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What are roles of flavonoids in the development of plant-cyanobacteria symbiosis ???

What are roles of flavonoids in the development of plant-cyanobacteria symbiosis ???. Gunnera and Nostoc’s Symbiosis. Gunnera manicata. Nostoc Punctiforme. Nitrogen Cycle. N 2. Denitrification. Biological Nitrogen Fixation. NO 3 -. slow. NH 4 +. Two months after inoculation. Control.

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What are roles of flavonoids in the development of plant-cyanobacteria symbiosis ???

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  1. What are roles of flavonoids in the development of plant-cyanobacteria symbiosis ???

  2. Gunnera and Nostoc’s Symbiosis Gunneramanicata Nostoc Punctiforme

  3. Nitrogen Cycle N2 Denitrification Biological Nitrogen Fixation NO3- slow NH4+

  4. Two months after inoculation Control

  5. Meeks & Elhai 2003 Hornworts (Anthrocerophyta) Bryophytes Azolla (Pterophyta) Water ferns Cycads (Cycadophyta) Gymnosperms

  6. CB colonize and penetrate glands produced below each leaf G. Manicata (Anthophyta) So What? Immature gland; 8-day old seeding Mucus covered gland Inoculation of gland with CB What’s so special about Gunnera’s relationship with CB?!! It’s the only intracellular endosymbiotic relationship known b/n angiosperm and cyanobacteria!!! How are Gunnera’s gland cells different from normal plant cells?

  7. BROAD IMPACT • Save the world and revolutionize agriculture!! Create productive symbiosis between cyanobacteria and crop plants!

  8. Bergman et al. 1992

  9. Trends in Plant Science, 10: 236-242

  10. N N - - starvation starvation Myb Myb - - transcription factor transcription factor Flavonoid Flavonoid synthesis synthesis Remobilization of nutrients Remobilization of nutrients from leaves to the stem from leaves to the stem High flavonoids at the gland gland primodia primodia inhibit inhibit auxin efflux auxin efflux Localized auxin accumulation Localized auxin accumulation Activation of cell division and gland development Activation of cell division and gland development 10 mM nitrate 0.1 mM nitrate

  11. Flavonoids

  12. Flavonoids • Inhibit Nostoc growth and hormogonia formation

  13. Test the hypothesis Trends in Plant Science, 10: 236-242

  14. Methods Create flavonoid deficient mutant in Gunnera !!!There’s a Problem!!! Gunnera’s genome has never been fully sequenced. How can I clone an enzyme when I don’t know it’s sequence? ANSWER Conserved regions across various species.

  15. Methods • Make primers using conserved regions residing in a wide range of dicots. > NCBI

  16. Methods • Align all of those various sequences and locate the conserved regions. > BIOBIKE

  17. F2 R1 421-444 1129-1154 Chalcone Synthase Sequence 913-941 352-376 0 F1 1190 R2 Conserved Regions

  18. Degenerate Primer F1-Outside Primer GCN ATH AAR GAR TGG GGB CAR CCH AA F2-INSIDE PRIMER GAC ATG CCY GGY … B= C or G or T D= A or G or T H= A or C or T K= G or T M= A or C N= A or C or G or T R= A or G S= C or G V= A or C or G W= A or T Y= C or T R1-OUTSIDE PRIMER ACV GTV ARN CCN … R2-INSIDE PRIMER GCN GGV CCD CCH …

  19. The Degenerate Primer GCA ATA AAA GAA TGG GGC CAA CCA AA C C G G G G C G T T T T

  20. Test the Primers PCR

  21. Procedures PCR Extracted DNA from Agarose Gel Ligation of DNA to pGEM-Easy Plasmid Transformation Sequence Clones

  22. Ligation of DNA to pGEM-Easy Plasmid LIGATION DNA PLASMID

  23. Transformation www.biochem.arizona.edu/.../Ligation-2.gif

  24. After Sequencing How many genes are there for CHS enzyme? GENE1 GENE2 GENE3 GENE4

  25. RNAi • Find Conserved Domains • Generate Primers for those regions. • Knock down expression of all sequence possessing homology to CHS. http://www.hhmi.org/biointeractive/rna/rnai/

  26. Near Future • Create full cDNA library enriched for genes expressed during and/or in early colonization of symbiont.

  27. Questions?

  28. 0.1mM 0.01mM 0.005mM Effect of the Flavoniod Naringenin (naringin) on Differentiation *Naringenin inhibited differentiation at conc. ≥ .01mM 0.001mM Control

  29. Flavonoids • polyphenolic compounds with 15 Carbon atoms, 2 benzene rings connected by a 3 carbon chain. • Flower pigments in angiosperms

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