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. 2013 Jan 8;111(3):489–497. doi: 10.1093/aob/mcs300

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

Katherine S Downes 1,*, Marnie E Light 2, Martin Pošta 3, Ladislav Kohout 3, Johannes van Staden 2
PMCID: PMC3579451  PMID: 23299994

Abstract

Background and Aims

A major germination-promoting chemical in smoke-water is 3-methyl-2H-furo[2,3-c]pyran-2-one (karrikinolide, KAR1). However, not all species that germinate in response to smoke-water are responsive to KAR1, 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 KAR1. 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, KAR1, 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 KAR1. 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

KAR1 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.

Keywords: Butenolide; cyanohydrin; germination; glyceronitrile; karrikinolide; smoke; 2,3-dihydroxypropanenitrile; 3-methyl-2H-furo[2,3-c]pyran-2-one; Gyrostemonaceae; Anigozanthos flavidus; Gyrostemon racemiger; Gyrostemon ramulosus

INTRODUCTION

In 1990, de Lange and Boucher reported the landmark discovery that aerosol smoke and aqueous smoke-water could promote the germination of Audouinia capitata, a rare South African species. Subsequently, smoke has been shown to promote the germination of many other species, many of which were previously difficult to germinate (Roche et al., 1997; Keeley and Fotheringham, 1998; Brown and Botha, 2004; Baker et al., 2005a). Smoke-stimulated germination has extensive implications for horticulture, weed control, conservation and restoration (Light and van Staden, 2004; Kulkarni et al., 2011).

Much effort has focused on determining the chemical(s) in smoke responsible for this germination stimulation (Baldwin et al., 1994; Van Staden et al., 1995; Light et al., 2009). In 2004, the butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one, was isolated from smoke-water (Flematti et al., 2004; Van Staden et al., 2004). This chemical, karrikinolide (KAR1; Flematti et al., 2009), has proved to be a major active germination-promoting agent in smoke (Light et al., 2009). Over 60 species in 29 different families are both smoke and KAR1 responsive (Chiwocha et al., 2009).

Interestingly, Tersonia cyathiflora, an Australian fire ephemeral in the Gyrostemonaceae with an obligate requirement for smoke to germinate, is unresponsive to KAR1 (Downes et al., 2010). Prior to this, it was assumed that KAR1 would induce germination in this smoke-responsive species (Fay and Christenhusz, 2010), as KAR1 was once considered by some to be the sole chemical responsible for all smoke-stimulated germination (Pausas and Keeley, 2009). Most members of the Gyrostemonaceae are fire ephemerals that germinate predominantly after fire, often in large numbers, and live for only a few years, thereafter persisting as seeds in the soil seed-bank until a subsequent fire (Bell et al., 1984; Baker et al., 2005a). In addition to T. cyathiflora, other fire ephemerals in the Gyrostemonaceae, including Gyrostemon racemiger and G. ramulosus, germinate in response to smoke following a period of burial (Baker et al., 2005a). However, the response of Gyrostemon seeds to KAR1 has not yet been tested.

Germination stimulation of T. cyathiflora by plant-derived smoke-water, but not KAR1, suggests that there may be other chemical(s) in smoke-water that promote the germination of certain species (Downes et al., 2010). Indeed, glyceronitrile (2,3-dihydroxypropanenitrile) was recently isolated from smoke-water and stimulates the germination of a number of species, including various Anigozanthos spp. that are also unresponsive to KAR1 (Flematti et al., 2011). This chemical contains nitrogen in addition to carbon, hydrogen and oxygen, and is proposed to operate through the release of cyanide (Flematti et al., 2011). It has, however, not yet been tested on any Gyrostemonaceae species.

Other chemicals that have been reported as germination-stimulating agents present in smoke, which have not previously been tested on the Gyrostemonaceae, are nitrogen oxides (Keeley and Fotheringham, 1997). The role of nitrogen oxides in smoke-stimulated germination has been questioned (Preston et al., 2004; Baldwin et al., 2005), partly because certain species responsive to nitrogen oxides also respond to the combustion of pure cellulose (Baldwin et al., 1994) and KAR1 (Flematti et al., 2004). Nevertheless, the discovery that glyceronitrile can promote the germination of some KAR1-responsive species highlights that the germination of certain species may be promoted by more than one chemical in smoke (Flematti et al., 2011).

The aims of this study were, first, to determine whether seeds of Gyrostemon racemiger and G. ramulosus respond to plant-derived smoke-water and/or KAR1; and, secondly, to test whether seeds of G. racemiger were stimulated to germinate by glyceronitrile, a newly isolated germinating-promoting chemical from smoke, or nitrogen oxides. Where seeds did not respond to either KAR1 or glyceronitrile, preliminary investigations were made into whether cellulose-derived smoke could stimulate the germination of these seeds. This was done to indicate whether the chemical(s) in smoke that promote the germination of these species was comprised of only carbon, hydrogen and oxygen.

