, Volume 218, Issue 4, pp 630–639 | Cite as

Dormancy termination of western white pine (Pinus monticola Dougl. Ex D. Don) seeds is associated with changes in abscisic acid metabolism

  • J. Allan Feurtado
  • Stephen J. Ambrose
  • Adrian J. Cutler
  • Andrew R. S. Ross
  • Suzanne R. Abrams
  • Allison R. Kermode
Original Article


Western white pine (Pinus monticola) seeds exhibit deep dormancy at maturity and seed populations require several months of moist chilling to reach their uppermost germination capacities. Abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA), 7′-hydroxy ABA (7′OH ABA) and ABA-glucose ester (ABA-GE), were quantified in western white pine seeds during dormancy breakage (moist chilling) and germination using an HPLC–tandem mass spectrometry method with multiple reaction monitoring and internal standards incorporating deuterium-labeled analogs. In the seed coat, ABA and metabolite levels were high in dry seeds, but declined precipitously during the pre-moist-chilling water soak to relatively low levels thereafter. In the embryo and megagametophyte, ABA levels decreased significantly during moist chilling, coincident with an increase in the germination capacity of seeds. ABA catabolism occurred via several routes, depending on the stage and the seed tissue. Moist chilling of seeds led to increases in PA and DPA levels in both the embryo and megagametophyte. Within the embryo, 7′OH ABA and ABA-GE also accumulated during moist chilling; however, 7′OH ABA peaked early in germination. Changes in ABA flux, i.e. shifts in the ratio between biosynthesis and catabolism, occurred at three distinct stages during the transition from dormant seed to seedling. During moist chilling, the relative rate of ABA catabolism exceeded ABA biosynthesis. This trend became even more pronounced during germination, and germination was also accompanied by a decrease in the ABA catabolites DPA and PA, presumably as a result of their further metabolism and/or leaching/transport. The transition from germination to post-germinative growth was accompanied by a shift toward ABA biosynthesis. Dormant imbibed seeds, kept in warm moist conditions for 30 days (after an initial 13 days of soaking), maintained high ABA levels, while the amounts of PA, 7′OH ABA, and DPA decreased or remained at steady-state levels. Thus, in the absence of conditions required to break dormancy there were no net changes in ABA biosynthesis and catabolism.


Abscisic acid metabolism Germination Seed dormancy Pinus 



abscisic acid


abscisic acid glucose ester


dihydrophaseic acid


7′-hydroxy abscisic acid


8′-hydroxy abscisic acid


multiple reaction monitoring


phaseic acid



We thank Dave Kolotelo and Dean Christianson of the BC Ministry of Forests, BC, Canada for helping obtain mature seed of western white pine and Mary Loewen and Tim Squires for their technical help at PBI, Saskatoon, Canada. We are also grateful to Marek Galka (PBI) for the synthesis of ABA-GE and 7′OH ABA and to Irina Zaharia (PBI) for the PA and DPA in labeled and unlabeled forms. This research was supported by Natural Sciences and Engineering Research Council of Canada (Strategic) and BC Ministry of Advanced Education, Training and Technology grants awarded to A.R.K and a Protein Engineering Network of Centres of Excellence (PENCE) grant awarded to A.R.K., S.R.A., A.R.S.R. and A.J.C., and other investigators. J.A.F. is a recipient of a Science Horizons Internship.


