Download Evaluation and Comparison of the GUS, LUC and GFP Reporter

Review Article
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, and A. R. van der Krol4
2
1
Lab. of Plant Cell Biology, Wageningen University, Arboretumlaan 4, 6703 BD, Wageningen, The Netherlands
Current address: Hubrecht Lab., Netherlands Inst. for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
3
Current address: Dept. of Plant Physiology, Univ. of Amsterdam, Kruislaan 318, 1098 Amsterdam, The Netherlands
4
Lab. of Plant Physiology, Wageningen University, Arboretumlaan 4, 6703 BD, Wageningen, The Netherlands
Received: August 2, 2002; Accepted: January 16, 2003
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 b-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.
Abbreviations:
GUS:
b-glucuronidase
LUC:
firefly luciferase
GFP:
green fluorescent protein
CLSM: confocal laser scanning microscope
the information on spatial distribution of a specific mRNA
within a tissue sample is also lost. For these reasons, numerous
efforts have been made to simplify the procedure by which
gene activity can be quantified, which has led to the development of different reporter systems. The term reporter gene is
here used for the combination of a promoter (most often from
the plant gene under study), combined with the coding sequence of a reporter protein. The reporter protein typically
has an activity that is easily quantified in vitro, or which can
be imaged in planta. In order to appreciate the potential of
the different reporter systems that have been developed, it is
necessary to recognise the difference between reporting the
intrinsic properties of the promoter and reporting intrinsic
properties of the RNA or protein of a gene. Here we will limit
ourselves to the application of reporters in promoter activity
analysis, but we will discuss how RNA or protein properties
may influence the outcome of these studies. There are three
aspects of promoter activity that can be studied: (1) expression level, (2) promoter activity dynamics and (3) spatial distribution of promoter activity. We compare the colorimetric
(GUS), bioluminescent (ff-LUC) and fluorescent (GFP) reporter
systems that are most commonly used in plant gene expression studies.
The Reporter Proteins
Introduction
The overall activity of a gene is determined by DNA transcription, mRNA processing, mRNA transport from the nucleus to
the cytoplasm, translation and sometimes also post-translational modifications of the protein. Although most of these
steps are common cellular activities, potentially each of these
steps can be regulated. Therefore, the relationship between
quantified mRNA and quantified protein level or activity
within a cell may not always be straightforward. Gene expression level is usually quantified by the specific mRNA steadystate pool in total RNA isolated from whole plants or plant tissues. The isolation of RNA is laborious, destructive and the efficiency of the isolation can be tissue-dependent. Only one
time point can be assayed from the same tissue sample, while
Plant biol. 5 (2003): 103 ± 115
Georg Thieme Verlag Stuttgart ´ New York
ISSN 1435-8603
b-Glucuronidase (GUS)
The GUS reporter gene is derived from the Escherichia coli gus
operon (also known as uid operon). The gusA reading frame of
this operon encodes for a 68 kD b-glucuronidase (GUS) protein
which, in its active form, assembles into a homo-tetramer
which can catalyze the hydrolysis of a wide variety of b-glucuronides and b-galacturonides. These substrates are formed
in most eukaryotic organisms to detoxify and excrete xenobiotic and endogenous metabolic waste products (Jefferson,
1987). Activity of GUS protein in plant tissues can be detected
with a histochemical chromogenic (colour) assay, in which 5bromo-4-chloro-3-indolyl b-D-glucuronide (X-Gluc) is used as
a substrate (Fig. 1 A). The original amino acid sequence of the
GUS protein contained two sites that can serve as donor site
for N-glycosylation in plants, causing significant reduction in
GUS enzyme activity in plants (Farrell and Beachy, 1990). These
amino acids have therefore been replaced by site-directed mutagenesis of the GUS coding sequence. The modifications had
no deleterious effect on the GUS activity.
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Fig. 1 Reporter protein activity. (A) GUS enzyme activity with chromogenic substrate
(e.g. 5-bromo-4-chloro-3-indolyl b-D-glucuronide; X-Gluc). Step 1: hydrolysis of the
X-gluc substrate by the GUS enzyme. Step 2:
dimerization of the Gluc product by reaction
with oxygen. (B) GUS enzyme activity with
fluorescent substrate (e.g. ImaGene Green,
Mol. Probes Inc.). Step 3: hydrolysis of the
X-Gluc substrate by the GUS enzyme. Step 4:
fluorescence of the released fluorescent Gluc
product. (C) Firefly LUC (ff-LUC) activity.
