D E PAR T M E N T O F P LAN T B I O L O G Y AN D B I O T E C H N O L O G Y
FACULTY OF LIFE S CIENC ES
UNIVERSITY OF COPENHAGEN
Unraveling the biosynthetic pathway of triterpenoid
saponins in Barbarea vulgaris
Jörg M. Augustin1, Vera Kuzina Poulsen1,2, Sven Bode Andersen2, Jens Kvist Nielsen3 and Søren Bak1
1Department
of Plant Biology and Biotechnology, 2Department of Agriculture and Ecology, 3Department of Basic Sciences and Environment, Faculty of Life Sciences, University of Copenhagen, Thorvaldensvej 40, DK-1871 Fredriksberg C, Denmark
INTRODUCTION
BIOCHEMISTRY
The Barbarea vulgaris – Phyllotreta nemorum model system
Proposed biosynthetic pathway of saponins in Barbarea vulgaris
The wild crucifer Barbarea vulgaris is polymorphic in respect to resistance towards the herbivorous pest Phyllotreta
nemorum. Thereby, the G-type is resistant to Phyllotreta larvae and adults. In contrary, both can feed on the P-type
without showing any decrease in their viability.
POLYMORPHIC:
P-type
pubescent
susceptible
G-type
glabrous
resistant
Barbarea vulgaris
(winter cress)
To identify metabolites responsible for the resistance,
an untargeted metabolomic investigation was performed. The
resistance of F2-population plants, gained from a cross between
G- and P-type, was correlated with their metabolite profile.
Four triterpenoid saponins were found to show the most
significant correlation with resistance.
CS (cycloartenol cyclase)
phytosterols
2,3-oxidosqualene
cycloartenol
βAS (β-amyrin synthase)
BvUGT1 catalyses the first glycosylation step
P450
P450
P450
Subsequently, to unravel the biosynthetic pathway of
saponins in Barbarea vulgaris, a strategy based on 454
pyrosequencing is pursued. Accordingly, transcriptomic data
was obtained from the resistant G-type and used for
Phyllotreta nemorum
comparative genomics towards Arabidopsis thaliana as well as
(flea beetle)
mapping of quantitative trait loci (QTL).
β-amyrin
Saponins are derived from the phytosterol pathway.
Enzymes belonging to the classes of oxidosqualenecyclases
(OSCs), cytochrome P450 monooxygenases (P450s) and
UDP-glycosyltransferases (UGTs) are expected to be
involved in their biosynthesis.
oleanolic
aldehyde
erythrodiol
oleanolic acid-O-glucoside
oleanolic acid
hederagenin-O-glucoside
P450
cyclisation - OSCs
oxidation - P450s
hederagenin
glycosylation - UGTs
Bv UGT1
BvUGT1
3-O-β-D-glucopyranosyl-hederagenin
TLC-scan of an BvUGT1 activity assays using [14C]UDP-Glucose and oleanolic acid or hederagnin as
substrates.
METABOLOMICS
3-O-β-D-glucopyranosyloleanolic acid
Experimental setup & data flow
Producing hybrids between P & G
UGT
BIOASSAYS
2
1
3
P
LC-MS
GROWTH & HARVEST
OF PLANT MATERIAL
METABOLITE
PROFILES
G
x
oleanolic acid cellobioside
SEPARATION & DETECTION
OF METABOLITES
EXTRACTION
6
5
4
hederagenin cellobioside
BvUGT1, isolated from a japanese Barbarea vulgaris
subspecies, was shown to catalyze the first 3-Oglucosylation of the saponin aglycons oleanolic acid and
hederagenin. Putative orthologs of this gene could be
found in both the G- and P-type.
GENOMICS
F1
DATA ANALYSIS
•
•
•
F2
DATA PROCESSING
MetAlign
Untargeted metabolite profiling by LC-MS
BLAST search 2:
against all Arabidopsis
thaliana proteins
Bioassays
29 putative OSC sequence fragments
269 putative P450 sequence fragments
137 putative UGT sequence fragments
verification if
no better hit
BLAST search 1:
against Arabidopsis thaliana
OSCs, P450s & UGTs
BLAST search 3:
against all Arabidopsis
thaliana cDNAs
verification if
no better hit
P and G
2
Amplitude, ×107
resistant F2 extremes
G9
contig/singlet
sequences
G10
G7
G8
G5
G6
G3
G4
G1
G2
Plant number
G-type (resistant)
n=20
P9
20
P10
0
P7
40
P8
60
P5
n=9
P6
susceptible F2 extremes
P3
P-type (susceptible)
n=20
1.5
8
7
6
5
4
3
50
P4
TIC
P1
TIC
2
Leaf
number
100
P2
low versus high resistant F2 plants
Larvae survival, %
P versus G type (parents)
n=9
60
1.5
15
F1
40
extracting
sequences
encoding for
OSCs, P450s
and UGTs
10
20
susc. F2 versus resist. F2
0.3
Intens.
