Enhanced polyhydroxybutyrate production in transgenic sugarcane

Polyhydroxybutyrate (PHB) is a bacterial polyester that has properties similar to some petrochemically produced plastics. Plant-based production has the potential to make this biorenewable plastic highly competitive with petrochemical-based plastics. We previously reported that transgenic sugarcane produced PHB at levels as high as 1.8% leaf dry weight without penalty to biomass accumulation, suggesting scope for improving PHB production in this species. In this study, we used different plant and viral promoters, in combination with multigene or single-gene constructs to increase PHB levels. Promoters tested included the maize and rice polyubiquitin promoters, the maize chlorophyll A/B-binding protein promoter and a Cavendish banana streak badnavirus promoter. At the seedling stage, the highest levels of polymer were produced in sugarcane plants when the Cavendish banana streak badnavirus promoter was used. However, in all cases, this promoter underwent silencing as the plants matured. The rice Ubi promoter enabled the production of PHB at levels similar to the maize Ubi promoter. The maize chlorophyll A/B-binding protein promoter enabled the production of PHB to levels as high as 4.8% of the leaf dry weight, which is approximately 2.5 times higher than previously reported levels in sugarcane. This is the first time that this promoter has been tested in sugarcane. The highest PHB-producing lines showed phenotypic differences to the wild-type parent, including reduced biomass and slight chlorosis.     

Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight

 Transgenic Arabidopsis thaliana (L.) Heynh. plants expressing the three enzymes encoding the biosynthetic route to polyhydroxybutyrate (PHB) are described. These plants accumulated more than 4% of their fresh weight (≈40% of their dry weight) in the form of PHB in leaf chloroplasts. These very high producers were obtained and identified following a novel strategy consisting of a rapid GC-MS analysis of a large number of transgenic Arabidopsis plants generated using a triple construct, thus allowing the parallel transfer of all three genes necessary for PHB synthesis in a single transformation event. The level of PHB produced was 4-fold greater than previously published values, thus demonstrating the large potential of plants to produce this renewable resource. However, the high levels of the polymer produced had severe effects on both plant development and metabolism. Stunted growth and a loss of fertility were observed in the high-producing lines. Analysis of the metabolite composition of these lines using a GC-MS method that we have newly developed showed that the accumulation of high levels of PHB was not accompanied by an appreciable change in either the composition or the amount of fatty acids. Substantial changes were, however, observed in the levels of various organic acids, amino acids, sugars and sugar alcohols. Received: 2 February 2000 / Accepted: 31 March 2000     

Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants

Selectable markers of bacterial origin such as the neomycin phosphotransferase type II gene, which can confer kanamycin resistance to transgenic plants, represent an invaluable tool for plant engineering. However, since all currently used antibiotic-resistance genes are of bacterial origin, there have been concerns about horizontal gene transfer from transgenic plants back to bacteria, which may result in antibiotic resistance. Here we characterize a plant gene, Atwbc19, the gene that encodes an Arabidopsis thaliana ATP binding cassette (ABC) transporter and confers antibiotic resistance to transgenic plants. The mechanism of resistance is novel, and the levels of resistance achieved are comparable to those attained through expression of bacterial antibiotic-resistance genes in transgenic tobacco using the CaMV 35S promoter. Because ABC transporters are endogenous to plants, the use of Atwbc19 as a selectable marker in transgenic plants may provide a practical alternative to current bacterial marker genes in terms of the risk for horizontal transfer of resistance genes.     

Isolation of cyanophycin from tobacco and potato plants with constitutive plastidic cphATe gene expression

A chimeric cyanophycin synthetase gene composed of the cphATe coding region from the cyanobacterium Thermosynechococcus elongatus BP-1, the constitutive 35S promoter and the plastid targeting sequence of the integral photosystem II protein PsbY was transferred to the tobacco variety Petit Havanna SRI and the commercial potato starch production variety Albatros. The resulting constitutive expression of cyanophycin synthetase leads to polymer contents in potato leaf chloroplasts of up to 35 mg/g dry weight and in tuber amyloplasts of up to 9 mg/g dry weight. Both transgenic tobacco and potato were used for the development of isolation methods applicable for large-scale extraction of the polymer. Two different procedures were developed which yielded polymer samples of 80 and 90% purity, respectively.     

