SIZ1mediated Sumoylation of ICE1 Controls CBF3DREB1A Expression and Freezing Tolerance in Arabidopsi

1
SIZ1-mediated Sumoylation of ICE1 Controls
CBF3/DREB1A Expression and Freezing Tolerance in
Arabidopsis
Miura et al. (2007) Plant Cell
2
ABSTRACT
Frosts cause substantial agricultural yield
losses, particularly if a freezing event occurs
during reproductive development. Low
temperatures also substantially limit the
geographic locations where crops can be grown.
SIZ1 is a SUMO E3 ligase that facilitates
conjugation of SUMO to protein substrates (Miura
et al., 2005). siz1-2 and siz1-3 T-DNA insertion
alleles that caused freezing and chilling
sensitivities were complemented genetically by
expressing SIZ1, indicating that the SIZ1 is a
controller of low temperature adaptation in
plants.   Cold-induced expression of CBF/DREB1,
particularly of CBF3/DREB1A, and of the regulon
genes was repressed by siz1. siz1 did not affect
expression of ICE1, which encodes a MYC
transcription factor that is a controller of
CBF3/DREB1A.
3
A K393R substitution in ICE1 (ICE1(K393R))
blocked SIZ1-mediated sumoylation in vitro and in
protoplasts identifying the K393 residue as the
principal site of SUMO conjugation.
SIZ1-dependent sumoylation of ICE1 in protoplasts
was moderately induced by cold. Sumoylation of
recombinant ICE1 reduced polyubiquitination of
the protein in vitro. ICE1(K393R) expression in
wild-type plants repressed cold-induced
CBF3/DREB1A expression and increased freezing
sensitivity. Furthermore, expression of
ICE1(K393R) induced transcript accumulation of
MYB15, which encodes a MYB transcription factor
that is a negative regulator of CBF/DREB1.
SIZ1-dependent sumoylation of ICE1 may activate
and/or stabilize the protein, facilitating
expression of CBF3/DREB1A and repression of
MYB15, leading to low temperature tolerance.
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Freezing Sensitivity of siz1 Plants
Temperature
23?C -2?C -4?C -6?C -8?C
-10?C
WT
-4Cn
Non-acclimated
siz1-2
siz1-3
siz1-2/SIZ1
WT
Cold-acclimated
siz1-2
siz1-2/vector
siz1-3
5
AtSIZ1 T-DNA Alleles
At5g60410 (AtSIZ1)
s
i
z
1
-
1
s
i
z
1
-
2
s
i
z
1
-
3
siz1-2
siz1-3
Col-0
AtSIZ1
Tublin
SAP
PHD
PINIT
SP-RING
SXS
873 aa
SIZ1 - SUMO E3 ligase
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Sumoylation Regulates Critical Biological/Biologic
al Functions in Animals
Transcription factor activity Subnuclear
localization DNA replication and repair Innate
immunity Nucleocytoplasmic targeting Agonist/ant
agonist of other peptide modification
processes Hormonal and environmental signaling
Substrates
SUMO
PIAS/Siz
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CBF/DREB1 and Regulon Expression Is Repressed by
siz1
400
COR15A
200
80
CBF1/ DREB1B
0
40
40
COR47
WT
0
20
160
CBF2/ DREB1C
siz1-2
Relative mRNA levels
0
80
60
KIN1
0
30
CBF3/ DREB1A
60
40
0
20
80
RD29A
0
0 3 6 12 24 h
40
0
0 3 6 12 24 h
8
AtSIZ1 SUMO E3 Ligase Activity
ScSIZ2
AtSIZ1

