Smoke

1. Investigation of the germination promoting compounds in smoke Gavin Flematti The University of Western Australia Smoke Emilio Ghisalberti (UWA) Kingsley Dixon (KPBG) Robert Trengove (Murdoch)

3. Solving the bushfire germination mystery……..

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

5. Smoke found to improve seed germination of many Australian native species

7. Sites with smoke-stimulated germination of wild species And the list of species includes…. lettuce, celery, parsley and others….

8. Apparatus for generating Plant Derived Smoke ‘Smoke-water’ ‘Aerosol Smoke’

9. Benefits for mining restoration Lateritic nickel project Mineral sands mining restoration - BUT… need 10 tonnes smoke water per hectare!

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)

11. Stylidium affine Lettuce (Grand Rapids) Conostylis aculeata The Needle in the Haystack - finding the chemical(s) in smoke responsible for germination • Bioassay - Test that uses a biological indicator for tracking the active components through separation steps

12. Isolation of Smoke Compounds Smoke Water

13. Isolation of Smoke Compounds Smoke Water Ether layer Ether extract Aqueous layer

14. Isolation of Smoke Compounds Smoke Water Ether extract Ether layer Aqueous layer

15. Isolation of Smoke Compounds Smoke Water Ether extract Ether layer NaHCO3 layer NaHCO3 soluble (stronger acids - COOH)

16. Neutral fraction (~35% by mass) Isolation of Smoke Compounds Smoke Water Ether extract Ether layer NaHCO3 soluble (stronger acids - COOH) NaOH soluble (weaker acids - phenols etc.) NaOHlayer -Activity found in the neutral fraction-

17. The Haystack!GC-MS chromatogram of neutral fraction

18. Lots of Chromatography! • Chromatography - Physical method of separating compounds Mobile phase Stationary phase Classical Liquid Chromatography

19. C18 silica Fraction 7 (Highest activity) 8 Fractions Test for Activity General separation scheme Alumina Neutral Fraction 15 Fractions

20. Typical germination of Grand Rapids lettuce when tested with C18 fractions

21. ~ 2 years later…Only 3 Compounds! UV recorded at 320nm 25% MeOH/water 3 2 1 • All had molecular formula C8H6O3 (mw=150) • Separated and tested for activity

22. Testing of the 3 compounds

23. Chemical structure proposed based on Nuclear Magnetic Resonance (NMR) and mass spectrometry (MS) data Identification of the 3 compounds

24. *”Karrikinolide” *‘karrik’ – Aboriginal word for smoke, -olide indicates lactone 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)

25. Germination of karrikinolide (2) with Grand Rapids Lettuce

26. Germination of karrikinolide (2) with confirming native species

27. karrikinolide Back to the haystack!

28. karrikinolide Expanded CDS-neutral fraction

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

30. Potent Germination Promoter What other species…? What other activity…? Is there a better (simpler) synthesis…? Are there active analogues…? What is the mode of action…?

31. Label Mode of Action?? Label = fluorescent Questions ??