Teresa Capell

Over-expression of the oat arginine decarboxylase cDNA in transgenic rice (Oryza sativa L.) affects normal development patterns in vitro and results in putrescine accumulation in transgenic plants

Abstract Transgenic rice (Oryza sativa L.) cell lines and plants expressing an oat arginine decarboxylase (Adc) cDNA under the control of the CaMV 35 S promoter were recovered using particle bombardment. Molecular analyses confirmed stable integration of the transgene and active transcription (mRNA). A four- to sevenfold increase in arginine decarboxylase (ADC) activity was observed in transformed plants compared to wild-type controls. Biochemical analysis of cellular polyamines (PAs) indicated up to fourfold increase in putrescine (Put) levels in transgenic callus and regenerated plants. This is the first report which demonstrates an increase in PA levels in plants engineered with the Adc gene. Implications of this increase are discussed in terms of development, physiology and nutrition. We observed a correlation between high levels of Adc gene expression and inability of callus tissue to develop normally into differentiated plants. This correlates well with reports in other species, in which perturbation of the PA pathway using genes involved in PA biosynthesis results in aberrant phenotypes. We have shown for the first time that PA biosynthesis can be manipulated in cereal species using genetic engineering.

Expression of a Heterologous S-Adenosylmethionine Decarboxylase cDNA in Plants Demonstrates That Changes in S-Adenosyl-L-Methionine Decarboxylase Activity Determine Levels of the Higher Polyamines Spermidine and Spermine

We posed the question of whether steady-state levels of the higher polyamines spermidine and spermine in plants can be influenced by overexpression of a heterologous cDNA involved in the later steps of the pathway, in the absence of any further manipulation of the two synthases that are also involved in their biosynthesis. Transgenic rice (Oryza sativa) plants engineered with the heterologous Datura stramonium S-adenosylmethionine decarboxylase (samdc) cDNA exhibited accumulation of the transgene steady-state mRNA. Transgene expression did not affect expression of the orthologoussamdc gene. Significant increases in SAMDC activity translated to a direct increase in the level of spermidine, but not spermine, in leaves. Seeds recovered from a number of plants exhibited significant increases in spermidine and spermine levels. We demonstrate that overexpression of the D. stramonium samdc cDNA in transgenic rice is sufficient for accumulation of spermidine in leaves and spermidine and spermine in seeds. These findings suggest that increases in enzyme activity in one of the two components of the later parts of the pathway leading to the higher polyamines is sufficient to alter their levels mostly in seeds and, to some extent, in vegetative tissue such as leaves. Implications of our results on the design of rational approaches for the modulation of the polyamine pathway in plants are discussed in the general framework of metabolic pathway engineering.

EU-OSTID: A collection of transposon insertional mutants for functional genomics in rice

A collection of 1373 unique flanking sequence tags (FSTs), generated from Ac/Ds and Ac transposon lines for reverse genetics studies, were produced in japonica and indica rice, respectively. The Ds and Ac FSTs together with the original T-DNAs were assigned a position in the rice genome sequence represented as assembled pseudomolecules, and found to be distributed evenly over the entire rice genome with a distinct bias for predicted gene-rich regions. The bias of the Ds and Ac transposon inserts for genes was exemplified by the presence of 59% of the inserts in genes annotated on the rice chromosomes and 41% present in genes transcribed as disclosed by their homology to cDNA clones. In a screen for inserts in a set of 75 well annotated transcription factors, including homeobox-containing genes, we found six Ac/Ds inserts. This high frequency of Ds and Ac inserts in genes suggests that saturated knockout mutagenesis in rice using this strategy will be efficient and possible with a lower number of inserts than expected. These FSTs and the corresponding plant lines are publicly available through OrygenesDB database and from the EU consortium members.

A transgenic rice cell lineage expressing the oat arginine decarboxylase (adc) cDNA constitutively accumulates putrescine in callus and seeds but not in vegetative tissues

We introduced the oat adc cDNA into rice under the control of the constitutive maize ubiquitin 1 promoter. We studied molecularly and biochemically sixteen independent transgenic plant lines. Significant increases in mRNA levels, ADC enzyme activity and polyamines were measured in transgenic callus. These increases were not maintained in vegetative tissue or seeds in regenerated plants, with the exception of one lineage. This particular lineage showed very significant increases in putrescine preferentially in seeds (up to 10 times compared to wild type and controls transformed with the hpt selectable marker alone). We have demonstrated that in cereals such as rice, over-expression of the oat adc cDNA results in increased accumulation of polyamines at different stages of development. We have also demonstrated that strong constitutive promoters, such as the maize ubiquitin 1 promoter, are sufficient to facilitate heritable high-level polyamine accumulation in seed. Our results demonstrate that by screening adequate numbers of independently derived transgenic plants, it is possible to identify those individuals which express a desired phenotype or genotype.

