analyses of the 1h-nmr spectral d

Page 1

JL Poëssel1*, MH Sauge2, MN Corre1, C Renaud3, M Gaudillère3, M Maucourt3, C Deborde3, C Dufour4, M Loonis4, JP Lacroze2, T Pascal1, A Moing3

1 Genetics and Breeding of Fruits and Vegetables, INRA, Domaine St Paul, Site AgroParc, 84914 Avignon, cedex 9, France2 Plants and cultural Systems in Horticulture, INRA, Domaine St Paul, Site AgroParc , 84914 Avignon cedex 9, France3 Plant Physiology and Biotechnology, UMR INRA/U. Bordeaux 1/U. V Segalen Bordeaux 2, BP 81, 33883 Villenave d'Ornon cedex, France4 Safety and Quality of Plant Products, UMR INRA/UAPV, 84914 Avignon cedex 9, France

* Contact: Jean-Luc.Poessel@avignon.inra.fr, Annick.Moing @bordeaux.inra.fr

Kaloshian I, Walling LL. 2005. Hemipterans as plant pathogens. Annual Review of Phytopathology 43: 491-521.

Karley, AJ, AF Douglas, WE Parker. 2002. Amino acid composition and nutritional quality of potato leaf phloem sap for aphids. Journal of Experimental Biology 205: 3009-3018.

Pascal T, F Pfeiffer, J Kervella, JP Lacroze, MH Sauge. 2002. Inheritance of green peach aphid resistance in the peach cultivar 'Rubira'. Plant Breeding 121 (5): 459-461.

Sauge MH, JP Lacroze, JL Poëssel, T Pascal, J Kervella. 2002. Induced resistance by Myzus persicae in the peach cultivar "Rubira". Entomologia Experimentalis et Applicata 102: 29-37.

Sauge MH, F Mus, JP Lacroze, T Pascal, J Kervella, JL Poëssel. 2006. Genotypic variation in induced resistance and induced susceptibility in the peach-Myzus persicae aphid system. Oikos, sous presse.

Thompson GA, Goggin FL. 2006. Transcriptomics and functional genomics of plant defence induction by phloem-feeding insects. Journal of Experimental Biology 57: 755-766.

4th International Conference on Plant Metabolomics, 7th April - 10th April 2006, Reading, Berkshire, United Kingdom

Context

Metabolic profiling of shoot apices infested by the peach-potato aphid

in susceptible and resistant peach cultivars

q Peach-potato aphid

• phloem feeding insect belonging to Hemiptera order

(aphids, whiteflies, planthoppers)

• most studied aphid species

q Plant/aphid relationships• a specialized, long lasting interaction, intermediate between

plant/herbivore and plant/pathogen systems

• several targeted biochemical studies of plant response

(Kaloshian and Walling 2005)

• emerging of transcriptomics of plant defense induction (Thompson and Goggin, 2006)

q Peach

• primary host for M. persicae

• model species for Rosaceae with genomic resources

(Genome Database for Rosaceae [GDR])

• different sources of resistance available

q Rubira: a red leaf peach cultivar• used as rootstock

• bearing a dominant gene of resistance to Myzus persicae

(Pascal et al, 2002)

Rubira q Induced resistance occurring two days after infestation

Induced resistance

after 48 h pre-infestation

0

20

40

60

80

100

0 24 48 72 96 120 144 168 Time (hours)

% a

ph

ids o

n p

lan

t

• Running away of aphids from Rubira

resistant plants within few days (antixenosis)

(Sauge et al 2002, 2006)

• Local reaction around feeding sites

on Rubira and a green-leaf resistant

Rubira hybrid

• observed resistances to all pesticides

• vector of viruses such as Plum Pox Virus

(Sharka, quarantine disease)

• and for many other crops as solanaceous vegetables

(secondary hosts)

•Development of durable-resistant cultivars to control

aphids and reduce the use of pesticides through a

better knowledge of peach/aphid relationships

q Objective

• a main threat for peach (Prunus persica)

(primary host)

