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“Glucosinolate diversity in New Zealand alpine Pachycladon "

1. “Glucosinolate diversity in New Zealand alpine Pachycladon ". Claudia Voelckel * 1 , M Reichelt 2 , PB Heenan 3 , PJ Lockhart 1. 2. October 07. *c.voelckel@massey.ac.nz. 3. Outline. 1. Pachycladon – A recent, endemic and alpine radiation.

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“Glucosinolate diversity in New Zealand alpine Pachycladon "

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  1. 1 “Glucosinolate diversity in New Zealand alpine Pachycladon" Claudia Voelckel *1, M Reichelt2, PB Heenan3, PJ Lockhart1 2 October 07 *c.voelckel@massey.ac.nz 3

  2. Outline 1. Pachycladon – Arecent, endemic and alpine radiation 2. Glucosinolates (GLS) – Metabolism and Diversity 3. Natural variation of glucosinolates in P. ensyii and P. fastigiata 4. Glucosinolate profiles across the Pachycladon radiation 5. Outlook Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook

  3. NZ Alpine Cress (Pachycladon, Brassicaceae) • 8 South Island species • recent radiation (< 1 mya) Greywacke clade: P. fastigiata, P. enysii, P. stellata Schist clade: P. novae-zealandiae, P. wallii Geological generalists: P. cheesemanii, P. exilis, P. latisiliqua S. Joly, unpublished super network Questions: Tools: • Ecological drivers of radiation? • Pathways and genes under selection? • transcript-, protein- and metabolite profiling • candidate gene studies • EST libraries and SNPs Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook

  4. (Aliphatic) Glucosinolates – Synthesis Methionine Chain elongation pathway Homomethionine (C3 GLS) GLS core pathway Methylthioalkyl GLS Dihomomethionine (C4 GLS) Methylsulfinylalkyl GLS Side chain modification Alkenyl GLS Hydroxalkyl GLS Hydroxalkenyl GLS Pachy intro GLS introP. enysii vs P. fastigiata Radiation profiling Outlook

  5. (Aliphatic) Glucosinolates – Synthesis and hydrolysis Methionine Chain elongation pathway Homomethionine (C3 GLS) GLS core pathway Methylthioalkyl GLS Dihomomethionine (C4 GLS) Methylsulfinylalkyl GLS Side chain modification Alkenyl GLS Hydroxalkyl GLS Hydroxalkenyl GLS GLS hydrolysis Thiocyanates Isothiocyanates Nitriles (Eithionitriles) Oxazolidine-2-thione Pachy intro GLS introP. enysii vs P. fastigiata Radiation profiling Outlook

  6. (Aliphatic) Glucosinolates – Synthesis and hydrolysis genes Methionine CYP79, CYP83, C-S lyase, SGT, SOT Chain elongation pathway Homomethionine (C3 GLS) GLS core pathway Methylthioalkyl GLS Dihomomethionine (C4 GLS) MAM, MAM-I, MAM-D, BCAT4 FMO Methylsulfinylalkyl GLS AOP2 AOP3 Side chain modification Alkenyl GLS Hydroxalkyl GLS GS-OH Hydroxalkenyl GLS GLS hydrolysis myrosinase ESP ESM1 Thiocyanates Isothiocyanates Nitriles (Eithionitriles) Oxazolidine-2-thione Pachy intro GLS introP. enysii vs P. fastigiata Radiation profiling Outlook

  7. P. enysii vs P. fastigiata – Sampling P. fastigiata P. enysii alpine (1485 m) high alpine (1885 m) glabrous hairy Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook

  8. Pe vs Pf – Predictions from microarray study – GLS synthesis Gene Regulation (log ratio) Prediction Test 0.80 0.94 0.71 0.73 0.75 Chain elongation MAM1(At5g23010) AtLeuD1 (At2g43100) AtLeuD2 (At3g58990) AtIMD1(At5g14200) AtIMD3 (At1g31180) More C4 GLS in P. enysii GLS (μmol/g dw) E1 E2 E3 F1 F2 F3 E F Side chain modification AOP2 (At4g03060) AOP3 (At4g03050) 1.27 2.31 More Alkenyl and Hydroxy-alkyl GLS in P. ensyii GLS (μmol/g dw) E1 E2 E3 F1 F2 F3 E F Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook

  9. Pe vs Pf – Predictions from microarray study – GLS hydrolysis Gene Regulation (log ratio) Prediction Test P. enysii • 6.29 • - 4.62 Nitriles inP. enysii Isothiocyanates in P. fastigiata Hydrolysis ESP (At1g54040) ESM 1 (At3g14210) HP (μ mol/g fw) P. fastigiata HP (μ mol/g fw) Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook

  10. P. enysii vs P. fastigiata – 5 chemotypes in the wild = Pachycladon chemotypes Arabidopsis ecotypes Allele configuration Elong/AOP CT1 e.g. Cape Verdi island (+11 more) 4/3 CT2 3/1 e.g. Wassilewskija (+ 1 more) e.g. Cape Verdi island (+11 more) 4/3 CT3 CT4 e.g. Canary islands (+11 more) 3/3 CT5 4Pentenyl 4MSOB 3MSOP 5MSOP 8MSOO 7MSOH 6MSOH 4MOI3M Allyl 4MTB 3 Butenyl 3MTP ? Landsberg 3/2 ? Dijon 4/2 ? Columbia 4/1 Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook

  11. P. enysii vs P. fastigiata – Chemotype frequencies Relative frequency E1 E2 E3 F1 F2 F3 • Each site dominated by one chemotype, except F3 • Local adaptation or drift? Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook

  12. Pachycladon radiation profiling • common garden study with • 5 species = 3 lineages Greywacke clade: P. fastigiata, P. enysii Schist clade: P. novae-zealandiae Geological generalists: P. cheesemanii, P. exilis • similar-aged plants harvested separately for roots and shoots • roots and shoots sub-sampled for transcript-, protein- and glucosinolate analysis • work in progress but glucosinolate data already available! Pachy intro GLS intro P. enysii vs P. fastigiataRadiation profiling Outlook

  13. Radiation profiling: Leaf GLS patterns ≠ phylogeny ≠ Pachy intro GLS intro P. enysii vs P. fastigiataRadiation profiling Outlook

  14. Radiation profiling: Root GLS patterns ≠ phylogeny ≠ Pachy intro GLS intro P. enysii vs P. fastigiataRadiation profiling Outlook

  15. Radiation profiling: zooming in on individual compounds • Pn+Pc make S-2-OH-3-butenyl whereas Pe+Pf+Px do not • leaves produce almost no indolyl GLS but roots of Pn+Pc produce 1MOI3M and 4MOI3M and Pf+Pn roots produce 4OHI3M • roots contain the MT precursors of the main leaf compounds (e.g. Pf+Px roots contain 3MTP while Pf+Px leaves contain 3MSOP) • Pn leaves+roots have highest 4MSOB levels (precursor of anticancer compound sulforaphane) Pachy intro GLS intro P. enysii vs P. fastigiataRadiation profiling Outlook

  16. Summary & outlook • GLS chemotype diversity in two species (Pe, Pf) • GLS profiles do not reflect phylogenetic relationships • GLS loci with differential evolution in Pachycladon: GS-Elong (MAM1), GS-AOP (AOP2), GS-OH, tryptophane-specific GLS genes • GS-OX (FMO) more active in the shoots • difference in GLS hydrolysis in Pe and Pf correlated with strong differential expression of ESP and ESM1 Next: • What’s driving within- and between species differences in GLS profiles? Stochastic processes or natural selection? • Why are there more of the non-oxidized GLS precursors in the roots? • Pe and Pf differ strongly in GLS hydrolysis – what about the other species? • Polymorphisms and/or molecular signatures of selection in ESP, ESM1, MAM1, AOP2? Links between biosynthetic and hydrolytic loci? • Cis- or trans-regulation responsible for differential expression of GLS genes? Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook

  17. Acknowledgements Allan Wilson Centre – Simon Joly, Richard Carter Landcare Research –Peter Heenan, Kerry Ford HortResearch –Bart Janssen MPI for Chemical Ecology – Michael Reichelt, Jonathan Gershenzon Funding –Marsden & Humboldt Foundation My New Zealand Humboldt hosts – Pete Lockhart & Trish McLanaghan YOU!

  18. Pe vs Pf – Predictions from microarray study – Flavonoids Gene Regulation (log ratio) Prediction Test Total quercetin glycosides (μg/mg dry weight) 1.14 1.54 Quercetin synthesis FLS (At5g63580) F3’H (At5g07990) More quercetin inP. enysii E1 E2 E3 F1 F2 F3 E F More sinapates in P. enysii Sinapate synthesis FAH1 (At4g36220) Dry weight-corrected peak areas of total cinnamic acid glycosides 0.88 E1 E2 E3 F1 F2 F3 E F Extra

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