PhD defense, Joel Fürstenberg-Hägg, 2014-02-21
•
3 gefällt mir•573 views
Cyanogenic glucosides (CNglcs) are phytoanticipins that are present in more than 2650 plant species. They play an important role in defense against generalist herbivory due to their bitter taste and their hydrolysis upon tissue disruption results in release of toxic hydrogen cyanide. Zygaena larvae (Zygaenidae, Lepidoptera) both sequester (take up and accumulate) the CNglcs linamarin and lotaustralin from their food plants (Fabacea) and biosynthesize them de novo from valine and isoleucine. The presented research demonstrates that de novo biosynthesis of CNglcs in Z. filipendulae is dependent on the CNglc levels in the food plant. Tight regulation of the de novo biosynthesis, both on the transcript and the enzyme steady-state level, allows precise control of ratios and content of CNglcs. De novo biosynthesis is shown to take place in close proximity to the storage chambers in the skin which enables rapid and continuous refill after usage in predator defense. During pupation, de novo biosynthesis is shut down and CNglcs slowly turned over. De novo biosynthesis of CNglcs in adult females is demonstrated and localized to the skin, while adult males do undexpectedly not de novo biosynthesize CNglcs. Adult females are shown to transfer CNglcs to the eggs following emergence from the pupa, providing protection against predation at this vulnerable developmental stage. It is finally speculated that de novo biosynthesis of CNglcs enabled colonization of and subsequently sequestration from the food plant, and has been maintained for fine-tuning of CNglc levels and composition for an optimized defense capacity.
Empfohlen
Empfohlen
Weitere ähnliche Inhalte
Was ist angesagt?
Ähnlich wie PhD defense, Joel Fürstenberg-Hägg, 2014-02-21
Ähnlich wie PhD defense, Joel Fürstenberg-Hägg, 2014-02-21 (20)
Kürzlich hochgeladen
Kürzlich hochgeladen (18)
PhD defense, Joel Fürstenberg-Hägg, 2014-02-21
- 1. February 21, 2014 Department of Plant and Environmental Sciences Interplay between De Novo Biosynthesis and Sequestration of Cyanogenic Glucosides in Arthropods PhD defense Joel Fürstenberg-Hägg
- 2. Joel Fürstenberg-Hägg, February 21, 2014
- 3. Feeding on plants increases species diversity in both hosts and herbivores Joel Fürstenberg-Hägg, February 21, 2014 Adapted from Fürstenberg-Hägg et al., 2013 Land plant diversity Time (million years ago) Feeding strategy diversity
- 4. VS Ongoing warfare Physical defense: eg. thorns, spines, trichomes Chemical defense: eg. phenolics, terpenes, alkaloids, cyangenic glucosides Joel Fürstenberg-Hägg, February 21, 2014 VS Physical defense: eg. strong skin, tough armour, camoflauge Chemical defense: eg. phenolics, alkaloids, cyanogenic glucosides
- 5. Strategy 1: De novo biosynthesis Joel Fürstenberg-Hägg, February 21, 2014 Production Storage
- 6. Strategy 2: Sequestration Uptake Joel Fürstenberg-Hägg, February 21, 2014 Accumulation Storage
- 7. To de novo biosynthesize or sequester? Controlled Cost efficient Joel Fürstenberg-Hägg, February 21, 2014
- 8. Zygaena filipendulae both de novo biosynthesize and sequester CNglcs Zygaena filipendulae Lotus corniculatus Joel Fürstenberg-Hägg, February 21, 2014
- 9. Zygaena filipendulae both de novo biosynthesize and sequester CNglcs CYP332A3 CYP736A2 Zygaena filipendulae Lotus corniculatus Joel Fürstenberg-Hägg, February 21, 2014 CYP405A2 UGT33A1 CYP79D3/D4 UGT85K2/K3 Jensen et al. 2011, Takos et al. 2011
- 10. Zygaena filipendulae both de novo biosynthesize and sequester CNglcs Zygaena filipendulae Lotus corniculatus Joel Fürstenberg-Hägg, February 21, 2014
- 11. CNglcs in the Zygaena life cycle Adapted from Zagrobelny et al, 2004 Joel Fürstenberg-Hägg, February 21, 2014
- 12. Aims of PhD project How is the pathway regulated? Where does biosynthesis take place? When does biosynthesize take place? Joel Fürstenberg-Hägg, February 21, 2014
- 13. Joel Fürstenberg-Hägg, February 21, 2014 Research paper I Chemical Defense Balanced by Sequestration and De Novo Biosynthesis in a Lepidopteran Specialist Fürstenberg-Hägg J, Zagrobelny M, Jørgensen K, Vogel H, Møller BL and Bak S Published in PLoS ONE
- 14. Larvae de novo biosynthesizing CNglcs experience retardation in growth Developmental studies Joel Fürstenberg-Hägg, February 21, 2014 cyanogenic (+)diet acyanogenic (–)diet L.c. 1 L.c. 0 Adapted from Zagrobelny et al., 2007
- 15. Differences in CNglc amounts and ratios are compensated for by de novo biosynthesis LC-MS Total amounts of CNglcs Composition of CNglcs Joel Fürstenberg-Hägg, February 21, 2014 L.c. 0 L.c. 1 L.c. 2 L.c. 0 L.c. 1 L.c. 2 Linamarin Lotaustralin Adapted from Zagrobelny et al., 2007
- 16. Larvae fed on acyanogenic plants have higher CNglc CNglc biosynthesis biosynthesis enzyme gene transcript steady-state levels levels qRT-PCR L7 larvae fed 2 weeks on cyanogenic or acyanogenic L.corniculatus plants 15 biological replicates Joel Fürstenberg-Hägg, February 21, 2014 Larval gene expression Western blot L5 larvae 10 biological replicates pooled
- 17. Joel Fürstenberg-Hägg, February 21, 2014 Larval anatomy Haemolymph
- 18. CNglc biosynthesis gene transcripts are localised in the fat body and skin qRT-PCR L7 larvae fed 2 weeks on acyanogenic L. corniculatus plants 10 biological replicates Joel Fürstenberg-Hägg, February 21, 2014 Larval tissues
- 19. How to make larval cross-sections Joel Fürstenberg-Hägg, February 21, 2014
- 20. CNglc biosynthesis gene transcripts are localized in fat body and integumental compartments in tube in situ PCR TRITC (rhodamine) labeling Joel Fürstenberg-Hägg, February 21, 2014 L2–L3 larvae, 2 weeks on acyanogenic L. corniculatus Sensory hair Small cavity Sensory hair Small cavity
- 21. CNglc biosynthesis enzymes are localised in the fat body and skin Immunolocalisation 2 weeks on acyanogenic L. corniculatus plants FITC (fluorescein) labeling Control CYP405A2 Joel Fürstenberg-Hägg, February 21, 2014 Small cavity Sensory hair Fat body Epidermis
- 22. Joel Fürstenberg-Hägg, February 21, 2014 Conclusions part I How is the pathway regulated? Regulation according to CNglc content in food-plant Gene transcript and enzyme steady-state level Where does biosynthesis take place? Biosynthesis in fat body and skin Secure rapid and continuous refill
- 23. Joel Fürstenberg-Hägg, February 21, 2014 Research paper II Transcriptional Regulation of De Novo Biosynthesis of Cyanogenic Glucosides throughout the Life-Cycle of the Burnet Moth Zygaena filipendulae (Lepidoptera) Fürstenberg-Hägg J, Zagrobelny M, Jørgensen K, Møller BL and Bak S Published in Insect Biochemistry and Molecular Biology
- 24. When are CNglcs de novo biosynthesized? Adapted from Zagrobelny et al, 2004 Joel Fürstenberg-Hägg, February 21, 2014
- 25. CNglcs may play different roles in imagines Calling Courting Nuptial gift CNglcs Joel Fürstenberg-Hägg, February 21, 2014 CNglcs CNglcs CNglcs Zagrobelny et al., 2007, 2014
- 26. Larvae contains more CNglcs than imagines ~5.00 mg Joel Fürstenberg-Hägg, February 21, 2014 ~2.25 mg –55 % Zagrobelny et al., 2007 Linamarin Lotaustralin
- 27. CNglcs are continuously turned over during pupation, preferentially lotaustralin? LC-MS 4 time points (1, 5, 10 and 15 days) 10 biological replicates Joel Fürstenberg-Hägg, February 21, 2014 Pupae
- 28. Biosynthesis of CNglcs is turned off during pupation qRT-PCR 4 time points (1, 5, 10 and 15 days), 6 + 2 biological replicates Female pupae Male pupae Joel Fürstenberg-Hägg, February 21, 2014
- 29. Female, but not male, imagines biosynthesize CNglcs Thin layer chromatography Val 14C and Ile 14C injection into imagines Joel Fürstenberg-Hägg, February 21, 2014
