Evaluation and Comparison of the GUS, LUC and GFP Reporter System for Gene Expression Studies in Plants

N. C. A. de Ruijter; J. Verhees; W. van Leeuwen; A. R. van der Krol

doi:10.1055/s-2003-40722

Plant Biology, Table of Contents

Plant Biol (Stuttg) 2003; 5(2): 103-115
DOI: 10.1055/s-2003-40722

Review Article

Georg Thieme Verlag Stuttgart · New York

Evaluation and Comparison of the GUS, LUC and GFP Reporter System for Gene Expression Studies in Plants

N. C. A. de Ruijter¹ , J. Verhees² , W. van Leeuwen³ , A. R. van der Krol⁴

¹Lab. of Plant Cell Biology, Wageningen University, Arboretumlaan 4, 6703 BD, Wageningen, The Netherlands
²Current address: Hubrecht Lab., Netherlands Inst. for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
³Current address: Dept. of Plant Physiology, Univ. of Amsterdam, Kruislaan 318, 1098 Amsterdam, The Netherlands
⁴Lab. of Plant Physiology, Wageningen University, Arboretumlaan 4, 6703 BD, Wageningen, The Netherlands

Abstract

The detailed analysis of the expression pattern of a plant gene can give important clues about its function in plant development, cell differentiation and defence reactions. Gene expression studies have been greatly facilitated by the employment of proteins like β-glucuronidase (GUS), green fluorescent protein (GFP), and firefly luciferase (LUC) as reporters of gene activity. The application of reporter genes in plants, specifically in the field of gene expression studies, has expanded over the years from a mere tool to quantify (trans) gene expression in tissue samples, to real-time imaging of in planta promoter dynamics. To correctly interpret the activity that is given by each reporter, it is important to have a good understanding of the intrinsic properties of the different reporter proteins. Here we discuss those properties of GUS, LUC and GFP that are of interest in gene expression studies.

Key words

Glucuronidase - luciferase - green fluorescent protein - reporter genes - gene expression

