, 200 differing by their Fe to Pi ratio, at pH 5.5. Twenty-four hours after transfer, the 201 seedlings were stained for GUS activity, vol.15

F. ,

, Increasing the Pi content reduces the expression of ALMT1. However, at the highest 204 Pi concentration tested (250 ?M), the expression of ALMT1 is still induced

, However, increasing the Fe content in a medium containing 250 ?M Pi, increases the 207 expression of ALMT1 (Figure 1b), vol.209, p.1

, It 212 prompted us to assay putative presence of iron in the agar using a quantification by 213 ICP-AES (Inductively Coupled Plasma -Absorption Emission Spectrometry). This 214 analysis revealed the presence of 38 ?g Fe, We observed that even without supplementing the -Pi growth medium with Fe

, To avoid the potential toxicity of a high 222 concentration of DFO that could interfere with gene expression, an alternative 223 approach was used to reduce bioavailable iron from the agar. The agar powder was 224 mixed with DFO and then washed (see Experimental procedures). We then tested 225 whether the level of expression of ALMT1 remained low. In -Pi plates made with this 226 washed, DFO-treated agar, the expression of ALMT1 is almost fully abolished, and 227 addition of 15 ?M Fe restored a high level of expression (Figure 2b). We conclude 228 that a strong chelator of Fe suppresses the expression of ALMT1, we reduced the availability of Fe in 218 the growth media using a siderophore

. Duan, Pho81/XPR1), a classical marker of the -Pi stress. SPX1 encodes a protein 234 regulating the activity of the transcription factor PHR1 -a master regulator of the -Pi 235 stress, and is frequently used as a marker of the -Pi transcriptional response. To 236 monitor its expression, we used WT seedlings containing the pSPX1::GUS marker 237, 2008.

. Balzergue, All 246 these observations were confirmed by qRT-PCR (Quantitative Reverse 247 Transcriptase-Polymerase Chain Reaction) (Figure 3b and Figure S2), and with the 248 PPsPase1 (pyrophosphatase [PPi]-specific phosphatase1) gene as an additional 597 bits using Zen black software (Zen black 2012 SP2 Version 11.0), then converted to 598 a maximal projection image. The average nuclei fluorescence intensities were 599 quantified using the Zen blue software, on an agar containing Fe, the expression 241 of ALMT1 is induced at pH 5.5 but not at pH 6.7, 2017.

B. Alonso, S. Chiarenza, H. Javot, and N. Léonhardt, CEA, cadarache) for experimental 607 helps and discussions

, This work was funded by CEA, Investissements d'avenir (DEMETERRES) and

R. Innovation,

X. W. and D. L. , were 610 funded by The National Natural Science Foundation of China

, Support for the microscopy equipment was provided by the Région Provence Alpes 612 Côte d'Azur, the Conseil General of Bouches du Rhône, the French Ministry of 613 Research, the CNRS and the Commissariat à l'Energie Atomique et aux Energies 614 Alternatives. The qRT-PCR machine was funded by Héliobiotech

, Picture of the root tip after the GUS staining, of WT seedlings carrying the 622 pALMT1::GUS marker. Seedlings were grown in low-Pi at pH 5.8 or 7.1, with or 623 without 15 ?M Fe

S. Figure,

, Expression (qRT-PCR) of ALMT1, SPX1 and PPsPase in seedlings roots

, Fe or Al added, transferred for 3 hours in the same original pH condition with or 634 without Al 3+ or Fe 2+ before extraction of root RNAs and qRT-PCR reactions. The 635 conditions for qRT-PCR reactions and the calculations of relative expression were as 636 in Figure 3 and Figure S2, p.1

, The GFP fluorescence was measured (a.u.) in nuclei at the root tip of pSTOP1::GFP-643 STOP1 seedlings

, Three-day-old seedlings were transferred 2h in -Pi plates buffered at the indicated 649 pH, containing 0 or 60 ?M Fe

, Picture of GFP fluorescence at the root tip, in seedlings transferred 2h in -Pi plates 651 without (left) or with (right) 60 ?M Fe

, Box plots indicate the median, the 25 th to 75 th percentiles (box edges) and the min to 654 max range (whiskers)

