Contents

1. Overexpression of the Heterotrimeric G-Protein a-Subunit Enhances Phytochrome-Mediated Inhibition of Hypocotyl Elongation in Arabidopsis

2. Contents 1. G-protein 2. Involvement of heterotrimeric G-protein in light signaling 3. Results 4. Conclusion

3. 1. G-protein : GTP-binding regulatory protein GT (trimeric protein) : Ga, Gb,g GM (monomeric protein) : small molecular high molecular G-Protein class Major families Gs family, Golf Gt (transducin) Go family Gz G4 family G12 family Trimeric G-proteins Ras family Rho family Rab family Arf family Ran family Monomeric G-protein : low molecular weight GH (glutaminase) Dynamin “Extra large” G-protein (XLaS) Monomeric G-protein : high molecular weight

4. Activation and inactivation cycles Trimeric G-proteins Monomeric G-proteins

5. Activation and inactivation cycles Trimeric G-proteins Monomeric G-proteins

6. G-protein-coupled Signal transduction events Trimeric G-proteins

7. G-protein-coupled Signal transduction events Monomeric G-proteins

8. Heterotrimeric G-protein in plants - Arabidopsis AtGPA1 : single Ga gene AGB1 : single Gb gene Gg-like : membrane associated single gene - AtGPA1 : 383 a.a. 45kDa : 36% identity and 73% similarity to Gai of mammals and Gat of vertebrates : conserved Arg residue – cholera toxic target site : wild spectrum detectable during development except in mature seed and vertually all parts - in rice : Ga antisense expressor line –dwarf phenotype : five alleles of dwarf1 (d1) : GA insensitive mutant : mutation in heterotrimeric Ga gene - Ga is involved in : Gibberellin induction of the a-amylase gene in oat aleurone cell : regulation of stomatal opening : pollen tube elongation in lily : light signaling pathway in tomato cells –aurea mutant

9. 2. Involvement of heterotrimeric G-protein in photychrome mediated signal transduction 1) phytochrome : photoreceptor in plants : R/FR light receptor

10. phyA is primary photoreceptor for FR light-mediated inhibition of hypocotyl elongation, induction of germination, induction of light regulated, FR light block of the greening response. phyB is primary photoreceptor for R light-mediated inhibition of hypocotyl elongation - FR light inhibition of hypocotyl elongation : phyA specific signaling fhy1-1, fhy3-1 : defective in phytochrome-mediated FR light inhibition of hypocotyl elongation : remain some phyA-mediated responses in FR light => phyA signal transduction is branched

11. 2) aurea mutant • contains less than 5% of the amount of type 1 phytochrome • found in wt seedlings • microinjection of phyA should rescued only phyA-regulated • processes Microinjection - GDPbS - Pertussis toxin - GTPgS - Cholera toxin Ref. : Cell 73, 937-952

12. 3) phytochrome A –mediated responses • CHS (chalcone synthase) : synthesis of anthocyanine • FNR (ferredoxin-NADP+-oxidoreductase • CAB (chlorophyll a/b-binding protein) • RBCS (Rubisco) Chloroplast development Anthocyanine biosynthesis Unit I CHS cGMP Pfr Ga FNR Chloroplast development Box II CAB RBCS Ca2+ CaM

13. 3. Results ◎ Construction of Ga-inducible Arabidopsis transgenic lines : under the control of a glucocorticoid-inducible promotor : Induce by exogeneously applied dexamethasone (DEX) - AtGPA1 wGa : wild-type full-length of AtGPA1 cGa : potential constitutive active form of AtGPA1 : Glu222Leu – disable the GTPase activity of Ga => locked as a active molecule VE : vector control

14. ◎ wGa and cGa Lines Overexpress Ga-Protein by DEX Induction Dark White light (Fig. 1) Dark White light active form GTP-binding form Effect of light on Ga expression

15. Ga Overexpression Results in Inhibition of Hypocotyl Elongation. (Fig. 2) (White light condition) • Inhibitoin of hypocotyl elongation • smaller cotyledone => Increased light sensitivity

