Genotoxicity testing of 3,4,5-trimethylfuran-2(5H)-one, a compound from plant-derived smoke with germination inhibitory activity.

Plant-derived smoke and certain smoke compounds improve seed germination and enhance seedling growth of many species. Thus, smoke-infused water and the active smoke-derived compounds have the potential to be used in different agricultural and horticultural applications. However, despite these interesting and potentially practical properties, it should also be ascertained whether such compounds may pose a health risk, particularly if they are to be used in the production of food or fodder crops. Amongst some of the aspects that would be important to understand are any possible genotoxic properties that the compounds may possess due to potential carry-over effects. Here, we report on a genotoxicity study of 3,4,5-trimethylfuran-2(5H)-one, a compound from plant-derived smoke previously shown to have germination inhibitory activity. Using two in vitro tests, namely the bacterial VITOTOX® test (with/without S9 metabolic activation) and the cytome assay on human C3A cells, no genotoxicity or toxicity was found. Furthermore, these results support a previous study where a related smoke-derived compound with germination promoting properties was investigated.

Structure-activity relationships of analogs of 3,4,5-trimethylfuran-2(5H)-one with germination inhibitory activities.

Smoke-derived butenolide compounds have, in recent years, been shown to be important germination signaling molecules, which also affect seedling growth. The butenolide 3,4,5-trimethylfuran-2(5H)-one was previously isolated from plant-derived smoke and was found to significantly reduce the effect on germination by the highly active promotor karrikinolide (KAR1, 3-methyl-2H-furo[2,3-c]pyran-2-one), another smoke-derived compound. In this study, 11 analogs of 3,4,5-trimethylfuran-2(5H)-one were synthesized and their effect on the germination of light-sensitive 'Grand Rapids' lettuce seeds (Lactua sativa cv. 'Grand Rapids') were evaluated. A concentration series (1mM-1µM) of the analogs were tested alone, or in combination with 0.01µM KAR1. Only two compounds were found to reduce the germination promotory effect of 0.01µM KAR1 in a similar manner as observed with 3,4,5-trimethylfuran-2(5H)-one, with activity ranging from 1mM to 10µM. Four compounds were found to have inhibitory activity at 1mM and 100µM. The retention of activity by some of the analogs may be useful for designing novel compounds with improved activity. Furthermore, understanding the structure-activity relationships of these compounds may be helpful in synthesizing molecular probes that can be used to further investigate the mechanism of action of these compounds in regulating seed germination.

Comparison of germination responses of Anigozanthos flavidus (Haemodoraceae), Gyrostemon racemiger and Gyrostemon ramulosus (Gyrostemonaceae) to smoke-water and the smoke-derived compounds karrikinolide (KAR1) and glyceronitrile.

BACKGROUND AND AIMS: A major germination-promoting chemical in smoke-water is 3-methyl-2H-furo[2,3-c]pyran-2-one (karrikinolide, KAR(1)). However, not all species that germinate in response to smoke-water are responsive to KAR(1), such as Tersonia cyathiflora (Gyrostemonaceae). In this study, a test was made of whether two Gyrostemon species (Gyrostemonaceae) that have previously been shown to respond to smoke-water, respond to KAR(1). If not, then the smoke-derived chemical that stimulates germination of these species is currently unknown. Recently, glyceronitrile was isolated from smoke-water and promoted the germination of certain Anigozanthos species (Haemodoraceae). Whether this chemical promotes Gyrostemon racemiger germination is also examined. Furthermore, an investigation was carried out into whether these species germinate in response to smoke-water derived from burning cellulose alone. METHODS: Gyrostemon racemiger and G. ramulosus seeds were buried after collection and retrieved in autumn the following year when dormancy was alleviated and seeds had become responsive to smoke-water. Anigozanthos flavidus seeds were after-ripened at 35 °C to alleviate dormancy. Gyrostemon and Anigozanthos seeds were then tested with 'Seed Starter' smoke-water, KAR(1), glyceronitrile and cellulose-derived smoke-water. KEY RESULTS: Although Gyrostemon racemiger, G. ramulosus and A. flavidus were all stimulated to germinate by 'Seed Starter' smoke-water, none of these species responded to KAR(1). Gyrostemon racemiger germination was not promoted by glyceronitrile. This is in contrast to A. flavidus, where glyceronitrile, at concentrations of 1-500 µm, promoted germination, although seedling growth was inhibited at ≥400 µm. Maximum A. flavidus germination occurred at glyceronitrile concentrations of 25-300 µm. Some Gyrostemon germination was promoted by cellulose-derived smoke-water. CONCLUSIONS: KAR(1) and glyceronitrile, chemicals in smoke-water that are known to stimulate germination in other species, did not promote the germination of G. racemiger. This suggests that other chemical(s) which promote germination are present in smoke, and may be derived from burning cellulose alone.

