Plant Cell-2009-Wang-2378-90
description
Transcript of Plant Cell-2009-Wang-2378-90
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Biochemical Insights on Degradation of Arabidopsis DELLAProteins Gained From a Cell-Free Assay System W
Feng Wang,a,b Danmeng Zhu,a,c,d Xi Huang,a,b Shuang Li,a,b Yinan Gong,a,b Qinfang Yao,e Xiangdong Fu,e
Liu-Min Fan,c and Xing Wang Denga,c,d,1
a National Institute of Biological Sciences, Zhongguancun Life Science Park, Beijing 102206, Chinab College of Life Sciences, Beijing Normal University, Beijing 100875, Chinac Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, National Laboratory of Protein Engineering
and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, Chinad Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104e State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese
Academy of Sciences, Beijing 100101, Peoples Republic of China
The phytohormone gibberellic acid (GA) regulates diverse aspects of plant growth and development. GA responses are
triggered by the degradation of DELLA proteins, which function as repressors in GA signaling pathways. Recent studies in
Arabidopsis thaliana and rice (Oryza sativa) have implied that the degradation of DELLA proteins occurred via the ubiquitin-
proteasome system. Here, we developed an Arabidopsis cell-free system to recapitulate DELLA protein degradation in vitro.
Using this cell-free system, we documented that Lys-29 of ubiquitin is the major site for ubiquitin chain formation to mediate
DELLA protein degradation. We also confirmed the specific roles of GA receptors and multisubunit E3 ligase components in
regulating DELLA protein degradation. In addition, blocking DELLA degradation with a PP1/PP2A phosphatase inhibitor in
our cell-free assay suggested that degradation of DELLA proteins required protein Ser/Thr dephosphorylation activity.
Furthermore, our data revealed that the LZ domain of Arabidopsis DELLA proteins is essential for both their stability and
activity. Thus, our in vitro degradation system provides biochemical insights into the regulation of DELLA protein
degradation. This in vitro assay system could be widely adapted for dissecting cellular signaling pathways in which
regulated proteolysis is a key recurrent theme.
INTRODUCTION
Responses to gibberellic acid (GA) play important roles in diverse
growth and developmental processes throughout the entire life
cycles of plants (Fleet and Sun, 2005). The DELLA proteins are
growth repressors first identified in Arabidopsis thaliana and
widely distributed in other crop plants, including rice (Oryza
sativa), barley (Hordeum vulgare), maize (Zea mays), wheat
(Triticum aestivum), and grape (Vitis vinfera) (Peng et al., 1997,
1999; Boss and Thomas, 2002; Chandler et al., 2002; Gubler
et al., 2002; Itoh et al., 2002). InArabidopsis, a family of five genes
encodes the DELLA proteins, including GA INSENSITIVE (GAI),
REPRESSOR OF ga1-3 (RGA), and three REPRESSOR OF ga1-
3-LIKE proteins (RGL1, RGL2, and RGL3). The five DELLA
proteins influence Arabidopsis growth and development with
both redundant and partially specialized functions. RGA and GAI
synergistically repress GA-regulated stem elongation, abaxial
trichome initiation, and leaf expansion (Dill and Sun, 2001; King
et al., 2001), while RGL1 and RGL2 are major players in seed
germination (Lee et al., 2002; Wen and Chang, 2002; Tyler et al.,
2004). Furthermore, RGA, RGL1, and RGL2 work in concert to
regulate floral development (Cheng et al., 2004; Tyler et al., 2004;
Yu et al., 2004). In addition, accumulating evidence suggests that
multiple environmental cues, including light (Achard et al., 2007;
Oh et al., 2007; de Lucas et al., 2008; Feng et al., 2008), cold
(Achard et al., 2008b), salt (Achard et al., 2006, 2008a; Magome
et al., 2008), and biotic stresses (Navarro et al., 2008), converge
on DELLA proteins to affect diverse aspects of plant growth and
development.
It was reported that the GA-induced degradation of DELLA
proteins relieved Arabidopsis growth restraint (Dill et al., 2001;
Silverstone et al., 2001). The kinetics of GA-induced degradation
vary among different DELLA proteins in Arabidopsis (Fu et al.,
2004).Moreover, theGA-induced degradation ofDELLAproteins
has also been reported in rice (Itoh et al., 2002) and barley (Fu
et al., 2002; Gubler et al., 2002). Based on the inhibition of DELLA
degradation by proteasome-specific inhibitors, it was generally
assumed that GA-induced degradation of DELLA proteins oc-
curs via the ubiquitin-26S proteasome system (Fu et al., 2002;
Feng et al., 2008).
As a major pathway for targeting protein degradation, the
ubiquitin-proteasome system plays essential roles in many cel-
lular processes in all eukaryotes (Hershko and Ciechanover,
1998; Pickart, 2001; Vierstra, 2003; Moon et al., 2004; Petroski
and Deshaies, 2005). Ubiquitin is attached to the substrate
through a cascade of three enzymes: ubiquitin-activating enzyme
(E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein
1 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Xing Wang Deng([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.108.065433
The Plant Cell, Vol. 21: 23782390, August 2009, www.plantcell.org 2009 American Society of Plant Biologists
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Figure 1. Cell-Free Degradation of Plant-Derived DELLA Proteins.
(A) and (B) Cell-free degradation of TAP-tagged RGA (A) and GAI (B) proteins. Protein extracts were prepared from 7-d-old 35S:TAP-RGA/rga-24 and
35S:TAP-GAI/gai-t6 seedlings and then incubated with or without MG132 over the indicated time course. RPN6 was used as a nondegraded loading
control. Start: time point zero in each degradation assay.
