Smoke-derived Compound Research Articles

A missense allele of KARRIKIN-INSENSITIVE2 impairs ligand-binding and downstream signaling in Arabidopsis thaliana

A smoke-derived compound, karrikin (KAR), and an endogenous but as yet unidentified KARRIKIN INSENSITIVE2 (KAI2) ligand (KL) have been identified as chemical cues in higher plants that impact on multiple aspects of growth and development. Genetic screening of light-signaling mutants in Arabidopsis thaliana has identified a mutant designated as ply2 (pleiotropic long hypocotyl2) that has pleiotropic light-response defects. In this study, we used positional cloning to identify the molecular lesion of ply2 as a missense mutation of KAI2/HYPOSENSITIVE TO LIGHT, which causes a single amino acid substitution, Ala219Val. Physiological analysis and genetic epistasis analysis with the KL-signaling components MORE AXILLARY GROWTH2 (MAX2) and SUPPRESSOR OF MAX2 1 suggested that the pleiotropic phenotypes of the ply2 mutant can be ascribed to a defect in KL-signaling. Molecular and biochemical analyses revealed that the mutant KAI2ply2 protein is impaired in its ligand-binding activity. In support of this conclusion, X-ray crystallography studies suggested that the KAI2ply2 mutation not only results in a narrowed entrance gate for the ligand but also alters the structural flexibility of the helical lid domains. We discuss the structural implications of the Ala219 residue with regard to ligand-specific binding and signaling of KAI2, together with potential functions of KL-signaling in the context of the light-regulatory network in Arabidopsis thaliana.

Synthesis of a 13C-labelled seed-germination inhibitor (3,4,5-trimethylfuran-2(5H)-one) for the mode of action elucidation

It has been found that the butenolide 3,4,5-trimethylfuran-2(5H)-one, isolated from plant-derived smoke, efficiently inhibits seed germination and significantly reduces the effect of the highly active germination promotor karrikinolide (KAR1, 3-methyl-2H-furo[2,3-c]pyran-2-one), another smoke-derived compound. This paper reports the synthesis of stable isotope-labelled 3,4,5-trimethylfuran-2(5H)-ones containing one and two 13C atoms for the identification of metabolic degradation products in order to provide insight into the mechanism of action.

Priming with smoke-derived karrikinolide enhances germination and transplant quality of immature and mature pepper seed lots

The aim of this study was to test the effect of the smoke-derived compound, karrikinolide (KAR1), and hydropriming treatments on the seed germination of pepper (Capsicum annuum L.). The effect of priming treatments on seedling emergence percentages and rates, seedling fresh and dry mass, catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity of immature and mature seed lots for four pepper cultivars (Kandil D., Surmeli, Yaglık and Corbacı) harvested in 2015 and 2016 were evaluated. Priming with 10−7M KAR1 enhanced seed germination and seedling emergence for seeds from both harvest periods, and was more influential on immature seeds than mature seeds. Specifically, priming with KAR1 improved germination by more than 20% in immature seeds, compared to around 10% in the mature seeds. The differences in seedling emergence were 18% and 22% in 2015, and 21% and 22% in 2016, respectively. Priming with KAR1 increased catalase activity, but reduced APX and SOD activity in both seed lots for cultivar Corbacı. Thus, results indicate that priming with KAR1 can be used as an enhancing treatment in the cultivation of pepper seeds, and that the benefit is greater in immature seeds than in mature seeds, regardless of the cultivar or year of harvest.

Enhancing Phytoremediation Potential of Pennisetum clandestinum Hochst in Cadmium-Contaminated Soil Using Smoke-Water and Smoke-Isolated Karrikinolide

The use of plant growth regulators (PGRs) and biostimulants to enhance phytoextraction is gaining popularity in phytoremediation technology. This study investigated the stimulatory effects of smoke-water (SW), a smoke-derived compound karrikinolide (KAR1) and other known plant growth regulators (PGRs) [gibberellic acid (GA3), kinetin (Kin) and indole-3-butyric acid (IBA)] to enhance the phytoextraction potential of Pennisetum clandestinum. Pennisetum clandestinum seedlings were grown for 10 weeks in vermiculite using Hoagland's nutrient solution and were treated with cadmium (Cd) (2, 5, and 10 mg L−1) and SW, KAR1 and PGRs. KAR1 exhibited positive effects on shoot and root dry weight (140 and 137 mg respectively) at the highest concentration of Cd (10 mg L−1) compared to all the other treatments. KAR1 and SW treatments used in the present study significantly improved the phytoextraction potential of P. clandestinum (602 and 575 mg kg−1 respectively) compared to the other tested PGRs. This is the first report on the use of SW and KAR1 to enhance phytoremediation potential in P. clandestinum. Further studies are needed to elucidate the exact mechanisms of smoke constituents involved in phytoextraction potential of plant species.

