Extraction buffer effects on detection of transient gene expression in white spruce

β-Glucuronidase (GUS) and luciferase (LUC) reporter genes were introduced into white spruce (Picea glauca [Moench] Voss) cultured cells via particle bombardment. Transient expression of these genes was evaluated by extracting the enzymes using 3 buffers. Different buffers resulted in significantly different sensitivities of GUS and LUC detection. In the case of cobombardment, the buffer that gave high levels of expression of one reporter gene did not necessarily result in a better detection of the second reporter gene. This study indicates that appropriate buffers should be used for maximum detection of reporter genes.     

Evaluation and Comparison of the GUS, LUC and GFP Reporter System for Gene Expression Studies in Plants

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 β-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.     

Post-transcriptional regulation in higher eukaryotes: the role of the reporter gene in controlling expression

We have investigated whether reporter genes influence cytoplasmic regulation of gene expression in tobacco and Chinese hamster ovary (CHO) cells. Two genes, uidA encoding beta-glucuronidase (GUS) from Escherichia coli and Luc, encoding firefly luciferase (LUC), were used to analyze the ability of a cap, polyadenylated tail, and the 5'- and 3'-untranslated regions (UTR) from tobacco mosaic virus (TMV) to regulate expression. The regulation associated with the 5' cap structure and the TMV 5'-UTR, both of which enhance translational efficiency, was reporter gene-independent. The poly(A) tail and the TMV 3'-UTR, which is functionally equivalent to a poly(A) tail, increase translational efficiency as well as mRNA stability. The regulation associated with these 3' ends was highly reporter gene-dependent; their effect on GUS expression was almost an order of magnitude greater than that on LUC expression. In tobacco, the tenfold reporter gene effect on poly(A) tail or TMV 3'-UTR function could not be explained by a differential impact on mRNA stability; GUS and LUC mRNA half-life increased only twofold when either the poly(A) tail or TMV 3'-UTR was present. In CHO cells, however, GUS mRNA was stabilized to a greater extent by a poly(A) tail or the TMV 3'-UTR than was LUC mRNA.     

Fusion genetic analysis of gibberellin signaling mutants

A fusion genetic strategy was used to identify gibberellin (GA) signaling mutants in transgenic Arabidopsis expressing the beta-glucuronidase (GUS) and firefly luciferase (LUC) reporter genes under control of the GA-responsive GASA1 promoter. Initial analyses determined the spatial and temporal patterns of reporter expression, and showed that reporter induction by GA was antagonized by ABA. gamma-Irradiated M2 progeny with altered reporter activities were identified by LUC bioimaging followed by GUS assays and northern hybridization of the endogenous GASA1 mRNA. Genetic analysis showed that three mutants, which overexpressed both reporters and endogenous GASA1, were caused by recessive (goe1 and goe2, for GASA over-expressed) and semi-dominant (goe3) mutations at different loci. These mutants are altered in their sensitivity to GA and the GA biosynthetic inhibitor paclobutrazol, and in the expression of several GA signaling related genes.     

Multiple GUS expression patterns of a single Arabidopsis gene

Ten independent transposant lines with gene or enhancer traps (ET) inserted into the same gene (At2g01170) were identified in Arabidopsis thaliana . Transposon insertions were confirmed for each line. Only three of five ET lines and only one of the five gene trap (GT) lines displayed uidA (GUS) staining. The GUS (β-glucuronidase) expression patterns of the ET lines were different in all three lines. In the GT line, the GUS expression was restricted to the vascular tissue under all conditions examined. The variation in ET GUS expression suggests that each ET was controlled by different enhancer elements or the different elements of the trapped locus may give rise to different GUS expression patterns. Of five GT lines, three have the GUS gene in the same orientation as the At2g01170 open reading frame, yet only one yielded GUS staining. Regardless of the insertion construct, only those transposants with an insertion at the 3' end of the gene yielded GUS staining. Some transposants displayed a longer root phenotype in the presence of kanamycin that was also observed in 3' insertion sites in At2g01170. Taken together, these data show that insertions in the 5' end of the gene disrupted expression and emphasise the complexity encountered with ET and GT constructs to characterise the expression patterns of genes of interest based solely on GUS expression patterns.     

Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis

The transient expression of foreign genes in the protoplasts of Porphyrayezoensis was examined using three recombinant vectors, pYez-Rub-GUS, pYez-Rub-GFP and pYez-Rub-LUC, which were constructed with the promoter sequence of the ribulose-bisphosphate-carboxylase / oxygenase (Rubisco) gene as a promoter and the bacterial β-glucuronidase (GUS), mutant of green fluorescent protein (S65T-GFP) and firefly luciferase (LUC) genes, respectively, as reporter genes. When the pYez-Rub-GUS was introduced into protoplasts by electroporation, cells stained dark blue by indigotin were observed after the histochemical GUS assay. GUS activity was also detected by quantitative enzyme assays with a chemiluminescent substrate. When the pYez-Rub-GFP was electroporated into protoplasts, the expression of GFP could be detected in vivo observations with fluorescence microscopy. However, the rates of gene expression cells to the total number of cells were different between the GUS and GFP genes. LUC activity was also detected by assay with a chemiluminescent substrate after the introduction of pYez-Rub-LUC into protoplasts, although the activity levels were considerably lower. Relatively high expression rates of introduced GUS genes were observed 3 to 5 days after electroporation. These results show that the promoter sequence of the chloroplast Rubisco gene functions as a promoter of foreign gene expression and that transient expression occurred in protoplasts of P. yezoensis after the introduction of foreign genes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Local and systemic induction of two defense-related subtilisin-like protease promoters in transgenic Arabidopsis plants. Luciferin induction of PR gene expression

Following a pathogenic attack, plants are able to mount a defense response with the coordinated activation of a battery of defense-related genes. In this study we have characterized the mode of expression of the P69B and P69C genes from tomato (Lycopersicon esculentum Mill.), which encodes two closely related subtilisin-like proteases associated with the defense response. We have compared the mode of gene regulation in heterologous transgenic Arabidopsis plants harboring promoter-beta-glucuronidase (GUS) and promoter-luciferase (LUC) gene fusions for these two genes. These studies revealed that the P69B and P69C promoters are induced by salicylic acid as well as during the course of both a compatible and an incompatible interaction with Pseudomonas syringae. Furthermore, P69B and P69C expression takes place in both the local and the distal (noninoculated) leaves upon inoculation with bacteria but following different and unique tissue-specific patterns of expression that are also different to that described for most other classical PR genes. Also, we report that luciferin, the substrate for the reporter luciferase (LUC) gene, is able to activate expression of PR genes, and this may pose a problem when using this gene reporter system in studies related to plant defense.     

Combination of the ALCR/alcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in Arabidopsis

The experimental control of gene expression in specific tissues or cells at defined time points is a useful tool for the analysis of gene function. GAL4/VP16-UAS enhancer trap lines can be used to selectively express genes in specific tissues or cells, and an ethanol-inducible system can help to control the time of expression. In this study, the combination of the two methods allowed the successful regulation of gene expression in both time and space. For this purpose, a binary vector, 962-UAS::GUS, was constructed in which the ALCR activator and β-glucuronidase (GUS) reporter gene were placed under the control of upstream activator sequence (UAS) elements and the alcA response element, respectively. Three different GAL4/VP16-UAS enhancer trap lines of Arabidopsis were transformed, resulting in transgenic plants in which GUS activity was detected only on ethanol induction and exclusively in the predicted tissues of the enhancer trap lines. As a library of different enhancer trap lines with distinct green fluorescent protein (GFP) patterns exist, transformation with a similar vector, in which GUS is replaced by another gene, would enable the control of the time and place of transgene expression. We have constructed two vectors for easy cloning of the gene of interest, one with a polylinker site and one that is compatible with the GATEWAY™ vector conversion system. The method can be extended to other species when enhancer trap lines become available.     

Gene and enhancer trap tagging of vascular-expressed genes in poplar trees

We report a gene discovery system for poplar trees based on gene and enhancer traps. Gene and enhancer trap vectors carrying the beta-glucuronidase (GUS) reporter gene were inserted into the poplar genome via Agrobacterium tumefaciens transformation, where they reveal the expression pattern of genes at or near the insertion sites. Because GUS expression phenotypes are dominant and are scored in primary transformants, this system does not require rounds of sexual recombination, a typical barrier to developmental genetic studies in trees. Gene and enhancer trap lines defining genes expressed during primary and secondary vascular development were identified and characterized. Collectively, the vascular gene expression patterns revealed that approximately 40% of genes expressed in leaves were expressed exclusively in the veins, indicating that a large set of genes is required for vascular development and function. Also, significant overlap was found between the sets of genes responsible for development and function of secondary vascular tissues of stems and primary vascular tissues in other organs of the plant, likely reflecting the common evolutionary origin of these tissues. Chromosomal DNA flanking insertion sites was amplified by thermal asymmetric interlaced PCR and sequenced and used to identify insertion sites by reference to the nascent Populus trichocarpa genome sequence. Extension of the system was demonstrated through isolation of full-length cDNAs for five genes of interest, including a new class of vascular-expressed gene tagged by enhancer trap line cET-1-pop1-145. Poplar gene and enhancer traps provide a new resource that allows plant biologists to directly reference the poplar genome sequence and identify novel genes of interest in forest biology.     

Mechanism of gene expression of Arabidopsis glutathione S-transferase, AtGST1, and AtGST11 in response to aluminum stress

The gene expression of two Al-induced Arabidopsis glutathione S-transferase genes, AtGST1 and AtGST11, was analyzed to investigate the mechanism underlying the response to Al stress. An approximately 1-kb DNA fragment of the 5'-upstream region of each gene was fused to a beta-glucuronidase (GUS) reporter gene (pAtGST1::GUS and pAtGST11::GUS) and introduced into Arabidopsis ecotype Landsberg erecta. The constructed transgenic lines showed a time-dependent gene expression to a different degree in the root and/or leaf by Al stress. The pAtGST1::GUS gene was induced after a short Al treatment (maximum expression after a 2-h exposure), while the pAtGST11::GUS gene was induced by a longer Al treatment (approximately 8 h for maximum expression). Since the gene expression was observed in the leaf when only the root was exposed to Al stress, a signaling system between the root and shoot was suggested in Al stress. A GUS staining experiment using an adult transgenic line carrying the pAtGST11::GUS gene supported this suggestion. Furthermore, Al treatment simultaneously with various Ca depleted conditions in root region enhanced the gene expression of the pAtGST11::GUS in the shoot region. This result suggested that the degree of Al toxicity in the root reflects the gene response of pAtGST11::GUS in the shoot via the deduced signaling system. Both transgenic lines also showed an increase of GUS activity after cold stress, heat stress, metal toxicity, and oxidative damages, suggesting a common induction mechanism in response to the tested stresses including Al stress.     

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of PCB phytoremediation

The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the beta-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants.