Christine A. Newell

Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids

The range of sap-sucking insect pests to which GNA, (the mannose specific lectin from snowdrops (Galanthus nivalis) has been shown to be insecticidal in artificial diets has been extended to include the peach potato aphid (Myzus persicae). A gene construct for constitutive expression of GNA from the CaMV35S gene promoter has been introduced into tobacco plants. A transgenic tobacco line which expresses high levels of GNA has been shown to have enhanced resistance toM. persicae in leaf disc and whole plant bioassays,demonstrating the potential for extending transgenic plant technology to the control of sap-sucking insect pests.

Transgenic potato plants with enhanced resistance to the peach-potato aphid Myzus persicae

Potato plants (Solanum tuberosum) cv. Desireé were transformed with the genes encoding the proteins bean chitinase (BCH), snowdrop lectin (GNA) and wheat α‐amylase inhibitor (WAI) under the control of the constitutive CaMV 35S promoter. Transgenic plants with detectable levels of foreign RNA were then selected for further characterisation with respect to protein expression levels by immunodot blot analysis using polyclonal antibodies raised against the respective protein. With the exception of WAI, plants expressing high levels of RNA, expressed correspondingly high levels of the foreign protein (1.5–2.0% of the total soluble protein). Although high levels of WAI mRNA were detected in some of the transformants, the protein could not be detected. On the bases of expression levels, two lines, designated PWG6#85 (transformed with the double construct WAI/GNA) and PBG6#47 (transformed with the double construct BCH/GNA), were selected for testing in aphid trials for enhanced levels of resistance.

The ancestral symbiont sensor kinase CSK links photosynthesis with gene expression in chloroplasts.

We describe a novel, typically prokaryotic, sensor kinase in chloroplasts of green plants. The gene for this chloroplast sensor kinase (CSK) is found in cyanobacteria, prokaryotes from which chloroplasts evolved. The CSK gene has moved, during evolution, from the ancestral chloroplast to the nuclear genomes of eukaryotic algae and green plants. The CSK protein is now synthesised in the cytosol of photosynthetic eukaryotes and imported into their chloroplasts as a protein precursor. In the model higher plant Arabidopsis thaliana, CSK is autophosphorylated and required for control of transcription of chloroplast genes by the redox state of an electron carrier connecting photosystems I and II. CSK therefore provides a redox regulatory mechanism that couples photosynthesis to gene expression. This mechanism is inherited directly from the cyanobacterial ancestor of chloroplasts, is intrinsic to chloroplasts, and is targeted to chloroplast genes.

Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleracea; effects of plant protease inhibitors in vitro and in vivo.

Three distinct digestive protease activities, with strongly alkaline pH optima, were identified in the gut of tomato moth (Lacanobia oleracea) larvae, and characterised using specific synthetic substrates and inhibitors. These were; a trypsin-like activity, a chymotrypsin-like activity specific for substrates and inhibitors containing more than one amino acid residue, and an elastase-like activity, accounting for 40%, 30% and 20% of overall proteolysis respectively. The protease activities differed in their sensitivities to inhibition by different plant protein protease inhibitors (PIs), as estimated by I(50) values. Soya bean Kunitz trypsin inhibitor (SKTI) was the only plant PI tested to inhibit all three digestive protease activities at concentrations <40 &mgr;g/ml (approx. 5x10(-6)M). Incorporation of SKTI into a potato leaf-based artificial diet at 2% of total protein, decreased larval survival and growth (by approx. 33% and 40% respectively after 21 days) and retarded development (by approx. 2 days). However, when SKTI was expressed in transgenic potato plants at approx. 0.5% of total protein, only marginal effects on L. oleracea larvae were observed, which decreased with time. Whilst the presence of SKTI in artificial diet increased endogenous larval trypsin-like activity by up to four-fold, no effects on this activity were observed in larvae feeding on transgenic plants.

Stable Plastid Transformation in Lettuce (Lactuca sativa L.)

Although plastid transformation in higher plants was first demonstrated in the early 1990s it is only recently that the technology is being extended to a broader range of species. To date, the production of fertile transplastomic plants has been reported for tobacco, tomato, petunia, soybean, cotton and Lesquerella fendleri (Brassicaceae). In this study we demonstrate a polyethylene glycol-mediated plastid transformation system for lettuce that generates fertile, homoplasmic, plastid-transformed lines. Transformation was achieved using a vector that targets genes to the trnA/trnI intergenic region of the lettuce plastid genome employing the aadA gene as a selectable marker against spectinomycin. Spectinomycin resistance and heterologous gene transcription were shown in T1 plants derived from self-pollinated primary regenerants demonstrating transmission of the plastid-encoded transgene to the first seed generation. Crossing with male sterile wild-type lettuce showed that spectinomycin resistance was not transmitted via pollen. Constructs containing the gfp gene showed plastid-based expression of green fluorescent protein. The lettuce plastid could have potential both as a production and a delivery system for edible human therapeutic proteins.

