| Abstract/Contents: | "Disruption of cellular membrane integrity, as a result of low temperature-induced water removal, is a primary cause of cold injury. The lipid bilayer (Fig 1) provides the necessary environment for proper functioning of membrane proteins. Membrane lipid 'fluidity' is thought to be one of the prerequisites for unimpaired survival at low temperature. Membrane fluidity is affected by the degree of unsaturation (i.e., number of double bonds) in the constituent fatty acid (FA) side-chains of membrane lipids. These double bonds induce "kinks" in the molecules (Fig. 2), thus resisting molecular compaction (Fig. 3), and maintaining fluidity. Therefore, increase in FA unsaturation reduces the temperature at which membranes undergo damaging, dehydration induced, phase transitions from a flexible to more rigid, gel-like, state. 'Midiron' and 'U3' bermudagrass were exposed to conditions that induce cold acclimation, and crown tissue from rhizomes was harvested for total membrane lipid isolation. Fatty acid analysis of total lipids revealed a significant increase in tri-unsaturated species (i.e., linolenic acid; C18:3), over shorter and saturated species, and an overall increase in the double-bond index. These changes were more pronounced in 'Midiron' (relatively cold tolerant) than in 'U3' (relatively cold sensitive) bermudagrass, during cold acclimation. Preliminary biochemical analysis of the isolated membrane lipids identified neutral lipids, glycolipids and phospholipids. At least four species of neutral lipids were detected, two of which were in abundance. Three glycolipids species were detected, in equimolar amounts. Phosphatidylcholine (PC), phosphatidylethanol-amine (PE), phosphatidyl glycerol (PG) and phosphatidylserine (PS) were the four phospholipids detected. Their relative abundance is PC >> PE = PG = PS. Exact species determination, quantification, and FA and intermolecular positional analysis during cold acclimation in the two cultivars is currently under investigation. Differential display is used to identify genes expressed in bermudagrass during cold acclimation. Comparisons of "displays" from 0, 12, 24, 48 and 72 hrs post-low temperature exposure to that from non-acclimating tissue over the same time period, allows for the identification of genes differentially expressed between treatments, timepoints and/or cultivars. The cDNA clones obtained represent both "RAPD"-type fragments, those with 10-mer/complimentary sequences on the ends, as well as gene fragments derived from amplifications utilizing both the anchor (e.g., T11AG or T11AC) and a specific 10-mer. The latter have an easily recognizable poly(A) addition signal, just 5' of the anchor primer sequence. The clones range in size from 300 to 450 bp. All were chosen as "up-regulated" genes from differential display gels. Searching DNA and protein databases with translations of the sequences failed to show significant homology to any previously cloned gene or protein/peptide sequence. This is to be expected for at least two reasons: (i) since the clones are biased toward the 3'-untranslated end of a gene transcript, protein databases or those containing gene sequences from genomic or random-primed cDNA libraries may not have sequence data for this region of any their clones; (ii) since, to date, few genes whose expression is regulated during cold acclimation have been cloned from any organism, such genes would be under-represented in current databases (i.e., we are cloning new, undiscovered genes)." |
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