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- Materials and Methods
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At the molecular level a range of mechanisms and signalling networks have been identified that regulate seed dormancy under laboratory conditions (Finch-Savage & Leubner-Metzger, 2006; Finkelstein et al., 2008; Nambara et al., 2010; Graeber et al., 2012). Manipulating seed dormancy states in the laboratory has further increased our understanding of how these molecular pathways and physiological components operate (Cadman et al., 2006; Finch-Savage et al., 2007). These regulatory mechanisms are dominated by a dynamic balance between the hormones abscisic acid (ABA) and gibberellins (GA), and the cohorts of genes that regulate their metabolism, perception and sensitivity via signalling networks considered central to dormancy and the control of germination completion (radical emergence through the seed coat; Finch-Savage & Leubner-Metzger, 2006; Nambara et al., 2010; Bassel et al., 2011; Morris et al., 2011; Dekkers et al., 2013).
However, a greater understanding of the annual pattern of changing dormancy status (dormancy cycling) in the natural environment is required to advance our understanding of soil seed bank dynamics in contexts such as the emergence timing of weed species for modelling weed/crop competition dynamics (Batlla et al., 2004), and the maintenance of species diversity and restoration of natural populations, especially in response to future climate change (Jump et al., 2009; Ooi et al., 2009). Eco-physiological studies on dormancy cycling in several species under field conditions have revealed seasonal changes in seed sensitivity to temperature, light, nitrate and GA leading to large differences in the germination response from month to month (Bouwmeester & Karssen, 1993a,b; Derkx & Karssen, 1993, 1994). This indicates that seeds are able to sense changes in the local soil seed bank environment and rapidly adjust their dormancy status in response.
In Arabidopsis, winter annual ecotypes such as Cape Verdi Isle (Cvi) normally germinate and establish in the autumn and overwinter as rosettes, before flowering and shedding seed in spring (Baskin & Baskin, 1972; Donohue, 2009; Footitt et al., 2011). Whereas, in the Arabidopsis summer annual Burren (Bur) seeds over winter in the soil before emerging in spring to flower over the summer period (Ratcliffe, 1976; Footitt et al., 2013). Before dispersal from the parent plant, the depth of seed dormancy is strongly affected by the maternal environment with nitrate, temperature and photoperiod having strong effects on dormancy (Alboresi et al., 2005; Donohue, 2009; Kendall & Penfield, 2012). Once dispersed seeds remain responsive at a molecular level in both the imbibed and dry state as dormancy changes (Rajjou et al., 2004; Cadman et al., 2006; Finch-Savage et al., 2007; Carrera et al., 2008; Arc et al., 2012).
In previous work we applied a molecular eco-physiological approach to the study of dormancy cycling in the field with two contrasting Arabidopsis ecotypes having winter and summer annual behaviour (Footitt et al., 2011, 2013). Seeds of both ecotypes were placed in soil in autumn to investigate the behaviour of seeds persisting in the seed bank. In this way we were able to show the temporal coordination of the major signalling networks that regulate seed dormancy in an ecological context. This highlighted that seeds in the seed bank are capable of adjusting the depth of dormancy through temporal sensing (identifying the correct season and climate space for emergence) and spatial sensing (identifying signals indicating suitable conditions to terminate dormancy and complete germination). Dormancy and the expression of dormancy-related genes were highly sensitive to the soil environment and molecular physiological changes could be equated to changes in sensitivity to soil temperature history, nitrate, light and GA. This shows dormancy to be a continuum with layers of dormancy being progressively removed by environmental signals until only light is required, in the absence of which seeds remain dormant and enter into another dormancy cycle as the seasons change (Footitt et al., 2011, 2013; Finch-Savage & Footitt, 2012).
Here we present an investigation of the response of dormant Cvi seeds buried in the soil seed bank at their natural dispersal time in spring. Following this spring dispersal into warm soils, seeds rapidly align their dormancy cycle to the prevailing soil environment with a rapid transition into the spatial sensing phase. This contrasts with ungerminated seeds remaining in the soil seed bank at the end of the season in the cold soils of autumn. These overwintering seeds enter a deep dormancy phase of temporal sensing before re-entering the shallow dormancy spatial sensing phase (Footitt et al., 2011). Following spring dispersal of Cvi, spatial sensing dominates resulting in rapid changes in gene expression compared to that seen in autumn buried Cvi. Differences in expression of the gene MOTHER OF FLOWERING TIME (MFT) in spring dispersed and overwintered (soil seed bank) seeds indicate an adaptive response to the spread of germination timing in the seed population. This and the role of DOG1 are discussed in the context of dormancy cycling behaviour in short-term and persistent seed banks of both winter and summer annual ecotypes.
