Metabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance

Rathinasabapathi, Bala; McCue, Kent F.; Gage, Douglas A.; Hanson, Andrew D.

doi:10.1007/bf00192524

Cited by 132 publications

(100 citation statements)

References 34 publications

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“…1H NMR spectroscopy study showed that rice plants incorporate exogenously-added betaine aldehyde from roots and convert it to glycinebetaine in the leaves (Table 1), to approximately 1 ~tmol g-1 fresh weight. Interestingly the rice plants showed resistance to betaine aldehyde and grew normally with 5 mM, a level that is quite toxic for tobacco seedlings (Rathinasabapathi et al, 1994). These results confirm our previous suggestion that rice plants possess an enzymatically active BADH (Ishitani et al, 1993).…”

Section: Resultssupporting

confidence: 88%

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Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes

Yokota²,

et al. 1997

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SummaryBetaine aldehyde dehydrogenase (BADH) catalyzes the last step in the plant biosynthetic pathway that leads to glycinebetaine. Rice plants (Oryza sativa L.}, albeit considered a typical non-glycinebetaine accumulating species, have been found to express this enzyme at low levels. This observation evokes an interest in phylogenic evolution of the enzyme in the plant kingdom. It is reported here that rice plants possess the ability to take up exogenously added betaine aldehyde through the roots and convert it to glycinebetaine, resulting in an enhanced salttolerance of the plants. A gene encoding a putative BADH from the rice genome was also cloned and sequenced. The gene was found to contain 14 introns, and the overall nucleotide sequence of the coding region is c. 78% identical to that of the barley BADH cDNA. Cloning of a partial BADH cDNA from rice was accomplished by reverse transcription-polymerase chain reaction (RT-PCR). The nucleotide sequence of the cloned fragment was found to be identical to the corresponding exon regions of the rice genomic BADH gene. The deduced amino acid sequences of rice and barley BADH both contain a C-terminal tripeptide SKL, a signal known to target preproteins to microbodies. This localization was confirmed by an immuno-gold labeling study of transgenic tobacco harboring barley cDNA, which showed BADH protein inside peroxisomes. Northern blot analysis revealed that the level of BADH mRNA is salt-inducible.

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Section: Resultssupporting

confidence: 88%

“…Betaine aldehyde is known to be toxic at high levels (Rathinasabapathi et al, 1994). Therefore, a low level of betaine aldehyde (1 mM) was given to the plants.…”

Section: Resultsmentioning

confidence: 99%

Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes

Yokota²,

et al. 1997

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“…Thus, when choline and glycine betaine aldehyde can be accumulated but not enzymatically oxidized to glycine betaine, the precursors for glycine betaine synthesis are highly toxic to the B. subtilis cells. Such a toxic effect of glycine betaine aldehyde has also been reported for the growth of plant cells (Nicotiana tabacum) incapable of glycine betaine synthesis (22,42). Glycine betaine aldehyde is a highly reactive compound that can readily form Schiff bases with free amino groups.…”

