JXB Advance Access published online on September 26, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm198
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RESEARCH PAPER |
On the role of abscisic acid in seed dormancy of red rice
1C.R.A. – Experimental Institute for Cereal Research, Via S. Protaso 302, I-29017 Fiorenzuola d'Arda (PC), Italy
2Dept Biologia delle Piante Agrarie – Università di Pisa, Viale delle Piagge 23, I-56124 Pisa, Italy
* To whom correspondence should be addressed. E-mail: agianinetti{at}tin.it
Received 26 June 2007; Revised 21 July 2007 Accepted 25 July 2007
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Abstract |
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Abscisic acid (ABA) is commonly assumed to be the primary effector of seed dormancy, but conclusive evidence for this role is lacking. This paper reports on the relationships occurring in red rice between ABA and seed dormancy. Content of free ABA in dry and imbibed caryopses, both dormant and after-ripened, the effects of inhibitors, and the ability of applied ABA to revert dormancy breakage were considered. The results indicate: (i) no direct correlation of ABA content with the dormancy status of the seed, either dry or imbibed; (ii) different sensitivity to ABA of non-dormant seed and seed that was forced to germinate by fluridone; and (iii) an inability of exogenous ABA to reinstate dormancy in fluridone-treated seed, even though applied at a pH which favoured high ABA accumulation. These considerations suggest that ABA is involved in regulating the first steps of germination, but unidentified developmental effectors that are specific to dormancy appear to stimulate ABA synthesis and to enforce the responsiveness to this phytohormone. These primary effectors appear physiologically to modulate dormancy and via ABA they effect the growth of the embryo. Therefore, it is suggested that ABA plays a key role in integrating the dormancy-specific developmental signals with the control of growth.
Key words: Abscisic acid, development arrest, Oryza sativa f. spontanea, pericarp splitting, red rice, seed dormancy, seedling growth
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Introduction |
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Seed dormancy occurs in a wide range of plants whose dispersal units either do not germinate, or germinate slowly, under conditions that are otherwise optimal for germination after dormancy has been removed (Bewley and Black, 1994; Bewley, 1997). Dormancy is also influenced by environmental clues (Bewley, 1997), but how these clues act and how the dormancy level is modulated have yet to be clarified (Bewley, 1997; Kucera et al., 2005). Among other modulation mechanisms, ABA has received a lot of attention (Gubler et al., 2005; Kermode, 2005; Kucera et al., 2005) and, in barley, it has been suggested that synthesis, catabolism, or removal of ABA, as well as sensitivity to ABA, concur to determine seed dormancy (Wang et al., 1995). ABA is known to regulate phase III water uptake in the metabolically active embryo (Bewley, 1997; Kucera et al., 2005) through its influence on water relations (Schopfer et al., 1979; Schopfer and Plachy, 1984; Ni and Bradford, 1992). Although there is considerable circumstantial evidence that ABA is involved in regulating the onset of dormancy and in maintaining the dormant state, there is a paucity of unequivocal evidence that, physiologically, ABA is in fact an important controlling factor in the dormancy of most seeds (Bewley, 1997).
Proof supporting the role of ABA in seed dormancy of many species is provided in a number of studies (Gubler et al., 2005; Kermode, 2005; Kucera et al., 2005): (i) exogenous ABA delays or blocks germination of seeds and embryos; (ii) in the immature seed, ABA maintains the embryo in a developing rather than germinating programme, so that precocious germination, i.e. ‘vivipary’, is inhibited; (iii) in addition to true ‘vivipary’, differences in susceptibility to pre-harvest sprouting have been linked to ABA content; (iv) the capability of the seed to synthesize ABA is necessary to acquire dormancy, so that dormancy is not established in seeds that are deficient in ABA because of mutation, transgenic modification, or chemical inhibition of ABA synthesis; (v) chemical inhibition of ABA synthesis also provokes germination of previously dormant seeds; (vi) on the other hand, overexpression of genes that increase ABA content also delays germination; (vii) soon after incubation at germination temperatures, a greater decrease of ABA occurs in non-dormant seeds as opposed to dormant ones.
However, a direct role of ABA in the physiological modulation of the dormancy level is questionable because: (i) vivipary is a phenomenon distinct from lack of dormancy in the mature seed and, in fact, it also occurs in non-dormant species such as maize (Bewley and Black, 1994); (ii) a number of plants have high ABA levels in the seed but show no dormancy (Bewley and Black, 1994); (iii) also in species with seed dormancy, inconsistent relationships between dormancy intensity in the mature grain and its ABA content have been observed (Kermode, 2005; Millar et al., 2006); (iv) fluridone, an inhibitor of ABA synthesis, was not effective when applied with some delay to the embryo excised from the almost mature sunflower seed (Le Page-Degivry and Garello, 1992), and, if applied alone, was only slightly promotive of germination in seeds of Pinus monticola (Feurtado et al., 2007); (v) during imbibition, the level of ABA also decreases in dormant seeds of many species (Bewley and Black, 1994; Poljakoff-Mayber et al., 2002; Ali-Rachedi et al., 2004; Millar et al., 2006; Feurtado et al., 2007); (vi) the pattern of de novo protein synthesis of non-dormant seeds incubated with ABA does not correspond to that of imbibed dormant seeds (Chibani et al., 2006).
