Supporting Information
Supplementary Figure Legends
Figure S 1. Overview of the experimental procedure and images of plants. A: Work flow of the differential
protein expression experiment (black arrows). The HA7-FIT overexpression line HA-FIT 8, wild-type Col-0 and
the fit knock-out line fit-3 were hydroponically grown in ¼-strength liquid Hoagland medium with 10 µM iron
(+Fe). The medium was refreshed every 7 days. After 5 weeks the plants were divided into two fractions, one
of which was supplied 10 µM iron (+Fe) medium and the other was supplied with medium devoid of iron (-Fe)
for one week. Total protein was extracted from the roots and 2-DGE was performed. In this overview only one
out of three biological replicates is represented. 2-DGE was performed in three technical replicates for each
biological replicate (n=9). The gels were stained with colloidal Coomassie and scanned. The images were
analyzed with the PDQuest v.8 software (Bio-Rad) and the resulting data were further analyzed with the NIA
Array Analysis Tool. Significantly regulated protein spots were excised from the gels and identified by mass
spectrometry. The pink arrows indicate the branching points to RNA analysis. RNA was extracted from the
same root samples as have been used for protein extraction and subjected to transcriptome and reverse
transcription-qPCR analysis. B-G: Photos of leaves of six week-old HA-FIT 8 (B, C), Col-0 (D, E) and fit-3 plants
(F, G) grown with sufficient iron supply (B, D, F) or under iron deficient conditions (C, E, G) during the last
week. H-M: Roots of six week-old HA-FIT 8 (H, I), Col-0 (J, K) and fit-3 plants (L, M) grown with sufficient iron
supply (H, J, L) or under iron deficient conditions (I, K, M) during the last week.
Figure S 2. Root iron content of six week-old Col-0, fit-3 and HA-FIT 8 plants that were grown under sufficient
iron supply (black bars) or under iron-deficient conditions (gray bars) during the 6th week. Different
characters represent significance in the respective comparison (p ≤ 0.05).
Figure S 3. Overview of the experimental setup of differential protein expression analysis. 2-D gels were made
from three independent biological replicates (B1 - B3) of roots from six week-old hydroponically grown
Arabidopsis plants that have been supplied with (+Fe) or without iron (-Fe) during the last week. Per biological
replicate three technical replicates (T1 - T3) were made. Gels of the corresponding biological and technical
replicates were grouped into replicate groups so each replicate group comprised nine gels.
Figure S 4. The artificial master gel representing the averaged intensities of all matched spots in the 2-DGE
analysis (top) and the detailed view of an example spot across all replicates in each replicate group with the
histogram of the individual spot values (bottom). The red dot marks the position of the example spot
(AT3G07720) in the master gel. The red arrow indicates the close view of the respective position of the
master gel in the bottom panel. The color of the bar under each individual spot represents the respective
replicate group as indicated in the legend.
Figure S 5. Overview of the numbers of identified proteins. Out of 1183 spots that have been analyzed by
PDQuest (Biorad) 266 were potentially regulated. The values of these 266 spots have been analyzed by PCA
and 72 of them were significantly regulated (p ≤ 0.05). In parallel to PCA pairwise comparisons between
different iron conditions within each line as well as between the lines at the same iron condition have been
performed. This resulted in 111 significantly regulated spots (p ≤ 0.05). All the 72 spots that had been found
by PCA were included in the resulting spots of the pairwise comparisons. The 111 regulated spots were
subjected to LC-ESI-MS/MS and 104 of these spots could be identified corresponding to 92 protein IDs.
Figure S 6. Master 2-D gel image with the SSPs of the regulated protein spots. Blue triangles mark the
positions of the regulated spots and the respective SSP is placed underneath each triangle.
Figure S 7. A: PCA scores-blot of the six replicate groups of the differential protein expression analysis. B:
Hierarchical clustering of the six replicate groups. PCA and Clustering were performed with the NIA Array
Analysis Tool [52].
