1. Carlene Ho & Ibraheem Dakilah
Complexes of MADS-box proteins are
sufficient to convert leaves into floral
Organs
Takashi Honma & Koji Goto
Carlene Ho & Ibraheem Dakilah

2. ABC Model
ABC Model specifies development of floral organ in four whorls in Arabidopsis
ABC model → specify development of four floral organs in concentric floral whorls
Sepals, petals, stamens, carpels
Different combinations of mads box proteins specify floral organs

3. MADS-box genes M - DNA binding domain I - Intervening region
K- Involved in protein-protein interactions
C - C-terminal domain
M → DNA Binding domain
I → also involved in prot-prot interactions but weakly
K → involved in interactions between proteins
C-term region → quite variable and, in some MADS domain proteins, is involved in transcriptional activation or multimeric complex formation
Mads-box proteins form dimers and bind to DNA regions called CaRG-boxes

4. ABC Model
Ectopic expression of combination of ABC genes do not turn vegetative leaves into floral organs
→ something else must modulate floral organ identity
In transgenic plants with PI fused with VP16, a transcr, activation domain, AP3:GUS is expressed all throughout the plant
→ Thus there might be a ternary factor that is flower specific, allowing AP3 to be expressed only in the flower

5. Hypothesis
Genes in the ABC model interact with a ternary factor which modulates DNA binding specificity and transcriptional activation
Another protein (MADS?)
Supplies a transcriptional activation domain to PI-AP3 complex
Expression is flower-specific
PI-AP3 are known to form a heterodimer; this autoregulates AP3
In transgenic plants with AP3 promotor fused with VP16, a transcr, activation domain, AP3 is expressed all throughout the plant
Thus there might be a ternary factor that is flower specific, allowing AP3 to be expressed only in the flower

6. Yeast two hybrid system
AD = activating domain (Gal4), BD = Binding domain (LexA)
Each one is fused to a different protein
Interaction = expression of reporter gene (lacZ) occurs (encodes β-galactosidase)
Yeast two hybrid system → used to determine if two proteins interact; bait encodes protein of interest + DNA binding domain, prey encodes the other protein of interest + activating domain. If these two proteins of interest interact, it will bring the binding domain and the activation domain together and you will get transcription of the reporter gene
Bait-LexA (bait = PI)
prey - Gal4 → if bait and prey interact, beta-gal will be expressed and cells will turn blue when expressed on X-gal containing plates

7. X-gal
Beta-galactosidase cleaves X-gal at the red line shown

8. X-Gal 5-bromo-4-chloro-3-hydroxyindole
Dimer of (1), which is an intense blue compound
Allows us to see if beta-galactosidase is active, and therefore if the proteins interact
X-gal is cleaved into galactose and then 1, which will dimerize into 2 and this is a blue product that is intensely blue
Gives us indication of which colonies are producing beta galactosidase, and this tells us if the two proteins we used as bait and prey are interacting.
Galactose is one of the biproducts

9. Yeast two hybrid system
No interaction = no expression
No blue colour!
No bidning, no blue colour, also i thnk the yeast die but i have to verify that
Lacz encodes beta galactosidase

10. Figure 1 a: cDNA selection
Clones selected: PI, SEP3 and AP1 and ATA20
Bound only when both PI and AP3 were present
Why are we only looking at proteins that interact only when both PI and AP3 are present?
Basically they took a flower cDNA library and screened for prey that resulted in blue colonies and did this with PI+AP3, PI alone and AP3 alone;
Only looked at ones where they bound to both
None of these bind to only PI or AP3
Why are we only looking at proteins that can interact only when both PI and AP3 are present?

11. Figure 1 a: cDNA selection
Clones selected: PI, SEP3 and AP1
We are looking for ternary factors that interact with the PI-AP3 complex!
ATA20 not studied because it is secreted specifically in the anthers
Basically they took a flower cDNA library and screened for prey that resulted in blue colonies and did this with PI+AP3, PI alone and AP3 alone;
Only looked at ones where they bound to both
None of these bind to only PI or AP3
Why are we only looking at proteins that can interact only when both PI and AP3 are present?

