1. SUMOylated SIZ1 may play an important role
for accumulation of SA

2. PART 1
SUMOylated SIZ1 protein (does not control or does control) its SUMO E3 ligase activity under the heat stress (or SA accumulation).
Alternatively, (Un)SUMOylated SIZ1 protein is involved in epigenetic traits.

3. The Material Information for studying SIZ1 epigenetics
BACKGROUND
Blocking of SIZ1 self-sumoylation exhibits a epigenetic trait. This trait seems to
be related with SA accumulation. Even though the same DNA (unSUMOylated
Form; K100R and K488R) transformed into siz1-2 mutant, the transplants are
shown various morphological phenotype. Some are similar to siz1-2, highly SA accumulated plants, some are similar to wild type complemented plants, lower level of SA, and others are in between these.

4. The Schematic SIZ1 Structure
sumo(K440R)
sumo(K100R)
sumo(K218R)
sumo(K450R)
sumo(K320R)
sumo(K488R)
sumo(K500R)
SAP
PHD
SP-RING
PINIT
SXS
Seven Predicted SUMOylation Motif.
The Designed Constructs
Binary vector construct backbone
PAtSIZ1
SIZ1
GFP
T ocs
1. siz1 (K100R)
2. siz1 (K320R)
3. siz1 (K450R)
4. siz1 (K488R)
5. siz1 (K500R)
6. siz1 (K100R/K488R) (=MD)
7. siz1 (K100R/K218R/K320R/K440R/K450R/K488R/K500R; all sites mut.) (= K7eaR) .

5. Selected T1 Plants K100R K320R K450R K488R K500R 34.7±4.3 28.3±6.13
35.7±3.13
32.9±4.85
32.2±6.41
Transgenic plants which are single amino acid mutation including K100R, K320R, K450R, K488R, and K500R represented to similar morphological phenotype to low level SA accumulation(=Wild Type). The western blotting for detecting SIZ1 protein was failed using GFP antibody, but it seems the proteins of SIZ1 to be expressed. One of the reason is that the morphological phenotypes are indicated proteins existence. If there is no protein , it would be the same to siz1-2 mutant background. The white number is the average height from matured plants. The photos are almost matured 8-week-old plants, in case of K500R plants are grown plants for 5 weeks.

6. Selected K7eaR T1 plants
These plants are selected from antibiotics (hygromycin) resistance. All plants are T1 generation and should be heterozygote. The plant are 4-week-old and growing under 16 h light /8 h dark at 22℃. The diagnostic PCR using genomic DNA indicated that all plants are contained a transgene in their chromosome.

7. Digoenestic PCR from K7eaR T1 plants
atsiz1-2 T-DNA
Digoenestic PCR from K7eaR T1 plants
SIZ1:GFP
primers binding region in the SIZ1 cDNA
atsiz1-2
Tall ; ± 4.0
middle; ± 2.1
Small ; ± 2.5
three groups(Cm)
The diagnostic PCR was done using genomic DNA and the primers can be designed to annealing on exons. If plants are the same to mutant background, the PCR products would not be amplified. Or if plants has transgene, SIZ1 cDNA, the PCR products should be amplified around 500 bp. Even tough there is no a photo for confirming the T-DNA insertion using LB(SALK T-DNA primer ) and siz1-2 F, all plants are amplified PCR products using those primer sets. This indicated that all plants are siz1-2 mutant background. Based on these results, all selected K7eaR plants were divided three different groups depending on average height at a matured adult stage. * this photo’s experiment was done 5 year ago.

8. Selected SIZ1(K100/488R; MD)T1 transgenic plants
Small
=siz1-2 mutant
Middle
Big=Wild type
These plants are 3 weeks old after selection using antibiotics (hygromycin). The growth condition is 16 h light /8 h dark at 22℃

9. The Diagnostic PCR for investigation of epigenetics
Example, 1 2 3 4 5 6 7 8
Primer sets 1 3 4 2
Salk LB & siz1-2 F
siz1-2 F and R 5 6 7 8
SIZ1 F and R
Diagnostic PCR using eight representative plants Diagnostic PCR was done only eight representative plants. The left photo is those plants and numbers and right panel is it’s diagnostic PCR result. The T1 plant (mother) was similar to siz1-2 plant
(red box from previous slide ) and T2 plants (its progeny ;a left photo) was shown various phenotypes. Siz1-2 mutant is a knockout mutant from SALK line. For diagnostic PCR(a right panel), the primers used for LB from SLAK line, siz1-2 F and siz1-2 R are from intron sequences which is not annealing to SIZ1 cDNA. This result indicated that all eight plants are siz1-2 knockout mutant and have transgene of SIZ1 cDNA even though the progeny from the same mother were shown different morphological
phenotypes.
Plan>> 1. Fix a homozygote line.
2. Check these pattern again (i.e. whether the same genetic background are shown different phenotypes or not.
3. qRT-PCR for checking expression level (SIZ1 and SID2) from its line of the progeny

