Download 1 - Plant Research International

1. Participant N° : P3
2. Address
School of Biological Sciences, Donnan Laboratories, The University of Liverpool, Liverpool. L69 7ZD,
United Kingdom.
Tel: 44 151794 3612, Fax: 44 151 794 3655, E-mail: h.a.collin@liverpool. ac.uk
3. Scientific team
Research leader:
Dr H. A. Collin
(8 person months in total)
Duties: Supervision of research, flavour compound analysis
Research scientists:
Dr M. G. Jones
(16 person months in total)
Duties: Plant biochemistry
Prof. A. B. Tomsett
(8 person months in total)
Duties: Plant molecular biology
Dr R. Cosstick
(4 person months in total)
Duties: Synthesis of pathway intermediates
Ms A. Tregova (Research Assistant)
(48 person months in total) Appointed from: May 2000
Duties: Differential display
Dr J. Hughes (Postdoctoral Research Assistant)
(48 person months) Appointed from: June 2000
Duties: Analysis of flavour precursor biosynthetic pathway
Mrs G. Rominez Van Der Werff
(Visiting Research Scientist from Peru - not funded by EU project, commenced visit in March 2000)
Duties : Initiation and maintenance of tissue cultures.
4. Contractual links to other participants
None.The School of Biological Sciences at the University of Liverpool, UK is independent.
5. Objectives
This group is primarily responsible for two objectives in the Sulphur Biochemistry task. The
first objective is to identify the intermediates on the pathway(s) leading to synthesis of alliin,
the major flavour precursor in garlic. This will involve detailed studies of the biochemical
pathways of sulphur compounds operating in garlic tissues, using specially synthesised
labelled precursors. The second objective is to identify genes with altered expression in
tissues with differences in levels of flavour compound pathway flux. A number of approaches
will be used for this, including differential display, cDNA library screening and related
techniques. Any genes isolated from this part of the project will be made available for the
genetic transformation programme in Task 2.
6. Workplan
Sulphur Biochemistry Workpackage: Synthesis pathway for precursors
Identification of the intermediates on the path(s) of synthesis will be by the use of pulse
labelling techniques. Path(s) of synthesis of secondary products, such as alliin, are usually
derived from a particular stage of intermediary metabolism. As illustrated in the bio-synthesis
of alliin the starting point of one pathway (1), dominant during bulb maturation, is serine and
for the other pathway (2), dominant during bulb storage and sprouting, the starting point is
glutathione. In the method of pulse labelling the plant material is exposed to the labelled
intermediate for a short time, which can be anything from 10 mins to 24 hrs, which is followed
by prolonged exposure to the unlabelled intermediate. During the period after the pulse, the
plant material is sampled and the intermediates and alliin separated by HPLC. The optimum
period of exposure to the radio-label and the frequency of the sampling after exposure needs
to be established for the garlic system.
Evidence for involvement of any intermediate in the pathway is through the appearance of
radioactivity in an intermediate and alliin, then subsequent decline as the radioactive
compounds are replaced by non radioactive sources. The importance of any intermediates
on a pathway is confirmed by chemical synthesis of the proposed intermediate incorporating a
radioactive label where possible, followed by exposure of the plant material to the radiolabelled intermediate and re-examination of the pattern of labelling. For examination of the
paths of synthesis of alliin, the two most useful sources of radioactivity are 14C and 35S which
will be employed as follows:
The most direct method of introducing a radioactive label into alliin via pathway 1 is to expose
a growing plant to 14C serine. If S-allyl cysteine, a supposed intermediate on pathway 1 does
become labelled, then this will be synthesised containing 14C and the experiment repeated for
confirmation.
The method of entry into pathway 2 will be by pulse labelling of the roots of rooted cloves in
nutrient medium containing 35SO4 followed by analysis of the pattern of labelling in the leaves.
Particular attention will be paid to the labelling in the S-allyl--glutamyl cysteine and S-allyl
cysteine. Leaves are used as the experimental material as this is the tissue where the most
active synthesis of alliin occurs.
Once the pathway has been outlined the next stage will be to examine sub-cellular
localisation. Isolated organelles will be treated in the same way as the intact leaf and
exposed to radioactive intermediates followed by extraction and HPLC analysis to establish
where in the cell the different pathways operate and if there is a site of synthesis with a
separate site of accumulation.
Sulphur Biochemistry Workpackage: Genes for CSO synthesis
The genes responsible for the pathway enzymes will be identified directly using the
techniques of differential display (P3). The added advantage of these studies is that it leads
into the possible genetic manipulation of the pathway. In the technique of differential display
mRNA is used to produce a large number of cDNAs, each of which is multiplied by PCR using
a single primer and the many amplified DNAs are then separated on a gel. The next stage is
to identify a situation in which alliin synthesis is stimulated in the intact plant, such as
exposure to high soil sulphur, high temperatures, drought or exposure to cadmium which
removes the glutathione. The differential display analysis is then repeated for these
conditions and the band pattern compared with the plants under standard conditions. New
bands, or those which are enhanced in the above conditions, are identified and assumed to
be related to the ability of the plant to show increased levels of alliin. Selected bands are
isolated and sequenced. In order to establish whether the bands are in any way related to the
pathway of alliin synthesis, the separated DNA will need to be incorporated into E.coli or
yeast and the protein expressed. These organisms may be incubated in the presence of an
intermediate on the path of alliin synthesis to see whether there is any conversion of the
intermediate into the next stage of the pathway.
7. Deliverables
Deliverable
DP. 8
DP. 16
DP. 23
DP. 24
DP. 29
DP. 33
DP. 35
DP. 36
Deliverable title
Delivery
date
Analytical methods for labelling and analysis.
12
Pathway intermediates identified.
24
Papers on alliin biosynthesis and sulphur partitioning
36
Genes for key CSO synthesis enzymes
36
Papers on the characterisation of key enzymes in alliin 48
biosynthesis and alliinase expression and the regulation of
sulphur biochemistry in garlic.
Paper on S pathway genes on the production of flavour 48
precursors in garlic
Paper on the regulation of the alliinase expression
36
Paper on the regulation of the sulphur biochemistry in 36
garlic
Delivered


Milestones
Identification of selected enzymes involved in the biosynthesis of alliin
.
ACHIEVED
Identification of genes responsible for selected enzymes involved in alliin biosynthesis
ACHIEVED
Continued identification of intermediates in the synthesis of alliin and isoallin
ACHIEVED
Research progress in year 3
Research was undertaken in the following three areas:
1. Identification of intermediates in the synthesis of alliin.
2. Analysis of enzymes in biosynthetic pathway leading to alliin synthesis
3. Identification of genes responsible for enzymes in the biosynthesis of alliin and the
expression of these genes in transformed cell cultures.
.
1. Identification of genes involved in syntheis of the flavour precursors 1.1.
1.1. Introduction
Cysteine synthesis is essential for growth and metabolism. It links carbon and
nitrogen metabolism and is a central precursor for all biological compounds that contain
reduced sulfur (Hofgen et al., 2001) including CSOs. Synthesis occurs in green and nongreen tissues (Barroso et al., 1998) in two sequential reactions. First, O-acetylserine (OAS),
the carbon precursor of cysteine, is synthesized from L-serine through addition of an acetyl
group from acetyl CoA by serine acetyltransferase (SAT) that is complex bound to cysteine
synthase (O-acetylserine (thiol) lyase; OAS-TL; CSase) (Zhu et al., 1998). Bound CSase acts
as a structural or regulatory subunit of SAT and is mostly inactive in protein synthesis (Wirtz
et al., 2001). Second, free CSase inserts sulfide into OAS. Thus for effective cysteine
synthesis to occur, an excess of free cysteine synthase is required ( Droux et al., 1998). It is
thought that in plant cells cysteine is synthesized in situ. Compartment specific SATases and
CSases have been localized in various plants (Urano et al., 2000).See Fig 1 for pathway
details.
Compartment specific SATases can be divided into two types: L-cysteine feedback
sensitive and insensitive (Noji et al., 1998). Interestingly these properties are also reflected at
the nucleotide level as illustrated by various sequence alignments (Urano et al., 2000).
SATases localized in chloroplasts and mitochondria are feedback insensitive. It is thought that
these isoforms are involved in cysteine synthesis only. In addition, glutathione and methionine
production in chloroplasts are likely to increase the demand for cysteine. In the cytoplasm the
cysteine biosynthetic pathway differs in that the intermediate OAS is engaged in two distinct
functions: cysteine synthesis and regulation of other sulfur responsive genes (Urano et al.,
2000). Thus SAT-C is L-cysteine feedback sensitive, although regulation can be overruled by
nutritional stress (Saito, 2000). Furthermore, sensitivity levels can vary between plant
species. For example, SAT-C isolated from Allium tuberosum (Chinese chive) showed a
significantly higher 50 % inhibition (IC50) value than SATases analysed from other plants.
Urano et al. (2000) indicated that this could explain the comparative high cysteine content
detected in Allium plants that might be required for CSO biosynthesis. It is not known why
cysteine synthases have acquired such broad substrate specificity, but it has prompted us to
examine their possible role in CSO biosynthesis.
1.2. Experimental
It is the aim of this work to isolate multiple SATases and CSases from a garlic
specific cDNA library with the objective to investigate the ability of each isoform to produce Sallyl-L-cysteine, the direct precursor of alliin.
1.3. Results
1.3.1.PCR amplification of CSase cDNA fragments from garlic callus, root, and leaf
Twenty full-length plant CSase cDNA nucleotide sequences were downloaded from
GenBank and aligned to design a range of degenerate PCR primer pairs with the objective to
RT-PCR cDNA fragments for more than one CSase isoform that can be used to enrich and
screen a cDNA library. First-strand RT-PCR reactions were conducted from total RNA
extracted from callus, leaf and root. Second strand synthesis reactions were primed
successfully with three degenerate primer pairs. The estimated molecular weight for a cDNA
fragment amplified with PCR primer pair CS1 was 685 bp, for primer pair CS2 617 bp, and for
primer pair CS5 582 bp. PCR products were resolved on a standard 2 % agarose gel.(Fig. 2)
PCR products of the expected size were excised from the gel, and among the seven
cDNA fragments analyzed, three distinctly different CSase cDNA fragments were recognized,
that when aligned fell into two contigs.
1.3.2. PCR amplification of SATase cDNA fragments from garlic clove, leaf, and root
Sixteen full-length plant SATase cDNA nucleotide sequences were downloaded from
GenBank and aligned. Two degenerate PCR primer pairs were designed with the objective to
RT-PCR one or more SATase cDNA fragment(s) that would be used to enrich and screen a
cDNA library. First-strand RT-PCR reactions were conducted from total RNA extracted from
clove, leaf and root. Second strand synthesis reactions were successfully primed with only
one degenerate primer. PCR products were resolved on a standard 2 % agarose gel ( Fig. 3).
Bands of the right size were excised from the gel, cloned sequenced and analyzed
1.3.3. PCR amplification of a S-allyl-cysteine synthase cDNA fragment from garlic
Dr. Jill Hughes assayed garlic leaf extract fractions for cysteine synthase activity by
HPLC analysis. Dr. Mark Wilkinson separated fractions that contained S-allyl-CSase activity
by polyacrylamide gel electrophoresis and partially sequenced a polypeptide fragment. The
data suggested that the amino acid composition of the analyzed peptide region is conserved
among plant CSases, including GCS1, GCS2, GCS3, three CSase isoforms detected in this
study. They showed the greatest similarity to RCS2 and RCS4, two recently identified CSases
from rice that potentially form a new CSase family (Nakamura et al., 1999). This clearly
indicates that the identified S-allyl-CSase is novel and could be related to the rice RCS2 and
RCS4 enzymes.
