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I

A DISSERTATION FOR THE DEGREE OF DOCTOR OF

PHILOSOPHY

Regulation of gibberellin signaling by E3 SUMO ligase

SIZ1 in Arabidopsis thaliana

BY

Sung-Il Kim

FEBRUARY 2015

MAJOR IN CROP SCIENCE AND BIOTECHNOLOGY

DEPARTMENT OF PLANT SCIENCE

COLLEGE OF AGRICULTURAL AND LIFE SCIENCES

THE GRADUATE SCHOOL OF SEOUL NATIONAL UNIVERSITY

II

ABSTRACT

Regulation of gibberellin signaling by E3 SUMO ligase

SIZ1 in Arabidopsis thaliana

BY

Sung-Il Kim

Gibberellins affect various plant development processes including germination,

cell division and elongation, and flowering. Here, I show that the E3 SUMO

ligase activity of AtSIZ1 regulates GA signaling in Arabidopsis by sumoylating

SLY1. SLY1 was less abundant in siz1-2 mutants than wild-type plants.

Transgenic sly1-13 mutants over-expressing SLY1 were phenotypically similar

to wild-type plants; however, sly1-13 plants over-expressing a mutated mSLY1

protein (K122R, a mutation at the sumoylation site) retained the mutant

dwarfing phenotype. Over-expression of SLY1 in sly1-13 mutants resulted in a

return of RGA (REPRESSOR of ga1-3) levels to wild-type levels, but RGA

accumulated to high levels in mutants over-expressing mSLY1. RGA was

clearly detected in Arabidopsis co-expressing AtSIZ1 and mSLY1, but not in

plants co-expressing AtSIZ1 and SLY1. In addition, sumoylated SLY1

III

interacted with RGA and SLY1 sumoylation was significantly increased by GA.

Taken together, my results indicate that AtSIZ1 positively controls GA signaling

through SLY1 sumoylation.

Keywords: AtSIZ1, E3 SUMO ligase, GA signaling, Gibberellin, RGA, SLY1,

SUMO, sumoylation

IV

CONTENTS

ABSTRACT

CONTENTS

LIST OF FIGURES

LIST OF TABLES

LIST OF ABBRIVIATIONS

LITERATURAL REVIEWS

1. GA signaling pathway.

2. SLY1, F-box protein that is involved in GA signaling.

3. DELLA, act to transcription repress of GA-responsive growth and

development.

4. SUMO, Small Ubiquitin like Modifier.

1. Consensus SUMO motif

2. E3 enzymes

3. The SUMOylation pathway

4. Components of the SUMO pathway in Arabidopsis

5. Role of the SUMO in plants

1. Responses to abiotic stress

2. Responses to hormone signaling

V

INTRODUCTION

MATERIALS AND METHODS

1. Plant materials and growth conditions

2. Effect on GA on seed germination and seedling growth

3. Construction of recombinant plasmids

4. Purification of recombinant proteins

5. In vitro binding assay

6. Subcellular Localization of Proteins

7. Yeast two-hybrid assays

8. Sumoylation assays

9. Production of transgenic Arabidopsis plants and complementation tests

10. Examination of SLY1 and RGA levels in siz1-2 and sly1-13 mutants

11. Examination of the effect of AtSIZ1 activity and SLY1 sumoylation on

RGA level

12. Interaction analysis between sumoylated SLY1 and RGA by in vitro

pull-down assay

13. Examination of the effect of SUMO protease on SLY1 level

14. Measurement of transcript levels of GA-responsive genes including

SLY1

15. Examination of AtSIZ1 transcript and protein levels

16. Hypocotyl length measurements

17. Antibody production

VI

RESULTS

1. Seed germination of the siz1-2 mutant is recovered by exogenous GA

2. SLY1 is sumoylated by E3 SUMO ligase AtSIZ1

3. SLY1 modification by SUMO promotes plant growth and development

4. siz1-2 mutant has short hypocotyls

5. SLY1 localization affect by exogenous GA

6. SLY1 is stabilized by AtSIZ1

7. RGA level is affected by both SLY1 and AtSIZ1

8. Sumoylated SLY1 interacts with RGA

9. Interactions between SLY1 and AtSIZ1 or ASK are strong and specific

10. Expression of GA signaling-related genes was not notably affected by

AtSIZ1

11. AtSIZ1 expression is regulated by GA signaling

12. Sumoylation of SLY1 is stimulated by GA supply

13. AtSIZ1 is not involved in regulation of GID1A activity

DISCUSSIONS

REFERENCES

ABSTRACT IN KOREAN

VII

LIST OF FIGURES

Figure 1. Schematic representation of activation of GA signaling in

Arabidopsis thaliana.

Figure 2. Role of SLY1 depending on GA.

Figure 3. SUMOylation pathway in Arabidopsis thaliana.

Figure 4. The roles of SUMOylation in Arabidopsis thaliana.

Figure 5. AtSIZ1 positively regulates seed germination.

Figure 6. AtSIZ1 physically interacts with SLY1.

Figure 7. Subcellular co-localization of AtSIZ1 and SLY1.

Figure 8. AtSIZ1 directly sumoylates SLY1 using SUMO1.

Figure 9. AtSIZ1 sumoylates SLY1 using SUMO1 in Arabidopsis.

Figure 10. Identification of the SLY1 sumoylation site.

Figure 11. sly1-13 mutant selection and SLY1 specific antibody

production.

Figure 12. Phenotypes of sly1-13 mutants over-expressing SLY1 or

mSLY1.

Figure 13. Hypocotyl lengths of siz1-2 mutants.

Figure 14. Subcellular co-localization of SLY1 and mSLY1 depending

on GA.

Figure 15. Effect of SUMOylation on the levels of SLY1.

Figure 16. Effect of AtSIZ1 on the levels of SLY1.

VIII

Figure 17. AtSIZ1 and GA effects on RGA level depending on SLY1 or

mSLY1.

Figure 18. AtSIZ1 negatively regulates RGA level through sumoylation

of SLY1.

Figure 19. RGA interacts with sumoylated SLY1.

Figure 20. Yeast two-hybrid analysis of interactions between mSLY1

and other proteins or between SLY2 and AtSIZ1.

Figure 21. Interactions between SLY1 or mSLY1 and ASK are strong.

Figure 22. Transcript levels of GA-synthetic and -responsive genes in

siz1-2 mutants.

Figure 23. Effect of GA treatment on relative levels of AtSIZ1 transcript

and protein.

Figure 24. Effect of GA on sumoylation of SLY1.

Figure 25. AtSIZ1 did not interact with GID1A.

Figure 26. Schematic representation of the possible mechanism of

activation of GA signaling by SLY1 sumoylation.

IX

LIST OF TABLES

Table 1. Arabidopsis proteins of the SUMO pathway

Table 2. List of primers used in this study

X

LIST OF ABBREVIATION

CHX Cycloheximide

DTT Dithiothreitol

YFP Yellow fluorescent protein

GST Glutathione S-transferase

MBP Maltose-binding protein

HA Haemagglutinin

MS Murashige and Skoog

35S Cauliflower mosaic virus promoter

AD Activation domain

BD DNA binding domain

E.coli Escherichia coli

IPTG Isopropyl-β-D-thiogalactoside

PMSF Phenylmethylsulphonyl fluoride

SUMO Small ubiquitin-related modifier

kDa Kilo Dalton

PCR Polymerase chain reaction

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

WT Wild type

cDNA Complementary DNA

RNA Ribonucleic acid

XI

DNA Deoxyribo nucleic acid

dNTP Deoxy nucleotide triphosphase

M Molar

mg Milligram

rpm Rotation per minute

ssDNA Single-stranded DNA

GID1 GA-insensitive dwarf1

SLY1 SLEEPY1

ABI Abscisic acid insensitive

DELLA DELLA protein

GA Gibberellic Acid

１

LITERATURAL REVIEWS

1. GA signaling pathway.

The plant hormone gibberellic acids (GAs) have a pronounced impact on plant

development. First identified in rice cultures infected with the fungus

Gibberella fujikuroi, important roles in diverse processes such as seed

germination, internode elongation, fruit formation, flower development and

control of flowering time are known by GA signaling (Davière and Achard,

2013). For example, Arabidopsis plants treated with GA grow taller, display a

light green color, early flowering and siliques with fewer seeds than wild type

plants (Davière and Achard, 2013). This broad spectrum of action has applied

GA into an important biotechnological tool both for improving field

productivity and for industrial application.

Gibberellins encompass plenty of chemically related compounds, of which only

amine or proportion, such as GA1, GA3, GA4 and GA7, are biologically active

(Olszewski et al., 2002). GA is adapted by the GA receptor GIBBERELLIN

INSENSITIVE DWARF 1(GID1). Structural analyses have displayed

conformation changes of GID1 after perceiving to bioactive GA as a key event

in GA signaling (Murase et al., 2008; Shimada et al., 2008). Single gid1

mutants normally develop, and only a triple knockout mutant (gid1a·b·c)

displays the dwarf phenotype characteristic of GA signaling deficiency

(Griffiths et al., 2006; Willige et al., 2007), it suggests that the three receptors in

２

Arabidopsis have overlapping biological functions. This is in agree with the

observation that the three paralogs are expressed in all tissues, but at different

levels (Griffiths et al., 2006; Nakajima et al., 2006).

The basis of the GA signaling are constituted by the GA-induced

conformational change in GID1a-c receptors. After binding, a pocket of GID1

locks in the bioactive GA molecule through its N-terminal region (Murase et al.,

2008). The biological importance of the N-terminal extension switch covering

the bioactive GA molecule relies on the interaction with the DELLA proteins, a

class of repressors (Griffiths et al., 2006). Binding to bioactive GA makes a

hydrophobic surface on the GID1-GA complex, which leads the interaction with

the DELLA proteins. In turn, this interaction further stabilizes the GID1-GA-

DELLA complex, and enhances its interaction with the F-box protein of the

ubiquitin E3 ligase SCF complex. Therefore, GA perceiving to GID1 finally led

to DELLA proteins ubiquitination and degradation via proteasome (Figure 1,

McGinnis et al., 2003; Sasaki et al., 2003; Dill et al., 2004; Fu et al., 2004).

３

DELLA proteins

E3 ubiquitin ligase

GA receptors

GA

SLY1/SNZ

RGA RGL2 RGL3GAI RGL1

Arabidopsis thaliana

Plant growth

GID1A GID1B GID1B

Figure 1. Schematic representation of activation of GA signaling in

Arabidopsis thaliana. GA first adapted GID1s are Arabidopsis GA receptors.

SLY1 or SNZ involves setting a SCF complex with DELLA proteins to plant

growth. GA: gibberellic acid.

４

2. SLY1, F-box protein that is involved in GA signaling.

The SCFSLY1 E3 ubiquitin ligase complex decreases DELLA repression by

targeting DELLA for destruction by the ubiquitin-proteasome pathway. The

SCF complex is composed of a Skp1 homolog termed ASK, a Cullin homolog,

an Rbx1 homolog, and an F-box protein that binds a specific target. Structure of

the quaternary complex contains Cul1, Rbx1, Skp1 and the F-box of Skp2, and

the structure of the Cul1–Rbx1 complex is revealed (Zheng et al., 2002). There

are 21 ASK family genes, five Cullins (CULs), two Rbx1 homologs, and 694 F-

box proteins in Arabidopsis (Gagne et al., 2002; Gray et al., 2002; Risseeuw et

al., 2003). The F-box protein binds to the Skp1 homolog of the complex

through direct protein-protein interaction via the F-box domain. The F-box

protein usually binds the target protein via the C-terminal domain (Dill et al.,

2004; Fu et al., 2004). Steber et al. (1998) first isolate GA response sleepy1

(SLY1) mutant then presents evidence that plant brassinosteroid (BR) hormones

rescues the germination phenotype of severe GA insensitive mutant sly1 as a

role in promoting germination (Steber et al., 2001). Positional cloning and gene

characterization of SLY1 revealed that it encodes a putative F-box protein. This

result suggests that SLY1 is the F-box subunit of an SCF E3 ubiquitin ligase

that regulates GA responses. Moreover, its substrates DELLA proteins also

identify (McGinnis et al., 2003). Yeast two-hybrid data indicate that the SLY1

protein can bind to DELLA protein via the C-terminal LSL domain (Dill et al.,

2004; Fu et al., 2004). GID2 is also identified as rice GA dependent F box

５

protein which is essential for GA mediated DELLA protein degradation.

Moreover, GID2 specifically interacts with a phosphorylated Slender Rice 1

(SLR1, Rice DELLA Homolog) leading the ubiquitin mediated degradation

(Gomi et al., 2004). Arabidopsis thaliana sleepy1gar2-1 (sly1gar2-1) mutant allele

encodes a mutant E138K of subunit on C terminal LSL domain as a gain of

function mutant. The DELLA target protein is more strongly interacted with

sly1gar2-1 than does SLY1. In addition, in Arabidopsis thaliana also, the strength

of the SCFSLY1–DELLA interaction is increased by phosphorylated target (Fu et

al., 2004). GA binding enhances the activity of the GA receptor GA-

INSENSITIVEDWARF1 (GID1) to bind DELLA proteins. Based on yeast

three-hybrid data, Arabidopsis GID1-GA binding to DELLA represents to

increase the affinity of the F-box protein SLY1 for DELLA (Griffiths et al.,

2006). Thus, GA increases SCFSLY1 binding to DELLA proteins, thereby

allowing SCFSLY1 to facilitate the polyubiquitination of DELLA protein.

Conjugation of four ubiquitin moieties to a target protein makes its recognition

and proteolysis by the 26S proteasome (Smalle and Vierstra, 2004). It appears

that DELLA is degraded via the ubiquitin-proteasome pathway, because both

mutations in SLY1 and 26S proteasome inhibitors result in stabilization and

increased level of DELLA protein in the presence of GA (Figure 2, McGinnis et

al., 2003; Dill et al., 2004; Fu et al., 2004). Overexpression of the SLY1

homolog SNEEZY (SNE)/SLY2 partially rescues most of sly1 phenotypes,

suggesting that SNE has similar function like a SLY1 (Ariizumi et al., 2011).

６

- GA

+ GASKP

GA

GA

DELLA GRASGA

DELLA

GR

AS

E2

Rbx

GA

DE

LL

A

GRAS

Ubi

26SProteosome

DELLA GRAS

PIF, JAZ, BZR, SCL, SPL,

ALC, MYC, EIN, IDD

Transcription

factor

Heterodimerization

Conformational change

Ubiquitination

GID1

GID1

GID1

GID

1

GID1

CULLIN

SL

Y1

LS

L

F b

ox

７

Figure 2. Role of SLY1 depending on GA. In GA treatment, GA adapted

GID1 has occurred conformational change to activated form then binds to

DELLA protein for its ubiquitination via SCFSLY1 complex. In no GA

conditions, DELLA proteins are hetero-dimerized with several transcription

factors involved in gibberellin, jasmonic acid, ethylene, brassinosteroid

signaling to repress activity.

８

3. DELLA, act to transcription repressor of GA-responsive growth and

development.

In Arabidopsis, DELLA proteins were first discovered in a genetic screen as a

GA insensitive mutant (gai-1) (Koornneef et al., 1985; Silverstone et al., 1997).

The gain-of function gai-1 mutant encodes a functional protein with a 17 amino

acids deletion that deletes a conserved 5 amino acids motif (DELLA) in the N-

terminal region (Peng and Harberd, 1993; Peng et al., 1997). Other GA-

insensitive alleles identified in wheat (Rht), rice (SLR) and maize (D8) also

encode non-functional proteins removing the DELLA domain (Fujioka et al.,

1988; Ikeda et al., 2001; Pearce et al., 2011). In contrast to other species

harboring a single copy gene, Arabidopsis encode 5 functionally redundant

DELLA proteins: GA INSENSITIVE1 (GAI), REPRESSOR OF ga1-3 (RGA)

and RGA-LIKE1, -2and -3 (RGL1, -2, -3) (Bolle, 2004). The cloning and

characterization of gai-1 and other GA-insensitive DELLA mutant represented

that the DELLA motif is essential for the interaction with the GID1 receptors

and DELLA degradation in the presence of GA (Peng and Harberd, 1993; Peng

et al., 1997; Dill et al., 2001; Willige et al., 2007). High GA levels enhance the

rapid degradation of DELLA proteins via the ubiquitin-proteasome pathway;

however GA-deficient mutants present increased levels of DELLA proteins

(Dill et al., 2001; Silverstone et al., 2001; Griffiths et al., 2006; Willige et al.,

2007). GA-insensitive DELLA mutants similar to some enhanced dark green

and dwarfed gibberellin-deficient mutants but differ from them in some aspects.

