SUPPORTING INFORMATION
Figure S1. Determination of Trx-isoform concentrations in the chloroplast stroma.
Chloroplasts were isolated from wild-type Arabidopsis leaves. Stromal proteins and
recombinant proteins were loaded on sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) in a dilution series and then immunodetected with
indicated antibodies. The amount of Trx isoform proteins was quantified on the western
blots by measuring the band intensity using LAS3000UVmini and Multi Gauge 3.1
Software (Fujifilm, Japan). For Trx f1 and Trx f2, all band signals containing minor
bands in the stromal protein were used to calculate the protein amount. Asterisks
indicate non-specific bands cross-reacting with Trx m3 or Trx y1 antibody.
Figure S2. Identification and characterization of the trx m2 T-DNA insertion
mutants.
(a) Schematic diagram of the structure of Trx m2 gene. The T-DNA insertion sites are
indicated in the diagram, and their nucleotide positions are described in the text. The
black box indicates the coding region. Black arrowheads indicate the position of primers
used for genotyping.
(b) Genotyping of the trx m2-1 (SALK_123570) and trx m2-2 (SAIL_100_E02) mutants.
Genomic DNA extracted from each individual was used as a template for PCR with
three primers, LP1, RP1, and LBb1.3 for the trx m2-1 mutant and LP2, RP2, and LB1
for the trx m2-2 mutant. The wild-type genome shows a band of approximately 1.1 kb,
whereas the T-DNA-inserted genome shows a band of approximately 0.5 kb for the trx
m2-1 mutant and 0.6 kb for the trx m2-2 mutant. Asterisk indicates non-specific
PCR-amplified band.
(c) Accumulation of Trx m proteins in the trx m mutants. Chloroplast stromal proteins
prepared from the wild-type, and the trx m single, double, and triple mutants were
immunodetected with specific antibodies against Trx m1, m2, m4, and CYP20-3
proteins. Protein samples corresponding to 10 µg stromal proteins (100%) were loaded
on each lane.
Figure S3. Screening of the Trx m RNAi transgenic plants.
(a) T1 plants transformed with the Trx m12 RNAi construct. (b) T1 plants transformed
with the Trx m124 RNAi construct. Transgenic plants were screened for
kanamycin-resistance. (c) Western blotting analysis of Trx m protein in the Trx m RNAi
plants. Total leaf proteins were extracted from the wild-type and the T1 generation of
Trx m RNAi transgenic plants and immunodetected with specific antibodies against Trx
m1, m2, m4, and CYP20-3 proteins. Protein samples corresponding to 50 µg total leaf
proteins (100%) were loaded on each lane.
Figure S4. Accumulation of reactive oxygen species (ROS) in the wild type and the
trx mutants.
Accumulation of hydroperoxide and superoxide was detected in 4-week-old wild type
plants and trx mutants. (A) Hydrogen peroxide (H2O2) levels were determined by
staining with 3’,3-diaminobenzidine (DAB). Aerial portions of the plants were detached
from the roots, vacuum-infiltrated with a solution containing 1 mg mL-1 DAB for 60
min, and incubated in the same solution under the dark, growth-light conditions (GL, 50
µmol photons m-2 s-1), or high-light conditions (HL, 800 µmol photons m-2 s-1) for 2 h.
(B) Superoxide anion radical (O2-) levels were determined by staining with nitroblue
tetrazolium (NBT). After incubation under the dark, growth-light conditions (GL) or
high-light conditions (HL) for 2 h, aerial portions of the plants were detached from the
roots, vacuum-infiltrated with a solution containing 1 mg mL-1 NTB for 10 min, and
incubated in the dark for 2 h. Stained leaves were boiled in 90% (v/v) ethanol for 5 min
and photographed. Similar results were obtained from three independent experiments.
Bar = 10 mm.
Table S1. Fresh weight, chlorophyll content and maximum quantum efficiency of
photosystem II (Fv/Fm) of the wild type, trx m and trx f1.1 mutants under
continuous light conditions.
Seedlings were grown under continuous light conditions (50 µmol photons m–2 s–1) for
4 weeks. The chlorophyll content was estimated by SPAD-502 Plus (Konica Minolta,
Japan). Each value is the mean ± standard deviation of 10 independent plants.
Table S2. Chlorophyll fluorescence parameters in the wild type and Trx m RNAi
plants.
Transgenic plants were screened for kanamycin resistance. Chlorophyll fluorescence
parameters were measured in Trx m RNAi lines after 30 min of dark adaptation. Actinic
light intensity was 70 µmol photons m-2 s-1. Each value in the wild type is the mean ±
standard deviation of 5 independent plants.
Table S3. Phenotypic analysis of the wild type, trx m124 and trxf1.1 mutants.
Seedlings were grown under continuous light conditions (50 µmol photons m–2 s–1). The
chlorophyll content was estimated by SPAD-502 Plus (Konica Minolta, Japan). Each
value is the mean ± standard deviation of 10 independent plants. DAS, days after
sowing
Table S4. Primers used in this study.