Chile (Capsicum annuum) plants transformed with the RB gene from Solanum bulbocastanum are resistant to Phytophthora capsici.

Phytophthora capsici is a soil borne pathogen, and is among the most destructive pathogens for Capsicum annuum (chile). P. capsici is known to cause diseases on all parts of the chile plants. Therefore, it requires independent resistance genes to control disease symptoms that are induced by each of the P. capsici strains. This requirement of multiple resistance genes to confer resistance to P. capsici, in chile makes breeding for resistance a daunting pursuit. Against this backdrop, a genetic engineering approach would be to introduce a broad host resistance gene into chile in order to protect it from different races of P. capsici. Notably, a broad host resistance gene RB from Solanum bulbocastanum has been shown to confer resistance to P. infestans in both S. tuberosum and S. lycopersicum. We agroinfiltrated the RB gene into the leaves of susceptible chile plants, demonstrating that the gene is also capable of lending resistance to P. capsici in chile. We introduced the RB gene into chile by developing an Agrobacterium tumefaciens mediated transformation system. The integration of the RB gene into the genome of the primary transformants and its subsequent transfer to the F1 generation was confirmed by genomic PCR using primers specific for the RB gene. A 3:1 ratio for the presence and absence of the RB gene was observed in the F1 progeny. In addition to showing resistance to P. capsici in a leaf inoculation experiment, about 30% of the F1 progeny also exhibited resistance to root inoculation. Our data, when taken together, suggests that the RB gene from S. bulbocastanum confers resistance against P. capsici in C. annuum, thereby demonstrating that the RB gene has an even broader host range than reported in the literature-both in terms of the host and the pathogen.

Distinct nodule and leaf functions of two different sucrose phosphate synthases in alfalfa.

MAIN CONCLUSION: In alfalfa, the B form of Sucrose phosphate synthase synthesizes sucrose in the leaves while the A form participates in regulatory cycles of synthesis/breakdown of sucrose/starch in the root nodules. Sucrose (Suc) is the major stable product of photosynthesis that is transported to all heterotrophic organs as a source of energy and carbon. The enzyme sucrose phosphate synthase (SPS) catalyzes the synthesis of Suc. Besides the leaves, SPS is also found in heterotrophic organs. There are two isoforms of SPS in alfalfa (Medicago sativa): SPSA and SPSB. While SPSA is expressed in the vasculature of all the organs and in the N2-fixing zone in the nodules, SPSB is exclusively expressed in the photosynthetic cells. Two classes of alfalfa transformants were produced, one with a gene construct consisting of the alfalfa SPSA promoter and the other with the SPSB promoter-both driving the maize SPS coding region-referred to as SPSA-ZmSPS and SPSB-ZmSPS, respectively. Both classes of transformants showed increased growth compared to control plants. The SPSB-ZmSPS transformants showed increased SPS protein levels and activity along with a significant increase in the Suc levels in the leaves. The SPSA-ZmSPS transformants showed an increase in the SPS protein level and enzyme activity both in the leaves and the nodules with no increase in Suc content in the leaves but a substantial increase in the nodules. Both SPSA and SPSB have unique roles in the nodules (sink) and leaves (source). SPSB is responsible for the synthesis of Suc in the photosynthetic cells and SPSA participates in a regulatory cycle in which Suc is simultaneously degraded and re-synthesized; both these functions contribute to plant growth in rhizobia nodulated alfalfa plants.

Comparison of alfalfa plants overexpressing glutamine synthetase with those overexpressing sucrose phosphate synthase demonstrates a signaling mechanism integrating carbon and nitrogen metabolism between the leaves and nodules.

Alfalfa, like other legumes, establishes a symbiotic relationship with the soil bacteria, Sinorhizobium meliloti, which results in the formation of the root nodules. Nodules contain the bacteria enclosed in a membrane-bound vesicle, the symbiosome where it fixes atmospheric N2 and converts it into ammonia using the bacterial enzyme, nitrogenase. The ammonia released into the cytoplasm from the symbiosome is assimilated into glutamine (Gln) using carbon skeletons produced by the metabolism of sucrose (Suc), which is imported into the nodules from the leaves. The key enzyme involved in the synthesis of Suc in the leaves is sucrose phosphate synthase (SPS) and glutamine synthetase (GS) is the enzyme with a role in ammonia assimilation in the root nodules. Alfalfa plants, overexpressing SPS or GS, or both showed increased growth and an increase in nodule function. The endogenous genes for the key enzymes in C/N metabolism showed increased expression in the nodules of both sets of transformants. Furthermore, the endogenous SPS and GS genes were also induced in the leaves and nodules of the transformants, irrespective of the transgene, suggesting that the two classes of plants share a common signaling pathway regulating C/N metabolism in the nodules. This study reaffirms the utility of the nodulated legume plant to study C/N interaction and the cross talk between the source and sink for C and N.