Figure S 8. Hierarchical clustering of all 2-D gels. The biological replicates are denominated B1 - B3 and the
technical replicates T1 - T3. Hierarchical clustering was performed with the NIA Array Analysis Tool [52].
Figure S 9. Confirmation of selected microarray results by reverse transcription-qPCR. Gene expression
analysis of eight iron-regulated genes. A: The mean expression values of the genes from RT-qPCR analysis. B:
Heatmaps of microarray and RT-qPCR-based gene expression data. Only the significant fold change values of
both analyses have been used to create the respective heatmaps. The heatmaps have been created with the
PermutMatrix v.1.9.3.software [95].
Figure S 10. Comparison of the regulation of identified proteins with their respective gene regulation in fit-3,
Col-0 and HA-FIT 8 under different iron conditions. The heatmaps have been created with the PermutMatrix
v.1.9.3 software [95].
Figure S 11. Tree view of GO terms (molecular functions) enriched in proteins that have been found in at least
two forms. The GO terms have been analyzed with the VirtualPlant 1.3 software [64]. The image was
generated by the software and further processed.
Figure S 12. Tree view of GO terms (molecular functions) enriched in genes/proteins with contradictory
regulation at the protein and the transcriptional level. The GO terms have been analyzed with the VirtualPlant
1.3 software [64]. The image was generated by the software and further processed.
Figure S 13. Tree view of GO terms (biological processes) enriched in proteins that have been found in at least
two forms. The GO terms have been analyzed with the VirtualPlant 1.3 software [64]. The image was
generated by the software and further processed.
Figure S 14. Tree view of GO terms (molecular functions) enriched in proteins that were down-regulated in fit3 compared to Col-0 and HA-FIT 8. The GO terms have been analyzed with the VirtualPlant 1.3 software [64].
The image was generated by the software and further processed.
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Supplementary Figures
Figure S 1. Overview of the experimental procedure and images of plants. A: Work flow of the differential protein expression experiment (black
arrows). The HA7-FIT overexpression line HA-FIT 8, wild-type Col-0 and the fit knock-out line fit-3 were hydroponically grown in ¼-strength liquid
Hoagland medium with 10 µM iron (+Fe). The medium was refreshed every 7 days. After 5 weeks the plants were divided into two fractions, one
of which was supplied 10 µM iron (+Fe) medium and the other was supplied with medium devoid of iron (-Fe) for one week. Total protein was
extracted from the roots and 2-DGE was performed. In this overview only one out of three biological replicates is represented. 2-DGE was
performed in three technical replicates for each biological replicate (n=9). The gels were stained with colloidal Coomassie and scanned. The
images were analyzed with the PDQuest v.8 software (Bio-Rad) and the resulting data were further analyzed with the NIA Array Analysis Tool.
Significantly regulated protein spots were excised from the gels and identified by mass spectrometry. The pink arrows indicate the branching
points to RNA analysis. RNA was extracted from the same root samples as have been used for protein extraction and subjected to transcriptome
and reverse transcription-qPCR analysis. B-G: Photos of leaves of six week-old HA-FIT 8 (B, C), Col-0 (D, E) and fit-3 plants (F, G) grown with
sufficient iron supply (B, D, F) or under iron deficient conditions (C, E, G) during the last week. H-M: Roots of six week-old HA-FIT 8 (H, I), Col-0 (J,
K) and fit-3 plants (L, M) grown with sufficient iron supply (H, J, L) or under iron deficient conditions (I, K, M) during the last week.
Figure S 2. Root iron content of six week-old Col-0, fit-3 and HA-FIT 8 plants that were grown under sufficient iron supply (black bars) or under
iron-deficient conditions (gray bars) during the 6th week. Different characters represent significance in the respective comparison (p ≤ 0.05).
