Manuel Dubald - Academia.edu (original) (raw)

Papers by Manuel Dubald

Plant Biotechnol J, 2007

Plant chloroplasts are promising vehicles for recombinant protein production, but the process of ... more Plant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli . This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco ( Nicotiana tabacum ) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.

Plant Molecular Biology, Nov 30, 1991

A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, h... more A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes from a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.

Plant Cell Reports, 1993

A regeneration and transformation protocol for ramie (Boehmeria nivea Gaud.) is presented. Regene... more A regeneration and transformation protocol for ramie (Boehmeria nivea Gaud.) is presented. Regeneration was obtained from leaf discs placed on solid B-5 medium (Gamborg et al. 1968) containing adequate concentrations of auxin and cytokinin. Co-cultivation of leaf discs with Agrobacterium tumefaciens and subsequent regeneration resulted in transgenic plants as shown by Southern blot and analysis of expression of the GUS-marker

Methods in Molecular Biology, 2009

Methods in Molecular Biology, 2014

The biotechnological potential of plastid genetic engineering has been illustrated in a limited n... more The biotechnological potential of plastid genetic engineering has been illustrated in a limited number of higher plant species. We have developed a reproducible method to generate plastid transformants in soybean (Glycine max), a crop of major agronomic importance. The transformation vectors are delivered to embryogenic cultures by the particle gun method and selection performed using the aadA antibiotic resistance gene. Homoplasmy is established rapidly in the selected events without the need for further selection or regeneration cycles, and genes of interest can be expressed at a high level in green tissues. This is a significant step toward the commercial application of this technology.

Transgenic Research, 2006

The stability of a plastid transgene has been evaluated in soybean transformants over six generat... more The stability of a plastid transgene has been evaluated in soybean transformants over six generations. These transformants had integrated the aadA selection cassette in the intergenic region between the rps12/7 and trnV genes. Three independent homoplasmic T0 transformation events were selected and ten plants from each event propagated to generation T5 in the absence of selection pressure. No transgene rearrangement nor wild-type plastome were detected in generation T5 by Southern blot analysis. All tested progenies were uniformly resistant to spectinomycin. Therefore, soybean transformants of generations T0 and T5 appear to be genetically and phenotypically identical.

Plant Science, 2002

Heliomicin (from Heliothis virescens ) and drosomycin (from Drosophila melanogaster ), two cystei... more Heliomicin (from Heliothis virescens ) and drosomycin (from Drosophila melanogaster ), two cystein-rich antifungal peptides are produced by insects in response to septic injury. Both exhibit in vitro a rather similar broad-spectrum activity on phytopathogens, but microscopic observations indicate different modes of action. Heliomicin and drosomycin have been expressed in transgenic tobacco under the control of strong constitutive promoters, and targeted to the apoplast using signal peptides of plant origin. A significant enrichment of well-cleaved peptides with the expected molecular weight has been detected in the intercellular space of transgenic leaves. The recombinant peptides, partially purified from transgenic tobacco, exhibited in vitro the same antifungal properties on germination of spores of Botrytis cinerea and germ tube elongation as the recombinant peptides produced in Saccharomyces cerevisiae . Heliomicin and drosomycin expressed in transgenic tobacco confer a minor but statistically significant enhanced resistance to the fungal pathogen Cercospora nicotianae. #

PLANT PHYSIOLOGY, 2009

Genetically engineered chloroplasts have an extraordinary capacity to accumulate recombinant prot... more Genetically engineered chloroplasts have an extraordinary capacity to accumulate recombinant proteins. We have investigated in tobacco (Nicotiana tabacum) the possible consequences of such additional products on several parameters of plant development and composition. Plastid transformants were analyzed that express abundantly either bacterial enzymes, alkaline phosphatase (PhoA-S and PhoA-L) and 4-hydroxyphenyl pyruvate dioxygenase (HPPD), or a green fluorescent protein (GFP). In leaves, the HPPD and GFP recombinant proteins are the major polypeptides and accumulate to higher levels than Rubisco. Nevertheless, these engineered metabolic sinks do not cause a measurable difference in growth rate or photosynthetic parameters. The total amino acid content of transgenic leaves is also not significantly affected, showing that plant cells have a limited protein biosynthetic capacity. Recombinant products are made at the expense of resident proteins. Rubisco, which constitutes the major leaf amino acid store, is the most clearly and strongly down-regulated plant protein. This reduction is even more dramatic under conditions of limited nitrogen supply, whereas recombinant proteins accumulate to even higher relative levels. These changes are regulated posttranscriptionally since transcript levels of resident plastid genes are not affected. Our results show that plants are able to produce massive amounts of recombinant proteins in chloroplasts without profound metabolic perturbation and that Rubisco, acting as a nitrogen buffer, is a key player in maintaining homeostasis and limiting pleiotropic effects.