MATERIALS AND METHODS

Seeds of Gyrostemon racemiger and G. ramulosus (Gyrostemonaceae) were collected along the Brand Highway north of Perth, Western Australia. Gyrostemon racemiger seeds were collected between Regens Ford and Cataby (30 °52·224′S 115 °37·421′E) on 21 November 2009 and G. ramulosus seeds were collected between Eneabba and Dongara (29 °17·000′S, 115 °00·508′E) on 24 December 2009. Within a week of collection, seeds were cleaned, placed in nylon mesh bags and buried approx. 1–2 cm beneath the soil surface at three sites within the source populations. Seeds of both species were exhumed on 5 April 2011. The seed burial pre-treatment was undertaken since germination response to smoke was previously shown to be enhanced in seeds of both these species following a period of soil burial and retrieval in autumn (Baker et al., 2005a).

Anigozanthos flavidus (Haemodoraceae) seeds were collected from Redmond State Forest (34 °51·512'S, 117 °33·234'E) on 2 March 2009 and stored at 15 °C. Preliminary trials indicated that freshly collected seeds were dormant and that after-ripening seeds at 35 °C alleviated dormancy and enabled seeds to respond to smoke-water. Grevillea leucopteris (Proteaceae) seeds were collected along the Brand Highway (29 °31·406'S, 115 °3·315'E) on 2 December 2010. Stylidium affine (Stylidiaceae) seeds were purchased from Nindethana Seed Service, Albany, Western Australia. These seeds were collected near Boddington, Western Australia in December 2007. Both G. leucopteris and S. affine seeds were stored at ambient room temperature prior to use.

Germination protocol

Seeds were surface sterilized prior to treatment to reduce fungal contamination. This involved placing seeds in 2 % sodium hypochlorite with a few drops of Tween-80 (polyoxyethylene sorbitan mono-oleate). The seeds and hypochlorite solution were shaken, placed under vacuum for 5 min, returned to normal air pressure for 5 min and placed under vacuum for a further 5 min. Seeds were then rinsed at least three times with sterile deionized water before being transferred to 9 cm Petri dishes. Within each Petri dish there were two pieces of Whatman No. 1 filter paper over three 4 cm2 pieces of ‘Vileda’ sponge. These were moistened with 10 mL of the test solution and sealed with Parafilm. Sterile deionized water was used as a control in all experiments. Seeds were incubated continuously in the test solutions unless otherwise specified. Each treatment comprised three replicate Petri dishes of 50 seeds. Seeds were incubated under a diurnal regime of 12 h light/12 h dark. Gyrostemon, Grevillea and Stylidium seeds were incubated at 20 °C, and Anigozanthos seeds were incubated at 15 °C. Seed germination was scored weekly for 4 weeks, unless noted otherwise.

Experiment 1: Gyrostemon and Anigozanthos response to smoke-water and KAR1

Gyrostemon racemiger, G. ramulosus and A. flavidus were initially tested with water (control), a 10 % (v/v) dilution of ‘Seed Starter’ smoke-water, 0·1 µm KAR1 and 0·01 µm KAR1. The A. flavidus seeds had been after-ripened at 35 °C for 20 weeks from November 2010. ‘Seed Starter’ smoke-water was purchased from Kings Park and Botanic Garden, Perth, Western Australia in 2003 and stored at 4 °C prior to use. It was produced by drawing smoke from the combustion of plant material though water (Tieu et al., 1999; Stevens et al., 2007). Before addition to the Petri dishes, the ‘Seed Starter’ smoke-water was filter sterilized through a 0·2 µm Acrodisc syringe filter. The diluted ‘Seed Starter’ had a pH of 4·1. KAR1 was synthesized from pyromeconic acid according to Flematti et al. (2005) and Light et al. (2010), and the 0·1 µm solution had a pH of 6·9.

Experiment 2: Gyrostemon and Anigozanthos response to glyceronitrile

The cyanohydrin, glyceronitrile (2,3-dihydroxypropanenitrile), was reported as a germination-promoting chemical found in plant-derived smoke-water (Flematti et al., 2011). Glyceronitrile was synthesized as a racemic mixture according to the method of Kopecký and Šmejkal (1984), and the identity of the compound was confirmed using nuclear magnetic resonance (NMR) spectroscopy. Initially 1 and 1000 µm solutions of glyceronitrile were tested on A. flavidus seeds after-ripened at 35 °C for 20 weeks from early November 2010. Subsequently, glyceronitrile was tested on A. flavidus (after-ripened for 26 weeks) and G. racemiger at a range of concentrations: 0 (control), 0·001, 0·01, 0·1, 1, 10, 100 and 1000 µm. Anigozanthos flavidus seeds were also tested with 1 µm glyceronitrile in the dark, by wrapping the Petri dishes in aluminium foil. Glyceronitrile (10 µm) had a pH of 5·2. The pH of glyceronitrile influences the amount of free cyanide produced, which is the proposed means by which glyceronitrile stimulates germination (Flematti et al., 2011).