12. Figure 1 a: cDNA selection
B. AG alone doesn’t interact with PI+AP3 but does with SEP3-MIK
Why use SEP-MIK?
WHY DO WE USE SEP-MIK?
Why does it still work?
C is important for transcriptional activity but not for binding; can still form a complex with AG “

13. Transactivation Assay
Deletion of C domain in SEP3 and AG results in severe decrease in transcriptional activity
The K2C domain retains activity
The K2C domain is sufficient for interaction with PI-AP3
AP1 and SEP3 have transcriptional activator domains, localized in the C domains and they can supply them to a complex of PI and AP3 or AG
Took these, fused cDNAs to binding domains and transformed them into yeast, and used AP3-K2C and SEP3-K2C
Beta-galactosidase was used with (ONPG), which turns yellow when cleaved (works in a similar way to X-gal)
ONPG

14. Figure 1 e, f: Interacting factor confirmed by co-immunoprecipitation:
Radiolabelled factor + HA tagged proteins
Interacting proteins precipitated by anti-HA antibodies
To test interaction of the proteins, they took radiolabelled SEP3 mixed with Haemagglutinin tagged proteins, and then precipitated with anti-HA antibody.
Bands separated for analysis by SDS-page, similar to electrophoresis but separates proteins based on size and charge.
Only the HA-protein complexes that interact are precipitated and shown by radioimaging. This is seen in the gel by a dark band.

15. Figure 1 e: e: interactions confirmed by co- immunoprecipitation:
PI-AP3 + AP1
Showing that PI-AP3 complex interacts with AP1.
AP1 forms a dimer and then this dimer binds to the CArG-box repeats (5'-CC (A/T)6 GG-3' ) within the protein

16. Figure 1 f: f:interactions confirmed by co- immunoprecipitation:
PI-AP3 + SEP3
AP1 + SEP3
AG + SEP3
Showing that PI-AP3 complex interacts with SEP3. AP1 and AG both respectively interact with SEP3.
SEP3 forms a dimer and then this dimer binds to the CArG-box repeats (5'-CC (A/T)6 GG-3' ) on the respective protein/ protein complex

17. Tetramers in whorls PI-AP3-AP1-SEP3 → second whorl
PI-AP3-SEP3-AG → third whorl
Showing that PI-AP3 complex interacts with SEP3. AP1 and AG both respectively interact with SEP3.
Suggested PI-AP3-AP1-SEP3 formation required for petal development
Suggested PI-AP3-SEP3-AG formation required for the formation of stamen
SEP3 forms a dimer and then this dimer binds to the CArG-box repeats (5'-CC (A/T)6 GG-3' ) on the respective protein/ protein complex. Formation of the tetramer increases DNA binding affinity to the CAr-G box.
Sources: Haugh G, Kunst L, Song L.

18. Transactivation domains
AP1/SEP3 add transcriptional-activator domains
AP1/ SEP3 transcriptionally active in yeast and onion epidermal cells
C-domain sufficient for transcriptional activation
C-domain most divergent among MADS-box proteins, what are the ramifications of this divergence?
AP1 transcriptional activity was moderate compared to that of SEP3 in both yeast and onion epidermal cells.
AP1 and SEP3 suggested to add transcriptional-activator domains not present on PI-AP3 or AG.
Deleting the C-domain (MIK) resulted in a significant decrease in the transcriptional activity whilst the deletion of the MADS domain K2-C resulted in a retention off transcriptional activity.
Some MADS proteins will have transcriptional activity whilst some will not.

19. Transactivation domains
Assay in onion epidermal show necessity of activation domain
PI-VP16 +AP3 → LUCIFERASE
SEP3→ LUCIFERASE
AP1→ LUCIFERASE
Within the Luciferase reporter gene assay containing CArG::LUC, luciferase was only expressed when the activation domain VP16 (a very strong transactivator from the herpes simplex virus, which causes gene transcription) was fused to
AP1 in the presence of AP3.
Relatively high transactivation observed in constitutively expressed AP1; SEP3 and somewhat in SEP1

20. Domains in MADS-box proteins
SEP3 and AP1 transactivation domains localized within C-domains
C-domains supplied PI-AP3/AG complex
Conserved in type-II
Yeast Transactivation assays demonstrated that the sufficient domain is the C-domain as the expression was almost null when this particular domain was deleted.
C-domain that were shown to be the primary transactivation domain were thought to be supplied to complexes of either PI-AP3 or AG.
These C-domains are of type II MADS box conserved domains. Type I MADS box proteins only have the SRF-MADS box like domain conserved in their class whilst type II contain an intervening (I), keratin-like (K) and C-domains (C)
Sources:

21. Modulating binding affinity
Tetramers suggested to increase DNA-binding affinity
Yeast MADS protein MCM1, Snapdragon floral MADS proteins
In-vivo assays to form transgenic lines
As mentioned previously the formation of the tetramers was suggested to increase the DNA binding of the transcription factors (classes A, B and C)
Previous studies showed that Yeast MADS protein MCM1, Antirrhinum floral MADS proteins modulate DNA binding affinity. In order to investigate this modulating quality of the MADS proteins in-vivo assays were performed. Initial experiment consisted of crossing AP3::GUS plants with constitutively expressed PI, AP3, AP1, SEP3 and combinations of these transgenes.

22. AP3::GUS assay AP3::GUS gene crossed into transgenic lines:
Constitutive expression of PI/AP3/AP1/SEP3
Combos of constitutively expressed transcription factors
GUS expression indicates transcriptional activation
AP3 is usually expressed in the 2nd and 3rd whorls. This assay was used to identify where the promoter of AP3 was activated by observing where the GUS is expressed, which would be seen as a light blue when reacted with X-gluc. Lines of arabidopsis with constitutively expressed A, B and C class genes and SEP as well as combinations of these genes were used in order to identify the interaction with AP3

23. AP3::GUS expressed in various tissues: 35S::PI;35S::AP3;35S::AP1 (c)
35S::PI;35S::AP3;35S::SEP3 (e)
Within the triply transgenic line constitutively expressing PI AP3 and AP1 GUS is observed in floral tissue as well as roots, cotyledons and cauline leaves. Whilst in the triply transgenic line constitutively expressing PI, AP3 and SEP3, GUS expression was found throughout the plant.

24. AP3::GUS expressed in floral organs:
35S::PI;35S::AP3 (a)
35S::AP1 (b)

25. AP3::GUS expressed in floral organs: 35S::SEP3 (d) 35S::PI 35S::AP3
GUS expression was localized to the floral organs, it should be noted that this is a severe phenotype of this transgene

26. Homodimers supply activation
AP SEP3
Homodimers provide activation site
Although monomers of either AP1 or SEP3 are sufficient to provide the activation domain to PI-AP3
Lol these look like apples

27. Tetramers - binding affinity
Formation of tetramers leads to an increase in DNA binding affinity

28. Phenotypic analysis
35S::SEP3 WT
Differences from WT?

29. Phenotypic analysis 35S::SEP3 Dwarf phenotype Curled leaves
Early flowering
Terminal flowers

30. Phenotypic analysis (b) 35S::PI;35S::AP3 Differences from WT?
(c) 35S::PI;35S::AP3;35S::SEP3

31. Phenotypic analysis (b) 35S::PI;35S::AP3 Curled leaves
Outer 2 whorls petals
Inner 2 whorls stamen
(c) 35S::PI;35S::AP3;35S::SEP3
1st true leaves → petaloid
Observations from PI/AP3 transgenic lines failed to convert vegetative leaves into floral organs however triply transgenic line of PI/AP3/SEP3 observed conversion from a vegetative identity to more of a floral one. PI-AP3-SEP3 suggested to be sufficient for this conversion, overexpression of SEP3 causes the SEP3 homodimer to replace the function of AP1-SEP3.

32. Phenotypic analysis
35S::PI;35S::AP3;35S::AP1
Differences from WT?

33. Phenotypic analysis 35S::PI;35S::AP3;35S::AP1
Cauline leaves → petaloid
AP1 ←can replace function→ SEP3
What does this suggest?
The conversion shown in this line suggests that the AP1 homodimer can replace the function of AP1-SEP3 resulting in a conversion to petaloid organs.
The functional redundancy observed from these results are indicative of the AP1-SEP superclade from which both proteins are members.

34. leaf phenotype (e) 35S::SEP3 (f) WT petal
(g) 35S::PI;35S::AP3;35S::SEP3
(h) 35S::PI;35S::AP3;35S::AP1
(e) irregular jigsaw puzzle shape with stomata interspersed between epidermal cells which is what would be seen in rosette and cauline leaves
(f) WT petal has conical ridged cells with no stomata
(g) petaloid rosette leaf
(h) petaloid cauline leaf
(g-h) show a change from the expected phenotype found in e to more like that of the petals in F.

