GUS Fusion Research Articles

Regulation of the expression of prtW::gusA fusions in Erwinia carotovora subsp. carotovora.

Erwinia carotovora subsp. carotovora, a Gram-negative phytopathogenic bacterium, secretes an extracellular metalloprotease, PrtW. Previous results demonstrated that protease activity is necessary for the normal progression of disease symptoms caused by this bacterium. The present study revealed that the prtW gene constitutes an independent transcriptional unit. It is demonstrated that introduction of the prtW(+) plasmid in trans into the prtW(-) mutant restores the protease activity in this strain. Gene fusions to the gusA (beta-glucuronidase) reporter were employed to analyse the transcription of prtW. The transcription of prtW is dependent on many environmental signals. When the bacteria were grown in the presence of potato extract, the expression of the protease gene was markedly higher at the beginning of the exponential phase of growth than that observed when cells were grown in the presence of polygalacturonate (PGA). Analysis of the promoter revealed that an essential regulatory region resided between 371 and 245 bp 5' of the translational start site. As this sequence showed no homology to the KdgR box it may be involved in the binding of an unknown negative regulator protein in E. carotovora subsp. carotovora. The differential responses of prtW expression to potato extract and to PGA appeared to be dependent on the KdgR repressor and the response regulator ExpA. According to the results presented here, it is conceivable that the multiple regulatory network allows flexibility in the expression of the prtW gene during different stages of infection.

The Rhizobium leguminosarum bv. viciae VF39 gamma-aminobutyrate (GABA) aminotransferase gene (gabT) is induced by GABA and highly expressed in bacteroids.

A Rhizobium leguminosarum bv. viciae VF39 gene (gabT) encoding a gamma-aminobutyrate (GABA) aminotransferase was identified, cloned and characterized. This gene is thought to be involved in GABA metabolism via the GABA shunt pathway, a theoretical bypass of the 2-oxoglutarate dehydrogenase complex. Mutants in gabT are still able to grow on GABA as a sole carbon and nitrogen source. 2-oxoglutarate-dependent GABA aminotransferase activity is absent in these mutants, while pyruvate-dependent activity remains unaffected. This indicates that at least two enzymes with different substrate specifities are involved in the GABA metabolism of R. leguminosarum bv. viciae VF39. The gabT promoter was cloned into a newly constructed, stable promoter-probe vector pJP2, suitable for the study of transcriptional GUS fusions in free-living bacteria and during symbiosis. Under free-living conditions the gabT promoter is induced by GABA and repressed by succinate. Transcriptional regulation is mediated by GabR in a repressor-like manner. During symbiosis with the pea host plant gabT is induced and highly expressed in the symbiotic zone. Nodules induced by gabT mutants, however, are still effective in nitrogen fixation.

A wound-inducible tobacco peroxidase gene expresses preferentially in the vascular system.

A tobacco peroxidase gene tpoxN1 was reported to be expressed within 1 h after wounding in leaves [Hiraga et al. (2000a) Plant Cell Physiol. 41: 165]. We describe here further results on the wound-induced tpoxN1 expression. The quick tpoxN1 induction occurred preferentially in stems and petioles, but was negligible in leaf blades even 8 h after wounding. Induced GUS activity was also detected rapidly after wounding in the stem of transgenic tobacco plants carrying the tpoxN1 promoter::GUS fusion gene, localized mainly in the vascular systems where it was maintained this level for 14 d or more. Strong GUS activity was also found in the petiole and veinlet as well as the epidermal tissue in the stem. Treatment of known inducers for wound-responsive genes such as jasmonate, 1-aminocyclopropane-1-carboxylate, spermine, phytohormones and other stress treatments did not enhance wound-induced tpoxN1 gene expression in stems at all, but rather repressed it in some cases. Studies using metabolic inhibitors suggested that phosphorylation and dephosphorylation of proteins together with de novo protein synthesis are likely to be involved in the wound-induced tpoxN1 expression as well as some other wound-responsive genes. Thus, tpoxN1 is a unique wound-inducible and possible wound-healing gene which is rapidly expressed being maintained for a long time in veins via an unknown wound-signaling pathway(s).

