WP4: Garlic sulphur biochemistry (P2,P3)

P2: HRI Wellesbourne Brian Thomas, Lol Trueman, Linda Brown, Brian

Smith, Gareth Griffiths

P3: The University of Liverpool, UK

Hamish Collin, Rick Cosstick, Brian Tomsett, Meriel Jones

Angela Tregova, Jill Hughes, Jon Milne Mark Wilkinson, Gloria van der Werff

WP4: Objectives

1. Identify intermediates in alliin biosynthetic pathway (P3)

2. Identify developmental control points on CSO synthesis and translocation (P2)

3. Identify genes with altered expression and/or involved in alliicin synthesis (P2,P3)

1. Identify intermediates in alliin biosynthetic pathway

Review knowledge of alliin biosynthesisBring improved HPLC methodology into

use in our laboratory Standards – purchase, synthesis, gifts, mass

spectrometry Gradient elution

Develop experimental protocols Tissue culture Garlic cloves

Biosynthetic pathway for garlic flavour precursors

SO42- SO3

2- S2- cysteine

glutathione(γ-glu-cys-gly)

S-methyl-γ-glu-cys

gly

S-methylcysteine

methiin

glu

trans-peptidase

oxidase

S-2-CP-γ-glu-cys

gly

S-trans-1-propenyl-γ-glu-cys

S-trans-1-propenylcysteineoxidase

trans-peptidaseglu

HCOOH

S-trans-1-propenylcysteine sulphoxide(isoalliin)

S-methylglutathioneS-(2-carboxypropyl)-glutathioneS-allylglutathione

S-allyl-γ-glu-cys

gly

S-allylcysteine

glu trans-peptidase

oxidase

S-allyl group(unknown sources)

valine & methacrylateserine

oxidase

S-allylcysteine

S-allyl-cysteine sulphoxide(alliin)

Biosynthetic capacity of garlic callus

alliin allyl cysteine isoalliin propiin propyl cysteineallyl thiol 10,1;10,1 10; 10,1propyl thiol 10; 1;10allyl cysteine 10;10,1propenyl cysteine 1;10,1propyl cysteine 10,1;10,1

Incubation for 5 days with 10mM or 1mM substrateIncubation for 12/15 days with 10mM or 1mM substrate

Conclusion:

These experiments suggest that in vivo the general reaction shown may occur:-

Alk(en)yl thiol Alk(en)yl cysteine

Alk(en)yl CSO

Glutathione-S-transferases

•Garlic leaf proteins - glutathione affinity matrix•Single step gives substantial purification

Fractions on SDS gel

25 kDa

substrate glutathionepropyl alcohol +allyl alcohol +methacrylic acid +allyl thiol +metabolite soup +allyl glutathionecarboxypropyl glutathionepropyl glutathione

No clear potential GST substrate

2. Identify developmental control points on CSO synthesis and translocation

Baseline data on garlic developmentResource allocation during

developmentDeveloped and tested theories:

Whether roots are an important source of S for developing bulbs

Whether CSOs are synthesised in leaves and transported to bulbs

Identify developmental control points on CSO synthesis and translocation

Growth studies of garlic (Messidrome, Printanor)

hydroponic versus pots

SO42-uptake using isotope labelling

effects of root and leaf removal

For controlled growth, greenhouse (and UK climate)

Measurements during growth

•Leaf number, bulb weight

•N, S, C, protein, CSO

Hydroponic vpot-grown Printanor - Leaf weight

0

5

10

15

20

25

0 50 100 150 200 250

Days after planting

Mea

n m

ass

of le

af (g

, n=3

)

Hydoponic-grown Printanor

Pot-grown Printanor

Hydroponic-grown garlic - comparison of bulb formation

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Days after planting

Fres

h w

eigh

t of c

love

Printanor clove

Messidrome Clove

Garlic growth and S partition

0.0

0.1

0.2

0.3

29 56 77 109 141 169 203Days after planting

Tota

l Su

lph

ur

Co

nte

nt

(g) Root

Leaf

Clove

0

500000

1000000

1500000

2000000

56 109 141 169 203Days after planting

CS

O c

on

ten

t Root

Leaf

Clove

1 2 3 4 1 2 3 4

Four stages in bulb development

Early growth phase: Day 0 – 40/70 Uses stored nutrients

Late growth phase: Day 40/70 - 150 roots, leaves grow rapidly

C, protein accumulate in leaves

S stored in roots

Four stages in bulb development

Bulb initiation: Day 150 – 200 temperature and day-length dependent S, N, C, protein and CSOs decline in

roots and leaves but accumulate in bulbs rise in CSO synthesis roots die

Four stages in bulb development

Bulb maturity: Day 200 Turgor loss as leaves and roots senesce S, N, C, protein fall in leaves, roots, and

rise in bulbs Neck closure and bulb matures.

