Alleviation of dormancy by reactive oxygen species in Bidens ... - Core

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South African Journal of Botany 76 (2010) 601 – 605 www.elsevier.com/locate/sajb

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Alleviation of dormancy by reactive oxygen species in Bidens pilosa L. seeds C. Whitaker a , R.P. Beckett a,⁎, F.V. Minibayeva b , I. Kranner c a

School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa b Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russian Federation c Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, United Kingdom Received 23 February 2010; received in revised form 19 April 2010; accepted 29 April 2010

Abstract Bidens pilosa L. is a weedy species in the Asteraceae producing dimorphic one-seeded fruits, with longer black seeds centrally situated in the capitulum, and shorter dormant brown peripheral seeds. While the outer seeds germinate readily after seed shedding, the shorter seeds possess various dormancy mechanisms, including requirements for after-ripening and red light. Here we show that applications of reactive oxygen species (ROS)-generating reagents can remove dormancy in the short seeds. In B. pilosa, reagents that generate hydroxyl radicals (UOH) and superoxide (O2U¯) partially replaced the requirement for after-ripening, while O2U¯ generation replaced the requirement for red light. Hence, ROS appear to be implicated in the alleviation of dormancy in the seeds of B. pilosa. © 2010 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Bidens; Dormancy; Fenton reagent; Methyl viologen; Reactive oxygen species; Seeds

1. Introduction Orthodox seeds enter a resting state at the end of development, which continues until external conditions create an environment that allows the seed to germinate (Finch-Savage and LeubnerMetzger, 2006; Kozlowski and Gunn, 1972). Such a seed is quiescent, but not necessarily dormant, which may be defined as the failure of an imbibed, viable seed to germinate under seemingly favorable conditions (Bewley, 1997). The block to germination may be either physical (e.g. a hard testa) or physiological (Finch-Savage and Leubner-Metzger, 2006; Finkelstein et al., 2008). Seed dormancy and subsequent germination are generally agreed to involve the modulation of the concentrations of, and sensitivity to, abscisic acid (ABA) and gibberellins (GA). ABA is involved in the induction of dormancy, while GA acts in the promotion of germination (Finch-Savage and Leubner-Metzger, 2006; Finkelstein et al., 2008). A recent model proposes that a heterodimeric protein complex exists that promotes germination, and that the abundance of one monomer is influenced by ABA and the ⁎ Corresponding author. Tel.: +27 33 2605141; fax: +27 33 2605105. E-mail address: [email protected] (R.P. Beckett).

other by GA (Penfield and King, 2009). This complex also regulates GA and ABA metabolism in seeds, and this creates feedback necessary to maintain the dormant and germinating states. One component of a dimeric complex has been proposed to be a phytochrome interaction factor, which may explain why for the seeds of some species under the canopy of trees, far-red (FR) light represses seed germination (Piskurewicz et al., 2009). ROS also appear to be involved in dormancy alleviation. Many studies over decades have shown that exogenous application of ROS and pro-oxidants can break dormancy in a variety of seeds (for review see El-Maarouf Bouteau and Bailly, 2008). Several recently published studies provide intriguing examples indicating how ROS may participate with hormones in the regulation of normal seed development. For example, in sunflower seeds after-ripening involves the progressive accumulation of ROS in the embryonic axes (Oracz et al., 2007). These ROS cause targeted changes in protein carbonylation patterns and gene expression, apparently leading to a loss of dormancy. Oracz et al. (2009) later showed that ethylene can also overcome the requirement for after-ripening in sunflower seeds, and ROS can also activate ERF1, a component of the ethylene signaling pathways. Further evidence for ROS in the control of dormancy come from the observation that

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C. Whitaker et al. / South African Journal of Botany 76 (2010) 601–605

