1. Volume 8, Issue 3, Pages 439-453 (March 2015)
Iron- and Ferritin-Dependent Reactive Oxygen Species Distribution: Impact on Arabidopsis Root System Architecture 
Guilhem Reyt, Soukaina Boudouf, Jossia Boucherez, Frédéric Gaymard, Jean-Francois Briat 
Molecular Plant 
Volume 8, Issue 3, Pages (March 2015)
DOI: /j.molp
Copyright © 2015 The Author Terms and Conditions

2. Figure 1
Ferritin Gene Expression in Arabidopsis Roots and Leaves in Response to Fe Excess.
Two-week-old plants are harvested before (0 h), and after (6 h, 9 h) adding 500 μM Fe(III)-EDTA in the medium.
(A–D) Ferritin transcripts abundance. RNA is extracted from leaves (A, B) or roots (C, D). Expression of AtFer1 (A, C), AtFer2, AtFer3, and AtFer4 (B, D) is analyzed by qRT-PCR. Mean values ± standard error are shown (n = 3). Asterisks indicate significant differences after 6 h of Fe excess (* if P < 0.05 and ** if P < 0.005; Student’s t-test)
(E) Ferritin protein abundance. Protein extracts (10 μg) of leaves or roots are separated by PAGE, blotted, and probed with ferritin antibodies. Loading is evaluated by Coomassie staining.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

3. Figure 2
Root Localization of GUS Expression Driven by AtFer1, AtFer3, or AtFer4 Promoters.
pAtFer::GUS lines are treated with Fe excess (500 μM of Fe(III)-EDTA) or not (50 μM of Fe(III)-EDTA) during 24 h before harvesting.
(A) GUS staining of whole root pAtFer::GUS lines.
(B) GUS staining of PRs and LRs, and on transversal thin cross-sections of mature roots or of emerging LR.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

4. Figure 3 Fe Content and Localization.
Fe content in roots (A) and leaves (B) from 2-week-old Col0 and fer1-3-4 plants, treated with 50 or 500 μM Fe(III)-EDTA during 48 h. Mean values ± standard error are shown (n ≥ 3). Asterisks indicate significant differences between the two genotypes (P < 0.05; Student’s t-test). Fe localization in roots by Perls-DAB staining the tip of PR and in young emerged LR (C) from Col0 plants grown at 50 μM Fe(III)-EDTA. Longitudinal (top) and transversal (bottom) cross-sections in the meristematic apical zone of roots from Col0 and fer1-3-4 plants (D) grown at 50 μM Fe(III)-EDTA. Transversal cross-sections of mature roots at different magnifications (E), from 2-week-old Col0 and fer1-3-4 plants, treated with 50 or 500 μM of Fe(III)-EDTA during 48 h. Arrows indicate Fe-rich structures.
c, cortex; end, endodermis; ep, epidermis; lrc, LR cap; p, pericycle.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

5. Figure 4
Fe-Mediated Oxidative Stress in Roots of Col0 and fer1-3-4 Plants.
Catalase activity (A) and malondialdehyde (MDA) content (B) of roots from Col0 and fer1-3-4 plants grown on MS/2 medium containing 50 μM Fe(III)-EDTA during 7 days, and then transferred on the same medium containing 50 or 250 μM Fe(III)-EDTA during 7 days. Mean values ± standard error are shown (n ≥ 5). Different letters (a, b, or c) indicate statistical differences (P < 0.05; Student’s t-test).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

