Seeds of Brassicaceae weeds have an inherent or ... - Oxford Journals

Annals of Botany 108: 933 –944, 2011 doi:10.1093/aob/mcr198, available online at www.aob.oxfordjournals.org

Seeds of Brassicaceae weeds have an inherent or inducible response to the germination stimulant karrikinolide Rowena L. Long 1,2,*, Jason C. Stevens 1,2, Erin M. Griffiths 2, Markus Adamek 1,2, Marta J. Gorecki 1,2, Stephen B. Powles 1 and David J. Merritt 1,2 1

School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley 6009, Western Australia, Australia and 2Kings Park and Botanic Garden, Fraser Ave, West Perth 6005, Western Australia, Australia * For correspondence. E-mail [email protected] Received: 8 April 2011 Returned for revision: 25 May 2011 Accepted: 10 June 2011 Published electronically: 5 August 2011

† Background and Aims Karrikinolide (KAR1) is a smoke-derived chemical that can trigger seeds to germinate. A potential application for KAR1 is for synchronizing the germination of weed seeds, thereby enhancing the efficiency of weed control efforts. Yet not all species germinate readily with KAR1, and it is not known whether seemingly non-responsive species can be induced to respond. Here a major agronomic weed family, the Brassicaceae, is used to test the hypothesis that a stimulatory response to KAR1 may be present in physiologically dormant seeds but may not be expressed under all circumstances. † Methods Seeds of eight Brassicaceae weed species (Brassica tournefortii, Raphanus raphanistrum, Sisymbrium orientale, S. erysimoides, Rapistrum rugosum, Lepidium africanum, Heliophila pusilla and Carrichtera annua) were tested for their response to 1 mM KAR1 when freshly collected and following simulated and natural dormancy alleviation, which included wet– dry cycling, dry after-ripening, cold and warm stratification and a 2 year seed burial trial. † Key Results Seven of the eight Brassicaceae species tested were stimulated to germinate with KAR1 when the seeds were fresh, and the remaining species became responsive to KAR1 following wet –dry cycling and dry after-ripening. Light influenced the germination response of seeds to KAR1, with the majority of species germinating better in darkness. Germination with and without KAR1 fluctuated seasonally throughout the seed burial trial. † Conclusions KAR1 responses are more complex than simply stating whether a species is responsive or nonresponsive; light and temperature conditions, dormancy state and seed lot all influence the sensitivity of seeds to KAR1, and a response to KAR1 can be induced. Three response types for generalizing KAR1 responses are proposed, namely inherent, inducible and undetected. Given that responses to KAR1 were either inherent or inducible in all 15 seed lots included in this study, the Brassicaceae may be an ideal target for future application of KAR1 in weed management. Key words: Brassicaceae, butenolide, germination, karrikinolide, karrikins, KAR1, physiological dormancy, seed dormancy, weeds.

IN T RO DU C T IO N The Brassicaceae family includes many important weed species that infest horticultural and field crops where they reduce crop yield and interfere with and contaminate crop harvest. Control of these Brassica weeds is essential in agricultural production (Cheam et al., 2008). Weediness in the Brassicaceae is facilitated by high fecundity and small seeds that have specific germination requirements and dormancy constraints – characteristics that favour the long-term persistence of at least part of the seed population (Lutman et al., 2002; Malik et al., 2010). At the time of seed dispersal, many Brassicaceae weeds exhibit physiological dormancy, which constrains their germination even when environmental conditions may be suitable for growth (Baskin and Baskin, 2001, 2006). Seeds in the soil seed bank vary in their dormancy state and emerge at different times (Chauhan et al., 2006), complicating weed management as each flush of germination must be controlled to minimize yield losses.

For physiological dormancy to be alleviated naturally, seeds may undergo wet– dry cycling (Merritt et al., 2007; Hoyle et al., 2008a), dry after-ripening (Foley, 1994) or periods of cold or warm stratification according to the species and seasonal conditions in the field (Noronha et al., 1997). Seeds may be dormant at maturity, and then modulate out of, and back into, dormancy in response to the humidity and temperature of their environment (Baskin and Baskin, 2006). Even when seeds are non-dormant, they may not germinate if the light or temperature conditions are not suitable for germination (Chauhan et al., 2006). Some Brassicaceae species require light for germination such as the crop species Brassica napus (Pekrun et al., 1997), whilst others like the agronomic weed Brassica tournefortii are negatively photoblastic and germinate preferentially in darkness (Chauhan et al., 2006). Taken together, the interaction of dormancy constraints, germination requirements and the varied light and temperature conditions provided by tillage and surface debris in agricultural environments favour the longterm persistence and nuisance of Brassicaceae weed seeds.

# The Author 2011. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected]

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Long et al. — Seeds of Brassicaceae weeds germinate with karrikinolide

To improve the efficiency of weed control efforts for Brassicaceae weeds, the inherent mechanisms that favour seed persistence can be exploited, such as by prematurely alleviating seed dormancy and triggering seeds to germinate synchronously. A long-term research goal has been to identify chemicals that could stimulate weed seeds to germinate on demand (Adkins and Peters, 2001; Daws et al., 2007; Stevens et al., 2007). One natural compound that shows promise as a germination stimulant for weed seeds is karrikinolide (KAR1; a butenolide), which is a biologically active component of smoke that was discovered by Flematti et al. (2004) (Daws et al., 2007; Stevens et al., 2007). The potential advantages of using KAR1 for managing weed seeds are 2-fold – an ability to bypass physiological dormancy (Stevens et al., 2007) and an ability to replace the light requirement for germination (Nelson et al., 2010). In an ideal situation, such attributes would benefit agriculture by promoting synchronous germination of weed seeds, thus depleting the soil seed bank and enabling fewer and more strategic weed control efforts. Investigations into the response of weed seeds to KAR1 shows that germination outcomes are not consistent, and it will be necessary to consider species and environmental influences to elicit the optimal germination response. Only some species can be triggered to germinate with KAR1, and the response may vary between populations of the same species (Stevens et al., 2007). Promisingly, all five Brassicaceae weeds reported on thus far were found to be responsive to KAR1, namely B. tournefortii, Raphanus raphanistrum and Sisymbrium orientale (Stevens et al., 2007), and Capsella bursa-pastoris and Sinapis alba (Daws et al., 2007). In the case of C. bursa-pastoris and S. alba, the degree of stimulation by KAR1 was temperature dependent (Daws et al., 2007), although it was not clear what, if any, temperatures were limiting to germination. Choosing the best time to apply KAR1 may also be influenced by seasonality; physiologically dormant seeds can cycle out of and into dormancy throughout the year and change in their germination response to smoke water (Baker et al., 2005), and the water content and any prior hydration can also influence whether seeds germinate in response to KAR1 (Long et al., 2010). A clearer understanding of how light, temperature and hydration combine to influence the receptivity of seeds to KAR1 is required. To assess the efficacy of KAR1 as a germination promoter for Brassicaceae weeds, three factors known to affect germination and dormancy were manipulated in this study, namely temperature, hydration and light. We reasoned that a stimulatory response to KAR1 may be present in physiologically dormant Brassicaceae species but may not be expressed under all circumstances. To test this hypothesis we first tested whether KAR1 could trigger the germination of a range of freshly collected seed lots of Brassicaceae weeds under different temperature and light/dark regimes. Further laboratory work aimed to simulate the environmental processes that could promote dormancy loss in physiologically dormant Brassicaceae, particularly in species that were unresponsive to KAR1 when freshly collected. Finally, a fieldbased seed burial trial was conducted to monitor changes in sensitivity of seeds to KAR1 during natural dormancy alleviation at different sites, and to validate the trends observed in laboratory-based experiments.

M AT E R I A L S A N D M E T H O D S Seed collections and storage

Seeds of eight Brassicaceae weed species were collected from across the Western Australian agricultural zone in 2008 and 2009 (Table 1), and the identities of species were verified by the Western Australian Herbarium. Multiple collections were made for some species to gauge the potential variability in KAR1 responses among seed lots of the same species, but correlations between site and KAR1 responses were not specifically tested. Following collection, seeds were dried at 15 % relative humidity (RH) and 15 8C for up to 1 month then stored in air-tight foil bags at 220 8C to minimize the effects of storage on dormancy and germination characteristics prior to testing.

Responses of freshly collected seeds to KAR1

After drying at 15 % RH and 15 8C for up to 1 month, and prior to storage at 220 8C, seeds of each seed lot were tested for their germination response to a range of temperatures, in 12-hourly alternating light or constant darkness, with and without 1 mM KAR1, to assess their initial dormancy state. TA B L E 1. Seeds of eight Brassicaceae weed species were collected at sites across Western Australia in 2008 and 2009 Species Brassica tournefortii

Seed lot A B C

Raphanus raphanistrum

A B C

Sisymbrium orientale

A B C

Sisymbrium erysimoides

A C

Heliophila pusilla

C

Lepidium africanum Rapistrum rugosum Carrichtera annua

C C C

Collection locality

GPS location Collection date

Eurady Station S27830′ 26′′ E114841′ 46′′ Cowella Rd S31812′ 43′′ E115835′ 34′′ Cowella Rd S31812′ 43′′ E115835′ 34′′ Avondale S32807′ 24′′ E116853′ 07′′ Beverley S32814′ 42′′ E117809′ 24′′ Merredin S31828′ 58′′ E118813′ 23′′ Avondale S32807′ 24′′ E116853′ 07′′ Moora S30828′ 58′′ E116802′ 03′′ Merredin S31830′ 01′′ E118812′ 55′′ Mullewa S28833′ 57′′ E115827′ 41′′ Lucky Well S29818′ 59′′ E116841′ 08′′ Perth S31858′ 03′′ E115850′ 14′′ Dalwallinu S30816′ 49′′ E116839′ 28′′ Dongara S29811′ 45′′ E114855′ 12′′ Watsonia S31817′ 57′′ E118841′ 35′′

5 October 2008 24 September 2008 19 October 2009 26 November 2008 15 November 2008 2 December 2009 26 November 2008 6 November 2008 2 December 2009 25 September 2008 26 November 2009 27 November 2009 21 October 2009 9 November 2009 10 March 2010

Seed lot ‘A’ refers to a collection that was large enough to provide seeds for ten retrieval times in the seed burial experiment, while seed lot ‘B’ refers to a collection that provided sufficient seeds for sampling at only five retrieval times. Seed lots marked ‘C’ were used in experiments other than the seed burial trial.


Field-based seed burial experiment

Seed burial trials were established for 2 years from December 2008 at three sites in Western Australia (Fig. 1): Perth (coast, S31857′ E115848′ , brown sand), Northam (100 km east of Perth, S31 845′ E116841′ , brown loamy sand) and Merredin (270 km east of Perth, S31830′ E118812′ , yellow-red sandy clay). Four species were included

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Seeds of a single seed lot of each of six species (Rapistrum rugosum, Raphanus raphanistrum, S. orientale, S. erysimoides, B. tournefortii and Heliophila pusilla) were treated with up to 3 months of (a) dry after-ripening; (b) wet– dry cycling; (c) a combination of both dry after-ripening and wet– dry cycling; (d ) cold stratification; or (e) warm stratification, to simulate environmental conditions that could promote dormancy loss in the field. For the dry after-ripening and wet– dry cycling treatments, replicates of 50 seeds, held within nylon bags, were dry-after-ripened at 50 % RH and 35 8C in electrical boxes ( polycarbonate electrical enclosure boxes; 270 × 190 × 100 mm; NHP Fibox, Perth, Australia) containing unsaturated LiCl solutions (320 g L21). At the start of the experiment and every 2 weeks thereafter, samples for the wet – dry cycling treatment were removed from the dry after-ripening environment and subjected to a wet – dry cycle. Each wet– dry cycle involved wetting seeds for 18 h at room temperature (21 8C) on filter paper moistened with deionized water, then drying the seeds for 6 h at 50 % RH and 20 8C before returning them to the dry after-ripening environment. Additionally, to test the combined effect of dry after-ripening and wet – dry cycling, some samples were subjected to 4 or 8 weeks of dry after-ripening before being transferred to the wet –dry cycling regime. For cold and warm stratification experiments, the bags of seeds were placed in individual Petri dishes containing water agar (1 %, w/v), wrapped in foil to omit light and incubated at either 5 8C (cold stratification) or 20 8C (warm stratification). For all treatments, three replicate samples were retrieved after 4, 8 and 12 weeks and tested at 20/10 8C (12-hourly alternating temperature) for their germination response with and without 1 mM KAR1 and light.

Rainfall

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Mimicking dormancy alleviation processes

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Constant temperatures of 10, 15, 20, 25, 30 and 35 8C, and 12-hourly alternating temperatures of 20/10 8C and 35/20 8C were tested, each in combination with 12-hourly alternating light (30 mmol m22 s21, fluorescent white light) or constant darkness. Three replicates of each seed lot were incubated in Petri dishes containing 1 % (w/v) water agar either with or without 1 mM KAR1, for each combination of light and temperature. For constant dark conditions, seeds were put in Petri dishes in a dark room then wrapped in two layers of foil to omit light. Raphanus raphanistrum seeds in individual silique segments were X-rayed to obtain filled, viable-looking seeds for germination testing. Germination was scored once after 21 d, and seeds for which the radicle had visibly protruded (.1 mm) were scored as ‘germinated’. Any seeds that did not germinate were inspected for obvious necrosis and squeezed with forceps to assess viability; seeds with firm white embryos were scored as viable.

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F I G . 1. Climate data for Perth, Northam and Merredin, where seed bags were buried for the period of December 2008 to December 2010: mean monthly temperature range and mean monthly rainfall, as indicated. ‘Total rainfall’ ¼ cumulative rainfall for the entire trial period; ‘No. of rain days’ ¼ total number of days on which ≥1 mm rainfall was recorded during the trial period; ‘No. of rain events’ ¼ number of rain events during the study period, where one ‘rain event’ was defined as a day or consecutive days of ≥1 mm rainfall, bounded by ≥1 day with zero rainfall. All data were taken from the Australian Government Bureau of Meteorology (2010).

in the Northam trial, namely S. orientale, S. erysimoides, R. raphanistrum and B. tournefortii, whilst only two species were included at the Perth and Merredin sites (R. raphanistrum and B. tournefortii). Each field site was divided into four blocks, and in each block bags of seeds of two seed lots per species were buried at 1 cm depth; in the case of S. erysimoides, only one seed lot, ‘A’, was included in the field trial (Table 1). For seed lot ‘A’ of each species ten bags were buried, whilst five bags were buried for the second seed lot, ‘B’. Seed bags were made of fibreglass fly-wire mesh and contained approx. 500 seeds in silique segments for R. raphanistrum, 1000 seeds of B. tournefortii, 2700

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seeds of S. erysimoides or 1400 seeds of S. orientale per bag; seeds of S. orientale and S. erysimoides were sealed in organza bags within the larger fibreglass bags due to the smaller seed dimensions. For seed lot ‘A’ of each species, one bag of seeds was retrieved from each block at each site in February, March, April, May, June, September and December 2009, and March, June and December 2010; bags of seed lot ‘B’ were retrieved in March, June, September and December 2009, and December 2010. Following retrieval, sub-samples of seeds were germinated in Petri dishes on water agar (1 %, w/v) with and without 1 mM KAR1, in both alternating light and constant dark conditions. Brassica tournefortii and R. raphanistrum were incubated at 15 8C constant temperature, and Sisymbrium spp. were incubated at constant 25 8C, in accordance with which temperature seemed optimal during initial germination tests. In the first 6 months, losses of seeds due to germination in the field were negligible, and 25 seeds from each bag were tested for each treatment; after 6 months, in-field germination led to a decline in the numbers of seeds retrieved, and so the total cohort of seeds from each bag was divided equally among treatments, and was .25 seeds per replicate in all cases. Germination, defined as .1 mm radicle protrusion, was scored 21 d after sowing.

Statistical analyses

Germination results were analysed using generalized linear modelling of binomial data with a logit link function, and stepwise addition of factors to simplify the model (GenStat 10th Edition; McCullagh and Nelder, 1989). Significance of factors was tested using a x2 test, and P-values are reported. Where relevant, treatment levels were compared using a twosample binomial test, and P-values reported.

RES ULT S Responses of freshly collected seeds to KAR1

Freshly collected seeds of 15 seed lots, constituting eight Brassicaceae weed species, were tested for their germination response to KAR1 under a range of temperature and light conditions. Seven of the eight Brassicaceae species tested were stimulated to germinate with 1 mM KAR1 at one or more of the tested temperature and light combinations (P , 0.001, Fig. 2). For some seed lots, the difference in germination response with and without KAR1 was as much as 95 %, for example B. tournefortii ‘C’ and L. africanum incubated in darkness at 15 8C (Fig. 2C, N). Heliophila pusilla was extremely dormant and did not germinate to ≥5 % under any of the conditions tested (Fig. 2L), and the following general findings apply to species and seed lots other than H. pusilla. Light strongly influenced the germination response for all species independently of KAR1 (P ≤ 0.001). Brassica tournefortii and R. raphanistrum germinated to the highest percentages, with and without KAR1, in constant darkness, whilst Sisymbrium spp., L. africanum and C. annua germinated to higher percentages when incubated in alternating light (P , 0.001).

Some species such as B. tournefortii were stimulated to germinate with KAR1 across a range of temperatures, whilst other species such as C. annua and L. africanum were stimulated by KAR1 to germinate beyond control levels only at select temperatures that may otherwise have been sub-optimal for germination. Indeed, KAR1 was more effective at some temperatures than others for B. tournefortii, R. raphanistrum (A), S. orientale (A), S. erysimoides, C. annua and L. africanum (KAR1 × temperature interaction, P , 0.001). The optimal temperature for germination with and without KAR1 was typically ≤20 8C for all species and seed lots, except for Sisymbrium spp., which germinated to higher percentages at 20– 30 8C in the light. With the exception of S. orientale, the trends in how seeds germinated in response to KAR1, temperature and light were consistent across all seed lots of a species irrespective of their collection source (Table 1), although the magnitude of the responses varied among seed lots (Fig. 2). For example, seeds of all three B. tournefortii seed lots germinated to a higher percentage in darkness, with KAR1 and at the lower temperatures (10 – 20 8C), but their particular germination profiles were also unique in that more seeds of seed lot ‘A’ germinated in darkness and alternating light than the other two seed lots. In the case of S. orientale, seed lots ‘B’ and ‘C’ were not stimulated to germinate with KAR1 relative to control levels (P ¼ 0.278 and P ¼ 0.926), but the positive stimulation of seed lot ‘A’ to germinate with KAR1 at 35/20 8C in the light (P , 0.001) indicated that this species could respond to KAR1 when fresh, even if particular seed lots did not. Mimicking the environment can induce a KAR1 response

Seeds of six species were subjected to combinations of dry after-ripening and wet – dry cycling for up to 3 months to assess whether these processes could alleviate dormancy and improve the germination response of seeds to KAR1. Species that were highly sensitive to KAR1 prior to dry after-ripening and wet– dry cycling retained a pattern of germinating best with KAR1 (Fig. 3). Overall, all six species tested germinated better with KAR1 than without it, although for S. orientale KAR1 was only beneficial in darkness (in light, P ¼ 0.365). Very few of the wet– dry cycling and dry after-ripening treatments improved the germination of seeds above control levels, and the only species for which a consistent improvement in germination was observed, with and without KAR1, during the 3 month trial was H. pusilla. Heliophila pusilla, which was initially unable to germinate under any combination of temperature, light and KAR1 treatments (Fig. 2L), became highly responsive to KAR1, particularly in darkness, after just 1 month of dry after-ripening or wet –dry cycling (P , 0.001, Fig. 3E). Wet –dry cycling and dry after-ripening progressively alleviated dormancy in this species, as seen by the gradual increase in germination without KAR1; after 3 months, 40– 60 % of seeds germinated, irrespective of KAR1 and light conditions, when the treatment included ≥1 month of wet –dry cycling. In contrast to the positive effects of wet – dry cycling and dry after-ripening on H. pusilla, the other species tested either did not respond to these treatments or in some cases the treatments inhibited germination. Rapistrum rugosum, which seemed

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F I G . 2. Germination of eight Brassicaceae weed species in response to 0 or 1 mM KAR1 (as indicated), with and without 12-h alternating light, at six constant temperatures (10–35 8C) and two alternating temperatures (20/10 8C and 35/20 8C). (A– C) Brassica tournefortii, (D– F) Raphanus raphanistrum, (G– I) Sisymbrium orientale, (J–K) S. erysimoides, (L) Heliophila pusilla, (M) Rapistrum rugosum, (N) Lepidium africanum and (O) Carrichtera annua. Seeds were tested within 1 month of harvest. n ¼ 150 seeds per treatment; vertical bars represent the 95 % confidence interval for binomial estimates.

dormant during initial testing (Fig. 2M), did not respond to wet – dry cycling or dry after-ripening, and germinated to a maximum of 20 % under all of the treatments tested. Wet-dry cycling noticeably impeded germination when

applied to B. tournefortii for ≥2 months, and to Raphanus raphanistrum for ≥1 month (P , 0.001). In a further experiment to test whether Brassicaceae seeds would change in dormancy state and sensitivity to KAR1

Long et al. — Seeds of Brassicaceae weeds germinate with karrikinolide 100

J S. erysimoides ‘A’

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Fig. 2 Continued

during mimicked environmental changes, seeds of the same six species (and seed lots) were stratified at 5 8C (cold stratification) or 20 8C (warm stratification) for up to 3 months (Fig. 4). Four species germinated to higher levels with KAR1 following warm stratification, namely B. tournefortii, R. raphanistrum, S. erysimoides and H. pusilla (P , 0.001). For these species, the highest germination response in both alternating light and darkness was typically recorded following 4 or 8 weeks of warm stratification; germination levels either stayed the same or declined following 12 weeks of stratification. Compared with warm stratification, cold stratification was generally less effective at alleviating dormancy and changing the response of seeds to KAR1. Germination following warm stratification was higher than that following cold stratification for all species (P , 0.001) except S. orientale (P ¼ 0.288); however, cold stratification did improve germination in two particular circumstances; more B. tournefortii seeds germinated with KAR1 (relative to time zero in darkness: P ¼ 0.027 at 2 months, P ¼ 0.002 at 3 months) and more seeds of R. raphanistrum germinated following up to 8 weeks of cold stratification when tested in alternating light (P , 0.016, irrespective of KAR1), and when tested without KAR1 in darkness (P , 0.014). In the

case of R. rugosum and S. erysimoides, fewer seeds germinated with KAR1 following cold stratification (P ,0.001). Dormancy state and KAR1 response vary seasonally and between sites

A 2 year seed burial trial aimed to assess whether germination of Brassicaceae weed seeds in response to KAR1 varies seasonally, and whether it depends on the particular seed lot and burial site. Overall, all four species were stimulated to germinate with KAR1 above control levels; however, the degree of stimulation was strongly seasonal, and more apparent when seeds were incubated in darkness than in alternating light [Figs 5 and 6; P , 0.001 for all factors and seed lots, except S. orientale ‘B’ for which light was not significant (P ¼ 0.158)]. Brassica tournefortii and R. raphanistrum were highly responsive to KAR1 when freshly collected and throughout the first few months of burial, whereas Sisymbrium spp. were initially very dormant, but became responsive to KAR1 within 2 months of burial. By the first winter season (June 2009), the relative benefit of KAR1 declined for the majority of seed lots, but was often restored


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F I G . 3. Germination response of six Brassicaceae weed species to combinations of 1– 3 months of dry after-ripening (DAR) and wet –dry cycling (WD), germinated with or without 1 mM KAR1 (as indicated), with and without 12-h alternating light at 20/10 8C. Germination was scored 21 d after sowing. (A) Brassica tournefortii, (B) Raphanus raphanistrum, (C) Sisymbrium orientale, (D) S. erysimoides, (E) Heliophila pusilla and (F) Rapistrum rugosum. * indicates significantly higher germination with KAR1 than without (P , 0.001); (*) indicates significantly higher germination without KAR1 than with (P , 0.001); numeric values above bars are P-values where P . 0.001 but , 0.05; n ¼ 150 seeds per treatment; vertical bars represent the 95 % confidence interval for binomial estimates.

as seeds cycled back into dormancy later in the year. In the first year of the trial, germinability varied seasonally and between species, peaking in autumn for S. erysimoides (April 2009), winter (June 2009) for B. tournefortii and S. orientale, and in spring (September 2009) for R. raphanistrum. In the second year, germinability for the majority of species and seed lots peaked in the winter (June 2010). Light during incubation reduced the stimulatory effect of KAR1 for all seed lots (light × KAR1 interaction, P ,0.001), except S. orientale ‘B’ (P ¼ 0.047). For B. tournefortii and R. raphanistrum, KAR1 stimulated as much as an extra 70 % of seeds to germinate in darkness in

the first and final summers of the trial (Fig. 6; December 2008 and 2010), whilst in alternating light the benefit of KAR1 reached a maximum of 40 % in the second year of the trial (Fig. 5). Seeds of Sisymbrium spp., which germinated more readily in light than in darkness overall, were also stimulated to germinate with KAR1 to the greatest degree in darkness. Interestingly, S. erysimoides became highly sensitive to KAR1 in the light after 2 months of burial, and retained this sensitivity throughout the trial, even though germination without KAR1 fluctuated seasonally. For S. orientale, KAR1 did not trigger more seeds to germinate in any season when the seeds were incubated in alternating light.

Long et al. — Seeds of Brassicaceae weeds germinate with karrikinolide Germination, darkness (%) Germination, 12-h light (%)

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3 months

Fig. 3 Continued

Of the four species included in the trial, B. tournefortii and R. raphanistrum were buried at each of three sites, enabling us to explore how subtle differences in location and climate can alter the response of seeds to KAR1 (see also Fig. 1). Both R. raphanistrum seed lots germinated best and similarly following burial at the drier sites of Northam and Merredin (compared with Perth, P , 0.001; compared with each other, seed lot ‘A’ P ¼ 0.072, seed lot ‘B’ P ¼ 0.135). The germination response of B. tournefortii seeds with and without KAR1 was also moderated by site differences, and, as with R. raphanistrum, germination was lowest following burial at the wettest site, Perth. Testing two seed lots for B. tournefortii, R. raphanistrum and S. orientale enabled us to assess how different seed populations vary in their response to the same environment. Although actual germination percentages varied between the paired seed lots (P , 0.001), key trends such as how seeds responded to KAR1 relative to control levels, with and without light, were consistent for each species.

DISCUSSION Seeds of all eight Brassicaceae species included in this study, encompassing 15 seed lots, were stimulated to germinate by KAR1 under at least some of the conditions tested, supporting our hypothesis that a stimulatory response to KAR1 may be present in physiologically dormant Brassicaceae species but may not be expressed under all circumstances. Previous reports of the germination responses of phylogenetically diverse taxa to KAR1 have concluded that species were either stimulated to germinate with KAR1 or were not responsive to it (e.g. Merritt et al., 2006; Daws et al., 2007; Stevens et al., 2007). In contrast, our results demonstrate that germination responses to KAR1 are more complex than stating whether a species is responsive or non-responsive. Indeed, in our study and those of others, light/dark conditions (Merritt et al.,

2006), dormancy state, seed lot (Stevens et al., 2007), temperature (Daws et al., 2007) and hydration state (Long et al., 2010) all influenced the sensitivity of seeds to KAR1, and we conclude that responses to KAR1 can be temporally and environmentally transient. Here we propose three response types for generalizing the germination responses of seeds to KAR1, then discuss how they apply to the results of our study. (1) Inherent: freshly collected seeds are sensitive to KAR1 when tested within 1 month of harvesting mature seeds, as defined by a statistically significant increase in germination with 1 mM KAR1 compared with a water control. This response is evident in seeds exposed to alternating light or darkness at one or more temperatures, with test conditions ideally including an alternating temperature regime that is relevant to the season in which seeds would naturally germinate. (2) Inducible: seeds become sensitive to KAR1 following dormancy release, as defined by a statistically significant increase in germination with 1 mM KAR1 compared with a water control. This response is evident in seeds exposed to alternating light or darkness at one or more temperatures, after the seeds have experienced dormancybreaking conditions through exposure to the natural environment (e.g. soil burial) or to simulated natural conditions in the laboratory (e.g. stratification, dry afterripening or wet – dry cycling). (3) Undetected: seed germination on 1 mM KAR1 media does not differ significantly from control levels under any of the conditions tested within a study.

The ‘inherent’ response type

Dormant seeds of many Brassicaceae weed species are responsive to KAR1 when freshly collected, as was found for


Germination (%)

Germination in 12-h alternating light 100 80 60

Germination in constant darkness

A B. tournefortii ‘C’ 5 °C strat, + KAR1 5 °C strat, – KAR1 20 °C strat, + KAR1 20 °C strat, – KAR1

40 20

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S. alba, were also found to be stimulated to germinate with 0.67 mM KAR1 by Daws et al. (2007). In our study we observed that KAR1 widened the range of temperature and light conditions in which fresh, dormant seeds of all inherently responsive seed lots would germinate. Moreover, the inherent response type persisted across different seed lots of a species, even when the actual germination percentages differed considerably between the seed collections.

Germination (%)

0 100

B Raphanus raphanistrum ‘C’

The ‘inducible’ response type

80

Dormant seeds that do not appear to be responsive to KAR1 when freshly collected can become responsive to KAR1 as dormancy is alleviated, as highlighted by the results for H. pusilla in our study. Even though seeds of H. pusilla were very dormant (,5 % germination, Fig. 2L) when freshly collected and could not be triggered to germinate with KAR1, periods of laboratory-based wet– dry cycling, after-ripening and warm stratification all induced seeds to become sensitive to KAR1 (Figs 3 and 4). Similarly, the Sisymbrium seed lots that were used in the seed burial trial were initially too dormant to germinate at 15 8C (,5 % germination, Fig. 2G, H), but were induced to germinate in response to KAR1 after being buried in the field for just 2 months. The notion that a response can be induced, or ‘switched on’, implies that it also can be switched off. Indeed, the germination response of the inherently responsive species B. tournefortii and R. raphanistrum to KAR1 was impeded by processes involving wet–dry cycling; germination with and without KAR1 was inhibited following simulated wet–dry cycling (Fig. 3), and under natural seed burial conditions at the wettest field site, Perth (Figs 1, 5 and 6). These results are supported by findings of Baker et al. (2005), in which the germination response of two Australian native fire ephemerals to smoke water fluctuated seasonally, and in the study of Long et al. (2010) in which seeds of B. tournefortii became less sensitive to KAR1 following hydration. Given this capacity to induce and negate germination responses to KAR1, it may be incorrect to assume that a species or seed lot is not able to respond to KAR1 just because it fails to germinate better with KAR1 when the seeds are fresh or tested under a narrow range of conditions. Natural and laboratory-controlled changes in dormancy state can induce seeds to respond to KAR1.

60 40 20

Germination (%)

0 100

C S. orientale ‘C’

80 60 40 20

Germination (%)

0 100

D S. erysimoides ‘C’

80 60 40 20

Germination (%)

0 100

E

H. pusilla

80 60 40 20

Germination (%)

0 100

F

Rapistrum rugosum

80 60 40 20

The ‘undetected’ response type

0 0

4

8

12

Weeks of stratification

0

4

8

12

Weeks of stratification

F I G . 4. Germination response of six Brassicaceae species to warm (20 8C) and cold (5 8C) stratification on 1 % (w/v) water agar media for up to 12 weeks. Stratified seeds were subsequently incubated at 20/10 8C for germination, with and without 1 mM KAR1 (as indicated). (A) Brassica tournefortii, (B) Raphanus raphanistrum, (C) Sisymbrium orientale, (D) S. erysimoides, (E) Heliophila pusilla and (F) Rapistrum rugosum. n ¼ 150 seeds per treatment; vertical bars represent the 95 % confidence interval for binomial estimates.

seven of the eight species included in this study, namely B. tournefortii, R. raphanistrum, S. orientale, S. erysimoides, L. africanum, R. rugosum and C. annua (Fig. 2). Two additional Brassicaceae species, C. bursa-pastoris and

An extension of the notion that seeds can be induced to germinate by KAR1 is that it is possible that the genetic mechanisms for responding to KAR1 may be present in a broader range of species than previously thought, but that the response may not be ‘switched on’ under all environmental conditions. Indeed, the biological activity of KAR1 extends beyond triggering germination to include photomorphogensis and seedling development (Chiwocha et al., 2009; Nelson et al., 2009, 2010). A recent molecular study of karrikin responses demonstrated that karrikin-induced changes in the transcriptome are dependent on the gene MAX2; MAX2 is required for karrikin responses and is present in diverse land plant species, including Pisum sativum which has no apparent physiological response to KAR1 (Nelson et al., 2011). Thus, rather


Germination, Northam (%)

Germination, Merredin (%) Germination, Northam (%)

Germination, Perth (%)

942 100 80

A B. tournefortii ‘A’


C R. raphanistrum ‘A’

F S. orientale ‘B’

G S. erysimoides ‘A’

D R. raphanistrum ‘B’

+ KAR1 12-h light – KAR1 12-h light

60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0

100

E S. orientale ‘A’

Dec- Jun- Dec- Jun- Dec08 09 09 10 10

80 60 40 20 0

Dec- Jun- Dec- Jun- Dec- Dec- Jun- Dec- Jun- DecDec- Jun- Dec- Jun- Dec08 09 09 10 10 08 09 09 10 10 08 09 09 10 10 F I G . 5. Germination in 12-h alternating light of seed lots recovered from seed burial trials at Perth, Northam and Merredin over a 2 year period. Seeds were germinated with or without 1 mM KAR1 (as indicated). Seed lots ‘A’ and ‘B’ of Brassica tournefortii (A, B) and Raphanus raphanistrum (C, D) were incubated at 15 8C constant temperature, whilst seed lots ‘A’ and ‘B’ of Sisymbrium orientale (E, F) and seed lot ‘A’ of S. erysimoides (G) were tested at 25 8C. n ≥100 seeds per treatment; vertical bars represent the 95 % confidence interval for binomial estimates.

than state that a species or seed lot is not responsive to KAR1 if it is not triggered to germinate above control levels, we propose that the karrikin response is as yet undetected. Even in this study we did not test every possible combination of seed dormancy state and germination conditions, and the transient nature of some responses indicates that sensitivity to KAR1 may be restricted to very particular conditions for some species. It is also still not known whether KAR1 only stimulates seeds that are non-dormant or marginally dormant to germinate, or if it can actively alleviate dormancy; crossover-type studies need to be conducted in which seeds are incubated with and without KAR1 media during the wetting and germination phases of stratification and wet – dry cycling processes. In this way, we may yet detect a role for KAR1 in overcoming dormancy in seemingly intractable species such as Lolium rigidum (Stevens et al., 2007).

Implications of this study

The finding that all 15 seed lots of Brassicaceae weeds were either inherently sensitive to KAR1 or could be induced to respond to it has clear implications for practice. First, we propose that the Brassicaceae family may be an ideal target for the future development of an agrochemical germination stimulant that incorporates KAR1. KAR1 can widen the range of temperature and light conditions in which seeds germinate, which could be instrumental in depleting the soil seed bank of seeds that would otherwise remain dormant or quiescent in the soil for ≥2 years, as was found in this study and others (Lutman et al., 2002; Telewski and Zeevaart, 2002). Given that seed lots from different collection sites differed in the magnitude of their response to KAR1, follow-up studies are required to investigate whether environmental

Germination, Northam (%)

Germination, Merredin (%) Germination, Northam (%)

Germination, Perth (%)

Long et al. — Seeds of Brassicaceae weeds germinate with karrikinolide A B. tournefortii ‘A’


C R. raphanistrum ‘A’

F S. orientale ‘B’

G S. erysimoides ‘A’

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D R. raphanistrum ‘B’

100 80 60 40 20 0

+ KAR1 darkness – KAR1 darkness

100 80 60 40 20 0 100 80 60 40 20 0 100

E S. orientale ‘A’

Dec- Jun- Dec- Jun- Dec08 09 09 10 10

80 60 40 20 0

Dec- Jun- Dec- Jun- Dec- Dec- Jun- Dec- Jun- DecDec- Jun- Dec- Jun- Dec08 09 09 10 10 08 09 09 10 10 08 09 09 10 10 F I G . 6. Germination in constant darkness of seed lots recovered from seed burial trials at Perth, Northam and Merredin over a 2 year period. Seeds were germinated with or without 1 mM KAR1 (as indicated). Seed lots ‘A’ and ‘B’ of Brassica tournefortii (A, B) and Raphanus raphanistrum (C, D) were incubated at 15 8C constant temperature, whereas seed lots ‘A’ and ‘B’ of Sisymbrium orientale (E, F) and seed lot ‘A’ of S. erysimoides (G) were tested at 25 8C. n ≥ 100 seeds per treatment; vertical bars represent the 95 % confidence interval for binomial estimates.

conditions that can influence seed dormancy whilst seeds mature on parent plants, such as temperature (Hoyle et al., 2008b), water supply and nutrient supply (Luzuriaga et al., 2006), also influence the response of seeds to KAR1. Our increasing awareness of the importance of light, temperature and dormancy state in mediating how much extra germination is afforded in the presence of KAR1 may allow us to time the application of KAR1 to maximize the germination of Brassicaceae weeds, and therefore the economic benefit of using it. Our findings also contribute to a growing base of theoretical knowledge about how seeds are triggered to germinate by KAR1. In some cases, KAR1 offers a more sensitive indicator of changes in dormancy state than germinating seeds without a stimulant, particularly when the majority of seeds in a population are deeply dormant and the only seeds to germinate

are those that are on KAR1 media. In other cases, KAR1 can mask subtle or seasonal changes in dormancy state; when the population of seeds is less dormant, response to KAR1 may be ‘maxed out’ at 100 % and only the germination response without KAR1 may vary. To what degree dormancy state and KAR1 response are inextricably linked is yet to be determined, but the non-parallel germination responses (i.e. with and without KAR1) to dormancy-alleviating processes in this study indicate that KAR1 may act at least partially independently of dormancy state. One final theoretical implication of this study is that the inherent and inducible responses of numerous wild Brassicaceae species bode well for future investigations of the genetic mechanisms underpinning KAR1 responses (e.g. Nelson et al., 2009). The model species for molecular studies, Arabidopsis thaliana, is in the same family as these

