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At least 811 records · Page 45Linked to original sources

PubMed1995

Reduction of coproporphyrinogen oxidase level by antisense RNA synthesis leads to deregulated gene expression of plastid proteins and affects the oxidative defense system.

A full-length cDNA sequence encoding coproporphyrinogen oxidase was inserted in inverse orientation behind a CaMV promoter and transferred to tobacco (Nicotiana tabacum) by standard transformation techniques. Transformants showed reduced coproporphyrinogen oxidase activity and accumulation of photosensitive coproporphyrin(ogen), indicating antisense RNA expression. An inverse correlation was observed between the level of coproporphyrinogen oxidase and transformant phenotype. The latter is characterized by a broad range of growth retardation and necrosis, indicating oxidative leaf damage. Coproporphyrinogen is an apparent chromophore and its excitation finally leads to the production of reactive oxygen. Evidence is presented that indicates a direct correlation between the accumulation of non-metabolized coproporphyrinogen and oxidative damage to cellular structural components. Enzymatic and non-enzymatic antioxidants were investigated. Whereas superoxide dismutase activity increased in transgenic plants, catalase and ascorbate peroxidase activity remained constant. Tocopherol, rather than carotene or zeaxanthin, seemed to be involved in detoxification, indicating the putative localization and allocation of coproporphyrinogen. Expression of coproporphyrinogen oxidase antisense RNA did not significantly influence the level of other enzymes in the chlorophyll metabolic pathway, but deregulated gene expression of nuclear encoded plastid proteins. Accumulation of coproporphyrinogen and/or the resulting effects, such as oxidative stress, impairs a plastid/nuclear signal which may adapt gene expression to the plastid state.

Antioxidants↗

PubMed1994

Influence of heterologous tobamovirus movement protein and chimeric-movement protein genes on cell-to-cell and long-distance movement.

Sunn-hemp mosaic tobamovirus (SHMV) moves slowly from cell to cell in Nicotiana tabacum cv. Xanthi, but fails to move long distance. To determine the role of the SHMV movement protein (MP) in cell-to-cell and long-distance movement in tobacco, the SHMV MP gene was inserted into a TMV-cDNA clone that had approximately the 5'-half of the endogenous MP gene deleted. RNA transcripts inoculated onto tobacco induced systemic infections by 8 days postinoculation. Sequence analysis of the MP genes from purified virus isolated from systemically infected leaf tissue indicated that chimeric SHMV/TMV MP genes had been generated through RNA-RNA recombination within the 3'-termini of the MP gene sequences. When exchanged for the MP gene of TMV, three of four chimeric MP genes analyzed provided long-distance movement function for the hybrid viruses in tobacco. Two of the three hybrid viruses that moved long distance showed enhanced cell-to-cell movement relative to a recombinant TMV that expressed the intact SHMV MP gene. These observations suggest that the C-terminus of the TMV MP contains a determinant that can influence cell-to-cell movement in tobacco. A recombinant virus, TLSM, that expressed the intact SHMV MP gene exhibited cell-to-cell movement that was intermediate to SHMV and TMV, but failed to produce coat protein and was defective in long-distance movement. To further examine the role of the SHMV MP gene in long-distance movement, transgenic N. tabacum cv. Xanthi that expressed the wild-type SHMV MP gene were generated and found to facilitate rapid and efficient long-distance movement of a TMV mutant that contained a dysfunctional MP gene. Therefore, the inability of SHMV to systemically infect tobacco is a function of virus components and sequences other than those encoded by the SHMV MP gene.

Base Sequence↗

PubMed1991

'Phytoantibodies': a general vector for the expression of immunoglobulin domains in transgenic plants.

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immunohistochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.

Agrobacterium tumefaciens↗

PubMed1995

The location of untranscribed DNA sequences within ras genes essential for eliciting plant growth suppression.

