Betaine Accumulation and [14C]Formate Metabolism in Water-stressed Barley Leaves

Barley ( Hordeum vulgare L.) plants at the three-leaf stage were water-stressed by flooding the rooting medium with polyethylene glycol 6000 with an osmotic potential of −19 bars, or by withholding water. While leaf water potential fell and leaf kill progressed, the betaine (trimethylglycine) content of the second leaf blade rose from about 0.4 micromole to about 1.5 micromoles in 4 days. The time course of betaine accumulation resembled that of proline accumulation. Choline levels in unstressed second leaf blades were low ( When [methyl- 14 C]choline was applied to second leaf blades of intact plants in the growth chamber, water-stressed plants metabolized 5 to 10 times more 14 C label to betaine than control plants during 22 hours. When infiltrated with tracer quantities of [ 14 C]formate and incubated for various times in darkness or light, segments cut from water-stressed leaf blades incorporated about 2- to 10-fold more 14 C into betaine than did segments from unstressed leaves. In segments from stressed leaves incubated with [ 14 C]formate for about 18 hours in darkness, betaine was always the principal 14 C-labeled soluble metabolite. This 14 C label was located exclusively in the N-methyl groups of betaine, demonstrating that reducing equivalents were available in stressed leaves for the reductive steps of methyl group biosynthesis from formate. Incorporation of 14 C from formate into choline was also increased in stressed leaf tissue, but choline was not a major product formed from [ 14 C]formate. These results are consistent with a net de novo synthesis of betaine from 1- and 2-carbon precursors during water stress, and indicate that the betaine so accumulated may be a metabolically inert end product.