6. Figure 5 Root Architecture Analysis.
(A) Root architecture from representative wild-type Col0 and fer1-3-4 plants grown on MS/2 medium containing 50 μM Fe(III)-EDTA during 7 days and then transferred on the same medium containing 50 or 500 μM Fe(III)-EDTA during 7 days.
(B) PR length of plants grown on MS/2 medium containing 50 μM Fe(III)-EDTA during 7 days and then transferred on the same medium containing 50, 150, 300, or 500 μM Fe(III)-EDTA during 7 days.
(C) Average length of LR on the same plants as in (B).
(D) LR density (number of emerged LR per centimeter of root branching zone) on the same plants as in (B).
(E) Percentage of LRP of the total LR initiation events within the LR branching zone on the same plants as in (B) but only transferred on a medium containing 50 or 500 μM Fe(III)-EDTA during 3 days. LR length (C), density (D), and proportion of aborted primordia (E) were measured only on LR formed below the location of the PR tip at the moment of the transfer.
Mean values ± standard error are shown (n ≥ 20 plants). Asterisks indicate significant differences between the two genotypes (P < 0.05; Student’s t-test).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

7. Figure 6
Meristem Size of PR and LR of Col0 and fer1-3-4 Plants Grown at 50 or 500 μM Fe-EDTA.
The meristem size of PR (A) or LR (B) was measured on plants grown on MS/2 medium containing 50 μM Fe(III)-EDTA during 7 days and then transferred on the same medium containing 50 or 500 μM Fe(III)-EDTA. For LR, the sizes were determined on the six youngest LR per plant longer than 1.5 mm. The size is obtained by measuring the distance between above the root cap and the transition zone, after propidium iodine staining. Mean values ± standard error are shown (n ≥ 18 plants). Different letters (a, b, or c) indicate statistical differences (P < 0.05; Student’s t-test).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

8. Figure 7 ROS Distribution in the PR Tips.
(A) 3′-(p-Hydroxyphenyl) fluorescein (HPF) and (B) DHE fluorescence photographs in the PR from wild-type Col0 and fer1-3-4 mutant grown on MS/2 medium containing 50 μM Fe(III)-EDTA during 7 days and then transferred on the same medium containing 50 or 500 μM Fe(III)-EDTA during 4 days.
(C) Overlay of the photographs presented in (A) and (B).
(D) Fluorescence intensity of HPF along PR tip.
(E) Staining intensity of DHE along PR tip.
Mean values ± standard error are shown (n ≥ 18 plants). Arrows indicate the transition zone.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

9. Figure 8 ROS Distribution in the LR Tips.
(A) 3′-(p-Hydroxyphenyl) fluorescein (HPF) and (B) DHE fluorescence photographs in the LR from Col0 and fer1-3-4 mutant grown as indicated in Figure 7.
(C) Overlay of the photographs presented in (A) and (B).
(D) Fluorescence intensity of HPF along the LR tip.
(E) Staining intensity of DHE along the LR tip.
Fluorescence intensity is measured on the six youngest LRs per plant longer than 1.5 mm. Mean values ± standard error are shown (n ≥ 18 plants). Arrows indicate the transition zone.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

10. Figure 9 Effects of ROS Level Changes on PR and LR Length.
PR length (A and B) and average LR length (C and D) of plants grown on MS/2 medium containing 50 μM Fe(III)-EDTA during 7 days and then transferred on the same medium containing 50 or 500 μM Fe(III)-EDTA supplemented with 100 μM potassium iodide (KI [A and C]) or 100 μM salicylhydroxamic acid (SHAM [B and D]) during 7 days. LR length (C, D) was measured only on LRs formed below the location of the PR tip at the moment of the transfer. Mean values ± standard error are shown (n ≤ 10 plants). Different letters (a, b, c, or d) indicate statistical differences (P < 0.05; Student’s t-test).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

11. Figure 10 Fe Regulation of Genes Controlling Meristem Activity.
Relative transcript abundance of UPB1 (A), SMR1 (B), SMR5 (C), and SMR7 (D) from roots of Col-0 and fer1-3-4 plants grown on MS/2 medium containing 50 μM Fe(III)-EDTA during 7 days and then transferred on the same medium containing 50 or 500 μM Fe(III)-EDTA for 9 h. Mean values ± standard error are shown (n = 4). Different letters (a, b, or c) indicate statistical differences (P < 0.05; Student’s t-test).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions