Transcript No Slide Title

Molecular analysis of flavour
biosynthesis in garlic
Angela Tregova
Jill Hughes, Jonothan Milne, Hamish Collin,
Meriel Jones, Rick Cosstick, Brian Tomsett
EU Framework 5
Garlic and Health
Objectives
• Identify genes coding for enzymes
involved in alliin biosynthesis
-
Novel enzymes
-
Known enzymes with novel functions
• Analysis of flavour precursors
Cysteine synthase (CSase)
L-Serine
Acetyl Co-A
SAT/CS
?
OAS
Free CS
Sulfide
Pyrazol
Allyl-mercaptan
Cyanide
-PA
S-allyl-L-Cysteine
Cysteine
Free
CAS/CS
3-Cyano-L-Ala
Clone cysteine synthase
• 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
Results
• 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
Northern blot analysis
1
2
3 4
5
gcs4
gcs3
gcs2
• The potential S-allyl CSase
and the SATase are
expressed in most tissues
examined.
• The cytosolic CSase is root
specific.
gsat1
18s
1.
2.
3.
4.
5.
7 degree C stored clove
RT stored clove
Sprouting clove
Leaf
Root
• Expression for the putative
plastidic CSase is uniformly
low.
Phylogenetic Tree
A. thaliana [8]
A. thaliana [1]
A. thaliana [9]
A. thaliana [7]
Watermelon
A. thaliana [5]
A. thaliana [2]
Spinach
A. thaliana [3, 10]
GCS3
A. thaliana [6]
GCS2
GCS4
RCS2
A. thaliana [4]
RCS4
GCS4 related to
two isoforms
identified from rice
that form a new
CSase family.
Expression of CSase and SAT
Transgenic tobacco BY2 cells and A. thaliana
RB
P
Garlic gene
t35S
alcR
palcA
pnos
nptII
T
palcA Garlic gene
Inducer
Transcription
Factor
T
Express
EtOH
ALCR
pAg7
ALCR
Garlic protein
LB
Transformation of tobacco
cells for protein expression
RB
t35S
Garlic gene
Transformed
sub-cultured
palcA
Untransformed
pnos
nptII
pAg7
Transformed
LB
Unexpected results
• SDS – PAGE
• No detectable increase in protein products
• Cysteine synthase assays
• No detectable increase in cysteine
• S-allylcysteine synthase assays (HPLC)
• No detectable S-allylcysteine synthase
• Northern blot analysis
• No detectable transgene expression
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!
In vitro protein biosynthesis
• Rapid Translation System RTS 100 E. coli
HY kit (Roche)
• Cell-free in vitro transcription/translation protein
expression system based on E. coli lysate
• Suggested by Rolf at February 2003 meeting
- thanks Rolf!
pIVEX expression vectors
Garlic genes:
5’
T7P
RBS
Garlic gene
His-tag
T7T
3’
His-tag
T7T
3’
gsat1
TAA
5’
T7P
RBS
Garlic gene
gcs2; gcs3
gcs4
• PCR cloning strategy to remove 5’ and 3’UTRs.
• Hi-fidelity PCR enzyme mix introduced 1 mutation into gsat1 and 2
mutations into gcs4.
• All mutations corrected.
In vitro cysteine biosynthesis
In vitro CSase activity
Results
µmol cys min-1 ml-1
35
30
• Background activity from E.
coli proteins subtracted
25
20
15
• All three genes gcs2 gcs3 gcs4
are functional to transcribe
and translate CSase
10
5
0
GCS2
GCS3
GCS4
Substrate: Na2S
• GCS4 shows the highest
activity in cysteine biosynthesis
Is GSC4 an S-allyl-CS?
35000
Results
30000
Peak area
25000
•
Background activity from E.
coli proteins subtracted
•
GCS4 functions as S-allylCSase
•
GCS2 and GCS3 can act
weakly as S-allyl-CSase
20000
15000
10000
5000
0
1GCS210
1 GCS3
10
GSC2
GCS3
Substrate: allyl mercaptan
1GCS4
10
GCS4
min
While this was going on …..
• Transformation of A. thaliana as in vivo strategy to
assess activity of GCS3, GSC4 and GSAT1
• Used constructs already created for transformation
of tobacco BY2 cells
• Used A. thaliana line containing:
• AlcR transcription factor on 35S promoter
• GUS reporter gene on AlcA promoter
• Checks for AlcR expression
• GUS and garlic transgenes only expressed in
presence of inducer (ethanol)
Transformation of A. thaliana
• Uses Agrobacterium tumefaciens
• Flower heads dipped into detergent and
bacterial mixture weekly for 3 weeks
• Allow seeds to set (~4 weeks)
• Collect seeds
• Used 432 plants per construct
• Several g seeds per construct
Screen seeds for transformants
• Kanamycin selection on phytogel plates
• ~200,000 seeds screened per construct
• Seedlings that survived transferred to soil
• When plants large enough, leaf DNA
preps screened for garlic transgene by
PCR
Results
• Transgenic A. thaliana
16 plants contain gcs4
7 plants contain gcs3
6 plants contain gsat1
• No obvious phenotype in non-induced
plants, as expected
• These transgenic plants (To) have been selffertilized to obtain seeds (T1)
The final step
Analyse T1 plants for:
• Presence of transgenes – PCR
• Expression of alcR – GUS staining
• Expression of transgenes - RT-PCR
• Activity of cysteine synthase - spectrophotometry
• Activity of S-allyl cysteine synthase - HPLC
A. thaliana HPLC profile
alliin
Young leaves
isoalliin
100
S-allylcysteine
80
mV
60
40
20
0
0
10
20
time (min)
30
Deliverables: by December 2003
• DP. 23 Papers on alliin biosynthesis and
sulphur partitioning
• Synthesis of alliin in garlic and onion tissue
cultures – draft on project website
• DP. 29 Papers on the characterisation of key
enzymes in alliin biosynthesis and alliinase
expression and the regulation of sulphur
biochemistry in garlic
• Functional analysis of a novel garlic cysteine
synthase in Arabidopsis thaliana
Deliverables: by December 2003
• DP. 33 Paper on S pathway genes on the
production of flavour precursors in garlic
• Biosynthesis of the flavour precursors of onion and
garlic, invited review for special issue on Sulphur
Metabolism in Plants, Journal of Experimental Botany
– in preparation
• DP. 36 Paper on the regulation of sulphur
biochemistry in garlic
• Induction of the pattern of flavour precursors in garlic
– in preparation
Other publications
• Poster presented at Seventh International
Congress of Plant Molecular Biology,
Barcelona, June 2003.
‘Molecular analysis of cysteine synthase and
allylcysteine synthase from garlic, and their
contributions to garlic flavour precursor
biosynthesis’