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We report the development of an efficient and reproducible genetic transformation system for the recalcitrant Spanish elite rice paella genotype, Bomba. Preconditioned embryos derived from dry seeds were bombarded with gold particles carrying a plasmid containing a screenable and a selectable marker. We confirmed integration and expression of hpt and gusA in the rice genome. Transformation frequency was ca: 10% in several independent experiments. We show Mendelian inheritance of the input transgenes and zygosity determination of the transgenic lines in the T1 generation. A unique and critical step for the regeneration of plants from transformed tissue was shading during the early stages of regeneration, combined with a specific cytokinin:auxin ration at the onset of shifting callus to regeneration media.

The first committed step in the endosperm starch biosynthetic pathway is catalyzed by the cytosolic glucose-1-phosphate adenylyl transferase (AGPase) comprising large and small subunits encoded by the OsAPL2 and OsAPS2b genes, respectively. OsAPL2 is expressed solely in the endosperm so we hypothesized that mutating this gene would block starch biosynthesis in the endosperm without affecting the leaves. We used CRISPR/Cas9 to create two heterozygous mutants, one with a severely truncated and nonfunctional AGPase and the other with a C-terminal structural modification causing a partial loss of activity. Unexpectedly, we observed starch depletion in the leaves of both mutants and a corresponding increase in the level of soluble sugars. This reflected the unanticipated expression of both OsAPL2 and OsAPS2b in the leaves, generating a complete ectopic AGPase in the leaf cytosol, and a corresponding decrease in the expression of the plastidial small subunit OsAPS2a that was only partially complemented by an increase in the expression of OsAPS1. The new cytosolic AGPase was not sufficient to compensate for the loss of plastidial AGPase, most likely because there is no wider starch biosynthesis pathway in the leaf cytosol and because pathway intermediates are not shuttled between the two compartments.

International audience; Cryphonectria parasitica (Murrill.) Barr is a necrotrophic fungus that affects European chestnut populations. In Catalonia (NE Spain), this fungus is widely spread affecting chestnut stands. In addition to vegetative compatibility (VC) types of the fungus, knowledge of the Cryphonectria hypovirus (CHV) occurrence is important to implement a biocontrol programme. We confirmed the presence of hypoviruses in several chestnut subpopulations of Catalonia, and we determined the VC types of CHV‐infected isolates. We also studied the nucleotide sequences of these hypoviruses from a variable region located in the hypovirus ORF‐A. Our results show a low occurrence of hypoviruses throughout C. parasitica populations in Catalonia, except for a few localized stands. From 312 sampled cankers, we obtained 179 white, pale orange or rickety isolates. In 35 of them, we detected CHV‐1. Infected isolates belonged to 5 of the 7 sampled subpopulations. We found 12 CHV‐1 haplotypes, based on the nucleotide sequence analysis. Most of the hypoviruses have the same nucleotide sequence or show high homology at nucleotide level with isolates previously included in CHV‐1 subtype‐I. However, we found that 3 haplotypes share at least 97% of their ORF‐A nucleotide sequence with CHV‐1 subtype‐E. Phylogenetic analysis grouped these 3 isolates in a different cluster than the other hypoviruses. These results suggest that multiple introductions of CHV‐1 have occurred recently in Catalonia. Dominant VC types of hypovirus‐infected isolates are EU‐1, EU‐2 and EU‐5, which are similar to those previously reported in this region.

The eight Millennium Development Goals (MDGs) are international development targets for the year 2015 that aim to achieve relative improvements in the standards of health, socioeconomic status and education in the world’s poorest countries. Many of the challenges addressed by the MDGs reflect the direct or indirect consequences of subsistence agriculture in the developing world, and hence, plant biotechnology has an important role to play in helping to achieve MDG targets. In this opinion article, we discuss each of the MDGs in turn, provide examples to show how plant biotechnology may be able to accelerate progress towards the stated MDG objectives, and offer our opinion on the likelihood of such technology being implemented. In combination with other strategies, plant biotechnology can make a contribution towards sustainable development in the future although the extent to which progress can be made in today’s political climate depends on how we deal with current barriers to adoption. Research in our laboratory is supported by Ministry of Science and Innovation-MICINN, Spain (Grant BFU2007-61413); European Research Council Advanced Grant BIOFORCE; Center Consolider, MICINN, Spain; COST Action FA0804, Associated Unit CAVA and SmartCell, FP7 Integrated project.

Arginine decarboxylase (ADC) is a key enzyme in plants that converts arginine into putrescine, an important mediator of abiotic stress tolerance. Adc genes have been isolated from a number of dicotyledonous plants but the oat and rice Adc genes are the only representatives of monocotyledonous species described thus far. Rice has a small family of Adc genes, and OsAdc1 expression has been shown to fluctuate under drought and chilling stress. We identified and characterized a second rice Adc gene (OsAdc2) which encodes a 629-amino-acid protein with a predicted molecular mass of 67 kDa. An unusual feature of the OsAdc2 gene is the presence of an intron and a short upstream open reading frame in the 5'-UTR. Sequence comparisons showed that OsAdc2 is more closely related to the oat Adc gene than to OsAdc1 or to its dicot homologs, and mRNA analysis showed that the two rice genes are also differently regulated. Whereas OsAdc1 is expressed in leaf, root and stem, OsAdc2 expression is restricted to stem tissue. Protein expression was investigated with specific antibodies against ADC1 and ADC2, corroborating the mRNA data. We discuss the expression profiles of OsAdc1 and OsAdc2 and potential functions for the two corresponding proteins.

We generated and characterized a transgenic wheat (Triticum aestivum L.) population expressing constitutively an oat arginine decarboxylase (ADC) cDNA. Transgenic wheat plants expressing the transgene had a significant increase in ADC activity with no concomitant changes in ODC activity. We measured 2-fold increases in putrescine, spermidine and spermine content in leaves of transgenic plants compared to wild-type. These plants also exhibited significant increases in the three polyamines in seeds (7-fold putrescine, 2.5-fold spermidine and 1.8-fold spermine). This significant polyamine accumulation was heritable in subsequent generations. In order to characterize molecularly this population we cloned a partial TaADC cDNA. This sequence was used as a probe to ascertain the presence of two distinct ADC transcripts in wheat. Further DNA analyses confirmed the presence of at least two distinct ADC genes in the wheat genome. Our results demonstrate that polyamine levels can be altered in transgenic wheat plants resulting in phenotypically normal and fertile plants. This transgenic wheat population will serve as useful germplasm to embark on further in depth studies to ascertain the role of polyamines in abiotic stress responses.

We investigated whether down-regulation of arginine decarboxylase (ADC) activity and concomitant changes in polyamine levels result in changes in the expression of downstream genes in the polyamine pathway. We generated transgenic rice (Oryza sativa L.) plants in which the rice adc gene was down-regulated by expression of its antisense oat (Avena sativa L.) ortholog. Plants expressed the oat mRNA adc transcript at different levels. The endogenous transcript was down-regulated in five out of eight plant lineages we studied in detail. Reduction in the steady-state rice adc mRNA levels resulted in a concomitant decrease in ADC activity. The putrescine and spermidine pool was significantly reduced in plants with lower ADC activity. Expression of the rice ornithine decarboxylase (odc), S-adenosylmethionine decarboxylase (samdc) and spermidine synthase (spd syn) transcripts was not affected. We demonstrate that even though levels of the key metabolites in the pathway were compromised, this did not influence steady-state transcription levels of the other genes involved in the pathway. Our results provide an insight into the different regulatory mechanisms that control gene expression in the polyamine biosynthetic pathway in plants by demonstrating that the endogenous pathway is uncoupled from manipulations that modulate polyamine levels by expression of orthologous transgenes.

