Introduction
A common understanding of the control of germination through dormancy is essential for effective communication between seed scientists whether they are ecologists, physiologists or molecular biologists. Vleeshouwers et al. (Reference Vleeshouwers, Bouwmeester and Karssen1995) realized that barriers between disciplines limited progress and through insightful conclusions in their paper ‘Redefining seed dormancy: an attempt to integrate physiology and ecology’, they did much to overcome these barriers at that time. However, times move on, understanding develops, and now there is a case for ‘Redefining seed dormancy as an integration of physiology, ecology and molecular biology’. Finch-Savage and Leubner-Metzger (Reference Finch-Savage and Leubner-Metzger2006) had this in mind when they extended and re-interpreted the definition of dormancy proposed by Vleeshouwers et al. (Reference Vleeshouwers, Bouwmeester and Karssen1995), by considering dormancy as a having a number of layers that must be removed, with the final layer of dormancy being synonymous with the stimulation/induction of germination. We write this opinion article to support this unifying understanding, and to counter the opinion expressed by Thompson and Ooi (Reference Thompson and Ooi2010). The latter argued against the view expressed by several speakers at the ISSS Seed Ecology Conference in Utah (2010) ‘that [physiological] dormancy is broken by light, smoke, nitrate or diurnal temperature alternations’. Finch-Savage and Leubner-Metzger (Reference Finch-Savage and Leubner-Metzger2006) identified a number of potential sources for this confusion in the literature that ‘has resulted in part from different views on dormancy, such as whether light terminates dormancy or induces germination’. Thompson and Ooi (Reference Thompson and Ooi2010) have perpetuated this confusion. We argue below, using accepted definitions of dormancy and evidence from physiology, ecology and molecular biology, that the regulation of germination is not through separate dormancy relief and stimulation of germination (sensu Thompson and Ooi, Reference Thompson and Ooi2010), but by a dormancy continuum. Our intention is to briefly show the evidence in a form that is accessible to seed scientists from different disciplines.
Thompson and Ooi (Reference Thompson and Ooi2010) state towards the end of their opinion paper that the right questions to be asked are ‘What factors control dormancy?’ and ‘Which cues promote germination once seeds are non-dormant?’. They insist that there is a distinction between cues that are responsible for dormancy alleviation and others that are germination stimulants. They suggest that without this distinction ‘results from experimental treatments on dormant seeds are often not clear and this can hinder subsequent research, by diverting attention away from developing the right questions’, i.e. those above. In contrast, Finch-Savage and Leubner-Metzger (Reference Finch-Savage and Leubner-Metzger2006) state:
A wide range of factors can alter dormancy in primary dormant seeds. However, there is an important distinction in the seeds' response to these factors. (1) There are factors that are related to slow seasonal change. These factors (e.g. temperature) are integrated over time to alter the depth of dormancy, and the sensitivity to other factors (e.g. light). (2) There are other factors that indicate in a more immediate way that conditions are suitable for germination (e.g. light), which could be considered to terminate dormancy and therefore induce germination. Each of these factors therefore remove successive blocks to germination, but this process usually needs to be carried out in a set order for it to work, i.e. in the process described light must come last to be effective.
Thus a dormancy continuum driven by environmental conditions in both directions is described, and when all layers are removed, germination occurs. This latter interpretation seems to be borne out by subsequent molecular ecophysiological studies of dormancy cycling in the laboratory (Cadman et al., Reference Cadman, Toorop, Hilhorst and Finch-Savage2006; Finch-Savage et al., Reference Finch-Savage, Cadman, Toorop, Lynn and Hilhorst2007) and field (Footitt et al., Reference Footitt, Douterelo-Soler, Clay and Finch-Savage2011) as we will discuss below.
Interestingly, despite the different views expressed, both these sets of authors support their arguments by quoting Vleeshouwers et al. (Reference Vleeshouwers, Bouwmeester and Karssen1995) and that ‘dormancy is a seed characteristic, the degree of which defines what conditions should be met to make the seed germinate’. Both argue that the key thing is that dormancy should not just be associated with the absence of germination, but rather as a characteristic of the seed that determines the conditions required for germination (Vleeshouwers et al., Reference Vleeshouwers, Bouwmeester and Karssen1995; Thompson, Reference Thompson2000; Fenner and Thompson, Reference Fenner and Thompson2005; Finch-Savage and Leubner-Metzger, Reference Finch-Savage and Leubner-Metzger2006). On this basis, Finch-Savage and Leubner-Metzger (Reference Finch-Savage and Leubner-Metzger2006) argue that ‘any environmental cue that alters the conditions required for germination is by definition altering dormancy. Also by extension, when the seed no longer requires specific environmental cues it is non-dormant’ and provide an argument that nitrate, light and alternating temperatures alter the conditions required for germination and so alter characteristics of the seed and therefore dormancy. Apparently in agreement, Thompson and Ooi (Reference Thompson and Ooi2010) state in the concluding paragraph of their opinion paper, after Vleeshouwers et al. (Reference Vleeshouwers, Bouwmeester and Karssen1995), ‘Thus a germination cue is a change in the environment that aligns that environment with germination requirements of the seed; dormancy breaking is a change in the seed that determines what those requirements are’. However, the confusion still remains.