９

First, they are genetically semi-dominant; second, they cannot be recovered by

exogenous GA application; third, bioactive GA level is increased in these

mutants (Koornneef et al., 1985; Dill et al., 2001). Therefore, absent of the

DELLA domain are insensitive to GA because they cannot interact with GID1

receptor even in the presence of high GA level. Together, these results suggest

that the high concentration of DELLA proteins in response to GA levels

facilitates the biological response to this hormone. Transcriptome experiments

in Arabidopsis reveal that GA controls numerous genes involved in a broad

range of biological processes (Willige et al., 2007; Zentella et al., 2007).

Nevertheless, the analysis of the primary protein sequence shows that the

DELLA proteins do not contain a canonical DNA binding domain typical of

transcription factors (Bolle, 2004). In concordance with this data, ChIP

experiments failed to consistently discover binding sites, even though binding to

some GA-related genes was observed (Zentella et al., 2007). These results

suggest that DELLA proteins do not act as direct transcriptional regulators but

instead control gene expression indirectly as co-factors. A ground breaking

observation on DELLA protein function came from the study of

PHYTOCHROME INTERACTING FACTOR (PIF) during photo-

morphogenesis. Two independent groups described the direct binding between

DELLA proteins and PIF transcription factors (de Lucas et al., 2008; Feng et al.,

2008). Interestingly, EMSA assay indicated that binding to DELLA proteins

destroyed PIF4 binding capacity to DNA, therefore blocking its activity (de

１０

Lucas et al., 2008). Based on these results it has been represented that DELLA

proteins control effector transcription factors through direct interaction (Daviere

et al., 2008). In support of this model, a little of interactions between DELLA

proteins and other transcription factors have been recently discovered (Hou et

al., 2010; Zhang et al., 2011; Hong et al., 2012). Recently, DELLA-dependent

growth regulation can be controlled independently of GA. when a proportion of

DELLAs is conjugated to the Small Ubiquitin-like Modifier (SUMO) protein,

the extent of conjugation increases during stress. SUMO-conjugated DELLA

binds to a SUMO interacting motif in GID1 this motif in a GA-independent

manner. The consequent sequestration of GID1 by SUMO-conjugated DELLAs

leads to an accumulation of non-SUMOylated DELLAs, resulting in

advantageous growth attenuate during stress (Conti et al., 2014).

4. SUMO, Small Ubiquitin like Modifier.

The process of SUMOylation is contained to all eukaryotes. In animals,

research has shown it to be essential in many cellular processes including

human diseases, oxygen and glucose deprivation, Alzheimer’s disease, cancer

(Sarge & Park-Sarge, 2009; Yang et al., 2008; Cimarosti et al., 2012; McMillan

et al., 2011; Morris et al., 2009). In plants the story is similar, in that

SUMOylation is necessary to be included in many ways in the cell. This

involves many stress responses (Elrouby & Coupland, 2010), and SUMO

paralogues have been discovered to have functions in plant development and

１１

defense through salicylic acid-regulated defense responses (van den Burg et al.,

2010). Indeed, the SUMO E3 ligase SIZ1 controls regulation on salicylic acid

mediated innate immunity in Arabidopsis, and this the same E3 ligase also has a

role in a broad range of processes such as cell growth and plant development

through salicylic acid (Miura & Hasegawa, 2010), phosphate deficiency

responses (Miura et al., 2005) and genetic regulation following drought stress

(Catala et al., 2007). Early in Short Days 4 (ESD4), a SUMO protease

contributes control of flowering time in Arabidopsis (Reeves et al., 2002;

Murtas et al., 2003). Genetic studies of SUMO conjugation mutants has shown

that SUMOylation of proteins, and with SUMO1 and SUMO2 in particular, is

necessary for Arabidopsis viability, Indeed, where SUMO-activating and

conjugating enzymes (SAE2 and SCE1) are lacking in null mutants, embryo

lethality is observed (Saracco et al., 2007). Finally, increase in SUMOylation

levels restrain abscisic acid (ABA)-governed inhibition of growth and enhance

the induction of ABA- and stress-responsive genes like RD29A (Lois, Lima&

Chua, 2003).

While ubiquitination and SUMOylation have many similarities, there are also

some clear differences. Like ubiquitin, SUMO is existed in all eukaryotic

kingdoms (Hanania et al., 1999) and well known, two systems have the same

ancestry, which may date back to ancient biosynthetic pathways of prokaryotes

(Hochstrasser, 2000). SUMO is highly conserved in all species from yeast to

humans (Müller et al., 2001). Approximately 18% sequence identity is observed

１２

in SUMO and ubiquitin even though they do share similar 3D structures with

the characteristic Ubfold (Bayer et al., 1998) present in both. SUMO has

numerous paralogues in both humans and plants, with at least three in

mammalian cells (SUMO1, 2, 3) and only one in Saccharomyces cerevisiae

(Smt3) (Johnson, 2004; Kerscher, 2007). In humans, SUMO1 and SUMO2/3

show the separated function from each other, as they conjugate with diverse

substrates, and SUMO 2/3 have been represented to have a role in

SUMOylation in cases where proteins have become damaged, such as

environmental stress (Saitoh & Hinchey, 2000). So far 8 SUMO orthologues

have been identified in Arabidopsis (Kurepa et al., 2003; Colby et al., 2006),

however only four of these have been identified thus far as having post-

translational modification function, SUMO 1, 2, 3 and 5 (Colby et al., 2006;

Budhiraja et al., 2009). Studies have shown that AtSUMO1 and AtSUMO2 are

well conserved in sites on the SUMO protein governing E1-activating enzyme

recognition, and E2-conjugating enzyme and SIM (SUMO interacting-motif)

non-covalent interactions, where AtSUMO3 is less conserved, and AtSUMO5

identifies the highest degree of variation. In addition, AtSUMO1/2 are

considerate to be necessary paralogues, as they are the most efficiently

conjugated isoforms, with AtSUMO3 less efficiently conjugated, and

AtSUMO5 even less so (Castaño-Miquel, Seguí & Lois, 2011).

4.1. Consensus SUMO motif

１３

A consensus motif for SUMOylation based on in target sequences was

identified as ΨKXE, where Ψ was any large, hydrophobic amino acid, K was

the target lysine, X was any amino acid, and E was a glutamic acid (Rodriguez

et al., 2001). In vitro this consensus was demonstrated to enhance SUMOylation,

however in vivo a nuclear localization signal was also needed for SUMOylation,

suggesting that the combination of these two might direct a protein to be

conjugated by SUMO (Rodriguez et al., 2001). In addition, the identification of

plasma membrane proteins that are SUMOylated highly indicates that nuclear

localization is not required for SUMOylation in all situations (Dai et al., 2009).

A second motif has been discovered in target proteins for SUMOylation named

as Phosphorylation Dependent SUMO Modification (PDSM). The motif is set

as ΨKXEXXSP in which the SUMO consensus sequence is distinguished by

two amino acids from a phosphorylatable serine followed by a proline

(Hietakangas et al., 2006). SUMOylation at the target lysine is blocked by

serine to alanine mutations of this motif, while constitutive SUMOylation is

occurred in serine to aspartic acid mutations, suggesting that phosphorylation of

a PDSM directly affects the SUMOylation status of the protein (Hietakangas et

al., 2006).

4.2. E3 Enzymes

Because only a single protein, UBC9, is chargeable for conjugating SUMO to

all of the target proteins in a cell, E3 ligases are thought to be needed to obtain

１４

target specificity in vivo. SUMO E3 ligases are identified by their ability to

increase SUMOylation of a substrate protein in vitro, and are usually required

for SUMOylation in vivo (Johnson and Gupta, 2001). The first E3 enzymes

characterized for the SUMOylation pathway are homologous to Protein

Inhibitor of Activated Stat (PIAS) proteins and contained a RING domain the

same to known ubiquitin E3 ligases (Johnson and Gupta, 2001; Sachdev et al.,

2001; Takahashi et al., 2001). SIZ proteins know as these E3 ligases in yeast

and Arabidopsis (Takahashi et al., 2001; Miura et al., 2005). SUMO E3 ligases

can be distinguished into two categories, the RING-type ligases and non-RING-

type ligases. One such example of a non-RING domain E3 ligase is the

polycomb protein Pc2, which plays to bind UBC9 and its substrate protein

CtBP together in a subnuclear structure known as the PcG complex in order to

increase SUMOylation (Kagey et al., 2003). In addition, the non-RING E3

ligase RanBP2 targets RanGAP1, the original SUMO target, for SUMOylation

by and crystallographic data identified that these non-RING domain E3 ligases

interact to a region of the UBC9 protein that is distinct from the binding domain

for RING E3 ligases (Pichler et al., 2002; Tatham et al., 2005). Three different

E3 ligases have been revealed to be capable of increasing in vitro SUMOylation

of the protein Mdm2, including two PIAS (RING-type) E3 ligases and RanBP2

(a non-RING) E3 ligase (Miyauchi et al., 2002). This suggests that a single

target can likely be played upon by multiple E3 ligases.

4.3. The SUMOylation pathway

１５

Sumoylation is a reversible modification which covalently conjugates SUMO to

a target protein followed by a cascade of enzyme reactions containing the steps

of E1-activation, E2-conjugation, E3-ligation and deconjugation (Bayer et al.,

1998). SUMO is matured through cleaving Gly-Gly motif in its C-terminus by

the specific Ulps (ubiquitin-like proteases) and then the presence of ATP

activates SUMO, heterodimeric E1 (SAE1, SAE2) catalyze the formation of

cysteine (C) residue in SAE2 through high-energy thioester bond. In the next

step, activated SUMO is moved to a cysteine residue of Conjugating enzyme E2

(SCE1) through transesterification played by SCE1. Subsequently, SUMO is

transferred to the target through an isopeptide linkage between C-terminal

glycine residue in SUMO and ε-amino group of the substrate lysine side chain

in the target protein’s sumoylation consensus motif. This step is catalyzed by E3

ligase enzyme although E3-independent transfer is possible. SUMO specific

proteases (ULPs) release the SUMO-substrate bonds to recycle free SUMO, as

well as participated in generating mature SUMO (Figure 3).

１６

SUMO-GGSUMO-GG~

SUMO-GG

SUMO-GG

SUMO-GG

Target

Ulp

SAE2

SAE1

ATG +

AMP SAE2

SAE1

SAE2

SAE1

SCE

SCE

SCE

K

TargetK

Ulp

E3 Ligase

E3 Ligase

Ma

tula

tio

n

Figure 3. SUMOylation pathway in Arabidopsis thaliana. Maturation, SUMO

isoforms are encoded as precursor proteins and are processed by SUMO-

specific cysteine proteases (ULP, ubiquitin-like protein-specific protease) with

SUMO peptidase activity. Maturation consists of carboxyl terminal truncation

to expose the di-glycine (GG) motif; Activation by E1, the SUMO carboxyl-

terminal glycine is linked to AMP (SUMO-AMP) catalyzed by the

heterodimeric E1 SUMO-activating enzymes 1 and 2 (SAE1 and SAE2) in an

ATP-dependent reaction. Subsequently, the glycine of SUMO is coupled to a

cysteine (C) residue in SAE2 via a high-energy thioester bond; Conjugation by

E2, SUMO is transferred to a cysteine residue of the E2 SUMO-conjugating

enzyme (SCE1) by transesterification catalyzed by SCE1; Ligation by E3,

１７

SUMO is transferred to the e-amino group of a lysine (K) side chain in the

sumoylation consensus motif (ψKXE/D. ψ, a large hydrophobic residue; X, any

amino acid; E/D, glutamic acid or aspartic acid) of the substrate protein,

forming an isopeptide bond between the carboxyl of glycine in SUMO and the

ε-amino of lysine in the substrate, a process that requires the E3 SUMO ligase

in vivo; Deconjugation, SUMO-specific proteases (ULP) with isopeptidase

activity cleave the isopeptide bond and SUMO is recycled through the

conjugation system.

１８

4.4. Components of the SUMO pathway in Arabidopsis

Arabidopsis genome analyses characterized the genes encoding eight SUMOs

(AtSUMO1-AtSUMO8) and one SUMO-like pseudogene (AtSUMO9), but

expression of only four paralogues (SUMO1/2, SUMO3 and 5) has been

confirmed (Kurepa et al., 2003). The SUMO E1 enzyme is a heterodimer

making of two small subunit (SAE1a, SAE1b) isoforms and one large subunit

SAE2. SAE1 isoforms, SAE1a and SAE1b share 80% identical amino acid

sequence, pronouncing functional redundancy (Saracco et al., 2007). The

SUMO E2 enzyme encoded by single copy of gene although, many Ubiquitin

E2 enzymes are encoded. Transcripts of SAE1a/b and SCE1 are abundantly

expressed in many tissue including seedling, cotyledon, root, stem, shoot tip,

leaf, flower and silique. In addition, SCE1 and SUMO are colocalized in

nucleus demonstrating that sumoylation reactions conduct in nucleus (Lois et al.,

2010). Null T-DNA insertion mutants of SAE2, SCE or both SUMO1 and

SUMO2 represented embryonic lethal, with restrained in early-stage embryos

(globular, heart, torpedo), indicating that sumoylation is necessary in

Arabidopsis (Saracco et al., 2007). Although there are many E3 ligases in

animal, fungi and yeast (Johnson and Gupta 2001, Kahyo et al., 2001, Rose and

Meier 2001, Pichler et al., 2002, Kagey et al., 2003), only two SUMO ligases

[AtSIZ1 (PIAS SUMOE3 ligase) and AtMMS21/HPY2 (Mms21 SUMO E3

ligase)] are identified in Arabidopsis. Loss of function mutants of SIZ1 and

HPY2 demonstrated pleiotropic phenotype indicating the importance of E3

１９

ligases in sumoylation pathway (Miura et al., 2005, Catala et al., 2007, Jin et al.,

2008, Huang et al., 2009, Ishida et al., 2009, Miura et al., 2010). Four of

Arabidopsis SUMO specific proteases (AtULPla, AtULP1c, AtULP1d and

AtESD4) are identified so far (Murtas et al., 2003, Colby et al., 2006, Conti et

al., 2008). These proteases not only have peptidase activity for cleavage of pre-

SUMO at the C-terminus to release the Gly-Gly motif, but also isopeptidase

activity for deconjugation of sumoylated protein to recycle pre-SUMO (Hay et

al., 2007). However, these proteases have a difference of SUMO isoform

discrimination and enzymatic activities (Table 1, Chosed et al., 2006, Colby et

al., 2006).

20

Table 1. Arabidopsis proteins of the SUMO pathway

Arabidopsis ATG number Location Description

SUMO isoforms

SUM1 At4g26840 Nucleus

SUM2 At5g55160 Nucleus Single mutants look like wild type; double mutant is embryo lethal.

SUM3 At5g55170

SUM4 At5g48710

SUM5 At2g32765

SUM6 At5g48700

SUM7 At5g55855

SUM8 N.A

SUM9 N.A

SUMO-activating enzyme E1

SAE1a At4g24940

SAE1b At5g50580

/At5g50680

SAE2 At2g21470 sae2 is embryo lethal

SUMO-conjugating enzyme E2

SCE At3g57870 sce is embryo lethal

E3 ligases

SIZ1 At5g60410 Nucleus Dwarfism, elevated SA level with increased PR1 expression and resistance to

bacterial pathogens, early flowering in short day, hypersensitivity to cold and

phosphorus-limited condition.

MMS21/HPY2 At3g15150 Nucleus Severe dwarf with short roots (fewer and shorter cells in the mature and

elongation zones), premature transition from mitotic cycle to endocycle.

21

Table 1. Continue.

SUMO-specific isopeptidases

ULP1a/ELS1 At3g06910 Cytosolic compartment Dwarfism, early flowering in short day.

ULP1b At4g00690

ULP1c/OTS2 At1g10570

ULP1d/OTS1 At1g60220 Single mutants look like wild type; double mutant is hypersensitive to salt

and plants flower early.

ESD4 At4g15880 Nucleus Early flowering in short day.

