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

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 and/or by suppressing its degradation.

Plants synthesize ethanolamine by direct decarboxylation of serine using a pyridoxal phosphate enzyme.

The established pathways from serine to ethanolamine are indirect and involve decarboxylation of phosphatidylserine. Here we show that plants can decarboxylate serine directly. Using a radioassay based on ethanolamine (Etn) formation, pyridoxal 5'-phosphate-dependent l-serine decarboxylase (SDC) activity was readily detected in soluble extracts from leaves of diverse species, including spinach, Arabidopsis, and rapeseed. A putative Arabidopsis SDC cDNA was identified by searching GenBank for sequences homologous to other amino acid decarboxylases and shown by expression in Escherichia coli to encode a soluble protein with SDC activity. This cDNA was further authenticated by complementing the Etn requirement of a yeast psd1 psd2 mutant. In a parallel approach, a cDNA was isolated from a rapeseed library by its ability to complement the Etn requirement of a yeast cho1 mutant and shown by expression in E. coli to specify SDC. The deduced Arabidopsis and rapeseed SDC polypeptides are 90% identical, lack obvious targeting signals, and belong to amino acid decarboxylase group II. Recombinant Arabidopsis SDC was shown to exist as a tetramer and to contain pyridoxal 5'-phosphate. It does not attack d-serine, l-phosphoserine, other l-amino acids, or phosphatidylserine and is not inhibited by Etn, choline, or their phosphoesters. As a soluble, pyridoxal 5'-phosphate enzyme, SDC contrasts sharply with phosphatidylserine decarboxylases, which are membrane proteins that have a pyruvoyl cofactor.

The S-methylmethionine cycle in angiosperms: ubiquity, antiquity and activity.

Angiosperms synthesize S-methylmethionine (SMM) from methionine (Met) and S-adenosylmethionine (AdoMet) in a unique reaction catalyzed by Met S-methyltransferase (MMT). SMM serves as methyl donor for Met synthesis from homocysteine, catalyzed by homocysteine S-methyltransferase (HMT). MMT and HMT together have been proposed to constitute a futile SMM cycle that stops the free Met pool from being depleted by an overshoot in AdoMet synthesis. Arabidopsis and maize have one MMT gene, and at least three HMT genes that belong to two anciently diverged classes and encode enzymes with distinct properties and expression patterns. SMM, and presumably its cycle, must therefore have originated before dicot and monocot lineages separated. Arabidopsis leaves, roots and developing seeds all express MMT and HMTs, and can metabolize [35S]Met to [35S]SMM and vice versa. The SMM cycle therefore operates throughout the plant. This appears to be a general feature of angiosperms, as digital gene expression profiles show that MMT and HMT are co-expressed in leaves, roots and reproductive tissues of maize and other species. An in silico model of the SMM cycle in mature Arabidopsis leaves was developed from radiotracer kinetic measurements and pool size data. This model indicates that the SMM cycle consumes half the AdoMet produced, and suggests that the cycle serves to stop accumulation of AdoMet, rather than to prevent depletion of free Met. Because plants lack the negative feedback loops that regulate AdoMet pool size in other eukaryotes, the SMM cycle may be the main mechanism whereby plants achieve short-term control of AdoMet level.

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.

Metabolic modeling identifies key constraints on an engineered glycine betaine synthesis pathway in tobacco.

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.

Biochemical evidence for two novel enzymes in the biosynthesis of 3-dimethylsulfoniopropionate in Spartina alterniflora.

3-Dimethylsulfoniopropionate (DMSP) is an osmoprotectant accumulated by the cordgrass Spartina alterniflora and other salt-tolerant plants. Previous in vivo isotope tracer and metabolic modeling studies demonstrated that S. alterniflora synthesizes DMSP via the route S-methyl-Met --> 3-dimethylsulfoniopropylamine (DMSP-amine) --> 3-dimethylsulfoniopropionaldehyde --> DMSP and indicated that the first reaction requires a far higher substrate concentration than the second to attain one-half-maximal rate. As neither of these reactions is known from other organisms, two novel enzymes are predicted. Two corresponding activities were identified in S. alterniflora leaf extracts using specific radioassays. The first, S-methyl-Met decarboxylase (SDC), strongly prefers the L-enantiomer of S-methyl-Met, is pyridoxal 5'-phosphate-dependent, generates equimolar amounts of CO(2) and DMSP-amine, and has a high apparent K(m) (approximately 18 mM) for its substrate. The second enzyme, DMSP-amine oxidase (DOX), requires O(2) for activity, shows an apparent K(m) for DMSP-amine of 1.8 mM, and is not accompanied by DMSP-amine dehydrogenase or transaminase activity. Very little SDC or DOX activity was found in grasses lacking DMSP. These data indicate that SDC and DOX are the predicted novel enzymes of DMSP synthesis.

cDNA cloning of phosphoethanolamine N-methyltransferase from spinach by complementation in Schizosaccharomyces pombe and characterization of the recombinant enzyme.

