Bacterial and Firefly Luciferase Genes in Transgenic Plants: Advantages and Disadvantages of a Reporter Gene
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DEVELOPMENTAL GENETICS 11:224-232 (1990)
Bacterial and Firefly Luciferase Genes in
Transgenic Plants: Advantages and Disadvantages
of a Reporter Gene
CSABA KONCZ, WILLIAM H.R. LANGRIDGE, OLOF OLSSON, JEFF SCHELL, AND
ALADAR A. SZALAY
Institute of Plant Physiology (C.K.),Biological Research Center, Hungarian Acadamy of Sciences, H-6701, Hungary;
Max-Planck-Znstitutfur Zuchtungsforschung (C.K.,J.S.), Koln 30, Federal Republic of Germany; Plant Molecular
Genetics and Plant Biotechnology Centre and Department of Cell Biology (W.H.R.L.,A.A.S.), Medical Sciences
Building, University of Alberta, Edmonton, Alberta, Canada; and Department of Plant Physiology (O.O.), University
of Umea, Umea, Sweden
ABSTRACT Genes encoding light-emitting therefore has been used widely a s a bioluminescent
luciferase were recently isolated from luminous indicator for metabolic assays (see below). The cata-
marine bacteria and fireflies. Expression of lu- lytic reaction is initiated by the formation of a n en-
ciferase genes in diverse organisms is a unique zyme-bound luciferin-adenylate. This is followed by a
way for studying gene expression by simple and change in protein conformation which provides a hy-
sensitive measurement of light. Recent advances in drophobic active site for deprotonation and hydroper-
application of luciferase reporter genes are re- oxide addition a t the C4 position of luciferin. Subse-
viewed and documented by examples of in vivo quent decarboxylation and splitting of the linear
visualization of their expression in transgenic peroxide leads to formation of COz, H20, AMP, and a n
plants. excited, dianionic form of oxyluciferin. In excess of sub-
strate the reaction produces a quick flash of light pro-
Key words: lux and luc reporter genes, light portional to the quantity of the enzyme. After the
emission, gene expression, single photon imaging flash, a n extended low-light emission occurs indicating
in vivo a slow-rate dissociation of the product. The quantum
yield of firefly luciferase is 0.88, the highest among
known luciferases.
INTRODUCTION All fireflies use the same substrates but diverse spe-
cies emit different colours of light varying from yellow
Light-emitting organisms attracted the attention of (582 nm) to green (522 nm). Low pH and divalent cat-
scientists throughout history from Caius Plinius (23- ions shift the light emission to red, indicating that the
79 A.D.) to E.N. Harvey [Harvey, 19571. The terms of conformational change of the enzyme and alterations
luciferase and luciferin were first applied by Dubois in in its structure play a n important role in determining
1885 to describe light-emitting proteins and their sub- the energy of the excited product and thus the colour of
strates extracted from jelly fishes. Luciferases and lu- emitted light [DeLuca, 1976, 19781. Recently, lu-
ciferins were purified and characterized from diverse ciferase cDNAs were cloned from the Japanese firely
species of fireflies, beetles, marine bacteria, molluscs, [Masuda et al., 19891 and from beetles [Wood et al.,
medusas, fishes, and earthworms. A schematic classi- 19891 which produce different colours of light when
fication of luciferins shows that most eukaryotic lu- expressed in E. coli. The identification of amino acid
ciferases use heterocyclic compounds as substrates exchanges between these enzymes should pinpoint pep-
while in marine bacteria and earthworms, photogenic tide domains involved in enzyme-substrate interaction,
substrates are aliphatic aldehydes [DeLuca, 1978; De- a s well as open the way to engineer novel luciferases.
Luca and McElroy, 19861. From an evolutionary point of view it is intriguing
that the reaction leading to luciferin adenylation is
FIREFLY LUCIFERASE analogous to those involved in the activation of amino
Luciferase isolated from the North American firefly,
Photinus pyralis (Photinus, 1uciferin:oxygen 4-oxy-
doreductase, EC. 1.13.12.7; 62 kD) catalyzes the OxY- h i v e d for publieation October 18,1989; accepted October 28,1989.
dative decarboxylation of luciferin, a 8hydroxy-
benzothiazole, to 0xyluciferin in the presence of ATP, Address reprint requests to Csaba Koncz, Max-Planck-Institut fiir
Mg2' and 02.The enzyme is specific for ATP and Ziichtungsforschung, D-5000 K6ln 30, Federal Republic of Germany.
O 1990 WILEY-LISS, INC.
LUCIFERASE GENES IN TRANSGENIC PLANTS
acids and fatty acids. Furthermore, luciferase can also of reduced FMN and long-chain aldehydes with molec-
catalyze the addition of adenylated dihydroluciferin to ular oxygen to yield FMN, H20, corresponding carbox-
CoA, in a reaction similar to that catalyzed by fatty ylic acids, and blue-green light (490 nm). The reaction
acyl CoA synthases. Since this reaction is CoA-specific, can be considered as a branch of an electron-transport
it is probably not a coincidence that the enzyme shows pathway which shunts electrons to oxygen a t the level
a significant homology to other CoA-specific enzymes, of flavin. The enzymatic reaction is unusual because it
such as plant 4-coumarate-CoA ligase [Schroeder, results in long-life intermediates. A key intermediate
19891. is an enzyme-bound 4a-hydroperoxide (FMN-OOH)
In Photinus the luciferase is encoded by a transcript whose reaction with the aldehyde probably leads to for-
of about 1,800 nt, which is synthesized from a single mation of a hydroxy-flavin emitter in its singulet ex-
copy luc gene containing six introns [deWet et al., 1985, cited state. Due to quick oxidation of free FMNH, and
19871. Localization of luciferase protein in photocytes to long-time relaxation of the enzyme from its altered
of firefly lantern, as well as in animal and plant cells, conformational state, only one catalytic cycle is possi-
indicated that the enzyme is targeted to peroxisomes ble. Therefore the light production is strictly propor-
by a peptide signal (PTS) consisting of the last three tional to the amount of enzyme in excess of FMNH,
C-terminal amino acids Lys-Ser-Leu [Keller et al., and aldehyde substrate. The quantum yield of bacterial
1987; Gould et al., 1987, 1989; Gould and Subramani, luciferase is 0.1, equivalent with 60 ATP per photon.