Arabidopsis lines with reduced transcript levels of NADPH oxidase (a membrane-bound enzyme complex involved in extracellular O2U¯ production) displayed reduced protein carbonylation and failed to after-ripen (Müller et al., 2009a). ROS are also important in endosperm weakening and radicle elongation. For example, in cress, electron paramagnetic resonance spectroscopy showed that polysaccharides are oxidized in vivo by the developmentally regulated action of apoplastic UOH in radicles and endosperm caps (Müller et al., 2009b). The production of UOH was inhibited by ABA, and this inhibition could be reversed by GAs. Although ROS produced at excess levels are clearly deleterious, it has been suggested that an “oxidative window” exists in which the generation of strictly regulated concentrations of ROS is required for germination (Bailly et al., 2008). The main aim of the present study was to investigate the potential of exogenous ROS such as hydrogen peroxide (H2O2), O2U¯ and UOH, to break dormancy in a weedy species. Here, the ability to force germination has potential applications in weed control, and could also be useful in the context of seed-banking and conservation. The model chosen for the present study was Bidens pilosa L., a weedy species in the Asteraceae (Forsyth and Brown, 1982). The plant produces dimorphic one-seeded fruits (referred to here as seeds for convenience). Longer black seeds are centrally situated in the capitulum, and are more numerous than the shorter, dormant, brown peripheral seeds. While the outer seeds germinate readily after imbibition, the shorter seeds possess more than one physiological dormancy mechanism. Dormancy may be partially overcome by storing the seeds for 14 days after collection (after-ripening) and then typically display c. 20% total germination (TG). For higher TG, the shorter seeds require red light in what appears to be a classical phytochromemediated dormancy-breaking mechanism (Amaral-Baroli and Takaki, 2001; Forsyth and Brown, 1982). Two sets of experiments were carried out. Firstly, it was determined whether ROS can replace the after-ripening requirement for moderate TG, and secondly, whether ROS can replace the requirement for red light for a more complete TG.

and 10 mg mL− 1 FeSO4, and 10% H2O2 and 30 mg mL− 1 FeSO4. 2.2. Alleviation of the requirement for after-ripening Seeds were collected, stored for 1 day, and then subjected to treatments as outlined in Table 1. In the first set of experiments the three controls used were first, seeds germinated immediately with no other treatment applied and second, seeds after-ripened for 14 days at 20 °C, and then germinated (“conventional dormancy-breaking treatment”), and third, seeds soaked for 6 h at 10 °C in distilled water, and then placed on moist filter paper as for the first control. All treatments comprised three independent replicates of 50 seeds, and all treatments were carried out in the dark, except for scoring TG which was carried out under a green safe light (0.3 µmol m− 2 s− 1). Treatment with MV solution was carried out by placing seeds in darkness at 10 °C for 3 h on a single layer of Whatman No. 1 filter paper in plastic Petri dishes (as recommend by Oracz et al., 2007). Treatment with H2O2 and Fenton reagent was done in the same way but for 6 h at room temperature. Seeds were rinsed three times in distilled H2O to remove ROS-generating reagents, rapidly transferred to new Petri dishes that contained moistened filter paper and were wrapped in aluminium foil. Dishes were placed in an incubator at 25 ± 1 °C, and TG monitored daily for 5 days. 2.3. Alleviation of the requirement for red light Seeds were after-ripened for 14 days at ambient temperature in the dark. The three controls were first, seeds placed on moist filter paper in the dark, second, seeds placed on moist filter paper and then exposed to red light (1.6 µmol m− 2 s− 1) for 15 min (dishes were open). For the third control, seeds were soaked for 6 h at 10 °C in distilled water, and then placed on moist filter paper as for the first control. After-ripened seeds were treated with ROS-generating reagents as above, and TG monitored daily for 5 days.

2. Materials and methods 2.4. Statistical analysis 2.1. Plant material and chemicals Seeds were collected close to the Life Sciences campus of the University of KwaZulu-Natal in Pietermaritzburg, South Africa. Seeds were sorted according to length, placed in brown paper bags inside a glass jar enclosed in aluminium foil, and stored in the dark at 20 °C until required. The short, dormant seeds were used for experimental purposes. Total germination was scored based on radicle protrusion. To generate O2U¯ radicals, methyl viologen dichloride hydrate (MV, Aldrich, St Louis) was used. While MV is often used to generate O2U¯ radicals in green tissues, effective generation of O2U¯ radicals in non-green tissues also occurs (Slooten et al., 1995). To generate UOH radicals, three concentrations of Fenton reagent were prepared, for convenience referred to by their H2O2 concentrations, containing 1% H2O2 and 3 mg mL− 1 FeSO4, 3% H2O2

Percentage germination data after 5 days were arcsine transformed and subjected to one-way ANOVA in combination with post-hoc comparison of means using the LSD test (SPSS 15 for Windows).

Table 1 Concentration and duration of treatment of B. pilosa seeds with ROS-generating compounds.