ULP2a At4g33620

ULP2b At1g09730

22

5. Role of the SUMO in plants

5.1. Responses to abiotic stress

Sumoylation acts essential role in various abiotic stress responses. The

accumulation of SUMO conjugates is induced by heat, cold, drought, salt,

exposure to excessive copper, incubation with H2O2 or ethanol, suggesting that

sumoylation involves in stress protection and recovery (Figure 4, Kurepa et

al.,2003, Miura et al., 2005, Yoo et al., 2006, Catala et al., 2007, Saracco et

al.,2007, Conti et al., 2008, Chen et al., 2011). Loss of function SIZ1 mutants

represented the phenotypes of short primary roots, increased number of lateral

roots, root hairs and higher anthocyanin accumulation sensitively in response to

pi starvation. The expression of low-Pi transcripts of AtIPS1 and AtRNS1 is

decreased in siz1-2 mutants. AtSIZ1 protein sumoylates the PHR1 (phosphate

starvation response 1) which is a transcriptional regulator of AtIPS1 and

AtRNS1 in vitro, implying that AtIPS1 and AtRNS1 are positively regulated

through sumoylation of PHR1 by AtSIZ1 (Miura et al., 2005). SUMO

conjugation is increased under drought stress. The expression of P5CS1, MYC2,

COR15A and KIN1 genes induced by drought stress is decreased in siz1-3

mutants. It is suggested that SIZ1 acts a crucial role in drought stress response

through the control of the genes expression (Catala et al., 2007). The siz1-3,

SIZ1 null mutant, is sensitive to cold stress, and the cold-induced accumulation

of sumo conjugates is decreased in the mutant, suggesting that SIZ1 acts an

essential role in cold tolerance. The expression of cold-induced genes such as

23

COR15A, COR47 and KIN1 is controlled by transcription factor ICE1 (inducer

of CBF/DREB1 expression 1). ICE1 is sumoylated by AtSIZ1 and its

modification inhibited the expression of MYB15, a negative regulator of

CBF3/DREB1A. This leads to the expression of CBF3/DREB1A and its

downstream genes, resulted in cold tolerance. SIZ1-dependent sumoylation of

ICE1 blocks its polyubiquitination and lead to increase ICE1 stability (Dong et

al., 2006, Miura et al., 2007, Ulrich et al., 2008). Sumoylation of AtHsfA2, heat

shock transcription factor, represses its activity and transcription of Psf101,

Hsf17.6 and Hsf17.4, HsfA2 target genes. Moreover, overexpression of SUMO

in seedlings reduces tolerance on repeated heat treatment, suggesting that

sumoylation might play negatively upon acquired thermo tolerance (Cohen-Peer

et al., 2010). Conversely, SIZ1 is a positive regulator of Salicylic acid (SA)-

independent basal thermo tolerance but not of acquired tolerance. It indicates

that the SIZ1-independent pathway involves in the acquired thermo tolerance

regulatory pathway (Yoo et al., 2006). Previous reports have been implied that

sumoylation is involved to salt stress response in Arabidopsis. OTS1/OTS2, salt

stress-related genes, double mutants represented enhanced salt sensitivity but

not the single ots1 or ots2 mutant. Sumo conjugation is increased much higher

in ost1ost2 double mutants compared with wild-type plants. OTS1-

overexpressing transgenic plants increase salt tolerance and reduce sumoylation,

but mutant overexpressing OST1 (C536S) protein disappearing sumo protease

activity failed to produce a salt tolerant phenotype. These results indicate that

24

OST1 and OST2 conduct as sumo deconjugation and required for salt tolerance

responses (Conti et al., 2008). SIZ1-mediated sumoylation also participates in

copper homeostasis and tolerance. Atsiz1 mutants represented hyposensitivity to

excessive copper and accumulated more copper in shoot tissue than wild type.

Excessive copper increased more sumo conjugates in wild type than in siz1

mutant, demonstrating that sumoylation might be involved in the response on

excessive copper. The expression of metal transporter genes of YELLOW

STRIPE-LIKE 1 (YSL1) and YSL3 is repressed under excess copper stress in the

siz1 mutant. The hyposensitivity to excess copper and abnormal distribution of

copper are decreased greatly in siz1ysl3-1 and slightly in the siz1ysl1 double

mutants. These results indicating that Cu-induced SIZ1 dependent sumoylation

participated in copper distribution and tolerance through transcriptional

repression of YSL1/3 (Chen et al., 2011).

5.2. Responses to hormone signaling

Sumoylation involved in modulation of ABA responses (Figure 4).

Overexpression of AtSUMO1/2 inhibits ABA-mediated root growth inhibition

while co-suppression of AtSCE1a displays no significant differences. In

addition, AtSUMO1- and AtSUMO2-overexpressing plants increase the

expression of stress-responsive genes, RDA29A and AtPLC1, indicating that

SUMO acts a role for ABA response (Lois et al., 2003). Atsiz1 mutants show

the ABA hyposensitivity phenotype that germination and seedling primary root

25

growth are restrained. ABI5 (ABA intensive 5), ABA-responsive transcription

factor, is sumoylated on Lys-391 by AtSIZ1. Expression of ABI5 (K391/R) in

abi5-4 resulted in greater sensitivity to ABA compared with ABI5 expression

suggesting that SIZ1-mediated sumoylation on ABI5 negatively controls ABA

signaling (Miura et al., 2009). Auxin signaling is also controlled by sumoylation.

The SIZ1 mutation caused the inhibition of primary root (PR) elongation and

the promotion of lateral root (LR) formation. However, similar root phenotypes

appear if Pi deficient wild-type seedlings are supplemented with auxin. N-1-

naphthylphthalamic acid (NPA), an auxin efflux inhibitor, reduces the Pi

starvation-induced lateral root formation in wild-type and siz1 seedling. Pi

starvation-induced Auxin accumulation in the PRs and LRs is faster in siz1 than

in wild-type seedlings. Moreover, expression of auxin-induced genes is higher

in siz1 compared to wild-type in response to Pi starvation. These results

suggesting that SIZ1 negatively controls Pi starvation-induced root architecture

remodeling through the regulation of auxin patterning (Miura et al., 2005, 2011).

Loss of SIZ1 function leads to accumulation of Salicylic acid (SA) and

constitutive Pathogenesis-related (PR) genes expression, resulting in increased

disease resistance to bacterial pathogens (Lee et al., 2007). These results

indicating SIZ1 regulates SA-mediated plant defense signaling.

26

Substrate

SUMO

Early Flowering(FLDK3R) → FLC↓)(siz1 mutant)

Defense (SA accumulation)(siz1 mutant)

ABA hypersensitivity(ABI5 (K391R) → hypersensitivity)(sce1 mutant)(siz1 mutant)

Dehydration and salt

hypersensitivity

(siz1 mutant, ots1 and ots2

mutant)starvation hypersensitivity (siz1 mutant )

Copper excess

Hyper thermosensitivity

(siz1 mutant)

Cold hypersensitivity

(ICE (K393R) →

CBF3/BREB1A↓)

(siz1 mutant)

Early Flowering(esd4 mutant)

DELLA protein stability

(ots1 and ots2 mutant)

Figure 4. The roles of SUMOylation in Arabidopsis thaliana. In Arabidopsis

thaliana, SUMOylation conducts several behavers involved in hormone

signaling and abiotic responses.

27

INTRODUCTION

Plant hormones control essential processes of plant development, including

germination, elongation growth, flowering time, floral development and

senescence (Vanstraelen and Benkove, 2012). GA is an important

phytohormone, regulating plant growth at various developmental stages.

Mutants deficient in GA biosynthesis or signaling are non-germinating or

poorly germinating, display dwarfism and are delayed in flowering or fail to

flower (Daviere and Achard, 2013).

GA signaling is regulated by the ubiquitin-mediated proteolysis pathway. Target

proteins are modified with ubiquitin, a process catalyzed by single polypeptide

or SCF (Skp, Cullin, F-box) complex E3 ubiquitin ligases; polyubiquitinated

proteins are degraded by the 26S proteosome complex. SCF complexes control

multiple signal transduction cascades and developmental processes (Hua and

Viestra, 2011). The F-box protein of the SCF complex plays a role in selecting

specific targets for 26S proteosomal proteolysis by polyubiquitination (Zheng et

al., 2002). In plants, SCF complexes also regulate a large number of regulatory

processes, including GA and auxin signaling (Gray et al., 2001; Richards et al.,

2001; McGinnis et al., 2003).

DELLA proteins are a subfamily of GRAS [GAI (GA INSENSITIVE), RGA

(REPRESSOR OF ga1-3), and SCARECROW] proteins, which act as putative

nuclear-localized transcription factors (Pysh et al., 1999). They contain a highly

28

conserved DELLA motif in the N-terminal that acts as a transcriptional

regulator (Silverstone et al., 1998; Peng et al., 1999; Itoh et al., 2005). The

Arabidopsis DELLA proteins GAI, RGA, and three RGA-LIKE proteins (RGL1,

RGL2, and RGL3) negatively regulate GA signaling. DELLA proteins directly

interact with SLY1 and disappear rapidly from the nucleus after ubiquitination,

probably due to proteolytic degradation by the 26S proteosome complex.

The Arabidopsis SLY1 gene encodes the F-box subunit that determines the

substrate specificity of the SCF complex (McGinnis et al., 2003; Dill et al.,

2004; Fu et al., 2004). SLY1 has three main domains (F-box, GGF, and LSL)

that act as a bridge between SKP and DELLA proteins. Numerous studies

demonstrate that SLY1 positively regulates growth by GA signaling; for

example, sly1 mutants show increased seed dormancy and increased sensitivity

to inhibition of seed germination by abscisic acid (ABA) (Strader et al., 2004).

In addition, sly1 plants are dwarfs, a phenotype that may be rescued by the

absence of DELLA repressors (Olszewski et al., 2002; Dill et al., 2004; Fu et al.,

2004). Moreover, average leaf cell number is decreased in sly1-10 plants and

increased in the quadruple-DELLA mutant gai-t6 rga-t2 rgl1-1 rgl2-1. The cell

division rate is also lower in sly1-10 and higher in quadruple-DELLA mutants

than in wild-type plants (Achard et al., 2009). These observations match earlier

data showing that SLY1 promotes growth by stimulating destruction of growth-

repressing DELLA proteins (Peng et al., 1997; Silverstone et al., 1998;

Silverstone et al., 2001; Olszewski et al., 2002). All of these results indicate that

29

SCFSLY1 positively regulates plant growth from germination to flowering by GA

signaling through the destruction of DELLA proteins.

SMALL UBIQUITIN-RELATED MODIFIER (SUMO) is a small peptide that

is covalently attached to lysine residues of target proteins by E3 SUMO ligase

via a reversible post-translational modification (Wilkinson et al., 2010). As a

protein modifier, SUMO controls various eukaryotic cellular processes such as

stress and defense responses, nitrogen metabolism, growth and the regulation of

flowering (Hotson et al., 2003; Kurepa et al., 2003; Lois et al., 2003; Murtas et

al., 2003; Miura et al., 2007; Catala et al., 2007; Lee et al., 2006, Conti et al.,

2008; Ishida et al., 2009, 2012; Zhang et al., 2013; Conti et al., 2014).

Modification of target proteins with SUMO is usually catalyzed by E3 SUMO

ligases, although conjugation of SUMO to target proteins can occur in the

absence of such ligases (Wilkinson et al., 2010). So far, numerous SUMO

conjugates have been identified, such as the nitrate reductases NIA1 and NIA2,

INDUCER OF CBF EXPRESSION 1 (ICE1), an R2R3-type transcription factor

MYB30 and FLOWERING LOCUS C (FLC) (Miller et al., 2010; Elrouby and

Coupland, 2010; Miura and Hasegawa, 2010; Park et al., 2011; Zheng et al.,

2012; Son et al., 2014). Very recently, it was reported that DELLA RGA is also

modified by SUMO, although the E3 SUMO ligase involved was not identified

(Conti et al., 2014).

AtSIZ1 regulates plant responses to nutrient deficiency and environmental

stress, and controls vegetative growth and development (Miura et al., 2007,

30

2010; Yoo et al., 2006; Catala et al., 2007; Lee et al., 2007; Garcia-Dominguez

et al., 2008; Jin et al., 2008; Miura and Ohta, 2010; Park et al., 2011; Son et al.,

2014). The siz1-2 mutant shows the dwarf phenotype common in plants

defective in GA or in GA signaling (Catala et al., 2007) and the sly1 mutant also

displays severe dark green dwarfism (McGinnis et al., 2003). On the basis of

these data, I speculated that GA signaling may be impaired in the siz1-2 mutant

and sumoylation might affect SLY1 activity.

Here, I show that SLY1 sumoylation by AtSIZ1 stabilizes and activates SLY1

and stimulates RGA degradation, thereby promoting SLY1-mediated plant

development. My results provide the first evidence in any organism that F-box

protein function can be regulated by sumoylation through the activity of E3

SUMO ligase.

31

MATERIALS AND METHODS

Plant materials and growth conditions

The Arabidopsis thaliana Columbia-0 ecotype (wild type; WT) and the T-DNA

insertion knock-out mutants siz1-2, sly1-13 and esd4 were used in this study.

The sly1-13 mutant has 12kb of T-DNA inserted 300 bases from the 3' end of

At4g24210. This mutant was obtained from ABRC (Salk_097559) and was

designated sly1-13 to distinguish it from other mutant alleles of sly1.

For plants grown on plates, seeds were surface-sterilized in commercial bleach

containing 5% sodium hypochlorite and 0.1% Triton X-100 solution for 10 min,

rinsed five times in sterilized water, and stratified at 4°C for 2 d in the dark.

Seeds were sown on agar plates containing Murashige and Skoog (MS) medium,

2% sucrose, and 0.8% agar, buffered to pH 5.7. For plants grown in soil, seeds

were directly sown in sterile vermiculite. All plants, including seedlings, were

grown at 22°C under a 16 hr light/8 hr dark cycle in a growth chamber.

Effect of GA on Seed Germination and Seedling Growth

To investigate the effect of ammonium ion on germination and seedling growth,

wild-type and siz1-2 mutant seeds were germinated on MS agar media. To

assess the effects of GA on germination and vegetative growth, wild-type and

siz1-2 mutant seeds were planted on MS agar media with or without 10 µM

GA3. One hundred seeds of each (wild-type and siz1-2 mutant) were examined.

32

Plants were grown at 22°C under a 16-h light/8-h dark cycle in a growth

chamber. The experiment was repeated three times.

Construction of recombinant plasmids

To produce His6-SLY1 and glutathione S-transferase (GST)-SLY1, the cDNA

sequence encoding full-length SLY1 was amplified by PCR with gene-specific

primers and inserted into the pET28a (Novagen) and pGEX4T-1 (Amersham

Biosciences) vectors. For maltose binding protein (MBP)-AtSIZ1, a cDNA

sequence encoding full-length AtSIZ1 was amplified by PCR with gene-specific

primers and inserted into the pMALc2 vector (New England Biolabs). To

produce GST-SLY1-HA, the cDNA sequence encoding full-length SLY1 was

amplified by PCR with a gene-specific primer and hemagglutinin (HA) primer

and inserted into the pGEX4T-1 vector. To produce the SLY1 mutant protein

GST-mSLY1 (K122R; the numbers indicate the position of the lysine in SLY1

that was mutated to arginine), GST-SLY1 was subjected to site-directed

mutagenesis using overlapping primers. The Arabidopsis SUMO1 full-length

cDNA sequence was amplified by PCR with gene-specific primers and inserted

into pET28a to produce the His6-AtSUMO1-GG, containing full-length

AtSUMO1 extended with GG at the 3’end.

All constructs were transformed into Escherichia coli BL21/DE3 (pLysS) cells.

The transformed cells were treated with isopropyl-D-thiogalactoside (IPTG) to

induce fusion protein expression. The sequences of the primers used in this

33

study are listed in Table 2. All constructs were verified by automatic DNA

sequencing to ensure that no mutations were introduced.

34

Table 2. List of primers used in this study.