A Mutation in the Arabidopsis KT2/KUP2 Potassium Transporter Gene Affects Shoot Cell Expansion

Potassium ions (K(+)) are the most abundant cations in plants and are necessary for cell growth. Arabidopsis shy3-1 mutant plants have a short hypocotyl, small leaves, and a short flowering stem, and these defects result from decreased cell expansion. The semidominant shy3-1 mutation changes an amino acid in KT2/KUP2, a K(+) transporter related to the Escherichia coli Kup protein. Second mutations in the KT2/KUP2/SHY3 gene, including presumed null mutations, suppress the shy3-1 phenotypes. Plants with these intragenic suppressor mutations appear similar to wild-type plants, suggesting that KT2/KUP2/SHY3 acts redundantly with other genes. Expression of the shy3-1 mutant version of KT2/KUP2/SHY3 in wild-type plants confers shy3-1-like phenotypes, indicating that shy3-1 probably either causes a gain of function or creates an interfering protein. The shy3-1 mutation does not eliminate the ability of the KT2/KUP2 cDNA to rescue the growth of a potassium transport-deficient E. coli mutant. A P(SHY3)::GUS fusion is expressed in growing portions of the plant. These results suggest that KT2/KUP2/SHY3 mediates K(+)-dependent cell expansion in growing tissues.

Divergent regulation of the HEMA gene family encoding glutamyl-tRNA reductase in Arabidopsis thaliana: expression of HEMA2 is regulated by sugars, but is independent of light and plastid signalling.

The synthesis of 5-aminolevulinic acid (ALA) is a key regulatory step for the production of hemes and chlorophyll via the tetrapyrrole synthesis pathway. The first enzyme committed to ALA synthesis is glutamyl-tRNA reductase encoded in Arabidopsis by a small family of nuclear-encoded HEMA genes. To better understand the regulation of the tetrapyrrole synthesis pathway we have made a detailed study of HEMA2 expression with transgenic Arabidopsis thaliana L. Col. plants carrying chimeric HEMA2 promoter:gusA fusion constructs. Our results show that the HEMA2 promoter directs expression predominantly to roots and flowers, but that HEMA2 is also expressed at low levels in photosynthetic tissues. Deletion analysis of the HEMA2 promoter indicates that a ca. 850 bp fragment immediately upstream of the HEMA2 coding region is sufficient to drive regulated gusA expression. In contrast to HEMA1, HEMA2 is not up-regulated by red, far-red, blue, UV or white light. In addition, elimination of a promotive plastid signal by Norflurazon-induced photobleaching of plastids had no effect on HEMA2 expression while being required for normal white-light induction of HEMA1. HEMA2 expression in the cotyledons is inhibited by the presence of sucrose or glucose, but not fructose, and this response is light-independent. HEMA1 expression in cotyledons is also inhibited by sugars, but in a strictly light-dependent manner. The roles of HEMA1 and HEMA2 in meeting cellular tetrapyrrole requirements are discussed.

T-DNA tagging in the model legume Medicago truncatula allows efficient gene discovery

The annual legume Medicago truncatula has been proposed as a model plant to study various aspects of legume biology including rhizobial and mycorrhizal symbiosis because it is well suited for the genetic analysis of these processes . To facilitate the characterization of M. truncatula genes participating in various developmental processes we have initiated an insertion mutagenesis program in this plant using three different T-DNAs as tags. To investigate which type of vector is the most suitable for mutagenesis we compared the behavior of these T-DNAs. One T-DNA vector was a derivative of pBin19 and plant selection was based on kanamycin resistance. The two other vectors carried T-DNA conferring Basta resistance in the transgenic plants. For each T-DNA type, we determined the copy number in the transgenic lines, the structure of the T-DNA loci and the sequences of the integration sites. The T-DNA derived from pBin19 generated complex T-DNA insertion patterns. The two others generally gave single copy T-DNA inserts that could result in gene fusions for the pGKB5 T-DNA. Analysis of the T-DNA borders revealed that several M. truncatula genes were tagged in these transgenic lines and in vivo gus fusions were also obtained. These results demonstrate that T-DNA tagging can efficiently be used in M. truncatula for gene discovery.

Subcellular localization of MURA and MURB proteins encoded by the maize MuDR transposon.

