1. Activation of a Floral Homeotic Gene in Arabidopsis
Maximilian A. Busch,* Kirsten Bomblies,* Detlef Weigel†
Presentation by Steven Wang and Clemens Langemeyer

2. Content: Introduction Objective(s) / Hypothesis What do we know?
Base of Knowledge
What is LEAFY?
What do they know?
Paper quotes / Sidenote
Homeotic gene expression
Methods
AG/LEAFY interaction
LFY:VP16
The second AG intron
Methods (continued)
Constructs
Staining
Results
Conclusion

3. Objective: Examine the interaction between homeotic gene AGAMOUS
and early action floral meristem-identity gene LEAFY with respect to the LFY mutant phenotype (leaves/shoots) and wild-type floral morphology.
Hypothesis: The predict that LFY will interact as a transcription factor,
inducing expression of AG and altering floral morphology-- not simply establishing a floral meristem.

4. What do we know? Classes of Homeotic Genes:
… responsible for “a distinct , overlapping pattern within the developing flower.”
Question: What is the AG mutant phenotype?
From last lecture: How and when does AG get expressed during floral development?

5. What do we know? Meristem-identity genes of interest:
LEAFY (LFY), APETALA1 (AP1)
Flowers do get produced by LFY and AP1 mutants despite early arising phenotypes.

6. Homeotic gene expression patterns
LEAFY is an early-acting, upstream regulator of flower-specific genes ABC that is thought to act primarily as an activator of AGAMOUS.
How does AG overcome the negative repression of both AP1/AP2 products?
Is LEAFY regulated in some way?
If so, how might that make the investigation of AG regulation difficult?
… Potential solutions?

7. What is LEAFY?
LEAFY is a loss-of-function (LoF) mutation resulting in a phenotype that replaces early flowers with shoots and leaves. LFY is suggested to affect the ABC genes responsible for wild-type floral development, notably--and the focus of this study--the MADS box gene AGAMOUS (AG).
Wild-type inflorescence
LEAFY inflorescence

8. What do they know? Observations: LEAFY
LFY / AG interactions are complex
AG RNA delayed in flowerlike structures
AG reaches WT levels eventually
Strong LFY mutants develop flowers…
Expressed ectopically (flowers/stem) in flowers of LFY:VP16
Flowers appear earlier in LFY:VP16 plants
LEAFY
Question: How is LFY thought to affect the regulation of AG?
Because it affects the production of AG
both piositively and negatively as AG is produced ectopically in mutant plants (negative), but also in flowers (positive)

9. From the paper...
“Control of floral identity by LFY can be genetically separated from a role of LFY in the later process of ABC regulation.”
“In plants that expressed an activated form of LFY protein [...] AG RNA was detected earlier, ectopically, and at elevated levels when compared to WT flowers…”
Temporal regulation of ABC by LFY specified by other gene products.
“LFY interaction with region- specific coregulators that restrict AG expression to a subset of LFY-expression cells.”
Question: What do they mean?
other gene products determine the regulatory effect of LFY protein on floral identity while also partaking in other processes
suggest temporal effects on LFY regulation
LFY interacts with localized regulators to determine level of AG expression in certain cells at certain times

10. Sidenote: Floricaula Snapdragon ortholog → Floricaula (FLO):
Demonstrates both positive and negative roles in regulation of the AG ortholog PLENA (PLE) due to non-autonomous activation--FLO and LFY are not regulating directly.
Question: Why bother identifying orthologous genes?

11. Interaction of LFY & AG
Research performed by Parcy et al. (1998) resulted in the development of an “active” LFY gene known as LFY:VP16--VP16 is a strong viral activation domain. The LFY gene has been modified for constitutive transcriptional-potential.
LFY binds to ds
oligonucleotide in the wild-type
They sought to determine whether LFY was affecting flower morphology by way of the ABC genes or specifying flower meristem fate.
Question: What would you expect to see if LFY only determine floral meristem fate? What would you expect to see if it affected flower morphology?
VP16
The reporter, GUS (β-glucuronidase) is activated when fused to a strong transcriptional activation domain.

12. VP16 inducing transcription:
Question: Potential downside to using this technique? Can you effectively compare results to wild type expression?

13. Agamous intronic enhancer regions
Hind III restriction fragment
EXON
3kb
EXON
… Enhancer ?
H Bc Sc Sn Sp N Xb Sn H2 Ss H3
Question: How can we locate the enhancer region?

14. Reporter construct minimal promoter AG restriction fragment GUS gene
A reporter gene encodes an enzyme with an easily assayable activity that is used to report on the transcriptional activity of a gene of interest. Using recombinant DNA methods, the original promoter of the reporter gene is removed and replaced by the promoter of the gene to be studied. However, the researchers were trying to figure out the effect of the enhancer so they used a minimal promoter. The new chimeric gene is introduced into an organism, and the expression of the gene of interest is monitored by assaying for the reporter gene product.

15. Staining of GUS
A widely used reporter gene in plants is the uidA, or gusA, gene that encodes the enzyme β-glucuronidase (GUS).
This enzyme can cleave the chromogenic (color- generating) substrate X-gluc (5-bromo-4-chloro-3-indolyl β-D-glucuronic acid)
Resulting in the production of an insoluble blue color in those plant cells displaying GUS activity.
Plant cells themselves do not contain any GUS activity, so the production of a blue color when stained with X-gluc in particular cells indicates the activity of the promoter that drives the transcription of the gusA-chimeric gene in that particular cell.

