Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants

BMC Biotechnology

David GJ Mann³  Laura L Abercrombie³  Mary R Rudis¹  Reggie J Millwood¹  John R Dunlap²  C Neal Stewart³ 

[1] Department of Plant Sciences, University of Tennessee, Knoxville, TN, 37996, USA

[2] Division of Biology, University of Tennessee, Knoxville, TN, 37996, USA

[3] BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA

关键词: Visual markers;    Transgenic plants;    Subcellular localization;    RFP;    Reporter genes;    Orange fluorescent protein;    OFP;    Marker genes;    GFP;    Fluorescent proteins;    Endoplasmic reticulum targeting;   

Others  :  1135192
DOI  :  10.1186/1472-6750-12-17

 received in 2012-01-11, accepted in 2012-04-25,  发布年份 2012

【 摘 要 】

Background

The expression of fluorescent protein (FP) genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully) appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their “brightness” and fluorescence, therefore, their usability as marker genes in transgenic plant tissues.

Results

The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER)-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed.

Conclusions

From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here.

【 授权许可】

   
2012 Mann et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Stewart CN: Go with the glow: fluorescent proteins to light transgenic organisms. Trends Biotechnol 2006, 24:155-162.
• [2]Shaner NC, Steinbach PA, Tsien RY: A guide to choosing fluorescent proteins. Nat Methods 2005, 2:905-909.
• [3]Shaner NC, Patterson GH, Davidson MW: Advances in fluorescent protein technology. J Cell Sci 2007, 120:4247-4260.
• [4]Held MA, Boulaflous A, Brandizzi F: Advances in fluorescent protein-based imaging for the analysis of plant endomembranes. Plant Physiol 2008, 147:1469-1481.
• [5]Nakagawa T, Suzuki T, Murata S, Nakamura S, Hino T, Maeo K, Tabata R, Kawai T, Tanaka K, Niwa Y, Watanabe Y, Nakamura K, Kimura T, Ishiguro S: Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci Biotechnol Biochem 2007, 71(8):2095-2100.
• [6]Curtis MD, Grossniklaus U: A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 2003, 133:462-469.
• [7]Goldman JJ, Hanna WW, Fleming G, Ozias-Akins P: Fertile transgenic pearl millet [Pennisetum glaucum (L.) R. Br.] plants recovered through microprojectile bombardment and phosphinothricin selection of apical meristem-, inflorescence-, and immature embryo-derived embryogenic tissues. Plant Cell Rep 2003, 21:999-1009.
• [8]Nishizawa K, Kita Y, Kitayama M, Ishimoto M: A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean. Plant Cell Rep 2006, 25:1355-1361.
• [9]Moon HS, Halfhill MD, Hudson LC, Millwood RJ, Stewart CN: Expression of green fluorescent protein in pollen of oilseed rape (Brassica napus L.) and its utility for assessing pollen movement in the field. Biotechnol J 2006, 1:1147-1152.
• [10]Hudson LC, Chamberlain D, Stewart CN: GFP-tagged pollen to monitor pollen flow of transgenic plants. Molecular Ecology Notes 2001, 1:321-324.
• [11]Furtado A, Henry RJ: The wheat Em promoter drives reporter gene expression in embryo and aleurone tissue of transgenic barley and rice. Plant Biotechnol J 2005, 3:421-434.
• [12]Furtado A, Henry R, Scott K, Meech S: The promoter of the asi gene directs expression in the maternal tissues of the seed in transgenic barley. Plant Mol Biol 2003, 52:787-800.
• [13]Cho M-J, Choi H-W, Jiang W, Ha CD, Lemaux PG: Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants. Physiol Plant 2002, 115:144-154.
• [14]Xiao K, Liu J, Dewbre G, Harrison M, Wang ZY: Isolation and characterization of root-specific phosphate transporter promoters from Medicago truncatula. Plant Biology 2006, 8:439-449.
• [15]Winicov I, Valliyodan B, Xue L, Hoober JK: The MsPRP2 promoter enables strong heterologous gene expression in a root-specific manner and is enhanced by overexpression of Alfin 1. Planta 2004, 219:925-935.
• [16]Chen A-P, Zhong N-Q, Qu Z-L, Wang F, Liu N, Xia G-X: Root and vascular tissue-specific expression of glycine-rich protein AtGRP9 and its interaction with AtCAD5, a cinnamyl alcohol dehydrogenase, in Arabidopsis thaliana. J Plant Res 2007, 120:337-343.
• [17]Freitas RL, Carvalho CM, Fietto LG, Loureiro ME, Almeida AM, Fontes EPB: Distinct repressing modules on the distal region of the SBP2 promoter contribute to its vascular tissue-specific expression in different vegetative organs. Plant Mol Biol 2007, 65:603-614.
• [18]Liu W, Mazarei M, Rudis MR, Fethe MH, Stewart CN: Rapid in vivo analysis of synthetic promoters for pathogen phytosensing. BMC Biotechnol 2011, 11:108. BioMed Central Full Text