19881. The aldehyde-binding site of the luciferase a-subunit
contains an essential sulfhydryl group close to the ap-
ANALOGIES BETWEEN EUKARYOTIC subunit interphase. The non-catalytic P-subunit is re-
LUMINESCENCE SYSTEMS quired for proper folding and conformational change of
the a-subunit during interaction with the flavin mol-
Formation of a linear peroxide at various positions of ecule. Structural mutations in both enzyme subunits,
eukaryotic luciferins appears to be a common event in as well as various flavin analogs, can alter the emis-
most luciferase reactions producing C02 and H20. Al-
sion spectra between 490 and 535 nm [Hastings, 1978;
though the excited oxyluciferin is always the primary Kurfiirst et al., 1984; Lee et al., 1988; Aboukhair et al.,
emitter it is common that the energy is transmitted to
1985; Paquette et al., 1988; Chen and Baldwin, 19891.
a secondary emitter, i.e., protein-bound flavin chro- Ln Photobacterium strains a lumazine protein forms
mophore. Luminescence systems are frequently regu- a complex with luciferase and, by energy transfer from
lated by the nerve net of organisms. When disturbed,
the flavin to the secondary emitter 6,7-dimethyl-8-(l1-
cypridinas produce a blue luminescence by ejecting lu- D-ribityll-lumazine, blue light (475 nm) is emitted [Lee
ciferase and luciferin from separate glands into seawa-
et al., 19891. In Vibrio fischeri strain Y-1 energy trans-
ter. A more complicated network, evolved in the antho- fer occurs from excited flavin intermediates to a flavin-
zoan coelenterate Renilla, involves four proteins. A containing secondary emitter protein, termed yellow
luciferin sulfokinase catalyzes the synthesis of lu-
fluorescent protein (YFP), causes a yellow shift (534
ciferin from luciferyl sulfate and 3',5'-diphosphoade-
nosine. Luciferin is oxydized to oxyluciferin and C02 nm) in the emission of light. In the presence of
NAD(P)H-FMN oxydoreductase, an enzyme supplying
by luciferase. The energy is transmitted to a luciferase- reduced FMN in luminous bacteria, the addition of
associated green fluorescent protein (GFP). The chro- YFP does not only shift the colour but also increases
mophore is enclosed in vesicles and emits green light
the intensity of total light emitted three- to four-fold
only when a luciferin-binding protein (BP-LH,) is ac- [Daubner et al., 19871. A further accessory enzyme of
tivated by binding of Ca2+ upon excitation of nerves bacterial bioluminescencesystems is a fatty acid reduc-
[Cormier, 19781. In the hydromedusa Aequorea both tase which is co-induced with the luciferase and recy-
luciferin and Ca2+ are bound by a single protein, ae- cles fatty acid products to substrate aldehydes (usually
quorin, that catalyzes the formation of oxyluciferin.
converts tetradecanonic acid to tetradecanal).
cDNA of blue light emitting aequorin has been isolated Bacterial luciferase genes were identified by trans-
.
. from Aequorea victoria and expressed in E. coli [Inouye
et al., 1986; Prasher et al., 19861.
poson insertional mutagenesis and by hybridization to
mixed oligonucleotide probes of known peptide se-
quences elas as et al., 1982; Cohn et al., 1983:~aldwin
BACTERIAL LUCIFERASES et al., 1984; Engebrecht and Silverman, 1984; Enge-
Luminous marine bacteria are ubiquitous and occur brecht et al., 1983; Delong et al., 19871. LuCDABE
either in planktonic forms or as symbionts within light genes are located in a single operon (R)in Vibrio
organs of fishes and squids. Luciferases from Vibrio harueyi, V. fischeri, and Photobacterium phosphoreum.
harveyi, V. fisheri, and Photobacterium phosphoreum L u C , D, and E encode fatty acid reductase (54 kD),
were extensively characterized [Ziegler and Baldwin, acyl-transferase (34 kD), and acyl-protein synthase (42
1981; Hastings and Nealson, 19771. kD) subunits of fatty acid reductase, respectively,
All bacterial luciferases are heterodimeric (ap), while the a- and P-subunits of luciferase (40 and 37 kD)
mixed function oxidases which catalyze the oxidation are synthesized from genes l u A and B [Cohn et al.,
