MAPK Phosphatase AP2C3 Induces Ectopic Proliferation of Epidermal Cells Leading to Stomata Development in Arabidopsis
Figure 8
AP2C3 overexpression induces cell proliferation.
(A) Analysis of CDK activity, CDK proteins and protein levels of CDKB1;1, RBR1, DPA, DPB, E2FB, E2FC, and PSTAIR in 35S::AP2C3-GFP (indicated as GFP) and 35S::AP2C3-HA (indicated as HA) lines compared with wild type plants (WT). CDK activity correlates with CDKB1;1 protein mobility shifts in SDS-PAGE of protein extracts isolated from both AP2C3oe lines (AP2C3 tagged with GFP and AP2C3 tagged with HA) compared with WT in seedlings 5 dpg; total protein extracts from: c - cotyledons, w – whole seedlings. Total CDKs were precipitated with p13Suc1 sepharose. Kinase activity was assayed on histone 1 substrate. Western blotting was performed with CDKB1;1, RBR1, DPA, DPB, E2FB, E2FC and PSTAIRE antibodies. Anti-PSTAIRE detects CDKA. (B) Induction of AP2C3 expression by estradiol affects abundance of E2FC protein. E2FC protein levels were detected by specific antibody in estradiol-induced AP2C3-Myc overexpressing seedlings 5 dpg. Seedlings were grown with (+) or without (−) 5 µM estradiol. AP2C3 induction demonstrated by Western blotting with anti-Myc antibody. (C) Western blotting of cell cycle marker proteins PSTAIRE, CDKB1;1 and E2FC in yda, ΔYDA and WT (whole seedlings 5 dpg). (D) Abundance of cell cycle marker proteins in AP2C3G163D overexpressing seedlings compared with WT, 5 dpg. AP2C3G163D is a mutant protein with ∼90% reduced phosphatase activity. Total protein extracts from: c - cotyledons, w – whole seedlings.