Search

Previous work has shown that tobacco (Nicotiana tabacum) plants engineered to express spinach choline monooxygenase in the chloroplast accumulate very little glycine betaine (GlyBet) unless supplied with choline (Cho). We therefore used metabolic modeling in conjunction with [(14)C]Cho labeling experiments and in vivo (31)P NMR analyses to define the constraints on GlyBet synthesis, and hence the processes likely to require further engineering. The [(14)C]Cho doses used were large enough to markedly perturb Cho and phosphocholine pool sizes, which enabled development and testing of models with rates dynamically responsive to pool sizes, permitting estimation of the kinetic properties of Cho metabolism enzymes and transport systems in vivo. This revealed that import of Cho into the chloroplast is a major constraint on GlyBet synthesis, the import rate being approximately 100-fold lower than the rates of Cho phosphorylation and transport into the vacuole, with which import competes. Simulation studies suggested that, were the chloroplast transport limitation corrected, additional engineering interventions would still be needed to achieve levels of GlyBet as high as those in plants that accumulate GlyBet naturally. This study reveals the rigidity of the Cho metabolism network and illustrates how computer modeling can help guide rational metabolic engineering design.

Choline monooxygenase (CMO) catalyzes the committing step in the synthesis of glycine betaine, an osmoprotectant accumulated by many plants in response to salinity and drought. To investigate how these stresses affect CMO expression, a spinach (Spinacia oleracea L., Chenopodiaceae) probe was used to isolate CMO cDNAs from sugar beet (Beta vulgaris L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced beet CMO amino acid sequence comprised a transit peptide and a 381-residue mature peptide that was 84% identical (97% similar) to that of spinach and that showed the same consensus motif for coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding motif was also present. When water was withheld, leaf relative water content declined to 59% and the levels of CMO mRNA, protein, and enzyme activity rose 3- to 5-fold; rewatering reversed these changes. After gradual salinization (NaCl:CaCl2 = 5.7:1, mol/mol), CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold at 400 mM salt, and returned to uninduced levels when salt was removed. Beet roots also expressed CMO, most strongly when salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a mononuclear Fe center, is inducibly expressed in roots as well as in leaves of Chenopodiaceae, and is not unique to this family.