Workpackage Number: WP4; Sulphur Biochemistry

Workpackage number: WP4; Sulphur biochemistry

Phase: 24-36 months

Start date: Month 0 (February 1 2000)

Completion date: Month 48 (January 31 2004)

Current status: Ongoing

Partners responsible: P2, P3

Person months per partner and total: P2: 66, P3: 132; total: 198

Already devoted person months per partner and total: P2:11.6, P3: 99; total: 110.6

Objectives

The overall aim of this task is to understand the rate-limiting processes in the accumulation of alliin and its subsequent conversion to allicin. These include the biochemical steps in alliin synthesis and alliicin formation as well as sulphur transport within the plant. Three distinct objectives will be addressed using different but complementary approaches.

· The first objective is to identify the intermediates on the pathway(s) leading to synthesis of the major flavour precursor in garlic, alliin. This will involve detailed studies the biochemical pathways of sulphur compounds operating in garlic tissues, using specially synthesised labeled precursors.

· The second objective is to identify the developmental control points of cysteine sulphoxide synthesis and translocation. We will accomplish this by profiling and tracking of sulphur components in leaf and clove tissues throughout their growth and development.

· The third objective is to identify genes with altered expression in tissues with differences in levels of flavour compound pathway flux and genes involved in the conversion of alliin to alliicin. 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.

Methodology and study materials

Intermediates on the path(s) of synthesis will be identified by pulse-labeling techniques. Two potential pathways exist. For one the entry is through serine and will be studied using direct labeling of leaves and other organs with 14C serine. For the other, entry may be through glutathione and labeling will be by feeding roots with 35SO4 followed by analysis of the pattern of labeling in the leaves. Once the pathway has been outlined the next stage will be to examine sub-cellular localisation.

The main sites of CSO synthesis within the whole plant and the stages of development where it occurs will be established by quantitative profiling of CSO and related compounds by HPLC, analysing leaf and bulb tissues during garlic growth in the field. Budgets will be constructed for individual and total sulphur compounds, along with other assimilates and quantitative information on the rates of synthesis and translocation of these compounds during the bulb cycle will be estimated. These studies will be complemented by pulse-labeling studies carried out in the glasshouse, in which whole plants will be labeled with 33SO4 at different stages during their development. Kinetic measurements on sulphur uptake and movement will indicate how transport patterns vary with development.

Genes encoding enzymes in the biosynthetic pathway of alliin will be isolated by differential display. Potential genes will be located based by correlation of their expression in a range of tissues and conditions that show large differences in alliin synthesis. Candidate sequences will be used to screen a cDNA library to obtain full-length genes. Gene function will be assessed by comparison with database sequences for known enzymes and be confirmed by assaying the enzymic properties of the encoded proteins when expressed in E.coli and/or yeast. Clones encoding alliinase will be isolated from a garlic cDNA library and sequenced to identify different members of the gene family. The expression of genes encoding alliin synthesising enzymes and alliinases will be determined in different tissues at different stages of the garlic bulb cycle. Any novel alliinase or other gene sequences will be made available to the transformation programme for over-expression or targeted antisense.

Deliverables

Deliverable /

Status

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) / 
DP. 24: Genes for key CSO synthesis enzymes (P2,P3) / 
DP. 29: Publication on the characterisation of key enzymes in alliin biosynthesis (P2, P3, P5) / 
DP. 35: Publication on the regulation of alliinase expression (P2, P3) / 
DP. 36: Publication on the regulation of sulphur biochemistry in garlic (P2, P3, P5) / 