We describe the development of a novel transformation system for pigeonpea (Cajanus cajan (L.) Millsp.) cell lines using particle bombardment. The aim of our study was not only to develop a transformation system for this important but recalcitrant species but to do so using a gene of interest and to study the behaviour of this transgene in a legume background. The gene we chose for our studies was the heterologous oat arginine decarboxylase cDNA. Transgenic lines engineered with this cDNA had a small, but significant increase in arginine decarboxylase activity (ADC; EC 4.1.1.17). We detected an increase in putrescine levels in all the lines we analysed irrespective of whether major or minor changes in ADC activity were observed. Spermidine and spermine levels were also increased in some of the lines. We confirmed that small changes in the adc message (mRNA) had a significant effect on enzyme activity and more importantly on polyamine levels. This is the first report describing modulation of a gene involved in a metabolic pathway in transgenic pigeonpea 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.

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

Metabolic engineering in plants can be used to increase the abundance of specific valuable metabolites, but single-point interventions generally do not improve the yields of target metabolites unless that product is immediately downstream of the intervention point and there is a plentiful supply of precursors. In many cases, an intervention is necessary at an early bottleneck, sometimes the first committed step in the pathway, but is often only successful in shifting the bottleneck downstream, sometimes also causing the accumulation of an undesirable metabolic intermediate. Occasionally it has been possible to induce multiple genes in a pathway by controlling the expression of a key regulator, such as a transcription factor, but this strategy is only possible if such master regulators exist and can be identified. A more robust approach is the simultaneous expression of multiple genes in the pathway, preferably representing every critical enzymatic step, therefore removing all bottlenecks and ensuring completely unrestricted metabolic flux. This approach requires the transfer of multiple enzyme-encoding genes to the recipient plant, which is achieved most efficiently if all genes are transferred at the same time. Here we review the state of the art in multigene transformation as applied to metabolic engineering in plants, highlighting some of the most significant recent advances in the field. Research at the Universitat de Lleida is supported by the Ministerio de Ciencia e Innovación (grants no. BFU2007-61413, BIO2011-23324, BIO02011-22525, PIM2010PKB-0074, Acciones complementarias BIO2007-30738-E and BIO2011-22525, and the Centre CONSOLIDER on Agrigenomics), the European Union Framework 7 Program (SmartCell Integrated Project 222716), the European Research Council IDEAS Advanced Grant Program (BIOFORCE) (to PC), the European Cooperation in Science and Technology (COST Action FA0804), and RecerCaixa.

Antioxidants are protective molecules that neutralize reactive oxygen species and prevent oxidative damage to cellular components such as membranes, proteins and nucleic acids, therefore reducing the rate of cell death and hence the effects of ageing and ageing-related diseases. The fortification of food with antioxidants represents an overlap between two diverse environments, namely fortification of staple foods with essential nutrients that happen to have antioxidant properties (e.g. vitamins C and E) and the fortification of luxury foods with healthpromoting but non-essential antioxidants such as flavonoids as part of the nutraceuticals/ functional foods industry. Although processed foods can be artificially fortified with vitamins, minerals and nutraceuticals, a more sustainable approach is to introduce the traits for such health-promoting compounds at source, an approach known as biofortification. Regardless of the target compound, the same challenges arise when considering the biofortification of plants with antioxidants, that is the need to modulate endogenous metabolic pathways to increase the production of specific antioxidants without affecting plant growth and development and without collateral effects on other metabolic pathways. These challenges become even more intricate as we move from the engineering of individual pathways to several pathways simultaneously. In this review, we consider the state of the art in antioxidant biofortification and discuss the challenges that remain to be overcome in the development of nutritionally complete and health-promoting functional foods. Research at the Universitat de Lleida is supported by MICINN, Spain (BFU2007-61413; BIO2011-23324; BIO02011-22525; PIM2010PKB-00746); Acciones complementarias, BIO2007- 30738-E, BIO2011-22525, MICINN, Spain; European Union Framework 7 Program-SmartCell Integrated Project 222716; European Union Framework 7 European Research Council IDEAS Advanced Grant (to PC) Program-BIOFORCE; COST Action FA0804: Molecular farming: plants as a production platform for high value proteins; Centre CONSOLIDER on Agrigenomics funded by MICINN, Spain.