  1. Abrams SR, Nelson K, Ambrose SJ (2003) Deuterated abscisic acid analogs for mass spectrometry and metabolism studies. J Labelled Cpds Radiopharmacol 46:273–283CrossRefGoogle Scholar
  2. Anderson HW, Wilson BC (1966) Improved stratification procedures for western white pine seed. Washington State Department of National Resources Report No. 8Google Scholar
  3. Balsevich JJ, Cutler AJ, Lamb N, Friesen LJ, Kurz EU, Perras MR, Abrams SR (1994) Response of cultured maize cells to (+)-abscisic acid, (−)-abscisic acid, and their metabolites. Plant Physiol 106:135–142PubMedGoogle Scholar
  4. Barthe P, Hogge LR, Abrams SR, Le Page-Degivry MT (1993) Metabolism of (+) abscisic acid to dihydrophaseic acid-4′-β-d-glucopyranoside by sunflower embryos. Phytochemistry 34:645–648CrossRefGoogle Scholar
  5. Bewley JD, Black M (1994) Seeds: physiology of development and germination, 2nd edn. Plenum Press, New YorkGoogle Scholar
  6. Bianco J, Garello G, Le Page-Degivry MT (1997) De novo ABA synthesis and expression of seed dormancy in a gymnosperm: Pseudotsuga menziesii. Plant Growth Regul 21:115–119CrossRefGoogle Scholar
  7. Carrier DJ, Kendall EJ, Bock CA, Cunningham JE, Dunstan DI (1999) Water content, lipid deposition, and (+)-abscisic acid content in developing white spruce seeds. J Exp Bot 50:1359–1364CrossRefGoogle Scholar
  8. Chiwocha S, von Aderkas P (2002) Endogenous levels of free and conjugated forms of auxin, cytokinins, and abscisic acid during seed development in Douglas fir. Plant Growth Regul 36:191–200CrossRefGoogle Scholar
  9. Chiwocha SDS, Abrams SR, Ambrose SJ, Cutler AJ, Loewen M, Ross ARS, Kermode AR (2003) A method for profiling classes of plant hormones and their metabolites using liquid chromatography–electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy in lettuce (Lactuca sativa L.) seeds. Plant J 35:405–417CrossRefPubMedGoogle Scholar
  10. Corbineau F, Bianco J, Garello G, Côme D (2002) Breakage of Pseudotsuga menziesii seed dormancy by cold treatment as related to changes in seed ABA sensitivity and ABA levels. Physiol Plant 114:313–319CrossRefPubMedGoogle Scholar
  11. Cutler AJ, Krochko JE (1999) Formation and breakdown of ABA. Trends Plant Sci 4:472–478PubMedGoogle Scholar
  12. Dumroese RK (2000) Germination-enhancing techniques for Pinus monticola seeds and speculation on seed dormancy. Seed Sci Technol 28:201–209Google Scholar
  13. Feurtado JA, Xia J-H, Ma Y, Kermode AR (2003) Increasing the temperature of the water soak preceding moist chilling promotes dormancy-termination in seeds of western white pine (Pinus monticola Dougl.). Seed Sci Technol 31:275–288Google Scholar
  14. Finkelstein RR, Rock CD (2002) Abscisic acid biosynthesis and response. In: Somerville CR, Meyerowitz EM (eds) The Arabidopsis Book. American Society of Plant Biologists, Rockville, MD. DOI 10.1199/tab.0058, Scholar
  15. Fins L, Byler J, Ferguson D, Harvey A, Mahalovich MF, McDonald G, Miller D, Schwandt J, Zack A (2002) Return of the giants: restoring western white pine to the Inland Northwest. J For 100:20–26Google Scholar
  16. Foley ME (2001) Seed dormancy: an update on terminology, physiological genetics, and quantitative trait loci regulating germinability. Weed Sci 49:305–317Google Scholar
  17. Gansel C (1986) Comparison of seed stratification methods for western white pine. Gen Tech Rep Rocky Mount 137:19–22Google Scholar
  18. Grappin P, Bouinot D, Sotta B, Miginiac E, Jullien M (2000) Control of seed dormancy in Nicotiana plumbaginifolia: post-imbibition abscisic acid synthesis imposes dormancy maintenance. Planta 210:279–285PubMedGoogle Scholar
  19. Hill RD, Liu J-H, Durnin D, Lamb N, Shaw A, Abrams SR (1995) Abscisic acid structure-activity relationships in barley aleurone layers and protoplasts: biological activity of optically active, oxygenated metabolites. Plant Physiol 108:573–579PubMedGoogle Scholar
  20. Hoff RJ (1987) Dormancy in Pinus monticola seed related to stratification time, seed coat, and genetics. Can J For Res 17:294–298Google Scholar
  21. Jacobsen JV, Pearce DW, Poole AT, Pharis RP, Mander LN (2002) Abscisic acid, phaseic acid and gibberellin contents associated with dormancy and germination in barley. Physiol Plant 115:428–441CrossRefPubMedGoogle Scholar
  22. Karssen CM, Brinkhorst-van der Swan DLC, Breekland AE, Koornneef M (1983) Induction of dormancy during seed development by endogenous abscisic acid: studies on abscisic acid deficient genotypes of Arabidopsis thaliana (L.) Heynh. Planta 157:158–165Google Scholar
  23. Kermode AR (1995) Regulatory mechanisms in the transition from seed development to germination: interactions between the embryo and the seed environment. In: Kigel J, Galili G (eds) Seed development and germination. Dekker, New York, pp 273–332Google Scholar
  24. Kleczkowski K, Schell J (1995) Phytohormone conjugates: nature and function. Crit Rev Plant Sci 14:283–298Google Scholar