Full Text

References

1 Aflalo C.. Biologically localized firefly luciferase: A tool to study cellular processes. Int. Rev. Cytol.. (1991); 130 267-323
2 Alwen A., Binto M. R. M., Vicente O., Heberle B. E.. Plant endogenous beta-glucuronidase activity: How to avoid interference with the use of the E. coli beta-glucuronidase as a reporter gene in transgenic plants. Transgenic Research. (1992); 1 (2) 63-70
3 Barnes W. M.. Variable patterns of expression of luciferase in transgenic tobacco leaves. Proc. Natl. Acad. Sci. USA. (1990); 87 9183-9187
4 Blundy K. S., Blunde M. A. C., Carter D., Wilson F., Park W. D., Burell M. M.. The expression of class I patatin gene fusions in transgenic potato varieties with both gene and cultivar. Plant Mol. Biol.. (1991); 16 153-160
5 Campisi L., Yang Y., Yi Y., Heilig E., Herman B., Cassista A., Allen D. W., Xiang H., Jack T.. Generation of enhancer trap lines in Arabidopsis and characterization of expression patterns in the inflorescence. The Plant Journal. (1999); 17 699-707
6 Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C.. Green fluorescent protein as a marker for gene expression. Science. (1994); 263 802-805
7 Chiu W. L., Niwa Y., Zeng W., Hirano T., Kobayashi H., Sheen J.. Engineered GFP as a vital reporter in plants. Current Biology. (1996); 6 325-330
8 Cody C. W., Prasher D. C., Westler W. M., Prendergast F. G., Ward W. W.. Understanding, improving and using green fluorescent proteins. Trends Biochemical. Sciences. (1993); 20 448-455
9 Cormack B. P., Valdivia R. H., Falkow S.. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. (1996); 173 (1) 33-38
10 Crameri A., Whitehorn E. A., Tate E., Stemmer W. P. C.. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnology. (1996); 14(3) 315-319
11 Dean C., Jones J., Favreau M., Dunsmuir P., Bedbrook J.. Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants. Nucleic Acids Research. (1988); 16 9267-9283
12 Delagrave S., Hawtin R. E., Silva C. M., Yang M. M., Youvan D. C.. Red-shifted excitation mutants of the green fluorescent protein. Bio/Technology. (1995); 13 151-154
13 DeLuca M. A., McElroy W. D.. Bioluminescence and chemiluminescence. DeLuca, M. A., ed. Methods in Enzymology. New York; Academic Press (1986): 3-15
14 Denburg J. L., Lee R. T., McElroy W. D.. Substrate-binding properties of firefly luciferase. Arch Biochem Biophys. (1969); 134 381-394
15 DeWet J. R., Wood K. V., Helinski. D. R., DeLuca. M.. Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. . Proc. Natl. Acad. Sci.. (1985); 82 7870-7873
16 Farrell L. B., Beachy R. N.. Manipulation of β-glucuronidase for use as a reporter in vacuolar targeting studies. Plant Mol. Biol.. (1990); 15 680-685
17 Federoff N., Smith D.. A versatile system for detecting transposition in Arabidopsis. . Plant Journal. (1993); 3 273-289
18 Fisk-Henry J., Dandekar-Abhaya M.. Nuclear localization of a foreign gene product in tobacco results in increased accumulation due to enhanced stability. Plant Science. (1998); 133 (2) 177-189
19 Flemming A. J., Manzara T., Gruissem W., Kuhlemeier C.. Fluorescent imaging of GUS activity and RT-PCR analysis of gene expression in the shoot apical meristem. The Plant Journal. (1996); 10 745-754
20 Ford S. R., Buck L. M., Leach F. R.. Does the sulfhydryl or the adenine moiety of CoA enhance firefly luciferase activity?. Biochim. Biophys. Acta. (1995); 1252 180-184
21 Forreiter C., Kirschner M., Nover L.. Stable transformation of an Arabidopsis cell suspension culture with firefly luciferase providing a cellular system for analysis of chaperone activity in vitro. . Plant Cell. (1997); 9 2171-2178
22 Gallie D. R., Feder J. N., Schimke R. T., Walbot V.. Post-transcriptional regulation in higher eukaryotes: The role of the reporter gene in controlling expression. Mol. Gen. Genet.. (1991); 228 258-264
23 Gould S. J., Subramani S.. Firefly luciferase as a tool in molecular and cell biology. Anal. Biochem.. (1988); 175 5-13
24 Goverse A., Biesheuvel J., Wijers G. J., Gommers F. J., Bakker F. J., Bakker J., Schots A., Helder J.. In planta monitoring of the activity of two constitutive promoters, CaMV 35 S and TR2í, in developing feeding cells induced by Globodera rostochiensis using green fluorescent protein in combination with confocal laser scanning microscopy. Physiological and Molecular Plant Pathology. (1998); 52 275-284