Following the above preliminary germination trial using a wide range of glyceronitrile concentrations, a fresh batch of A. flavidus seeds from the same seed lot were after-ripened for 18 weeks from November 2011 and the seeds were incubated in the following concentrations of glyceronitrile: 0 (control), 5, 10, 25, 50, 75, 100, 200, 300, 400 and 500 µm. Seeds were also incubated with 10 µm glyceronitrile for 24 h and thereafter transferred to fresh Petri dishes containing 10 mL of deionized water, to determine whether the different duration of exposure to glyceronitrile influenced germination levels. At the completion of the germination trial, the lengths of 15 random seedlings per Petri dish in each of the five highest glyceronitrile concentrations were measured to determine whether higher concentrations inhibited seedling growth.

Experiment 3: Gyrostemon response to other chemicals present in smoke or smoke-water

Gyrostemon racemiger seeds were also incubated in 0·1 µm KAR4 (3,7-dimethyl-2H-furo[2,3-c]pyran-2-one), and a combination of 10 µm glyceronitrile and 0·1 µm KAR1. A ‘Seed Starter’ smoke-water treatment was also included to verify the continued responsiveness of the seeds. KAR4 is another karrikin in smoke-water that has been shown to stimulate the germination of certain species including Lactuca sativa (Apiaceae) and Solanum orbiculatum (Solanaceae; Flematti et al., 2007, 2009). It was synthesized from maltol according to Flematti et al. (2007), and the 0·1 µm solution had a pH of 6·3.

Gyrostemon ramulosus seeds were incubated in 10, 100 and 1000 µm sodium nitroprusside (SNP; Ajax Chemicals, Auburn, Australia). Fresh solutions of SNP were prepared just before the start of the experiment since SNP decomposes in the light to produce both cyanide and nitrogen oxides (Beligni and Lamattina, 2000; Bethke et al., 2006). Cyanide is released by glyceronitrile and promotes germination in certain species (Flematti et al., 2011), and nitrogen oxides have also been shown to stimulate the germination of certain smoke-responsive seeds (Keeley and Fotheringham, 1997). The ‘Seed Starter’ smoke-water was included to verify the continued responsiveness of the seeds.

Experiment 4: Gyrostemon and Anigozanthos response to cellulose-derived smoke-water

To determine whether Gyrostemon seeds were stimulated to germinate by chemical(s) in smoke containing only carbon, hydrogen and oxygen, cellulose-derived smoke-water was produced. Whatman No. 1 filter paper (150 sheets of 9 cm diameter, approx. 80 g in weight), as used by Flematti et al. (2004), was burnt in a bee smoker and the smoke was bubbled into 0·5 L of sterile deionized water for 12·5 min. The smoke-water was filter sterilized through a 0·2 µm Acrodisc syringe filter and diluted to 0·1, 1 and 10 % (v/v). The pH of the 10 % cellulose-derived smoke-water was 3·6. The two Gyrostemon species were incubated in these solutions, along with water (control) and 10 % (v/v) ‘Seed Starter’ smoke-water. Since ‘Seed Starter’ is produced by the combustion of plant material (Tieu et al., 1999; Stevens et al., 2007), it would contain compounds with elements in addition to carbon, hydrogen and oxygen.

Anigozanthos flavidus seeds after-ripened at 35 °C for 16 weeks from May 2011 were also incubated with water (control) and three concentrations of the cellulose-derived smoke-water to verify that this smoke-water did not contain nitrogen, as this species does not germinate in response to smoke-water created from carbon, hydrogen and oxygen only (Flematti et al., 2011).

Experiment 5: verification of chemical activity using Stylidium affine and Grevillea leucopteris

Germination trials were performed on S. affine and G. leucopteris seeds to authenticate the activity of KAR1 and to test the effectiveness of the cellulose-derived smoke-water. Seeds of S. affine were previously shown to germinate in response to KAR1 (Flematti et al., 2004; Downes et al., 2010) and, although the responsiveness of G. leucopteris to smoke or KAR1 has not yet been tested, a number of other Grevillea species are smoke and/or KAR1 responsive (Roche et al., 1997; Downes et al., 2010).

Previously, seeds from the same S. affine seed lot germinated to low levels in the light (Downes et al., 2010) and were, therefore, pre-treated to alleviate dormancy. This pre-treatment involved placing seeds on two pieces of Whatman No. 1 filter paper in each of three 9 cm Petri dishes, moistening them with 5 mL of deionized water, and then sealing the Petri dishes with Parafilm and placing them in a 20 °C incubator. After 24 h, the Parafilm was removed, and the top piece of filter paper and the seeds were placed on the lid of the Petri dish and returned to the 20 °C incubator to dry for 24 h. The seeds were then transferred to 35 °C for the remaining 5 d of the week. This weekly wet/dry and shifting temperature regime was repeated on these seeds a further three times.

Grevillea leucopteris and pre-treated S. affine seeds were tested with water (control), a 10 % (v/v) dilution of ‘Seed Starter’ smoke-water, 0·1 µm KAR1, 10 µm glyceronitrile and 10 % (v/v) cellulose-derived smoke-water. The G. leucopteris and S. affine trials commenced in July and October 2011 and ran for 4 and 12 weeks, respectively.

Experiment 6: comparison of Stylidium affine germination between smoke-water derived from cellulose and hay

Seeds of S. affine were used to compare the germination of a KAR1-responsive species with cellulose-derived smoke-water vs. hay-derived smoke-water. For pre-treatment, S. affine seeds were placed in a 35 °C oven for 2 weeks, then, for the subsequent 4 weeks, seeds were placed in a 20 °C incubator (90–95 % humidity) for 48 h before transfer to the 35 °C oven (15–20 % humidity) for the remainder of each week.

Hay-derived smoke-water was produced by burning oaten hay, as used by Stevens et al. (2007), in a bee smoker and bubbling the smoke into 0·5 L of sterile deionized water for 12 min. Markings with Thermochrom® chromatic thermometer crayons (Faber-Castell, Nürnberg, Germany), which change colour when particular temperatures are attained, indicated that the maximum temperature in the bee smoker during burning was between 350 and 500 °C. The hay-derived smoke-water was filter sterilized through a 0·2 µm Acrodisc syringe filter and diluted to 1, 5, 10 and 20 % (v/v). Cellulose-derived smoke-water, as produced in Experiment 4 and stored at 4 °C, was diluted to 1, 5, 10, 20 and 30 % (v/v). Pre-treated S. affine seeds were tested with the hay- and cellulose-derived smoke-water solutions, as well as deionized water (control) and 10 % (v/v) ‘Seed Starter’ smoke-water. This experiment commenced in February 2012 and cumulative germination was scored over 12 weeks.

Statistical analysis

Comparisons were made using analysis of variance (ANOVA). Prior to analysis, percentage data were converted to a value between 0 and 1, and arcsine square-root transformed. When germination was absent from all replicates of a treatment, that treatment was excluded from analysis to satisfy the ANOVA assumption of equal variances. Fisher's protected l.s.d. test was used as a post-hoc test. Treatments were regarded as significantly different at P < 0·05. Statistical analyses were performed in Genstat versions 13 and 14 (VSN International, Oxford, UK).

RESULTS

Gyrostemon racemiger, G. ramulosus and A. flavidus seeds were promoted to germinate by ‘Seed Starter’ smoke-water but not KAR1, at the concentrations tested (Table 1). Germination of G. racemiger seeds was negligible (<1 %) across the range of glyceronitrile concentrations tested (Table 2). In contrast, glyceronitrile promoted A. flavidus germination at concentrations of 1–500 µm (Tables 2 and 3), though seedling length was inhibited by glyceronitrile concentrations of ≥400 µm (Fig. 1). Maximum A. flavidus germination was attained at glyceronitrile concentrations of 25–300 µm (Table 3). Anigozanthos flavidus seeds after-ripened for 26 weeks germinated to similar levels at 10 and 100 µm glyceronitrile (Table 2). However, seeds after-ripened for a shorter period (18 weeks) produced lower germination at 10 µm than at 100 µm (Table 3). Germination of A. flavidus in 1 µm glyceronitrile was higher in seeds incubated in constant darkness than in those incubated under an alternating light/dark regime (Table 2). There was no difference in final germination between seeds incubated in 10 µm glyceronitrile for 24 h (71·3 ± 3·5 %) compared with those incubated in glyceronitrile for 4 weeks (continuous exposure) (F1,4 = 0·81, P = 0·418; Table 3).

Table 1.

Germination (%, mean ± s.e.) of Gyrostemon racemiger, G. ramulosus and Anigozanthos flavidus seeds in response to water, smoke-water and KAR1

Gyrostemon racemiger Gyrostemon ramulosus Anigozanthos flavidus
Control 0 0 15·3 ± 2·4a
Smoke-water (‘Seed Starter’ 10 %) 46·0 ± 7·6 38·0 ± 7·2 84·0 ± 3·5b
KAR1 0·01 µm 0 0 18·7 ± 2·4a
KAR1 0·1 µm 0 0 15·3 ± 1·8a
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Prior to testing, seeds of both Gyrostemon species had been buried for >1 year and exhumed in autumn, while A. flavidus seeds had been after-ripened for 20 weeks at 35 °C. Gyrostemon and Anigozanthos seeds were incubated at 20 and 15 °C, respectively. All seeds were exposed to 12 h light/12 h darkness daily. Different letters indicate significant differences (P < 0·05) in germination response within a species, between treatments.

