Arabidopsis cell fate

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Arabidopsis cell fate
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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)

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Auxin distribution (DR5, an ARF -auxin response
element)
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Auxin transport
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Basal/root development
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monopteros ARF auxin response factor
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Bodenlos and Monopteros
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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

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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 35SGFP-BDL B,E GFPnuclear signal (positive
control) C,F GFPnuclear export
transient expression in parsley protoplasts
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BDL binds to MP (ARF5)

• 1) BDLMP C-terminus
• 2) GNOMMP C-terminus (negative control)
• 3) BDLBDL (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

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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

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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

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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

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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??