Spatial and temporal patterns of green fluorescent protein (GFP) fluorescence during leaf canopy development in transgenic oilseed rape,<Emphasis Type="Italic"> Brassica napus</Emphasis> L.

The green fluorescent protein (GFP) holds promise as a field-level transgene marker. One obstacle to the use of GFP is fluorescence variability observed within leaf canopies. In growth chamber and field experiments, GFP fluorescence in transgenic oilseed rape (Brassica napus) was shown to be variable at each leaf position over time and among different leaves on the same plant. A leaf had its highest GFP fluorescence after emergence and, subsequently, its fluorescence intensity decreased. GFP fluorescence intensity was directly correlated with the concentration of soluble protein. The concentration of the genetically linked recombinant Bacillus thuringiensis (Bt) cry1Ac endotoxin protein also was examined, and GFP fluorescence was positively correlated with Bt throughout development. The results show that GFP can be used as an accurate transgene marker but that aspects of plant developmental should be taken into account when interpreting fluorescence measurements.Communicated by M.C. Jordan     

A simple and sensitive high-throughput GFP screening in woody and herbaceous plants

Green fluorescent protein (GFP) has been used widely as a powerful bioluminescent reporter, but its visualization by existing methods in tissues or whole plants and its utilization for high-throughput screening remains challenging in many species. Here, we report a fluorescence image analyzer-based method for GFP detection and its utility for high-throughput screening of transformed plants. Of three detection methods tested, the Typhoon fluorescence scanner was able to detect GFP fluorescence in all Arabidopsis thaliana tissues and apple leaves, while regular fluorescence microscopy detected it only in Arabidopsis flowers and siliques but barely in the leaves of either Arabidopsis or apple. The hand-held UV illumination method failed in all tissues of both species. Additionally, the Typhoon imager was able to detect GFP fluorescence in both green and non-green tissues of Arabidopsis seedlings as well as in imbibed seeds, qualifying it as a high-throughput screening tool, which was further demonstrated by screening the seedlings of primary transformed T₀ seeds. Of the 30,000 germinating Arabidopsis seedlings screened, at least 69 GFP-positive lines were identified, accounting for an approximately 0.23% transformation efficiency. About 14,000 seedlings grown in 16 Petri plates could be screened within an hour, making the screening process significantly more efficient and robust than any other existing high-throughput screening method for transgenic plants.     

A Rapid and Sensitive Fluorimetric Protocol for the Quantification of Green Fluorescent Protein in Soluble Protein Extracts from Transgenic Arabidopsis

Green fluorescent protein (GFP) is a popular qualitative reporter protein used to study different aspects of plant biology. However, to be used as a reliable quantitative reporter in expression studies using fluorescence based assays, methods to eliminate interfering endogenous molecules must be considered. Therefore, a standard curve based solid phase fluorescent immunoassay that eliminates the effects of interfering endogenous molecules was developed to quantify the GFP levels in soluble green extracts prepared from plants. Microtiter plates coated with anti-GFP were used to capture GFP from soluble plant extracts, interfering endogenous molecules was eliminated by washing without disturbing the anti-GFP binding of GFP, and then the fluorescence intensity of bound GFP was measured using a spectrofluorometer. We report in this study the use of this method to quantify the expression levels of soluble modified GFP in transgenic Arabidopsis thaliana.     

Sample topography and position within plant body influence the detection of the intensity of green fluorescent protein fluorescence in the leaves of transgenic tobacco plants

