1. Activation of a Floral Homeotic Gene in Arabidopsis
Maximilian A. Busch, Kirsten Bomblies, Detlef Weigel
Biol 433 Tutorial 7 Presented by
Vincent Zheng and
Sebastian Wels-Lopez
Mar 6, 2018

2. LEAFY GENE http://dev.biologists.org/content/130/16/3735
To start with, question: what’s the difference between Columbia ecotype, and leafy-12 mutants?
These two were used to generate lines.
Flowers were replaced by leaves with associated shoots
Sepal, petal, stamen and carpel are gone, only leaf-like structures present.
Leafy gene is one of the floral meristem-identity genes.
Inactivation and overexpression of leafy gene will cause opposite phenotypes
They have made an example of the snapdragon lfy ortholog Floricaula(FLO) which also have both positive and negative roles in regulation of AG ortholog PLENA (PLE).
The non autonomously activation of plena by floricaula indicates that leafy may not regulate agamous directly.
However, throughout this paper, it’s been testified that leafy protein can form a DNA-protein complex with leaf-responsive enhancer in AG, which indicates that leafy is a direct upstream regulator of floral genes.

3. ABC floral-organ identity model
After 2 days of a flower has been initiated, the ABC model are expressed in distinct and overlapping patterns within the developing flower
In this paper, the focus was on C. AG gene specifies the development of stamen and carpel
Dr. George Haughn, BIOL 433, Spring 2018

4. MADS protein structure
In Arabidopsis, C function is specified by the MADS box gene AGARMOUS (AG)
Floral organ-identity MADS genes are DNA binding proteins that interact with other polypeptides.
Dr. George Haughn, BIOL 433, Spring 2018

5. AG expression in wild type
During floral organ development, AG is normally expressed in the center, the third and fourth whorls.
Although lfy mutation will cause loss of AG expression, and was formerly believed to be an upstream regulator of AG. but not all flowers of leafy mutants are replaced by leaves and shoots. Older flowers of lfy mutants will eventually show wild-type floral structure. Those indicates the true relation of leafy gene and AG expression are much more complex.
This paper aims to investigate the correlation between leafy gene and AG expression.
Dr. George Haughn, BIOL 433, Spring 2018

6. Activated form of LFY protein, LFY:VP16
Mature virions of herpes simplex virus type 1 contain an activating factor that primes transcription from the five virally encoded immediate early (IE) genes. This activator is specified by a 65-kD polypeptide termed VP16.
LFY:VP16, a fusion of LFY to the strong activation domain from the viral transcription factor VP16 is expressed under the control of normal LFY regulatory sequences. They form a chimeric protein.
If LFY acts only to specify flower meristem fate, LFY:VP16 might affect the initial establishment of flower primordia, but not downstream events such as specification of floral organ identity.
In this case, VP16 fuses with Leafy and strongly activate and increase the expression of AG RNA transcription. So that AG RNA can be detect earlier and ectopically.
Diagram of LFY:VP16 transgenes. Exons are indicated by rectangles. Sequences coding for the VP16 activation domain are shown in black.
The reason why Leafy:VP16 is used is because the experiment cut the enhancer region so small, that there’s not much left for protein binding, a strong activator is needed to show that leafy gene is playing an important role in the expression of AG gene.
This will be explained later.

7. β-glucuronidase (GUS) reporter system
AG sequence fragment minimal promoter GUS gene
AG sequence fragment was cut using various restriction enzyme
This sequence functions as a enhancer region, if it binds with an activator, they the minimal promoter which is a cauliflower mosaic virus promoter 46 bp upstream of GUS gene will also be activated, the GUS gene will be expressed. The intensity of the GUS gene expression is reflecting the AG RNA being transcribed. Which means, the stronger GUS expression, the stronger enhancement AG sequence fragment can provide.
Question: the advantage of GUS reporter system?
The absence of GUS activity in plants, that’s a key advantage, without background interference, the GUS activity can be accurately measured even in single cells.

8. Examples of GUS expression
In figure 1B. Examples of weak, intermediate, and strong GUS staining in whole apices of different reporter lines (from left to right, MX68 line 38; KB31 line VIIC1; KB31 line VIIC7). A mid–stage 3 flower is indicated in each apex. Faint staining in the left apex is indicated by arrowheads.

