1. Complexes of MADS-box proteins are sufficient to convert leaves into floral organs
Authors: Takashi Honma & Koji Goto
Presented by: Claire Yang and Athena Wong

2. MADS Box Type What is MADS Box?
A conserved sequence motif (around 56 aa) found in the DNA binding domains of MADS-box family of transcription factors
In plants, there are two classes of MADS-domain proteins based on sequence similarity outside of MADS domain
Type I and Type II
In this paper we will focus on Type II MADS box type, which have this MIKC structure

3. Type II MADS box Conserved Domain Structures (MIKC)
“Carboxyl Terminal Domain” = acts as a transcriptional activation domain in SOME members
AP1, AP3, PI and AG are members of type II!!
Plant MADS proteins consist of 4 domains:
“MADS (M)” Domain = a highly conserved DNA- binding domain, a major determinant in DNA binding specificity
“Intervening Domain” = (involved in dimerization specificity)
“Keratin-like Domain” = involved in protein to protein interactions (dimerization)
“Carboxyl Terminal Domain” = acts as a transcriptional activation domain in SOME members of the type II MADS box protein

4. 3D Protein Structure CArG Box = CC[A/T]6GG)
Members of the MADs box family have this kind of 3D protein structure
Interactions of amphipathic alpha helices allow for two proteins w/ similar structures to interact
In vitro, MADS box type II TFs are found to form homo or heterodimers (in which their K domains align)
And the MADS domain complexes recognizes
palindromic sequence (CC[A/T]6GG) in DNA “CArG box”
CArG Box = CC[A/T]6GG)

5. ABC Model Hypothesis Homo- or Heterodimers
ABC model suggests that MADS proteins interact in different combinations to activate and target different genes in each whorl
What is the problem with this hypothesis?
Do we know that these are the only TFs to play a role in floral organ identity?
We do not know how homologous TFs are regulated, and if they play whorl-specific functions

6. Ectopic Expression of ABC genes in Vegetative Tissue
Furthermore, expression of ABC genes ectopically, does not result in the conversion of vegetative leaves -> floral organs
See normal phenotype
Why did we see leaves turn into flowers if we have expression of all the ABC genes?
Suggest that other unknown factors are required for organ identity
35S::AP3, 35S::PI, 35S: AP1, 35S::AP2, 35S::AG

7. Reporter Construct or AP3 promoter GUS/LUC DNA GUS/LUC mRNA
Β-glucuronidase
Luciferase
Reporter constructs are used to measure the expression level of a particular gene or determine the subcellular localization of specific protein
With recombinant DNA techniques, a reporter gene can be fused to the regulatory region of the gene of interest, in this case gene AP3, generate reporter construct
A reporter gene can be GUS or LUC …

8. AP3 and PI Expression in Transgenic Plants (35S::AP3, 35S::PI)
One study narrows down on the what these “unknown factors” are by focusing on AP3 and PI expression in 35S::AP3, 35S::PI transgenic plants
So where do we expect to see AP3 and PI expression in 35S::AP3, 35S::PI transgenic plants?
There should be AP3 and PI expression everywhere (from the leaves to the flowers) due to the constitutive 35S promoter

9. AP3::GUS in Transgenic Plants (35S::AP3, 35S:PI)
AP3 Promoter
GUS reporter -> blue color
(AP3 Promoter)
AP3::GUS fusion gene (which is a AP3 promoter fused to a GUS gene)
When this was introduced into the plants
We can only see GUS expression in flowers
How is the AP3::GUS fusion gene different from a regular AP3 gene?
Only differs in GUS gene
Does anyone have a guess to as why we see this expression pattern?
AP3::GUS Fusion Gene

10. AP3::GUS in Transgenic Plants (35S::AP3, 35S:PI)
AP3 Promoter
GUS reporter
-> blue color
AP3 Promoter
Thus, this suggests that there is some kind of unknown ternary factor that interacts w/ the AP3-PI complex that localizes the expression of GUS in only flowers
A simple way this could happen is if this ternary factor has flower specific expression
Why didn’t they do a PI::GUS Fusion Gene?
AP3::GUS Fusion Gene

