Hamish Collin, Rick Cosstick, Meriel Jones, Brian Tomsett

Jill Hughes and Angela Jill Hughes and Angela TregovaTregova

Acknowledgement: Mark Wilkinson, protein purification facilities

Molecular Analysis of Flavour biosynthesis in garlic

To refresh your memories …The search for genes involved in flavour biosynthesis:Partial sequences obtained for potential:

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

Investigation of intermediates in the biosynthetic pathway:Gradient hplc method brought into use

Improved resolutionSeparation of gamma-glutamyl compounds

possible

Since Spring 2002 …1. Expression of garlic sequences for cysteine

synthase and serine acetyl transferase to obtain protein – to test activity

2. Analysis of expression of CSases and SATase in garlic tissues

3. Inter-clove variation in flavour precursors

4. Preliminary analysis for glutathione-S-transferases – another route for flavour biosynthesis?

1. Expression of CSase and SATase

Tobacco cell suspension culture• Plant cell system• Regulated, high level of expression using the alc system

alcRP T

ALCR

Transcription Factor

EtOH

Inducer

ALCR

Garlic geneT

Express

Garlic protein

palcA

Transformation of tobacco cells for protein expression

RB LBt35S palcA pAg7nptIIpnosGarlic gene

Transformed Untransformed Transformed sub-cultured

Transformation of tobacco cells for protein expression

• The tobacco cells will be multiplied in liquid culture

• Induce protein expression

• Assay for CSase and SATase activity

• Determine kinetic parameters – Km, Vmax

2. Expression of GCS4 in garlic

1. 7 degree C stored clove

2. RT stored clove

3. Sprouting clove

4. Leaf

5. Root

1 2 3 4 5 The potential S-allyl-cysteine synthase is expressed in most tissues examined.

Quantitation of this result, and Northern analysis of the three further cysteine synthase and one serine-acetyl transferase will be carried out.

3. Inter-clove variation in flavour compoundsFurther investigation of the biosynthetic

pathway

• Inter-clove variation

• Effect of low temperature storage

• Statistical analysis of data

Result: Hplc profiles differed significantly • clove location • bulb storage conditions

Inter-clove variationRepresentative HPLC traces of Outer

Cloves

0.00 10.00 20.00 30.00 40.00

0

20

40

60

80

100

0.00 10.00 20.00 30.00 40.00

0

20

40

60

80

100

Bulbs stored at room temperature

Bulbs stored at 40C for 6 weeks

Alliin

Isoalliin

GLUAC

isoGLUAC

Isoalliin

Alliin

isoGLUAC

GLUAC

Inter-clove variationIn summary: which changes are significant?

Alliin level is influenced by location.

Isoalliin, GLUAC and isoGLUAC are influenced by both location and storage temperature

Location within bulb

Cold storage

Location and storage

P values location storage within bulb temperature

peak1peak2

alliinisoalliin

peak5

peak6peak7GLUACisoGLUACpeak10peak11

4. Glutathione-S-transferases

SO42- SO3

2- SO22- 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

Allyl group(unknown sources)

valine & methacrylateserine

oxidase

S-allylcysteine

S-allyl-cysteine sulphoxide(alliin)

Glutathione-S-transferases

•This family of enzymes is widespread in plants

•The Arabidopsis genome has approx. 50 GSTs

•Extensive literature on GST biochemistry and analysis

•GSTs conjugate glutathione to many compounds

Glutathione-S-transferase•Garlic leaf proteins - glutathione affinity matrix

•Single step gives substantial purification

5.81 5.96

Fractions on SDS gel

25 kDa

Fractions on 2D gel

Mol wt

pI

Glutathione-S-transferase• 1-chloro-2,4-dinitrobenzene - GST activity present

• Potential substrates and products were incubated with total protein or the GST fraction

• Products were analysed by hplcNo clear potential GST substrate

substrate glutathionepropyl alcohol +allyl alcohol +methacrylic acid +allyl thiol +metabolite soup +allyl glutathionecarboxypropyl glutathionepropyl glutathione * synthesised in Liverpool

**

Deliverables

Genes for CSO synthesis enzymes (36m)Genes for CSO synthesis enzymes (36m)

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

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

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