Promoter strength influences polyamine metabolism and morphogenic capacity in transgenic rice tissues expressing the oat adc cDNA constitutively

We analyzed molecularly and biochemically a series of transgenic rice lines expressing the oat adc (arginine decarboxylase) cDNA under the control of the constitutive maize ubiquitin 1 promoter. We established baseline biochemical parameters to elucidate the role of polyamines (PAs) during morphogenesis. We measured mRNA levels, ADC enzyme activity and cellular PAs in dedifferentiated callus. Polyamine levels were also quantified in two subsequent developmental stages – regenerating tissue and differentiated shoots. We observed significant (P<0.05) differences in the levels of individual PAs at the three developmental stages. The amounts of putrescine (Put) and spermidine (Spd) in dedifferentiated transgenic callus were lower than those in the wild type or in hpt (hygromycin resistant)-controls, whereas the amount of spermine (Spm) was increased up to two-fold. In regenerating tissue, this trend was reversed, with significantly higher levels of Put and Spd (P<0.05), and lower levels of Spm (P<0.05) compared to non-transformed or hpt-control tissues at the same developmental stage. In differentiated shoots, there was a general increase in PA levels, with significant increases in Put, Spd, and Spm (P<0.05); on occasion reaching six times the level observed in wild type and hpt-control tissues. These results contrast those we reported previously using the weaker CaMV 35S promoter driving adc expression. mRNA measurements and ADC enzyme activity were consistently higher (P<0.01) in all tissues expressing pUbiadcs compared to equivalent tissues engineered with 35Sadc. Our findings are consistent with a threshold model which postulates that high adc expression leading to production of Put above a basal level is necessary to generate a big enough metabolic pool to trigger PA flux through the pathway leading to an increase in the concentration of Spd and Spm. This can be best accomplished by a strong constitutive promoter driving adc. We discuss our results in the context of flux through the PA pathway and its impact on morphogenesis.

Over-expression of a cDNA for human ornithine decarboxylase in transgenic rice plants alters the polyamine pool in a tissue-specific manner.

Abstract. We investigated how over-expression of a cDNA for human ornithine decarboxylase (odc) affects the polyamine pools in transgenic rice. We further investigated tissue-specific expression patterns and product accumulation levels of the transgene driven by either constitutive or seed-specific promoters. Our results indicate that: (1) whereas the expression of a heterologous arginine decarboxylase (adc) cDNA in rice resulted in increased putrescine and spermine levels only in seeds, plants engineered to express odc cDNA exhibited significant changes in the levels of all three major polyamines in seeds and also in vegetative tissues (leaves and roots); (2) there was no linear correlation between odc mRNA levels, ODC enzyme activity and polyamine accumulation, suggesting that control of the polyamine pathway in plants is more complex than in mammalian systems; (3) ODC activity and polyamine changes varied in different tissues, indicating that the pathway is regulated in a tissue-specific manner. Our results suggest that ODC rather than ADC is responsible for the regulation of putrescine synthesis in plants.

Simultaneous reduction of the activity of two related enzymes, involved in early steps of the polyamine biosynthetic pathway, by a single antisense cDNA in transgenic rice

Abstract. Transgenic rice cell lines transformed with a heterologous cDNA derived from the arginine decarboxylase gene of oat, in an antisense orientation, exhibited significant (P<0.05) down-regulation of the activity of the endogenous arginine and ornithine decarboxylases, compared to wild type and controls transformed only with the selectable marker (hpt). Changes in enzyme activity were reflected in a marked decrease in the level of putrescine (P<0.001) and spermidine (P<0.01) but not spermine (P>0.05) in the majority of cell lines analyzed. In agreement with previous results, we confirmed that cell lines with low levels of polyamines exhibited normal morphogenic responses. In vegetative tissue at the whole plant level no significant variation (P>0.05) in polyamine levels was observed. However, we measured significant reductions (P<0.001) in putrescine levels in seeds derived from three out of five plants analyzed in detail. Thus, simultaneous reduction of the activity of the two alternative enzymes in the early steps of the polyamine pathway results in significant reduction in end-product accumulation in the seeds of transgenic plants.

Transgenic Rice as a Vehicle for the Production of the Industrial Enzyme Transglutaminase

Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases has increased the demand for an inexpensive, efficient and safe source of recombinant enzyme. We explored the use of plant-based systems for the production of this important industrial enzyme. Transgenic rice plants engineered with a rat prostate transglutaminase (rTGp), driven by the strong constitutive maize-1 ubiquitin promoter and its first intron, were shown to express the recombinant enzyme at the mRNA and protein levels. The Ca2+ dependence of the recombinant enzyme was confirmed by the biotin-labelled cadaverine-incorporation assay. In this communication we report the molecular and biochemical characterisation of transgenic plants expressing rTGp and this sets the stage for establishing a bioreactor system for the production of transglutaminases in plants.

Building bridges: an integrated strategy for sustainable food production throughout the value chain

The food production and processing value chain is under pressure from all sides—increasing demand driven by a growing and more affluent population; dwindling resources caused by urbanization, land erosion, pollution and competing agriculture such as biofuels; and increasing constraints on production methods driven by consumers and regulators demanding higher quality, reduced chemical use, and most of all environmentally beneficial practices ‘from farm to fork’. This pressure can only be addressed by developing efficient and sustainable agricultural practices that are harmonized throughout the value chain, so that renewable resources can be exploited without damaging the environment. Bridges must, therefore, be built between the diverse areas within the food production and processing value chain, including bridges between different stages of production, between currently unlinked agronomic practices, and between the different levels and areas of research to achieve joined-up thinking within the industry, so that the wider impact of different technologies, practices and materials on productivity and sustainability is understood at the local, regional, national and global scales. In this article, we consider the challenges at different stages and levels of the value chain and how new technologies and strategies could be used to build bridges and achieve more sustainable food/feed production in the future.

Producing transglutaminases by molecular farming in plants: Minireview article

Summary.Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities, both in terms of biology and biotechnology, is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases in research, medicine and industry has increased the demand for an inexpensive, efficient and safe source of recombinant enzymes. We describe initial results concerning the production of a mammalian transglutaminase in transgenic rice plants as a first step towards the large-scale molecular farming of this enzyme.