L-ascorbic acid (vitamin C) is an antioxidant and electron donor whose metabolism in plants isunder strict feedback control. The factors that influence L-ascorbic acid accumulation in staplecrops are only partially understood. One way to gain insight into the regulation of L-ascorbic acidmetabolism is to investigate the endogenous pathways in various genetic backgrounds and char-acterize their interactions with transgenes encoding relevant enzymes. In an initial step, we inves-tigated the developmental profile of L-ascorbic acid accumulation in the endosperm of threediverse maize genotypes and a transgenic line expressing rice dehydroascorbate reductase, whichenhances L-ascorbic acid recycling. We determined the transcript levels of all the key genes in theL-ascorbic acid metabolic pathways as well as the specific levels of ascorbic acid and dehy-droascorbate. L-ascorbic acid levels were high 20 days after pollination and declined thereafter. Wefound significant genotype-dependent variations in the transcript levels of some genes, with par-ticular complexity in the ascorbic acid recycling pathway. Our data will help to elucidate the com-plex mechanisms underlying the regulation of L-ascorbic acid metabolism in plants, particularlythe impact of genetic background on the strict regulation of ascorbic acid metabolism inendosperm cells. This study was supported by the Ministerio de Ciencia e Innovación, Spain (BIO2007-61413 and BIO2011-22525),Proyecto Consolider-Ingenio 2010 Centre Agrigenomica CSD2007-00036, RecerCaixa, and European ResearchCouncil Advanced Grant (BIOFORCE) to PC.

Genetically engineered (GE) crops can be used as part of a combined strategy to address food insecurity, which is defined as a lack of sustainable access to safe and nutritious food. In this article, we discuss the causes and consequences of food insecurity in the developing world, and the indirect economic impact on industrialized countries. We dissect the healthcare costs and lost productivity caused by food insecurity, and evaluate the relative merits of different intervention programs including supplementation, fortification and the deployment of GE crops with higher yields and enhanced nutritional properties. We provide clear evidence for the numerous potential benefits of GE crops, particularly for small-scale and subsistence farmers. GE crops with enhanced yields and nutritional properties constitute a vital component of any comprehensive strategy to tackle poverty, hunger and malnutrition in developing countries and thus reduce the global negative economic effects of food insecurity. Research at the Universitat de Lleida is supported by MICINN, Spain (BFU2007-61413; BIO2011-23324; BIO02011-22525; PIM2010PKB-00746); European Union Framework 7 Program-SmartCell Integrated Project 222716; European Union Framework 7 European Research Council IDEAS Advanced Grant (to PC) Program-BIOFORCE; COST Action FA0804: Molecular farming: plants as a production platform for high value proteins; COST Action FA1006: Plant Engine: Plant metabolic engineering for high value products; Centre CONSOLIDER on Agrigenomics funded by MICINN, Spain and RecerCaixa.

Malnutrition is a prevalent and entrenched global socioeconomic challenge that reflects the combined impact of poverty, poor access to food, inefficient food distribution infrastructure, and an over-reliance on subsistence mono-agriculture. The dependence on staple cereals lacking many essential nutrients means that malnutrition is endemic in developing countries. Most individuals lack diverse diets and are therefore exposed to nutrient deficiencies. Plant biotechnology could play a major role in combating malnutrition through the engineering of nutritionally enhanced crops. In this article, we discuss different approaches that can enhance the nutritional content of staple crops by genetic engineering (GE) as well as the functionality and safety assessments required before nutritionally enhanced GE crops can be deployed in the field. We also consider major constraints that hinder the adoption of GE technology at different levels and suggest policies that could be adopted to accelerate the deployment of nutritionally enhanced GE crops within a multicomponent strategy to combat malnutrition.

