Supplemental Figure 1: Increased Fe deficiency gene expression in roots of nas4x-2

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1 Supplemental Figure 1: Increased Fe deficiency gene expression in roots of nas4x-2 IRT1, FRO2 and FIT expression levels in roots of the wild-type, nas4x- 1 and nas4x-2, showing that in both nas mutants the three genes were induced compared to the wild-type. Plants were grown in a hydroponic system under Fe-sufficient conditions up to the LVS. Gene expression was determined by reverse transcription-qpcr. The result has been reproduced in three independent experiments. 1

2 Supplemental Figure 2: Fe accumulated in leaves of nas4x-2 but not in leaves of frd3 nas4x-2 Perls Fe staining of middle-aged leaves, confirming that frd3 nas4x-2 plants did not accumulate Fe in the leaves. Fe deposits are observed in nas4x-2 leaf veins and hydathodes, shown with higher magnification in the circles (compare with Fig. 6); bar = 1 cm. 2

3 Supplemental Figure 3: Fe accumulated in roots of nas4x-2 and frd3 nas4x-2 Plant material was harvested from plants in the RS. A, Perls Fe staining of roots, showing Fe accumulation in the steles of roots that have a frd3 background. Note that Fe accumulated in frd3 nas4x-2 roots, indicating that the transport of Fe towards the central stele was not affected by loss of NA; bar = 1 cm B, Fe contents per g dry weight of roots, demonstrating a high Fe content in roots with a frd3 background (error bars are standard deviations; n= 4). Numbers 1-6 indicate p < 0.05 in the comparisons 1 nas4x-2 vs WT; 2 frd3 vs. WT; 3 frd3 nas4x-2 vs. WT; 4 frd3 vs. nas4x-2; 5 frd3 nas4x-2 vs. nas4x-2; 6 frd3 nas4x-2 vs. frd3. 3

4 Supplemental Figure 4: Malate, 2-oxoglutarate and succinate contents were not affected by frd3 and nas4x-2 mutations Contents of malate, 2-oxoglutarate and succinate in the xylem saps, showing no significant changes between the mutants and wild type; error bars are standard deviations; n= 5; p<

5 Supplemental Figure 5: Perls Fe staining signals in sections of wild-type and nas4x-2 leaves Enlargement of Fig. 6B; Perls Fe staining signals in 7 µm sections of wild-type and nas4x-2 young and middle-aged leaves, showing Fe deposits in the phloem of the vessels in nas4x-2 (indicated by arrows); Xy = xylem; Ph = phloem; bar = 50 µm. 5

6 Supplemental Figure 6: nas4x-2 plants were late-flowering Percentage of nas4x-2 and wild-type plants in the flowering stage during a growth period of up to ten weeks; n=

7 Supplemental Figure 7: NAS genes were not expressed in wild-type carpels and stamens NAS1, NAS2, NAS3 and NAS4 expression in sepals, petals, carpels and stamens of wild-type plants, showing that only NAS3 was expressed in flowers and only in sepals and petals. Gene expression was determined by reverse transcription-qpcr; error bars are standard deviations; n= 3. 7

8 Supplemental Figure 8: nas4x-2 sterility was rescued by Fe and a combination of Fe and Zn spraying Images of nas4x-2 flowering plants that were not treated with fertilizer as control (co), sprayed with 0.1% ZnSO 4 (+ Zn), 0.05% Fe EDDHA (+ Fe) and a combination of 0.1% ZnSO 4 and 0.05% Fe EDDHA (+ Zn/Fe). The arrows point to developed siliques. The numbers indicate the number of rescued plants producing siliques out of five tested plants. 8