Annals of Botany 111: 489–497, 2013 doi:10.1093/aob/mcs300, available online at www.aob.oxfordjournals.org

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

Katherine S. Downes1,*, Marnie E. Light2, Martin Posˇta3, Ladislav Kohout3 and Johannes van Staden2 1Department of Environment and Agriculture, Curtin University, PO Box U1987, Perth, WA 6845, Australia, 2Research Centre for Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa and 3Institute of Organic Chemistry and Biochemistry, AS CR, v.v.i., Flemingovo na´m. 2., 166 10, Prague 6, Czech Republic * For correspondence. E-mail [email protected]

Received: 25 September 2012 Revision requested: 30 October 2012 Accepted: 6 December 2012 Published electronically: 8 January 2013 Downloaded from

† 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 re- sponsive to KAR1, such as 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 http://aob.oxfordjournals.org/ 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 8C 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 by guest on March 25, 2014 ‘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 mM, promoted germination, although seedling growth was inhibited at ≥400 mM. Maximum A. flavidus ger- mination occurred at glyceronitrile concentrations of 25–300 mM. 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.

Key words: 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 2004, the butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one, In 1990, de Lange and Boucher reported the landmark discovery was isolated from smoke-water (Flematti et al., 2004; that aerosol smoke and aqueous smoke-water could promote Van Staden et al., 2004). This chemical, karrikinolide the germination of Audouinia capitata, a rare South African (KAR1; Flematti et al.,2009), has proved to be a major species. Subsequently, smoke has been shown to promote the active germination-promoting agent in smoke (Light et al., germination of many other species, many of which were previ- 2009). Over 60 species in 29 different families are both ously difficult to germinate (Roche et al., 1997; Keeley and smoke and KAR1 responsive (Chiwocha et al., 2009). Fotheringham, 1998; Brown and Botha, 2004; Baker et al., Interestingly, Tersonia cyathiflora, an Australian fire ephem- 2005a). Smoke-stimulated germination has extensive implica- eral in the Gyrostemonaceae with an obligate requirement for tions for horticulture, weed control, conservation and restoration smoke to germinate, is unresponsive to KAR1 (Downes et al., (Light and van Staden, 2004; Kulkarni et al., 2011). 2010). Prior to this, it was assumed that KAR1 would induce Much effort has focused on determining the chemical(s) in germination in this smoke-responsive species (Fay and smoke responsible for this germination stimulation (Baldwin Christenhusz, 2010), as KAR1 was once considered by some et al., 1994; Van Staden et al., 1995; Light et al., 2009). to be the sole chemical responsible for all smoke-stimulated

# The Author 2013. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 490 Downes et al. — Germination responses to smoke-water, KAR1 and glyceronitrile germination (Pausas and Keeley, 2009). Most members of the populations. Seeds of both species were exhumed on 5 April Gyrostemonaceae are fire ephemerals that germinate predom- 2011. The seed burial pre-treatment was undertaken since inantly after fire, often in large numbers, and live for only a germination response to smoke was previously shown to be few years, thereafter persisting as seeds in the soil seed-bank enhanced in seeds of both these species following a period until a subsequent fire (Bell et al., 1984; Baker et al., of soil burial and retrieval in autumn (Baker et al., 2005a). 2005a). In addition to T. cyathiflora, other fire ephemerals in Anigozanthos flavidus (Haemodoraceae) seeds were collected the Gyrostemonaceae, including Gyrostemon racemiger and from Redmond State Forest (34 851.512’S, 117 833.234’E) on 2 G. ramulosus, germinate in response to smoke following a March 2009 and stored at 15 8C. Preliminary trials indicated period of burial (Baker et al., 2005a). However, the response that freshly collected seeds were dormant and that after- of Gyrostemon seeds to KAR1 has not yet been tested. ripening seeds at 35 8C alleviated dormancy and enabled Germination stimulation of T. cyathiflora by plant-derived seeds to respond to smoke-water. Grevillea leucopteris smoke-water, but not KAR1, suggests that there may be (Proteaceae) seeds were collected along the Brand Highway other chemical(s) in smoke-water that promote the germination (29 831.406’S, 115 83.315’E) on 2 December 2010. Stylidium of certain species (Downes et al., 2010). Indeed, glyceronitrile affine (Stylidiaceae) seeds were purchased from Nindethana (2,3-dihydroxypropanenitrile) was recently isolated from Seed Service, Albany, . These seeds were smoke-water and stimulates the germination of a number of collected near Boddington, Western Australia in December species, including various Anigozanthos spp. that are also un- 2007. Both G. leucopteris and S. affine seeds were stored at responsive to KAR1 (Flematti et al., 2011). This chemical con- ambient room temperature prior to use. Downloaded from tains nitrogen in addition to carbon, hydrogen and oxygen, and is proposed to operate through the release of cyanide (Flematti Germination protocol et al., 2011). It has, however, not yet been tested on any Gyrostemonaceae species. Seeds were surface sterilized prior to treatment to reduce Other chemicals that have been reported as germination- fungal contamination. This involved placing seeds in 2 % stimulating agents present in smoke, which have not previous- sodium hypochlorite with a few drops of Tween-80 (polyoxy- http://aob.oxfordjournals.org/ ly been tested on the Gyrostemonaceae, are nitrogen oxides ethylene sorbitan mono-oleate). The seeds and hypochlorite (Keeley and Fotheringham, 1997). The role of nitrogen solution were shaken, placed under vacuum for 5 min, returned oxides in smoke-stimulated germination has been questioned to normal air pressure for 5 min and placed under vacuum for a (Preston et al., 2004; Baldwin et al., 2005), partly because further 5 min. Seeds were then rinsed at least three times with certain species responsive to nitrogen oxides also respond to sterile deionized water before being transferred to 9 cm Petri the combustion of pure cellulose (Baldwin et al., 1994) and dishes. Within each Petri dish there were two pieces of 2 KAR1 (Flematti et al., 2004). Nevertheless, the discovery Whatman No. 1 filter paper over three 4 cm pieces of