Molecular aspects of the antagonistic interaction of smoke-derived butenolides on the germination process of Grand Rapids lettuce (Lactuca sativa) achenes.

Smoke-derived compounds provide a strong chemical signal to seeds in the soil seed bank, allowing them to take advantage of the germination niche created by the occurrence of fire. The germination stimulatory activity of smoke can largely be attributed to karrikinolide (KAR(1) ), while a related compound, trimethylbutenolide (TMB), has been shown to have an inhibitory effect on germination. The aim of this study was to characterize the interaction of these potent fire-generated compounds. Dose-response analysis, leaching tests and a detailed transcriptome study were performed using highly KAR(1) -sensitive lettuce (Lactuca sativa cv 'Grand Rapids') achenes. Dose-response analysis demonstrated that the compounds are not competitors and TMB modulates germination in a concentration-dependent manner. The transcriptome analysis revealed a contrasting expression pattern induced by the compounds. KAR(1) suppressed, while TMB up-regulated ABA, seed maturation and dormancy-related transcripts. The effect of TMB was reversed by leaching the compound, while the KAR(1) effect was only reversible by leaching within the first 2 h of KAR(1) treatment. Our findings suggest that the compounds may act in concert for germination-related signaling. After the occurrence of fire, sufficient rainfall would contribute to post-germination seedling recruitment by reducing the concentration of the inhibitory compound.

Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR1.

BACKGROUND: Smoke released from burning vegetation functions as an important environmental signal promoting the germination of many plant species following a fire. It not only promotes the germination of species from fire-prone habitats, but several species from non-fire-prone areas also respond, including some crops. The germination stimulatory activity can largely be attributed to the presence of a highly active butenolide compound, 3-methyl-2H-furo[2,3-c]pyran-2-one (referred to as karrikin 1 or KAR1), that has previously been isolated from plant-derived smoke. Several hypotheses have arisen regarding the molecular background of smoke and KAR1 action. RESULTS: In this paper we demonstrate that although smoke-water and KAR1 treatment of maize kernels result in a similar physiological response, the gene expression and the protein ubiquitination patterns are quite different. Treatment with smoke-water enhanced the ubiquitination of proteins and activated protein-degradation-related genes. This effect was completely absent from KAR1-treated kernels, in which a specific aquaporin gene was distinctly upregulated. CONCLUSIONS: Our findings indicate that the array of bioactive compounds present in smoke-water form an environmental signal that may act together in germination stimulation. It is highly possible that the smoke/KAR1 'signal' is perceived by a receptor that is shared with the signal transduction system implied in perceiving environmental cues (especially stresses and light), or some kind of specialized receptor exists in fire-prone plant species which diverged from a more general one present in a common ancestor, and also found in non fire-prone plants allowing for a somewhat weaker but still significant response. Besides their obvious use in agricultural practices, smoke and KAR1 can be used in studies to gain further insight into the transcriptional changes during germination.

The fire ephemeral Tersonia cyathiflora (Gyrostemonaceae) germinates in response to smoke but not the butenolide 3-methyl-2H-furo[2,3-c]pyran-2-one.

BACKGROUND AND AIMS: Tersonia cyathiflora (Gyrostemonaceae) is a fire ephemeral with an obligate requirement for smoke to germinate. Whether it is stimulated to germinate by 3-methyl-2H-furo[2,3-c]pyran-2-one (karrikinolide, KAR(1)), the butenolide isolated from smoke that stimulates the germination of many other smoke-responsive species, is tested. METHODS: Seeds of T. cyathiflora were buried in autumn following collection and were exhumed 1 year later, as this alleviates dormancy and enables seeds to germinate in response to smoke-water. Exhumed seeds were tested with smoke-water and KAR(1). Fresh preparations of these solutions were again tested on seeds exhumed 2 months later under a broader range of conditions. They were also tested on Grevillea eriostachya (Proteaceae) and Stylidium affine (Stylidiaceae) to confirm the activity of KAR(1). KEY RESULTS: T. cyathiflora seeds germinated in response to smoke-water but not to KAR(1). In contrast, G. eriostachya and S. affine germinated in response to both smoke-water and KAR(1). CONCLUSIONS: Although many smoke-responsive seeds germinate in the presence of KAR(1), this does not apply universally. This suggests that other chemical(s) in smoke-water may play an important role in stimulating the germination of certain species.

