Phosphorylation of the Tomato Β-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 death ADI3 VIRAL INDUCED GENE SILENCING (VIGS) Devarenne et al. (2006) EMBO J. How does Adi3 Regulate cell death? Silencing the expression of Adi3 causes spontaneous cell death in tomato

(β)(β) (γ)(γ) (α)(α) SNF1 Related Protein Kinase Complex SNF (Sucrose Non Fermenting) AMPK (AMP Dependent-Kinase) AKIN/SNRK Adi3 Adi3 interacts with the tomato SnRK complex The SnRK complex is highly conserved across eukaryotes MASTER REGULATOR OF ENERGY HOMEOSTASIS

(β)(β) (γ)(γ) (α)(α) ? METABOLIC HOMEOSTASIS STRESS SIGNALING ? The SnRK complex (Mammals vs. Plants) Adi3 Cell Death Regulation GERMINATION IN SRUCROSE

(β)(β) (γ)(γ) (α)(α) Adi3 Adi3 phosphorylates β at Ser26 P 32 (β)(β) Adi3 (β)(β) Ser26Ala Replacing serine 26 with alanine blocks phosphorylation by Adi3. ? Ser26Ala

(β)(β) Experimental design Karlheinz Knoch (β)(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 Does phosphorylation of β- play a role in SnRK function in vivo?

(β)(β) Transgenic plants express β-GFP Karlheinz Knoch (β)(β) Ala GFP A. 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)(β) (γ)(γ) (α)(α) Adi3 SnRK complex regulation.