A
0
12
24
36
(h)
CaRLK1
CaAct
B
0
BY
2
4
6
0
RLKox
2
4
6
(d)
CaRLK1
NtGLB1
NtEF1α
Figure S1. (A) Expression of CaRLK1 mRNA in response to hypoxia as induced by submergence. The effect of
submergence was analysed by the harvesting of whole plant samples every 12 h after the treatment. A timecourse experiment was performed after the treatment with sterile distilled water. The relative levels of CaRLK1
and Capsicum annuum actin (CaAct) transcripts were determined by semi-quantitative RT-PCR. (B) The
ectopic expression of the CaRLK1 gene regulates amino acid biosynthesis-related genes. Samples were
harvested every two d during suspension culturing. Day 0 represents the first day of cell transfer. Levels of
NtGLB1 and Nicotiana tobaccum elongation factor 1 alpha (NtEF1α) transcripts were determined by semiquantitative RT-PCR. The NtEF1α transcripts were amplified as a loading control. One representative
experiment is shown in the figure. This analysis was performed with appropriate primers (Table S1).
Figure S1
A
150
50
AA (μmol·g-1 DW)
40
BY
BY
RLKox
RLKox
100
30
20
50
10
0
Pro
Asn
Gly
Tyr
Cys
Ser
Glu
Asp
Phe
Met
Ile
Leu
Val
Thr
His
Trp
B
Lys
Arg
0
Gln
Dissolved Oxygen (mg·L-1)
8
6
4
2
Figure S2. (A) Quantification of free amino acids. Each
composition was determined using Hitachi’s Amino Acid
Analyzer. Error bars represent the standard deviation from the
mean (n = 2). (B) Validation of the hypoxic conditions that
were induced by the suspension cell culture (A). Dissolved
oxygen concentrations in liquid culture medium were analysed
after 7 d of culturing. Error bars represent the standard error
from the mean (n = 3). The differences between the three
conditions are significant at P<0.05.
0
Figure S2
B
A
BY
NtRBOHD
43 KDa
RLKox
BY
95
43
RLKox
200 μm
Figure S3. (A) NtRBOHD protein levels were increased in the RLKox cells. The proteins were extracted,
resolved by SDS-PAGE, and analysed by western blot with the anti-NtRBOHD antibody, which was
directed against 2 peptides of the NtRBOHD protein, as described in Simon-Plas et al. (2002). A total
of 25 μg of protein was loaded. A protein band (43 KDa) that was used as a loading control was
detected non-specifically. The molecular mass markers that are indicated on the right sides denote
the molecular masses in kDa. (B) Detection of hydrogen peroxide by DAB staining in N. tabacumoriginated cells. Red bar indicates length.
Figure S3
WT
RLKox 3
Figure S4. Recovery phenotype of wild-type N. benthamiana and
transgenic plants. Four-week-old seedlings were flooded for 8 d
and photographed on d 25 after the termination of the flood
treatment.
Figure S4