Abstract
The immunohistochemical technique enables us to visualize phytohormones, for example, abscisic acid (ABA) in plant cells. The distributions and levels of ABA would fluctuate dramatically in organs or tissues in response to environmental changes or developmental stages. The visualization of the spatial changes of ABA is crucial for understanding its physiological function. Here we present the procedures of ABA immunostaining of the seeds of a terrestrial orchid, Cypripedium formosanum using fluorochrome and immunocolloid gold labelings. Unlike the young and fresh tissues, desiccated mature seeds with strong hydrophobic characteristics of seed coats require a longer duration of prefixation and subsequent processing steps. To prevent artifactitious signals from nonspecific binding of antibodies or autofluorescence from lignified cell wall, procedures including pretreating antibodies, quenching the autofluorescence by Toluidine blue O (TBO) staining, and setting careful control staining conditions need to be implemented. Proper selection of a narrow band-pass filter is also necessary to optimize the detection of emission from conjugated fluorochrome to minimize the autofluorescence from the specimen. Though our protocol uses orchid seeds for demonstrating a complete ABA immunostaining procedure, it can be adapted for other plant seeds, or the hard and dry tissues.
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References
Taiz L, Zeiger E (2010) Plant physiology, 5th edn. Sinauer Associates, Sunderland
Zavala ME, Brandon DL (1983) Localization of a phytohormone using immunocytochemistry. J Cell Biol 97:1235–1239
Eberle J, Arnscheidt A, Klix D, Weiler EW (1986) Monoclonal antibodies to plant growth regulators. III. Zeatinriboside and dihydrozeatinriboside. Plant Physiol 81:516–521
Aloni1 R, Langhans M, Aloni E, Dreieicher E, Ullrich CI (2005) Root-synthesized cytokinin in Arabidopsis is distributed in the shoot by the transpiration stream. J Exp Bot 56:1535–1544
Sotta B, Sossountzov L, Maldiney R, Sabbagh I et al (1985) Abscisic acid localization by light microscope immunohisto-chemistry in Chenopodium polyspermum L. J Histochem Cytochem 33:201–208
Ohmiya A, Hayashi T, Kakiuchi N (1990) Immuno-gold localization of indole-3-acetic acid in peach seedlings. Plant Cell Physiol 31:711–715
Ohmiya A, Hayashi T (1992) Immuno-gold localization of IAA in leaf cells of Prunus persica at different stages of development. Physiol Plant 85:439–445
Nambara E, Marion-Poll A (2005) Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol 56:165–185
Harris MJ, Outlaw WH, Mertens JRR, Weiler EW (1988) Water-stress-induced changes in the abscisic acid content of guard cells and other cells of Vicia faba L. leaves as determined by enzyme-amplified immunoassay. Proc Natl Acad Sci U S A 85:2584–2588
Christmann A, Hoffmann T, Teplova I, Grill E, Muller A (2005) Generation of active pools of abscisic acid revealed by in vivo imaging of water-stressed Arabidopsis. Plant Physiol 137:209–219
Schraut D, Ullrich CI, Hartung W (2004) Lateral ABA transport in maize roots (Zea mays): visualization by immunolocalization. J Exp Bot 55:1635–1641
Bewley JD, Black M (1985) Seeds: physiology of the development and germination. Plenum, NY
Koornneef M, Hanhart CJ, Hilhorst HWM, Karssen CM (1989) In vivo inhibition of seed development and reserve protein accumulation in recombinants of abscisic acid biosynthesis and responsiveness mutants in Arabidopsis thaliana. Plant Physiol 90:463–469
Peng YB, Zou C, Wang DH, Gong HQ et al (2006) Preferential localization of abscisic acid in primordial and nursing cells of reproductive organs of Arabidopsis and cucumber. New Phytol 170:459–466
Wachter R, Langhans M, Aloni R, Gotz S et al (2003) Vascularization, high-volume solution flow, and localized roles for enzymes of sucrose metabolism during tumorigenesis by Agrobacterium tumefaciens L. Plant Physiol 133:1024–1037
Sossountzov L, Sotta B, Maldiney R, Sabbagh I, Miginiac E (1986) Immunoelectron-microscopy localization of abscisic acid with colloidal gold on Lowicryl-embedded tissues of Chenopodium polyspermum L. Planta 168:471–481
Betrand S, Benhamou N (1992) Immunogold localization of free abscisic acid in tomato root cells. Can J Bot 70:1001–1011
Weiler EW (1982) An enzyme-immunoassay for cis-(+)-abscisic acid. Physiol Plant 54:510–514
Pastor A, Cortadellas N, Alegre L (1995) Immunolocalization of abscisic acid by monoclonal antibodies in Lavandula stoechas L. leaves. Plant Growth Regul 16:287–292
Welbaum GE, Pavel EW, Hill DR, Gunatilaka MK, Grayson RL (2000) Compartmentation of abscisic acid in developing muskmelon (Cucumis melo L.) seeds. In: Black M, Bradford KJ, Vazquez-Ramos J (eds) Seed biology: advances and applications, Proceedings of 6th International Workshop on Seeds. CABI, Merida, Mexico, pp 85–100
Arditti J, Ghani AKAR (2000) Numerical and physical properties of orchid seeds and their biological implications. New Phytol 145:367–421
Lee YI, Lee N, Yeung EC, Chung MC (2005) Embryo development of Cypripedium formosanum in relation to seed germination in vitro. J Am Soc Hortic Sci 130:747–753
Van der Kinderen G (1987) Abscisic acid in terrestrial orchid seeds: a possible impact on their germination. Lindleyana 2:84–87
Lee YI, Lu CF, Chung MC, Yeung EC, Lee N (2007) Changes in endogenous abscisic acid levels and asymbiotic seed germination of a terrestrial orchid, Calanthe tricarinata Lindl. J Am Soc Hortic Sci 132:246–252
Walker-Simmons M (1987) ABA levels and sensitivity in the developing embryos of sprouting and susceptible cultivars. Plant Physiol 84:61–66
Li X, Lamotte CE, Stewart CR, Cloud NP et al (1992) Determination of IAA and ABA in the some plant sample by a widely applicable method using GC-MS with selected ion monitoring. J Plant Growth Regul 11:55–65
Fong F, Smith JD, Koehler DE (1983) Early events in maize seed development. Plant Physiol 73:899–901
Biggs AR (1985) Detection of impervious tissue in tree bark with selective histochemistry and fluorescence microscopy. Stain Technol 60:299–304
Acknowledgments
This research was supported by National Science Council, Taiwan, ROC (NSC 97-2313-B-178-001), to Yung-I Lee and by Academia Sinica, Taiwan, ROC, to Mei-Chu Chung. We also thank Dr. Wann-Neng Jane, Miss Mei-Jane Fang, and Miss Yi-Jia Chou (Plant Cell Biology Core lab, IPMB) for the help in the immunogold labeling and the use of a TEM and a CLSM.
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Lee, YI., Chung, MC. (2015). Abscisic Acid Immunostaining. In: Yeung, E., Stasolla, C., Sumner, M., Huang, B. (eds) Plant Microtechniques and Protocols. Springer, Cham. https://doi.org/10.1007/978-3-319-19944-3_15
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DOI: https://doi.org/10.1007/978-3-319-19944-3_15
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