1. Hypocotyl Elongation in Arabidopsis
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
Monomeric G-protein
: high molecular weight
GH (glutaminase)
Dynamin
“Extra large” G-protein
(XLaS)

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. 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,

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

13. 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

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)
Dark
Light
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. ◎ 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)

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

21. 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

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. 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

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 Ga
phenotype 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 not involved 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-Mediated B Light Inhibition of Hypocotyl Elongation
Ga는 CRY1-mediated pathway에 관여하지 않고 다른 photoreceptor와 관여하여 B light effect를 보인다.

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