Angela Tregova and Jill Hughes

1

Molecular Analysis of Flavour biosynthesis in
garlic

• Angela Tregova and Jill Hughes

Hamish Collin, Rick Cosstick, Meriel Jones, Brian
Tomsett
Acknowledgement Mark Wilkinson, protein
purification facilities
2
Biosynthetic Pathway
SO42-
SO32-
serine
S2-
cysteine
Allyl group (source ?)
serine
valine methacrylate
glutathione
S-allylglutathione
gly
S-methylglutathione
S-(2-carboxypropyl)-glutathione
S-allyl-?-glu-cys
gly
gly
S-methyl-?-glu-cys
S-2-CP-?-glu-cys
glu
HCOOH
trans- peptidase
glu
trans- peptidase
S-trans-1-propenyl-?-glu-cys
S-allylcysteine
trans- peptidase
glu
oxidase
S-methylcysteine
S-trans-1-propenylcysteine
oxidase
S-allyl-cysteine sulphoxide (alliin)
oxidase
methiin
S-trans-1-propenylcysteine sulphoxide (isoalliin)
3
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 synthases
• 1 cytosolic cysteine synthase
• 1 S-allyl cysteine synthase
• 1 cytosolic serine acetyl transferase

4
Key observation

• Callus converts
• allyl thiol to allyl cysteine alliin
• CH2CHCH2-SH ( O-acetyl-serine ?)
• CH2CHCH2-S-CH2CHNH2COOH
• CH2CHCH2-S-CH2CHNH2COOH
• But not allyl alcohol
• CH2CHCH2-OH

But this is not species-specific
5
Allyl Cysteine Synthase?
Cysteine synthase
Sulphide O-Acetyl Serine
Cysteine Allyl thiol O-Acetyl Serine
Allyl Cysteine
Allyl Cysteine synthase
Is there a specific cysteine synthase
homologue? Cysteine synthases do a range of
reactions in other organisms
6
Protein purification Ion Exchange chromatography
Garlic leaves were fractionated with ammonium
sulphate then separated by ion-exchange
chromatography.
Many fractions show cysteine synthase activity
Only a few fractions show allyl cysteine synthase
activity
7
Protein purification Hydrophobic Interaction
Chromatography
Allyl cysteine synthase and cysteine synthase
activity co-elute
Cysteine production was assayed colorimetrically
and allyl cysteine by HPLC
8
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
 
9
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)
10
Probe for S-allyl-CSase

• cDNA fragments PCR amplified with degenerate
  primer A I from the cDNA library
• Peptide sequences
• FLGVMPSHYSI
• YLGADLALTDT
• ANPGAHYA

Peptide 1 2 3
A B C D E F G H I
. to find the gene and related genes
11
AllylCSase aligns with rice sequences
Partial protein sequences relative to Arabidopsis
(C) sequence
RCS2 IGLVLVAVQ-KGYRFIAVMPAKYSLDKQMLLRFLGAELILTDPA-
IGFNGMMDKVEEL RCS4 IGVAYNALL-KGYRFVAVMPAEYSLDKQML
LTYLGAEVILTDPT-LGFQGQ-LDKVEQI GCS4 IALAYI-GLKKGYKF
LGVMPSHYSIERRMLLKYLGADLALTD-TNLGFKG-VLDKVAEL I
KGY F VMP YS MLL LGA LTD GF G
DKV
12
Proposed Serine Pathway

• Important enzymes
• 1 SAT/CS complex
• 2 Free CSase
• 3 S-allyl-CSase ?
• 4 Oxidase

Cysteine
Sulfide
Acetyl CoA
2
L-Serine
OAS
1
Allyl-source
3
S-allyl-L-Cysteine
4
Alliin
13
cDNA library screening

• gsat1 - cytosolic SATase
• gcs1 - putative plastidic CSase
  (pseudogene)
• gcs2 - putative plastidic CSase
• gcs3 - cytosolic CSase
• gcs4 - putative S-allyl-CSase

14
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

15
Northern blot analysis

• 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.

