Expression Of GUS Activity Research Articles

Features of the hmg 1 subfamily of genes encoding HMG-CoA reductase in potato

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the beta-glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R2 pollen of R1 progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.

Transient expression of gus gene in intact seed embryos of Indica rice after electroporation-mediated gene delivery

Two-day-old germinating intact seed embryos of Oryza sativa variety Basmati 370 were electroporated with a view to examine suitability of this system for gene delivery. The experiments were done with a plasmid having gus gene under the control of CaMV 35S promoter. Spectrofluorophotometric GUS assay revealed high activity of the introduced gene when embryos were given three electrical pulses at 1600 V cm(-1) and 100 μF capacitance with a pulse length of 75 ms. Additionally, histochemical localization of GUS activity in seedlings and various organs such as leaves, coleoptiles and roots was also done. Expression of GUS activity was studied up to 15 days and found to be organ-specific, thereby showing that embryos can indeed serve as efficient recipient system. Use of cycloheximide revealed that GUS activity appears as a result of early protein synthesis after electroporation and is substantially stable in vivo.

Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase.

Phage T7 RNA polymerase has been used extensively in Escherichia coli for high-level expression of selected genes placed under the control of the phage T7 gene 10 promoter. We have constructed an analogous system for use in plastids of higher plants. A T7 RNA polymerase chimeric gene containing a cauliflower mosaic virus 35S promoter and a tobacco ribulose-bisphosphate carboxylase/oxygenase small-subunit chloroplast transit-peptide sequence was introduced into tobacco by nuclear transformation. Stable plastid transformation of tobacco expressing the T7 RNA polymerase activity with a T7 promoter/beta-glucuronidase (GUS) reporter gene construct resulted in expression of GUS mRNA and enzyme activity in all tissues examined. Expression of GUS activity was extremely high in mature leaves, moderate in young leaves and petals, and low in stems, roots, and developing seeds. Plastid transformation of wild-type tobacco with the same chimeric GUS gene resulted in undetectable levels of GUS mRNA and enzyme activity. Genetic crosses demonstrated that a silent T7/GUS reporter gene could be activated in the F1 generation by transmission of an active nuclear T7 RNA polymerase gene from the male parent.

Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

We developed a polyethylene glycol (PEG)-mediated direct DNA transfer method from intact Saccharomyces cerevisiae spheroplasts into Arabidopsis thaliana protoplasts. To monitor the DNA transfer from yeast to plant cells, beta-glucuronidase (GUS) reporter gene in which a plant intron was inserted was used as a reporter. This intron-GUS reporter gene on a 2 microns-based plasmid vector was not expressed in yeast transformants, while it expressed GUS activity when the plasmid DNA was introduced into plant cells. When a mixture of 1 x 10(8) of S. cerevisiae spheroplasts harboring the plasmid and 2 x 10(6) of A. thaliana protoplasts was treated with PEG and high pH-high Ca2+ solution (0.4 M mannitol, 50 mM CaCl2, 50 mM glycine-NaOH pH 10.5), GUS activity was detected in the extract of the plant cells after a three-day culture. The GUS activity was higher than that of a reconstitution experiment in which the mixture of 1 x 10(8) of S. cerevisiae spheroplasts which did not carry the reporter gene, 2 x 10(6) of A. thaliana protoplasts and the same amount of the reporter plasmid DNA as that contained in 1 x 10(8) of S. cerevisiae spheroplasts, was treated with PEG and high pH-high Ca2+ solution. Moreover, the GUS gene expression was resistant to micrococcal nuclease treatment before and during PEG treatment. From these results, we concluded that plasmid DNA can be directly transferred from intact yeast spheroplasts to plant protoplasts by a nuclease-resistant process, possibly by the cell fusion.

High frequency shoot regeneration and Agrobacterium-mediated transformation of chrysanthemum ( Dendranthema grandiflora)

