Fig 1: Auxin activity increases in csn5a-1 following 7 d heat treatment: (a) Relative integrated density in the roots of DR5::N7-VENUS and csn5a-1 × DR5::N7-VENUS, 3 h after 7 d heat treatment. (b) Relative integrated density of DR5::N7-VENUS and csn5a-1 × DR5::N7-VENUS, 3 d after 7 d heat treatment. Cell wall was stained with propidium iodide. (c) White dots represent the area used to quantify DR5 expression levels. Scale bars, 20 µm. Error bars represent SEM of biological replicates (n = 4–7). Student’s t test * p < 0.05.
Fig 2: Image and graph of abaxial epidermal cell size in 6th true leaf of plants 30 DAS having undergone 7 d heat treatment: Cell size increases in csn5a-1 after 7 d heat treatment. (a) Differential interference contrast (DIC) image of abaxial epidermal cell after 7 d heat treatment of 30 DAS csn1-10 (left), Col-0 (middle), and csn5a-1 (right). Cells colored in different color are most likely representing, 2n (green), 4n (orange), 8n (red), and stomata (yellow). Scale bar, 100 µm. (b) Graph displaying average abaxial cell size in µm2. (c) Graph displaying the number of abaxial cells per 6th true leaf. (d) Graph displaying area of 6th true leaf. Error bars represent SEM of biological replicates (n = 2–4). Student’s t test ** p < 0.01; *** p < 0.001.
Fig 3: Difference in the percentage of nuclear ploidy after 7 d heat treatment in csn5a-1 30 DAS. (a) Percentage of diploid, tetraploid, and octaploid nuclei in 30 DAS Col-0, csn5a-1, and csn1-10 seedlings following 7 d heat treatment showing increase in the ploidy of csn5a-1 following heat treatment. (b) Graph showing increase in the level of higher ploidy (4n and 8n) of csn5a-1 (middle) following heat treatment whereas Col-0 (left) and csn1-10 (right) does not show increase in higher ploidy (4n and 8n). Error bars represent SEM of biological replicates (n = 3). Student’s t test * p < 0.05; ** p < 0.01.
Fig 4: Increased growth of csn5a-1 following 7 d heat treatment is due to an increase in deneddylation activity (against CUL1) and auxin response. (a) Immunoblot using equal protein concentrations with CSN5 antibodies did not show any change in the protein band of csn5a-1 after heat treatment. (b) Immunoblot using equal protein concentrations with CUL3 antibodies did not show any change in cullin neddylation/deneddylation (93/85 kDa) ratio in either in csn5a-1 or Col-0 after heat treatment. (c) Immunoblot using equal protein concentrations with CUL1 showed increase in deneddylation activity of csn5a-1 and Col-0. Rubisco large subunit stained with ponceau is used as loading control. NT, non-treated. (d) Expression of auxin responsive genes (SAUR19 (Small auxin-up RNA 19) and EXPA4 (Expansin A4) of which the expression was downregulated in csn5a-1 control plants compared to WT (Col-0) control plants increases after heat treatment. Expression of WT (Col-0) control plant is taken as the baseline (0) in the log2 fold change. Error bars represent SEM of biological replicates (n = 3–5). Student’s t test * p < 0.05; ** p < 0.01.
Fig 5: Phenomics study showing time series of relative fluorescence decline ratio in steady-state (Rfd_Lss) for the photo-morphogenesis repressor mutants following heat stress (2 h, 44 °C, 14–21 DAS). (a) Rfd_Lss increases significantly in csn5a-1 after 7 d heat stress and continues to be higher for 11D before getting equal to csn5a-1 control. (b) Rfd_Lss increases in Col-0 after 7 d heat stress but becomes equal to Col-0 control after 4D. (c) Rfd_Lss increases in csn1-10 after 7 d heat stress but becomes equal to csn1-10 control after 4D. (d) Rfd_Lss increases in csn5b-1 after 7 d heat stress but becomes equal to csn5b-1 control after 4D. (e) Rfd_Lss does not change in cop1-4 after 7 d heat stress. (f) Rfd_Lss does not change in cop1-4 after 7 d heat stress. Error bars represent SEM of biological replicates (n = 4). Student’s t test * p < 0.05; ** p < 0.01; *** p < 0.001.
Supplier Page from Abcam for Anti-CSN5 antibody