In most organisms that accumulate glycinebetaine, this compound is synthesized from choline in two oxidation steps, via the intermediate betaine aldehyde.

Four genes have been identified in Escherichia coli which confer osmotolerance (ability to grow in the presence of greater than 0.6 M NaCl) when choline is supplied to the growth medium; betA, betB, betI and betT (Styrvold et al, 1986; Lamark et al, 1991; 1996). These genes encode choline dehydrogenase [EC 1.1.99.1] (betA), betaine aldehyde dehydrogenase [EC 1.2.1.8] (betB), a choline-sensing repressor of the bet regulon (betI), and a high affinity choline transporter (betT) (Lamark et al, 1991; 1996). The entire bet gene cluster has been sequenced and the promoter regions characterized (Lamark et al, 1991; 1996). The bet genes are regulated at the transcriptional level by glycine betaine, temperature, osmolarity, oxygen and choline (Eshoo, 1988; Lamark et al, 1991; 1996).

The E. coli choline dehydrogenase (CDH) is a flavoprotein and may contain an N-terminal FAD-binding region. The E. coli CDH is oxygen-dependent, appears to be independent of soluble cofactors, is membrane bound and probably electron-transfer-linked (Lamark et al, 1991). When the enzyme is solubilized, phenazine methosulfate (PMS) can be used as artificial electron acceptor (Styrvold et al, 1986):

CDH also catalyzes the oxidation of betaine aldehyde to glycinebetaine in vitro (Styrvold et al, 1986; Lamark et al, 1991), but it is not certain that this reaction occurs in vivo, as CDH has a lower affinity for betaine aldehyde than betaine aldehyde dehydrogenase. Due to the O₂ requirement of CDH, E. coli can utilize choline only under aerobic growth conditions (Landfald and Strom, 1986). ArcA is known to control the expression of a number of oxygen-inducible genes of E. coli, including the bet genes (Lamark et al, 1996).

The betaine aldehyde dehydrogenase (BADH) of E. coli is a soluble enzyme with a high affinity for betaine aldehyde and has a strong preference for NAD⁺ as electron acceptor (Boyd et al, 1991):

An alternative choline oxidizing enzyme has been characterized in the gram-positive soil bacteria Arthrobacter pascens and A. globoformis. Here choline oxidase (COX) (E.C. 1.1.3.17), a soluble enzyme that does not require cofactors, uses O₂ as the primary electron acceptor (Ikuta et al, 1977ab; Rozwadowski et al, 1991):

The COX of Arthobacter species (~ 66 kDa) appears to be immunologically related to the COX of Alcaligenes sp. (Rozwadowski et al, 1991). Like CDH, COX is also capable of oxidizing betaine aldehyde (K_m for betaine aldehyde = 8.7 mM; cf. K_m for choline = 1.2 mM) (Ikuta et al, 1977ab):

The choline oxidase gene of the soil bacterium Arthrobacter globiformis has been designated codA (Hayashi et al, 1997). Rozwadowski et al (1991) note that although choline oxidase can catalyze both steps of the pathway, the presence of BADH may be advantageous because this enzyme has a 17- to 28-fold lower K_m for betaine aldehyde than COX, and generates NAD(P)H rather than H₂O₂. Betaine aldehyde is highly reactive and its accumulation can result in toxicity (Boch et al, 1996).

Although COX does not confer osmotolerance in the Arthobacter species from which it is derived (the enzyme appears to play a role in catabolism of choline in Arthobacter species), when the cox gene is expressed in an E. coli mutant that is defective in glycinebetaine synthesis, this confers glycinebetaine accumulation and osmotolerance in the presence of an exogenous supply of choline or betaine aldehyde (Rozwadowski et al, 1991).

In Bacillus subtilis, synthesis of glycine betaine from choline or betaine aldehyde confers considerable osmotic stress tolerance in high-osmolarity medium (Boch et al, 1996). Using E. coli betBA deletion mutants defective in the glycinebetaine synthesis pathway, two genes (gbsA and gbsB) encoding the Bacillus subtilis choline-glycinebetaine synthesis pathway were cloned by functional complementation (Boch et al, 1996). Whereas the deduced gbsA gene product exhibited strong sequence identity to nonspecialized aldehyde dehydrogenases, including the BADH from E. coli, the deduced gbsB gene product (~ 43 kDa) shows significant similarity to the family of type III alcohol dehydrogenases (Boch et al, 1996). This family of proteins comprises NAD/NADP-dependent alcohol dehydrogenases from several microorganisms (Boch et al, 1996). The deduced GbsB protein exhibits no homology to the membrane-bound FAD-containing CDH (BetA) of E. coli, or the COX of Arthrobacter pascens, implying that Bacillus subtilis may possess a novel mechanism of choline oxidation.