Butenolides from plant-derived smoke: natural plant-growth regulators with antagonistic actions on seed germination.

Smoke plays an intriguing role in promoting the germination of seeds of many species following a fire. Recently, a bicyclic compound containing a condensed butenolide moiety, 3-methyl-2H-furo[2,3-c]pyran-2-one (1), was reported as a potent germination promoter from plant-derived smoke. In this study, a related butenolide, 3,4,5-trimethylfuran-2(5H)-one (2), which inhibits germination and significantly reduces the effect of 1 when applied simultaneously, was also isolated from plant-derived smoke. The interaction of these compounds with opposing actions on seed germination may have important ecological implications in a post-fire environment and could be useful molecules for understanding the events involved in breaking seed dormancy and promoting seed germination.

Stress-related genes define essential steps in the response of maize seedlings to smoke-water.

Smoke from burning vegetation is widely recognised as a germination cue for seed germination and recent reports suggest that smoke treatments can improve seedling vigour also. We investigated the effect of smoke-water on seedling vigour and changes of the global transcriptome in the early post-germination phase in maize. Application of smoke-water improved the germination characteristics and seedling vigour. The transcriptional response of embryos and emerging radicles 24 and 48 h after the onset of smoke treatment was investigated. The microarray study revealed a number of smoke-responsive genes amongst which stress- and abscisic acid (ABA)-related genes were over-represented. The global promoter analysis of the smoke-responsive genes revealed a tight correlation with the results obtained from Gene Ontology annotations. This concerted over-expression shows that smoke treatment induces stress and ABA-related responses in the early post-germination phase which leads to better adaptation to environmental stress factors occurring during germination, eventually resulting in greater seedling vigour.

The polyacetylene falcarindiol with COX-1 activity isolated from Aegopodium podagraria L.

Extracts of Aegopodium podagraria L. were screened in vitro for cyclooxygenase-1 (COX-1) inhibitory activity. The isolation of the active compound falcarindiol was achieved by bioassay-guided fractionation. The identification of the active compound was confirmed by (1)H NMR and (13)C NMR. The IC(50)-value of falcarindiol was 0.3 microM in the COX-1 assay. A quantitative determination of the seasonal variation in the content of falcarindiol in different plant parts was carried out by HPLC analysis. The flowers from Aegopodium podagraria collected in June 2006 had the highest concentration of falcarindiol (88 mg/g plant material).

Genetic toxicity testing of 3-methyl-2H-furo[2,3-c]pyran-2-one, an important biologically active compound from plant-derived smoke.

Aerosol smoke, aqueous smoke solutions and 3-methyl-2H-furo[2,3-c]pyran-2-one isolated from plant-derived smoke stimulate seed germination and enhance seedling vigour of many species. Consequently, smoke technology has important potential applications in agriculture, horticulture and other environmental sectors. However, use of 3-methyl-2H-furo[2,3-c]pyran-2-one as a germination promoter and/or growth stimulant is only possible provided it can be shown that it is not hazardous, i.e. not toxic or genotoxic. We therefore performed an investigation to evaluate the genotoxic effects of this compound in five strains of Salmonella typhimurium in the Ames test (1.5microg/plate to 7.5ng/plate), and in the VITOTOX test (1500-0.045ppb). In all tests performed, no mutagenic activity was induced by 3-methyl-2H-furo[2,3-c]pyran-2-one, with or without S9 metabolic activation. Due to the potential wide use of this smoke-derived compound, it is of importance that no toxic and genotoxic effects were observed at the concentrations tested.

Formation of a seed germination promoter from carbohydrates and amino acids.

The ability of plant-derived smoke to act as a germination cue in many species has led to widespread interest in this aspect of seed biology. Recently, 3-methyl-2H-furo[2,3-c]pyran-2-one was identified as the main germination cue from smoke. Here, we report on the formation of this compound from reactions of sugars with amino acids. Heating proteins or amino acids with sugars at 180 degrees C for 30 min produces water soluble extracts that promote germination. High-performance liquid chromatography indicated that the active compound(s) derived from these reactions coeluted with the active fraction from a smoke solution. Gas chromatography-mass spectroscopy showed that the active constituent is identical to the germination cue from plant-derived smoke. The results presented in this paper provide evidence for the formation of the major germination cue found in smoke from ubiquitously occurring organic compounds.