(C) and (D) Half-life plot for cell-free degradation of TAP-RGA and TAP-GAI in (A) and (B), respectively.
Cell-Free Degradation of DELLA Proteins 2379
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ligase (E3). This ubiquitin moiety then serves as the signal for
recognition by the 26S proteasome, which degrades the sub-
strates into small peptides and amino acids. Among the three
enzymes, E3 ligases largely dictate the substrate specificities,
thus constituting the key regulatory components in the ubiquitin-
proteasome pathway. The SCF complexes, a large group of
multisubunit RING domain E3 ligases, are the most extensively
characterized E3 family (Petroski and Deshaies, 2005). They are
named after three subunits: Skp1, Cullin (CUL), and F-box
protein (Moon et al., 2004). In this complex, the CUL subunit
serves as a scaffold protein that binds the linker protein SKP1 to
its N-terminal domain. The SKP1 protein in turn interacts with the
F-box protein, which directly recruits the specific substrate to the
SCF E3 complex (Zheng et al., 2002). The important role of SCF
E3 ligases in plant development, especially in phytohormone
signaling pathways, has been well established (Sullivan et al.,
2003; Vierstra, 2003; Thomann et al., 2005; Stone and Callis,
2007).
DELLA proteins accumulate to high levels in Arabidopsis and
rice mutants that have defects in the F-box genes sly1 and gid2,
respectively (McGinnis et al., 2003; Sasaki et al., 2003; Strader
et al., 2004). These recessive mutants, sly1-10 and gid2, exhibit
GA-insensitive dwarf phenotypes and can be suppressed by
loss-of-functionmutations in DELLA proteins (Steber et al., 1998;
McGinnis et al., 2003; Sasaki et al., 2003). By contrast, a sly1
gain-of function allele, sly1-d, causes reduced levels of RGA
proteins, and strong interactions between DELLA proteins and
sly1-d can be observed in vitro (Dill et al., 2001, 2004; Fu et al.,
2004). Furthermore, physical interactions between SLY1/GID2
and the SCF components, SKPs, and DELLA proteins were
detected through yeast two-hybrid and immunoprecipitation
assays. Together, these results suggest that these F-box
Figure 1. (continued).
(E) Cell-free degradation of all five DELLA proteins. Wild-type plants expressing 35S promoterdriven full-length RGA, GAI, RGL1, RGL2, and RGL3
transgenes tagged with TAP at the N terminus were used to perform the degradation assays.
(F) Degradation of TAP-RGA in wild-type extracts. TAP-RGA was enriched from 7-d-old 35S:TAP-RGA/rga-24 seedlings by affinity matrix and then
incubated in wild-type extracts with or without MG132.
(G) Half-life plot for cell-free degradation of TAP-RGA in (F).
Figure 2. Cell-Free Degradation of Recombinant DELLA Proteins Expressed in E. coli.
(A) and (B) Degradation of recombinant MBP-RGA (A) and MBP-GAI (B) in the cell-free system. MBP-RGA and MBP-GAI were expressed and purified
from E. coli and then added to wild-type Landsberg erecta (Ler) extracts.
(C) and (D) Half-life plot for cell-free degradation of MBP-RGA and MBP-GAI in (A) and (B), respectively.
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proteins may be components of functional SCF E3 ligase com-
plexes regulating the stability of DELLA proteins (Sasaki et al.,
2003; Dill et al., 2004; Fu et al., 2004).
Although the recent data indicated that the proteasome-
dependent degradation of DELLA proteins plays an important
role inGA-triggeredplant growth regulation, detailed knowledgeof
thebiochemicalmechanismunderlying this degradationprocess is
still lacking. Here, we report the development of an Arabidopsis
cell-free assay system for reconstituting DELLA protein degrada-
tion in vitro. Using this system, we investigated the linkage spec-
ificity of the ubiquitin chains that facilitate degradation of DELLA
proteins. We also combined genetic and biochemical analyses to
verify the roles of individual proteins that have been implicated by
previous studies. Moreover, we found that inhibitors of PP1/PP2A
phosphatases can efficiently block DELLA protein degradation in
our cell-free system, suggesting that phosphatase activity may be
involved in regulating the stability of DELLA proteins. Domain dis-
section analysis revealed that the LZ (Leu heptad repeat) domain is
required for regulating degradation and molecular functions of
DELLA proteins.
RESULTS
The Five DELLA Proteins Exhibit Distinct Degradation
Kinetics in the Cell-Free System
To explore the biochemical factors and activities that regulate
the degradation of DELLA proteins during plant growth and
development, we developed a cell-free system that can reca-
pitulate the regulation of DELLA protein stability. We investi-
gated the degradation of DELLA proteins in cell-free extracts
prepared from transgenic Arabidopsis plants expressing 35S
promoterdriven N-terminal tandem affinity purification (TAP)
tagged full-length RGA andGAI transgenes in their correspond-
ing rga-24 and gai-t6 mutant backgrounds, in the presence or
absence of 40 mM MG132 based on a modified approach
reported previously (Naidoo et al., 1999; Osterlund et al., 2000;
Yanagisawa et al., 2003). Our protein gel blots showed that the
TAP-tagged RGA and GAI proteins degraded rapidly in the
samples without MG132, and only trace amounts of proteins
were observed after 2 h (Figures 1A and 1B). Degradation of
these proteins was significantly delayed by MG132 treatment,
consistent with the notion that DELLA proteins are degraded by
the 26S proteasome. Quantification of the degradation kinetics
showed that MG132 treatment significantly prolonged the half-
life (t1/2) of both the TAP-RGA and TAP-GAI proteins (at least
sixfold longer, compared with non-MG132 treated samples)
(Figures 1C and 1D). Using a similar approach, we confirmed
that all five DELLA proteins (TAP-tagged RGA, GAI, RGL1,
RGL2, and RGL3) were degraded in our cell-free system in a
similar 26S proteasomedependent manner (Figure 1E). This
result is consistent with the previous in vivo degradation studies
(Feng et al., 2008).