H2O2 Fenton reagent (% refers to H2O2 concentration) Methyl viologen

Concentrations

Duration of treatment

1, 3, 10% 1, 3, 10%

6h 6h

0.1 mM, 0.01 mM, 0.001 mM

3h


3. Results Freshly collected short seeds displayed c. 3% TG after 5 days (“first control” in Fig. 1a, c and e), and after-ripening for 14 days increased TG to c. 20% (“second control” in Fig. 1a, c and e). The response of freshly collected seeds soaked for 6 h at 10 °C (“third control”) did not differ statistically from that of the first control, and are not shown. Treatment of seeds with all concentrations of MV and with 1% Fenton reagent significantly (P b 0.05) overcame the requirement for after-ripening (Fig. 1a and e), while H2O2 and higher concentrations of Fenton reagent did not affect TG. However, while naturally after-ripened seeds displayed almost maximum TG after 1 day, MV and Fenton reagent-treated seeds needed 2 to 3 days for maximal TG. Short seeds after-ripened for 14 days and then kept in the dark displayed c. 20% TG (“first control” in Fig. 1b, d and f), while seeds after-ripened for 14 days and then exposed to red light for 15 min displayed c. 70% TG after 5 days (“second

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control in Fig. 1b, d and f). The response of after-ripened seed soaked for 6 h at 10 °C (“third control”) did not differ statistically from that of the first control, and are not shown. Treatment of seeds with all concentrations of MV increased TG, significantly so for 0.01 mM (P b 0.05, Fig. 1b). Seeds exposed to red light displayed almost maximum TG after 1 day, while MV-treated seeds needed 2 to 3 days. Treatment with H2O2 appeared to either damage the seeds or deepen their dormancy, because TG was lower at all concentrations used than that of the “first control”, and was at best only 1%. Fenton reagent was much less inhibitory, but either had no effect or decreased TG compared with the “first control” (Fig. 1f). 4. Discussion Results presented here show that applications of ROSgenerating reagents can partially substitute for after-ripening and red light, and can release dormancy in the short seeds of

Fig. 1. Total germination (TG) in fresh Bidens pilosa seeds that normally require after-ripening (a, c and e) and in after-ripened seeds that require red light (b, d and f). In a, c and e, the “first control” comprised fresh seeds placed on moist filter paper in the dark immediately after collection with no other treatment (squares) and the “second control” seeds after-ripened for 14 days (circles). In b, d and f, the “first control” comprised after-ripened seeds placed on moist filter paper in the dark (squares), and the “second control” comprised after-ripened seeds placed on moist filter paper and then exposed to red light for 15 min (circles). Methyl viologen (a and b) was supplied at 0.001 mM (triangles), 0.01 mM (stars) and 0.1 mM (diamonds). Hydrogen peroxide (c and d) was supplied at 1% (triangles), 3% (stars) and 10% (diamonds). Fenton reagent (e and f) was supplied with H2O2 at concentrations of 1% (triangles), 3% (stars) and 10% (diamonds). Values are given ±SE (n = 3 replicates of 50 seeds).

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B. pilosa. Specifically, UOH and O2U¯-generating reagents partially replaced the requirement for after-ripening (Fig. 1a and e), and the O2U¯ radical partially replaced the requirement for red light (Fig. 1b). The absence of an effect of imbibition at 10 °C suggests that the effects of ROS-generating solutions were not simply the result of chilling or imbibition. In this study, H2O2 tended to inhibit rather than promote TG (Fig. 1c and d). This was somewhat unexpected, because H2O2 has been used to break dormancy in seeds of several species including rice (Naredo et al., 1998) (3.4% H2O2), Tripsacum dactyloides (Huarte and Garcia, 2009) (2.8% H2O2) and sunflower (e.g., Oracz et al., 2009) (1.7% H2O2), the latter representing another species in the Asteraceae family. Even in recalcitrant sweet chestnut seeds, concentrations of H2O2 up to 1 M (3.4%) improved TG, particularly in seeds subjected to mild desiccation (Roach et al., 2010). In B. pilosa seeds, higher concentrations of Fenton reagent decreased TG (Fig. 1c), which could be associated with the higher H2O2 concentrations present in the solutions. While the treatment times and concentrations of H2O2 used here were similar to those used in other studies, lower or higher concentrations or shorter or longer treatment times may still be found to have dormancy-breaking effects. However, in B. pilosa, reagents that generate UOH and O2U¯ can remove blocks to germination, indicating that ROS other than H2O2 are implicated in the control of dormancy in this species. After-ripening can be defined as the requirement for a period of dry storage at room temperature of freshly harvested, mature seeds, before germination can occur, and is rather common (Finch-Savage and Leubner-Metzger, 2006). The present study demonstrates that exogenous application of MV and Fenton reagent can replace the after-ripening requirement for moderate TG. As discussed earlier, a recent study suggested that in sunflower seeds after-ripening involves a progressive accumulation of ROS in the embryonic axes (Oracz et al., 2007), causing targeted changes in protein carbonylation patterns and gene expression, which lead to germination. Moreover, O2U¯ and H2O2 have been suggested to be involved in a ROS-hormone signaling network that involves ethylene and is required for dormancy release (Oracz et al., 2009). Although after-ripened seeds of B. pilosa germinated faster than those treated with ROS (Fig. 1a and e), ROS-generating reagents may break dormancy by changing protein carbonylation patterns and gene expression in this species in the same way as in sunflower seeds. After-ripened seeds of many species still possess moderate dormancy. To germinate, some, such as B. pilosa, require red light, which is perceived by a phytochrome system (Finkelstein et al., 2008). The requirement of B. pilosa seeds for red light was partially overcome by treatment with reagents that generate O2U¯ radicals, but not H2O2 or UOH (Fig. 1b, d and f). One component of the dimeric protein complex thought to regulate dormancy has been proposed to be a phytochrome interaction factor (Penfield and King, 2009). Recent research suggests that inhibition of germination in the absence of red light involves stabilization of DELLA proteins, which suppress transcription of proteins necessary for germination to occur (Piskurewicz et al., 2009). Interestingly, DELLA proteins have also been implicated in regulating the levels of ROS in plant leaves and

