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

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)

Electrospinning of diphenylalanine nanotubes

Electrospinning from concentrated diphenylalanine solutions in a low-boiling-point solvent results in tubes that are chemically identical to self-assembled tubes, but show different morphologies, especially extreme lengths. Electrospinning of tubes offers more possibilities for manipulation, for example, bridging electrodes in parallel orientation, a possible patterning strategy for electrospun material

Partial rescue of the <i>f6</i>′<i>h1-5</i> growth defect in alkaline medium by co-cultivation with wild-type plants.

<p><i>F6</i>′<i>H1</i> knock-out plants (<i>f6</i>′<i>h1–5</i>) 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 <i>A. thaliana</i> Col-0 plants (wt) (bottom row). Experiments were repeated three times independently with nearly identical outcome.</p

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

<p>(A) Optical spectra of solutions (CH₃CN) of ligands esculetin (<b>1</b>), fraxetin (<b>2</b>) and scopoletin (<b>3</b>), 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: <b>1</b> (blue); <b>1</b>+ NEt₃ (orange); <b>1</b>+ NEt₃+ FeCl₂ (green); <b>1</b>+ NEt₃+ FeCl₃ (red); FeCl₃ (black); FeCl₂ (magenta). Center: <b>2</b> (blue); <b>2</b>+ NEt₃ (orange); <b>2</b>+ NEt₃+ FeCl₂ (green); <b>2</b>+ NEt₃+ FeCl₃ (red). Right: <b>3</b> (blue); <b>3</b>+ NEt₃ (orange); <b>3</b>+ NEt₃+ FeCl₂ (green); <b>3</b>+ 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 <i>m/z</i> 316.02.</p

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

<p>Structures of coumarins and their respective glycosides investigated in this study.</p

Metabolic changes in <i>A. thaliana</i> roots upon cultivation in hydroponic medium without added Fe as identified by GC-MS metabolite profiling.

<p>Polar metabolites were extracted with methanol, derivatized and subjected to GC-MS analysis. Listed are compounds that showed a significant (P<0.05, Student’s t-test) difference (>1.5-fold) in three independent biological experiments. Entries are ordered according to their maximum relative abundance under –Fe conditions; n. i.: not unambiguously identified (no comparison with authentic standard), RI: retention index, R-Match: reverse match (only values above 800 are given).</p

<i>A. thaliana</i> Col-0 plants were exposed to two different conditions causing Fe deficiency.

<p>(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.</p

Metabolic changes in <i>A. thaliana</i> roots upon cultivation in hydroponic medium with alkaline pH as identified by GC-MS metabolite profiling.

<p>Polar metabolites were extracted with methanol, derivatized and subjected to GC-MS analysis. Listed are compounds that showed a significant (P<0.05, Student’s t-test) difference (>1.5-fold) in three independent biological experiments. Entries are ordered according to their maximum relative abundance under alkaline conditions; n. i.: not unambiguously identified (no comparison with authentic standard), RI: retention index, R-Match: reverse match (only values above 800 are given).</p