GUS Fusion Gene Research Articles

The tobacco extensin gene Ext 1.4 is expressed in cells submitted to mechanical constraints and in cells proliferating under hormone control

The promoter region of a Nicotiana tabacum extensin gene (Ext 1.4) was studied in tobacco transgenic plants carrying Ext 1.4/GUS (beta-glucuronidase) chimeric genes. The pattern of expression could be defined and cis-regulatory elements were localized in small regions of the promoter. In healthy plants, expression was essentially found in cells under mechanical stress, that is at the emergence of lateral roots, at the junction between stem and petiole and at the fusion of carpels. In roots of germinating plantlets, expression was found in the piliferous zone. In flowers, expression was found on the one hand in the placenta, in the locular tissue of ovaries and in the zone of carpel fusion, and on the other hand in the connective tissue of anthers, in mature and in germinating pollen. A developmental regulation during seed germination, where the gene fusion is transiently expressed in the endosperm and in the root tip before its expression becomes similar to that found in mature plants has also been shown. The expression of the Ext 1.4/GUS chimeric gene was also induced during cell proliferation under hormone control, for example, in response to Agrobacterium tumefaciens infection and in calli. However, when organogenesis occurred under hormone control, expression was never found in root or shoot primordia. Cis-regulatory elements important for expression of the Ext 1.4/GUS gene fusion in germinating seeds, in mature plants or in proliferating cells have been localized in the proximal promoter region whereas enhancer elements have been located further upstream.

Arabidopsis Rho-related GTPases: differential gene expression in pollen and polar localization in fission yeast.

The Rho small GTP-binding proteins are versatile, conserved molecular switches in eukaryotic signal transduction. Plants contain a unique subfamily of Rho-GTPases called Rop (Rho-related GTPases from plants). Our previous studies involving injection of antibodies indicated that the pea Rop GTPase Rop1Ps is critical for pollen tube growth. In this study we show that overexpression of an apparent Arabidopsis ortholog of Rop1Ps, Rop1At, induces isotropic cell growth in fission yeast (Schizosaccharomyces pombe) and that green fluorescence protein-tagged Rop1At displays polar localization to the site of growth in yeast. We found that Rop1At and two other Arabidopsis Rops, Rop3At and Rop5At, are all expressed in mature pollen. All three pollen Rops fall into the same subgroup as Rop1Ps and diverge from those Rops that are not expressed in mature pollen, suggesting a coupling of the structural conservation of Rop GTPases to their gene expression in pollen. However, pollen-specific transcript accumulation for Rop1At is much higher than that for Rop3At and Rop5At. Furthermore, Rop1At is specifically expressed in anthers, whereas Rop3At and Rop5At are also expressed in vegetative tissues. In transgenic plants containing the Rop1At promoter:GUS fusion gene, GUS is specifically expressed in mature pollen and pollen tubes. We propose that Rop1At may play a predominant role in the regulation of polarized cell growth in pollen, whereas its close relatives Rop3At and Rop5At may be functionally redundant to Rop1At in pollen.

The homeobox gene NTH23 of tobacco is expressed in the basal region of leaf primordia

We reported isolation and characterization of a homeobox gene from tobacco, NTH23. The homeodomain structure of NTH23 was highly homologous to the same regions of class 2 genes of the KN1-type homeobox (sharing more than 85% amino acid identity), but was less similar to class 1 genes of KN1-type. RNA gel blot analysis revealed that NTH23 was expressed in all organs we tested although the gene is primarily expressed in young leaves. To determine more precisely the spatial expression pattern of NTH23 in tobacco, a chimeric NTH23::GUS fusion gene was introduced into tobacco. The signal of GUS activity was observed at the basal part of leaf blade primordia in the NTH23::GUS transgenic tobacco plants. This observation suggests the possibility that NTH23 may be important for the lateral growth of leaf blades.

Analysis of T-DNA-mediated translational beta-glucuronidase gene fusions.