± Infestation by

20 wingless adult aphids

per plant

• no settlement of aphids

• induced resistance

• development of aphid colonies

• induced susceptibility

48 hours

• Sampling: “i” or “c” shoot apices

• Lyophilisation

• 25 plants per condition

pooled in 5 replicates

Metabolic profiling

Rubira

resistant

genotype

“R”

GF305

susceptible

genotype

“ S ”

q 1D 1H NMR non-targeted profiling

of polar metabolites

q HPLC targeted analyses

5

17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 8 0 7 0 6 0

24 0

20 0

16 0

12 0

8 0

4 0

0

Ch

lo

rog

en

ic

ac

id

Int

ern

al

sta

nda

rd

2 9

8

4

6

7

Ethanol/H2O 70/30, 4°C

Ethanol/H2O 70/30, 4°C

Ethanol/H2O 70/30, 4°C

Ethanol/H2O 70/30, 4°C

Ethanol/H2O serie,

80°C

Extraction

inverse phase

inverse phase

inverse phase

anion exchange

anion exchange

Separation

Secondary

metabolites

Primary

metabolites

Metabolites

fluorescenceamino acids

UV absorbancephenolic

compounds

UV absorbancecyanogenic

compounds

conductivityorganic acids

PADsoluble

carbohydrates

Detection

Analytical strategy of shoot apices profiling

Representative 1H-NMR spectra of polar extracts

and assigned resonances

Resistant cultivar

Co

ntr

ol

Infe

sted

Pru

nasin

Fe

rulic a

cid

+

Chlo

rogen

ic a

cid

Chlo

rogen

ic a

cid

Glu

cose

Malic

acid

Pru

nasin

Sucrose

Quin

ic a

cid

Aspa

rag

ine

Pru

nasin

Pru

nasin

Ino

sito

l

Chlo

rogen

ic a

cid

Aspa

rag

ine

Citric

ac

id

Citric

ac

id

Arg

inin

e

Ala

nin

e

Resid

ual e

tha

nol

Aspa

rtic

acid

Malic

acid

Cho

line

Glu

cose

Arg

inin

eIn

osito

l

Malic

acid

Glu

tam

ic a

cid

+M

alic

acid

+P

roline

Succin

ic a

cid

La

ctic a

cid

+Th

reonin

e

Valine

Iso

leuc

ine

Iso

leuc

ine +

Leu

cin

e

Acetic

acid

Quin

ic a

cid

Arg

inin

e

Chlo

rgenic

acid

+Q

uin

ic a

cid

+

Glu

tam

ic a

cid

+

Pro

line

Chemical shift (ppm)

x8x2

• 22 metabolites identified

• several unknown resonances

Results1H NMR HPLC

Analyses of the 1H-NMR spectral

signatures

Targeted analyses

- 0.4

- 0.3

- 0.2

- 0.1

0

0.1

0.2

0.3

0.4

012345678910

p pm

load

ing

s

PC1

• Loadings

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

01234567891 0

ppm

load

ing

s

PC2

265 variables issued from the spectral signature:

- 0.03 ppm spectral domains - normalized /sum of signal

Chem ical sh ift (p pm )

q Data reduction

• Scoresq Multivariate analysis: PCA (cov)

-400

-300

-200

-100

0

100

200

300

400

-400 -300 -200 -100 0 100 200 300 400

principal component 1 (63%)

pri

nc

ipal

co

mp

on

en

t 2

(10

%)

Rc

Ri

Sc Si

• Discriminant spectral

domains on PC1

Hyd roxycinnamic acid s

(c hloroge nic acid)

Quinic acid+ u nknow n

Citric acid

Asparagine

Proline+unk now n

Acetic ac id

Unkn ow ns …

Comparative

metabolomics of

resistant /

susceptible cvs after

infestation

Targeted HPLC analyses

Multivariate analysis on 43 metabolites

• Loadings

g lu

seras p

as n

gly

g lnh is

thre

arg

a lagab a

protyr

val ile u

le u

lys

ph e

ch lo

U-HCA1

DiCQ

Q3G pCCQ

K3G

U-HCA2

U-HCA3U-HCA4

pru n qu in

ace t

U-Acet- Lsuc cfum

ox ac it

iso c

ino s

sorb

U-so rb- L

fru

su c

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

-0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5

PC1

PC

3

Univariate analyses

• Discriminant compounds

Hydroxycinnamic acids

Several amino acidsSucrose…

• Scores

q PCA (cor)

