The class of small molecules known as "compatible osmolytes" includes certain amino acids (notably proline), quaternary ammonium compounds (e.g. glycinebetaine, prolinebetaine, B-alaninebetaine, and choline-O-sulfate), and the tertiary sulfonium compound 3-dimethylsulfoniopropionate (DMSP). The quaternary ammonium compounds and DMSP are derived from amino acid precursors.

These compounds share the property of being uncharged at neutral pH, and are of high solubility in water (Ballantyne and Chamberlin, 1994). Moreover, at high concentrations they have little or no perturbing effect on macromolecule-solvent interactions (Yancey et al, 1982; Low, 1985; Somero, 1986; Timasheff, 1993; Yancey, 1994).

Unlike perturbing solutes (such as inorganic ions) which readily enter the hydration sphere of proteins, favoring unfolding, compatible osmolytes tend to be excluded from the hydration sphere of proteins and stabilize folded protein structures (Low, 1985). These compounds are thought to play a pivotal role in plant cytoplasmic osmotic adjustment in response to osmotic stresses (Wyn Jones et al, 1977).

Proline accumulation is a common metabolic responses of higher plants to water deficits, and salinity stress, and has been the subject of numerous reviews over the last 20 years (see e.g. Stewart and Larher, 1980; Thompson, 1980; Stewart, 1981; Hanson and Hitz, 1982; Rhodes, 1987; Delauney and Verma, 1993; Samaras et al, 1995; Taylor, 1996; Rhodes et al, 1999).

This highly water soluble imino acid is accumulated by leaves of many halophytic higher plant species grown in saline environments (Stewart and Lee, 1974; Treichel, 1975; Briens and Larher, 1982), in leaf tissues and shoot apical meristems of plants experiencing water stress (Barnett and Naylor, 1966; Boggess et al, 1976; Jones et al, 1980), in desiccating pollen (Hong-qi et al, 1982; Lansac et al, 1996), in root apical regions growing at low water potentials (Voetberg and Sharp, 1991), and in suspension cultured plant cells adapted to water stress (Tal and Katz, 1980; Handa et al, 1986; Rhodes et al, 1986), or NaCl stress (Katz and Tal, 1980; Tal and Katz, 1980; Treichel, 1986; Binzel et al, 1987; Rhodes and Handa, 1989; Thomas et al, 1992).

Proline protects membranes and proteins against the adverse effects of high concentrations of inorganic ions and temperature extremes (Pollard and Wyn Jones, 1979; Paleg et al, 1981; Nash et al, 1982; Paleg et al, 1984; Brady et al, 1984; Gibson et al, 1984; Rudolph et al, 1986; Santarius, 1992; Santoro et al, 1992). Proline may also function as a protein-compatible hydrotrope (Srinivas and Balasubramanian, 1995), and as a hydroxyl radical scavenger (Smirnoff and Cumbes, 1989).

The proline accumulated in response to water stress or salinity stress in plants is primarily localized in the cytosol (Leigh et al, 1981; Ketchum et al, 1991; Pahlich et al, 1983).

In cell cultures of tobacco adapted to 428 mM NaCl, proline represents over 80% of the free amino acid pool (Rhodes and Handa, 1989). Assuming uniform distribution of the proline in total intracellular water volume this amino acid is present at levels in excess of 129 mM (Binzel et al, 1987). If confined to the cytoplasm, however, the concentration of proline could exceed 200 mM in these cells and therefore contribute substantially to cytoplasmic osmotic adjustment (Binzel et al, 1987). Similarly, the cytosolic proline concentration of salt stressed Distichlis spicata cells (treated with 200 mM NaCl) is estimated to be >230 mM (Ketchum et al, 1991).

In the apical millimeter of maize roots, proline represents a major solute, reaching concentrations of 120 mM in roots growing at a water potential of -1.6 MPa (Voetberg and Sharp, 1991). The accumulated proline accounts for a significant fraction (~50%) of the osmotic adjustment in this region (Voetberg and Sharp, 1991). Proline accumulation in maize root apical meristems in response to water deficits involves increased proline deposition to the growing region, and appears to require abscisic acid (ABA) (Ober and Sharp, 1994; Sharp et al, 1994).

Although maize roots are known to synthesize proline (Oaks et al, 1970), at present it is unclear whether increased deposition of proline in the apical region is a consequence of increased transport to the apex via the phloem, or de novo synthesis of proline in the apex (Voetberg and Sharp, 1991).

Exogenously supplied proline is osmoprotective for bacteria, facilitating growth in highly saline environments (Csonka, 1989; Strom et al, 1983; Csonka and Hanson, 1991; Yancey, 1994). Accumulation of proline in the cytoplasm is accompanied by a reduction in the concentrations of less compatible solutes and an increase in cytosolic water volume (Cayley et al, 1991; 1992).

Exogenously supplied proline can also be osmoprotective (Tal and Katz, 1980; Wyn Jones and Gorham, 1983; Handa et al, 1986; Lone et al, 1987) and cryoprotective (Withers and King, 1979; van Swaaji et al, 1985; Duncan and Widholm, 1987; Songstad et al, 1990; Santarius, 1992) to higher plant cells.

Increased osmotolerance of bacteria has been achieved by proline over-production caused by altered feedback inhibition of the proline biosynthesis pathway (Csonka, 1981; Smith, 1985).

Selection for hydroxyproline-resistant mutants of barley and winter wheat has succeeded in identifying lines that accumulate greater quantities of proline than wild-type (Kueh and Bright, 1981; Dorffling et al, 1993). However, it appears that the concentrations of proline accumulated by these mutants may be an order of magnitude smaller than required to produce a significant physiological effect on osmotic stress tolerance (Lone et al, 1987). In winter wheat the hydroxyproline-resistant lines are significantly more frost tolerant than wild-type (Dorffling et al, 1993). Salt tolerant and polyethylene glycol resistant mutants of Nicotiana plumbaginifolia have been derived from protoplast culture and appear to have enhanced proline accumulation in comparison to wild-type (Sumaryati et al, 1992).

Proline synthesis is implicated as a mechanism of alleviating cytoplasmic acidosis, and may maintain NADP⁺/NADPH ratios at values compatible with metabolism (Hare and Cress, 1997). Rapid catabolism of proline upon relief of stress may provide reducing equivalents that support mitochondrial oxidative phosphorylation and the generation of ATP for recovery from stress and repair of stress-induced damage (Hare and Cress, 1997; Hare et al, 1998).

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