Three random translational beta-glucuronidase (gus) gene fusions were previously obtained in Arabidopsis thaliana, using Agrobacterium-mediated transfer of a gus coding sequence without promoter and ATG initiation site. These were analysed by IPCR amplification of the sequence upstream of gus and nucleotide sequence analysis. In one instance, the gus sequence was fused, in inverse orientation, to the nos promoter sequence of a truncated tandem T-DNA copy and translated from a spurious ATG in this sequence. In the second transgenic line, the gus gene was fused to A. thaliana DNA, 27 bp downstream an ATG. In this line, a large deletion occurred at the target site of the T-DNA. In the third line, gus is fused in frame to a plant DNA sequence after the eighth codon of an open reading frame encoding a protein of 619 amino acids. This protein has significant homology with animal and plant (receptor) serine/threonine protein kinases. The twelve subdomains essential for kinase activity are conserved. The presence of a potential signal peptide and a membrane-spanning domain suggests that it may be a receptor kinase. These data confirm that plant genes can be tagged as functional translational gene fusions.

Cooperation of two distinct cis‐acting elements is necessary for the S phase‐specific activation of the wheat histone H3 promoter

We have previously shown that the proximal promoter region (−185 to +57) of the wheat histone H3 gene (TH012) is sufficient for regulating S phase‐specific expression of a reporter GUS gene. To define the cis‐acting element(s) responsible for S phase‐specific expression, GUS fusion genes under the control of wild‐type or variously mutated H3 promoters were stably introduced into cultured rice Oc cells and their temporal expression was analyzed during the cell cycle by quantitative S1 analysis. The S phase‐specific expression of the full‐sized promoter (−1716 to +52) was significantly impaired by short internal deletions disrupting the type I element from −175 to −158 (CCACGTCACCaATCCGCG), composed of the Hex (CCACG‐TCA) and reverse‐oriented Oct (GATCCGCG) motifs. Moreover, the H3 proximal promoters (−184 to +52) harboring base‐substitution mutations in either or both of the Hex and Oct motifs could no longer activate gene expression during the S phase. These results indicate that the type I element is the first cis‐acting element identified responsible for the S phase‐specific expression of plant histone genes. Results also suggested the presence of a redundant cis‐acting element(s) responsible for S phase‐specific expression in the H3 far‐upstream region (−1716 to −185).

Chloroplast-dependent and light-independent expression of the tobacco rbcS promoter–GUS chimeric gene in black spruce

The tobacco rbcS (ribulose bisphosphate carboxylase small subunit) promoter, fused to the β-glucuronidase (GUS) reporter gene, was delivered to black spruce (Piceamariana (Mill.) BSP) tissues via microprojectile DNA bombardment, and its regulation was studied. The expression of the tobacco rbcS promoter–GUS chimeric gene was dependent on the presence of chloroplasts in black spruce tissues, as demonstrated in two ways: (i) there was no GUS activity expressed in zygotic embryos where no chloroplasts were observed, whereas it was expressed in light- and dark-grown seedlings that contained mature or immature chloroplasts; (ii) a herbicide, Norflurazon, destroyed chloroplast structure in seedlings and inhibited the expression of the tobacco rbcS promoter–GUS chimeric gene. A control chimeric gene, the cauliflower mosaic virus (CaMV) 35S promoter–GUS fusion gene was not inhibited by Norflurazon. Unlike in angiosperms, light had no effect on the expression of tobacco rbcS promoter–GUS chimeric gene. Both light- and dark-grown seedlings showed GUS activity, and expression in dark-grown seedlings was not enhanced by light. These results suggest that the tissue-specific regulation of the rbcS promoter may be conserved between angiosperms and conifers, but that the light regulation of this promoter may not be conserved.

Analysis of the promoter of an abscisic acid responsive late embryogenesis abundant gene of Arabidopsis thaliana