Table 2.

Germination (%, mean ± s.e.) of Anigozanthos flavidus and Gyrostemon racemiger seeds following incubation for 4 weeks in water and different concentrations of glyceronitrile ranging from 0·001 to 1000 µm at 15 and 20 °C, respectively

Glyceronitrile concentration (μm) Gyrostemon racemiger Anigozanthos flavidus
Control 0 32·0 ± 4·0a
0·001 0·7 ± 0·7 34·0 ± 4·2a
0·01 0 40·7 ± 4·1a
0·1 0 33·3 ± 3·5a
1 0 64·0 ± 4·0b
10 0·7 ± 0·7 90·0 ± 1·2cd
100 0 93·3 ± 3·5d
1000 0 0·7 ± 0·7e
1 (dark)* – 79·3 ± 3·3c
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Seeds were exposed to 12 h light/12 h darkness daily except for the treatment marked with * which was incubated in darkness. Prior to treatment, A. flavidus seeds were after-ripened at 35 °C for 26 weeks and G. racemiger seeds were buried and exhumed in autumn. Different letters indicate significant differences (P < 0·05) in germination response between glyceronitrile concentrations within a species.

Table 3.

Germination (%, mean ± s.e.) of Anigozanthos flavidus seeds following 18 weeks of after-ripening at 35 °C, and incubation at 15 °C for 4 weeks in water and glyceronitrile at concentrations ranging from 5 to 500 µm

Glyceronitrile concentration (μm) Anigozanthos flavidus
Control 27·3 ± 2·4a
5 78·0 ± 2·0b
10 77·3 ± 5·8b
25 88·7 ± 0·7cd
50 94·7 ± 0·7de
75 92·7 ± 1·3de
100 93·3 ± 2·4de
200 94·7 ± 2·4de
300 95·3 ± 1·3e
400 82·7 ± 0·7bc
500 80·7 ± 5·9bc
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Seeds were exposed to 12 h light/12 h darkness daily. Different letters indicate significant differences (P < 0·05) in germination response between glyceronitrile concentrations.

Fig. 1.

Fig. 1.

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Mean length (± s.e.) of Anigozanthos flavidus seedlings at glyceronitrile concentrations between 100 and 500 µm after 4 weeks of imbibition (n = 45). Seeds were incubated at 15 °C under a daily 12 h light/12 h dark regime. Seeds were after-ripened for 18 weeks at 35 °C prior to incubation. Different letters indicate significant differences (P < 0·05) in seedling length at different glyceronitrile concentrations.

Neither 0·1 µm KAR4, nor a combination of 0·1 µm KAR1 and 10 µm glyceronitrile, stimulated >1 % of G. racemiger seeds to germinate, even though seeds were responsive to ‘Seed Starter’ smoke-water (Table 4). Germination of G. ramulosus seeds was promoted by ‘Seed Starter’ smoke-water at the time of testing with SNP, but SNP did not promote any germination at the concentrations tested (Table 5).

Table 4.

Germination (%, mean ± s.e.) of Gyrostemon racemiger seeds following incubation at 20 °C in different smoke-derived chemicals (KAR4, a combination of glyceronitrile and KAR1, and smoke-water) for 4 weeks under a daily 12 h light/12 h dark regime

Treatment Gyrostemon racemiger
KAR4 0·1 µm 0·7 ± 0·7
Glyceronitrile 10 µm + KAR1 0·1 µm 0
Smoke-water (‘Seed Starter’ 10 %) 42·7 ± 4·4
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Table 5.

Germination (%, mean ± s.e.) of Gyrostemon ramulosus seeds following incubation at 20 °C for 4 weeks under a daily 12 h light/12 h dark regime at three concentrations of the nitrogen oxide and cyanide-releasing compound sodium nitroprusside (SNP), and smoke-water to verify seed responsiveness to smoke at the time of SNP testing

Treatment Gyrostemon ramulosus
SNP 10 µm 0
SNP 100 µm 0
SNP 1000 µm 0
Smoke-water (‘Seed Starter’ 10 %) 25·3 ± 1·8
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Gyrostemon racemiger and G. ramulosus seeds were promoted to germinate by the 1 and 10 % concentrations of cellulose-derived smoke-water, though to lower levels than the ‘Seed Starter’ smoke-water (Table 6). Anigozanthos flavidus seeds did not germinate to significantly higher levels in cellulose-derived smoke-water than in water (F3,8 = 3·98, P = 0·053; Table 6).

Table 6.