To avoid possible artifacts caused by the transgenic plants, we
next tested the degradation of plant-derived DELLA proteins in
wild-type cell-free extracts in vitro. To accomplish this, we first
affinity enriched TAP-RGA proteins from 35S:TAP-RGA/rga-24
transgenic plants using anti-Myc antibody-conjugated beads
and then added the precipitates to wild-type extracts. Our
protein gel blots showed that the degradation of plant-derived
TAP-RGA proteins was similar in wild-type extracts (although
slightly faster) compared with transgenic extracts and effectively
blocked by MG132 (Figures 1F and 1G).
Degradation of Recombinant RGA and GAI Is Blocked by
MG132 in Vitro
We next asked whether our cell-free extracts could degrade
recombinant DELLA proteins purified from Escherichia coli. As
shown in Figures 2A and 2B, we found that recombinant maltose
binding protein (MBP)-tagged RGA and GAI were degraded in
wild-type Arabidopsis extracts over similar time courses as
observed for plant-derived DELLA proteins (Figure 1). Moreover,
MG132 reduced the degradation rates of recombinant RGA and
GAI by sevenfold to eightfold (Figures 2C and 2D). Notably, 10
Figure 3. Cell-Free Degradation of DELLA Proteins Is ATP Dependent.
(A) Degradation of plant-derived TAP-RGA is sensitive to apyrase. Protein extracts were prepared from 7-d-old 35S:TAP-RGA/rga-24. The amounts of
added apyrase are indicated.
(B) MBP-RGA degradation requires ATP. Protein extracts were prepared in buffer containing ATP (+ATP, as in other cell-free degradation tests) or
lacking ATP (ATP, with or without added apyrase). Recombinant MBP-RGA proteins were expressed and purified from E. coli and then added to thedegradation extracts.
Cell-Free Degradation of DELLA Proteins 2381
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mM MG132 blocked MBP-RGA degradation with a similar effi-
ciency as 40 mM MG132 blocked MBP-GAI degradation, imply-
ing that DELLA proteins may have different sensitivities to
MG132 for proteasome-dependent degradation. Furthermore,
quantification of the degradation kinetics suggested that
MBP-RGA is degraded faster than MBP-GAI (Figures 2C and
2D), consistent with a previous in vivo observation (Fu et al.,
2004). We also observed that degradation of both TAP-RGA and
MBP-RGA was blocked by Paclobutrazol, a GA biosynthesis
inhibitor (see Supplemental Figure 1 online), indicating that their
degradation required GA. Thus, we concluded that we success-
fully reconstituted a cell-free system for analyzing DELLA protein
degradation in vitro.
ATP Is Required for DELLA Protein Degradation in the
Cell-Free System
The requirement for ATP is an important characteristic of the
ubiquitin-proteasomal proteolysis pathway (Pickart, 2004). To
determine whether ATP is essential for degradation of DELLA
proteins in our cell-free system, we treated our plant extracts
with apyrase, an ATPase, and then monitored TAP-RGA degra-
dation over the same time course as shown in Figure 1. We
observed that apyrase prevented TAP-RGA degradation in a
dose-dependent manner (Figure 3A), supporting the necessity
forATP inDELLAdegradation.Consistentwith theATP-dependent
degradation of endogenous TAP-RGA, exogenousMBP-RGAwas
Figure 4. DELLA Protein Degradation Is Ubiquitin Dependent.
(A) and (B) TAP-RGA degradation was delayed by exogenously added K0 (no Lys ubiquitin) and 3KTR Lys-29, Lys-48, and Lys-63 mutated to Arg
ubiquitin mutants.
(C) Protein blot showing that TAP-RGA degradation can be delayed by 3KTR ubiquitin but not by K48/63R (both Lys-48 and Lys-63 mutated to Arg)
ubiquitin.
(D) and (E) Protein blot showing that TAP-RGA degradation can be delayed by K29R but not by other single-Lys mutant ubiquitins.
(F) Protein blot showing that TAP-RGA degradation can be delayed by K0 ubiquitin but not by Lys-29-only ubiquitin.
Each mutant ubiquitin was applied at 1 mg/mL, except for K0 ubiquitin as indicated in (A).
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degraded slowly in the absence of ATP (Figure 3B). Moreover,
MBP-RGA degradation was completely blocked by the addi-
tion of 10 units of apyrase to extraction buffer lacking ATP
(Figure 3B).
Lys-29 of Ubiquitin Is the Predominant Lys Residue
Responsible for DELLA Protein Degradation
Proteasome-dependent degradation is always mediated by
ubiquitination of substrate proteins (Vierstra, 2003; Moon et al.,
2004). Since polyubiquitinated DELLA proteins were detected in
the presence of proteasomal inhibitors after GA treatment
(Sasaki et al., 2003; Feng et al., 2008), we tested whether
functional ubiquitin is involved in DELLA cell-free degradation.