Purpose Gene Vector Forward primer (5’ -> 3’) Reverse primer (5’ -> 3’)

Real time

qRT-PCR

SLY1 tgatctgtactcgtcactggacta tggattctggaagaggtctcttag

RGA tgtaatctggtggcttgtgaaggt tgccaacccaacatcaaacatcca

GAI ccggagattttcactgtggt aactcggtcaggtccatcac

GA2OX2 aaggtgttggagatggttgc gaagacccgccgtgttatta

GA3OX1 tgccttccaaatctcaaacc gccagtgatggtgaaacctt

SLY2 aaccaagttcccgtgttcag cacaacggaacgaagagaca

AtSIZ1 atagcgcctctgggaatcat gccttgtcttgtctactgtcattcatac

TUB cgaaaacgctgacgagtgta ccttgggaatgggataaggt

Mutant

selection

SLY1 attccacatgaagcgcagtac tggttacggcgataatgattc

RGA aaacctttttcatgaaattatcgc taccgtggattcgttgctaac

Border attttgccgatttcggaac

35

In vitro pull

down assay

SLY1 pGEX4T-1 tctacgaattcatgaagcgcagtactaccgactct tctacctcgagttatttggattctggaagaggtct

AtSIZ1 pMAL-C2X tctacgaattcatggatttggaagctaat tctactctagactcagaatccgagtcaatgga

GID1A pGEX4T-1 gtgacagaattcatggctgcgagcgatgaagtta gtgacagtcgacacattccgcgtttacaaacgcc

Yeast two

hybrid assay

SLY1, mSLY1 pGBT8 tctaggaattcatgaagcgcagtactaccgac tctagctgcagtttggattctggaagaggtctc

SLY2 pGBT8 tctaggaattcatgtcgtcggagaaacgtgtagg tctagctgcaggacaacgttaacgggcttccg

RGA pGAD424 tctaggtcgacatgaagagagatcatcaccaattc tctagctgcagtcagtacgccgccgtcgagagttt

AtSIZ1 pGAD424 tctacgaattcatggatttggaagctaat tctagcccgggctcagaatccgagtcaatgga

GID1A pGBT8 gtgacagaattcatggctgcgagcgatgaagtta gtgacagtcgacacattccgcgtttacaaacgcc

SKP1 pGAD424 ctctaggaattcatgtctgcgaagaagattgtgt ctctagctgcagttcaaaagcccattggttctct

ASK2 pGAD424 tctaggaattcatgtcgacggtgagaaaaatcac tctagctgcagttcaaacgcccactgattctcacgg

ASK3 pGAD424 tctaggtcgacatggcagaaacgaagaagatgatc tctagctgcagctcgaacgcccacctgttctcattgc

ASK4 pGAD424 tctaggtcgacatggcagaaacgaagaagatgatc tctagctgcagctcgaacgcccacttgttctcatt

36

ASK11 pGAD424 tctaggaattcatgtcttcgaagatgatcgtgttg tctagctgcagttcaaaagcccattgattctcctt

ASK13 pGAD424 tctaggaattcatgtcgaagatggttatgttgctg tctagctgcagttcaaaagcccattgattctcctt

In vitro

sumoylation

assay

AtSUMO1-

GG

pET28a tctacgaattcatgtctgcaaaccaggaggaagac tctacctcgagttagccaccagtctgatggagcat

Site-specific

mutagenesis

SLY1

(K122R)

pGEX4T-1 gatcgatcgaattcatgaagcgcagtactaccga

atgcatgcctcgagttatttggattctggaagaggtc

tcttagtgaaactcatcttctcgtagtatcgaattgag

agaagagaaagagtgagtttaagctcgtcacgacc

gaacctagcacgcgg

Production

of transgenic

plants

SLY1-

FLAG3,

mSLY1-

FLAG3

pBA002 tctacctcgagatgaagcgcagtactaccgactct

tctacacgcgtttacttgtcatcgtcatccttgtagtcc

atcttgtcatcgtcatccttgtagtccatcttgtcatcg

tcatccttgtagtccattttggattctggaagaggtct

ctt

37

In vivo

infiltration

HA3-SUMO1 pBA002 tctacacgcgtatgtctgcaaaccaggaggaa tctagactagtgccaccagtctgatggagcatc

RGA-MYC6 pBA002 ctacgtcgacatgaagagagatcatcaccaattc tctacactagttcacaagtcctcttcagaa

HA3-AtSIZ1 pBA002 atctacacgcgtatggatttggaagctaattgta atctacggtaccaactccggtgtcttgtctgatg

AtSIZ1-

MYC6

pBA002 gatctcatatgatggatttggaagctaattgtaa gactgcgaattcctcagaatccgagtcaatggag

Sub-cellular

localization

SLY1

pEarlyGate

102

atgaagcgcagtactaccgac tttggattctggaagaggtctc

AtSIZ1

pEarlyGate

104

atggatttggaagctaattgtaa ctcagaatccgagtcaatggag

38

Purification of recombinant proteins

All recombinant proteins were expressed in E. coli strain BL21 and purified

according to the manufacturer’s instructions. Briefly, for purification of His6-

AtSAE1b, His6-AtSAE2, His6-AtSCE1, His6-AtSUMO1-GG, and His6-SLY1,

bacteria were lysed in 50 mM NaH2PO4 (pH8.0), 300 mM NaCl, 1% Triton X-

100, 1 mM imidazole, 5 mM Dithiothreitol (DTT), 2 mM phenyl

methanesulfonyl fluoride (PMSF), and proteinase inhibitor cocktail (Roche),

and purified on nickel-nitrilotriacetate (Ni2+-NTA) resins (Qiagen).

For GST-SLY1, GST-mSLY1 and GST-SLY1-HA purification, bacteria were

lysed in PBS buffer (pH 7.5) containing 1% Triton X-100, 2 mM PMSF, and

proteinase inhibitor cocktail (Roche), and purified on glutathione resins

(Pharmacia).

For MBP and MBP-AtSIZ1 purification, bacteria were lysed in 20 mM Tris-

HCl (pH 7.4), 200 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1%

Triton X-100, and 2 mM PMSF containing proteinase inhibitor cocktail (Roche),

and purified on amylose resins (New England BioLabs). Protein concentrations

were determined by the Bradford assay (Bio-Rad).

In vitro binding assay

To examine in vitro binding of MBP-AtSIZ1 to His6-SLY1, 2 µg of full-length

MBP-AtSIZ1 and 2 µg of full-length His6-SLY1 were added to 1 ml of binding

buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% Triton X-100, 0.2%

39

glycerol, 0.5 mM beta-mercaptoethanol). After incubation at 25°C for 2 hr, the

reaction mixtures were incubated with an amylose resin for 2 hr before washing

six times with buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% Triton

X-100).

Absorbed proteins were analyzed using 11% sodium dodecyl sulfate (SDS)-

polyacrylamide gel electrophoresis (PAGE) and detected by western blotting

with anti-His antibody (0.4 μgml−1; Santa Cruz Biotechnology).

For in vitro binding analysis of MBP-AtSIZ1 to GST-SLY1 or GST-GID1A, 2

µg of full-length MBP-AtSIZ1 and 2 µg of full-length His6-SLY1 or GST-

GID1A were added to 1 ml of binding buffer, as described above. After

incubation at 25°C for 2 hr, reaction mixtures were incubated with glutathione

resin for 1 hr before washing six times with the same buffer. Absorbed proteins

were analyzed using 11% SDS-PAGE and detected by western blotting with

anti-GST antibody (0.4 μgml−1; Santa Cruz Biotechnology).

Subcellular Localization of Proteins

The YFP and CFP coding sequences were fused in-frame to the 5’-end of

AtSIZ1 and 3’-end of SLY1 using pEarlyGate 104 and pEarlyGate 102,

respectively. Both fusion genes were expressed from the 35S promoter. The

epidermis of the inner surface of onion scales was bombarded with three

micrograms of each plasmid using a biolistic particle gun (Bio-Rad). Transient

40

expression in bombarded tissues was visualized using confocal microscopy after

14 h incubation in darkness.

Yeast two-hybrid assays

Yeast two-hybrid assays were performed using the GAL4-based two-hybrid

system (Clontech). Full-length AtSIZ1, RGA, SLY1, mSLY1, SLY2, ASK1, ASK2,

ASK3, ASK4, ASK11, ASK13 and GID1A cDNA sequences were cloned into

pGAD424 containing an activating domain (AD) and pGBT8 containing a

binding domain (BD) (Clontech) to generate the constructs AD-AtSIZ1, AD-

RGA, AD-ASK1, AD-ASK2, AD-ASK3, AD-ASK4, AD-ASK11, AD-ASK13, BD-

SLY1, BD-mSLY1, BD-SLY2 and BD-GID1A.

All constructs were transformed into yeast strain AH109 using the lithium

acetate method. Yeast cells were grown on minimal medium (Leu/Trp).

Transformants were plated onto minimal medium (Leu/Trp/His/10 mM 3-

AT (3-amino-1, 2, 4-triazole)) to test the interactions between AtSIZ1 and SLY1

(or mSLY1), AtSIZ1 and SLY2, and ASK proteins and SLY1 (or mSLY1).

Sumoylation assays

In vitro sumoylation assays were performed in 30 µl of reaction buffer (20 mM

Hepes (pH 7.5), 5 mM MgCl2, 2 mM ATP) with 50 ng His6-AtSAE1b, 50 ng

His6-AtSAE2, 50 ng His6-AtSCE1, 8 µg His6-AtSUMO1-GG and 100 ng GST-

SLY (or GST-mSLY1), with or without 500 ng MBP-AtSIZ1. After incubation

41

for 3 hr at 30°C, the reaction mixtures were separated by 11% SDS-PAGE.

Sumoylated GST-SLY1 was detected by western blotting with anti-GST

antibody. To identify the sumoylation site in SLY1, GST-mSLY1 was added to

the reaction mixtures instead of GST-SLY1. The reactions and subsequent steps

were performed as described above.

For in vivo sumoylation assays, siz1-2 plants over-expressing HA3-AtSUMO1-

GG were produced by transforming mutant plants with 35S-HA3-AtSUMO1-GG.

Following this initial plant transformation, Agrobacterium transformed with

35S-SLY1-FLAG3 and 35S-Myc6-AtSIZ1 was infiltrated into siz1-2 plants over-

expressing HA3-AtSUMO1-GG. After 3 d, the total proteins were extracted

from each sample and immunoprecipitated with anti-HA antibody (1 μgml−1,

Santa Cruz Biotechnology) in a buffer containing 50 mM Tris (pH 8.0), 150

mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA, 1 mM PMSF, and protease

inhibitor cocktail (Promega). Sumoylated SLY1 was detected by western

blotting with anti-FLAG antibody (0.5 μgml−1, Sigma-Aldrich) after

immunoprecipitation.

To investigate the effect of GA on sumoylation of SLY in plant, transgenic

plants over-expressing His6-strep-AtSUMO1-GG were first produced by

transforming wild type Arabidopsis with 35S-His6-Strep-AtSUMO1-GG. Next,

the transgenic plants were infiltrated with Agrobacterium transformed with 35S-

SLY1-FLAG3 and 35S-HA3-AtSIZ1. After 2 d, the infiltrated plants were treated

with 100 μM GA and then further incubated for 3 hr. Total proteins were

42

extracted from each sample and purified on Ni2+-NTA resins in buffer

containing 10 mM Tris (pH 8.0), 100 mM Na2HPO4, 1% triton X-100, 8M urea,

10 mM imidazole and protease inhibitor cocktail. Purified proteins were

separated by 10% SDS-PAGE and sumoylated SLY1 was detected by western

blotting with anti-FLAG antibody.

Production of transgenic Arabidopsis plants and complementation tests

To produce plants over-expressing SLY1 or mSLY1 (K122R), the

corresponding full-length cDNA sequences were amplified by PCR using a

forward primer and a reverse primer tagged with FLAG3 and inserted into the

plant expression vector pBA002. Recombinant plasmids, 35S-SLY1-FLAG3 and

35S-mSLY1-FLAG3, were introduced into wild-type Arabidopsis by floral

dipping, and transformants were selected on MS media containing BASTA (50

μgml−1).

To introduce 35S-SLY1-FLAG3 and 35S-mSLY1-FLAG3 into sly1-13 mutants,

transgenic plants were crossed with sly1-13 mutants and BASTA-resistant

offspring were selected. T3 homozygote lines were used for complementation

analysis. To examine SLY1-FLAG3 or mSLY1-FLAG3 expression, total proteins

were extracted from leaves of wild-type plants, sly1-13 mutants and sly1-13

mutants over-expressing SLY1-FLAG3 or mSLY1-FLAG3. SLY1-FLAG3 or

mSLY1-FLAG3 levels were estimated by western blotting with anti-FLAG

antibody.

43

Examination of SLY1 and RGA levels in siz1-2 and sly1-13 mutants

To examine relative levels of SLY1 in wild-type and siz1-2 plants, total proteins

were extracted from the leaves of wild-type and siz1-2 plants grown for 15 d on

MS media. Following 11% SDS-PAGE, levels of SLY1 were examined by

western blotting with anti-SLY1 antibody. The level of SLY1 was also

examined in cycloheximide-treated samples. Wild-type and siz1-2 plants grown

on MS media for 15 d were treated with 100 μM cycloheximide, and the

samples were collected at the indicated time points. Total proteins were

extracted from each sample, and the SLY1 was detected by western blotting

with anti-SLY1 antibody.

To determine levels of RGA, total proteins were extracted from the leaves of

wild-type, sly1-13, siz1-2, 35S-SLY1-FLAG3/sly1-13, and 35S-mSLY1-

FLAG3/sly1-13 plants grown for 15 d on MS media. Following 11% SDS-PAGE,

the levels of the RGA were examined by western blotting with anti-RGA

antibody.

Examination of the effect of AtSIZ1 activity and SLY1 sumoylation on

RGA level

To investigate whether SLY1 sumoylation affected RGA stability in

Arabidopsis, wild-type plants were infiltrated with different combinations of

Agrobacterium transformed with 35S-SLY1-FLAG3, 35S-mSLY1-FLAG3 and

35S-RGA-Myc6 constructs. After 2 d, the total proteins were extracted from each

44

sample, and SLY1-FLAG3, SLY1-mFLAG3 and RGA-Myc6 were detected by

western blotting with anti-FLAG and anti-Myc antibodies, respectively.

To check the effect of E3 ligase activity of AtSIZ1 on RGA level through SLY1

sumoylation, wild-type and siz1-2 plants were infiltrated with different

combinations of Agrobacterium transformed with 35S-HA3-AtSIZ1, 35S-SLY1-

FLAG3, 35S-mSLY1-FLAG3 and 35S-RGA-Myc6. After 2 d, total proteins were

extracted from each sample and separated by 11% SDS-PAGE. HA3-AtSIZ1,

SLY1-FLAG3, SLY1-mFLAG3 and RGA-Myc6 were detected by western

blotting with anti-HA, anti-FLAG and anti-Myc antibodies, as appropriate.

Interaction analysis between sumoylated SLY1 and RGA by in vitro pull-

down assay

To check the effect of SUMO conjugation on the interaction between SLY1 and

RGA, an in vitro sumoylation assay was performed as described above, and

sumoylated SLY1 was then detected by western blotting with anti-GST

antibody. The reaction products were mixed with purified MBP or MBP-RGA

and pulled down with amylose resin. After elution, GST-SLY1 and sumoylated

GST-SLY1 were detected by western blotting with anti-GST antibody.

Examination of the effect of SUMO protease on SLY1 level

To check the effect of SUMO protease on SLY1 stability, wild-type and esd4

plants were grown on MS media for 15 d. After harvesting the samples, total

45

proteins were extracted with 2x protein loading buffer. Proteins were separated

by 11% SDS-PAGE and SLY1 level was examined by western blotting with

anti-SLY1 antibody.

Measurement of transcript levels of GA-responsive genes including SLY1

Wild-type plants and siz1-2 mutants were grown on plates containing MS

medium for 15 d. Total RNA was extracted from the leaves of wild-type and

siz1-2 plants, quantified and divided into equal amounts. First-strand cDNA was

synthesized from 5 μg total RNA using an iScript cDNA Synthesis Kit (Bio-

Rad). An equal volume of cDNA was amplified by real-time qRT-PCR (MyiQ,

Bio-Rad), according to the manufacturer’s protocol.