PLANT PHYSIOLOGY, 2004

Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential ant... more Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential antioxidant components of both human and animal diets. Because of their potential health benefits, there is a considerable interest in plants with increased or customized vitamin E content. Here, we have explored a new strategy to reach this goal. In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids. In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E. Therefore, we intended to increase the flux of these two compounds by deriving their synthesis directly at the level of prephenate. This was achieved by the expression of the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene in tobacco (Nicotiana tabacum) plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence. A massive accumulation of tocotrienols was observed in leaves. These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines. An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed. This work demonstrates that the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants. ; fax 33-438 -78 -50 -91. Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/

Plant Molecular Biology, 2005

We describe the generation of fertile and homoplasmic soybean plastid transformants, expressing t... more We describe the generation of fertile and homoplasmic soybean plastid transformants, expressing the Bacillus thuringiensis insecticidal protoxin Cry1Ab. Transgenes were targeted in the intergenic region of Glycine max plastome, between the rps12/7 and trnV genes and selection was carried out using the aadA gene encoding spectinomycin resistance. Molecular analysis confirmed the integration of the cry1Ab and aadA expression cassettes at the expected location in the soybean plastome, and the transmission of the transgenes to the next generation. Western blot analyses showed that the Cry1Ab protoxin is highly expressed in leaves, stems and seeds, but not in roots. Its expression confers strong insecticidal activity to the generated transgenic soybean, as exemplified with velvetbean caterpillar (Anticarsia gemmatalis) w This manuscript is dedicated to the memory of Fabien Goutorbe. 58:659-668 Ó Springer 2005

Plant Molecular Biology, 1991

A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, h... more A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes from a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.

Plant Cell Reports, 1993

A regeneration and transformation protocol for ramie (Boehmeria nivea Gaud.) is presented. Regene... more A regeneration and transformation protocol for ramie (Boehmeria nivea Gaud.) is presented. Regeneration was obtained from leaf discs placed on solid B-5 medium (Gamborg ct a1.1968) containing adequate concentrations of auxin and cytokinin. Co-cultivation of leaf discs with Agrobacterium tumefaciens and subsequent regeneration resulted in transgenic plants as shown by Southern blot and analysis of expression of the GUS-marker gene.

Plant Biotechnology Journal, 2007

Plant chloroplasts are promising vehicles for recombinant protein production, but the process of ... more Plant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli . This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco ( Nicotiana tabacum ) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.

Plant Biotechnology Journal, 2007

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is part of the biosynthetic pathway leading to p... more Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is part of the biosynthetic pathway leading to plastoquinone and vitamin E. This enzyme is also the molecular target of various new bleaching herbicides for which genetically engineered tolerant crops are being developed. We have expressed a sensitive bacterial hppd gene from Pseudomonas fluorescens in plastid transformants of tobacco and soybean and characterized in detail the recombinant lines. HPPD accumulates to approximately 5% of total soluble protein in transgenic chloroplasts of both species. As a result, the soybean and tobacco plastid transformants acquire a strong herbicide tolerance, performing better than nuclear transformants. In contrast, the over-expression of HPPD has no significant impact on the vitamin E content of leaves or seeds, quantitatively or qualitatively. A new strategy is presented and exemplified in tobacco which allows the rapid generation of antibiotic marker-free plastid transformants containing the herbicide tolerance gene only. This work reports, for the first time, the plastome engineering for herbicide tolerance in a major agronomic crop, and a technology leading to marker-free lines for this trait.

Plant Biotechnol J, 2007

Plant chloroplasts are promising vehicles for recombinant protein production, but the process of ... more Plant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli . This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco ( Nicotiana tabacum ) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.

Plant Molecular Biology, Nov 30, 1991

A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, h... more A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes from a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.

Plant Cell Reports, 1993

A regeneration and transformation protocol for ramie (Boehmeria nivea Gaud.) is presented. Regene... more A regeneration and transformation protocol for ramie (Boehmeria nivea Gaud.) is presented. Regeneration was obtained from leaf discs placed on solid B-5 medium (Gamborg et al. 1968) containing adequate concentrations of auxin and cytokinin. Co-cultivation of leaf discs with Agrobacterium tumefaciens and subsequent regeneration resulted in transgenic plants as shown by Southern blot and analysis of expression of the GUS-marker

Methods in Molecular Biology, 2009

Methods in Molecular Biology, 2014

The biotechnological potential of plastid genetic engineering has been illustrated in a limited n... more The biotechnological potential of plastid genetic engineering has been illustrated in a limited number of higher plant species. We have developed a reproducible method to generate plastid transformants in soybean (Glycine max), a crop of major agronomic importance. The transformation vectors are delivered to embryogenic cultures by the particle gun method and selection performed using the aadA antibiotic resistance gene. Homoplasmy is established rapidly in the selected events without the need for further selection or regeneration cycles, and genes of interest can be expressed at a high level in green tissues. This is a significant step toward the commercial application of this technology.

Transgenic Research, 2006

The stability of a plastid transgene has been evaluated in soybean transformants over six generat... more The stability of a plastid transgene has been evaluated in soybean transformants over six generations. These transformants had integrated the aadA selection cassette in the intergenic region between the rps12/7 and trnV genes. Three independent homoplasmic T0 transformation events were selected and ten plants from each event propagated to generation T5 in the absence of selection pressure. No transgene rearrangement nor wild-type plastome were detected in generation T5 by Southern blot analysis. All tested progenies were uniformly resistant to spectinomycin. Therefore, soybean transformants of generations T0 and T5 appear to be genetically and phenotypically identical.

Plant Science, 2002

Heliomicin (from Heliothis virescens ) and drosomycin (from Drosophila melanogaster ), two cystei... more Heliomicin (from Heliothis virescens ) and drosomycin (from Drosophila melanogaster ), two cystein-rich antifungal peptides are produced by insects in response to septic injury. Both exhibit in vitro a rather similar broad-spectrum activity on phytopathogens, but microscopic observations indicate different modes of action. Heliomicin and drosomycin have been expressed in transgenic tobacco under the control of strong constitutive promoters, and targeted to the apoplast using signal peptides of plant origin. A significant enrichment of well-cleaved peptides with the expected molecular weight has been detected in the intercellular space of transgenic leaves. The recombinant peptides, partially purified from transgenic tobacco, exhibited in vitro the same antifungal properties on germination of spores of Botrytis cinerea and germ tube elongation as the recombinant peptides produced in Saccharomyces cerevisiae . Heliomicin and drosomycin expressed in transgenic tobacco confer a minor but statistically significant enhanced resistance to the fungal pathogen Cercospora nicotianae. #

PLANT PHYSIOLOGY, 2009

Genetically engineered chloroplasts have an extraordinary capacity to accumulate recombinant prot... more Genetically engineered chloroplasts have an extraordinary capacity to accumulate recombinant proteins. We have investigated in tobacco (Nicotiana tabacum) the possible consequences of such additional products on several parameters of plant development and composition. Plastid transformants were analyzed that express abundantly either bacterial enzymes, alkaline phosphatase (PhoA-S and PhoA-L) and 4-hydroxyphenyl pyruvate dioxygenase (HPPD), or a green fluorescent protein (GFP). In leaves, the HPPD and GFP recombinant proteins are the major polypeptides and accumulate to higher levels than Rubisco. Nevertheless, these engineered metabolic sinks do not cause a measurable difference in growth rate or photosynthetic parameters. The total amino acid content of transgenic leaves is also not significantly affected, showing that plant cells have a limited protein biosynthetic capacity. Recombinant products are made at the expense of resident proteins. Rubisco, which constitutes the major leaf amino acid store, is the most clearly and strongly down-regulated plant protein. This reduction is even more dramatic under conditions of limited nitrogen supply, whereas recombinant proteins accumulate to even higher relative levels. These changes are regulated posttranscriptionally since transcript levels of resident plastid genes are not affected. Our results show that plants are able to produce massive amounts of recombinant proteins in chloroplasts without profound metabolic perturbation and that Rubisco, acting as a nitrogen buffer, is a key player in maintaining homeostasis and limiting pleiotropic effects.