Chemically inducible expression of the PHB biosynthetic pathway in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Arabidopsis plants were transformed with a multi-gene construct for expression of the polyhydroxybutyrate (PHB) biosynthetic pathway containing a gene switch that can be activated by commercially available non-steroidal ecdysone analogs approved for use on some crops as pesticides. T(1) progeny of transgenic Arabidopsis plants were isolated and screened for PHB production in the presence of ecdysone analogs. T(2) progeny derived from selected T(1) lines were subjected to further analysis by comparing PHB production levels prior to treatment with inducing agent and 21 days after initiation of induction. Significant PHB production was delayed in many of the engineered plants until after induction. PHB levels of up to 14.3% PHB per unit dry weight were observed in young leaves harvested from engineered T(2) plants after applications of the commercial ecdysone analog Mimic. PHB in older leaves reached levels of up to 7% PHB per unit dry weight. This study represents a first step towards engineering a chemically inducible gene switch for PHB production in plants using inducing agents that are approved for field use.     

Inducible cross-tolerance to herbicides in transgenic potato plants with the rat CYP1A1 gene

A gene of the enzyme involved in xenobiotic metabolism in mammalian liver was introduced into potato to confer inducible herbicide tolerance. A rat cytochrome P450 monooxygenase, CYP1A1 cDNA, was kept under the control of the tobacco PR1a promoter in order to apply the system of chemical inducible expression using the plant activator Benzothiadiazole (BTH). Transgenic plants were obtained based on the kanamycin resistance test and PCR analysis. Northern-blot analysis revealed the accumulation of mRNA corresponding to rat CYP1A1 in the transgenic plants treated with BTH (3.0 μmol/pot), whereas no accumulation of the corresponding mRNA occurred without BTH treatment. These transgenic plants also produced a protein corresponding to CYP1A1 in the leaves by BTH treatment. The transgenic plants with BTH application showed a much-higher tolerance to the phenylurea herbicides chlortoluron and methabenzthiazuron than non-transgenic plants. These findings indicated that the ability of metabolizing the two herbicides to less-toxic derivatives was displayed in the transgenic plants after BTH treatment. Transgenic plants harboring the CYP1A1 cDNA fused with the yeast P450 reductase (YR) gene under the control of PR1a were also produced. Although the plants showed a lower expression level of the fused gene than transgenic plants with CYP1A1 cDNA alone, they were tolerant to herbicides. These facts suggested that the CYP1A1 enzyme fused with YR showed a higher specific activity than CYP1A1 alone. This study demonstrated that the mammalian cDNA for the de-toxification enzyme of herbicides under the control of the PR1a promoter conferred chemical-inducible herbicide tolerance on potato. Received: 15 March 2001 / Accepted: 14 June 2001     

A critical review on the improvement of photosynthetic carbon assimilation in C3 plants using genetic engineering

Global warming is one of the most serious challenges facing us today. It may be linked to the increase in atmospheric CO2 and other greenhouse gases (GHGs), leading to a rise in sea level, notable shifts in ecosystems, and in the frequency and intensity of wild fires. There is a strong interest in stabilizing the atmospheric concentration of CO2 and other GHGs by decreasing carbon emission and/or increasing carbon sequestration. Biotic sequestration is an important and effective strategy to mitigate the effects of rising atmospheric CO2 concentrations by increasing carbon sequestration and storage capacity of ecosystems using plant photosynthesis and by decreasing carbon emission using biofuel rather than fossil fuel. Improvement of photosynthetic carbon assimilation, using transgenic engineering, potentially provides a set of available and effective tools for enhancing plant carbon sequestration. In this review, firstly different biological methods of CO2 assimilation in C3, C4 and CAM plants are introduced and three types of C4 pathways which have high photosynthetic performance and have evolved as CO2 pumps are briefly summarized. Then (i) the improvement of photosynthetic carbon assimilation of C3 plants by transgenic engineering using non-C4 genes, and (ii) the overexpression of individual or multiple C4 cycle photosynthetic genes (PEPC, PPDK, PCK, NADP-ME and NADP-MDH) in transgenic C3 plants (e.g. tobacco, potato, rice and Arabidopsis) are highlighted. Some transgenic C3 plants (e.g. tobacco, rice and Arabidopsis) overexpressing the FBP/SBPase, ictB and cytochrome c6 genes showed positive effects on photosynthetic efficiency and growth characteristics. However, over the last 28 years, efforts to overexpress individual, double or multiple C4 enzymes in C3 plants like tobacco, potato, rice, and Arabidopsis have produced mixed results that do not confirm or eliminate the possibility of improving photosynthesis of C3 plants by this approach. Finally, a prospect is provided on the challenges of enhancing carbon assimilation of C3 plants using transgenic engineering in the face of global warming, and the trends of the most promising approaches to improving the photosynthetic performance of C3 plants.     

