flora 25 may 2009tonelli.ppt

7/29/2019 Flora 25 May 2009Tonelli.ppt

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FLORA Final scientific meeting

Istanbul (TR), 25-26 May 2009

Department of Biomolecular Sciences and Biotechnology

Dr. Katia PetroniFunctional Genomics Group, Prof. Chiara Tonelli

Production of model foodsin the FLORA project


2/44

Production of model foods

Maize lines with different types and amount of flavonoids


3/44

Maize seeds with high levels of

Hydroxycinnamates

Anthocyanins

Flavonols

Phlobaphenes


4/44

Phenylalanin

PAL

Cinnamic acid

LDOX

4-coumaric acid

4-coumaroil-CoA

C4H

4CL

CHI (CHI 1)

CHS (C2)

Naringenin chalcone

NaringeninF3H

DFR

(A1)

Dihydrokaempferol

Vacuolar glycosilated

anthocyanins

UFGT

GST

Dihydroquercetin

F3H

DFR (A1)

Pelargonidin Cyanidin

Regulation of anthocyanin biosynthesis in maize seeds

bHLH

MYBC1/Pl1

R1/B1

C3H OMT4CL CCRCADHydroxy-

cinnamates

FLAVONOLSFLSKaempferol

Quercetin


5/44

Tissue specificity ofbHLHand MYB regulatory genesin maize seeds

PericarpPericarp

Seed

Aleurone

(R1+C1)

Scutellum

(Hopi1+C1)

endosperm

(B1+Pl1)

Anthocyanins

(P1)

Phlobaphenes

Procissi et al(1997) Plant Cell 9:1547


6/44

Phenylalanin

PAL

Cinnamic acid

4-coumaric acid

4-coumaroil-CoA

C4H

4CL

CHI (CHI 1)

CHS (C2)

Naringenin chalcone

Naringenin

Regulation of phlobaphene biosynthesis in maize seeds

DFR (A1)

Phlobaphenes

P1C3H OMT4CL CCRCAD

Hydroxy-

cinnamatesMYB


7/44

Phenylalanin

PAL

Cinnamic acid

LDOX

4-coumaric acid

4-coumaroil-CoA

C4H

4CL

CHI (CHI 1)

CHS (C2)

Naringenin chalcone

NaringeninF3H

DFR

(A1)

Dihydrokaempferol

UFGT

GST

Dihydroquercetin

F3H

DFR (A1)


bHLH

MYBC1

R1


cinnamates


Quercetin


8/44

Phenylalanin

PAL

Cinnamic acid

4-coumaric acid

4-coumaroil-CoA

C4H

4CL


bHLH

MYBC1/Pl1

R1/B1


cinnamates

LDOX

CHI (CHI 1)

CHS (C2)

Naringenin chalcone

NaringeninF3H

DFR

(A1)

Dihydrokaempferol

Vacuolar glycosilated

anthocyanins

UFGT

GST

Dihydroquercetin

F3H

DFR (A1)

Pelargonidin Cyanidin


Quercetin


9/44

X

Regulatory genes of anthocyanin biosynthesis were introgressedin isogenic high-producing lines by classical breeding

R1C1

B1Pl1P1


10/44

Available maize lines

r1 c1 R1 C1 (ACR)

115 Kg 115 Kg

anthocyaninscontrol

p1 P1

phlobaphenescontrol

b1 pl1 B1 Pl1

2XanthocyaninscontrolW23/B73W22/DK

W22/B73

r1 a1

flavonolscontrolW22

145 Kg 145 Kg

145 Kg 145 Kg 13 Kg 18 Kg


11/44

Maize seeds for anthocyanin-rich diets

r1c1 R1 C1 (ACR)

2005 18 Kg 18 Kg

2006 10 Kg 10 Kg

b1pl1 B1 Pl1

2005 10 Kg

2007 2.1 Kg 2.1 Kg

Effects on-myocardial infarction

- vascular reactivity

in WP3 (Michel de Lorgeril)

Effects on

-adipogenesis- oxidative stress

in WP3 (Marco Giorgio)

30% reduction ofdamaged cardiac tissue


12/44

A. f lavus A. parasi ticus F. verti ci l l oid es F. pr olif eratum

Aflatoxin B1

Aflatoxin B2Aflatoxin B1

Aflatoxin B2

Aflatoxin G1

Aflatoxin G2

Fumonisin B1

Fumonisin B2

Fumonisin B3

Aflatoxin B1 and G1 are

1. mutagenic (chromosomal aberrations)

2. carcinogenic (liver, kidney)

Fumonisin B1, B2 and B3 are

carcinogenic (liver, kidney)

teratogenic (spina bifida)

Mycotoxins are natural by-products produced by

Fumonisin B1

Fumonisin B2

Fumonisin B3

Asperg i l lus Fusar ium

Prevalent in northern France Prevalent in Italy


13/44

Fungi can infect maize seedsfrom pre-harvest to post-harvest storage

Mechanical injuryand damage by pests(e.g. corn borer)


14/44

0

2

4

6

8

10

12

14

16

2005 2006 2007 2008

r1 control

ACR-anthocyanins

0

2

4

6

8

10

12

14

16

2005 2006 2007 2008

b pl control

B Pl-2X anthocyanins

0

2

4

6

8

10

12

14

16

2005 2006 2007 2008

p control

P-phlobaphenes

FumonisinB1(p

pm)

EU Rule 1126/20074 ppm maximum levels in maize seeds

Anthocyanins and phlobaphenes reduce mycotoxin infections


15/44

Colored maize can be a safer/healthier source of food

corn silage


16/44

maize for polenta

and for humans

( )


17/44

seeds

flour

polenta

Use of colored maize for human nutrition (polenta)

colorless colored colored

(selected lines)


18/44

Anthocyanin-rich extracts are obtained from

Grape 730 mg/Kg

Blueberry 900-4.500 mg/Kg

Blackberry and blackcurrant 600-3.500 mg/Kg

Selected B1 Pl1 maize lines 1.650-5.300 mg/kgcan be an economic alternative

Anthocyaninextraction

Productionof pills


19/44


Arabidopsis transgenic lines with high levels of

- anthocyanins

- flavonols

- hydroxycinnamates



20/44

Arabidopsis lines rich in

Hydroxycinnamatestt4 mutant

Ectopic expression ofA. majus regulatory genes Anthocyanins

in all organs and tissues

Ectopic expression ofA. majus regulatory genes

in tt3 mutants

Flavonolsin all organs and tissues


21/44

Col

pACT::ROS + 35S::DEL13-12 pACT::ROS + 35S::DEL14-10

35S::DEL (donor C. Martin)

ROSEA1 was sufficient to activate anthocyanin synthesis in 4 day old seedlings

ROSEA

ROSEA+DELILA

pACT::ROS 13-9pACT::ROS 1-8


22/44

Col

pACT::ROS 13-9

pACT::ROS + 35S::DEL13-12 pACT::ROS + 35S::DEL14-10

35S::DEL

ROSEA

pACT::ROS 1-8

ROSEA+DELILA

Anthocyanin accumulation in 14 day old seedlings


23/44

0

20

40

60

80

100

120

140

Quantitative analysis of anthocyanin accumulation in 14 day old seedlings

Relativeanthocyanin/gf.w

t.

Col 35S::DEL pACT::ROS 35S::DEL+pACT::ROS


24/44

Light regime to enhance flavonoid accumulation in Arabidopsis leaves

Dim light High-light UV-B light

tt3-1

Flavonols(quercetin)

ttg1-1

Hydroxycinnamates

Dpttg1

Higher levels ofhydroxycinnamates

Columbia, Landsberg

Phenylpropanoid profiling in WP2 (H.-P. Mock)


25/44

0

5

10

15

20

25

30

35

dim light high light UV-B

Area*10-6i

n1gfw

0

50

100

150

200

250

300


Ar

ea*10-6i

n1gfw

0

5

10

15

20

25

30


Area*10-6i

n1g

fw

0

20

40

60

80

100

120


Area*10-6i

n1g

fw

peak b (275 nm, unknown)

peak l (264/342 nm, Km-rha-rha)peak g ( 328 nm, unknown)

peak d (278 nm, unknown)

Clear influence of the light conditions (particularly UV-B treatment) and of thegenotype on the accumulation of the different compounds

Data from Silke Peterek and Hans-Peter Mock

Col

Ler

ttg1-1

tt3-1

ttg1-1 tt3-1

pttg1


26/44


Effect of UV-B light on flavonoid content and gene expressionin tomato fruits of commercial hybrids


Arabidopsis transgenic lines with high levels of

- anthocyanins

- flavonols

- hydroxycinnamates


27/44

(quercetin rutinoside)