Germination (%, mean ± s.e.) of the two Gyrostemon species and Anigozanthos flavidus in response to different concentrations of cellulose-derived smoke-water

Cellulose derived smoke-water dilution (%, v/v) Gyrostemon racemiger Gyrostemon ramulosus Anigozanthos flavidus
Control 0 0 25·3 ± 4·4a
0·1 0·7 ± 0·7a 0 34·0 ± 7·9a
1 3·3 ± 1·8ab 4·7 ± 1·3a 25·3 ± 2·4a
10 9·3 ± 2·9b 15·3 ± 2·9b 47·3 ± 5·5a
Smoke-water (‘Seed Starter’ 10 %) 42·7 ± 4·4c 25·3 ± 1·8c –
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Gyrostemon and Anigozanthos seeds were incubated for 4 weeks under a daily 12 h light/12 h dark regime at 20 and 15 °C, respectively. Different letters indicate significantly different (P < 0·05) responses to the treatments within each species.

Stylidium affine and G. leucopteris germination were promoted by both ‘Seed Starter’ smoke-water and KAR1 (Table 7). Neither glyceronitrile nor the 10 % (v/v) cellulose-derived smoke-water stimulated S. affine germination. Grevillea leucopteris seeds were also unresponsive to 10 µm glyceronitrile, but germination with the 10 % (v/v) cellulose-derived smoke-water was similar to that with the ‘Seed Starter’ smoke-water.

Table 7.

Germination (%, mean ± s.e.) of Stylidium affine and Grevillea leucopteris seeds following incubation at 20 °C for 12 and 4 weeks respectively, under a daily 12 h light/12 h dark regime

Treatment Stylidium affine Grevillea leucopteris
Control 4·7 ± 2·4a 7·3 ± 0·7a
Smoke-water (‘Seed Starter’ 10 %) 38·7 ± 2·4b 82·0 ± 1·2b
KAR1 0·1 µm 48·7 ± 1·8b 90·0 ± 2·3c
Glyceronitrile 10 µm 5·3 ± 2·4a 10·7 ± 2·9a
Cellulose-derived smoke-water 10 % 10·7 ± 2·7a 75·3 ± 1·3b
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Different letters indicate significantly different (P < 0·05) responses to the treatments within each species.

Both cellulose-derived and hay-derived smoke-water stimulated germination of the second batch of S. affine seeds that were pre-treated differently (Fig. 2). Overall, S. affine germination was generally higher in the plant-derived than the cellulose-derived smoke-water.

Fig. 2.

Fig. 2.

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Germination of Stylidium affine seeds imbibed in water and smoke-water (sw), derived from burning filter paper (cellulose) or oaten hay (plant material), and incubated at 20 °C for 12 weeks under a daily 12 h light/12 h dark regime. Values are means (± s.e.) and different letters indicate significant differences (P < 0·05) in germination between the different treatments.

DISCUSSION

The discovery that the fire ephemeral T. cyathiflora (Gyrostemonaceae) required smoke-water to germinate, yet was unresponsive to KAR1, highlighted the possibility that there may be additional chemical(s) in smoke-water that promote germination (Downes et al., 2010). Similarly, results from this study have shown that other species from this family, namely G. racemiger and G. ramulosus, also respond to smoke-water but not to KAR1. The lack of germination response to KAR1 in these two Gyrostemon species cannot be attributed to the inactivity of this chemical since it stimulated the germination of S. affine and G. leucopteris.

Glyceronitrile, another germination-stimulating chemical recently isolated from smoke (Flematti et al., 2011), was also tested at a range of concentrations on smoke (‘Seed Starter’)-responsive G. racemiger seeds, but did not promote any germination. Hence there may be yet further germination-promoting chemicals present in smoke-water. Despite the lack of germination response to glyceronitrile in G. racemiger, glyceronitrile stimulated A. flavidus germination. Germination of this species was stimulated at glyceronitrile concentrations of 1–500 µm, and germination over 4 weeks was optimal at 25–300 µm. The glyceronitrile concentrations that promote Anigozanthos manglesii and Rhodocoma arida germination fall within this range (Flematti et al., 2011). Furthermore, the concentrations of glyceronitrile in two samples of 10 % smoke-water (the dilution of smoke-water typically used to promote Anigozanthos germination) were 19 and 30 µm (Flematti et al., 2011), which are also within the range of concentrations found to promote germination. Although 400 and 500 µm glyceronitrile promoted A. flavidus germination over 4 weeks of incubation, germination percentages were lower and radicles were shorter than at lower glyceronitrile concentrations. Shorter radicles may be the result of delayed germination and/or suppressed radicle growth. In Arabidopsis, potassium cyanide concentrations >300 µm inhibit the onset of germination (Bethke et al., 2006).

The promotion of A. flavidus germination by glyceronitrile here contrasts with the lack of stimulation by either smoke-water or glyceronitrile in this species in a previous study, although glyceronitrile was shown to stimulate the germination of three other Anigozanthos species (Flematti et al., 2011). This difference can possibly be attributed to the dormancy state of the seeds. Changing responsiveness to smoke with changes in seed dormancy has been reported in a number of species (Baker et al., 2005b; Merritt et al., 2007). Prior to this study, trials were undertaken to determine the duration of after-ripening required to enable A. flavidus seeds to become smoke responsive. In A. manglesii, following extended after-ripening of seeds under laboratory conditions, seeds germinate to high levels of germination in water alone and have a reduced requirement for smoke to promote germination (K. S. Downes, unpubl. res.). Similarly, in Arabidopsis, KAR1 was less effective at promoting germination with extended after-ripening (Nelson et al., 2009).