25 Groskreutz D. J., Sherf B. A., Wood K. V., Schenborn E. T.. Increased expression and convenience with the new pGL3 luciferase reporter vectors. Promega Notes. (1995); 50 2-8
26 Hall A., Kozma-Bognar L., Toth R., Nagy F., Millar A. J.. Conditional Circadian Regulation of PHYTOCHROME A . Gene Expression. Plant Physiology. (2001); 127 (4) 1808-1818
27 Hanson R. M., Köhler R. H.. GFP imaging: methodology and application to investigate cellular compartmentation in plants. J. Exp. Bot.. (2001); 52 529-539
28 Haseloff J., Amos B.. GFP in plants. Trends in Genetics. (1995); 11 328-329
29 Haseloff J., Siemering K. R., Prasher D. C.. Removal of cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl Acad. Sci USA. (1997); 94 2122-2127
30 Heim H., Cubitt A. B., Tsien R. Y.. Improved green fluorescence. Nature. (1995); 373 663-664
31 Heim R., Prasher D. C., Tsien R. Y.. Wavelength mutations and post translational autooxidation of green fluorescent protein. Proc. Natl Acad. Sci USA. (1994); 91(26) 12,501-12,504
32 Herrera-Estrella L., León P., Olson O., Teeri T. H.. Reporter genes for plants. Gelvin, S. B. and Schilperoort, R. A., eds. Plant Molecular Biology Manual, Vol. C2. London; Kluwer Academic Publishers (1994): 1-32
33 Hideg E., Kobayashi M., Inaba H.. Delayed fluorescence and ultraweak light emission from isolated chloroplasts (comparison of emission spectra and concentration dependence). Plant Cell Physiol.. (1992); 33(6) 689-693
34 Ishitani M., Xiong L., Stevenson B., Zhu J. K.. Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: interactions and convergence of abscisic acid dependent and abscisic-independent pathways. The Plant Cell. (1997); 9 1935-1949
35 Jefferson R. A., Kavanagh T. A., Bevan M. W.. GUS fusions: glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal. (1987); 6 3901-3907
36 Jorda L., Vera P.. Local and systemic induction of two defense-related subtilisin-like protease promoters in transgenic Arabidopsis plants. Luciferin induction of PR gene expression. Plant Physiology. (2000); 124 (3) 1049-1057
37 Kay S. A., Millar A., Smith K. W., Anderson S. L., Brandes C., Hall J. C.. Video imaging of regulated firefly luciferase activity in transgenic plants and Drosophila. . Promega Notes. (1994); 49 22-27
38 Kerbundit H., De Greve F., Deboeck, van Montagu M., Heernalseens J.. In vivo random β-glucuronidase gene fusions in Arabidopsis thaliana . PNAS. (1991); 88 5212-5216
39 Kindon C. W., Ling R., Callis J.. Engineering in vivo instability of firefly luciferase and E. coli glucuronidase in higher plants using recognition elements from the ubiquitin pathway. Plant Mol. Biol.. (1998); 37 337-347
40 Kohler R. H., Ziphel W. R., Webb W. W., Hanson M. R.. The green fluorescent protein as a marker to visualize plant mitochondria in vivo. The Plant Journal. (1997); 11(3) 613-621
41 Kost B., Schnorf M., Potrykus I., Neuhous G.. Non-destructive detection of firefly luciferase (LUC) activity in single plant cells using a cooled, slow-scan CCD camera and an optimized assay. The Plant Journal. (1995); 8 155-166
42 Kosugi S., Ohashi V., Nakjima K., Arai V.. An improved assay for β-glucuronidase in transformed cells: Methanol almost completely suppresses a putative endogenous B-glucuronidase activity. Plant Sci.. (1990); 70 133-140
43 Krol van der A. R., van Poecke M. P., Vorst O. F. J., Voogt C., van Leeuwen W., Borst-Vrensen T. W. M., Takatsuji H., van der Plas L. H. W.. Developmental and wound-, cold, desiccation-, ultraviolet B-stress induced modulations in the expression of the petunia zinc finger transcription factor Gene ZPT2-2. Plant Physiology. (1999); 121 1153-1162
44 Leclerc G. M., Bookcfor F. R., Faught J., Frawleay L. S.. Development of a destabilized firefly luciferase enzyme for measurement of gene expression. BioTechniques. (2000); 29 590-601
45 Leeuwen van W.. Transgene expression in plants: position induced spatial and temporal variations of luciferase expression. Wageningen University, Department of Plant Physiology: Thesis. (2001)
46 Leeuwen van W., Hagendoorn M. J. M., Ruttink T., van Poecke R., van der Plas L. H. W., van der Krol A. R.. The use of the luciferase reporter system for in planta gene expression studies. Plant Mol. Biol. Reporter. (2000); 18 (2) 143a-143t