-

-
207
SubstrateScCdc3 SUMO ScSmt3 E1
ScAos1/ScUba2 E2 ScUbc9 E3
AtSIZ1 or ScSIZ2
112
79
ScCdc3
WB T7 (ScCdc3)
Yeast proteins courtesy of Professor Y. Kikuchi
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AtSIZ1 Is Localized to Nuclear Foci
Transmitted Light
Merge
AtSIZ1GFP
NLSRFP
23 C
AtSIZGFP suppresses siz1
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AtSIZ1 Controls ICE1 Activity through
Sumoylation?
AtSIZ1 (SUMO E3)
HOS1 (Ub E3)
S
ICE1 (MYC)
S
MYB15
K393
CBF1/2/DREB1B/C
SNOW Box
ICE Box
CBF3/DREB1A
CRT/DRE
COR15A, COR47, KIN1
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ICE1 Is Sumoylated by AtSIZ1 In Vitro
-
-
AtSIZ1, E1, E2, SUMO



-
-

-
ICE1

-
-
-

ICE1 (K393R)

121
74
ICE1
Substrate ICE1 SUMO ScSmt3 E1
ScAos1/ScUba2 E2 ScUbc9 E3
AtSIZ1
WB T7 (T7-ICE1)
Yeast proteins courtesy of Professor Y. Kikuchi
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ICE1 Sumoylation by SIZ1 in Protoplasts
Protoplast (T7SUMO1, HAICE1)
WT, 23C
WT, 0C
siz1-2, 0C
ICE1 (K393R)
ICE1 (K393R)
ICE1 (K393R)
ICE1
ICE1
ICE1
T7-SUMO
IP T7
HA-ICE1
81
48
IP T7, WB HA
T7-SUMO (11 kDa)
WB HA
Cold induced sumoylation SIZ1 mediated
HA-ICE1 (53 kDa)
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K393R in ICE1 Causes Freezing Sensitivity
-4C -5 C -6 C -7 C -8 C
WT
100
WT
siz1-2
siz1-2
80
ICE1oe
ICE1oe
3
60
8
ICE1
Survival ratio ()
12
ICE1 -8
40
6
ICE1 K393R
7
20
ICE1 K393R-8
8
Vector
0
-4
-6
-8
-10
Vector
Temerature (ºC)
14
K393R Reduces Cold-induced CBF/DREB1 and Regulon
Expression
300
100
CBF1/DREB1B
CBF2/DREB1C
150
50
0
0
6
200
CBF3/DREB1A
MYB15
Relative mRNA levels
100
3
0
0
80
40
COR47
COR15A
40
20
0
0
0 3 6 12 24
0 3 6 12 24
0 3 6 12 24
0 3 6 12 24
0 3 6 12 24
0 3 6 12 24
ICE1 K393R-8
ICE1 K393R-8
ICE1-8
Vector
ICE1-8
Vector
Time of cold treatment (h)
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ICE1 Sumoylation ICE1 Reduces Poly-ubiquitination
and Proteasome Targeting by HOS1
Sumoylation
Sumoylation
Ubiquitination
Ubiquitination
ICE1 K393R
ICE1 K393R
ICE1 K393R
ICE1 K393R
ICE1
ICE1
ICE1
ICE1
116
81
ICE1
16
AtSIZ1 Control ICE1 through Sumoylation
SIZ1 (SUMO E3)
HOS1 (Ub E3)
S
ICE1 (MYC)
S
K393
MYB15
CBF1/DREB1B CBF2/DREB1C
MYB Box
ICE Box
CBF3/DREB1A
CRT/DRE
COR15A, COR47, KIN1
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AtSIZ1 SUMO E3 Ligase

• AtSIZ1 is a SUMO E3 ligase that facilitates
  sumoylation and regulates cold signaling through
  ICE1 and then CBF/DREB1
• AtSIZ1 transcription factor substrates include
• PHR1 Pi starvation signaling
• ICE1 - cold signaling
• ABI5 - ABA signaling
• FLD - flowering time

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Sumoylation in Plant Stress Responses Ray Bressan
Researchers
Collaborators
Jian-Kang Zhu, University of Arizona Muppala
Reddy, Central Salt and Marine Institute,
Guajarat, India
Kenji Miura Jingbo Jin Chan Yul Yoo Ana
Rus Altan Sharkhuu
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Frosts cause substantial agricultural yield
losses, particularly if a freezing event occurs
during reproductive development. Low
temperatures also substantially limit the
geographic locations where crops can be grown.
20
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