Ps Pf Pe

Pe

Pe

Pe

Pn Px

Pl

Pw

Pc

9 6 2

14

1

7

3 10

16

11

4

“Glucosinolate diversity in New Zealand alpine

Pachycladon"

October 07

Claudia Voelckel *1, M Reichelt2, PB Heenan3, PJ Lockhart1

*c.voelckel@massey.ac.nz

1

3

2

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S. Joly, unpublished super network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

20

40

60

E1 E2 E3 F1 F2 F3 E F

C3 C4

0

22

44

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

0

2

4

6

8

10

12

14

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

0.2

0.4

0.6

0.8

1

E1 E2 E3 F1 F2 F3

CT

5

CT

2

CT

3

CT

4

CT

1

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!

0

8

16

24

E1 E2 E3 F1 F2 F3 E F

To

tal q

ue

rce

tin g

lyco

sid

es

(μg

/mg

dry

we

igh

t)000.0E+0

90.0E+3

180.0E+3

E1 E2 E3 F1 F2 F3 E FDry

we

igh

t-co

rre

cte

d p

ea

k a

rea

s o

f to

tal c

inn

am

ic a

cid

gly

cosi

de

s

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