The N-methylation of phosphoethanolamine is the committing step in choline biogenesis in plants and is catalyzed by S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferase (PEAMT, EC ). A spinach PEAMT cDNA was isolated by functional complementation of a Schizosaccharomyces pombe cho2(-) mutant and was shown to encode a protein with PEAMT activity and without ethanolamine- or phosphatidylethanolamine N-methyltransferase activity. The PEAMT cDNA specifies a 494-residue polypeptide comprising two similar, tandem methyltransferase domains, implying that PEAMT arose by gene duplication and fusion. Data base searches suggested that PEAMTs with the same tandem structure are widespread among flowering plants. Size exclusion chromatography of the recombinant enzyme indicates that it exists as a monomer. PEAMT catalyzes not only the first N-methylation of phosphoethanolamine but also the two subsequent N-methylations, yielding phosphocholine. Monomethyl- and dimethylphosphoethanolamine are detected as reaction intermediates. A truncated PEAMT lacking the C-terminal methyltransferase domain catalyzes only the first methylation. Phosphocholine inhibits both the wild type and the truncated enzyme, although the latter is less sensitive. Salinization of spinach plants increases PEAMT mRNA abundance and enzyme activity in leaves by about 10-fold, consistent with the high demand in stressed plants for choline to support glycine betaine synthesis.

Radiotracer and computer modeling evidence that phospho-base methylation is the main route of choline synthesis in tobacco.

Among flowering plants, the synthesis of choline (Cho) from ethanolamine (EA) can potentially occur via three parallel, interconnected pathways involving methylation of free bases, phospho-bases, or phosphatidyl-bases. We investigated which pathways operate in tobacco (Nicotiana tabacum L.) because previous work has shown that the endogenous Cho supply limits accumulation of glycine betaine in transgenic tobacco plants engineered to convert Cho to glycine betaine. The kinetics of metabolite labeling were monitored in leaf discs supplied with [(33)P]phospho-EA, [(33)P]phospho-monomethylethanolamine, or [(14)C]formate, and the data were subjected to computer modeling. Because partial hydrolysis of phospho-bases occurred in the apoplast, modeling of phospho-base metabolism required consideration of the re-entry of [(33)P]phosphate into the network. Modeling of [(14)C]formate metabolism required consideration of the labeling of the EA and methyl moieties of Cho. Results supported the following conclusions: (a) The first methylation step occurs solely at the phospho-base level; (b) the second and third methylations occur mainly (83%-92% and 65%-85%, respectively) at the phospho-base level, with the remainder occurring at the phosphatidyl-base level; and (c) free Cho originates predominantly from phosphatidylcholine rather than from phospho-Cho. This study illustrates how computer modeling of radiotracer data, in conjunction with information on chemical pool sizes, can provide a coherent, quantitative picture of fluxes within a complex metabolic network.

Plant one-carbon metabolism and its engineering.

The metabolism of one-carbon (C1) units is vital to plants. It involves unique enzymes and takes place in four subcellular compartments. Plant C1 biochemistry has remained relatively unexplored, partly because of the low abundance or the lability of many of its enzymes and intermediates. Fortunately, DNA sequence databases now make it easier to characterize known C1 enzymes and to discover new ones, to identify pathways that might carry high C1 fluxes, and to use engineering to redirect C1 fluxes and to understand their control better.

Characterization and functional expression of cDNAs encoding methionine-sensitive and -insensitive homocysteine S-methyltransferases from Arabidopsis.