Introduction of bacterial metabolism into higher plants by polycistronic transgene expression

Multiple-gene transformation is required to improve or change plant metabolisms effectively; but this many-step procedure is time-consuming and costing. We succeeded in the metabolic engineering of tobacco plants by introducing multiple genes as a bacteria-type operon into a plastid genome. The tobacco plastid was transformed with a polycistron consisting of three bacterial genes for the biosynthesis of a biodegradable polyester, polyhydroxybutyrate (PHB). Accumulation of PHB in the leaves of the transgenic tobacco indicated that the introduced genes were polycistronically expressed. This "phyto-fermentation" system can be used in plant production of various chemical commodities and pharmaceuticals.     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.     

Isolation and characterization of plant N-acetyl glucosaminyltransferase I (GntI) cDNA sequences. Functional analyses in the Arabidopsis cgl mutant and in antisense plants

We report on the isolation and characterization of full-length cDNA sequences coding for N-acetylglucosaminyltransferase I (GnTI) from potato (Solanum tuberosum L.), tobacco (Nicotiana tabacum L.), and Arabidopsis. The deduced polypeptide sequences show highest homology among the solanaceous species (93% identity between potato and tobacco compared with about 75% with Arabidopsis) but share only weak homology with human GnTI (35% identity). In contrast to the corresponding enzymes from animals, all plant GnTI sequences identified are characterized by a much shorter hydrophobic membrane anchor and contain one putative N-glycosylation site that is conserved in potato and tobacco, but differs in Arabidopsis. Southern-blot analyses revealed that GntI behaves as a single-copy gene. Northern-blot analyses showed that GntI-mRNA expression is largely constitutive. Arabidopsis cgl mutants deficient in GnTI activity also possess GntI mRNA, indicating that they result from point mutations. GntI-expression constructs were tested for the ability to relieve the GnTI block in protoplasts of the Arabidopsis cgl mutant and used to obtain transgenic potato and tobacco plants that display a substantial reduction of complex glycan patterns. The latter observation indicates that production of heterologous glycoproteins with little or no antigenic glycans can be achieved in whole plants, and not in just Arabidopsis, using antisense technology.     

Wound-inducible and organ-specific expression of ORF13 from Agrobacterium rhizogenes 8196 T-DNA in transgenic tobacco plants

Tissue-specific expression of the ORF13 promoter from Agrobacterium rhizogenes 8196 was assessed throughout the development of transgenic tobacco plants using a GUS reporter gene. ORF13 exhibited high activity in roots but with different patterns of expression. The activity of the ORF13 promoter in vascular tissues increased from the base to the tip of the stem. The ORF13 promoter is wound inducible in a limited area adjacent to the wound site. The time course of wound induction of ORF13 in transgenic tobacco containing an ORF13 promoter-GUS translational fusion was similar to that previously described for genes involved in plant defense responses. A series of 5′ deletions of the ORF13 promoter fused to the β-glucuronidase gene was examined for expression in roots and leaves of transgenic plants. Cis-acting elements that modulate quantitative expression of the transgene after wounding were detected.     

Genetic engineering of glycinebetaine production toward enhancing stress tolerance in plants: metabolic limitations

Glycinebetaine (betaine) affords osmoprotection in bacteria, plants and animals, and protects cell components against harsh conditions in vitro. This and a compelling body of other evidence have encouraged the engineering of betaine production in plants lacking it. We have installed the metabolic step for oxidation of choline, a ubiquitous substance, to betaine in three diverse species, Arabidopsis, Brassica napus, and tobacco (Nicotiana tabacum), by constitutive expression of a bacterial choline oxidase gene. The highest levels of betaine in independent transgenics were 18.6, 12.8, and 13 micromol g(-1) dry weight, respectively, values 10- to 20-fold lower than the levels found in natural betaine producers. However, choline-fed transgenic plants synthesized substantially more betaine. Increasing the choline supplementation further enhanced betaine synthesis, up to 613 micromol g(-1) dry weight in Arabidopsis, 250 micromol g(-1) dry weight in B. napus, and 80 micromol g(-1) dry weight in tobacco. These studies demonstrate the need to enhance the endogenous choline supply to support accumulation of physiologically relevant amounts of betaine. A moderate stress tolerance was noted in some but not all betaine-producing transgenic lines based on relative shoot growth. Furthermore, the responses to stresses such as salinity, drought, and freezing were variable among the three species.     