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.

Seed germination of the carnivorous plantByblis gigantea(Byblidaceae) is cued by warm stratification and karrikinolide

Byblidaceae is one of the most poorly studied carnivorous plant families, with seed dormancy and germination biology remaining unresolved. This knowledge deficit has significant conservation and management implications, particularly as the most southerly distributed species, the south-west Western Australian endemic Byblis gigantea, is listed as critically endangered. This study examined the ecophysiology of seed dormancy and germination in B. gigantea in concert with a study of the population dynamics of a single plant community. Mass seedling emergence and plant establishment were observed after a wildfire in 2007 in the natural population, followed by a rapid decline in mature individuals (91%) over a 4-year monitoring period, with almost no inter-fire recruitment (1.4% of all emergence) observed. Seeds possessed a fully developed embryo, and the germination characteristics of fresh seeds classified them as showing physiological dormancy. Seed dormancy was partially alleviated by warm stratification (30 °C) for 8 weeks prior to incubation at 15 °C, with c. 40% germination observed. With the additional exposure of seeds to the germination-active chemical in smoke, karrikinolide, the germination of warm-stratified seeds increased to 89%. Seeds also displayed orthodox storage behaviour and appeared to be amenable to long-term seed banking for conservation. These results present the first observation of the stimulation of the germination of a carnivorous plant by a smoke-derived compound. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 143–152.

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.

Prior hydration of Brassica tournefortii seeds reduces the stimulatory effect of karrikinolide on germination and increases seed sensitivity to abscisic acid

The smoke-derived compound karrikinolide (KAR(1)) shows significant potential as a trigger for the synchronous germination of seeds in a variety of plant-management contexts, from weed seeds in paddocks, to native seeds when restoring degraded lands. Understanding how KAR(1) interacts with seed physiology is a necessary precursor to the development of the compound as an efficient and effective management tool. This study tested the ability of KAR(1) to stimulate germination of seeds of the global agronomic weed Brassica tournefortii, at different hydration states, to gain insight into how the timing of KAR(1) applications in the field should be managed relative to rain events. Seeds of B. tournefortii were brought to five different hydration states [equilibrated at 15 % relative humidity (RH), 47 % RH, 96 % RH, fully imbibed, or re-dried to 15 % RH following maximum imbibition] then exposed to 1 nm or 1 microm KAR(1) for one of five durations (3 min, 1 h, 24 h, 14 d or no exposure). Dry seeds with no history of imbibition were the most sensitive to KAR(1); sensitivity was lower in seeds that were fully imbibed or fully imbibed then re-dried. In addition, reduced sensitivity to KAR(1) was associated with an increased sensitivity to exogenously applied abscisic acid (ABA). Seed water content and history of imbibition were found to significantly influence whether seeds germinate in response to KAR(1). To optimize the germination response of seeds, KAR(1) should be applied to dry seeds, when sensitivity to ABA is minimized.

The potential of the smoke-derived compound 3-methyl-2H-furo[2,3-c]pyran-2-one as a priming agent for tomato seeds

AbstractThe stimulatory role of 3-methyl-2H-furo[2,3-c]pyran-2-one, a smoke-derived butenolide, on germination and post-germination events is well documented. Previous studies have involved germinating seeds in the continuous presence of the compound. However, commercial growers cannot exploit the potential of the butenolide for large-scale production of crops due to limited availability and environmental constraints. The present study was undertaken to assess the potential of the butenolide as a priming agent of tomato (Solanum esculentumMill.) seeds. Flow cytometry data revealed that butenolide-primed seeds had a higher percentage of nuclei at the 4C stage than water-primed seeds. Emergence of the radicle was much faster in the primed seeds. After 36 h of imbibition, a higher percentage of the butenolide-primed seeds (22%) exhibited radicle emergence compared to the water-primed seeds (12%). While butenolide-primed seeds initially germinated more rapidly than either water-primed or unprimed seeds in a 48-h period, water-imbibed seeds reached a similar germination level as the butenolide-primed seeds by 60 h of incubation. The butenolide-primed seeds produced significantly (P ≤ 0.05) more vigorous seedlings than water-primed seeds or seeds in the continuous presence of either water or butenolide. A gradual decrease in the seedling vigour index was recorded for both water and butenolide-primed seeds with increased seed storage at room temperature. Nevertheless, the vigour index was significantly greater in the butenolide-primed seeds than the water-primed seeds. Vigour indices were significantly (P ≤ 0.05) higher for the butenolide-primed seeds under various stress conditions (salinity, osmoticum or temperature) compared to control or water-primed seeds. Results of the present study suggest that the butenolide is a good seed-priming agent. Additionally, primed seeds retained the promotive effect for a considerable time. This was also the case for tomato seeds under various simulated field stress conditions.