In order to generate a short S-allyl-CSase cDNA fragment required for successful cDNA
library screening nine degenerate PCR primers were designed that were used in combination
with library specific 5’ and 3’ sequencing primers. Among the seven cDNA fragments
generated by these primers,only one fragment was confirmed as S-allyl-Csase.(Fig 4)
Interestingly, this fragment had been amplified with a degenerate primer optimized for the rice
RCS2 sequence . The deduced amino acid sequence shows that this cDNA fragment shares
62 – 65 % homology with the RCS4 and 58 – 60 % homology with the RCS2 polypeptide
sequences providing further evidence that these enzymes could be functionally related.
1.3.4. Detection and sequence analysis for plural CSase isoforms - clones GCS1,
GCS2, GCS3 and GCS4
This section describes the isolation and analysis of four full-length CSase cDNAs
coding for two potentially chloroplastic (GCS1, GCS2), one cytosolic (GCS3), and a potential
S-allyl-CSase (GCS4).
First, a plasmid cDNA library was constructed from combined total RNA extracted
from garlic clove, leaf and root organs that was enriched and screened separately for multiple
cysteine synthase isoforms. Clones GCS1 and GCS2, two potential chloroplastic isoforms
(see below) were isolated from a plasmid cDNA library that had been enriched with a PCR
product amplified from cDNA fragment leaf 2 - 3. Clone GCS4, a potential S-allyl-CSase was
isolated from a plasmid cDNA library that had been enriched with a PCR product derived from
a S-allyl-CSase specific cDNA fragment. Among the 3.6 x 103 colonies screened, twelve
identical full-length cDNAs were identified.
Second, the complete cDNA nucleotide sequences for clones GCS1, GCS2, GCS3
and GCS4 were analyzed. The assumption that GCS1 and GCS2 represent two different
genes within the garlic genome, i.e. GCS1 did not arise from a library construction error, is
supported by the fact that the 5’ and 3’ UTRs of both clones are not identical. GCS3 consists
of an ORF of 969 base pairs. There are two in-frame transcription termination sequences
within the 5’ UTR and one within the 3’ UTR confirming the isolation of a full-length cDNA. In
comparison, clone GCS4, i.e. the potential S-allyl-CSase contains an ORF of 1005 base pairs
that is not surrounded by in-frame termination sequences. However, the cDNA for clone
GCS4 is full-length. It contains a 5’ adenine rich UTR and a translation initiation motif that
closely resembles the consensus sequence for the plant gene initiation motif AACAATGGC
(Luetcke et al., 1987). The potential translation initiation motifs for GCS1, GCS2, GCS3 and
GCS4 are as follows:
consensus sequence
GCS1
GCS2
GCS3
GCS4
AACAATGGC
CGCCATGGC
CGCCATGGC
AGAAATGGG
GAAGATGGG.
Furthermore, all four isolated cDNAs contain potential polyadenylation sites. In plants
this site generally consists of AATAAA, is surrounded by guanine or cytosine residues and is
usually located 20 nucleotides from the translation termination codon (Mantell et al., 1985).
The potential polyadenylation site for GCS3 and GCS4, i.e. AATAAA, is found 63 base pairs
downstream from the translation termination codon. The potential polyadenylation site for
GCS2 is located 132 base pairs downstream from the translation termination codon and
consists of AAATTA instead of AATAAA.
Finally, the deduced amino acid sequences for clone GCS1, GCS2, GCS3 and GCS4
were analyzed. We predicted that GCS1 codes for a non-functional and GCS2 for a functional
putative chloroplastic CSase. The immature GCS2 polypeptide consists of 383 amino acids
with a calculated molecular mass of 40.615 kDa. The mature polypeptide consists of 332
amino acids with a calculated molecular mass of 34.922 kDa. In contrast, the polypeptides for
GCS3 and GCS4 do not contain any transit peptides and are therefore active in the cytosol.