11. AP3::GUS in Transgenic Plants (35S::AP3, 35S:PI-VP16)
Everywhere (vegetative structures & floral organs)
VP16
AP3 Promoter
VP16 = Transcriptional activation domain (Herpes simplex virus)
When a VP16 (very active Transcriptional activation domain) is fused with PI
AP3 interact w/ PI-VP16
We see GUS expressed everywhere
Suggests that the AP3-PI complex is found everywhere in plant (from vegetative structures to floral organs) but doesn’t allow transcription of GUS usually everywhere, only in the floral organs
This suggests that not only does the “unknown” ternary factor have flower specific expression
But also supplies the AP3-PI complex with a transcriptional activation domain
AP3::GUS Fusion Gene

12. How did they find & identify the ternary factor?
Hypothesis:
There is a floral specific ternary factor needed for the activation of PI-AP3 complex. Q:
How did they find & identify the ternary factor?
Thus, there must be some protein that interacts w/ PI and/or AP3
DNA flower library screening via Yeast-Two Hybrid System
Confirmation w/ B-gal Assay

13. A molecular genetic approach to analyze protein-protein interactions
Screening a Flower Complementary DNA library using the Yeast Two-Hybrid System
A molecular genetic approach to analyze protein-protein interactions
So with all that info, our paper tried to find this “ternary factor” by screening a flower complementary DNA library using the Yeast-two hybrid system

14. Yeast Two-Hybrid System
Prey A
Protein of interest = “Bait” & is fused to a DNA binding domain
Proteins we want to test, to see if they bind to bait = “Prey” & are fused to a transcriptional activation domain

15. Yeast Two-Hybrid System
If a prey protein does not bind to the bait = will not activate transcription of the reporter gene (HIS)
Any prey protein that does bind to the bait = will activate transcription of the reporter gene
prey

16. Yeast Two-Hybrid System
PREY PLASMID
Prey
(Lex A)
(AP3 or PI)
cDNA library on pACT2
First step
Construct a bait and prey plastids
Each type of plastid contains a selectable markers (amino acid TRP, and LEU)

17. Yeast Two-Hybrid System
(Positive control)
PREY PLASMID LIBRARY
PI as prey
AP3 bait
Prey 1
Prey 2
PI bait
Prey 3
Prey n
PI and AP3 used as bait (find if there is anything that interacts w/ either of them or both of them)

18. Yeast Two-Hybrid System
Transfected cells are plated on media lacking leucine, tryptophan and histidine
Selection in media w/o leucine or tryptophan
Yeast w/ mutation in genes required for leucine, tryptophan and histidine biosynthesis)

19. Yeast Two-Hybrid System
Can’t grow on medium lacking His, Leu, and Trp
NO LacZ
HIS and LacZ Genes
Grow on medium lacking His, Leu, and Trp
LacZ (B-galactosidase bacterial gene)
Interacts w/ substrate X-gal -> blue
HIS and LacZ Genes

20. Yeast Two-Hybrid System
340 positive colonies from 5.9 x 10^7 clones
Sequenced 170 of these clones, clones that appeared more than twice, then identified..
19 clones of PI
18 clones of SEP3
15 clones of AP1
4 clones of ATA20
MADS box transcription factor (whorls #2-4)
Growth on medium lacking His, Leu, and Trp
HIS and LacZ Genes
AG?
Can anyone tell me what kind of protein ATA20 is?
Anther specific secreted protein
Putative = Don’t really know the function of it
BUT know that it is similar to a cell wall protein in Petunias
Assumed not involved in floral organ specificity so it was OMITTED
Also notice that AG was not fished out, why?
Suggest that AG doesn’t directly interact w/ AP3-PI complex