16. Ga Overexpression Results in Inhibition of Hypocotyl Elongation. (Fig. 2) Light Dark Light ~60% Hypocotyl elongation inhibition ~50% reduced chlorophyll content

17. Fluence Responses of Ga Overexpressor Seedlings. (Fig. 3)

18. Stomata Cell Differentiation in the Hypocotyl Epidermis. Smaller cotyledon, short hypocotyl burrowed cell counting ? : reduced cell number? : reduced cell elongation? protruding cell counting Hypocotyl epidermal cell (Fig. 4) - protruding cell - burrowed edll : stomata

19. protruding cell - no difference between VE and Ga overexpression plants in cell number - length was reduced about one-half than VE burrowed cell - increase of (stomata structure)/(burrowed cell) wGa : 5.0 ± 1.1 cGa : 5.5 ± 0.7 control : 1.1 ± 0.3 ◎ factors to stimulate stomatal differentiation • Ethylene : influence phytohormene • Light : high photon flux -> increase stomata differentiation - Inhibition of hypocotyl epidermal cell (protruding cell) elongation - Increase of stomatal structure per hypocotyl epidermal cell (burrowed cell)

20. (Fig. 4) VE protruding cell stomatal structure burrowed cell cGa wGa

21. Responsiveness of the Ga Overexpressors to Exogenous Application of GA3. rice Ga-knockout / antisence mutrants : dwarfism, GA-insensitive similar phenotype GA-insensitive short phenotype? Ga-overexpression At plats Test the GA-insensitivity of Ga-overexpressor transgenic At

22. (Fig. 5) Ga overexpression dose not affect the gibberllic acid stimulation of hypocotyl elongation

23. Light dependent phenotype of Ga-overexpression plants Light source specificity? Blue, Red, Far-Red

24. (Fig. 5) Ga modulates signals from both B and R/FR light response Analyze the effect of Ga-overexpression on phyA,phyB and CRY1 signal transduction

25. phyA null mutant Ga↑ X Dependence of the Ga Overexpression Phenotype on the phyA Signaling Pathway. (Fig. 6) FR If there is no inhibition of hypocotyl elongation Ga가 overexpression 되어도 Functional phyA가 있어야 FR Light-mediated inhibition of Hypocotyl elongation이 일어난다

26. (Fig. 6) Ga Could Be Involved in a Branch of the phyA-Mediated FR Light Signal Transduction Pathway fhy1-1, fhy3-1, fin219 : phytochromeA down steam signaling mutants fhy1-1 x Ga↑ : no Ga↑ effect under FR light fhy3-1 x Ga↑ Normal Ga↑ effect under FR light fin219 x Ga↑ FR phyA FHY1 phenotype Ga R

27. The Effect of Ga Overexpression on R Light Inhibition of Hypocotyl Elongation Requires Functional phyB. (Fig. 7) Ga Overexpressors Required Functional phyB for Their Enhancement of the R Light–Mediated Inhibition of Hypocotyl Elongation R light phyB is notinvolved in the corresponding FR effect of the Ga overexpressors FR light

28. The EODFR Response in Ga-Overexpressing Lines. (Fig. 8) EOD FR reponse : end-of-day FR pulse at seed sowing : elongated hypocotyl, stem, internode : phyB specific signal transduction Ga는 phyB-mediated EOD FR response에는 관여하지 않는다.

29. The Effect of Ga Overexpression on B Light Inhibition of Hypocotyl Elongation Does Not Require Functional CRY1. B light FR light Ga Overexpression Does Not Affect CRY1-MediatedB Light Inhibition of Hypocotyl Elongation Ga는 CRY1-mediated pathway에 관여하지 않고 다른 photoreceptor와 관여하여 B light effect를 보인다.

30. Conclusion FR R EODR/FR phyA phyB Ga FHY3, FIN219 FHY1 Regulation of hypocotyl elongation Regulation of EODFR response