Late embryogenesis abundant (lea) proteins are a diverse group of proteins present in many mono- and dicotyledonous plants. The genes encoding lea proteins are expressed in the embryo during the late stages of seed development. However, expression can also be induced in immature seeds and vegetative tissues by abscisic acid (ABA). Lea genes thus provide a model with which to study tissue-specific, developmental and hormonal regulation of expression. We used the β-glucuronidase ( iudA) reporter gene (gus) to identify functional domains in the promoter of a lea gene of Arabidopsis thaliana (AtEm1). We found that a promoter fragment extending from −182 bp to +72 bp is sufficient to direct gus expression to embryos and pollen of transgenic tobacco. Gus expression in embryos and pollen was developmentally regulated, being expressed during the late stages of seed and anther development. Comparison of different deletion constructs showed that in both tissues promoter sequences between −1443 bp and −430 bp had no effect on the level of gus expression, whilst the region between −430 bp and −182 bp is necessary for full level expression. The response to ABA was studied in seedlings of transgenic tobacco transformed with a gus gene fusion construct containing −1443 bp to +72 bp of promoter. Treatment with 50 μM ABA resulted in a 3–4-fold increase in GUS activity, indicating that ABA induction of AtEm1 acts at least in part at the level of transcription. Conserved regulatory elements were identified in the promoter of AtEm1 by sequence analysis. The possible role of these elements, and the significance of the observed gus expression in pollen are discussed.

The spinach AtpC and AtpD genes contain elements for light-regulated, plastid-dependent and organ-specific expression in the vicinity of the transcription start sites.

Run-on assays with isolated nuclei demonstrate that the transcription rates of AtpC and AtpD (gene products: the CF1 subunits gamma and delta of the chloroplast ATP synthase) are comparable in spinach seedlings. However, chimeric GUS gene fusions with 5'-flanking regions of the AtpC gene direct an approximately 10-fold lower GUS level in transgenic tobacco compared with equivalent fragments from the AtpD gene. Both promoters contain sequences in the vicinity of the respective TATA boxes, which are sufficient to direct light-regulated, plastid-dependent and organ-specific expression of the GUS gene. In contrast, the upstream regions of both promoters differ the higher GUS level directed by the AtpD promoter is caused by enhancer-like elements located upstream of the region involved in the regulated expression, while nucleotides upstream of -73 in the AtpC promoter contribute relatively little to the promoter activity. 5'-Deletion analyses and site-directed mutagenesis studies indicated that the -73/-48 bp AtpC region contains cis-elements crucial for this regulated expression. If five nucleotides within this region (-59/-55) are exchanged, the GUS gene is constitutively expressed and the activity in etiolated seedlings, in seedlings with photobleached plastids and in roots increases to the level detectable in green cotyledons. It is concluded that signal transduction pathways from different regulators converge prior to gene regulation and that these five nucleotides are part of a cis-element which functions as a repressor in darkness, in tissues with impaired plastids and in roots.

Competition for nodule occupancy on Phaseolus vulgaris by Rhizobium etli and Rhizobium tropici strains can be efficiently monitored in an ultisol during the early stages of growth using a constitutive GUS gene fusion

The GUS gene fusion (pKW107) was transferred into 4 Rhizobium phaseoli strains (KIM5s, CIAT 895, CIAT 7202, CIAT 151) and 1 Rhizobium tropici strain (CIAT 899) in order to determine the nodulation competitiveness of these strains on common bean ( Phaseolus vulgaris) in soil. DNA was fingerprinted with the different mutants to verify correct integration of the glucuronidase gene in the strains' genomes. Co-inoculation trials in growth pouches proved that the GUS + mutant strains were unaltered in their nodulation capabilities. Plant studies in a non-sterile ultisol examined the value of GUS-marked Rhizobium strains for monitoring nodule occupancy under tropical conditions. At 14 and 21 days after planting (DAP), blue nodule color allowed consistently clear and reliable differentiation between nodules formed by GUS + rhizobia and indigenous soil rhizobia. At 30 DAP, however, expression of the GUS trait was inconsistent. Older nodules formed by the GUS + rhizobia were only a faint green after incubation in detection buffer, probably because of the restriction of gusA gene expression in the construct to free-living rhizobia. Nodules formed by GUS-harboring inocula strains were differentiated from ineffective green nodules formed by native rhizobia in 30 DAP nodules in microplates by crushing nodules before adding detection buffer. Data obtained by use of the GUS gene fusion were confirmed by data derived using the ELISA technique and antibiotic resistance markers. The GUS gene fusion was found to be a powerful tool to examine competitiveness of inocula strains in a tropical soil.

The steady-state mRNA levels for thylakoid proteins exhibit coordinate diurnal regulation.