Using our cell-free system, we compared the degradation of
plant-derived TAP-RGA in wild-type extracts supplemented
with intact ubiquitin or a series of ubiquitins mutated in different
Lys residues critical for ubiquitin polymerization. As shown in
Figure 4A and Supplemental Figure 2A online, the addition of K0
ubiquitin (which has all seven Lys residues mutated to Arg res-
idues) retarded TAP-RGA degradation in a dosage-dependent
manner. To further investigate the linkage specificity of the
ubiquitin chain, we next asked which ubiquitin Lys residues
were needed for DELLA protein degradation. Interestingly, we
found that the addition of 3KTR (Lys-29, Lys-48, and Lys-63
mutated to Arg residues) slowed TAP-RGA degradation asmuch
as K0 ubiquitin (Figure 4B; see Supplemental Figure 2B online).
By contrast, addition of ubiquitin with both Lys-48 and Lys-63
mutated to Arg residues had almost no observable effect on
DELLA degradation compared with 3KTR (Figure 4C; see Sup-
plemental Figure 2C online). Moreover, ubiquitin in which Lys-29
was mutated slowed TAP-RGA degradation, whereas mutant
ubiquitins in which Lys-48 or Lys-63 were mutated had no effect
(Figures 4D and 4E; see Supplemental Figures 2D and 2E online).
By contrast, the K29R ubiquitin mutation had no effect on the
cell-free degradation of HY5, a well-established proteasomal
substrate (see Supplemental Figure 3 online). In addition, we
found that Lys-29-only ubiquitin had much less effect on the
degradation of TAP-RGA compared with K0 ubiquitin in our cell-
free system (Figure 4F; seeSupplemental Figure 2F online). Thus,
our data suggested that Lys-29 is a dominant site responsible for
polyubiquitin chain formation and subsequent degradation of
DELLA proteins. It is worth noting that 3KTR ubiquitin can
stabilize TAP-RGA more efficiently than K29R ubiquitin in our
cell-free system (Figure 4D). This suggests that Lys-48, the
conventional site for proteolysis, and Lys-63 may function syn-
ergistically with Lys-29 in mediating DELLA protein degradation,
albeit playing a more minor role.
Genetic Requirements for DELLA Protein Degradation
Previous studies have documented that the degradation of RGA
and GAI is abolished in both the GA receptor mutants and sly1-
10, a mutant with a lesion in the F-box protein that acts as the
adaptor for the SCF E3 ligase directly targeting DELLA proteins
for degradation (Dill et al., 2004; Fu et al., 2004). The availability of
these GA-related mutants allowed us to examine the genetic
requirements for DELLA protein degradation in vitro. We pre-
pared extracts from gid1b/c and sly1 mutants, respectively, to
test MBP-RGA degradation in cell-free extracts. Consistent with
the previous studies, the degradation of MBP-RGA was delayed
in gid1b/c extracts and almost completely blocked in sly1-10
extracts (Figures 5A and 5B). Moreover, we also demonstrated
that DELLA degradation in sly1-10 extracts was facilitated by
supplementation with purified myc-SLY1 proteins (see Supple-
mental Figure 4 online). In addition, degradation of MBP-RGA
Figure 5. Genetic Requirements for DELLA Protein Degradation.
(A) and (B) Loss-of-function gid1 and sly1 mutants delayed MBP-RGA degradation in the cell-free system. Recombinant MBP-RGA was added to the
total protein extracts prepared from 7-d-old seedlings of Columbia-0 (Col-0) and gid1b/c double mutants in (A) and Col-0 and sly1-10 mutants in (B).
(C) Cell-free degradation of MBP-RGA was promoted in sly1-d. Recombinant MBP-RGA protein (200 ng/100 mL total cell-free extracts) was added to
the extracts prepared from 7-d-old Ler and sly1-d (with or without MG132 treatment).
Cell-Free Degradation of DELLA Proteins 2383
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was accelerated in the extracts of a gain-of-function mutant,
sly1-d, and this process was efficiently blocked by proteasome-
specific inhibitors (Figure 5C). These data also supported the
essential roles of GA receptors and SLY1 in mediating DELLA
degradation.
CUL1-Based SCFSLY1 E3 Ligase Activity Is Required for
DELLA Protein Degradation
SLY1 is the F-box protein that associates with the SCF (Skp/CUL/
F-box) E3 ubiquitin ligase responsible for turnover of DELLA
proteins (Fu et al., 2004;Gomi et al., 2004). In the SCFE3complex,
CUL1 serves as a scaffold and is involved in multiple signaling
pathways (Petroski and Deshaies, 2005). To assess the contribu-
tion of CUL1-containing SCF complexes to the degradation of
DELLA proteins, we performed cell-free degradation assays of
recombinant MBP-RGA using extracts prepared from cul1-6, a
viable and fertile allele of CUL1 (Moon et al., 2007). As shown in
Figure 6A, we found that MBP-RGA was degraded much more
slowly in cul1-6 extracts than in wild-type extracts. To test further
whether in vivo degradation of DELLA proteins is affected by this
cul1mutation, we treated Arabidopsis seedlings with 100 mMGA3and monitored endogenous RGA abundance over time by protein
blot analysis. In linewith our cell-free degradation result, the in vivo
degradation of RGA was also significantly delayed in the cul1-6
mutant (Figure 6B). Taken together, our results suggest that the
CUL1-based SCFSLY1 E3 complex is essential for GA-mediated
degradation of DELLA proteins.
Role of Protein Phosphorylation and Dephosphorylation in
Cell-Free Degradation of DELLA Proteins
It was reported that phosphorylation and dephosphorylation
events are likely involved in the regulation of DELLA protein
Figure 6. A Point Mutation in the CUL1 Gene Delays the Degradation of DELLA Protein Both in Vitro and in Vivo.
