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)

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.

Solving the bushfire germination mystery……..

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.

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

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

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

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

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

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)

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

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

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

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

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

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-

The Haystack! GC-MS chromatogram of neutral fraction

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

General separation scheme Alumina Neutral Fraction 15 Fractions Fraction 7 (Highest activity) C18 silica 8 Fractions Test for Activity 1000 24cm filter papers burnt = 4 kg cellulose

Typical germination of Grand Rapids lettuce when tested with C18 fractions

~ 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

Testing of the 3 compounds

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

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)

Germination of karrikinolide (2) with Grand Rapids Lettuce

Germination of karrikinolide (2) with confirming native species

Back to the haystack! karrikinolide

Expanded CDS-neutral fraction karrikinolide

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

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…?

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 ??