HA-FIT 8 +Fe
Col-0 +Fe
fit-3 +Fe
HA-FIT 8 -Fe
Col-0 -Fe
fit-3 -Fe
Figure S 3. Overview of the experimental setup of differential protein expression analysis. 2-D gels were made from three independent
biological replicates (B1 - B3) of roots from six week-old hydroponically grown Arabidopsis plants that have been supplied with (+Fe) or without
iron (-Fe) during the last week. Per biological replicate three technical replicates (T1 - T3) were made. Gels of the corresponding biological and
technical replicates were grouped into replicate groups so each replicate group comprised nine gels.
Figure S 4. The artificial master gel representing the averaged intensities of all matched spots in the 2-DGE analysis (top) and the detailed view
of an example spot across all replicates in each replicate group with the histogram of the individual spot values (bottom). The red dot marks the
position of the example spot (AT3G07720) in the master gel. The red arrow indicates the close view of the respective position of the master gel
in the bottom panel. The color of the bar under each individual spot represents the respective replicate group as indicated in the legend.
Figure S 5. Overview of the numbers of identified proteins. Out of 1183 spots that have been analyzed by PDQuest (Biorad) 266 were potentially
regulated. The values of these 266 spots have been analyzed by PCA and 72 of them were significantly regulated (p ≤ 0.05). In parallel to PCA
pairwise comparisons between different iron conditions within each line as well as between the lines at the same iron condition have been
performed. This resulted in 111 significantly regulated spots (p ≤ 0.05). All the 72 spots that had been found by PCA were included in the
resulting spots of the pairwise comparisons. The 111 regulated spots were subjected to LC-ESI-MS/MS and 104 of these spots could be identified
corresponding to 92 protein IDs.
Figure S 6. Master 2-D gel image with the SSPs of the regulated protein spots. Blue triangles mark the positions of the regulated spots and the
respective SSP is placed underneath each triangle.
Figure S 7. A: PCA scores-blot of the six replicate groups of the differential protein expression analysis. B: Hierarchical clustering of the six
replicate groups. PCA and Clustering were performed with the NIA Array Analysis Tool [52].
Figure S 8. Hierarchical clustering of all 2-D gels. The biological replicates are denominated B1 - B3 and the technical replicates T1 - T3.
Hierarchical clustering was performed with the NIA Array Analysis Tool [52].
Figure S 9. Confirmation of selected microarray results by reverse transcription-qPCR. Gene expression analysis of eight iron-regulated genes. A:
The mean expression values of the genes from RT-qPCR analysis. B: Heatmaps of microarray and RT-qPCR-based gene expression data. Only the
significant fold change values of both analyses have been used to create the respective heatmaps. The heatmaps have been created with the
PermutMatrix v.1.9.3.software [95].
Figure S 10. Comparison of the regulation of identified proteins with their respective gene regulation in fit-3, Col-0 and HA-FIT 8 under different
iron conditions. The heatmaps have been created with the PermutMatrix v.1.9.3 software [95].
Figure S 11. Tree view of GO terms (molecular functions) enriched in proteins that have been found in at least two forms. The GO terms have
been analyzed with the VirtualPlant 1.3 software [64]. The image was generated by the software and further processed.
Figure S 12. Tree view of GO terms (molecular functions) enriched in genes/proteins with contradictory regulation at the protein and the
transcriptional level. The GO terms have been analyzed with the VirtualPlant 1.3 software [64]. The image was generated by the software and
further processed.
Figure S 13. Tree view of GO terms (biological processes) enriched in proteins that have been found in at least two forms. The GO terms have
been analyzed with the VirtualPlant 1.3 software [64]. The image was generated by the software and further processed.
Figure S 14. Tree view of GO terms (molecular functions) enriched in proteins that were down-regulated in fit-3 compared to Col-0 and HA-FIT 8.
The GO terms have been analyzed with the VirtualPlant 1.3 software [64]. The image was generated by the software and further processed.
Figure S 15. Tree view of GO terms (biological processes) enriched in proteins that were down-regulated in fit-3 compared to Col-0 and HA-FIT 8.
The GO terms have been analyzed with the VirtualPlant 1.3 software [64]. The image was generated by the software and further processed.