16. Figure 1A.
Reporter constructs carrying AG genomic fragments in front of a minimal promoter driving GUS
- F (forward): 5’ end closest to promoter. R (reverse): 3’
-activity range: primary transformants that showed no, weak, intermediate, strong staining due to GUS expression
-wildtype: “++” shows GUS expression during stage 3 of flower development

17. Figure 1A. Continued
lfy-: GUS expression in a lfy-12(null allele) background
LFY:VP6 : whether or not GUS expression increased in LFY:VP6
n: number of lines analysed for each construct
n/d stands for not determined

18. Figure 2. - Shoot apical meristem indicated by *
- Numbers indicate stage of flower development
- p: pedicels
- Orange: weak staining
Pink/purple: strong staining

19. KB9
Why did the researchers use the same enhancer twice indicated by Forward and Reverse?
What conclusion can be drawn from the results?

20. KB9
The reverse enhancer showed higher GUS expression even when it is shorter than the forward enhancer.
As a result, the researchers focused on the 3’ enhancers.

21. KB9 reporter construct
The KB9 reporter responded to changes in LFY activity in a similar fashion as endogenous AG.

22. KB14 reporter construct
The non-overlapping fragments in the above reporter construct both conferred GUS expression in the center of young flowers also resembling endogenous AG.
This indicates there are two redundant enhancers that could drive GUS expression.

23. KB21 reporter construct

24. KB18 reporter construct
GUS is ectopically expressed in the outer whorls but much less so in the floral stem or pedicel of LFY:VP16 flowers.

25. Results from reporter constructs
1. There are synergistic enhancers that mediate the action of LFY-independent activators of AG because smaller reporters were more sensitive to loss of LFY activity.

26. Results from reporter constructs
1. There are synergistic enhancers that mediate the action of LFY-independent activators of AG because smaller reporters were more sensitive to loss of LFY activity.
2. There appear to be cryptic regulatory elements that have overlapping roles in repressing AG in different regions of the flowers.
In F GUS expression is seen consistently throughout the first whorl and the pedicel.
In contrast, L shows GUS expression heavily in the first whorl but none in the stem or pedicel

27. LFY:VP16 and LFY response elements
If LFY binds to the AG sequence then AG expression is increased.
Where does LFY bind?
How can we find it?

28. LFY:VP16 and LFY response elements
If LFY binds to the AG sequence then AG expression is increased.
Where does LFY bind?
How can we find it?

29. LFY:VP16 and LFY response elements
KB24 and KB28 both showed no staining(no GUS expression) in a wild-type and lfy- background showed increased GUS expression when introduced into LFY:VP16.

30. Figure 4A. -Free refers to 160bp fragments from the AG 3’ enhancer.
-LFY shows from 0 to high levels of LFY protein added.
What does the result show?
What is your conclusion?
Alpha refers to one DNA-protein complex. Beta represents a second slowly migrating complex.
There are two LFY binding sites within this 160bp fragment.

31. Figure 4A.
The researchers then went on to refer the two LFY binding sites as AG I and AG II

32. Figure 4B.
What does the result show?
What is your conclusion?

33. Figure 4B.
The m1 is a 2-bp mutation which eliminates in vitro binding of LFY
The m2 is a single base mutation that does not appear to affect LFY binding.
Can you explain why?

34. Figure 4C.
Because the m1 mutation affects the consensus sequence it renders the binding site useless.
The m2 mutation, however, rests in between the consensus sequence and thus does not affect binding of LFY.

35. Figure 3. KB45 has the AG I site deleted
KB46 has both AG I and AG II sites deleted
MX68 AG I m1 & AG II m1
MX100 AG I m2 & AG II m2
m1 interferes with in vitro binding of LFY, eliminating in vivo activity of 3’ enhancer
m2 does not affect in vitro binding of LFY.

36. Confirmation of results, Conclusion
As a result of the LFY binding sites observed in vitro, they sought to confirm their findings by crossing several MX68 lines to LFY:VP16.
Question: What would you expect to see in the cross progeny?
Unlike the complimenting they observed in control reporter KB31 or even smaller WT seq fragments such as KB24 and KB28, they observed no increase regardless of the introduction of LFY:VP16.
Therefore, LEAFY is crucial for transcriptional activation of AGAMOUS.

37. Future research Identifying coregulators
AG activation in subset of LFY cells (as result of VP16 ectopic AG)
Further research into the negative controls of AP1/AP2, etc. and their relation to AG.

38. Works Cited
Busch, M. A., Weigel, D., Parcy, F., Lee, I., & Nilsson, O. (1998). A genetic framework for floral patterning. Nature, 395(6702), doi: / Pelaz, S., Ditta, G. S., Baumann, E., Yanofsky, M. F., & Wisman, E. (2000). B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature, 405(6783), doi: / Pidkowich, M. S., Klenz, J. E., & Haughn, G. W. (1999). The making of a flower: Control of floral meristem identity in arabidopsis. LONDON: Elsevier Ltd. doi: /S (98) Schultz, E. A., & Haughn, G. W. (1991). LEAFY, a homeotic gene that regulates inflorescence development in arabidopsis. The Plant Cell, 3(8), doi: /tpc