Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono - and dicotyledonous plant cells ( plants / protoplasts / mutagenesis / reporter gene / blue fluorescent protein ) CHRISTOPH REICHEL

The expression of the jellyfish green fluorescent protein (GFP) in plants was analyzed by transient expression in protoplasts from Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, and Zea mays. Expression of GFP was only observed with a mutated cDNA, from which a recently described cryptic splice site had been removed. However, detectable levels of green fluorescence were only emitted from a small number of protoplasts. Therefore, other mutations in the GFP cDNA leading to single-amino acid exchanges in the chromophore region, which had been previously studied in Escherichia coli, were tested in order to improve the sensitivity of this marker protein. Of the mutations tested so far, the exchange of GFP amino acid tyrosine 66 to histidine (Y66H) led to detection of blue fluorescence in plant protoplasts, while the exchange of amino acid serine 65 to cysteine (S65C) and threonine (S65T) increased the intensity of green fluorescence drastically, thereby significantly raising the detection level for GFP. For GFP S65C, the detectable number of green fluorescing tobacco (BY-2) protoplasts was raised up to 19-fold, while the fluorimetricly determined fluorescence was raised by at least 2 orders of magnitude. A powerful tool for the rapid analysis of promoters is the use of marker genes for which expression can be easily monitored by autoradiography (NPT II, CAT), light emission (LUC, GUS), or color production (GUS). Commonly used reporter genes are CAT, NPT II, GUS, and LUC, of which GUS and LUC are of special interest since their assays do not involve any radioactivity (1). However, none of these reporter genes allows convenient, noninvasive in vivo detection of the respective enzyme in intact plant cells. An attractive alternative turned out to be the green fluorescent protein (GFP) from the jellyfishAequorea victoria. Use of this marker protein has been described for Escherichia coli, Caenorhabditis elegans, Drosophila melanogaster, yeast, and HeLa cells (2-5). Detection of GFP is noninvasive and nondestructive, which is a clear advantage over formerly used reporter genes such as f-glucuronidase or firefly luciferase (6, 7). Illumination of GFP with long-wave UV light (395 nm) or blue light (475 nm) leads to bright green fluorescence (510 nm) without any need for additional substrates, since chromophore formation and light emission are intrinsic properties of this marker protein (8). Expression and detection of wild-type GFP in maize and sweet orange (Citrus sinensis) protoplasts using constructs driven by a heat-shock promoter or by a constitutive promoter (9-11), using a potato virus X expression system in Nicotiana clevelandii and Nicotiana benthamiana plants (12, 13), or using The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tobacco mosaic virus in N. benthamiana and tobacco protoplasts (14, 15), has been described recently. However, in our hands expression of a wild-type GFP cDNA driven by a CaMV 35S promoter was neither detectable in transgenic tobacco plants nor in transient expression studies in protoplasts from Arabidopsis thaliana, Nicotiana tabacum, and Hordeum vulgare. Recently it has been described that the A. victoria wild-type GFP mRNA can be aberrantly processed in plant cells, due to the recognition of internal cryptic splice sites leading to inefficient expression of the GFP. Mutation of a cryptic splice site improves expression of GFP in cells from transgenic A. thaliana plants significantly (J. Haseloff, K. Siemering, D. Prasher, and S. Hodge, personal communication; ref. 16). Moreover, mutagenesis of the GFP cDNA in Escherichia coli has led to changes in the fluorescence properties of this new marker protein. A number of GFP amino acid exchange mutants have been isolated. They exhibit modifications in their excitation and emission spectra (17-21). Of these reported mutations, at least three are of great interest for expression in plant cells. GFP mutation Y66H (19) was shown to have a shifted emission peak leading to blue fluorescence. Analysis of a second set of GFP mutations, S65C and S65T (20, 21), revealed increased excitation and emission values, which might significantly improve brightness of green fluorescence also in plant cells. In order to evaluate marker gene expression in plants, plant protoplasts provide a powerful tool. Transient gene expression studies in protoplasts from a variety of plant species and organs have been used widely as a rapid and powerful method to monitor gene expression and to analyze expression levels of different marker gene constructs (22-24). The investigation of several GFP derivatives by transient gene expression in protoplasts from widely used plant species would thus provide valuable information about the potential of mutations in the GFP cDNA to improve the brightness of green fluorescence and to alter the GFP emission spectrum. Here we report transient expression studies of GFP genes, driven by the CaMV 35S promoter, in protoplasts of various monoand dicotyledonous plant species. The cDNA of these GFP constructs was mutated to abolish aberrant splicing in plant cells. Additionally, this cDNA was modified to introduce single amino acid changes into the GFP chromophore region, leading to significantly improved brightness of green fluoresAbbreviations: GFP, green fluorescent protein; CaMV, cauliflower mosaic virus; CAT, chloramphenicol acetyltransferase; GUS, 3-glucuronidase; LUC, firefly luciferase; NPT II, neomycin phosphotransferase II. *To whom reprint requests should be addressed. e-mail: [email protected]. tPresent address: Hoechst-Schering AgrEvo GmbH, Forschung Biochemie H872N, Postfach 800320, D-65926 Frankfurt, Germany.