Red rice (Oryza sativa f. spontanea) is a weedy rice that, given the complete lack of germination in the dormant seed of some accessions, has been proposed as a model plant to elucidate the mechanisms of dormancy in grasses (Cohn, 1996; Chao et al., 2005). As in cultivated rice and many other plant species, dormancy of red rice is commonly removed by dry after-ripening the seed (Footitt and Cohn, 1992; Bewley, 1997; Gianinetti et al., 2007).
The aim of this work was to evaluate the role of ABA in red rice dormancy (primary dormancy, that is the dormancy shown by the mature seed at dispersal time, was considered here). Both the effects of natural removal of dormancy (dry after-ripening) and of chemicals interfering with ABA synthesis were considered. Finally, the capability of applied ABA to compensate the dormancy-releasing action of the ABA synthesis inhibitor fluridone was tested.
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Plant materials
Seed from a population of straw-hulled red rice from Vercelli (Italy) was multiplied in a single paddy plot close to Vercelli in 2000 and 2001. The seed (botanically, the dispersal units are spikelets, i.e. caryopses covered by the hull; Ellis et al., 1985) was harvested when showing shattering capability. Harvest was performed by gentle hand threshing in 2000 (also slightly immature spikeletes, that are not yet easily shattered, but having firm endosperm, were collected), and by hand shattering in 2001 (only easily shattering grains were collected). After harvest the spikelets were dried for 1 d at 35 °C and stored at –18 °C to preserve dormancy. Samples of the seed were after-ripened in closed containers at 30 °C for different times (either after or before manual dehulling). Some of the dormant seed harvested in 2000 was manually dehulled and caryopses were visually grouped into six classes of colour (A–F) that were tested and after-ripened separately. Seed harvested in 2001 was more uniform (classes C–E) and thus was not subdivided in colour classes. Moisture content of the dried spikelets at the start of experiments was 13.5±0.04% in 2000 and 10.2±0.15% in 2001 (fresh weight basis). No significant differences were observed among caryopses of the different colour classes.
Additionally, eight pots of red rice were grown in Fiorenzuola (Italy) to measure the moisture content of the caryopses of different colours at physiological maturity. In this experiment, over six dates during maturation (24 August to 17 September, 2001), a total of 878 caryopses were harvested, dehulled and immediately weighed (±0.1 mg). Caryopses of the different classes of colour were put together, in groups of about 20, and dried at 80 °C for 7 d to determine dry weight. Moisture content was calculated on a fresh weight basis. The relative frequencies of the six colour classes gradually moved from higher relative abundance of the less coloured classes (classes A–C) to the most coloured ones (classes E and F) over sampling dates.
Chemicals
Fluridone, amitrole, and tungstate are inhibitors of ABA synthesis (Matusova et al., 2005). Gibberellic acid is considered an antagonist of ABA (Kucera et al., 2005). Stock solutions were prepared by dissolving fluridone (Duchefa, Haarlem, The Netherlands), racemic (±)-ABA, and amitrole (3-amino-1,2,4-triazole; Sigma, St Louis, MO, USA) in dimethylsulphoxide (DMSO; Sigma) to a concentration of 0.1, 0.1, and 0.2 M, respectively. Gibberellic acid (GA3 
50%; Sigma) was dissolved in 10:90 (v/v) DMSO/water to 0.1 M. Sodium tungstate (BDH, Poole, Dorset, UK) was dissolved in water to 0.1 M. Fresh solutions of D-(+)-glucose (BDH) were prepared directly. When pH buffering of the incubation medium was required, 1,2,3,4-butanetetracarboxylic acid (BTCA; Aldrich, Steinheim, Germany) that provides pH buffering over a wide physiological range (Dean, 1985) was used; pH was adjusted with freshly prepared 5 M NaOH.
Germination tests
Two stages of germination were recorded: (i) pericarp splitting, the first visible sign of germination (Karssen, 1976; Schopfer et al., 1979; Footitt and Cohn, 1995; Gianinetti et al., 2007); and (ii) first growth stage (S1) (Counce et al., 2000), recorded when rootlet or coleoptile were 1 mm (minimal visible seedling growth). Although the latter is the conventional stage at which germination is recorded, protrusion of the radicle (or coleoptile in the case of rice) is the visible evidence that germination has actually occurred (Bewley and Black, 1994; Kucera et al., 2005). Indeed, stage S1 includes some early seedling growth that makes germination easier to see (Schopfer et al., 1979; Gianinetti et al., 2007). Therefore, the seed can show different responses at the two stages when post-germinative seedling growth, rather than germination sensu strictu, is effected (Karssen, 1976; Schopfer et al., 1979; Footitt and Cohn, 1995; Gianinetti et al., 2007). Pericarp splitting was determined, with the aid of a magnifier, as the opening of the red pericarp covering the swelling embryo axis into two lips, disclosing the underlying tissues. Seedlings attaining growth stage S1 were always discharged after recording. At the end of each of the tests described below, non-germinated caryopses were transferred to plastic Petri dishes (90 mm diameter; Bibby Sterilin, Stone, Staffs, UK) with two sheets of filter paper (90 mm diameter; Schleicher & Schuell, Dassel, Germany) and 4 ml water, and assayed for viability by longitudinally cutting the endosperm from the distal edge to the proximity of the embryo with a surgical blade (Gianinetti et al., 2007). In addition, 50 µl ethanol was added to the incubation medium (1.25% final concentration). Caryopses that had not attained growth stage S1 in 1 week of incubation at 30 °C were considered not vital.