The Expression of GUS Gene Driven by T-cyt Promoter in Transgenic Tobacco andPotato

The location of GUS gene expression under control of T-cyt gene (gene 4 of T- DNA coding isopenteryl transferase) 5′ region in transgenic tobacco (Nicotiana tabacum cv. W38) and potato (Solanum tuberosum L, cv. Desiree) plants was examined with biochemical assays. The results showed differential distribution in various organs and different cell types. The highest levels of GUS activity were found in tobacco stem where axillary bud was initiated and potato buds on tubers. Moreover, the expression of T-cyt promoter/GUS was found to be inducible in transgenic tobacco stem with cytokinin rather than auxin treatment. Additionally, the level of expression was high in the wounded leaf of transgenic potato. It was suggested that T-cyt promoter may be selectively induced by some exogenous plant hormones.     

Polyhydroxybutyrate synthesis in transgenic flax

Flax (Linum usitatissimum L.) is an annual plant species widely cultivated in temperate climates for bast fibres and linseed oil. Apart from traditional textile use, the fibres are fast becoming an integral part of new composite materials utilized in automobile and constructive industry. Especially attractive for environmental safety demands are biodegradable and renewable biocomposities based on polyhydroxybutyrate (PHB) polymer as a matrix and reinforced with the flax fibres. Manufacturing of PHB by bacteria fermentation is however substantially more expansive as compared to technologies producing conventional plastics. We report for the first time generation of transgenic plants which produce both components of flax/PHB composites, i.e. the fibres and the thermoplastic matrix in the same plant organ of a crop. The flax (cv. Nike) plants were transformed using constructs bearing either single cDNA, encoding the beta-ketothiolase enzyme (C plants), or all three of the genes necessary for poly-beta-hydroxybutyrate (PHB) synthesis (M plants). Both constructs contained a plastidial targeting sequence. The amount of PHB produced by the transgenic plants was up to over 70-fold higher than in wild-type plants, when analysed using the gas chromatography/mass spectrometry (GC-MS method). The PHB accumulation in plastids caused change both in their shape and size. The use of a stem-specific promoter for transgene expression protected the transgenic plant from growth retardation and also provided higher PHB synthesis than in the case of constructs governed by the 35S CaMV constitutive promoter. None toxic effects that could lead to stunted growth or the loss of fertility were observed, when 14-3-3 promoter was used as the stem-specific. Significant modifications in stem mechanical properties were accompanied to the PHB accumulation in growing cell of fibres in the transgenic plants. The Young's modulus E, the average measure of stem tissues resistance to tensile loads increased up to twice in M plants as compared to a single gene transformed ones. However, a wide range of E values, from 24.1 to 54.4 MPa, was observed in dependence of tested strain. Potential commercial significance of the genetic manipulation approach enabling synthesis of thermoplastic in crops cultivated for fibres is discussed.     

Constitutive expression of the <Emphasis Type="Italic">ARGOS</Emphasis> gene driven by dahlia mosaic virus promoter in tobacco plants

The auxin-inducible gene ARGOS from Arabidopsis thaliana is expressed in growing tissues and controls the plant organ size by regulating cell proliferation and meristematic competence. The promoter of the dahlia (Dahlia pinnata Cav.) mosaic virus (DMV) resembles the well-known cauliflower mosaic virus 35S promoter but shows a higher activity in transgenic tobacco plants (Nicotiana tabacum L.). We obtained transgenic tobacco plants expressing the Arabidopsis ARGOS gene under the control of the DMV promoter. Several of the T0 generation plants exhibited an accelerated transition to flowering, a slight increase in flower size, and a significant increase in the leaf size. The T1 transgenic plants were characterized by faster growth, the increased leaf size, and somewhat enlarged flowers as compared with control plants. These phenotypic traits, as well as stability and inheritance of the transgene were demonstrated also in T2 transgenic plants.