226 KONCZ ET AL.
1985; Johnston et al., 1986; Haygood and Cohn, 1986; aldehydes can be monitored by firefly and bacterial
Miyamoto et al., 1985, 1987, 1988; Foran and Brown, luciferases. Special features of other luminescent pro-
1988; Illarionov et al., 1988; Mancini et al., 19881. In teins, such as aequorin also allow one to measure
Photobacteriumphosphoreum an additional gene, luxF, Ca2+-mediatedreactions. The range of in vitro assays
was identified between luxB and E and shown to result extends from clinical, microbial detection of pathogens
from a duplication of the luxB gene [Soly et al., 19881. to biochemical assays of enzymes, cofactors, and sub-
Homology between luxA and luxB suggests that they strates, to mutagenicity tests, to detection of steroid
also evolved by an earlier gene duplication event [Bald- hormones and insect pheromones, and to the measure-
win et al., 19791. Although overall homology exists be- ment of membrane transport and organellar functions
tween lux genes of diverse species, protein complemen- [Weinhausen and De Luca, 1982; Campbell et al., 1985;
tation studies between luciferase subunits of Vibrw Ulitzur, 1986; Kricka, 19881. Recent advances in selec-
and Photobacterium species demonstrated significant tive modification of a reactive sulfhydryl group of bac-
differences [Meighen and Bartlet, 19801. In Vibrio fis- terial luciferase and in the synthesis of firefly lucifer-
cheri gene luxI, located immediately upstream of l a c , ins derivatized a t the 6-position led to the general
belongs to the 1uxCDABE operon. A gene located fur- application of luciferases in immunoassays, protein im-
ther upstream, luxR, constitutes an independent op- muno-blotting, and non-radioactive nucleic acid hy-
eron (L) which is transcribed in the opposite direction. bridization [Baldwin et al., 1986; Haubner and Geiger,
Expression of lux operons is regulated a t the levels of 19881.
both transcription and translation. l w R encodes a pos-
itive regulatory protein while lux1 is responsible for the LUCIFERASE REPORTER GENES
synthesis of an autoinducer, N-(P-ketocaproy1)-homo- It has been realized early that a number of sensitive
serine lactone. lux gene expression correlates with the assays, such as determination of the concentration of
density of cell cultures. Initially the luxR protein is O,, anaesthetics, antibiotics, mutagens, etc., can be
constitutively produced and the luxCDABE operon is carried out in vivo by expression of luciferases in living
expressed only a t a low level. At higher cell densities cells [Hastings and Nealson, 19771.
an increased concentration of luxR protein, bound to Cloning of luciferase genes opened the way to novel
autoinducer, leads to further activation of the 1uxICD- applications in molecular biology. Gene fusion is a gen-
ABE operon and to a burst of autoinducer synthesis eral approach to study the temporal and spatial regu-
and light production. Later on the concentration of lation of gene expression and to delineate regulatory
luxR protein becomes limiting because the luxR pro- DNA sequences both in procaryotic and eucaryotic or-
tein-autoinducer complex inhibits the translation of ganisms. Reporter genes, such as P-galactosidase (lac),
luxR transcript. lux operons are also regulated by cat- p-glucuronidase (gus), chloramphenicol acetyltrans-
abolic repression because their promoters contain ferase (cat), and aminoglycoside phosphotransferase
cAMP/CRP binding sites [Dunlap and Greenberg, (aph(3')II),are fused to transcriptional regulatory ele-
1985; Dunlap, 1989; Engebrecht and Silverman, 1987; ments by construction of chimaeric genes which are
Devine et al., 19881. At low 0, concentrations the syn- then transformed into cells of target organisms. The
thesis of luciferase is limited in Vibrio harveyi and expression of reporter gene fusions is followed in tran-
Photobacterium leiognathi. In contrast, the synthesis of sient assays or in stable transformants either by in
luciferase is not influenced by oxygen in Vibrio fischeri vitro enzyme assays or by histological staining. Alter-
and Photobacterium phosphoreum which results in ac- natively, promoterless reporter genes are linked to the
cumulation of luciferase at low 0, tensions. Low osmo- ends of transposable elements such that their insertion
larity stimulates, whereas high iron concentrations re- into genes will generate transcriptional or transla-
press bacterial luminescence [Dunlap, 1985; Haygood tional gene fusions.
and Nealson, 19851. Engebrecht et al. [I9851 used the latter technique for
isolation of gene fusions to the luxCDABE operon by
mini-Mu transposon insertional mutagenesis in bacte-
LUCIFERASE ASSAYS ria and demonstrated that light production provides a
Light can be monitored visually, photographically, simple and sensitive in vivo indicator of gene expres-
or electronically at different sensitivities. A great va- sion. A systematic development of luciferase gene con-
riety of methods for detection and measurement of bi- structs and light assays followed this initial report. A
oluminescence have been described [Van Dyke, 19851. similar transposon, Tn4431, and diverse plasmid con-
Following pioneering work by several laboratories on structs were designed to visualize gene expression in
the purification and immobilization of luminescent en- Xanthomonas during its pathogenic invasion of plant
zymes, luciferases found a wide range of applications in tissues [Shaw et al., 1986, 1987, 19881. Expression of
most areas of life sciences. A particular advantage of luxAB genes in E. coli was successfully visualized by
luciferase assays is their ease, sensitivity, and effi- exogenous addition of n-decanal demonstrating that
ciency. Practically any reaction which can be linked to volatile aldehyde substrates of bacterial luciferase are
measurement of ATP, NAD(P), FMN, fatty acids, or promptly taken up by living cells and that luxAB struc-
LUCIFERASE GENES IN TRANSGENIC PLANTS
tural genes are as effective reporters as the full-length Luxp subunits has been confirmed in plants, there was
lux operon [Baldwin et al., 19861. Subsequently expres- some question regarding protease sensitivity of the cat-
sion of luxAB genes was demonstrated in filamentous alytic a-subunit. Furthermore, inefficient in vitro as-
cyanobacteria [Schmetterer et al., 19861, in Bacillus sembly of separately folded luciferase subunits led to
subtilis [Karp, 19891, and in Pseudomonas, Agrobacte- the assumption that coordinate folding of subunits dur-
rium, and Rhizobium [Legocki et al., 1986; Boivin et al., ing translation in bacteria would be essential. Since
19881. Various vectors were constructed for the study of transcription and translation are coupled in bacteria,
promoters and transcription terminators in bacteria this model predicted poor assembly of bacterial lu-
[Carmi et al., 1987; Peabody et al., 19891. In order to ciferase in eukaryotic systems. Other concerns pre-
monitor gene activation in symbiotic bacteroids of dicted that the concentration of FMN would be limiting
rhizobia, luxAB genes were fused to a regulated pro- because FMN is enzyme-bound or enclosed in cell com-
moter of nifD [O'Kane et al., 19881. This offered a good partments in eucaryotes. In contrast, the firefly lu-
model system to adjust several methods for visualiza- ciferase substrate ATP is ubiquitous in eukaryotic
tion of light-emitting tissues and cells inside the plant cells. Comparisons of kinetic parameters and quantum
body (Fig. 1). yields of luciferases also favoured the firefly enzyme
Expression and stability of firefly luciferase in E. which needs only one ATP per emitted photon in con-
coli has been explored by the synthesis of N-terminal trast to the requirement of 60 ATP for the bacterial
Cro-luciferase fusion proteins by using partial luc enzyme. On the other hand, it is known that luciferin is
cDNA clones. However, attempts to monitor light- taken up inefficiently by living cells. In order to en-
emission mediated by the firefly enzymes in vivo failed hance the uptake, cells have to be treated by DMSO,
because bacteria did not take up the luciferin substrate low pH, and high concentrations of luciferin, all of
[deWet et al., 19851. which reduce their viability. Furthermore, the trans-
port of the firefly enzyme to peroxisomes further re-
EXPRESSION OF LUCIFERASE GENES IN duces the availability of the substrate.