3GT

RT

RutinKaempferol

rutinoside

Isoquercetin

4-coumaroil-CoA

CH I

CHS

Naringenin chalcone

Naringenin

F3H

Dihydrokaempferol Dihydroquercetin

F3H

FLS

Kaempferol

+ 3X Malonil-CoA

Quercetin

FLS

3GT

RT

Flavonoid biosynthesis in tomato

Prevalently lycopene (carotenoids)No anthocyanins in commercial varieties

Flavonoids 5-10 mg/Kg, but present only in peel

Muir et al., (2001) Nature Biotechnology 19: 470-474


28/44

3GT

RT

RutinKaempferol

rutinoside

Isoquercetin

4-coumaroil-CoA

CH I

CHS

Naringenin chalcone

Naringenin

F3H

Dihydrokaempferol Dihydroquercetin

F3H

FLS

Kaempferol

+ 3X Malonil-CoA

Quercetin

FLS

3GT

RT

Flavonoid biosynthesis in tomato

CHS

CHI

FLS

Muir et al., (2001) Nature Biotechnology 19: 470-474


29/44

Solar radiation Solar radiation without UV-B

UV-B

Effect of UV-B depletion on flavonoid content of tomato fruits


30/44

Maturegreen

Turning Red ripe

Analysis of flesh and peel

Analysis of three ripening stages

Flavonoid quantification Analysis of gene expression

G d fl id l d i l d fl h


31/44

4-Coumaroyl CoA3X Malonil CoA

CHS

Naringenin Chalcone

CHI

Naringenin

F3H

Dihydrokaempferol DihydroquercetinF3H

Kaempferol

FLS

Quercetin

FLS

Kaempferol-3-rutinoside

3GTRT

Rutin

3GT

RT

Isoquercitin

Genes and flavonoids analysed in peel and flesh


32/44

Low lycopene contentEsperanza

sun lightNo UV-B

Giuntini et al. (2008) J. Agric. Food Chem. 56: 5905-15

High lycopene contentDRW 5981

UV-B depletion enhanced flavonoid content in Esperanza fruits


33/44

sun light

No UV-B

Low lycopene content

Esperanza

High lycopene content

DRW 5981

Giuntini et al. (2008) J. Agric. Food Chem. 56: 5905-15

CHS and CHI differently respond to UV-B depletion in DRW and Esp

ff f d l k d h


34/44

Money-Maker wild-type

high pigment-1HP1 gene is homologous toAtDDB1 gene, negative regulator of light signal transduction

photomorphogenic mutant

Effect of UV-B depletion on Money-Maker and hp-1

In hp 1 peel flavonoid genes are earlier and higher activated


35/44

MG TU RR

HP1

negativeregulator

wild-type high pigm ent-1

CHS

CHI

F3H

F3H

FL S

CHS

CHI

F3H

F3H

FLS

Sun light

1

10

100

1000

buccia verde buccia invaiata buccia rossa

0,1

1

10

100


1

10

100


0,1

1

10

100


1

10

100

1000


Logaritmic scale Logaritmic scale

PEEL

In hp-1 peel flavonoid genes are earlier and higher activated

10

100

1000

0,1

1

10

100


1

10

100


0,1

1

10

100


1

10

100

1000


MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

In hp-1 peel flavonoids are earlier accumulated


36/44

X 3

CHS

CHI

F3H

FLS

F3H

wild-type

mg/100gD.W.

mg

/100gD.W.

mg/100gD.W

.

CHS

high pigm ent-1

mg/100gD.W.

mg/100gD.W.

mg/100gD.W

.

PEEL

Sun light

Naringenin-Calcone

0

100

200

300

400


Quercetina

0

2

4

6


Rutina

0

40

80

120

160


Naringenin-Calcone

0

100

200

300

400


Quercetina

0

2

4

6

8


Rutina

0

50

100

150


In hp-1 peel flavonoids are earlier accumulated

Naringenin chalcone Naringenin chalcone

QuercetinQuercetin

Rutin

3GT

RT Rutin

3GT

RT

MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

CHI

F3H

FLS

F3H

In wt peel activation of flavonoid genes is UV-B dependent at MG stage


37/44

wild-type

CHI

F3H

FLS

F3H

mg/100gD.W.

wild-type

mg/100gD.W.

mg/100gD.W.