Sc Si

Ri

Rc

-12

-10

-8

-6

-4

-2

0

2

4

6

8

10

12

-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12

PC1 (54%)

PC

3 (8

%)

Similarities and differences between S and R for infestation response

æ3 83aa

ä3

020

Targeted HPLC: overview on 43 metabolites

79%

21%(9/43)

Susceptible

72%

28%

(31/43)

Resistant

10 aa

7 phenolics

Targeted HPLC: primary metabolites

Effect of infestation

q Amino acids: contrasted responses in the resistant cultivar

Glutamine

CC ii0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Susceptible Resis tant

mg

.g D

W-1

¬¬¬

¬

Phenylalanine

CC ii0.0

0.2

0.4

0.6

0.8

1.0

Susceptible Resis tant

mg

.g D

W-1

¬¬¬

NS

q Carbohydrates: a general decrease in the resistant cultivar

q Organic acids: varying responses in the resistant cultivar

Targeted HPLC: primary metabolites

Effect of infestation

Oxalate

C Ci i

0. 0

0. 2

0. 4

0. ,6

Susce ptibl e Resistant

mg

.g D

W-1

¬¬¬NS

Quinate

C Ci i0, 0

2, 0

4, 0

6, 0

8, 0

10,0

Susce ptibl e Resistant

mg

.g D

W-1

¬¬¬

NS

Suc rose

CC ii0

2

4

6

8

10

Susce ptibl e Resistant

mg

.g D

W-1

¬¬¬NS

Sorbitol

CC ii0

2

4

6

8

Susce ptibl e Resistant

mg

.g D

W-1

¬¬¬NS

c: control

i: infested

Targeted HPLC: correlations between 43 metabolites

Rubira (R) response to infestation

Coordinated response for several amino acids, organic acids and phenolics?

0.96

0.80

-0.80

0.84

0.88

0.92

-0.96

-0.92

-0.88

-0.84

R

asp

glu ser asn

gly gln

his

thre

arg

ala

ga

ba

pro

tyr

va

l

ileu

leu

lys

ph

e

ch

lo

UH

CA

1D

iCQ

Q3

G

pC

CQ

K3

G

UH

CA

2

UH

CA

3

UH

CA

4

pru

na

s

qu

inacet

eq

Ace

t*su

ccin

ma

l

fum

ox

al

cit is

oC

it

ino

s

sor

b

eq

So

rL

fru

su

cg

luc

aspgluserasn

glyglnhisthre

argala

gabapro

tyrvalileuleu

lysphechloUHCA1

DiCQQ3G

pCCQK3G

UHCA2

UHCA3UHCA4prunas

quinacet

eqAcetLsuccinmal

fumoxalcit

isoCit

inossorb

eqSorLfru

sucgluc

Correlation matrix

• No significant changes of

chlorogenic acid content

Targeted HPLC: secondary metabolites

q Phenolics: divergent responses in the resistant cultivar

• Increased level of 3,5 dicaffeoylquinic

acid in the resistant cultivar

Effect of infestation

Chlorogenic acid

CC ii0

2

4

6

8

10

12

14

Susceptible Resistant

mg

.g D

W-1

NS

NS

3,5 dicaffeoylquinic acid

C Ci i0

1

2

3

4

5

6

7

8

9

10

Susceptible Resistant

mg

.g D

W-1

NS

***

Rubira (R) response to infestation:

which metabolic pathways involved?

