1'ou say you donJt 6efieve? Wfiat do you caffit when you sow a tiny seedandare convincedthat a pfant wiffgrow? - Elizabeth York- Contents Abstract . , .. vii Declaration .. ,,., , ,........... .. ix Acknowledgements ,, ,, , .. , x Publications from this Thesis ,, , ", .. ,., , xii Patents from this Thesis ,,,'' ,, .. ',. xii Conference Contributions ' xiii Related Publications .................................................... .. xiv List of Figures , xv List of Tables , ,,,. xviii List of Abbreviations ,,, ,, ,,, ,. xix 1 Introduction ,,,, 1 1.1 SMOKE AS A GERMINATION CUE .. ,,,, .. ,,,,, .. , .. , , . , 1 1.2 AIMS AND OBJECTIVES , '.. , , . 1 1.3 GENERAL OVERVIEW ,, " , .. , .. , 2 2 Literature Review ,",,,,", 4 2.1 THE ROLE OF FIRE IN SEED GERMINATION .. ,,,,.,,,,. ,4 2.1.1 Fire in mediterranean-type regions ', .. ,, , , 4 2,1.2 Post-fire regeneration. ,,,, .. , , . , , , , 5 2,1.3 Effects of fire on germination .,,, , , . 7 2,1,3.1 Physical effects of fire on germination .. ,," .. ,.,. 8 2.1,3.2 Chemical effects of fire on germination ., ,, .. ,., 11 2.2 GERMINATION RESPONSES TO SMOKE., , '" ., , 16 2.2.1 The discovery of smoke as a germination cue, ,,., .. , , .. ,, 16 2.2.2 Studies on South African species. ,.,, .. , ,,,,., 17 2.2,3 Studies on Australian species "",., ,"," ".,." 20 2.2.4 StUdies on species from other regions. , ,,.,, 22 2.2.5 Responses of vegetable seeds ., .. ' .. , ,', , , 23 2.2.6 Responses of weed species .. ,,,.,, 24 2.2.7 General comments and considerations ., .. ,,, .. , .. ,,, 25 2.2.7.1 Concentration effects .. ,", ,., 25 2.2.7.2 Experimental considerations ,,,,,,, 26 2.2,7.3 Physiological and environmental effects ,,, .. ,, 27 2.2.8 The interaction of smoke and heat, ,, ,,,,,,, 29 \ 2.3 SOURCES OF SMOKE ., , .. , .. ,, .. ,., .. ,, 35 2.3,1 Chemical components of smoke ,, .. " ,, 35 iii Contents 2.3.2 Methods of smoke treatments 36 2.3.2.1 Aerosol smoke and smoked media . 37 2.3.2.2 Aqueous smoke solutions 39 2.3.3 Commercial smoke extracts 42 2.3.3.1 Smoke in the food industry 42 2.3.3.2 Commercial smoke products for seeds 43 2.4 MODE OF ACTION OF SMOKE-STIMULATED GERMINATION 44 2.4.1 Scarification vs a signal-mediated mechanism 45 2.4.2 Stimulatory chemicals in smoke 47 2.4.3 The interaction of smoke and plant growth regulators 48 . 2.4.4 Possible modes of action " 52 2.5 ADDITIONAL BIOLOGICAL RESPONSES TO SMOKE 55 2.5.1 Antimicrobial activity 56 2.5.2 Antifungal activity 56 2.5.3 Dormancy-breaking in bulbs 57 2.5.4 Flowering 58 2.5.5 Seedling vigour 59 2.5.6 Root development 60 2.5.7 Somatic embryogenesis 61 2.6 POTENTIAL APPLICATIONS OF SMOKE TECHNOLOGY 61 2.6.1 Horticulture 62 2.6.2 Agriculture 63 2.6.3 Smoke as a primer or pre-treatment 63 2.6.4 Weed control , 65 2.6.5 Ecological studies using smoke 66 2.6.6 Habitat restoration and conservation 67 2.7 SUMMARY , 70 3 The Lettuce Seed Bioassay 71 3.1 INTRODUCTION 71 3.2 MATERIALS AND METHODS 75 3.2.1 Lettuce seed bioassay 75 3.2.2 Statistical analysis 76 3.3 RESULTS AND DISCUSSION 76 3.3.1 Response to smoke treatments 76 3.3.2 Effect of red light and far-red light on smoke-treated seeds 79 3.3.3 Interaction of plant hormones with smoke 81 3.4 SUMMARY 86 IV Contents 4 Dual Nature of the Regulation of Germination by Smoke 87 4.1 INTRODUCTION 87 4.2 MATERIALS AND METHODS 88 4.2.1 Smoke pulse treatments 88 4.2.2 Storage treatments. : 88 4.3 RESULTS AND DISCUSSION 89 4.3.1 Smoke pulse treatments 89 4.3.2 Storage treatments 91 4.3.3 Dual nature of germination regulation 92 4.4 SUMMARY 95 5 Is Nitric Oxide the Active Compound in Smoke? 96 5.1 INTRODUCTION 96 5.2 . MATERIALS AND METHODS 98 5.3 RESULTS AND DISCUSSION 99 5.4 SUMMARY 101 6 Isolation of the Germination Cue from Smoke 102 6.1 INTRODUCTION 102 6.1.1 The chemical nature of the germination cue 102 6.1.2 Isolation of the active components 103 6.2 MATERIALS AND METHODS 105 6.2.1 Isolation of the germination promoter 105 6.2.2 GC-MS analysis of the active constituent 105 6.2.3 Structure elucidation 107 6.3 RESULTS AND DISCUSSION 107 6.3.1 Isolation and structure elucidation 107 6.3.2 Germination activity 109 6.3.3 Recent studies with 3-methyl-2H-furo[2,3-cjpyran-2-one _ 111 . 