10. Additional lines for MD (T2 generation) Transgenic Plants#4
#76
#49
#34
#72 #1
The plants are growing for 3 to 4 weeks under 16 h light /8 h dark at 22 ℃. These mother plants(T1) are one copy inserted transgenic plants and these photos are T2 generation. As expected, these plants are various morphological phenotype. To confirm existence of SIZ1 cDNA in these genome, it needs to do diagnostic PCRs like previous slides and for checking either one copy insertion line, it is necessary to count a segregation ratio. After this job, it will be tested SIZ1 mRNA expression for SIZ1 expression and SID2 mRNA expression for SA biosynthesis or accumulation by qRT-PCR. It would be a marker of co-relation between SIZ1 expression and SID2 expression.

11. Un-SUMOylated SIZ1 still interact with SUMO1 non-covalently
NYFP-SUMO1
SIZ1-CYFP
SUMO1
SIZ1WT
SUMO1 ΔGG
SIZ1WT
SUMO1
SIZ1K7eaR
SUMO1 ΔGG
SIZ1K7eaR
N-terminus YFP was fused to N-terminus of SUMO1 in-frame translationally and C-terminus YFP was fused to C-terminus of SIZ1 wild type and mutant (K7eaR; which cannot be sumoylated from). The YFP signal from BiFC indicated that SIZ1 protein and SUMO1 protein are interacted each other. And mutated protein can be expressed in vivo and may play some specific roles.

12. Plan of study 1. To confirm that SIZ1 is related with epigenetics.
- un-sumoylated SIZ1 protein expression in Arabidopsis (K7eaR and MD) does not be shown
a consistent morphological phenotype in every generation based on its transgenic plants pool.
- This phenotype ratio is not followed a Mendelian segregation rule. (approximately, 7(wild type, big):1(middle):2(mutant, small))
>>To decide one copy inserted line by a Southern blotting.
>>To fix and keep a homozygote of one copy inserted lines
>>To check a insertion locus by TAIL PCR
- These clones, K7eaR and MD, are also transformed into Col-0 wild type and check phenotype of these T1 plants.
(I already transformed into Col-0 (T0) and am waiting for harvesting seeds (T1))
2. To establish that SIZ1 protein plays a specific role for modulating SA accumulation directly or indirectly.
>> Check the expression level of SIZ1 and SID2 by qRT-PCR using fixed homozygote lines.
and make a relationship between expression level and morphological phenotype (SA level).
3. To establish a SIZ1 working model
3-1. If SIZ1 protein modify Chromatin structure.
>>To investigate whether this protein has DNA binding activity and can modulate a specific gene expression
as SIZ1 is a member of PIAS.
>>To test interaction between SIZ1 protein and SID promoter DNA or it’s mRNA
or >>To test interaction between SIZ1 and Histone (H3 or H4)
3-2. If SIZ1 protein modifying target proteins such as SID sumoylation as a SUMO E3 ligase.
>> Testing the SUMOylation of SID2 or possible target protein such as PAD4 etc.
( SID2 protein doesn;’t sumoylate when Jung Hoon did in vitro SUMOylation assay.
>> To consider other targets (such as PAD4) through searching interacting partners using yeast two hybrid.
( Jung Hoon is testing PAD4 sumoylation in vitro and I am testing self activity between BD-SIZ1 and AD vector for Y2H )

13. Figure2 SIZ1 protein can be sumolyation.
Figure1 SIZ1 protein may be regulated by post-translational modification.
Heat stress
0.25
0.5 1 3 6 hr
qRT-PCR (RT-PCR) (transcript)
Western blotting (protein)
SUMO-induced conjugates (E3 ligase activity)
Figure2 SIZ1 protein can be sumolyation.
In vivo/ in vitro sumoylation
Figure 3 Un-sumoylated SIZ1 protein (doesn’t) affact to SUMO E3 ligase activity
Using the Wild type complemented transgenic plants,
Heat treatment
0 15 min 30min 1hr 2hr 3hr
Transcript level (SIZ1)
other heat shock treatment marker.
Western analysis. (SIZ1 protein level and other protein marker such as HSP101)
In case of drought stresses, the transcript level doesn’t changed.
If the transcript level was changed, the microarray results exhibited.
Catala et al (2008) group is showed that the siz1 may be regulated the post translation level in directly.
SUMO-conjugates
under the heat shock using transgenic plants
which are mutated at SUMOylation sites

14. For SA (in)dependent approach/ abiotic stresses related approach,
Figure 4-1 Transgenic plants’ morphological phenotype
and stress responsibility such as heat shock.
Morphological phenotype and stress responses
For SA dependent approach,
Figure 4-2 Transgenic plants’ morphological phenotype
Morphological phenotype
SID2 transcript level and SA content