The Promoter of the Gene for Plastidic Glutamine Synthetase (GS2) fromRice Is Developmentally Regulated and Exhibits Substrate-Induced Expression in Transgenic Tobacco Plants

A 1.3-kb fragment from the 5'-flanking region of the RGS-38gene, which encodes the plastidic glutamine synthetase in Oryzasativa L., was fused to a ß-glucuronidase (GUS) reportergene and introduced into Nicotiana tabacum by Agrobacterium-mediatedtransformation. The promoter directed GUS expression, both inleaves and in roots, and the expression of GUS was regulatedby light. The GUS activity was high in the mature leaves ofthe transgenic tobacco plants, in marked contrast to the activityof the GS1 promoter. The GS2 promoter also responded to externallyapplied ammonia, as is the case for the GS1 promoter. Theseresults suggest that the cis-acting regulatory elements thatcontrol the response to ammonia, a substrate for glutamine synthetase,are located within a 1.3-kb region of the promoter. (Received October 1, 1991; Accepted January 20, 1992)     

Expression and Promoter Activity of Genes for Isozymes of Horseradish Peroxidase

The expression and promoter activity of genes for isozymes ofhorseradish peroxidase, namely, prxCla, prxClb, prxC2 and prxC3,were studied. Organ-specific expression of these genes in horseradishplants was examined by Northern blot analysis. The group ofprxCl genes was expressed mostly in stems, while prxC2 and prxC3were expressed to a greater extent in roots. Hardly any expressionof any of the genes was detected in leaves. In transient-expressionassays with tobacco protoplasts, about 500 bp of the 5'-noncodingregions of each of the genes, ligated to the gene for ß-glucuronidase(GUS), exhibited significant promoter activity. In particular,the fragments extending from the initiation codon of the prxC2gene to –529 bp and –1 kbp supported high levelsof GUS activity, which were 4.4 and 11.4 times respectively,the activity observed under control of the 35S promoter fromcauliflower mosaic virus (CaMV). Conserved enhancer sequencesof human genes were found in the 5'-flanking region of prxC2,and deletion of the regions that contained the enhancer sequencesreduced the GUS activity. High levels of GUS activity were observedin transgenic tobacco plants that contained 1 kbp of the 5'flanking region of prxC2 fused to the GUS gene. GUS activitywas diminished when deletion from the 5' end extended as faras the CAAT box. No significant organ-specific expression ofGUS was observed with any such deletion. (Received April 15, 1992; Accepted September 11, 1992)     

Ethylene-Induced Gene Expression of Osmotin-Like Protein, a Neutral Isoform of Tobacco PR-5, is Mediated by the AGCCGCC eft-Sequence

Osmotin-like protein (OLP) is a neutral isoform in the group5 pathogenesis-related (PR) tobacco proteins. The OLP gene,like the basic PR protein genes, is constitutively expressedin tobacco roots and cultured cells. OLP is not naturally presentin intact healthy leaves, but ethylene treatment induces a highaccumulation there. To study the mechanism of OLP gene expressionas induced by ethylene, we cloned the gene from Nicotiana sylvestris,an ancestor of N. tabacum. Sequence analysis showed that ithas no intron and that its promoter region contains two AGCCGCCsequences that are conserved in most basic PR-protein genes.The function of the AGCCGCC sequences in transgenic tobaccoplants that harbor the wild and mutated OLP promoter::GUS fusiongenes was analyzed. Mutation in the AGCCGCC sequences clearlyinhibited the GUS expression induced by ethylene, indicativethat the AGCCGCC sequence(s) is a DNA element(s) responsiveto ethylene. An EREBP2 protein, isolated as one of the proteinsbinding the AGCCGCC sequence of the tobacco rß-1,3-glucanasegene, also was found to bind to the AGCCGCC sequence(s) of OLPgene. These results suggest that the ethylene-induced expressionof OLP is regulated by trans-acting factor(s) common to basicPR-proteins. (Received November 13, 1995; Accepted January 17, 1996)     

Use of the rice sucrose synthase-1 promoter to direct phloem-specific expression of {beta}-glucuronidase and snowdrop lectin genes in transgenic tobacco plants

The promoter region from the rice sucrose synthase-1 gene (RSs1)was fused with coding sequences for ß-glucuronidase(GUS) and snowdrop (Galanthus nivalis) lectin (GNA). Tobaccoplants were transformed with these chimaenc genes in order todetermine the expression pattern directed by the RSs1 promoter.Histochemical and immunochemical assays demonstrated that theexpression of both GUS and GNA was restricted to phloem tissue,and was not observed in any other tissues. This phloem-specificexpression pattern was consistent in stem, leaf and root, andin different transgenic plants. Chimaeric genes of RSs 1-GUSand RSs1 GNA were stably inherited in T1 plants. In addition,GNA was detected by immunological assay in the honeydew producedby peach potato aphids (Myzus persicae) feeding on RSs1-GNAtransgenic tobacco plants. This provided direct evidence thatGNA was not only expressed in the phloem tissue, but was alsopresent in the phloem sap of transgenic tobacco plants. TheRSs1 promoter can thus be used to direct expression of an insecticidalprotein, such as GNA, in transgenic plants to control phloemsap-feeding insect pests. Key words: Rice sucrose synthase-1 promoter, phloemspecific, transgenic plants, ß-glucuronidase, Galanthus nivalis agglutinin, gene expression     

The Mode of Expression and Promoter Analysis of the arcA Gene, an Auxin-Regulated Gene in Tobacco BY-2 Cells

The arcA, a member of the G protein rß-subunit family,was isolated from tobacco BY-2 cells as an auxin-responsivegene. Characterization of arcA, which should help to elucidatethe function of the gene product in the plant cells, was performedwith emphasis on the mode of expression and the analysis ofits promoter. Accumulation of the arcA message was detectedonly after treatments with auxins and not after treatments withother phytohormones or CdCl₂, implying that responsiveness ofarcA was exclusive to auxin. The putative arcA promoter regionwas fused to a reporter gene for rß-glucuronidase(GUS), and transient expression was analyzed in tobacco BY-2cells. Two series of arcA promoter/GUS chimeric genes were constructed.One consisted of a set of 5' nested deletions of the arcA promoterconnected to the gene for GUS and the other consisted of a varietyof the arcA promoter fragments fused to a minimal promoter-GUSconstruct. The results indicated that the promoter sequencecovering four sets of direct repeats (– 562 to –167)was necessary for the sufficient response of arcA promoter toauxin in BY-2 cells. Moreover, irrespective of auxin treatment,elevated activity of GUS driven by this promoter fragment wasdetected, a result that implies that this region behaves anenhancer in BY-2 cells. (Received September 30, 1995; Accepted March 1, 1996)     

Regulated Expression of a Gene-Fusion Product Derived from the Gene for Phytochrome I from Pisum sativum and the uidA Gene from E. coli in Transgenic Petunia hybrida

The 5'-upstream region (2.4 kb) of the gene for phytochromeI from Pisum sativum (phyl) was fused to the uidA gene fromEscherichia coli that encodes ß-glucuronidase (GUS).The resulting PHY-GUS fusion was introduced into Petunia hybridaand was used as a reporter of the expression of the phyI genewhich was recognized by GUS activity. The PHY-GUS fusion wasexpressed at a relatively high level when transgenic plantswere grown in the dark, while leaves and stems of light-grownplants showed background activity. Flowers of light-grown plantswere shown to have significant levels of GUS activity but rootsdid not have such activity. When light-grown transgenic plantswere transferred to the dark, they expressed the activity atlevels that corresponded to those of dark-grown plants. Lighttreatment prior to growth in darkness revealed red/far-red reversibilityof recovery of the activity. Thus, the 2.4-kb fragment fromthe 5' region of the phyI gene carries the information necessaryfor the light-repressible autoregulation. (Received March 30, 1991; Accepted May 20, 1991)     

Transient Expression of Exogenous DNA in Intact, Viable Wheat Embryos Following Particle Bombardment

Expression of foreign DNA has been detected in intact, germinatingwheat embryos (Triticum aestivum L.) following bombardment withtungsten particles complexed with a reporter gene encoding thebacterial enzyme ß-glucuronidase (‘GUS’:E.C.3.2.1.31). Expression was detected in situ in individualcells and groups of cells, by supplying the germinating embryoswith the chromogenic substrate of the GUS enzyme, ‘X-gluc’.Expression was dependent on the presence of a constitutive plantpromoter, the Cauliflower Mosaic Virus ‘35S’ promoter,fused to the GUS structural coding sequence. The relative simplicityof this technique recommends its future use for the assay ofregulatory elements which control the spatial and temporal specificityof genes expressed during embryo development. Key words: Transient expression, particle bombardment, wheat embryo, Triticum aestivum L.     

Experimentally Synthesized Plant Chimeras 3. Qualitative and Quantitative Characteristics of the Flowers of Interspecific Nicotiana Chimeras

The flowers from a series of interspecific periclinal chimerasbetween Nicotiana glauca Grahm. and N. tabacum Su/su L. werequantitatively and qualitatively compared with the flowers oftheir component species and the sexual hybrid. Results indicatethat the epidermal component of the chimeral flowers has thegreatest influence on flower morphology and that each histogenicarrangement results in a unique flower morphology. When comparedto interspecific hybrids a greater diversity of flower shapes,sizes and colours exists with periclinal chimeras, demonstratingthat the experimental synthesis of chimeras between morphologicallydistinct components can be an important source of new phenotypes. Nicotiana glauca Grahm., Nicotiana tabacum L., tobacco, chimeras, graft chimeras, floral morphology, flowers     

The Promoter of the Gene for Glutamine Synthetase from Rice Shows Organ-Specific and Substrate-Induced Expression in Transgenic Tobacco Plants

A 2-kb fragment from the 5'-flanking region of the RGS-28 gene,which encodes the cytosolic glutamine synthetase in Oryza sativaL., was fused to a ß-glucuronidase (GUS) reportergene and introduced into Nicotiana tabacum by Agrobacterium-mediatedtransformation. The promoter was predominantly active in theleaves of transgenic plants, as it is in authentic rice plants.The promoter also responded to externally applied ammonium ions.It is suggested that the cis-acting regulatory elements responsiblefor the recognition of the leaf as a site of synthesis and ofammonia, a substrate for glutamine synthetase, are located withina 2-kb region of the promoter. (Received October 15, 1990; Accepted January 11, 1991)     

High-efficiency <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation of heat inducible sHSP18.2-GUS in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 μM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42°C heat treatment, and the expressed GUS specific activity was 7–26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed.     

Maternal Effects of mto1 Mutation, That Causes Overaccumulation of Soluble Methionine, on the Expression of a Soybean {beta}Conglycinin Gene Promoter-GUS Fusion in Transgenic Arabidopsis thaliana

ß-Conglycinin, the 7S seed storage protein of soybean(Glycine max [L.] Merr.), is comprised mainly of three subunits,designated , ' and ß. Expression of the gene encodingthe ß subunit is unique because its expression hasbeen shown to be down-regulated by exogenously applied L-methioninein immature soybean cotyledon cultures in vitro. Arabidopsisthaliana strain carrying a mto1-1 mutation overaccumulates solublemethionine. By using this mutant, we analyzed the effects ofmethionine on expression of the ß subunit gene invivo. Reciprocal crosses were made between the mto1-1 mutantand a transgenic A. thaliana strain, designated SNTß3,which carries a ß-glucuronidase (GUS) reporter geneunder the control of the promoter region of the ßsubunit gene. Analysis of GUS activity in F₁ seeds indicatedthat the GUS activity was dramatically repressed when the mto1-1mutant plants were used as female parents. We constructed astrain which carries both the transgene and mto1-1 mutationin the homozygous state. Analyses of the GUS activity in seedsof this double homozygous strain indicated that the GUS activitywas repressed to 2.5% of control by introduction of the mto1-1mutation. These results indicate that the ß subunitgene promoter activity in seeds is down-regulated by maternalgenotype and suggest that soluble methionine, or its mobilemetabolite, is translocated from mother plants to repress ßsubunit gene expression in seeds. ⁵Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan ⁶Present address: Department of Biotechnology, Faculty of Agriculture,The University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan     

Expression of an ABA-induced gene of tomato in transgenic tobacco during periods of water deficit

The gene le25 is an abscisic acid (ABA)-induced gene of tomatowhich is expressed both in wilted vegetative organs and developingseeds. Spatial and temporal expression was analysed in tobaccoplants transformed with a chimeric gene in which 5'-upstreamDNA sequences of le25 were fused to the E. coli uidA gene, whichencodes ß-glucuronidase (GUS). Histochemical stainingrevealed that GUS was expressed in all tissues of vegetativeorgans in response to water deficit. Exogenous ABA induced expressionto a lesser extent, even though ABA content was the same asdroughtstressed leaves, indicating a difference in responseto endogenous ABA compared to exogenous ABA. Water-deficit-inducedGUS expression in floral tissues was examined in pre-anthesisfloral buds from four different stages (I–IV; 11, 16,33, 49 mm bud length, respectively). While non-stressed floralorgans showed no GUS activity except in pollen at stages IIIand IV, GUS activity was water-deficit-induced in sepals ofall stages, petals of stage II, and stigmas of stage II andIII. In seeds, GUS activity was detected in both the embryoand endosperm at 15 d post-anthesis, which coincided with alarge increase in the concentration of ABA in the seed. In transgenicplants, the le25 5'-flanking DNA drove expression of GUS duringwater deficit in two modes: non-tissue-specific expression invegetative organs, and tissue-specific expression in reproductiveorgans. The location of GUS activity indicated that ABA concentrationis elevated throughout the tissues of the leaf during periodsof water deficit. Key words: Tomato, ABA, drought stress, lea gene, water deficit     

Different Expression Patterns of the Promoters of the NgrolB and NgrolC Genes during the Development of Tobacco Genetic Tumors

Hybrids (F1) between Nicotiana glauca and N. langsdorffii areprone to develop tumorous tissues on normaltype F1 tissues,namely, genetic tumors. To investigate the patterns of expressionof Ngrol genes during the development of genetic tumors, weperformed an analysis of transgenic genetic tumors that harboredthe promoters of the NgrolB and NgrolC genes fused to a reportergene for rß-glucuronidase (GUS) using a tumorization-redifferentiationsystem derived from F1 plants in vitro. Histochemical analysis of the expression of NgrolB-GUS in normal-typeF1 transgenic plants revealed GUS activity in meristematic zones,while in NgrolC-GUS transformed plants the activity was detectedmainly in the vascular systems of various organs. Tumorous tissues,which arose spontaneously as a consequence of aging or wereinduced by cutting, showed high levels of GUS expression underthe control of promoters of both the NgrolB and the NgrolC gene.Time course analysis during tumorization that followed cuttingof leaves of normal-type F1 plants showed clearly that NgrolB-GUSwas expressed in all dividing cells in the cut region after3 days. By contrast, the expression of NgrolC-GUS was detectedin organized tissues, such as procambium in teratomatous tumors,7–10 days after cutting treatment. During redifferentiationfrom genetic tumors to normal-type plants, the expression ofGUS under control of both Ngrol promoters decreased and expressionresembled that in normal-type tissues. These results suggestthe possibility that the Ngrol genes might be involved in formationof genetic tumors and, moreover, that the expression of NgrolBmight be linked to mitosis and while that of NgrolC might berelated to differentiation of tissues, such as the vascularsystem, in F1 plants. (Received January 19, 1996; Accepted March 24, 1996)     

Expression of gus in somatic embryo cultures of black spruce after microprojectile bombardment

Immature embryos (stage I) and cotyledonary somatic embryos(stage III) of black spruce [Picea mariana (Mill) B.S.P.] werebombarded with tungsten particles coated with a gene constructcontaining the fusion of gus:: nptll. GUS (ß-glucuronidase)activity was monitored histochemically with X-gluc giving ablue colour where transient gene expression was detected inthe bombarded tissues. A high transient expression of gus wasobserved in stage I embryo cultures 2 d after bombardment (202GUS foci per 300 mg tissue). GUS activity had substantiallydiminished in this material 14 d after bombardment, when grownin liquid LP maintenance medium containing BA (4.4µM),2,4-D (9µM) and 1% sucrose. However, when stage I embryoswere cultured on LP maturation medium containing BA (40 µM),IBA (1 µM), 3.4% sucrose and 0.8% agar, GUS activity after2 d was 335 GUS foci per 300 mg tissue, and the activity wasdetected until 30 d after bombardment. With stage III somaticembryos cultured on LP maintenance medium, 92% showed GUS activity2d after bombardment (16 GUS foci per embryo), and 31 % showedactivity 30 d after bombardment (4 GUS foci per embryo). GUSactivity was still evident in 12% of the embryos (2 GUS fociper embryo) 45 d after bombardment. Key words: Black spruce, gus = E. coli geneuid A encoding ß-glucuronidase, nptll = gene encoding neomycin phos-photransferase, somatic embryos