- Top of page
- Materials and Methods
- Supporting Information
We have used the Arabidopsis accession Cvi because it has become an accepted model for dormancy studies and we have previously shown that it exhibits the lifecycle of a winter annual in UK soil conditions (Footitt et al., 2011). Nevertheless having been collected in the Cape Verde Islands it may not be naturally adapted to these conditions. Here we show the response of primary dormant seeds of this ecotype buried in the soil seed bank in spring, the natural shedding and dispersal season for winter annuals (Baskin & Baskin, 1972; Donohue, 2009). The physiological changes in the dormancy status of these seeds in response to warm spring soil are broadly similar to those reported for the same seed lot overwintered following burial in autumn (Footitt et al., 2011). However, changes in gene expression identify more subtle differences in the interpretation of environmental signals linked to spatial sensing in these two cohorts of seeds. Understanding the behaviour of the whole seed population (existing seed bank and newly dispersed) has wide significance because it determines the timing and spread of new entrants to natural plant communities or crop competing weed populations.
Change in dormancy status
Upon burial in spring, dormancy immediately decreased. At this time soil temperature was increasing and soil moisture content was between 11% and 17%. Under these dry conditions dry after ripening may have played a role in these shallow dormant seeds. By contrast, the deep dormancy of over-wintered seeds was lost more rapidly in spring than could be accounted for by dry after-ripening and appeared to have occurred in the imbibed state (Footitt et al., 2011). However, high-temperature thermodormancy decreased and GA sensitivity increased in a broadly similar fashion in both seed cohorts, revealing dormancy to be lowest around August. In both cohorts there was a subsequent rapid induction of deep dormancy in the month following peak germination potential. Overall these responses are consistent with other Arabidopsis field and glasshouse studies (Baskin & Baskin, 1972, 1983; Derkx & Karssen, 1994; Footitt et al., 2011, 2013). We consider below the regulation of germination potential at a molecular level as suggested by seasonal changes in the transcription of key genes with known functions in the control of dormancy.
The role of DOG1
The DOG1 locus has the strongest dormancy association in QTL analyses (Bentsink et al., 2006). Its expression is not directly associated with [ABA], but may enhance ABA sensitivity (Footitt et al., 2011). In the field, expression of DOG1 greatly increased as soil temperature decreased in the autumn in both spring and autumn burials of Cvi and in the summer annual Bur buried in autumn (Footitt et al., 2011, 2013; Fig. 7). In Cvi (winter annual) this change is positively correlated with dormancy, but it is not in Bur (summer annual) indicating subtle differences between ecotypes that readily cycle into deep dormancy (Cvi) compared to those adapted to a shallow dormancy cycle (Bur). This is consistent with the idea that accumulation and retention of DOG1 protein via temperature sensing is directly related to the depth and persistence of dormancy (Nakabayashi et al., 2012; Footitt et al., 2013). Although experimental verification will be required, we propose that DOG1 is part of a thermal sensing mechanism that measures the passage of time (temporal sensing) with the accumulation of DOG1 serving to represent accumulated thermal time to regulate the depth and persistence of dormancy.