We investigated the effect of methyl jasmonate (MeJa) treatment on the expression of two genes in the rice polyamine biosynthesis pathway and on the polyamine content in wild type plants and transgenic rice plants expressing a Datura stramonium (Ds) Adc cDNA, the latter accumulating up to three-fold the normal level of putrescine. Exogenous MeJa transiently inhibited the expression of OsAdc1, OsSamdc and Spermidine synthase (OsSpds) genes in the polyamine biosynthesis pathway, probably through transcriptional repression. There was also a similar negative impact on the DsAdc transgene in transgenic plants, even though a constitutive promoter was used to drive transgene expression. The free putrescine content was reduced significantly in the leaves of both wild type and transgenic plants in response to MeJa, although the magnitude of the effect was greater in wild type plants. We discuss our findings with respect to the previously proposed threshold model of polyamine metabolism in plants subjected to abiotic stress.

Vitamin deficiency affects up to 50% of the world's population, disproportionately impacting on developing countries where populations endure monotonous, cereal-rich diets. Transgenic plants offer an effective way to increase the vitamin content of staple crops, but thus far it has only been possible to enhance individual vitamins. We created elite inbred South African transgenic corn plants in which the levels of 3 vitamins were increased specifically in the endosperm through the simultaneous modification of 3 separate metabolic pathways. The transgenic kernels contained 169-fold the normal amount of β-carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate. Levels of engineered vitamins remained stable at least through to the T3 homozygous generation. This achievement, which vastly exceeds any realized thus far by conventional breeding alone, opens the way for the development of nutritionally complete cereals to benefit the world's poorest people.

Polyamines are known to play important roles in plant stress tolerance but it has been difficult to determine precise functions for each type of polyamine and their interrelationships. To dissect the roles of putrescine from the higher polyamines spermidine and spermine, we generated transgenic rice plants constitutively expressing a heterologous S-adenosylmethionine decarboxylase (SAMDC) gene from Datura stramonium so that spermidine and spermine levels could be investigated while maintaining a constant putrescine pool. Whereas transgenic plants expressing arginine decarboxylase (ADC) produced higher levels of putrescine, spermidine and spermine, and were protected from drought stress, transgenic plants expressing SAMDC produced normal levels of putrescine and showed drought symptoms typical of wild type plants under stress, but the transgenic plants showed a much more robust recovery on return to normal conditions (90% full recovery compared to 25% partial recovery for wild type plants). At the molecular level, both wild type and transgenic plants showed transient reductions in the levels of endogenous ADC1 and SAMDC mRNA, but only wild type plants showed a spike in putrescine levels under stress. In transgenic plants, there was no spike in putrescine but a smooth increase in spermine levels at the expense of spermidine. These results confirm and extend the threshold model for polyamine activity in drought stress, and attribute individual roles to putrescine, spermidine and spermine.

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.

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 Ca(2+) dependence of the recombinant enzyme was confirmed by the biotin-labelled cadaverine-incorporation assay. In this communication we report the molecular and biochemical characterization of transgenic plants expressing rTGp and this sets the stage for establishing a bioreactor system for the production of transglutaminases in plants.

We have generated transgenic rice plants expressing the Datura stramonium adc gene and investigated their response to drought stress. We monitored the steady-state mRNA levels of genes involved in polyamine biosynthesis ( Datura adc , rice adc , and rice samdc ) and polyamine levels. Wild-type plants responded to the onset of drought stress by increasing endogenous putrescine levels, but this was insufficient to trigger the conversion of putrescine into spermidine and spermine (the agents that are believed to protect plants under stress). In contrast, transgenic plants expressing Datura adc produced much higher levels of putrescine under stress, promoting spermidine and spermine synthesis and ultimately protecting the plants from drought. We demonstrate clearly that the manipulation of polyamine biosynthesis in plants can produce drought-tolerant germplasm, and we propose a model consistent with the role of polyamines in the protection of plants against abiotic stress.