47 Leeuwen van W., Mlynarova L., Nap J. P., van der Plas L. H. W., van der Krol A. R.. The effect of MAR elements on variation in spatial and temporal regulation of transgene expression. Plant Mol. Biology. (2001a); 47 543-554
48 Leeuwen van W., Ruttink T., Borst-Vrenssen A. W. M., van der Plas L. H. W., van der Krol A. R.. Characterization of position induced spatial and temporal regulation of transgene promoter activity in plants. J. Exp. Bot.. (2001b); 52 (358) 1-11
49 Leffel S. M., Mabon S. A., Stewart Jr C. N.. Applications of green fluorescent protein in plants. BioTechniques. (1997); 23 (5) 912-916
50 Li X., Zhao X., Fang Y., Jiang X. F., Duong T., Fan C., Huang C.-C., Kain S. R.. Generation of Destabilized Green Fluorescent Protein as a transcription reporter. The Journal of Biol. Chemistry. (1998); 273 34970-34975
51 Liu J., Wang Y., Szalay A. A., Escher A.. Visualising and quantifying protein secretion using a renilla luciferase-GFP fusion protein. Luminescence. (2000); 15 45-49
52 Loew D., Braendle K., Nover L., Forreiter C.. Cytosolic heat stress proteins Hsp17.7 class I and Hsp 17.3 class II of tomato act as molecular chaperones in vivo. . Planta. (2000); 211 575-582
53 Lonsdale D. M., Lindup S., Moisan L. J., Harvey A. J.. Using firefly luciferase to identify the transition from transient to stable expression in bombarded wheat scutellar tissue. Physiol. Plant.. (1998); 102 447-453
54 Lonsdale D. M., Moisan L. J., Harvey A. J.. The effect of altered codon usage on luciferase activity in tobacco maize and wheat. Plant Cell Rep.. (1998); 17 396-399
55 Luehrsen K. R., Walbot V.. Firefly luciferase as a reporter for plant gene expression. Promega Notes. (1993); 44 24-29
56 Matsumura I., Wallingford J. B., Surana N. K., Vize P. D., Ellington A. D.. Directed evolution of the surface chemistry of the reporter enzyme glucuronidase. Nature Biotechnology. (1999); 17 696-701
57 Michels A. A., Nguyen V. T., Konings A. W. T., Kampinga H. H., Bensaude O.. Thermostability of a nuclear-targeted luciferase expressed in mammalian cells. Destabilizing influence of the intranuclear microenvironment. Eur. J. Biochem.. (1995); 234 382-389
58 Millar A. J., Carre I. A., Strayer C. A., Chua N. H., Kay S. A.. Circadian clock mutants in Arabidopsis identified by luciferase imaging. Science. (1995); 267 1161-1166
59 Millar A. J., Short S. R., Chua N. H., Kay S. A.. A novel circadian phenotype based on firefly luciferase expression in transgenic plants. Plant Cell. (1992a); 4 1075-1087
60 Millar A. J., Short S. R., Hiratsuka K., Chua N. H., Kay S. A.. Firefly luciferase as a reporter of regulated gene expression in higher plants. Plant Mol. Biol. Reporter. (1992 b); 10 (4) 324-337
61 Mlynárová L., Jansen R. C., Conner A. J., Stiekema W. J., Nap J. P.. The MAR mediated reduction in position effect can be uncoupled from copy number dependent expression in transgenic plants. Plant Cell. (1995); 7 599-609
62 Mlynárová L., Loonen A., Heidens J., Jansen R. C., Keizer P. C., Stiekema W. J., Nap J. P.. Reduced position effect in mature transgenic plants conferred by the chicken lysozyme matrix associated region. Plant Cell. (1994); 6 417-426
63 Mudge S. R., Birch R. G.. T-DNA tagging and characterisation of a novel meristem-specific promoter from tobacco. . Australian Journal of Plant Physiology. (1998); 25 (6) 637-643
64 Nass N., Scheel D.. Enhanced luciferin entry causes rapid wound-induced light emission in plants expression high levels of luciferase. Planta. (2001); 212 149-154
65 Neidz R. P., Sussman M. R., Satterlee J. S.. Green fluorescent protein: an in vivo reporter of plant gene expression. Plant Cell Reports. (1995); 14 403-497
66 Neumann K., Droege L. W., Koehne S., Broer I.. Heat treatment results in a loss of transgene encoded activities in several tobacco lines. Plant Physiology. (1997); 115 939-947
67 Newman T. C., Ohme-Takagi M., Taylor C. B., Green P. J.. DST sequences, highly conserved among plant SAUR genes, target reporter transcripts for rapid decay in tobacco. . Plant Cell. (1993); 5 701-714
68 Ohme T. M., Taylor C. B., Newman T. C., Green P. J.. The effect of sequences with high AU content on mRNA stability in tobacco. . Proc Nat. Acad. Sci. USA.. (1993); 90 (24) 11811-11815