that glyceronitrile can promote the germination of some ‘Vileda’ sponge. These were moistened with 10 mL of the by guest on March 25, 2014 KAR1-responsive species highlights that the germination of test solution and sealed with Parafilm. Sterile deionized certain species may be promoted by more than one chemical water was used as a control in all experiments. Seeds were in smoke (Flematti et al., 2011). incubated continuously in the test solutions unless otherwise The aims of this study were, first, to determine whether specified. Each treatment comprised three replicate Petri seeds of Gyrostemon racemiger and G. ramulosus respond to dishes of 50 seeds. Seeds were incubated under a diurnal plant-derived smoke-water and/or KAR1; and, secondly, to regime of 12 h light/12 h dark. Gyrostemon, Grevillea and test whether seeds of G. racemiger were stimulated to germin- Stylidium seeds were incubated at 20 8C, and Anigozanthos ate by glyceronitrile, a newly isolated germinating-promoting seeds were incubated at 15 8C. Seed germination was scored chemical from smoke, or nitrogen oxides. Where seeds did weekly for 4 weeks, unless noted otherwise. not respond to either KAR1 or glyceronitrile, preliminary investigations were made into whether cellulose-derived Experiment 1: Gyrostemon and Anigozanthos response to smoke could stimulate the germination of these seeds. This smoke-water and KAR was done to indicate whether the chemical(s) in smoke that 1 promote the germination of these species was comprised of Gyrostemon racemiger, G. ramulosus and A. flavidus were only carbon, hydrogen and oxygen. initially tested with water (control), a 10 % (v/v) dilution of ‘Seed Starter’ smoke-water, 0.1 mM KAR1 and 0.01 mM KAR . The A. flavidus seeds had been after-ripened at 35 8C MATERIALS AND METHODS 1 for 20 weeks from November 2010. ‘Seed Starter’ smoke- Seeds of Gyrostemon racemiger and G. ramulosus water was purchased from Kings Park and Botanic Garden, (Gyrostemonaceae) were collected along the Brand Highway Perth, Western Australia in 2003 and stored at 4 8C prior to north of Perth, Western Australia. Gyrostemon racemiger use. It was produced by drawing smoke from the combustion seeds were collected between Regens Ford and Cataby (30 of plant material though water (Tieu et al., 1999; Stevens 852.224′S 115 837.421′E) on 21 November 2009 and et al., 2007). Before addition to the Petri dishes, the ‘Seed G. ramulosus seeds were collected between Eneabba and Starter’ smoke-water was filter sterilized through a 0.2 mm Dongara (29 817.000′S, 115 800.508′E) on 24 December Acrodisc syringe filter. The diluted ‘Seed Starter’ had a pH 2009. Within a week of collection, seeds were cleaned, of 4.1. KAR1 was synthesized from pyromeconic acid accord- placed in nylon mesh bags and buried approx. 1–2 cm ing to Flematti et al. (2005) and Light et al. (2010), and the beneath the soil surface at three sites within the source 0.1 mM solution had a pH of 6.9. Downes et al. — Germination responses to smoke-water, KAR1 and glyceronitrile 491

Experiment 2: Gyrostemon and Anigozanthos response to The ‘Seed Starter’ smoke-water was included to verify the glyceronitrile continued responsiveness of the seeds. The cyanohydrin, glyceronitrile (2,3-dihydroxypropanenitrile), was reported as a germination-promoting chemical found Experiment 4: and response to in plant-derived smoke-water (Flematti et al., 2011). Gyrostemon Anigozanthos cellulose-derived smoke-water Glyceronitrile was synthesized as a racemic mixture according to the method of Kopecky´ and Sˇmejkal (1984), and the iden- To determine whether Gyrostemon seeds were stimulated to tity of the compound was confirmed using nuclear magnetic germinate by chemical(s) in smoke containing only carbon, resonance (NMR) spectroscopy. Initially 1 and 1000 mM hydrogen and oxygen, cellulose-derived smoke-water was pro- solutions of glyceronitrile were tested on A. flavidus seeds duced. Whatman No. 1 filter paper (150 sheets of 9 cm diam- after-ripened at 35 8C for 20 weeks from early November eter, approx. 80 g in weight), as used by Flematti et al. (2004), 2010. Subsequently, glyceronitrile was tested on A. flavidus was burnt in a bee smoker and the smoke was bubbled into (after-ripened for 26 weeks) and G. racemiger at a range of 0.5 L of sterile deionized water for 12.5 min. The smoke-water concentrations: 0 (control), 0.001, 0.01, 0.1, 1, 10, 100 and was filter sterilized through a 0.2 mm Acrodisc syringe filter 1000 mM. Anigozanthos flavidus seeds were also tested with and diluted to 0.1, 1 and 10 % (v/v). The pH of the 10 % 1 mM glyceronitrile in the dark, by wrapping the Petri dishes cellulose-derived smoke-water was 3.6. The two Gyrostemon in aluminium foil. Glyceronitrile (10 mM)hadapHof5.2. species were incubated in these solutions, along with water The pH of glyceronitrile influences the amount of free (control) and 10 % (v/v) ‘Seed Starter’ smoke-water. Since Downloaded from cyanide produced, which is the proposed means by which ‘Seed Starter’ is produced by the combustion of plant material glyceronitrile stimulates germination (Flematti et al., 2011). (Tieu et al., 1999; Stevens et al., 2007), it would contain com- Following the above preliminary germination trial using a pounds with elements in addition to carbon, hydrogen and wide range of glyceronitrile concentrations, a fresh batch of oxygen.

