Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves

Abstract

In barley, glycine betaine is a metabolic end product accumulated by wilted leaves; betaine accumulation involves acceleration of de novo synthesis from serine, via ethanolamine, N-methylethanolamines, choline, and betaine aldehyde (Hanson, Scott 1980 Plant Physiol 66: 342-348). Because in animals and microorganisms the N-methylation of ethanolamine involves phosphatide intermediates, and because in barley, wilting markedly increases the rate of methylation of ethanolamine to choline, the labeling of phosphatides was followed after supplying [(14)C]ethanolamine to attached leaf blades of turgid and wilted barley plants. The kinetics of labeling of phosphatidylcholine and betaine showed that phosphatidylcholine became labeled 2.5-fold faster in wilted than in turgid leaves, and that after short incubations, phosphatidylcholine was always more heavily labeled than betaine. In pulse-chase experiments with wilted leaves, label from [(14)C]ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When [(14)C]monomethylethanolamine was supplied to wilted leaves, phosphatidylcholine was initially more heavily labeled than betaine. These results are qualitatively consistent with a precursor-to-product relationship between phosphatidylcholine and betaine.The following experiments, in which tracer amounts of [(14)C]ethanolamine or [(14)C]formate were supplied to wilted barley leaves, implicated phosphoryl and phosphatidyl bases as intermediates in the methylation steps between ethanolamine and phosphatidylcholine. Label from both [(14)C]ethanolamine and [(14)C]formate entered phosphorylmonomethylethanolamine and phosphorylcholine very rapidly; these phosphoryl bases were the most heavily labeled products at 15 to 30 minutes after label addition and lost label rapidly as the fed (14)C-labeled precursor was depleted. Phosphatidylmonomethylethanolamine and phosphatidylcholine were also significantly labeled from [(14)C]ethanolamine and [(14)C]formate at early times; the corresponding free bases and nucleotide bases were not. Addition of a trapping pool of phosphorylcholine reduced [(14)C]ethanolamine conversion to both phosphatidylcholine and betaine, and resulted in accumulation of label in the trap.A computer model of the synthesis of betaine via phosphatidylcholine was developed from (14)C kinetic data. The model indicates that about 20% of the total leaf phosphatidylcholine behaves as an intermediate in betaine biosynthesis and that a marked decrease (>/=2-fold) in the half-life of this metabolically active phosphatidylcholine fraction accompanies wilting. Dual labeling experiments with [(14)C]choline and [(3)H]glycerol confirmed that the half-life of the choline portion of phosphatidylcholine falls by a factor of about 2 in wilted leaves.