Summary
A fluorescently labeled phenylalkylamine, DM-Bodipy PAA, was used as a probe for the in vivo detection of ion channels in embryonic and nonembryonic tissues of sunflower. Zygotic embryos, somatic embryos, callus, leaves, roots, and shoots were analysed. Fluorescence intensity in the tissues was determined with cytofluorometry and confocal microscopy. DM-Bodipy PAA intensively labeled the protoderm and epidermis cells in both zygotic and somatic embryos. Callus cultures exhibited labeling on sites where somatic embryos developed. Labeling was, however, very weak in leaves, shoots, and roots, except in the root cap and in the epidermis of the root. Considering that the location of phenylalkylamine binding sites is related to the distribution of ion channels in both animal and plant cells, the high intensity of labeling observed in the protoderm and epidermis of zygotic and somatic embryos as well as in protoderm, epidermis, and caps of root tips, is consistent with the role these tissues may play in ion exchange with the environment.
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Andrejauskas E, Hertel R, Marmé D (1985) Specific binding of the calcium antagonist [3H] verapamil to membrane fractions from plants. J Biol Chem 260: 5411–5414
Cho HT, Hong YN (1996) Effect of calcium channel blockers on the IAA-induced cell elongation of sunflower hypocotyl segments. J Plant Physiol 149: 377–383
Graziana A, Fosset M, Ranjeva R, Hetherington AM, Lazdunski M (1988) Ca2+channel inhibitors that bind to plant cell membranes block Ca2+ entry into protoplasts. Biochemistry 27: 764–768
Harvey HJ, Venis MA, Trewavas AJ (1989) Partial purification of a protein from maize (Zea mays) coleoptile membranes binding the Ca2+-channel antagonist verapamil. Biochem J 257: 95–100
Knaus HG, Moshammer T, Kang HC, Haugland RP, Glossmann H (1992) A unique fluorescent phenylalkylamine probe for L-type Ca2+ channels. J Biol Chem 267: 2179–2189
Laux T, Jürgens G (1997) Embryogenesis: a new start in life. Plant Cell 9: 989–1000
Maathuis FJM, Ichida AM, Sanders D, Schroeder JI (1997) Roles of higher plant K+ channels. Plant Physiol 114: 1141–1149
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15: 473–497
Norris DK, Bradford HF (1985) On the specificity of verapamil as a calcium channel-blocker. Biochem Pharmacol 34: 1953–1956
Pélissier P, Bouchefra O, Pépin R, Freyssinet G (1990) Production of isolated somatic embryos from sunflower thin cell layers. Plant Cell Rep 9: 47–50
Piñeros M, Tester M (1997) Characterisation of the high-affinity verapamil binding site in a plant plasma membrane Ca2+-selective channel. J Membr Biol 157: 139–145
Shabala SN, Newman IA, Morris J (1997) Oscillations in H+ and Ca2+ ion fluxes around the elongation region of corn roots and effects of external pH. Plant Physiol 113: 111–118
Sterk P, Booij H, Schellekens GA, Van Kammen A, De Vries S (1991) Cell-specific expression of the carrot EP2 lipid transfer protein. Plant Cell 3: 907–921
Terry BR, Findlay GP, Tyerman SD (1992) Direct effects of Ca2+-channel blockers on plasma membrane cation channels ofAmaranthus tricolor protoplasts. J Exp Bot 43: 1457–1473
Thomine S, Zimmermann S, van Duijn B, Barbier-Brygoo H, Guern J (1994) Calcium channel antagonists induce direct inhibition of outward rectifying potassium channel in tobacco protoplasts. FEBS Lett 340: 45–50
Thuleau P, Graziana A, Canut H, Ranjeva R (1990) A 75-kDa polypeptide, located primarily at the plasma membrane of carrot cell-suspension cultures, is photoaffinity labeled by the calcium channel blocker LU 49888. Proc Natl Acad Sci USA 87: 10000–10004
Timmers ACJ, Reiss HD, Boshung J, Traxel K, Schel JHN (1996) Localization of calcium during somatic embryogenesis of carrot (Daucuscarota L.). Protoplasma 190: 107–118
Vallée N, Brière C, Petitprez M, Souvré A, Alibert G (1997) Studies on ion channel antagonist-binding sites in sunflower protoplasts. FEBS Lett 411: 115–118
Vroemen CW, Langeveld S, Mayer U, Ripper G, Jürgens G, Van Kammen A, De Vries SC (1996) Pattern formation in theArabidopsis embryo revealed by position-specific lipid transfer protein gene expression. Plant Cell 8: 783–791
Ward JM, Pei Z-M, Schroeder JI (1995) Roles of ion channels in initiation of signal transduction in higher plants. Plant Cell 7: 833–844
XuHan X, Van Lammeren AAM (1997) Structural analysis of embryogenesis and endosperm formation in celery-leafed buttercup (Ranunculus sceleratus L.). Acta Bot Neerl 46: 291–301
—, Souvré A, Granier C, Petiprez M (1995) An improved immunolabeling method for microtubular cytoskeleton in poplar free nuclear endosperm. Biotech Histochem 70: 252–257
Yeung EC, Meinke DW (1993) Embryogenesis in angiosperms: development of the suspensor. Plant Cell 5: 1371–1381
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XuHan, X., Brière, C., Vallée, N. et al. In vivo labeling of sunflower embryonic tissues by fluorescently labeled phenylalkylamine. Protoplasma 210, 52–58 (1999). https://doi.org/10.1007/BF01314955
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DOI: https://doi.org/10.1007/BF01314955