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 biosynthesis  Bring 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

serine SO 4 2 SO 3 2 S-allyl group (unknown sources) S-allylglutathione gly S 2 valine & methacrylate cysteine glutathione (γ-glu-cys-gly) S-(2-carboxypropyl)-glutathione S-allyl-γ-glu-cys glu trans peptidase S-allylcysteine S-allylcysteine oxidase oxidase S-allyl-cysteine sulphoxide (alliin) S-methylglutathione S-2-CP-γ-glu-cys gly gly S-methyl-γ-glu-cys HCOOH S trans -1-propenyl-γ-glu-cys glu trans peptidase S trans -1-propenylcysteine oxidase S trans -1-propenylcysteine sulphoxide (isoalliin) glu trans peptidase S-methylcysteine oxidase methiin

Biosynthetic capacity of garlic callus

allyl thiol propyl thiol propyl cysteine alliin 10,1 ; allyl cysteine propenyl cysteine 10 ; 10,1 allyl cysteine isoalliin 10,1 ; 10,1 1 ; 10,1 propiin 10 ; propyl cysteine 1 ; 10 10,1 ; 10,1 Incubation for 5 days with 10mM or 1mM substrate Incubation for 12/15 days with 10mM or 1mM substrate

Conclusion:

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

propyl alcohol allyl alcohol methacrylic acid allyl thiol metabolite soup allyl glutathione carboxypropyl glutathione propyl glutathione

glutathione

+ + + + + No clear potential GST substrate

2. Identify developmental control points on CSO synthesis and translocation

 Baseline data on garlic development  Resource allocation during development  Developed 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  SO 4 2 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

Garlic growth and S partition

Hydroponic v pot-grown Printanor - Leaf weight

25 20 15 10 5 0 0 Hydoponic-grown Printanor Pot-grown Printanor 1 2 50 100 150

Days after planting

3 4 200 250 0.3

0.2

Root Leaf Clove 0.1

0.0

29 56 77 109 141

Days after planting

169 203 100 80 60 40 20 0 0 160

Hydroponic-grown garlic - comparison of bulb formation

140 Printanor clove Messidrome Clove 120 1 50 2 100 150

Days after planting

3 4 200 250 2000000 1500000 1000000 500000 0 Root Leaf Clove 56 109 141 169

Days after planting

203

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, 34/32 S ratio (delta value)

Sulfur labelling design

Distribution and remobilization of sulphur taken up early * * * * * * * * * * * Distribution and remobilization of sulphur taken up late * * * * * * * * * * *

Growth pattern in Year 2 experiment

34 S 32 S 200 150 100 50 0 0 25 Clove Leaf Root 50 75 100 125

Days after planting

150 175 200 225

A B

34 S 32 S

Year 3 hydroponic garlic

250 200 150 100 50 0

Sulpur accumulation in system A plants

Clove Leaf Root Total

Date

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

250 200 150 100 50 0 Bulb Leaf Root 200 150 100 50 0 Bulb Leaf Root 34 32 32 34 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

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 hydroponically  at start of bulbing, remove most of either roots or leaves  compare data from this and end-point

120 100 80 60 40 20 0 400 300 200 100 0 Clove Clove

Fresh weight

Leaf

Dry Weight

Leaf Early Control Leafless Rootless Late Control Root Early Control Leafless Rootless Late Control Root Severe virus infection during growth

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 recovery

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

1 0.8

0.6

0.4

0.2

0 08/02/01 10/03/01 Bulb Leaf 09/04/01 09/05/01 08/06/01

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 garlic

 Two 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

0.7

0.6

0.5

0.4

0.3

0.2

cysteine 0.1

syntase activity 0 allyl cysteine synthase activity

Phenyl sepharose fractionation Fraction

Sequence of peptides from this protein …….FLGVMPSHYSIE………. YLGADLALTDTN………… SANPGAHYATTGP………….

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)

Northern blot analysis

1 2 3 4 5

gcs4 gcs3 gcs2 gsat1 18s

1.

2.

3.

4.

5.

7 degree C stored clove RT stored clove Sprouting clove Leaf 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.

Expression of

gcs2 gcs3 gcs4

in vitro

In vitro CSase activity

35 30 25 20 15 10 5 0 Substrate: Na 2 S

GCS2 GCS3 GCS4

35000 30000 25000 20000 15000 10000 Substrate: allyl mercaptan 5000 0

GCS2 GCS3 GCS3 Results GCS4 GCS4

min • 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

Transformation of with garlic genes

Arabidopsis

 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 T 1 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 Background line After induction with ethanol

Some plants show activity of the inducible GUS transgene

A. thaliana

containing

gcs3

or

gcs4

RT-PCR for gcs4 transgene expression 1.6 kbp 1.0 kbp uninduced induced

Some plants show expression of the inducible gcs4 transgene

Arabidopsis

with garlic genes

 A. thaliana containing gcs3 or gcs4  Plants did not show a phenotype

GCS4 line 2

0.60

0.50

0.40

0.30

0.20

0.10

0.00

none silenced express 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)

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 – to Phytochemistry submitted  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