How can this be resolved?
It seems to all boil down to the question: what do we mean when we say ‘dormancy is a seed characteristic, the degree of which defines what conditions should be met to make the seed germinate’? The authors in both cases agree in this definition by Vleeshouwers et al. (Reference Vleeshouwers, Bouwmeester and Karssen1995) made in point 2 of their summary. The difference comes in point 4 of the summary, which states ‘it is argued that the process of dormancy should be clearly distinguished from the germination process itself. It is stated that as yet only temperature has been shown to alter the degree of dormancy in seeds. Factors like light and nitrate are often indispensible for germination, but only by promoting the germination process itself, not by mitigating the requirements for germination’. Perhaps the key phrase here is ‘as yet only temperature’, i.e. in 1995, and another is the phrase in the concluding paragraph of Thompson and Ooi (Reference Thompson and Ooi2010) ‘dormancy breaking is a change in the seed’. We therefore concentrate below on the following criteria: (1) do light and nitrate ‘mitigate the requirements for germination’, i.e. alter dormancy; and (2) do light and nitrate result in a 'change in the seed’? If these criteria are satisfied, then by the agreed definitions light and nitrate are breaking dormancy.
Physiological evidence since 1995
Finch-Savage and Leubner-Metzger (Reference Finch-Savage and Leubner-Metzger2006) summarized the physiological evidence in the following way. Nitrate:
… exogenous nitrate can affect the requirement for light to promote A. thaliana seed germination (Batak et al., Reference Batak, Devic, Giba, Grubisi, Poff and Konjevic2002), and the initial level of dormancy in the seed is influenced by the nitrate regime fed to the mother plant (Alboresi et al., Reference Alboresi, Gestin, Leydecker, Bedu, Meyer and Truong2005). Therefore nitrate affects the requirements for germination and so could be said to directly affect dormancy rather than just promote germination.
Light:
Light has both been considered to stimulate germination (e.g. Vleeshouwers et al., Reference Vleeshouwers, Bouwmeester and Karssen1995) and to terminate dormancy (e.g. Benech-Arnold et al., Reference Benech-Arnold, Sanchez, Forcella, Kruk and Ghersa2000; Batlla et al., Reference Batlla, Kruk and Benech-Arnold2004). To some extent, this depends on where one chooses to draw the line between the processes of dormancy and germination. In this review [i.e. Finch-Savage and Leubner-Metzger, Reference Finch-Savage and Leubner-Metzger2006], we have used the definition above that dormancy is a seed characteristic which defines the conditions required for germination and therefore any cue that widens the environmental requirements for germination should be regarded as a dormancy release factor. Following this argument, exposure to light changes the seed so that it can germinate in darkness and is therefore the last step in the dormancy-breaking process, rather than the first step in the germination process (Bewley and Black, Reference Bewley and Black1994; Pons, Reference Pons2000; Leubner-Metzger, Reference Leubner-Metzger2003). This light effect (red light via phytochrome) can also be reversed in some cases by far-red light, until the seed is committed to the process of germination (Casal and Sanchez, Reference Casal and Sanchez1998; Sanchez and Mella, Reference Sanchez and Mella2004). In seeds with coat dormancy, it is thought that light and GA can both release (coat) dormancy and promote germination (e.g. Casal and Sanchez, Reference Casal and Sanchez1998; Leubner-Metzger, Reference Leubner-Metzger2001; Leubner-Metzger and Meins, Reference Leubner-Metzger and Meins2001; Sanchez and Mella, Reference Sanchez and Mella2004; Kucera et al., Reference Kucera, Cohn and Leubner-Metzger2005).