21. Colony Lift B-Gal Assay
SEP3, AP1, & PI etc.
HIS and LacZ Genes
LacZ (encodes B-galactosidase protein)
Used as a reporter
Interacts w/ substrate X-gal -> blue precipitate
To further confirm interactions in yeast
Re-transformed 3 independent colonies onto the plates auxotrophic for Leu, Trp, Histidine & let them grow for a few days
Again these colonies that grew, tell us that there is interaction btwn the bait and prey protein
Which allows for the transcription of these genes
One of the genes is the bacterial LacZ gene, encodes a B-galactosidase protein that interacts w/ substrate X-gal to produce a blue precipitate
This is very similar to GUC gene in Plants as I mentioned before
Performed a colony lift B-Gal Assay
Put NC filter on top of plate of yeast colonies (1-2 mins)
Transfer NC filter to petri dish containing a-factor, colony side up, incubate 30mins
Then freeze w/ liquid nitrogen for 10 sec to permeabilize the membrane and allow entry of X-gal (the substrate for b-galactosidase)
Allow filter to warm up and then incubate w/ Z buffer and X gal overnight
Blue color starts to appear after few hours

22. Colony Lift B-Gal Assay
SEP3, AP1, & PI
(Prey interacts w/ bait)
Colony side up in petri dish w/ a-factor
(Prey doesn’t interact w/ bait)
So after the colonies were plated and grown for a few days
We put NC filter on top of plate of yeast colonies (1-2 mins)
Next we take the NC filter and but it in a petri dish containing a-factor, colony side up
Cover w/ parafilm and incubate for ~2.5h
Then freeze w/ liquid nitrogen for 10 sec to permeabilize the membrane and allow entry of X-gal (the substrate for b-galactosidase)
Allow filter to warm up and then incubate w/ Z buffer and X gal overnight
Blue color starts to appear after few hours
A Dohlman Lab Protocol

23. Results of Yeast-Two Hybrid Sys & B-Gal Assay
(Positive control)
Results:
See both SEP3 and AP1 primarily interacting w/ the PI-AP3 complex, as shown by yeast two hybrid and further confirmed by B-Gal Assay
SEP3 and AP1 do not interact with PI or AP3 alone
Confirm

24. Results of Yeast-Two Hybrid Sys & B-Gal Assay (AP3-PI complex and AG)
(Negative control = make sure SEP3-MIK doesn’t interact w/ AP3-PI complex on its own)
“Carboxyl Terminal Domain” = acts as a transcriptional activation domain in SOME members
Focusing on and further examining the interaction btwn AP3-PI complex and AG
Found that indeed AG does not interact with the AP-PI complex directly as shown in both tests, confirms our predictions from before
When an AG prey and SEP-MIK prey are used
We see that AG interacts with the AP3-PI complex
Suggests that SEP3 mediates the interaction btwn AP3-PI and AG
Also examined interaction btwn AP1 and SEP3

25. Results of Yeast-Two Hybrid Sys & B-Gal Assay (AP3-PI complex and AP1)
(Prey)
SEP3 and AP1 can survive as bait w/o interacting w/ prey
SEP3-MIK and AP1-MIK cannot
“Carboxyl Terminal Domain” = acts as a transcriptional activation domain in SOME members
Also further examined interactions btwn AP3-PI complex and AP1
Found that AP1 and SEP3 interact w/ each other
Found that full length SEP3 and AP1 can survive on bait vector w/o any prey
But when using C-domain delete AP1 and SEP3 = not able to survive alone
Observation suggests that AP1 AND SEP3 already have transcriptional activation domains in the “C” domain, that allow the bait itself activate the transcription of the amino acids and allow the yeast colonies to survive

26. How did they confirm that?
Hypothesis:
There are interactions between PI-AP3-AP1, PI-AP3-SEP3, AP1-SEP3 and AG-SEP3. Q:
How did they confirm that?