A. flavidus seeds from the same seed lot were after-ripened for Anigozanthos flavidus seeds after-ripened at 35 8C for http://aob.oxfordjournals.org/ 18 weeks from November 2011 and the seeds were incubated 16 weeks from May 2011 were also incubated with water in the following concentrations of glyceronitrile: 0 (control), (control) and three concentrations of the cellulose-derived 5, 10, 25, 50, 75, 100, 200, 300, 400 and 500 mM. Seeds were smoke-water to verify that this smoke-water did not contain also incubated with 10 mM glyceronitrile for 24 h and thereafter nitrogen, as this species does not germinate in response to transferred to fresh Petri dishes containing 10 mL of deionized smoke-water created from carbon, hydrogen and oxygen only water, to determine whether the different duration of exposure (Flematti et al., 2011). to glyceronitrile influenced germination levels. At the comple- tion of the germination trial, the lengths of 15 random seedlings per Petri dish in each of the five highest glyceronitrile concen- Experiment 5: verification of chemical activity using Stylidium by guest on March 25, 2014 trations were measured to determine whether higher concentra- affine and Grevillea leucopteris tions inhibited seedling growth. 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. Experiment 3: Gyrostemon response to other chemicals present in Seeds of S. affine were previously shown to germinate in smoke or smoke-water response to KAR1 (Flematti et al., 2004; Downes et al., Gyrostemon racemiger seeds were also incubated in 0.1 mM 2010) and, although the responsiveness of G. leucopteris to KAR4 (3,7-dimethyl-2H-furo[2,3-c]pyran-2-one), and a com- smoke or KAR1 has not yet been tested, a number of other bination of 10 mM glyceronitrile and 0.1 mM KAR1. A ‘Seed Grevillea species are smoke and/or KAR1 responsive (Roche Starter’ smoke-water treatment was also included to verify et al., 1997; Downes et al., 2010). the continued responsiveness of the seeds. KAR4 is another Previously, seeds from the same S. affine seed lot germi- karrikin in smoke-water that has been shown to stimulate the nated to low levels in the light (Downes et al., 2010) and germination of certain species including Lactuca sativa were, therefore, pre-treated to alleviate dormancy. This pre- (Apiaceae) and Solanum orbiculatum (Solanaceae; Flematti treatment involved placing seeds on two pieces of Whatman et al., 2007, 2009). It was synthesized from maltol according No. 1 filter paper in each of three 9 cm Petri dishes, moistening to Flematti et al. (2007), and the 0.1 mM solution had a pH them with 5 mL of deionized water, and then sealing the Petri of 6.3. dishes with Parafilm and placing them in a 20 8C incubator. Gyrostemon ramulosus seeds were incubated in 10, 100 and After 24 h, the Parafilm was removed, and the top piece of 1000 mM sodium nitroprusside (SNP; Ajax Chemicals, filter paper and the seeds were placed on the lid of the Petri Auburn, Australia). Fresh solutions of SNP were prepared dish and returned to the 20 8C incubator to dry for 24 h. The just before the start of the experiment since SNP decomposes seeds were then transferred to 35 8C for the remaining 5 d of in the light to produce both cyanide and nitrogen oxides the week. This weekly wet/dry and shifting temperature (Beligni and Lamattina, 2000; Bethke et al., 2006). Cyanide regime was repeated on these seeds a further three times. is released by glyceronitrile and promotes germination in Grevillea leucopteris and pre-treated S. affine seeds were certain species (Flematti et al., 2011), and nitrogen oxides tested with water (control), a 10 % (v/v) dilution of ‘Seed have also been shown to stimulate the germination of certain Starter’ smoke-water, 0.1 mM KAR1,10mM glyceronitrile and smoke-responsive seeds (Keeley and Fotheringham, 1997). 10 % (v/v) cellulose-derived smoke-water. The G. leucopteris 492 Downes et al. — Germination responses to smoke-water, KAR1 and glyceronitrile and S. affine trials commenced in July and October 2011 and ran Statistical analyses were performed in Genstat versions 13 for 4 and 12 weeks, respectively. and 14 (VSN International, Oxford, UK).