To establish after-ripening curves (dry after-ripening, 30 °C) for both spikelets and dehulled caryopses, germination treatments were performed in square (10x10 cm) Petri dishes (Bibby Sterilin) with 12 ml of water, maintained inclined in humidity boxes during incubation at 30 °C for 14 d. Twenty spikelets, or caryopses, were placed in each dish between two layers of Kleenex facial tissue. Depending on the experiment, a total of 140 grains or 120 caryopses was tested for germination in each treatment. Germination was recorded weekly. As seedling growth stage S1 closely follows pericarp splitting in seed incubated in the absence of inhibitors (Footitt and Cohn, 1995), seedling growth stage S1 was used to mark germination in this experiment. Total viability averaged 97±1%.
Experiments with inhibitors and gibberellic acid were performed in disposable round Petri dishes using only dehulled caryopses. For each treatment, replicated dishes were prepared by placing 20 caryopses on two circles of filter paper with 5 ml of incubation medium. A total of 200 caryopses were counted for germination in each treatment. The appropriate medium was prepared by diluting the stock solution of each inhibitor in water or 20 mM BTCA buffer at the required pH. Petri dishes were maintained flat in humidity boxes during incubation at 30 °C for 14 d; thereafter, non-germinated caryopses were assayed for viability. Germination was recorded after 1 d of imbibition and then every other day. The effect of glucose was tested in the same conditions, but the caryopses were pre-imbibed for 1 d in water, transferred to 2% glucose for 1 d, and then transferred to water again.
To determine sensitivity to ABA in both non-dormant and fluridone-treated dormant caryopses, the same incubation system as described above for inhibitors was used, with caryopses being rinsed, blotted, and transferred to new Petri dishes with fresh incubation solutions 1 d after the start of the experiment, and then every other day. One hundred caryopses were tested for germination in each treatment. At the end of the 14 d test, non-germinated caryopses were rinsed, blotted, and transferred to Petri dishes with water; if necessary, further rinses were performed. After that, non-germinated caryopses were assayed for viability: it averaged 98±1% for dormant and 99±1% for non-dormant caryopses.
To assay the effect of pH on ABA capability to revert fluridone-induced germination, the same experimental system as for tests with inhibitors was used for dormant caryopses, but incubation media were buffered at pH 4.4 and 6.8 with 20 mM BTCA. Depending on the experiment, a total of 60 or 200 caryopses was tested for germination in each treatment. One day after the start of the experiment, and then every other day, the caryopses were transferred to new Petri dishes with fresh incubation solutions. The volume of 5 M NaOH required to adjust medium pH was measured and osmolarities of unbuffered control and pH 4.4 medium were adjusted to that of the medium at pH 6.8 by adding NaCl. Universal indicator paper (Riedel-de Haën, Seeize, Germany) was used to check that the pH in the disposable dishes did not show visible change with respect to the original buffer solution. Preliminary experiments showed that the use of a traditional buffer system like citrate/phosphate over long incubation times was particularly troublesome because of the growth of micro-organisms (moulds at pH 4.4 and bacteria at pH 6.8) notwithstanding the frequent changes of dish and even if the caryopses were rinsed at each dish change (not shown). In BTCA-buffered solutions no visible growth of micro-organisms was observed and the need to rinse the caryopses was overcome. Viability averaged 99±1% in all treatments.
The effect of medium pH on the content of ABA in the embryo and endosperm (de-embryonated caryopsis) was tested in 20 mM BTCA buffer either alone or in the presence of 10 µM fluridone or 10 µM fluridone and 10 µM ABA. Caryopses were incubated in disposable round Petri dishes (two dishes with 10 caryopses each, for every test). Ungerminated caryopses were collected after 7 d of incubation (dishes and incubation solutions were changed at 2, 4 and 6 d) and ABA extracts were immediately prepared as below. All the unanalysed caryopses were verified to be viable. Three-way ANOVA (fixed factors: pH, test solution, and tissue) and Tukey test were used to evaluate the significance of the differences. In performing ANOVA, data were transformed as X1/2+(X+1)1/2 to compensate for the unequal variances that were proportional to the magnitude of the means (Zar, 1999).
Determination of ABA
Abscisic acid (free form) was determined in crude aqueous extracts of single grains by solid phase radioimmunoassay (RIA) using the monoclonal antibody DBPA-1 (Vernieri et al., 1989), which proved to be highly specific for S(+)-ABA (Walker-Simmons et al., 1991). Single caryopses (or embryos, endosperms, and hulls) were weighed, squashed in a 1.5 ml Eppendorf tube, frozen in liquid nitrogen, then thawed and extracted with 0.5 ml of distilled water overnight at +5 °C in the dark. Thereafter, 50 µl aliquots were assayed in triplicate by RIA. Validation of RIA results on rice seed tissues by HPLC fractionation of crude extracts and by internal standardization experiments was as previously described (Ikeda et al., 2002).