Step 5: in the absence of CoA the protein is inactivated by complex formation with oxyluciferin and the reaction is non-enzymatic (the
so-called flash reaction). Step 6: in the presence of CoA, the luciferase protein is rapidly
released from the complex, resulting in an
enzymatic reaction. (D) GFP fluorescent activity. Step 7: Formation of the chromophore requires molecular oxygen. Step 8: fluorescence
of GFP. Wild type GFP absorbs light mainly at
395 nm (UV) and to a lesser extent at 475 nm
(blue), while emission of the fluorescent light
is at 509 nm. Several mutated versions of the
wild type GFP have been made with changed
excitation and emission wavelength (see
Table 1).
Fig. 2 A shows the steps of the GUS staining assay. There are
two steps in the procedure that can lower the resolution of
the assay: (1) diffusion of the GUS enzyme before protein
cross-linking by a fixation step (Fig. 2 A, step 2) and (2) diffusion of the reaction product before oxidation into the insoluble
blue precipitate (Fig. 2 A, step 3). In order to obtain rapid penetration of the fixative, a mild detergent may be used. However,
this may also increase the amount of leakage from the cells.
The use of a strong fixative can result in rapid protein crosslinking, preventing leakage from the cells, but may also result
in inactivation of GUS activity. A formaldehyde-insensitive
GUS derivative has been developed that allows for the usage
of rigorous and therefore rapid fixation conditions, thus inhibiting leakage and diffusion of GUS protein from the cells (Matsumura et al., 1999). The GUS reporter protein can also use
substrates that yield a fluorescent molecule (Fig. 1 B). These
substrates are mainly used in in vitro assays to quantify the
amount of GUS protein in plant extracts. However, fluorescent
probes have also been developed for the histochemical detection of GUS activity within tissue sections (e.g. ImaGene green
from Molecular Probes Inc.). The steps of fluorescent labelling
of tissues expressing a GUS reporter gene are outlined in
Fig. 2 B. The main advantage over the chromogenic assay is
that GUS expression can potentially be reported in vivo, allowing re-growth of the plant material after monitoring of GUS activity. The fluorogenic GUS substrate can access living cells
since the lipophilic probe can pass membranes of plant cells
(Flemming et al., 1996). In practice, GUS staining is most reli-
able when applied to fixed tissue samples or small intact seedlings. The ease of blue staining by GUS reporter activity has
made this reporter an extremely useful tool for localizing the
activity of a promoter fragment with cellular resolution in
plants.
Firefly luciferase (ff-LUC)
Proteins that show bioluminescence activity have been named
luciferase, regardless of the biochemical reaction that is catalysed by the protein. The luciferases from bacteria (LUX), firefly (ff-LUC) or the soft coral Renilla (r-LUC) all have different
structures and utilise different types of substrates. Similarly,
the substrates that are used by the different types of luciferases are all named luciferin, regardless of the chemical structure of the compound. The term luciferase or luciferin should
therefore either be well defined by context or otherwise indexed to refer to a specific type of enzyme or substrate. Here,
we will only discuss the properties of the firefly luciferase,
which is mostly used in plant research.
The luciferase (ff-LUC) gene from the firefly Photinus pyralis
was cloned in 1985 (DeWet et al., 1985) and was quickly adapted as a reporter of promoter activity in many different systems
including bacteria, animal and plant cells (reviewed by Gould
and Subramani, 1988). The gene encodes a single active polypeptide of 62 kD (ff-LUC) which, in a reaction with its substrates firefly luciferin, ATP and oxygen, causes the release of
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Table 1 Different versions of GUS, ff-LUC and GFP reporter proteins
Reporter
Type
Reference
T50 mRNA
T50 protein
GUS
wild type
modified for expression
1
19, 76
n. r.
~ 120 min (67)
formaldehyde sensitivity modified
specific instability modified A
56
18, 22, 39, 67, 76
n. r.
~ 220 min (22)
~ 40 min (67)
n. r.
days (19),
> 4 h (76)
n. r.
~ 9 h (76)
wild type
modified for expression
15
23, 25, 53, 54, 69, 80, 85
~ 45 min (22)
~ 108 min (22)
specific instability modified A
22, 39, 44, 86, 90
wild type
modified for expression
excitation modified B
emission modified C
specific instability modified A
6, 79
28, 29, 82
50, 88
7, 9,, 31, 40
7, 9, 10, 70, 82
LUC
GFP
~ 24 min (22)
n. r.
n. r.
n. r.
n. r.
n. r.