Experiment 6: comparison of Stylidium affine germination RESULTS between smoke-water derived from cellulose and hay Gyrostemon racemiger, G. ramulosus and A. flavidus seeds Seeds of S. affine were used to compare the germination of a were promoted to germinate by ‘Seed Starter’ smoke-water KAR1-responsive species with cellulose-derived smoke-water but not KAR1, at the concentrations tested (Table 1). vs. hay-derived smoke-water. For pre-treatment, S. affine Germination of G. racemiger seeds was negligible (,1%) seeds were placed in a 35 8C oven for 2 weeks, then, for the across the range of glyceronitrile concentrations tested subsequent 4 weeks, seeds were placed in a 20 8C incubator (Table 2). In contrast, glyceronitrile promoted A. flavidus (90–95 % humidity) for 48 h before transfer to the 35 8C germination at concentrations of 1–500 mM (Tables 2 and 3), oven (15–20 % humidity) for the remainder of each week. though seedling length was inhibited by glyceronitrile Hay-derived smoke-water was produced by burning oaten concentrations of ≥400 mM (Fig. 1). Maximum A. flavidus hay, as used by Stevens et al. (2007), in a bee smoker and bub- germination was attained at glyceronitrile concentrations of bling the smoke into 0.5 L of sterile deionized water for 25–300 mM (Table 3). Anigozanthos flavidus seeds after- 12 min. Markings with Thermochromw chromatic thermom- ripened for 26 weeks germinated to similar levels at 10 and eter crayons (Faber-Castell, Nu¨rnberg, Germany), which 100 mM glyceronitrile (Table 2). However, seeds after-ripened Downloaded from change colour when particular temperatures are attained, indi- for a shorter period (18 weeks) produced lower germination at cated that the maximum temperature in the bee smoker during 10 mM than at 100 mM (Table 3). Germination of A. flavidus in burning was between 350 and 500 8C. The hay-derived smoke- 1 mM glyceronitrile was higher in seeds incubated in constant water was filter sterilized through a 0.2 mm Acrodisc syringe darkness than in those incubated under an alternating light/ filter and diluted to 1, 5, 10 and 20 % (v/v). Cellulose- dark regime (Table 2). There was no difference in final http://aob.oxfordjournals.org/ derived smoke-water, as produced in Experiment 4 and germination between seeds incubated in 10 mM glyceronitrile stored at 4 8C, was diluted to 1, 5, 10, 20 and 30 % (v/v). for 24 h (71.3 + 3.5 %) compared with those incubated in gly- Pre-treated S. affine seeds were tested with the hay- and ceronitrile for 4 weeks (continuous exposure) (F1,4 ¼ 0.81, cellulose-derived smoke-water solutions, as well as deionized P ¼ 0.418; Table 3). water (control) and 10 % (v/v) ‘Seed Starter’ smoke-water. Neither 0.1 mM KAR4, nor a combination of 0.1 mM KAR1 This experiment commenced in February 2012 and cumulative and 10 mM glyceronitrile, stimulated .1% of G. racemiger germination was scored over 12 weeks. 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- by guest on March 25, 2014 Statistical analysis water at the time of testing with SNP, but SNP did not Comparisons were made using analysis of variance promote any germination at the concentrations tested (ANOVA). Prior to analysis, percentage data were converted (Table 5). to a value between 0 and 1, and arcsine square-root trans- Gyrostemon racemiger and G. ramulosus seeds were pro- formed. When germination was absent from all replicates of moted to germinate by the 1 and 10 % concentrations of a treatment, that treatment was excluded from analysis to satisfy the ANOVA assumption of equal variances. Fisher’s ABLE Germination (%, mean + s.e.) of protected l.s.d. test was used as a post-hoc test. Treatments T 2. Anigozanthos flavidus and seeds following incubation for 4 were regarded as significantly different at P , 0.05. Gyrostemon racemiger weeks in water and different concentrations of glyceronitrile ranging from 0.001to1000mM at 15 and 20 8C, respectively TABLE 1. Germination (%, mean + s.e.) of Gyrostemon racemiger, G. ramulosus and Anigozanthos flavidus seeds in response to Glyceronitrile concentration Gyrostemon Anigozanthos (mM) racemiger flavidus water, smoke-water and KAR1 Control 0 32.0 + 4.0a Gyrostemon Gyrostemon Anigozanthos 0.001 0.7 + 0.734.0 + 4.2a racemiger ramulosus flavidus 0.01 0 40.7 + 4.1a 0.1033.3 + 3.5a Control 0 0 15.3 + 2.4a 1064.0 + 4.0b Smoke-water 46.0 + 7.638.0 + 7.284.0 + 3.5b 10 0.7 + 0.790.0 + 1.2cd (‘Seed Starter’ 10 %) 100 0 93.3 + 3.5d a e KAR1 0.01 mM 0018.7 + 2.4 1000 0 0.7 + 0.7 a c KAR1 0.1 mM 0015.3 + 1.8 1 (dark)* – 79.3 + 3.3

Prior to testing, seeds of both Gyrostemon species had been buried for .1 Seeds were exposed to 12 h light/12 h darkness daily except for the year and exhumed in autumn, while A. flavidus seeds had been after-ripened treatment marked with * which was incubated in darkness. Prior to treatment, for 20 weeks at 35 8C. Gyrostemon and Anigozanthos seeds were incubated at A. flavidus seeds were after-ripened at 35 8C for 26 weeks and G. racemiger 20 and 15 8C, respectively. All seeds were exposed to 12 h light/12 h seeds were buried and exhumed in autumn. Different letters indicate darkness daily. Different letters indicate significant differences (P , 0.05) in significant differences (P , 0.05) in germination response between germination response within a species, between treatments. glyceronitrile concentrations within a species. Downes et al. — Germination responses to smoke-water, KAR1 and glyceronitrile 493

