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

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

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 Monomeric G-protein : high molecular weight GH (glutaminase) Dynamin “Extra large” G-protein (XLaS)

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

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

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

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

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

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

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

contains less than 5% of the amount of type 1 phytochrome 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

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 cGMP Unit I CHS Anthocyanine biosynthesis Pfr Ga FNR Chloroplast development Box II CAB RBCS Ca2+ CaM

3. Results ◎ Construction of Ga-inducible Arabidopsis transgenic lines - AtGPA1 : under the control of a glucocorticoid-inducible promotor : Induce by exogeneously applied dexamethasone (DEX) 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

◎ 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

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

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

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

? 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

◎ factors to stimulate stomatal differentiation 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)

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

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

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

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

(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

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

(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 Ga phenotype R

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 not involved in the corresponding FR effect of the Ga overexpressors FR light

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.

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-Mediated B Light Inhibition of Hypocotyl Elongation Ga는 CRY1-mediated pathway에 관여하지 않고 다른 photoreceptor와 관여하여 B light effect를 보인다.

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