Choline import into chloroplasts limits glycine betaine synthesis in tobacco: analysis of plants engineered with a chloroplastic or a cytosolic pathway.
The biosynthesis of the osmoprotectant glycine betaine (GlyBet) is a target for metabolic engineering to enhance stress resistance in crops. Certain plants synthesize GlyBet in chloroplasts via a two-step oxidation of choline (Cho). In previous work, a chloroplastic GlyBet synthesis pathway was inserted into tobacco (which lacks GlyBet) by expressing spinach choline monooxygenase (CMO). The transformants had low CMO enzyme activity, and produced little GlyBet (less than or = 70 nmol g(-1) fresh wt). In this study, transformants with up to 100-fold higher CMO activity showed no further increase in GlyBet. In contrast, tobacco expressing a cytosolic GlyBet synthesis pathway accumulated significantly more GlyBet (430 nmol g(-1) fresh wt), suggesting that subcellular localization influences pathway flux. Modeling of the labeling kinetics of Cho metabolites observed when [14C]Cho was supplied to engineered plants demonstrated that Cho import into chloroplasts indeed limits the flux to GlyBet in the chloroplastic pathway. A high-activity Cho transporter in the chloroplast envelope may therefore be an integral part of the GlyBet synthesis pathway in species that accumulate GlyBet naturally, and hence a target for future engineering.
Nuccio
M L
ML
Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611-0690, USA.
McNeil
S D
SD
Ziemak
M J
MJ
Hanson
A D
AD
Jain
R K
RK
Selvaraj
G
G
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism; D001622:Betaine / Q000378:metabolism*; D050728:Betaine-Aldehyde Dehydrogenase; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism; D002736:Chloroplasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002794:Choline / Q000378:metabolism* / Q000494:pharmacology; D003198:Computer Simulation; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism*; D005818:Genetic Engineering; D015151:Immunoblotting; D007700:Kinetics; D026901:Membrane Transport Proteins; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D012313:RNA / Q000235:genetics / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology; D014026:Nicotiana / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; C065637:choline transporter; D001622:Betaine; D012313:RNA; C105448:choline monooxygenase; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D010105:Oxygenases; D000445:Aldehyde Oxidoreductases; D050728:Betaine-Aldehyde Dehydrogenase; D002794:Choline