TABLE 3. Germination (%, mean + s.e.) of Anigozanthos TABLE 4. Germination (%, mean + s.e.) of Gyrostemon flavidus seeds following 18 weeks of after-ripening at 35 8C, and racemiger seeds following incubation at 20 8C in different incubation at 15 8C for 4 weeks in water and glyceronitrile at smoke-derived chemicals (KAR4, a combination of glyceronitrile concentrations ranging from 5 to 500 mM and KAR1, and smoke-water) for 4 weeks under a daily 12 h light/12 h dark regime Glyceronitrile concentration (mM) Anigozanthos flavidus Treatment Gyrostemon racemiger Control 27.3 + 2.4a . . b 5780 + 2 0 KAR 0.1 mM 0.7 + 0.7 . . b 4 10 77 3 + 5 8 Glyceronitrile 10 mM + KAR 0.1 mM 0 cd 1 25 88.7 + 0.7 Smoke-water (‘Seed Starter’ 10 %) 42.7 + 4.4 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 TABLE 5. Germination (%, mean + s.e.) of Gyrostemon 400 82.7 + 0.7bc ramulosus seeds following incubation at 20 8C for 4 weeks under 500 80.7 + 5.9bc a daily 12 h light/12 h dark regime at three concentrations of the nitrogen oxide and cyanide-releasing compound sodium Seeds were exposed to 12 h light/12 h darkness daily. Different letters

nitroprusside (SNP), and smoke-water to verify seed Downloaded from . indicate significant differences (P , 0 05) in germination response between responsiveness to smoke at the time of SNP testing glyceronitrile concentrations. Treatment Gyrostemon ramulosus

18 SNP 10 mM 0

a SNP 100 mM 0 http://aob.oxfordjournals.org/ 16 ab SNP 1000 mM 0 Smoke-water (‘Seed Starter’ 10 %) 25.3 + 1.8 14 b

12

10 were pre-treated differently (Fig. 2). Overall, S. affine germin- ation was generally higher in the plant-derived than the 8 cellulose-derived smoke-water. 6 by guest on March 25, 2014 Seedling length (mm) 4 c c DISCUSSION 2 The discovery that the fire ephemeral T. cyathiflora 0 (Gyrostemonaceae) required smoke-water to germinate, yet 100 200 300 400 500 was unresponsive to KAR1, highlighted the possibility that Glyceronitrile concentration (µM) there may be additional chemical(s) in smoke-water that

F IG. 1. Mean length (+ s.e.) of Anigozanthos flavidus seedlings at glycero- promote germination (Downes et al., 2010). Similarly, nitrile concentrations between 100 and 500 mM after 4 weeks of imbibition results from this study have shown that other species from (n ¼ 45). Seeds were incubated at 15 8C under a daily 12 h light/12 h dark this family, namely G. racemiger and G. ramulosus, also regime. Seeds were after-ripened for 18 weeks at 35 8C prior to incubation. respond to smoke-water but not to KAR1. The lack of germin- Different letters indicate significant differences (P , 0.05) in seedling length at different glyceronitrile concentrations. ation 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. cellulose-derived smoke-water, though to lower levels than the Glyceronitrile, another germination-stimulating chemical ‘Seed Starter’ smoke-water (Table 6). Anigozanthos flavidus recently isolated from smoke (Flematti et al., 2011), was seeds did not germinate to significantly higher levels in also tested at a range of concentrations on smoke (‘Seed cellulose-derived smoke-water than in water (F3,8 ¼ 3.98, Starter’)-responsive G. racemiger seeds, but did not promote P ¼ 0.053; Table 6). any germination. Hence there may be yet further germination- Stylidium affine and G. leucopteris germination were pro- promoting chemicals present in smoke-water. Despite the lack moted by both ‘Seed Starter’ smoke-water and KAR1 of germination response to glyceronitrile in G. racemiger, gly- (Table 7). Neither glyceronitrile nor the 10 % (v/v) cellulose- ceronitrile stimulated A. flavidus germination. Germination of derived smoke-water stimulated S. affine germination. this species was stimulated at glyceronitrile concentrations of Grevillea leucopteris seeds were also unresponsive to 10 mM 1–500 mM, and germination over 4 weeks was optimal at glyceronitrile, but germination with the 10 % (v/v) cellulose- 25–300 mM. The glyceronitrile concentrations that promote derived smoke-water was similar to that with the ‘Seed Anigozanthos manglesii and Rhodocoma arida germination Starter’ smoke-water. fall within this range (Flematti et al., 2011). Furthermore, Both cellulose-derived and hay-derived smoke-water stimu- the concentrations of glyceronitrile in two samples of 10 % lated germination of the second batch of S. affine seeds that smoke-water (the dilution of smoke-water typically used to 494 Downes et al. — Germination responses to smoke-water, KAR1 and glyceronitrile

TABLE 6. Germination (%, mean + s.e.) of the two Gyrostemon 60 f species and Anigozanthos flavidus in response to different f concentrations of cellulose-derived smoke-water 50 ef

