2. Arabidopsis cell fate

4. 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)

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

6. Auxin transport

7. Basal/root development

8. monopteros: ARF auxin response factor

9. Bodenlos and Monopteros

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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??