Section: Vol 178 1996 Glycine Betaine Synthesis In B Subtilis 5127mentioning

confidence: 86%

Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes

et al. 1996

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Synthesis of the osmoprotectant glycine betaine from the exogenously provided precursor choline or glycine betaine aldehyde confers considerable osmotic stress tolerance to Bacillus subtilis in high-osmolarity media. Using an Escherichia coli mutant (betBA) defective in the glycine betaine synthesis enzymes, we cloned by functional complementation the genes that are required for the synthesis of the osmoprotectant glycine betaine in B. subtilis. The DNA sequence of a 4.1-kb segment from the cloned chromosomal B. subtilis DNA was established, and two genes (gbsA and gbsB) whose products were essential for glycine betaine biosynthesis and osmoprotection were identified. The gbsA and gbsB genes are transcribed in the same direction, are separated by a short intergenic region, and are likely to form an operon. The deduced gbsA gene product exhibits strong sequence identity with members of a superfamily of specialized and nonspecialized aldehyde dehydrogenases. This superfamily comprises glycine betaine aldehyde dehydrogenases from bacteria and plants with known involvement in the cellular adaptation to high-osmolarity stress and drought. The deduced gbsB gene product shows significant similarity to the family of type III alcohol dehydrogenases. B. subtilis mutants with defects in the chromosomal gbsAB genes were constructed by marker replacement, and the growth properties of these mutant strains in high-osmolarity medium were analyzed. Deletion of the gbsAB genes destroyed the cholineglycine betaine synthesis pathway and abolished the ability of B. subtilis to deal effectively with high-osmolarity stress in choline-or glycine betaine aldehyde-containing medium. Uptake of radiolabelled choline was unaltered in the gbsAB mutant strain. The continued intracellular accumulation of choline or glycine betaine aldehyde in a strain lacking the glycine betaine-biosynthetic enzymes strongly interfered with the growth of B. subtilis, even in medium of moderate osmolarity. A single transcription initiation site for gbsAB was detected by high-resolution primer extension analysis. gbsAB transcription was initiated from a promoter with close homology to A -dependent promoters and was stimulated by the presence of choline in the growth medium.

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“…The spinach betaine aldehyde dehydrogenase (BADH) gene has been developed as a plant-derived selectable marker to transform chloroplast genomes [4]. The selection process involves conversion of toxic betaine aldehyde (BA) by the chloroplast-localized BADH enzyme to non-toxic glycine betaine, which also serves as an osmoprotectant [35]. Because the BADH enzyme is present only in chloroplasts of a few plant species adapted to dry and saline environments [35,36], it is suitable as a selectable marker in many crop plants.…”

Section: Engineering the Chloroplast Genome Without Antibiotic Resistmentioning

confidence: 99%

“…The selection process involves conversion of toxic betaine aldehyde (BA) by the chloroplast-localized BADH enzyme to non-toxic glycine betaine, which also serves as an osmoprotectant [35]. Because the BADH enzyme is present only in chloroplasts of a few plant species adapted to dry and saline environments [35,36], it is suitable as a selectable marker in many crop plants. The transformation study showed that BA selection was 25-fold more efficient than spectinomycin, exhibiting rapid regeneration of transgenic shoots within two weeks.…”

Section: Engineering the Chloroplast Genome Without Antibiotic Resistmentioning

confidence: 99%

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

Daniell

Khan

Allison

2002

Trends in Plant Science

324

153

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Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.Chloroplast transformation is an environmentally friendly approach to plant genetic engineering that minimizes out-crossing of transgenes to related weeds or crops [1,2] and reduces the potential toxicity of transgenic pollen to non-target insects [3]. Because the plastid genome is highly polyploid, transformation of chloroplasts permits the introduction of thousands of copies of foreign genes per plant cell, and generates extraordinarily high levels of foreign protein [3]. Chloroplast transformation vectors use two targeting sequences that flank the foreign genes and insert them, through homologous recombination, at a precise, predetermined location in the organelle genome (Fig. 1). This results in uniform transgene expression among transgenic lines and eliminates the 'position effect' often observed in nuclear transgenic plants. Gene silencing, frequently observed in nuclear transgenic plants, has not been observed in genetically engineered chloroplasts. The ability to express foreign proteins at high levels in chloroplasts and chromoplasts, and to engineer foreign genes without the use of antibiotic resistant genes [4,5],make this compartment ideal for the development of edible vaccines [6]. Moreover, the ability of chloroplasts to form disulfide bonds and to fold human proteins has opened the door to high-level production of biopharmaceuticals in plants [7]. Furthermore, foreign proteins observed to be toxic in the cytosol are non-toxic when accumulated within transgenic chloroplasts [6,8]. Chloroplast and nuclear genetic engineering are compared in Table 1.

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Metabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance

Cited by 132 publications

References 34 publications

Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes

Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes

Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

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