Abwehr Bei Blattkäfern Mit Eigenen Und Pflanzlichen Giften

Mit der Kraft der zwei Herzen – Abwehr bei Blattkäfern mit eigenen und pflanzlichen Giften

2. Jahrestreffen der Senior Expert Chemists 0.7 - 09. Mai 2009 in Hanau

Wilhelm Boland, MPI für Chemische Ökologie, Jena

A. Burse, S. Discher, K.Tolzin, M. Kunert, R. Kirsch, A. Strauss, J. Svoboda & J. Pasteels Leaf Beetles: Living Jewels

• Diverse and ornamental insect family • 35,000 described species • 2,500 genera

• Subtribe Chrysomelina is adapted to special host plants (ca. 3000 species; monophagous or narrow oligophagous)

Salicacea Betulaceae Brassicaceae Leaf beetles: a liftetime defended

C HO

O H

H O R

H O O R H O : H HO N H H O H O O O H O H H HO N H O H R = H O O R = 3-nitro -p rop io nic a cid Chrysomela populi H H R = H other species: R = 3-nitro -p rop io nic a cid other species: • benzaldehyde other species: • cardenolides • mandelonitrile • curcubitacins • esters of phenylethanol • cardenolides • salicin • juglone • polyoxygenated steroids • (11Z)-eicosenyl acetate • curcubitacins • iridoids • anthraquinones Chrysomela populi: defense in action !

1 cm Defense mechanism of exocrine glands

Architecture and function of the defensive system:

Opening of reservoir Tubercle

Gland reservoir

Gland cells Muscle strand

Larvae attacked by e.g. ants, spiders, birds Gland reservoir with attached gland cells Defensive compounds

Sequestrierung Sequestration O R und R and GlcO esterificationVeresterung von O ofde de novo novo glukosylierteGlycosylated Esterifiedveresterte leaf synthesizedsynthetisierten Blattalkoholeleaf alcohols Blattalkoholealcohols productsProdukten HO O

OGlc OH

SequestrationSequestrierung SalicinSalicin SalicylaldehydSalicylaldehyde

O De novo OH de novo Synthesis, Synthese O Seques- OGlc tration ? 8-Hydroxygeraniol-8- - IridoidIridoid 8-OO--βß--DD--glukosidglucoside

Termonia et al., 2001 Esters from the defensive secretion of C. lapponica

Monosaccharides (e.g. glucose)

R= H or CH3

O O R R O O acylated monosaccharides O O (e.g. O R O

O H O Relative abundance Relative H O HO OH HO OH H OH H H

SPME analysis Non-volatile fraction, (derivat. with MSTFA) Retention time The long way from the leaf to the gland

gland and reservoir

Malpighian tubules

gut

Transport Transport system I system II

gut hemolymph reservoir food

feces Transport C H O H O H CH 2O H system III H O O H O

H O O H HO H H

H Sequestration of Phytogenic Precursors: Salicin

reservoir H O H O C HO

O G lc O H O H Glc glucosidase oxidase

gland

diffusion active transport by membrane-bound hemolymph transport proteins ?

H O C H O H gut H CH 2O H 2 H O O glucosidase ? H O food H O O H HO H H

H membrane-bound transport proteins involved ??? Thioglycosides as Glycomimics

O H H O H C O 2 H O H O X Thioglycosides: O H

X = O salicin X = S thiosalicin  exhibit unique structural similarity to the natural substrates  may adopt the same bioactive conformations

 exhibit exceptional chemical and biological stability

 ideal tools to study transport phenomena of glycosidically bound compounds ! Application of thioglycosides

• larvae fed for certain intervals

• Secretion collected with glass capillaries and quantified (II)

• Haemolymph collected after killing and dissecting legs

glas capillary

• Impregnation of leafs with thioglycosides: (I) (II) Ranunculus repens (H. marginella) Populus canadensis (C.populi, Sample preparation: P. laticolis, P. vitellinae) Salix caprea (x alba) (C. lapponica) sample + 40 µL MeOH/water (1:1), centrifuged  LC-MS „Sequestration“ of Thiosalicin"

2000 HHO O H C H 2OH H O secretion S hemolymphhemolymph H O O H SGlc 1500 H O H H

H 1000

500 fold 300

4 concentration [nmol/mg] concentration 0 2 4 8 16 32 48 time [h] Sequestration of salicin in Chrysomela populi Thioglucosides as mechanistic probes

HO OH

SGlc SGlc SGlc SGlc OH SGal OH

SGlc SGlc SGlc SGlc SGlc SGlc Kuhn et al., PNAS 2004, 101, 13809. Sequestration of iridoid precursors ? gut glandular reservoir O OH OH O cyclase O glucosidase oxidase O OGlc OH isomerase 8-hydroxy- 8-hydroxy-geraniol geranylglucoside iridoid

food mevalonate pathway location ?

HO OH

SGlc SGlc SGlc SGlc OH SGal OH

SGlc SGlc SGlc SGlc SGlc SGlc Kuhn et al., PNAS 2004, 101, 13809. Selectivity of thioglycoside-transport

1400 HO OH H. marginella SGlc P. laticollis 1200 SGlc C. populi P. vitellinae 1000 C. lapponica

800

SGlc

600 HO HO SGlc SGal

GlcS 400 SGlc

concentration [nmol/mg] concentration 200

n.t. n.t. n.t. 0 Selectivity of individual transport Systems; discovery of a network

Precision control unit Positioning for injection: of apillary maximum amount 69 nL

capillary

Transport Transport system I system II

food gut hemolymph reservoir

Kuhn et al., PNAS 2004, 101(38), 13808 feces Transport system III Selectivity of individual transport Systems; microinjection experiments: secretion Phaedon cochleariae

100 H O H O H H O 80 H O H O S H

[%] 60 O H H 40 H thiosalicin 20

relative composition composition relative 0 H O H

(S ) H O 1 h H O 6 h H O (S ) S (S ) 24 h H O H (S ) (E ) 48 h H H

H O (E ) Chrysomela populi 8-OH-Ger-S-glucoside 100

80 H HO (S ) H 60 H O O (S ) (S ) 40 H O H S 20 H O H (S) H 0 PheEt-S-glucoside 1 h 6 h 24 h

48 h relative composition [%] composition relative Selectivity of individual transport Systems; microinjection experiments: frass Phaedon cochleariae

60 H O H O H H O H O 40 H O S H O H H H 20 thiosalicin

[%] 0 H O H

(S ) H O 1 h H O 6 h H O (S ) S (S )

24 h H O H (S ) (E ) relative composition composition relative 48 h H H

H O (E ) Chrysomela populi 8-OH-Ger-S-glucoside 60 50 H HO (S ) H 40 H O 30 O (S ) (S ) 20 H O H S H O H (S) 10 H 0 PheEt-S-glucoside 1 h 6 h 24 h

48 h relative composition [%] composition relative Selectivity of individual transport Systems: in vitro gut incubation

H O H O H H O Phaedon cochleariae H O 60 H O S H O H H H 50 thiosalicin 40

H O H

30 (S ) H O H O H O (S ) S (S ) 20 H O H (S ) (E ) H H

10 H O (E ) relative composition [%) composition relative 0 8-OH-Ger-S-glucoside

H HO (S ) H H O O (S ) (S ) H O H S H O H (S) H Relative concentration of the glucoside mix [%] in the PheEt-S-glucoside gut after in vitro incubation Selectivity of individual transport Systems: in vitro gut incubation

H O H O H H O Chrysomela populi H O 45 H O S H O H 40 H H 35 thiosalicin 30

25 H O H

(S ) H O 20 H O H O (S ) S (S ) 15 H O H (S ) (E ) H H 10 H O (E )

