Presentation on theme: "Figure S1 BY RLKox 0 2 4 6 0 2 4 6 (d) B CaRLK1 NtGLB1 NtEF1α 0 12 24 36 (h) A CaRLK1 CaAct Figure S1. (A) Expression of CaRLK1 mRNA in response to hypoxia."— Presentation transcript:
Figure S1 BY RLKox 0 2 4 6 0 2 4 6 (d) B CaRLK1 NtGLB1 NtEF1α 0 12 24 36 (h) A CaRLK1 CaAct 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 time- course 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 semi-quantitative 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 S2 AA (μmol·g -1 DW) 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. A B Dissolved Oxygen (mg·L -1 )
Figure S3 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. tabacum-originated cells. Red bar indicates length. NtRBOHD 43 KDa BY RLKox 95 43 A B BY RLKox 200 μm
WT RLKox 3 Figure S4 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.