roots (Achard et al., 2008). Hence, it will be interesting in the future to use the advantages of the B. pilosa system to further study the interaction between O2U¯ and DELLA proteins in dormancy breaking. However, as for after-ripening, O2U¯ treated seeds germinated slower than those exposed to red light (Fig. 1b), again suggesting that O2U¯ and red light may alleviate dormancy in different ways. Forsyth and Brown (1982) showed that scarification can replace the requirement for red light in B. pilosa. High H2O2 concentrations may have the potential to break physical dormancy by oxidative damage to the seed coat. However, in the present study, neither H2O2 on its own or in the Fenton reagent could overcome the need for red light, arguing against scarification by H2O2. It has been suggested that seed dormancy is the best weapon that weeds possess for their continued survival (Cohn, 2002). Knowledge of how to break dormancy may have important benefits in weed control in the future. Results presented here suggest that ROS-generating compounds are potential candidates that can be used to alleviate dormancy. Acknowledgements Funding by the Leverhulme Trust (grant F/00 731/C to IK), the Russian Foundation for Basic Research (09-04-01394 to FVM), and the University of KwaZulu-Natal Research Fund (to RPB) is acknowledged. The Millennium Seed Bank Project was supported by the Millennium Commission, The Wellcome Trust, Orange Plc. and Defra. The Royal Botanic Gardens, Kew receive grant-in-aid from Defra. References Amaral-Baroli, A., Takaki, K., 2001. Phytochrome controls achene germination in Bidens pilosa L (Asteraceae) by very low fluence response. Brazilian Archives of Biology and Technology 44, 121–124. Achard, P., Renou, J.-P., Berthomé, R., Harberd, N.P., Genschik, P., 2008. Plant DELLAs restrain growth and promote survival of adversity by reducing the levels of reactive oxygen species. Current Biology 18, 656–660. Bailly, C., El-Maarouf-Bouteau, H., Corbineau, F., 2008. From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology. Comptes Rendus Biologies 331, 806–814. Bewley, J.D., 1997. Seed germination and dormancy. The Plant Cell 9, 1055–1066. Cohn, M.A., 2002. Reflections on investigating dormancy-breaking chemicals — a balance of logic and luck. Weed Science 50, 261–266. El-Maarouf Bouteau, H., Bailly, C., 2008. Oxidative signaling in seed germination and dormancy. Plant Signaling and Behaviour 3, 175–182. Finch-Savage, W.E., Leubner-Metzger, G., 2006. Seed dormancy and the control of germination. The New Phytologist 171, 501–523. Finkelstein, R., Reeves, W., Ariizumi, T., Steber, C., 2008. Molecular aspects of seed dormancy. Annual Review of Plant Biology 59, 387–415. Forsyth, C., Brown, N.A.C., 1982. Germination of dimorphic fruits of Bidens pilosa L. The New Phytologist 90, 151–164. Huarte, R., Garcia, M.D., 2009. Tripsacum dactyloides (L.) L. (Poaceae) caryopsis dormancy and germination responses to scarification, hydrogen peroxide and phytohormones. Seed Science and Technology 37, 544–553. Kozlowski, T.T., Gunn, C.R., 1972. Importance and characteristics of seeds. In: Kozlowski, T.T. (Ed.), Seed Biology I. Importance, Development, and Germination. Academic Press, pp. 1–18. Müller, K., Carstens, A.C., Linkies, A., Torres, M.A., Leubner-Metzger, G., 2009a. The NADPH-oxidase AtrbohB plays a role in Arabidopsis seed afterripening. The New Phytologist 184, 885–897.

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