In response to salinity and drought, some higher plants accumulate the secondary metabolite glycinebetaine which functions as a compatible osmolyte (Rhodes and Hanson, 1993). Choline, a precursor of glycinebetaine (Rhodes and Hanson, 1993), is also a component of a primary metabolite phosphatidylcholine, an ubiquitous membrane phospholipid (Moore, 1982). In leaves of the glycinebetaine accumulator spinach, choline is synthesized as phosphocholine (PCho) and PCho is synthesized by three sequential N-methylations of phosphoethanolamine (PEA) ~ phosphomethyl-EA (PMEA) ~ phosphodimethyl-EA (PDEA) ~ PCho. The methyl group donor is Sadenosyl- L-methionine, SAM (Summers and Weretilnyk, 1993). The enzyme SAM: PMEA N-methyltransferase (PMEAMeT) is suggested to N-methylate PMEA ~ PDEA ~ PCho (Weretilnyk and Summers, 1992). A four-step strategy was developed for the partial purification of PMEAMeT on the basis of PMEA-dependent methylations (PMEAMeT activity) that involved the extraction of soluble leaf protein, ammonium sulfate precipitation, and column chromatography on DEAE Sepharose, Phenyl Sepharose and High Q Anion matrices. PDEA-dependent methylating activity co-purified with PMEAMeT activity which suggested PMEAMeT may N-methylate PMEA ~ PDEA ~ PCho. PMEAMeT was purified 43-fold and has specific activities of 14.7 and 18.0 nmol•min-1•mg-1 protein with PMEA and PDEA as substrates, respectively. Thin layer chromatography was used to identify the reaction products formed during the 30 minute assay incubation: with PMEA as the substrate, PDEA and PCho were detected in a ratio of 9: 1 as products; and with PDEA as the substrate, PCho was detected as the only product. PMEAMeT was estimated to have a native molecular mass of 76 kDa by HPLC gel filtration chromatography. Both PMEA and PDEA N-methylating activities have an alkaline pH optimum between 8.5 and 9.0 in 0.1 M Tris-HCl buffer. Neither activity was iii affected by the omission of Na2-EDT A from the assay. The addition of 10 mM Mg2+ to the assay inhibited PMEA and PDEA-dependent methylation by approximately 49% and 32%, respectively; whereas, the addition of 1 and 10 mM Mn2+ to the assay completely inhibited both activities. Both activities were inhibited by the reaction products S-adenosyl-Lhomocysteine by over 90% at 0.2 mM and PCho by approximately 80% at 10 mM. Of the products ofPCho hydrolysis, choline inhibited PMEA-dependent methylation by 10% at 10 mM; whereas, Pi inhibited PMEA and PDEA-dependent methylation by 38 and 19%, respectively at 10 mM. The compatible osmolyte glycinebetaine inhibited PMEA and PDEA-dependent methylation by between 20 and 30% at 140 mM; however, the inibition of PMEA-dependent methylation can be partly accounted for by the presence of cr ions in the assay. If present at these concentrations in the same subcellular compartment, these metabolites could serve as regulators ofPMEAMeT activities in vivo. Study of PMEAMeT contributed to identifying the number of enzymes that Nmethylate PEA ~ PMEA ~ PDEA ~ PCho and possible regulatory metabolites for choline biosynthesis in vivo. These data are pertinent to basic research and also to genetic-engineering studies aimed at introducing the glycinebetaine-accumulating trait into crop plants as an approach to enhancing osmotic-stress resistance. / Thesis / Master of Science (MSc)

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A four-year drought, increasing population and shifting climate has spurred water conservation practices within Virginia. Creeping bentgrass (<i>Agrostis palustris</i> "L93"), Kentucky bluegrass (<i>Poa pratensis </i>"Midnight"), and tall fescue (<i>Festuca arundinacea</i>) Dominion blend were evaluated under deficit irrigation and upon exogenous application of plant growth stimulants (PGS), seaweed extract (SWE) + humic acid (HA), glycinebetaine (GB) and a commercial SWE product (PP). The objectives were to determine crop coefficients (K_c) for creeping bentgrass fairways and tall fescue home lawns, to determine if PGS application allowed for more water conservation, and to determine if they impacted physiological function and/or root morphology. A preliminary greenhouse experiment was conducted with creeping bentgrass and Kentucky bluegrass irrigated with 100%, 85% and 70% of evapotranspiration (ET). The study determined that an additional deficit irrigation level should be included for the field study and that GB application and 100% and 85% ET irrigation level produced the greatest creeping bentgrass root mass. The two–year field study evaluated creeping bentgrass and tall fescue. Tall fescue home lawns could be irrigated every five days with a K_c of 0.55 or once a week with a K_c of 0.70. Creeping bentgrass fairways could be irrigated every four days with a K_c of 0.85. Glycinebetaine application increased bentgrass rooting after planting and showed osmoprotectant properties. Another greenhouse study evaluated five GB rates on bentgrass and tall fescue. No differences were found between the five rates and concluded that the rate utilized in the field study may be appropriate for turfgrass application. / Master of Science