27. C0-immunoprecipitation
centrifugation
Proteins
Protein complex
Antibody
Beads coupled with antibody
As the yeast two hybrid system, we know that the PI-AP3 complex primarily interacts with AP1 and SEP3, and SEP3 mediates the interaction between PI-AP3 complex and AG.
How to further confirm the results,
Co-immunoprecipitation (Figure 1e,f) experiments to isolated protein of interest based on p-p interaction
Separated by SDS-PAGE
Identified by western blot
Q: Why red also been precipitated?
Precipitation 1 2 3 4

28. Co-immunoprecipitation of MADS protein
To confirm:
PI-AP3-AP1, PI-AP3-SEP3, AP1-SEP3 and AG-SEP3
AP1 or SEP3 were radiolabeled for further identification process
Detected by radio-imaging analyser
Background
haemagglutinin（HA）- tagged and precipated with anti-HA antibody
HA -- glycoprotein that can bind to antibody

29. C0-immunoprecipitation
Haemagglutinin HA
Radiolabeled protein
centrifugation
Proteins
HA tagged Pro
Protein complex
HA Antibody
Beads coupled with antibody
As the yeast two hybrid system, we know that the PI-AP3 complex primarily interacts with AP1 and SEP3, and SEP3 mediates the interaction between PI-AP3 complex and AG.
How to further confirm the results,
Co-immunoprecipitation (Figure 1e,f) experiments to isolated protein of interest based on p-p interaction
AP1 or SEP3 were radiolabeled for further identification process
haemagglutinin（HA）- tagged and precipated with anti-HA antibody
HA -- glycoprotein that can bind to antibody
Separated by SDS-PAGE 1 2 3 4

30. Co-immunoprecipitation of MADS protein
The results confirm interactions of PI-AP3-AP1, PI-AP3-SEP3, AP1-SEP3 and AG-SEP3
The most likely tetramer in the second whorl:
The most likely tetramer in the third whorl:
PI-AP3-AP1-SEP3
PI-AP3-SEP3-AG
Detected by radio-imaging analyser
Background
How to demonstrate Hypothesis: AP1 and SEP3 have transcription activation domains and provide to PI-AP3 and AG which do not have themAP1 and SEP3 have transcription activation domains and provide to PI-AP3 and AG which do not have them
MADS box proteins make a dimer to bind to the CarG box
Tetramer enhance binding affinity

31. Hypothesis:
AP1 and SEP3 have transcription activation domains and provide to PI-AP3 and AG which do not have them. Q:
How did they test that?

32. Transcriptional Assay of MADS protein
In yeast
MADS protein cDNA
UAS
lacZ
co-transform
GAL4 DNA-binding domain
pAS2-1
β -gal
Yeast strain YRG-2
How to prove that AP1and SEP3 have transcriptional activation domain?
TEST: measure the transcriptional activity of MADS box proteins in yeast and plant cells (table 1) using transactivation assay
For yeast: MADS protein cDNA + GAL4 DNA-binding domain; co-transformed; (truncated MADS pro; grow at 22; beta-gel at 30
GAL4-VP16 is an unusually potent transcriptional activator
Recent work has defined a class of transcriptional activators, members of which activate transcription in yeast, plant, insect and mammalian cells. These proteins contain two parts: one directs DNA binding and the other, called the activating region, presumably interacts with some component of the transcriptional machinery. Activating regions are typically acidic and require some poorly-understood aspect of structure, probably at least in part an alpha-helix. Here we describe a new member of this class, formed by fusing a DNA-binding fragment of the yeast activator GAL4 to a highly acidic portion of the herpes simplex virus protein VP16 (ref. 11; also called Vmw65). VP16 activates transcription of immediate early viral genes by using its amino-terminal sequences to attach to one or more host-encoded proteins that recognise DNA sequences in their promoters. We show that the hybrid protein (GAL4-VP16) activates transcription unusually efficiently in mammalian cells when bound close to, or at large distances from the gene. We suggest that the activating region of VP16 may be near-maximally potent and that it is not coincidental that such a strong activator is encoded by a virus.

33. Transcriptional Assay of MADS protein
In Onion epidermal cells
dual-luciferase reporter system
35S
MADS
CarG box
35S mini
LUC
effector
reporter
35S
RLUC
For onion epidermal cell: 35S promoter-driven cDNA with MADS (effector) + CArG::LUC (reporter) cotransfered into onion epidermal cell using Bio-Rad delivery system; internal control; dual-luciferase reporter system (a primer report of interest can be normalized against a control to increase the precision )
These enzymes differ in their substrate and cofactor requirements.
Firefly luciferase uses luciferin in the presence of oxygen, ATP and magnesium to produce light,
while Renilla luciferase requires only coelenterazine and oxygen.
Firefly luciferase produces a greenish yellow light in the 550–570nm range. Renilla luciferase produces a blue light of 480nm. These enzymes can be used in dual-reporter assays due to their differences in substrate requirements and light output.
control

