WP4: Garlic sulphur biochemistry

1. WP4: Garlic sulphur biochemistry • P3: The University of Liverpool, UK Angela Tregova reported by Brian Tomsett Hamish A Collin, Meriel G Jones, Rick Cosstick, Jill Hughes, Gloria van der Werff

2. Possible Molecular Approaches signal ?? Cysteine Alk(en)yl CSOs • Genes only expressed when signal is present • Genes always expressed, but differentially processed • Genes always expressed, protein expression differential • Genes and Protein always expressed What is the signal?

3. Sulphur metabolism SO4 SO3 S Serine Methionine Cysteine O-acetyl serine Alk(en)yl CSOs

4. Differential display - 3 • Five arbitrary 20-mer primers (3 - 7) • H = Normal S L = Low S • Duplicate PCR reactions • Boxes – differential expression

5. Differential display to identify genes - 5 • Changing S status has both increased and decreased levels of bands • Bands from long-term Low S and Normal S, give more bands than short-term changes • The identity of these bands tested by extraction from gel, Re-PCR, cloning and sequencing

6. Differential display - problems 1 • Extraction from gel - bands highly heterogeneous • Re-PCR - high No PCR cycles increases contamination • Cloning - which product or all products from PCR? • Sequencing - not all bands can be sequenced. Even then - not all bands will be differentially expressed

7. Verifying differential expression by Northern analysis RNA samples from garlic callus grown on low S for 2 weeks (LS) or normal S for 2 weeks (NS) False positive result LS NS

8. Verifying differential expression by Northern analysis LS NS RNA samples from garlic callus grown on low S for 2 weeks (LS) or normal S for 2 weeks (NS) Arrows show hybridisation with a differential cDNA in low S total RNA

9. The Way Forward • Define conditions for secondary Sulphur metabolism • Consider alternative strategies • Differential display • cDNA subtractive hybridisation • cDNA arrays • proteomics These will depend on clues from the biochemistry