Hamish Collin, Rick Cosstick, Meriel Jones, Brian Tomsett

Angela Tregova and Jill Hughes

Acknowledgement: Mark Wilkinson, protein purification facilities

Molecular Analysis of Flavour biosynthesis in garlic

Biosynthetic Pathway SO42-

SO32-

S2-cysteine

glutathione

S-methyl-γ-glu-cys

gly

S-methylcysteine

methiin

glutrans-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)-glutathione

valine & methacrylate

S-allylglutathione

S-allyl-γ-glu-cys

gly

glutrans-peptidase

Allyl group(source ?)

serine

oxidase

S-allylcysteine

S-allyl-cysteine sulphoxide(alliin)

serine

What we have done……

• Investigation of intermediates in the pathway

• Identification of key compounds

• Purification of a key enzyme• Allylcysteine synthase

• The search for genes involved in flavour biosynthesis:

2 chloroplastic cysteine synthases1 cytosolic cysteine synthase1 S-allyl cysteine synthase+ 1 cytosolic serine acetyl transferase

Key observation

Callus convertsallyl thiol to allyl cysteine & alliin

CH2CHCH2-SH (+ O-acetyl-serine ?)

CH2CHCH2-S-CH2CHNH2COOH

CH2CHCH2-S-CH2CHNH2COOH

But not allyl alcoholCH2CHCH2-OH

=

O

XXBut this is not species-specific

Allyl Cysteine Synthase?

Is there a specific cysteine synthase homologue?

Cysteine synthases do a range of reactions

in other organisms

Sulphide + O-Acetyl Serine Cysteine

Allyl thiol + O-Acetyl Serine Allyl Cysteine

Cysteine synthase

Allyl Cysteine synthase

Cysteine synthase activity. Q-Sepharose. 7.11.01

00.05

0.10.15

0.20.25

0.3

Fraction

OD

cysteine synthaseactivity

Many fractions show cysteine synthase activity

Protein purification: Ion Exchange chromatography

Garlic leaves were fractionated with ammonium sulphate then separated by ion-exchange chromatography.

Only a few fractions show allyl cysteine synthase activity

Protein purification: Hydrophobic Interaction Chromatography

Allyl cysteine synthase and cysteine synthase activity co-elute

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

Cysteine production was assayed colorimetrically and allyl cysteine by HPLC

Protein purification

  SDS-PAGE shows a distinct band in the allyl cysteine synthase active fractions at approx. 34 kd

Molecular weight consistent with plant cysteine synthase monomers found previously Fractions 26 27 28 29 30

34000

What is the Enzyme?

Extract 34000 band and digest with trypsin

- the resultant peptides separated by preparative HPLC

Three selected peptides were sequenced:-

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

A simple BLAST search of these peptides in the protein database shows most similarity to a cysteine synthase from Oryza sativa (Rice)

Probe for S-allyl-CSase

A B C D E F G H I

Peptide 1 2 3cDNA fragments PCR amplified with degenerate primer A – I from the cDNA library

Peptide sequences:

1. FLGVMPSHYSI

2. YLGADLALTDT

3. ANPGAHYA

…. to find the gene and related genes

AllylCSase aligns with rice sequences

Partial protein sequences relative to Arabidopsis (C) sequence

RCS2 IGLVLVAVQ-KGYRFIAVMPAKYSLDKQMLLRFLGAELILTDPA-IGFNG—MMDKVEELRCS4 IGVAYNALL-KGYRFVAVMPAEYSLDKQMLLTYLGAEVILTDPT-LGFQGQ-LDKVEQIGCS4 IALAYI-GLKKGYKFLGVMPSHYSIERRMLLKYLGADLALTD-TNLGFKG-VLDKVAEL

I KGY F VMP YS MLL LGA LTD GF G DKV

Proposed Serine Pathway

Important enzymes:

1 SAT/CS complex

2 Free CSase

3 S-allyl-CSase + ?

4 OxidaseL-Serine OASAllyl-source

S-allyl-L-Cysteine

Sulfide

Alliin

Cysteine

1

2

3

4

Acetyl CoA

cDNA library screening

gsat1 - cytosolic SATase

gcs1 - putative plastidic CSase

(pseudogene)

gcs2 - putative plastidic CSase

gcs3 - cytosolic CSase

gcs4 - putative S-allyl-CSase

What next ?

• Where are the genes expressed in garlic?• Northerns

• Does the gene encode allylcysteine synthase?• How do we prove it?

• What does it do in planta?• Transformation

Northern blot analysis1 2 3 4 5

gcs4

gcs3

gcs2

gsat1

18s

1. 7 degree C stored clove

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

S-allyl CSase and

the SATase

are expressed in most tissues examined.

The cytosolic CSase is root specific.

Expression for the putative plastidic CSase is uniformly low.

Is this allylcysteine synthase?

Proof requires expression of the gene and phenotypic testing

• Garlic? This would be best but…..time ?

• E. coli? Does it function alone? In vitro testing only?

• Heterologous plant system? Time ? Arabidopsis ?

• Plant tissue culture? Quick and could form complexes allowing tests in planta

Ideal Choice ?

A quick assessment could mean that we can plan the alternative

If we use ethanol-regulated expression, then we can test the effect on the cellular phenotype of the expression of the

allylcysteine synthase vs. its absence !

Why ethanol-regulated expression?

alcR transgene tpCAMV35S palcAt

AlcR

AlcR EcDNA

E

+ Ethanol

alc is a simple two component system

Does it work?

Real time Luciferase Imagingin Arabidopsis

wt

AGS

LUC 1-12

1 hour before induction

wt

AGS

LUC 1-12

Time of induction

wt

AGS

LUC 1-12

30 minutes after induction

wt

AGS

LUC 1-12

1 hour after induction

wt

AGS

LUC 1-12

1.5 hour after induction

wt

AGS

LUC 1-12

2 hours after induction

wt

AGS

LUC 1-12

2.5 hours after induction

wt

AGS

LUC 1-12

3 hours after induction

wt

AGS

LUC 1-12

3.5 hours after induction

wt

AGS

LUC 1-12

4 hours after induction

wt

AGS

LUC 1-12

4.5 hours after induction

wt

AGS

LUC 1-12

5 hours after induction

wt

AGS

LUC 1-12

6 hours after induction

wt

AGS

LUC 1-12

7 hours after induction

wt

AGS

LUC 1-12

7.5 hours after induction

wt

AGS

LUC 1-12

8 hours after induction

wt

AGS

LUC 1-12

11 hours after induction

wt

AGS

LUC 1-12

13 hours after induction

wt

AGS

LUC 1-12

Real time ArabidopsisLuciferase Imaging

Time of induction

wt

AGS

LUC 1-12

8 hours after induction

wt

AGS

LUC 1-12

Functional analysis of plant cell cycle genes

At progeny of AmcycA20 x alcRalcAGUS

A B C D E F

G H I J K L

A B C D E F

G H I J K L

Induced

Non-induced

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 A B C D E F G H I J K L

1.6 kb 1 kb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 A B C D E F G H I J K L

1 kb

500 bp

A B C D E F G H I J K L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1 kb

500 bp

AmcycA20 PCR

GUS PCR

alcR PCR

RT-PCR of AmcycA20 & controls

1 2 3 4 5 6 7 8 9 10 11 12 13 14

A B C D E A B C D E

1 kb

plus RT

1 kb

1 2 3 4 5 6 7 8

A B C D E

Induced RNA minus RT

Total RNA extractedfrom plants ofA = cyclin A20B = HA-tagged cyclin A20 C = siblingD = wild typeE = AGS-1-3

Total DNA extracted fromInduced plants ofA = cyclin A20B = HA-tagged cyclin A20 C = sibling (cyc+;GUS-)D = wild typeE = AGS-1-3 13 = A.majus genomic DNA

There is no DNA contamination

1 2 3 4 5 6 7 8 9 10 11 12 13 14

A B C D E A B C D E

1 kb

Induced plants

Uninduced plants

cycA20 message is specific to

induced plants containing both

T-DNAs

Western Blots of HA tagged cycA20 WT I N WT I N

Probe = antibody to HA tag

48 kDaHA-CycA20

Phenotypic analysis Rosette leaves

1 2

Leaf cell density, primary leaf area, rosette leaf number, trichomes and flowering time.

Plants were grown for six weeks.

Vertically grown A.thaliana plants, growing in a tissue culture square plate.

Root growth experiments (after 15 days) and fresh weight measurements (after four weeks).

Fresh Weight

0

20

40

60

80

100

WT AGS SIBLING CYCA20-HA CYCA20

NON-INDUCED INDUCED

Root Length – AmcycA20 expressionWT 25 26 27 28 29 30 31 32 AGS

G A A

Root length

0

1

2

3

4

5

6

7

8

9

0 5 10 15

Days growth

Ro

ot

len

gth

(c

m)

WT

AGS

SIBLING

CYCA20-HA

CYCA20

WT

AGS

SIBLING

CYCA20-HA

CYCA20

Leaf number and area

sibling

Cyclin A20

Leaf number remains constantafter AMcycA20 expression

Leaf area is bigger after induction in

AmcycA20 expressing lines

Minus ethanol

Minus ethanol

Plus ethanol

Plus ethanol

Leaf Area

0.00

0.50

1.00

1.50

2.00

2.50

WT AGS Sibling CycA20-HA CycA20

Non-induced Induced

Cell Size and density

HA-tagged cyclin A20

Uninduced Induced

There appear to be less cells per unit area - cells are larger

Trichomes on rosette leaves

Flowering time of cycA20 and controls

05

10152025

WT AGS Sibling CycA20 CycA20

Non-induced Induced

Day

s

Mean flowering time (days) of twenty seedlings of each of HA-tagged cyclin A20, cyclin A20, wild type (Columbia), AGS-1-3 and sibling plants in comparison between non-induced and induced conditions. The plants were induced after 5 days of germination, when the plants reached the 2 leaf-stage, and were checked regularly until the appearance of the first visible flower bud.