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Results |
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After-ripening
Dry after-ripening of fully dormant red rice caryopses is commonly accomplished by storing dry spikelets at a fixed temperature (usually 30 °C), and dehulling them to obtain naked caryopses just before germination tests (Footitt and Cohn, 1992, 1995). However, as in this work some experiments required classification of caryopses according to their colour at harvest, and because after-ripening alters the colour of the caryopses, dehulling before after-ripening was adopted in such cases. A comparison of the results obtained with the two methods is shown in Fig. 1 for the unsorted seed, indicating a modest diversity between them. Such a difference is significant for after-ripening to germination percentages around 50%, so that dehulling before after-ripening increases fastness of after-ripening, reducing tAR(50) of about 3 d. However, no substantial diversity in the after-ripening curve was observed, and then this effect is not relevant if samples prepared with the same method are compared.
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ABA in the dry grain
Seed harvested in 2000 was dehulled and subdivided into six classes by colour (Fig. 2). ABA content was significantly dependent on the colour class (Fig. 3A). Indeed, the pot experiment in 2001 showed that grain moisture content at harvest is dependent on colour class as well (Fig. 4), so that reddening of the caryopsis on the plant corresponded to decreasing moisture and ABA content. They are, of course, only correlative processes; in fact, when seed was harvested and artificially dried in a short time, to a uniform moisture content, the caryopsis colour was not substantially affected. Despite this, such an association could be usefully employed to reduce unexplained variability in grain ABA content. In this way, it could be observed that dry after-ripening the dormant seed for 42 d rendered it fully germinating (Fig. 3B), but did not affect ABA content in any class of seed (Fig. 3A). In addition, median after-ripening time, tAR(50), which is an index of dormancy (Roberts, 1961), did not correlate with differences in ABA content among the colour classes (Fig. 3C).
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The proportion of ABA in the three different parts of the spikelet (hull, embryo, endosperm) was the same in colour class A (highest ABA content) as well as in colour class F (lowest ABA content) (Fig. 5). This indicates that, although the embryo showed a higher ABA content (Fig. 5), the ABA reduction during maturation occurred with the same intensity in the different tissues. Analogously, the distribution of ABA in the three tissues was the same in both dormant and fully germinating seed (Fig. 6). In addition, the ABA content was roughly proportional to the weight of each tissue with respect to the whole spikelet, though the embryo showed a significantly higher proportion of ABA both in the dormant and the germinating caryopses (Fig. 6). These results support the suggestion that the ABA content of the caryopsis can be used as a representative measure of relative ABA level over different tissues, at least when comparing after-ripening times. Similar values were reported by Qin et al. (1990) for non-dormant caryopses of japonica rice. Relevant differences, however, occurred in red rice between caryopses of diverse degrees of maturation (Fig. 5).
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Seed imbibition
During imbibition of naked caryopses from the 2001 harvest (Fig. 7A), differences in ABA levels were observed between dormant and non-dormant (fully after-ripened) caryopses after 12 h. To reduce variability in the ABA content and then to highlight variations in ABA levels linked to imbibition, caryopses of colour-class E (harvest 2000) were also tested. This experiment confirmed that ABA levels in dormant and non-dormant caryopses differed after 12 h of imbibition (Fig. 7B). It also showed that a peak of ABA could be detected for both dormant and non-dormant caryopses at 12 h. However, when testing the seed harvested in 2001, no peak was detected within the time the non-dormant seed attained full germination, although a peak of ABA evidently occurred later (Fig. 7A). Therefore, the presence of a peak of ABA early during imbibition was linked to the harvest lot but not to the dormancy status. Although the absolute amounts of ABA in the dry grain as well as in caryopses imbibed for 24 h varied greatly between the two harvest lots, a large amount of ABA was observed in non-dormant caryopses of both lots, also after germination occurred (Fig. 7). Moreover, a stable level of ABA (0.4–0.8 ng caryopsis–1) was reached in imbibed dormant caryopses of the 2001 seed lot (Fig. 7A) as well in caryopses of colour classes A and F (seed lot 2000) after some days of incubation (not shown). Interestingly, this ABA level was lower than (harvest 2001; Fig. 7A) or similar to (harvest 2000; Fig. 7B) the ABA level observed in the germinating non-dormant seed.
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Effect of chemicals
Glucose had no effect on the dormant seed (Table 1). The effect of inhibitors of ABA synthesis (fluridone, amitrole, tungstate) and of gibberellin on germination (Table 1) and on the ABA level (Fig. 8) were tested. A reduced level of ABA at 12 h of imbibition was confirmed in non-dormant compared with dormant caryopses, whereas an intermediate behaviour was shown at such time for all the chemical-treated caryopses (Fig. 8). However, after 4 d of incubation (96 h), when the non-dormant seed had already germinated but the fluridone-treated dormant seed had not (Table 1), a significant reduction in ABA content, below that of untreated dormant seed, was shown for fluridone-treated dormant seed. At the same time, an increase in the ABA level was observed for dormant caryopses treated with amitrole, tungstate, and gibberellin (Fig. 8), with such treatments being ineffective in breaking dormancy (Table 1). Neither did germination occur if tungstate, gibberellin, and amitrole were mixed, but full germination occurred if fluridone was included in the mixture too (not shown). Norflurazon (80% p.a.; Novartis Crop Protection AG, Basel, Switzerland) was as effective as fluridone in breaking dormancy, but longer times or higher concentrations were necessary to induce 100% germination (not shown). A longer experiment was set up to compare effects of fluridone (effective in breaking dormancy) with amitrole (ineffective) on dormant caryopses (Fig. 9). Again, amitrole showed no significant effect on ABA levels of dormant caryopses, whereas the effect of fluridone was confirmed.