Sequence analysis of three short polypeptide fragments indicated that the detected CSase
isoform might be related to the rice RCS2 and RCS4 enzymes. In order to gain more
information an identity count was conducted from amino acid residue 102 – 455 (relative to
the A. thaliana amino acid sequence) according to the BLOSUM 62 Matrix (http://wwwlehre.img.bio.uni-goettingen.de/edu/Bio_Inf/MolBiol/blosum62.htm
The results illustrated that there was relatively little similarity between the S-allyl-CSase
GCS4 and CSase isoform A, B, and C from various sources. In contrast, the similarity
between GCS4 and RCS2 / RCS4 was relatively high. These differences suggest that the
CSases may each perform a different function
1.3.5. Detection and sequence analysis for a cytosolic SATase
A plasmid cDNA library was constructed from combined total RNA extracted from
garlic clove, leaf and root organs. An aliquot of the cDNA library was enriched with a PCR
product derived from a cDNA fragment (see Fig. 2.9) with the aim to detect at least one
SATase isoform. Among the 1.6 x 104 colonies screened one full-length SATase cDNA was
identified.
2. In vivo expression and analysis of allyl cysteine synthase gene
2.1. Introduction
Screening for a novel CSase that exhibits S-allyl-CSase activity has let to the
isolation of four clones encoding cysteine synthase.
1. GCS1 – a potential pseudogene
2. GCS2 – a potential choroplastic type
3. GCS3 – a cytosolic type
4. GCS4 – a potential S-allyl-CSase.
In addition, a cDNA that codes for a cytosolic SATase i.e. clone GSAT1 was also
isolated.. The cDNA, however, contains an in-frame stop codon, hence is either transcribed
into a truncated, non-functional protein, or may represent a novel selenoprotein. A functional
analysis for the gcs4 and gsat1 cDNAs was carried out by in vivo expression, HPLC and
Northern blot analysis.
For the in vivo studies we decided to transform the gcs4 and gsat1 cDNAs into a
tobacco cell-suspension culture by Agrobacterium-mediated gene transfer.
2.2. Experimental
To investigate the functions of the gcs4 and gsat1 gene products we decided to take
advantage of the alcR gene switch. This chemically inducible promoter system was
developed in our laboratory by Caddick et al. (1998) and is ideal for plant expression studies,
i.e. it is highly inducible with ethanol a low phytotoxic chemical, shows low background
activity, is not activatable by endogenous plant transcription factors, and expression is
reversible after approx. nine days. The gene switch consists of two components that form part
of the ethanol utilization regulon found in the ascomycete fungus Aspergillus nidulans. The
first component alcR codes for the trans-acting transcription factor ALCR and is constitutively
expressed from the cauliflower mosaic virus 35S promoter. In the presence of certain
chemicals, e.g. ethanol, the zinc DNA-binding protein ALCR is activated and binds to the
second switch component, the highly inducible alcA promoter that is linked to a gene of
interest. In Aspergillus binding activates the alcohol dehydrogenase gene (Kulmburg et al.
1992; Felenbok, 1991).
However, plant tissue cultures contain endogenous ethanol at levels high enough to
activate constitutive transgene expression. To overcome this problem John Dunnan et al.
(personal communication) developed a system similar to the recently published SMGD
(steroid mediated gene transfer) method (Rebuffat et al., 2001). SMGD had been developed
to overcome the barrier of the nuclear envelope in mammalian cell transformation. In
principle, a steroid receptor backbone is crosslinked to a transgene. Glucocorticoid receptors
located in the cytoplasm recognize the steroid and binding triggers translocation of the steroid
-receptor complexinto the nucleus. John Doonan and his team have fused the cytosolic
glucocorticoid receptors to the ALCR protein. As a consequence the fusion protein remains in
the cytoplasm. When activated with a suitable steroid, e.g. dexamethasone, the transcription
factor migrates to the nucleus where it interacts with endogenous ethanol and consequently
binds to the alcA promoter inducing trans-gene expression. John Doonan kindly provided the
tobacco cell suspension culture harboring the modified ALCR protein.
2.3. Results
2.3.1. Construction of binary plasmids pGPTV-GCS3, pGPTV-GCS4 and pGPTV-GSAT1
Cloning strategies were designed to insert the gcs4 and gsat cDNAs into the
expression system. This utilised a series of vectors already available in our laboratory
(Caddick et al 1998)and taking into account internal endonuclease restriction sites in the
gsat1 Currently the construct have been inserted into cells suspensions of tobacco. The
synthesis and expression of the enzymes in these cells is being assessed.