34. MIK
K2C
Control (mean value): yeast(only GAL4-binding domain) onion(vector only contain 35S promoter and nos terminator)
Result:
Compare with mean - No (PI, AP3, AG)/ moderate/ strong (AP1, SEP3) activity
In both yeast & onion cell
In yeast, C domian - transcriptional activity
In plant (onion) PI+PI(PI-VP16)+AP3 can activite reporter gene
Interpretation: AP1 & SEP3 have transcriptional-activator domains and within C domian
C domain is the most divergent region among the plant MADS proteins

35. Up To Now... Co-immunoprecipitation further confirmed that
SEP3 and AP1 interact w/ AP3-PI complex
SEP3 and AP1 interact w/ each other
SEP3 interacts with AG
Transactivation Assay
SEP3 and AP1 have transcriptional activator domains w/i C region
AP3, PI and AG do not
Both SEP3 and AP1 have
Flower-specific expression
Transcriptional activator domain
Interact w/ AP3-PI
PI-AP3 form a heterodimer to bind to the CarG box. I.e. AP3 promoter
Yeast Two Hybrid System and B- Gal Assay
Both SEP3 and AP1 interact w/ AP3-PI
SEP3 and AP1 interact directly
SEP3 mediates AG and AP3-PI interaction
Full length SEP3 and AP1 bait plasmids can survive w/o interacting w/ prey
Summary of what we already know
Two in vivo assays to examine the quaternary complexes function in vivo to control floral organ type:
(Fig2)Cross AP3::GUS gene into 35S::PI/AP3/AP1/SEP3 plants or other combinations GUS reporter system
Result: AP3::GUS expression in various tissues of both triply transgenic plants; other transgenic plants only expressed in the floral organs
Interpretation: the ternary complex PI-AP3-AP1 and PI-AP3-SEP3 are sufficient to activate the AP3 promoter
Monomer -> dimer (increase DNA-binding affinity )
(Fig3)Examined the phenotype of the triply transgenic plant and the quadruple transgenic plant
Result: triply & quadruply transgenic plants show phenotype---vegetative leaves were transformed into floral organs
Cryo-scanning electron micrograph (cryo-SEM)

36. Q:
After the in vitro experiments, what can we do to further understand the function of MADS protein complexes?
In vivo tests...

37. AP3::GUS Expression in the Transgenic Plants
Why in vivo?
Examine the modulating ability of the complexes of Arabidopsis MADS proteins
AP3::GUS plants have a 600-base-pair region of the AP3 promoter
Cross AP3::GUS gene into 35S promoter transgenic plants
GUS reporter gene
Enzyme β-glucuronidase
There is no detectable GUS activity in plants

38. AP3::GUS Expression in the Transgenic Plants
flower
Floral bud
35S::PI; 35S::AP3
35S::AP1
35S::SEP3
In a, b, d transgenic plants GUS activity was only observed in flowers and floral buds
D is a more severe phenotype-dwarf: curved leaves, early flowering and terminal flowers
Sufficient

39. AP3::GUS Expression in the Transgenic Plants
C represents 35S::PI;35S::AP3;35S::AP1 triply transgenic plants
E represents 35S::PI;35S::AP3;35S::SEP3 triply transgenic plants
35S::SEP3
Q8. what explanation do the authors give for the ability of the ternary complex to activate AP3 expression and specify floral organ type?
In c, GUS activity was observed not only in floral organs, but also in roots, cotyledons, rosette, and cauline leaves
In e, GUS activity within whole plant
C & E suggest that ternary complex, PI-AP-AP3 and PI-AP3-SEP3, are sufficient to activate the AP3 promoter
AP1 and SEP3 provide activation domain to PI-AP3 complex
(As both AP1 and SEP3 form homodimers (data not shown), dimers probably provide the activation domain and then tetramers increase the DNA- binding affinity, although monomers of AP1 and SEP3 are sufficient to supply the activation domain to PI - AP3)