PLANT PHYSIOLOGY, 2004

Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential ant... more Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential antioxidant components of both human and animal diets. Because of their potential health benefits, there is a considerable interest in plants with increased or customized vitamin E content. Here, we have explored a new strategy to reach this goal. In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids. In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E. Therefore, we intended to increase the flux of these two compounds by deriving their synthesis directly at the level of prephenate. This was achieved by the expression of the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene in tobacco (Nicotiana tabacum) plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence. A massive accumulation of tocotrienols was observed in leaves. These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines. An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed. This work demonstrates that the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants. ; fax 33-438 -78 -50 -91. Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/

Plant Molecular Biology, 2005

We describe the generation of fertile and homoplasmic soybean plastid transformants, expressing t... more We describe the generation of fertile and homoplasmic soybean plastid transformants, expressing the Bacillus thuringiensis insecticidal protoxin Cry1Ab. Transgenes were targeted in the intergenic region of Glycine max plastome, between the rps12/7 and trnV genes and selection was carried out using the aadA gene encoding spectinomycin resistance. Molecular analysis confirmed the integration of the cry1Ab and aadA expression cassettes at the expected location in the soybean plastome, and the transmission of the transgenes to the next generation. Western blot analyses showed that the Cry1Ab protoxin is highly expressed in leaves, stems and seeds, but not in roots. Its expression confers strong insecticidal activity to the generated transgenic soybean, as exemplified with velvetbean caterpillar (Anticarsia gemmatalis) w This manuscript is dedicated to the memory of Fabien Goutorbe. 58:659-668 Ó Springer 2005

Plant Molecular Biology, 1991

A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, h... more A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes from a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.

Plant Cell Reports, 1993

A regeneration and transformation protocol for ramie (Boehmeria nivea Gaud.) is presented. Regene... more A regeneration and transformation protocol for ramie (Boehmeria nivea Gaud.) is presented. Regeneration was obtained from leaf discs placed on solid B-5 medium (Gamborg ct a1.1968) containing adequate concentrations of auxin and cytokinin. Co-cultivation of leaf discs with Agrobacterium tumefaciens and subsequent regeneration resulted in transgenic plants as shown by Southern blot and analysis of expression of the GUS-marker gene.

Plant Biotechnology Journal, 2007

Plant chloroplasts are promising vehicles for recombinant protein production, but the process of ... more Plant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli . This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco ( Nicotiana tabacum ) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.

Plant Biotechnology Journal, 2007

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is part of the biosynthetic pathway leading to p... more Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is part of the biosynthetic pathway leading to plastoquinone and vitamin E. This enzyme is also the molecular target of various new bleaching herbicides for which genetically engineered tolerant crops are being developed. We have expressed a sensitive bacterial hppd gene from Pseudomonas fluorescens in plastid transformants of tobacco and soybean and characterized in detail the recombinant lines. HPPD accumulates to approximately 5% of total soluble protein in transgenic chloroplasts of both species. As a result, the soybean and tobacco plastid transformants acquire a strong herbicide tolerance, performing better than nuclear transformants. In contrast, the over-expression of HPPD has no significant impact on the vitamin E content of leaves or seeds, quantitatively or qualitatively. A new strategy is presented and exemplified in tobacco which allows the rapid generation of antibiotic marker-free plastid transformants containing the herbicide tolerance gene only. This work reports, for the first time, the plastome engineering for herbicide tolerance in a major agronomic crop, and a technology leading to marker-free lines for this trait.