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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: [email protected], 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