(A) Cell-free degradation of MBP-RGA was delayed in cul1 mutant extracts. MBP-RGA proteins were added to extracts prepared from 7-d-old Col-0
and cul1-6 seedlings.
(B) In vivo degradation of RGA was delayed in cul1 mutant seedlings. Seven-day-old Col-0 and cul1-6 seedlings were treated with 100 mM GA3.
Seedlings were harvested at the indicated times, and endogenous RGA was analyzed by protein gel blots using anti-RGA antibody.
Figure 7. Effects of Protein Kinase and Phosphatase Inhibitors on DELLA Protein Degradation in the Cell-Free System.
In vitro degradation assays were performed in the presence of the kinase inhibitors staurosporine in (A), AG555 in (B), and the phosphatase inhibitor
Microsystin-LR in (C).
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degradation (Fu et al., 2002; Sasaki et al., 2003; Gomi et al., 2004;
Hussain et al., 2005, 2007). To test directly their possible effects
on the stability of DELLA proteins in our cell-free system, we
added staurosporine, a broad-range protein kinase inhibitor, and
AG555, a potent Tyr kinase inhibitor, to wild-type Arabidopsis
protein extracts containing MBP-RGA purified from E. coli. As
shown in Figures 7A and 7B, neither of these drugs had any
obvious effect on DELLA protein degradation. By contrast,
treatment with Microcystin-LR, a PP1/PP2A-type phosphatase
inhibitor, markedly inhibited the degradation of MBP-RGA com-
pared with the mock sample (Figure 7C). To exclude possible
nonspecific effects ofMicrocystin-LR on the ubiquitin-proteasome
system, we tested the effect of Microcystin-LR on the degradation
of HY5 in our cell-free system. As shown in Supplemental Figure 5
online, Microcystin-LR did not affect the degradation of HY5,
supporting the specificity of its effects on DELLA degradation.
Together, our results suggest that a dephosphorylation event may
be a prerequisite for the degradation of Arabidopsis DELLA pro-
teins, which is consistent with a previous observation on DELLA
protein degradation in tobacco (Nicotiana tabacum) BY2 cells
(Hussain et al., 2005).
Domain Dissection for the Regulation of DELLA Protein
Stability in the Cell-Free System
It was shown that mutant derivatives of rice GFP-SLR protein (a
homolog of Arabidopsis DELLA) lacking the DELLA or Leu
heptad repeat (LZ) domains consistently stayed in the nucleus
even when GA was applied (Itoh et al., 2002). To gain further
insights into the structure-function relationships of Arabidopsis
DELLA proteins, we tested the degradation of a series of
truncated and internal deletion derivatives of RGA (Figure 8A).
Interestingly, deletion of the N-terminal 108 amino acids en-
compassing the DELLA and VHYNPmotifs (RGAnN108) slowed
RGA degradation (Figure 8B; see Supplemental Figure 6A
online). Similar kinetics were seen in the degradation of RGA
lacking the LZ domain (Figure 8C; see Supplemental Figure 6B
online), a highly conserved domain among plant species (see
Figure 8. The N-Terminal and LZ Domain of RGA Are Involved in Regulating Degradation of DELLA Proteins.
(A) Schematic diagram of RGA and RGA deletion derivatives.
(B) and (C) Cell-free degradation of MBP-RGA, MBP-RGAnN108 (deletion of amino acid residues 1 to 108), and MBP-RGAnLZ (deletion of amino acid
residues 220 to 257). Equal amounts of full-length (F.L.) and nN108 in (B) or nLZ in (C) RGA were added to extracts prepared from 7-d-old wild-type
seedlings.
(D)Cell-free degradation of MBP-RGAnN108nLZ (deletion of both N108 and the LZ domain) with or without MG132. Equal amounts ofnN108nLZ and
F.L. RGA were added to extracts prepared from 7-d-old wild-type seedlings.
Cell-Free Degradation of DELLA Proteins 2385
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Supplemental Figure 7 online). Furthermore, recombinant DELLA
protein lacking both the N108 and LZ domains was completely
stabilized inour cell-freeextracts (Figure8D).Moreover, compared
with RGAnN108, RGAnLZ was more stable in Paclobutrazol-
treated extracts (see Supplemental Figure 8 online). Thus, our
results suggest that in addition to the N-terminal regulatory do-
main, the LZ domain is also involved in regulating DELLA protein
stability.
The LZ Domain of DELLA Proteins Is Essential for Their
Stability and Activity
To investigate further the function of the LZ domain of DELLA
proteins in vivo, we generated transgenic lines expressing 35S:
TAP-RGA and 35S:TAP-RGAnLZ in the della pentuple mutant
background (in which all five DELLA genes are defective). Using
these lines, we found that in both continuous dark andwhite light,
TAP-RGA protein was degraded upon GA3 induction and stabi-
lized byMG132 treatment, while TAP-RGAnLZ protein remained
stable in both cases (Figure 9A; see Supplemental Figure 9
online). Again, our data demonstrate that the LZ domain was
indeed required for DELLA degradation.
Since hypocotyl elongation of seedlings was shown to be
promoted by GA and repressed by DELLA proteins in red light
(Feng et al., 2008), we next compared hypocotyl lengths of wild-
type, della global mutant, 35S:TAP-RGA/della, and 35S:TAP-
RGAnLZ/della seedlings grown in red light. As shown in Figures
9B and 9C, TAP-RGA, but not TAP-RGAnLZ, can rescue the
long hypocotyl phenotype caused by the loss of function of all
five DELLA proteins. Therefore, our data indicate the LZ domain
is not only required for the regulation of DELLA protein stability
but also essential for its activity in vivo.
