Dias nummer 1

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Department of Plant Biology Biotechnology

Department of Agriculture Ecology
Department of Basic Sciences Environment
Ecometabolomic Approach for Identification of
Defensive Compounds in Plants towards Herbivorous
Insects
Vera Kuzina Poulsen (vkp_at_life.ku.dk)
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Interactions between Barbarea flea beetles
P-type pubescent susceptible G-type
glabrous resistant
POLYMORPHIC
to flea beetle attack
Barbarea vulgaris (winter cress)
Phyllotreta nemorum (flea beetle)
Untargeted metabolic profiling involves the
comparison of metabolomes (collection of all
metabolites in an organism) between P-
G-types to find differences in their metabolite
profiles. Those differences may be correlated
with the resistance of Barbarea towards flea
beetles.
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Winter cress (Barbarea vulgaris)

• Brassicaceae weed of European origin naturalized
  in North America, Africa, Australia, North
  Zealand Japan
• Potential as an oil crop for use in Northern
  latitudes
• Used for salads or garnishes as medicinal plant
  (anti-inflammatory)
• Resistant to many insects fungi
• Saponins are possible defensive compounds against
  flea beetles

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Distribution of Barbarea vulgaris in Denmark
50 km
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Bioassays which plant is resistant / susceptible
?

• Bioassays with flea beetles

• Bioassays with flea
• beetle larvae

resistant
susc.
resistant
susc.
72 h with 5 larvae
susc.
resistant
resistant
susc.
16 h
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Survival of flea beetle larvae on P, G, F1 F2
plants
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Experimental setup data flow in metabolic
profiling
BIOASSAYS
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1
3
LC-MS
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5
4
MetAlign
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Metabolite profiling by LC-MS MetAlign
What is the difference in LC-MS profiles of P G?
P-type
n20
G-type
n20
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Data processing
Available Metabolomics Software Packages
Software URL Reference BinBase http/
/fiehnlab.ucdavis.edu/projects/binbase_setupx/ Fie
hn et al., 2005 MetAlign http//www.pri.wur.nl/
UK/products/MetAlign/ Tikunov et al., 2005
MET-IDEA http//www.noble.org/plantbio/MS/index.
html Broeckling et al., 2006 MZmine
http//mzmine.sourceforge.net/ Katajamaa
Oresic, 2005 XCMS http//metlin.scripps.edu/dow
nload/ Smith et al., 2006
MetAlign
- software for untargeted profiling of complex
mixtures of compounds

• reveals compounds that are relevantly changed
• allows background subtraction, alignment,
  normalization statistical analysis
• to reveal statistically significant
  accumulating metabolites in large LC-MS datasets

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MetAlign interface
A Baseline noise reduction
Define what a mass peak is search all data
B Scaling (normalization) alignment
Align the same peaks in all data files
C Statistics
Define selection criteria to filter out relevant
mass peaks
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Metabolic profiling by LC-MS MetAlign
- detecting differences in LC-MS profiles
Low versus high resistant F2 plants
P versus G type
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Distribution of saponins in F2 plants
2.5 2.0 1.5 1.0 0.5 0
Amplitude, 107
0-30 larvae survived
33-87 larvae survived
90-100 larvae survived
F2 plants
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Correlation analysis
Hederagenin cellobioside
Oleanolic acid cellobioside
x represents 1 plant
1. m/z 819
2. m/z 817
3. m/z 803
4. m/z 819
Metabolite concent- ration ln(y1)
Ret.time 42,5 min
Ret.time 39,6 min
Ret.time 42,0 min
Ret.time 46,1 min
Metabolites negatively correlated to
larvae survival
Larvae survival ln(x1)
Relative abundance
MS spectra
MS2 spectra
Mass-to-charge ratio
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Cluster analysis of metabolites
UPGMA clustering with distances 1- R2 of 150 most
correlated with resistance metabolites
1st 2nd most negatively correlated metabolites
hederagenin cellobioside
oleanolic acid cellobioside
Metabolites
Average distance between clusters
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Cluster analysis of metabolites
F2 plants
Two-way hierarchical clustering

• Metabolites as well as plants are divided
• into clusters of similar patterns
• Metabolite levels are represented by a vector
• Similarity scores are calculated for all
  possible
• pairs of metabolites (angle between vectors)
• Most similar patterns are joined in a tree

Metabolites
1st 2nd most negatively correlated metabolites
oleanolic acid cellobioside
hederagenin cellobioside
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Principal Component Analysis
Relationships between plants metabolites
Data plotted as a swarm of points in the variable
space, revealing a hidden trend. PC-system
consists of a number of ortho- gonal PCs, each
lying along a maximum variance direction in
decreasing order.
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Conclusions

• Proved concept
• Discovery of metabolites that confer resistance
  of Barbarea against flea beetles
• 2 known saponins 2 unknown
  saponins

Hederagenin cellobioside - like
MNa819 Glucose in a different position?
Hederagenin cellobioside - like
MNa817 Additional double bond in the
sceleton ?
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Present future work

• DEFENSIVE COMPOUNDS - chemical structure
  biological activity
• QTL-mapping number chromosomal localization
  of genes for resistance
• GENES associated with resistance Barbarea
  transcriptome is sequenced!
• EXPRESSION of Barbarea resistance genes in other
  plants
•  

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Acknowledgements
Department of Plant Biology Biotechnology
Department of Natural Sciences Environment
Carl Erik Olsen Claus Ekstrøm Jens Kvist Nielsen
Susanne Bidstrup Søren Bak
Department of Agriculture Ecology
Sven Bode Andersen