The specific primers and template cDNA were combined with 25 µl of iQ

SYBR Green Super Mix (Bio-Rad), and the reactions were performed under the

following thermal conditions: 50°C for 2 min; 95°C for 10 min; 40 cycles of

95°C for 15 sec and 60°C for 1 min. The CT values obtained for target genes

were normalized to the CT value for tubulin, and the data were analyzed using

iCycler IQ software (Bio-Rad). PCR primers were designed using Primer3

(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi), and their specificity was

verified by cloning into the pGEM T-Easy vector (Promega) and sequencing

using an ABI 3730xl DNA Analyzer (Applied Biosystems).The primer sets used

in these studies are listed in Table 2.

46

Examination of AtSIZ1 transcript and protein levels

Wild-type and sly1-13 mutant plants were grown on plates containing MS

medium for 15 d and treated with 100 μM GA3 for 12 hr. Samples were

harvested at the indicated time points, and total RNA and protein were isolated

from each sample. AtSIZ1 transcript levels were determined by real time qRT-

PCR with gene-specific primers, as described above. The primer sets used in

these studies are listed in Table 2.

To evaluate relative AtSIZ1 protein levels in wild-type and siz1-2 mutant plants,

total proteins were separated by 11% SDS-PAGE and the level of AtSIZ1 was

determined by western blotting with anti-AtSIZ1 antibody.

Hypocotyl length measurements

Seeds of wild-type, siz1-2 and sly1-13 plants were germinated in Petri dishes

containing MS medium and grown for 7 d under long day conditions. Seedlings

were harvested from each Petridish, and scanned at 600 dots per inch.

Hypocotyl lengths were measured from the resulting images using Image J

software. Values of hypocotyl length are the means ± SD of three independent

experiments, each containing at least 20 seedlings.

Antibody production

To produce full-length or deletion mutant proteins, SLY1 and RGA cDNA

sequences, encoding either the full-length or truncated gene, were amplified

47

using PCR with gene-specific primers and inserted into pET28a to produce

His6-SLY1 and His6-RGA-C. His6-RGA-C contained amino acids 418587 of

RGA. After transformation of E. coli BL21/DE3 (pLysS) cells with

recombinant plasmids, the cells were treated with IPTG to induce recombinant

protein production. The recombinant His6-SLY1 and His6-RGA-C proteins were

purified using a Ni2+-NTA affinity column, and the protein concentrations were

determined. Anti-SLY1 and anti-RGA antibodies were produced by

subcutaneous injection of proteins into rabbits, and used for the analysis of

SLY1 and RGA levels.

48

RESULTS

Seed germination of the siz1-2 mutant is recovered by exogenous GA

Arabidopsis siz1-2 mutants display dwarfism, early flowering, and abnormal

seed development. A recent study showed that the phenotypes of the siz1-2

mutants were recovered to wild-type phenotypes by exogenous ammonium

treatment (Park et al., 2011). My further investigation revealed that siz1-2

mutants germinated unevenly and their germination ratio was relatively low

compared to that of wild-type plants (Figure 5). GA positively regulates seed

germination. Therefore, I investigated whether treatment with exogenous GA

could recover germination and vegetative growth of the siz1-2 mutants. The

results showed that germination of siz1-2 mutants was completely recovered by

treatment with 10 µM GA3 (Figure 5).

49

0

12

24

36

48

60

72

No treatment GA (10uM)

WT siz1-2(1) siz1-2(2) WT siz1-2(hr)(hr)

0

12

24

36

48

60

72

WT siz1-2

No treatment GA (10uM)

WT siz1-2

WT

No treatment

siz1-2

GA treatment (uM)

0 10

Ge

rmin

ation

(%

)

0

20

40

60

80

100

120

(A) (B)

(C) (D)

GA (10uM)

Figure 5. AtSIZ1 positively regulates seed germination. (A) Germination of

wild-type plants and siz1-2 mutants was tested on MS media containing 10 uM

GA3 for short time as indicated. Seed dormancy of siz1-2 mutants was totally

overcome by treatment with GA although seed dormancy increased in siz1-2

mutant. (B) Some of the seeds shown in (A) were chosen and photographed. (C)

Wild-type plants and siz1-2 mutants were also germinated on MS media

50

containing 10 uM GA3 for 10 days. Vegetative growth of siz1-2 mutants was

recovered to wild-type by treatment with GA3. (D) 23% of the seeds of siz1-2

mutants germinated. Black bar, wild-type plants; white bar, siz1-2 mutants.

51

SLY1 is sumoylated by E3 SUMO ligase AtSIZ1

Abnormal GA signaling by the loss of SLY1 function results in defective

growth, resulting in phenotypes such as severe dwarfism and low fertility

(McGinnis et al., 2003). Arabidopsis siz1-2 mutants were reported to also

display dwarfism and abnormal seed development, thus GA signaling may be

disrupted in the siz1-2 mutant (Catala et al., 2007) due to reduced SLY1 activity

or stability. To test this, I investigated whether there is an interaction between

AtSIZ1 and SLY1.

Two recombinant plasmids were constructed: maltose binding protein (MBP)-

tagged AtSIZ1 and histidine-tagged SLY1. Over-expression of tagged proteins

in Escherichia coli was induced by IPTG treatment and proteins were purified

with amylose and nickel resins, respectively (Figure 6A). After purification, I

conducted in vitro pull-down assays for His6-SLY1 using MBP or MBP-AtSIZ1.

As expected, I observed a strong interaction between AtSIZ1 and SLY1 (Figure

6A). A yeast two-hybrid assay was performed to confirm this interaction. The

full-length cDNAs of AtSIZ1 and SLY1 were cloned into yeast expression

vectors. After introducing the constructs into yeast strain AH109, I examined

the protein interactions. Consistent with the results of the pull-down assay, I

found that AtSIZ1 strongly interacted with SLY1 (Figure 6B).

This strong interaction between AtSIZ1 and SLY1 suggested they might co-

localize in cells. I therefore examined their subcellular localization, transiently

expressing SLY1 tagged with cyan fluorescent protein (CFP) and AtSIZ1 tagged

52

with yellow fluorescent protein (YFP) in onion epidermal cells, a model system

for the processes in Arabidopsis cells. I observed localization of both proteins in

the nucleus, whether expressed separately or together (Figure 7).

The direct interaction between SLY1 and AtSIZ1 suggested that AtSIZ1 may

act as an E3 SUMO ligase for SLY1. To determine whether this notion is

correct, I produced the recombinant proteins MBP-AtSIZ1 and GST-SLY1

(Figure 8A). In vitro sumoylation assays revealed that GST-SLY1 was

sumoylated by AtSIZ1, and that sumoylation was dependent on E1 and E2

activities (Figure 8B).

To test whether AtSIZ1 could function as an E3 SUMO ligase for SLY1 in

plants, 35S-Myc6-AtSIZ1 and 35S-SLY1-FLAG3 constructs were co-infiltrated

into the leaves of siz1-2 mutants over-expressing HA3-AtSUMO1-GG.

Expression of the recombinant proteins, HA3-AtSUMO1-GG, SLY1-FLAG3

and Myc6-AtSIZ1, was detected by western blotting using anti-HA, anti-FLAG

and anti-Myc antibodies, respectively (Figure 9A-C). Sumoylation of SLY1 was

examined by western blotting with anti-FLAG antibody following

immunoprecipitation with anti-HA antibody. Notably, sumoylated SLY1 was

detected only in siz1-2 mutants that had been infiltrated with 35S-Myc6-AtSIZ1

(Figure 9D). No sumoylated SLY1 was observed in mutants that had not been

infiltrated.

Next, I tried to identify the SLY1 sumoylation site. The predicted amino acid

sequence of SLY1 contained a putative sumoylation site (ψKXE) located at

53

lysine 122 (K122; Figure 10A and B). Therefore, a derivative sequence was

generated containing the mutation K122R. The mutated protein was over-

expressed in E. coli and purified with glutathione affinity columns. In vitro

sumoylation assays using SLY1 (GST-SLY1) and mutated SLY1 (GST-mSLY1

(K122R)) showed that GST-mSLY1 was not modified with SUMO, whereas

GST-SLY1 was clearly sumoylated (Figure 10C). These results indicate that

K122 is the principal SUMO conjugation site in SLY1.

54

4 3

21

1: AD/BD

2: AD-AtSIZ1/BD

3: BD-SLY1

4: AD-AtSIZ1/BD-SLY1

Leu-/Trp-

Inp

ut

MB

P

MB

P-A

tSIZ

1

His

6-S

LY1

MB

P+H

is6-S

LY1

MB

P-A

tSIZ

1+

His

6-S

LY1

(A) (B)

Leu-/Trp-/His-

(20mM 3-AT)His6-SLY1

100

70

5040

30

20

(SDS-PAGE) (Pull-down)

Figure 6. AtSIZ1 physically interacts with SLY1. (A) In vitro pull-down

assay of SLY1 with full-length AtSIZ1. MBP and MBP-AtSIZ1 (bait) and His6-

SLY1 (prey) were purified using affinity chromatography (left). His6-SLY1

absorbed with MBP or MBP-AtSIZ1 was detected by western blotting with

anti-His antibody (middle). After pull-down, MBP and MBP-AtSIZ1 were also

detected by western blotting with anti-MBP antibody (right). (B) Yeast two-

hybrid analysis of AtSIZ1 and SLY1. Full-length AtSIZ1 and SLY1 cDNAs

were fused to sequences encoding the Gal4 activation domain (AD) and the

Gal4 DNA-binding domain (BD) in pGAD424 and pGBT8, respectively.

Constructs were transformed into the yeast strain AH109. Numbers indicate

yeast cells transformed with only pGAD424 and pGBT8 vectors or recombinant

plasmids. Transformants were plated onto minimal media, -Leu/-Trp and -Leu/

-Trp/-His (20 mM 3-AT), and incubated for 4 d.

55

CFP YFP Merged Dic

Figure 10. Subcellular co-localization of AtSIZ1 and SLY1. AtSIZ1 and

SLY1 co-localize in the nucleus of onion epidermal cells. YFP-AtSIZ1 and

SLY1-CFP refer to YFP and CFP fusion to the N-terminal of AtSIZ1 and the C-

terminal of SLY1, respectively. Dic: Differential interference contrast. Bar = 20

μm.

56

His6-AtSUMO1-GG

GST-SLY1

GST-SLY1-SUMO1

(kDa)

100

70

140

35

50

H

is6-A

tSA

E1+ 2

His

6-A

tSU

MO

1-G

G

GS

T-S

LY1

His

6-A

tUB

C9

MB

P-A

tSIZ

1

50

35

25

70

100

140

(kDa)

His6-AtSAE1+2 (E1)His6-AtUBC9 (E2)MBP-AtSIZ1 (E3)GST-SLY1

(A) (B)

Figure 8. AtSIZ1 directly sumoylates SLY1 using SUMO1. (A) His6-

AtSAE1b, His6-AtSAE2, His6-AtUBC9, MBP-AtSIZ1, His6-AtSUMO1-GG,

and GST-SLY1 purified from E.coli were separated by 11% SDS-PAGE. (B)

E3 ligase activity of AtSIZ1 for SLY1 was assayed in the presence or absence

of His6-AtSAE1+2, His6-AtUBC9, MBP-AtSIZ1, His6-AtSUMO1-GG and

GST-SLY1. After the reaction, sumoylated SLY1 was detected by western

blotting with anti-GST antibody.

57

35

25

5070

20

35

25

5070

20

(kDa)(kDa)

HA3-AtSUMO1-

GGSLY1-FLAG3

siz

1-2

SLY1-FLAG3

siz1-2/35S-HA3-AtSUMO1-GG

35

25

50

70

20

(kDa)

Sumoylated SLY1-FLAG3

IgG100

70

140240

50

(kDa)

Myc6-AtSIZ1

*

*

Myc6-AtSIZ1

siz1-2/35S-HA3-AtSUMO1-GG

siz1-2/35S-HA3-AtSUMO1-GG

siz1-2/35S-HA3-AtSUMO1-GG

SLY1-FLAG3

Myc6-AtSIZ1

SLY1-FLAG3

Myc6-AtSIZ1

SLY1-FLAG3

Myc6-AtSIZ1

(A) (B)

(C) (D)

Figure 9. AtSIZ1 sumoylates SLY1 using SUMO1 in Arabidopsis.

Sumoylation of SLY1 was also analyzed in Arabidopsis. siz1-2 mutants over-

expressing HA3-AtSUMO1-GG were infiltrated with or without 35S-SLY1-

FLAG3 and 35S-Myc6-AtSIZ1, as indicated. After incubation for 3 d, SLY1-

FLAG3, HA3-AtSUMO1-GG and Myc6-AtSIZ1 were detected by western

blotting with anti-FLAG (A), anti-HA (B) and anti-Myc (C) antibodies, as

appropriate. Asterisk indicates non-specific bands. (D) Sumoylated SLY1-

FLAG3 was detected by western blotting with anti-FLAG antibody following

immunoprecipitation with anti-HA antibody. IgG: immunoglobulin G. non-

specific bands were indicated by asterisk.

58

WT

(SLY1)

GST-SLY1

GST-SLY1-AtSUMO1-GG

70

35

50

(kDa)

MKRSTTDSDLAGDAHNETNKKMKSTEEEEI 30

GFSNLDENLVYEVLKHVDAKTLAMSSCVSK 60

IWHKTAQDERLWELICTRHWTNIGCGQNQL 90

RSVVLALGGFRRLHSLYLWPLSKPNPRARF 120

GKDELKLTLSLLSIRYYEKMSFTKRPLPES 150

K 151

GST-SLY1

K122

GST

K122R

(mSLY1)

His6-AtSUMO1-GG

His6-AtSAE1+2 (E1)His6-AtUBC9 (E2)MBP-AtSIZ1 (E3)GST-SLY1(or GST-mSLY1)

F-Box LSL

(A)

(B)

(C)

Figure 10. Identification of the SLY1 sumoylation site. (A) Predicted amino

acid sequence of the SLY1 protein. A putative sumoylation site (ψKXE),

identified using the SUMOplot Analysis Program, is indicated in yellow box

and F-box and LSL domains are represented in bold type and underlined,

respectively. (B) Schematic diagram of the recombinant GST-SLY1 protein.

The F-box, LSL and putative sumoylation site (K122) are indicated. (C) In vitro

59

sumoylation assays. Recombinant GST-SLY1 and GST-mSLY1 were over-

expressed in E. coli and purified using a glutathione affinity column. The

reaction mixture contained E1 (His6-AtSAE1b and His6-AtSAE2), E2 (His6-

AtSCE1), His6-AtSUMO1-GG and GST-SLY1 (or GST-mSLY1), without ()

or with (+) E3 (MBP-AtSIZ1). The mutant protein mSLY1 has an amino acid

substitution at the residue predicted to be the SUMO conjugation SLY1 site, as

indicated. After the reaction, sumoylated SLY1 protein was detected by western

blotting with anti-GST antibody.

60

SLY1 modification by SUMO promotes plant growth and development

The Arabidopsis thaliana T-DNA insertion knock-out mutant sly1-13 was

obtained from ABRC (Salk_097559) and named it sly1-13 to distinguish it from

other mutant alleles of sly1. The sly1-13 mutant has 12kb of T-DNA inserted

300 bases from the 3' end of At4g24210 (Figure 11A). This result was

confirmed by PCR using Salk_097559 line specific left, right and border

primers (Data not shown). The sly1-13 mutants had the same phenotypes as

other sly1 loss of function mutants, sly1-2, sly1-10 (Figure 11B). I first

produced anti-SLY1 antibody and examined its specificity by western blotting,

and found that SLY1 was clearly detected in wild-type but not in sly1-13 plants

(Figure 11C), indicating that the antibody specifically interacts with SLY1.

One of the notable phenotypes of sly1 mutants is severe dwarfism (Steber et al.,

1998). Considering the function of SLY1 in plant growth and my SLY1

sumoylation data, I therefore examined the effect of sumoylation on SLY1

activity. I produced transgenic sly1-13 mutants over-expressing SLY1-FLAG3 or

mSLY1-FLAG3, and measured their growth. Growth of sly1-13 mutants over-

expressing wild-type protein was restored to wild-type (Figure 12A). By

contrast, growth of sly1-13 mutants over-expressing the mutated protein

resembled sly1-13 mutants. To determine if these phenotypes were caused by

differential expression of SLY1-FLAG3 and mSLY1-FLAG3, I analyzed levels of

SLY1-FLAG3 and mSLY1-FLAG3 in transgenic sly1-13 mutants and found that

SLY1-FLAG3 and mSLY1-FLAG3 were highly expressed (Figure 12B).

