Post on 01-Jan-2016
description
Phosphorylation of the TomatoΒ-Subunit of the SnRK Complex by Adi3 and its
Possible Role in Sugar Signaling
JULIAN AVILA-PACHECO
Protein Phosphorylation
One of the most ubiquitous types of post-translational modifications
Ser, Thr, Tyr
Protein Phosphorylation
STRUCTURE
ACTIVITY
LOCALIZATION
PROTEIN STABILITY
INTERACTION PARTNERS
Adi3
(i.e. VIGS)
Adi3
Adi3 is a kinase involved in cell deathADI3 VIRAL INDUCED
GENE SILENCING (VIGS)
Dev
aren
ne e
t al.
(200
6) E
MB
O J
.
How does Adi3Regulate cell death?
Silencing the expression of Adi3 causes spontaneous cell death in tomato
(β) (γ)
(α)
SNF1 Related Protein Kinase Complex
SNF (Sucrose Non Fermenting)
AMPK (AMP Dependent-Kinase)
AKIN/SNRKAdi3
Adi3 interacts with the tomato SnRK complex
The SnRK complex is highly conserved across eukaryotes
MASTER REGULATOR OF ENERGY HOMEOSTASIS
(β) (γ)
(α)?
METABOLICHOMEOSTASIS
STRESSSIGNALING
?
The SnRK complex (Mammals vs. Plants)
Adi3 Cell Death Regulation
GERMINATIONIN SRUCROSE
(β) (γ)
(α)
Adi3
Adi3 phosphorylates β at Ser26
P32
(β)
Adi3
(β)
Adi3
Ser26Ala
Replacing serine 26 with alanine blocks phosphorylation by Adi3.
?
Ser26Ala
(β)
Experimental design
Karlheinz Knoch
(β)Ala
- Generate two transgenic plants over-expressing β and βAla using the plant model organism Arabidopsis thaliana using Agrobacterium mediated transformation.- Assess cell death phenotypes- Estimate germination efficiency under high, normal, or low concentration of sucrose.
GFP GFP
Does phosphorylation of β- play a role in SnRK function in vivo?
(β)
Transgenic plants express β-GFP
Karlheinz Knoch
(β)Ala
GFP GFPA. thaliana
WT β βAla
< β-GPF
α-GFP
The expression of β-subunit in transgenic plants was tested using western blot with α-GFP Antibodies.
Transgenic plant cell death phenotype
wt
β
βAla - Line 1
βAla – Line 2
No significant differences in plant development or viability were observed.
Transgenic germination on sucrose
0mM 29mM 200mM
WT, β, βAla
wt
β
βAla1
βAla2
Transgenic germination on high sucrose
200mM
wt
β
βAla1
βAla2
High sucrose inhibits cotyledon expansion
β over-expression causes aberrant phenotypes
Conclusions
- We successfully produced Arabidopsis plants over-expressing wild type and phosphorylation deficient β.
- No cell death related phenotype was observed in phosphorylation deficient β Plants.
- Plants over-expressing β phosphorylation deficient mutants appear unaffected by germination inhibition in sucrose.
(β) (γ)
(α)
Adi3
SnRK complex regulation.