3. Separation of flavour precursors and intermediates by HPLC
3.1. Introduction
The method of of separation of flavour precursors used earlier was perfectly
adequate for the separation of the flavour precursors but not the gamma glutamyl cysteine
sulphoxides, which may be important storage compounds or even intermediates in precursor
synthesis. The method involving heptane sulphonic acid used currently in the project was
attempted and although it gave a good separation of the flavour precursors the content of the
mobile phase meant that identification of the separated compounds could not be achieved by
mass spectrometry. A simpler mobile phase was required to enable the HPLC to be combined
with the mass spectrometry.
3.2. Experimental
A simple HCl:Acetonitrile gradient was developed for the HPLC which allowed
simultaneous identification of alkyl cysteine sulphoxides and gammaglutamyl peptides.
3.3. Results
A typical HPLC profile of an extract of garlic clove is shown in Fig. 5. The profile
shows a good separation of alliin and isoalliin and gamma-glutamylallylcysteine and γglutamylisoallylcysteine and other γ-glutamyl compounds which have yet to be identified.
Retention times have been identified for γ-glutamyl cysteine, methionine, ethyl cysteine,
methylglutathione , 3-carboxypropyl cysteine, carboxypropylcystine,2-carboxypropylcysteine,
allylcysteine, butyl cysteine sulphoxide, 1,2 dicarboxyglutathione, propyl cysteine,
propylcysteine, γ glutamylallylcysteine, butyl cysteine, and propylglutathione.
4. Feeding intermediates of flavour precursors to tissue cultures
4.1. Introduction
The aim of the precursor feeding was to elucidate the biosynthetic pathway for the
synthesis of the flavour precursor allyl cysteine sulphoxide ((+)-S-2-propenyl-L-cysteine
sulphoxide, alliin) in Allium sativum. The tissue chosen for these studies was garlic callus.
Onion callus had been used previously for precursor feeding experiments in this laboratory
since it is relatively easy to introduce the substrate into this intact tissue since it is friable and
has a limited waxy outer layer. The advantages of using an intact tissue is the containment of
the vacuolar alliinase enzyme which would normally breakdown any alk(en)yl cysteine
sulphoxide products. The presence alliinase makes the analysis of this secondary pathway
more difficult since any injury to the tissue results in the degradation of the final product alliin.
Early HPLC analysis of the undifferentiated garlic callus showed little background interference
and no alliin was present. The limitations of this tissue are that the substrate may not be able
to get into this (or any) tissue type, or that the enzymes that utilise the substrates may not be
present.
Undifferentiated garlic callus was used for the earlier precursor feeding studies. The
callus was exposed to a wide range of possible precursors and intermediates and their
products analysed by HPLC. Particular focus was placed on the general conversion of
alk(en)yl thiols to their corresponding cysteine conjugates and cysteine sulphoxides since
limited evidence suggested that serine together with an allyl source was the starting point for
the path of alliin production in vegetatively vigorous tissue.The evidence for this conversion is
that purified cysteine synthase enzymes have been shown to convert allyl thiol to allyl
cysteine (Ikegami,et al 1988). In addition differentiating tissue cultures of garlic exposed to
allyl cysteine are able to oxidise this intermediate to alliin (Oshumi, Hayashi and 1993)..
Exposure of the undifferentiated garlic tissue to allyl thiol led to the accumulation of
both allyl cysteine and alliin while allyl cysteine was readily oxidized to alliin. Although the
amounts of propiin are generally very low in garlic the callus was still capable of making the
conversion from propyl thiol to propyl cysteine and propiin. (second year report). Interestingly
onion callus, which does not normally accumulate alliin formed alliin when exposed to either
allyl thiol or allyl cysteine. Propenyl cysteine and propyl cysteine were oxidized to their
respective iso alliin and propiin end products. Most of these conversions occurred after 5
days incubation.