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.

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 compared to 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.

We used the polyamine biosynthetic pathway and rice as a relevant model to understand the genetic basis of variation in endogenous levels of metabolites and key enzymes involved in the pathway. Wild-type tissues and also tissues containing a commonly used selectable marker gene were employed. We detected a wide variation in levels of arginine decarboxylase activity and in the three polyamines, putrescine, spermidine and spermine, in different tissues and varieties, but this was not dependent on the presence of the selectable marker. A more-extensive profile of enzyme activities (ADC, ODC, SAMDC, DAO and PAO) and polyamine levels in different tissues was generated in two different varieties. Our results indicate that genetic background is important in terms of the basal levels of metabolites and enzyme activity, particularly in situations in which we aim to engineer metabolic pathways that are also encoded by homologous endogenous genes. We did not find any evidence that the presence of a selectable marker in any way influences enzyme activity or metabolite levels.

Transgenic rice (Oryza sativa L.) cell lines engineered with the pea diamine oxidase cDNA in antisense orientation under the control of two different promoters were recovered using particle bombardment. Plasmids p35Sdaoa and pEdaoa contained the pea damine oxidase cDNA driven by the CaMV35S and the pea ENOD12 nodulin promoter, respectively. Molecular analyses confirmed stable integration of the transgene and active transcription (mRNA) with both promoters driving the gene of interest. We observed a two-fold decrease in diamine oxidase activity (P < 0.01) in transformed callus lines expressing p35Sdaoa compared to wild-type or lines expressing the selectable marker gene alone (hpt). In cell lines transformed with pEdaoa, a 2.5-fold reduction in enzyme activity was measured (P < 0.01). Biochemical analysis of cellular polyamines from p35Sdaoa transformants indicated up to a 6-fold increase in putrescine levels (P < 0.001). Concentrations of higher polyamines also increased, with spermidine and spermine levels increasing 8- (P < 0.05) and 3.5-fold (P < 0.01) respectively. A maximum of 2.5-fold increase in putrescine (P < 0.001) and spermidine (P < 0.001) was measured in lines transformed with pEdaoa. No significant variation (P > 0.05) was observed in spermine levels in pEdaoa transformants. We demonstrate for the first time the down-regulation of an endogenous enzyme involved in polyamine metabolism in plants accompanied by a concomitant increase in the concentration of putrescine and spermidine.

Transgenic rice cell lines transformed with a heterologous cDNA derived from the arginine decarboxylase gene of oat, in an antisense orientation, exhibited significant (P0.05) 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 (P0.001) and spermidine (P0.01) but not spermine (P0.05) 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 (P0.05) in polyamine levels was observed. However, we measured significant reductions (P0.001) 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.

Multigene transformation (MGT) is becoming routine in plant biotechnology as researchers seek to generate more complex and ambitious phenotypes in transgenic plants. Every nuclear transgene requires its own promoter, so when coordinated expression is required, the introduction of multiple genes leads inevitably to two opposing strategies: different promoters may be used for each transgene, or the same promoter may be used over and over again. In the former case, there may be a shortage of different promoters with matching activities, but repetitious promoter use may in some cases have a negative impact on transgene stability and expression. Using illustrative case studies, we discuss promoter deployment strategies in transgenic plants that increase the likelihood of successful and stable multiple transgene expression. Work in our laboratory is supported by the Ministry of Science and Innovation, Spain (BFU2007-61413 and BIO2007-30738-E), a European Research Council Advanced Grant (BIOFORCE) to PC, and the CONSOLIDER Agrigenomics program funded by MICINN, Spain