Supporting Information Figs S1–S7 and Tables S1–S3
Fig. S1 Unrooted phylogram for all Arabidopsis MT protein amino acid sequences.
Phylogenetic analysis of amino acid sequences of Arabidopsis MTs were performed using
Clustal X (2.0) for multiple sequence alignment and TreeView X for tree visualization. The tree is drawn to scale, with scale bar representing nucleotide substitutions per site.

Bgl II restriction site
Fig. S2 Alignment of DNA sequences from the MT1a and MT1c RT-PCR amplification products.
Nucleotide sequence alignment of MT1a and MT1c cDNAs amplified with primers #1 and #2 by RT-PCR. The box at 225-230 shows the nucleotide change in the 3’ UTR sequence of
MT1c that creates a Bgl II restriction site, which is absent in MT1a . This polymorphism was used to discriminate between MT1a and MT1c RNA expression in the mt1a-2 and quad-MT mutants. The nucleotide sequence alignment was performed using the online software
MultAlin (multiple sequence alignment by Florence Corpet).

mt1a-2 mt2a-1 mt2b-1 mt3-1 quad
(a)
100
80
60
**
40
20
0
0 0.02
0.04
0.06
0.08
0.1
Paraquat (µM)
(b)
Paraquat (
0 wt (Col-0)
100

Fig. S3 Response of wild type and MT-deficient plants to paraquat.
(a) Wild type (filled circles) and quad-MT mutant (open circles) Arabidopsis seedlings were grown for 8 d on MS medium containing increasing concentrations of paraquat. The control treatment was standard MS medium containing 0.05 µM CuSO
4
and 15 µM ZnSO
4
. Root length was measured after 8 d; results are the means ± SE of 50 seedlings from each genotype. Significant differences from the wild type were determined by Student’s t test and are indicated by one ( P < 0.05) or two ( P < 0.01) asterisks. (b) Fully expanded leaves were detached from soil-grown wild type and MT-deficient mutant Arabidopsis plants and placed on half-strength solidified MS medium without sucrose. A 2 µL drop of 100 µM paraquat dissolved in water or water (0) was placed on each leaf. Leaves were incubated under a transparent cover to maintain high humidity for 2 d before photographs were taken.

800
600
400 wt quad
200
0
0 1 2 3
Days in the dark
4 5
Fig. S4 Chlorophyll content in detached leaves during dark-induced leaf senescence.
Fully expanded leaves were excised from 4-wk old wild type and quad-MT mutant plants and incubated in the dark for 5 d. Chlorophyll content was measured each day. Results are the means ± SE of 4 replicates. There were no significant differences between the wild type and
MT-deficient mutant.

wt (Col-0) mt1a-2 mt2a-1 mt2b-1 mt3-1 quad
Fig. S5. Response of wild type and MT-deficient Arabidopsis plants to Alternaria
brassicicola.
Fully expanded leaves were excised from wild type and MT-deficient mutant plants and inoculated with a 5 µl drop containing spores of Alternaria brassicicola (5

10 5 spores ml -1 ).
Photographs were taken 6 d after inoculation.

wt (Col-0) mt2a-1 mt1a-2 mt2b-1 mt3-1 quad
Fig. S6. Response of wild type and MT-deficient plants to Botrytis cinerea.
Four week-old soil-grown wild type and MT-deficient mutant Arabidopsis plants were sprayinoculated with a suspension of Botrytis cinerea spores (3

10
5
spores ml
-1
). Photographs of representative plants were taken 6 d after inoculation.

wt (Col-0) mt1a-2 mt2a-1 mt2b-1 mt3-1 quad
Fig. S7. Response of wild type and MT-deficient plants to Pseudomonas syringae.
Fully expanded leaves of 4 wk-old soil-grown wild type and MT-deficient mutant Arabidopsis plants were infiltrated with the bacterial strain Pseudomonas syringae pv tomato DC3000. Plants were inoculated by pressing a 1-ml syringe without a needle against the abaxial side of the leaves and forcing the bacterial suspension through the stomata into the intercellular spaces.
Photographs of representative plants were taken 3 d after inoculation.

