Figure 4. Conditional molecular phenotypes are linked with protein degradation and deactivation of lt-degron fusion proteins.

(a) Conditional accumulation of K2:GFP fusion protein in whole seedlings and (b) functionality of K2:GFP in root tips. Also Arg-initiated K2:GFP is conditionally depleted (Supplementary Fig. 4c). Scale bar, 50 μm. (c) In vivo hydrolase activity of transgenic ProUBQ10::K2:GUS compared with Pro35S::GUS, overexpression lines and the WT constitutively grown at permissive or restrictive temperature. Scale bar, 1 cm. (d) K2:GUS protein levels of seedlings constitutively grown at the indicated temperatures. Membranes were immunostained with α-HA antibody followed by a second staining with α-GUS antibody after stripping. The asterisk indicates K2:GUS protein. (e) Quantitative GUS assays of plants constitutively grown at 13 versus 29 °C. Six biological replicates were performed for K2:GUS and three biological replicates for WT and 35S::GUS. All samples were measured as three technical replicates. P values for Student's t-test (two-sided t-test, type 3) are K2:GUS: 6.44766 × 10⁻⁷ (***); 35S::GUS: 0.004365663 (**). All error bars in this panel represent s.d., asterisk: significant. (f) Western blots of ProUBQ10::K2:GUS seedlings constitutively grown at ambient temperature (room temperature (RT)) shifted to permissive or restrictive temperature. (g) Quantitation of enzyme activity of f. Seedlings were grown under standard long-day conditions (16-h light, 8-h dark, 21 °C) aseptically on MS plates. One-week-old seedlings were shifted to permissive or restrictive conditions, respectively. In all, 5–10 individual seedlings were collected and pooled every 4 h. Three biological replicates were performed with two technical replicates each. All error bars in this panel represent s.d. (h) Degron tuning assay monitoring K2:GUS activity through temperature shift. Plants expressing ProUBQ10::K2:GUS were grown at cold (permissive) or warm (restrictive) temperature and then shifted to the respective opposite temperature and back over the course of 10 days. Samples were taken every 24 h, followed by a temperature shift of 2.8 °C. Activity of the K2:GUS fusion protein clearly follows the temperature shift. The numbers next to the data points represent the sampling temperature, n=3. All error bars in this panel represent s.d. MS analysis of K2:GUS populations is documented in Supplementary Fig. 7. (i) K2:TEV in stably transformed Arabidopsis plants. Western blot of material collected from seedlings grown at permissive and restrictive temperatures. Equal loading was further confirmed by staining of blotted membranes with Coomassie Brilliant Blue G225 after immunostaining or α-PSTAIRE antibody detecting CYCLIN-DEPENDENT KINASE A;1 (CDKA;1). For transcript levels under restrictive and permissive conditions, see Supplementary Fig 4.