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A wide range of dormancy mechanisms have evolved in higher plants to regulate germination of their seeds. Physiological seed dormancy is present throughout higher plants, and has a profound impact on the timing and periodicity of seedling emergence and, more widely, on the structure and development of plant communities across all major climatic regions (Baskin & Baskin, 1998). Consequently, the induction and loss of dormancy are triggered by diverse environmental cues activated through many different physiological mechanisms. The role of dormancy is, however, the same across all species – to spread germination across time but in synchrony with seasonal cycles to avoid unfavourable weather, maximize competitive advantage and ensure the establishment of plants (Baskin & Baskin, 1998). This conserved role suggests there may be a common underlying molecular mechanism. Studies with model species that have comparatively shallow dormancy have identified a number of genes involved in the regulation of dormancy and germination (Bewley, 1997; Holdsworth et al., 1999; Bentsink & Koornneef, 2002; Koornneef et al., 2002). However, there have been very few molecular studies in more deeply dormant species in which moist seeds require prolonged exposure to low temperature to release dormancy and induce germination. To address this, the presence and expression patterns of putative dormancy regulating gene homologues were investigated in the deeply dormant seeds of Prunus avium (wild cherry) exposed to dormancy-regulating temperatures.
The dormancy mechanisms in P. avium, like those of other species with deeply dormant seeds, appear to be complex. The embryo is dormant and the surrounding structures (endosperm, seed coat and endocarp) also impose some control (coat-enhanced dormancy). Embryo dormancy is deemed to be present when the embryo fails to grow after the removal of the tissues that enclose it. In P. avium, embryo dormancy is thought to occur early in development before seeds are fully mature, but shortly after seeds acquire the ability to germinate (Jensen & Eriksen, 2001). At this stage, growth can occur in excised embryos, but they produce physiologically dwarf plants and other abnormalities (Tukey, 1934). As development continues, the potential for embryo growth decreases (Abou-Zeid, 1972; Abou-Zeid & Gruppe, 1972; Jensen & Eriksen, 2001). Embryos extracted from fully mature seeds have very little capacity for growth, but this increases progressively when moist seeds are exposed to low temperature (Pollock & Olney, 1959). There are reports in the literature of variable potential for radicle growth between seed lots in the absence of low-temperature treatment (Michalska, 1982), but this may have resulted from using seeds that are not fully mature. Due to its fleshy fruit, the extent of seed maturity at harvest in P. avium is difficult to judge, and this can greatly influence the extent of seed dormancy and its response to low-temperature treatment (Finch-Savage et al., 2002). The presence of surrounding tissues further delays germination during low temperature treatment. A period of higher temperature (20–25°C) for 2 wk or more can re-impose full dormancy at any time before complete loss of dormancy in individual seeds (Suszka, 1962, 1967; Suszka et al., 1996). When dormancy is lost in any one seed, these same temperatures will increase the germination rate, as there is no separate treatment required for the induction of germination in this species. This well characterized system of progressive loss of primary dormancy and germination in the population, and then the induction of secondary dormancy as ambient temperatures rise in late spring, is thought to result in the spread of germination within and across years under natural conditions (Suszka, 1990).
At the molecular level there is growing, but still limited, understanding of how dormancy is imposed in maturing seeds, maintained, cycled, or broken. Studies in Arabidopsis thaliana have demonstrated the importance of two closely related protein phosphatase 2C genes in imposing primary dormancy during seed maturation: ABI1 and ABI2 (abscisic acid-insensitive; Leung et al., 1994, 1997; Meyer et al., 1994; Rodriguez et al., 1998; Merlot et al., 2001); and a B3-type transcription factor, ABI3 (Giraudat et al., 1992), which is homologous to the maize Viviparous1 gene (Vp1, McCarty et al., 1991). The ABI3 or Vp1 gene has been studied extensively in a number of species in which its expression has been closely linked to primary dormancy. The expression of Vp1 in Avena fatua (wild oat) seeds has also been linked to environmentally imposed or secondary dormancy (Holdsworth et al., 1999). Early germination in several species has been shown to involve expression of an ACONITASE (aconitate hydratase, citrate hydro-lyase) gene (Hayashi et al., 1995; Peyret et al., 1995; Eastmond & Graham, 2001). Developmental changes are linked to changes in de novo protein synthesis. In plants, eIF3 is an essential multiprotein complex for the initiation of protein synthesis (reviewed by Hershey & Merrick, 2000; Hinnebusch, 2000). Subunit 8 of the eIF3 complex (nomenclature according to Browning et al., 2001; homologous to mammalian p110 and yeast NIP1 or eIF3c) has also been linked to interaction with the COP9 signalosome, and may have multiple complex roles in the control of plant development (Karniol & Chamovitz, 2000; Kim et al. 2001; Seeger et al., 2001; Yahalom et al., 2001).