Sulfur uptake and distribution in more detail

grow hydroponically

use isotope labelled sulfur stable heavy isotope sulfur-34

Measure total S, 34/32S ratio (delta value)

0

50

100

150

200

0 25 50 75 100 125 150 175 200 225

Days after planting

Fre

sh w

eig

ht

(g) Clove

Leaf

Root

Distribution and remobilizationof sulphur taken up early

Distribution and remobilizationof sulphur taken up late

* * * * * * * * * * *

* * * * * * * * * * *

34S32S

A

B

Growth pattern in Year 2 experiment

Sulfur labelling design

Sulpur accumulation in system A plants

0

50

100

150

200

250

05/0

4/02

12/0

4/02

19/0

4/02

26/0

4/02

03/0

5/02

10/0

5/02

17/0

5/02

24/0

5/02

31/0

5/02

07/0

6/02

14/0

6/02

21/0

6/02

28/0

6/02

05/0

7/02

12/0

7/02

Date

Tota

l m

ass

in m

g

Clove

Leaf

Root

Total

34S 32S

Year 3 hydroponic garlic

0

50

100

150

200

25005

/04/

02

19/0

4/02

03/0

5/02

17/0

5/02

31/0

5/02

14/0

6/02

28/0

6/02

12/0

7/02

26/0

7/02

d v

alu

e

Bulb

Leaf

Root

0

50

100

150

200

05/0

4/02

19/0

4/02

03/0

5/02

17/0

5/02

31/0

5/02

14/0

6/02

28/0

6/02

12/0

7/02

26/0

7/02

d v

alu

e

Bulb

Leaf

Root

A: 34S then 32S B: 32S then 34S

S pools in root, leaf, bulb increase while root takes up S

After S uptake by roots cease, it is exported to bulb

Roots therefore appear an important S source for the bulb

3234 3432

Effects of root and leaf removal on bulbing

To test: Are roots an important source of S for

bulbs? Are all CSOs synthesised in leaves and

transported to bulbs?plants grown hydroponicallyat start of bulbing, remove most of

either roots or leavescompare data from this and end-point

Dry Weight

020406080

100120

Clove Leaf Root

Ma

ss

in g

Early Control

Leafless

Rootless

Late Control

Fresh weight

0

100

200

300

400

Clove Leaf Root

Ma

ss

in g

Early Control

Leafless

Rootless

Late Control

Normal development:

bulb: x10 fold mass increase

leaf: x 2.5 fold mass increase

root: unchanged

Leaves removed:

bulb: 0.5 mass

leaf: mass almost fully recovers

roots: 0.5 mass

Roots removed:

bulb: mass almost unaffected

leaf: x 3.5 fold mass increase

roots: no recoverySevere virus infection during growth

Measurements on S being done

3. Identify genes with altered expression and/or involved in alliicin synthesis

Alliinase

Other genes from earlier part of biosynthetic pathway cysteine synthase serine acetyl transferase

Alliinase – sequence obtained

Clustering of alliinase fragments from leaf (l) and bulb(b)

97% identity among all clones

Relative alliinase expression during development

0

0.2

0.4

0.6

0.8

1

08/02/01 10/03/01 09/04/01 09/05/01 08/06/01Rel

ativ

e al

liin

ase

exp

ress

ion

Bulb

Leaf

Other genes in biosynthetic pathway

Identify genes coding for enzymes involved in alliin biosynthesis

- Novel enzymes

- Known enzymes with novel functions

Evidence from literature and tissue culture experiments for synthesis of cysteine derivatives by cysteine synthase several CSase genes in all plants

including S-allyl cysteine

Isolation of cysteine synthases from garlicTwo strategies:

Screening a garlic cDNA library for sequences with homology to known CSase

Identify a protein with S-allyl CSase activity and screen garlic cDNA library for it

Confirm function of CSase genes through expression of the protein

Purification of an allyl cysteine synthase from garlic leaves

Phenyl sepharose fractionation

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1 3 5 7 9

11

13

15

17

19

21

23

25

27

29

31

33

35

37

39

Fraction

OD

55

0

cysteinesyntase activity

allyl cysteinesynthaseactivity

…….FLGVMPSHYSIE………. YLGADLALTDTN………… SANPGAHYATTGP………….

Sequence of peptides from this protein

Obtained CSase and SATase from garlic

Five full-length cDNAs isolated and sequenced: GSAT1 – cytosolic SATase GCS1 – potential plastidic CSase

(contains frameshift - pseudogene ?)