17
18
19
20
13
14
15
16
21
22
23
24
10
11
12
7
8
5
6
9
2
3
4
Primer # Name
1 MT1 -5’
MT1 -3’
MT1c
MT1c
-5’
-3’
MT2a -5’
MT2a -3’
MT2b -5'
MT2b -3'
MT3 -5’
MT3 -3’
5-TDNA
Tag3-LB
MT2a -5’
MT2 a-3’
MT2b -5’
MT2b -3’
ACT2/8 -5’
ACT2/8 -3’
AtMT1acF
AtMT1acR
AtMT1cF
AtMT1cR
ACT2F
ACT2R
Table S1 Oligonucleotide primers used in this study
Oligonucleotide sequence (5’
→ 3’)
GAGAAGTAAGAGAAATGGCA
GGCAGGTCTTTATTCCAAC
CAGCATGGTCTCAAACCAAGGA
TACATATACTATTTATGATTTAGGTTACA
CCAAGTGGACGACTGAGGTT
CAAACGAGACGTTGGAGACTT
TCTAGAGAATTCATGTCTTGCTGTGGTGG
TGACCCGGGTCATTTGCAGGTACAAGG
CTCTGGATCCATGTCAAGCAACTGCGGA
CTCTAAGCTTTTAGTTGGGGCAGCAAGT
CGTCCGCAATGTGTTATTAAGTTGTCTA
CTACAAATTGCCTTTTCTTATCGAC
CTCTGGATCCATGTCTTGCTGTGGAGGA
CTCTAAGCTTTCACTTGCAGGTGCAAGG
GGCGCCTCGAGATCATCTTCATAAATCT
GGCGCGAATTCAGCAAGAAAGAAGCAAACT
CATCATGGTGTCATGGTTGG
CATGAGGTAATCAGTAAGGTCACG
AGAGAAGTAAGAGAAATGGCAG
GTTTGATCCACAGCTACAGT
ATCAGCATGGTCTCAAACCAAGGA
ACAGCAGGAACAAGTCAAGCAAG
TGCCAATCTACGAGGGTTTC
TCTCTTACAATTTCCCGCTCTG

Table S2 Development of MT-deficient mutants
Mutant
Singles mt1a-2 mt2a-1 mt2b-1 mt3-1
Cross/backcross description
Backcrossed 3x
Backcrossed 2x
Backcrossed 3x
Backcrossed 3x
Cross between non-backcrossed mt1a-2 and mt2a-1
Doubles mt1a-2/mt2a-1 mt1a-2/mt2b-1 mt1a-2/mt3-1 mt2a-1/mt2b-1 mt2a-1/mt3-1 m2b-1/mt3-1
Triples mt1a-2/mt2a-1/mt2b-1 mt1a-2/mt2a-1/mt3-1 mt1a-2/mt2b-1/mt3-1 mt2a-1/mt2b-1/mt3-1
Quadruple mt1a-2/mt2a-1/mt2b-1/mt3-1
Cross between mt1a-2 and mt2b-1 both backcrossed 2x
Cross between mt1a-2 and mt3-1 both backcrossed 3x
Cross between non-backcrossed mt2a-1 and mt2b-1
Cross between non-backcrossed mt2a-1 and mt3-1
Cross between mt2b-1 and mt3-1 both backcrossed 3x
Cross between non-backcrossed mt1a-2/mt2b-1 and mt2a-1
Rescued from F2 progeny of a cross between mt1a-
2/mt2a-1/mt2b-1 and mt1a-2/mt2b-1/mt3-1
Cross between non-backcrossed mt1a-2/mt2b-1 and mt3-1
Rescued from F2 progeny of a cross between mt1a-
2/mt2a-1/mt2b-1 and mt1a-2/mt2b-1/mt3-1
Cross between mt1a-2/mt2a-1/mt2b-1 and mt1a-2/mt2b-
1/mt3-1
Backcrossed 3x

Table S3 Germination rate of seeds collected from plants grown under control and Cu deficiency during reproductive stage
Condition
Control
-Cu
Genotype wt quad wt quad
Germination rate (%)
98.8 ± 1.1
98.8 ± 1.1
100
2.2 ± 2.2
Wild type and quad-MT mutant Arabidopsis plants were grown in a hydroponic system with
0.12 μM CuSO
4 under 12-h light photoperiod for 6 wk until the first flower opened. The photoperiod was changed to 16-h light and plants were grown under either Cu-sufficient conditions (control) or transferred to medium without any CuSO
4
(-Cu). Seeds were collected at
12 wk. Seeds were germinated on ¼ Hoagland’s medium plates containing 0.12 μM CuSO
4,
1% sucrose (w/v) and 1% agar (w/v). Germination was determined as emergence of the radicle at 43 h. Germination rate was calculated from means of 3 individual seed batches with 30 seeds per batch.