Choline Import into Chloroplasts Limits Glycine Betaine Synthesis in Tobacco: Analysis of Plants Engineered with a Chloroplastic or a Cytosolic Pathway

Nuccio, Michael L.; McNeil, Scott D.; Ziemak, Michael J.; Hanson, Andrew D.; Jain, R. K.; Selvaraj, Gopalan

doi:10.1006/mben.2000.0158

Cited by 65 publications

(51 citation statements)

References 55 publications

(87 reference statements)

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“…The latter finding is consistent with the view that phosphatidylcholine synthesis is governed downstream of Cho and phosphocholine formation, by the rate of the phosphocholine cytidylyltransferase reaction (30). As predicted by metabolic modeling (19,24), the expanded Cho pool drives a higher flux through the transgenic GlyBet synthesis pathway. That this flux is not even higher can be explained by additional constraints on the operation of the transgenic pathway in tobacco, particularly a low capacity to import Cho into the chloroplast from its site of synthesis in the cytosol (19,24).…”

Section: Discussionsupporting

confidence: 89%

“…As predicted by metabolic modeling (19,24), the expanded Cho pool drives a higher flux through the transgenic GlyBet synthesis pathway. That this flux is not even higher can be explained by additional constraints on the operation of the transgenic pathway in tobacco, particularly a low capacity to import Cho into the chloroplast from its site of synthesis in the cytosol (19,24). It should be noted that this constraint is missing from plants engineered with a cytosolic Cho 3 GlyBet pathway by using bacterial Cho-oxidizing enzymes (3,5,19), so that overexpressing PEAMT in such plants could lead to higher GlyBet levels than those we attained.…”

Section: Discussionmentioning

confidence: 80%

“…The PEAMT coding sequence was amplified by PCR, using Pfu polymerase (Stratagene) with the forward primer 5Ј-AGCTTCTAGAT T T T TACA ACA AT TACCA ACA ACA-ACA A ACA ACA A ACA ACAT TACA AT TACTAT T TAC-A AT TACA A A A ATGGCCGCT TCAGCTATGG-3Ј, the reverse primer 5Ј-ACGTGGATCCTCACAT T T TCT TG-GCAATG-3Ј, and pREP3-PEAMT (12) as template. The forward primer includes the tobacco mosaic virus ⍀ sequence (18) and alters the sequence context of the start codon as described (19). The reaction product was ligated to pFMV as an XbaI͞ BamHI fragment to yield pFMV-PEAMT.…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant spinach PEAMT with a C-terminal histidine tag was produced by using the pET28b vector (Novagen), purified by Ni 2ϩ affinity chromatography, and used to raise rabbit Abs. Immunoblotting was as described (19); the anti-PEAMT serum was diluted 1:1000; no crossreaction with tobacco PEAMT was detectable.…”

Section: Construction Of Plants Expressing a Glybet Synthesis Pathwaymentioning

confidence: 99%

“…This construct was used to transform tobacco plants that were coexpressing spinach CMO (19) and beet BADH (20) and so contained a transgenic GlyBet pathway in their chloroplasts. Glyphosate-resistant transformants were identified that had levels of spinach PEAMT protein close to those in spinach itself ( Fig.…”

Section: Peamt Overexpression Enhances Cho Levelsmentioning

confidence: 99%

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Enhanced synthesis of choline and glycine betaine in transgenic tobacco plants that overexpress phosphoethanolamine N -methyltransferase

McNeil

Nuccio

Ziemak

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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176

132

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Choline (Cho) is the precursor of the osmoprotectant glycine betaine and is itself an essential nutrient for humans. Metabolic engineering of Cho biosynthesis in plants could therefore enhance both their resistance to osmotic stresses (drought and salinity) and their nutritional value. The key enzyme of the plant Cho-synthesis pathway is phosphoethanolamine N-methyltransferase, which catalyzes all three of the methylations required to convert phosphoethanolamine to phosphocholine. We show here that overexpressing this enzyme in transgenic tobacco increased the levels of phosphocholine by 5-fold and free Cho by 50-fold without affecting phosphatidylcholine content or growth. Moreover, the expanded Cho pool led to a 30-fold increase in synthesis of glycine betaine via an engineered glycine betaine pathway. Supplying the transgenics with the Cho precursor ethanolamine (EA) further enhanced Cho levels even though the supplied EA was extensively catabolized. These latter results establish that there is further scope for improving Cho synthesis by engineering an increased endogenous supply of EA and suggest that this could be achieved by enhancing EA synthesis and͞or by suppressing its degradation.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 80%

Section: Methodsmentioning

confidence: 99%

Section: Construction Of Plants Expressing a Glybet Synthesis Pathwaymentioning

confidence: 99%

Section: Peamt Overexpression Enhances Cho Levelsmentioning

confidence: 99%

See 3 more Smart Citations

Enhanced synthesis of choline and glycine betaine in transgenic tobacco plants that overexpress phosphoethanolamine N -methyltransferase

McNeil

Nuccio

Ziemak

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

176

132

View full text Add to dashboard Cite

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Producing Biopharmaceuticals in the Desert: Building an Abiotic Stress Tolerance in Plants for Salt, Heat, and Drought

Gepstein¹,

Grover²,

Blumwald³

2005

Modern Biopharmaceuticals

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Functional Genomics of Drought Tolerance in Crops: Engineering Transcriptional Regulators and Pathways

Rathinasabapathi

2012

Improving Crop Resistance to Abiotic Stress

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Drought stress is a major limitation for crop productivity worldwide, especially in irrigated agriculture. One solution to this problem is the use of cultivars that are tolerant to drought. However, conventional breeding to improve drought tolerance has proven difficult because drought and dehydration tolerance in plants are not fully understood. As a result of research during the past two decades, networks of genes participating in plant adaptations to drought stress have been identified. In this chapter, I present this research progress and indicate the use of specific genes to engineer crops for improved drought tolerance.

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Choline Import into Chloroplasts Limits Glycine Betaine Synthesis in Tobacco: Analysis of Plants Engineered with a Chloroplastic or a Cytosolic Pathway

Cited by 65 publications

References 55 publications

Enhanced synthesis of choline and glycine betaine in transgenic tobacco plants that overexpress phosphoethanolamine N -methyltransferase

Enhanced synthesis of choline and glycine betaine in transgenic tobacco plants that overexpress phosphoethanolamine N -methyltransferase

Producing Biopharmaceuticals in the Desert: Building an Abiotic Stress Tolerance in Plants for Salt, Heat, and Drought

Functional Genomics of Drought Tolerance in Crops: Engineering Transcriptional Regulators and Pathways

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