The compatible solute glycine betaine accumulates in many plants including spinach (Spinacea oleracea) under conditions of water deficit stress. The precursor to glycine betaine is choline, a ubiquitous metabolite in plants as a component of phosphotidylcholine. In spinach choline is synthesized from phosphocholine, a product of three sequential N-methylations of phosphoethanolamine catalysed by the cytosolic enzyme S-adenosyl-L-methionine: phosphoethanolamine-N-methyltransferase (PEAMT). PEAMT activity shows diurnal changes with peak activity at the end of the photoperiod and a decrease overnight. The activity of this enzyme is up-regulated 2 to 3-fold in salt-stressed plants relative to unstressed plants. The objective of this thesis is to determine how PEAMT activity is regulated in vivo. Thus, PEAMT activity, protein and transcript levels were quantified in spinach leaves from plants subjected to different light and salinity conditions. A spinach PEAMT eDNA sequence was used to over-express recombinant PEAMT in the protein expression vector pET30a (+). The presence of a polyhistidine-tag on the overexpressed protein allowed for purification by a cobalt metal affinity column. The affinity purified protein was used to produce polyclonal antibodies for immunoblot hybridization analysis. For these studies, PEAMT protein was first immunoaffinity purified from soluble extracts prepared from leaves and then the protein subjected to electrophoresis by SDS-p AGE. Enzyme assays and immunoblot analysis show PEAMT activity and protein levels increase and become relatively constant in leaves of plants exposed to continuous light. In continuous darkness, PEAMT activity and protein levels decrease and remain low and constant. Thus the pattern of changes in PEAMT activity levels are associated with changes in PEAMT protein levels. In contrast, Northern blot hybridizations show that under conditions of constant light, peamt transcript levels undergo cyclical changes with peak levels at 20 and 40 h and troughs at 28 and 52 h after the continuous light treatment was imposed. These peaks coincide with the dark and light cycles of the normal photoperiod. The same cyclical changes in peamt transcript levels was seen for plants transferred from a normal photoperiod to continuous darkness. Since these changes persist in the absence of a day/night cue we conclude that peamt transcript levels are circadian-regulated. The peamt transcript levels of control unstressed and salt-stressed plants also show circadian rhythms, however the levels found in salt-stressed plants were 0.5 to 2-fold higher than the controls. Therefore, while salinization of plants increases peamt transcript abundance, it does not alter the circadian rhythm that transcripts of this gene display. Changes in PEAMT activity and protein levels are likely controlled by other as yet unknown post-translational mechanisms, processes that override and obscure operation of a circadian rhythm in regulating the level of peamt transcripts. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22462 |
| Date | 09 1900 |
| Creators | Drebenstedt, Martina |
| Contributors | Weretilnyk, Elizabeth, Science |
| Source Sets | McMaster University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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