Plant Transformation Technology Developments and Applications

Plant transformation has its roots in the research on Agrobacterium that was being undertaken in the early 1980s. The last two decades have seen significant developments in plant transformation technology, such that a large number of transgenic crop plants have now been released for commercial production. Advances in the technology have been due to development of a range of Agrobacterium-mediated and direct DNA delivery techniques, along with appropriate tissue culture techniques for regenerating whole plants from plant cells or tissues in a large number of species. In addition, parallel developments in molecular biology have greatly extended the range of investigations to which plant transformation technology can be applied. Research in plant transformation is concentrating now not so much on the introduction of DNA into plant cells, but rather more on the problems associated with stable integration and reliable expression of the DNA once it has been integrated.

Independent and Parallel Recruitment of Preexisting Mechanisms Underlying C4 Photosynthesis

Recurrent evolution of C4 photosynthesis is due to conserved regulatory sequences that localize photosynthetic enzymes. C4 photosynthesis allows increased photosynthetic efficiency because carbon dioxide (CO2) is concentrated around the key enzyme RuBisCO. Leaves of C4 plants exhibit modified biochemistry, cell biology, and leaf development, but despite this complexity, C4 photosynthesis has evolved independently in at least 45 lineages of plants. We found that two independent lineages of C4 plant, whose last common ancestor predates the divergence of monocotyledons and dicotyledons about 180 million years ago, show conserved mechanisms controlling the expression of genes important for release of CO2 around RuBisCO in bundle sheath (BS) cells. Orthologous genes from monocotyledonous and dicotyledonous C3 species also contained conserved regulatory elements that conferred BS specificity when placed into C4 species. We conclude that these conserved functional genetic elements likely facilitated the repeated evolution of C4 photosynthesis.

Influence of plant development and environment on transgene expression in potato and consequences for insect resistance

Clonal replicates of different transformed potato plants expressing transgene constructs containing the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, and sequences encoding the plant defensive proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA), and bean chitinase (BCH) were propagated in tissue culture. Plants were grown to maturity, at first under controlled environmental conditions, and later in the glasshouse. For a given transgene product, protein accumulation was found to vary between the different lines of clonal replicates (where each line was derived from a single primary transformant plant), as expected. However, variability was also found to exist within each line of clonal replicates, comparable to the variation of mean expression levels observed between the different clonal lines. Levels of GNA, accumulated in different parts of a transgenic potato plant, also showed variation but to a lesser extent than plant–plant variation in expression. With the majority of the clonal lines investigated, accumulation of the transgene product was found to increase as the potato plant developed, with maximum levels found in mature plants. The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level of GNA present in the plants, and that conditions under which the plants were grown affect the levels of GNA expression and subsequent levels of insect resistance.

Expression and characterisation of single-chain antibody fragments produced in transgenic plants against the organic herbicides atrazine and paraquat

Single-chain antibody fragments (scAbs), which have a human C-kappa constant domain and a hexa-histidine tail attached to the carboxy terminus of the single-chain Fv (ScFv) fragments to facilitate purification, have been raised against the herbicides paraquat and atrazine and expressed in transgenic Nicotiana tabacum cv. Samsun NN. Prior to purification, the anti-atrazine scAb is expressed as up to 0.014% of soluble leaf protein and has a binding profile in ELISA, against an atrazine-bovine serum albumin (BSA) conjugate, similar to that of the scAb produced in Escherichia coli. Competition ELISA has shown that the plant-derived scAb also recognises free atrazine. Following antibody affinity purification to isolate dimers, the affinity for immobilised antigen approaches that of the parental monoclonal antibody. This was confirmed by surface plasmon resonance analysis. The purified scAb also recognises related triazine herbicides. When isolated from cell-suspension cultures, the anti-paraquat scAb binds to a paraquat conjugate in a concentration-dependent manner, with a profile similar to the parental monoclonal antibody. This is the first demonstration that functional scAbs against organic pollutants can be produced in transgenic plants and that the scAbs may be appropriate for the development of immunoassay-based detection systems.

Plastid stromules are induced by stress treatments acting through abscisic acid.

Stromules are highly dynamic stroma-filled tubules that extend from the surface of all plastid types in all multi-cellular plants examined to date. The stromule frequency (percentage of plastids with stromules) has generally been regarded as characteristic of the cell and tissue type. However, the present study shows that various stress treatments, including drought and salt stress, are able to induce stromule formation in the epidermal cells of tobacco hypocotyls and the root hairs of wheat seedlings. Application of abscisic acid (ABA) to tobacco and wheat seedlings induced stromule formation very effectively, and application of abamine, a specific inhibitor of ABA synthesis, prevented stromule induction by mannitol. Stromule induction by ABA was dependent on cytosolic protein synthesis, but not plastid protein synthesis. Stromules were more abundant in dark-grown seedlings than in light-grown seedlings, and the stromule frequency was increased by transfer of light-grown seedlings to the dark and decreased by illumination of dark-grown seedlings. Stromule formation was sensitive to red and far-red light, but not to blue light. Stromules were induced by treatment with ACC (1-aminocyclopropane-1-carboxylic acid), the first committed ethylene precursor, and by treatment with methyl jasmonate, but disappeared upon treatment of seedlings with salicylate. These observations indicate that abiotic, and most probably biotic, stresses are able to induce the formation of stromules in tobacco and wheat seedlings.