Three heterologous ras DNA-coding sequences and their deletion derivatives were introduced into plant cells to investigate the role of the ras-coding sequences, especially conserved regions, in eliciting growth inhibition. All three ras-coding sequences caused a similar inhibition of plant cell growth, and it was the conserved coding regions which were responsible for this inhibitory effect. The 493 bp conserved region within the v-Ha-ras-coding sequence was studied further, and was shown to be responsible for the inhibitory effect. This region is conserved (over 44%) among the three ras genes studied and encodes a catalytic region of the Ras protein. Small deletions at either the 5' or 3' end of this 493 bp sequence could abolish or dramatically reduce the inhibitory effect. A 36 bp region at the 5' end of the 493 bp region was found to be highly conserved between v-Ha-ras and eight different plant ras or ras-related genes based upon analysis of published sequences. Small deletions affecting this highly conserved 36 bp region completely abolished the inhibitory effect, while deletion of a similar number of base pairs in adjacent regions did not. These results indicate that plant growth inhibition by ras DNA requires small regions at both ends of the 493 bp conserved region.

Base Sequence↗

PubMed1999

Apple chlorotic leaf spot virus 50 kDa protein is targeted to plasmodesmata and accumulates in sieve elements in transgenic plant leaves.

We investigated the in situ localization of the 50 kDa protein encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) genome which is thought to be a movement protein. In immunogold electron microscopy of ACLSV-infected Chenopodium quinoa leaves, the 50 kDa protein was localized on plasmodesmata and nearby cytoplasm. Observation of transgenic Nicotiana occidentalis leaves expressing the 50 kDa protein fused to enhanced green fluorescent protein (EGFP) by fluorescence and confocal laser scanning microscopes revealed that green fluorescence was observed as spots on the cell wall or strands passing through the cell wall of several cell types, i.e., epidermal, palisade and spongy mesophyll and collenchyma cells. In transverse and longitudinal sections of leaf veins of transgenic plants showed that the 50K-EGFP fusion accumulated in sieve elements and formed an extensive interconnecting network of threadlike structure. These results indicated that ACLSV 50 kDa protein can target plasmodesmata and traffic into sieve elements.

Chenopodiaceae↗

PubMed1997

Improving scFv antibody expression levels in the plant cytosol.

Expression of single-chain antibody fragments (scFvs) in the plant cytosol is often cumbersome. It was unexpectedly shown that addition at the C-terminus of the ER retention signal KDEL resulted in significantly improved expression levels. In this report the cytosolic location of the scFv-CK was confirmed, excluding possible mistranslocation to other subcellular compartments. It was shown that expression of several other scFvs was also improved in tobacco protoplasts. In addition expression was improved in transgenic potato. Changing from KDEL to KDEI did not affect the enhanced protein expression level. Addition of the KDEL motif is a simple and straightforward tool to stabilize in planta cytosolic expression of many scFvs.

Carboxylic Ester Hydrolases↗

PubMed2001

Edited transcripts compete with unedited mRNAs for trans-acting editing factors in higher plant chloroplasts.

Chloroplast RNA transcripts of vascular plants undergo C to U editing at approximately 30 sites, but there is no consensus sequence that identifies a C to be edited. Both sequences closely surrounding an edited C and unidentified site-specific trans-acting factors have been shown to be important for editing. The ability of an already edited transgenic sequence to bind and thus titrate a trans-acting editing factor was evaluated for two editing sites, ndhF and rpoB site 2. The U-containing rpoB transcripts did not affect editing of the endogenous rpoB transcripts, likely because the comparable C-containing transcripts containing 27 nucleotides surrounding the edited C were only 20% edited, indicating a low affinity of a trans-factor for this length of edited sequence. Surprisingly, U-containing ndhF transgene transcripts reduced endogenous ndhF transcript editing to the same degree as a C-containing transgene transcript. This indicates that the C target of editing is not a critical recognition feature for the site-specific trans-acting factor.

Binding Sites↗

PubMed1999

Radiation hazard caused by the Chernobyl accident in inhabited areas of Ukraine can be monitored by transgenic plants.

The genetic impact of the 1986 accident at the Ukraine Chernobyl Nuclear Power Plant (NPP) on populations of living organisms has yet to be fully assessed. Monitoring of the genotoxicity of polluted soils is a key element in the disaster management program. We used Arabidopsis thaliana and Nicotiana tabacum plants transgenic for a reporter gene revealing homologous recombination to study the genetic effects of chronic low-dose radiation stemming from the soil in inhabited areas of Ukraine where contamination by the accident ranges from 1 to 40 Ci/km2. We noted a significant dose-dependent increase of homologous recombination in plants cultivated in the affected inhabited areas, proving the persistently high genotoxicity of the radioactively contaminated soils.