9. Examples of GUS expression
In Figure 2. Apices were stained for GUS activity with X-gluc (5- bromo-4-chloro-indolyl- -D-glucuronic acid), embedded, sectioned, and viewed under dark- field illumination. Weak staining appears orange, and strong staining pink to purple. Asterisks indicate shoot apical meristems, numbers indicate stages of flower development

10. Inflorescence SEM Dr. George Haughn, BIOL 433, Spring 2018
The pictures in figure 2 is produced by a thin section through a developing inflorescence which we just learned yesterday.
Dr. George Haughn, BIOL 433, Spring 2018

11. Figure 1. A This chart shows a lot of info in a very compact space:
Due to the limitation of space, they only discuss the lines marked in bold.
Each row shows a section of the 3kb Hind III restriction fragment in the 2nd intron of AG. Thick sections are parts that were kept, and thin sections are bits they chopped out.
The bottom indicates what restriction enzymes they used to make the various cuts
1st column: orientation of fragment relative to minimal promoter; F = 5’ end closest to promoter, R = 3’ end closer to promoter
2nd column: typical range of GUS staining, none/weak/intermediate/strong
3rd/4th column: how strongly a particular line stained when compared to a wildtype plant, or lfy- plant at stage 3 of flower development.
5th column: whether or not the GUS activity increased when using LFY:VP16
6th: sample size
Now we’ll take a look at some of the various different constructs they used:

12. KB9 KB9 is a whole Hind III with no extra parts cut out.
Reacted to changes in LFY activity the same way endogenous AG would, which shows that somewhere in this fragment there are sequences that are LFY-responsive.
In another experiment, where they did not have the KB9 fragment and simply used GUS and the AG promoter, there was no response to using LFY:VP16. This showed that this fragment is also likely to be responsible for most if not all of the AG response to LFY.
Building on top of that, an important part is that in the leafy- mutant, GUS expression was delayed and reduced, but in LFY:VP16 plants, GUS was expressed earlier and there was far more expression.
Difference is that for one of them the promoter was closer to the 5’ end (top), or close to 3’ end (bottom)
As seen in the activity range diagram, there was stronger activity when the promoter was closer to the 3’ end, which is the first reason as to why they would later focus on the 3’ enhancers

13. KB14
This line combined with KB31 are two different non-overlapping segments that both showed GUS expression in young flowers, similar to the way a wildtype flower would. They both also exhibited higher GUS expression levels with LFY:VP16.
Together this shows that there are at least 2 redundant enhancer sequences that can drive GUS expression in wt flowers

14. KB31
Although both KB14 and KB31 both show low GUS expression in leafy- mutants, and greater GUS expression in LFY:VP16, the KB31 line shows a stronger activity range consistent with what we saw earlier, where the 3’ promoters seem to have greater levels of GUS expression. Also the staining looks much more intense compared to KB14

15. KB21
In this line, we see another experiment where they tried to leave sections from the 5’ and 3’ end.
The reason they do so many different lines is that ultimately they want to find out which areas in the hind III fragment are needed for GUS to be expressed. Compare to KB20 above, in KB20 there is no GUS expression even in wildtype, and in KB21 which examines almost the opposite areas, there is GUS expression, which is very good evidence that the missing gap isn’t needed

16. KB18
In KB18 they found a small fragment that has very low GUS expression in the leafy- plant, but still shows large GUS expression in LFY:VP16, the next several lines are based on similar fragments.
Having small fragments like this that are very sensitive to loss of LFY activity suggests that there appear to be other synergistic enhancers in the hind III fragment that don’t rely on LFY for AG activation. Otherwise you would see similar low expression of GUS in larger lines like KB9.

17. KB30
By further deletions progressively from KB31, the activity of GUS expression is negligible even in wild type, thus they don’t measure GUS expression in lfy mutant for the following experiments.
Now wild type becomes the background control group instead of fly mutants. The GUS expression in KB30 is very weak to almost undetectable, but when introduce LFY:VP16, the reporter activity is reactivated.
This is suggesting that leafy is definitely involved in regulation of AG expression directly.

18. KB24 and KB28
Then they start to wonder how Leafy as a protein can bind to a DNA sequence in this fragment.
They cut KB30 into two smaller fragment and find out they share a common region of 230 base pairs.
Genetic analysis has done, then they start to investigate on biochemical side. They test if Leafy protein can bind to the 3’ AG enhancer they find.
Using immunoprecipitation of DNA-protein complexes followed by electromobility shift assays

19. Mapping of LFY binding sites in AG
The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid.
Results show there are two DNA-protein complexes, at lower concentration, alpha complex is seen, and at higher concentration, there is a beta complex.
two binding sites for Leafy protein, they define them as AG I and AG II, only 31 bp apart, and they are indeed located in the 3’ AG enhancer region

20. Sequences of AP1 and two AG sites
They compare the two AG binding sites with previously defined Leafy binding site in promoter of the AP1 gene. It turns out that their sequences are very similar.
AP1, AG I and AG II have a consensus sequences. They also find out that Leafy bind both AG sites with similar affinity, but can’t bind either AG site as strong as it binds to AP1 site.

