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PubMed1981

Mendelian transmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens.

Insertion of the bacterial transposon Tn7 was used to obtain mutants of an octopine Ti plasmid. Crown gall tumours induced on tobacco by an Agrobacterium tumefaciens strain carrying a particular mutant Ti plasmid (pGV2100) were found to give rise to shoots. These shoots were grown in vitro and one of them (rGV-1) was found to contain the T-DNA specific enzyme lysopine dehydrogenase (LpDH) and to form roots. After transfer to soil, rGV-1 developed into a morphologically and functionally normal tobacco plant. All cells of the regenerant and of vegetatively produced offspring were shown, by cloning of leaf protoplasts, to contain T-DNA and LpDH activity, rGV-1 and vegetatively produced offspring flowered normally. Plantlets obtained from haploid anther cultures were tested for LpDH activity. Forty-one percent of these plantlets were LpDH positive. Moreover, both self-pollination of rGV-1 and crosses between rGV-1 and normal tobacco plants showed that the LpDH character was transmitted both through the pollen and through the eggs of rGV-1 as a single dominant factor with Mendelian segregation ratios typical for monohybrid crosses. By repeated selfing, homozygous plants were obtained which bred true with respect to LpDH. The importance of these findings with respect to the use of Agrobacterium tumefaciens and Ti plasmids for genetic engineering in plants is discussed.

Crosses, Genetic↗
PubMed2006

Review: Genetically modified plants for the promotion of human health.

Plants are attractive biological resources because of their ability to produce a huge variety of chemical compounds, and the familiarity of production in even the most rural settings. Genetic engineering gives plants additional characteristics and value for cultivation and post-harvest. Genetically modified (GM) plants of the "first generation" were conferred with traits beneficial to producers, whereas GM plants in subsequent "generations" are intended to provide beneficial traits for consumers. Golden Rice is a promising example of a GM plant in the second generation, and has overcome a number of obstacles for practical use. Furthermore, consumer-acceptable plants with health-promoting properties that are genetically modified using native genes are being developed. The emerging technology of metabolomics will also support the commercial realization of GM plants by providing comprehensive analyzes of plant biochemical components.

Carotenoids↗
PubMed2004

Controlled glycosylation of therapeutic antibodies in plants.

Recombinant therapeutic monoclonal antibodies (mAb) can be expressed, assembled, and glycosylated in plants. Transgenic plants, producing anti-rabies mAb and anti-colorectal cancer mAb, were obtained from Agrobacterium-mediated transformation. The heavy chain (HC) of anti-rabies mAb was fused to the Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal whereas the HC of anti-colorectal cancer mAb was not fused to the KDEL sequence. Gel release of glycans and detection by high-performance liquid chromatography (HPLC), together with computer assisted analysis and matrix-assisted laser desorption/ionization time-of-flight (MALD-TOF) mass spectrometry, revealed that the plant-derived anti-rabies mAb with KDEL contained mainly oligomannose type N-glycans while the plant-derived anti-colorectal cancer mAb carried mainly biantennary glycans with and without a pentose sugar, that is thought to be xylose. This finding indicates that the KDEL sequence can affect the N-glycosylation processing of antibody in plant cells. The plant-derived mAbs with addition of a KDEL sequence did not contain any of the known antigenic glycan epitopes that are frequently found in other plant glycans or in mammalian-derived mAbs. The altered glycosylation on both plant-derived mAbs did not affect the activities that are required for therapy. These results indicate that plant genetic engineering could provide an effective and inexpensive means to control the glycosylation of therapeutic proteins such as mAbs, by the addition of a KDEL signal as a regulatory element.

Animals↗
PubMed2026

Transforming Curcuma longa leaf waste into cellulose scaffolds.