MuDR controls transposition of the Mu transposable element family in Zea mays L. It produces two major transcripts: mudrA and mudrB, mudrA encodes the MURA transposase, but no specific function has been ascribed to mudrB, which lacks strong homology to known genes. Using transient expression assays in onion epidermal cells, we defined three monopartite nuclear localization signals (NLSs) of MURA; each was functionally sufficient for nuclear targeting of MURA:GUS fusion proteins. Interestingly, one NLS (NLS-A3) is produced by the splicing of the third intron. In contrast, there were no clear NLS in MURB, and the major form of MURB aggregated in the cytoplasm. Self-interaction of MURA and of MURB was also shown in a yeast two-hybrid assay. To test whether interactions of MURA and MURB can occur at the level of protein translocation into the nucleus, a cytoplasmically localized MURB:GFP was co-expressed with MURA or with the GUS fusion proteins. Co-expression did not change the localization pattern of either MURA or MURB; MURA and MURB do not detectably interact in a yeast two-hybrid assay. These results suggest that MURA and MURB do not mutually affect their localization, at least in the forms examined here.

T-DNA tagging of the translation initiation factor eIF-4A1 of Arabidopsis thaliana

A transgenic Arabidopsis thaliana line, generated using a T-DNA vector carrying a promoterless gus reporter gene, showed intense GUS expression in young leaves and rapidly growing stem tissues. The gus fusion has tagged the 3′ region of the gene encoding the A. thaliana eukaryotic translation initiation factor eIF-4A1. Comparison of the genomic and cDNA sequence shows that the eIF-4A1 gene contains four introns. Three introns interrupt the translated region, whereas the largest intron splits the 5′-untranslated region. In plants homozygous for the T-DNA markers, the eIF-4A1 and gus genes are expressed as different mature mRNAs. The gus gene is possibly expressed from a cryptic promoter downstream the eIF-4A1 gene.

Selection of several classes of mimosine-degradation-defective Tn3Hogus-insertion mutants of Rhizobium sp. strain TAL1145 on the basis of mimosine-inducible GUS activity

Rhizobium sp. strain TAL1145 that nodulates Leucaena leucocephala degrades mimosine, a toxin produced by this tree legume. A cosmid clone, pUHR263, containing approximately 25 kb cloned DNA was isolated by plating Escherichia coli cells containing the cosmid clone library of TAL1145 on a minimal medium in which 3-hydroxy-4-pyridone (HP), a degradation product of mimosine, was used as the source of nitrogen. Cosmid pUHR263 was mutagenized by random insertions of Tn3Hogus, a transposon that makes transcriptional gus fusions when it is inserted in a gene in the correct orientation. Various pUHR263::Tn3Hogus derivatives that showed mimosine-inducible or mimosine-repressible GUS activities when transferred to the Rhizobium sp. strain TAL1145 were selected. Mutants of TAL1145 were constructed by transferring these Tn3Hogus insertions into the TAL1145 chromosome through double-homologous recombination. These mutants were classified into five classes on the basis of defects in mimosine degradation. The growth of these mutants was inhibited to different extents by mimosine applied to the growth medium. Mimosine forms a red-colored Fe-mimosine complex when FeCI3 is added to the medium. The inhibitory effect of Fe-mimosine on growth of the mutants was much less than that of mimosine.

The 5' untranslated region of the At-P5R gene is involved in both transcriptional and post-transcriptional regulation.

The steady-state level of transcripts coding for the pyrroline-5-carboxylate reductase of Arabidopsis (At-P5R) increased under salt and heat stress, mainly because of an enhanced mRNA stability. However, the At-P5R protein level was not induced, and its translation was inhibited at initiation stage and probably also at later stages. Replacement of the 5' untranslated region (5'UTR) and beta-glucuronidase (gus) fusion analysis revealed that the first 92 bp region of the At-P5R 5'UTR was sufficient to mediate transcript stabilization and translation inhibition during salt and heat stresses. Furthermore, the first 92 bp region of the At-P5R 5'UTR was also involved in transcription efficiency in a promoter-dependent manner. The results demonstrated that the stress regulation of At-P5R is complex and involves the 5'UTR which acts at three levels, partly in opposing directions.

