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

Gunnera and Nostoc’s Symbiosis

Nostoc PunctiformeGunnera manicata

NO3-

NH4+slow

N2Denitrification

BiologicalNitrogenFixation

Nitrogen Cycle

Two months after inoculation Control

Meeks & Elhai 2003

Hornworts(Anthrocerophyta)

Bryophytes

Azolla (Pterophyta)

Water ferns

Cycads (Cycadophyta)

Gymnosperms

G. Manicata(Anthophyta)

CB colonize and CB colonize and penetrate glands penetrate glands produced below produced below each leafeach leaf

Immature gland; 8-day old seeding

Mucus covered gland

Inoculation of gland with CBWhat’s so special about Gunnera’s What’s so special about Gunnera’s

relationship with CB?!!relationship with CB?!!

It’s the only intracellular It’s the only intracellular endosymbiotic relationship endosymbiotic relationship known b/n angiosperm and known b/n angiosperm and

cyanobacteria!!!cyanobacteria!!!

How are Gunnera’s gland cells different from normal plant cells?

BROAD IMPACT

• Save the world and revolutionize agriculture!!

Create productive Create productive symbiosis between symbiosis between cyanobacteria and crop cyanobacteria and crop plants!plants!

Bergman et al. 1992

Trends in Plant Science, 10: 236-242

N-starvation

Myb -transcription factor

Flavonoid synthesis

gland primodia inhibit

auxin efflux

Localized auxin accumulation

Activation of cell division and gland development

Remobilization of nutrients

from leaves to the stem

N-starvation

Myb -transcription factor

Flavonoid synthesis

High flavonoids at the

gland primodia inhibit

auxin efflux

Localized auxin accumulation

Activation of cell division and gland development

Remobilization of nutrients

from leaves to the stem

10 mM nitrate

0.1 mM nitrate

Flavonoids

Flavonoids

• Inhibit Nostoc growth and hormogonia formation

Test the hypothesis

Trends in Plant Science, 10: 236-242

MethodsCreate 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?

ANSWERConserved regions across various species.

Methods• Make primers using conserved regions

residing in a wide range of dicots.> NCBI

Methods• Align all of those various sequences and locate

the conserved regions.> BIOBIKE

Conserved Regions

01190

352-376

1129-1154421-444

913-941

F1

R1F2

R2

Chalcone Synthase Sequence

Degenerate PrimerF1-Outside PrimerGCN ATH AAR GAR TGG GGB CAR CCH AA

F2-INSIDE PRIMER

GAC ATG CCY GGY …

R1-OUTSIDE PRIMER

ACV GTV ARN CCN …

R2-INSIDE PRIMER

GCN GGV CCD CCH …

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

The Degenerate Primer

GCA ATA AAA GAA TGG GGC CAA CCA AA C C G G G G C G T T T T

Test the PrimersPCR

ProceduresPCR

Extracted DNA from Agarose Gel

Ligation of DNA to

pGEM-Easy Plasmid

Transformation

Sequence Clones

Ligation of DNA to pGEM-Easy Plasmid

PLASMID

DNA

LIGATION

Transformation

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

After Sequencing

How many genes are there for CHS enzyme?

GENE1 GENE2 GENE3 GENE4

RNAi

1. Find Conserved Domains2. Generate Primers for those regions.3. Knock down expression of all sequence

possessing homology to CHS.

http://www.hhmi.org/biointeractive/rna/rnai/

Near Future

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

Questions?

Effect of the Flavoniod Naringenin (naringin) on Differentiation

Control

0.1mM 0.005mM0.01mM

0.001mM

*Naringenin inhibiteddifferentiation at conc. ≥ .01mM

Flavonoids

• polyphenolic compounds with 15 Carbon atoms, 2 benzene rings connected by a 3 carbon chain.

• Flower pigments in angiosperms