Arabidopsis↗

PubMed2001

Assembly and plasma membrane targeting of recombinant immunoglobulin chains in plants with a murine immunoglobulin transmembrane sequence.

The cDNA encoding a full-length murine immunoglobulin gamma 1 heavy chain with its native leader sequence, transmembrane and intracellular domains was introduced into transgenic plants. Transformed plants expressed the recombinant polypeptide, but, in contrast to plants expressing the heavy chain without transmembrane sequence, the protein appeared to be associated with a plant cell membrane. Extraction of the membrane-associated heavy chain required the presence of a non-ionic detergent, and immunofluorescence studies of protoplasts demonstrated surface expression of membrane Ig heavy chain on up to 40% of the cells from a transgenic leaf. In plants expressing both the membrane Ig heavy chain and its partner light chain, functional antibody was also localised to the plant cell membrane and retention of the heavy chain at this site appeared to have no effect on the efficiency of antibody assembly. This approach of localising and accumulating recombinant antibody in cell membranes may have a number of applications, including passive immunisation against plant pathogens.

Amino Acid Sequence↗

PubMed1995

Simultaneous targeting of pea glutathione reductase and of a bacterial fusion protein to chloroplasts and mitochondria in transgenic tobacco.

N-terminal presequences from cDNAs encoding mitochondrion- or chloroplast-specific proteins are able, with variable efficiencies, to target preproteins to their respective organelles. In the few cases studied in which a nuclear-encoded protein is found in both these organelles, each compartment-specific isoform is encoded by a separate gene. Glutathione reductase (GR) from peas is encoded by a single nuclear gene and yet GR is distributed between chloroplasts, mitochondria and the cytosol. Previous sequence analysis of a full-length GR cDNA revealed the presence of a putative plastid transit peptide. However, expression of this cDNA in transgenic tobacco resulted in substantially elevated GR activities in both chloroplasts and mitochondria in four independent lines examined. There was no effect on expression of the endogenous tobacco GR genes. Replacement of the GR presequence with presequences from pea rbcS (chloroplast) and Nicotiana plumbaginifolia Mn-SOD (mitochondrion) resulted in targeting of GR only into the appropriate organelle. Expression of a fusion protein between the amino terminal region of GR and phosphinothricin acetyl transferase resulted in targeting of the foreign protein to chloroplasts and mitochondria. Thus, the pea GR presequence is capable of co-targeting this enzyme or a foreign protein to chloroplasts and mitochondria in vivo. This is the first example of co-targeting by a higher plant preprotein.

Acetyltransferases↗

PubMed2000

Sense transformation reveals a novel role for class I beta-1, 3-glucanase in tobacco seed germination.

'Coat-enhanced' seed dormancy of many dicotyledonous species, including tobacco, is released during after-ripening. Rupture of the endosperm, which is the limiting step in tobacco seed germination, is preceded by induction of class I beta-1,3-glucanase (betaGLU I) in the micropylar endosperm where the radicle will penetrate. Treating after-ripened tobacco seeds with abscisic acid (ABA) delays endosperm rupture and inhibits betaGLU I induction. Sense transformation with a chimeric ABA-inducible betaGLU I transgene resulted in over-expression of betaGLU I in seeds and promoted endosperm rupture of mature seeds and of ABA-treated after-ripened seeds. Taken together, these results provide direct evidence that betaGLU I contributes to endosperm rupture. Over-expression of betaGLU I during germination also replaced the effects of after-ripening on endosperm rupture. This suggests that regulation of betaGLU I by ABA signalling pathways might have a key role in after-ripening.

Abscisic Acid↗

PubMed2000

Transient expression and heat-stress-induced co-aggregation of endogenous and heterologous small heat-stress proteins in tobacco protoplasts.

