The ocs element in the soybean GH2/4 promoter is activated by both active and inactive auxin and salicylic acid analogues

The octopine synthase (ocs or ocs-like) element has been previously reported to be responsive to the plant hormones, auxin, salicylic acid, and methyl jasmonate. Using transient assays with carrot protoplasts, we have demonstrated that an ocs element from the soybean auxin-inducible GH2/4 promoter is not only activated by strong auxins (i.e, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, -naphthalene acetic acid) and salicylic acid, but also by weak auxin analogues (-naphthalene acetic acid), inactive auxin analogs (i.e., 2,3-dichlorophenoxyacetic acid, 2,4,6-trichlorophenoxyacetic acid), and inactive salicylic acid analogs (3-hydroxybenzoic acid and 4-hydroxybenzoic acid). Our results indicate that the ocs element in the GH2/4 promoter is not selectively induced by plant hormones and might function similarly to tandem AP-1 sites in some animal glutathione S-transferase (GST) genes. The ocs element, like the AP-1 sites in animal GST promoters, may be induced not only by certain hormones but also by some non-hormonal stress-inducing or electrophilic agents.Abbreviations GST glutathione S-transferase - MUG 4-methyl-umbelliferyl-glucuronide - GUS -glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,3-D 2,3-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid - NAA naphthalene acetic acid - SA salicylic acid - SARE putative salicylic acid-responsive element - BA benzoic acid - UTR untranslated region - nos nopaline synthase - ocs octopine synthase - mas mannopine synthase - ocs element-(–)46 CaMV 35S promoter-GUS reporter gene: the ocs element fused to a minimal –46 cauliflower mosaic virus 35S promoter fused to a GUS reporter gene with a 3 nos untranslated region     

Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants

A genomic clone encoding the potato homolog of the yeast ubiquitin-ribosomal protein fusion gene ubi3 was isolated and characterized. Chimeric genes containing the ubi3 promoter (920 bp of 5 to the ubiquitin start codon) were constructed in which the reporter gene -glucuronidase (GUS) was either fused directly to the promoter, or introduced as a translational fusion to the ubiquitin-coding region. After introduction into the potato by Agrobacterium-mediated transformation, GUS activities were measured in leaves and in tubers of transgenic clones. GUS activity was 5- to 10-fold higher in clones expressing the ubiquitin-GUS translational fusion than in clones containing GUS fused directly to the ubi3 promoter. For both types of constructs, GUS activity was highest in meristematic leaves and declined during leaf expansion, then rose again to near the meristematic levels during senescence. GUS activity in tubers was similar to that in young leaves. In contrast to the native ubi3 genes, the chimeric ubi3-GUS transgenes were not activated in the tuber by wounding.     

Agrobacterium rhizogenes-mediated DNA transfer inPinus halepensis Mill.

Agrobacterium rhizogenes strain LBA9402 was used to transformPinus halepensis embryos, seedlings and shoots. Mature embryos exhibited susceptibility to the agrobacterium as monitored by -glucurortidase (GUS) expression, with more than 85% showing considerable transient GUS expression in the radicle. GUS expression was also observed in cotyledons, but at a lower rate of about 24% of the embryos (1–5 spots/embryo). Stable transformation was evidenced by the regeneration of GUS-expressing roots and calli from infectedP. halepensis seedlings. Inoculum injections into intact seedling hypocotyls induced callus and root formation at the wound sites in 64% of the seedlings. Dipping seedling cuttings in a bacterial suspension resulted in adventitious root formation in 7I% of the seedling cuttings, all of which expressed GUS activity. Adventitious shoots, that were induced on 2.5-year-old seedlings by pruning and spraying with 6-benzylaminopurine, were infected by injecting of bacterial suspension into their basal side. Two months later, adventitious roots and root primordia regenerated in 74% and 40% of 2- and 5-month-old shoots, respectively. Non-transformed shoots, either without or with auxin application, failed to form roots. Polymerase chain reaction and Southern blot analyses confirmed theuidA-transgenic nature of the root and callus, as well as the presence ofrolC androlB genes in roots from infectedP. halepensis seedlings.Abbreviations BA 6-benzylaminopurine - NOS nopaline synthase - PCR polymerise chain reaction - EtOH ethanol - GUS -glucuronidase - NPTII neomycin phosphotransferase II - CaMV cauliflower mosaic virus - X-gluc 5-bromo-4-chloro-3-indolyl -D-glucuronic acid     

Semi-constitutive expression of an Arabidopsis thaliana α-tubulin gene

In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple -tubulin and -tubulin genes. Previous evidence suggested that the TUA2 -tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5-flanking DNA fused to the -glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active.     