M. Test,

*. P<0, 0001; NS, not significant 655 (P>0.05); number of nuclei per condition: a), pp.329-346

, Non-transgenic WT seedlings were grown on a -Pi-Fe plate made with the DFO-agar

, Three days old seedlings were treated 6 h (a) or 4 h (b), without or with 60, 125 or 667 250 ?M (a) or 250 ?M (b) MG132, and photographed as in Figure 4b. a and b 668 represent two independent experiments, vol.100

, Figure S7 671 Aluminum stimulates the expression of pALMT1

, made with a washed DFO-674 treated agar, supplemented or not with 15 ?M Al 3+ , before GUS staining, Seedlings were grown four days on a -Pi medium pH 5, vol.5

. Bar, , vol.1

, Figure S8

, Al 3+ promotes the accumulation of GFP-STOP1 in root nuclei

, The GFP fluorescence was measured (a.u.) in nuclei at the root tip of pSTOP1::GFP-682 STOP1 seedlings

, Three-day-old seedlings were transferred 2h in -Pi plates buffered at the indicated 688 pH, containing 0 or 30 ?M Al 3+

, Box plots indicate the median, the 25 th to 75 th percentiles (box edges) and the min to 691 max range (whiskers)

M. Test,

*. , , pp.315-348

, WT and the als3 mutant seedlings carrying the pSTOP1::GFP-STOP1 construct 697 were grown 3 days on a -Pi-Fe plate, transferred to -Pi or -Pi-Fe plates for 2 h, and 698 GFP-fluorescence was pictured by confocal microscopy, vol.100

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, Table S1: Al, Fe and P content (?g/100 mg agar or agarose, p.840

, Seedlings were grown 5 days (for the GUS staining experiment) or 3.5 days (for the 872 qRT-PCR experiment) on a medium not supplemented with Pi and Fe

, The GFP fluorescence was measured (a.u.) in nuclei at the root tip of pSTOP1::GFP-879 STOP1 seedlings

, Top: three-day-old seedlings were transferred 2 h in -Pi plates with the indicated 881 concentration of Fe. Bottom: representative pictures used for measurements, p.882

, b) MG132 promotes the accumulation of GFP-STOP1 in nuclei of the root tip

, under low-Pi without Fe or Al added. Then, they were 886 treated 4 h with or without 250 ?M MG132 before photographed with the confocal 887 microscope. One representative picture is shown for each condition (see Figure S6 888 for additional pictures), Seedlings carrying the pSTOP1::GFP-STOP1 reporter were grown 3 days in agar 885 (Agar Sigma A1296, Table S1)

, Left: three-day-old seedlings were transferred for the indicated time in -Pi plates 891 containing 0 or 60 ?M Fe. Right: representative pictures used for measurements

, Top: three-day-old seedlings were transferred 2 h in -Pi plates buffered at the 893 indicated pH, containing 0 or 60 ?M Fe. Bottom: representative pictures used for 894 measurements

, Box plots indicate the median, the 25 th to 75 th percentiles (box edges) and the min to 897 max range (whiskers)

M. Test,

*. P<0,

*. P&lt;0, 001; *P<0.5; NS, 898 not significant (P>0.05); number of nuclei per condition: a) 94-154; b), p.87

, Al 3+ promotes the accumulation of GFP-STOP1 in root nuclei

, The GFP fluorescence was measured (a.u.) in nuclei at the root tip of pSTOP1::GFP-905 STOP1 seedlings

, Top: three-day-old seedlings were transferred 2 h in -Pi plates with the indicated 907 concentration of Al 3+ . Bottom: representative pictures used for measurements

, Left: three-day-old seedlings were transferred for the indicated time in -Pi plates 909 containing 0 or 30 ?M Al 3+ . Right: representative pictures used for measurements

, Top: three-day-old seedlings were transferred 2 h in -Pi plates buffered at the 911 indicated pH, containing 0 or 30 ?M Al 3+ . Bottom: representative pictures used for 912 measurements

, Box plots indicate the median, the 25 th to 75 th percentiles (box edges) and the min to 914 max range (whiskers)

M. Test,

*. P&lt;0,

. **p&lt;0, 01; NS, not 915 significant (P>0.05); number of nuclei per condition: a) 80-143; b) 102-125; c), p.86