Metabolome analysis of Arabidopsis thaliana roots identifies a key metabolic pathway for iron acquisition

Abstract

Fe deficiency compromises both human health and plant productivity. Thus, it is important to understand plant Fe acquisition strategies for the development of crop plants which are more Fe-efficient under Fe-limited conditions, such as alkaline soils, and have higher Fe density in their edible tissues. Root secretion of phenolic compounds has long been hypothesized to be a component of the reduction strategy of Fe acquisition in non-graminaceous plants. We therefore subjected roots of Arabidopsis thaliana plants grown under Fe-replete and Fe-deplete conditions to comprehensive metabolome analysis by gas chromatography-mass spectrometry and ultra-pressure liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. Scopoletin and other coumarins were found among the metabolites showing the strongest response to two different Fe-limited conditions, the cultivation in Fe-free medium and in medium with an alkaline pH. A coumarin biosynthesis mutant defective in ortho-hydroxylation of cinnamic acids was unable to grow on alkaline soil in the absence of Fe fertilization. Co-cultivation with wild-type plants partially rescued the Fe deficiency phenotype indicating a contribution of extracellular coumarins to Fe solubilization. Indeed, coumarins were detected in root exudates of wild-type plants. Direct infusion mass spectrometry as well as UV/vis spectroscopy indicated that coumarins are acting both as reductants of Fe(III) and as ligands of Fe(II).

Figure 1. A. thaliana Col-0 plants were exposed to two different conditions causing Fe deficiency.

(A) Plants were grown hydroponically in 1/10 Hoagland Solution for six weeks. Plants were cultivated either at a pH of 5.7 with Fe-HBED as Fe source (Control), at a pH of 7.7 with Fe-HBED as Fe source (pH 7.7), or the final two weeks at a pH of 5.7 without Fe-HBED (−Fe). (B) Fe concentrations in roots (blue bars) and shoots (red bars) were determined by ICP-OES. The means of three independent biological experiments are displayed. Error bars indicate standard deviation. Significant differences to plants grown under control conditions were determined by Student’s t-test, *P<0.05.

Figure 2. The root metabolome changes triggered by two different conditions of Fe deficiency partially overlap.

Venn diagrams display the total number of significant metabolic changes detected by GC-MS (A) and UPLC-ESI-QTOF-MS (B) when comparing growth in the presence of Fe with growth in Fe-free medium for two weeks, or growth at pH 5.7 with growth at pH 7.7. Detailed analysis focused on the metabolites and features (m/z – retention time pairs) that were shared by the two conditions of Fe deficiency (see Tables 1 and 2, Tables S1 and S2 in File S6).

Figure 3. Citrate and malate showed strong increases of root concentrations in response to Fe deficiency.

A. thaliana Col-0 plants were cultivated hydroponically and in separate sets of experiments exposed to two different conditions of Fe deficiency. First, root metabolomes of plants cultivated in control conditions (+Fe, white bars) were compared to those grown in Fe-free medium for two weeks (−Fe, dark grey bars). Second, plants were cultivated at pH 5.7 (light grey bars) and at pH 7.7 (black bars). Shown is the quantification of citrate and malate, the most prominent changes detected by GC-MS (see Tables 1 and 2). The means of three independent biological experiments are displayed. Error bars indicate standard deviation. Significant differences between respective control and Fe deficiency conditions were determined by Student’s t-test, *P<0.05.

Figure 4. Coumarins were among the compounds most strongly responding to Fe deficiency as identified in UPLC-ESI-QTOF-MS-generated metabolite profiles of A. thaliana wild-type roots.

The coumarin response to Fe deficiency is illustrated here by showing peak areas of the quantifier ions (ESI+ ionization mode, see Table S1 in File S6) in metabolite profiles of A. thaliana Col-0 plants cultivated either at pH 5.7 (light grey bars) or at pH 7.7 (black bars). Identification was based on a comparison with authentic standards. The means of three independent biological experiments are displayed. Error bars indicate standard deviation. Significant differences between control (pH 5.7) and Fe deficiency (pH 7.7) conditions were determined by Student’s t-test, *P<0.05.

Figure 5. Structures of coumarins and their respective glycosides investigated in this study.

Figure 6. Growth of coumarin-deficient f6′h1 T-DNA insertion mutants is strongly impaired under Fe-limited alkaline conditions.

(A) A. thaliana wild type plants (wt) and f6′h1 T-DNA insertion mutants f6′h1-1 and f6′h1-5 were grown on normal soil (left) and on soil alkalinized through the addition of CaO (right). (B) Overexpression of F6′H1 under control of the 35S promoter in f6′h1-5 fully rescues the mutant phenotype on alkaline soil. Shown are two independent transgenic lines grown alongside the wild type and the f6′h1-5 mutant on alkaline soil. (C) The mutant phenotype on alkaline soil can be partially rescued by Fe fertilization through watering with 0.5% Fetrilon (Fe-EDTA solution). (D) Col-0 wild type plants (wt, left) and f6′h1-5 mutant plants (right) were cultivated for six weeks in hydroponic medium with a pH of 7.7 instead of 5.7. All experiments were repeated three times independently with nearly identical outcome. The individual plants shown represent the range of phenotypes observed.

Figure 7. Partial rescue of the f6′h1-5 growth defect in alkaline medium by co-cultivation with wild-type plants.

F6′H1 knock-out plants (f6′h1–5) were co-cultivated for four weeks in alkaline Hoagland’s solution (pH 7.7) either with plants of the same mutant genotype (top row) or with A. thaliana Col-0 plants (wt) (bottom row). Experiments were repeated three times independently with nearly identical outcome.

Figure 8. UV/vis spectroscopy and direct-infusion ESI-QTOF-MS demonstrate reduction of Fe(III) by coumarins and the formation of Fe(II)-coumarin complexes in vitro.

(A) Optical spectra of solutions (CH₃CN) of ligands esculetin (1), fraxetin (2) and scopoletin (3), of iron salts, of mixtures of ligands and 2 equiv of NEt₃, and of mixtures containing 3 equiv of ligand, 6 equiv of NEt₃, and 1 equiv of FeCl₂ or FeCl₃. Left: 1 (blue); 1+ NEt₃ (orange); 1+ NEt₃+ FeCl₂ (green); 1+ NEt₃+ FeCl₃ (red); FeCl₃ (black); FeCl₂ (magenta). Center: 2 (blue); 2+ NEt₃ (orange); 2+ NEt₃+ FeCl₂ (green); 2+ NEt₃+ FeCl₃ (red). Right: 3 (blue); 3+ NEt₃ (orange); 3+ NEt₃+ FeCl₂ (green); 3+ NEt₃+ FeCl₃ (red). (B) Interpretation of the UV/vis spectra depicted in (A). (C) Results of direct-infusion ESI-QTOF-MS of a mixture of Fe(II) with synthesized and purified scopoletin. (D) MS-MS spectrum of the main signal at m/z 316.02.