Unraveling the biosynthetic pathway of triterpenoid saponins in Barbarea vulgaris

1. LARVAE SURVIVAL 0 – 30 % GROWTH & HARVEST OF PLANT MATERIAL SEPARATION & DETECTION OF METABOLITES EXTRACTION 33 – 87 % 90 – 100 % DATA ANALYSIS METABOLITE PROFILES DATA PROCESSING ≤ 10 11-30 31-50 51-70 71-90 ≥ 91 P G x F1 F2 1 -5 0 5 0.3 0.2 0.1 0 -0.1 -0.2 Metabolite: mass_ret.time QTLs related to resistance resistance- correlated metabolites Plant: number_insect survival (0 to 30) 5 0 -5 3 1 2 4 resistant plants PC2 2 susceptible plants -0.2 -0.1 0 0.1 0.2 0.3 PC1 Unraveling the biosynthetic pathway of triterpenoid saponins in Barbarea vulgaris 7.65 5.07 0.3 0.2 0 0 0 10 10 20 20 30 30 40 40 50 50 Time (min) Time (min) 1 2 3 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. Saponins arederivedfromthephytosterolpathway. Enzymes belongingtotheclassesofoxidosqualenecyclases(OSCs), cytochrome P450 monooxygenases (P450s) and UDP-glycosyltransferases (UGTs) areexpectedtobeinvolved in theirbiosynthesis. To identify metabolites responsiblefor 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. 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 comparative genomics towards Arabidopsis thaliana as well as mapping of quantitative trait loci (QTL). POLYMORPHIC: P-type pubescent susceptible G-type glabrous resistant BvUGT1 catalyses the first glycosylation step Barbarea vulgaris (winter cress) Phyllotreta nemorum (flea beetle) METABOLOMICS TLC-scan of an BvUGT1 activity assays using [14C]-UDP-Glucose and oleanolic acid or hederagnin as substrates. Experimental setup & data flow Producing hybrids between P & G BvUGT1, isolatedfrom a japaneseBarbareavulgarissubspecies, was showntocatalyzethefirst 3-O-glucosylation ofthesaponinaglyconsoleanolicacidandhederagenin. Putative orthologsofthisgenecouldbefound in boththeG-andP-type. BIOASSAYS 2 1 3 LC-MS GENOMICS 6 5 4 • 29 putative OSC sequence fragments • 269 putative P450 sequence fragments • 137 putative UGT sequence fragments MetAlign BLAST search 2: against all Arabidopsis thaliana proteins BLAST search 3: against all Arabidopsis thaliana cDNAs BLAST search 1: againstArabidopsisthaliana OSCs, P450s & UGTs verification if no better hit verification if no better hit Untargeted metabolite profiling by LC-MS Bioassays Leaf number P and G P versus G type (parents) low versus high resistant F2 plants 8 contig/singlet sequences 7 6 TIC TIC 100 Larvae survival, % P-type (susceptible) susceptible F2 extremes 50 5 2 n=20 n=9 60 4 1.5 0 P1 P2 P3 P4 P5 3 P6 P7 P8 P9 G1 40 P10 G2 G3 G4 G5 G6 G7 G8 G9 G10 Amplitude, ×107 Plant number 20 extracting sequences encoding for OSCs, P450s and UGTs G-type (resistant) resistant F2 extremes F1 2 n=9 15 n=20 60 1.5 40 10 20 PversusG susc. F2 versus resist. F2 5 pyrosequencing dataset Number of plants 0 29,369 contigs F2 10 40 5 RNA 454 pyrosequencing cDNA 262,843 reads 20 Correlation and co-variation between metabolite composition, metabolite profile and resistance identification of molecular markers for QTL mapping 0 Larvae survival, % G-type (resistant) 33,708 singlets F2 plants unknown saponin 1 unknown saponin 2 metabolite concentration ln(y+1) metabolites hederagenin cellobioside oleanolic acid cellobioside 2 oleanolic acid-O-glucoside hederagenin-O-glucoside 1 larvae survival ln(x+1) 4 correlation analysis principal component analysis two-way hierarchical clustering BvUGT1 CONCLUSIONS:- an untargeted metabolomics approach identified four saponins as most correlating with resistance to herbivory in Barbarea vulgaris - pyrosequencingprovided 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 forhomologsofenzymesknowntocatalyzesimilarstepsofrelatedpathways in thepyrosequencingdataset - setupandscreenmicrosomepreparationsfor relevant activities& identificationofenzymes in activefractionsby MALDI-TOF-MS - studies of metabolite profile co-segregation with QTLs - exploitationofthesyntenytoArabidopsisthalianafor QTL saturationmapping - Use knowledge of the saponin pathway for bioengineering or molecular breeding of crop plants with increased anti-insecticidal properties