Regulation of HEMA1 expression by phytochrome and a plastid signal during de‐etiolation in Arabidopsis thaliana

The synthesis of 5-aminolevulinic acid (ALA) is the rate-limiting step for the formation of all plant tetrapyrroles, including chlorophyll and heme, and regulation of ALA synthesis is therefore critical to plant development. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of this pathway and is encoded by a small family of nuclear HEMA genes. Here, we have used transgenic Arabidopsis (Arabidopsis thaliana L. Col) lines expressing chimeric HEMA1 promoter:gusA fusion genes, combined with RNA gel blot analyses, to characterise the light-mediated regulation of the Arabidopsis HEMA1 gene during de-etiolation. HEMA1 was expressed strongly, but not exclusively, in photosynthetic tissues and was shown to be light regulated at the transcriptional level by the phytochrome family of photoreceptors acting in both the far-red high irradiance and low fluence response modes. The HEMA2 gene, which is expressed only in roots of seedlings, was not light regulated. Analysis of truncated HEMA1 promoter constructs demonstrated that a -199/+252 promoter fragment was sufficient to confer full light-responsiveness to gusA expression. This fragment contained GT-1/I-box and CCA-1 binding sites that are implicated as the light-responsive cis elements. Both the full-length and truncated HEMA1 promoters required the presence of intact chloroplasts for full expression, consistent with previous indications that light and plastid factor signals converge to co-ordinately regulate expression of photosynthesis-related nuclear genes. These results provide the most comprehensive analysis to date of the light-regulation of a tetrapyrrole biosynthetic gene and support a direct link between regulation of HEMA1 transcription and chlorophyll accumulation during seedling de-etiolation.

High-yield expression of a viral peptide vaccine in transgenic plants

A high-yield production of a peptide vaccine in transgenic plants is described here. A 21-mer peptide, which confers protection to dogs against challenge with virulent canine parvovirus, has been expressed in transgenic plants as an amino-terminal translational fusion with the GUS gene. Transformants were selected on the basis of their GUS activities, showing expression levels of the recombinant protein up to 3% of the total leaf soluble protein, a production yield comparable to that obtained with the same epitope expressed by chimeric plant viruses. The immunogenicity of the plant-derived peptide was demonstrated in mice immunized either intraperitoneally or orally with transgenic plant extracts, providing the suitability of the GUS fusions approach for low-cost production of peptide vaccines.

Sugar-responsible elements in the promoter of a gene for beta-amylase of sweet potato.

Expression of genes coding for sporamin and beta-amylase, the two most abundant proteins in storage roots of sweet potato, is coordinately inducible in atypical vegetative tissues by sugars. A sweet potato gene for beta-amylase (beta-Amy) with introns as well as a beta-Amy::GUS fusion gene composed of the beta-Amy promoter and the GUS coding sequence, both showed sugar-inducible expression in leaves of transgenic tobacco which occurred via a hexokinase-independent pathway. Analyses using various 5'-terminal and internal deletions of the beta-Amy promoter indicated that truncated promoters of beta-Amy containing a sequence between -901 and -820, relative to the transcription start site, and the basic promoter region can confer sugar-inducible expression. This 82 bp region contained the TGGACGG sequence that plays an essential role in the sugar-inducible expression of the truncated promoter of the sporamin gene. Deletion or base substitutions of this element in the truncated beta-Amy promoter abolished the sugar-inducible expression, the results suggesting that the TGGACGG element plays an important role in the coordinate induction of expression of genes for beta-amylase and sporamin by sugars.

Gibbestatin B inhibits the GA-induced expression of α-amylase expression in cereal seeds

The expression of α-amylase in aleurone layers of barley is known to be induced by gibberellin A3 (GA). In the present study, gibbestatin B (GNB) was isolated from Streptomyces sp. C-39 as an inhibitor of the GA-induced expression of α-amylase in barley and rice, with IC 50 values of 125 and 70 μM, respectively. GNB suppressed accumulation of GA-induced barley high-p I type B and rice RAmy1A α-amylase transcripts. However, GNB showed no inhibitory activity on GUS expression in transgenic tobacco harboring the auxin-inducible par B promoter:: GUS fusion gene. The transcription of an abscisic acid (ABA)-inducible gene, HVA1, was unaffected by GNB. In addition, GNB prevented aleurone cells from cell death induced by GA. In tobacco and Arabidopsis plants, GNB suppressed the germination and retarded the growth of seedlings without toxicity. The growth of gai, spy and abi mutants was also retarded by GNB. Normal plants treated with GA-biosynthesis inhibitors and GA-defective and GA-signaling mutants normally have dwarf dark green leaves. However, dwarfed healthy green leaves were observed in normal plants treated with GNB. GA-induced stem elongation of plants was also detected in the presence of GNB. These analyses indicate that GNB inhibits the GA-induced expression of α-amylase by regulating one of the steps involved in ABA signaling, but not by acting as a weak ABA analog.

