B. Glycine Betaine Accumulation in Microorganisms
Glycine betaine is one of the most widespread osmotic solutes in microorganisms, particularly in
halophilic eubacteria. Glycine betaine functions as a compatible solute, enabling them to withstand the
high osmotic potential (OP) of their surrounding medium [55]. In cyanobacteria GB prevents the in-
hibitory effects of high salt levels on enzyme activity [56]. Only three bacterial groups are reported to syn-
thesize betaine de novo; these are cyanobacteria [57–59], anaerobic photosynthetic bacteria (Ectohiorho-
dospira), and Actinopolyspora halophila[60]. Transport systems for GB are reported for Escherichia
coli,Salmonella typhimurium, and Klebsiella pneumoniae[61];Rhizobium meliloti[62];Rhodobacter
sphaeroides[63]; and Azospirillum brasilense[64].
Osmoprotective effects of endogenously synthesized and exogenously supplied GB are reported for
bacteria [65– 67] and cyanobacteria [68]. Addition of GB to the plating medium restored the ability of E.
colito form colonies under otherwise toxic osmotic conditions [69]. A number of halophilic bacteria ac-
cumulate GB from the medium [70]. Under salt stress, cyanobacteria [71], halophilic bacteria
[18,19,72,73], nonhalophilic eubacteria [74,75], E. coli[76], halotolerant microorganisms [77], and green
algae [8,66,78,79] accumulate GB. Intracellular GB concentrations can vary from 0.5 to 2.0 M in
halophilic eubacteria [58,73]. Glycine betaine protects the respiratory system against salt damage in mod-
erately halophilic bacteria [80].
II. ANALYTICAL METHODOLOGY
The quantitative determination of betaines (fully N-methylated amino acids) has research implications for
both plant and animal systems. Both chromatographic and spectrometric methods (see Refs. 4 and 8 for
earlier reviews on these methods) are discussed in this chapter. This review is intended to provide a quick
reference to researchers in finding suitable tools for their specific research. We focus only on GB analy-
sis during this review. Glycine betaine analysis involves three important steps: sample extraction, purifi-
cation, and final quantification (Figure 1).
A. Extraction
Quantification of GB in vivo is possible [43,81]; however, most studies of GB are performed on extracts.
Glycine betaine is soluble in water (160 g/100 g water), methanol (55 g/100 g solvent), and ethanol (8.7
g/100 g solvent) but sparingly soluble in ether or chloroform. Thus, betaines are extracted with hot aque-
ous alcohol [82], methanol [35,83], or methanol-chloroform-water (MCW) mixtures [84–86]. The pro-
portion of methanol-chloroform-water used in previous studies varied with plant materials. A ratio of
12:5:3 has been widely used. An optimal solvent system is one that renders the best recovery and highest
GLYCINE BETAINE IN STRESS RESISTANCE 883
TABLE 2 Glycine Betaine Levels Under Stress in Various Crop Plants
Betaine levels (mol g^1 dwt)Crop species Stress type Control Stress References
Wheat Salinity 20 a 170 [40]
Salinity 40 [41]
Leymus sabulosus Salinity
(250 mM NaCl) 8.0b 28 b [42]
Barley Salinity 20 a 170 [40]
Sugarbeet
Spinach Salinity 150 a [43]
Halophyrum mucronatum Salinity 100 [44]
Suaeda fruticosa Salinity 200 [44]
Haloxylon recurvum Salinity 600 [44]
Amaranthus tricolor Salinity 80 260 [45]
aConverted from fresh weight values and expressed as dry weight values assuming that fresh weight/dry weight ratio is about
- bConcentration in mM of leaf sap.