Heat-stress granules (HSG) are highly ordered, cytoplasmic chaperone complexes found in all heat-stressed plant cells. We have developed an experimental system involving expression of cytosolic class I and class II small heat-stress proteins (Hsps) of pea, Arabidopsis and tomato in tobacco protoplasts to study the structural prerequisites for the assembly of HSG or HSG-like complexes. Class I and class II small Hsps formed class-specific dodecamers of 210-280 kDa, which, upon heat stress, were incorporated into HSG complexes. Interestingly, class II dodecamers alone could form HSG-like complexes (auto-aggregation), whereas class I dodecamers could do so only in the presence of class II proteins (recruitment). By analysing C-terminal deletion forms of Hsp17 class II, we obtained evidence that the intact C-terminus is critical for the oligomerization state, for the heat-stress-induced auto-aggregation and for recruitment of class I proteins. The class-specific formation of dimers as a prerequisite for oligomerization was analysed by the yeast two-hybrid system. In the presence of the endogenous (tobacco) set of heat-stress-induced proteins, all heterologous class I and class II proteins were incorporated into HSG complexes, whose ultrastructure was different from that of complexes formed by class I and class II proteins alone. Although other, more distantly related, members of the Hsp20 family, i.e. the plastidic pea Hsp21, the Drosophila Hsp23 and the mouse Hsp25, were well expressed in tobacco protoplasts and formed homo-oligomers of 200-700 kDa, none of them could be recruited to HSG complexes.

Amino Acid Sequence↗

PubMed1997

Two mRNAs are transcribed from banana bunchy top virus DNA-1.

We have mapped the mRNA transcripts of banana bunchy top virus (BBTV) DNA-1. Northern hybridization and 3' RACE analysis identified two poly-adenylated RNAs associated with BBTV DNA-1. Previously, one major ORF in the virion sense of DNA-1 had been identified, which encoded a putative replication protein (Rep). An mRNA was identified in BBTV infected bananas that was clearly transcribed from this Rep ORF. Further, a second transcript was identified which mapped to an ORF completely within the Rep ORF. This encoded a putative 5 kDa protein of unknown function. Both these transcripts were also identified in a tobacco plant that had been transformed with Agrobacterium tumefaciens harbouring a binary construct containing the Rep ORF from BBTV DNA-1. This Rep ORF was inserted 3' of a cauliflower mosaic virus 35S promoter and 5' of a vegetable storage protein terminator. The transcripts mapped from these tobacco plants were identical at the 3' end to the transcripts from BBTV infected banana plants. The site of polyadenylation for the Rep ORF was at base 963 immediately 3' of the translational stop codon confirming that the polyadenylation signals for this transcript were all within the ORF. However, the internal ORF had a large untranslated region of 272 bases with its site of polyadenylation at nucleotide 803 and a polyadenylation signal 3' of the translational stop codon. A possible upstream termination signal (A/TTGTAA) was identified and was conserved within BBTV DNA-1 sequences from different international isolates.

Agrobacterium tumefaciens↗

PubMed2000

Post-transcriptional maturation of the S receptor kinase of Brassica correlates with co-expression of the S-locus glycoprotein in the stigmas of two Brassica strains and in transgenic tobacco plants.

The S-locus-encoded S receptor kinase (SRK) is an intrinsic plasma membrane protein that is viewed as the primary stigma determinant of specificity in the self-incompatibility response of Brassica spp. We analyzed two self-compatible mutant strains that express low levels of the S-locus glycoprotein (SLG), a cell wall-localized protein also encoded at the S locus that is coordinately expressed with SRK. We found that mutant stigmas synthesized wild-type levels of SRK transcripts but failed to produce SRK protein at any of the developmental stages analyzed. Furthermore, SRK was shown to form aberrant high-molecular mass aggregates when expressed alone in transgenic tobacco (Nicotiana tabacum) plants. This aggregation was prevented in tobacco plants that co-expressed SRK and SLG, but not in tobacco plants that co-expressed SRK and SLR1, an SLG-related secreted protein not encoded at the S locus. In analyses of protein extracts under reducing and non-reducing conditions, evidence of intermolecular association was obtained only for SLG, a fraction of which formed disulfide-linked oligomers and was membrane associated. The data indicate that, at least in plants carrying the S haplotypes we analyzed, SRK is an inherently unstable protein and that SLG facilitates its accumulation to physiologically relevant levels in Brassica stigmas.

Brassica↗

PubMed1998

Application of the human hepatitis B virus core antigen from transgenic tobacco plants for serological diagnosis.

