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Emilio Ghisalberti (UWA) Kingsley Dixon (KPBG) Robert Trengove (Murdoch) Investigation of the germination promoting compounds in smoke Gavin Flematti The University of Western Australia Slide 2 Slide 3 Solving the bushfire germination mystery.. Slide 4 Smoke as a germination cue In 1990, researchers in South Africa identified smoke as the key agent for stimulating germination of a threatened fynbos species Audouinia capitata (Bruniaceae) Researchers at Kings Park and Botanic Garden (Western Australia) have since identified smoke as the key element for promoting the germination of many Australian native seeds Slide 5 Smoke found to improve seed germination of many Australian native species Slide 6 Slide 7 Sites with smoke-stimulated germination of wild species And the list of species includes. lettuce, celery, parsley and others. Slide 8 Apparatus for generating Plant Derived Smoke Smoke-water Aerosol Smoke Slide 9 Benefits for mining restoration Lateritic nickel project Mineral sands mining restoration - BUT need 10 tonnes smoke water per hectare! Slide 10 Attempts to identify the active compound(s) Baldwin et al. (1994) identified 71 compounds and tested a total of 233 Dixon et al. (1997) identified a further 43 compounds Researchers in South Africa (1995) and California (1997) have also identified compounds in smoke Importantly, burning cellulose produces the active compound(s) Slide 11 Lettuce (Grand Rapids) The Needle in the Haystack - finding the chemical(s) in smoke responsible for germination Conostylis aculeata Stylidium affine Bioassay - Test that uses a biological indicator for tracking the active components through separation steps Slide 12 Isolation of Smoke Compounds Smoke Water Slide 13 Isolation of Smoke Compounds Ether extract Smoke Water Ether layer Aqueous layer Slide 14 Isolation of Smoke Compounds Ether extract Smoke Water Ether layer Aqueous layer Slide 15 Isolation of Smoke Compounds NaHCO 3 soluble (stronger acids - COOH) Ether extract Smoke Water Ether layer NaHCO 3 layer Slide 16 Isolation of Smoke Compounds NaHCO 3 soluble (stronger acids - COOH) Ether extract NaOH soluble (weaker acids - phenols etc.) Neutral fraction (~35% by mass) Smoke Water Ether layer NaOH layer -Activity found in the neutral fraction- Slide 17 The Haystack! GC-MS chromatogram of neutral fraction Slide 18 Lots of Chromatography! Chromatography - Physical method of separating compounds Classical Liquid Chromatography Stationary phase Mobile phase Slide 19 General separation scheme Alumina Neutral Fraction 15 Fractions C 18 silica 8 Fractions Fraction 7 (Highest activity) Test for Activity Slide 20 Typical germination of Grand Rapids lettuce when tested with C 18 fractions Slide 21 ~ 2 years laterOnly 3 Compounds! All had molecular formula C 8 H 6 O 3 (mw=150) Separated and tested for activity 1 2 3 UV recorded at 320nm 25% MeOH/water Slide 22 Testing of the 3 compounds Slide 23 Identification of the 3 compounds Chemical structure proposed based on Nuclear Magnetic Resonance (NMR) and mass spectrometry (MS) data Slide 24 Synthesis of the active compound (2) IUPAC name: 3-methyl-2H-furo[2,3-c]pyran-2-one (2) Prepared in ~5% yield from Kojic acid (4) *karrik Aboriginal word for smoke, -olide indicates lactone *Karrikinolide Slide 25 Germination of karrikinolide (2) with Grand Rapids Lettuce Slide 26 Germination of karrikinolide (2) with confirming native species Slide 27 Back to the haystack! karrikinolide Slide 28 Expanded CDS-neutral fraction karrikinolide Slide 29 Summary First chemical identified from smoke that promotes germination Novel structure new to science Devised a method of synthesis Bioassay data demonstrating effect with species from Australia, South Africa and North America Potential uses in agriculture, horticulture, land restoration etc Compared with smoke water, karrikinolide operates at < 1 g per hectare Slide 30 What other species? What other activity? Is there a better (simpler) synthesis? Are there active analogues? What is the mode of action? Potent Germination Promoter Slide 31 Mode of Action?? Label Label = fluorescent Questions ??