DISCUSSION
In plants, the DELLA proteins are known to be fundamental
repressors in GA signaling pathways and also serve as integra-
tors of other phytohormone and environmental signals to regu-
late many aspects of growth and development (Jiang and Fu,
2007). GA responses are triggered by turning off the growth
repression caused by DELLA proteins, and a key event in this
derepression process is the 26S proteasomedependent deg-
radation of DELLA proteins (Sun and Gubler, 2004; Stone and
Callis, 2007). In this report, we successfully reconstituted DELLA
protein degradation in anArabidopsis cell-free system and built a
platform for further uncovering the mechanism underlying the
regulation of their stability.
Polyubiquitination of proteins is crucial for proteasomal rec-
ognition and subsequent degradation. This chain elongation
process requires the modification of specific Lys residues in
each successive ubiquitin moiety. Previous studies showed that
all seven Lys residues of ubiquitin can be used for chain forma-
tion in yeast (Peng et al., 2003). Different linkage specificity
results in distinct topologies and functions of ubiquitin chains.
Although chains linked through ubiquitin Lys-48 usually function
as the signal for proteasome-dependent degradation (Kerscher
et al., 2006), recent studies showed that human APC/C, a
multisubunit E3, triggers substrate degradation by preferentially
assembling Lys-11linked rather than Lys-48linked ubiquitin
chains (Jin et al., 2008). Thus, other linkage specificities can also
mediate protein degradation by the 26S proteasome pathway.
This hypothesis was further confirmed by the finding that all the
Lys linkages of ubiquitin, except Lys-63, can induce proteasomal
degradation in yeast (Xu et al., 2009). A more recent study in
Arabidopsis using a combination of TAP and mass spectrometry
techniques suggested that the topology of ubiquitin chains can
be highly complex in plants (Saracco et al., 2009). In this study,
we used the cell-free degradation of TAP-RGA to test the
competitive efficiencies of exogenously added mutant ubiqui-
tins. Our data showed that exogenous application of ubiquitin
mutants with Lys-48 to Arg (K48R) or both Lys-48 and Lys-63
mutated to Arg residues (K48/63R) had no observable effect on
DELLA degradation, whereas the 3KTR (Lys-29, Lys-48, and
Lys-63 mutated to arginines) ubiquitin mutant had similar effi-
ciency at blocking degradation as the ubiquitin with all seven Lys
residues mutated to Arg (K0). We also noted that the K29R single
mutant was less efficient than the 3KTR mutant ubiquitin (Figure
4; see Supplemental Figure 3 online). Taken together, our results
Figure 9. The LZ domain of DELLA Protein Is Necessary for Both Protein
Stability and Activity.
(A) Levels of TAP-RGA and TAP-RGAnLZ in 7-d-old 35S:TAP-RGA/della
and 35S:TAP-RGAnLZ/della seedlings grown in continuous white light. The
seedlings were treated with 50 mM MG132 and 100 mM GA3 as indicated.
(B) Morphology of 7-d-old red lightgrown wild-type, 35S:TAP-RGA/
della, and 35S:TAP-RGAnLZ/della mutant seedlings. Bar = 5 mm.
(C) Comparison of hypocotyl lengths of red lightgrown seedlings
(mean 6 SE, n = 20). della refers to the rga-t2 gai-t6 rgl1-1 rgl2-1 rgl3-1
pentuple mutant.
2386 The Plant Cell
-
suggest that although Lys-48 is the canonical site for signaling
degradation, Lys-29linked ubiquitin chains seem to be the
major linkage triggering degradation of DELLA proteins. Similar
observations have been made with the UFD pathway (ubiquitin
fusion degradation) in yeast, in which Lys-29 is more dominantly
used than Lys-48 in forming ubiquitin isopeptide bonds (Johnson
et al., 1995; Koegl et al., 1999).
Consistent with previous studies in rice (Itoh et al., 2002), our
domain dissection analysis in the Arabidopsis cell-free system
revealed that the LZ domain is important for the stability of DELLA
proteins.Moreover, taking advantage of the phenotypic analysis of
transgenic lines in thedellapentuplemutant background,we found
that the LZ domain is also essential for the growth-suppressive
function of DELLAproteins. The LZ domain is conserved in all plant
DELLA proteins, and yeast two-hybrid studies suggested that it is
required for dimerization of rice DELLA proteins (Itoh et al., 2002).
Consistent with our findings, a recent study reported that DELLA
proteins can inactivate PIF proteins by binding to them through the
LZ domain (de Lucas et al., 2008). These observations raise the
possibility that absence of the LZ domainmay abolish the ability of
DELLA proteins to interact with transcription factors that function
as positive regulators of plant growth, thus compromising the
growth-suppressive function of the DELLA proteins.
A large number of mutants have been generated and identified
over the past decades, making Arabidopsis a powerful model
system for studying gibberellin signal transduction and the
ubiquitin-proteasome pathway. A major benefit of this cell-free
degradation system is that it allows us to test the requirements
and roles of both known and unknown regulators of DELLA
protein degradation. Using our system, we provided direct
biochemical evidence that the GA receptors and the F-box
protein SLY1 are involved in DELLA degradation, thus corrobo-
rating and substantiating previous genetic studies. In addition,
although SLY1/GID2 have been shown to be associated with
CUL1 in vivo (Fu et al., 2004; Gomi et al., 2004), CUL1 has not
been directly demonstrated to be functionally important for
SCFSLY1/GID2 activity. In this report, we provided both in vitro
and in vivo degradation results showing that the biological
activity of the CUL1-based SCFSLY1/GID2 E3 complex is essential
for regulating the stability of DELLA proteins.