  25. Kong L, Yeung EC (1995) Effects of silver nitrate and polyethylene glycol on white spruce (Picea glauca) somatic embryo development: enhancing cotyledonary embryo formation and endogenous ABA content. Physiol Plant 93:298–304CrossRefGoogle Scholar
  26. Larsen JA (1925) Methods of stimulating germination of western white pine seed. J Agric Res 31:889–899Google Scholar
  27. Leadem C (1996) A guide to the biology and use of forest tree seeds. British Columbia Ministry of Forests, Victoria, BCGoogle Scholar
  28. Le Page-Degivry MT (1970) Acide abscissique et domance chez les embryons de Taxus baccata. C R Acad Sci Ser D 271:482–484Google Scholar
  29. Le Page-Degivry MT, Garello G (1992) In situ abscisic acid synthesis. A requirement for induction of embryo dormancy in Helianthus annus. Plant Physiol 98:1386–1390Google Scholar
  30. Le Page-Degivry MT, Bulard C, Milborrow BV (1969) Mise en evidence de l’acide (+) abscissique chez une Gymnosperme. C R Acad Sci Ser D 269:2534–2536Google Scholar
  31. Le Page-Degivry MT, Garello G, Barthe P (1997) Changes in abscisic acid biosynthesis and catabolism during dormancy breaking in Fagus sylvatica embryo. J Plant Growth Regul 16:57–61Google Scholar
  32. Martínez-Honduvilla CJ, Santos-Ruiz A (1978) Germination inhibitors in the pine seed coat. Planta 141:141–144Google Scholar
  33. Nelson LAK, Shaw AC, Abrams SR (1991) Synthesis of (+)-, (−)-, and (±)-7′-hydroxy abscisic acid. Tetrahedron 47:3259-3270CrossRefGoogle Scholar
  34. Owens JN, Molder M (1977) Seed-cone differentiation and sexual reproduction in western white pine (Pinus monticola). Can J Bot 55:2574–2590Google Scholar
  35. Owens JN, Catalano G, Bennett JS (2001) The pollination mechanism of western white pine. Can J For Res 31:1731–1741CrossRefGoogle Scholar
  36. Phillips J, Artsaenko O, Fiedler U, Horstmann C, Mock HP, Müntz K, Conrad U (1997) Seed-specific immunomodulation of abscisic acid activity induces a developmental switch. EMBO J 16:4489–4496PubMedGoogle Scholar
  37. Pitel JA, Wang BSP (1985) Physical and chemical treatments to improve laboratory germination of western white pine seeds. Can J For Res 15:1187–1190Google Scholar
  38. Schmitz N, Abrams SR, Kermode AR (2000) Changes in abscisic acid content and embryo sensitivity to (+)-abscisic acid during dormancy termination of yellow-cedar seeds. J Exp Bot 51:1159–1162CrossRefPubMedGoogle Scholar
  39. Schmitz N, Abrams SR, Kermode AR (2002) Changes in ABA turnover and sensitivity that accompany dormancy termination of yellow-cedar (Chamaecyparis nootkatensis) seeds. J Exp Bot 53:89–101CrossRefPubMedGoogle Scholar
  40. Setter TL, Brenner ML, Brun WA, Krick TP (1981) Identification of a dihydrophaseic acid aldopyranoside from soybean tissue. Plant Physiol 68:93–95Google Scholar
  41. Stasolla C, Kong L, Yeung E, Thorpe TA (2002) Maturation of somatic embryos in conifers: morphogenesis, physiology, biochemistry, and molecular biology. In Vitro Cell Dev Biol Plant 38:93–105CrossRefGoogle Scholar
  42. Tillberg E (1992) Effect of light on abscisic acid content in photosensitive Scots pine (Pinus sylvestris L.) seed. Plant Growth Regul 11:147–152Google Scholar
  43. Wang M, Heimovaara-Dijkstra S, Van Duijn B (1995) Modulation of germination of embryos isolated from dormant and non-dormant barley grains by manipulation of endogenous abscisic acid. Planta 195:586–592Google Scholar
  44. Works DW, Boyd RJ (1972) Using infrared irradiation to decrease germination time and to increase percent germination in various species of western conifer trees. Trans Am Soc Agric Eng 15:760–762Google Scholar
  45. Yoshioka T, Endo T, Satoh S (1998) Restoration of seed germination at supraoptimal temperature by fluridone, an inhibitor of abscisic acid biosynthesis. Plant Cell Physiol 39:307–312Google Scholar
  46. Zeevaart JAD (1999) Abscisic acid metabolism and its regulation. In: Hooykaas PJJ, Hall KR, Libbenga KR (eds) Biochemistry and molecular biology of plant hormones. Elsevier, The Netherlands, pp 189–207Google Scholar
  47. Zhou R, Squires TM, Ambrose SJ, Abrams SR, Ross ARS, Cutler AJ (2003) Rapid extraction of abscisic acid and its metabolites for liquid chromatography-tandem mass spectrometry. J Chromatogr A 1010:75–85CrossRefPubMedGoogle Scholar
  48. Zou J, Abrams GD, Barton DL, Taylor DC, Pomeroy MK, Abrams SR (1995) Induction of lipid and oleosin biosynthesis by (+)-abscisic acid and its metabolites in microspore-derived embryos of Brassica napus L. cv. Reston. Plant Physiol 108:563–571PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • J. Allan Feurtado
    • 1
  • Stephen J. Ambrose
    • 2
  • Adrian J. Cutler
    • 2
  • Andrew R. S. Ross
    • 2
  • Suzanne R. Abrams
    • 2
  • Allison R. Kermode
    • 1
  1. 1.Department of Biological SciencesSimon Fraser UniversityBurnabyCanada
  2. 2.Plant Biotechnology InstituteNational Research Council of CanadaSaskatoonCanada

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