Journal of Experimental Botany, Vol. 51, No. 347, pp. 1159-1162,
June 2000
© 2000 Oxford University Press
Short Communication Papers |
Changes in abscisic acid content and embryo sensitivity to (+)-abscisic acid during the termination of dormancy of yellow cedar seeds
1 Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
2 Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada
Received 16 December 1999; Accepted 15 February 2000
Abstract
Yellow cedar seeds are dormant at maturity. The abscisic acid (ABA) content of the embryo (but not the megagametophyte) decreased approximately 2-fold following exposure of seeds to a dormancy-breaking treatment; this process was also accompanied by a 10-fold lowered sensitivity of the embryo to S-(+)-ABA. A decline in ABA within the seed is not sufficient for dormancy breakage; reduced embryo sensitivity to ABA is also required.
Key words: Coat-imposed dormancy, embryo, megagametophyte, abscisic acid, seed germination, yellow cedar.
Introduction
Yellow cedar (Chamaecyparis nootkatensis [D. Don] Spach) inhabits a narrow coastal band extending from Prince William Sound in Alaska to southern Oregon, with some small isolated stands outside its main area of distribution. Increased harvesting of trees at high elevations has led to a greater demand for yellow cedar stock for reforestation. After pollination, cones and seed take between 1.5 and 2 years to mature depending upon the elevation and environmental factors such as temperature (reviewed in Owens and Molder, 1984
). Following dispersal from the parent tree, yellow cedar seeds are unable to germinate and most require a year to undergo moist chilling and break dormancy (Pawuk, 1993).
The dormancy mechanism of yellow cedar is complex; it is primarily but not exclusively coat-imposed (Ren and Kermode, 1999). Embryo immaturity at the metabolic and physiological levels is not a contributing factor (Xia and Kermode, 1999). Leachable inhibitors supplied by the megagametophyte appear to play a role in maintaining the embryo in a dormant state (Ren and Kermode, 1999). In addition, the megagametophyte also plays a role as a mechanical barrier to prevent radicle protrusion, a factor which may also involve regulation by ABA and other hormones such as gibberellins (through regulation of cell wall rigidity) (Ren and Kermode, 1999).
Here the role of ABA in dormancy maintenance is investigated by quantifying changes in ABA in the embryo and megagametophyte at different times throughout an effective dormancy-breaking treatment. Changes in embryo sensitivity to S-(+)-ABA were also examined.
Materials and methods
Seed materials and dormancy-breaking and control treatments
Mature yellow cedar seeds of seedlot 30156 (previously collected from natural stands by MacMillan Bloedel and obtained from the Tree Seed Centre in Surrey, BC, Canada) were used for all analyses because of their high viability. For dormancy termination, seeds were subjected to a 72 h running water imbibition at 22±1 °C and then incubated in near darkness at 25 °C for 4 weeks (warm moist treatment), then transferred to 4 °C for 8 weeks (moist chilling). Throughout the warm and cold treatments, seeds were kept moist as outlined previously (Ren and Kermode, 1999). After the 87 d dormancy-breaking treatment, seeds were placed in germination conditions (30 °C days, 20 °C nights with an 8 h photoperiod; light intensity at 25 µmol m-2 s-1, PAR 400–700 nm) (Ren and Kermode, 1999). A control treatment was carried out in which seeds were subjected to the 3 d soak and 4-week warm, moist period; however the subsequent 8 weeks of moist chilling was substituted with an equivalent period in warm, moist conditions (25 °C). No germination of the whole seed was elicited by the control treatment.
ABA extraction
At different time points throughout the dormancy-breaking treatment, embryos and megagametophytes were dissected from seeds, flash frozen in liquid N2 and stored at -80 °C until needed. The frozen tissues were ground in a tissue homogenizer in 1.5 ml 95% isopropanol containing 5% glacial acetic acid and allowed to extract overnight on a shaker (50 RPM) in the dark at 23 °C. Five ng of an internal standard (d6-ABA: 3', 5', 5', 7', 7', 7'-ABA) was added prior to overnight extraction. Extraction of ABA was as outlined previously (Qi et al., 1998). The resulting residue was dissolved in 200 µl MeOH, placed in a vial and dried under N2. All samples were stored at -20 °C prior to GCMS analysis. All data points were based on three replicates of 15 seed parts each.
ABA quantification by GCMS
Diazomethane was used to methylate the samples, following the addition of methanol/ethyl acetate. The reaction was allowed to proceed for 30 min, after which the samples were dried in a fume cupboard for several hours. After reconstituting in ethylacetate, samples were introduced into the gas chromatography column and GCMS was performed as outlined previously (Holbrook et al., 1992).
Germination of isolated embryos to determine sensitivity to S-(+)-ABA
At different time points throughout the dormancy-breaking treatment, embryos were dissected from seeds and then placed in germination conditions in water, or in different concentrations of S-(+)-ABA. The embryos (3 replicates of 10 embryos each) were placed in 9 cm Petri dishes on one layer of Whatman no. 1 filter paper pre-wetted with 5 ml of water or ABA solution (10-7, 10-6 and 10-5 M). Solutions of S-(+)-ABA were prepared by dissolving known weights (2–4 mg) into 1 ml methanol and then diluting them further with the appropriate amount of sterile water. All solutions, including the control, contained equal amounts of methanol and were adjusted to a pH of 5.5. Germination was monitored every 3 d for a total of 12 d. Germination conditions were 26 °C with a 16 h photoperiod; light intensity at 100 µmol m-2 s-1, PAR 400–700 nm. All solutions were replaced on day 6. Germination was defined as elongation of the radicle, which was also accompanied by the opening and greening of the cotyledons.
