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
Trimeric G-proteins
Monomeric G-protein
: low molecular weight
Monomeric G-protein
: high molecular weight
Major families
Gs family, Golf
Gt (transducin)
Go family
Gz
G4 family
G12 family
Ras family
Rho family
Rab family
Arf family
Ran family
GH (glutaminase)
Dynamin
“Extra large” G-protein
(XLaS)
Activation and inactivation cycles
Trimeric G-proteins
Monomeric G-proteins
Activation and inactivation cycles
Monomeric G-proteins
Trimeric 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
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
Chloroplast
CAB (chlorophyll a/b-binding protein)
development
RBCS (Rubisco)
Unit I
cGMP
Pfr
Ga
CHS
Anthocyanine
biosynthesis
FNR
Ca2+
CaM
Box II
CAB
RBCS
Chloroplast
development
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
(Fig. 4)
Hypocotyl epidermal cell
- protruding cell
- burrowed edll : stomata
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
1.
2.
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
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 X
Ga↑
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↑
fin219 x Ga↑
FR
phyA
Normal Ga↑ effect
under FR light
FHY1
Ga
R
phenotype
The Effect of Ga Overexpression on R Light Inhibition of
Hypocotyl Elongation Requires Functional phyB.
(Fig. 7)
R light
FR light
Ga 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 Ga overexpressors
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
FR
R
phyA
FHY3, FIN219
EODR/FR
phyB
FHY1
Ga
Regulation of
hypocotyl elongation
Regulation of
EODFR response