Engineering Plant One-Carbon
(1-C) Metabolism
David Rhodes
Purdue University
Primary and secondary metabolism intersect in the
one-carbon (C1) area, with primary metabolism supplying most
of the C1 units and competing with secondary metabolism for
their use. This competition is potentially severe
because secondary products such as lignin, alkaloids and
glycine betaine require massive amounts of C1 units.
Many current metabolic engineering projects aim to change
levels of these products, or entail reducing the supply of C1
units. It is therefore essential to understand how C1
metabolism is regulated at the metabolic and gene levels so as
to successfully engineer C1 supply to match demand. Our
project aims to acquire this understanding. Specific
objectives are: (1) to clone complete suites of C1 genes from
maize and tobacco, and to incorporate them into DNA arrays;
(2) to use sense and antisense approaches as well as mutants
to engineer alterations in C1 unit supply and demand; and
(3) to quantify the impacts of these alterations on gene
expression (using DNA arrays), and on metabolic fluxes (by
combining radio- and stable isotope labeling, MS, NMR and
computer modeling).
Four findings from Year 1 are summarized. All were
unexpected and have implications for engineering C1
metabolism: (1) Unlike other eukaryotes, plants have
methylenetetrahydrofolate reductases that use NADH rather than
NADPH as reductant, and are not allosterically inhibited by
AdoMet. (2) DNA arrays show that formate dehydrogenase
and a cluster of enzymes for methyl group synthesis and
transfer are more highly expressed in roots than leaves.
(3) Metabolic flux analysis and modeling of tobacco engineered
to convert choline to glycine betaine suggests a crucial role
for a chloroplast choline transporter. (4) Plants have
an unsuspected source of formate ö the irreversible
hydrolysis 10-formyltetrahydrofolate, via an enzyme previously
known only in prokaryotes. The first and last of these
findings depended on genomics-based approaches, and illustrate
the value of bioinformatics in metabolic engineering.
Significant findings from Year 2 include: (1) Confirmation
of the crucial role for a chloroplast choline transporter in
conversion of choline to glycine betaine by metabolic flux
analysis and modeling of transgenic tobacco expressing choline
monooxygenase and betaine aldehyde dehydrogenase in the
chloroplast, or choline oxidase and betaine aldehyde
dehydrogenase in the cystosol. (2) Analysis of
14C-formaldehyde and 14C-serine metabolism in leaves of near-isogenic
maize lines differing for alternative alleles of a single
locus conferring glycine betaine accumulation (Bet1/Bet1) or
lack thereof (bet1/bet1), show markedly different fluxes of
radiolabel into choline moieties under salinity stress.
Despite these large differences in flow of C1 units into
choline moieties, no significant differences between near-isogenic
maize lines were found in the mRNA transcript abundances of
any of the C1 enzymes, with the single exception of
phosphoethanolamine-N-methyltransferase, which shows a modest
2-fold down-regulation in the glycine betaine-deficient
(bet1/bet1) line. The latter result suggests control of
C1 flux into choline moieties primarily by
post-transcriptional mechanisms.
Tobacco lines expressing antisense
methylenetetrahydrofolate reductase, antisense S-formylglutathione
hydrolase, and formate dehydrogenase have been derived and are
currently being characterized for C1 gene expression and
metabolic fluxes. A dynamic kinetic model of the
intersecting transmethylation, methionine salvage and S-methylmethionine
cycles has been developed, and is being used to explore the
effects of altering one or more enzyme levels on metabolism of
U-13C5-methionine.
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