1. Smoke 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)

2. Fire plays an important role in the regeneration of growth in many plant communities
After a fire event has occurred a remarkable regeneration of growth appears of the native species, in particular species that were perhaps not present before the fire event
The Heat associated with fire was always thought responsible for breaking open the seed coat. Gases in smoke such as ethylene had been shown to stimulate flowering especially in some fire orchids.
A mixture of smoke stimulated species following a > summer bushfire and winter rains - pink flower is a native parakeelya > (Calandrinia polyandra) and all the other little plants are also smoke > stimulated.

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
Wasn’t until 1990 that de lange and Boucher identified smoke as the key agent for promoting germination of ….
Since then research at Kings Park has identified smoke as the key agent.

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

6. White flower is NSW flannel flower (Actinotus helianthii)
Pink one is star flower (Calytrix fraseri)
yellowish thing is Blueboy a relative of banksia plants and hugely wild picked - smoke has broken the dormancy of this species.
And kangaroo paw

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. 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
Lettuce (Grand Rapids)
Conostylis aculeata
Stylidium affine

12. Isolation of Smoke Compounds
Smoke Water
Stronger acids- carboxylic acids
Weaker acids- phenols etc.
Left with neutral fraction

13. Isolation of Smoke Compounds
Smoke Water
Ether layer
Ether extract
Aqueous layer
Stronger acids- carboxylic acids
Weaker acids- phenols etc.
Left with neutral fraction

14. Isolation of Smoke Compounds
Smoke Water
Ether extract
Ether
layer
Stronger acids- carboxylic acids
Weaker acids- phenols etc.
Left with neutral fraction
Aqueous
layer

15. Isolation of Smoke Compounds
Smoke Water
Ether extract
Ether layer
NaHCO3 layer
NaHCO3 soluble
(stronger acids
- COOH)
Stronger acids- carboxylic acids
Weaker acids- phenols etc.
Left with neutral fraction

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

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

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

19. General separation scheme
Alumina
Neutral Fraction
15 Fractions
Fraction 7
(Highest activity)
C18 silica
8 Fractions
Test for Activity
cm filter papers burnt = 4 kg cellulose

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

24. Synthesis of the active compound (2)
*‘karrik’ – Aboriginal word for smoke, -olide indicates lactone
*”Karrikinolide”
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. Back to the haystack!
karrikinolide

28. Expanded CDS-neutral fraction
karrikinolide

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. Mode of Action?? Questions ?? Label Label = fluorescent
Biotin/ avidin system
photoaffinity = azide group which is activated by UV light to react with groups in the active site
In addition, look at interaction with GA’s and ABA
Plus the powerful resources available with Arabidopsis
Questions ??