1. Structural and functional studies of Arabidopsis SIZ1(Cheong et al., 2009) Specific domain structures control ABA, SA, and stress mediated SIZ1 phenotypes Background PART 2

2. Structural Domain of SIZ1 and its point mutations SAP LxxVL PHD C3CHC3 PINIT PIIT SP-RING CCHC3 SXS hhxSxSaaa SIZ1 WT SAP LxxAA SIZ1 sap PHD C3YHC3 SIZ1 phd SP-RING CAHC3 SIZ1 sp-ring PINIT PAAT SIZ1 pinit SXS hhxAxAaaa SIZ1 sxs

3. The PHD domain of SIZ1 may have a plant specific role involving light and sugar signaling

4. The PHD mutation(C134Y) is shown long hypocotyl elongation SIZ1 phd SIZ1 WT Col-0 siz1-2 3% D-Glucose Col-0 SIZ1/ SIZ1 phd (phd Col-o ) 2% Sucrose The mutation of SIZ1 PHD domain dominantly and negatively works for hypocotyls elongation. When carbohydrate was supplied to seedling early growth, the hypocotyls from mutant (SIZ1 phd ) are longer than wild type. This phenotype was representative even Col-0 wild type background (phd col-0 ) which has SIZ1 protein (wild type) is normally expressed. The seedlings are 7 days old and grown under 16 hr light/8 hr dark at 22 ℃ using indicated carbohydrate. One another thing, this phenotype is not shown under constitutive dark (next slide)

5. 0% Sugar 2% Sucrose Light Dark The hypocotyl phenotype is dependent on light and sugar

6. Catala et al., 2007 SIZ1 is expressed in all tissues and all developmental stage but not in hypocotyl. At5g60410 (SIZ1)At5g60400 β -Glucoronidase (GUS) SIZ1 promoter (3650bp)

7. Dominant and Negative effect of C134Y(phd mutation) All seeds from phd Col-0 plants exhibit bright color both heterozygote (T1 generation) and homozygote (since T2 generation). All plants are grown under the same condition at the same time. Seeds were harvested from the matured plants. Photo was taken T3 seeds which harvested a pool from T2 plants. The number from 1 to 4 are indicated individual T1 line.

8. PHY A (or PHY B) may be regulated by PHD region of SIZ1 Background :: 1. the KO mutant of these photo-receptor are exhibit a long hypocotyl and bright seed color. 2. These proteins are formed the same structural feature, nuclear body. 3. These proteins are regulated by post-translational modification such as ubiqutination, phosphorylation. The Scenario>>> SIZ1(PHD) PHYA/PHYB activity Subcellular localization Protein stability Accelerating other modification (such as conformational change or dimerization) If the PHD region of SIZ1 works as a scaffold protein of PHYA, PHYA’s activity may be regulated by SIZ1 due to either SUMOylation or not. Alternatively, the PHYA(PHYB) target proteins such as PKSs can be also regulated by SIZ1

9. 3. Determine sub-cellular localization and interaction between SIZ1 and PHY A. ; BiFC (or Y2H) between SIZ1 and PhyA(wild type and sumoylation site mutants). ; identify the interaction domain with SIZ1 or SUMOylation site of PhyA. 1.Determine the phenotype of double mutation at K100/K488 ; To observe the morphological phenotypes of transgenic plants including the possibility of epigenetic trait ; To test SUMO binding and auto-sumoylation. ; To test SUMO E3 ligase activity Study plan and Ongoing works 4. Identify the relationship between SIZ1 regulation modules(activity, stability) and sumoylation of target protein such as PhyA. ; hypocotyl phenotype investigation using double(or triple) mutants such as phyA X siz1-2 (X SIZ1 phd ) 2. Determine the hypocotyl phenotype of transgenic plants, SIZ1 dphd which has no PHD domain like mammalian PIAS s or yeast SIZ1. ;SIZ1 dphd (T2 plants and waiting for T3 seeds for homozygote pool.) >> T1 and T2(homozygote) plants are the same morphological phenotype to siz1-2 mutant. >> Determine SIZ1 phd (C134Y) is either a gain of function mutant (neotropism) or a regulator

10. AtSOS1 C-term can be phosphorlation by MPK3 PART 3

11. AtSOS1 C-term protein can be phosphorlation by MPK3 MPK3 SOS1 C-term Ca 2+ + + + + + - + - - + - + + - - (a positive control for MPK substrate) MBP 1. Putative MPK phosphorlyation sites in SOS1 C-terminus part are S925, S930, and S1136. In addition, S1138 could be MPKs phosphorylation site as well as SOS2 phosphorylation site. Identify the phosphorylation site by kinase assay using mutant proteins. 3. Interaction test with MPKs and SOS1 2. Biological function investigation of SOS1 phosphorylation by MPK3 using null mutant both mpk3 and sos2 after designing the constitutively phosphorylated and (or) unphosphorylated form from #1 result. such as responsiveness under the salt stress. By Prof. Chung lab. MPK3 SOS1-C term

12. SOS1 several mutants are still phosphoylated by MPK3 MPK6MPK4 WT SOS1-CD2 GST MPK3 SOS1-C S986AWTS1054AS1136AS1138AS1136/ 1138A WT GST SOS1-CD2 SOS1-C * In vitro kinase assay Recombinant proteins, wild type (WT) and mutant (S986A, S1054A, S1136A, S1138A, S1136/1138A) are purified from E.coli. GST was used as a negarive control. Wild type and mutants are still phosphorylated by MPK3 but MPK4 and MPK 6 are not phosphorylated even WT. Upper panel is the scheme of the recombinant protein of SOS1. Only cloned SOS1-CD2 and tested phosphorylation. The result indicated that there was no phosphorylation in the CD2 fragment. (* is un-washed E-Coli protein during purification and is phosphorylated ). In case of S1054A, the protein used smaller than other and showed lower intensity. In case of S1136 A and S1138A are used the same amount to WT and S986A and showed lower intensity. This is speculated that one of these should be a phosphorylation site. In addition, another phosphorylation site should be predicted. To consider these points, other possible sites are doing site directed mutageneses and making double or triple mutants. GST SOS1-C SOS1-CD1 SOS1-CD2 SOS1-CD3 454611 4541146* 610 857 855

13. AtSOS1 protein can interact with MPK3(?) SOS1MPKs If interaction, MPKs>> MPK1, MPK2, MPK3, MPK4, MPK5, MPK6, MPK9 To show that Still now, Nub-SOS1 C-term was cloned and is doing a transformation using yeast cell. After selecting the transformed cells, the growth assay will be done. Like this, the growth assay will be confirmed using SOS1 full form clone..

14. Thank You