BACKGROUND AND OBJECTIVES: The aim was to produce HBcAg from plants more cheaply than can be done by other currently available means, and to apply such antigen to immunoassay procedures for pretransfusion testing of donor blood. MATERIALS AND METHODS: Transgenic Nicotiana tabacum cv. SR-1 plants expressing the human hepatitis B virus (HBV) core antigen (HBcAg) gene were generated by Agrobacterium-mediated transformation. The recombinant product, called tHBcAg, can assemble itself into a spherical particle with a diameter of 25 to 30 nm, and can maintain two antigenic determinants of HBcAg, namely HBc/alpha and HBc/beta. Partly purified tHBcAg was used in the hemagglutination-inhibition (HI) test, as routinely used by the Japanese Blood Center, to test a panel of 524 blood units taken from HBV-positive donors. RESULTS: In the HI test, tHBcAg showed serologic properties comparable to that from Escherichia coli, the standard antigen used in the Japanese Blood Center. CONCLUSIONS: Transgenic plants can produce reagents for serologic testing and perhaps even such medical materials as oral vaccines.

Agrobacterium tumefaciens↗

PubMed2000

Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate 5-hydroxylase.

Ferulate 5-hydroxylase (F5H) is a cytochrome P450-dependent monooxygenase that catalyses the hydroxylation of ferulic acid, coniferaldehyde and coniferyl alcohol in the pathways leading to sinapic acid and syringyl lignin biosynthesis. Earlier studies in Arabidopsis have demonstrated that F5H over-expression increases lignin syringyl monomer content and abolishes the tissue-specificity of its deposition. To determine whether this enzyme has a similar regulatory role in plants that undergo secondary growth, we over-expressed the F5H gene in tobacco and poplar. In tobacco, over-expression of F5H under the control of the cauliflower mosaic virus 35S promoter increased lignin syringyl monomer content in petioles, but had no detectable effect on lignification in stems. By contrast, when the cinnamate 4-hydroxylase (C4H) promoter was used to drive F5H expression, there was a significant increase in stem lignin syringyl monomer content. Yields of thioglycolic acid and Klason lignin in C4H-F5H lines were lower than in the wild-type, suggesting that F5H over-expression leads to a reduced deposition or an altered extractability of lignin in the transgenic plants. Histochemical analysis suggested that the novel lignin in C4H-F5H transgenic lines was altered in its content of hydroxycinnamyl aldehydes. Transgenic poplar trees carrying the C4H-F5H transgene also displayed enhanced lignin syringyl monomer content. Taken together, these data show that hydroxylation of guaiacyl-substituted lignin precursors controls lignin monomer composition in woody plants, and that F5H over-expression is a viable metabolic engineering strategy for modifying lignin biosynthesis in forest species.

Arabidopsis↗

PubMed1995

Replicase-mediated resistance to alfalfa mosaic virus.

Tobacco plants transformed with the P1 and P2 replicase genes of alfalfa mosaic virus (AIMV) have been shown to produce functional replicase proteins, permitting their infection with AIMV inocula lacking the genome segments encoding P1 and P2, respectively. To see whether expression of a mutant P2 protein would interfere with the assembly of a functional replicase complex, tobacco plants were transformed with modified P2 genes. When plants were transformed with a P2 gene encoding an N-terminally truncated protein which mimicked the tobacco mosaic virus 54K protein, no resistance was observed with 10 independent lines of transformants. Similarly, when the GDD motif in the full-length P2 protein was changed into VDD, no resistance was observed in 14 transgenic lines. However, when the GDD motif was changed into GGD (5 lines), GVD (15 lines), or DDD (13 lines), 20 to 30% of the transgenic lines showed a high level of resistance to AIMV infection. This resistance was effective to inoculum concentrations of 10 to 25 micrograms/ml of virus and 100 micrograms/ml of viral RNA, causing severe necrosis of control plants. For all transgenic lines, the expression of the transgenes was analyzed at the RNA level. With the GGD, GVD, and DDD mutants, resistance was generally observed in plants with a relatively high expression level. This indicates that the resistance is due to the mutant replicase rather than to an RNA-mediated cosuppression phenomenon.

Alfalfa mosaic virus↗

PubMed2004

The nutritional fortification of cereals.

The low micronutrient content of cereals requires the fortification of food and biofortification of plants. Many laboratories are currently pursuing biofortification using breeding and genetic modification, but progress is challenged by technical hurdles and our understanding of physiological processes. Recent studies have largely been confined to the improvement of levels of iron, zinc, some vitamins and a variety of essential amino acids. Progress has been made in the accumulation of iron, zinc, and vitamins A and E in genetically modified plants. For future success in this area, many more studies will be required on the physiology of ion uptake and on the transport of vitamin precursors.

Animal Feed↗