5 relative composition [%) composition relative 0 8-OH-Ger-S-glucoside

H HO (S ) H H O O (S ) (S ) H O H S H O H (S) H Relative concentration of the glucoside mix [%] in the PheEt-S-glucoside gut after in vitro incubation. Selective Glucosylation of d5-8-OH-Ger

70 Feeding or Injection: hemolymph 60 faeces d -Lin-8-OH and 50 glucosidase 5 d5-Ger-8-OH (1:1) 40

30 Glc (%) of total Glc‘stotalof(%) Glc

- NO glucosylation of O - d

8 20 5-Lin-8-OH

- Ger

- 10 5

C H 2 O H d O H 0

C H O H C H 2O H 2

LC-MS analysis after feeding or injection; larvae feeding on leaves Glucoside transport in leaf beetle larvae hc mt ds fb

df df

gut glucosidase

fg mg hg

hc hemocoel, mt Malpighian tubules, ds defensive system, fb fat body, fg foregut, mg midgut, hg hindgut defense aglycon glucoside Release of defensive Early Iridoid H C secretion from a dorsal 3 CHO gland CHO Biosynthesis

Fat body

OHO + Gland NADPH+H +O2 OGlcO Geraniol de novo biosynthesis proceeds via the 6 + NADP +H2O PPi mevalonate pathway 5 2H2O OH GDP HMGR is the most important enzyme GDP(C10) OH in the early biosynthetic sequence 4

?-D-Glu 7 IDP(C5)IDP DMADPDMADP(C5) HMGR is often the key regulatory H O 3 2 1. 2 enzyme Mevalonate OH Inhibition by late metabolites know ((?-)) 2 NADP++CoASH OGlc HMGR OHOH 2 NADPH+2H+

OGlc  HMGR could maintain OGlc Fat body HMG -CoA homoeostasis between sequestered OH OH 1 and de novo produced metabolites OH Acetyl -CoA OH Gut lumen Acetoacetyl -CoA The HMG-CoA reductase of P. cochleariae

. Eukaryotic HMGRs (classI): integrale membrane glycoproteins of the endoplasmatic reticulum . Screening by a homology based PCR approach

SwissPfam Transmembrane domaine (AA 1-378) Linker region (AA 379-448) Catalytic domaine (AA 449-842) entry from Ips pini HMGR (866 residues)

P. cochleariae sequence P. cochleariae sequence (AA 210-490) (AA 624-755) Homologous to HMGR with: Homologous to HMGR with:

Organism HMGCR Similarity Identity Gaped AA Similarity Identity Gaped AA AA residues Ips pini 866 48% 31% 4 91% 83% 0

Dendroctonus jeffreyi 846 46% 31% 3 90% 80% 0

Drosophila melanogaster 920 51% 35% 12 80% 65% 0

Blattella germanica 856 47% 32% 33 95% 86% 0

Agrotis ypsilon 833 40% 24% 6 79% 62% 0

Homo sapiens 888 58% 39% 1 89% 70% 0 HMGR expression in different tissues

• Identification and isolation of the putative HMGR sequence from fat body of P. cochleariae  2,664-bp ORF (888 AS, 97.1 kDa)

De novo Sequestration

PhaedonPhaedon cochleariaecochleariae GastrophysaGastrophysa viridula viridula ChrysomelaChrysomela populi populi

gene gene

HMGR HMGR HMGR

relative to gut gut toto relative relative

normalized normalized

expression expression

Fold Fold

Head Gut Malpig. Fat Glands Head Gut Malpig. Fat Glands Head Gut Malpig. Fat Glands tubules body tubules body tubules body HMGR enzyme activity in different tissues

Phaedon cochleariae 140 Chrysomela populi Enzyme assay 120 14

100 • C labeled HMG CoA as reductase reductase substrate

80  14 CoA

CoA DL-3-[Glutaryl-3- C]-)

