Isolde Saalbach

Ectopic Expression of an Amino Acid Transporter (VfAAP1) in Seeds of Vicia narbonensis and Pea Increases Storage Proteins

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.

Increasing amino acid supply in pea embryos reveals specific interactions of N and C metabolism, and highlights the importance of mitochondrial metabolism

SUMMARY The application of nitrogen to legumes regulates seed metabolism and composition. We recently showed that the seed-specific overexpression of amino acid permease VfAAP1 increases amino acid supply, and the levels of N and protein in the seeds. Two consecutive field trials using Pisum sativum AAP1 lines confirmed increases in the levels of N and globulin in seed; however, compensatory changes of sucrose/starch and individual seed weight were also observed. We present a comprehensive analysis of AAP1 seeds using combinatorial transcript and metabolite profiling to monitor the effects of nitrogen supply on seed metabolism. AAP1 seeds have increased amino acids and stimulated gene expression associated with storage protein synthesis, maturation, deposition and vesicle trafficking. Transcript/metabolite changes reveal the channelling of surplus N into the transient storage pools asparagine and arginine, indicating that asparagine synthase is transcriptionally activated by high N levels and/or C limitation. Increased C-acceptor demand for amino acid synthesis, resulting from elevated levels of N in seeds, initiates sucrose mobilization and sucrose-dependent pathways via sucrose synthase, glycolysis and the TCA cycle. The AAP1 seeds display a limitation in C, which leads to the catabolism of arginine, glutamic acid and methionine to putrescine, beta-alanine and succinate. Mitochondria are involved in the coordination of C/N metabolism, with branched-chain amino acid catabolism and a gamma-amino-butyric acid shunt. AAP1 seeds contain higher levels of ABA, which is possibly involved in storage-associated gene expression and the N-dependent stimulation of sucrose mobilization, indicating that a signalling network of C, N and ABA is operating during seed maturation. These results demonstrate that legume seeds have a high capacity to regulate N:C ratios, and highlight the importance of mitochondria in the control of N-C balance and amino acid homeostasis.

The Role of Proteolysis in the Processing and Assembly of 11S Seed Globulins

11S seed storage proteins are synthesized as precursors that are cleaved post-translationally in storage vacuoles by an asparaginyl endopeptidase. To study the specificity of the reaction catalyzed by this asparaginyl endopeptidase, we prepared a series of octapeptides and mutant legumin B and G4 glycinin subunits. These contained amino acid mutations in the region surrounding the cleavage site. The endopeptidase had an absolute specificity for Asn on the N-terminal side of the severed peptide bond but exhibited little specificity for amino acids on the C-terminal side. The ability of unmodified and modified subunits to assemble into hexamers after post-translational modification was evaluated. Cleavage of subunits in trimers is required for hexamer assembly in vitro. Products from a mutant gene encoding a noncleavable prolegumin subunit (LeBΔN281) accumulated as trimers in seed of transgenic tobacco, but products from the unmodified prolegumin B gene accumulated as hexamers. Therefore, the asparaginyl endopeptidase is required for hexamer assembly.

Increasing Sucrose Uptake Capacity of Wheat Grains Stimulates Storage Protein Synthesis

Increasing grain sink strength by improving assimilate uptake capacity could be a promising approach toward getting higher yield. The barley (Hordeum vulgare) sucrose transporter HvSUT1 (SUT) was expressed under control of the endosperm-specific Hordein B1 promoter (HO). Compared with the wild type, transgenic HOSUT grains take up more sucrose (Suc) in vitro, showing that the transgene is functional. Grain Suc levels are not altered, indicating that Suc fluxes are influenced rather than steady-state levels. HOSUT grains have increased percentages of total nitrogen and prolamins, which is reflected in increased levels of phenylalanine, tyrosine, tryptophan, isoleucine, and leucine at late grain development. Transcript profiling indicates specific stimulation of prolamin gene expression at the onset of storage phase. Changes in gene expression and metabolite levels related to carbon metabolism and amino acid biosynthesis suggest deregulated carbon-nitrogen balance, which together indicate carbon sufficiency and relative depletion of nitrogen. Genes, deregulated together with prolamin genes, might represent candidates, which respond positively to assimilate supply and are related to sugar-starch metabolism, cytokinin and brassinosteroid functions, cell proliferation, and sugar/abscisic acid signaling. Genes showing inverse expression patterns represent potential negative regulators. It is concluded that HvSUT1 overexpression increases grain protein content but also deregulates the metabolic status of wheat (Triticum aestivum) grains, accompanied by up-regulated gene expression of positive and negative regulators related to sugar signaling and assimilate supply. In HOSUT grains, alternating stimulation of positive and negative regulators causes oscillatory patterns of gene expression and highlights the capacity and great flexibility to adjust wheat grain storage metabolism in response to metabolic alterations.

A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertlrolletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes

The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and β-glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R2 progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUCl2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants.