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In the experiment reported in Fig. 9, after imbibition with fluridone, ABA slowly decreased to about 0.12 ng caryopsis–1 at 7 d of imbibition (Fig. 9A). Caryopses that had already germinated (Fig. 9B) were excluded from ABA determination, thus this ABA level is that immediately preceding germination of most of the caryopses and, because of the very gradual decrease at 7 d, 0.12 ng caryopsis–1 represents the best approximation to the minimal level of ABA preventing germination itself. After such a threshold in ABA level was reached, full seed germination occurred within 2 d. Complete germination was also obtained when dormant caryopses pre-imbibed in water for one to several weeks were transferred to Petri dishes with fluridone, though germination was anticipated by about 1 d (not shown).
Sensitivity to ABA
Figure 10 shows the effect of the level of exogenous ABA on the progress of germination through pericarp splitting and seedling growth stage S1. In non-dormant seed (Fig. 10A), the pericarp splitting was reached within 1 d of incubation at all but the highest ABA concentration (1 mM), which had a delayed germination. A greater effect of ABA on the seedling growth stage S1 was observed (Fig. 10B): a delay was seen in the presence of 10 µM ABA and a strong to complete reduction occurred at the higher concentrations. At 100 µM, only removing ABA from the incubation medium restored full germination, whilst at 1 mM a second change of medium was needed before seedling growth could occur, and slow germination was observed even after three changes of the medium. This suggests that some residual ABA was retained in the seed after the first rinse and also that some persistent block to germination was induced. Fluridone-treated dormant caryopses, although fully germinating after about 1 week of incubation, were more sensitive to ABA than non-dormant caryopses; pericarp splitting was already reduced at 100 µM, and in 1 mM ABA it occurred only after removal of ABA from the medium (Fig. 10C). Growth of the seedling to 1 mm was still more sensitive, with full inhibition recorded from 10 µM ABA upwards (Fig. 10D). A further delay was observed at 100 µM ABA and, as in the non-dormant seed, a second change of medium was required before the block to seedling growth stage S1 by 1 mM ABA could be partially removed, confirming retention of ABA in the seed after the first rinse. However, by contrast with what was observed for non-dormant caryopses incubated with 1 mM ABA, no delay of seedling growth stage S1 occurred in these caryopses after the second transfer to water, suggesting that some production of endogenous ABA could be necessary to permit such a slowdown. Thus, although non-dormant caryopses were less sensitive to ABA, the persistent effect of 1 mM ABA in the non-dormant seed represented an exception to the general trend, and seems be due to a post-germination effect of exogenous ABA that requires the synthesis of endogenous ABA. In all cases, when dormant seed was treated with fluridone, the effect of the inhibitor, i.e. dormancy breaking, remained after two subsequent incubations in water, suggesting that its action, and probably its presence in the seed, persists after the removal of exogenous supply.
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The effect of pH
The effect of medium pH on the effectiveness of exogenous ABA to revert breaking of dormancy by fluridone in dormant seed was verified at close-to-neutral and acidic pH values (Fig. 11). The minimal level of ABA that in the previous trial had shown some relevant effect (i.e. 10 µM for seedling growth in fluridone-treated dormant seed) was used in this experiment. The incubation media were all kept at the same low osmolarity, calculated to be about 100 mOsm l–1. This value approximately corresponds to a water potential of –0.25 MPa, a level that has a negligible effect on the germination behaviour of after-ripened red rice seed (Gianinetti and Cohn, 2007).
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As observed in the previous experiment, in the unbuffered medium, fluridone-induced seedling growth (stage S1), but not pericarp splitting, was suppressed by 10 µM ABA (Fig. 11). However, at pH 6.8 the ability of ABA to revert fluridone-induced seedling growth was strongly reduced. On the contrary, at pH 4.4 both pericarp splitting and growth stage S1 were strongly reduced by ABA. Complete germination of viable seed (99%) was confirmed after transfer to water in all samples (not shown).
To verify the effect of pH on the penetration of exogenous ABA, in a parallel experiment free ABA was measured in ungerminated caryopses at 7 d of incubation (Table 2). Measures of ABA in the embryo and endosperm of seed treated with fluridone and ABA (Table 2) showed that at pH 4.4 there was a significant increase in ABA concentration both in the embryo and endosperm as compared with pH 6.8. Weights of embryos, endosperms, and whole caryopses averaged 1.5±0.3 mg, 27.5±1.9 mg, and 29.0±1.9 mg (mean±standard deviation), respectively. The present data (Table 2) indicate a greater accumulation of ABA in the embryo than in the endosperm, consistent with the higher proportion of symplastic volume of the former (Bruggeman et al., 2001). In fact, because of the ion-trap mechanism, the phytohormone accumulates in the cellular symplast (Bruggeman et al., 2001). As a consequence, in the untreated seed, at pH 4.4 almost all the ABA should accumulate in the symplast, whereas at pH 6.8, the apoplastic concentration of ABA should be about one-third of the symplastic concentration (assuming a symplastic pH value of 7.3; Footitt and Cohn, 1992). Thus, the higher concentrations of ABA at pH 6.8 were consistent with the additional accumulation of ABA that is predicted to occur in the apoplast at this pH.