35. Phenotypic analysis 35S::PI;35S::AP3;35S::AG;35S::SEP3
Differences from WT?

36. Phenotypic analysis 35S::PI;35S::AP3;35S::AG;35S::SEP3
Cauline leaves--> staminoid organs
Floral organs→ stamen/staminoid organs

37. Phenotypic analysis Cauline leaf of quadruply transgenic plant
(m) filament, (n) cauline leaf of transgenic plants (o) basal region of cauline leaf
(k) WT anther (l) cauline leaf of quadruply transgenic plants
(j) staminoid cauline leaf of the quadruply transgenic line
(k,m,o)SEM of WT anther, filament and basal region of cauline leaf
(n) transformed cauline leaves exhibit morphology similar to that of WT anthers and filaments with a change in the shaping of the epidermal cells and overall organ morphology

38. Phenotypic analysis Flowers of 35S::PI;35S::AP3;35S::AG
Sablowski R 2007
(p) staminoid flowers of the quadruply transgenic lines
(q) 35S::PI;35S::AP3;35S::AG petaloid first whorl organs are partially converted to stamenoid organs. SEP3 is not distinctly expressed within the first whorl which is why a clear conversion into petal organs is not observed.
Suggesting that PI-AP3-SEP3-AG is sufficient for conversion of cauline leaves into staminoid organs. Floral identity is independent of floral meristem.
Flowers of 35S::PI;35S::AP3;35S::AG
Flowers of quadruply transgenic plant
WT flower

39. Conclusions SEP3 interacts with B and C class gene products
PI-AP3-SEP3/PI-AP3-AP1 sufficient for leaves → petaloid
PI-AP3-SEP3-AG complex sufficient for cauline → stamenoid
Floral identity independent of floral meristem

40. Conclusions PI-AP3 gene target expressed in PI-AP3 + SEP3/AP1
PI, AP3 and AG absent of transcriptional activators
SEP3, AP1 act as transcriptional activators
Sep mutant similar phenotype to bc double mutant
SEP3 mutants had a subtle phenotype but in combo with sep1 and sep2 mutants they show a similar phenotype to mutants of the B and C class of genes. This result is observed due to the functional redundancy of the SEP proteins, similarly able to provide transactivation domains just like SEP3 due to SEP1 and SEP2 showing transcriptional activity in onion epidermal cells. SEP1/2 are expressed in whorls 1-4, alongside the functional redundancy shared with SEP3 suggests that SEP confers floral functionality on B and C genes by forming complexes of their gene products.

41. Question
What changes to the ABC model would you propose after this experiment?
So since SEP3 interacts with B and C class genes, maybe we could throw them in a separate class since it operates in all 4 whorls
Provides a transcriptional activation domain to all 4 whorls
SEP3 binds to the gene products above and adds an activation domain to allow the target genes to be transcribed
The formation of the tetramer increases DNA binding affinity to the target genes leading to the development of the distinct organs

42. Revised Model SEP added as E class genes in ABC model
Provide transactivational domains to ternary and quaternary complexes
The revised model includes the SEP function in the three inner whorls represented redundantly by SEP1/2/3. The floral quartet model implies that the MADS-box proteins act as tetrameric complexes in order to specifically bind and, at the same time, to transcriptionally activate the
target genes.
Sources:

43. References
Gramzow, L., & Theissen, G. (2010). A hitchhikers guide to the MADS world of plants. Genome Biology, 11(6), doi: /gb
Ma, H. (2005). Molecular Genetic Analyses Of Microsporogenesis And Microgametogenesis In Flowering Plants. Annual Review of Plant Biology, 56(1), doi: /annurev.arplant
Honma, T., & Goto, K. (2001). Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature, 409(6819), doi: /
Robles, P., & Pelaz, S. (2005). Flower and fruit development in Arabidopsis thaliana. The International Journal of Developmental Biology, 49(5-6), doi: /ijdb pr
Sablowski, R. (2007). Flowering and determinacy in Arabidopsis. Journal of Experimental Botany,58(5), doi: /jxb/erm002