Plants synthesize S-methylmethionine (SMM) from S-adenosylmethionine (AdoMet), and methionine (Met) by a unique reaction and, like other organisms, use SMM as a methyl donor for Met synthesis from homocysteine (Hcy). These reactions comprise the SMM cycle. Two Arabidopsis cDNAs specifying enzymes that mediate the SMM --> Met reaction (SMM:Hcy S-methyltransferase, HMT) were identified by homology and authenticated by complementing an Escherichia coli yagD mutant and by detecting HMT activity in complemented cells. Gel blot analyses indicate that these enzymes, AtHMT-1 and -2, are encoded by single copy genes. The deduced polypeptides are similar in size (36 kDa), share a zinc-binding motif, lack obvious targeting sequences, and are 55% identical to each other. The recombinant enzymes exist as monomers. AtHMT-1 and -2 both utilize l-SMM or (S,S)-AdoMet as a methyl donor in vitro and have higher affinities for SMM. Both enzymes also use either methyl donor in vivo because both restore the ability to utilize AdoMet or SMM to a yeast HMT mutant. However, AtHMT-1 is strongly inhibited by Met, whereas AtHMT-2 is not, a difference that could be crucial to the control of flux through the HMT reaction and the SMM cycle. Plant HMT is known to transfer the pro-R methyl group of SMM. This enabled us to use recombinant AtHMT-1 to establish that the other enzyme of the SMM cycle, AdoMet:Met S-methyltransferase, introduces the pro-S methyl group. These opposing stereoselectivities suggest a way to measure in vivo flux through the SMM cycle.

Isolation, characterization, and functional expression of cDNAs encoding NADH-dependent methylenetetrahydrofolate reductase from higher plants.

Methylenetetrahydrofolate reductase (MTHFR) is the least understood enzyme of folate-mediated one-carbon metabolism in plants. Genomics-based approaches were used to identify one maize and two Arabidopsis cDNAs specifying proteins homologous to MTHFRs from other organisms. These cDNAs encode functional MTHFRs, as evidenced by their ability to complement a yeast met12 met13 mutant, and by the presence of MTHFR activity in extracts of complemented yeast cells. Deduced sequence analysis shows that the plant MTHFR polypeptides are of similar size (66 kDa) and domain structure to other eukaryotic MTHFRs, and lack obvious targeting sequences. Southern analyses and genomic evidence indicate that Arabidopsis has two MTHFR genes and that maize has at least two. A carboxyl-terminal polyhistidine tag was added to one Arabidopsis MTHFR, and used to purify the enzyme 640-fold to apparent homogeneity. Size exclusion chromatography and denaturing gel electrophoresis of the recombinant enzyme indicate that it exists as a dimer of approximately 66-kDa subunits. Unlike mammalian MTHFR, the plant enzymes strongly prefer NADH to NADPH, and are not inhibited by S-adenosylmethionine. An NADH-dependent MTHFR reaction could be reversible in plant cytosol, where the NADH/NAD ratio is 10(-3). Consistent with this, leaf tissues metabolized [methyl-(14)C]methyltetrahydrofolate to serine, sugars, and starch. A reversible MTHFR reaction would obviate the need for inhibition by S-adenosylmethionine to prevent excessive conversion of methylene- to methyltetrahydrofolate.

S-methylmethionine plays a major role in phloem sulfur transport and is synthesized by a novel type of methyltransferase.

All flowering plants produce S-methylmethionine (SMM) from Met and have a separate mechanism to convert SMM back to Met. The functions of SMM and the reasons for its interconversion with Met are not known. In this study, by using the aphid stylet collection method together with mass spectral and radiolabeling analyses, we established that l-SMM is a major constituent of the phloem sap moving to wheat ears. The SMM level in the phloem ( approximately 2% of free amino acids) was 1.5-fold that of glutathione, indicating that SMM could contribute approximately half the sulfur needed for grain protein synthesis. Similarly, l-SMM was a prominently labeled product in phloem exudates obtained by EDTA treatment of detached leaves from plants of the Poaceae, Fabaceae, Asteraceae, Brassicaceae, and Cucurbitaceae that were given l-(35)S-Met. cDNA clones for the enzyme that catalyzes SMM synthesis (S-adenosylMet:Met S-methyltransferase; EC 2.1.1.12) were isolated from Wollastonia biflora, maize, and Arabidopsis. The deduced amino acid sequences revealed the expected methyltransferase domain ( approximately 300 residues at the N terminus), plus an 800-residue C-terminal region sharing significant similarity with aminotransferases and other pyridoxal 5'-phosphate-dependent enzymes. These results indicate that SMM has a previously unrecognized but often major role in sulfur transport in flowering plants and that evolution of SMM synthesis in this group involved a gene fusion event. The resulting bipartite enzyme is unlike any other known methyltransferase.

Metabolic engineering of plants for osmotic stress resistance.

