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Phosphorylation of the Tomato

Phosphorylation of the Tomato Β -Subunit of the SnRK Complex by Adi3 and its Possible Role in Sugar Signaling. JULIAN AVILA-PACHECO. Protein Phosphorylation. Ser, Thr , Tyr. One of the most ubiquitous types of post-translational modifications. Protein Phosphorylation. STRUCTURE.

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Phosphorylation of the Tomato

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  1. Phosphorylation of the Tomato Β-Subunit of the SnRK Complex by Adi3 and its Possible Role in Sugar Signaling JULIAN AVILA-PACHECO

  2. Protein Phosphorylation Ser, Thr, Tyr One of the most ubiquitous types of post-translational modifications

  3. Protein Phosphorylation STRUCTURE INTERACTION PARTNERS ACTIVITY PROTEIN STABILITY LOCALIZATION

  4. Adi3 is a kinase involved in cell death ADI3 VIRAL INDUCED GENE SILENCING (VIGS) (i.e. VIGS) Adi3 Adi3 Devarenne et al. (2006) EMBO J. How does Adi3 Regulate cell death? Silencing the expression of Adi3 causes spontaneous cell death in tomato

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

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

  7. Adi3 phosphorylates β at Ser26 Ser26Ala Ser26Ala Adi3 (β) (γ) (β) (α) Adi3 Adi3 (β) ? Replacing serine 26 with alanine blocks phosphorylation by Adi3. P32

  8. Experimental design Does phosphorylation of β- play a role in SnRK function in vivo? Ala (β) GFP (β) GFP KarlheinzKnoch • 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.

  9. Transgenic plants express β-GFP Ala A. thaliana (β) GFP (β) GFP βAla WT β < β-GPF α-GFP KarlheinzKnoch The expression of β-subunit in transgenic plants was tested using western blot with α-GFP Antibodies.

  10. Transgenic plant cell death phenotype wt β βAla - Line 1 βAla – Line 2 No significant differences in plant development or viability were observed.

  11. Transgenic germination on sucrose WT, β, βAla 0mM 200mM 29mM wt β βAla1 βAla2

  12. Transgenic germination on high sucrose wt High sucrose inhibits cotyledon expansion β βAla1 βAla2 β over-expression causes aberrant phenotypes 200mM

  13. 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.

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