Glyceronitrile stimulates germination via the release of cyanide (Flematti et al., 2011). Sodium nitroprusside (SNP) releases both cyanide and nitrogen oxides (Beligni and Lamattina, 2000; Bethke et al., 2006) but did not stimulate the germination of G. ramulosus. This correlates with the finding that the closely related species, G. racemiger was unreceptive to glyceronitrile. The lack of G. ramulosus germination is unlikely to be due to inappropriate SNP concentrations as such concentrations have induced the germination of other glyceronitrile-responsive species (K. S. Downes, unpubl. res.).

Sodium nitroprusside was tested because it releases nitrogen oxide (NO), as well as cyanide (Beligni and Lamattina, 2000). However, subsequent research has shown that many SNP-responsive species actually respond to the cyanide in an NO-dependent manner (Bethke et al., 2006). Thus, NO may be involved in cyanide-promoted germination, which requires further investigation in glyceronitrile-stimulated species. However, G. racemiger was unresponsive to glyceronitrile at concentrations that stimulated A. flavidus to germinate. The actual amounts of NO released by the SNP concentrations tested are very low (Preston et al., 2004; Bethke et al., 2006), and such concentrations did not stimulate G. ramulosus germination. Further research is required, however, to determine whether G. ramulosus is responsive to higher levels of nitrogen oxides. Higher concentrations of nitrogen oxides promote germination of the smoke-responsive Californian fire follower, Emmenanthe penduliflora (Keeley and Fotheringham, 1997; Fotheringham and Keeley, 2005). Evidence for such levels of nitrogen oxides in smoke-water has not been found (Doherty and Cohn, 2000; Preston et al., 2004), and they are therefore unlikely to be the chemical in smoke-water that promotes Gyrostemon germination. Nevertheless, nitrogen oxides may still play an ecological role in stimulating germination as these gases are produced during fires (Andreae and Merlet, 2001).

Preliminary results suggest that at least one compound in smoke-water that stimulates G. ramulosus and G. racemiger germination is comprised of carbon, hydrogen and oxygen only, since smoke-water prepared by the combustion of cellulose promoted germination. This finding must be treated tentatively however, because although A. flavidus seeds were not stimulated to germinate by cellulose-derived smoke-water, the P-value was close to 0·05. Anigozanthos flavidus seeds were used to test whether the cellulose-derived smoke-water contained nitrogenous compounds because it responds to glyceronitrile, which contains nitrogen in addition to carbon, hydrogen and oxygen. Indeed, Whatman No. 1 filter paper contains a number of trace elements, including 23 µg g−1 nitrogen (Anonymous, 2004). There is a very slight chance that this trace nitrogen could ‘contaminate’ the smoke-water, especially as chemicals in smoke stimulate germination at very low levels (Flematti et al., 2004) and glyceronitrile promoted A. flavidus germination at concentrations as low as 1 µm. However, Whatman No. 1 filter paper has been used elsewhere to produce smoke-water containing compounds consisting of only carbon, hydrogen and oxygen (Flematti et al., 2004, 2011).

Although cellulose-derived smoke-water stimulated some Gyrostemon germination, higher levels of germination were stimulated by plant-derived ‘Seed Starter’ smoke-water. This might indicate that compounds comprised of elements other than carbon, hydrogen and oxygen are more effective at stimulating germination. Alternatively, the optimum concentration of the cellulose-derived smoke-water might not have been tested or the combustion conditions, such as temperature, might have been less efficient at producing the active chemical(s), or possibly more efficient at generating inhibitory compounds (Light et al., 2010). Stylidium affine, which responds to KAR1, a compound comprised of only carbon, hydrogen and oxygen (Flematti et al., 2004), but not glyceronitrile, generally had higher germination with the hay-derived smoke-water than with the cellulose-derived smoke-water. It is important to note, however, that due to differences in the combustion conditions between the cellulose- and hay-derived smoke-water, direct comparisons were not made between corresponding dilutions of these two batches of smoke-water. Nicotiana attenuata, which is also KAR1 responsive (Flematti et al., 2004), is stimulated to germinate by both cellulose-derived and plant-derived smoke-water, but to higher levels in the latter (Baldwin et al., 1994). Unless S. affine or N. attenuata also respond to chemicals in smoke-water other than karrikins, these examples highlight that species which require smoke-derived compounds comprised of carbon, hydrogen and oxygen only may have higher germination in plant- than in cellulose-derived smoke-water.

