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Complexes of MADS-box proteins are sufficient to convert leaves into floral Organs

This study explores the role of MADS-box proteins in the development of floral organs in Arabidopsis. The ABC model specifies the development of floral organs in four whorls, and genetic interactions between the PI-AP3 complex and other proteins are investigated. The study also examines transactivation domains and modulation of DNA binding affinity. Ectopic expression of certain combinations of ABC genes is found to be insufficient to convert vegetative leaves into floral organs, suggesting the involvement of other factors. Phenotypic analyses of transgenic lines further support the role of MADS-box proteins in floral development.

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Complexes of MADS-box proteins are sufficient to convert leaves into floral Organs

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  1. 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

  3. MADS-box genes M - DNA binding domain I - Intervening region K- Involved in protein-protein interactions C - C-terminal domain

  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

  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

  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)

  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

  9. Yeast two hybrid system No interaction = no expression No blue colour!

  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?

  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

  12. Figure 1 a: cDNA selection B. AG alone doesn’t interact with PI+AP3 but does with SEP3-MIK Why use SEP-MIK?

  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 ONPG

  14. Figure 1 e, f: Interacting factor confirmed by co-immunoprecipitation: • Radiolabelled factor + HA tagged proteins • Interacting proteins precipitated by anti-HA antibodies

  15. Figure 1 e: e: interactions confirmed by co-immunoprecipitation: • PI-AP3 + AP1

  16. Figure 1 f: f:interactions confirmed by co-immunoprecipitation: • PI-AP3 + SEP3 • AP1 + SEP3 • AG + SEP3

  17. Tetramers in whorls • PI-AP3-AP1-SEP3 → second whorl • PI-AP3-SEP3-AG → third whorl Sources: https://www.researchgate.net/figure/The-ABC-model-A-The-ABC-functions-are-indicated-as-boxes-with-the-Arabidopsis-genes_fig2_7876365; 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?

  19. Transactivation domains Assay in onion epidermal show necessity of activation domain • PI-VP16 +AP3 → LUCIFERASE • SEP3→ LUCIFERASE • AP1→ LUCIFERASE

  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 Sources: https://www.researchgate.net/figure/Primary-and-domain-structure-of-MADS-domain-proteins-a-Sequence-logo-of-the-MADS_fig1_44851007

  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

  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

  23. AP3::GUS expressed in various tissues: • 35S::PI;35S::AP3;35S::AP1 (c) • 35S::PI;35S::AP3;35S::SEP3 (e)

  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

  26. Homodimers supply activation AP1 SEP3 Homodimers provide activation site

  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 Differences from WT?

  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

  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?

  34. leaf phenotype (e) 35S::SEP3 (f) WT petal (g) 35S::PI;35S::AP3;35S::SEP3 (h) 35S::PI;35S::AP3;35S::AP1

  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

  38. Phenotypic analysis 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

  41. Question What changes to the ABC model would you propose after this experiment?

  42. Revised Model • SEP added as E class genes in ABC model • Provide transactivational domains to ternary and quaternary complexes Sources:https://www.researchgate.net/figure/The-ABC-model-A-The-ABC-functions-are-indicated-as-boxes-with-the-Arabidopsis-genes_fig2_7876365

  43. References Gramzow, L., & Theissen, G. (2010). A hitchhikers guide to the MADS world of plants. Genome Biology,11(6), 214. doi:10.1186/gb-2010-11-6-214 Ma, H. (2005). Molecular Genetic Analyses Of Microsporogenesis And Microgametogenesis In Flowering Plants. Annual Review of Plant Biology,56(1), 393-434. doi:10.1146/annurev.arplant.55.031903.141717 Honma, T., & Goto, K. (2001). Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature,409(6819), 525-529. doi:10.1038/35054083 Robles, P., & Pelaz, S. (2005). Flower and fruit development in Arabidopsis thaliana. The International Journal of Developmental Biology,49(5-6), 633-643. doi:10.1387/ijdb.052020pr Sablowski, R. (2007). Flowering and determinacy in Arabidopsis. Journal of Experimental Botany,58(5), 899-907. doi:10.1093/jxb/erm002

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