Advertisement

Basic Techniques to Assess Seed Germination Responses to Abiotic Stress in Arabidopsis thaliana

  • Urszula Piskurewicz
  • Luis Lopez-MolinaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1398)

Abstract

The model organism Arabidopsis thaliana has been extensively used to unmask the molecular genetic signaling pathways controlling seed germination in plants. In Arabidopsis, the normal seed to seedling developmental transition involves testa rupture soon followed by endosperm rupture, radicle elongation, root hair formation, cotyledon expansion, and greening. Here we detail a number of basic procedures to assess Arabidopsis seed germination in response to different light (red and far-red pulses), temperature (seed thermoinhibition), and water potential (osmotic stress) environmental conditions. We also discuss the role of the endosperm and how its germination-repressive activity can be monitored genetically by means of a seed coat bedding assay. Finally we detail how to evaluate germination responses to changes in gibberellin (GA) and abscisic acid (ABA) levels by manipulating pharmacologically the germination medium.

Key words

Seed germination Testa rupture Endosperm rupture Seed coat bedding assay Osmotic stress Thermoinhibition Gibberellins Abscisic acid 

References

  1. 1.
    Belin C, Lopez-Molina L (2008) Arabidopsis seed germination responses to osmotic stress involve the chromatin modifier PICKLE. Plant Signal Behav 3(7):478–479PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Muller K, Tintelnot S, Leubner-Metzger G (2006) Endosperm-limited Brassicaceae seed germination: abscisic acid inhibits embryo-induced endosperm weakening of Lepidium sativum (cress) and endosperm rupture of cress and Arabidopsis thaliana. Plant Cell Physiol 47(7):864–877CrossRefPubMedGoogle Scholar
  3. 3.
    Piskurewicz U, Jikumaru Y, Kinoshita N, Nambara E, Kamiya Y, Lopez-Molina L (2008) The gibberellic acid signaling repressor RGL2 inhibits Arabidopsis seed germination by stimulating abscisic acid synthesis and ABI5 activity. Plant Cell 20(10):2729–2745. doi: 10.1105/tpc.108.061515 PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Lopez-Molina L, Mongrand S, Chua NH (2001) A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis. Proc Natl Acad Sci USA 98(8):4782–4787PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Lopez-Molina L, Mongrand S, McLachlin DT, Chait BT, Chua NH (2002) ABI5 acts downstream of ABI3 to execute an ABA-dependent growth arrest during germination. Plant J 32(3):317–328CrossRefPubMedGoogle Scholar
  6. 6.
    Nambara E, Marion-Poll A (2005) Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol 56:165–185CrossRefPubMedGoogle Scholar
  7. 7.
    Olszewski N, Sun TP, Gubler F (2002) Gibberellin signaling: biosynthesis, catabolism, and response pathways. Plant Cell 14(Suppl):S61–S80PubMedCentralPubMedGoogle Scholar
  8. 8.
    Bentsink L, Koornneef M (2009) Seed dormancy and germination. In: The Arabidopsis book. The American Society of Plant Biologists, pp 1–18. doi:10.1199/tab.0119Google Scholar
  9. 9.
    Finkelstein R, Reeves W, Ariizumi T, Steber C (2008) Molecular aspects of seed dormancy. Annu Rev Plant Biol 59:387–415. doi: 10.1146/annurev.arplant.59.032607.092740 CrossRefPubMedGoogle Scholar
  10. 10.
    Holdsworth MJ, Bentsink L, Soppe WJ (2008) Molecular networks regulating Arabidopsis seed maturation, after-ripening, dormancy and germination. New Phytol 179(1):33–54, doi:NPH2437 [pii] 10.1111/j.1469-8137.2008.02437.xCrossRefPubMedGoogle Scholar
  11. 11.
    Lee KP, Piskurewicz U, Tureckova V, Carat S, Chappuis R, Strnad M, Fankhauser C, Lopez-Molina L (2012) Spatially and genetically distinct control of seed germination by phytochromes A and B. Genes Dev 26(17):1984–1996. doi: 10.1101/gad.194266.112 PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Shinomura T, Nagatani A, Chory J, Furuya M (1994) The induction of seed germination in Arabidopsis thaliana Is regulated principally by phytochrome B and secondarily by phytochrome A. Plant Physiol 104(2):363–371, doi:104/2/363 [pii]PubMedCentralPubMedGoogle Scholar
  13. 13.
