members of Chenopodiaceae [117,118], Amaranthaceae [119,120], and Gramineae [32,47]. In spinach
leaves, the majority of the BADH activity is located in the chloroplast stroma [8,121]; however, in mem-
bers of Gramineae, BADH may be peroxisomal [47]. Substantial levels of BADH were reported in roots
of sugar beet [117] and in the etiolated leaves of barley [29]. Nevertheless, GB synthesis has not been de-
tected in sugar beet roots [21]. Thus it is not certain that GB synthesis can occur outside chloroplasts and
organs other than leaves. BADH enzyme has been purified to homogeneity from spinach [122,123] and
amaranth [119,120]. BADH is a dimer with subunits of 60 kDa and has a native molecular mass of 125
kDa. The spinach and amaranth BADH has a pH optimum of about 8.6 and optimum temperature of
around 50°C. BADH is activated by relatively low concentrations of K, sucrose, and proline but is in-
hibited by NH 4 , Na, and high concentrations of GB [120].
B. Biosynthetic Pathway(s) of Choline (GB Precursor)
The biosynthetic pathway of choline has been studied in GB-accumulating (e.g., sugar beet, spinach, and
barley) and GB-nonaccumulating species (e.g., carrot, Lemna, soybean cell cultures, and castor bean en-
dosperm). In higher plants, choline is synthesized from serine via ethanolamine [36]. Choline biosynthe-
sis involves three parallel, interconnected series of N-methylation reactions at the free base, phospho base,
or phosphytidyl base level (Figure 3). The predominant routes have been reviewed by Rhodes and Han-
son [8]. Ethanolamine kinase and three S-adenosylmethionine phospho bases, N-methyl transferases, are
reported to catalyze the methylation of phosphoethanolamine [124]. The regulatory step for choline syn-
thesis is the enzyme catalyzing the first N-methylation of phosphoethanolamine, which is stimulated by
light and also is triggered by osmotic stress [125,126].
C. Regulation of Glycine Betaine Synthesis
Glycine betaine synthesis is regulated at the biosynthetic level and is proportional to the severity of salin-
ity or water deficits [21,127]. In vivo radiotracer studies indicate that enhanced GB synthesis is accom-
panied by a higher rate of choline synthesis followed by oxidation of choline to GB [8]. Consistent with
these in vivo data, activities of both CMO and BADH increase under salinity and water stress. Choline
monooxygenase in sugar beet and Amaranthus caudatusincreased severalfold in response to osmotic
stress [114]. Similarly, BADH levels increased two- to fourfold in leaves of sugar beet when plants were
exposed to NaCl (500 mM) salinity [117,128]. The increase of BADH activity in sugar beet leaves and
roots under salinity is paralleled by an increase in levels of translatable BADH messenger RNA (mRNA).
GLYCINE BETAINE IN STRESS RESISTANCE 889
Figure 2 The biosynthesis of glycine betaine from choline in higher plants.