Development of a simple particle bombardment device for gene transfer into plant cells

A simple particle bombardment device was designed, constructed and shown to be efficient for the delivery of DNA into plant cells. High levels of transient -glucuronidase expression were observed in alfalfa suspension-cultured cells and embryogenic soybean suspension-cultured cells. Expression of -glucuronidase in alfalfa suspension-cultured cells was used to optimize the bombardment conditions for the device. Transient gene expression in alfalfa was found to be dependent on the state of the target tissue, the size of particles employed, the helium pressure used to accelerate the particles and the distance travel led by the tungsten particles carrying DNA.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MS Murashige & Skoog (1962) medium     

Expression of an auxin-inducible promoter of tobacco in Arabidopsis thaliana

The expression of the auxin-inducible Nt103-1 gene of tobacco was studied in Arabidopsis thaliana. For this purpose we introduced a gene fusion between the promoter of the gene and the -glucuronidase reporter gene (GUS) into Arabidopsis thaliana. The expression and location of GUS activity were studied histochemically in time and after incubation of seedlings on medium containing auxins or other compounds. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and 1-naphthylacetic acid (1-NAA) were able to induce GUS activity in the root tips of transgenic seedlings. The auxin transport inhibitor 2,3,5-triiodobenzoic acid was able to induce GUS activity not only in the root tip, but also in other parts of the root. Induction by the inactive auxin analog 3,5-dichlorophenoxyacetic acid was much weaker. Compounds like glutathione and the heavy metal CuSO₄ were weak inducers. GUS activity observed after induction by glutathione was located in the transition zone. Salicylic acid and compounds increasing the concentration of hydrogen peroxide in the cell were also very well able to induce GUS activity in the roots. The possible involvement of hydrogen peroxide as a second messenger in the pathway leading to the induction of the Nt103-1 promoter is discussed.     

Endophytic bacteria expressing β-glucuronidase cause false positives in transformation of Dioscorea species

Summary False positive transformants obtained during plant transformation experiments on species of the monocotyledonous genus Dioscorea (yam) are described. The false positive results were found to be due to endophytic bacteria which exist within aseptically micropropagated shoot cultures and which express -glucuronidase (GUS). The bacteria were isolated and identified as two species of Curtobacterium. The expression of GUS in these organisms was found to be induced by a variety of glucuronide substrates. The induction of GUS activity in the bacteria can be inhibited by chloramphenicol, tetracycline, ticarcillin and sodium azide. Implications of these results for use of the gus gene in plant transformation work are discussed.Abbreviations DTT Dithiothreitol - EDTA Ethylenediamine tetra-acetic acid - ELISA Enzyme-linked immuno-sorbent assay - GUS -glucuronidase - LB Luria Bertani - MUG 4-Methylumbelliferyl--D-glucuronide - PNPG p-nitrophenyl--D-glucuronide - PVP Polyvinylpyrrolidone - SDS Sodium dodecyl sulphate - TAE Tris-acetate-EDTA buffer - X-Gluc 5-Bromo-4-chloro-3-indolyl--D-glucuronide     

rol genes of Agrobacterium rhizogenes cucumopine strain: sequence,effects and pattern of expression

By sequencing the central region of the cucumopine-type T-DNA of Agrobacterium rhizogenes strain 2659, we identified three open reading frames homologous, to different extents, to ORFs 10, 11 and 12 (rolA, B and C) of the agropine-type (1855) T-DNA. Recombinant Agrobacterium strains encompassing the ORFs of 2659 T-DNA-which we refer to as rol, and -were utilized to infect carrot discs and to obtain transgenic tobacco plants, in order to compare the morphogenetic capabilities to those of the 1855 rol genes. Moreover, a long segment of the 5 non-coding region of rol and rol was fused to the GUS reporter gene and the pattern of expression and the responsiveness to auxin of the constructs was analysed in transgenic tobacco. Differences in the auxin requirement for root induction between the 2659 rol genes and their respective 1855 counterparts were pinpointed. These differences are not due to gene regulation and presumably reflect functional differences in the proteins encoded. Differences were also observed in the pattern of expression of rol in roots of transgenic plants, as compared to rolB. In addition, the pattern of expression of rol-GUS construct in roots was found to be analogous to that observed for a construct driven by two of the five regulatory domains of the rolB promoter.     

Evidence for non-proteinaceous inhibitor(s) of β-glucuronidase in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>L.) leaf and root tissues

The GUS gene of E. coli, encoding -glucuronidase, has been widely used as a reporter gene in plant transformation. However, -glucuronidase activity in transgenic wheat leaf or root tissue is rarely observed or reported. To address this question, we investigated three wheat lines transformed with the GUS reporter gene. We found all three lines expressed GUS mRNA as well as -glucuronidase protein in their leaf and root tissues as detected by RNA gel blot, ELISA, and immunoblot analyses. However, -glucuronidase enzyme activity was only detected in pollen grains from the transgenic plants. Fluorometric and histochemical assays performed in the presence of wheat tissue extracts indicated that wheat leaf and root tissues contain inhibitor(s) of -glucuronidase activity, but pollen grains contain much lower concentrations. Further characterizations indicated that the inhibitor(s) is of low molecular weight (<10 kDa) and is non-proteinaceous.     

Upstream non-coding region which confers polar expression to Ri plasmid root inducing gene rolB

Summary Root differentiation could be elicited on carrot discs by transformation with the agropine Ri plasmid rolB gene cloned in the binary vector Bin19, provided two conditions were met. Firstly, an adequate auxin supply had to be provided. This was achieved by co-inoculation with a strain carrying only the auxin synthetic genes of the T_R-DNA. Most of the resulting roots were then shown to harbour only rolB and no aux genes. Secondly, an extended non-coding region (1200 bp) at the 5 end of rolB had to be included in the construction. A shorter (300 bp) 5 region, including TATA and CCAAT boxes, was not sufficient to trigger root differentiation. Both the extended (B1185) and reduced (B310) 5 regions of rolB were then cloned upstream of the -glucuronidase (GUS) reporter gene and infections carried out both on the apical and on the basal side of carrot discs. Strong expression of GUS, visualized histochemically as an intense blue colouring of transformed cells was observed with B1185-GUS constructions on the apical side of the discs. Only occasionally could coloured cells be observed on the basal side of the discs with B1185-GUS and on both apical and basal sides with B310-GUS constructions. Strong GUS expression was, on the contrary, achieved on cells of both auxin-rich (apical) and auxin-depleted (basal) sides of the discs with the strong constitutive viral promoter, CaMV35S. These results indicate the presence of an upstream regulatory region which confers polar expression to the rolB gene and suggest a role for auxin in its activation.     

The promoter of the Arabidopsis thaliana SUC2 sucrose-H+ symporter gene directs expression of β-glucuronidase to the phloem: Evidence for phloem loading and unloading by SUC2

The Arabidopsis thaliana (L.) Heynh. SUC2 gene encodes a plasma-membrane sucrose-H⁺ symporter. The DNA sequence of the SUC2 promoter has been determined. Using a translational fusion of this promoter to the N-terminus of -glucuronidase (GUS) and the GUS histochemical assay, the tissue specificity of the SUC2 promoter was studied in Arabidopsis plants transformed with this fusion construct. The SUC2 promoter directed expression of GUS activity with high specificity to the phloem of all green tissues of Arabidopsis such as rosette leaves, stems, and sepals. During leaf development the expression of SUC2-GUS activity was first seen in the tips of young rosette leaves. In older leaves and during their concomitant sink/source transition, expression proceeded from the tips to the bases of the leaves, indicating that expression of the SUC2 sucrose-H⁺ symporter is tightly coupled to the source-strength of Arabidopsis leaves. Expression of SUC2-GUS activity was also seen, however, in sink tissues such as roots and developing Arabidopsis pods, suggesting that the product of the SUC2 gene might not only be important for phloem loading, but also for phloem unloading. A possible regulatory effect of carbohydrates (glucose and sucrose) on the activity of the SUC2 promoter was studied and excluded, both in excised leaves and young seedlings of transgenic Arabidopsis plants. The overall pattern of SUC2-GUS expression correlated well with that of the Arabidopsis thaliana AHA3 plasma-membrane H⁺ -ATPase which is also expressed in the phloem and most likely represents the primary pump generating the energy for secondary active transporters such as SUC2.Abbreviations GUS -glucuronidase - MS Murashige & Skocgmedium - X-Gluc 5-bromo-4-chloro-3-indolyl--d-glucuronic acid Accession number for SUC2-promoter sequences: The DNA sequence data reported in this paper will appear in the EMBL, GenBank, and DDBJ nucleotide sequence databases under the accession number X79702 (AtSUC2 promoter sequence)We want to thank Günther Peissig for growing the Arabidopsis thaliana plants. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 43/C5) and a grant to N.S. from the Bundesministerium für Forschung und Technologie.     

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

Rol genes alter hormonal requirements for protoplast growth and modify the expression of an auxin responsive promoter

Summary Growth characteristics of tobacco protoplasts containing rolA linked to its own promoter, or the rolB, or rolC genes of Agrobacterium rhizogenes linked to the Cauliflower Mosaic Virus 35S RNA promoter were compared with those from untransformed plants. RolA protoplasts require auxin and cytokinin for callus formation. Protoplasts overexpressing rolB and C form callus in the absence of exogenously applied auxin and cytokinin, respectively. Long term callus growth requires auxin, but the requirement for cytokinin is not critical. Optimal transient expression of an auxin responsive promoter element occurred at lower external levels of auxin in rolB and rolC protoplasts compared with untransformed protoplasts. Addition of putrescine was required for auxin responsive transient gene expression in rolA protoplasts suggesting that polyamines, or their products affect gene expression in rolA plants.Abbreviations T-DNA transferred DNA - TL-DNA left transferred DNA - NAA naphthalene acetic acid - PEG polyethylene glycol - GUS glucuronidase - CaMV cauliflower mosaic virus     

Leaf developmental stage and tissue location affect the detection of β-glucuronidase in transgenic tobacco plants

Expression in Nicotiana tabaccum L. plants containing the -glucuronidase (GUS) gene under the control of the 35S (CaMV promoter) was affected by tissue type and ontogenic development of the leaves. GUS activity in ontogenetically younger leaves was 1003–1022 nmol 7-hydroxy-4-methylcoumarin (MU) formed mg^–1 (protein) min^–1 and in ontogenetically older leaves was only 140–198 nmol (MU) mg^–1 (protein) min^–1.     

Carrot (Daucus carota) hypocotyl transformation usingAgrobacterium tumefaciens

Summary Daucus carota hypocotyl sections were transformed withAgrobacterium tumefaciens LBA4404 containing CaMV 35S promoter, -glucuronidase coding sequence and the nopaline synthase (Nos) poly adenylation sequences in Bin 19. Sliced sterile seedling hypocotyl segments were preincubated for 2 days, co-cultivated withAgrobacterium for an additional 2 days, and then transferred to medium containing 100ug/ml of kanamycin and 400ug/ml carbenicillin. In 6 weeks kanamycin resistant calli were obtained in 5.8% of the explants from one variety. Calli were subcultured on solid medium, and in 4 weeks introduced into suspension culture. NPTII and Southern blot analysis confirmed that three selected lines were transformed with 1–3 copies of the GUSII construction. GUS activity in transformants was 5 to 250 fold over background.Abbreviations NPT II neomycin phosphotransferase II - Nos nopaline synthase - GUS -glucuronidase - CaMV cauliflower mosaic virus - 2,4-D 2,4 dichlorophenoxyacetic acid - PMSF phenylmethylsulfonyl fluoride     

Developmental,hormonal, and pathogenesis-related regulation of the tobacco class I β-1,3-glucanase B promoter

The class I -1,3-glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis-related regulation. The tobacco enzymes are encoded by a small gene family with members derived from ancestors related to the present-day species Nicotiana sylvestris and N. tomentosiformis. We studied the expression in transgenic tobacco plants of a chimeric -glucuronidase (GUS) reporter gene fused to 1.6 kb of upstream sequence of the tobacco class I -1,3-glucanase B (GLB) gene, which is of N. tomentosiformis origin. Expression of the GUS reporter gene and the accumulation of class I -1,3-glucanase and its mRNA showed very similar patterns of regulation. In young seedlings the reporter gene was expressed in the roots. In mature tobacco plants it was preferentially expressed in lower leaves and roots and was induced in leaves by ethylene treatment and by infection with tobacco mosaic virus (TMV). Furthermore, it was down-regulated in cultured leaf discs by combinations of the hormones auxin and cytokinin. Histological studies of GUS activity showed that the GLB promoter shows highly localized expression in roots of seedlings. It is also expressed in a ring of cells around necrotic lesions induced by TMV infection, but not in cells immediately adjacent to the lesions or in the lesions themselves. The results of deletion analyses suggest that multiple positive and negative elements in the GLB promoter regulate its activity. The region from –1452 to –1193 containing two copies of the heptanucleotide AGCCGCC, which is highly conserved in plant-stress and defense-related genes, is necessary for high level expression in leaves. Additional regions important for organ-specific and regulated expression were: –568 to –402 for ethylene induction of leaves; –402 to –211 for expression in lower leaves and cultured leaf discs and for TMV induction of leaves; and –211 to –60 for expression in roots.     

Transgene Expression in the Vegetative Tissues of Apple Driven by the Vascular-specific rolC and CoYMV Promoters

The ability of the heterologous promoters, rolCP and CoYMVP, to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill.) has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Replicate plants of each transgenic clone were propagated in soil to a uniform size and samples of leaf, petiole, stem, and root were taken for the measurement of -glucuronidase (GUS) activity by fluorometric assay. The levels of expression were compared with those in tissues of a representative clone containing the CaMV 35S promoter. These quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The CoYMV promoter was slightly more active than the rolC promoter, although both expressed gusA at a lower level than the CaMV 35S promoter. In clones containing the rolC promoter with multiple transgene loci, expression values were generally among the highest or lowest in the range. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with a chromogenic substrate. This analysis demonstrated that with both the rolC and CoYMV promoters the reporter gene activity was primarily localised to vascular tissues, particularly the phloem. Our results indicate that both promoters would be suitable to drive the expression of transgenes to combat pests and diseases of apple that are dependent on interaction with the phloem.     

Spatial and temporal expression patterns directed by theAgrobacterium tumefaciens T-DNA gene 5 promoter during somatic embryogenesis in carrot

We have analysed the patterns of expression of a gene encoding -glucuronidase (GUS) fused to the promoter of theAgrobacterium tumefaciens T-DNA gene 5 during embryogenesis in carrot,Daucus carota L. Gene expression was monitored by a histochemical assay of -glucuronidase activity. The gene 5 promoter, although of bacterial origin, conferred expression upon the marker gene in all stages of embryo development. The patterns of expression however, differed between embryos in different stages of development. In the globular stage expression was confined to the basal part of the embryo, suggesting that the promoter is sensitive to regulatory functions active in the primary establishment of polarity in the radially symmetric globular embryo. In the heart and torpedo stages of development GUS expression was high in the entire embryonic axis, but not in the cotyledons. During germination expression was reduced in the elongating hypocotyl and radicle, and high levels of expression were detected only in the shoot and root apices. Among the transformed cell lines analysed, one was found that showed an aberrant pattern of GUS expression during embryogenesis, in that expression in the upper part of the embryo was undetectable, and expression was restricted to the root apex in later stages of development. This difference in organ specificity of expression is likely due to a large deletion of the promoter.     

Assessment of microinjection for introducing DNA into uninuclear microspores of rapeseed

Approximately 2,000 embryogenic uninuclear microspores of rapeseed (Brassica napus) cv. Topas were intranuclearly injected with a chimaeric -glucuronidase (Escherichia coli Uid A) gene. Stable integration had not occurred among 55 plants that were regenerated. Coinjection of the dye Lucifer Yellow and detection of injected DNA by the polymerase chain reaction revealed high frequencies of transfer. However, the amount of DNA injected was less than 20 copies, which may have been insufficient for stable transformation of microspores.Abbreviations PCR polymerase chain reaction - GUS -glucuronidase