40. Phenotype of Triply Transgenic Plants
35S::SEP3
35S::PI; 35S::AP3
35S::PI;35S::AP3;35S::AP1
35S::PI;35S::AP3;35S::SEP3
Genotype and phenotype
A. dwarf: curved leaves, early flowering
B. curved leaves , flower: petal petal stamen stamen
A & B fail to (vegetative cells → flower)
Ap1 mutant → no petal
C. first true leave → petaloid (PI-AP3-SEP3 sufficient)
Ap1 for petal, when SEP3 over express, replace AP1-SEP3
D. CL → petaloid
S: stamens
1,2,3,4 true leaves with the order of development
TF: terminal flowers
CL: cauline leaves

41. Phenotype of Triply Transgenic Plants
35S::SEP3 rosette leaves
Compare g with e, f, what do we see?
Wild type petal
* Cryo-scanning electron micrograph (Cryo-SEM)
Cryo-scanning electron micrograph (Cryo-SEM) of the adaxial surface of a 35S::SEP3 rosette and cauline leaves
E similar to WT, the epidermis of vegetative leaves consists of irregular “jigsaw-puzzle-shaped” cells with interspersed stomata
F is WT petal, petal epidermis consists of conical rigged cells and lacks stomata

42. Phenotype of Triply Transgenic Plants
35S::SEP3
35S::PI;35S::AP3;35S::SEP3
35S::PI;35S::AP3;35S::AP1
WT petal
E similar to WT
F is WT petal
D&H : Leave → petal like
AP1 homodimer can function as AP1-SEP3.
Functional redundancy of AP1 & SEP3

43. Phenotype of Quadruply Transgenic Plants
35S::SEP3 (severe);35S::AG no growth, no progeny
So use 35S::SEP3 (intermediate);35S::AG
Quadruply transgenic plant (not only all floral organs but also CL are transformed into stamens or stamiond organs)
CL: cauline leaves
F: lateral flowers
White arrow point to trichomes (CL->stamens or stamoid organ)
L & N (CL-> anther & filament)
35S::PI;35S::AP3;35S::AG; 35S::SEP3

44. Flowers of Triply and Quadruply Transgenic Plants
P & q
p CL & flower → stemoid organs
Asterisks show petaloid 1st whorl organs which often incompletely converted into staminod organs (SEP3 not in 1st whorl)
PI-AP3-AG-SEP3 activity is sufficient for the conversion of leaves into staminoid organs.
35S::PI;35S::AP3;35S::AG; 35S::SEP3
35S::PI;35S::AP3;35S::AG

45. Summary: SEP Genes and SEP Proteins
Interacts with PI - AP3
Mediate between PI - AP3 and AG
PI-AP3-SEP3 is sufficient to transform leaves into petal
PI-AP3-SEP3-AG is sufficient to transform leaves into stamen
Provide activation domain
SEP
Sep 1, 2, 3 triple mutants
Q10. general role of SEP gene in floral development.
What is the triple mutant looks like? Similar to b,c double mutant
Sep genes making Sep proteins that supply transcription activator domain to the class b,c gene products
Provide flower-specific activity

46. Summary: Triply and Quadruply Transgenic Plants -> Stamenoid organs
AP3 PI
AP3 PI
AND
Vegetative leaves
-> Petaloid organs
AP1
SEP3
AP3 PI
Vegetative leaves
-> Stamenoid organs AG
SEP3

47. Summary: ABC Model & SEP
AP3 PI
AP3 PI
AP1
SEP3 AG
SEP3
SEP3 interacts w/ AP1 and AP3-PI complex to specify 2nd whorl (petal)
SEP3 mediates interaction of AG and AP3-PI complex to specify 3rd whorl (stamen)
Flower-specific activity of SEP3 restricts the action of ABC genes to flower
These findings indicate that the formation of ternary and quaternary complexes of ABC proteins may be the molecular basis of ABC model
Instead of dimer complexes as previously thought

48. References
Honma, T. and Goto, K. (2001). Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature, 409(6819), pp
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