Figure 7. Seed responses (molecular and physiological) to environmental sensing (temporal and spatial) drive progress through the annual dormancy cycle to time the key lifecycle transition from seed to seedling (germination) in Arabidopsis. The schematic summarises data from the present study, Footitt et al. (2011, 2011) and illustrates how subtle differences in the coordination of these responses can result in winter and summer annual lifecycles of Arabidopsis ecotypes. These studies have also shown how hormone metabolism and signalling networks operate to put into effect these responses to the soil environment. In the schematic the winter annual ecotype Cvi is represented by the blue bars and the summer annual ecotype Bur by the orange bars. The height of each bar indicates the amplitude of the response measured across the seasons. Temperature represents the annual fluctuation in soil temperature at seed depth. Seed shedding times and emergence timing are based on the field observations. The short-term seed bank is made up of those seeds that are shed from the mother plant, enter the soil, lose dormancy and germinate in the soil in one dormancy cycle so contributing to the next sequential generation. The persistent seed bank represents seeds that pass through more than one annual dormancy cycle thus dispersing seeds in time. Temporal sensing shows the major changes in molecular (DOG1 & MFT) and physiological markers for dormancy. Depth of dormancy is related to the time to 50% after-ripening (AR50; Cvi) and Ψb (base water potential; Bur) in seeds. The thermal germination window represents that period when as a result of declining thermodormancy the thermal base line for germination and soil temperature overlap, so permitting germination and seedling emergence if other environmental requirements are met (water, light, nitrate). Spatial sensing shows when seeds exhibit increased germination potential as reflected by sensitivity to environmental signals such as light (PHYA) and nitrate (CIPK23), which inform the seed as to its position in relation to the soil surface and existing vegetation.
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The role of MFT
MFT is a member of the phosphatidylethanolamine-binding protein (PEBP) family, which includes flowering time regulators FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1). The MFT gene is downregulated by ABI3 and upregulated by the DELLAs, RGA2 and RGL2, and by ABI5, the latter being in turn downregulated by MFT (Xi et al., 2010). It has therefore been assigned the role of negatively regulating ABA signalling to promote embryo growth during germination. However, this is difficult to reconcile with our field observations in Cvi (overwintered and newly sown), where MFT expression was negatively correlated to germination potential (P < 0.05 at 5 and 10°C) and so increased with dormancy. However, despite these correlations there was, unlike DOG1, a significant difference in gene expression in May–August between the overwintered and newly dispersed seeds (Figs 3, 7). In overwintered seeds MFT expression declined to low levels by May, but was relatively high in the newly dispersed seeds and initially remained high before declining to its lowest level in August (Fig. 7). These differences may change with different soil conditions and the resulting germination timing differences in the two cohorts may contribute to bet hedging for the population as a whole.
The subsequent increase in MFT expression with declining soil temperature is consistent with observations in wheat where MFT expression increased > 10-fold in embryos matured at 13°C compared to 25°C (Nakamura et al., 2011). Cold conditions lead to increased dormancy in both wheat (Nakamura et al., 2011) and Cvi seeds (Fig. 1; Footitt et al., 2011). However, in the low dormancy summer annual ecotype Bur, which naturally enters the cold soil seed bank in autumn, MFT and soil temperature are positively correlated (Footitt et al., 2013). Furthermore, MFT expression is closely associated with high germination potential and positively correlated with the genes involved in spatial sensing in Bur (data S1 in Footitt et al., 2013; Fig. 7). This is consistent with the suggestion that MFT has a duel role in promoting both primary dormancy and germination potential in after-ripened seeds (Vaistij et al., 2013) and may be part of a temperature sensitive signalling system (Nakamura et al., 2011).
Control of germination potential following dispersal
From spring sowing to maximum germination potential in August seeds were more nitrate sensitive than overwintered seeds in which sensitivity had previously been reduced by exposure to low temperatures (Footitt et al., 2011). Nitrate induces expression of the ABA catabolism gene, CYP707A2 (Matakiadis et al., 2009). However, seed nitrate is rapidly lost in the soil (Derkx & Karssen, 1993) and nitrate uptake from the soil would be required by seeds to enhance ABA catabolism via the action of CYP707A2 (Matakiadis et al., 2009). A reduction in [ABA] in conjunction with the observed increase in both GA3ox1 (GA biosynthesis) and GID1A (GA receptor) expression results in the increased germination potential upon exposure to light in the spatial sensing phase of the dormancy cycle that removes the final block to germination. Of the three members of the GID receptor family, GA signalling via GID1A & C is required for germination in Arabidopsis with only GID1A exhibiting a strong expression pattern during dormancy cycling in the laboratory (see dataset S2 in Footitt et al., 2011) (Cadman et al., 2006; Voegele et al., 2011). In both the newly dispersed and overwintered seeds GID1A and ABI2 expression peaked when GA sensitivity was greatest (Figs 1, 5; Footitt et al., 2011). This in conjunction with increased GA3ox1 expression in the light (Cadman et al., 2006) explains the nitrate enhancement of germination in the light when dormancy levels are low (Hilhorst & Karssen, 1988). Interestingly the initial transient GID1A peak in June is matched by the SNF1-related protein kinase, SnrK 2.1 which may antagonise GA sensitivity at this point. This initial transient increase in the expression of genes linked to both dormancy promotion (ABI3 and SnRK2.1) and dormancy relief (CYP707A2, GID1A and GA3ox1) following spring sowing suggest that seed behaviour is not fully determined during seed development, but that it also relies upon initial sensing of the environment following dispersal.