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The interaction of pH with the ability of ABA to revert fluridone action was further studied in a longer experiment (Fig. 12). Over 36 d of incubation, pericarp splitting was attained by almost all the caryopses at both pH 6.8 and pH 4.4 as well as in the unbuffered control, although at pH 4.4 a relevant delay was observed (Fig. 12A). Seedling growth stage S1 showed a stronger response to ABA (Fig. 12B); almost complete blockage occurred at pH 4.4 as well as in the unbuffered control, whereas only a partial reduction in fluridone-induced seedling growth was observed at pH 6.8. The similar response of seedling growth at pH 4.4 and in the unbuffered control is consistent with an acidic pH of the embryo apoplast in the unbuffered control as well. On the other hand, the greater effectiveness of ABA in controlling pericarp splitting at pH 4.4 suggests a bottleneck for entry of the chemical into the seed (Karssen, 1976; Cohn et al., 1987).
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This work provides a uniquely detailed picture of the relationships between seed dormancy and ABA in a model plant (Chao et al., 2005). In this sense, a useful trait of red rice is that the red colour of the caryopsis gradually appears during maturation (Fig. 2). The close association between the colour of the caryopsis and the content of ABA (Fig. 3) makes it possible to sort the seed for colour to reduce the variability in ABA concentration due to differences in ripening.
ABA content of the dry seed
Analogously to barley and oat (Jacobsen et al., 2002; Poljakoff-Mayber et al., 2002), as well as cultivated rice (Qin et al., 1990) also in red rice the embryo shows a higher concentration of ABA than the endosperm (Fig. 5). However, by contrast to oat (Poljakoff-Mayber et al., 2002), in the rice caryopsis most of the ABA is in the endosperm (Fig. 6; Qin et al., 1990). In any case, consistent with reports for oat as well as barley and Arabidopsis (Jacobsen et al., 2002; Poljakoff-Mayber et al., 2002; Millar et al., 2006) the amount of ABA in the dry seed was not associated with the intensity of dormancy (Fig. 3). Neither was the difference in embryo's ABA between caryopses of different degrees of maturation related to dormancy (compare Figs 5 and 3C). Indeed, Karssen et al. (1983) showed that embryonic (endogenous) ABA controls dormancy inception in Arabidopsis. However, having observed that ABA levels decreased in wild type at the end of seed maturation while dormancy increased, those same authors suggested that ABA is necessary to induce a state of arrest but thereafter may not be responsible for the physiological control required to maintain dormancy. It has been proposed that ABA is the main factor involved in rice seed dormancy and both natural and artificial breaking of dormancy would be proportional to the degradation of endogenous ABA (Hayashi, 1987). However, the present results support the view that dormancy breaking by dry after-ripening does not act via modifications in ABA content. Thus, consistent with the indications of Jacobsen et al. (2002) for barley, something other than absolute content of ABA appears to distinguish dormant from after-ripened seed.
ABA in imbibing caryopses
Studies on many species indicate that the synthesis of ABA after imbibition is a feature of the dormant seed (Kermode, 2005). However, it can occur in non-dormant seed as well (Fig. 7A, B; Bewley and Black, 1994). Indeed, after-ripening involves the development of the ability to reduce the amount of ABA quickly following hydration (Fig. 7A, B; Jacobsen et al., 2002; Kermode, 2005; Millar et al., 2006). In our view, however, this change in ABA turnover can be seen as a consequence of dormancy removal by after-ripening, and does not appear to support the view that ABA is the cause of dormancy.
Moreover, after some days of incubation, free ABA in the dormant seed can decrease below any level previously observed in the non-dormant germinating seed before and during pericarp splitting (Fig. 7A). Hence, the level of ABA is not directly linked to the dormancy status even during imbibition. Indeed, the amount of ABA is not likely to be immediately indicative of changes in dormancy status, although more intense ABA metabolic flux has been suggested to be a superior indicator of dormancy termination (Kermode, 2005; Millar et al., 2006). However, a central role for ABA metabolism in the physiological modulation of dormancy can only be invoked if ABA itself has been proved to bear that role.
The effects of chemicals
Dormant red rice caryopses are not induced to germinate by glucose (Table 1). Thus, assuming that exogenous glucose can be taken up by the embryo of ungerminated caryopses (Abdul-Baki, 1969) a block in reserve mobilization does not seem to be the basis of dormancy in red rice. On the other hand, dormant seed was induced to germinate by fluridone after an almost complete depletion of endogenous ABA, whereas the inability of the other chemicals (GA3, tungstate, and amitrole) to break dormancy was indeed linked to their inefficacy in reducing the ABA content. However, fluridone-induced germination occurred only after ABA content was reduced well below the level observed in after-ripened, non-dormant caryopses, and this required several days. Germination induced by fluridone was thus notably delayed with respect to the after-ripened caryopses. Similarly, a minimal ABA threshold level, rather than a direct proportionality between ABA levels and the intensity of dormancy, has been invoked to be necessary for maintenance of seed dormancy homeostasis in Pinus monticola (Feurtado et al., 2007). Although some level of ABA appears to be necessary to maintain the seed in a state of dormancy, this phytohormone cannot be considered the primary cause of dormancy in the imbibed seed.