A series of further experiments were undertaken to build on the results so far
obtained. Callus was exposed to an enlarged range of single substrates at 1 and 10mM
concentration (serine, cysteine, glutathione, 2-carboxy propyl cysteine, 3-carboxy propyl
cysteine, allyl alcohol, propyl alcohol, vinylacetic acid, methacrylic acid and cystathione) and
reduced times of incubation. (1 and 4 days). Analysis of this experiment was halted when it
became apparent that the callus was very variable such that supposed undifferentiated callus
showed signs of differentiation, thus invalidating the significance of any results obtained.
(Burgaud,Gravot, Milesi and Gontier, 2001). This variation in callus has been reduced by
selecting one rapidly growing callus as a source for all subsequent subcultures. The aim was
to repeat the precursor feeding with garlic callus grown from this single selected clone so as
to establish whether the serine pathway was functional in garlic.
4.2. Experimental
Undifferentiated garlic callus was maintained on a phytogel medium and a sulphur
depleted medium (all sulphate compounds replaced with chloride). A range of potential
precursors, synthesised or purchased were incorporated in callus medium prior to incubation.
The production of any alk(en)yl cysteine conjugate and/or alk(en)yl cysteine sulphoxide was
measured by HPLC after extraction of the callus. The precursors used were allyl thiol, propyl
thiol, allyl cysteine and propyl cysteine all at a concentration of 10 mM. The tissue was
sampled at 2, 6 and 13 days.
4.3. Results
There was an increase in alliin, propiin, allyl cysteine and propyl cysteine
when the relevant intermediates were used which indicated the network nature of the
metabolic pathways.There were no obvious differences between the sampling times. It
appeared that thiols provided an intermediate for the synthesis of alliin or propiin confirming
the possibility of the existence of this pathway. (Table 1).
5. Induction of enzymes involved in alliin and isoalliin synthesis
5.1. Introduction
In an attempt to induce the pathways of synthesis of the flavour precursors so as to
identify specific enzymes associated with these pathways, a number of possible inductive
situations were investigated . These included location within the bulb ie clove variation , the
storage temperature of the garlic bulb and the effect of differentiation on the garlic callus. The
effect of clove variation and storage temperature were combined in one experiment. Portions
of undifferentiated callus were placed on a hormone free medium for six weeks then extracted
and analysed by HPLC.
5.2. Experimental
Garlic bulbs were maintained at room temperature and at 4oC for six weeks then the
tissue extracted and analysed by HPLC.
5.3. Results
The results of the temperature treatment are presented in Fig. 6. The major change
was the effect of temperature on the flavour precursors. The greatest change was the large
increase in the concentration of isoalliin and the reduction in glutamylallylcysteine and
isoglutamylallylcysteine. Although the results are not shown the differentiated tissue shows a
large increase in isoalliin from very low or non existent levels in the undifferentiated callus.
Both the temperature effect and the differentiation in the callus provide inductive situations
with which to examine the expression of the enzymes involved in the synthesis of isoalliin.
6. Role of glutathione in synthesis of alliin and isoalliin
6.1. Introduction
Glutathione-S-transferases (GSTs) are known to conjugate glutathione to xenobiotics
(Marrs 1996) and may also be involved with conjugation of glutathione to endogenous
substrates to form secondary products such as alliin. The proposed intermediateds
allylglutathione, carboxypropylglutathione and methylglutathione are all glutathione
conjugates.
6.2. Experimental
To investigate the position of glutathione in the biosynthetic pathway, an attempt was
made to isolate glutathione –S-transferase from garlic. Soluble proteins were absorbed onto a
glutathione affinity matrix, and after elution with glutathione one dimensional SDS protein
electrophoresis 1DSDS gels showed only one major band (approximate molecular weight of
25000). Two main components could be resolved after separation on a two dimensional
protein gel. A range of potential endogenous substrates and products either synthesised or
obtained commercially, were incubated with total soluble protein or the GST fraction from the
affinity column. The purpose was to identify a potential substrate whose structure would
indicate a compound likely to be part of the alliin biosynthetic pathway. The products were
analyzed by HPLC. Enriched protein was shown to contain GST activity using 1-chloro-2,4dinitrobenzene (CDNB) as substrate. (Fig.7)
6.3. Results
Unfortunately the enzyme fraction showed no activity towards substrates that were
candidates for precursors of γ glutamyl peptides.
