1
Method S1. Phenotype of bzip19-1 mutant on different type of Zn-deficient media
2
Preparation of growth media
3
MGRL-based Zn-deficient medium was modified using general agar for plant growth
4
(Wako Pure Chemical Industries) instead of purified agar (Nacalai tesque). Composition
5
of MS-based Zn-deficient media was 20 mM NH4NO3, 19 mM KNO3, 3 mM
6
CaCl2•2H2O, 1.5 mM MgSO4•7H2O, 1.2 mM KH2PO4, 100 M H3BO3, 100 M
7
MnSO4•4H2O, 100 M Na2•EDTA, 100 M FeSO4•7H2O, 5 M KI, 1 M
8
Na2MoO4•2H2O, 110 nM CoCl2•6H2O, 100 nM CuSO4•5H2O, 2.3 mM MES-KOH,
9
pH5.7, 1.0% (w/v) sucrose, and 1.2% agar. General agar or purified agar was used to
10
solidify the media.
11
Elemental analysis of the growth media
12
Small fragments of the growth media (8 cm2) were excised from unused plates. They
13
were dried at 60°C for 2 days and then digested with ultrapure HNO3. Digestion and
14
elemental analysis were performed as described in “Determination of Zn contents in
15
plant materials” in the section of Experimental procedure.
1
1
Method S2. iTRAQ analysis
2
Sample preparation for iTRAQ reagents
3
Roots (~0.4 g fresh weight) were homogenized with buffer A (50 mM HEPES-KOH,
4
pH 7.5, 5 mM EDTA, 400 mM sucrose, protease inhibitor cocktail). The homogenates
5
were centrifuged at 1,000 × g at 4°C for 20 min, and the supernatants were centrifuged
6
at 8,000 × g at 4°C for 20 min. The su
7
for 60 min to prepare the microsomal fractions. The pellets were washed by bufferA
8
twice under the same condition and then dissolved in 30 µl of iTRAQ buffer (Applied
9
Biosystems). The protein concentration was determined using NanoDrop (Thermo
10
Scientific). These iTRAQ analyses were performed with eight independent biological
11
replicates.
12
Peptide labeling with iTRAQ reagents
13
Aliquots of 20 µl of microsomal protein fraction (2.5 mg ml-1) were reduced with
14
Tris-(2-carboxyethyl) phosphine at 60°C for 60 min and then alkalized with methyl
15
methanethiosulfonate at room temperature for 10 min. Samples were digested with 10
g at 4°C
2
1
µl of trypsin (1 mg ml-1) at 37°C for 16 h. The peptides from Col-0 grown on Basal
2
medium, Col-0 grown on Zn0 medium, bzip19-1 mutant grown on Basal medium, and
3
bzip19-1 mutant grown on Zn0 medium were labeled with the iTRAQ-114, 115, 116,
4
and 117 reagents, respectively, at room temperature for 60 min. Mixed peptides were
5
manually separated with 25, 50, 75, 100, 200, 350, and 1,000 mM KCl using strong
6
cation exchange (Applied Biosystems) and then desalted on Sep-Pak C18 Cartridges
7
(Waters). The labeled peptides were concentrated in a vacuum concentrator.
8
Mass spectrometric analysis
9
iTRAQ analysis was performed using an LTQ Orbitrap XL-HTC-PAL-Paradigm MS4
10
system. The iTRAQ labeled peptides were loaded onto the column (75-μm internal
11
diameter, 15 cm, L-Column; CERI) using a Paradigm MS4 HPLC pump (Michrom
12
BioResources) and an HTC-PAL autosampler (CTC Analytics). Buffers were 0.1% (v/v)
13
acetic acid and 2% (v/v) acetonitrile in water (A) and 0.1% (v/v) acetic acid and 90%
14
(v/v) acetonitrile in water (B). A linear gradient from 5% to 45% B for 70 min was
15
applied, and peptides eluted from the column were introduced directly into an LTQ
16
Orbitrap XL mass spectrometer (Thermo Scientific) at a flow rate of 200 nl min-1 and a
3
1
spray voltage of 2.0 kV. The range of MS scan was m/z 450–1,500 and the top three
2
peaks were subjected to MS/MS analysis. The obtained spectra were compared against
3
data in The Arabidopsis Information Resource (TAIR10; http://www.arabidopsis.org/)
4
using the MASCOT server (version 2.4; Matrix Science) and Proteome Discoverer
5
(version 1.3; Thermo Scientific) with the following search parameters: threshold cut-off
6
at 0.05 in the ion-score cut-off; protein identification cut-off set to two assigned spectra
7
per predicted protein; peptide tolerance at 10 ppm; MS/MS tolerance at ± 0.2 Da;
8
peptide charge of 2+ or 3+; trypsin as the enzyme and allowing up to one missed
9
cleavage; iTRAQ label and methyl methanethiosulfonate on cysteines as a fixed
10
modification; and oxidation on methionine as a variable modification. iTRAQ data for
11
eight biological replicates were analyzed by MASCOT, and only the data with FDR <
12
1% were used for subsequent analyses. Only proteins that were identified more than
13
five times in eight independent experiments were considered. The P-values were
14
calculated by assuming that measurements approximated to a normal distribution.
4