An efficient, high-frequency transformation protocol was developed for the commercial chrysanthemum cultivar ‘Iridon’. Regeneration protocols were also developed for two other commercial cultivars, ‘Hekla’ and ‘Polaris’. All protocols utilized embryogenesis media composed of the basal medium of Murashige and Skoog (MS) supplemented with 11.5 μM indoleacetic acid and 0.5 or 1.0 μM benzyladenine. Regenerationof shoots from cultivar ‘Iridon’ was obtained with continuous culture on these media until shoots were transferred to rooting medium at approximately 6 weeks. By contrast, shoot regeneration from ‘Hekla’ explants required transfer to hormone-free medium 2 weeks after initial culturing. ‘Polaris’ regeneration required transfer of explants to hormone-free medium when shoot primordia first developed as well as continuous culture of explants in the absence of blue wavelengths of light. Shoots from all cultivars were rooted on 25% MS medium with 0.5 μM naphthaleneacetic acid. Three wild type strains of Agrobacterium tumefaciens (Ach5, A281, Chry5) were evaluated for tumor production on the three cultivars. Chry5 and A281 were significantly more virulent on all three cultivars than was Ach5. Transformed plants of ‘Iridon’ were obtained using Agrobacterium strain EHA105, a disarmed version of A281. Explants were transformed with two plasmids, pBI121, which encodes kanamycin resistance and β-glucuronidase (GUS) activity, and pBI121 containing the nucleocapsid gene of a highly virulent isolate of tomato spotted wilt virus (TSWV). Transformed shoots regenerated and rooted on medium containing 50 μg/ml kanamycin. Vegetatively propagated progeny of transformed plants were identified which expressed GUS activity and which contained multiple copies of the TSWV nucleocapsid gene.

Asymmetric somatic hybridization between tomato (Lycopersicon esculentum Mill) and gamma-irradiated potato (Solanum tuberosum L.): a quantitative analysis

We analyzed 110 asymmetric fusion products, obtained by fusion of hygromycin-resistant tomato protoplasts and gamma-irradiated kanamycin-resistant potato protoplasts that expressed β-glucuronidase (GUS). The fusion products were selected for resistance to both antibiotics, and were subsequently analyzed for their shoot regeneration potential, GUS activity, expression of two potato isoenzymes, chloroplast type, total genomic DNA content, and relative genomic composition. No viable plants could be obtained and the calli were highly polypoid. All hybrids expressed GUS activity, whereas they displayed a large variation with respect to the other traits.

Agrobacterium tumefaciens-mediated expression ofgusA in maize tissues

To develop a system forAgrobacterium-mediated transformation of maize (Zea mays L.), we have investigated histochemically the transient expression of β-glucuronidase (GUS) activity in maize seedling tissue segments using binary vectors that allow minimal (pKIWI105 and pCNL1) or undetectable (p35S-GUS-INT and pCNL56) levels of GUS activity inA. tumefaciens. Tissue segments from three- to five-day-old sterile seedlings of maize genotype A188 were inoculated withA. tumefaciens. Four days after inoculation, transient expression of GUS activity was found in mesocotyl segments originating from the intercalary meristem region. This GUS activity was specific to the vascular cylinder and was not found in the internal cortical or epidermal layers, nor was it found in mature mesocotyl tissue (segments 5 mm below the coleoptilar node). Transient GUS activity was also detected in leaf and coleoptile tissues of shoot segments, but not in the shoot apexper se or in leaves younger than the first leaf. Maize tissues inoculated withA. tumefaciens strains that harbourgusA-containing binary vectors but no Ti-plasmid did not show GUS activity, supporting evidence from previous work thatvir gene activity was essential for the observed GUS activity.A. tumefaciens strains containing different types of Ti-plasmids were also tested. A strain harbouring an agropine-type Ti-plasmid was the most effective for expressing GUS activity in mesocotyl segments, whereas a strain harboring a nopaline-type Ti-plasmid was most effective for expression of GUS activity in the apical meristem-containing segment. These results indicate that different interactions occurred between the differentA. tumefaciens strains and the susceptible plant tissues. Maize genotype specificity for GUS activity in mesocotyl tissues was observed; variations in the cocultivation medium had a profound effect on the frequency of expression of GUS activity.

Expression of the E. coli β-glucuronidase gene in the light leaf spot pathogen Pyrenopeziza brassicae and its use as a reporter gene to study developmental interactions in fungi

Co-transformation using the hygromycin-resistant plasmid pAN7-1 for transformation selection was used to transform spores of both mating types of the light leaf spot pathogen P. brassicae with the chimeric β-glucuronidase (GUS) vector pNOM102. 78 and 90% of the hygromycin-resistant transformants of strains JH26 (MAT1-2) and NH10 (MAT1-1) respectively expressed GUS activity and 30% of these transformants showed high levels of GUS activity. Quantitative analysis of GUS expression by measurement of the rate of hydrolysis of the fluorogenic substrate 4-methylumbelliferyl glucuronide revealed that the wild type produces no detectable activity and that 15 transformants of each mating type displayed a 500-fold range of activity. Ten of the transformants were analysed further to establish mitotic stability of GUS, the ability to pathogenize the plant and an ability to undergo sexual morphogenesis when crossed with a compatible mating-type partner. All of the transformants tested were fertile when crossed with a compatible mating-type partner. Two out of the transformants tested had lost their ability to express GUS activity on continuous sub-culturing. All of the NH10 (MAT1-1) GUS transformants were able to produce extracellular proteolytic activity and were able to pathogenize the plant. However three JH26 (MAT1-2) GUS transformants displayed reduced proteolytic activity and one (JH26pNOM102/13) produced no extracellular protease in vitro and had lost the ability to produce pathogenic symptoms in planta. GUS transformants of P. brassicae were shown to stain blue with the chromogenic substrate 5-bromo-4-chloro-3-indolyl β- d -glucuronide. This staining reaction enabled differentiation between both spores and mycelium during sexual development when a wild type of either mating type was crossed with a GUS transformant of the opposite mating type. Such preliminary studies demonstrate the relative usefulness of the GUS reporter system for the analysis of an otherwise indistinguishable mating interaction and suggest that it will be a valuable reporter system for analysis of the temporal and spatial expression of developmentally regulated genes in this economically important plant pathogen.

