Figure 4. Opposite effects of GA and ABA on APC/C^TE-mediated degradation of R10.

(a) Seeds of WT, te and OE17 were imbibed for 24 h, then treated for 3 h with 100 μM GA3. Western blot analysis shows the corresponding levels of R10 protein in WT, te and OE17 seeds. (b) 100 μM GA₃ treatment for 3 h induces degradation of R10 in WT, but not in te plants. (c) 10 μM ABA treatment for 3 h induces accumulation of R10 protein in WT plants. (d) Treatment for 3 h with 100 μM GA₃ induces degradation of R10-GFP in the R10-GFP overexpression lines. (e) Treatment for 3 h with 10 μM ABA induces accumulation of R10-GFP protein in the R10-GFP overexpression lines. (f) A unit of 100 μM ABA but not GA₃ treatment for 12 h reduced the expression level of OsPYL/RCAR10 in both WT and te plants. Student's t-test analysis indicated a significant difference (*P<0.05, **P<0.01). (g) Degradation kinetics analysis of R10 protein. Seeds of WT and te were imbibed for 24 h, then treated with 100 μM GA3. The relative abundance of R10 over the time course was determined based on the R10/HSP82 ratios. (h) Induced degradation of R10 by 100 μM GA₃ was repressed gradually by application of increased amounts of ABA in WT plants. (i) Induced accumulation of R10 by 10 μM ABA was reduced by application of increased amounts of GA₃ in WT plants. (j) Co-IP assay showing the quantities of TE and R10 proteins pulled down from the extracts of 1-week-old OE17 plants treated with 10 μM ABA or 100 μM GA₃ by equal amounts of His-agarose. ‘Eluate' shows the quantities of TE and R10 proteins that were not pulled down by His-agarose. ‘Total' shows the starting quantities of TE and R10 proteins that were used in the Co-IP assay. The ‘α-HSP82' signals in a–e, h and i show that roughly equal amounts of total plant extracts were used. Values are means±s.d. (n=3 replicates) in f and g. Note: The seeds or seedlings were pre-treated for 1 h with 1 mM cycloheximide before phytohormone treatment in a–e and g–i.