Steady-state mRNA levels for thylakoid proteins were analysed in spinach cotyledons under diurnally changing light conditions. Most fluctuate considerably throughout the day, while the levels of others show only low amplitude or no oscillation. Levels of mRNAs coding for proteins that belong to the same multiprotein complex generally oscillate in parallel and exhibit maxima that are specific for that complex: mRNAs for photosystem I proteins appear prior to those for photosystem II polypeptides and these again prior to mRNAs for the three polypeptides constituting the oxygen-evolving complex. For the mRNAs that change with high amplitudes (e.g. those for LHCP or the 20 kDa apoprotein of the CP24 complex) oscillations have also been found under constant conditions, indicating that a circadian oscillator is involved. Transgenic tobacco seedlings harbouring chimeric GUS gene fusions with 5'-flanking sequences from the spinach genes Lhcb, PsaF and AtpD (encoding a light-harvesting chlorophyll a/b apoprotein of photosystem II, subunit 3 of photosystem I and subunit delta of the plastid ATP synthase, respectively) confirm that the differences in the amplitudes as well as the timepoints of maximum mRNA accumulation are perceived via cis-regulatory elements upstream of the respective ATG codons.

Involvement of Ca2+ signalling in the sugar‐inducible expression of genes coding for sporamin and β‐amylase of sweet potato

SummaryGenes coding for sporamin and β‐amylase of sweet potato are inducible not only by high levels of metabolizable sugars, such as sucrose, but also by a low concentration of polygalacturonic acid (PGA). Calmodulin inhibitors and EGTA inhibited both the PGA‐inducible and the sucrose‐inducible accumulation of mRNAs for sporamin and β‐amylase in sweet potato. Calmodulin inhibitors, EGTA and La3+, also inhibited the sucrose‐inducible expression, in leaves of transgenic tobacco, of a fusion gene, β‐Amy:GUS, which consists of the promoter of the β‐amylase gene and the coding sequence for β‐glucuronidase. The sucrose‐inducible expression of the β‐Amy:GUS fusion gene was also inhibited by two inhibitors of Ca2+ channels, diltiazem and nicardipine. These results suggest that the sugar‐inducible expression of genes for sporamin and β‐amylase involves, at least in part, Ca2+‐mediated signalling, and that the cytosolic free Ca2+ may mediate cross‐talk between signals related to carbohydrate metabolism and other stimuli. Treatment of coelenterazine‐loaded leaf discs of tobacco expressing a Ca2+‐binding photoprotein, aequorin, with 0.2 M sucrose for 24 h significantly reduced the level of luminescence that could be induced by cold shock, as compared to cold shock‐induced luminescence in coelenterazine‐loaded leaf discs treated with water. Repression of cold shock‐induced luminescence was due to the conversion of holoaequorin to apoaequorin during the treatment with sucrose. Treatment of coelenterazine‐loaded leaf discs with a 0.2 M solution of glucose or fructose, but not of mannitol or sorbitol, also reduced the cold shock‐induced luminescence. It is suggested that non‐synchronous increases in cytosolic level of free Ca2+ occur in leaf discs during treatment with high levels of metabolizable sugars.

Pathogen, salicylic acid and developmental dependent expression of a beta-1,3-glucanase/GUS gene fusion in transgenic tobacco plants.

The 5' flanking region of a gene encoding an acidic beta-1,3-glucanase from Nicotiana tabacum was isolated and characterized. A chimeric gene composed of 1759 bp of the promoter sequence from the PR-2 gene was fused to the beta-glucuronidase (GUS) coding region and used to transform tobacco. Transcriptional activation of the PR-2 promoter was investigated in response to inoculation with tobacco mosaic virus (TMV), after treatment of leaves with salicylic acid (SA), and in specific tissues during the normal development of healthy plants. In TMV-inoculated transgenic plants, GUS activity was induced locally around necrotic viral lesions and systemically in uninoculated leaves. GUS activity was also induced by treatment of leaves with SA. The chimeric gene was expressed in floral organs of healthy plants and in newly germinated seedlings. Analyses of a series of 5' deletions of the glucanase promoter indicated that the cis-acting elements necessary for induction by all these signals are localized in the region between -321 bp and -607 bp upstream of the transcription start site.

The GUS gene fusion system (Escherichia coli beta-D-glucuronidase gene), a useful tool in studies of root colonization by Fusarium oxysporum.