Expression of a phosphoenolpyruvate carboxylase promoter from Mesembryanthemum crystallinum is not salt-inducible in mature transgenic tobacco

The 5' flanking region of a salt-stress-inducible, CAM-specific phosphoenolpyruvate carboxylase (PEPC) gene from the facultative halophyte Mesembryanthemum crystallinum, was fused to the beta-glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum SR1. The Ppc1 promoter displayed high levels of expression in transgenic tobacco quantitatively and qualitatively similar to a full-length 35S CaMV-GUS construct. Histochemical assays revealed that the full-length Ppc1-GUS fusions expressed GUS activity in all tissues except in root tips. While tobacco is capable of utilizing the Ppc1 cis-acting regulatory regions from M. crystallinum to yield high levels of constitutive expression, this glycophyte fails to direct a stress-inducible pattern of gene expression typical of this promoter in its native, facultative halophytic host.

Interest in and limits to the utilization of reporter genes for the analysis of transcriptional regulation of nitrate reductase.

Reporter gene techniques and mutant analysis were used to identify the molecular basis of the regulation of the expression of nitrate reductase (NR) by nitrate and nitrate-, or ammonium-derived metabolites (N-metabolites), in the true diploïd species Nicotiana plumbaginifolia and in the amphidiploïd species Nicotiana tabacum. The N. plumbaginifolia mutant E23 results from the insertion of a Tnt1-like retrotransposon (Tnp2) in the first exon of the single-copy nia gene, which encodes nitrate reductase. One of the resulting transcripts ends in the 5' LTR (long terminal repeat) sequence of this retrotransposon, and another one in the 3' LTR. Nitrate and N-metabolites modulate the expression of these truncated transcripts, indicating that intron splicing and termination processes are not essential to these regulatory events. A GUS reporter sequence was transcriptionally linked to the promoter of the nia-1 gene of N. tabacum. This fusion was functional in transient expression assays done with protoplasts derived from mesophyll cells of N. tabacum. However none of the regulatory mechanisms known to affect steady-state levels of the nia-1 transcript were operative under these experimental conditions. Transgenic plants carrying either this fusion or translational fusions of GUS linked to the promoter of either the nia-1 or nia-2 gene of N. tabacum were obtained by Agrobacterium-mediated transfer. A low proportion of the transgenic plants (22 out of 105 independent transformants) expressed GUS activity although at a low level. Only 4 plants exhibited a detectable level of GUS mRNA. The concentration of this mRNA increased significantly in an NR-deficient background, indicating regulation by N-metabolites. Only 2 plants, however, showed regulation (induction) by nitrate. Attempts to use aux2 or nptII reporter sequences linked to either the nia-1 or nia-2 promoter as marker genes for the selection of regulatory mutants of the nitrate assimilation pathway were unsuccessful because of our inability to isolate transgenic plants in which these reporter genes were properly regulated by nitrate. The implications of these results are discussed.

Use of a nuclear‐targeted β‐glucuronidase fusion protein to demonstrate vegetative cell‐specific gene expression in developing pollen

SummaryTo examine the site of expression of the tomato anther‐specific gene, LAT52, in the developing male gametophyte, the LAT52 gene promoter was fused to a nuclear‐targeted version of the β‐glucuronidase (GUS) gene and introduced into tobacco. Transformed plants expressing GUS activity showed nuclear localization of the GUS reaction product to the vegetative cell of the pollen grain. No staining or localization was detected in the generative cell, at pollen maturation or during pollen tube growth in vitro. These results clearly demonstrate differential gene expression within the male gametophyte, and highlight regulatory events which determine the differing fates of the vegetative and generative cells following microspore mitosis.

