De novo biosyntheses versus sequestration in leaf beetles a mechanistic approach by stable isotopes and molecular biology

Transcription

1 De novo biosyntheses versus sequestration in leaf beetles a mechanistic approach by stable isotopes and molecular biology Astrid Søe, Antje Burse, Stefan Bartram, Wilhelm Boland Max Planck Institute for Chemical Ecology, Germany (Jena, GASIR meeting, 11th ct. 2005)

2 Secretion of defense compounds Predator H 3 C CH De novo production in insects? CH Sequestration of plant substances? CH 3

3 Biosynthesis of monoterpenes in the glandular reservoir

4 Biosynthesis of monoterpenes in the glandular reservoir Glc H 8-Hydroxygeraniol-8--β-D-glucoside H 2 β-d-glucose Glandular cells H 8-Hydroxygeraniol H ½ 2 H 2 Reservoir 8-xogeranial H 3 C CH CH Plagiodial CH 3 H 3 C CH CH 3 CH Chrysomelidial

5 Terpene biosynthesis Pyruvate Acetyl-CoA HMG CoA reductase Mevalonic acid IPP (C5) ( ) IPP (C5) DMAPP (C5) GPP synthase MVA-pathway (in cytosol) GPP (C10) Phosphatase Cytochrome P-450 oxygenase β-d-glucosidase Insect de novo 8-Hydroxygeraniolglucoside Methylerythritol-4-P fosmidomycin Deoxy-D-xylulose-5-P (DX) Glyceraldehyde-3-P MEP-pathway (in plastids) Plant de novo (insect sequestered)

6 Questions Are precursors for defensive monoterpenes de novo synthesized or sequestered? Where (in the larvae) are they synthesized?

7 HMG CoA reductase expression Relative unit in % Phaedon cochleariae Gastrophysa viridula

8 Catalytic activity of GPP synthase in fat body tissue + IPP DMAPP (C 5 ) + 14 C-IPP (C 5 ) 14 C-GPP (C 10 ) 14 C-FPP (C 15 ) + IPP GGPP (C 20 ) ADC1 B, raga 10mL tube (AXEL\ D) Radio-GC cps G F P. cochleariae Bequerel ADC1 B, raga 10mL tube (D:\HPCHEM\1\DATA\AXEL\ D) cps 34 min (terpene secreting species) H C. populi (species sequestering plant aromatic compounds) 25 uv 100 TCD1 B, (AXEL\ D) Geraniol Farnesol min Standard min

9 13 C labeling experiments 13 C-DX JA 1. Addition of isotopically labeled compunds ( 13 C-DX or 13 C-Glc) to plants 2. Induction of terpenoid biosynthesis by jasmonic acid (JA) 3. Allow larvae to feed 4. Collect defensive secretion and volatiles Soe, Bartram, Gatto and Boland (2004) Isotopes Environ. Health Stud. 40, Define 13 C/ 12 C-ratios in volatiles and secretion by GC-C-IRMS and in solid samples by EA-IRMS H H 13CH 2 13 CH 3 13 C DX: 13CH HC H C Glc: H 13CH H 13CH 13 CH H D D H H

10 Leaf volatiles from Rumex obtusifolius 1500 Non-labeled 13 C-DX 13 C-Glc Plant volatiles δ 13 C [ ] Retention time [min] 1: cimene, 2: E-β-caryophyllene, 3: Bergamotene, 4: C 16 H 24, 5: TMTT, 6: C 20 H 30

11 Insect monoterpene and plant bulk material INSECT SECRETIN PLANT MATERIAL INSECT SECRETIN PLANT MATERIAL 800 Gastrophysa viridula on Rumex obtusifolius Phaedon cochleariae on Brassica rapa H 600 Glc δ 13 C [ ] Phaedon cochleariae on Brassica oleracae Phaedon cochleariae on Amoraciae rusticana Non-labeled 13C-DX 13C-Glc Non-labeled 13C-DX 13C-Glc Non-labeled 13C-DX 13C-Glc Non-labeled 13C-DX 13C-Glc De novo biosynthesis

12 The picture is different in another leaf beetle larvae INSECT PLANT Plagiodera versicolora on Salix fragilis Chrysomelidial JA treatment H 2 treatment δ 13 C [ ] Plagiodial JA treatm ent H 2 treatment Non-labeled 13C-DX 13C-Glc Non-labeled 13C-DX 13C-Glc The larvae seem to utilize plant precursors for their own defense H Glc?