1. 7 degree C stored clove
2. RT stored clove
3. Sprouting clove
4. Leaf
5. Root

16
Is this allylcysteine synthase?
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 !

• 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

17
Why ethanol-regulated expression?
cDNA
alcR
transgene
t
pCAMV35S
palcA
t
alc is a simple two component system
18
Does it work?
Real time Luciferase Imagingin Arabidopsis
19
LUC 1-12
wt AGS
20
LUC 1-12
wt AGS
1 hour before induction
21
LUC 1-12
wt AGS
Time of induction
22
LUC 1-12
wt AGS
30 minutes after induction
23
LUC 1-12
wt AGS
1 hour after induction
24
LUC 1-12
wt AGS
1.5 hour after induction
25
LUC 1-12
wt AGS
2 hours after induction
26
LUC 1-12
wt AGS
2.5 hours after induction
27
LUC 1-12
wt AGS
3 hours after induction
28
LUC 1-12
wt AGS
3.5 hours after induction
29
LUC 1-12
wt AGS
4 hours after induction
30
LUC 1-12
wt AGS
4.5 hours after induction
31
LUC 1-12
wt AGS
5 hours after induction
32
LUC 1-12
wt AGS
6 hours after induction
33
LUC 1-12
wt AGS
7 hours after induction
34
LUC 1-12
wt AGS
7.5 hours after induction
35
LUC 1-12
wt AGS
8 hours after induction
36
LUC 1-12
wt AGS
11 hours after induction
37
LUC 1-12
wt AGS
13 hours after induction
38
Real time ArabidopsisLuciferase Imaging
39
LUC 1-12
wt AGS
Time of induction
40
LUC 1-12
wt AGS
8 hours after induction
41
Functional analysis of plant cell cycle genes
42
At progeny of AmcycA20 x alcRalcAGUS
AmcycA20 PCR
GUS PCR
alcR PCR
43
RT-PCR of AmcycA20 controls
plus RT
Induced plants
Total RNA extracted from plants of A cyclin
A20 B HA-tagged cyclin A20 C sibling D
wild type E AGS-1-3
Induced RNA minus RT
A B C D E
1 2 3 4 5 6 7 8
Uninduced plants
1 kb
Total DNA extracted from Induced plants of A
cyclin A20 B HA-tagged cyclin A20 C sibling
(cycGUS-) D wild type E AGS-1-3 13
A.majus genomic DNA
There is no DNA contamination
cycA20 message is specific to induced plants
containing both T-DNAs
44
Western Blots of HA tagged cycA20
WT I N
WT I N
48 kDa HA-CycA20
Probe antibody to HA tag
45
Phenotypic analysis
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).
46
Fresh Weight
47
Root Length AmcycA20 expression
WT 25 26 27 28 29 30 31 32 AGS
G A A
48
Root length
49
Leaf number and area
Leaf number remains constant after AMcycA20
expression
Plus ethanol
Minus ethanol
Leaf area is bigger after induction in AmcycA20
expressing lines
Minus ethanol
Plus ethanol
50
Leaf Area
51
Cell Size and density
There appear to be less cells per unit area -
cells are larger
52
Trichomes on rosette leaves
53
Flowering time of cycA20 and controls
Days
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.
54
Tobacco transformation for protein expression
kanR
55
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?

56
Diagnostic PCRs for transgenic BY2 lines
1 2 3 4 5 6 7
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
PCR primers 1.palcA forward 2. t35S reverse
57
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.

58
Northern blot analysis

• 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.

gcs3
gcs4
gsat1
Garlic RNA
Tobacco RNA
59
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?

60
Is alcR expressed?
RT-PCR results
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

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

61
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?

62
alcR expression in the new cell-line
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!

Positive RT-PCR controls using degenerate primers
that anneal to SAT.
63
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)
64
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)
65
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)
66
Future ?

• The longer route looks more attractive !
• E. coli his-tagged protein
• purification
• assay in vitro
• Arabidopsis - test for expression
• assay in vivo
• phenotype

67
Expression of a wheat CSase in tobacco

1. Transgenic tobacco shows 2-fold higher Cys
   content.
2. SO2 fumigation increased thiol levels.

68
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)

69
Thanks to ..

• Liverpool
• Angela Tregova
• Jill Hughes
• Piyarat Parinyapong
• Hairul Roslan
• Chris Wood
• Mike White
• Mark Caddick
• Brian Tomsett

• Jealotts Hill
• Jackie Paine
• Mary Knight
• Susan Wright
• Justin Sweetman
• Alberto Martinez
• Wolfgang Schuch
• Andy Greenland
• Ian Jepson
• ICI Agrochemicals
• ICI Seeds
• Zeneca Seeds
• Zeneca Agrochemicals
• Syngenta

JIC John Doonan and his
lab Funding BBSRC EU FP5 Garlic Health