Sensitivity to exogenous ABA
Both for the non-dormant and the fluridone-treated dormant caryopses, the attainment of seedling growth stage S1 was highly responsive to exogenous ABA and inversely related to the applied concentration of ABA (Fig. 10B, D) as observed in non-dormant wheat at a similar stage (Morris et al., 1989). In dormant caryopses that had been induced to germinate by fluridone, pericarp splitting was also prevented by the application of exogenous ABA, although quite higher, non-physiological, concentrations of ABA were required than was needed to prevent seedling growth stage S1. By contrast, in after-ripened caryopses, pericarp splitting was actually unaffected by ABA. Thus, in non-dormant caryopses ABA blocked only post-germinative growth. Indeed, also in non-dormant dicots, ABA does not affect the kinetics of testa rupture, whereas further growth is highly responsive to ABA inhibition (Karssen, 1976; Schopfer et al., 1979; Schopfer and Plachy, 1984; Leubner-Metzger, 2003).
The fact that pericarp splitting was responsive to different concentrations of exogenous ABA in dormant seed treated with fluridone (Fig. 10) provided an indirect confirmation that ABA can enter the caryopsis before the pericarp is split. Although extra-embryonic tissues may slow the movement of exogenous ABA during imbibition, this phytohormone is absorbed by intact seeds and is then active on the embryo (Karssen, 1976; Morris et al., 1989). This tenet is further supported by data confirming that exogenous ABA accumulates in the seed before pericarp splitting at both neutral and acidic pH (Table 2).
The ability of ABA to block the promotion of germination induced by fluridone was also observed in seeds of Nicotiana plumbaginifolia (Grappin et al., 2000) and in almost mature embryos of Helianthus annuus (Le Page-Degivry et al., 1990). However, the attainment of pericarp splitting and growth stage S1 was more sensitive to exogenous ABA in caryopses induced to germinate by fluridone than in caryopses that had been after-ripened (Fig. 10). This greater responsiveness to ABA demonstrates that ABA sensitivity is a dormancy-related trait that is removed by dry after-ripening but not by fluridone. This difference indicates that dormancy involves at least one factor, additional to ABA, that modulates the sensitivity to ABA and that, evidently, is not affected by fluridone.
The ABA effect is dependent upon sensitivity to ABA (Ni and Bradford, 1992) and in a number of species it has been demonstrated that such responsiveness is related to the degree of dormancy (Walker-Simmons, 1987; Morris et al., 1989; Ried and Walker-Simmons, 1990; Wang et al., 1995; Le Page-Degivry et al., 1996; Grappin et al., 2000; Schmitz et al., 2002). However, many factors either inducing or breaking dormancy show increased or decreased efficacy, respectively, in relation to the level of dormancy itself (Vegis, 1964; Bewley and Black, 1994; Finch-Savage et al., 2007). It is then at least questionable to state that ABA is the primary cause of dormancy whereas changes in the responsiveness to other factors are effects of dormancy. In addition, ABA responsiveness appears to be directly related to the capacity to synthesize ABA (Le Page-Degivry et al., 1996; Grappin et al., 2000; Ali-Rachedi et al., 2004) and to reduced ABA catabolism (Jacobsen et al., 2002; Schmitz et al., 2002; Kermode, 2005; Millar et al., 2006; Feurtado et al., 2007). Thus, both ABA turnover and sensitivity appear to be modulated by dormancy.
The effect of pH
Fluridone was effective in depleting almost all endogenous ABA in 7 d (Fig. 9A; Table 2), and inducing germination (Table 1; Figs 9B, 11, 12). Incubation in the presence of exogenous ABA increased the amount of the phytohormone in the caryopsis to well above that in the untreated control at both pH values (Table 2). The greater proportion of ABA in the endosperm of treated seed was probably due to extracellular diffusion of the ABA provided exogenously. The pH of the incubation medium showed a relevant effect on the accumulation of exogenous ABA. Indeed, weak acids (like ABA) have low ability to penetrate through the seed coats at pH values higher than their pK because the movement of the undissociated form of weak acids into seeds is not favoured (Cohn et al., 1987). Thus, the greater total amount of exogenous ABA entering the seed at pH 4.4 (Table 2) suggests that this effect of pH was related to permeation through the seed coat (the pK of ABA is 4.75; Bruggeman et al., 2001). On the other hand, the higher embryo/endosperm ratio of ABA accumulation at pH 4.4 compared with pH 6.8 (Table 2) confirms that, because of the ion-trap mechanism (Bruggeman et al., 2001), the symplastic accumulation of exogenous ABA is also favoured at pH 4.4. The summed effect resulted in a high concentration of exogenous ABA in the embryo of caryopses incubated at pH 4.4.
However, even at the more acidic pH, which favoured the accumulation of exogenous ABA into the embryo and endosperm above the levels of the untreated control (Table 2), ABA was not able to revert the dormancy-breaking effect of fluridone (Fig. 12A). In addition, although this seed showed an ABA concentration that was comparable to that of the dry seed (compare Table 2 with Fig. 5), almost immediate seedling growth occurred following transfer to water (Fig. 12B). By contrast, dormant caryopses directly imbibed in the presence of fluridone needed about 1 week to deplete endogenous ABA and germinate (Fig. 9). Note that they took 5–6 d if they were pre-imbibed in water for some days before the application of fluridone (not shown). Inability of ABA to fully restore dormancy in the presence of fluridone suggests that some other factor effects dormancy, and that, like ABA, this factor is also depleted by fluridone.