Future tests could include the response of Gyrostemon species to other karrikins. Although G. racemiger did not germinate in response to either KAR1 (at 0·01 or 0·1 µm) or KAR4 (at 0·1 µm), there are at least four other karrikins present in smoke-water (Flematti et al., 2009). To date, only species responsive to KAR1 have shown a response to any of the other karrikins. However, this may simply be because these other karrikins have only been tested on a limited number of species [S. orbiculatum, L. sativa, Emmenanthe penduliflora and Arabidopsis thaliana (Ler)], all of which are KAR1 responsive (Flematti et al., 2007, 2009; Nelson et al., 2009). KAR3 in particular should be tested as, other than KAR1, only KAR3 was present in the smoke-water tested at sufficient levels to contribute to the germination of S. orbiculatum (Flematti et al., 2009). This does not necessarily preclude the potential role of other karrikins in smoke, as the source material and combustion conditions, such as temperature or duration of smoke capture, can influence the quantities of different chemicals present in aqueous smoke solutions (Guillén and Ibargoitia, 1996; Flematti et al., 2009). Also different species show different sensitivities to the various karrikins (Nelson et al., 2009). For example, in A. thaliana (Ler), KAR2 was the most active of the four karrikins tested (KAR1–KAR4; Nelson et al., 2009), whereas KAR1 and KAR3 were the most active karrikins tested on S. orbiculatum (Flematti et al., 2007, 2009).

A chemical feature of the Gyrostemonaceae that may influence germination is the presence of glucosinolates in plant tissue and seeds. Glucosinolates hydrolyse to form isothiocyanates that operate in herbivory defence (Halkier and Gershenzon, 2006), and also inhibit germination (Al-Khatib et al., 1997). Interestingly, there are similarities between the production of glucosinolates, and cyanogenic glycosides that release cyanide (Halkier and Gershenzon, 2006). All 17 families in the Brassicales order, including the Gyrostemonaceae, produce glucosinolates (Rodman et al., 1998; Fay and Christenhusz, 2010). Most families in the Brassicales have not yet been tested for KAR1 responsiveness, apart from the Brassicaceae. In contrast to the Gyrostemonaceae species tested, the Brassicaceae include a number of KAR1-responsive species (Stevens et al., 2007; Nelson et al., 2009; Long et al., 2011). Differences in the germination requirements of taxa between these two families may have arisen because the lineages of these families diverged >90 million years ago (Beilstein et al., 2010). In addition, the Gyrostemonaceae arose in the Late Cretaceous and thus is a much older family than the Brassicaceae, which arose in the Eocene (Beilstein et al., 2010). As these two families arose under different environmental pressures, they may have perhaps developed responses to different chemicals in smoke to cue germination. KAR1 stimulates the germination of a number of Brassicaceae species, but the chemical(s) in smoke-water that stimulates Gyrostemonaceae germination is yet to be found.

Whether the smoke-stimulated germination response is an ancient trait or has arisen from convergent evolution has been debated (Pausas and Keeley, 2009). Both smoke and KAR1 responsiveness are phylogenetically widespread (Chiwocha et al., 2009; Pausas and Keeley, 2009) which gives some support to this being an ancient trait. The existence of species that respond to smoke but not KAR1, such as T. cyathiflora (Downes et al., 2010), several Anigozanthos species, R. arida (Flematti et al., 2011), Gyrostemon racemiger and G. ramulosus, confirms that the way in which smoke stimulates germination is not universal and highlights that KAR1 responsiveness cannot be assumed of all smoke-responsive species. Nevertheless, this does not preclude KAR1 responsiveness as an ancient trait as it may have been subsequently lost in certain species. The existence of chemicals in smoke other than KAR1 that stimulate germination, such as glyceronitrile (Flematti et al., 2011), nitrogen oxides (Keeley and Fotheringham, 1997) and an unknown chemical that stimulates Gyrostemon germination, also suggest the operation of convergent evolution in smoke-promoted germination.

Gyrostemon racemiger is similar to A. flavidus in that both Australian endemic species germinate in response to smoke but not KAR1, which promotes the germination of a wide range of other smoke-responsive species (Chiwocha et al., 2009). However they are phylogenetically distant (different orders) and differ in their response to glyceronitrile. Glyceronitrile, also derived from smoke-water, and consisting of nitrogen in addition to carbon, hydrogen and oxygen, promotes A. flavidus germination, whereas G. racemiger does not germinate in response to this chemical but to an as yet unidentified compound in smoke-water possibly consisting of only carbon, hydrogen and oxygen.

ACKNOWLEDGEMENTS

Thanks to Peter Downes for assistance with preparing the smoke-water and to Professor Caroline Gross for the idea of creating smoke using a bee smoker for my fourth year project at the University of New England in 1998. Seeds were collected with permission from the Western Australian Department of Environment and Conservation. M.E.L. and J.V.S. gratefully acknowledge the support of the National Research Foundation, Pretoria, and the University of KwaZulu-Natal.

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