The effect of the type of leaf tissue selected for the study of green fluorescent protein (GFP) fluorescence intensity was investigated here using the T(1) generation of transgenic tobacco expressing the m-gfp5-ER gene. The fluorescence of GFP was detected by fluorescence binocular microscope coupled with the CCD camera and quantified by means of image analyses using the Lucia((R)) software. Mean brightness values from various leaf tissues were compared. First, an original data revealing the significant differences in the fluorescence intensity between the abaxial and adaxial surfaces are given. Stronger signal was detected on the abaxial side. Subsequently, the effect of the tissue location within the leaf surface was investigated and higher fluorescence was detected on the samples detached from leaf tips. Finally, the effect of the physiological age of leaves was studied using the in vitro clonally propagated plants. Leaves from the analogous positions within the plant body of three clones were investigated. The decrease in the fluorescence towards the plant top (youngest leaves) was observed in all studied plants. Surprisingly, the variability of the fluorescence within the clones of studied genotype was high enough to conclude, that the fluorescence of each individual is unique and affected by particular genotype and environment. Our study showed that the origin of leaf tissue selected for the GFP quantification is crucial and that the fluctuations in the fluorescence intensity should be taken into account when comparing the GFP fluorescence patterns of different plants. Moreover, the degree of fluorescence variability seems to be individually affected.     

Limited utility of blue fluorescent protein (BFP) in monitoring plant virus movement

While the green fluorescent protein (GFP) is a routinely used marker gene in higher plants, there are only a few data concerning the use of blue fluorescent protein (BFP). These proteins together are used for dual colour tagging experiments in various biological systems; however, the benefits of this technique in plant virology have not been exploited yet. In this work, our aim was to determine whether the BFP is a suitable second marker in conjunction with GFP for following the progress of virus infection. Nicotiana clevelandii, N. benthamiana and N. tabacum cv. Xanthi-nc plants were infected with potato virus X vector carrying the GFP or the Y66H type BFP gene. While GFP was brightly fluorescent in all species, the fluorescence intensity of BFP varied widely, from the bright fluorescence observed in N. clevelandii to the absence of fluorescence in N. tabacum cv. Xanthi-nc. Since at even mild acidic pH BFP rapidly fades, the more acidic cytosol of N. tabacum could be responsible for impaired in vivo fluorescence. After infiltration of the infected leaves of N. clevelandii with pH 5 phosphate buffer, the fluorescence faded thus confirming this situation.     

Patterns of green fluorescent protein expression in transgenic plants

Modified forms of genes encoding green fluorescent protein (GFP) can be macroscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP visualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing transgenic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice.     

Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny

Several modifications of a wild-type green fluorescent protein (GFP) gene were combined into a single construct, driven by the ubi-1 promoter and intron region, and transformed into maize. Green fluorescence, indicative of GFP expression, was observed in stably transformed callus as well as in leaves and roots of regenerated plants and their progeny. Cell wall autofluorescence made GFP expression difficult to observe in sections of leaves and roots. However, staining sections with toluidine blue allowed detection of GFP in transgenic tissue. Bright GFP fluorescence was observed in approximately 50% of the pollen of transgenic plants. These results suggest that GFP can be used as a reporter gene in transgenic maize; however, further modification, i.e., to alter the emission spectra, would increase its utility. Received: 17 December 1997 / Revision received: 6 March 1998 / Accepted: 20 March 1998     

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.     

‘Fukusensor:’ a genetically engineered plant for reporting DNA damage in response to gamma radiation

Transgenic plants can be designed to be ‘phytosensors’ for detection of environmental contaminants and pathogens. In this study, we describe the design and testing of a radiation phytosensor in the form of green fluorescence protein (GFP)‐transgenic Arabidopsis plant utilizing a DNA repair deficiency mutant background as a host. Mutant lines of Arabidopsis AtATM (At3g48190), which are hypersensitive to gamma irradiation, were used to generate stable GFP transgenic plants in which a gfp gene was under the control of a strong constitutive CaMV 35S promoter. Mutant and nonmutant genetic background transgenic plants were treated with 0, 1, 5, 10 and 100 Gy radiation doses, respectively, using a Co‐60 source. After 1 week, the GFP expression levels were drastically reduced in young leaves of mutant background plants (treated by 10 and 100 Gy), whereas there were scant visible differences in the fluorescence of the nonmutant background plants. These early results indicate that transgenic plants could serve in a relevant sensor system to report radiation dose and the biological effects to organisms in response to radionuclide contamination.     

Correlated expression of gfp and Bt cry1Ac gene facilitates quantification of transgenic hybridization between Brassicas

Gene flow from transgenic oilseed rape (BRASSICA NAPUS) might not be avoidable, thus, it is important to detect and quantify hybridization events with its relatives in real time. Data are presented showing the correlation between genetically linked green fluorescent protein (GFP) with BACILLUS THURINGIENSIS (Bt) CRY1AC gene expression in hybrids formed between transgenic B. NAPUS "Westar" and a wild Chinese accession of wild mustard (B. JUNCEA) and hybridization between transgenic B. NAPUS and a conspecific Chinese landrace oilseed rape. Hybrids were obtained either by spontaneous hybridization in the field or by hand-crossing in a greenhouse. In all cases, transgenic hybrids were selected by GFP fluorescence among seedlings originating from seeds harvested from B. JUNCEA and the Chinese oilseed rape plants. Transgenicity was confirmed by PCR detection of transgenes. GFP fluorescence was easily and rapidly detected in the hybrids under greenhouse and field conditions. Results showed that both GFP fluorescence and Bt protein synthesis decreased as either plant or leaf aged, and GFP fluorescence intensity was closely correlated with Bt protein concentration during the entire vegetative lifetime in hybrids. These findings allow the use of GFP fluorescence as an accurate tool to detect gene-flow in time in the field and to conveniently estimate BT CRY1AC expression in hybrids on-the-plant.     

Observer-based online compensation of inner filter effect in monitoring fluorescence of GFP-expressing plant cell cultures

The green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria is a very useful reporter for real-time bioprocess sensing. GFP culture fluorescence is a composite signal that can be influenced by factors such as culture autofluorescence, inner filter effect (IFE), and photobleaching. These factors complicate accurate estimation of GFP concentrations from the culture fluorescence. IFE is especially problematic when using GFP in monitoring transgenic plant cell suspension cultures, due to the aggregated nature of the cells and the high biomass concentration in these culture systems. Reported approaches for online compensation of IFE in monitoring culture NADH fluorescence or bioluminescence require online measurement of biomass density or culture turbidity/optical density, in addition to fluorescence/bioluminescence measurement. In this study, culture GFP fluorescence was used successfully to estimate GFP concentration and other important states in bioreactor culture of transgenic tobacco cells, while the influences of IFE and culture autofluorescence were rectified without the need for an additional biomass sensor. This was achieved by setting up a novel model-based state observer. First, we developed an improved model for a backscatter fluorescence probe that takes into account the influence of IFE and autofluorescence on reporting culture GFP concentration from online fluorescence. The state observer was then established using the extended Kalman filter (EKF), based on the fluorescence probe model, a dynamic state model of the plant cell bioreactor, and online GFP fluorescence measurement. Several versions of the observer were introduced to address practical requirements associated with monitoring GFP fluorescence of plant cell cultures. The proposed approach offers an effective means for online compensation of IFE to enable quantitative interpretation of the culture fluorescence signals for accurate reporting of GFP or GFP-fusion protein expression.     

Green fluorescent protein as a signal for protein-protein interactions.

Green fluorescent protein (GFP) is autofluorescent. This property has made GFP useful in monitoring in vivo activities such as gene expression and protein localization. We find that GFP can be used in vitro to reveal and characterize protein-protein interactions. The interaction between the S-peptide and S-protein fragments of ribonuclease A was chosen as a model system. GFP-tagged S-peptide was produced, and the interaction of this fusion protein with S-protein was analyzed by two distinct methods: fluorescence gel retardation and fluorescence polarization. The fluorescence gel retardation assay is a rapid method to demonstrate the existence of a protein-protein interaction and to estimate the dissociation constant (Kd) of the resulting complex. The fluorescence polarization assay is an accurate method to evaluate Kd in a specified homogeneous solution and can be adapted for the high-throughput screening of protein or peptide libraries. These two methods are powerful new tools to probe protein-protein interactions.     

Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants

Green fluorescent protein (GFP) has emerged as a powerful new tool in a variety of organisms. An engineered sGFP(S65T) sequence containing optimized codons of highly expressed eukaryotic proteins has provided up to 100-fold brighter fluorescence signals than the original jellyfish GFP sequence in plant and mammalian cells. It would be useful to establish a non-invasive, quantitative detection system which is optimized for S65T-type GFP, one of the brightest chromophore mutants among the various GFPs. We demonstrate here that highly fluorescent transgenic Arabidopsis can be generated, and the fluorescence intensity of whole plants can be measured under non-disruptive, sterile conditions using a quantitative fluorescent imaging system with blue laser excitation. Homozygous plants can be distinguished from heterozygous plants and fully fertile progenies can be obtained from the analyzed plants. In the case of cultured tobacco cells, GFP-positive cells can be quantitatively distinguished from non-transformed cells under non-selective conditions. This system will be useful in applications such as mutant screening, analysis of whole-body phenomena, including gene silencing and quantitative assessments of colonies from microorganisms to cultured eukaryotic cells. To facilitate the elucidation of protein targeting and organelle biogenesis in planta, we also generated transgenic Arabidopsis that stably express the plastid- or mitochondria-targeted sGFP(S65T). Etioplasts in dark-grown cotyledons and mitochondria in dry seed embryos could be visualized for the first time in transgenic Arabidopsis plants under normal growing conditions.     

Plasmodesmata transport of GFP alone or fused to potato virus X TGBp1 is diffusion driven

Summary. Plasmodesmata (Pd) provide a pathway for exchanging various macromolecules between neighboring plant cells. Researchers routinely characterize the mobility of the green-fluorescent protein (GFP) and GFP fusions through Pd by calculating the proportion of sites in bombarded leaves which show fluorescence in multiple cell clusters (% movement). Here, the Arrhenius equation was used to describe the temperature dependence of GFP and GFP-TGBp1 (potato virus X triple gene block protein1) movement, using % movement values, and to calculate the activation energy for protein transport. The resulting low activation energy indicates GFP and GFP-TGBp1 movement are diffusion driven. Furthermore, GFP movement is inversely proportional to the leaf surface area of expanding leaves. The increase in leaf area results mainly from cell expansion during the sink–source transition. The increasing cell size results in lower Pd density, which decreases the probability that a GFP attains an open Pd by diffusion. The decline in GFP movement as leaf area expands indicates that, in addition to GFP diffusion through Pd, attaining an open Pd by undirected diffusion might be limiting for Pd transport. In summary, this report provides a new quantitative method for studying Pd conductivity. Correspondence: Jeanmarie Verchot Lubicz, Department of Entomology and Plant Pathology, 127 Noble Research Center, Oklahoma State University, Stillwater, OK 74078, U.S.A.     

绿色荧光蛋白基因作为报告基因在水稻基因转化中的应用研究

本研究中 ,构建了含有编码绿色荧光蛋白的改进型基因质粒pJPM5。用基因枪法分别把pJPM5和另一带有绿色荧光蛋白基因的质粒pSBG70 0转入水稻TNG6 7愈伤组织。用South ern杂交法证实了转基因的存在 ,而且表明多数转基因植株含有 1到 8个拷贝的转基因。取 2个月的转基因植株上的叶片用于分析绿色荧光蛋白基因表达。用SLM - 80 0 0荧光分析仪定量测定绿色荧光蛋白。多数转基因植株具有很高的绿色荧光蛋白信号。虽然水稻植株有少量自发荧光 ,但是绿色荧光蛋白基因表达出的绿色荧光蛋白信号比植株的自发荧光强得多 ,其测定不会受自发荧光的太大影响。在荧光显微镜下观察到了绿色荧光蛋白基因的表达。借助观察分析绿色荧光蛋白基因的瞬时表达 ,本研究还发现基因枪法转化中 ,如果两枪的气压为90 0psi& 135 0psi,比两枪的气压都为 90 0psi或者 135 0psi更好 ,因其能使质粒进入更多的细胞。研究结果表明 ,绿色荧光蛋白基因可以作为水稻 (甚至小麦、玉米 )转基因研究中的报告基因。研究还显示 ,MAR序列能明显增强绿色荧光蛋白基因的表达能力 (这一结果在另文讨论 ) .