61

Col 0 sly1-13

(SALK_097559)

sly1-13

1

SLY1

Tubulin

WT sly1-13

sly1-10

(8 amino acid deletion)

(E138K)

sly1-d

(A)

(B) (C)

105 561 930

SLY1 gene (At4g24210)

Figure 11. sly1-13 mutant selection and SLY1 specific antibody production.

(A) Schematic diagram of SLY1 (At4G24210) gene. Gain of function

polymorphism (sly1-d) is indicated as allow and loss of function polymorphism

(sly1-10 and sly1-13) is represented by allow head. (B) Phenotype of wild type

(Col0) and selected sly1-13 mutant plants. (C) Determination of the specificity

of anti-SLY1 antibody. Total proteins were extracted from wild-type and sly1-13

plants grown for 15 d and SLY1 was detected by western blotting with anti-

SLY1 antibody; tubulin was used as a loading control. Tubulin was used as a

loading control.

62

WT

SLY1-FLAG3 or

mSLY1-FLAG3

Tubulin

WT

(A)

(B)

Figure 12. Phenotypes of sly1-13 mutants over-expressing SLY1 or mSLY1.

Arabidopsis plants were transformed with 35S-SLY1-FLAG3 or 35S-mSLY1-

FLAG3 constructs, and then transgenic plants were crossed with sly1-13 mutants.

(A) Transgenic sly1-13 mutants over-expressing SLY1-FLAG3 or mSLY1-

FLAG3 were selected, and their growth was examined 28 d after sowing. (B)

SLY1-FLAG3 or mSLY1-FLAG3 expression was evaluated in transgenic sly1-

13 mutants using western blotting with anti-FLAG antibody. Tubulin was used

as a loading control.

63

siz1-2 mutant has short hypocotyls

RGA and GAI are the main repressors of hypocotyl growth and stem elongation

in Arabidopsis (Peng et al., 1997; Dill et al., 2001). In addition, SLY1 positively

controls the effect of GA on cell expansion and division (Achard et al., 2009).

All sly1-13 mutants so far described display dwarfism, resembling the siz1-2

mutant phenotype; however, growth defects of sly1-13 mutants are more severe

than those of siz1-2 plants. I found that loss of AtSIZ1 caused SLY1 degradation

and RGA accumulation, suggesting that hypocotyl elongation is repressed in the

siz1-2 mutant. I therefore measured hypocotyl lengths of wild-type, siz1-2 and

sly1-13 plants grown in light or dark conditions. As expected, compared with

sly1-13 mutants, siz1-2 mutants showed long hypocotyl phenotypes under both

conditions (Figure 13A and B), although hypocotyls of siz1-2 mutants were still

shorter than those of wild-type plants.

64

Hypocoty

lle

ngth

(cm

)

0

2

4

6

8

10

12

14

Light Dark

Light

Dark

(A) (B)

WT siz1-2 sly1-13

Figure 13. Hypocotyl lengths of siz1-2 mutants. Seedlings were grown for 7 d

under light or dark conditions. (A) Hypocotyl phenotypes of wild-type, siz1-2

and sly1-13 plants. (B) Mean hypocotyl lengths of the seedlings grown in the

growth conditions indicated in (A). Error bars indicate standard deviations.

65

SLY1 localization affect by exogenous GA.

I showed that SLY1 was SUMOylated by the E3 SUMO ligase, SIZ1. This post

translational modification suggested that SLY1 might be changed its functional

roles. To test this hypothesis, I first check SLY1 and mSLY1 subcellular

localization, transiently expressing SLY1 and mSLY1 tagged with green

fluorescent protein (GFP) with or without exogenous GA in onion epidermal

cells, a model system for the processes in Arabidopsis cells. I observed

localization of both proteins in the nucleus, whether expressed in the same

organelles or different. Subcellular localization results showed that mSLY1

tagged with GFP localization was not changed by exogenous GA3 (Figure 14).

However, diffused form of SLY1 tagged with GFP localization was different to

speckled form by exogenous GA3.

66

None

100μM GA

SLY1-GFP

mSLY1-GFP

None

100μM GA

(A)

(B)

Figure 14. Subcellular co-localization of SLY1 and mSLY1 depending on

GA. SLY1 and mSLY1 localize in the nucleus of onion epidermal cells. SLY1-

GFP and mSLY1-GFP refer to GFP fusion to the C-terminal of SLY1. 100 μM

GA3 was treated for 3 hour with MS media. Dic: Differential interference

contrast. Bar = 20 μm.

67

SLY1 is stabilized by AtSIZ1

Many proteins are post-translationally modified by small or large

molecules before becoming stable. In eukaryotes, various target proteins are

stabilized by sumoylation. To investigate whether SLY1 stability is regulated by

AtSIZ1 activity, I analyzed the levels of SLY1 in wild-type and siz1-2 plants

using western blotting with anti-SLY1 antibody, and found that the level of

SLY1 was significantly lower in the siz1-2 mutants (Figure 15A, upper panel).

In addition, I examined the effect of SUMO protease on SLY1 stability. SLY1

level was evaluated in SUMO protease mutant esd4. As expected, the SLY

amount was significantly high in esd4 mutants compared to wild-type plants

(Figure 15A, lower panel), indicating that SLY1 is stabilized by sumoylation. I

used real-time qRT-PCR to examine SLY1 transcript levels in the same samples

and found similar levels of transcript in wild-type and siz1-2 plants (Figure

15B).

Many proteins are post-translationally modified by small or large molecules

before becoming stable. In eukaryotes, various target proteins are stabilized by

modification by SUMO. To investigate whether SLY1 stability is regulated by

AtSIZ1 activity, I first performed a cell-free degradation assay using total

protein extracts, prepared from leaves of wild-type and siz1-2 plants, and a

recombinant protein, GST-SLY1-HA, over-expressed in E.coli and purified on a

glutathione column. Purified GST-SLY1-HA was mixed with the extracts and its

degradation examined using western blotting with an anti-HA antibody. I found

68

GST-SLY1-HA decayed much faster in the presence of protein extracts from

siz1-2 than in extracts from wild-type plants (Figure 16A and B).

Next, the decay of SLY1 was evaluated in the presence of cycloheximide to

compare the degradation rates between wild-type and siz1-2 plants. The

degradation of SLY1 was much faster in siz1-2 plants than in wild-type plants

(Figure 16C and D), indicating that SLY1 is stabilized by AtSIZ1.

69

WT siz1-2

SLY1

Tubulin

Rela

tive t

ranscript

level

0

0.01

0.02

0.03

0.04

0.05

WT

WT esd4

SLY1

Tubulin siz1-2

(A) (B)

Figure 15. Effect of SUMOylation on the levels of SLY1. (A) Examination of

SLY1 level in siz1-2 mutant (upper panel). Total proteins were extracted from

wild-type and siz1-2 plants grown for 15 d and SLY1 was detected by western

blotting with anti-SLY1 antibody. Effect of Arabidopsis SUMO protease on the

SLY1 level (lower panel). Total proteins were extracted from wild-type and

esd4 plants grown for 15 d. SLY1 was detected by western blotting with anti-

SLY1 antibody. Tubulin was used as a loading control. (B) Effect of AtSIZ1 on

the transcript level of SLY1. Total RNA was isolated from the same samples and

the levels of SLY1 transcript were examined by real-time qRT-PCR with gene-

specific primers. Results are expressed as means ± S.D. (n = 3).

70

WT

siz1-2

0 0.5 1 2 5 8 (hr)

0 0.5 1 2 5 8 (hr)

SLY1

Tubulin

SLY1 Rela

tive level (S

LY

1)

1.2

0.2

0.4

0.6

0.8

1.0

0

Tubulin

GST-SLY1-HA

BIP

Col0

siz1-3

0 0.5 1 1.5 2 3 4 6 8 (hr)

0 30 60 90 120 180 240 360 480

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Col0

siz1-3

GST-SLY1-HA

BIP

Rela

tive

level (G

ST

-SLY

1-H

A)

1.2

0.2

0.4

0.6

0.8

1.0

0

Time (hr)

0 0.5 1 1.5 2 3 4 6 8

Time (hr)

0 0.5 1 2 4 8

0 0.5 1 1.5 2 3 4 6 8 (hr)

(A) (B)

(C) (D)

Figure 16. Effect of AtSIZ1 on the levels of SLY1. (A) cell-free degradation

of SLY1. Purified GST-SLY1-HA was mixed with extracts from wild-type or

siz1-2 plants. After incubation at room temperature for 8 h, samples were

collected at the times indicated and levels of GST-SLY1-HA were examined

using western blotting with anti-HA antibody. Binding Protein (BiP) in the

extracts is shown as loading controls. (B) GST-SLY1-HA levels during

degradation depicted in graph form. Relative levels of GST-SLY1-HA shown in

(C) were normalized to numerical values, based on a value of 1.0 for the protein

levels at time 0. Closed and open circles indicate GST-SLY1-HA extracted

from wild-type and siz1-2 plants, respectively. (C) Total proteins were extracted

71

from wild-type and siz1-2 plants treated with 100 μM cycloheximide for

indicated length of time and SLY1 was detected by western blotting with anti-

SLY1 antibody; tubulin was used as a loading control. (F) SLY1 levels during

degradation were also depicted in graph form. Relative levels of SLY1 shown in

(D) were normalized to numerical values, based on a value of 1.0 for the protein

levels at time 0. Closed and open circles indicate SLY1 in wild-type and siz1-2

plants, respectively.

72

RGA level is affected by both SLY1 and AtSIZ1

In Arabidopsis, SLY1 recognizes target DELLA proteins, including RGA, and

induces their degradation by the 26S proteasome complex. Therefore, I initially

examined the effect of SLY1 sumoylation on RGA levels using bacterial

infiltration of Arabidopsis. To examine the effect of AtSIZ1 on RGA levels

through SLY1 sumoylation, I again used bacterial infiltration of Arabidopsis.

The constructs 35S-HA3-AtSIZ1, 35S-SLY1-FLAG3, 35S-mSLY1-FLAG3 and

35S-RGA-Myc6 were co-infiltrated into the leaves of wild-type and siz1-2 plants,

and I used western blotting with anti-Myc antibody to detect the level of RGA-

Myc6 (Figure 17A-a). RGA-Myc6 levels were decreased in both wild-type

plants and siz1-2 mutant plants infiltrated with 35S-HA3-AtSIZ1 due to the

presence of endogenous SLY1. As expected, co-infiltration with 35S-HA3-

AtSIZ1 and 35S-SLY1-FLAG3 resulted in a significant decrease in RGA-Myc6

levels in both wild-type and siz1-2 plants; in fact, RGA-Myc6 could not be

detected under this condition. However, RGA was clearly detected in both wild-

type and siz1-2 plants co-infiltrated with 35S-HA3-AtSIZ1 and 35S-mSLY1-

FLAG3, although the levels were relatively low compared with those of

untreated plants. Western blotting also clearly detected the recombinant proteins

HA3-AtSIZ1, SLY1-FLAG3 and mSLY1-FLAG3. To confirm lower level RGA-

Myc6 level in SLY1-FLAG3 infiltrated plant, I check RNA level of RGA-Myc6

in the same samples, but RNA levels were not changed in all samples (Figure

17-b).

73

After then, I needed to clarify that mSLY1 affected by exogenous GA and 26S

proteosomal degradation pathway. To check these idea, I performed similar test

that co-infiltration with 35S-HA3-AtSIZ1, 35S-SLY1-FLAG3, 35S-mSLY1-FLAG3

and 35S-RGA-Myc6 into the leaves as indicated compositions with or without

GA and MG132. The relative levels of RGA were much higher in non-

exogenous GA treated mSLY1 and MG132 treated combinations. In contrary,

RGA levels were low in non-exogenous GA treated SLY1 and both of

exogenous GA treated combination even with mSLY1 (Figure 17B).

To check these results in transgenic plants, I used siz1-2 and transgenic sly1-13

mutants over-expressing SLY1 or mSLY1 to examine the effect of SLY1

sumoylation on RGA stability. The relative levels of RGA in extracts of total

proteins from wild-type, sly1-13, siz1-2, 35S-SLY1-FLAG3/sly1-13, and 35S-

mSLY1-FLAG3/sly1-13 plants were assessed by western blotting with anti-RGA

antibody. As expected, the levels of RGA were much higher in sly1-13, siz1-2,

and 35S-mSLY1-FLAG3/sly1-13 plants than in wild-type and 35S-SLY1-

FLAG3/sly1-13 plants (Figure 18A). This result indicates that RGA is stabilized

in siz1-2 mutants and 35S-mSLY1-FLAG3/sly1-13 plants.

The RGA levels were similar in wild-type and 35S-SLY1-FLAG3/sly1-13 plants.

However, its level was relatively low in siz1-2 plants compared with sly1-13

plants (Figure 18A), which explains why the dwarfing phenotype is less severe

in siz1-2 than in sly1-13.

To confirm SIZ1 direct effect on RGA level through SLY1 or mSLY1, I used

74

transgenic wild-type and siz1-3 mutants over-expressing SLY1 or mSLY1 to

observe the RGA stability. The relative levels of RGA from Wild type, mSLY1-

FLAG3/Col 0, SLY1-FLAG3/siz1-3 were low than siz1-3 and mSLY1-

FLAG3/siz1-3 but SLY1-FLAG3/Col 0 was not detected (Figure 18B).

75

RGA-Myc6

SLY1-FLAG3 or

mSLY1-FLAG3

HA3-AtSIZ1

Tubulin

WT

*

RGA-Myc6

Tubulin

(RT-PCR)

GA

MG132No infiltra

tion

(Immunoblot analysis)

siz1-2

RGA-Myc6

SLY1-FLAG3

mSLY1-FLAG3

HA3-AtSIZ1

RGA-Myc6

SLY1-FLAG3

mSLY1-FLAG3

HA3-AtSIZ1

RGA-Myc6

SLY1-FLAG3 or

mSLY1-FLAG3

HA3-AtSIZ1

Tubulin

*

(A)

(B)

76

Figure 17. AtSIZ1 and GA effects on RGA level depending on SLY1 or

mSLY1. (A, upper panel) Arabidopsis thaliana Col0 and siz1-2 plants were

infiltrated with 35S-HA3-AtSIZ1, 35S-SLY1-FLAG3, 35S-mSLY1-FLAG3 and

35S-RGA-Myc6, as indicated. After 3 days incubation, HA3-AtSIZ1, SLY1-

FLAG3, mSLY1-FLAG3 and RGA-Myc6 proteins were detected using western

blotting with anti-HA, anti-FLAG and anti-Myc antibodies, as appropriate.

Tubulin was used as a loading control. (A-lower panel) Total RNA was

extracted from the same samples and expression level of 35S-RGA-Myc6 was

determined by real-time RT-PCR with gene and tag-specific primers. Tubulin

was used as a loading control. (B) Arabidopsis thaliana Col0 plants were

infiltrated as indicated combination. 20mM MG132 was treated for 16hr and

10uM GA3 was treated for 6 hours as indicated, respectively. Each proteins

were also detected using western blotting with anti-HA, anti-FLAG and anti-

Myc antibodies, as appropriate. Tubulin was used as a loading control.

77

rga

Tubulin

RGA

Col0 siz1-3

Tubulin

RGA

SLY1-FLAG3 or

mSLY1-FLAG3

*

(A)

(B)

WT siz1-2

78

Figure 18. AtSIZ1 negatively regulates RGA level through sumoylation of

SLY1. (A) Compared RGA level in siz1-2, sly1-13 mutants and sly1-13

harboring overexpressed SLY1 or mSLY1 plants. Total proteins were extracted

from leaves of 15-d-old wild-type, siz1-2, sly1-13, 35S-SLY1-FLAG3/sly1-13,

35S-mSLY1-FLAG3/sly1-13 and rga (negative control) plants. Following 11%

SDS-PAGE, the levels of RGA were examined by western blotting with anti-

RGA antibody. Tubulin was used as a loading control. (B) RGA level in Col0

and siz1-3 mutants harboring overexpressed SLY1 or mSLY1 plants. Total

proteins were extracted from leaves of 15-d-old plants. The levels of RGA were

examined by western blotting with anti-RGA antibody. The overexpressed

proteins were detected by western blotting with anti-FLAG antibody. Tubulin

was used as a loading control. Non-specific bands were indicated by asterisk.