In the extreme halophytic phototrophic bacterium Ectothiorhodospira halochloris, glycinebetaine is not synthesized from choline, but rather by direct N-methylation of the amino acid, glycine (Galinksi and Truper, 1994; Nyyssola et al, 2000; 2001):

(where SAM = S-adenosylmethionine, and SAHC = S-adenosylhomocysteine).

These three N-methylations are catalyzed by two methyltransferases, glycine sarcosine methyltransferase and sarcosine dimethylglycine methyltransferase, with partially overlapping substrate specificity. The methyltransferases have been successfully expressed in E. coli, where they confer betaine accumulation and improved salt tolerance.

A pathway of choline oxidation similar to Arthobacter sp. has been described for the fungus Cylindrocarpon didymum M-1. A choline oxidase (a dimer of two subunits of MWt. 64 kDa, containing two mol of FAD per mol of enzyme), utilizes O₂ as electron acceptor and generates H₂O₂ as product. Like the Arthobacter enzyme, the fungal enzyme is also capable of oxidizing betaine aldehyde (K_m for betaine aldehyde = 5.8 mM, cf. K_m for choline = 1.3 mM), but occurs together with a NAD⁺-dependent BADH (Yamada et al, 1979; Tani et al, 1977; 1979).

In mammals, where glycinebetaine plays an important role as a compatible solute, particularly in the kidney (Bagnasco et al, 1986; Garcia-Perez and Burg, 1991ab), the pathway of glycinebetaine synthesis resembles that in E. coli, and is catalyzed by a particulate, mitochondrial CDH and a soluble BADH (Chern and Pietruszko, 1995). As with the E. coli enzyme, the CDH of mammals can utilize PMS as an artificial electron acceptor.

In the most extensively studied group of glycinebetaine-accumulating higher plants, the Chenopodiaceae, glycinebetaine is synthesized from choline in a two-step oxidation catalyzed by a ferredoxin (Fd)-dependent choline monooxygenase (CMO) (Brouquisse et al, 1989) and a betaine aldehyde dehydrogenase (BADH) [EC 1.2.1.8] with a strong preference for NAD⁺ (Weretilnyk and Hanson, 1989; Rhodes and Hanson, 1993). ¹⁸O₂ tracer studies confirm that CMO utilizes molecular oxygen as a substrate (Lerma et al, 1988). The immediate product of the CMO reaction may be betaine aldehyde hydrate [(CH₃)₃N⁺-CH₂-CH(OH)₂] which is in spontaneous equilibrium with betaine aldehyde [(CH₃)₃N⁺-CH₂-CH=O] (Lerma et al, 1988):

Both CMO and BADH enzymes are predominantly localized in the chloroplast stroma of chenopods (Weigel et al, 1986; 1988; Brouquisse et al, 1989), although in spinach leaves approximately 10% of the BADH may exist as a cytosolic isoform (Weretilnyk and Hanson, 1988). Glycinebetaine is also localized predominantly in the chloroplasts of salinized spinach leaves, where it provides osmotic adjustment (Robinson and Jones, 1986). The spinach CMO is not membrane bound, requires ferredoxin and Mg²⁺, and unlike the bacterial choline oxidizing enzymes (CDH and COX) does not appear to catalyze the oxidation of betaine aldehyde to glycinebetaine (Brouquisse et al, 1989).

Both CMO (Burnet et al, 1995) and BADH (Weretilnyk and Hanson, 1989) have been purified to homogeneity from spinach. CMO appears to be a homodimer (or possibly homotrimer) of subunits of MWt 42.9 kDa (Burnet et al, 1995; Rathinasabapathi et al, 1997), whereas BADH is a homodimer with a subunit MWt of ~ 60 kDa (Weretilnyk and Hanson, 1989).

cDNAs encoding BADH have been isolated from spinach (Weretilnyk and Hanson, 1990) and sugar beet (McCue and Hanson, 1992a). The spinach and sugarbeet BADHs show significant sequence identity to the human and bacterial BADHs described above (Boyd et al, 1991; Chern and Pietruszko, 1995). The spinach BADH has an unusual chloroplast transit target sequence (Rathinasabapathi et al, 1994). The chenopod BADH cDNAs have facilitated cloning of BADHs from a number of other higher plant sources (reviewed in Rhodes and Hanson, 1993), including several monocotyledons (barley, rice and sorghum) (Ishitai et al, 1995; Nakamura et al, 1997; Wood et al, 1996). In leaves of both chenopods and grasses BADH transcripts and enzyme level increase in response to salinity stress (Arakawa et al, 1990; McCue and Hanson, 1992a). A biochemical signal from the roots is implicated in this response in sugar beet (McCue and Hanson, 1992b). Transgenic tobacco plants over-expressing chloroplast localized BADH have been obtained (Rathinasabapathi et al, 1994). However, these are unable to accumulate glycinebetaine in the absence of exogenously supplied betaine aldehyde because tobacco lacks CMO (Rathinasabapathi et al, 1994).

It should be cautioned, however, that BADH may be a general aldehyde dehydrogenase, acting on other aldehyde substrates in addition to betaine aldehyde (Trossat et al, 1997). BADH will utilize 3-dimethylsulfoniopropionaldehyde, an intermediate in DMSP synthesis, and certain aldehydes (3-aminopropionaldehyde and 4-aminobutyraldehyde) involved in polyamine metabolism (Trossat et al, 1997). Transgenic plants engineered for BADH expression (Holmstrom et al, 1994; Rathinasabapathi et al, 1994) may possibly exhibit phenotypes related to perturbed polyamine metabolism in addition to phenotypes attributable to alterations in the glycinebetaine synthesis pathway (Trossat et al, 1997). The multiple substrate specificities of BADH may explain the occurrence of BADH in plants that do not accumulate glycinebetaine (Ishitani et al, 1993; Weretilnyk et al, 1989), and in organs of glycinebetaine-accumulating plants which do not contain glycinebetaine (e.g. roots of cereals) (Ishitani et al, 1995).

In monocotylednous plants, recent studies suggest that BADH is localized in peroxisomes (Nakamura et al, 1997). All monocotyledenous BADHs have a C-terminal tripeptide (SKL) that is known to be a signal for targeting preproteins to microbodies (Nakamura et al, 1997). This raises the question of whether the subcellular localization of the choline oxidation pathway is the same in grasses as in chenopods. If glycinebetaine synthesis occurs in the peroxisome in grasses, then CMO is unlikely to be the choline-oxidizing enzyme of grasses, because its Fd requirement would not be met in the peroxisome (Russell et al, 1998).

A cDNA encoding CMO has recently been cloned from spinach (Rathinasabapathi et al, 1997). The cDNA sequence confirms that CMO is an Fe-S protein containing one [2Fe-2S] cluster per subunit, and that CMO has a typical chloroplast transit peptide sequence (Rathinasabapathi et al, 1997). Spinach CMO shows no homology to any of the other known choline oxidizing enzymes (CDH and COX) (Rathinasabapathi et al, 1997). The activity of CMO rises incrementally in response to salinity levels (Brouquisse et al, 1989). This is likely the result of increased transcription (Rathinasabapathi et al, 1997). The magnitude of salt induction of CMO mRNA is comparable to that reported for BADH (Rathinasabapathi et al, 1997). The spinach CMO has been used to clone the sugar beet CMO, and to detect salt-inducible CMO mRNA in leaves of Amaranthus caudatus (Amaranthaceae) (Russell et al, 1998).

As noted above, in bacteria such as Escherichia coli, choline is an osmoprotectant only because it is oxidized to glycinebetaine (Lamark et al, 1991; 1996). The entire E. coli bet gene cluster has been introduced into the freshwater cyanobacterium Synechococcus and has been shown to confer glycinebetaine accumulation and increased salt tolerance, due in part to stabilization of Photosystem II (PS-II) (Nomura et al, 1995; 1998). However, this increased salt tolerance appears to depend on the presence of an exogenous supply of choline. Transgenic tobacco plants expressing the E. coli betA gene encoding choline dehydrogenase alone or in combination with the E. coli betB gene encoding betaine aldehyde dehydrogenase, accumulate glycinebetaine and exhibit increased tolerance to salt stress, enhanced recovery from photoinhibition caused by high light and salt stress, and improved tolerance to photoinhibition under low temperature conditions, partly due to improved protection of the photosynthetic apparatus (Holmstrom et al, 2000).

