Abstract
To a great extent, the cellular compartmentalization and molecular interactions are indicative of the function of a protein. The development of simple and efficient tools for testing the subcellular location of proteins is indispensable to elucidate the function of genes in plants. In this report, we assessed the feasibility ofAgrobacterium-mediated transformation of hydroponically grown roots to follow intracellular targeting of proteins fused to green fluorescent protein (GFP). We developed a simple in planta assay for subcellular localization of proteins inArabidopsis roots via transient transformation and tested this method by expressing a GFP fusion of a known nuclear protein, IQD1. Visualization of transiently expressed GFP fusion proteins in roots by means of confocal microscopy is superior to the analysis of green tissues because the roots are virtually transparent and free of chlorophyll autofluorescence.
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Abbreviations
- GFP:
-
green fluorescent protein
- PAR:
-
photosynthetic active radiation
- PI:
-
propidium iodide
References
Abel S and Theologis A (1994) Transient transformation ofArabidopsis leaf protoplasts: a versatile experimental system to study gene expression. Plant J 5: 421–427.
Chalfie M, Tu Y, Euskirchen G, Ward WW, and Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263: 802–805.
Christou P (1996) Transformation technology. Trends Plant Sci 1: 423–431.
Chung MH, Chen MK, and Pan SM (2000) Floral spray transformation can efficiently generateArabidopsis transgenic plants. Transgenic Res 9: 471–476.
Clough SJ and Bent AF (1998) Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana. Plant J 16: 735–743.
Cutler SR, Ehrhardt DW, Griffitts JS, and Somerville CR (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells ofArabidopsis at a high frequency. Proc Natl Acad Sci USA 97: 3718–3723.
Day RN, Periasamy A, and Schaufele F (2001) Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus. Methods 25: 4–18.
Fehr M, Ehrhardt DW, Lalonde S, and Frommer WB (2004) Minimally invasive dynamic imaging of ions and metabolites in living cells. Curr Opin Plant Biol 7: 345–351.
Fehr M, Okumoto S, Deuschle K, Lager I, Looger LL, Persson J, Kozhukh L, Lalonde S, and Frommer WB (2005) Development and use of fluorescent nanosensors for metabolite imaging in living cells. Biochem Soc Trans 33: 287–290.
Haseloff J, Siemering KR, Prasher DC, and Hodge S (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenicArabidopsis plants brightly. Proc Natl Acad Sci USA 94: 2122–2127.
Janssen BJ and Gardner RC (1990) Localized transient expression of GUS in leaf discs following cocultivation withAgrobacterium. Plant Mol Biol 14: 61–72.
Kapila J, De Rycke R, Van Montagu M, and Angenon G (1997) AnAgrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122: 101–108.
Klein KK, Salmon RE, and Larmond ME (1979) A linear programming model for determining the optimal level of low glucosinolate rapeseed meal in diets of growing turkeys. Can J Agric Econ: 61–73.
Klein T, Fromm M, Weissinger A, Tomes D, Schaaf S, Sletten M, and Sanford J (1987) High velocity microprojectiles for delivering nucleic acids into living cells. Nature 327: 70–73.
Levy M, Wang Q, Kaspi R, Parrella MP, and Abel S (2005) IQD1, a novel nuclear calmodulin-binding protein modulates glucosinolates accumulation and plant defense responses in Arabidopsis. Plant J 43: 79–96.
Misteli T and Spector DL (1997) Applications of the green fluorescent protein in cell biology and biotechnology. Nat Biotechnol 15: 961–964.
Rachmilevitch S, Cousins AB, and Bloom AJ (2004) Nitrate assimilation in plant shoots depends on photorespiration. Proc Natl Acad Sci USA 101: 11506–11510.
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Levy, M., Rachmilevitch, S. & Abel, S. TransientAgrobacterium-mediated gene expression in theArabidopsis hydroponics root system for subcellular localization studies. Plant Mol Biol Rep 23, 179–184 (2005). https://doi.org/10.1007/BF02772708
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DOI: https://doi.org/10.1007/BF02772708