Accumulation and nuclear targeting of BnC24, a Brassica napus ribosomal protein corresponding to a mRNA accumulating in response to cold treatment

Previously, we described two cDNA clones corresponding to a new family of cold regulated genes, pBnC24a and pBnC24b homologous to the human BBC1 (Breast Basic Conserved) gene. In order to further analyze the function and regulation of these two genes we have prepared antibodies against a recombinant fusion protein, MBP–BnC24A and used them to study expression at the protein level. In contrast to the increased mRNA accumulation induced by cold, immuno-blot analysis showed that the quantity of the BnC24 protein was not correlated with the accumulation of BnC24 transcripts and is identical in both control and low temperature treated (4°C) plants, suggesting a translational or post-translational regulation. This was confirmed by overexpressing BnC24 in transgenic Arabidopsis thaliana plants. Despite a substantial increase in mRNA, the BnC24 protein level is unchanged. In addition, we demonstrated by subcellular fractionation and immunodetection that a significant fraction of BnC24 protein is located in the nucleus. Using a GUS fusion construction and biolistic experiments it was found that a portion of the amino terminal region is sufficient to target this protein to the nucleus. These results are consistent with the recent finding that BBC1 and its homologues code for the ribosomal large subunit protein L13. In addition they illustrate the difficulty of correlating accumulation of a given mRNA in response to a given stimulus with a biologically significant role in the adaptation to a new environment.

Possible involvement of protein phosphorylation in the wound-responsive expression of Arabidopsis plastid ω-3 fatty acid desaturase gene

The plastid ω-3 fatty acid desaturase (FAD7) catalyzes the conversion of linoleic acid to linolenic acid. Wounding enhances the expression of the FAD7 gene in leaves and induces its expression in stems and roots. The wound-induced expression of the FAD7 promoter was investigated in transgenic tobacco plants carrying the −825 Arabidopsis FAD7 promoter::β-glucuronidase ( GUS) fusion gene. The protein kinase inhibitor, staurosporine, and the protein phosphatase inhibitor, calyculin A, suppressed the wound induction of the FAD7 gene in stems. A tobacco mitogen-activated protein kinase (WIPK) was rapidly activated upon wounding not only in leaves but also in stems and roots, indicating that WIPK probably mediates the wound signals in most vegetative organs. The FAD7 promoter:: GUS fusion gene was introduced into the transgenic tobacco plants in which the wipk gene was expressed constitutively at a high level or into the transgenic plants in which the wipk gene was suppressed possibly due to the transgene-induced gene silencing. The wound-induced expression of the FAD7 gene in stems was enhanced in the former transgenic tobacco plants and suppressed in the latter plants. These results suggest that the wound activation of the FAD7 promoter depends on both protein phosphorylation and dephosphorylation events especially in stems, and also that WIPK is involved in such signaling cascades.

The essential Mcm7 protein PROLIFERA is localized to the nucleus of dividing cells during the G(1) phase and is required maternally for early Arabidopsis development.

PROLIFERA (PRL) encodes a homologue of the DNA replication licensing factor Mcm7, a highly conserved protein found in all eukaryotes. Insertions in the PROLIFERA gene are lethal, resulting in decreased transmission through the female gametophyte, and homozygous embryonic lethality. We show here that PROLIFERA is specifically expressed in populations of dividing cells in sporophytic tissues of the plant body, such as the palisade layer of the leaf and founder cells of initiating flower primordia. Gene fusions with the green fluorescent protein (GFP) reveal that the PROLIFERA protein accumulates during the G(1) phase of the cell cycle, and is transiently localized to the nucleus. During mitosis, the fusion protein rapidly disappears, returning to daughter nuclei during G(1). PROLIFERA::GUS fusions are strongly expressed in the central cell nucleus of mature megagametophytes, which have a variety of arrest points reflecting a leaky lethality. Expression is also observed in the endosperm of mutant prl embryo sacs that arrest following fertilization. Crosses with wild-type pollen result in occasional embryonic lethals that also stain for GUS activity. In contrast, embryos resulting from crosses of wild-type carpels with PRL::GUS pollen do not stain and are phenotypically normal. In situ hybridization of GUS fusion RNA indicates transcription is equivalent from maternally and paternally derived alleles, so that accumulation of maternally derived gametophytic protein is likely to be responsible for the 'maternal' effect.