GCS2 – chloroplastic CSase GCS3 – cytosolic CSase GCS4 – S-allyl-CSase (based on protein

isolated)

1 2 3 4 5

gcs4

gcs3

gcs2

gsat1

18s

1. 7 degree C stored clove

2. RT stored clove3. Sprouting clove4. Leaf5. Root

• The potential S-allyl CSase gcs4 and the SATase gsat1 are expressed in most tissues examined.

• The cytosolic CSase gcs3 is root specific.

• Expression for the putative plastidic CSase gcs2 is uniformly low.

Northern blot analysis

Results

• Background activity from E. coli proteins subtracted

• All three genes gcs2 gcs3 gcs4 are functional to transcribe and translate CSase

• GCS4 shows the highest activity in cysteine biosynthesis

• GCS4 functions as S-allyl-CSase

• GCS2 and GCS3 can act weakly as S-allyl-CSase

Pea

k ar

ea

In vitro CSase activity

0

5

10

15

20

25

30

35

µm

ol c

ys

min

-1 m

l-1

Substrate: Na2S

GCS2 GCS3 GCS4

0

5000

10000

15000

20000

25000

30000

35000

GCS2 GCS3 GCS4 0 10 0 10 0 10 min

Substrate: allyl mercaptan

GCS2 GCS3 GCS4

Expression of gcs2 gcs3 gcs4 in vitro

Transformation of Arabidopsis with garlic genes

Transformed with gcs3, gcs4, gsat1 Plants also carry GUS reporter gene Expression should not be constitutive Expression of both garlic and GUS genes are induced by

ethanol Seed produced from plants carrying each transgene has

been analysed (ie T1 plants) PCR to detect transgenes in genome RT-PCR and staining (for GUS) to detect expression of transgenes Spectrophotometric and hplc analysis for cysteine and allyl

cysteine

A. thaliana containing gcs3 or gcs4

Histochemical staining for GUS

Uninduced After induction with ethanol

Background line Some plants show activity of the inducible GUS transgene

A. thaliana containing gcs3 or gcs4

GCS4-2-MGCS4-2-JGCS4-2-IGCS4-2-GGCS4 – FGCS4-2-EGCS4-2-DGCS4-2-CGCS4-2-BGCS4-2-AAGS1-3Control

1.6 kbp1.0 kbp

GCS4-2-BGCS4-2-AAGS1-3GCS4-2-IGCS4-2-GGCS4-2-FGCS4-2-EGCS4-2-DGCS4-2-BGCS4-2-AAGS1-3Control

RT-PCR for gcs4 transgene expression

uninduced induced

Some plants show expression of the inducible gcs4 transgene

Arabidopsis with garlic genes

A. thaliana containing gcs3 or gcs4Plants did not show a phenotype

GCS4 line 2

0.00

0.10

0.20

0.30

0.40

0.50

0.60

µmo

l/cys

tein

e/m

in/m

g t

ota

l p

rote

in

AGS1-3 (un)

AGS1-3 (in)

GCS4-2-D (un)

GCS4-2-D (in)

GCS4-2 silenced (un)

GCS4-2 silenced (in)

GCS2-2 expressed (un)

GCS4-2 expressed (in)

none silenced express

TIP and Annual reports

TIP Completed by P2 and P3

Fourth Annual report Completed by P2 and P3

Final report Being written by P2 Completed by P3

Deliverables

DP. 8: Analytical methods for labeling and analysis (P2, P3)DP. 9: A cDNA library from garlic (P2)DP. 16: Pathway intermediates identified (P3)DP. 17: First sulphur budget for garlic (P2)DP. 18: Clones for alliinase (P2) DP. 23: Publication on alliin biosynthesis and sulphur partitioning (P2, P3)

Synthesis of alliin in garlic and onion tissue cultures – submitted to Phytochemistry

DP. 24: Genes for key CSO synthesis enzymes (P2,P3) DP. 29 Papers on the characterisation of key enzymes in alliin biosynthesis and alliinase expression and the regulation of sulphur biochemistry in garlic (P2, P3, P5)

Functional analysis of a novel garlic cysteine synthase in Arabidopsis thaliana – being written

Deliverables:

DP. 33 Paper on S pathway genes on the production of flavour precursors in garlic Biosynthesis of the flavour precursors of onion and garlic –

submitted to Journal of Experimental Botany

DP. 35 Publication on the regulation of alliinase expression (P2)

DP. 36 Paper on the regulation of sulphur biochemistry in garlic Effect of storage on the flavour precursors in garlic – being

written