AGROBACTER1UM-MED1ATED TRANSFORMATION OF APPLE

Two strains of Agrobacterium tumefaciens carrying a disarmed Ti-binary vector, pZA-7, were used as vectors for transformation of apple leaf segments, EHA101:pZA-7 carries a helper plasmid derived from pTiB0542, and C58Z707:pZA-7 carries a helper plasmid derived from pTiC58. The binary vector provides two selection markers, neomycin phosphotransferase (nptII) and hygromycin resistance genes, and a screening marker, β-glucuronidase (GUS) gene. Preliminary experiments were conducted to determine the effect of different concentrations of kanamycin, carbenicillin, and cefotaxime on regeneration of apple leaf sections and inhibition of A. tumefaciens strains. In vitro-derived leaf sections of `Royal Gala' apple were grown on a regeneration medium containing thidiazuron and NAA; these were then dipped into a suspension culture of A. tumefaciens and transferred to a fresh regeneration medium. Callus lines exhibiting kanamycin and hygromycin resistance were obtained mostly with agrobacterium strain EHA101:pZA-7. Expression of GUS activity was also determined in putative transformed calli. Southern blot analysis was used for confirming integrative transformation in transgenic lines.

Promoters of auxin-induced genes from tobacco can lead to auxin-inducible and root tip-specific expression

In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells. The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment. In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35. To further study the regulation of the expression of these genes their 5' regions were translationally fused with the beta-D-glucuronidase reporter gene (GUS). The GNT1 5' region led to GUS expression only in the root tips of transgenic plants. By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5' regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity. The homology of the 103-like genes with other auxin-regulated genes is evaluated.

Silicon carbide fiber-mediated DNA delivery into plant cells

Silicon carbide fiber-mediated delivery of DNA into intact plant cells was investigated. Black Mexican Sweet (BMS) maize (Zea mays) and tobacco (Nicotiana tabacum) suspension culture cells were vortexed in the presence of liquid medium, plasmid DNA encoding β-glucuronidase (GUS), and silicon carbide fibers. Penetration of BMS cells by the silicon carbide fibers was observed by scanning electron microscopy of vortexed cells. Following fiber and DNA treatment, BMS cells transiently expressed GUS activity at a mean frequency of 139.5 units (one unit = one blue cell or one colony of blue cells) per sample. Treated tobacco cells expressed an average of 373 GUS units per sample. Untreated controls did not exhibit GUS activity. These results indicate that the silicon carbide fibers-vortex procedure can be used to rapidly and inexpensively deliver foreign DNA into intact plant cells for investigations of transient gene expression.

Construction and Expression in Tobacco of a β-Glucuronidase (GUS) Reporter Gene Containing an Intron Within the Coding Sequence

An Intron-GUS reporter gene which contains a modified intron of the castor bean catalase gene within the N-terminal part of the β-glucuronidase (GUS) coding sequence was constructed. When the Intron-GUS reporter gene placed downstream of the cauliflower mosaic virus (CaMV) 35S promoter (35S-Intron-GUS) was introduced into protoplasts of tobacco suspension-cultured cells by electroporation, expression of GUS activity was observed suggesting splicing of the intron occured in tobacco cells

Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation

Agrobacterium tumefaciens is a commonly used tool for transforming dicotyledonous plants. The underlying mechanism of transformation however is not very well understood. One problem complicating the analysis of this mechanism is the fact that most indicator genes are already active in Agrobacterium, thereby preventing the precise determination of timing and localisation of T-DNA transfer to plant cells. In order to overcome this obstacle a modified prokaryotic indicator gene was constructed. The expression of this indicator gene and its use in analysing early events in Agrobacterium-mediated plant transformation are described. A portable intron, derived from a plant intron, was introduced into the beta-glucuronidase (GUS) gene. In transgenic plants containing this chimaeric gene the intron is spliced efficiently, giving rise to GUS enzymatic activity. Mapping of the splice junction indicates the exact removal of the intron. No GUS activity is detected in agrobacteria containing this construct due to the lack of a eukaryotic splicing apparatus in prokaryotes. Early phases after transformation of Arabidopsis cotyledon explants were analysed using this GUS-intron chimaeric gene showing that as early as 36 h after Agrobacterium infection significant GUS activity is detected. In vivo GUS staining of transformed cells clearly shows that quickly proliferating calli expressing GUS activity are formed, mainly at the cut surface. Minor transformation events occur however throughout the whole cotyledon. These data indicate that Agrobacterium-mediated T-DNA transfer to plants is much more efficient than has been judged from experiments where selection is applied immediately. The intron-containing GUS gene can be used as an optimised marker gene in transient and stable transformation experiments.