The constant dearth of transplantable tissues and organs in India required the development of substitute biomaterials for tissue engineering. Plant-based decellularized scaffolds have become attractive options because of their abundance, ethical acceptability, architectural diversity, and lower risks of zoonotic transmission. Curcuma longa leaves were investigated in this study as a possible source of cellulose-based scaffolding for use in biomedical applications. After cuticle removal, an immersion decellularization technique utilizing sodium dodecyl sulphate (SDS) and triton-X-100 was developed to successfully remove cellular and nuclear material while maintaining leaf parenchyma architecture. Histology, DAPI staining, scanning electron microscopy, and a notable decrease in leftover DNA content all demonstrated efficient decellularization. When contrasted with native leaves, the resultant decellularized C. longa leaf scaffolds showed significant increase in porosity, water vapor transmission rate and swelling percent, and significantly lower contact angle with an optimum surface roughness promoting cell adhesion. Mechanical test manifest higher tensile strength with decreased stiffness. Fourier transform infrared spectra of leaf scaffold reveals persistence of different components except cuticle but the intensity of different peaks was decreased. The leaf scaffolds showed superior hemocompatibility and excellent compatibility with Madin-Darby canine kidney cells (MDCK) which is demonstrated by cell attachment and proliferation. MTT assay of seeded scaffold showed significantly higher metabolically active cell. In vivo subcutaneous implantation of decellularized scaffolds showed host tissue incorporation, accumulation of collagen, and neovascularization. C. longa leaf scaffolds can be utilized as cost effective and sustainable biomaterials for soft tissue engineering and regenerative medicine.

Curcuma↗
PubMedDate not supplied

Differential induction of glutathione transferases and glucosyltransferases in wheat, maize and Arabidopsis thaliana by herbicide safeners.

By learning lessons from weed science we have adopted three approaches to make plants more effective in phytoremediation: (1) The application of functional genomics to identify key components involved in the detoxification of, or tolerance to, xenobiotics for use in subsequent genetic engineering/breeding programmes. (2) The rational metabolic engineering of plants through the use of forced evolution of protective enzymes, or alternatively transgenesis of detoxification pathways. (3) The use of chemical treatments which protect plants from herbicide injury. In this paper we examine the regulation of the xenome by herbicide safeners, which are chemicals widely used in crop protection due to their ability to enhance herbicide selectivity in cereals. We demonstrate that these chemicals act to enhance two major groups of phase 2 detoxification enzymes, notably the glutathione transferases and glucosyltransferases, in both cereals and the model plant Arabidopsis thaliana, with the safeners acting in a chemical- and species-specific manner. Our results demonstrate that by choosing the right combination of safener and plant it should be possible to enhance the tolerance of diverse plants to a wide range of xenobiotics including pollutants.

Arabidopsis↗
PubMed1995

Immunotherapy of plant viral diseases.

The stable expression of antibodies in plants is one recent strategy for the unconventional control of plant viruses that is undergoing development. The advantages of this approach are its wide applicability and intrinsic safety; however, to be successful, the 'genetic immunization' of plants requires careful antibody design, efficient expression and targeting to appropriate cell compartments.

Antibodies, Viral↗
PubMed1986

Transformation of the genomic expression of plant cells.

Agrobacterium-induced transformation of plant cells results from integration of T-DNA of the Ti or Ri plasmids into the genome of susceptible plants. Expression of T-DNA genes induces physiological changes in transformed cells which modify normal plant development to produce proliferations characteristic of crown gall and hairy root diseases. Understanding of the molecular basis of the transformation events associated with these examples of naturally occurring genetic engineering of plant cells, has stimulated efforts to construct vectors for transferring specific genes into plants. Vector construction has progressed from the use of wild-type Ti plasmids, giving phenotypically abnormal regenerated plants, to non-oncogenic plasmids. The range of vectors now available should enable useful foreign genes to be inserted into a range of dicotyledons and monocotyledons without impairing normal plant development.

Arginine↗
PubMed1990

Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements.

The CaMV 35S and Ti plasmid mannopine synthetase (mas) promoters are commonly used by plant genetic engineers. To combine their useful properties, we constructed hybrid promoters incorporating elements from both. These promoters were spliced to the beta-glucuronidase reporter gene and introduced into tobacco and tomato plants by Agrobacterium cocultivation. T1 and T2 transgenic plant populations transformed with different constructs were assayed for the marker enzyme. Comparisons were made based on the range of expression levels found for each promoter construct. We found that a hybrid promoter incorporating the mas region from +65 to -301 and the 35S enhancer region from -90 to -941 had new and interesting properties. This promoter, called Mac, expressed gus at a level three to five times that expressed by a double 35S promoter in the leaves, and 10 to 15 times in hypocotyls and roots. The Mac promoter, however, showed only marginal wound inducibility. Five- to seven-fold wound induction required the presence of the region from -301 to -613 of mas. Reiteration of the 35S enhancer region, from -90 to -430, behind the 35S TATA box region or the mas +65 to -301 region had a smaller effect on expression, ranging from equal to twice the level of the single enhancer control.

