(a)
Col: TcNRAMP4
4-1 4-2 WT
Col: TcNRAMP4
4-1 4-2 WT
NRAMP4 actin
(b)
Ws _ev
Ws _ TcNRAMP4 -6
(c)
80 tubulin
3221
4522
NRAMP4
2511
954700
NRAMP4/ tubulin
0,78
211
Col _WT 4-1 4-2 Ws _ev 4-6
60
40
20
(d)
0 10 30 300
CdCl
2
(µM) ZnSO
4
(µM)
Col _WT 4-1 4-2 Ws _ev 4-6
300
200
100
90
60
30
Zn Cd
Supporting Information
Fig. S3 Expression of TcNRAMP4 in A. thaliana accessions Columbia ( Col ) and Wassilewskija ( Ws ) does not modify Zn or Cd sensitivity nor their content.
(a) Semi-quantitative RT-PCR of TcNRAMP4 and Actin (control) expression in the Col 35S::TcNRAMP4 lines ( 4-1 and 4-2 ) and WT. (b) Quantitative RT-PCR analysis of TcNRAMP4 and tubulin (control) expression in Ws
35S::TcNRAMP4 line 4-6 and empty vector control. (c) Root length of the
35S::TcNRAMP4 lines and the control lines grown vertically for 11 d on ABIS medium containing 50 µM FeHBED. The medium was supplemented with 0,
10 and 10 µM CdCl
2
, or 300 µM ZnSO
4
. Values are the average of measurements on 10-12 roots and bars represent SD. (d) Zn and Cd contents in shoots of the 35S::TcNRAMP4 lines and the control lines grown on control
ABIS medium (Zn content) and ABIS with 3
µM CdCl
2
(Cd content).
Metal content determination (different from the method used in the principle manuscript)
Seedlings were grown vertically for 12 d on plates containing ABIS medium (1% agar), supplemented or not with 30 µM CdCl
2 or 300 µM ZnCl
2
. Roots and shoots were harvested separately and their metal content was measured as described in Lanquar et al.
(2005). The dry weight (DW) of the samples was measured after drying at 60
°C for 3 d. The dried samples were digested in a mixture of 1 ml of 70% nitric acid and 5 ml ultrapure H
2
O in a MARS X microwave (CEMCorporation, Matthews, NC, USA)) at
210
°C at 24 bar for 20 min. After dilution in trace-metal-free water, the metal content of the samples was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) using a Vista-MPX,
(Varian, Australia)).