There are several possible reasons why the phosphatase
inhibitor but not the kinase inhibitors blocked the degradation
of recombinant DELLA proteins purified from E. coli in our cell-
free assays. First, we may have failed to detect kinase activity
toward the recombinant DELLA proteins in our assay system.
This is entirely possible as any cell-free assay system comeswith
its limitations, and ours is no exception. It is also possible that
bacteria may have kinases that already modified the recombi-
nant DELLA proteins. Alternatively, it may also be possible that
DELLA proteins themselves might not be directly phosphor-
ylated and dephosphorylated. Instead, because the degradation
of DELLA proteins most likely requires other regulators, it is
possible that DELLA degradation may be influenced by the
phosphorylation status of one of these proteins. This raises the
possibility that there is a new layer of the fine regulation of DELLA
protein degradation.
Taken together, knowledge gained from our in vitro studies,
combined with previous genetic and in vivo data, allowed us to
place the various signaling components into a biochemical
framework. We envisage that our cell-free degradation system
might be widely adapted for studying other phytohormone or
cellular signaling pathways, in which regulated protein degrada-
tion seems to be a recurrent theme.
METHODS
Plant Materials and Growth Conditions
The wild-type plants used in this study were of the Col-0 and Ler ecotypes.
The 35S:TAP-RGA/rga-24, 35S:TAP-GAI/gai-t6, 35S:TAP-RGA/Ler, 35S:
TAP-GAI/Ler, 35S:TAP-RGL1/Ler, 35S:TAP-RGL2/Ler, 35S:TAP-RGL3/
Ler, gid1b/c, della pentuple mutant, sly1-d, sly1-10, and cul1-6 used in
the study were described previously (Dill et al., 2004; Fu et al., 2004; Moon
et al., 2007; Feng et al., 2008). Seeds were surface sterilized and cold
treated for 4 d at 48Cbefore theywere sown. Seedlingswere grownonsolid
Murashige and Skoog medium at 228C under continuous white light or
darkness for 7 d before harvest, unless otherwise stated.
Plasmid Construction and Generation of Transgenic Arabidopsis
thaliana Plants
For purification of recombinant MBP-DELLA proteins, BamHI/SalI DNA
fragments encoding full-length RGA coding sequence, RGAnN108 (de-
letion of amino acid residues 1 to 108), RGAnLZ (deletion of amino acid
residues 220 to 257), RGAnN108nLZ, and EcoRI/SalI DNA fragments
encoding full-lengthGAI coding sequencewere cloned into the pMal-C2X
vector (NEB). The gene-specific primers used for those clones are listed in
Supplemental Table 1 online. The MBP-tagged fusion proteins were
expressed in the Escherichia coli strain BL21 (DE3) and purified with
amylase resin (NEB) according to the manufacturers instructions. To
construct 35S:TAP-RGAnLZ, a fragment containing RGAnLZ was in-
serted into the SacII and AscI site of pENTR/D-TOPO (Invitrogen). This
fragment was then subcloned into the pN-TAP binary vector using the
Gateway cloning system (Rubio et al., 2005). Finally, 35S:TAP-RGA and
35S:TAP-RGAnLZ fragments were introduced into the della pentuple
mutant, a loss-of-function mutant for all five DELLA proteins (Feng et al.,
2008), by the flower dip method, and the transgenic plants were selected
on Murashige and Skoog plates with 100 mg/L of gentamycin (Amer-
esco). The expression level of TAP-tagged DELLA proteins in total protein
extracts from seedlings of each independent transgenic linewas checked
by protein blots using anti-Myc antibody (Sigma-Aldrich).
For generation of pSLY1:myc-SLY1, the cauliflower mosaic virus 35S
promoter of p35S:SLY1 vector (Fu et al., 2004) was replacedwith theDNA
fragment 3 kb upstream of the SLY1 transcripts start site, which was
amplified using the Expand Long Template PCR System (Roche Applied
Science) and the primers PS-F (59-CCGCTCGAGTTCCGACAGAACC-
GATGCGCACTCGCAT-39) and PS-R (59-CTCCTAGGGTGGAATC-
GATTGCGAAGATCAATCA-39). The PCR product of 33myc coding
sequence was amplified using primers myc-F (59-CTCCTAGGATGGAA-
CAAAAGTTGATTTCTGAAG-39) and myc-R (59-GACGCATGCGGTT-
CAAGTCTTCTTCTGA-39) and then cloned into the AvrII/SphI site. SLY1
coding sequence was also cloned in frame into the SphI/BamHI site. The
DNA sequences of pSLY1:myc-SLY1 construct were confirmed before
plant transformation.