ADP-glucose pyrophosphorylase-deficient pea embryos reveal specific transcriptional and metabolic changes of carbon-nitrogen metabolism and stress responses.

We present a comprehensive analysis of ADP-glucose pyrophosphorylase (AGP)-repressed pea (Pisum sativum) seeds using transcript and metabolite profiling to monitor the effects that reduced carbon flow into starch has on carbon-nitrogen metabolism and related pathways. Changed patterns of transcripts and metabolites suggest that AGP repression causes sugar accumulation and stimulates carbohydrate oxidation via glycolysis, tricarboxylic acid cycle, and mitochondrial respiration. Enhanced provision of precursors such as acetyl-coenzyme A and organic acids apparently support other pathways and activate amino acid and storage protein biosynthesis as well as pathways fed by cytosolic acetyl-coenzyme A, such as cysteine biosynthesis and fatty acid elongation/metabolism. As a consequence, the resulting higher nitrogen (N) demand depletes transient N storage pools, specifically asparagine and arginine, and leads to N limitation. Moreover, increased sugar accumulation appears to stimulate cytokinin-mediated cell proliferation pathways. In addition, the deregulation of starch biosynthesis resulted in indirect changes, such as increased mitochondrial metabolism and osmotic stress. The combined effect of these changes is an enhanced generation of reactive oxygen species coupled with an up-regulation of energy-dissipating, reactive oxygen species protection, and defense genes. Transcriptional activation of mitogen-activated protein kinase pathways and oxylipin synthesis indicates an additional activation of stress signaling pathways. AGP-repressed embryos contain higher levels of jasmonate derivatives; however, this increase is preferentially in nonactive forms. The results suggest that, although metabolic/osmotic alterations in iAGP pea seeds result in multiple stress responses, pea seeds have effective mechanisms to circumvent stress signaling under conditions in which excessive stress responses and/or cellular damage could prematurely initiate senescence or apoptosis.

Stable Expression of the Sulphur-rich 2S Albumin Gene in Transgenic Vicia narbonensis Increases the Methionine Content of Seeds

Summary Both the narbon bean ( Vicia narbonensis ) and tobacco plants were transformed using Agrobacterium tumefaciens strains containing binary vectors with the gene coding for the methionine-rich 2S albumin of Brazil nuts under the control of either the Cauliflower Mosaic Virus 35S promoter or the legumin B4 promoter from V. faba . Stable integration in the genome was verified by both Southern blotting and segregation analysis. Under the control of the viral promoter, 2S albumin was produced in a variety of tissues at a relatively low level in both species. The LeB4 promoter conferred seed-specific high level expression in V. narbonensis and low level seed-specific expression in tobacco. A total of 50 independent transgenic narbon bean lines containing the LeB4-2S albumin construct have so far been isolated. Eighteen of these lines were further characterised and several stable homozygous lines established. Comparisons of homozygous and heterozygous progeny in these strains revealed that a doubling of the number of genes resulted in an approximate doubling in the amount of gene product. The absolute protein levels reached varied between lines. Amino acid analysis revealed an approximate 3 fold increase in the methionine content of seed protein in one R 0 line. Further analysis of these lines is aimed at determining how to achieve high level production of foreign proteins in seeds and more specifically at improving the nutritional quality of the seed protein of legumes.

Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens

BackgroundCoccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds.ResultsUsing the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability). Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts.ConclusionThe results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market.

Seed specific expression of the 2S albumin gene from Brazil nut (Bertholletia excelsa) in transgenicVicia narbonensis

In order to evaluate the possibility of enhancing the sulphur amino acids in grain legumes, we have transferred the gene for the methionine-rich 2S albumin from Brazil nut (Bertholletia excelsa) controlled by a seed-specific promoter intoVicia narbonensis. The coding region of the 2S albumin gene that has been completely synthesized was fused to the seed-specific leguminB4 promoter fromVicia faba. Transgenic lines ofV. narbonensis were generated viaAgrobacterium-mediated gene analysis of leaves, stems, roots and seeds of four R0 plants revealed that the expression of the foreign 2S albumin gene occurred in a seed-specific manner and that the foreign protein is present at levels ranging from 1% to 4.8% of total SDS-soluble seed protein. Since the current protocol for transformation ofV. narbonensis has not yet been published, a detailed description of the method is given.

High-level expression of a single-chain Fv fragment (scFv) antibody in transgenic pea seeds

Summary The field pea(Pisum sativumL.)is a protein-rich crop and pea seeds are well suited for the production of recombinant proteins. Here we show, that recombinant antibodies can be accumulated in transgenic pea seeds in a homozygous transgenic line up to 2 percnt; of total soluble seed protein. The expression was controlled by the seed-specific USP promoter and the transgenic single-chain Fv antibody protein was retended in the endoplasmic reticulum. The stable inheritance, shown by investigation of the high-level accumulation in the R3 offspring is another important feature of this new antibody production system. The suitability of transgenic pea seeds as an economically potent production system for recombinant antibodies is clearly demonstrated by our results.