Reporter Genes In Transgenic Plants Research Articles

Physiological and transcriptional responses of Baccharis halimifolia to the explosive “composition B” (RDX/TNT) in amended soil

Unexploded explosives that include royal demolition explosive (RDX) and trinitrotoluene (TNT) cause environmental concerns for surrounding ecosystems. Baccharis halimifolia is a plant species in the sunflower family that grows naturally near munitions sites on contaminated soils, indicating that it might have tolerance to explosives. B. halimifolia plants were grown on 100, 300, and 750 mg kg(-1) of soil amended with composition B (Comp B) explosive, a mixture of royal demolition explosive and trinitrotoluene. These concentrations are environmentally relevant to such munitions sites. The purpose of the experiment was to mimic contaminated sites to assess the plant's physiological response and uptake of explosives and to identify upregulated genes in response to explosives in order to better understand how this species copes with explosives. Stomatal conductance was not significantly reduced in any treatments. However, net photosynthesis, absorbed photons, and chlorophyll were significantly reduced in all treatments relative to the control plants. The dark-adapted parameter of photosynthesis was reduced only in the 750 mg kg(-1) Comp B treatment. Thus, we observed partial physiological tolerance to Comp B in B. halimifolia plants. We identified and cloned 11 B. halimifolia gene candidates that were orthologous to explosive-responsive genes previously identified in Arabidopsis and poplar. Nine of those genes showed more than 90% similarity to Conyza canadensis (horseweed), which is the closest relative with significant available genomics resources. The expression patterns of these genes were studied using quantitative real-time PCR. Three genes were transcriptionally upregulated in Comp B treatments, and the Cytb6f gene was found to be highly active in all the tested concentrations of Comp B. These three newly identified candidate genes of this explosives-tolerant plant species can be potentially exploited for uses in phytoremediation by overexpressing these genes in transgenic plants and, similarly, by using promoters or variants of promoters from these genes fused to reporter genes in transgenic plants for making phytosensors to report the localized presence of explosives in contaminated soils.

A Thermostable β-Glucuronidase Obtained by Directed Evolution as a Reporter Gene in Transgenic Plants

A β-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable β-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo β-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic β-glucuronidase activity, which is a welcome improvement in its use as a reporter.

Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: How to avoid interference with the Gus transgene in transformation studies

The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed ( Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24 °C, 38 °C and 55 °C) were investigated. To avoid this problem in microspores, two incubation temperatures (38 °C and 55 °C) at different periods after GUS assay (4, 24 and 48 h) and in the presence of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4 h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55 °C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2 μM gibberellic acid.

Polyubiquitin Promoter-Based Binary Vectors for Overexpression and Gene Silencing in Lotus japonicus

In this study, we compared the transcriptional activities between Cauliflower mosaic virus (CaMV)35S promoter and polyubiquitin (Ljubq1) promoter from Lotus japonicus using beta-glucuronidase (gus) reporter gene in transgenic plants of L. japonicus. The promoter analysis demonstrated that the Ljubq1 promoter possessed higher activity than the CaMV35S promoter in leaves, stems, roots, nodules, and pollen. Finally, we created GATEWAY conversion technology-compatible binary vectors for over-expression and RNA interference under the Ljubq1 promoter. These materials could provide alternative choice for studies in L. japonicus.

PSAT RNA Interference Vectors: A Modular Series for Multiple Gene Down-Regulation in Plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.

The promoter of the TLC1.1 retrotransposon from Solanum chilense is activated by multiple stress-related signaling molecules

The LTR retrotransposons are the most abundant mobile elements in the plant genome and seem to play an important role in genome reorganization induced by environmental challenges. Their success in this function depends on the ability of their promoters to respond to different signaling pathways that regulate plant adaptation to biotic and abiotic stresses. The promoter of the TLC1.1 retrotransposon from Solanum chilense contains two primary ethylene-responsive elements (PERE boxes) that are essential for its response to ethylene and for the stress-induced expression. Here, we describe that a 270 bp fragment (P270), derivative of this retroelement promoter, is also able to activate the transcription of the GUS reporter gene in transgenic plants in response to salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (MeJA), hydrogen peroxide (H2O2) and the synthetic auxin 2,4-D. PERE box-dependent and independent routes are involved in the response of P270 to these signal molecules. MeJA, H2O2 and 2,4-D activate this promoter through cis-acting elements other than PERE boxes, whereas ABA and SA act via a PERE box-independent pathway but require this element for maximal activation. Three putative cis-acting elements MRE, GCN4 and GT1/TCA identified in the P270 promoter may be involved in the PERE box-independent activation pathway. These results suggest that the promoter of TLC1.1 may act as an integrator of different signal transduction pathways, allowing this member of the TLC1 retrotransposon family to be activated in response to multiples challenges.