TRANSGENIC PLANTS
Application of luciferase genes in eucaryotes is still ADVANCES
in its infancy, but will certainly lead to a burst of novel Recent developments in the use of both reporter
experimental systems soon. Among eukaryotic organ-
genes provided some answers to the initial questions.
isms the expression of luciferase genes was first dem- The firefly luciferase gene has been used successfully
onstrated in plants. Linked to the promoter of the Cau-
for the analysis of promoters [Ow et al., 19871, tran-
liflower Mosaic Virus 35s transcript, the firefly scription terminator signals, and translational en-
luciferase gene was introduced into carrot protoplasts
hancer elements in plants [Gallie et al., 19891. The luc
by electroporation and into tobacco by Agrobacterium- gene was also exploited to optimize transient assays
mediated transformation. In addition to in vitro mea-
and to confirm stable transformation in plants [Ballas
surements of luciferase activity in transient assays, the
et al., 1988; Gupta et al., 1988; Ellis et al., 1989; Ko-
luciferase-mediated light emission was visualized by
mari, 19891, mammalian cells [deWet et al., 1987;
autoradiography of cell suspensions and by contact ex- Nguyen et al., 1988; Maxwell and Maxwell, 1988;
posure of a transgenic plant to a photographic emulsion
Williams et al., 19891, Dictyostelium [Howard et al.,
[Ow et al., 19861. 19881 and transgenic mice [DiLella et al., 19881. Inser-
To study the expression of bacterial luciferase genes
tion of the luc gene into a vaccinia virus genome illus-
in plants, luxA and l u B coding sequences were dis- trated possible applications of the luc reporter gene for
sected from the lux operon and fused separately to 1'
the monitoring of viral gene expression and virus dis-
and 2' promoters of mannopine synthase genes. lux semination in cell cultures and in tissues of infected
gene expression vectors were transformed into carrot animals [Rodriguez et al., 19881. The sensitivity of the
and tobacco cells. Light emission and detection of lu-
in vitro luciferase assay was estimated to be 100- to
._ ciferase subunits showed that assembly of functional
luciferase occurred in the cytoplasm of transformed
1,000-fold higher than that of P-galactosidase or CAT.
When luciferin was supplied in low pH buffer with
plant cells [Koncz et al., 19871. DMSO, this sensitivity permitted photographic detec-
tion of light in bacterial colonies as well as in animal
PROBLEMS and in plant tissues. Computer-enhanced video imag-
These initial reports incited much debate about fa- ing of individual cells was also achieved [Wood and
vourable and disadvantageous traits of both lu- DeLuca, 1987; Gallie et al., 1989; Maly et al., 19881.
ciferases. It was initially thought that the bacterial A step forward in the systematic development of bac-
system would not be useful for application in the eu- terial luciferase reporter genes involved a functional
karyotic cells because expression of two genes is re- analysis of luxA and luxB coding sequences. It was
quired for the synthesis of an active enzyme. Although demonstrated that, although the N-terminal domain of
the stability of individually synthesized Luxa and the a-subunit is required for enzyme activity, both 5'
228 KONCZ ET AL.
and 3' transcriptional and translational fusions to the
luxA gene can be generated [Olsson et al., 19891. luxA
and luxB genes introduced into bacteria on separate
plasmids expressed active luciferase in a n amount
equal to that produced by cells carrying linked luxAB
genes [Gupta et al., 1985; Olsson et al., 19881. In cells in
which the P-subunit is present in excess, light produr-
tion is correlated with the limiting concentration of the
a-subunit, indicating that luxA alone can be employed
as a reporter gene. A similar analysis showed that
transcriptional and translational gene fusions can also
be generated by using both 5' and 3' ends of the luxB
gene [Sugihara and Baldwin, 19881.
These observations led to the construction of 1uxA-B
and luxB-A gene fusions encoding functional mono-
meric bacterial luciferases [Olsson et al., 1989; Boylan
et al., 19891 which were expressed in bacterial, yeast,
and plant cells. Analysis of the correlation between
structure and activity of monomeric luciferases indi-
cated that the length of the interconnecting peptide
region plays an important role in determining proper
folding of the fusion enzymes. Luciferases displaying
different thermal stability and light emission proper-
ties were crystallized for comparison of their structure
to that of the native enzyme [Swanson et al., 1985;
Escher et al., unpublished].
It was demonstrated that both binary and fused lux
genes can be used for precise and reproducible in vitro
assays and for in vivo visualization of temporal and
spatial regulation of gene expression in plants [Lang-
ridge et al., 19891 (Fig. 2). Recently, due to a great
interest in the identification and isolation of plant reg-
ulatory genes, we developed a technology to screen
large numbers of plants expressing luciferase reporter
gene fusions. T-DNA vectors carrying a t their integra-
tion borders either fused l w A B or promoterless luxA
and linked chimaeric luxB gene cassettes were intro-
duced into tobacco and Arabidopsis by Agrobacterium-
mediated transformation. Since the T-DNA is fre-
quently integrated into transcribed loci of the plant
genome [Koncz et al., 19891, transcriptional lux gene
fusions can thus be isolated a t high frequencies. The
expression of T-DNA-tagged genes can be followed con-
tinuously during plant development by in vivo visual-
ization of the expression of lux gene fusions in diverse
tissues of transgenic plants (Fig. 2) [Redei GP, Univ.
Missouri, personal comm.].
PROSPECTIVES
The results described above clearly demonstrate that
both luciferase reporter gene systems can contribute
very significantly to various fields of molecular and Fig. 1. Visualization of bioluminescence in soybean nodules and in
cellular biology. Some trends for the improvement of single nodule cells infected by Bradyrhizobium japonicum containing
a nifD-lwAB gene fusion. Top (left): Nodules under normal illumi-
luciferase reporter genes and for their application are nation. Top (right): Bioluminescence recorded on IS0 400 .Ekta-
already predictable. Deletion analysis of the firefly luc chrome film. Middle: Nodule cells under normal illumination. Bot-
gene showed that the removal of C terminal amino tom: Visual image of light-emitting cells recorded by LDG low-light
acids does not influence enzyme activity but converts video microscope system [O'Kane et al., 19881.LUCIFERASE GENES IN TRANSGENIC PLANTS
Fig. 2.230 KONCZ ET AL.
Fig. 2. Visualization of lux gene expression in transgenic plants, Baldwin TO, Holzman TF, Holzman R (1986): Active center-based
organs, and tissues by a photon-counting video camera-photomulti- immuno-assay approach using bacterial luciferase. In DeLuca M,
plier system. P a n e l A: Light image. P a n e l B: Recorded image of light McElroy WD (eds): "Bioluminescence and Chemiluminescence."
emission. Top: Tobacco leaf expressing 1wA and luxB genes driven by Methods Enzymol 133:248-264.
1' and 2' promoters of mannopine synthase genes (left) and leaf of a Ballas N, Zakai N, Friedberg D, Loyter A (1988): Linear forms of
non-transformed tobacco plant (right). Middle: Stem section of a to- plasmid DNA are superior to supercoiled structures as active tem-
bacco plant expressing mas promoter-lux gene fusions. Bottom: Ex- plates for gene expression in plant protoplasts. Plant Mol Biol 11:
pression of a lux gene fusion in a transgenic Arabidopsis thaliana 517-527.
plant. Belas R, Mileham A, Cohn D, Hilmen M, Simon M, Silverman M
(1982): Bacterial bioluminescence: Isolation and expression of the
luciferase genes from Vibrw harveyi. Science 218:791-792.
Boivin R, Chalifour F-P, Dion P (1988): Construction of a Tn5 deriv-
ative encoding bioluminescence and its introduction in Pseudorno-
the peroxisomal luciferase to a cytoplasmic enzyme; nns, Agrobacteriurn and Rhizobium. Mol Gen Genet 213:50-55.
Boylan M, Pelletier J , Meighen EA (1989): Fused bacterial luciferase
thus the need for the transport of luciferin to peroxi- subunits catalyze light emission in eucaryotes and procaryotes. J
somes can be overcome [Could et al., 19891. The perox- Biol Chem 264:1915-1918.
isomal targeting signals identified in luciferases can Campbell AK, Hallett MB, Weeks I (1985): Chemiluminescence a s a n
moreover be employed to transport other proteins into analytical tool in cell biology and medicine. In Glick D (ed): Meth-
ods in Biochem Anal 31:317-416.
peroxisomes. Genes for beetle luciferases responsible Carmi OA, Stewart GSAB, Ulitzur S, Kuhn J (1987): Use of bacterial
for the emission of light of different colours could pro- luciferase to establish a promoter probe vehicle capable of non-
vide tools for the simultaneous monitoring of the ex- destructive real-time analysis of gene expression in Bacillus ssp. J
pression of different genes in a single cell. Inactive mo- Baeteriol 169:2165-2170.
nomeric bacterial luciferases carrying recognition sites Chen LH, Baldwin TO (1989): Random and site-directed mutagenesis
of bacterial luciferase: Investigation of the aldehyde binding site.
for specific proteases in between the fused subunits Biochemistry 28:2684-2689.
could be used for sensitive detection of proteases within Cohn DH, Ogden RC, Abelson J N , Baldwin TO, Nealson KH, Simon
cells or during fermentation in vivo. Both enzymes can MI, Mileham AJ (1983): Cloning of the Vibrw h r v e y i luciferase
be applied to measure thermal denaturation of proteins genes: Use of a synthetic oligonucleotide probe. Proc Natl Acad Sci
USA 80:120-123.
in diverse tissues during heat-shock. By addition of
Cohn DH, Mileham AJ, Simon MI, Nealson KH, Rausch SK, Bonam
appropriate signal peptides, luciferases could probably D, Baldwin TO (1985): Nucleotide sequence of the IuxA gene of
be transported into cell organelles such as chloroplasts Vibrio harveyi and the complete amino acid sequence of the ol sub-
or mitochondria for monitoring ATP-consuming syn- unit of bacterial luciferase. J Biol Chern 260:6139-6146.
thesis or electron transport. Production of luciferase- Cormier MJ (1978): Applications of Renilla bioluminescence: An in-
troduction. In DeLuca M (ed): "Bioluminescence and Chemilumi-
fused antibodies may play an important role for diverse nescence." Methods Enzymol 57:237-244.
aspects of immunology. Fusion of luciferases to chro- Daubner SC, Astorga AH, Leisman GB, Baldwin TO (1987): Yellow
mosomal proteins can lead to unique approaches in cy- light emission of Vibrw ficheri strain Y1: Purification and charac-
tology. The binary luxA-luxB system may find an im- terization of the energy-accepting yellow fluorescent protein. Proc
mediate application for the visualization of cell fusion Natl Acad Sci USA 84:8912-8916.
Delong EF, Steinhauer D, Israel A, Nealson KH (1987): Isolation of
and fertilization events or help in hybrid seed produc- the lux genes from Photobacterium lewgnathi and expression in
tion. Both binary and monocistronic luciferase genes Escherichia coli. Gene 54:203-210.
can be employed to monitor virus-cell interactions and DeLuca M (1976): Firefly luciferase. Adv Enzymol 44:37-68.
virus or virus-induced gene expression. Furthermore, DeLuca M (1978): "Bioluminescence and Chemiluminescence." Meth-
luciferase gene fusions could be constructed to detect ods Enzymol 57.
site-specific recombination or translocation events in a DeLuca M, McElroy WD (1986): "Bioluminescence and Chemilumi-
nescence." Methods Enzymol 133.
cell population. Expression of proteins associated with Devine J H , Countryman C, Baldwin TO (1988): Nucleotide sequence
the bacterial luciferase may also provide in vivo assays of the 1wR and lux1 genes and structure of the primary regulatory
for selection of fatty-acid- or riboflavin-overproducing region of the lux regulon of Vibrio fischeri ATCC 7744. Biochemis-
organisms. This prospective list of applications can try 27:837-842.
probably be extended, but our intention was simply to deWet JR, Wood KV, Helsinki DR, DeLuca M (1985): Cloning of fire-
fly luciferase cDNA and expression of active luciferase in Escher-
illustrate the potential of this reporter gene system. ichia coli. Proc Natl Acad Sci USA 82:7870-7873.
deWet JR, Wood KV, DeLuca M, Helsinki DR, Subramani S (1987):
Firefly luciferase gene: Structure and expression in mammalian
REFERENCES cells. Mol Cell Biol 7:725-737.
Aboukhair NK, Ziegler MM, Baldwin TO (1985): Bacterial luciferase: DiLella AG, Hope DA, Chen H, Trumbauer M, Schwartz RJ, Smith
Demonstration of a catalytically- competent
- altered conformational RG (1988): Utility of firefly luciferase a s a reporter gene for pro-
state follow?ng a single turnover. Biochemistry 24:3942-3947. moter activity in transgenic mice. Nucleic Acids Res 16:4159.
Baldwin TO, Ziegler MM, Powers DA (1979): Covalent structure of Dunlap PV (1985): Osmotic control of bioluminescence and growth in
subunits of, bacterial luciferase: NH,-terminal sequence demon- Photobacterium lewgnathi from ponyfish light organs. Arch Micro-
strates subunit homology. Proc Natl Acad Sci USA 76:4887-4889. biol 141:44-50.
Baldwin TO, Berends T, Bunch TA, Holzman TF, Rausch SK, Sha- Dunlap PV, Greenberg E P (1985): Control of Vibrw fscheri lumines-
mansky L, Treat ML, Ziegler MM (1984): Cloning of luciferase cence gene expression in Escherichia coli by cyclic AMP and cyclic
structural genes from Vibrw harveyi and expression of biolumines- AMP receptor protein. J Bacteriol 164:45-50.
cence in Escherichia coli. Biochemistry 23:3663-3667. Dunlap PV (1989): Regulation of luminescence by cyclic AMP in cya-
. .-LUCIFERASE GENES IN TRANSGENIC PLANTS 231
like and crp-like mutants of Vibrio fischeri. J Bacteriol 171:1199- Komari T (1989): Transformation of callus cultures of nine plant spe-
1202. cies mediated by Agrobacterium. Plant Sci 60:223-229.
Ellis D, Roberts D, Sutton B, Lazaroff W, Webb D, Flinn B (1989): Koncz C, Olsson 0 , Langridge WHR, Schell J , Szalay AA (1987):
Transformation of white spruce and other conifer species by Agro- Expression and assembly of functional bacterial luciferase in
bacterium tunaefaciens. Plant Cell Rep 8:16-20. plants. Proc Natl Aoad Sci USA 84:131-135.
Engebrecht J, Nealson K, Silverman M (1983): Bacterial biolumines- Koncz C, Martini N, Mayerhofer R, Kona-Kalman ZS, Korber H,
cence: Isolation and genetic analysis of functions from Vibrio fis- Redei GP, Schell J (1989) High-frequency T-DNA-mediated gene
cheri. Cell 32773-781. tagging in plants. Proc Natl Acad Sci USA 86:8467-8471.
Engebrecht J, Silverman M (1984): Identification of genes and gene Kricka LJ (1988): Clinical and biochemical applications of luciferases
products necessary for bacterial bioluminescence. Proc Natl Acad and luciferins. Anal Biochem 175:14-21.
Sci USA 81:4154-4158. Kurfiirst M, Ghisla S, Hastings W (1984): Characterization and pos-
Engebrecht J , Simon M, Silverman M (1985): Measuring gene expres- tulated structure of the primary emitter in the bacterial luciferase
sion with light. Science 227:1345-1347. reaction. Proc Natl Acad Sci USA 81:2990-2994.