Tobacco transformation for protein expression

RB t35S palcAGarlic gene LB pAg7nptIIpnos

Transformed Untransformed Transformed

kanR

Tobacco transformation for protein expression

• The tobacco cells can be multiplied in liquid culture

• Induce protein expression

• Determine whether –SH content of cells has increased

• Assay for allylCSase activity

• Use HPLC to look for allylcysteine and ….?

Does tobacco possess an oxidase to make alliin?

Diagnostic PCRs for transgenic BY2 lines

1 2 3 4 5 6 7

PCR primers:

1.palcA forward

2. t35S reverse

Lane 1 = untransformed BY2

Lane 2 = gcs3 plasmid control

Lane 3 = gcs3 transformant

Lane 4 = gcs4 plasmid control

Lane 5 = gcs4 transformant

Lane 6 = gsat1 plasmid control

Lane 7 = gsat1 transformant

However…….

Cysteine synthase assays

No detectable increase in cysteine. Time course assays and assay optimisations failed.

S-allyl-cysteine synthase assay (HPLC)

No detectable levels of S-allyl-cysteine.

Northern blot analysis

gcs3

gcs4

gsat1

Garlic RNA Tobacco RNA

RNA extracted from transgenic tobacco cells after 1, 3 and 6 days induction.

Northern blots show no transgene expression, except gcs3 that was detected after several days induction.

Why are the transgenes not expressed?

Are there mistakes in the binary constructs?Re-sequencing verified correct assemblies.

Is the alcR cDNA present in the tobacco cell-line? alcR confirmed by PCR.

Is alcR expressed?

Is alcR expressed?

1 2 3 4 Lane 1 = alcR control (genomic DNA)

Lane 2 = gcs3 BY-2 transformant

Lane 3 = gcs4 BY-2 transformant

Lane 4 = gsat1 BY-2 transformant

RT-PCR results:

No alcR expression detected in any of the transformed cell lines!

Repeat BY-2 transformation

New BY-2 cell-lines from the John Innes Centre

Transformations have been repeated and we

are currently waiting for new transformants to grow

But is alcR expressed in the new cell-line?

alcR expression in the new cell-line

Positive RT-PCR controls using degenerate primers that anneal to SAT.

1 2 3 RT-PCR results:Lane 1 = No RT control

Lane 2 = alcR control (genomic DNA)

Lane 3 = alcR expression in the new cells

Again, no detectable alcR expression in the new cell line!

RT-PCRs using a highly sensitive detection

1 2 3 4 5 6 7 8 9

RT-PCR results:

Lane 1 = untransformed BY-2

Lane 2 = gcs3 BY-2 transformant

Lane 3 = gcs4 BY-2 transformant

Lane 4 = gsat1 BY-2 transformant

Lane 5-8 = No RT controls

Lane 9 = alcR control (genomic DNA)

RT-PCRs using a highly sensitive detection

1 2 3 4 5 6 7 8 9

RT-PCR results:

Lane 1 = untransformed BY-2

Lane 2 = gcs3 BY-2 transformant

Lane 3 = gcs4 BY-2 transformant

Lane 4 = gsat1 BY-2 transformant

Lane 5-8 = No RT controls

Lane 9 = alcR control (genomic DNA)

RT-PCRs using a highly sensitive detection

1 2 3 4 5 6 7 8 9

RT-PCR results:

Lane 1 = untransformed BY-2

Lane 2 = gcs3 BY-2 transformant

Lane 3 = gcs4 BY-2 transformant

Lane 4 = gsat1 BY-2 transformant

Lane 5-8 = No RT controls

Lane 9 = alcR control (genomic DNA)

Future ?

The longer route looks more attractive !• E. coli – his-tagged protein

purification

assay in vitro

• Arabidopsis - test for expression

assay in vivo

phenotype

Expression of a wheat CSase in tobacco

A. Transgenic tobacco shows 2-fold higher Cys content.

B. SO2 fumigation increased thiol levels.

Deliverables

• Genes for CSO synthesis enzymes (36m)

• Publication on regulation of S biochemistry in garlic (36m)

• Paper on characterising enzymes in alliin biosynthesis, and alliinase expression, and regulation of sulphur biochemistry in garlic (48m)

• Paper on S pathway genes on production of flavour precursors in garlic (48m)

Thanks to ……..

LiverpoolAngela TregovaJill Hughes

Piyarat Parinyapong

Hairul Roslan

Chris WoodMike White

Mark CaddickBrian Tomsett

Jealott’s HillJackie PaineMary KnightSusan WrightJustin SweetmanAlberto MartinezWolfgang SchuchAndy GreenlandIan Jepson

ICI AgrochemicalsICI SeedsZeneca SeedsZeneca AgrochemicalsSyngenta

JICJohn Doonan and his lab

FundingBBSRCEU FP5 Garlic &

Health