Gene silencing using a heat-inducible RNAi system in Arabidopsis

Controlling gene expression during plant development is an efficient tool to explore gene function. In this paper, we describe a gene expression system driven by a heat-shock gene promoter ( HSP18.2), to trigger the expression of an intron-containing inverted-repeat. RNA interference became a powerful way for gene functional analysis by reverse genetic approaches. However, constitutive gene silencing cannot be used with genes involved in fundamental processes such as embryo viability. Inducible promoters provide an alternative approach for temporal and spatial gene expression control and we described here a new system, complementary to those using chemical gene inducers. To evaluate the efficiency of this system, RNA corresponding to the phytoene desaturase gene of Arabidopsis thaliana was used as a reporter gene in transgenic plants and a comparative study was performed using either the CaMV35S constitutive promoter or the HSP18.2 inducible promoter.

Significance of the expression of the CER6 condensing enzyme for cuticular wax production in Arabidopsis.

To learn more about the role of the CER6 condensing enzyme in Arabidopsis surface wax production, we determined CER6 transcription domains and the timing of CER6 transcription in vegetative and reproductive structures from juvenile, mature, and senescing tissues. We found that CER6 is highly transcribed throughout development, exclusively in the epidermal cells in all tissues examined. The only exception to the epidermal expression was observed in anthers nearing maturity, in which CER6 mRNA was localized in the tapetum. To determine if environmental factors such as light and water deficit, which are known to stimulate wax accumulation, induce CER6 transcription, we examined the effects of these factors on CER6 transcript abundance. Our results demonstrate that light is essential for CER6 transcription, and that osmotic stress and the presence of abscisic acid enhance CER6 transcript accumulation. CER6 promoter-directed expression of the beta-glucuronidase reporter gene in transgenic plants demonstrated that the CER6 promoter was highly effective in directing epidermis-specific expression in Arabidopsis and tobacco (Nicotiana tabacum). Furthermore, CER6 promoter-driven CER6 overexpression resulted in increased wax deposition in Arabidopsis stems. These experiments indicate that the expression level of CER6 in the epidermis is one of the factors controlling wax accumulation on Arabidopsis stems.

The expression of small heat shock proteins in seeds responds to discrete developmental signals and suggests a general protective role in desiccation tolerance.

To learn more about the function and regulation of small heat shock proteins (sHSPs) during seed development, we studied sHSP expression in wild-type and seed maturation mutants of Arabidopsis by western analysis and using an HSP17.4 promoter-driven beta-glucuronidase (GUS) reporter gene in transgenic plants. In the absence of stress, GUS activity increases during development until the entire embryo is stained before desiccation. Heat-stressed embryos stained for GUS at all stages, including early stages that showed no detectable HSP17. 4::GUS activity without heat. Examination of HSP17.4 expression in seeds of the transcriptional activator mutants abi3-6, fus3-3 (AIMS no. CS8014/N8014), and lec1-2 (AIMS no. CS2922/N2922) showed that protein and HSP17.4::GUS activity were highly reduced in fus3-3 and lec1-2 and undetectable in abi3-6 seeds. In contrast, heat-stressed abi3-6, fus3-3, and lec1-2 seeds stained for GUS activity throughout the embryo. These data indicate that there is distinct developmental and stress regulation of HSP17.4, and imply that ABI3 activates HSP17.4 transcription during development. Quantitation of sHSP protein in desiccation-intolerant seeds of abi3-6, fus3-3, lec1-2, and line24 showed that all had <2% of wild-type HSP17.4 levels. In contrast, the desiccation-tolerant but embryo-defective mutants emb266 (AIMS no. CS3049/N3049) and lec2-1 (AIMS no. CS2728/N2728) had wild-type levels of HSP17.4. These data correlate a reduction in sHSPs with desiccation intolerance and suggest that sHSPs have a general protective role throughout the seed.

