Figure 5.

APC/C^TE Mediates the Opposite Effects of ABA and GA on the Degradation of OsSHR1 and MOC1.

(A) Co-IP assay shows that His-agarose simultaneously pulls down TE and OsSHR1 from the OE17 but not te plant extracts by specifically binding to the His epitope in the N terminus of TE. “α-His” indicates detected TE proteins by the α-His antibody.

(B) In vitro pull-down assay shows that MBP-TE-His pulls down OsSHR1 from the te plant extracts. WT, wild type.

(C) In vitro ubiquitination assay of MBP-OsSHR1-His by the wild-type and te plant extracts.

(D) Immunoblot analysis shows the levels of OsSHR1 protein in the shoot bases of wild-type, te, OE17, OE21, and OE22 plants. “α-HSP82” indicates that roughly equal amounts of total plant extracts were used.

(E) Cell-free degradation assay shows the stability of MBP-OsSHR1-His protein in wild-type and te plant extracts or MBP-OsSHR1-mD-His protein in wild-type extracts with or without 50 μM of proteasome inhibitor MG132. “Input” shows the initiation amounts of MBP-OsSHR1-His proteins.

(F) and (G) The mRNA levels (F) of OsSHR1 in 6-d–old wild type treated with 1 μM of ABA, 1 μM of GA₃ or no phytohormone treatment (CK) for 3 dor protein levels (G) of OsSHR1 in 6-d–old wild type, te and OE17 treated with 1 μM of ABA, 1 μM of GA₃ or no phytohormone treatment (CK) for 3 d. Values are means ±sd (n = 3 biological replicates) and *P < 0.001, Student’s t test (two-tailed) in (F). “α-HSP82” indicates that roughly equal amounts of total plant extracts were used in (G). NS, not significant.

(H) Immunoblot analysis showing the quantities of MOC1 proteins in 1-month–old wild-type, te, and OE17 plants treated with 1 μM of ABA, 1 μM of GA₃, or no phytohormone treatment (CK) for 3 d. “α-HSP82” indicates that roughly equal amounts of total plant extracts were used.