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, People’s 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 xingwang.deng@yale.edu.The 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(xingwang.deng@yale.edu).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.108.065433

The Plant Cell, Vol. 21: 2378–2390, August 2009, www.plantcell.org ã 2009 American Society of Plant Biologists

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

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 promoter–driven 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.

2380 The Plant Cell

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

promoter–driven 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 proteasome–dependent 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 the

degradation extracts.

Cell-Free Degradation of DELLA Proteins 2381

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).

2382 The Plant Cell

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

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 mMGA3

and 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).

2384 The Plant Cell

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

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 proteasome–dependent 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-11–linked rather than Lys-48–linked 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 light–grown wild-type, 35S:TAP-RGA/

della, and 35S:TAP-RGAnLZ/della mutant seedlings. Bar = 5 mm.

(C) Comparison of hypocotyl lengths of red light–grown 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-29–linked 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 manufacturer’s 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. coli–purified 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, People’s 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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2390 The Plant Cell

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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References /content/21/8/2378.full.html#ref-list-1

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