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. 2020 Dec 2;40(1):e104273. doi: 10.15252/embj.2019104273

Figure 4. ChHFR1 and AtHFR1 proteins show different stability in shade.

Figure 4

  1. Expression of HFR1 and protein levels of HFR1‐3xHA in seedlings of hfr1>pAt:ChHFR1 (line #22) and hfr1>pAt:AtHFR1 (line #13). Seedlings were grown for 7 days in continuous W (~ 20 µmol/m2·s1) after which they were incubated for 3 h in high W (~ 100 µmol/m2·s1) and then either kept at high W or transferred to W + FR (R:FR, 0.06) for 3 or 6 h, as indicated in the cartoon at the top. Relative HFR1 transcript levels, normalized to UBQ10, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22 grown for 3 h under W + FR. Relative protein levels, normalized to actin, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22. Samples were collected at data points marked in the cartoon with asterisks.
  2. Cartoon of constructs containing ChHFR1 or AtHFR1 under the 35S promoter used for transient expression of transgenes in N. benthamiana leaves.
  3. Relative HFR1 transcript levels transiently expressed in tobacco leaves, normalized to the GFP, are the means ± SE of three independent biological replicates (left). Relative HFR1 protein levels, normalized to the GFP levels, are the means ± SE of four independent biological replicates (right). In (A) and (C), asterisks mark significant differences (Student's t‐test: *P‐value < 0.05, **P‐value < 0.01) between the indicated pairs.
  4. Degradation of ChHFR1 (35S:ChHFR1) and AtHFR1 (35S:AtHFR1) in tobacco leaf disks treated with cycloheximide (CHX, 100 µM) for the indicated times. Tobacco plants were kept under high W (~ 200 µmol/m2·s1) for 3 days after agroinfiltration and then leaf circles were treated with W + FR (R:FR, 0.2) and CHX. Relative HFR1 protein levels (ChHFR1, blue bars; AtHFR1, red bars), normalized to the GFP levels, are the means ± SE of four biological replicates relative to data point 0, taken as 1 for each line. Asterisks mark significant differences (2‐way ANOVA: *P‐value < 0.05) between ChHFR1 and AtHFR1 at the same time point.

Source data are available online for this figure.