69 Ow D. W., Wood K. V., DeLuca M., DeWey J. R., Helinski D. R., Howell S. H.. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Proc. Natl. Acad. Sci.. (1986); 84 4870-4874
70 Pang S.- Z., DeBoer D. L., Wan Y.- C., Ye G., Layton J. G., Neher M. K., Armstrong C. L., Fry J. E., Hinchee M. A. W., Fromm M. E.. An improved green fluorescent protein gene as a vital marker in plants. Plant Physiology. (1996); 112 893-900
71 Plautz J. D., Strayer C. A., Kay S. A.. Automated recording of luciferase reported gene transcription in living seedlings and fruitflies. TopCount Topics TCA-025 Packard Instrument Company. (1996)
72 Plegt L., Bino R. J.. β-glucuronidase activity during development of the male gametophyte from transgenic and non-transgenic plants. Mol. Gen. Genet. (1989); 216 321-327
73 Quaedvlieg N. E. M., Schlaman H. R. M., Admiraal P. C., Wijting S. E., Stougaard J., Spaink H. P.. Fusions between green fluorescent protein and Glucuronidase as sensitive and vital bifunctional reporters in plants. Plant Mol. Biol.. (1998); 37 715-727
74 Quandt H. J., Broer I., Puhler A.. Tissue specific activity and light dependent regulation of a soyben rbcS promoter in transgenic tobacco plants monitored with the firefly luciferase gene. Plant Science. (1992); 82 59-70
75 Remans T., Schenk P. M., Manners J. M., Grof C. P. L., Elliot A. R.. A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65 T) in transgenic plants. Plant Mol. Biol. Reporter. (1999); 17 385-395
76 Sawant-Samir V., Kiran K., Singh P. K., Tuli R.. Sequence architecture downstream of the initiator codon enhances gene expression and protein stability in plants. Plant Physiology. (2001); 126 (4) 1630-1636
77 Schneider M., Ow D. W., Howell S. H.. The in vivo pattern of firefly luciferase expression in transgenic plants. Plant Mol. Biol.. (1990); 14 935-947
78 Selinger H. H., McElroy W. D.. The colors of firefly luminescence: enzyme configuration and species specificity. Proc. Natl Acad. of Sci.. (1964); S2 75-81
79 Sheen J., Hwang S. B., Niwa Y., Kobayashi H., Galbraith D. W.. Green-fluorescent protein as a new vital marker in plant cells. Plant Journal. (1995); 8 777-784
80 Sherf B. A., Wood K. V.. Luminometry for in vivo and in vitro reporting of firefly luciferase. Promega Notes. (1993); 44 18-23
81 Sherf B. A., Wood K. V.. Firefly luciferase engineered for improved genetic reporting. Promega Notes. (1994); 49 14-21
82 Siemering K. R., Golbik R., Sever R., Haseloff J.. Mutations that suppress the thermosensitivity of green fluorescent protein. Current Biology. (1996); 6 (12) 1653-1663
83 Stewart Jr. C. N.. The untility of green fluorescent protein in transgenic plants. Plant Cell Reports. (2001); 20 376-382
84 Sundaresan P., Springer T., Volpe S., Jones H. J., Dean C., Martienssen R.. Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. Genes and Development. (1995); 9 1797-1810
85 Tanguay R. L., Gallie D. R.. The effect of the length of the 3′untranslated region on expression in plants. FEBS Letters. (1996); 394 285-288
86 Thompson J. F., Hayes L. S., Lloyd D. B.. Modulation of firefly luciferase stability and impact on studies of gene regulation. Gene. (1991); 103 171-177
87 Toonen M. A. J., Verhees J. A., Schmidt E. D. L., van Kammen A., de Vries S. C.. AtLTPI luciferase expression during carrot somatic embryogenesis. Plant J.. (1997); 12 (5) 1213-1221
88 Tricas J. A., Pinto R., Britton W. J.. Destabilized green fluorescent protein for monitoring transient changes in mycobacterial gene expression. Research in Microbiology. (2002); 153 379-383
89 Wood K. V.. Marker proteins for gene expression. Current Opinion in Biotechnology. (1995); 6 50-58
90 Worley C. K., Zenser N., Ramos J., Rouse D., Leyser O., Theologis A., Callis J.. Degradation of Aus/IAA proteins is essential for normal auxin signalling. The Plant Journal. (2000); 21 553-562
91 Xiong L. M., David L., Stevenson B.. High throughput screening of signal transduction mutants with luciferase imaging. Plant Mol. Biol. Reporter. (1999); 17 (2) 159-170

A. R. van der Krol

Lab. of Plant Physiology
Wageningen University

Arboretumlaan 4

6703 BD Wageningen

The Netherlands

Email: sander.vanderkrol@wur.nl

Section Editor: L. A. C. J. Voesenek