- 30. Where are CNglcs located in the female? LC-MS Female imaginal tissues 8 biological replicates Joel Fürstenberg-Hägg, February 21, 2014 Female imaginal tissues
- 31. CNglc biosynthesis in female imagines take place mainly in the integument qRT-PCR Virgin and mated females and males, 6 biological replicates each Joel Fürstenberg-Hägg, February 21, 2014 Female imaginal tissues
- 32. CNglcs appear to be transferred into eggs following emergence from pupa LC-MS Eggs dissected from pupae and imagines, and laid eggs 5 biological replicates CNglcs in eggs Joel Fürstenberg-Hägg, February 21, 2014
- 33. No CNglcs are biosynthesized in embryos qRT-PCR Eggs and larvae fed on cyanogenic L. corniculatus plants Joel Fürstenberg-Hägg, February 21, 2014 Eggs and larvae eggs
- 34. Conclusions part II How are CNglc levels regulated during pupation? Decline in CNglcs at beginning of pupation Shift in ratios from 1:1 to 3:1 No biosynthesis during pupation How are CNglc levels regulated in imagines? Sexual differences CNglcs in abdomen, eggs, head and thorax Biosynthesis in abdominal integument CNglcs reallocated to eggs Biosynthesis initiated in hatched larvae Joel Fürstenberg-Hägg, February 21, 2014
- 35. When are CNglcs de novo biosynthesized? Adapted from Zagrobelny et al, 2004 Joel Fürstenberg-Hägg, February 21, 2014
- 36. Concluding remarks De novo biosynthesis of CNglcs is dynamic and regulated according to demand Diet Tissue Development Sex De novo biosynthesis probably evolved prior to sequestration and has been kept to secure and fine-tune CNglc homeostasis… Joel Fürstenberg-Hägg, February 21, 2014
- 37. Joel Fürstenberg-Hägg, February 21, 2014 Perspectives How to avoid autotoxicity? Why no biosynthesis in male imagines? How are CNglcs transported? Can other species both sequester and biosynthesize defense compounds? How have the biosynthetic enzymes evolved?
Hinweis der Redaktion
- Thanks Title Broad introduction to plant-insect interactions Start by showing you this Introduction to plant-insect interactions and chemical defenses Results and conclusions from two research papers Round up with concluding remarks and perspectives
- This is a picture from northern Sweden where I am from – beautiful Huge diversity of plants, you see grasses, bushes, trees, many different species If you was to wander in this beautiful forest, you would discover a huge diversity of insects eating these plants But earth was not always like this
- Plants and arthropods have coexisted for > 400 MY, and their interactions is one reason why we see this extreme plant and herbivore diversity today. Schematic representation show increased plant diversity over time: mosses, ferns, conifers and more recently burst in flowering angiosperms Insects has also diversified along with the plants Remember that beautiful picture of northern Sweden, looks calm and peaceful, but if you scratch the surface there is actually a war going on between plants and insects, this war is increasing the diversity in both host and herbivores
- Insects have evolved mechanisms to tolerate, excrete avoid these chemicals In some specialised species, evolved to use plants own chemicals for their own use Insects caught in the middle, also have to defend against predators Biosynthesize Or can sequester!
- Production Storage
- Uptake Accumulation Storage Know how to handle => evolved after biosynthesis Cannot evolve uptake if no way to handle them Cannot evolve mechanism to handle them if not exposed to them
- In general insects use one strategy Biosynthesis: controlled Sequestration: cost efficient => idea: biosynthesis lost after evolution of sequestration One exception, one insect family has retained both strategies: Zf Focus of PhD: why? How regulated?