Cloning, Molecular↗
PubMed2002

Expression and inheritance of nine transgenes in rice.

A total of 66 transgenic rice cell lines were produced by simultaneously transforming rice callus with nine different plasmids/genes. PCR analysis indicated that the co-transformation frequency of each gene was about 70%. All the cell lines carried at least three genes and 11 cell lines carried all nine genes. Thirty-two fertile transgenic plants (R0) were generated from the transgenic cell lines and seeds of 32 transgenic R1 lines and 5 R2 lines were harvested and analyzed for gene inheritance and protein expression. Progeny segregation analysis indicated that the multiple transgenes were integrated into the same locus of the rice genome, resulting in a 3:1 segregation ratio of the transgenes. Expression analysis of all nine transgenes revealed that the transgenes were expressed in all generations (R0, R1, and R2) and about half of the transgenes from each line were expressed. The expression of one transgene appears to have no effect on the expression of another transgene. Among the 66 cell lines, six lines (9.1%) expressed seven or eight transgenes out of the nine transformed genes. All together, our results showed that multiple genes could be delivered into rice cells simultaneously and cell lines expressing multiple genes could be generated. The results and procedures reported here should be useful in designing multi-plasmid transformation experiments such as those required for plant metabolic engineering.

Acetyltransferases↗
PubMed2004

[The application of chloroplast genome transformation in higher plant].

The chloroplast genome transformation of higher plant has become the research hotspot of plant genetic engineering with several advantages over nuclear genetic engineering, and it has become into a powerful approach for both basic and applied research. This paper presents a brief summary for the principle and methods of chloroplast genome transformation in higher plant. The particular emphasis was placed on its application in the basic and applied research. These applications include the studies of rearrangement of Rubisco and the structure, transcription, translation and RNA editing of genes in chloroplast; the producing of antibodies, vaccines, poly-beta-hydroxybutyrate and bio-elastic protein using chloroplast as bioreactor; the generating of transgenic plants with resistance to insect, disease, herbicide and drought; and the decreasing the gene flow of transgenic plants.

Chloroplasts↗
PubMed1996

Biological control of plant root pathogens.

Rhizobacteria introduced to control soil-borne root diseases must establish metabolically active populations that mediate protection either by direct antagonism of pathogens or by stimulation of host plant defenses. Recent interest has focused on the genetic and biochemical basis of disease control and the influence of environmental factors on the expression and activity of biocontrol mechanisms. The cloning and sequencing of genes involved in the production of microbial metabolites playing key roles in plant defense opens new possibilities for improving the performance of biocontrol agents.

