Plant Molecular Biology 36: 521–528, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
Soluble, highly ﬂuorescent variants of green ﬂuorescent protein (GFP) for
use in higher plants
Seth J. Davis & Richard D. Vierstra
Department of Horticulture and Laboratory of Genetics, University of Wisconsin-Madison, 1575 Linden Drive,
Madison, WI 53706, USA (
author for correspondence)
Received 25 June 1997; accepted in revised form 15 September 1997
Key words: green ﬂuorescent protein, reporter gene, dual localization, dominant genetic marker, Arabidopsis
thaliana, transient expression
Green ﬂuorescent protein(GFP) from Aequorea victoria has rapidly become a standard reporter in many biological
systems. However, the use of GFP in higher plants has been limited by aberrant splicing of the corresponding
mRNA and by protein insolubility. It has been shown that GFP can be expressed in Arabidopsis thaliana after
altering the codon usage in the region that is incorrectly spliced, but the ﬂuorescence signal is weak, possibly
due to aggregation of the encoded protein. Through site-directed mutagenesis, we have generated a more soluble
version of the codon-modiﬁed GFP called soluble-modiﬁed GFP (smGFP). The excitation and emission spectra
for this protein are nearly identical to wild-type GFP. When introduced into A. thaliana, greater ﬂuorescence was
observed compared to the codon-modiﬁed GFP, implying that smGFP is ‘brighter’ because more of it is present in
a soluble and functional form. Using the smGFP template, two spectral variants were created, a soluble-modiﬁed
red-shiftedGFP(smRS-GFP)and a soluble-modiﬁedblue-ﬂuorescentprotein(smBFP). The increased ﬂuorescence
output of smGFP will further the use of this reporter in higher plants. In addition, the distinct spectral characters of
smRS-GFP and smBFP should allow for dual monitoring of gene expression, protein localization, and detection of
in vivo protein-protein interactions.
Reporter proteins have been recruited to monitor a
variety of cellular events which would be difﬁcult or
impossible to observe otherwise. In plants, the two
most widely used reporters are
luciferase [19, 24]. These reporters have been instru-
mental in many studies, but each has limitations. The
most notable is that neither allows for convenient non-
invasive in vivo analysis,becausebothrequireanadded
substrate to monitor reporter activity.
Recently a new reporter, green ﬂuorescent pro-
tein (GFP) isolated from the jellyﬁsh Aequorea vic-
toria, has been described which is not limited in this
The nucleotide sequence data reported are in the EMBL, Gen-
Bank and DDBJ Nucleotide Sequence Databases under the acces-
sion numbers U70495 (smGFP), U70496 (smRS-GFP) and U70497
respect . GFP ﬂuorophore is assembled by the
self-catalyzed covalent modiﬁcation of amino acids
Ser-Tyr-Gly at positions 65–67 to form a cyclized p-
hydroxybenzylidene-imidazolidinone species [8, 15].
When the wild-type ﬂuorophore is excited with either
UV or blue light (maximally at 396 nm or 475 nm), it
emits green ﬂuorescence (maximally at 508 nm) .
The intrinsicﬂuorescenceof GFP allows for noninvas-
ive detection without the introduction of cofactors or
the destruction of the biological sample [3, 8]. This
reporter has been shown to function in a wide range of
species, including higher plants [5, 12, 20, 29].
The initial reports of GFP activity in plant tissues
involved either transient expression in protoplasts [11,
18, 22, 28]ortheexpressionofGFPfromcytoplasmic-
RNA-virus vectors [1, 17]. However, stable expres-
sion in transgenic plants had been unsuccessful. This
problem was partially solved by Haseloff et al.who