Sulfur biochemistry of garlic: the biosynthesis of flavour precursors

Hamish A Collin, Jill M Hughes, Angela Tregova,Jonathan GC Milne, Gloria van der Werff, Mark Wilkinson, Rick Cosstick, Meriel G Jones and A Brian TomsettThe School of Biological Sciences, The University of Liverpool

Laurence Trueman, Tim Crowther, Linda Brown and Brian ThomasWarwick HRI, The University of Warwick, Wellesbourne, UK

Project objectives: Garlic flavour

Improved understanding of S allocation and translocation during garlic development

Identify genes and intermediates involved in alliicin synthesis

For controlled growth in the UK climate - hydroponic and pot culture in a glasshouse

Measurements during growth

•Leaf number, bulb weight

•N, S, C, protein, CSO

•SO42-uptake using

stable isotope labelling

Improved understanding of S allocation and translocation during garlic development

Hydroponic vpot-grown Printanor - Leaf weight

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Hydoponic-grown Printanor

Pot-grown Printanor

Hydroponic-grown garlic - comparison of bulb formation

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Printanor clove

Messidrome Clove

Garlic growth and S partition

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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

Bulb initiation: Day 150 – 200 S, N, C, protein and CSOs decline in roots and leaves but

accumulate in bulbs rise in CSO synthesis

Bulb maturity: Day 200 turgor loss as leaves and roots senesce S, N, C, protein falls in leaves, roots, and rises 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)

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Distribution and remobilizationof sulphur taken up early

Distribution and remobilizationof sulphur taken up late

* * * * * * * * * * *

* * * * * * * * * * *

34S32S

A

B

Growth pattern in earlier experiment

Sulfur labelling design

Sulpur accumulation in system A plants

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Hydroponic garlic in isotopically labelled sulfur

Sulphur accumulation in system A plants (34S then 32S)

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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

To identify genes and intermediates in flavour precursor biosynthesis

Alliinase

Other genes from earlier part of biosynthetic pathway

Sequence obtained

Relative alliinase expression during development

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Bulb

Leaf

Alliinase

Biosynthetic pathway for garlic flavour precursors

SO42- SO3

2- S2- cysteine

glutathione(γ-glu-cys-gly)

S-methyl-γ-glu-cys

gly

S-methylcysteine

S-methylcysteine sulphoxide(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 source)

valine & methacrylateserine

oxidase

S-allylcysteine

S-allyl-cysteine sulphoxide(alliin) Lancaster and Shaw 1989; Granroth

1970

Is cysteine synthase involved in garlic flavour precursor biosynthesis?

O-acetyl serine + sulphide cysteine

cytoplasmic, plastid and mitochondrial forms

non-protein amino acids synthesised

Non-protein aminoacid synthesis by CSases

serine   SAT/CSase Complex   O-acetyl serine   H2S CH2=CH-CH2-SH methyl-SH 3,4-dihydroxy-

pyrazole Free CSase pyridine     L-cysteine S-allyl-L-cysteine S-methyl- mimosine -pyrazol-

1-yl alanine L-cysteine    Free CAS HCN  

3-cyano-L-ala

watermelonMimosa pudica

CSase cysteine synthase; CAS -cyanoalanine

synthase

Pea (Pisum sativum)

Leucaena leucocephala

watermelon

Leucaena leucocephala

Lathyrus latifolius

Ikegami and Murakoshi 1994; Warrilow and Hawkesford 2002

Biosynthetic capacity of garlic callus

allyl cysteine alliin isoalliin propyl cysteine propiinallyl thiol 10; 10,1 10,1;10,1 allyl cysteine 10;10,1propenyl cysteine 1;10,1propyl thiol 1;10 10;propyl 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 reactions shown may occur:-

alk(en)yl thiol alk(en)yl cysteine alk(en)yl CSO

Isolation of cysteine synthases from garlicStrategy:

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

Screening using homology to known CSases

Three full-length sequences from garlic cDNA library GCS1, GCS2

GCS1 – frameshift; truncated 202 aa, 22 kDa

GCS2 – 332 aa, 35 kDa51 aa predicted transit peptide - plastid

GCS3323 aa, 34 kDaNo transit peptide - cytosol

Purification of an allyl cysteine synthase from garlic leaves

Phenyl sepharose fractionation

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cysteinesyntase activity

allyl cysteinesynthaseactivity

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

Sequence of peptides from this protein

34 kDa

Obtained CSase from garlic

Four full-length cDNAs isolated and sequenced:

GCS1 – potential plastidic CSase (frameshift)

GCS2 – potential plastidic CSase GCS3 – potential cytosolic CSaseGCS4 – potential S-allyl-CSase (based on

protein data)

Phylogenetic tree of garlic cysteine synthases

 

    

Spinach

A. thaliana [3, 10]

A. thaliana [6]

GCS2

A. thaliana [4]

RCS4RCS2

GCS4

GCS3

A. thaliana [2]

A. thaliana [5]

Watermelon

A. thaliana [1]

A. thaliana [8]A. thaliana [9]

A. thaliana [7]

50 changes

PAUP version 4.0b 10

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1 2 3 4 5

gcs4

gcs3

gcs2

18s

1. 7o stored clove

2. 20o stored clove3. Sprouting clove4. Leaf5. Root

• Low expression of putative plastidic CSase gcs2

• Root expression of cytosolic CSase gcs3

• Most tissues expressed potential S-allyl CSase gcs4

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

In vitro CSase activity

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GCS2 GCS3 GCS4

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Substrate: allyl mercaptan

GCS2 GCS3 GCS4

Expression of gcs2 gcs3 gcs4 in vitro

Pea

k ar

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SummaryS allocation and re-mobilisation during garlic

development Alliinase

Sequence obtained Expression during development

Could a cysteine synthase be involved in flavour precursor biosynthesis in garlic?

Sequences of three cysteine synthases obtained, all expressed in garlic Functional in vitro

cysteine synthesis – GCS2, GCS3, GCS4S-allyl cysteine synthesis – GCS4

Role in planta?

Acknowledgements

The Garlic and Health project partners

EU FP5 Quality of Life program: Garlic and Health project QLK1-CT-1999-00498