An integrated role for ABA action
Taken together the present results confirm the importance of ABA in the seed dormancy of red rice, but do not support ABA as the key factor in the physiological maintenance of dormancy. Indeed, ABA appears to mediate dormancy in concert with other endogenous constituents (Morris et al., 1989), and the regulation of seed developmental processes involves cross-talk between different interacting components, with both ABA-dependent and ABA-independent steps (Wang et al., 1998; Kermode, 2005; Kucera et al., 2005). In particular, gibberellin promotes germination and antagonizes the action of ABA during such a phase (Kucera et al., 2005). Physiologically, the accumulation of active gibberellin in the embryo mostly occurs only after ABA content has decreased (Gubler et al., 2005). Thus, the response of dormant caryopses to exogenous GA3 (Fig. 8) suggests that the seed was in a physiological state predisposed to the action of ABA, and not of gibberellin. In fact, application of GA3 was counteracted by an increment in the ABA content, rather than causing a decline in ABA as could be expected (Kucera et al., 2005). A high ratio in the ABA/GA balance is indeed favoured in dormant seeds (Kucera et al., 2005; Finch-Savage and Leubner-Metzger, 2006).
In the course of germination, at least during a narrow developmental window, the embryo remains competent to respond to ABA with growth arrest (Schopfer et al., 1979; Lopez-Molina et al., 2001). Thus, the ability of ABA to modulate embryo growth extends to the growth of the seedling, and ABA appears to be more efficient as an early growth inhibitor than as an inhibitor of germination (Lopez-Molina et al., 2001). Indeed, ABA provokes a cascade of up- and down-regulation of proteins (Chibani et al., 2006), but its primary action in controlling germination rates and percentages consists in preventing cell wall loosening by reducing the extensibility of the cell wall and increasing the minimum turgor threshold for cell expansion (Schopfer and Plachy, 1985; Welbaum et al., 1990). Analogously, the role of ABA in seed dormancy appears to be to limit weakening of the structures surrounding the embryo, cell wall loosening, and radicle extension, which are the later events associated with the breaking of seed dormancy (Bewley, 1997). On the contrary, gibberellin increases the growth potential of the embryo and is necessary to overcome the mechanical restraint conferred by the seed-covering layers by weakening of the tissues surrounding the embryo (Kucera et al., 2005; Finch-Savage and Leubner-Metzger, 2006). Various mechanisms have been suggested to explain these effects (Bewley, 1997; Kermode, 2005; Kucera et al., 2005; Finch-Savage and Leubner-Metzger, 2006). The net result is, however, that ABA prevents the embryo from entering the growth phase of germination (Bewley, 1997). Hence, ABA appears to act as a growth regulator also in the context of dormancy and germination, and not as a dormancy-specific factor. Similarly, the control of development in immature cereal embryos has been suggested not to be mediated directly by ABA, but continued embryo development is nonetheless possible because the phytohormone inhibits water uptake and then prevents precocious germination (Morris et al., 1988).
We therefore deem that unidentified physiological modulators that are specific to dormancy stimulate ABA synthesis and, most importantly, enforce the responsiveness to this growth inhibitory stress hormone. Thus, through it, they affect the growth of the embryo and its speed. One modulator, depleted by fluridone, would explain the inability of ABA to revert fluridone promotion of germination; the other, unaffected by fluridone, would explain the greater sensitivity to ABA of the seed that is induced to germinate by fluridone. Hyper-sensitivity to ABA would then slow down or block the kinetics of germination by mediating the control of expansion growth. Indeed, key protein kinases mediating the control of responses to ABA during seed germination, dormancy, and seedling growth have recently been identified in Arabidopsis thaliana (Fujii et al., 2007). Dormancy-specific factors are also evidently required to predispose the embryo to respond to ABA with the pattern of protein synthesis that is proper for this developmental stage (Chibani et al., 2006). Figure 13 shows the working scheme for the proposed mechanism of dormancy modulation, including a feedback mechanism that can account for the reinforcement of dormancy that occurs in response to unfavourable germination conditions (Bewley and Black, 1994; Gianinetti and Cohn, 2007) as well as the key role of ABA in the initiation of seed dormancy (Karssen et al., 1983). In addition, the present model considers that non-physiological interventions that force the dormant seed to germinate, like depletion of ABA by fluridone, may represent shortcuts that bypass the physiological breakage of dormancy normally carried out by dry after-ripening, without fully terminating the developmental programme associated with dormancy (Fig. 13). Further studies will extend this model to include the antagonism between ABA and gibberellin as well as the removal of covering layer restraints.
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ABA is confirmed to be a necessary but not sufficient factor of seed dormancy, and, at least in red rice, it is not a physiological modulator of the dormancy status. Instead, ABA sensitivity and then any biological role of the phytohormone into dormancy seem to be expressed conditionally to some, unknown, determining factors. Therefore, we propose that ABA plays a key role in integrating the dormancy-specific developmental signal(s) with the control of growth, and in this way it assists, but does not direct, the physiological modulation of dormancy.
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