Results and discussion
A treatment comprised of 4 weeks of warm, moist conditions followed by 8 weeks of moist chilling is effective for terminating dormancy of yellow cedar seeds. Following this ‘stratification’ treatment, germination of yellow cedar seeds of seed lot 30156 is typically 80–90% after 30 d in germination conditions (Ren and Kermode, 1999). Moist chilling alone is relatively ineffective in breaking dormancy and promotes only ~25% germination.
Abscisic acid (ABA) content of the embryo and megagametophyte was determined before, during and after dormancy termination (Fig. 1A, B). Within the embryos of yellow cedar seeds, ABA decreased with increasing time of exposure to the dormancy-breaking treatment (Fig. 1A). Following exposure of seeds to the complete dormancy-breaking treatment, there was an approximate 2-fold reduction of ABA in embryos (as compared to that in embryos of 3 d soaked dormant seed); the amount of ABA in embryos did not change significantly following transfer of the seeds to germination conditions for 4 d, although there was a slight reduction. In contrast, subjecting seed to a control treatment (in which seeds were maintained in warm, moist conditions at 25 °C for 12 weeks) did not lead to any substantial decrease of ABA within the embryo (Fig. 1A). The ABA content of the megagametophyte remained relatively constant during the dormancy-breaking treatment and even increased in the germinating seed (Fig. 1B).
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Embryos (excised from seeds at the same times throughout the dormancy-breaking treatment) were examined with respect to their sensitivity to S-(+)-ABA (Fig. 2
) by monitoring their capacity to germinate in different concentrations of the hormone. Embryos isolated from mature dormant seeds (subjected to only a 3 d soak) (Fig. 2A) were sensitive to all concentrations of S-(+)-ABA tested except 10-7 M; 10-6 M ABA led to a near complete inhibition of germination during the 12 d study period. Embryos excised from seeds that had been subjected to longer lengths of the dormancy-breaking treatment became increasingly less sensitive to 10-5 M and 10-6 M ABA (Fig. 2B–E). Following exposure of seeds to the full dormancy-breaking treatment (4 weeks warm and 8 weeks moist chilling), the embryos exhibited an approximate 10-fold lowered sensitivity to S-(+)-ABA as compared to embryos excised from mature seeds that received only the 3 d water soak (Fig. 2E).
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It was concluded that dormancy termination of yellow cedar (and hence, an increased capacity for germination of the whole seed) may require a decline in the embryo's sensitivity to ABA. Dormancy termination was accompanied by a moderate decrease in ABA within the embryo. The major decline in ABA within the embryo occurred after a 4-week exposure of mature seeds to warm, moist conditions, i.e. before the seeds had been exposed to a sufficient length of the dormancy-breaking treatment to elicit high germinability. Nonetheless, the decrease in ABA within the embryo may still be important for dormancy termination. For example, embryo ABA declined further when seeds were subsequently exposed to 8 weeks of moist chilling in contrast to that in seeds maintained in warm, moist conditions for a further 8 weeks (after which seeds remain dormant). ABA within the megagametophyte of yellow cedar seeds showed no significant change. In other seeds, there is evidence to support the contention that a decline in ABA alone is insufficient to break dormancy (reviewed in Bewley and Black, 1994
).
Notes
3 To whom correspondence should be addressed. Fax: +1 604 291 3496. E-mail:kermode{at}sfu.ca 
References
Bewley JD, Black M.1994. Seeds: physiology of development and germination, 2nd edn. New York: Plenum Press.
Holbrook LA, Magus JR, Taylor DC.1992. Abscisic acid induction of elongase activity, biosynthesis and accumulation of very long chain mono-unsaturated fatty acids and oil body proteins in microspore-derived embryos of Brassica napus L. cv. Reston. Plant Science 84, 99–115.
Owens JN, Molder M.1984. The reproductive cycles of western red cypress and yellow cypress. Victoria, BC, Canada: BC Ministry of Forests.
Pawuk WH.1993. Germination of Alaska cedar seeds. Tree Planters' Notes 44, 21–24.
Qi Q, Rose PA, Abrams GD, Taylor DC, Abrams SR, Cutler AJ.1998. (+)-Abscisic acid metabolism, 3-ketoacyl-coenzyme A synthase gene expression and very-long-chain mono-unsaturated fatty acid biosynthesis in Brassica napus embryos. Plant Physiology 117, 979–987.
Ren C, Kermode AR.1999. Analyses to determine the role of the megagametophyte and other seed tissues in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds: morphological, cellular and physiological changes following moist chilling and during germination. Journal of Experimental Botany 50, 1403–1419.
Xia JH, Kermode AR.1999. Analyses to determine the role of embryo immaturity in dormancy maintenance of yellow-cedar (Chamaecyparis nootkatensis) seeds: synthesis and accumulation of storage proteins and proteins implicated in desiccation tolerance. Journal of Experimental Botany 50, 107–118.
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