 = Achieved  = delayed  = future deliverable

Milestones

Year 1 Milestones

Status

Measurement of alkyl cysteine sulphoxides, pathway intermediates and gamma-glutamyl peptides in bulbing and sprouting plants completed. Optimal conditions for pulse labeling of alliin established(P3). / 
Experimental material planted and harvests begun (P2). / 
Differential display of tissues exposed to different cultural conditions complete. (P3). / 
cDNA library from actively synthesising tissues constructed (P2). / 
Year 2 Milestones
Radiolabelled intermediates identified using HPLC and HPLC-MS in comparison with chemically synthesised compounds (P3). / 
First year harvests and analysis completed (P2). / 
Second year experimental material planted. (P2). / 
Differential display sequences and isolated and reanalysed by northern blots and in situ hybridisation, where possible, with other Allium sp. (P3) / 
cDNA clones encoding alliinase isolated and sequenced (P2). / 
Year 3 Milestones
Analysis of pattern of labeling in later stage intermediates completed (P3). / 
Analysis of second-year field experiment completed (P2). / 
Whole plant labeling studies completed (P2). / 
Publication on the developmental control of alliin synthesis submitted (P2). / 
Purification of PCR amplified cDNA and sequencing completed (P3). / 
Expression studies on alliinase clones initiated (P2). / 
Year 4 Milestones
Hypotheses on limiting steps in alliin synthesis tested. (P2, P3). / 
PCR products, and full-length cDNA clones isolated (P2, P3). / 
Heterologous expression in E. coli and/or yeast completed. (P3). / 

 = Achieved  = delayed  = future milestone

Progress during the first reporting period.

Good progress has been made during the first reporting period. All of the milestones were met in full and the two deliverables scheduled for the first year were delivered in full and on time. Progress against the relevant sections of the "Methodology and Study Materials" section is summarised below. Full details are given in the reports from Participants P2 and P3.

"Intermediates on the path(s) of synthesis will be identified by pulse-labeling techniques. Two potential pathways exist. For one the entry is through serine and will be studied using direct labeling of leaves and other organs with 14C serine. For the other, entry may be through glutathione and labeling will be by feeding roots with 35SO4 followed by analysis of the pattern of labeling in the leaves."

The flavour precursors alliin, isoalliin and methiin have been synthesised and also several possible intermediates in the path of synthesis of these compound. The identity of these compounds has been established by mass spectroscopy (MS) and nuclear magnetic resonance spectroscopy (NMR). Further compounds have been purchased, or obtained as gifts from members of the EU Garlic and Health consortium. The HPLC behaviour of these compounds has been established in our analytical system.

The method of extraction from garlic tissues, and separation by HPLC of the flavour precursors and possible intermediates has been simplified and the repeatability of the methods confirmed.

The formation of alliin by garlic tissue cultures from the intermediates, allyl cysteine and allyl thiol, indicated that callus was a suitable tissue to study the path of synthesis of alliin, the major flavour precursor. Synthesis of isoalliin by callus after incubation with carboxypropyl cysteine showed that the tissue was also capable of synthesising isoalliin and could therefore be used to study the synthesis of this minor flavour precursor in garlic.

"The main sites of CSO synthesis within the whole plant and the stages of development where it occurs will be established by quantitative profiling of CSO and related compounds by HPLC, analysing leaf and bulb tissues during garlic growth in the field."

The need to profile sulphur accumulation and re-mobilisation in different tissues of garlic required the development of a cultivation method that allows sulphur inputs to be managed and labeled sulphur to be added or removed from the growing garlic. This has been achieved by growing the garlic varieties in a hydroponic system rather than in soil. During the reporting period a new hydroponic system has been developed and tested by growing garlic plants successfully to the bulbing stage. Test Cysteine sulphoxide determinations have been carried out on cloves from a single bulb grown hydroponically using the HPLC methods developed by partner P2. Alliin is the major CSO at nearly 80% of the total CSOs in garlic clove tissues.

"Genes encoding enzymes in the biosynthetic pathway of alliin will be isolated by differential display. Potential genes will be located based by correlation of their expression in a range of tissues and conditions that show large differences in alliin synthesis."

