SIZ1mediated Sumoylation of ICE1 Controls CBF3DREB1A Expression and Freezing Tolerance in Arabidopsi - PowerPoint PPT Presentation

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


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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.
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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
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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
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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)
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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
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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.
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Student Date Name Time Title of
Presentation April 23 Christian
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23 Darla 1245-100 An expression atlas of rice
mRNAs and small RNAs April 23 Nanette
100-115 Biochemical features of maize tissues
with different capacities to regenerate
plants April 25 Joëlle 1230-1245 Plastoglobules
a new address for targeting recombinant proteins
in the chloroplast April 25 Dustin 1245-100 The
use of transgenic plants in the bioremediation of
soils contaminated with trace elements April
25 Steven 100-115 Shining light on signaling
and metabolic networks by genetically encoded
biosensors