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WP4: Garlic sulphur biochemistry

This report from The University of Liverpool explores the molecular mechanisms of sulphur metabolism in garlic, focusing on differentially expressed genes under varying sulphur conditions. It highlights methodologies such as differential display, cDNA subtraction, and proteomics to unravel the complexities of gene expression influenced by sulphur status. The study reveals that genes can be expressed under specific signalling conditions, and the potential effects of sulphur levels on plant metabolism are examined. Various experimental approaches and the challenges encountered in gene expression verification are also discussed.

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WP4: Garlic sulphur biochemistry

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  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

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