Germination repression: the role of DELLAs
Countering the increasing potential to germinate in the soil, are a series of genes that repress germination potential in the absence of light. The DELLA family are major repressors of germination potential and GA signalling (Daviere et al., 2008). Here the expression of RGA2 and RGL2 were highest immediately before and after the largest changes in germination potential. RGA2 had a single peak in both newly dispersed and overwintered seeds, whereas RGL2 had two peaks in the former, but only one in the latter. This indicates that the role of DELLAs in spatial sensing may be more responsive to the environment in those seeds dispersed to the seed bank in the spring. At the same time DELLAs also depress metabolic activity in seeds by repressing genes related to carbohydrate, lipid and protein metabolism, potentially acting in concert with ABI4 (Cao et al., 2006; Penfield et al., 2006). They also repress genes involved in cell wall loosening and organisation of the cytoskeleton, events crucial for endosperm weakening and radical extension during germination.
Germination repression: the role of PIFs
The Phytochrome Interacting Factor (PIF) PIL5 has two peaks of expression in both newly dispersed and overwintered seeds. PIL5 upregulates the GA receptor GID1A and ABI5 and downregulates ABI4 and CYP707A2 (Oh et al., 2009); expression patterns and correlation analysis of newly dispersed seeds (but not in overwintered seeds) support this for GID1A and ABI5. However, PIL5 is not correlated with ABI4 expression in spring-dispersed seeds and is positively correlated in overwintered seeds. Furthermore, ABI4 – a repressor of CYP707A2 expression (Shu et al., 2013) – is significantly positively correlated with this gene in both newly sown and overwintered seeds. There was also no correlation in either study between PIL5 and the expression patterns of either GA3ox1 or GA2ox2, which are negatively and positively regulated (respectively) by PIL5 (Oh et al., 2009). This highlights the complex system of positive and negative control operating over time in the soil seed bank that is not always revealed in mutant and single time-point laboratory studies.
Germination derepression: the role of PIFs
The PIF family transcription factor SPT, is highly expressed in both studies when germination potential is highest. SPT has an important role in the regulation of primary dormancy and is reported to repress ABI4, GA3ox1 and MFT, and induce ABI5 expression (Penfield et al., 2005; Vaistij et al., 2013). Here we found that the correlation between SPT expression and these genes is more complex in the soil seed bank. SPT is positively correlated with ABI4 in both newly dispersed and overwintered seeds (Table 1; and Footitt et al., 2011). The same is true for GA3ox1 in overwintered seeds, but not newly dispersed seeds. The correlation with ABI5 is negative, but not significantly so. This again serves to emphasise the complex interactions in progress in the integration of the temporal and spatial sensing pathways that are influenced by seed history.
Table 1. Linear correlation coefficients of expression comparisons between SPT, MFT and selected genes in buried seeds recovered from the field
| ||Arabidopsis ecotype and burial cycle|
|Cvi spring||Cvi autumn||Bur autumn|
|Gene correlations with SPT|
| ABI4 ||0.949b||0.7182b||0.862a|
| ABI5 ||−0.592||−0.334||0.603c|
| PYR1 ||0.947b||0.600c||0.764b|
| CYP707A2 ||0.760c||0.722b||0.767b|
| GA3ox1 ||−0.249||0.727b||0.846a|
| MFT ||−0.642||−0.850a||0.678c|
|Gene correlations with MFT|
| ABI3 ||0.694d||0.731b||0.806b|
| ABI5 ||−0.001||0.229||0.732c|
| RGA2 ||−0.328||−0.681c||0.746b|
| RGL2 ||0.019||−0.728b||0.736b|
This complex cycle of gene induction and repression by DELLA and PIF gene families is rapidly removed by interaction with the GID protein-GA complex and PHYB. The PIF family members are repressed by binding to RGL2 and RGA to form an inactive complex (Gallego-Bartolome et al., 2010). PIF proteins are released when the GID protein-GA complex binds DELLA proteins. This leads to DELLA degradation by the proteosome and the targeting of released PIL5 and SPT to the proteasome by PHYB in the light (Daviere et al., 2008); we show that this removes the final layer of dormancy resulting in germination. Phytochromes have an important role in the control of seasonal germination timing (Donohue et al., 2012). The magnitude of the transcriptional response to light is seen in GA3ox1 where transcript amounts increased c. 600-fold when shallow dormant seeds were exposed to 5 min of red light (Cadman et al., 2006), whereas deeply dormant seeds are unresponsive to light potentially via the action of PHYA which has a strong dormancy-associated expression pattern (Footitt et al., 2013) and contributes to dormancy in cold-matured seeds (Donohue et al., 2008; Heschel et al., 2008; Dechaine et al., 2009).