79

Sumoylated SLY1 interacts with RGA

Over-expression of mSLY1 in sly1-13 mutant plants does not induce RGA

degradation and cannot rescue the dwarfing phenotype, which suggests that

SLY1 sumoylation is important for the interaction with, and degradation of

RGA. I thus investigated the interaction between sumoylated SLY1 and RGA

by in vitro pull down. An in vitro sumoylation reaction was first performed with

purified GST-SLY1 and sumoylation machinery. After detection of sumoylated

GST-SLY1 by western blotting with anti-GST antibody (Figure 19A), the

remaining reaction products were pulled down with MBP or MBP-RGA, and

sumoylated SLY1 was then detected by western blotting with anti-GST

antibody. The results showed that sumoylated GST-SLY1 interacted with MBP-

RGA (Figure 19B).

80

Reaction 1

Re

action 2

MB

P +

Re

action 1

MB

P +

Re

action 2

MB

P-R

GA

+ R

eaction 1

MB

P-R

GA

+ R

eaction 2

GST-SLY1

Sumoylated

GST-SLY1

100

70

50

(kDa)

GST-SLY1

Sumoylated

GST-SLY1

100

70

50

(kDa)His6-AtSUMO1-GG

His6-AtSAE1+2 (E1)His6-AtUBC9 (E2)MBP-AtSIZ1 (E3)GST-SLY1

(A) (B)

Figure 19. RGA interacts with sumoylated SLY1. (A) In vitro sumoylation

reactions were carried out as indicated, and GST-SLY1 and sumoylated GST-

SLY1 levels were investigated by western blotting with anti-GST antibody. (B)

Reaction mixtures in (A) were pulled down with MBP or MBP-RGA. GST-

SLY1 and sumoylated GST-SLY1 were detected by western blotting with anti-

GST antibody.

81

Interactions between SLY1 and AtSIZ1 or ASK are strong and specific

In vitro sumoylation assays showed that mSLY1 was not sumoylated by AtSIZ1.

In addition, bacterial infiltration assays in Arabidopsis demonstrated that SLY1

controls the level of RGA after sumoylation. However, the possibility remains

that mSLY1 has no effect on RGA stability because mSLY1 does not interact

with AtSIZ1. I thus performed a yeast two-hybrid assay to examine whether

mSLY1 interacts directly with AtSIZ1 and found evidence for such an

interaction (Figure 20A). These results were supported by in vivo

immunoprecipitation analysis with anti-FLAG and western blotting anti-HA

from 35S-HA3-AtSIZ1, 35S-SLY1-FLAG3 and 35S-mSLY1-FLAG3 co infiltrated

Arabidopsis leaves. In Arabidopsis result also showed that SIZ1 could interact

with SLY1 and mSLY1 also (Figure 20B and C).

A previous study demonstrated that SNE/SLY2 (SNEEZY/SLEEPY2), a SLY1

homolog, is involved in degrading RGA, GAI and RGL2 (RGA-like 2)

(Ariizumi and Steber, 2011). I therefore investigated whether SLY2 is activated

or stabilized by sumoylation through AtSIZ1 activity, using a yeast two-hybrid

assay to look for possible interactions between AtSIZ1 and SLY2. The results

showed that AtSIZ1 did not interact with SLY2 (Figure 20A).

Because SLY1 interacts with Arabidopsis SKP1-like (ASK) protein to form the

SCF complex required for degradation of its target DELLA proteins, I used

yeast two-hybrid assays to examine further the effect of the SLY1 sumoylation

site on the interaction of SLY1 with ASK proteins. I tested six ASK proteins,

82

ASK1, ASK2, ASK3, ASK4, ASK11 and ASK13, and found that all interacted

with both SLY1 and mSLY1 (Figure 21).

83

-Leu/-Trp/-His

(10mM 3-AT)

1

2

3

4 5

8

7

6

1 : AD/BD2 : AD-AtSIZ1/BD3 : AD/BD-SLY14 : AD/BD-mSLY15 : AD/BD-SLY26 : AD-AtSIZ1/BD-SLY17 : AD-AtSIZ1/BD-mSLY18 : AD-AtSIZ1/BD-SLY2

-Leu/-Trp

(A)

IPHA3-AtSIZ1SLY1-FLAG3

mSLY1-FLAG3

Anti-HA

1 2Tota

l pro

tein

1

Anti-FLAG

Tota

l pro

tein

2

25

70

50

35

20

1 2(B) (C)

SLY1-FLAG3 or

mSLY1-FLAG3

HA3-AtSIZ1

Tubulin

IgG

*

Figure 20. Yeast two-hybrid analysis of interactions between mSLY1 and

other proteins or between SLY2 and AtSIZ1. (A) Full-length AtSIZ1 was

fused to sequences encoding the Gal4 activation domain (AD) in pGAD424.

SLY1, mSLY1, and SLY2 cDNAs were fused to sequences encoding the Gal4

84

DNA-binding domain (BD) in pGBT8. (B) Transient expression of HA3-

AtSIZ1 and SLY1-FLAG3 or mSLY1-FLAG3 in Col0 plants. (C) HA3-AtSIZ1

was detected by western blotting with anti-HA antibody following

immunoprecipitation with anti-FLAG antibody. IgG: immunoglobulin G. non-

specific bands were indicated by asterisk.

85

-Leu/-Trp/-His

(10mM 3-AT)

BD

BD-SLY1

BD-mSLY1

-Leu/-Trp

Figure 21. Interactions between SLY1 or mSLY1 and ASK are strong. Full-

length sequences of the Arabidopsis ASK genes ASK1, ASK2, ASK3, ASK4,

ASK11 and ASK13 were fused to sequences encoding the Gal4 activation

domain (AD) in pGAD424. SLY1 and mSLY1 cDNAs were fused to sequences

encoding the Gal4 DNA-binding domain (BD) in pGBT8. Constructs were

transformed into the yeast strain AH109. Numbers indicate the yeast cells

transformed with only pGAD424 and pGBT8 vectors or with recombinant

plasmids. Transformants were plated onto minimal media, Leu/Trp and

Leu/Trp/His (10 mM 3-AT), and incubated for 4 d.

86

Expression of GA signaling-related genes was not notably affected by

AtSIZ1

I have shown that AtSIZ1 is an E3 SUMO ligase for SLY1, suggesting that the

defective growth of siz1-2 mutants may be caused in part by low level and

activity of SLY1. However, it is also possible that dwarfism in siz1-2 mutants

results from a change in gene expression. I therefore measured transcript levels

of GA-responsive genes (SLY1 and SLY2), a GA-biosynthetic gene (GA3ox1), a

GA-deactivating gene (GA2ox2) and DELLA protein genes (RGA and GAI) in

siz1-2 mutants. Transcript levels of these genes were determined, using real-

time qRT-PCR, in samples of total RNA isolated from both wild-type and siz1-2

mutants. The level of SLY1 transcript did not greatly change in siz1-2 mutants,

relative to levels in wild-type plants (Figure 15B). Expression of RGA and GAI

was very slightly increased in siz1-2 mutants compared with wild-type plants

(Figure 22); however, there were no obvious differences in expression of

GA2ox2 and GA3ox1 between wild-type and siz1-2 plants (Figure 22).

Expression patterns of SLY2 were also very similar in wild-type and siz1-2

plants, although expression was somewhat higher in the siz1-2 mutants (Figure

22).

87

Rela

tive t

ranscript

level

0

0.02

0.04

0.06

0.08

Rela

tive t

ranscript

level

0

0.05

0.10

0.15

0.20

0.25RGA

WT siz1-2

GA2ox2

SLY2

Rela

tive t

ranscript

level

0

0.02

0.04

0.06

0.08

0.10

0.12

Rela

tive t

ranscript

level

0

0.02

0.04

0.06

0.08

0.10

Rela

tive t

ranscript

level

0

0.02

0.04

0.06

0.08

WT siz1-2

WT siz1-2WT siz1-2

WT siz1-2

GAI

GA3ox1

88

Figure 22. Transcript levels of GA-synthetic and -responsive genes in siz1-2

mutants. Total RNA was isolated from 15-d-old wild-type and siz1-2 mutant

plants. Transcript levels were examined using real-time qRT-PCR with gene-

specific primers. Results are expressed as means ± S.D. (n = 3). Abbreviations:

RGA: Repressor of ga1-3; GAI: GA insensitive; GA2ox2: GIBBERELLIN 2-

OXIDASE 2; GA3ox1: GIBBERELLIN 3-OXIDASE 1; SLY2: SLEEPY2.

89

AtSIZ1 expression is regulated by GA signaling

Our results show that SLY1-mediated GA signaling is tightly controlled by

AtSIZ1. To determine the effect of GA signaling at the level of AtSIZ1, I used

real-time qRT-PCR to investigate AtSIZ1 expression in sly1-13 mutants and

found a low level of transcript compared with wild-type plants (Figure 23A). In

addition, an exogenous application of GA induced AtSIZ1 transcription in wild-

type plants but not in sly1-13 mutants (Figure 23A), although the transcript

levels decreased after 3 hr. I also used western blotting with anti-AtSIZ1

antibody to analyze the level of AtSIZ1 in sly1-13 mutants. There was a similar

pattern also detected in AtSIZ1 expression; in wild-type plants, the level of

AtSIZ1 increased and then decreased after 3 hr (Figure 23B).

90

Rela

tive tra

nscript le

vel

AtSIZ1

Tubulin

0 1 3 6 0 1 3 6 0 (h)

WT

00.020.040.060.080.100.120.140.160.180.20

0 1 3 6 (h)

GA (100 mM)

(A)

(B)sly1-13

Figure 23. Effect of GA treatment on relative levels of AtSIZ1 transcript

and protein. Wild-type and sly1-13 plants were treated with 100 uM GA3

during vegetative growth and samples were collected at the indicated time

points. (A) Total RNA was extracted from samples and expression level of

AtSIZ1 was determined by real-time qRT-PCR with gene-specific primers.

Filled circle: wild-type; Open circle: sly1-13 mutants. (B) Total proteins were

91

extracted from the same samples and level of AtSIZ1 was estimated by western

blotting with anti-AtSIZ1 antibody. Tubulin was used as a loading control.

Sumoylation of SLY1 is stimulated by GA supply

The results of development assays using transgenic sly1-13 mutants (Figure

12A) strongly suggest that SLY1 must be modified by SUMO to be in an active

form. The results also imply that GA can stimulate sumoylation of SLY1 to

activate and stabilize this protein. I therefore examined whether SLY1

modification with SUMO is stimulated by GA. To do this, I used bacterial

infiltration of Arabidopsis. The constructs 35S-HA3-AtSIZ1, 35S-SLY1-FLAG3,

and 35S-His6-Strep-AtSUMO1-GG were co-infiltrated into the leaves of wild-

type and siz1-2 plants. Concurrent with infiltration, the leaves were treated with

100 µM GA3. First, I examined the expression of recombinant proteins HA3-

AtSIZ1, SLY1-FLAG3 and His6-Strep-AtSUMO1-GG by western blotting and

found that they were clearly detected (Figure 24A). Next, I purified His6-Strep-

AtSUMO1-GG-modified proteins with nickel columns and evaluated His6-

Strep-AtSUMO1-GG-modified SLY1-FLAG3 by western blotting with anti-

FLAG antibody. The result showed that the level of His6-Strep-AtSUMO1-GG-

modified SLY1-FLAG3 was very high in GA-supplied samples whereas it was

very low in non-treated samples (Figure 24B), indicating that the modification

of SLY1-FLAG3 with His6-Strep-AtSUMO1-GG was stimulated by

exogenously supplied GA.

92

HA3-AtSIZ1

SLY1-FLAG3

His6-Strep-AtSUMO1-GG

*

100µM GA

SLY1-FLAG3

His6-Strep-AtSUMO1-GG

HA3-AtSIZ1

1 2 3 4

(A) (B)

Anti-Flag

Anti-SUMO1

1007050

35

2520

240

15

1 2 3 4

His6-Strep-AtSUMO1-GG

SUMO1 conjugates

Figure 24. Effect of GA on sumoylation of SLY1. (A) The effect of GA on

sumoylation of SLY1 was examined by bacterial infiltration. Transgenic plants

over-expressing His6-strep-AtSUMO1-GG were infiltrated with Agrobacterium

transformed with 35S-SLY1-FLAG3 and 35S-HA3-AtSIZ1, as indicated. After 2

d, 100 μM GA3 was added as indicated. Recombinant proteins, HA3-AtSIZ1,

SLY1-FLAG3 and His6-Strep-AtSUMO1-GG, were detected by western

blotting with anti-HA, anti-FLAG and anti-Strep antibodies, as appropriate. (B)

His6-strep-AtSUMO1-GG-conugated proteins were purified from total proteins

using Ni2+

-NTA resins and sumoylated SLY1 was detected by western blotting

with anti-FLAG (upper panel) or anti-SUMO1 (lower panel) antibody.

93

AtSIZ1 is not involved in regulation of GID1A activity

GA signaling begins with the binding of the receptor protein GID1A (GA

INSENSITIVE DWARF1A) to GA; therefore, I investigated whether AtSIZ1

can modulate the function of GID1A. I used in vitro pull-down and yeast two-

hybrid assays to test the interaction between AtSIZ1 and GID1A because the

predicted amino acid sequence of GID1A contains three putative sumoylation

sites (Figure 25A). For in vitro pull-down assays, recombinant proteins, MBP,

MBP-AtSIZ1, GST-GID1A and GST-SLY1, were prepared (Figure 25B-a), and

then GST-GID1A and GST-SLY1 were pulled-down using MBP or MBP-

AtSIZ1. AtSIZ1 did not interact with GID1A, but a clear interaction between

AtSIZ1 and SLY1 was observed (Figure 25B-b). I also tested this interaction

using a yeast two-hybrid assay; consistent with the results of the pull-down

assay, AtSIZ1 did not interact with GID1A (Figure 25C).

94

MAASDEVNLI ESRTVVPLNT WVLISNFKVA YNILRRPDGT 40

FNRHLAEYLD RKVTANANPV DGVFSFDVLI DRRINLLSRV 80

YRPAYADQEQ PPSILDLEKP VDGDIVPVIL FFHGGSFAHS 120

SANSAIYDTL CRRLVGLCKC VVVSVNYRRA PENPYPCAYD 160

DGWIALNWVN SRSWLKSKKD SKVHIFLAGD SSGGNIAHNV 200

ALRAGESGID VLGNILLNPM FGGNERTESE KSLDGKYFVT 240

VRDRDWYWKA FLPEGEDREH PACNPFSPRG KSLEGVSFPK 280

SLVVVAGLDL IRDWQLAYAE GLKKAGQEVK LMHLEKATVG 320

FYLLPNNNHF HNVMDEISAF VNAEC 345

4 1

23

1: AD/BD

2: AD-AtSIZ1/BD

3: BD-GID1A

4: AD-AtSIZ1/BD-GID1A

Leu-/Trp-/His-

(10mM 3-AT)

100

70

50

140

MB

P+

GS

T-G

ID1

A

Input

(GS

T-G

ID1A

)

GST-SLY1

MB

P-A

tSIZ

1+

GS

T-G

ID1A

Re

sin

+G

ST

-GID

1A

Input

(GS

T-S

LY

1)

MB

P-A

tSIZ

1+

GS

T-S

LY

1GST-GID1A

100

70

50

140

MB

P

MB

P-A

tSIZ

1

GS

T-G

ID1A

GS

T-S

LY

1

(kDa)(kDa)

Leu-/Trp- Leu-/Trp-/His-

(1mM 3-AT)

(A)

(B)

(C)

95

Figure 25. AtSIZ1 did not interact with GID1A. (A) Predicted amino acid

sequence of SLY1. Potential sumoylation sites (ψKXE), identified using the

SUMOplot Analysis Program, are indicated as bold type and underlined. (B) In

vitro pull-down assay of SLY1 with full-length AtSIZ1. MBP and MBP-AtSIZ1

baits and GST-SLY1 and GST-GID1A preys were purified using affinity

chromatography (left). GST-SLY1 and GST-GID1A, absorbed with MBP or

MBP-AtSIZ1, were detected using western blotting with an anti-GST antibody

(right). (C) Yeast two-hybrid analysis of the interaction between AtSIZ1 and

GID1A. Full-length AtSIZ1 was fused to a sequence encoding the Gal4

activation domain (AD) in pGAD424. GID1A cDNA was fused to a sequence

encoding the Gal4 DNA-binding domain (BD) in pGBT8. The constructs were

transformed into the yeast strain AH109. Numbers indicate the yeast cells

transformed with only pGAD424 and pGBT8 vectors or with recombinant

plasmids. Transformants were plated onto minimal media, Leu/Trp and

Leu/Trp/His (1 and 10 mM 3-AT), and incubated for 4 d.