The aim of the present study was to gain insight into the molecular control of dormancy in the deeply dormant seeds of P. avium by investigating expression of P. avium homologues of the dormancy-inducing genes ABI1/2 and ABI3 during low-temperature treatment. Their temporal expression patterns were compared to those of ACO1 and eIF3 SUBUNIT 8 as likely indicators of germination. The results were consistent with a role for ABI1/ABI2 and ABI3 homologues in primary dormancy of P. avium axes, but not secondary dormancy, as control of germination appeared overridden by the tissues surrounding the embryo.
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The germination behaviour of P. avium seeds used in the present study was consistent with that reported elsewhere, showing an absolute requirement for low temperature treatment and the induction of secondary dormancy by higher temperatures (Suszka, 1962, 1967; Suszka et al., 1996; Jensen & Eriksen, 2001; Finch-Savage et al., 2002). Fully cold-treated (nondormant) seeds will germinate at a wide range of temperatures, including 3 and 20°C as used here, without the need for further induction treatments. However, if the seeds are not fully cold-treated, significant exposure to the higher of these temperatures will induce secondary dormancy that is manifested by reversing any previous cold treatment. Thus secondary dormant seeds require the full period of cold treatment characteristic of primary dormant seeds (Fig. 2b). Previous work has shown this cycle can be repeated several times (Suszka, 1962, 1967; Suszka et al., 1996). Four genes likely to be involved in inducing dormancy and permitting germination were studied in embryonic axes excised from seeds exposed to these dormancy-breaking and dormancy-inducing temperature treatments.
The genes studied were shown to be highly similar in structure and genomic copy number to their counterparts in other, more studied, angiosperms. The similarity levels of Pa-PP2C1 to the A. thaliana ABI1- and ABI2-coding regions are equally high, with no obvious homology in the N-terminal 60 amino acids, followed by 45% identity and 58% similarity over the remainder of the protein. We cannot therefore define Pa-PP2C1 as a specific homologue of either ABI1 or ABI2.
The expression of the selected genes during loss of primary dormancy and germination closely mimics that expected from studies of their counterparts in other species. However, none of them revealed expression patterns suggestive of specific induction or repression reflecting initiation of the nondormant state. This is of particular note in the case of Pa-PP2C1 and Pa-ABI3, as the products of these genes are known to be important in imposing embryo dormancy in other systems (McCarty et al., 1991; Giraudat et al., 1992; Leung et al., 1994; Meyer et al., 1994; Leung et al., 1997; Rodriguez et al., 1998; Merlot et al., 2001), but transcript levels were low when 99% of seeds were still dormant (Fig. 2). This observation generates two hypotheses. Transcriptional analysis does not directly reflect the levels of active protein product, and it may be the case that Pa-ABI3 and Pa-PP2C1 activity declined to some dormancy-releasing level concomitant with the observed germinability of seed batches shown in Fig. 2. More likely, perhaps, is that embryo dormancy had indeed been lost some weeks before dormancy of the intact seed was broken. In this case dormancy would have been maintained through control exerted by the surrounding tissues (endosperm and seed coat). This second hypothesis is consistent with earlier morphological observations (Pollock & Olney, 1959; Michalska, 1982) that report a variable but enhanced capacity for root and leaf growth in isolated P. avium embryos after only 2–4 wk storage at 3°C.
Control levels of both Pa-ABI3 and Pa-PP2C1 transcripts also fell during storage at 20°C, but not to the levels induced by chilling, suggesting that some transcriptional activity continued at these loci at 20°C, or that the transcripts were more stable. However, inducing secondary dormancy by warming the seeds to 20°C after 6 or 9 wk chilling did not return transcript levels to control values (Fig. 3a,b). Again, this observation is consistent with a model of embryo dormancy having been lost after limited exposure to dormancy-breaking temperatures and subsequent control of germination during both continued primary dormancy and induction of secondary dormancy residing in the tissues surrounding the embryo. Involvement of an abi3 homologue in imposing secondary dormancy has been suggested only in whole seeds of A. fatua (Holdsworth et al., 1999).
