found in at least three samples with RA > 0.05%. Differential enrichment was calculated using a negative binomial generalized log-linear model at an FDR-adjusted p value of 0.05. (B) Family-level taxonomic classification of deASVs in f6’h1 plants growing on IS. Colors indicate if deASVs were enriched or depleted in f6’h1 compared with Col-0. Hypergeometric enrichment test was performed to determine if each family was over- or under-represented in deASV list compared with all detected ASVs. Red asterisks indicate significance with FDR-adjusted p values. (C) Sample-wise aggregated relative abundance of the top three families most significantly over-represented in deASVs: Burkholderiaceae, Rhizobiaceae, and Streptomycetaceae. Each data point represents the average RA aggregated at the family level in a single sample. Significance between genotypes in each soil was determined by Wilcoxon ranked sum test. (D) Overnight growth of Burkholderiaceae bacterial strains in the presence of scopoletin or fraxetin. Optical density (OD) of cultures was normalized to the OD of each strain in the absence of coumarins. Significant differences (p ≤ 0.05 by Tukey’s HSD) in growth compared with the control are indicated for scopoletin (S^∗) and fraxetin (F^∗) to the right of each strain. Data are averages of 2–4 experiments, each with 2–3 technical replicates, per strain. ^∗, ^∗∗, and ^∗∗∗ in (B) and (C) indicate p ≤ 0.05, 0.01, and 0.001, respectively. See also Figures S2 and S3.

Figure 3

Taxonomically Diverse Root Commensals Improve Iron-Limiting Plant Performance (A) Phylogenetic tree of 115-strain SynCom derived from At-RSPHERE culture collection (Bai et al., 2015) used for microbiota reconstitution. Red arrows indicate strains used in (D). (B) Representative images of plants grown for 2 weeks on media containing available (avFe) and unavailable (unavFe) forms of iron inoculated with live SynCom or heat-killed control. (C) SFW and total chlorophyll quantification of Col-0 plants after 2 weeks of growth on indicated iron conditions. Data are pooled from three experiments with avFe and unavFe: n = 42–54 plants per condition, and chlorophyll measured in pooled samples, n = 13–15 per group. Insufficient iron data are from one experiment, n = 18 plants. Letters indicate significant pairwise differences between groups (p-adj ≤ 0.05 by Dunn’s pairwise comparison with Bonferroni correction for SFW, and Tukey’s HSD corrected for multiple comparisons for chlorophyll content). (D) Iron-limiting growth rescue activity of SynCom strains in mono-association. SFW was measured and plotted as percent growth rescue of bacteria-inoculated plants on unavFe compared with the growth deficit between sterile plants on avFe versus unavFe. Black and red lines indicate 0% (axenic plants on unavFe) and 100% growth rescue (axenic plants on avFe), respectively. Data are pooled from 1–4 experiments per strain and normalized to respective sterile controls (n = 18 plants per experiment). Asterisks indicate significance from sterile plants by Wilcoxon ranked sum test with FDR adjustment (^∗, ^∗∗, and ^∗∗∗ indicate p-adj ≤ 0.05, 0.01, and 0.001, respectively). See also Figure S4.

Figure 4

Microbiota-Mediated Plant Growth Rescue Occurs via the Reductive Import of Iron (A and B) (A) SFW and (B) leaf chlorophyll content of indicated mutants in the reductive import of iron pathway grown on unavFe media inoculated with heat-killed or live bacterial SynCom. Total chlorophyll content was measured in pooled leaf samples from six plants. Data are from two independent experiments per genotype (n = 36 plants, 6 chlorophyll samples). Each experiment included Col-0 control (n = 90 plants, 18 chlorophyll samples). Asterisks indicate significance between heat-killed- and live SynCom-inoculated groups by Wilcoxon ranked sum test for SFW and Student’s t test for chlorophyll content (^∗, ^∗∗, and ^∗∗∗ indicate p ≤ 0.05, 0.01, and 0.001, respectively). See also Figure S5.

Figure 5

Plant Biosynthesis and Secretion of Fraxetin Is Necessary for Microbiota-Mediated Growth Rescue (A and B) (A) SFW and (B) leaf chlorophyll content of indicated coumarin biosynthesis and export mutants grown on unavFe media inoculated with heat-killed or live bacterial SynCom. SFW data are from two experiments (n = 36 plants). Chlorophyll content is from one experiment (n = 3 pooled leaf samples). Asterisks indicate significance between heat-killed- and live SynCom-inoculated groups by Wilcoxon ranked sum test for SFW and Student’s t test for chlorophyll content (^∗, ^∗∗, and ^∗∗∗ indicate p ≤ 0.05, 0.01, and 0.001, respectively). See also Figure S5.

Figure 6

Supplementation with Fraxetin Restores Microbiota-Mediated Growth Rescue of f6’h1 and s8h Plants (A and B) (A) SFW and (B) leaf chlorophyll content of Col-0 plants, and f6’h1 plants grown on unavFe supplemented with 50 μM scopoletin and/or fraxetin and inoculated with heat-killed or live SynCom. (C) SFW of Col-0, and f6’h1 and s8h plants grown on unavFe supplemented with 50 μM scopoletin and fraxetin and inoculated with heat-killed or live SynCom. Data in (A) and (B) are from two experiments (n = 30–42 plants, 6 pooled leaf chlorophyll samples). Data in (C) are from a single experiment (n = 18 plants). Letters indicate significant pairwise differences between groups (p-adj ≤ 0.05 by Dunn’s pairwise comparison with Bonferroni correction for SFW, and Tukey’s HSD corrected for multiple comparisons for chlorophyll content). See also Figure S5.

Figure 7

A Bacterial SynCom Improves Plant Iron Nutrition, Relieves the Iron Deficiency Response, and Modulates a Subset of Defense Genes in a Coumarin-Dependent Manner (A) Shoot iron content of Col-0 and f6’h1 plants grown on avFe and unavFe media with a live SynCom or heat-killed control measured by ICP-MS (n = 3–4 pooled plant samples per group). (B) PCA ordination of sample distances between root transcriptional profiles of Col-0 and f6’h1 plants grown for 1 week on avFe or unavFe media inoculated with a live SynCom or heat-killed control. Data are from two pooled experiments (n = 6 samples pooled from 6 plant roots each). (C) Heatmap of median-centered Z scores for 2,440 DEGs identified across samples, arranged by k-means clustering. Significantly enriched iron homeostasis-related and defense-related GO terms of pertinent clusters are indicated on right of heatmap. GO analysis was performed by comparing the indicated DEG cluster to the whole transcriptome (p-adj ≤ 0.05). (D) Expression of select iron deficiency response marker genes. Data are log₂-transformed, normalized counts. Letters in (A) and (D) indicate significant pairwise differences between groups (p-adj ≤ 0.05 by Tukey’s HSD corrected for multiple comparisons). See also Figures S6 and S7.