• [19]Nelson BK, Cai X, Nebenführ A: A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 2007, 51:1126-1136.
• [20]Dietrich C, Maiss E: Red fluorescent protein DsRed from Discosoma sp. as a reporter protein in higher plants. Biotechniques 2002, 32:288-290.
• [21]Jach G, Binot E, Frings S, Luxa K, Schell J: Use of red fluorescent protein from Discosoma sp. (dsRED) as a reporter for plant gene expression. Plant J 2001, 28:483-491.
• [22]Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY: A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 2002, 99:7877-7882.
• [23]Shaner NC, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY: Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 2004, 22:1567-1572.
• [24]Alieva NO, Konzen KA, Field SF, Meleshkevitch EA, Hunt ME, Beltran-Ramirez V, Miller DJ, Wiedenmann J, Salih A, Matz MV: Diversity and evolution of coral fluorescent proteins. PLoS One 2008, 3(7):e2680.
• [25]Mathur J, Radhamony R, Sinclair AM, Donoso A, Dunn N, Roach E, Radford D, Mohaghegh PSM, Logan DC, Kokolic K, Mathur N: mEosFP-based green-to-red photoconvertible subcellular probes for plants. Plant Physiol 2010, 154:1573-1587.
• [26]Haseloff J, Siemering KR, Prasher DC, Hodge S: Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci U S A 1997, 94:2122-2127.
• [27]Mann DGJ, LaFayette PR, Abercrombie LL, King ZR, Mazarei M, Halter MC, Poovaiah CR, Baxter H, Shen H, Dixon RA, Parrott WA, Stewart CN: Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatumL.) and other monocot species. Plant Biotechnol J 2012, 10:226-236.
• [28]Mann DJG, King ZR, Liu W, Joyce BL, Percifield RJ, Hawkins JS, LaFayette PR, Artelt BJ, Burris JN, Mazarei M, Bennetzen JL, Parrott WA, Stewart CN: Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation. BMC Biotechnol 2011, 11:74. BioMed Central Full Text
• [29]Burris JN, Mann DJG, Joyce BL, Stewart CN: An improved tissue culture system for embyrogenic callus production and plant regeneration in switchgrass (Panicum virgatum L.). BioEnergy Res 2009, 2:267-274.
• [30]Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, Dawson J, Desai N, Hill M, Kadwell S, Launis K, Lewis K: Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technology 1993, 11:194-200.
• [31]Wiedenmann J, Schenk A, Röcker C, Girod A, Spindler K-D, Nienhaus GU: A far-red fluorescent protein with fast maturation and reduced oligomerization tendency. from Entacmaea quadricolor (Anthozoa, Actinaria). Proc Natl Acad Sci U S A 2002, 99:11646-11651.
• [32]Kooshki M, Mentewab A, Stewart CN: Pathogen inducible reporting in transgenic tobacco using a GFP construct. Plant Sci 2003, 165:213-219.
• [33]Mazarei M, Teplova I, Hajimorad MR, Stewart CN: Pathogen phytosensing: plants to report plant pathogens. Sensors 2008, 8:2628-2641.
• [34]Spiegel H, Schillberg S, Sack M, Holzem A, Nähring J, Monecke M, Liao Y-C, Fischer R: Accumulation of antibody fusion proteins in the cytoplasm and ER of plant cells. Plant Sci 1999, 149:63-71.
• [35]Schouten A, Roosien J, Engelen FA, de (Ineke) Jong GAM, (Tanja) Borst-Vrenssen AWM, Zilverentant JF, Bosch D, Stiekema WJ, Gommers FJ, Schots A, Bakker J: The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and thesecretory pathway in transgenic tobacco. Plant Mol Biol 1996, 30:781-793.
• [36]Conrad U, Fiedler U: Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Mol Biol 1998, 38:101-109.
• [37]Ma JK-C, Drake PMW, Christou P: Genetic modification: the production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 2003, 4:794-805.
• [38]Goulet C, Khalf M, Sainsbury F, D’Aoust M-A, Michaud D: A protease activity–depleted environment for heterologous proteins migrating towards the leaf cell apoplast. Plant Biotechnol J 2012, 10:83-94.
• [39]Richardson SM, Wheelan SJ, Yarrington RM, Boeke JD: GeneDesign: rapid, automated design of multikilobase synthetic genes. Genome Res 2006, 16:550-556.
• [40]Jayaraj S, Reid R, Santi DV: GeMS: an advanced software package for designing synthetic genes. Nucleic Acids Res 2005, 33(9):3011-3016.
• [41]Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT: A simple and general-method for transferring genes into plants. Science 1985, 227:1229-1231.
• [42]Clough SJ, Bent AF: Floral dip: a simplified method for Agrobacterium-mediatedtransformation of Arabidopsis thaliana. Plant J 1998, 16:735-743.
• [43]Millwood RJ, Halfhill MD, Harkins D, Russotti R, Stewart CN: Instrumentation and methodology for quantifying GFP fluorescence in intact plant organs. Biotechniques 2003, 34:638-643.