Rosette iron deficiency transcript and microRNA profiling reveals links between copper and iron homeostasis in Arabidopsis thaliana

Abstract

Iron (Fe) is an essential plant micronutrient, and its deficiency limits plant growth and development on alkaline soils. Under Fe deficiency, plant responses include up-regulation of genes involved in Fe uptake from the soil. However, little is known about shoot responses to Fe deficiency. Using microarrays to probe gene expression in Kas-1 and Tsu-1 ecotypes of Arabidopsis thaliana, and comparison with existing Col-0 data, revealed conserved rosette gene expression responses to Fe deficiency. Fe-regulated genes included known metal homeostasis-related genes, and a number of genes of unknown function. Several genes responded to Fe deficiency in both roots and rosettes. Fe deficiency led to up-regulation of Cu,Zn superoxide dismutase (SOD) genes CSD1 and CSD2, and down-regulation of FeSOD genes FSD1 and FSD2. Eight microRNAs were found to respond to Fe deficiency. Three of these (miR397a, miR398a, and miR398b/c) are known to regulate transcripts of Cu-containing proteins, and were down-regulated by Fe deficiency, suggesting that they could be involved in plant adaptation to Fe limitation. Indeed, Fe deficiency led to accumulation of Cu in rosettes, prior to any detectable decrease in Fe concentration. ccs1 mutants that lack functional Cu,ZnSOD proteins were prone to greater oxidative stress under Fe deficiency, indicating that increased Cu concentration under Fe limitation has an important role in oxidative stress prevention. The present results show that Cu accumulation, microRNA regulation, and associated differential expression of Fe and CuSOD genes are coordinated responses to Fe limitation.

Fig. 1.

Three-way Venn diagrams of expression of Fe-regulated genes in rosettes. Numbers represent counts of up-regulated or down-regulated genes in Col-0, Tsu-1, and Kas-1 ecotypes under control or Fe deficiency conditions.

Fig. 2.

Venn diagrams of genes regulated in both rosettes and roots of Arabidopsis in response to Fe deficiency. Numbers represent the counts of genes (A) up-regulated or (B) down-regulated in both tissues specifically in Col-0, Tsu-1, and Kas-1 ecotypes. (C) Four-way Venn diagram of genes that were Fe regulated in any of the Kas-1, Tsu-1, or Col-0 ecotypes.

Fig. 3.

Time course of expression of metal homeostasis-related genes in Kas-1 and Tsu-1 ecotypes. (A) OPT3, (B) FRO3, (C) NRAMP4, (D) COPT2 in rosettes, and (E) COPT2 in roots. n=3 ±SD. *Denotes statistical significance for Kas-1, + denotes statistical significance for Tsu-1 (P < 0.05) between treatments at each time point. +Fe, 50 µM Fe; –Fe, no added Fe.

Fig. 4.

Relative changes (–Fe/+Fe) in expression for miRNAs in Kas-1 and Tsu-1 roots and rosettes in response to Fe deficiency. (A) miR172c, (B) miR397a, (C) miR165, (D) miR166a, (E) miR158a, (F) miR163, (G) miR398a, and (H) miR398b/c. n=3 ±SD. 50 µM Fe; –Fe, no added Fe.

Fig. 5.

Fe deficiency regulation of SOD genes in Kas-1 and Tsu-1 rosettes. (A) CSD1, (B) CSD2, (C) FSD1, and (D) FSD2. n=3 ±SD. *Denotes statistical significance for Kas-1, + denotes statistical significance for Tsu-1 (P < 0.05) between treatments at each time point. +Fe, 50 µM Fe; –Fe, no added Fe.

Fig. 6.

Time course changes in metal concentration of Arabidopsis Kas-1, Tsu-1, and Col-0 ecotypes in response to Fe deficiency. Iron concentration in (A) rosettes, (B) roots; Cu concentration in (C) rosettes, (D) roots; Zn concentration in (E) roots, and (F) rosettes. n=3; +Fe, 50 µM Fe; –Fe, no added Fe.

Fig. 7.

Responses to Fe and/or Cu deficiency in Col-0 rosettes. Changes in (A) Fe and (B) Cu concentration. (C) Changes in miR398a and miR398b/c abundance. – Fe, no added Fe; –Cu no added Cu; –Fe–Cu, omission of both.

Fig. 8.

Fe and/or Cu regulation of gene expression in Col-0 rosettes. (A) FRO3, (B) COPT2, (C) FER1, (D) CSD1, (E) CSD2, and (F) FSD1; n=3 ±SD. Different letters denote statistical significance by ANOVA (P < 0.05), followed by Duncan’s test. Ctrl, 50 µM Fe and 0.5 µM Cu; – Fe, no added Fe and 0.5 µM Cu; –Cu, 50 µM Fe and no added Cu; –Fe–Cu, omission of both.

Fig. 9.

Lipid peroxidation in rosettes of Arabidopsis plants after 3 d growth on –Fe–Cu, or –Fe–Cu solution. n=3 ±SD. Different letters denote statistical significance by ANOVA (P < 0.05), followed by Duncan’s test. +Fe, 50 µM Fe and 0.5 µM Cu; – Fe, no added Fe and 0.5 µM Cu; –Cu, 50 µM Fe and no added Cu; –Fe–Cu, omission of both.