Anti-Bacterial Agents↗
PubMed2004

Overexpression of a cyanobacterial phosphoenolpyruvate carboxylase with diminished sensitivity to feedback inhibition in Arabidopsis changes amino acid metabolism.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Synechococcus vulcanus (SvPEPC) is a unique enzyme, being almost insensitive to feedback inhibition at neutral pH. In order to assess its usefulness in metabolic engineering of plants, SvPEPC was expressed in Arabidopsis thaliana (L.) Heynh. under the control of the cauliflower mosaic virus 35S promoter. About one-third of the transformants of the T1 generation showed severe visible phenotypes such as leaf bleaching and were infertile when grown on soil. However, no such phenotype was observed with Arabidopsis transformed with Zea mays L. PEPC (ZmPEPC) for C4 photosynthesis, which is normally sensitive to a feedback inhibitor, L-malate. For the SvPEPC transformants of the T2 generation, which had been derived from fertile T1 transformants, three kinds of phenotype were observed when plants were grown on an agar medium containing sucrose: Type-I plants showed poor growth and a block in true leaf development; Type-II plants produced a few true leaves, which were partially bleached; Type-III plants were apparently normal. In Type-I plants, total PEPC activity was increased about 2-fold over the control plant but there was no such increase in Type-III plants. The phenotypes of Type-I plants were rescued when the sucrose-containing agar medium was supplemented with aromatic amino acids. Measurement of the free amino acid content in whole seedlings of Type-I transformants revealed that the levels of the aromatic amino acids Phe and Tyr were lowered significantly as compared with the control plants. In contrast, the levels of several amino acids of the aspartic and glutamic families, such as Asn, Gln and Arg, were markedly enhanced (4- to 8-fold per plant fresh weight). However, when the medium was supplemented with aromatic amino acids, the levels of Asn, Gln, and Arg decreased to levels slightly higher than those of control plants, accompanied by growth recovery. Taken together, it can be envisaged that SvPEPC is capable of efficiently exerting its activity in the plant cell environment so as to cause imbalance between aromatic and non-aromatic amino acid syntheses. The growth inhibition of Type-I plants was presumed to be primarily due to a decreased availability of phosphoenolpyruvate, one of the precursors for the shikimate pathway for the synthesis of aromatic amino acids and phenylpropanoids. The possible usefulness of SvPEPC as one of the key components for installing the C4-like pathway is proposed.

Amino Acids↗
PubMed2003

Isolation and identification of a super strong plant promoter from cotton leaf curl Multan virus.

The activity of the C1 and the V1 gene promoter of cotton leaf curl Multan virus (CLCuMV) was investigated in transgenic plants with the gus gene as a reporter gene. Quantitative GUS activity analysis of the transgenic plant leaves showed the average activity of the CLCuMV C1 gene promoter was 3- to 5-fold higher than that of the CaMV 35S promoter, with maximal expression being 10-fold higher. CLCuMV V1 gene promoter activity was only about 1/10th that of the CaMV 35S promoter in the absence of trans-activator C2. Histochemical GUS staining of the transgenic plants indicated that the CLCuMV C1 gene promoter was active in leaves, stems, roots and almost all reproductive organs. Functional analysis of promoter 5'-deletion series indicated that promoter activity of a 257 nucleotide fragment (-257 to the transcription initiation site) and a 241 nucleotide fragment (-241 to the transcription initiation site) were 5-fold and 2-fold stronger than that of the full-length CLCuMV C1 promoter respectively. These results demonstrate that the CLCuMV C1 promoter is a super-strong near-constitutive promoter in plants and has great application potential for plant genetic engineering studies.

Base Sequence↗
PubMed2002

In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria.

Interkingdom gene transfer is limited by a combination of physical, biological, and genetic barriers. The results of greenhouse experiments involving transplastomic plants (genetically engineered chloroplast genomes) cocolonized by pathogenic and opportunistic soil bacteria demonstrated that these barriers could be eliminated. The Acinetobacter sp. strain BD413, which is outfitted with homologous sequences to chloroplastic genes, coinfected a transplastomic tobacco plant with Ralstonia solanacearum and was transformed by the plant's transgene (aadA) containing resistance to spectinomycin and streptomycin. However, no transformants were observed when the homologous sequences were omitted from the Acinetobacter sp. strain. Detectable gene transfer from these transgenic plants to bacteria were dependent on gene copy number, bacterial competence, and the presence of homologous sequences. Our data suggest that by selecting plant transgene sequences that are nonhomologous to bacterial sequences, plant biotechnologists could restore the genetic barrier to transgene transfer to bacteria.

Acinetobacter↗
PubMed1990

Altered gene expression in plants due to trans interactions between homologous genes.

We have attempted to explain co-suppression in terms of a hypothesis whereby homologous sequences are able to interact somatically in trans, in a manner influenced by sequence context or location. We have speculated that there might be mechanistic similarities between co-suppression and some other trans interaction and epigenetic phenomena in plants, fungi and animals; and that it might be the sequence context in which a gene lies, or its location in the genome that influences the likelihood that it will participate in these phenomena. We suspect that elucidation of the mechanisms behind these phenomena will play a role in developing a better understanding of the relationship between nuclear architecture and gene expression. This, in turn, will be helpful in understanding the developmental regulatory mechanisms that exert control over somatic trans interactions in plants, and perhaps in understanding some aspects of the basis of cellular differentiation in development.