References
Barroso, C., Vega, J.M. and Gotor, C. (1998). The role of roots in cysteine biosynthesis by
Arabidopsis thaliana. J. Physiol. Biochem. 54: 189 – 94.
Bourgaud, F., Gravot, A., Milesi, S. and Gontier, E (2001) Production of plant secondary
metabolites :a historical perspective Plant Science , 161, 839-51.
Caddick, M.X., Greenland, A.J. Jepson, I., Krause, K.P., Qu, N., Riddell, K. V., Salter, M.G.,
Schuch, W., Sonnewald, U. and Tomsett, A.B. (1998) An ethanol inducible gene switch for
plants used to manipulate carbon metabolism. Nat. Biotechn., 16, 177-80.
Droux, M.,Ruffet, M.L., Douce, R. and Job, D. (1998) Interactions between serine
acetyltransferase and O-acetylserine (thio) lyase in higher plants. Structural and kinetic
properties of the free and bound enzymes. European J. Biochem. 255, 235-45.
Hofgen, R., Kreft, O., Willmitzer, L. and Hesse, H. (2001). Manipulation of thiol contents in
plants. Amino Acids, 20: 291 – 299.
Ikegami, F., Takayama, K., Tajima,, C. and Murakoshi, I (1988) Purification and properties of
B-cyano-L-alanine synthase from Spinacia oleracea. Phytochem., 27, 2011-2016.
Kulmburg,P., Sequeval, D., Lenouvel, F., Mathieu, M., and Felenbok, B. (1992) Identification
of the promoter region involved in the autoregulation of the transcriptional activator ALCR in
Aspergillus nidulans. Mol. Cell Biology 12, 1932-1939.
Luetcke, H.A., Chow, K.C., Mickel, F.S., Moss, K.A., Kern, H.F. and Scheele, G.A.
(1987).Selection of AUG initiation codons differs in plants and animals. EMBO Journal, 6, 4348.
Mantell, S.H., Matthews, J.A. McKee, R.A. (1985). Principles of plant biotechnology-an
introductionto genetic engineering in plants. Blackwell Scientific Publications.
Nakumara, T., Yamaguchi, Y., and Sano, H. (1999). Four rice genes encoding cysteine
synthase: isolation and differential response to sulfur, nitrogen and light. Gene 229, 161.
Noji, M., Inoue, K., Kimura, N., Gouda, A. and Saito, K. (1998). Isoform-dependent
differences in feedback regulation and subcellular localization of serine acetyltransferase
involved in cysteine biosynthesis from Arabidopsis thaliana. The J. Biol. Chem. 273: 32739–
2745.
Ohsumi, C., Hayashi, T. and Sano, K. (1993). Formation of alliin in the culture tissues of
Allium Sativum. Oxidation of S-allyl-L-cysteine. Phytochem. 33: 107–11.
Rebuffat, A., Bernasconi, A., Ceppi, M., Wehrli, H., Vercas, S.B., Ibrahim,M., Frey, B.M., Frey,
F.J.and Rusconi, S. (2001) Selective enhancement of gene transfer by steroid –mediated
gene delivery. Nat. Biotech. 19, 1155-157.
Saito, K.( 2000) Regulation of sulphate transport and synthesis of sulfur- containing amino
acids. Current Opinions in Plant Biology. 3, 188-195.
Wirtz, M., Berkowitz,, O., Droux, D. and Hell, R. (2001). Three Arabdopsis Genes encoding
proteins with differential activities for cysteine synthase and B-cyanoalanine synthase. Plant
Cell Physiol. 41, 465-476.
Urano, Y., Manabe, T., Noji, M. and Saito, K. (2000). Molecular cloning and functional
characterization of cDNAs encoding cysteine synthase and serine acetyltransferase that may
be responsible for high cellular cysteine content in Allium tuberosum. Gene 257: 269–77.
Zhu, X., Yamaguchi, T. and Masada, M. (1998) Complexes of serine acetyltransferase and
isoenzymes of cysteine synthase in spinach leaves. Biosc. Biotechnol. Biochem. 62, 947-52.
Appendix