21. KB45
KB30
Then they try to delete AG I site, and create KB45, the GUS expression can still be observed weakly and can only be detected in whole mount, in C shown here as a sectioned tissue, no GUS activity can be seen.
They LFY:VP16 is introduced, and the GUS expression increased, but compared to KB30 which have the binding site AG I, the expression is greatly reduced even in presence of LFY:VP16.
This results indicate that both binding sites are required for 3’ enhancer to function normally, and the weakened GUS expression suggests Leafy binding sites are only partially redundant.
They propose one rationale for this is because the two binding sites are only 31 bp apart, they may have some cooperative interaction, but this have not been proofed.

22. KB46
Then they delete both binding sites, and resulting KB46 showed a even weaker response than single deletion in KB45.
They introduce LFY:VP16, the GUS expression is not increased.
This suggests the loss of enhancer activity can’t be rescued even with the strong activator.

23. Sequences of AP1 and two AG sites and mutation
To test if the loss of enhancer activity is really due to mutation in the binding sites, they perform another mutation m1 and m2 in the consensus sequence of AG I and AG II
mutation 1 is 2-bp mutation in the binding sites which has been known to bind with Leafy in AP1 gene
Mutation 2 is 1-bp mutation which is known to be in the consensus sequence, but don’t bind with Leafy in AP1 gene.

24. Mapping of AG I and AG II binding sites
From the assay, both AG I and AG II mutation 2 sequence can bind with Leafy protein. And neither of them can bind with leafy protein in mutation 1.
This means mutation in non-binding sequence will not affect Leafy binding. This is true for AG I and AG II as well as AP1 gene.

25. KB68
They put mutation 1 sequence back to 3’ enhancer and generate MX68.
It turns out that MX68 have very weak GUS expression, and can’t be restored by introducing LFY:VP16.

26. n/d
KB100
In contrast, they put mutation 2 back to 3’ enhancer, and it turns out the GUS expression is very strong even in wild type. They do 73 samples to test that, and there is no need to introduce LFY:VP16 to confirm. Since it will only express even stronger.

27. Summary What did we learn? Existence of several AG enhancer regions
Specific AG1 and AG2 binding sites
LFY binds directly to these sites to promote AG expression
How are we sure that we learned what we learned?
Lack of other genes in LFY’s gene family
What comes next?
Determining the sequence or other coregulators that affect AG
Before this research, all we really knew was that LFY was somehow a regulator of AG, and that it could be both a positive and negative regulator because when there is no LFY, AG expression can be greatly reduced, but at the same time in flowers that develop later on, we can see AG expression once more.
We had no idea if LFY was an indirect factor where LFY would activate protein X which would then in turn activate AG, or if LFY would activate AG directly.
By testing several lines we figured out there are several AG enhancers, some of which are LFY-independant, but they also found a small section in the Hind III fragment that contained two similar binding sites, AG1 and AG2. We now know that LFY is directly responsible for AG activation because of the in vitro experiments that Vincent talked about earlier which showed that FLY binds directly to DNA
We can be extra confident in this because LFY is not part of any gene family, meaning that there is only 1 homolog per haploid genome. This means that there is little concern that a related gene of the same family could bind to the same binding sites that LFY was found to bind to. Furthermore the levels of LFY binding to AG1 and AG2 in vitro was also very similar to expression of GUS in vivo.
Some previous studies have shown negative control of AG by genes such as AP1, APETALA2, LEUNIG, or CURLY LEAF, but none of the reporters in this experiment showed an AG expression pattern that resembled plants mutant for some of these negative regulators, which suggests that repression and activation are closely linked.
Knowing that LFY directly activates AG paves the way for future studies to determine what some of the coregulators might be responsible for AG expression besides LFY.

28. References
Hellman, L. M. L. L. M. L., & Fried, M. M. G. (2007). Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein- Nucleic Acid Interactions. Nature Protocols, 2(8), 1849–
Jefferson, R. A. (1989). The GUS reporter gene system. Nature, 342(6251), 837–838.
Parcy, F., Nilsson, O., Busch, M. A., Lee, I., & Weigel, D. (1998). A genetic framework for floral patterning. Nature, 395(6702), 561–566.
Triezenberg, S. J., Kingsbury, R. C., & McKnight, S. L. (1988). Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes & Development, 2(6), 718–29.
Weigel, D., & Meyerowitz, E. M. (1993). Activation of floral homeotic genes in Arabidopsis. Science (New York, N.Y.), 261(5129), 1723–6.