13 Secretion from the DX labeling experiments Secretion from Gastrophysa and Phaedon (on Rumex and Brassica) 3000 Secretion from Plagiodera versicolora (on Salix) δ 13 C [ ] δ 13 C [ ] Non-labeled 13C-DX 13C-Glc DX-painted Glc-painted 0 Non-labeled 13C-DX 13C-Glc DX-painted Glc-painted fos-dx-paint Thus, the larvae cannot use the DX directly, but metabolic compounds from the plant

14 Conclusions The precursor (8-hydroxy-geraniol-glucoside) is present and produced in the fatbody of the de novo producing species. The isotopically labeled terpene precursor, 13 C-DX, readily entered the plant biosynthesis. The species, G. viridula and P. cochleariae, did not seem to take up any precursors for their defense. However, the species, Pl. versicolora and Phr. laticolis did take up precursors from their host plants (sequestration). By blocking the MEP-pathway it was shown that DX could not directly be used by the larvae, but had to be metabolized by the plants. The plant precursor could be 8-hydroxy-geraniol-glucoside (or a structurally related terpene-glucoside).

15 Thanks to: Prof. Wilhelm Boland Prof. Jacques Pasteels Antje Burse, Stefan Bartram, Nathalie Gatto, Sabrina Discher, Lihua Nie, Karla Tolzin, Janine Seyfferth, Maritta Kunert & Angelika Berg all members of the Department Bioorganic Chemistry And thank you for your attention!

16 Measurement method Injector Reference C 2 GCT GC Combustion Cu, 850 C Source IRMS

17 Defense strategies in Chrysomelina larven De novo synthesis of iridoid monoterpenes gut H glandular reservoir H cyclase 8-hydroxygeranylglucoside Glc? food? glucosidase? H 8-hydroxy-geraniol 8-hydroxy-geranylglucoside oxidase isomerase iridoid MVA Sequestration of salicylaldehyde gut H glandular reservoir H salicin Glc? food? glucosidase saligenin H oxidase H salicylaldehyde Sequestration and esterification of leaf alcohols gut glandular H amino acids reservoir Glc leaf alcohol glucosidase butyryl transferase + H R leaf alcohol? glucoside leaf alcohol butyric acid leaf alcohol ester? food R

18 Terpenoid biosynthesis pathways glucose glycolysis MEP pathway CH P- H glyceraldehyde-3-p DXP-synthase pyruvate C 2 H C -P H deoxy-d-xylulose-5-p (DX-P) DXP-reductoisomerase Chloroplast H H C -P H methylerythritol-4-p monoterpenes (C 10 ) δ 13 C~ -31 diterpenes (C 20 ) isopentenyl diphosphate (IDP) pyruvate CH C 2 pyruvatedehydrogenase S-CoA acetyl-coa Cytosole H 3 x AcCoA HC H ; d13c~31 mevalonic acid -PP sesquiterpenes (C 15 ) δ 13 C~ -38 steroids (C 30 ) ~4 13 C-depletion MVA pathway Jux, A.; Gleixner, G. and Boland, W. (2001). Angew. Chem. Int. Ed. 40 (11), CH 2 H 1. glucoside cleavage CH 1. cyclisation 2. oxidation 2. isomerisation CH Glc glucoside of 8-hydroxy-geraniol CH chrysomelidial

19 Evolution of chemical defense strategies in leaf beetles CH 2 H Glc Termonia et al., PNAS 2001 C. interrupta* C. knabi* C. mainensis* C. lapponica-que C. lapponica-mc C. lapponica-cze C. lapponica-finl C. sp* C. aenicollis* C. littorea* C. falsa* C. walshi* C. interna* C. salicivorax C. scripta C. laurentia C. confluens C. schaefferi C. collaris C. cuprea C. populi C. tremulae C. 20punctata L. aenea Pl. versicolora P. vitellinae P. tibialis P. laticolis Ph. coclearae G. viridula G. cyanea Pl. viridipennis 3. Esterification of host plant derived leaf alcohols with de novo synthesised butyric acids 2. Sequestration of aromatic aldehydes H 1. De novo synthesis of iridoid monoterpenes

20 Detection of glucosidically bound 8-hydroxygeraniol I. De novo synthesis HPLC-MS Protein (BCA) [mg/ml] Not detectable 1.7 Malpigh. tub. N. d. 3.1 Head N. d. 3.5 Detectable 1.5 Gut N.d. 5.6 Malpigh. tub. N. d. 4.1 Head N. d. 2.9 Fat body N. d. 1.5 P. cochleariae Tissue H C. populi [M-H2+NH4] H H H H Gut Fat body