96

DISCUSSION

This study highlights the notion that SLY1-mediated plant development is

positively regulated by sumoylation via the E3 SUMO ligase activity of AtSIZ1.

Various GA-dependent development processes (Richards et al., 2001; Hedden,

2003) are controlled by DELLA proteins, and their repressor activities are

modulated by phosphorylation and ubiquitination via the E3 ligase activity of

SCFSLY1 (Sasaki et al., 2003; Dill et al., 2004; Gomi et al., 2004; Itoh et al.,

2005). Such observations have led researchers to focus on SLY1 and DELLA

proteins to elucidate GA signaling. Very recently, Conti et al. (2014) reported

that DELLA protein-mediated growth can be regulated by sumoylation.

Sumoylation is an essential mechanism for regulating developmental processes

in eukaryotic cells (Praefcke et al., 2012; Park and Yun, 2013) and therefore, an

increasing number of studies on the function of E3 SUMO ligase have been

performed since SIZ1 was first identified in (Miura et al., 2007, 2010; Yoo et al.,

2006; Catala et al., 2007; Lee et al., 2007; Garcia-Dominguez et al., 2008; Jin et

al., 2008; Miura and Ohta, 2010; Park et al., 2011; Son et al., 2014). One of the

most prominent features of Arabidopsis siz1-2 mutants is their dwarf phenotype

(Catala et al., 2007), suggesting impairment of the GA signaling pathways

responsible for elongation and growth. This hypothesis is supported by the

severe, dark green dwarfism observed in sly1 mutants (McGinnis et al., 2003).

Therefore, I investigated whether an AtSIZ1 mutation affects GA signaling by

97

interrupting SLY1-mediated DELLA protein degradation.

Seed germination test was the most certain way to confirm that siz1-2 mutants

whether affected on GA signaling or not. Seed germination vigorosity and

radical emergency results show that siz1-2 mutants germination rate were

rescued by 10 uM exogenous GA3 (Figure 5). These results suggested that one

of GA signaling protein could affect by SIZ1 to SUMOylate for post

translational modification.

AtSIZ1 acts as an E3 SUMO ligase on various target proteins, including

NIA1/NIA2, ICE1, GTE3, and FLC (Miura et al., 2007; Garcia-Dominguez et

al., 2008; Park et al., 2011; Son et al., 2014). I investigated whether SLY1 is

also a target of AtSIZ1 in nucleus. My various sumoylation and localization

assays showed that SLY1 is directly sumoylated by AtSIZ1 in vitro and vivo

(Figure 6, Figure 7, Figure 8 and Figure 9). It is known that sumoylation

stabilizes target proteins (Praefcke et al., 2012; Park and Yun, 2013); for

example, nitrate reductase decays more quickly in siz1-2 mutants than wild-type

Arabidopsis (Park et al., 2011) and RGA is more highly accumulated in the

SUMO protease ots1 ots2 mutants than wild-type Arabidopsis (Conti et al.,

2014). My data showed lower levels of SLY1 protein in siz1-2 mutants than in

wild-type plants (Figure 15A, upper panel), despite similar levels of transcript

(Figure 15B) and faster degradation of SLY1 protein in siz1-2 mutants than in

wild-type plants (Figure 16A-D), strongly indicating that SLY1 protein is

stabilized by sumoylation through AtSIZ1 activity.

98

The PML gene of acute promyelocytic leukemia (APL) encodes a cell growth

and tumor suppressor. PML localizes to discrete nuclear bodies that is regulated

by SUMO3 in human cell (Fu et al., 2005). I could hypnotize that SUMOylation

of SLY1 and GA signal might affect on SLY1 localization. To reveal that SLY1

and mSLY1 tagged GFP were test with or without exogenous GA its subcellular

localization. No difference in localization in mSLY1 tagged GFP but diffused

form of SLY1 tagged GFP was changed speckled form by exogenous GA,

which suggest that structural changes derived in SUMOylation site mutation

and exogenous GA could affect SLY1 localization (Figure 14).

In addition, previous studies have shown that SUMO conjugation modulates the

activity of target proteins in yeast, animals and plants (Praefcke et al., 2012;

Park and Yun, 2013); for example, the activities of nitrate reductases increased

significantly following sumoylation (Park et al., 2011). I thus speculated that

SLY1 activity must be affected by SUMO attachment. I identified the SLY1

sumoylation site (Figure 10) and examined the effect of SUMO conjugation on

SLY1 function using transgenic sly1-13 mutants over-expressing SLY1 or

mSLY1. Over-expression of SLY1 restored the growth and development of sly1-

13 mutants to wild-type levels but over-expression of mSLY1 did not (Figure

12A). The result suggests that SLY1 is modified by SUMO before it can

function to degrade DELLA proteins and activate GA signaling. This scenario is

also supported by my observation that sumoylation of SLY1 is induced by GA

(Figure 24B).

99

GA signaling is negatively regulated by the activity of DELLA proteins, which

are degraded by the 26S proteasome complex after polyubiquitination by

SCFSLY1 (Dill et al., 2004). My results showed that the stability and activity of

SLY1 were positively regulated by sumoylation through AtSIZ1 activity (Figure

9A-D, Figure 12A, Figure 15 and Figure 16), suggesting that the levels of

DELLA proteins, including RGA, would be affected. Bacterial infiltration

assays in Arabidopsis revealed that over-expression of AtSIZ1 alone was

sufficient to cause RGA degradation (Figure 17A). Moreover, co-expression of

AtSIZ1 and SLY1 resulted in no detectable level of RGA, although RGA was

clearly detected when AtSIZ1 and mSLY1 were co-expressed (Figure 17A);

mSLY1 alone had no effect on RGA levels (Figure 15A). In a complete

inversion of the SLY1 expression pattern, the level of RGA was much higher in

siz1-2 than in wild-type plants (Figure 18A). RGA accumulated to a high level

in transgenic sly1-13 mutants over-expressing mSLY1, as it did in siz1-2

mutants, but the level of RGA in transgenic sly1-13 mutants over-expressing

SLY1 was as low as that observed in wild-type plants (Figure 18A). These data

indicate that SLY1 is positively regulated by AtSIZ1 and SLY1-mediated RGA

degradation is stimulated by SLY1 sumoylation through the E3 ligase activity of

AtSIZ1 (Figure 26). In addition, the low level of RGA in the siz1-2 mutants

relative to sly1-13 (Figure 18A) explains the less severe growth defects of the

siz1-2 mutants.

Accumulation of DELLA repressors in the absence of SLY1 inhibits cell

100

elongation and division, leading to the suppression of hypocotyl growth (Peng

et al., 1997; Dill et al., 2001; Achard et al., 2009). In the current study,

hypocotyls of the siz1-2 mutants were shorter than those of wild-type plants but

longer than those of the sly1-13 mutants (Figure 13). In addition, the level of

SLY1 was much lower in siz1-2 than in wild-type plants (Figure 15A, upper

panel) and RGA levels were low in siz1-2 relative to those in the sly1-13

mutants (Figure 18A). These results indicate that the relatively long hypocotyls

of siz1-2, compared with sly1-13 mutants, result from the relatively low level of

RGA in these mutants and that AtSIZ1 regulates SLY1- and DELLA protein-

mediated hypocotyl growth.

The results of transgenic assays in which over-expression of SLY1 rescued the

growth of sly1-13 mutants but over-expression of mSLY1 did not (Figure 12)

suggest that SLY1 must be sumoylated to exert its function as a component of

the SCF complex, which led us to examine whether there was a direct

interaction between RGA and sumoylated SLY1. A pull-down assay using in

vitro sumoylation reaction products demonstrated that both sumoylated and

non-sumoylated SLY1 strongly interacted with RGA (Figure 19B). Moreover, a

bacterial infiltration assay using Arabidopsis clearly showed that the level of

sumoylated SLY1 was greatly increased by GA treatment (Figure 24B),

demonstrating that GA indeed stimulates the sumoylation of SLY1 by AtSIZ1

and the function of SLY1 must be activated by sumoylation. In other words, this

result demonstrates that GA positively controls plant growth and development

101

through stimulation of SLY1 sumoylation by AtSIZ1. Therefore, the phenotype

of sly1-13 mutants over-expressing mSLY1 must be caused by inactivation of

mSLY1 due to non-sumoylation rather than the loss of RGA function.

The Arabidopsis genome contains another SLY1 homolog, SLY2. SLY2 (SNE)

also directly binds to RGA protein, as well as the cullin subunit of the SCF E3

complex, suggesting that SLY2 forms a functional SCF E3 ubiquitin ligase

complex that negatively regulates the subset of DELLA proteins regulated by

SLY1 (Ariizumi et al., 2011). SLY2 over-expression partially rescues the

germination, dwarfism and infertility of the sly1-10 mutant by reducing the

accumulation of DELLA proteins RGA and GAI (Strader et al., 2004; Fu et al.,

2004; Sun, 2010; Ariizumi et al., 2011). This observation suggests that the

function of SLY2 may also be regulated by AtSIZ1. However, the current results

showed that AtSIZ1 did not interact with SLY2 (Figure 20A), indicating that

AtSIZ1-medited GA signaling is SLY1-specific.

Another possible reason for the failure of mSLY1 over-expression to rescue the

growth phenotype of sly1-13 mutant is that mSLY1 is unable to interact with

ASK proteins and thereby form the SCF complex. However, yeast two-hybrid

assays showed strong interactions between mSLY1 and all of the ASK proteins

tested (Figure 21), indicating that mSLY1 is capable of forming the E3 ubiquitin

ligase SCFmSLY1 complex.

All of these data indicate that dwarfism in the siz1-2 mutant results from the

loss of AtSIZ1 activity, which leads to low SLY1 levels or activity, although the

102

growth defect may be caused by changes in the expression of GA synthesis and

response genes. However, I found very similar expression patterns of such

genes in both wild-type and siz1-2 mutant plants, although the absolute

transcript levels were slightly different (Figure 22). This result suggests that

AtSIZ1 mainly regulates SLY1 levels post-translationally and that the growth

defect of siz1-2 mutants is caused by low activity and level of SLY1.

Finally, I investigated whether GA plays a role in regulating AtSIZ1 levels. I

detected low levels of AtSIZ1 transcript in sly1-13 mutants compared with wild-

type plants and found that the transcript and protein levels of AtSIZ1 increased

after exogenous application of GA (Figure 23). This result indicates that the

level of AtSIZ1 is controlled by GA signaling, particularly SLY1-mediated GA

signaling, at both the transcriptional and translational levels.

Proteolytic turnover of GA signaling-associated proteins can be regulated by

sumoylation as well as ubiquitination. Recent data (Conti et al., 2014) show that

RGA is sumoylated in a GA-independent manner although its E3 SUMO ligase

has not yet been identified, and SUMO-conjugated RGA directly binds to

GID1A, which blocks the interaction between GID1A and the remaining non-

sumoylated RGA. This interaction causes the accumulation of RGA, resulting

in growth inhibition. My finding demonstrates that SLY1 is directly sumoylated

by E3 SUMO ligase AtSIZ1 and subsequently induces the degradation of its

target protein RGA in an AtSIZ1-dependent manner (Figure 26). In addition,

exogenous GA stimulated SLY1 sumoylation (figure 24), leading to activation

103

of SLY1 function. On the contrary, GA induced the disappearance of

sumoylated and non-sumoylated RGA, although it has not yet been revealed

whether the desumoylation of RGA is primarily required for the degradation of

RGA. Both studies clearly provide evidence that the function and stability of

GA signaling-associated proteins can be regulated by sumoylation in the

presence of GA.

It is well-known that F-box proteins are substrate-recruiting subunits of SCF-

type E3 ubiquitin ligases. To date, a large number of F-box proteins have been

identified in plants and human (Gray et al., 2001; Richards et al., 2001;

McGinnis et al., 2003; Nelson et al., 2013). However, the regulatory

mechanisms underlying the activity of F-box proteins remain unclear. My

results demonstrate that SLY1 function is tightly regulated by sumoylation. This

is the first report demonstrating that the activity of an F-box protein is

controlled by sumoylation. I thus expect that my results will help to elucidate

the mechanism by which functional SCF complexes are produced according to

the requirements of the cell.

In conclusion, I found that GA stimulates the sumoylation of SLY1 through the

E3 SUMO ligase activity of AtSIZ1, indicating that sumoylation is an important

post-translational modification that plays a role in the regulation of GA

signaling by directly modulating the stability and activity of SLY1 protein. My

results provide the first biochemical evidence that sumoylation is a critical

protein modification required for the regulation of SLY1 function, and that

104

sumoylated SLY1 induces degradation of its target proteins. My study is also

the first to report that F-box protein function is regulated by sumoylation,

suggesting that the regulation of plant and animal development by SCF

complexes is regulated by sumoylation of F-box proteins through E3 SUMO

ligase activity.

105

SLY1-SUMO

AtSIZ1( )

SKP

SLY1/SUMO Induction

Ub26S proteasome complex

(stability/activity (up))

GA-GID1A-RGA

Cullin

RGA(DELLAs)

GA signaling(Plant growth)(Development)

Cell division andexpansion(up)

SCFSLY1-SUMO

GA

-GID1A-GA

RGA

GA signaling

GID1A(DELLAs)

RGA-SUMO

SUMO

(stability (up))

E3 SUMO ligase (?)GA

SUMO

RGA-SUMO-GID1AGA-GID1A

GID1A

Figure 26. Schematic representation of the possible mechanism of

activation of GA signaling by SLY1 sumoylation. GA and SLY1 positively

regulate AtSIZ1 expression. SLY1 is sumoylated by AtSIZ1, becoming the

active form. Sumoylated SLY1 forms the SCFSLY1-SUMO

complex, directly

interacts with DELLA proteins, including RGA, and ubiquitinates them.

Polyubiquitinated DELLA proteins are degraded by the 26S proteasome

complex. Finally, plant cells divide and expand, which leads to growth and

development. RGA is also sumoylated by E1- and E2-dependent manner and

then stabilized and may be able to directly inhibit GA signaling. Sumoylated

RGA can also form a complex with GID1A, which results in the accumulation

of non-sumoylated RGA, leading to the blocking of GA signaling. GA:

gibberellic acid; Ub: ubiquitin.

106

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ABSTRACT IN KOREAN

지베렐린은 종자 발아, 세포 분열과 신장 그리고 개화까지, 다양한

식물발달단계에 영향을 미치는 식물 호르몬이다. 본 연구에서는 E3

수모화 효소인 AtSIZ1 단백질이 애기장대의 지베렐린 신호전달체계를

SLY1 단백질의 수모화를 통하여 조절한다는 것을 밝혀냈다. SLY1

단백질은 야생형에 비해 siz1-2 돌연변이체에서 적은 양이 존재하였다.

sly1-13 돌연변이체에 SLY1 단백질 과다발현을 시킨 형질전환체는

야생형과 비슷한 표현형을 나타냈으나 수모화 조절부위 돌연변이

형의 mSLY1 단백질이 과다 발현되는 형질전환체에서는 돌연변이체

본연의 난쟁이 표현형이 나타났다. sly1-13 돌연변이체에 SLY1 단백질

과다발현을 시킨 형질전환체에서는 RGA 단백질의 양 역시 야생형과

같은 수준으로 나타났지만 mSLY1 과다 발현체에서는 돌연변이체처럼

높은 양이 존재하였다. RGA 단백질은 mSLY1과 AtSIZ1 단백질이 함께

발현되는 조건에서도 분해되지 않았으나, SLY1과 AtSIZ1 단백질이

함께 발현되는 조건에서는 다량이 분해되었다. 게다가, 수모화된

130

SLY1은 RGA 단백질과 상호작용 할 수 있으며, SLY1의 수모화는

지베렐린에 의해 매우 증가하였다. 정리하자면, 본 연구는 AtSIZ1이

SLY1의 수모화를 통하여 지베렐린 신호전달체계를 조절한다는 것을

암시한다.