drogenase (BADH, EC 1.2.1.8) (Figure 2) [8]. Immunological studies suggest that the pathway is com-
mon in angiosperms. Glycine betaine–accumulating species from several distantly related families of di-
cotyledons (Spinacia oleracea,Amaranthus caudatus,Convolvulus arvensis, and Lycium ferocissimum)
have expressed immunologically related BADH enzymes [24]. The nonaccumulating species in Magno-
liaandSoulangianahave small amounts of GB and express a 63-kDa protein that cross-reacted with an-
tibodies of BADH from spinach (S. oleracea).
The first step in GB synthesis is catalyzed by ferredoxin-dependent choline monooxygenase [109,110]
that converts choline to the hydrate form, which is the dominant form (99%) in aquous extracts. CMO is
an unusual ferrodoxin-dependent enzyme that is unique to plants and has been purified only from spinach
[111,112]. CMO expression is reported only in Amaranthaceae and Chenopodiaceae [113,114] and has not
been reported in monocots [36]. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (PAGE) and
matrix-assisted laser desorption ionization of MS showed the presence of a polypeptide with a subunit
molecular mass of 45 kDa, suggesting that CMO is a homodimer of identical subunits [111,112], each of
which has a Rieske-type [2Fe-2S] cluster [113,114]. CMO is located in the chloroplast stroma [110], has a
pH optimum close to 8, and is strongly stimulated by light and Mg^2 [109,115].
The second step in GB synthesis is catalyzed by a pyridine nucleotide–dependent betaine aldehyde
dehydrogenase and has a strong preference for NAD[8,24,29,116]. BADH enzyme activity is found in
888 SUBBARAO ET AL.
Precipitation with
either periodide
or reineckate or
Dragendorff
reagent
TLC and TLE
Pyrolysis-GC/FID
GC/FID upon
derivatization
HPLC/UV
(^1) H NMR and (^13) C
NMR
FAB-MS
PD-MS
TABLE 3 Advantages and Disadvantages Associated with Various Analytical Techniques for Glycine
Betaine Analysis
Method Advantages Disadvantages
Moderate Sensitivity
Specific
Sensitive (2 nmol limit)
Moderate sensitivity
Versatile
Selective and specific
Convenient
Sensitive (sensitivity equal to or
better than pyrolysis GC using
low UV, greater when
derivatized)
Easy to automate and suitable for
large number of samples
Specific
Nondestructive, suitable for
analysis in vivo
Extremely sensitive (MDL 0.05
nmol)
Specific
Rapid
Simple sample preparation
Sensitive for choline analysis
Nonspecific
Sensitivity varies (2.5 nmol using methyl orange
reagent, 85 nmol using Dragendroff reagent)
Lack of precision, semiquantitative
Limited separation efficiency
Tedious, a complete standard curve being required
for every plate
Not very specific
Costly (pyrolysis probe)
Derivatization is required
Not well characterized for betaine analysis
Not very sensitive (MDL—85 nmol of 90 Hz^1 H
NMR)
Costly (instrument)
Expensive (equipment and stable isotope labeled
GB)
Less sensitive for GB than FAB-MS
Expensive (equipment and stable isotope labeled
GB)