Figure 2.

TINY Is Phosphorylated and Stabilized by BIN2.

(A) and (B) BL responses of tiny mutants. Seven-day-old plants grown on 1/2 LS medium with or without 50 nM BL under light (A). Hypocotyl length was measured using ImageJ (B). Data represent mean and sd from three biological replicates (n = 3); each replicate contained 12 to 15 plants. *P < 0.05, **P < 0.01; two-way analysis of variance. WT, wild type.

(C) TINY-FLAG protein decreased after BL treatment. XVE:TINY-FLAG transgenic lines were treated with indicated concentration of BL and 10 μM β-estradiol for 7 d. Samples were collected to detect TINY protein with anti-FLAG antibody. WT, wild type.

(D) and (E) CHX treatment for TINY degradation. XVE:TINY-FLAG line 6 was grown on β-estradiol plates for 7 d. The seedlings were treated with 400 μM CHX in the absence or presence of 100 nM BL for the indicated time. Tissues were collected for immunoblotting analysis, and TINY-FLAG protein was detected with anti-FLAG antibody (D). Protein level was normalized with loading control and treatment at 0 h (E).

(F) TINY was phosphorylated in plants. TINY-FLAG protein was immunoprecipitated from 7-d-old TINY-OE seedlings (line 3 was used for most experiments unless otherwise specified) and treated with CIP. Phosphorylated TINY (P) was indicated.

(G) BIN2 phosphorylated TINY. MBP-TINY, but not MBP, was phosphorylated by BIN2 in the in vitro kinase assay. BIN2 phosphorylation of TINY was inhibited by indicated concentration of bikinin.

(H) TINY interacted with BIN2 in by BiFC. Cotransformation of TINY-cYFP and BIN2-nYFP led to the reconstitution of YFP activity in N. benthamiana nucleus under YFP filter, whereas coexpression of TINY-cYFP and nYFP or BIN2-nYFP and cYFP did not produce any positive YFP signal.

(I) Co-IP assay showed TINY and BIN2 interaction. TINY-FLAG and BIN2-GFP as well as control vectors were cotransformed into Arabidopsis protoplasts overnight. After 20 μM MG132 treatment for 1 h, protoplasts were collected and protein was immunoprecipitated with anti-FLAG and detected with anti-FLAG and anti-GFP antibodies.

(J) Phenotype of 3-week old bin2-1 TINY:TINY-FLAG double mutant (TINY:TINY-FLAG line 2 was used for crossing) in F1 generation (top). bin2-1 background was genotyped (Yan et al., 2009): BIN2 DNA was amplified by PCR (middle) and digested with XhoI (bottom).

(K) TINY protein level in bin2-1/BIN2-1 TINY:TINY-FLAG double mutant from (J).

(L) Phenotype of 4-week-old bin2-3 bil1 bil2 TINY:TINY-FLAG homozygous mutant (TINY:TINY-FLAG line 2 was used for crossing). Wassilewskija (WS) ecotype is used as control. WT, wild type.

(M) TINY protein level in bin2-3 bil1 bil2 TINY:TINY-FLAG from (L). WS, Wassilewskija; WT, wild type.