Genes encoding critical steps in the synthesis of osmoprotectant compounds are now being expressed in transgenic plants. These plants generally accumulate low levels of osmoprotectants and have increased stress tolerance. The next priority is therefore to engineer greater osmoprotectant synthesis without detriment to the rest of metabolism. This will require manipulation of multiple genes, guided by thorough analysis of metabolite fluxes and pool sizes.

Dimethylsulfoniopropionate biosynthesis in Spartina alterniflora1. Evidence that S-methylmethionine and dimethylsulfoniopropylamine are intermediates.

The osmoprotectant 3-dimethylsulfoniopropionate (DMSP) occurs in Gramineae and Compositae, but its synthesis has been studied only in the latter. The DMSP synthesis pathway was therefore investigated in the salt marsh grass Spartina alterniflora Loisel. Leaf tissue metabolized supplied [35S]methionine (Met) to S-methyl-l-Met (SMM), 3-dimethylsulfoniopropylamine (DMSP-amine), and DMSP. The 35S-labeling kinetics of SMM and DMSP-amine indicated that they were intermediates and, consistent with this, the dimethylsulfonium moiety of SMM was shown by stable isotope labeling to be incorporated as a unit into DMSP. The identity of DMSP-amine, a novel natural product, was confirmed by both chemical and mass-spectral methods. S. alterniflora readily converted supplied [35S]SMM to DMSP-amine and DMSP, and also readily converted supplied [35S]DMSP-amine to DMSP; grasses that lack DMSP did neither. A small amount of label was detected in 3-dimethylsulfoniopropionaldehyde (DMSP-ald) when [35S]SMM or [35S]DMSP-amine was given. These results are consistent with the operation of the pathway Met --> SMM --> DMSP-amine --> DMSP-ald --> DMSP, which differs from that found in Compositae by the presence of a free DMSP-amine intermediate. This dissimilarity suggests that DMSP synthesis evolved independently in Gramineae and Compositae.

Osmotic stress induces expression of choline monooxygenase in sugar beet and amaranth.

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.

Salinity promotes accumulation of 3-dimethylsulfoniopropionate and its precursor S-methylmethionine in chloroplasts.

Wollastonia biflora (L.) DC. plants accumulate the osmoprotectant 3-dimethylsulfoniopropionate (DMSP), particularly when salinized. DMSP is known to be synthesized in the chloroplast from S-methylmethionine (SMM) imported from the cytosol, but the sizes of the chloroplastic and extrachloroplastic pools of these compounds are unknown. We therefore determined DMSP and SMM in mesophyll protoplasts and chloroplasts. Salinization with 30% (v/v) artificial seawater increased protoplast DMSP levels from 4.6 to 6.0 mumol mg-1 chlorophyll (Chl), and chloroplast levels from 0.9 to 1.9 mumol mg-1 Chl. The latter are minimum values because intact chloroplasts leaked DMSP during isolation. Correcting for this leakage, it was estimated that in vivo about one-half of the DMSP is chloroplastic and that stromal DMSP concentrations in control and salinized plants are about 60 and 130 mM, respectively. Such concentrations would contribute significantly to chloroplast osmoregulation and could protect photosynthetic processes from stress injury. SMM levels were measured using a novel mass-spectrometric method. About 40% of the SMM was located in the chloroplast in unsalinized W. biflora plants, as was about 80% in salinized plants; the chloroplastic pool in both cases was approximately 0.1 mumol mg-1 Chl. In contrast, > or = 85% of the SMM was extrachloroplastic in pea (Pisum sativum L.) and spinach (Spinacia oleracea L.), which lack DMSP. DMSP synthesis may be associated with enhanced accumulation of SMM in the chloroplasm.

The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase.

