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

OAS

Cysteine

SAT/CS

Free CS

3-Cyano-L-Ala

Free CAS/CS

Acetyl Co-A

Sulfide

Cyanide

Allyl-mercaptanPyrazol

-PA S-allyl-L-Cysteine

?

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

gsat1

18s

1. 7 degree C stored clove

2. RT stored clove3. Sprouting clove4. Leaf5. Root

• The potential 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.

Phylogenetic Tree

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]GCS4 related to two isoforms identified from rice that form a new CSase family.

Expression of CSase and SAT

alcRP T

ALCR

Transcription Factor

EtOH

Inducer

ALCR

Garlic geneT

Express

Garlic protein

palcA

Transgenic tobacco BY2 cells and A. thaliana

LBt35S palcA pAg7nptIIpnosRB Garlic

gene

Transformation of tobacco cells for protein expression

LBt35S palcA pAg7nptIIpnosRB Garlic

gene

Transformed Untransformed Transformed sub-cultured

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?

1 2 3 4

RT-PCR results: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

T7P

5’ RBS Garlic gene His-tag T7T 3’

T7P

RBS Garlic gene T7T 3’5’

Garlic genes:

gsat1

gcs2; gcs3gcs4

His-tag

TAA

• 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

0

5

10

15

20

25

30

35

µm

ol c

ys

min

-1 m

l-1

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

Substrate: Na2S

GCS2 GCS3 GCS4

Is GSC4 an S-allyl-CS?

Results

• Background activity from E. coli proteins subtracted

• GCS4 functions as S-allyl-CSase

• GCS2 and GCS3 can act weakly as S-allyl-CSase

Substrate: allyl mercaptan

0

5000

10000

15000

20000

25000

30000

35000

GCS2 GCS3 GCS4

Pe

ak

are

a

1 10 1 10 1 10 min GSC2 GCS3 GCS4

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. thaliana16 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 self-

fertilized 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

0 10 20 30

0

20

40

60

80

100

mV

time (min)

alliin

isoalliin

S-allylcysteine

Young leaves

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

• 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

Deliverables: by December 2003

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’