Malate

Fumarate

Succinate

CitrateCitrateIsocitrateIsocitrate

TCA Cycle

Threonine

AspartateAspartatedecrease after infestation

Pyruvate

Phenylalanine TyrosineLeucineValineIsoleucine

Lysine

ThreonineThreonine DeaminaseDeaminase

Malic Enzyme

PALPAL

Shikimate

pathway

Quinate

Branched-chain

amino-acid

pathways

ChlorogenicChlorogenic acidacid

CCTCCT

OtherOther phenolicsphenolics

FlavonoidsFlavonoids

BCATsBCATs

3,5 3,5 DiCQDiCQ

+ + QuinateQuinatePrunasin

Aromatic secondary metabolites

PhenolicsPhenolicsCyanogenicCyanogenic compoundscompounds

NADPH

increase after infestation

no change

Some hypotheses on mechanisms

involved in resistance…

Effectors of induced

resistance?

HypersensitivityHypersensitivity

oxalate decreaseoxalate decrease(oxalate oxidase activity

generating H2O2?)

cell death could

impede aphid feeding

Oxalate

C i

0,0

0,2

0,4

0,6

Resistant

mg

.g D

W-1

¬¬¬

local symptoms

around feeding sites

reddish spots

Nutritionally imbalanced diet?Nutritionally imbalanced diet?

T

i0,0

0,2

0,4

0,6

0,8

1,0

1,2

mg.g

MS

-1

¬¬¬

decrease in glutaminedecrease in glutamine

a key factor for

Myzus persicae development

(Karley et al, 2002)

tissue plasmolysis could

hamper aphid feeding

Osmotic stressOsmotic stress

prolineproline and and

sorbitolsorbitol decreasesdecreases

ciwilting symptoms

Toxic or repulsive compounds?Toxic or repulsive compounds?

OH

HO2C

OCO

OH

OCO

OH

OH

OH

OH

1 3

54

increase in phenolicsincrease in phenolics

preliminary results show

repulsive effect and toxicity of

phenolics identified in Rubira apex

on M. persicae reared on synthetic

diet

Discussion

References

Conclusions and perspectives

Perspectives

q Transcriptomics

• Comparison with metabolite responses

q Metabolomics

• Analyses of non-polar compounds

• Analyses of volatiles (M Staudt, CNRS, Montpellier)

• Time course and tissue localisation of infestation responses

OH

HO2C

OCO

OH

OCO

OH

OH

OH

OH

1 3

5

4

q Phenolics

• Isolation of candidate compounds for toxicity or

repulsive effects on M. persicae

• Metabolism and regulation of candidate compounds

?

Choice test

die

t 1

die

t 2

Conclusions

q First metabolomic approach of plant / phloem feeding insect

interactions

• towards system biology approach

q Contrasted responses in susceptible and resistant genotypes

• high metabolic changes associated with induced resistance

q Several metabolite families and metabolic pathways involved

• primary and secondary metabolites

Experiment design

Material and methods

5

17016015014013012011010090807060

280

240

200

160

120

80

40

0

Ch

loro

gen

ica

cid

Inte

rn

al

sta

nd

ard

32 9

8

4

6

7

q Identification of unknown phenolicsby HPLC coupled to negative-electrospray

mass and diode array detections

330 nm

Targeted HPLC: secondary metabolites

Two main esters of caffeic acid:

chlorogenic acid (5 caffeoylquinic acid) and 3,5 dicaffeoylquinic acid

[M – ArCHCHCO] -

200 300 400 500 m/z0

100

%

353

191

179

135

515

375

537

[M – H] -

[M + Na – 2H] -

[quinic acid – H] -

[Caffeic acid – CO2] -

[Caffeic acid – H] -

200 250 300 350 400 nm

nm

0

100

%

328 nm

218 nm

242 nm

OH

HO2 C

OCO

OH

OCO

OH

OH

OH

OH

1 3

54

OH

HO2C

O

OH

OCO

OH

OH

H

1 3

54

OH

HO2 C

O

OH

O C O

OH

OH

H

1 3

54

5 identified compounds

+ 4 unknown HCAs

pC

CQ

DiC

QU

-HC

A-1

U-H

CA

4

U-H

CA

-3

Q3

G

K3

G U-H

CA

-2

Rubira: a peach cultivar

resistant to Myzus persicae

Myzus persicae Sulzer

peach-potato aphid

Prunus persica / Myzus persicae

a model system to study plant

resistance to phloem feeding insects