    Shinomura T, Nagatani A, Hanzawa H, Kubota M, Watanabe M, Furuya M (1996) Action spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thaliana. Proc Natl Acad Sci USA 93(15):8129–8133PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Oh E, Kim J, Park E, Kim J-I, Kang C, Choi G (2004) PIL5, a phytochrome-interacting basic helix-loop-helix protein, is a key negative regulator of seed germination in Arabidopsis thaliana. Plant Cell 16(11):3045–3058. doi: 10.1105/tpc.104.025163 PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Bethke PC, Libourel IG, Aoyama N, Chung YY, Still DW, Jones RL (2007) The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy. Plant Physiol 143(3):1173–1188PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Lee KP, Piskurewicz U, Tureckova V, Strnad M, Lopez-Molina L (2010) A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds. Proc Natl Acad Sci USA 107(44):19108–19113. doi: 10.1073/pnas.1012896107 PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Lee KP, Lopez-Molina L (2013) A seed coat bedding assay to genetically explore in vitro how the endosperm controls seed germination in Arabidopsis thaliana. J Vis Exp 9(81):e50732Google Scholar
  18. 18.
    Finkelstein RR, Lynch TJ (2000) Abscisic acid inhibition of radicle emergence but not seedling growth is suppressed by sugars. Plant Physiol 122(4):1179–1186PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Lopez-Molina L, Chua NH (2000) A null mutation in a bZIP factor confers ABA-insensitivity in Arabidopsis thaliana. Plant Cell Physiol 41(5):541–547CrossRefPubMedGoogle Scholar
  20. 20.
    Piskurewicz U, Tureckova V, Lacombe E, Lopez-Molina L (2009) Far-red light inhibits germination through DELLA-dependent stimulation of ABA synthesis and ABI3 activity. EMBO J 28(15):2259–2271. doi: 10.1038/emboj.2009.170 PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Saito S, Okamoto M, Shinoda S, Kushiro T, Koshiba T, Kamiya Y, Hirai N, Todoroki Y, Sakata K, Nambara E, Mizutani M (2006) A plant growth retardant, uniconazole, is a potent inhibitor of ABA catabolism in Arabidopsis. Biosci Biotechnol Biochem 70(7):1731–1739CrossRefPubMedGoogle Scholar
  22. 22.
    Piskurewicz U, Lopez-Molina L (2011) Isolation of genetic material from Arabidopsis seeds. Methods Mol Biol 773:151–164. doi: 10.1007/978-1-61779-231-1_10 CrossRefPubMedGoogle Scholar
  23. 23.
    Vicient CM, Delseny M (1999) Isolation of total RNA from Arabidopsis thaliana seeds. Anal Biochem 268(2):412–413CrossRefPubMedGoogle Scholar
  24. 24.
    Toh S, Imamura A, Watanabe A, Nakabayashi K, Okamoto M, Jikumaru Y, Hanada A, Aso Y, Ishiyama K, Tamura N, Iuchi S, Kobayashi M, Yamaguchi S, Kamiya Y, Nambara E, Kawakami N (2008) High temperature-induced abscisic acid biosynthesis and its role in the inhibition of gibberellin action in Arabidopsis seeds. Plant Physiol 146(3):1368–1385. doi: 10.1104/pp. 107.113738
  25. 25.
    Kendall SL, Hellwege A, Marriot P, Whalley C, Graham IA, Penfield S (2011) Induction of dormancy in Arabidopsis summer annuals requires parallel regulation of DOG1 and hormone metabolism by low temperature and CBF transcription factors. Plant Cell 23(7):2568–2580. doi: 10.1105/tpc.111.087643 PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Endo A, Tatematsu K, Hanada K, Duermeyer L, Okamoto M, Yonekura-Sakakibara K, Saito K, Toyoda T, Kawakami N, Kamiya Y, Seki M, Nambara E (2012) Tissue-specific transcriptome analysis reveals cell wall metabolism, flavonol biosynthesis and defense responses are activated in the endosperm of germinating Arabidopsis thaliana seeds. Plant Cell Physiol 53(1):16–27, doi:pcr171 [pii] 10.1093/pcp/pcr171CrossRefPubMedGoogle Scholar
  27. 27.
    Penfield S, Li Y, Gilday AD, Graham S, Graham IA (2006) Arabidopsis ABA INSENSITIVE4 regulates lipid mobilization in the embryo and reveals repression of seed germination by the endosperm. Plant Cell 18(8):1887–1899PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Tatematsu K, Nakabayashi K, Kamiya Y, Nambara E (2008) Transcription factor AtTCP14 regulates embryonic growth potential during seed germination in Arabidopsis thaliana. Plant J 53(1):42–52, doi:TPJ3308 [pii] 10.1111/j.1365-313X.2007.03308.xCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Plant BiologyUniversity of GenevaGeneva 4Switzerland

Personalised recommendations