Engineering Plant One-Carbon Metabolism
Primary and secondary metabolism intersect in the one-carbon (C1) area. Primary metabolism supplies most of the C1 units and competes with secondary metabolism for their use. This competition is potentially severe because secondary products such as lignin, alkaloids, and glycine betaine (GlyBet) require massive amounts of C1 units. Towards the goal of understanding how C1 metabolism is regulated at the metabolic and gene levels so as to successfully engineer C1 supply to match demand, we have: (1) cloned complete suites of C1 genes from maize and tobacco, and incorporated them into DNA arrays; (2) prepared antisense constructs and mutants engineered with alterations in C1 unit supply and demand; and (3) have quantified the impacts of these alterations on gene expression (using DNA arrays), and on metabolic fluxes (by combining isotope labeling, MS, NMR and computer modeling). Metabolic flux analysis and modeling in tobacco engineered for GlyBet synthesis by expressing choline oxidizing enzymes in either the chloroplast or cytosol, has shown that the choline biosynthesis network is rigid, and tends to resist large changes in C1 demand. A major constraint on engineering enhanced flux to GlyBet in tobacco is a low capacity of choline transport across the chloroplast envelope. Maize and sorghum mutants defective in GlyBet synthesis show greatly reduced flux of C1 units into choline in comparison to GlyBet-accumulating wildtypes, but this is not associated with altered expression of any of the C1 genes. Control of C1 flux to choline in tobacco, maize and sorghum appears to reside primarily at the level of N-methylation of phosphoethanolamine. A candidate signal for the control of this flux is the pool size of phosphocholine which down-regulates and feedback inhibits phosphoethanolamine N-methyltransferase. Methionine S-methyltransferase (MMT) catalyzes the synthesis of S-methylmethionine (SMM) from methionine (Met) and S-adenosylmethionine (AdoMet). SMM can be reconverted to Met by donating a methyl group to homocysteine, and concurrent operation of this reaction and that mediated by MMT sets up the SMM cycle. The genes encoding the enzymes of the SMM cycle were cloned and characterized during this project. SMM has been hypothesized to be essential as a methyl donor or as a transport form of sulfur, and the SMM cycle has been hypothesized to guard against depletion of the free Met pool by excess AdoMet synthesis, or to regulate AdoMet level and hence the AdoMet/S-adenosylhomocysteine ratio (the methylation ratio). To test these hypotheses, we isolated insertional mmt mutants of Arabidopsis and maize. Both mutants lacked the capacity to produce SMM and thus had no SMM cycle. They nevertheless grew and reproduced normally and the seeds of the Arabidopsis mutant had normal sulfur contents. These findings rule out an indispensable role for SMM as a methyl donor or in sulfur transport. The Arabidopsis mutant had significantly higher AdoMet a nd lower S-adenosylhomocysteine (AdoHCy) levels than the wild type, and consequently a higher methylation ratio (20 vs. 14). Free Met and thiol pools were unaltered in this mutant, although there was a 50% decrease in free threonine (Thr) and changes in other amino acids. These data indicate that the SMM cycle contributes to regulation of AdoMet levels rather than preventing depletion of free Met. Since AdoMet activates Thr synthase, that Thr level was not higher but lower in the mmt mutant implies that AdoMet is sequestered away from Thr synthase, which is chloroplastic. Results obtained with the Arabidopsis mmt mutant and wildtype have been integrated into a metabolic model of the intersecting methylation, SMM, and methionine salvage cycles. This model adequately accounts for the steady-state pool sizes of Met, SMM, AdoMet and AdoHCy in wildtype, and the small changes in AdoMet and AdoHCy levels associated with knockout of MMT. This model is now being used to predict the time-course of changes in AdoMet, Met, AdoHCy, and SMM mass isotopomers when Arabidopsis is fed with 13C5-Met at different doses, and to evaluate the metabolic consequences of knockout of adenosine kinase or AdoHCy hydrolase in Arabidopsis.
- Research Organization:
- David Rhodes/Purdue University (US)
- Sponsoring Organization:
- USDOE Office of Energy Research (ER) (US)
- DOE Contract Number:
- FG02-99ER20344
- OSTI ID:
- 836736
- Resource Relation:
- Other Information: PBD: 9 Feb 2005
- Country of Publication:
- United States
- Language:
- English
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