Regulation of the Arabidopsis GSK3-like Kinase ... - Molecular Plant

Peng et al. d BIN2 Regulation in Brassinosteroid Signaling | 341
Figure 2. BRZ Enhances the Protein Stability of BIN2.
(A) A gBIN2:GFP transgenic line responds to BL treatment whereas
a strongly dwarfed gbin2-1:GFP line is BL-insensitive. From left to
right are gbin2-1:GFP (top panel) and gBIN2:GFP (lower panel)
transgenic seedlings grown on 1 lM BL-containing medium for
0, 3, 6, and 9 d.
(B) BRZ treatment significantly increases the steady-state level of
BIN2:GFP but has a little effect on bin2-1:GFP. The top panel is
the result of the IP/Western analysis while the lower panel shows
the effect of BRZ treatment on the morphology of the selected
transgenic lines.
seedlings. Consistent with the morphological change, the BRZ
treatment significantly increased the amount of BIN2:GFP but
had little effect on the accumulation of bin2-1:GFP.
Next, we tested whether the BRZ-stabilized BIN2:GFP protein
can be depleted by BL treatment. The BRZ-treated gBIN2:GFP
seedlings were transferred to liquid medium containing different
concentrations of BL and incubated for different periods of
time. As shown in Figure 3A and 3B, the BRZ-stabilized BIN2:GFP
is rapidly depleted after 20–30 min treatment with 0.1 or 1 lM
BL, whereas similar treatments had little effect on the abundance
of the bin2-1:GFP protein (Figure 3C and 3D).
To determine whether other plant hormones could induce
a similar depletion of the Arabidopsis GSK3 kinase, we transferred
the BRZ-treated gBIN2:GFP seedlings to liquid medium
containing various other plant hormones at concentrations
known to inhibit root growth of wild-type Arabidopsis seedlings
(Li et al., 2001). The treated seedlings were collected after
30 min incubation for extraction of total proteins and the
amount of BIN2:GFP fusion proteins was analyzed by the IP/
Western blotting method. As shown in Figure 4, none of
the hormone treatments resulted in significant depletion of
the BRZ-stabilized BIN2:GFP fusion proteins. We thus concluded
that the steady-state level of BIN2 is specifically regulated
by the plant steroid hormone.
The BL-Induced BIN2 Depletion Requires a Functional
BRI1 Receptor
To test whether the observed BL-induced BIN2 depletion is mediated
by the Arabidopsis BR receptor BRI1, we crossed the
gBIN2:GFP transgene into bri1-9—a previously characterized
weak bri1 mutant that is caused by ER retention of a structurally
defective but biochemically competent BRI1 (Jin et al., 2007).
TheresultinggBIN2:GFPbri1-9transgenicseedlingsweregrown
side by side with the parental gBIN2:GFP seedlings on synthetic
medium containing 2 lM BRZ for 2 weeks. The seedlings were
then transferred to liquid medium containing 1 lM BL and collected
after 30 min incubation for the IP/Western blotting analysis.AsshowninFigure5,theamountofBIN2:GFPinBRZ-treated
gBIN2:GFP bri1-9 seedlings is similar to that of the BRZ-treated
gBIN2:GFP plants and that further treatment of 1 lM BL did not
lead to BIN2:GFP depletion, indicating that a functional BR receptor
is required for the BL-induced BIN2 depletion.
The BL-Induced BIN2 Depletion Involves Proteasome-
Mediated Protein Degradation
Proteasome-mediated protein degradation is an important
regulatory mechanism for a wide variety of plant signaling
pathways (Smalle and Vierstra, 2004), including the BRsignaling
process (He et al., 2002; Yin et al., 2002). To directly
test whether the BL-induced BIN2 depletion is caused by
proteasome-mediated protein degradation, we first grew
gBIN2:GFP transgenic seedlings on BRZ-containing medium
for 14 days and then transferred them to liquid medium containing
1 lM BL and/or 10 lM MG132—a well known proteasome
inhibitor (Rock et al., 1994). The seedlings were collected
after 30 min incubation for the IP/Western blotting analysis. As
shown in Figure 6, MG132 treatment effectively blocked the
BL-induced depletion of BIN2:GFP, whereas a similar treatment
with a cocktail of common protease inhibitors had no effect on
BIN2 stability. This result not only ruled out the possibility that
the BL-induced depletion of BIN2 is due to low immunoprecipitation
efficiency of the wild-type BIN2:GFP fusion protein by
the polyclonal GFP antiserum, but also confirmed that a proteasome-mediated
protein degradation process is involved in
the BL-induced BIN2 disapperance.
The BL-Induced BIN2 Degradation Correlated Well
with the BL-Induced Reduction in BIN2 Activity
To test whether the BL-induced BIN2 degradation is responsible
for the previously observed BL-induced reduction in BIN2
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