- Model organism, biosynthesize lin and lot de novo and sequester from food-plant Lotus corniculatus
- Biosynthetic pathway Zf and Lc Amino acid (Val or Ile) enzymes take both substrates! Aldoxime Nitrile Convergent evolution Newly discovered, nothing known of when where and how it is expressed
- Degradation => keton and HCN Remove stabilizing glucose moiety => α-hydroxynitrile Cleave binding => Keto compound cytotoxic, HCN binds to cytochrome oxidase and inhibit cell respiration chain
- What we know: Zygaena, moth, complete metamorphosis, it passes several larval stages, enters pupation and finally emerges as an adult (imago) 7 larval stages, separated by molting (change to larger skin), fourth larval stage enter diapause reduce 70 % metabolism and sleep through winter, wake up in spring CNglcs found throughout the life-cycle, used as defense Stored throughout the larva, especially in storage compartments in the skin, form which CNglcs can be released as droplets Biosynthesis and sequestration only in larva Mating communication in imagines Nuptial gift transferred from male to female
- Eg. How the pathway is expressed depending on food source. What happens when fed on cyanogenic vs acyanogenic Lotus? CNglcs throughout larva, biosynthesis everywhere or specialized tissues? CNglcs present throughout the life cycle Biosynthesized at particular stages? Sexual difference?
- Answer two question: How is the pathway regulated? Where does biosynthesis take place?
- Cyanogenesis is polymorphic and varies between and within plant species depending on developmental stages, tissues and environmental conditions
- Polymorphism in plants Lc 0 – botanical garden, Lc 1 – Tåstrup, Lc 2 – Kvistgård Compensation/fine-tuning in insects – 50/50 lin/lot
- Regulation on transcriptional and enzym steady-state level Cyanogenic plants, still upregulated => still biosynthesizing although they can sequester Zf has the ability to downregulate the genes when they can sequester
- Where does de novo biosynthesis of CNglcs take place in larvae? Integument – outer protection, cuticle and proteins Sensory hairs – reacting to outer stimuli Large cavities – weak irritation Small cavities – harsh irritation Epidermis – mono cell layer, excreting cuticle during molting Fat body – main energy storage organ and major metabolic organ Gut – uptake of nutrients and CNglcs Malpighian tubules – detoxification and excretion Haemolymph – blood and lymph circulation system
- September, frozen larvae, cryotome, freezing medium => brittle, fell apart Resin embedment (plastic), 1 cm => 1 million sections, beautiful, 2 days, RNA and ezyme degradation Vibratome, 5 % agarose embedment, 30 min in fridge, 10 x thicker, young larva but low expression
- Amplifying genes of interest by PCR on sections Gene specific rhodamin labelling
- Autofluorescent hairs
- When fed on acyanogenic: high expression Cyanogenic: low expression
- Biosynthesis in larvae CNglcs present throughout the life-cycle Biosynthesis at certain stages?
- Larval biosynthesis known half a century (50 years) Found throughout the life-cycle Function as defense compounds in most life-stages Proposed to play additional roles in imagines
- Female calling, attract males Male courting, presenting CNglcs Female accessing quality of male Transfer of CNglcs to female and eggs, 30 % body mass Advantage to imagines to biosynthesize CNglcs
- What happens with CNglc de novo biosynthesis during pupation?
- Puapating larva taken out of cocoon Decline day 1-10, levels do not reach levels in imagines But, ratios are the same as in adults
- Expression in abdomen, fat body, head and thorax, integument UGT involved in other processes Indicates biosynthesis in female imagines => Confirm biosynthesis
- 1-4 reps, 2 sexes, controls: separation of substrate and CNglcs produced by Lotus Lin and lot Females Not males Males replenish reserves after mating? 2 representative mated males (12 more) Why? Protect eggs? Only females => results from females
- Found in eggs, abdomen, head and thorax Biosynthesis in these tissues?
- Pathway expressed in abdomen, baseline expression in eggs, head and thorax Biosynthesis in abdomen, transport of CNglcs to developing eggs (Inner organs pulled out from abdomen, only integument left => similar localization as in larva) CNglc levels throughout development?
- Stable in pupa, increase after emergence Biosynthesized by embryo or transported from female abdomen?
- Transferred from female abdomen into eggs following emergence Biosynthesis turned on in hatched larvae Larvae hatched on acyanogenic plants do not survive ~1 w old L1 larvae survive on acyanogenic plants No biosynthesis in diapaused larvae, 70 % less metabolism
- CNglc biosynthesis is shut down during pupation Biosynthesis is turned on in females prior to eclosion Localized to integument No CNglc biosynthesis in embryos CNglcs are transferred into eggs following eclosion
- Zf able to downregulate the pathway when it can sequester