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) : G, G,

GM (monomeric protein) : small molecularhigh molecular

G-Protein class Major families

Trimeric G-proteins Gs family, GolfGt (transducin)Go familyGzG4 familyG12 family

Monomeric G-protein: low molecular weight

Ras familyRho familyRab familyArf familyRan family

Monomeric G-protein: high molecular weight

GH (glutaminase)Dynamin“Extra large” G-protein (XLS)

Activation and inactivation cycles

Monomeric G-proteins

Trimeric G-proteins

Activation and inactivation cycles

Monomeric G-proteins Trimeric G-proteins

G-protein-coupled Signal transduction events

Trimeric G-proteins

Monomeric G-proteins

G-protein-coupled Signal transduction events

Heterotrimeric G-protein in plants

- Arabidopsis AtGPA1 : single G geneAGB1 : single G geneG-like : membrane associated single gene

- AtGPA1

: 383 a.a. 45kDa: 36% identity and 73% similarity to Gi 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 Ggene

- G is involved in

: Gibberellin induction of the -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

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

Ref. : Cell 73, 937-952

Microinjection

- GDPS- Pertussis toxin- GTPS- Cholera toxin

3) phytochrome A –mediated responses

- CHS (chalcone synthase) : synthesis of anthocyanine- FNR (ferredoxin-NADP+-oxidoreductase- CAB (chlorophyll a/b-binding protein)- RBCS (Rubisco)

Chloroplastdevelopment

Pfr G

cGMP

Ca2+

Unit I

Box II

CHS

FNR

CABRBCS

CaM

Anthocyaninebiosynthesis

Chloroplastdevelopment

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 moleculeVE : vector control

◎ wG and cG Lines Overexpress G-Protein by DEX Induction

Dark White light

Dark

White light

active formGTP-binding form

Effect of light on Gexpression

(Fig. 1)

G Overexpression Results in Inhibition of Hypocotyl Elongation.

(White light condition)

- Inhibitoin of hypocotyl elongation- smaller cotyledone

(Fig. 2)

=> Increased light sensitivity

G Overexpression Results in Inhibition of Hypocotyl Elongation.

Dark Light Light

~60% Hypocotyl elongation inhibition~50% reduced chlorophyll content

(Fig. 2)

Fluence Responses of G Overexpressor Seedlings.

(Fig. 3)

Stomata Cell Differentiation in the Hypocotyl Epidermis.

(Fig. 4)

Smaller cotyledon, short hypocotyl

: reduced cell number?: reduced cell elongation?

?

Hypocotyl epidermal cell

- protruding cell - burrowed edll : stomata

protruding cell counting

burrowed cell counting

protruding cell

- no difference between VE and G overexpression plants in cell number- length was reduced about one-half than VE

burrowed cell

- increase of (stomata structure)/(burrowed cell)wG : 5.0 ± 1.1cG : 5.5 ± 0.7control : 1.1 ± 0.3

◎ factors to stimulate stomatal differentiation1. Ethylene : influence phytohormene2. 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)

- Inhibition of hypocotyl epidermal cell (protruding cell) elongation- Increase of stomatal structure per hypocotyl epidermal cell (burrowed cell)

VE

wG cG

protruding cell

burrowed cell

stomatal structure

(Fig. 4)

Responsiveness of the G Overexpressors to Exogenous

Application of GA3.

rice G-knockout / antisencemutrants

G-overexpression At plats

: dwarfism, GA-insensitive

similar phenotype

GA-insensitive short phenotype?

Test the GA-insensitivity of Ga-overexpressor transgenic At

(Fig. 5)

Goverexpression 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)

G modulates signals from both B and R/FR light response

Analyze the effect of G-overexpression on phyA, phyB and CRY1 signal transduction

Dependence of the G Overexpression Phenotype on the phyA Signaling

Pathway.

If there is no inhibition of hypocotyl elongation

phyA null mutant G↑X

FR

G 가 overexpression 되어도Functional phyA 가 있어야 FR Light-mediated inhibition of

Hypocotyl elongation 이 일어난다

(Fig. 6)

G 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 G↑ fin219 x G↑

Normal G↑ effect under FR light

FR phyA

G phenotypeR

FHY1

(Fig. 6)

The Effect of G Overexpression on R Light Inhibition of Hypocotyl Elongation Requires

Functional phyB.

G Overexpressors Required Functional

phyB for Their Enhancement of the R

Light–Mediated Inhibition of Hypocotyl

Elongation

phyB is not involved in the corresponding FR

effect of the G overexpressors

R light

FR light

(Fig. 7)

The EODFR Response in G-Overexpressing Lines.

EOD FR reponse: end-of-day FR pulse at seed sowing: elongated hypocotyl, stem, internode: phyB specific signal transduction

G 는 phyB-mediated EOD FR response 에는 관여하지 않는다 .

(Fig. 8)

The Effect of G Overexpression on B Light Inhibition of Hypocotyl Elongation Does Not

Require Functional CRY1.

G Overexpression Does Not Affect CRY1-Mediated B Light Inhibition of Hypocotyl

Elongation

B light FR light

G 는 CRY1-mediated pathway 에 관여하지 않고 다른 photoreceptor 와 관여하여 B light effect 를

보인다 .

G

Conclusion

FR

phyA

FHY1FHY3, FIN219

Regulation of hypocotyl elongation

R

phyB

Regulation of EODFR response

EODR/FR