Wt PEELCHS

CHI

F3H

F3H

FL S

Sun light

No UV-B

RNA Flavonoids

Naringenin-Calcone

0

100

200

300

400


Quercetina

0

2

4

6


Rutina

0

20

40

60


0,1

1

10

100

1000


0,01

0,1

1

10


1

10

100


1

10

100

1000


Logaritmic scale

0,1

1

10

100


Naringenin chalcone

CHS

Quercetin

Rutin

3GT

RT

MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

In wt peel activation of flavonoid genes is UV B dependent at MG stage

In hp-1 peel UV-B depletion has no effect on flavonoid content


38/44

CHS

CHI

F3H

F3H

FLS

CHS

mg/100gD.W.

mg/100gD.W.

mg/100gD.W

.

hp-1 PEEL

Sun light

No UV-B

RNA Flavonoids

Naringenin-Calcone

0

100

200

300

400


Quercetina

0

2

4

6

8


Rutina

0

50

100

150


0,01

0,1

1

10


1

10

100


0,1

1

10

100


1

10

100

1000


Logaritmic scale

1

10

100

1000


In hp 1 peel UV B depletion has no effect on flavonoid content

MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

Naringenin chalcone

Quercetin

Rutin

CHI

F3H

FLS

F3H

3GT

RT

MG TU RR

MG TU RR

MG TU RR

Effect of UV-B depletion on flavonoid gene expression in wild-type and hp-1 flesh


39/44

wild-type

CHS

CHI

F3H

F3H

FL S

high pigm ent-1

CHS

CHI

F3H

F3H

FL S

0,01

0,1

1

10

100

polpa verde polpa invaiata polpa rossa

0,01

0,1

1

10


0,01

0,1

1

10

polpa ve rde polpa inva ia ta polpa rossa

0,01

0,1

1

10

polpa ve rde polpa invaiata polpa rossa

0,01

0,1

1

10

100

polpa ve rde polpa i nvaia ta po lpa rossa

0,01

0,1

1

10

100


0,01

0,1

1

10


0,01

0,1

1

10


0,01

0,1

1

10


0,01

0,1

1

10

100


Sun light

No UV-B

FLESH

Logaritmic scale Logaritmic scale

Effect of UV B depletion on flavonoid gene expression in wild type and hp 1 flesh

MG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RRMG TU RR

MG TU RR

MG TU RR

MG TU RR

MG TU RR

Effect of UV-B depletion on flavonoid content in wild-type and hp-1 flesh


40/44

CHI

F3H

FLS

F3H

CHS

Naringenin-Calcone

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4


Quercetina

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8


Rutina

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8


Naringenin-Calcone

0

1

2

3

4

5

6

7


Quercetina

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8


Rutina

0,0

0,5

1,0

1,5

2,0

2,5

3,0


mg/100gD.W.

mg/100gD.W.

mg/100gD.W.

mg/100gD.W.

mg/100gD.W.

mg/100

gD.W.

wild-type high pigm ent-1

Sun light

No UV-B

X 3

X 3

X 8

FLESH

Effect of UV B depletion on flavonoid content in wild type and hp 1 flesh

Naringenin chalcone

Quercetin

Rutin

3GT

RT

CHI

F3H

FLS

F3H

Naringenin chalcone

Quercetin

Rutin

3GT

RT

CHS

MG TU RR

MG TU RR

MG TU RR MG TU RR

MG TU RR

MG TU RR

C l i


41/44

Conclusions

UV-B depletiona) reduced flavonoid content in DRW fruits (open-field cultivation)

b) enhanced flavonoid content in Esperanza fruits (UV-B opaque plastic covers)

In hp-1 peel and fleshthe earlier and higher activation of flavonoids was correlated with the earlier and

higher activation of flavonoid genes

The activation of structural genes is UV-B dependent mostly at MG stage

UV-B depletiona) reduced flavonoid content in wild-type peel

b) increased flavonoid content in hp-1 flesh

d ti


42/44

Analysis of putative regulatory genes of flavonoidbiosynthesis in tomato

Effect of supplementary UV-B radiation

a) on MG fruits in post-harvest conditions

b) on fruits during growth in greenhouse

and pespectives


43/44

Acknowledgments

FLORA partners:

Cathie Martin (UK)

Eugenio Butelli (UK)

Hans-Peter Mock (DE)

Silke Peterek (DE)

Michel De Lorgeril (FR)

Marie-Claire Toufektsian (FR)

Marco Giorgio (I)

Lucilla Titta (I)

Chiara Tonelli

Roberto Pilu

Eleonora Cominelli

Valentina Calvenzani

Moira Martinelli

AU

0,000

0,002

0,004

0,006

0,008

0,010

0,012

0,014

Minutes

5 ,0 0 1 0, 00 1 5, 00 2 0, 00 2 5, 00 3 0, 00 3 5, 00 4 0, 00 4 5, 00 5 0, 00 5 5, 00 6 0, 00

Anna RanieriValerio Lazzeri

Deborah Giuntini

Gianni Galaverna

Chiara DallAstaUNIVERSITDI PARMA


44/44

flora 25 may 2009tonelli.ppt

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