Certain plants produce glycine betaine (GlyBet) in the chloroplast by a two-step oxidation of choline. Introducing GlyBet accumulation into plants that lack it is a well-established target for metabolic engineering because GlyBet can lessen damage from osmotic stress. The first step in GlyBet synthesis is catalyzed by choline mono-oxygenase (CMO), a stromal enzyme with a Rieske-type [2Fe-2S] center. The absence of CMO is the primary constraint on GlyBet production in GlyBet-deficient plants such as tobacco, but the endogenous choline supply is also potentially problematic. To investigate this, we constructed transgenic tobacco plants that constitutively express a spinach CMO cDNA. The CMO protein was correctly compartmented in chloroplasts and was enzymatically active, showing that its [2Fe-2S] cluster had been inserted. Salinization increased CMO protein levels, apparently via a post-transcriptional mechanism, to as high as 10% of that in salinized spinach. However, the GlyBet contents of CMO+ plants were very low (0.02-0.05 mumol g-1 fresh weight) in both unstressed and salinized conditions. Experiments with [14C]GlyBet demonstrated that this was not due to GlyBet catabolism. When CMO+ plants were supplied in culture with 5 mM choline or phosphocholine, their choline and GlyBet levels increased by at least 30-fold. The choline precursors mono- and dimethylethanolamine also enhanced choline and GlyBet levels but ethanolamine did not, pointing to a major constraint on flux to choline at the first methylation step in its synthesis. The extractable activity of the enzyme mediating this step in tobacco was only 3% that of spinach. We conclude that in GlyBet-deficient plants engineered with choline-oxidizing genes, the size of the free choline pool and the metabolic flux to choline need to be increased to attain GlyBet levels as high as those in natural accumulators.

A new route for synthesis of dimethylsulphoniopropionate in marine algae.

The 3-dimethylsulphoniopropionate (DMSP) produced by marine algae is the main biogenic precursor of atmospheric dimethylsulphide (DMS). This biogenic DMS, formed by bacterial and algal degradation of DMSP, contributes about 1.5 x 10(13) g of sulphur to the atmosphere annually, and plays a major part in the global sulphur cycle, in cloud formation and potentially in climate regulation. Although DMSP biosynthesis has been partially elucidated in a higher plant, nothing is known about how algae make DMSP except that the whole molecule is derived from methionine. Here we use in vivo isotope labelling to demonstrate that DMSP synthesis in the green macroalga Enteromorpha intestinalis proceeds by a route entirely distinct from that in higher plants. From methionine, the steps are transamination, reduction and S-methylation to give the novel sulphonium compound 4-dimethylsulphonio-2-hydroxybutyrate (DMSHB), which is oxidatively decarboxylated to DMSP. The key intermediate DMSHB was also identified in three diverse phytoplankton species, indicating that the same pathway operates in other algal classes that are important sources of DMS. The fact that a transamination initiates this pathway could help explain how algal DMSP (and thereby DMS) production is enhanced by nitrogen deficiency.

Choline monooxygenase, an unusual iron-sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants: prosthetic group characterization and cDNA cloning.

Plants synthesize the osmoprotectant glycine betaine via the route choline --> betaine aldehyde --> glycine betaine. In spinach, the first step is catalyzed by choline monooxygenase (CMO), a ferredoxin-dependent stromal enzyme that has been hypothesized to be an oligomer of identical subunits and to be an Fe-S protein. Analysis by HPLC and matrix-assisted laser desorption ionization MS confirmed that native CMO contains only one type of subunit (Mr 42,864). Determination of acid-labile sulfur and nonheme iron demonstrated that there is one [2Fe-2S] cluster per subunit, and EPR spectral data indicated that this cluster is of the Rieske type--i.e., coordinated by two Cys and two His ligands. A full-length CMO cDNA (1,622 bp) was cloned from spinach using a probe generated by PCR amplification for which the primers were based on internal peptide sequences. The ORF encoded a 440-amino acid polypeptide that included a 60-residue transit peptide. The deduced amino acid sequence included two Cys-His pairs spaced 16 residues apart, a motif characteristic of Rieske-type Fe-S proteins. Larger regions that included this motif also showed some sequence similarity (approximately 40%) to Rieske-type proteins, particularly bacterial oxygenases. Otherwise there was very little similarity between CMO and proteins from plants or other organisms. RNA and immunoblot analyses showed that the expression of CMO in leaves increased several-fold during salinization. We conclude that CMO is a stress-inducible representative of a new class of plant oxygenases.

Transgenically Expressed Betaine Aldehyde Dehydrogenase Efficiently Catalyzes Oxidation of Dimethylsulfoniopropionaldehyde and [omega]-Aminoaldehydes.

Tobacco (Nicotianum tabacum L.) plants engineered to express a sugar beet (Beta vulgaris L.) betaine aldehyde dehydrogenase (BADH) cDNA acquired not only BADH activity, but also three other aldehyde dehydrogenase activities (those measured with 3-dimethylsulfoniopropionaldehyde, 3-aminopropionaldehyde, and 4-aminobutyraldehyde, all of which are natural products). This shows that BADH is not, as believed up to now, a substrate-specific enzyme and that its role may not be limited to glycine betaine synthesis.