Maintaining shallow dormancy (ABA signalling pathway)
Underlying the regulation of germination potential is the operation of ABA signalling that operates via low-sensitivity (shallow dormancy) and high-sensitivity (deep dormancy) pathways (Footitt et al., 2011). From June to August dormancy is controlled by the low-sensitivity pathway after which dormancy rapidly increased in the field. During this period expression of genes for ABA catabolism CYP707A2, the ABA receptor PYR1, the repressor of ABA signalling ABI2, and the ABA-induced transcription factor ABI4, all decreased except when germination potential was high in August (Figs 4, 5). This is consistent with seeds overwintered in field soils where expression of these genes was also higher when germination potential was maximal in June–July (Footitt et al., 2011).
Entry into deep dormancy
As autumn soil temperature decreased, the onset of deep dormancy occurred in newly dispersed and overwintered seeds. As dormancy (AR50 values) increased so did expression of the ABA receptor gene PYL7 and the SNF1-related protein kinases, SnrK 2.1 & 2.4. The latter are positive regulators of ABA signalling acting downstream of ABA receptors to activate transcriptional regulators (Nambara et al., 2010). Expression of these SnrK2 protein kinases are inversely related to soil temperature in Cvi whereas in the Bur accession SnrK 2.1 is inversely [ABA] and SnrK 2.4 is positively related to temperature, indicating that as ABA does not increase as deep dormancy develops these genes may be central to controlling ABA sensitivity in response to seasonal changes in soil temperature (Footitt et al., 2011, 2013). Hormone synthesis responds to the changing conditions with a sustained increase in expression of NCED6 (ABA biosynthesis) and GA2ox2 (GA catabolism) in both newly dispersed and overwintered seeds consistent with increasing dormancy. At the same time both ABI3 and ABI5 increased consistent with their roles in blocking the seed/seedling transition (Nambara et al., 2010; Graeber et al., 2012).
In the work presented, analysis of the Arabidopsis winter annual ecotype Cvi introduced into the soil seed bank at their natural time of dispersal in spring has revealed their physiological and transcriptional response on introduction to a warming soil seed bank. In Fig. 7 we contrast this to overwintered seeds of Cvi (Footitt et al., 2011) and seeds of the summer annual ecotype Bur (Footitt et al., 2013) to identify differences between seeds forming the short-term and persistent seed bank. The schematic illustrates how subtle differences in the temporal expression of key genes linked to temporal and spatial sensing may determine germination patterns resulting in winter and summer annual lifecycles. Seeds passing through the shallow dormancy cycle of the short-term seed bank (Cvi spring and Bur) show rapid responses to environmental change in the spatial sensing phase of the cycle manifested in rapid changes in germination potential. We suggest deep dormancy cycling in the persistent seed bank (overwintered Cvi) dampened these physiological responses through temporal sensing and thermal time measurement via DOG1. In each case seeds germinate when the thermal window permitting germination coincides with ambient temperature and spatial environmental signals (e.g. nitrate concentrations and light intensities). High seed sensitivity to these environmental signals (e.g. temperature, nitrate, light) occur when the expression of key repressing genes (e.g. DOG1, CIPK23, PHYA, respectively) are at their lowest. Subtle differences in the response of seeds from the persistent and short-term seed bank will contribute to bet hedging against variable soil conditions.