Biochemical characterization and expression of RLK4, a receptor-like kinase from Arabidopsis thaliana

The Arabidopsis RLK4 gene encodes a Ser/Thr receptor-like protein kinase. A lysine to glutamic acid substitution in an essential subdomain of the RLK4 catalytic domain, results in an inactive protein kinase. Transphosphorylation experiments, using the inactive form of RLK4 as a substrate for an active form, indicated that autophosphorylation could proceed via an intermolecular mechanism. Multiple sites are phosphorylated at the catalytic domain of RLK4, as indicated by two-dimensional (2D) thin-layer electrophoresis and chromatography. To analyze the expression pattern of RLK4, we used the RLK4 promoter to drive the expression of the β- glucuronidase ( GUS) reporter gene in transgenic plants. GUS activity was observed in the shoot apex and roots. In roots, the GUS staining pattern was limited to the lateral roots and at the root-hypocotyl transition zone. Analysis of an RLK4 mutant line did not show any difference in the phenotype with respect to the wild type. The expression of other related RLKs with similar expression patterns suggest there is functional redundancy for RLK4.

The Arabidopsis ERECTA gene is expressed in the shoot apical meristem and organ primordia.

In Arabidopsis thaliana (L.) Heynh, the mutation in ERECTA is known to confer a compact inflorescence by a reduction in the lengths of internodes and pedicels. We analyzed the expression pattern of this gene during plant development. In situ hybridization and histochemical analysis using transgenic plants carrying chimeric gene fusions, with the ERECTA promoter fused to the beta-glucuronidase (GUS) gene, showed that ERECTA was predominantly expressed in the shoot apical meristems and organ primordia. ERECTA expression in the shoot apical meristem was weak early in plant development but increased with the transition from the vegetative to the reproductive growth phase. ERECTA was also strongly expressed in organ primordia and immature organs but weakly in mature organs. Thus, ERECTA was expressed in a cell-specific and developmentally regulated manner. In order to identify the regulatory mechanism responsible for the expression pattern of ERECTA, the cis-acting regions in the ERECTA promoter were defined by study of the expression of the chimeric genes that consist of the 5'- or internal deleted promoter and a GUS reporter gene in transgenic plants. The results showed that the essential cis-regulatory elements governing the spatially and temporally specific expression of ERECTA are located between positions -462 and -228 bp and between positions -228 and -153 bp with respect to the transcriptional initiation site.

A transcription activation system for regulated gene expression in transgenic plants.

A widely applicable promoter system is described that allows a gene of interest to be activated in specific plant tissues after a cross between defined transgenic lines. The promoter, pOp, consists of lac operators cloned upstream of a minimal promoter. No expression was detected from this promoter when placed upstream of a beta-glucuronidase (GUS) reporter gene in transgenic plants. Transcription from the promoter was activated by crossing reporter plants with activator lines that expressed a chimeric transcription factor, LhG4. This factor comprised transcription-activation domain-II from Gal4 of Saccharomyces cerevisiae fused to a mutant lac-repressor that binds its operator with increased affinity. When LhG4 was expressed from the CaMV 35S promoter, the spatial and quantitative expression characteristics of the 35S promoter were exhibited by the GUS reporter. The LhG4/pOp system may be used to study toxic or deleterious gene products, to coordinate the expression of multiple gene products, to restrict transgene phenotypes to the F1 generation, and to generate hybrid seed. The LhG4 system offers spatially regulated gene expression in the tissues of whole plants growing under normal conditions without the need for external intervention. It complements inducible expression systems that offer temporal control of gene expression in tissues that can be treated with inducing chemicals.

Characterization of Δ 1-pyrroline-5-carboxylate synthetase gene promoter in transgenic Arabidopsis thaliana subjected to water stress

Many plants accumulate proline in response to osmotic stress. Δ 1-pyrroline-5-carboxylate synthetase (P5CS) is the rate limiting enzyme in proline biosynthesis in plants. The promoter of an Arabidopsis P5CS gene was isolated and analyzed using a β-glucuronidase (GUS) reporter gene in transgenic plants. NaCl stress induced GUS activity to the maximum level within 3 h while dehydration continued to enhance the GUS activity up to 24 h. The P5CS gene promoter was found to contain two transcription start sites, and dehydration stimulated the transcription predominantly from the downstream site. Abscisic acid (ABA) failed to induce the P5CS gene expression although ABA is known to increase during water stress. These observations suggest that induction of the P5CS gene is a specific response of plants to salt and water stresses.

Promoter/leader deletion analysis and plant expression vectors with the figwort mosaic virus (FMV) full length transcript (FLt) promoter containing single or double enhancer domains.