6.4 SUMMARY : : 113 6.5 COLLABORATION 113 7 Formation of the Germination Promote~. from Carbohydrates and Amino Acids 114 7.1 INTRODUCTION 114 7.1.1 Common constituents for smoke production 114 7.1.2 Heating plant material 115 7.1.3 Maillard reactions 116 v Contents 7.2 MATERIALS AND METHODS 117 7.2.1 Instrumentation 117 7.2.2 Lettuce seed bioassay 118 7.2.3 Chemicals 118 7.2.4 Reactions of proteins with glucose 119 7.2.5 Reactions of amino acids with glucose 119 7.2.6 Variation of the amino acid-to-glucose ratio 119 7.2.7 Variation of reaction conditions 119 7.2.8 Reaction with different sugars 120 7.2.9 HPLC and GC-MS analysis 120 7.3 RESULTS AND DISCUSSION 121 7.3.1 Reactions of proteins with glucose 121 7.3.2 Reactions of amino acids with glucose 122 7.3.3 Variation of the amino acid-to-glucose ratio 124 7.3.4 Variation of reaction conditions 124 7.3.5 Reaction with different sugars 127 7.3.6 HPLC and GC-MS analysis 129 7.4 SUMMARy 132 7.5 COLLABORATION 132 8 General Conclusions 133 9 References 135 Appendix 1 List of species which show a germination response to smoke treatments ................... A1-1 Appendix 2 List of species which do not show a germination response to smoke treatments A2-1 VI Abstract The intriguing role of smoke in stimulating seed germination was first highlighted by a study on Audouinia capitata, a threatened fynbos species. Further studies on South African fynbos, Californian chaparral and Australian species have illustrated the widespread ability of smoke to promote germination of many species from fire-prone areas. Interestingly, a variety of species from fire-free habitats also respond positively to smoke treatments. Consequently, aerosol smoke and smoke solutions can potentially be used for a variety of applications related to seed technology. These include uses in horticulture, agriculture, ecological management, habitat restoration and conservation. Various investigations were conducted, on different aspects relating to smoke­ stimulated seed germination, in an attempt to gain a better understanding of the action of smoke as a germination cue, and to isolate and characterise the compound(s) responsible for the stimulatory action of smoke on germination. Importantly, Grand Rapids lettuce seeds, which have a light-requirement for germination, are able to germinate within 24 h in the dark when treated with smoke solutions. Thus, Grand Rapids lettuce seeds were used as a biological test system, and formed an integral role in the experiments conducted. Pulse-treatments of Grand Rapids lettuce seeds with smoke solutions revealed that there is a required threshold level of the active compound(s) in the seed. The retention of the germination cue by the seeds, and the reversal of the inhibitory effects of high concentrations of smoke after rinsing, suggest a dual regulatory role for smoke. This competitive interaction, in which the germination promoter(s) cannot be leached while the inhibitory compound(s) can, may be important in post-fire environments. It may provide seeds with a mechanism to prevent germination until sufficient rainfall has leached the inhibitory compound(s) away from the seeds, thereafter allowing the stimulatory compound(s), which are active over a broad concentration range, to promote germination. Following the suggestion that smoke-stimulated seed germination is due to nitric oxide (NO), the effects of two NO-releasing compounds, N-tert-butyl-a-phenylnitrone (PBN) vii Abstract and sodium nitroprusside (SNP), on the germination of Grand Rapids lettuce seeds were examined. Neither PBN nor SNP stimulated seed germination in the dark. Additionally, the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5­ tetramethylimidazoline-1-oxyl-3-oxide potassium (c-PTIO) was unable to reduce germination in response to a smoke solution. These results suggested that NO is unlikely to be responsible for the enhanced germination of Grand Rapids lettuce seeds by smoke solutions. ), A highly active butenolide compound, 3-methyl-2H-furo[2,3-c]pyran-2-one (CaH60 3 that stimulates germination of Grand Rapids lettuce seeds in the dark, was isolated from plant-derived smoke water using bioactivity-guided fractionation. Purification steps included liquid-liquid partitioning, vacuum liquid chromatography, and HPLC fractionation. The active fractions from the final HPLC step were analysed using NMR and GC-MS. It was found that the compound promoted the germination of Grand Rapids lettuce seed over a wide range of concentrations, and at a concentration as low as 10-9 M. Further experiments showed that 3-methyl-2H-furo[2,3-c]pyran-2-one could also be formed during Maillard reactions between sugars and amino acids. Heating proteins or amino acids with sugars at 180 ·C for 30 min produced water-soluble extracts that promoted the germination of Grand Rapids lettuce seeds in the dark. Using HPLC fractionation, it was demonstrated that the active compound(s) formed during these reactions co-eluted with the active fraction from a smoke extract. Analysis using GC-MS showed that the active constituent is identical to the germination cue from plant-derived smoke. The major germination cue found in smoke can therefore be formed from ubiquitously occurring organic compounds.