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

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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.