The plant-pathogenic fungus Fusarium oxysporum was successfully transformed with the beta-D-glucuronidase gene from Escherichia coli (gusA) (GUS system) in combination with the gene for nitrate reductase (niaD) as the selectable marker. The frequency of cotransformation, as determined by GUS expression on plates containing medium supplemented with 5-bromo-4-chloro-3-indolyl glucuronide (GUS+), was very high (up to 75%). Southern hybridization analyses of GUS+ transformants revealed that single or multiple copies of the gusA gene were integrated into the genomes. High levels of GUS activity are expressed in some transformants, but activity in F. oxysporum does not appear to be correlated with the copy number of the gusA gene. Since the highest activity was found in a transformant with a single copy, it can be assumed that sequence elements of F. oxysporum integrated upstream of the gene can act as a promoter or enhancer. Expression of the gusA gene was also detected during growth of the fungus in plants, indicating that the GUS system can be used as a sensitive and easy reporter gene assay in F. oxysporum.

Genetic transformation by Agrobacterium tumefaciens in the interspecific hybrid Helianthus annuus × Helianthus tuberosus

A genetic transformation method has been developed in the interspecific hybrid Helianthus annuus × Helianthus tuberosus using Agrobacterium tumefaciens. Leaf explants of a clone with an efficient tissue culture regeneration capacity were inoculated with A. tumefaciens carrying a disarmed Ti plasmid containing the Cauliflower Mosaic Virus (CaMV) 35S- GUS fusion gene with the nopaline sinthase (NOS) neomycin phosphotransferase II ( NPT II) gene. On selection medium containing 25 mg/l of kanamycin, the inoculated leaf explants formed meristematic centers with buds and embryo-like structures that successively developed into putative transformed shoots, when transferred onto medium without growth regulators. Under suitable conditions, three days of cocultivation on medium containing BAP and NAA, the highest transformation frequency was 5.4%. Histochemical staining for the β-glucuronidase ( GUS) activity provided evidence for transformation in different tissues and organs of transgenic plants. Integration of foreign DNA into genomic H. annuus × H. tuberosus DNA was demonstrated by Southern analysis.

Nodulation competitiveness of Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici strains measured by glucuronidase (gus) gene fusion

The nodulation competitiveness of 17 Rhizobium leguminosarum bv. phaseoli and 3 R. tropici strains was analysed in growth pouches, at pH 5.2 and 6.4. All 20 strains were coinoculated with a gus+ strain of R. leguminosarum bv. phaseoli strain KIM5s. The gus+ phenotype, carrying the glucuronidase gene, was used to type nodules directly in the growth pouches. Nodule occupancy ranged from 4% for the least competitive to 96% for the most competitive R. leguminosarum bv. phaseoli strain. The R. tropici strains showed low rates of nodule occupancy at pH 6.4 but their competitiveness improved significantly under acid conditions. CIAT 895 was the only R. leguminosarum bv. phaseoli strain that was less competitive (P<0.05) at the lower pH. The competitiveness of all the other R. leguminosarum bv. phaseoli strains was unaffected by pH. Various physiological and genetic properties of the strains were analysed in search of correlations with nodulation competitiveness. Hybridisation patterns with three different DNA probes (nif KDH, common nod genes, and hup genes) and the metabolism of 53 different C sources were compared. No general correlations were found between hybridisation or growth pattern and competitiveness. The less competitive R. tropici strains had a unique DNA hybridisation pattern and were not able to use shikimate, ferulate, coumarate, or asparagine as C sources. Most of the less competitive R. leguminosarum bv. phaseoli strains could not metabolize either ferulate or coumarate. This might indicate a relationship between nodulation competitiveness and the ability to degrade aromatic compounds.

