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In secondary leaves from spinach plants pretreated in vermiculite for 24 h with 300 mM NaCl, glycinebetaine accumulated at a rate of circa 0.16 μmol 100 μg Chl d (2 μmol g FW d), about three times the rate of control plants. The soluble carbohydrate and free amino acid contents did not increase significantly following salinisation until after 4 d when the relative growth rate also decreased. Leaf proline levels remained very low throughout the experimental period. K on a tissue water basis remained constant at 200 mM while Cl and Na levels increased linearly to reach 175 and 100 mM respectively after 5 d of saline treatment. The osmotic pressure of leaf tissue also increased from 300 to 500 mosmol kg. These experimental conditions were considered suitable to study glycinebetaine biosynthesis and its induction by salinity in the absence of marked growth inhibition or metabolic disturbance. Radioactive labelled [C]serine, ethanolamine and choline (all 1 μmol, 13.3 MBq in 10 μl) were fed to detached secondary leaves via the petiole 24 h after the exposure of plants to salt. The rate of isotope incorporation into water soluble products, lipids and residue was measured over a further 24 h. The major metabolic fate of exogenous [C]choline and [C]ethanolamine was incorporation into glycinebetaine while less C-label was found in phosphatidyl choline and phosphatidyl ethanolamine. Incorporation rates were identical in control and salinised leaves and were adequate to account for observed values of glycinebetaine accumulation previously reported in spinach. In contrast the labelling of glycinebetaine from [C]serine was twice as great in salinated plants as in the controls. These results, together with short term labelling experiment with [C]ethanolamine using leaf slices, were consistent with the formation of glycinebetaine via serine, ethanolamine and its methylated derivatives to choline with some control being exerted at the serine level. However a flux through the phosphorylated intermediates is not excluded. From a consideration of these results and the published data on barley subjected to water stress (Hanson and Scott, 1980 Plant Physiol. 66, 342-348) there appear to be significant differences in the biosynthetic pathways in spinach and barley. [ABSTRACT FROM AUTHOR]