Relationship between Phospholipid Breakdown and Freezing Injury in a Cell Wall-Less Mutant of Chlamydomonas reinhardii

Abstract

The effects of freezing and thawing on a cell walless mutant (CW15+) of Chiamydomonas reinhardii were investigated by monitoring enzyme release, cell viability, cell ultrastructure, and lipid composition. Cells suspended in Eugkena gracilis medium were extremely susceptible to freezing injury, the median lethal temperature in the presence of extracellular ice being -5.3°C. Cell damage was associated with a release of intracellular enzymes and massive breakdown of ceDular organization. Changes in phosphoUpid fatty acid composition consistent with either a peroxidation process or phospholipase A2 activity were evident, but the time course of these changes showed clearly that alterations in phospholipid fatty acid composition were a secondary, pathological event and not the the primary cause of freeze-thaw injury in Chlamydomonas reinhardii CW15+.It is now generally accepted that an early event during freezing injury is an alteration in the structure and function of cellular membranes. The precise nature of this damage is, however, far from understood.In many plant cell types, alterations in phospholipid composition are observed following freezing and thawing (1 1, 15, 18, 20, 23, 26-30), and in some chilling-sensitive plants a similar degradation of phospholipids accompanies damage (24,25). These changes in phospholipid composition are consistent with the activation of intracellular phospholipases. It is not clear, however, whether the observed changes in cellular lipids are the primary cause of freezing injury or merely a secondary pathological event, although Yoshida (27-29) has proposed that activation of phospholipase D is the specific mechanism of freezing injury in woody plant cells.In this study, we have examined the relationship between freezing injury and phospholipid composition in the unicellular green alga Chlamydomonas reinhardii. We have used a cell wallless mutant (CW15+) because in this organism the plasma membrane is exposed directly to the suspending medium, and this avoids any complicating effects of an external cell wall (7 Enzyme Assay. Loss of membrane integrity following freezing and thawing was determined by measuring the release of the cytoplasmic enzyme GOT.' The activity of GOT in cell-free supernatants and cell sonicates was assayed by the method of Schmidt and Schmidt (17). Enzyme loss to the supernatant was expressed as a percentage of the total intracellular enzyme activity.Electron Microscopy. Thin-section electron microscopy was carried out as previously described (10).Lipid Extraction and Analysis. Cells were harvested by centrifugation and the total lipids extracted with excess methanol-chloroform (2). Purified lipids were stored in chloroform containing 0.0 1% 2,6-di-tert-butyl-p-cresol (BHT) as antioxidant, under nitrogen at -50°C.Phospholipids and other polar lipids were separated from neutral lipids, sterols, carotenoids, and porphyrins by chromatography on prepacked silica gel columns (Sep-paks; Waters Associates). Purity of this polar lipid fraction was checked ...

Alterations of Intracellular pH in Response to Low Temperature Stresses

“…Exposure of cortical tissues of non-acclimated woody plants to lethal freezing temperatures results in a drastic degradation of membrane phospholipids via an activation of phospholipase D (EC 3.1.4.4) (Yoshida and Sakai 1974). This phenomenon is also observed in many herbaceous species (Wilson and Rinne 1976, Clark et a/. 1982, Sikorska and Kacperska 1982, Borochov et a1.1987.…”

Protein and lipid dynamics in photosynthetic thylakoid membranes investigated by in-situ solid-state NMR

“…The first is a cell wall-less mutant [25] used as reference, used as a parental strain for the isolation of a mutant depleted in Zea epoxidase (ZE) [26]. Both strains were cultivated in Erlenmeyer flasks with liquid tris-acetate phosphate (TAP) medium, at 100 rpm agitation and 21°C in a growth chamber.…”

Lipid storage compounds in raw activated sludge microorganisms for biofuels and oleochemicals production

“…It can cause alterations in phospholipid composition of the cells due to either peroxidation or phospholipase activity. 40 Furthermore, the accelerated solvent extraction was conducted at 100 uC using methanol as one of the extraction solvents. At this condition, significant production of FAMEs was observed (data not shown).…”