The boundaries required for maximal expression from the promoter/leader region of the full length transcript of figwort mosaic virus (FLt promoter) coupled to reporter genes were defined by 5' and 3' deletion analyses. In transient expression assays using protoplasts of Nicotiana edwardsonii, a 314 bp FLt promoter fragment sequence (-249 to +65 from the transcription start site) was sufficient for strong expression activity. Plant expression vectors developed with modified FLt promoters were tested with GUS or CAT as reporter genes in transgenic plants. The FLt promoter is a strong constitutive promoter, with strength comparable to or greater than that of the CaMV 35S promoter. The FLt promoter with its double enhancer domain linked to GUS or CAT reporter genes provides an average 4-fold greater activity than the FLt promoter with a single enhancer domain (-55 to -249 bp upstream fragment) in tests with transgenic plants and in protoplast transient expression assays.

In situ detection of expression of thegus reporter gene in transgenic plants: ten years of blue genes

Among the methods now available to localize the sites of gene expression in plant materials, reporter genes based on thegus (uidA) gene ofEscherichia coli, which encodes a β-glucuronidase (E.C. 3.2.1.31; GUS), have been the most widely used during the last ten years. The apparent simplicity of the histochemical GUS assay has been a major factor in the increase in articles usinggus genes. However, over the last four years, there have been occasional reports expressing doubts concerning the specificity of the observed localizations based on discrepancies between results obtained with GUS histochemistry and immunocytochemistry and/orin situ hybridization. This brief review compares the results obtained with immunocytochemistry with those obtained with various GUS substrates for histochemical studies. Certain sources of artefact are discussed, as are the limits that should be imposed on interpretation of GUS histochemistry results at the organ, tissue and cell levels.

Jellyfish green fluorescent protein as a reporter for virus infections.

The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Production of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV-illuminated plants in a darkened room. The PVX.GFP-infected tissue has been analysed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of extracts from infected plants. To illustrate the potential application of this reporter gene in virological studies a derivative of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell-to-cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue.

Auxin-Regulated Transcription

We have cloned and sequenced a number of auxin-responsive cDNAs and their corresponding genes from soybean and Arabidopsis. Each of these genes, with the exception of GH2/4, is transcriptionally regulated specifically by auxins within minutes after hormone application. The auxin-responsive mRNAs are induced some 3-60-fold depending on the type of mRNA analysed, the tissue examined, the dose and duration of auxin application, and the manipulation of the organ tested. Some of the mRNAs show rapid turnover kinetics. The mRNAs show distinct patterns of organ-specific, tissue-specific, and developmental-specific expression. The promoters of the auxin-responsive genes have been fused to the E. coli uidA gene which encodes β-glucuronidase (GUS) and transferred into tobacco and/or Arabidopsis via Agrobacterium T-DNA. These promoters and parts of these promoters have been used to follow the expression patterns and auxin-inducibility of the reporter genes in transgenic plants. We are attempting to identify minimal auxin-responsive elements and gravity-responsive elements within these promoters. We have also fused the auxin-inducible promoters to bacterial genes that encode cytokinin and auxin biosynthetic or conjugating enzymes to study the effects of organ, tissue, and developmental-specific expression of cytokinins and auxins on plant growth, development, and physiology.

Analysis of regulatory elements involved in the induction of two tobacco genes by salicylate treatment and virus infection.

Tobacco genes encoding the PR-1a protein and a glycine-rich protein are expressed after treatment of plants with salicylate or infection with tobacco mosaic virus. Upstream sequences of these genes were fused to reporter genes, and these constructs were used to transform tobacco. Upstream sequences of the PR-1a gene of 689 base pairs or longer were sufficient for induction of the reporter gene in tobacco mosaic virus-inoculated leaves, systemically induced leaves from infected plants, and leaves treated with salicylate. No such induction was found with upstream sequences of 643 base pairs or shorter of the PR-1a gene. When the PR-1a upstream sequence from nucleotides -625 to -902 was fused to the cauliflower mosaic virus 35S core promoter, a construct was obtained that conferred tobacco mosaic virus and salicylate inducibility to the reporter gene in transgenic plants. This confirmed the localization of tobacco mosaic virus- and salicylate-responsive elements between positions -643 and -689 in the PR-1a promoter. With the glycine-rich protein gene, an upstream sequence of 645 base pairs was sufficient for tobacco mosaic virus and salicylate inducibility of the reporter gene, whereas constructs containing 400 base pairs or fewer of the glycine-rich protein promoter were largely inactive.