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Overexpression of the Heterotrimeric G-Protein a-Subunit Enhances Phytochrome-Mediated Inhibition of
Hypocotyl Elongation in Arabidopsis
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Contents
1. G-protein
2. Involvement of heterotrimeric G-protein in light signaling
3. Results
4. Conclusion
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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)
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Activation and inactivation cycles
Monomeric G-proteins
Trimeric G-proteins
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Activation and inactivation cycles
Monomeric G-proteins Trimeric G-proteins
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G-protein-coupled Signal transduction events
Trimeric G-proteins
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Monomeric G-proteins
G-protein-coupled Signal transduction events
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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
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2. Involvement of heterotrimeric G-protein in photychrome mediated signal transduction
1) phytochrome
: photoreceptor in plants: R/FR light receptor
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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
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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
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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
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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
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◎ 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)
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G Overexpression Results in Inhibition of Hypocotyl Elongation.
(White light condition)
- Inhibitoin of hypocotyl elongation- smaller cotyledone
(Fig. 2)
=> Increased light sensitivity
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G Overexpression Results in Inhibition of Hypocotyl Elongation.
Dark Light Light
~60% Hypocotyl elongation inhibition~50% reduced chlorophyll content
(Fig. 2)
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Fluence Responses of G Overexpressor Seedlings.
(Fig. 3)
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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
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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)
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VE
wG cG
protruding cell
burrowed cell
stomatal structure
(Fig. 4)
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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
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(Fig. 5)
Goverexpression dose not affect the gibberllic acid stimulation of hypocotyl
elongation
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Light dependent phenotype of Ga-overexpression plants
Light source specificity?
Blue, Red, Far-Red
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(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
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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)
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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)
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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)
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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)
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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 .
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G
Conclusion
FR
phyA
FHY1FHY3, FIN219
Regulation of hypocotyl elongation
R
phyB
Regulation of EODFR response
EODR/FR