Engebrecht J , Silverman M (1987): Nucleotide sequence of the regu- Longridge WHR, Fitzgerald KJ, K o n a C, Schell J, Sealay AA (1989)
latory locus controlling expression of bacterial genes for biolumi- Dual promoter of Agrobacterium tumefaciens mannopine synthase
nescence. Nucleic Acids Res 15:10455-10467. genes is regulated by plant growth hormones. Proc Natl Acad Sci
Foran DR, Brown WM (1988): Nucleotide sequence of the luxA and USA 86:3213-3223.
IuxB genes of bioluminescent marine bacterium Vibrw fischeri. Nu- Lee J , O'Kane DJ, Gibson BG (1988): Dynamic fluorescence proper-
cleic Acids Res 16:777. ties of bacterial luciferase intermediates. Biochemistry 27:4862-
Gallie DR, Lucas WJ, Walbot V (1989): Visualizing mRNA expression 4870.
in plant protoplasts: Factors influencing efficient mRNA uptake Lee J, O'Kane DJ, Gibson BG (1989): Bioluminescence spectral and
and translation. Plant Cell 1:301-311. fluorescence dynamics study of the interaction of lumazine protein
Gould SJ, Keller GA, Subramani S (1987): Identification of a perox- with the intermediates of bacterial luciferase bioluminescence. Bio-
isomal targeting signal a t the carboxy terminus of firefly lu- chemistry 28:4263-4271.
ciferase. J Cell Biol 105:2923-2931. Legocki RP, Legocki M, Baldwin TO, Szalay AA (1986): Biolumines-
Gould SJ, Subramani S (1988): Firefly luciferase as a tool in molec- cence in soybean root nodules: Demonstration of a general approach
ular and cell biology. Anal Biochem 175:5-13. to assay gene expression in vivo by using bacterial luciferase. Proc
Gould SJ, Keller GA, Hosken N, Wilkinson J , Subramani S (1989): A Natl Acad Sci USA 83:9080-9084.
conserved tripeptide sorts proteins into peroxisomes. J Cell Biol Maly FE, Urwyler A, Rolli HP, Dahinden CA, DeWeck LA (1988): A
108:1657-1664. single-photon imaging system for the simultaneous quantitation of
Gupta SC, O'Brien D, Hastings JW (1985): Expression of the cloned luminescence emission from multiple samples. Anal Biochem 168:
subunits of bacterial luciferase from separate replicons. Biochem 462-469.
Biophys Res Commun 127:1007-1011. Mancini JA, Boylan M, Soly RR, Graham AF, Meighen EA (1988):
Gupta PK, Dandekar AM, Durzan DJ (1988): Somatic proembryo for- Cloning and expression of the Photobacterium phosphoreum lumi-
mation and transient expression of a luciferase gene in douglas fir nescence system demonstrates a unique lux gene organization. J
and loblolly pine protoplasts. Plant Sci 58:85-92. Biol Chem 263:14308-14314.
Harvey EN (1957): "A History of Luminescence From Earliest Time Masuda T, Tatsumi H, Nakano E (1989): Cloning and sequence anal-
Until 1900." Philadelphia: Philosophical Society of Pennsylvania. ysis of cDNA of a Japanese firefly, Luciola crmiata. Gene 77:265-
Hastings WJ, Nealson KH (1977): Bacterial bioluminescence. Annu 270.
Rev Microbiol 31:549-595. Maxwell IH, Maxwell F (1988): Electroporation of mammalian cells
Hastings WJ (1978): Bacterial bioluminescence: An overview. In De- with a firefly luciferase expression plasmid: Kinetics of transient
Luca M (ed): "Bioluminescence and Chemiluminescence." Methods expression differ markedly among cell types. DNA 7:557-562.
Enzymol57:125-135. Meighen EA, Bartlet I(1980): Complication of subunits from different
Haubner R, Geiger R (1988): A sensitive, bioluminescent enhanced bacterial luciferases. J Biol Chem 255:11181-11187.
detection method for DNA dot-hybridization. Nucleic Acids Res 16: Miyamoto C, Graham AD, Boylan M, Evans JR, Hasel KW, Meighen
1213. EA, Graham AF (1985): Polycistronic mRNA codes for polypeptides
Haygood MG, Nealson KH (1985): Mechanism of iron regulation of of the Vibrw hurvey luminescence system. J Bacteriol 161:995-
luminescence in Vibrw fischeri. J Bacteriol 162:209-216. 1001.
Haygood MG, Cob:, DM (1986): Luciferase genes cloned from the un- Miyamoto C, Byers D, Graham AF, Meighen EA (1987): Expression of
culturable luminous bacteroid symbiont of the Caribbean flash- bioluminescence by Escherichia coli containing- recombinant Vibrio
light fish, Kryptophanaron alfredi. Gene 45203-209. hurvey DNA. J ~ a c t e r i o 169:247-253.
l
Howard PK, Ahern KG, Firtel RA (1988): Establishment of a tran- Miyamoto C, Graham AF, Meighen EA (1988): Nucleotide sequence of
sient expression system for Dictyostelium discoideum. Nucleic Ac- the l w C gene and the upstream DNA from the bioluminescent
ids Res 16:2613-2623. system of Vibrio hurveyi. Nucleic Acids Res 16:1551-1562.
Illarionov BA, Protopopova MV, Karginov VA, Mertvetsov NP, Gitel- Nguyen VT, Morange M, Bensaude 0 (1988): Firefly luminescence
son J I (1988): Nucleotide sequence of part of Photobacterium lei- assays using scintillation counters for quantitation in transfected
ognathi lux region. Nucleic Acids Res 16:9855. mammalian cells. Anal Biochem 171:404-408.