Further to this, dark stratification is often used to alleviate physiological dormancy (e.g. Goggin et al., Reference Goggin, Steadman and Powles2008, Reference Goggin, Powles, Toorop and Steadman2011; Long et al., Reference Long, Stevens, Griffiths, Adamek, Powles and Merritt2011) and the presence of white light actively inhibits dormancy alleviation; the quality and quantity of light received is therefore critical for seeds undergoing ‘ecologically relevant’ dormancy alleviation.
Molecular biological evidence
One of the findings that has come to the forefront relatively recently at the molecular level is that dormancy induction (prevention of germination) and dormancy relief are both under negative regulation. There are proteins, PP2Cs and DELLAs, that block the downstream gene expression that results in dormancy induction or dormancy relief, respectively (e.g. Cutler et al., Reference Cutler, Rodriguez, Finkelstein and Abrams2010; Sun and Gubler, Reference Sun and Gubler2004, respectively). These negative regulators must be removed before these processes can be completed. A balance of the hormones abscisic acid (ABA; inhibits completion of germination) and gibberellic acid (GA; promotes completion of germination), in response to environmental signals, influences these negative regulators. This balance mechanism is summarized in Fig. 1.
A schematic summary of ABA and GA metabolism and signalling genes implicated in the regulation of seed dormancy where environmental signals influence the relative balance of dormancy induction and relief. When induction or relief is induced, the pathways indicated in red dominate. ● Active GA levels increase just before radical emergence, suggesting that it plays a key role in the regulation of germination; the key stages of GA metabolism during this process have been described (Ogawa et al., Reference Ogawa, Hanada, Yamauchi, Kuwahara, Kamiya and Yamaguchi2003). GA3-oxidase is responsible for the final step in GA biosynthesis to produce active GAs. Subsequent degradation is via GA2-oxidase. ● Key genes responsible for ABA biosynthesis, degradation and conjugation during Arabidopsis seed germination have been identified and investigated (Müller et al., Reference Müller, Tintelnot and Leubner-Metzger2006; Okamoto et al., Reference Okamoto, Kuwahara, Seo, Kushiro, Asami, Hirai, Kamiya, Koshiba and Nambara2006; Penfield et al., Reference Penfield, Li, Gilday, Graham and Graham2006; Holdsworth et al., Reference Holdsworth, Bentsink and Soppe2008; Piskurewicz et al., Reference Piskurewicz, Jikumaru, Kinoshita, Nambara, Kamiya and Lopez-Molina2008). NCEDs are the primary regulatory step in ABA synthesis; subsequent inactivation is by hydroxylation (CYP707) or conjugation with sugars (Nambara et al., Reference Nambara, Okamoto, Tatematsu, Yano, Seo and Kamiya2010). The balance of these processes regulates ABA content. ● Hormone signalling, especially that resulting from the dynamic balance of the hormones abscisic acid (ABA) and gibberellic acid (GA), is a key component of the interacting networks regulating germination (Kucera et al., Reference Hartweck2005; Holdsworth et al., Reference Holdsworth, Bentsink and Soppe2008). ● Understanding of ABA signal transduction is developing rapidly, and a model has recently emerged in which PYR/PYL/RCAR receptors bind to ABA to remove the repression by PP2cs of downstream signalling via SnRK2s to ABRE-binding transcription factors (ABI3, ABI4, ABI5) (Cutler et al., Reference Cutler, Rodriguez, Finkelstein and Abrams2010). ● On the other side of this balance, DELLA proteins repress GA responses and therefore germination potential (Sun and Gubler, Reference Sun and Gubler2004). DELLAs are degraded to remove this repression on forming a complex with GA and GID1 receptors (Hartweck, Reference Hartweck2008). ● These signalling pathways are influenced by a diverse range of environmental signals as germination potential increases, principally temperature and light. ● The two phytochrome-interacting bHLH transcription factors, PIL5 and SPT are important components of the response via GA to environmental signals. This figure was first published in PNAS (Footitt et al., Reference Footitt, Douterelo-Soler, Clay and Finch-Savage2011).
As we begin to understand this negative form of regulation, it becomes clear that there is no distinction between dormancy relief and stimulation of germination. You could try and read this into the bifurcated mechanism illustrated in Fig. 1, but everything seems to suggest that both sides interact. Recently, Footitt et al. (Reference Footitt, Douterelo-Soler, Clay and Finch-Savage2011) have shown under ecologically relevant conditions, namely in seeds undergoing dormancy cycling in the soil seed bank, that these processes are seasonally separated over the annual cycle in such a way that emphasis on one side or the other of the balance changes with the seasons (see Fig. 6 in Footitt et al., Reference Footitt, Douterelo-Soler, Clay and Finch-Savage2011).
Vleeshouwers et al. (Reference Vleeshouwers, Bouwmeester and Karssen1995) say ‘we reserve the term dormancy for a block or blocks within the seed that prevent germination, and distinguish it from the absence of factors required to evoke germination’. Within this definition the negative regulation by PP2Cs and DELLAs can be seen as blocks to dormancy induction and dormancy relief, respectively. So what effect do light and nitrate have on these negative regulators (blocks)?
Light
The action of light is to promote the expression of GA3ox1 (e.g. Cadman et al., Reference Cadman, Toorop, Hilhorst and Finch-Savage2006), the key gene responsible for synthesis of GA. The effect of this in the mechanism summarized in Fig. 1 is to increase GA and therefore remove the DELLA repression (block) of GA signalling and metabolism to relieve dormancy, which results in the completion of germination.
Nitrate
The action of nitrate is to promote the expression of CYP707A2 (Alboresi et al., Reference Alboresi, Gestin, Leydecker, Bedu, Meyer and Truong2005; Matakiadis et al., Reference Matakiadis, Alboresi, Jikumaru, Tatematsu, Pichon, Renou, Kamiya, Nambara and Truong2009), the key gene responsible for ABA catabolism. The effect of this in the mechanism summarized in Fig. 1 is to reduce ABA and therefore enhance the repression (block) of ABA signalling and thus reduce dormancy. It was observed in the soil seed bank that nitrate sensitivity only increased following increased expression of the nitrate transporter, NRT1.1, which coincided with increased CYP707A2 expression (Footitt et al., Reference Footitt, Douterelo-Soler, Clay and Finch-Savage2011).
Conclusions from physiology and molecular biology
Physiological studies (post 1995) now clearly show that light and nitrate break dormancy. Molecular biological studies have progressed further to underpin this physiology. Therefore, if we consider the criteria raised above; first, do light and nitrate ‘mitigate the requirements for germination’? The answer is clearly ‘yes’ as the negative repressors PP2Cs and DELLAs determine the requirements for germination and these are altered by nitrate and light, respectively. For the second question, do they result in a ‘change in the seed’? The answer is also ‘yes’ since they are clearly responsible for a cascade of events (gene expression, etc.) in the seed (Fig. 1). Thus, by the agreed definition ‘dormancy breaking is a change in the seed’, light and nitrate are clearly breaking dormancy. The alternative view of them being a germination cue, defined as ‘a change in the environment that aligns the environment with the germination requirement of the seeds’, is not now relevant to light and nitrate. We feel these same arguments can be made for alternating temperatures and smoke (karrikinolide), but wished to keep this opinion article short and focused. However, the question remains: are there any germination cues or does germination just go to completion when all layers of dormancy have been removed by changes in the seed? Possibly water, but not oxygen as this can have specific effects and anoxia is essential for germination in some species (Probert and Benchley, Reference Probert and Benchley1999), as pointed out by Thompson and Ooi (Reference Thompson and Ooi2010).
Further discussion
Thompson and Ooi (Reference Thompson and Ooi2010) make valid points about problems in practice concerning use of applied treatments (such as smoke) in restoration, i.e. they may be applied at the wrong time when seeds are not sensitive due to a lack of understanding by the practitioner. However, we do not think their approach is helpful. The problems are more likely avoided if dormancy is considered as a continuous variable and dormancy breaking cues have to come in the correct order for the completion of germination sensu Finch-Savage and Leubner-Metzger (Reference Finch-Savage and Leubner-Metzger2006). Thompson and Ooi (Reference Thompson and Ooi2010) suggest that the problem is solved when the right questions are asked: ‘What factors control dormancy?’ and ‘Which cues promote germination once seeds are non-dormant?’. However, the problem is not solved by making this distinction between dormancy and germination, but from understanding that the seed germinates in the right season, in a favourable place under suitable conditions because of a complex set of responses to a range of different environmental signals required to fully relieve dormancy, i.e. smoke has to be applied at the right time when these other criteria have been satisfied.
In conclusion, Thompson and Ooi (Reference Thompson and Ooi2010) provide an excellently crafted and compelling set of words, but without facts to convince the reader. We hope to have shown that physiological and molecular understanding can help to resolve these otherwise philosophical debates that can lead to confusion. There is no clear distinction between the ecological, physiological and molecular biological understanding of seed behaviour, and it is only the separation of these communities that has brought about some of the difficulties. We should embrace the avalanche of new information that makes seed science so interesting at the moment, but interpret it in a framework that is relevant to all disciplines.