Analysis of the Rhizobium leguminosarum siderophore-uptake gene fhuA: differential expression in free-living bacteria and nitrogen-fixing bacteroids and distribution of an fhuA pseudogene in different strains.

A mutation was isolated in the Rhizobium leguminosarum gene fhuA, which appears to specify the outer-membrane receptor for the siderophore vicibactin. The mutant was defective in iron uptake and accumulated the siderophore vicibactin in the extracellular medium. Expression of fhuA was regulated by Fe3+, transcription being higher in iron-depleted cells. Transcription of fhuA was independent of a functional copy of rpol, a neighbouring gene that specifies a putative ECF sigma factor of RNA polymerase and which is involved in siderophore production in Rhizobium. Mutations in fhuA did not detectably affect symbiotic N2 fixation on peas. An fhuA::gus fusion was expressed by bacteria in the meristematic zone of pea nodules but not in mature bacteroids. Some other strains of R. leguminosarum also contain a pseudogene version of fhuA. The sequences of some of these and the 'real' fhuA genes were determined.

Matrix polysaccharide precursors in Arabidopsis cell walls are synthesized by alternate pathways with organ-specific expression patterns.

The expression pattern of the single-copy gene UDP-glucose dehydrogenase (Ugd) was analysed in transgenic Arabidopsis plants by promoter:GUS and GFP fusions, Western blots, activity assays and histochemical activity staining. The enzyme oxidizes UDP-glucose to UDP-glucuronic acid and thus directs carbohydrates irreversibly into a cell wall-specific pool of nucleotide sugars. UDP-glucuronic acid is the central intermediate in the interconversion pathway to other nucleotide sugars, including the UDP-derivatives of arabinose, xylose, apiose and galacturonic acid which account for half the biomass of a typical Arabidopsis leaf cell wall. These activated sugars are needed as substrates for the biosynthesis of matrix polysaccharide polymers. In plants up to 5 days old the Ugd gene is strongly expressed in young roots, but very little in hypocotyls. Older plants show a more uniform expression pattern with a preference for the vascular system. A complex expression pattern was observed in flowers with high activity in the stamen, stigma and nectaries. Meristems in the leaf axil of rosette and inflorescence leaves exhibit a high level of activity of the Ugd gene. Although many of the growing tissues show high activity levels of the Ugd gene, others such as the hypocotyl and the cotyledons of young seedlings do not. Instead these tissues efficiently incorporate 3H-inositol into their cell walls. This indicates the biosynthesis of UDP-glucuronic acid through an alternative pathway via the oxidation of inositol to glucuronic acid and subsequent activation to the nucleotide sugar. The data strongly suggest two alternative pathways for matrix polysaccharide precursors with spatial and developmental regulation.

Intron-mediated enhancement of gene expression independent of unique intron sequences and splicing.

Either of the first two introns of the Arabidopsis tryptophan pathway gene PAT1 elevates mRNA accumulation from a PAT1:beta-glucuronidase (GUS) fusion roughly 5-fold without affecting the rate of PAT1:GUS transcription. To further explore the mechanism of this intron-mediated enhancement of gene expression, we wanted to determine whether splicing or specific intron sequences were necessary. In-frame derivatives of PAT1 intron 1, whose splicing was prevented by a point mutation or large deletions, were able to increase mRNA accumulation from a PAT1:GUS fusion, demonstrating that splicing per se is not required. Furthermore, each of a series of introns containing overlapping deletions that together span PAT1 intron 1 increased PAT1:GUS mRNA accumulation as much as the full-length intron did, indicating that all intron sequences are individually dispensable for this phenomenon. These results eliminate the simple idea that this intron stimulates mRNA accumulation via a unique RNA-stabilizing sequence or through the completed act of splicing. However, they are consistent with a possible role for redundant intron sequence elements or an association of the pre-mRNA with the spliceosome.