Germination and post-germination response of Acacia seeds to smoke-water and butenolide, a smoke-derived compound

Fire plays a critical role in breaking hard-seeded dormancy and establishing seedlings of several Acacia species in arid and semi-arid regions. Numerous studies have reported an increase in seedling densities of some Acacias after fire without identifying the exact cause. However, it is generally believed and speculated that the effect is largely physical and heat related. Recent studies have revealed that smoke generated from wildfires has the ability to stimulate seed germination and also to improve seedling vigour. This stimulatory characteristic of smoke has now been confirmed by the isolation of a butenolide (3-methyl-2 H-furo[2,3- c]pyran-2-one) from plant smoke that actively promotes germination of many plant species. The species of Acacia investigated were A. hebeclada (deciduous shrub), A. mearnsii (invasive tree, native to Australia) and A. robusta (deciduous tree). Seeds of A. hebeclada germinated under different light conditions with smoke-derived butenolide solution (10 −7 M), exhibited a significantly ( p<0.05) greater germination percentage than untreated seeds. Whereas A. mearnsii seeds exposed to constant dark conditions showed a significantly ( p<0.05) better germination percentage than the control. However, there was a non-significant improvement for A. robusta seeds. All three species responded positively to the butenolide treatment (10 −7 M) after incubating for 10 days under constant dark conditions at 25±0.5 °C, achieving a higher vigour index and seedling mass in comparison to the controls. Smoke-water (1:500) had an intermediate effect on these species. This study shows that the butenolide, isolated from smoke, may have a significant effect on the post-fire seedling ecology of Acacias.

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

Aerosol smoke, aqueous smoke solutions and 3-methyl-2 H-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-2 H-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 (1.5 μg/plate to 7.5 ng/plate), and in the VITOTOX ® test (1500–0.045 ppb). In all tests performed, no mutagenic activity was induced by 3-methyl-2 H-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.

Post-germination effects of the smoke-derived compound 3-methyl-2 H-furo[2,3- c]pyran-2-one, and its potential as a preconditioning agent

The newly identified compound from plant-derived smoke, 3-methyl-2 H-furo[2,3- c]pyran-2-one (butenolide), is generating much interest in seed biology. The present study reports on the effects of this new butenolide on the germination, and post-germination, of seeds of three vegetable crops (tomato, okra and bean) and maize. Seeds of all the vegetable crops showed a significant improvement ( p < 0.05) in seedling vigour and growth in comparison to controls when treated with this compound. Results showed that the butenolide-treated (10 −7 M) maize kernels achieved better percentage germination with higher vigour indices than untreated kernels. Eight-day-old butenolide and smoke water-treated maize seedlings developed more roots in comparison to the control seedlings. Maize seeds soaked in a solution of butenolide (10 −7 M) for an hour prior to planting, significantly improved root and shoot fresh and dry weights after 30 days. The number of leaves, shoot height and percentage survival were also greater in butenolide-treated plants. Although this compound was previously isolated for its effective promotion of germination, studies now show that this smoke-derived compound exhibits dynamic stimulatory effects at post-germination levels, and could be used as a plant growth promoter. Furthermore, results from these experiments suggest the possible use of 3-methyl-2 H-furo[2,3- c]pyran-2-one as a seed priming or preconditioning agent for better stand establishment of maize plants.

Dark conditioning, cold stratification and a smoke-derived compound enhance the germination of Eucomis autumnalis subsp. autumnalis seeds

Seed germination requirements are important for the conservation and propagation of Eucomis autumnalis subsp. autumnalis, a threatened, yet widely utilized medicinal plant in southern Africa. In this study, conditions to maximize germination were investigated. The seeds of E. autumnalis were subjected to various environmental factors such as constant and alternating temperature regimes, cold stratification, temperature shifts and light/dark conditions. Effects of smoke–water and the newly identified smoke-compound (3-methyl-2 H-furo[2,3- c]pyran-2-one) were tested on germination. At 20 °C and alternating temperature (30/15 °C) under a 16 : 8 h light / dark regime, germination was improved. The highest percentage germination (100%) was achieved with a mean germination time (MGT) of 30.6 ± 1.3 days, when the seeds were subjected to constant dark at room temperature (25 ± 0.5 °C). This indicates the negative photoblastic characteristic of E. autumnalis seeds. The species is associated with high-altitude regions. Cold stratification (5 °C) for 45 days significantly improved the percentage germination (90%) as compared to non-stratified controls (40%) when subsequently incubated at 25 ± 0.5 °C under a 16 : 8 h light / dark regime. Seeds treated with 3-methyl-2 H-furo[2,3- c]pyran-2-one and incubated in constant light or a 16 : 8 h light : dark photoperiod germinated better (86.7% and 66.7%, respectively) compared to the controls (55% and 40%, respectively). This smoke-derived compound substituted for the dark and cold stratification requirements for the germination of E. autumnalis seeds.