Acyltransferases↗
PubMed1996

The catalytic cysteine and histidine in the plant acyl-acyl carrier protein thioesterases.

The plant acyl-acyl carrier protein (acyl-ACP) thioesterases (TEs) play an essential role in chain termination during de novo fatty acid synthesis and are of biochemical interest because of their utilities in the genetic engineering of plant seed oils. Biochemical data have shown the possible involvement of an active-site cysteine and a histidine in catalysis, suggesting that these enzymes activate the hydrolysis of the thioester bond using the same basic catalytic machinery as those of proteases and lipases. To identify the cysteine and histidine residues that are critical in catalysis we substituted, in a 12:0 ACP TE (Uc FatB1), a conserved cysteine (Cys-320) to an Ala or a Ser, and three conserved histidines (His-140, His-285, and His-345) to an Ala or an Arg. Each Ala mutation caused a substantial loss of enzyme activity. However, only C320A and H285A completely inactivated the enzyme, indicating that these two residues are essential for catalysis. Considerable activity (>60%) still remained when Cys-320 was converted to a Ser, but this mutant (C320S) displayed a reversed sensitivity toward thiol or serine hydroxyl inhibitors compared with the wild-type enzyme. A pH optimal study demonstrates that while the wild-type enzyme has the highest activity between pH 8.5 and 9.5, the mutant H285A shows a shifted optimum to higher pH and a significant increase of activity around pH 12. This result suggests that Arg-285 (pKa 12) is deprotonated at high pH, thus partially mimicking the role of His-285 for proton abstraction in the wild-type enzyme. We conclude that the Cys-320 of the wild-type enzyme and Ser-320 of the mutant enzyme can attack the thioester bond of the substrate 12:0 ACP, assisted by His-285. Because plant TEs are highly conserved in length and sequence and the residues investigated here are completely conserved in all available TEs, it is reasonable to believe that homologues of Cys-320 and His-285 are present in the active sites of all plant acyl-ACP TEs.

Amino Acid Sequence↗
PubMed2005

Molecular genetic evidence for the role of SGT1 in the intramolecular complementation of Bs2 protein activity in Nicotiana benthamiana.

Pepper plants (Capsicum annuum) containing the Bs2 resistance gene are resistant to strains of Xanthomonas campestris pv vesicatoria (Xcv) expressing the bacterial effector protein AvrBs2. AvrBs2 is delivered directly to the plant cell via the type III protein secretion system (TTSS) of Xcv. Upon recognition of AvrBs2 by plants expressing the Bs2 gene, a signal transduction cascade is activated leading to a bacterial disease resistance response. Here, we describe a novel pathosystem that consists of epitope-tagged Bs2-expressing transgenic Nicotiana benthamiana plants and engineered strains of Pseudomonas syringae pv tabaci that deliver the effector domain of the Xcv AvrBs2 protein via the TTSS of P. syringae. This pathosystem has allowed us to exploit N. benthamiana as a model host plant to use Agrobacterium tumefaciens-mediated transient protein expression in conjunction with virus-induced gene silencing to validate genes and to identify protein interactions required for the expression of plant host resistance. In this study, we demonstrate that two genes, NbSGT1 and NbNPK1, are required for the Bs2/AvrBs2-mediated resistance responses but that NbRAR1 is not. Protein localization studies in these plants indicate that full-length Bs2 is primarily localized in the plant cytoplasm. Three protein domains of Bs2 have been identified: the N terminus, a central nucleotide binding site, and a C-terminal Leu-rich repeat (LRR). Co-immunoprecipitation studies demonstrate that separate epitope-tagged Bs2 domain constructs interact in trans specifically in the plant cell. Co-immunoprecipitation studies also demonstrate that an NbSGT1-dependent intramolecular interaction is required for Bs2 function. Additionally, Bs2 has been shown to associate with SGT1 via the LRR domain of Bs2. These data suggest a role for SGT1 in the proper folding of Bs2 or the formation of a Bs2-SGT1-containing protein complex that is required for the expression of bacterial disease resistance.

Arabidopsis Proteins↗