Threshold Change in Expression of GFP-FABD2 Fusion Protein During Development of Arabidopsis thaliana Leaves

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Threshold Change in Expression of GFP-FABD2 Fusion Protein During Development of Arabidopsis thaliana Leaves

One of the most important technical challenges in cell biology is visualization of the actin cytoskeleton. The widely used GFP-FABD2 fusion protein is a helpful tool for investigating actin architecture in living plants. Here we report our attempt to visualize F-actin in mature leaves of transgenic Arabidopsis thaliana. With a confocal microscope we observed loss of GFP fluorescence in mature Arabidopsis leaves between 19 and 21 days of development. As this pattern was characteristic of all investigated plants and dependent on the age of the plants, we performed precise expression studies at the mRNA (semiquantitative PCR) and protein (Western blot) levels. Our results clearly show a sudden decrease of GFP-FABD2 expression in Arabidopsis leaves after the third week of growth. This means that transgenic Arabidopsis bearing the GFP-FABD2 construct is not a good model system for visualization of the actin cytoskeleton in leaves of mature plants.

Adai A, Johnson C, Mlotshwa S, Archer-Evans S, Manocha V, Vance V, and Sundaresan V. 2005. Computational prediction of miRNAs in Arabidopsis thaliana. Genome Research 15: 78-91;

Balandin T, and Castresana C. 1997. Silencing of α-1,3-glucanase transgene is overcome during seed formation. Plant Molecular Biology 34: 125-137.

de Borne F D, Vincentz M, Chupeau Y, and Vaucheret H. 1994. Co-suppression of nitrate reductase host genes and transgenes in transgenic tobacco plants. Molecular Genetics and Genomics 243: 613-621.

de Carvalho F, Gheysen G, Kushnir S, van Montagu M, Inze D, and Castresana C. 1992. Suppression of 3-1,3-glucanase transgene expression in homozygous plants. The EMBO Journal 11: 2595-2602.

Dehio C, and Schell J. 1994. Identification of plant genetic loci involved in a posttranscriptional mechanism for meiotically reversible transgene silencing. Proceedings of the National Academy of Sciences 91: 5538-5542.

Elmayan T, and Vaucheret H. 1996. Expression of single copies of a strong expressed 35S transgene can be silenced post-transcriptionally. The Plant Journal 9: 787-797.

Hamilton A, Voinnet O, Chappell L, and Baulcombe D. 2002. Two classes of short interfering RNA in RNA silencing. The EMBO Journal 21: 4671-4679.

Holweg CL. 2006. Living markers for actin block myosin-dependent motility of plant organelles and auxin. Cell Motility and the Cytoskeleton 64: 69-81.

Krzeszowiec W, Rajwa R, Dobrucki J, and Gabryś H. 2007. Actin cytoskeleton in Arabidopsis thaliana under blue and red light. Biology of the Cell 99: 251-260

Nagata T, Okada K, Kawazu T, and Takebe I. 1987. Cauliflower mosaic virus 35 S promoter directs S phase specific expression in plant cells. Molecular Genetics and Genomics 207: 242-244.

Napoli C, Lemieux C, and Jorgensen R. 1990. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. The Plant Cell 2: 279-289.

Odell JT., Nagy F, and Chua N-H. 1985. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810-812.

Scheid OM, Paszkowski J, and Potrykus I. 1991. Reversible inactivation of transgene in Arabidopsis thaliana.Molecular Genetics and Genomics 228: 104-112.

Sheahan M, Staiger C J, Rose R.J, and McCurdy DW. 2004. A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells. Plant Physiology 136: 3968-3978.

Schubert D, Lechtenberg B, Forsbach A, Gils M, Bahadur S, and Schmidt R. 2004. Silencing in Arabidopsis T-DNA transformants: the predominant role of a gene-specific RNA sensing mechanism versus position effects. The Plant Cell 16: 2561-2572.

Smith CJS, Watson CF, Bird CR, Ray J, Schuch W, and Grierson D. 1990. Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants. Molecular Genetics and Genomics 224: 477-481.

Sunilkumar G, Mohr LA, Lopata-Finch E, Emani C, and Rathore KS. 2002. Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP. Plant Molecular Biology 50: 463-474.

Que Q, Wang H-Y, English JJ, and Jorgensen RA. 1997. The frequency and degree of cosuppression by sense chalcone synthase transgenes are dependent on transgene promoter strength and are reduced by premature nonsense codons in the transgene coding sequence. The Plant Cell 9: 1357-1368.

van Gestel K, Le J, and Verbelen JP. 2001. A comparison of F-actin labeling methods for light microscopy in different plant specimens: multiple techniques supplement each other. Micron 32: 571-578.

Vaucheret H. 2006. Post-transcriptional small RNA pathways in plants: mechanisms and regulations. Genes and Development 20: 759-771.

Voigt B, Timmers CJ, Samaj J, Muller J, Baluska F, and Menzel D. 2005. GFP-FABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of Arabidopsis seedlings. European Journal of Cell Biology 84: 595-608.

Voinnet O, Vain P, Angell S, and Baulcombe DC. 1998. Systemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA. The Cell 95: 177-187.

Wang YS, Motes CM., Mohamalawari DR, and Blancaflor EB. 2004. Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots. Cell Motility and the Cytoskeleton 59: 79-93.

Wang YS, Yoo CM, and Blancaflor EB. 2007. Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2. New Phytologist 177: 525-536.

Wille AW, and Lucas WJ. 1984 Ultrastructural and histochemical studies on guard cells. Planta 160: 129-142.

Williamson JD, Hirsh-Wyncott ME, Larkins BA, and Gelvin SB. 1989. Differential accumulation of a transcript driven by the CaMV 35S promoter in transgenic tobacco. Plant Physiology 90: 1570-1576.

Wilsen KL, Lovy-Wheeler A, Voigt B, Menzel D, Kunkel JG, and Hepler PK. 2006. Imaging the actin cytoskeleton in growing pollen tubes. Sexual Plant Reproduction 19: 51-62.

Acta Biologica Cracoviensia s. Botanica

The Journal of Polish Academy of Sciences

Journal Information

IMPACT FACTOR 2016: 0.491
5-year IMPACT FACTOR: 0.787

CiteScore 2016: 0.51

SCImago Journal Rank (SJR) 2016: 0.242
Source Normalized Impact per Paper (SNIP) 2016: 0.264

Ministry of Science and Higher Education: 20 points


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