Increased sensitivity to iron deficiency in Arabidopsis thaliana overaccumulating nicotianamine

Abstract

Nicotianamine (NA) is a non-protein amino acid derivative synthesized from S-adenosyl L-methionine able to bind several metal ions such as iron, copper, manganese, zinc, or nickel. In plants, NA appears to be involved in iron availability and is essential for the plant to complete its biological cycle. In graminaceous plants, NA is also the precursor in the biosynthesis of phytosiderophores. Arabidopsis lines accumulating 4- and 100-fold more NA than wild-type plants were used in order to evaluate the impact of such an NA overaccumulation on iron homeostasis. The expression of iron-regulated genes including the IRT1/FRO2 iron uptake system is highly induced at the transcript level under both iron-sufficient and iron-deficient conditions. Nevertheless, NA overaccumulation does not interfere with the iron uptake mechanisms since the iron levels are similar in the NA-overaccumulating line and wild-type plants in both roots and leaves under both sufficient and deficient conditions. This observation also suggests that the translocation of iron from the root to the shoot is not affected in the NA-overaccumulating line. However, NA overaccumulation triggers an enhanced sensitivity to iron starvation, associated with a decrease in iron availability. This study draws attention to a particular phenotype where NA in excess paradoxically leads to iron deficiency, probably because of an increase of the NA apoplastic pool sequestering iron. This finding strengthens the notion that extracellular NA in the apoplast could be a major checkpoint to control plant iron homeostasis.

Fig. 1.

Induction of iron uptake mechanisms in Arabidopsis lines overaccumulating NA. Wild-type plants (Columbia ecotype Col-0, black bars) and plants with a 4-fold (K8, grey bars) or a 100-fold (K1, white bars) increase in their NA content were sown on half-strength MS medium and after 5 d transferred to an iron-sufficient medium [50 μM Fe(III)-EDTA] for 12 d. Real-time RT-PCR determination of the relative transcript levels corresponding to the genes involved in the iron uptake, AtIRT1 and AtFRO2 (A), or in the transcriptional regulation of the iron starvation response, AtFIT1 (bHLH29), bHLH38, and bHLH39 (B). Error bars represent the SE of four repetitions. IRT1 protein accumulation in roots (C) was detected using an IRT1 affinity-purified peptide antibody (upper panel). The Coomassie staining shows equal loading (lower panel). Root Fe(III) chelate reductase activity (D) was performed on a mix of five plantlets. Error bars represent the SE of four repetitions.

Fig. 2.

The NA-overaccumulating line is highly sensitive to iron deficiency. (A) The phenotype of Col-0 plants and K1 plants overaccumulating NA sown and transferred after 5 d to an iron-deficient medium (no iron added) for 12 d. (B) Fresh weight and total chlorophyll content (C) of the entire rosette part of 10 wild-type plants (black bars) or 10 NA-overaccumulating plants (white bars) grown under iron-deficient conditions as described above. (D) Kinetics of the root growth of wild-type plants (filled squares) or the NA-overaccumulating line (open squares) transferred 5 d after germination on half-strength MS medium without iron added. Error bars represent the SE of 10 repetitions.

Fig. 3.

Overinduction of iron uptake mechanisms upon iron starvation in plants overaccumulating NA. Col-0 plants (black bars) and K1 plants overaccumulating NA (white bars) were sown for germination on half-strength MS medium and were transferred after 5 d to an iron-deficient medium (no iron added) for 12 d. Real-time RT-PCR determination of FIT1, bHLH38, and bHLH39 (A) or IRT1 and FRO2 (B) transcript levels in roots of iron-starved plants. Error bars represent the SE of four repetitions. IRT1 protein accumulation (C) was detected in a crude protein extract from roots by using an IRT1 affinity-purified peptide antibody. The Coomassie staining shows equal loading. Root Fe(III) chelate reductase activity (D) was determined on a mix of five plantlets. Error bars represent the the SE of four repetitions.

Fig. 4.

Metal contents in an Arabidopsis line overaccumulating NA. Col-0 plants (black bars) and plants overaccumulating NA (white bars) were sown on half-strength MS medium and after 5 d transferred to an iron-sufficient [50 μM Fe(III)-EDTA] or iron-depleted (no iron added) medium for 12 d. Iron (A), copper (B), zinc (C), and manganese (D) concentrations were determined by atomic absorption spectrometry in roots and rosettes. Error bars represent the SE of four repetitions. Asterisks above the bars indicate significant differences (P <0.01; Student's test) between genotypes within the same organs under the same treatment.

Fig. 5.

Decreased water-extractable iron in the NA-overaccumulating line. Col-0 plants and plants overaccumulating NA were sown on half-strength MS medium and were transferred after 5 d to an iron-sufficient [50 μM Fe(III)-EDTA] or iron-deficient medium (no iron added) for an additional 12 d (as indicated below the graph). Soluble iron was extracted in water, then soluble iron (supernatant, grey bars) and the remaining iron (pellet, white bars) were measured by absorbance of Fe²⁺-o-phenanthroline using thioglycolic acid as the reducing agent. Error bars represent the SE of four repetitions. Differences were found to be highly significant (P <0.01; Student's test) between the soluble pools of Col-0 and the K1 line under iron-sufficient condition as well as between the K1 line soluble pools. Differences between the soluble pools of Col-0 and the K1 line under iron starvation are significant (P <0.05; Student's test).