- -

HMGHMG pmol/mg/60min pmol/mg/60min 40 • Fat body 30 times higher

20 activity compared to gut

Activity Activity

0 Fat body Gut Malpigh. tubules

 Enzyme activity shows the same pattern like transcript abundance  Involvement of fat body tissue in production of iridoid precursors Identification of 8-hydroxygeraniol-8-O-β- D-glucoside in fat body tissue

349,9 IS 100 + [M+NH4] 100

135,0 -H2O H O H

H O Abundance 50 -H2O H O 153,0 314,9 H O O

H O H Relative Relative

H H 296,9 -H2O

H O + 50 [M] -H2O 332,9 107,1107.1 279,2 261,0 350,9 181,1 231,2 360,7 391,2 413,1 451,4 Relative Abundance Relative 134,3 488,1 0 50 100 150 200 250 300 350 400 450 500 m/z

0 IS = thioglucoside as internal standard 0 2 4 6 8 10 12 14 16 18 20 Time (min) Heterologous expression – catalytic domain

N C

Cloning into Transformation in different Purification by Ni-NTA expression vector E. coli strains

P D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 M kDa 150 100 75

Enzyme assay Regulation of HMGR

Heterologous expressed protein Raw enzyme extract of fat body tissue

(IC50~1.2 mM)

• 8-Hydroxygeraniol acts as a competitive inhibitor of HMGR • Activity is modulated by iridoid precursors Homology 3D-modelling of HMGR

• Based on 3D-structure of the catalytic domain of human HMGR • Homotetrameric Structure

• H-bond and hydrophobic interactions between the monomers of the protein and 8-hydroxygeraniol

• S255, D338 from monomer 1

• N441 from monomer 2

• Interaction of 8-hydroxygeraniol corresponds to the position of HMG within the active site

Wolfgang Brandt - Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Halle/Saale, Germany Release of defensive Biosynthesis of Terpenoids

H 3C secretion from a dorsal C H O in Leaf Beetle Larvae gland C H O

O Fat Body: NADPH+H++O Gland 2 O 1. Early terpenoid biosynthesis Geraniol 6 + (geraniol) NADP +H2O PPi 5 2. -Functionalisation (P450 ??) 2H2O O H (8-hydroxygeraniol) GDP(C10) O H 3. Glucosyltransfer 4 (8-Glc-geraniol) β-D-Glu 7 IDP(C5) DMADP(C5) 3 H2O 2 Glandular reservoir: Mevalonate 1. Glucosidase O H 2. Oxidase (―) 2 NADP++CoASH O G lc 3. Cyclase HMGR

O H 2 NADPH+2H+ 4. Isomerase

O G lc Fat body HMG-CoA The hemolymph level of 8-Glc O H 1 controls HMGR activity in

Acetyl-CoA the fat body. O H Acetoacetyl-CoA Gut lumen Burse et al., Insect Biochem. & Mol. Biol. 2008 Chemical defense in Chrysomelinae leaf beetles: Host shift requires adaptation of transporters

Habit Host Salicylaldehyde Ester (Oxidase) (Transferase)

(Finl) Finland Salix borealis +++ + (MC) France Salix borealis +++ +++ (Cze) Czech Republic Betula pendula - +++

Betula

Predator Parasyrphus pendula nigritarsis larva

Chrysomela lapponica Salix borealis Host plant shift Uptake of salicin and Uptake of leaf leaf alcohols alcohols The Glucoside-based Transport-Network

Why Glucosides ?

► Polar compounds, unable to penetrate membranes (common in food) ► Require transport systems ► Specific transport allows specific addressing of organs or organells (different selectivity of local transport systems allows import/export) ► Can be safely circulated in the hemolymph ► Easily cleaved into glucose and the aglycone in the „target organ or cell“ (no energy required) ► Aglycons are processed to the defensive compounds and utilized

► Glucoside transporters may have general significance (e.g. steroids) Thank you !

Prof. J. Pasteels, Brussels

Stefan Bartram Angelika Berg Antje Burse Sabrina Discher Jürgen Kuhn Eva Petterson Astrid Soe Karla Tolzin-Banasch Max Planck Society