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Arabidopsis cell fate

Arabidopsis cell fate. Auxin gradients are formed and maintained by the PIN transporters. PIN7 early apical side of basal cell (2-cell stage) then switches to basal side of suspensor cells PIN1 throughout cell early then localized to basal side of provascular cells

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Arabidopsis cell fate

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  1. Arabidopsis cell fate

  2. Auxin gradients are formed and maintained by the PIN transporters • PIN7 early apical side of basal cell (2-cell stage) then switches to basal side of suspensor cells • PIN1 throughout cell early then localized to basal side of provascular cells • PIN4 hypophyseal lineage and vascular initials • PIN3 root pole (heart stage)

  3. Auxin distribution (DR5, an ARF -auxin response element)

  4. Auxin transport

  5. Basal/root development

  6. monopteros: ARF auxin response factor

  7. Bodenlos and Monopteros

  8. BDL is IAA12, a gene involved in auxin response • bld1 is a GA transition resulting in a Pro74Ser exchange • Pro74 is in the highly conserved domain II • comparable mutations have been found in homologs IAA3 (shy2-2) and IAA7 (Axr2-1) • This mutation increases stability of the proteins!! • Transformation with BDL does not rescue the phenotype • The phenotype is dependent on copy number • domains III and IV are involved in homomeric and heteromeric interactions

  9. BDL is nuclear-localized (GFP fusion) • GFP-BDL is strongly localized to punctate locations within the nucleus • If BDL is truncated (66-72aa) it becomes localized equally in the nucleus and cytoplasm (data not shown) A,D 35S::GFP-BDL B,E GFP+nuclear signal (positive control) C,F GFP+nuclear export transient expression in parsley protoplasts

  10. BDL binds to MP (ARF5) 1) BDL+MP C-terminus 2) GNOM+MP C-terminus (negative control) 3) BDL+BDL (homomer) 4) empty vector (negative control) Yeast two-hybrid system 2 part to the GAL-4 (beta-galactosidase) activator Half is one protein, half to the other If they interact  transcription and beta-galactosidase activity

  11. BDL and MP are co-localized (most of the time) in situ (RNA) not protein • A-D BDL • E-H MP • I BDL • J-L MP • Neither is expressed in the lens-shaped cell or hypophysis where they presumably are active

  12. in situ hybrization • Complemetary oligonucleotide to desired RNA • Tag with small molecule or protein • Enyzme linked target binds tag • Chemical reaction at enzyme causes "detection" of the RNA

  13. Summary • PIN transporter cause accumulation of auxin in forming root pole • ARFs are presumably required for differentiation • AUX/IAA (BDL/MP) heteromer inhibits ARF (sets up and maintains meristem identity) • auxin activates SCF-TIR (E3-ligase  proteasome) • SCF-TIR causes degradation of ARF • ARF transcription repression is released • Cell undergoes terminal differentiation

  14. Conclusions • Possible mode of action: • Transport from site of synthesis to site of action (like short-root)? • Activation of auxin response gene that mediates response in adjacent cell??

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