Differential display analysis had suggested that P. avium ACONITASE and eukaryotic INITITAION FACTOR 3 SUBUNIT 8 gene expression were elevated in nondormant embryos relative to dormant and germinated embryos. On this basis, it was hypothesized that an elevation of expression concomitant with increasing nondormancy might be observed on the basis that entering and maintaining the nondormant state might require greater demands on energy reserves than primary dormancy (Fig. 2). However, analysis of ungerminated embryonic axes stored at 3°C for 6, 9, 12, 15 and 17 wk did not indicate any increased demand for the aconitase enzyme before a significant increase in expression in visibly germinated embryos, as expected from published studies (Hayashi et al., 1995; Peyret et al., 1995; Eastmond & Graham, 2001; Fig. 3c).
Expression of Pa-AC01 initially declined during storage at 20°C and later regained the higher expression shown in newly imbibed seeds. Some evidence of a peak in Pa-ACO1 expression shortly after the shift to 3°C was revealed (Fig. 3c). Expression of Pa-eIF3 SUBUNIT 8 rose between imbibition and the next sampling point after 2 wk at 20°C. Expression fell during chilling at 3°C, and was only significantly induced in visibly germinated embryos with an increased demand for protein synthesis (Fig. 3d). Expression remained significantly higher in control seeds stored at 20°C, suggesting that Pa-eIF3 SUBUNIT 8 is more actively transcribed or more stable in moist, primary dormant seeds than in those in which dormancy-breaking temperatures have been experienced. These findings are reminiscent of the rates of protein synthesis in whole P. avium seeds described by Michalski (1982). Michalski used 14C-leucine incorporation to monitor protein synthesis, and suggested a higher rate in seeds stored at 20°C than in those stored at 3°C.
eIF3 SUBUNIT 8 transcript in secondary dormant embryos induced by a period at 20°C did not increase; levels continued to fall rather than rising to control values (Fig. 3d). Wrzeœniewski (1985) demonstrated an increase in respiratory rate in cherry embryos on interruption of dormancy breaking with thermal increases to 20°C which returned to baseline within 1 wk. This burst in respiration has been associated with the onset of secondary dormancy, and may be expected to involve ACONITASE and/or eIF3 SUBUNIT 8 expression. As transcript levels were not measured in this study until 3 wk after the thermal shift to 20°C, no information on this possibility was recovered.
The function of eIF3 SUBUNIT 8 is complex and may involve both protein synthesis and degradation (Karniol et al., 1998; Kim et al., 2001; Seeger et al., 2001; Yahalom et al., 2001), and the results presented might suggest that it has an active role in maintaining the state of primary dormancy. These data suggest it has a function in mature P. avium embryos which is lost if they are exposed to dormancy-breaking conditions for even the relatively short period of 6 wk. The discrepancy between quantitative reverse-transcription PCR and expression patterns suggested by differential display analysis for these genes was probably caused by the nonquantitative nature of the differential display assay. It should be noted, however, that aconitase is a dual function protein which also functions as a translational regulator of iron-responsive element-containing transcripts (Haile et al., 1992; Peyret et al., 1995; Beinert et al., 1996), so varying iron availability may also have influenced levels of this transcript.
This study has provided an insight into the molecular mechanisms involved in the control of dormancy and early germination of P. avium embryos. The low genomic copy number of all genes studied here gives us confidence that the quantitative PCR approach detected transcripts from all relevant P. avium paralogues. The finding that homologues of ABI1/ABI2 and ABI3 are expressed in mature P. avium embryonic axes supports physiological evidence for dormancy in the embryo, and is the first molecular evidence for the existence of embryo dormancy in P. avium. Embryo dormancy therefore appears to be lost some time before the whole seed becomes nondormant, and is clearly separated from expression of the germination-indicator genes ACONITASE and eukaryotic INITIATION FACTOR 3 SUBUNIT 8. These findings emphasize the likely importance of the endosperm and seed coat in controlling germinability; indeed, they imply that secondary dormancy in P. avium seeds may not involve dormancy in the embryonic axis, but rather may prove to be entirely controlled by the surrounding endosperm and seed coat. This finding may demonstrate a major difference in dormancy cycling strategies between the woody P. avium and annual A. fatua in which a link between whole-seed ABI3 expression and secondary dormancy has been made (Holdsworth et al., 1999). It will be of interest to use the quantitative PCR methods developed here to monitor the expression of these indicator genes in P. avium endosperm and seed coat during dormancy-breaking treatments.