x10 8
Number of plants
P versus G
1.5
1.0
10
0.5
5
0.2
0.0
0
10
20
30
40
50
Time (min)
0
0
10
10
20
20
30
30
40
40
50 Time (min)
50
5
pyrosequencing dataset
0
F2
40
Time [min]
RNA
20
0
Correlation and co-variation
between metabolite composition, metabolite profile and resistance
≤ 10
11-30
31-50
51-70
71-90
454 pyrosequencing
cDNA
G-type (resistant)
33,708 singlets
identification
of molecular
markers for
QTL mapping
F2 plants
LARVAE
SURVIVAL
unknown
saponin 2
0 – 30 %
33 – 87 %
-5
90 – 100 %
0
0.3
7.65
0.2
5
QTLs related
to resistance
Metabolite: mass_ret.time
Plant: number_insect survival (0 to 30)
resistancecorrelated
metabolites
1
2
3
4
5
6
7
8
1
5
oleanolic acid
cellobioside
5.07
0
3
12
4
0
0
-0.1
1
2
3
-5
-0.2
larvae survival ln(x+1)
-0.2
two-way hierarchical
clustering
p10m50-112
-3
0
2
3
p11m48-81
p41m49-350
p14m47-319
p10m50-433
12
13
14
p14m47-130
p11m59-54
p40m49-220
24
26
28
30
p14m48-163
p10m50-222 p38m49-196
p32m48-172
p14m47-119
39
41
43
p41m49-302 p14m47-276
p41m49-124
p41m60-492
50
p38m49-195
1
4
correlation analysis
-10
0.1
PC2
hederagenin
cellobioside
metabolites
metabolite concentration ln(y+1)
262,843 reads
≥ 91
Larvae survival, %
unknown
saponin 1
29,369 contigs
-0.1
0
PC1
0.1
0.2
principal component
analysis
0.3
59
61
64
66
70
74
78
p10m50-127
p41m60-130 p35m62-280
p32m48-185
p11m59-66
p35m62-278
p15m60-75
p15m60-76 p10m50-126
83
p15m60-292
0
4
7
8
p41m60-215
p10m50-301
p14m48-88
p14m47-309
15
19
20
25
26
30
35
38
40
45
47
p38m49-135
p14m48-227
p38m49-136
p35m62-139
p38m48-104
p16m49-341
p10m50-164
p38m48-200
p14m48-256 p38m49-275
p40m49-255
p14m48-394
53
p14m59-383
60
62
p41m60-194
p10m50-88
73
p40m49-354
81
p14m47-273
87
p10m50-247
94
p38m48-391
99
103
p40m49-178
p11m48-391
0
13
16
19
0
5
p38m49-189
p10m50-343
0
p11m59-146
18
19
p11m59-251
p40m49-266
10
p14m59-110
36
p40m49-265
18
p35m62-112
49
52
p38m49-191
p41m60-212
25
p10m50-98
68
74
83
86
91
102
105
108
p41m60-102
p41m60-70
p15m60-198
p11m59-215
p14m47-434
p14m59-340
p38m48-321
p11m48-307
121
p40m49-229
131
p40m49-231
p11m59-148
p41m49-311
p14m47-337
p35m62-365
25
27
p38m48-111
p35m62-138
33
34
37
38
p14m47-106
p14m48-238
p40m49-183
p10m50-468
46
48
52
55
59
60
64
p11m48-141
p41m49-227
p10m50-369
p35m62-369
p14m47-223
p40m49-117
p10m50-305
70
p10m50-461
74
78
79
p15m60-103
p41m49-436
p14m59-309
163
p14m47-215
85
p10m50-230
182
p14m59-270
2
33
37
39
40
p14m48-244
p11m48-234
p14m47-210
p35m62-214
0
6
p14m59-227
p14m47-296
15
p35m62-109
20
p14m59-244
24
p10m50-155
29
33
56
p10m50-213
62
64
p40m49-294
p11m48-221
70
p40m49-418 p40m49-419
87
p38m48-307 p14m47-245 50
0
p35m62-166
4
p14m47-326
8
p40m49-180
21
p14m48-318
26
p35m62-176
0
p41m49-125
7
p40m49-466
12
p10m50-107
23
p41m49-285
2
33
p41m60-87
p14m59-372
38
p14m47-212
30
p40m49-475
p41m60-126
43
p10m50-224
34
p40m49-162
51
p41m49-112
66
p40m49-221
53
p10m50-401
p41m49-115
CONCLUSIONS:
- an untargeted metabolomics approach identified four saponins as most correlating with resistance to herbivory in Barbarea vulgaris
- pyrosequencing provided sequence information about homlogs of genes of interest as well as SSRs for QTL mapping
PERSPECTIVES:
- Search for genes involved in saponin biosynthesis by:
- screening for homologs of enzymes known to catalyze similar steps of related pathways in the pyrosequencing dataset
- setup and screen microsome preparations for relevant activities & identification of enzymes in active fractions by MALDI-TOF-MS
- studies of metabolite profile co-segregation with QTLs
- exploitation of the synteny to Arabidopsis thaliana for QTL saturation mapping
- Use knowledge of the saponin pathway for bioengineering or molecular breeding of crop plants with increased anti-insecticidal properties