We have established a differential display method that displays a highly reproducible banding pattern from independent RNA extractions. Several experimental difficulties were identified and overcome successfully. We have used both cut leaf sections and garlic tissue culture as sources of mRNA. In order to establish conditions under which tissues will differentially synthesise alliin it has been necessary to establish cultures of garlic growing in vitro. This has been achieved. Callus cultures of variety Printanor have been initiated and are being maintained regularly. In addition, attempts were made to initiate cultures from variety Messidrôme but progress has been slow. Recently the inhibition on root production has been overcome.

"Clones encoding alliinase will be isolated from a garlic cDNA library and sequenced to identify different members of the gene family."

In order to isolate alliinase gene sequences from garlic it is necessary to construct a cDNA library from tissues actively expressing the enzyme. Two cDNA libraries have been produced, one from bulb and one from leaf material. Both libraries have in excess of 2x106 plaque forming units and an estimated size range of 400 - 3000+ base pairs. These will be used to isolate alliinase sequences from garlic and are available for the isolation of full-length clones of genes isolated in differential display experiments.

Progress during the second reporting period.

Good progress continues to be made in the work package. All of the milestones and deliverables for the second year were achieved. Detailed accounts of the work in this workpackage are contained in the individual reports from partners P2 and P3. The following summarised progress against the Methodology and Study Materials.

"Intermediates on the path(s) of synthesis will be identified by pulse-labeling techniques."

Exposure of the garlic tissue to allyl thiol led to the accumulation of both allyl cysteine and alliin. Allyl cysteine, when taken up by the tissue culture, 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. Under low sulphur conditions only the final stage of the pathway is found after feeding the same intermediates i.e. alliin, isoalliin or propiin. 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 isoalliin and propiin end products in both garlic and onion callus. Most of these conversions occurred after 5 days incubation.

For the system studied the generalised reaction of: alk(en)yl thiol - alk(en)yl cysteine - alk(en)yl cysteine sulphoxide can be carried out by callus. This may indicate that a generalised alk(en)yl cysteine synthase and alk(en)yl cysteine oxidase enzyme(s) exist in garlic and onion tissues. These feeding experiments have given new insights into potential key steps in the synthesis of alliin, in particular indication the potential importance of cysteine synthase in the pathway.

To profile sulphur accumulation and re-mobilisation in different tissues of garlic we grew garlic plants hydroponically in a defined medium using pot-grown plants as a control. Plants were been harvested at regular intervals and the fresh weight, dry weight, total sulphur, total nitrogen, total carbon, total solubilised protein and the cysteine sulphoxide profile determined for clove leaf and root. Measurements were made on two genotypes, Printanor and Messidrome. This data has allowed us to build up for the first time, a picture of the dynamic uptake, allocation and redistribution of carbon, nitrogen and sulphur through the garlic life cycle. We can identify four stages of development from clove planting. The first a period of slow growth and establishment sustained by resources within the clove. The second is a period of rapid and active growth of leaves and root. It is during this phase that the bulk of sulphur and nitrogen is taken up by the plant and the bulk of CSOs are formed. The third phase is bulb formation, during which sulphur (including CSOs) and nitrogen are redistributed to the new cloves in the bulb. Additional carbon continues to be assimilated during this period and accumulates in the bulb. The last phase is maturation where the leaves senesce and die and bulb formation is complete. We will look in more detail at the redistribution of assimilates in the next year.

Following on from the previous year, a differential display method that displayed a highly reproducible banding pattern from independent RNA extractions was developed. Twelve differentially expressed cDNAs were identified that showed no function in flavour precursor biosynthesis. We concluded that a different approach was essential to continue this work successfully. A major objective of the workpackage is to identify key enzyme(s) in the pathway(s) leading to alliin biosynthesis. Based on a comprehensive review of recent literature and the feeding experiments described above the hypothesis that cysteine synthase (CSase) and serine acetyltransferase (SATase) are key enzymes in flavour precursor biosynthesis was developed and work put in place to test this.