When the codA gene [encoding choline oxidase [EC 1.1.3.17], derived from the soil bacterium Arthrobacter globiformis] is transformed into Arabidopsis thaliana, this confers glycinebetaine accumulation and increased salt, light, heat and cold stress tolerance (Alia et al, 1998a; 1998b; 1999; Hayashi et al, 1997; 1998; Hayashi and Murata, 1998). The increased salt and light stress tolerance of the transgenic plants is associated with maintenance of PS-II activity or enhanced recovery of the PS-II complex from the photo-inactivated state (Hayashi et al, 1997; Alia et al, 1999). Transformation of Arabdiopsis with the codA gene modestly increased the levels of H₂O₂ (Alia et al, 1999). The activity of ascorbate peroxidase and catalase were also elevated in the transgenic plants, suggesting that H₂O₂ produced by choline oxidase in the transformed plants might have stimulated the expression of H₂O₂ scavenging enzymes (Alia et al, 1999).

Transformation of rice (Oryza sativa) with the codA gene targeted to the chloroplasts or cytosol, respectively, produced levels of glycinebetaine of from 1 and 5 umol umol.g^-1 Fw of leaf tissue, respectively (Sakamoto et al, 1998). Rice plants expressing choline oxidase in the chloroplast were more tolerant to inactivation of photosynthesis under salt stress and low-temperature stress than transgenic plants expressing the enzyme in the cytoplasm (Sakamoto et al, 1998; 2000; Sakamoto and Murata, 2000, 2001).

In transgenic tobacco expressing spinach choline monooxygenase (CMO) in the chloroplast, levels of glycinebetaine achieved are only <0.1 umol.g^-1 Fw, unless the plants are supplied with exogenous choline, or its precursors mono- and dimethylethanolamine (Nuccio et al, 1998). This suggest that in glycinebetaine-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 glycinebetaine levels as high as those in natural accumulators (Nuccio et al, 1998; 1999). [Similar results were obtained with three diverse species, Arabidopsis thaliana, Brassica napus, and tobacco (Nicotiana tabacum), by constitutive expression of the bacterial COX gene, suggesting the need to enhance the endogenous choline supply to support accumulation of physiologically relevant amounts of glycinebetaine (Huang et al, 2000). Huang et al (2000) note that a moderately increased stress tolerance was observed in some but not all betaine-producing transgenic lines.] McNeil et al (2000) conclude from analyses of ¹⁴C-choline metabolism in transgenic tobacco expressing CMO in the chloroplast, that a major constraint on glycinebetaine synthesis is the low capacity of the chloroplast envelope for transport of choline. The chloroplast-localized CMO does not compete effectively with choline kinase for the low cytosolic choline concentrations (McNeil et al, 2000; Nuccio et al, 2000). Tobacco plants that overexpress phosphoethanolamine N-methyltransferase in the cytosol and both CMO and BADH in the chloroplast show enhanced glycine betaine synthesis in comparison to CMO+ and BADH+ transgenics (McNeil et al, 2001). However, levels of glycine betaine accumulated are still well below those of natural glycine betaine-accumulators. Tobacco plants that overexpress phosphoethanolamine N-methyltransferase in the cytosol and both CMO and BADH in the chloroplast, predominantly accumulate choline and P-choline, suggesting that choline import from the cytosol to the chloroplast still remains a serious constraint on flux from choline to glycine betaine (McNeil et al, 2001; Rontein et al, 2002).

In maize, a number of naturally occurring glycinebetaine-deficient inbred lines have been identified (Brunk et al, 1989). These lack the ability to accumulate glycinebetaine in leaf tissue in response to either salinity stress or water deficits due to a single recessive gene (bet1) which has been mapped to the short arm of chromosome 3 near the centromere (Rhodes et al, 1993; Yang et al, 1995). Glycinebetaine deficiency in maize is associated with an inability to oxidize ¹⁴C-choline to ¹⁴C-glycinebetaine, but an unimpaired ability to oxidize ²H₃-betaine aldehyde to ²H₃-glycinebetaine, suggesting a lesion at the CMO step in the biosynthetic pathway, or equivalent choline oxidizing activity (Lerma et al, 1991). Homozygous glycinebetaine-deficient maize lines appear to be more salt-sensitive than near-isogenic homozygous glycinebetaine-containing lines (Saneoka et al, 1995), and exhibit less membrane injury and less damage to PS-II in response to heat stress (Yang et al, 1996). Glycinebetaine-deficient maize lines exhibit a significantly elevated pool of free choline; however, choline does not accumulate to levels equal to the glycinebetaine level of glycinebetaine-containing lines, suggesting that choline must down-regulate its own synthesis (Yang et al, 1995). Consistent with this, glycinebetaine-deficient lines of maize exhibit an elevated level of serine in comparison to homozygous glycinebetaine-containing lines (Yang et al, 1995).

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