Inouye S, Sakaki Y, Goto T, Tsuji FI (1986): Expression of apoae- O'Kane DJ, Lingle WL, Wampler JE, Legocki M, Legocki PR, Szalay
quorin complementary DNA in Escherichia coli. Biochemistry 25: AA (1988): Visualization of bioluminescence a s a marker of gene
8425-8429. expression in Rhizobium-infected nodules. Plant Mol Biol 10:387-
Johnston TC,Thompson RB, Baldwin TO (1986): Nucleotide sequence 399.
of the luxB gene of Vibrio hurveyi and the complete amino acid Olsson 0 , Koncz C, Szalay AA (1988): The use of the luxA gene of the
sequence of the f3 subunit of bacterial luciferase. J Biol Chem 261: bacterial luciferase operoe a s a reporter gene. Mol Gen Genet 215:
4805-4811. 1-9.
Karp M (1989): Expression of bacterial luciferase genes from Vibrw Olsson 0 , Escher A, Sandberg G, Schell J , Koncz C, Szalay AA (1989):
harveyi in Bacillus subtilis and in Escherichia coli. BBA 1007:84- Engineering of monomeric bacterial luciferases by fusion of luxA
90. and IuxB genes in Vibrio harueyi. Gene 81:335-347.
Keller GA, Gould S, DeLuca M, Subramani S (1987): Firefly lu- Ow DW, Wood KV, DeLuca M, deWet JR, Helsinki DR, Howell SH
ciferase is targeted to peroxisomes in mammalian cells. Proc Natl (1986): Transient and stable expression of the firefly luciferase gene
Acad Sci USA 84:3264-3268. in plant cells and transgenic plants. Science 234:856-859.232 KONCZ ET AL.
Ow DW, Jacobs J , Howell SH (1987): Functional regions of the Cau- a non-pathogenic mutant and cloning of a locus for pathogenicity.
liflower Mosaic Virus 35s RNA promoter determined by use of the Mol Plant Microb Interact 1:39-45.
firefly luciferase gene as a reporter of promoter activity. Proc Natl Soly RR, Mancini JA, Ferri SR, Boylan M, Meighen EA (1988): A new
Acad Sci USA 84:4870-4874. lux gene in bioluminescent bacteria codes for a protein homologous
Paquette 0 , Fried A, Tu SC (1988): Delineation of bacterial luciferase to the bacterial luciferase subunits. Biochem Biophys Res Commun
aldehyde site by bifunctional labeling reagents. Arch Biochem Bic- 155:351-358.
phys 264:392-399. Sugihara J , Baldwin TO (1988): Effects of 3' end deletions from the
Peabody DS, Andrews CL, Escudero KW, Devine JH, Baldwin TO, Vibrw harveyi lwcB gene on luciferase subunit folding and enzyme
Bear DG (1989): A plasmid vector and quantitative techniques for assembly: Generation of temperature-sensitive polypeptide folding
the study of transcription termination in Escherichia coli using bac- mutants. Biochemistry 27:2872-2880.
terial luciferase. Gene 75:289-296. Swanson R, Weaver LH, Remington ST,Matthews BW, Baldwin TO
Prasher DC, McCahn RO, Cormier MJ (1986): Isolation and expres- (1985): Crystals of luciferase from Vibrin hnrueyi. J Biol Chem 260:
sion of a cDNA for aequorin, the Ca2+-activated photoprotein from 1287-1289.
Aequorea victoria. In DeLuca M, McElroy WD (eds): "Biolumines- Ulitzur S (1986): Determination of antibiotic activities with the aid of
cence and Chemiluminescence." Methods Enzymol 133288-297. luminous bacteria. In DeLuca M, McElroy WD (eds): "Biolumines-
Rodriguez J F , Rodriguez D, Rodriguez JR, McGowan EB, Estehan M cence and Chemiluminescence." Methods Enzymol 133:275-284.
(1988): Expression of the firefly luciferase gene in vaccinia virus: A VanDyke K (1985): "Bioluminescence and Chemiluminescence: In-
highly sensitive gene marker to follow virus dissemination in tis- struments and Applications." Boca Raton, Florida: CRC Press.
sues of infected animals. Proc Natl Acad Sci USA 85:1667-1671. Weinhausen G, DeLuca M (1982): Bioluminescent assays of picomole
Schmetterer G, Wolk PC, Elhai J (1986): Expression of luciferases levels of various metabolites using immobilized enzymes. Anal Bio-
from Vibrw harvey and Vibrw F c h e r i in filamentous cyanobacte- chem 127:380-388.
ria. J Bacteriol 167:411-414. Williams TM, Burlein JE, Ogden S, Kricka LJ,Kant J A (1989): Ad-
Schroeder J (1989): Protein sequence homology between plant 4 vantages of firefly luciferase as a reporter gene: Application to the
coumarate coenzyme A ligase and firefly luciferase. Nucleic Acids interleukin-2 gene promoter. Anal Biochem 176:28-32.
Res 17:460. Wood KV, DeLuca M (1987): Photographic detection of luminescence
Shaw J J , Kado CI (1986): Development of a Vibrio bioluminescence
in Escherichia coli containing the gene for firefly luciferase. Anal
gene-set to monitor phytopathogenic bacteria during the ongoing
Biochem 161:501-507.
disease process in a n o n d i s ~ p t i v emanner. Biotechnology 4:560-
Wood KV, Lam AY, Seliger HH, McElroy WD (1989): Complementary
564.
DNA encoding click beetle luciferases can elicit bioluminescence of
Shaw J J , Rogowsky P, Close TJ, Kado CI (1987): Working with bac-
different colours. Science 244700-702.
terial bioluminescence. Plant Mol Biol Rep 5225-236.
Shaw J J , Settles LG, Kado CI (1988): Transposon Tn4431 mutagen- Ziegler MM,Baldwin TO (1981): Biochemistry of bacterial biolumi-
esis of Xanthomnas campestris pv. campestris: Characterization of nescence. Curr Top Bioenerg 12:65-113.You can also read