~ 4 h (46)
~ 4 min (at 40 8C; 57)
~ 13 min (44, 90)
n. r.
~ 18 h (50)
n. r.
n. r.
~ 2 h (50)
Modified for expression: modifications aimed at improved expression in plant
cells. A Modifications that are specifically aimed at decreasing the stability of
the mRNA or protein, in order to increase time resolution of reporter activity.
B
Modifications that affect the excitation spectrum of GFP. C Modifications that
affect the emission spectrum of GFP. T50: half-life time (mRNA or protein) of the
reporter. n. r.: not reported.
Green fluorescent protein
Reporter Turnover Effects
Green fluorescent protein (GFP) is a 27-kD protein from the jellyfish Aequorea victoria that due to its unique structure, shows
a bright green fluorescence when folded correctly and illuminated with UV or blue light (Fig. 1 D; Chalfie et al., 1994). For
effective expression in plants, the GFP coding sequence was
adapted to remove regions with high AT content (cryptic intron sequences and splice sites) and a cryptic nuclear import
signal (Haseloff and Amos, 1995, Sheen et al., 1995, Siemering
et al., 1996, Haseloff et al., 1997). The accumulation of unmodified GFP within the nucleus has an adverse effect on plant
growth, possibly by increasing the chance of free radical formation by GFP that can cause DNA damage. Mutation of the
amino acid (aa) sequence inside the chromophore resulted in
the production of GFP variants that contain higher emission
levels and/or shifted excitation and emission spectra. Variants
of GFP with an absorption peak at 490 nm made GFP more
suitable for excitation with the 488 nm line of visible lasers
used in confocal laser scanning microscopes (CLSM) and analysis with standard FITC filters (excitation 450 ± 490/DM 510/
emission 520 nm) in wide field fluorescence microscopy (see
Table 1). When argon laser sources are used, as in confocal laser scanning microscopy (CLSM), the use of a GFP form with a
shift in the excitation wavelength to blue light is preferable.
Mutations outside the chromophore produced mutants that
show an increased fluorescence when excited with UV light
and mutants with an equalised excitation peak, one for UV
and one for blue light (Table 1). The GFP fluorescent properties
are not hindered by most N- or C-terminal peptide or protein
fusions. Chimeric GFP with specific targeting sequences are
used to visualise the sub-cellular localisation of different proteins within a cell (Kohler et al., 1997; Haseloff et al., 1997,
Hanson and Köhler, 2001). The properties of different forms of
GFP proteins has been reviewed by Haseloff and Amos (1995),
Neidz et al. (1995), Leffel et al. (1997), Stewart (2001) and Hanson and Köhler (2001).
Whether reporter activity is detectable within a plant cell or
tissue depends on the promoter strength, duration of promoter
activity, stability of the reporter mRNA, stability and activity of
the reporter protein, the intrinsic background level within a
tissue, and reporter signal detection techniques. From the moment synthesis stops, it takes approximately three times the
half-life of the mRNA (or the protein) to reduce the level to
90 % of the initial steady state level. Therefore, the shorter the
reporter mRNA and reporter protein half-life time, the more
closely that reporter will reflect transcriptional activity of the
gene (see also Wood, 1995). The mRNA stability is influenced
by the AU content (Ohme et al., 1993) and specific 5¢leader
and/or 3¢tail sequences which may be included into a chimeric
reporter gene. Potential post-translational regulation of expression encoded within the amino acid (aa) sequence of a
protein may be transferred to the reporter by an in-frame fusion of two coding DNA sequences in the reporter gene. For instance, analysis of highly abundant proteins in plants shows
the presence of a conserved structure at position 4 ± 11 of the
protein aa sequences. Adaptation of the N-terminal sequence
of the GUS protein to this structure resulted in a 30 ± 40fold increase in activity compared to wild type GUS enzyme activity
in transient assays (Sawant-Samir et al., 2001). Protein stability may also depend on tissue or cell type and may even vary
with subcellular localisation (Fisk-Henry and Dandekar-Abhaya, 1998, Michels et al., 1995). Destabilisation of the reporter
mRNA or protein may increase the time resolution of promoter
activity. For this purpose, several destabilized versions of GUS,
ff-LUC and GFP have been constructed (see Table 1). A destabilised version of ff-LUC may be redundant when protein activity
is not regenerated. Thus, in the absence of CoA, ff-LUC half-life
time is determined by the rate of the reaction with its substrates (van Leeuwen et al., 2000). As the detection of GFP fluorescence is a function of the amount of GFP and the level of
background fluorescence of plant cell components, the fluo-