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weeds, and this will hopefully facilitate the extension of mechanistic studies to wild species. AC KN OW LED GEMEN T S We thank Simone Tausche, Stephen Long, Pippa Michael, Tom North and Dean Carter, Anthony Robinson, Michael Blair, Matthew Harrod, Alan Harrod, Michalie Foley, Mark Waters, Winslow Briggs and the Kings Park Writing Group for their assistance. Gavin Flematti and Adrian Scaffidi (UWA Department of Chemistry) synthesized the KAR1 chemical used in this study. This research was supported by an Australian Research Council Linkage Grant (LP 0776951). L I T E R AT U R E CI T E D Adkins SW, Peters NCB. 2001. Smoke derived from burnt vegetation stimulates germination of arable weeds. Seed Science Research 11: 213– 222. Australian Government Bureau of Meteorology. 2010. Climate data online. http://www.bom.gov.au/climate/data. Accessed 4 Dec, 2010. Baker KS, Steadman KJ, Plummer JA, Merritt DJ, Dixon KW. 2005. The changing window of conditions that promotes germination of two fire ephemerals, Actinotus leucocephalus (Apiaceae) and Tersonia cyathiflora (Gyrostemonaceae). Annals of Botany 96: 1225–1236. Baskin CC, Baskin JM. 2001. Seeds: ecology, biogeogrphy, and evolution of dormancy and germination. San Diego: Academic Press. Baskin CC, Baskin JM. 2006. The natural history of soil seed banks of arable land. Weed Science 54: 549– 557. Chauhan BS, Gill G, Preston C. 2006. African mustard (Brassica tournefortii) germination in southern Australia. Weed Science 54: 891–897. Cheam AH, Storrie AM, Koetz EA, Holding DJ, Bowcher AJ, Barker JA. 2008. Managing wild radish and other brassicaceous weeds in Australian cropping systems. Adelaide, Australia: CRC for Australian Weed Management. Chiwocha SDS, Dixon KW, Flematti GR, et al. 2009. Karrikins: a new family of plant growth regulators in smoke. Plant Science 177: 252–256. Daws MI, Davies J, Pritchard HW, Brown NAC, Van Staden J. 2007. Butenolide from plant-derived smoke enhances germination and seedling growth of arable weed species. Plant Growth Regulation 51: 73– 82. Flematti GR, Ghisalberti EL, Dixon KW, Trengove RD. 2004. A compound from smoke that promotes seed germination. Science 305: 977. Foley ME. 1994. Temperature and water status of seed affect afterripening in wild oat (Avena fatua). Weed Science 42: 200 –204. Hoyle GL, Daws MI, Steadman KJ, Adkins SW. 2008a. Mimicking a semiarid tropical environment achieves dormancy alleviation for seeds of Australian native Goodeniaceae and Asteraceae. Annals of Botany 101: 701–708.

Hoyle GL, Daws MI, Steadman KJ, Adkins SW. 2008b. Pre- and postharvest influences on seed dormancy status of an Australian Goodeniaceae species, Goodenia fascicularis. Annals of Botany 102: 93–101. Long RL, Williams K, Griffiths EM, et al. 2010. Prior hydration of Brassica tournefortii seeds reduces the stimulatory effect of karrikinolide on germination and increases seed sensitivity to abscisic acid. Annals of Botany 105: 1063– 1070. Lutman PJW, Cussans JW, Wright KJ, Wilson BJ, Wright GM, Lawson HM. 2002. The persistence of seeds of 16 weed species over six years in two arable fields. Weed Research 42: 231–241. Luzuriaga AL, Escudero A, Perez-Garcia F. 2006. Environmental maternal effects on seed morphology and germination in Sinapis arvensis (Cruciferae). Weed Research 46: 163– 174. Malik MS, Norsworthy JK, Riley MB, Bridges W. 2010. Temperature and light requirements for wild radish (Raphanus raphanistrum) germination over a 12-month period following maturation. Weed Science 58: 136– 140. McCullagh P, Nelder JA. 1989. Generalized linear models. Boca Raton, FL: CRC Press. Merritt DJ, Kristiansen M, Flematti GR, et al. 2006. Effects of a butenolide present in smoke on light-mediated germination of Australian Asteraceae. Seed Science Research 16: 29–35. Merritt DJ, Turner SR, Clarke S, Dixon KW. 2007. Seed dormancy and germination stimulation syndromes for Australian temperate species. Australian Journal of Botany 55: 336–344. Nelson DC, Riseborough J-A, Flematti GR, et al. 2009. Karrikins discovered in smoke trigger Arabidopsis seed germination by a mechanism requiring gibberellic acid synthesis and light. Plant Physiology 149: 863–873. Nelson DC, Flematti GR, Riseborough J-A, Ghisalberti EL, Dixon KW, Smith SM. 2010. Karrikins enhance light responses during germination and seedling development in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 107: 7095–7100. Nelson DC, Flematti GR, Riseborough J-A, Ghisalberti EL, Dixon KW, Smith SM. 2011. The F-box protein MAX2 has dual roles in karrikin and strigolactone signalling in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 108: 8897–8902. Noronha A, Andersson L, Milberg P. 1997. Rate of change in dormancy level and light requirment in weed seeds during stratification. Annals of Botany 80: 795–801. Pekrun C, Lutman PJW, Baeumer K. 1997. Germination behaviour of dormant oilseed rape seeds in relation to temperature. Weed Research 37: 419 –431. Stevens JC, Merritt DJ, Flematti GR, Ghisalberti EL, Dixon KW. 2007. Seed germination of agricultural weeds is promoted by the butenolide 3-methyl-2H-furo[2,3-c]pyran-2-one under laboratory and field conditions. Plant and Soil 298: 113– 124. Telewski FW, Zeevaart JAD. 2002. The 120-yr period for Dr. Beal’s seed viability experiment. American Journal of Botany 89: 1285– 1288.