“glucosinolate diversity in new zealand alpine pachycladon" october 07 claudia voelckel * 1,...
TRANSCRIPT
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“Glucosinolate diversity in New Zealand alpine
Pachycladon"
October 07
Claudia Voelckel *1, M Reichelt2, PB Heenan3, PJ Lockhart1
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1. Pachycladon – A recent, 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
Outline
NZ Alpine Cress (Pachycladon, Brassicaceae)
8 South Island species recent radiation (< 1 mya)
Ecological drivers of radiation? Pathways and genes under selection?
Questions: transcript-, protein- and metabolite profiling candidate gene studies EST libraries and SNPs
Tools:
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S. Joly, unpublished super network
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
Methionine Chain elongation pathway
Homomethionine (C3 GLS)Dihomomethionine(C4 GLS)
Methylthioalkyl GLS
Methylsulfinylalkyl GLS
Alkenyl GLS Hydroxalkyl GLS
Hydroxalkenyl GLS
GLS core pathway
Sid
e c
hain
mod
ifica
tion
(Aliphatic) Glucosinolates – Synthesis
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
Methionine Chain elongation pathway
Homomethionine (C3 GLS)Dihomomethionine(C4 GLS)
Methylthioalkyl GLS
Methylsulfinylalkyl GLS
Alkenyl GLS Hydroxalkyl GLS
Hydroxalkenyl GLS
GLS core pathway
GLS hydrolysis
Thiocyanates Nitriles (Eithionitriles)
Isothiocyanates Oxazolidine-2-thione
Sid
e c
hain
mod
ifica
tion
(Aliphatic) Glucosinolates – Synthesis and hydrolysis
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
Methionine Chain elongation pathway
Homomethionine (C3 GLS)Dihomomethionine(C4 GLS)
Methylthioalkyl GLS
Methylsulfinylalkyl GLS
Alkenyl GLS Hydroxalkyl GLS
Hydroxalkenyl GLS
GLS core pathway
GLS hydrolysis
Thiocyanates Nitriles (Eithionitriles)
Isothiocyanates Oxazolidine-2-thione
Sid
e c
hain
mod
ifica
tion
(Aliphatic) Glucosinolates – Synthesis and hydrolysis genes
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
MAM, MAM-I, MAM-D, BCAT4
CYP79, CYP83, C-S lyase, SGT, SOT
FMO
AOP2 AOP3
GS-OH
myrosinase
ESM1 ESP
P. enysiiP. fastigiata
alpine (1485 m) high alpine (1885 m)glabrous hairy
P. enysii vs P. fastigiata – Sampling
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
Gene Prediction
Chain elongation
MAM1(At5g23010)AtLeuD1 (At2g43100)AtLeuD2 (At3g58990)AtIMD1(At5g14200)AtIMD3 (At1g31180)
Side chain modificationAOP2 (At4g03060)AOP3 (At4g03050)
Regulation (log ratio)
0.800.940.710.730.75
1.272.31
More C4 GLS in P. enysii
More Alkenyl and Hydroxy-alkyl GLS in P. ensyii
0
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40
60
E1 E2 E3 F1 F2 F3 E F
C3 C4
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66
E1 E2 E3 F1 F2 F3 E F
Methylsulfinyl Alkenyl
Test
GLS
(μ
mo
l/g d
w)
GLS
(μ
mo
l/g d
w)
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
Pe vs Pf – Predictions from microarray study – GLS synthesis
E1 E2 E3 F1 F2 F3 E F
E1 E2 E3 F1 F2 F3 E F
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Isothiocyanates Nitriles/Epithionitriles
Allyl 3MTP
01234567
Isothiocyanates Nitriles/Epithionitriles
3MSOP
Hydrolysis
ESP (At1g54040)
ESM 1 (At3g14210)
6.29
- 4.62
Nitriles in P. enysii
Isothiocyanatesin P. fastigiata
P. enysii
P. fastigiata
HP
(μ
mo
l/g f
w)
HP
(μ
mo
l/g f
w)
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
Gene Prediction Regulation (log ratio)
Test
Pe vs Pf – Predictions from microarray study – GLS hydrolysis
3MS
OP
4MS
OB
7MS
OH
5MS
OP
4Pen
teny
l
4MO
I3M
6MS
OH
3MT
P
4MT
B
8MS
OO
3 B
uten
yl
Ally
l
CT1
CT2
CT3
CT4
CT5
Allele configuration
Arabidopsis ecotypes
Elong/AOP
4/3
3/1
4/3
3/3
e.g. Cape Verdi island (+11 more)
e.g. Canary islands (+11 more)
e.g. Cape Verdi island (+11 more)
e.g. Wassilewskija (+ 1 more)
Landsberg DijonColumbia
3/2 4/24/1
Pachycladon chemotypes
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
P. enysii vs P. fastigiata – 5 chemotypes in the wild
=
? ??
Each site dominated by one chemotype, except F3
Local adaptation or drift?
Re
lativ
e f
req
ue
ncy
0
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E1 E2 E3 F1 F2 F3
CT
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CT
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CT
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Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
F3F1 F2E3E1 E2
P. enysii vs P. fastigiata – Chemotype frequencies
similar-aged plants harvested separately for roots and shoots
Greywacke clade: P. fastigiata, P. enysii
Schist clade: P. novae-zealandiae
Geological generalists: P. cheesemanii, P. exilis
common garden study with 5 species = 3 lineages
roots and shoots sub-sampled for transcript-, protein- and glucosinolate analysis
work in progress but glucosinolate data already available!
Pachycladon radiation profiling
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
Radiation profiling: Leaf GLS patterns ≠ phylogeny
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
≠
Radiation profiling: Root GLS patterns ≠ phylogeny
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
≠
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
Pn leaves+roots have highest 4MSOB levels (precursor of anticancer compound sulforaphane)
roots contain the MT precursors of the main leaf compounds (e.g. Pf+Px roots contain 3MTP while Pf+Px leaves contain 3MSOP)
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
Radiation profiling: zooming in on individual compounds
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?
Cis- or trans-regulation responsible for differential expression of GLS genes?
Polymorphisms and/or molecular signatures of selection in ESP, ESM1, MAM1, AOP2? Links between biosynthetic and hydrolytic loci?
Pe and Pf differ strongly in GLS hydrolysis – what about the other species?
Why are there more of the non-oxidized GLS precursors in the roots?
Summary & outlook
Pachy intro GLS intro P. enysii vs P. fastigiata Radiation profiling Outlook
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!
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To
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lyco
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(μg
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we
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90.0E+3
180.0E+3
E1 E2 E3 F1 F2 F3 E FDry
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Gene Prediction Regulation (log ratio)
Test
Quercetin synthesisFLS (At5g63580)F3’H (At5g07990)
1.141.54
More quercetin in P. enysii
Sinapate synthesisFAH1 (At4g36220) 0.88 More sinapates in
P. enysii
Extra
E1 E2 E3 F1 F2 F3 E F
E1 E2 E3 F1 F2 F3 E F
Pe vs Pf – Predictions from microarray study – Flavonoids