A Comparitive Biochemical Study of the Enzymes of Choline Synthesis in Several Dicotyledon Families

Abstract

Choline is universal among plants as phosphatidylcholine and in many plants it serves as a precursor for the compatible osmolytes glycine betaine and choline-𝘖-sulphate. In spinach, choline is synthesized by the sequential 𝘕-methylation of phosphoethanolamine (PEA) → phosphomethylethanolamine (PMEA) → phosphodimethylethanolamine (PDEA) → phosphocholine (PCho) and PCho is hydrolyzed to choline. The objective of this biochemical survey was to determine whether the activities of enzyme(s) converting PEA to PCho could be found in leaves of diverse plants and, if so, if common regulatory properties could be identified. Leaf tissue was harvested from 14 diverse dicot plants. Enzyme activities for the three sequential 𝘕-methylations were quantified in vitro and for representative assays using PEA as substrate, reaction products were identified by thin layer chromatography. Extracts of all plants tested could metabolize PEA to PMEA, the rate of conversion varied from 0.04 to 25 nmol · min⁻¹ · g⁻¹ Fresh wt for soybean and cotton, respectively. In vitro PMEA → PDEA and PDEA → PCho rates varied between the plant species tested. Both steps were highest in sugar beet (22 and 24 nmol · min⁻¹ · g⁻¹ Fresh wt for · PMEA → PDEA and PDEA → PCho, respectively) to below detection limits for soybean (<0.03 nmol· min⁻¹ · g⁻¹ Fresh wt). Upon dark exposure, PEA → PMEA reaction rates were reduced from the light period levels but not in all cases was the activity reduced beyond the level that could be attributed to a general loss in plant vigour. The reduction in 𝘕-methylation capacity was most pronounced in plants with the highest levels of PEA → PMEA activity, with reduction greatest in sugar beet > amaranth > spinach. Thus PEA → PMEA is catalyzed by a light-regulated enzyme in many but apparently not all dicot plants. Datko and Mudd (1988a) have proposed that PEA → PMEA conversion is ubiquitous among plants and the enzyme responsible catalyzes a committing step for PCho synthesis. PEAMeT activity was found in leaf extracts of 10 additional dicot species. The level of in vitro activity and light regulation is not equivalent among all plants examined. If in vitro rates faithfully reflect the in vivo capacity to synthesize choline, why the variability among plants with respect to their capacity to synthesize a universal metabolite? Alternate routes of PCho/phosphatidyl choline synthesis may explain these differences (Datko and Mudd, 1988a,b; Hanson and Rhodes, 1983; Hitz 𝘦𝘵 𝘢𝘭., 1981; Weretilnyk and Summers, 1992). Interestingly, the plants with the highest rates of PEA → PMEA activity, (cotton, sugar beet, amaranth, sunflower, spinach and statice) are all documented glycine betaine accumulating species. These plants may have higher rates of PEA methylation in order to meet requirements for osmolyte synthesis. Thus, it would be interesting to see if PEA metabolism to PCho is up-regulated in response to osmotic stress.