Protein Extraction and Cell-Free Degradation
Arabidopsis seedlings were harvested and ground into fine powder in
liquid nitrogen. Total proteins were subsequently extracted in degrada-
tion buffer containing 25 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM
MgCl2, 4 mM PMSF, 5 mMDTT, and 10mMATP as previously described
(Naidoo et al., 1999; Osterlund et al., 2000; Yanagisawa et al., 2003). Cell
Cell-Free Degradation of DELLA Proteins 2387
-
debris was removed by two 10-min centrifugations at 17,000g in 48C. The
supernatant was collected and protein concentration was determined by
the Bio-Rad protein assay. The total protein extracts prepared from
gid1b/c, sly1-10, sly1-d, and cul1-6mutants and correspondingwild-type
control seedlingswere adjusted to equal concentration in the degradation
buffer for each assay. Then, MG132 (Calbiochem), staurosporine (Sigma-
Aldrich), AG555 (Calbiochem), microsystin-LR (Sigma-Aldrich), apyrase
(Sigma-Aldrich), and ubiquitin Lys mutants (BostonBiochem) were selec-
tively added to various in vitro degradation assays as indicated. For plant-
derived TAP-DELLA protein degradation, the extracts of TAP-tagged
DELLA transgenic lines were used. For E. colipurified MBP-DELLA
protein degradation, 100 ng of recombinant MBP-DELLA protein was
incubated in 100-mL extracts (containing 500 mg total proteins) for the
individual assays, unless otherwise stated. All the mock controls used an
equal amount of solvents for each drug. The extracts were incubated at
228C, samples were taken at indicated intervals for determination of
DELLA protein abundance by immunoblots, and the results were quan-
tified using Quantity One software (Bio-Rad).
In Vivo Immunoprecipitation
Seven-day-old 35S:TAP-RGA/rga-24 seedlings were extracted in an
extraction buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 10 mM MgCl,
1 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, and 13 Roche protease
inhibitor cocktail). The final protein concentration was adjusted to 1 mg/
mL, and then myc affinity matrix (Covance) was incubated with the
extracts at 48C for 4 h with gentle rotation. After incubation, myc-affinity
matrix was recovered by centrifugation and thoroughly washed and then
added to degradation extracts for cell-free degradation assays.
Immunoblot Analysis and Antibodies
All the harvested protein samples were resolved on 8% SDS-PAGE and
transferred to polyvinylidene fluoride membranes. Anti-myc monoclonal
antibody at 1:1000 dilution (Sigma-Aldrich) and affinity-purified anti-
regulatory particle non-ATPase 6 (RPN6) polyclonal antibody (1:3000
dilution) were used as primary antibodies (Kwok et al., 1999). MBP-
DELLA proteins were detected by anti-MBP monoclonal antibody at
1:5000 dilution (NEB). Immunoblots were detected using peroxidase-
conjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich) at both
1:8000 dilutions and ECL Plus reagent (GE Healthcare).
Other primary antibodies used in this study included anti-HY5 at 1:1000
dilution (Osterlund et al., 2000) and anti-RGA at 1:500 dilution (Feng et al.,
2008).
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Ge-
nome Initiative or GenBank/EMBL/Swiss-Prot databases under the fol-
lowing accession numbers: CUL1 (At4g02570), SLY1 (At4g24210),
GID1A (At3g05120), GID1B (At3g63010), GID1C (At5g27320), RGA
(At2g01570), GAI (At1g14920), RGL1 (At1g66350), RGL2 (At3g03450),
RGL3 (At5g17490),SLR1 (Q 7G7 J 6),SLN1 (Q 8W127),DWRF8 (Q 9S T
4 8), RHT1 (Q 9 S T 5 9), and RPN6 (At1g29150).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Cell-Free Degradation of DELLA Protein
Was Blocked by PAC Treatment.
Supplemental Figure 2. Quantifications of the Percentages of TAP-
RGA Remaining in Cell-Free Extracts.
Supplemental Figure 3. Application of Exogenous K29R Ubiquitin
Has No Effect on HY5 Cell-Free Degradation.
Supplemental Figure 4. Myc-SLY1 Facilitates Cell-Free Degradation
of MBP-RGA Protein in sly1-10 Mutant Extracts.
Supplemental Figure 5. Microcystin-LR Has No Effect on HY5
Degradation in the Cell-Free System.
Supplemental Figure 6. Quantifications of Percentages of MBP-RGA
Protein Remaining in the Cell-Free Extracts.
Supplemental Figure 7. Alignment of the Conserved Leucine Heptad
Repeat Motif of DELLA Homologs in Higher Plants.
Supplemental Figure 8. PAC Treatment Delays the in Vitro Degra-
dation of RGAnN108 and RGAnLZ to Different Extents.
Supplemental Figure 9. Expressions and GA Responses of TAP-
RGA and TAP-RGAnLZ in Continuous White Light and Dark.
Supplemental Table 1. Summary of Primers for Generating RGA and
GAI Constructs.
ACKNOWLEDGMENTS
We thank Tai-ping Sun for providing sly1-d and sly1-10 mutants and
Mark Estelle for providing cul1-6 seeds. We also thank Suhua Feng for
kindly providing useful transgenic lines, Haiyang Wang and William
Terzaghi for critical comments on the manuscript, and all the former and
present members of the Deng lab in the National Institute of Biological
Sciences for their helpful discussions. This study was supported by
National 863 High-Tech Project of National Ministry of Science and
Technology, Peoples Republic of China (2003AA 210070), the Beijing
municipal government commission of science and technology, and the
National Natural Science Foundation of China (90717112).
Received December 31, 2008; revised July 2, 2009; accepted August 12,
2009; published August 28, 2009.
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DOI 10.1105/tpc.108.065433; originally published online August 28, 2009; 2009;21;2378-2390Plant Cell
Fan and Xing Wang DengFeng Wang, Danmeng Zhu, Xi Huang, Shuang Li, Yinan Gong, Qinfang Yao, Xiangdong Fu, Liu-Min
Assay System DELLA Proteins Gained From a Cell-FreeArabidopsisBiochemical Insights on Degradation of
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Supplemental Data http://www.plantcell.org/content/suppl/2009/08/13/tpc.108.065433.DC1.html
References http://www.plantcell.org/content/21/8/2378.full.html#ref-list-1
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