Cellulose derived 40 smoke-water dilution Gyrostemon Gyrostemon Anigozanthos de de de (%, v/v) racemiger ramulosus flavidus 30 cd Control 0 0 25.3 + 4.4a cd 0.10.7 + 0.7a 034.0 + 7.9a 20 13.3 + 1.8ab 4.7 + 1.3a 25.3 + 2.4a bc 10 9.3 + 2.9b 15.3 + 2.9b 47.3 + 5.5a Germination (%) . . c . . c Smoke-water (‘Seed 42 7 + 4 4 25 3 + 1 8 – 10 ab Starter’ 10 %) a 0 Gyrostemon and Anigozanthos seeds were incubated for 4 weeks under a daily 12 h light/12 h dark regime at 20 and 15 8C, respectively. Different letters indicate significantly different (P , 0.05) responses to the treatments within each species.

1 % oaten5 % hayoaten sw hay sw Downloaded from 10 % oaten20 % hay oaten10 sw% hay‘Seed sw Starter’

1 % cellulose-derived5 % cellulose-derived sw sw TABLE 7. Germination (%, mean + s.e.) of Stylidium affine and Deionized water (control)10 % cellulose-derived20 % cellulose-derived30 % cellulose-derived sw sw sw Grevillea leucopteris seeds following incubation at 20 8C for 12 Source and cencentration of smoke water and 4 weeks respectively, under a daily 12 h light/12 h dark regime F IG. 2. Germination of Stylidium affine seeds imbibed in water and smoke-

water (sw), derived from burning filter paper (cellulose) or oaten hay (plant http://aob.oxfordjournals.org/ material), and incubated at 20 8C for 12 weeks under a daily 12 h light/12 h Stylidium Grevillea dark regime. Values are means (+ s.e.) and different letters indicate signifi- Treatment affine leucopteris cant differences (P , 0.05) in germination between the different treatments. Control 4.7 + 2.4a 7.3 + 0.7a Smoke-water (‘Seed Starter’ 10 %) 38.7 + 2.4b 82.0 + 1.2b . . . b . . c KAR1 0 1 mM 48 7 + 1 8 90 0 + 2 3 reduced requirement for smoke to promote germination (K. S. . . a . . a Glyceronitrile 10 mM 5 3 + 2 4 10 7 + 2 9 Downes, unpubl. res.). Similarly, in Arabidopsis, KAR was Cellulose-derived smoke-water 10.7 + 2.7a 75.3 + 1.3b 1 10 % less effective at promoting germination with extended after-

ripening (Nelson et al., 2009). by guest on March 25, 2014 Different letters indicate significantly different (P , 0.05) responses to the Glyceronitrile stimulates germination via the release of treatments within each species. 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 promote Anigozanthos germination) were 19 and 30 mM the germination of G. ramulosus. This correlates with the (Flematti et al., 2011), which are also within the range of con- finding that the closely related species, G. racemiger was unre- centrations found to promote germination. Although 400 and ceptive to glyceronitrile. The lack of G. ramulosus germination 500 mM glyceronitrile promoted A. flavidus germination over is unlikely to be due to inappropriate SNP concentrations as 4 weeks of incubation, germination percentages were lower such concentrations have induced the germination of other and radicles were shorter than at lower glyceronitrile concen- glyceronitrile-responsive species (K. S. Downes, unpubl. res.). trations. Shorter radicles may be the result of delayed germin- Sodium nitroprusside was tested because it releases nitrogen ation and/or suppressed radicle growth. In Arabidopsis, oxide (NO), as well as cyanide (Beligni and Lamattina, potassium cyanide concentrations .300 mM inhibit the onset 2000). However, subsequent research has shown that many of germination (Bethke et al., 2006). SNP-responsive species actually respond to the cyanide in an The promotion of A. flavidus germination by glyceronitrile NO-dependent manner (Bethke et al.,2006). Thus, NO may here contrasts with the lack of stimulation by either smoke- be involved in cyanide-promoted germination, which requires water or glyceronitrile in this species in a previous study, al- further investigation in glyceronitrile-stimulated species. though glyceronitrile was shown to stimulate the germination However, G. racemiger was unresponsive to glyceronitrile at of three other Anigozanthos species (Flematti et al., 2011). concentrations that stimulated A. flavidus to germinate. The This difference can possibly be attributed to the dormancy actual amounts of NO released by the SNP concentrations state of the seeds. Changing responsiveness to smoke with tested are very low (Preston et al., 2004; Bethke et al., 2006), changes in seed dormancy has been reported in a number of and such concentrations did not stimulate G. ramulosus germin- species (Baker et al., 2005b; Merritt et al., 2007). Prior to ation. Further research is required, however, to determine this study, trials were undertaken to determine the duration whether G. ramulosus is responsive to higher levels of nitrogen of after-ripening required to enable A. flavidus seeds to oxides. Higher concentrations of nitrogen oxides promote become smoke responsive. In A. manglesii, following extended germination of the smoke-responsive Californian fire follower, after-ripening of seeds under laboratory conditions, seeds ger- Emmenanthe penduliflora (Keeley and Fotheringham, 1997; minate to high levels of germination in water alone and have a Fotheringham and Keeley, 2005). Evidence for such levels of Downes et al. — Germination responses to smoke-water, KAR1 and glyceronitrile 495 nitrogen oxides in smoke-water has not been found (Doherty smoke-water (Flematti et al., 2009). To date, only species re- and Cohn, 2000; Preston et al.,2004), and they are therefore un- sponsive to KAR1 have shown a response to any of the other likely to be the chemical in smoke-water that promotes karrikins. However, this may simply be because these other Gyrostemon germination. Nevertheless, nitrogen oxides may karrikins have only been tested on a limited number of still play an ecological role in stimulating germination as these species [S. orbiculatum, L. sativa, Emmenanthe penduliflora gases are produced during fires (Andreae and Merlet, 2001). and Arabidopsis thaliana (Ler)], all of which are KAR1 re- Preliminary results suggest that at least one compound in sponsive (Flematti et al., 2007, 2009; Nelson et al., 2009). smoke-water that stimulates G. ramulosus and G. racemiger ger- KAR3 in particular should be tested as, other than KAR1, mination is comprised of carbon, hydrogen and oxygen only, only KAR3 was present in the smoke-water tested at sufficient since smoke-water prepared by the combustion of cellulose pro- levels to contribute to the germination of S. orbiculatum moted germination. This finding must be treated tentatively (Flematti et al., 2009). This does not necessarily preclude however, because although A. flavidus seeds were not stimulated the potential role of other karrikins in smoke, as the source ma- to germinate by cellulose-derived smoke-water, the P-value was terial and combustion conditions, such as temperature or dur- close to 0.05. Anigozanthos flavidus seeds were used to test ation of smoke capture, can influence the quantities of whether the cellulose-derived smoke-water contained nitrogen- different chemicals present in aqueous smoke solutions ous compounds because it responds to glyceronitrile, which (Guille´n and Ibargoitia, 1996; Flematti et al., 2009). Also contains nitrogen in addition to carbon, hydrogen and oxygen. different species show different sensitivities to the various kar-