An intragenic approach to confer glyphosate resistance in chile (Capsicum annuum) by introducing an in vitro mutagenized chile EPSPS gene encoding for a glyphosate resistant EPSPS protein.

Chile pepper (Capsicum annuum) is an important high valued crop worldwide, and when grown on a large scale has problems with weeds. One important herbicide used is glyphosate. Glyphosate inactivates the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the synthesis of aromatic amino acids. A transgenic approach towards making glyphosate resistant plants, entails introducing copies of a gene encoding for glyphosate-resistant EPSPS enzyme into the plant. The main objective of our work was to use an intragenic approach to confer resistance to glyphosate in chile which would require using only chile genes for transformation including the selectable marker. Tobacco was used as the transgenic system to identify different gene constructs that would allow for the development of the intragenic system for chile, since chile transformation is inefficient. An EPSPS gene was isolated from chile and mutagenized to introduce substitutions that are known to make the encoded enzyme resistant to glyphosate. The promoter for EPSPS gene was isolated from chile and the mutagenized chile EPSPS cDNA was engineered behind both the CaMV35S promoter and the EPSPS promoter. The leaves from the transformants were checked for resistance to glyphosate using a cut leaf assay. In tobacco, though both gene constructs exhibited some degree of resistance to glyphosate, the construct with the CaMV35S promoter was more effective and as such chile was transformed with this gene construct. The chile transformants showed resistance to low concentrations of glyphosate. Furthermore, preliminary studies showed that the mutated EPSPS gene driven by the CaMV35S promoter could be used as a selectable marker for transformation. We have shown that an intragenic approach can be used to confer glyphosate-resistance in chile. However, we need a stronger chile promoter and a mutated chile gene that encodes for a more glyphosate resistant EPSPS protein.

Transgenic alfalfa (Medicago sativa) with increased sucrose phosphate synthase activity shows enhanced growth when grown under N2-fixing conditions.

MAIN CONCLUSION: Overexpression of SPS in alfalfa is accompanied by early flowering, increased plant growth and an increase in elemental N and protein content when grown under N2-fixing conditions. Sucrose phosphate synthase (SPS; EC 2.3.1.14) is the key enzyme in the synthesis of sucrose in plants. The outcome of overexpression of SPS in different plants using transgenic approaches has been quite varied, but the general consensus is that increased SPS activity is associated with the production of new sinks and increased sink strength. In legumes, the root nodule is a strong C sink and in this study our objective was to see how increasing SPS activity in a legume would affect nodule number and function. Here we have transformed alfalfa (Medicago sativa, cv. Regen SY), with a maize SPS gene driven by the constitutive CaMV35S promoter. Our results showed that overexpression of SPS in alfalfa, is accompanied by an increase in nodule number and mass and an overall increase in nitrogenase activity at the whole plant level. The nodules exhibited an increase in the level of key enzymes contributing to N assimilation including glutamine synthetase and asparagine synthetase. Moreover, the stems of the transformants showed higher level of the transport amino acids, Asx, indicating increased export of N from the nodules. The transformants exhibited a dramatic increase in growth both of the shoots and roots, and earlier flowering time, leading to increased yields. Moreover, the transformants showed an increase in elemental N and protein content. The overall conclusion is that increased SPS activity improves the N status and plant performance, suggesting that the availability of more C in the form of sucrose enhances N acquisition and assimilation in the nodules.

Impact of concurrent overexpression of cytosolic glutamine synthetase (GS1) and sucrose phosphate synthase (SPS) on growth and development in transgenic tobacco.

MAIN CONCLUSION: The outcome of simultaneously increasing SPS and GS activities in transgenic tobacco, suggests that sucrose is the major determinant of growth and development, and is not affected by changes in N assimilation. Carbon (C) and nitrogen (N) are the major components required for plant growth and the metabolic pathways for C and N assimilation are very closely interlinked. Maintaining an appropriate balance or ratio of sugar to nitrogen metabolites in the cell, is important for the regulation of plant growth and development. To understand how C and N metabolism interact, we manipulated the expression of key genes in C and N metabolism individually and concurrently and checked for the repercussions. Transgenic tobacco plants with a cytosolic soybean glutamine synthetase (GS1) gene and a sucrose phosphate synthase (SPS) gene from maize, both driven by the CaMV 35S promoter were produced. Co-transformants, with both the transgenes were produced by sexual crosses. While GS is the key enzyme in N assimilation, involved in the synthesis of glutamine, SPS plays a key role in C metabolism by catalyzing the synthesis of sucrose. Moreover, to check if nitrate has any role in this interaction, the plants were grown under both low and high nitrogen. The SPS enzyme activity in the SPS and SPS/GS1 co-transformants were the same under both nitrogen regimens. However, the GS activity was lower in the co-transformants compared to the GS1 transformants, specifically under low nitrogen conditions. The GS1/SPS transformants showed a phenotype similar to the SPS transformants, suggesting that sucrose is the major determinant of growth and development in tobacco, and its effect is only marginally affected by increased N assimilation. Sucrose may be functioning in a metabolic capacity or as a signaling molecule.

The 5' untranslated region of the soybean cytosolic glutamine synthetase ß(1) gene contains prokaryotic translation initiation signals and acts as a translational enhancer in plants.

Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In plants, it occurs as two major isoforms, a cytosolic form (GS(1)) and a nuclear encoded chloroplastic form. The focus of this paper is to determine the role of the 5'UTR of a GS(1) gene. GS(1) gene constructs with and without its 5' and 3' UTRs, driven by a constitutive promoter, were agroinfiltrated into tobacco leaves and the tissues were analyzed for both transgene transcript and protein accumulation. The constructs were also tested in an in vitro transcription/translation system and in Escherichia coli. Our results showed that while the 3'UTR functioned in the destabilization of the transcript, the 5'UTR acted as a translation enhancer in plant cells but not in the in vitro translation system. The 5'UTR of the GS(1) gene when placed in front of a reporter gene (uidA), showed a 20-fold increase in the level of GUS expression in agroinfiltrated leaves when compared to the same gene construct without the 5'UTR. The 5'UTR-mediated translational enhancement is probably another step in the regulation of GS in plants. The presence of the GS(1) 5'UTR in front of the GS(1) coding region allowed for its translation in E. coli suggesting the commonality of the translation initiation mechanism for this gene between plants and bacteria.

The 3' untranslated region of the two cytosolic glutamine synthetase (GS(1)) genes in alfalfa (Medicago sativa) regulates transcript stability in response to glutamine.

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia with glutamate to produce glutamine. The GS enzyme is located either in the chloroplast (GS(2)) or in the cytoplasm (GS(1)). GS(1) is encoded by a small gene family and the members exhibit differential expression pattern mostly attributed to transcriptional regulation. Based on our recent finding that a soybean GS(1) gene, Gmglnß ( 1 ) is subject to its 3'UTR-mediated post-transcriptional regulation as a transgene in alfalfa (Medicago sativa) we have raised the question of whether the 3'UTR-mediated transcript destabilization is a more universal phenomenon. Gene constructs consisting of the CaMV35S promoter driving the reporter gene, GUS, followed by the 3'UTRs of the two alfalfa GS(1) genes, MsGSa and MsGSb, were introduced into alfalfa and tobacco. The analysis of these transformants suggests that while both the 3'UTRs promote transcript turnover, the MsGSb 3'UTR is more effective than the MsGSa 3'UTR. However, both the 3'UTRs along with Gmglnß ( 1 ) 3'UTR respond to nitrate as a trigger in transcript turnover. More detailed analysis points to glutamine rather than nitrate as the mediator of transcript turnover. Our data suggests that the 3'UTR-mediated regulation of GS(1) genes at the level of transcript turnover is probably universal and is used for fine-tuning the expression in keeping with the availability of the substrates.

Nodule-enhanced expression of a sucrose phosphate synthase gene member (MsSPSA) has a role in carbon and nitrogen metabolism in the nodules of alfalfa (Medicago sativa L.).

Sucrose phosphate synthase (SPS) catalyzes the first step in the synthesis of sucrose in photosynthetic tissues. We characterized the expression of three different isoforms of SPS belonging to two different SPS gene families in alfalfa (Medicago sativa L.), a previously identified SPS (MsSPSA) and two novel isoforms belonging to class B (MsSPSB and MsSPSB3). While MsSPSA showed nodule-enhanced expression, both MsSPSB genes exhibited leaf-enhanced expression. Alfalfa leaf and nodule SPS enzymes showed differences in chromatographic and electrophoretic migration and differences in V (max) and allosteric regulation. The root nodules in legume plants are a strong sink for photosynthates with its need for ATP, reducing power and carbon skeletons for dinitrogen fixation and ammonia assimilation. The expression of genes encoding SPS and other key enzymes in sucrose metabolism, sucrose phosphate phosphatase and sucrose synthase, was analyzed in the leaves and nodules of plants inoculated with Sinorhizobium meliloti. Based on the expression pattern of these genes, the properties of the SPS isoforms and the concentration of starch and soluble sugars in nodules induced by a wild type and a nitrogen fixation deficient strain, we propose that SPS has an important role in the control of carbon flux into different metabolic pathways in the symbiotic nodules.

The 3' untranslated region of a soybean cytosolic glutamine synthetase (GS1) affects transcript stability and protein accumulation in transgenic alfalfa.

Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). Glutamine synthetase 1 is regulated in different plants at the transcriptional level and there are some reports of regulation at the level of protein stability. Here we present data that clearly establish that GS1 in plants is also regulated at the level of transcript turnover and at the translational level. Using a Glycine max (soybean) GS1 transgene, with and without its 3' untranslated region (UTR), driven by the constitutive CaMV 35S promoter in Medicago sativa (alfalfa) and Nicotiana tabacum (tobacco), we show that the 3' UTR plays a major role in both transcript turnover and translation repression in both the leaves and the nodules. Our data suggest that the 3' UTR mediated turnover of the transcript is regulated by a nitrogen metabolite or carbon/nitrogen ratios. We also show that the 3' UTR of the gene for the soybean GS1 confers post-transcriptional regulation on a reporter gene. Our dissection of post-transcriptional and translational levels of regulation of GS in plants shows that the situation in plants strongly resembles that in other organisms where GS is regulated at almost all levels. Multistep regulation of GS shows the high priority given by organisms to regulating and ensuring optimal control of nitrogen substrates and preventing overproduction of glutamine and drainage of the glutamate pool.

Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively over-expressing a cytosolic glutamine synthetase gene.

Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS1 gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS1 polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22-24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants.

Cytosolic glutamine synthetase in soybean is encoded by a multigene family, and the members are regulated in an organ-specific and developmental manner.

Gln synthetase (GS) is the key enzyme in N metabolism and it catalyzes the synthesis of Gln from glutamic acid, ATP, and NH4+. There are two major isoforms of GS in plants, a cytosolic form (GS1) and a chloroplastic form (GS2). In leaves, GS2 functions to assimilate ammonia produced by nitrate reduction and photorespiration, and GS1 is the major isoform assimilating NH3 produced by all other metabolic processes, including symbiotic N2 fixation in the nodules. GS1 is encoded by a small multigene family in soybean (Glycine max), and cDNA clones for the different members have been isolated. Based on sequence divergence in the 3'-untranslated region, three distinct classes of GS1 genes have been identified (alpha, beta, and gamma). Genomic Southern analysis and analysis of hybrid-select translation products suggest that each class has two distinct members. The alpha forms are the major isoforms in the cotyledons and young roots. The beta forms, although constitutive in their expression pattern, are ammonia inducible and show high expression in N2-fixing nodules. The gamma1 gene appears to be more nodule specific, whereas the gamma2 gene member, although nodule enhanced, is also expressed in the cotyledons and flowers. The two members of the alpha and beta class of GS1 genes show subtle differences in the expression pattern. Analysis of the promoter regions of the gamma1 and gamma2 genes show sequence conservation around the TATA box but complete divergence in the rest of the promoter region. We postulate that each member of the three GS1 gene classes may be derived from the two ancestral genomes from which the allotetraploid soybean was derived.