Histological analysis of the expression of Agrobacterium rhizogenes rolB‐GUS gene fusions in transgenic tobacco

summaryThe gene rolB from the T‐DNA of Agrobacterium rhizogenes is capable of directing differentiation of roots in transformed plant cells. A detailed histological analysis of the expression of this gene in transgenic tobacco plants is presented here. Gene fusions between the promoter of rolB and the GUS reporter gene (encoding E. coliβ‐glucuronidase) were utilized: tissue and cell specific GUS activity as driven by a long (1185 base pairs) and a short (306 bp) version of the rolB promoter was visualized histochemically during different phases of plant development, from embryo to flowering. The extended (1185bp) rolB promoter is under developmental control and is specifically active in the initial cells of all types of meristems and in the vascular system (phloem, phloem parenchyma, xylem parenchyma, pericycle and pericycle‐like cells) of mature organs as well as in the central cylinder of the embryo. In contrast, the 306 bp deletion of the promoter is unable to drive expression in menstematic initials but is still active in the vascular system of the seedling.

Functional analysis of the Sesbania rostrata leghemoglobin glb3 gene 5'-upstream region in transgenic Lotus corniculatus and Nicotiana tabacum plants.

Expression of the Sesbania rostrata leghemoglobin glb3 gene was analyzed in transgenic Lotus corniculatus and tobacco plants harboring chimeric glb3-uidA (gus) gene fusions to identify cis-acting elements involved in nodule-specific gene expression and general transcriptional control. A 1.9-kilobase fragment of the glb3 5'-upstream region was found to direct a high level of nodule-specific beta-glucuronidase (GUS) activity in L. corniculatus, restricted to the Rhizobium-infected cells of the nodules. The same fragment directed a low level of GUS activity in tobacco, restricted primarily to the roots and to phloem cells of the stem and petiole vascular system. A deletion analysis revealed that the region between coordinates -429 and -48 relative to the ATG was sufficient for nodule-specific expression. Replacement of the -161 to -48 region, containing the glb3 CAAT and TATA boxes, with the heterologous truncated promoters delta-p35S and delta-pnos resulted in a loss of nodule specificity and reduction of GUS activity in L. corniculatus but a significant increase in tobacco, primarily in the roots. The same fragment could not direct nodule-specific expression when fused to a heterologous enhancer in cis. This region contains DNA sequences required, but not sufficient, for nodule-specific expression in L. corniculatus that function poorly or may be involved in promoter silencing in tobacco. By fusing further upstream fragments to the delta-p35S and delta-pnos promoters, two positive regulatory regions were delimited between coordinates -1601 and -670, as well as -429 and -162. The former region appears to function as a general enhancer because it significantly increased promoter activity in both orientations in L. corniculatus and tobacco. The latter region could enhance gene expression in both orientations in tobacco, but only in the correct orientation in L. corniculatus. These results show that efficient expression of the S. rostrata glb3 gene in nodules is mediated by an ATG-proximal, tissue-specific element, as well as further 5'-upstream positive elements; that the S. rostrata glb3 promoter is induced in a nodule-specific fashion in the heterologous legume L. corniculatus, suggesting a high degree of conservation of the relevant regulatory signals; and that the S. rostrata lb promoter is not silent in the nonlegume tobacco, but is expressed primarily in the roots.

An improved assay for β-glucuronidase in transformed cells: Methanol almost completely suppresses a putative endogenous β-glucuronidase activity

The bacterial β-glucuronidase (GUS) gene is often introduced into plants as a reporter gene fused to a promoter because of advantages over other reporter genes. However, many plants have non-neglegible levels of endogenous GUS or GUS-like activity which can interfere with the activity originating from the introduced GUS gene, especially if this level is a low one. To eliminate the endogenous GUS activity, we studied the effect of some water-soluble organic solvents on decreasing it and found that addition of methanol at 20% volume to a GUS reaction mixture was very effective, lowering the endogenous activity to 0–15% of the origin in all plant extracts tested. Moreover, methanol at this concentration enhanced by 1.4-fold the activity originating from the introduced GUS gene or GUS purified from E. coli. This method enabled us to determine GUS gene expression more reliably, after introduction of GUS gene fusion into rice protoplasts. In histochemical GUS analysis of transgenic tobacco, this method was also effective for suppressing the endogenous GUS activity.

A non-destructive assay for GUS in the media of plant tissue cultures

β-glucuronidase (GUS) can be assayed in the spent media of plant tissues transformed with some GUS gene fusions (Jefferson, 1988). This approach is based on the presence of GUS in the media of transformed plant tissues expressing the gene and can be used to monitor the progress of transformation without destruction of the tissue under study.