Indeed, Whatman No. 1 filter paper contains a number of rikins (Nelson et al., 2009). For example, in A. thaliana (Ler), Downloaded from 21 trace elements, including 23 mgg nitrogen (Anonymous, KAR2 was the most active of the four karrikins tested (KAR1 – 2004). There is a very slight chance that this trace nitrogen KAR4; Nelson et al., 2009), whereas KAR1 and KAR3 were could ‘contaminate’ the smoke-water, especially as chemicals the most active karrikins tested on S. orbiculatum (Flematti in smoke stimulate germination at very low levels (Flematti et al., 2007, 2009). et al.,2004) and glyceronitrile promoted A. flavidus germination A chemical feature of the Gyrostemonaceae that may http://aob.oxfordjournals.org/ at concentrations as low as 1 mM. However, Whatman No. 1 influence germination is the presence of glucosinolates in filter paper has been used elsewhere to produce smoke-water plant tissue and seeds. Glucosinolates hydrolyse to form iso- containing compounds consisting of only carbon, hydrogen thiocyanates that operate in herbivory defence (Halkier and and oxygen (Flematti et al.,2004, 2011). Gershenzon, 2006), and also inhibit germination (Al-Khatib Although cellulose-derived smoke-water stimulated some et al., 1997). Interestingly, there are similarities between the Gyrostemon germination, higher levels of germination were production of glucosinolates, and cyanogenic glycosides that stimulated by plant-derived ‘Seed Starter’ smoke-water. This release cyanide (Halkier and Gershenzon, 2006). All 17 families might indicate that compounds comprised of elements other in the order, including the Gyrostemonaceae,

than carbon, hydrogen and oxygen are more effective at stimu- produce glucosinolates (Rodman et al.,1998; Fay and by guest on March 25, 2014 lating germination. Alternatively, the optimum concentration Christenhusz, 2010). Most families in the Brassicales have not of the cellulose-derived smoke-water might not have been yet been tested for KAR1 responsiveness, apart from the tested or the combustion conditions, such as temperature, . In contrast to the Gyrostemonaceae species might have been less efficient at producing the active chemi- tested, the Brassicaceae include a number of KAR1-responsive cal(s), or possibly more efficient at generating inhibitory com- species (Stevens et al., 2007; Nelson et al., 2009; Long et al., pounds (Light et al., 2010). Stylidium affine, which responds to 2011). Differences in the germination requirements of taxa KAR1, a compound comprised of only carbon, hydrogen and between these two families may have arisen because the oxygen (Flematti et al., 2004), but not glyceronitrile, generally lineages of these families diverged .90 million years ago had higher germination with the hay-derived smoke-water than (Beilstein et al.,2010). In addition, the Gyrostemonaceae with the cellulose-derived smoke-water. It is important to note, arose in the Late Cretaceous and thus is a much older family however, that due to differences in the combustion conditions than the Brassicaceae, which arose in the Eocene (Beilstein between the cellulose- and hay-derived smoke-water, direct et al.,2010). As these two families arose under different comparisons were not made between corresponding dilutions environmental pressures, they may have perhaps developed of these two batches of smoke-water. Nicotiana attenuata, responses to different chemicals in smoke to cue germination. which is also KAR1 responsive (Flematti et al., 2004), is KAR1 stimulates the germination of a number of Brassicaceae stimulated to germinate by both cellulose-derived and plant- species, but the chemical(s) in smoke-water that stimulates derived smoke-water, but to higher levels in the latter Gyrostemonaceae germination is yet to be found. (Baldwin et al., 1994). Unless S. affine or N. attenuata also Whether the smoke-stimulated germination response is an respond to chemicals in smoke-water other than karrikins, ancient trait or has arisen from convergent evolution has these examples highlight that species which require smoke- been debated (Pausas and Keeley, 2009). Both smoke and derived compounds comprised of carbon, hydrogen and KAR1 responsiveness are phylogenetically widespread oxygen only may have higher germination in plant- than in (Chiwocha et al., 2009; Pausas and Keeley, 2009) which cellulose-derived smoke-water. gives some support to this being an ancient trait. The existence Future tests could include the response of Gyrostemon of species that respond to smoke but not KAR1, such as species to other karrikins. Although G. racemiger did not ger- T. cyathiflora (Downes et al., 2010), several Anigozanthos minate in response to either KAR1 (at 0.01 or 0.1 mM) or KAR4 species, R. arida (Flematti et al., 2011), Gyrostemon racemi- (at 0.1 mM), there are at least four other karrikins present in ger and G. ramulosus, confirms that the way in which smoke 496 Downes et al. — Germination responses to smoke-water, KAR1 and glyceronitrile stimulates germination is not universal and highlights that Bell DT, Hopkins AJM, Pate JS. 1984. Fire in the kwongan. In: Pate JS, KAR responsiveness cannot be assumed of all smoke- Beard JS. eds. Kwongan: plant life of the sandplain. Nedlands: 1 University of Western Australia Press, 178–204. responsive species. Nevertheless, this does not preclude Bethke PC, Libourel IGL, Reino¨hl V, Jones RL. 2006. Sodium nitroprus- KAR1 responsiveness as an ancient trait as it may have been side, cyanide, nitrite, and nitrate break Arabidopsis seed dormancy in a subsequently lost in certain species. The existence of chemi- nitric oxide-dependent manner. Planta 223: 805–812. Brown NAC, Botha PA. 2004. Smoke seed germination studies and a guide to cals in smoke other than KAR1 that stimulate germination, such as glyceronitrile (Flematti et al., 2011), nitrogen oxides seed propagation of from the major families of the Cape Floristic Region, South Africa. South African Journal of Botany 70: 559–581. (Keeley and Fotheringham, 1997) and an unknown chemical Chiwocha SDS, Dixon KW, Flematti GR, et al. 2009. Karrikins: a new that stimulates Gyrostemon germination, also suggest the oper- family of plant growth regulators in smoke. Plant Science 177: 252–256. ation of convergent evolution in smoke-promoted germination. Doherty LC, Cohn MA. 2000. Seed dormancy in red rice (Oryza sativa). XI. Gyrostemon racemiger is similar to A. flavidus in that both Commercial liquid smoke elicits germination. Seed Science Research 10: 415–421. Australian endemic species germinate in response to smoke Downes KS, Lamont BB, Light ME, Van Staden J. 2010. The fire ephemeral but not KAR1, which promotes the germination of a wide Tersonia cyathiflora (Gyrostemonaceae) germinates in response to smoke range of other smoke-responsive species (Chiwocha et al., but not the butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one. Annals of 2009). However they are phylogenetically distant (different Botany 106: 381–384. orders) and differ in their response to glyceronitrile. Fay MF, Christenhusz MJM. 2010. Brassicales – an order of plants charac- terised by shared chemistry. Curtis’s Botanical Magazine 27: 165–196. Glyceronitrile, also derived from smoke-water, and consisting Flematti GR, Ghisalberti EL, Dixon KW, Trengove RD. 2004. A compound of nitrogen in addition to carbon, hydrogen and oxygen, from smoke that promotes seed germination. Science 305: 977. Downloaded from promotes A. flavidus germination, whereas G. racemiger Flematti GR, Ghisalberti EL, Dixon KW, Trengove RD. 2005. Synthesis of does not germinate in response to this chemical but to an as the seed germination stimulant 3-methyl-2H-furo[2,3-c]pyran-2-one. Tetrahedron Letters 46: 5719–5721. yet unidentified compound in smoke-water possibly consisting Flematti GR, Goddard-Borger ED, Merritt DJ, Ghisalberti EL, Dixon of only carbon, hydrogen and oxygen. KW, Trengove RD. 2007. Preparation of 2H-furo[2,3-c]pyran-2-one derivatives and evaluation of their germination-promoting activity. Journal of Agricultural and Food Chemistry 55: 2189–2194. http://aob.oxfordjournals.org/ Flematti GR, Ghisalberti EL, Dixon KW, Trengrove RD. 2009. ACKNOWLEDGEMENTS Identification of alkyl substituted 2H-furo[2,3-c]pyran-2-ones as germin- ation stimulants present in smoke. Journal of Agricultural and Food Thanks to Peter Downes for assistance with preparing the Chemistry 57: 9475–9480. smoke-water and to Professor Caroline Gross for the idea of Flematti GR, Merritt DJ, Piggott MJ, et al. 2011. Burning vegetation pro- creating smoke using a bee smoker for my fourth year duces cyanohydrins that liberate cyanide and stimulate seed germination. project at the University of New England in 1998. Seeds Nature Communications 2: 360. Fotheringham CJ, Keeley JE. 2005. NO news is no new news. Seed Science were collected with permission from the Western Australian Research 15: 367–371.

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