Localization of Spin Labels in Oat Leaf Protoplasts

Abstract

An assay based on light-mediated oxidation was used to determine whether specific spin labels were partitioned throughout the protoplast or retained in the plasmalemma of Avena sativa L. cv. Garry and Park. Many classes of spin label were tested, including phospholipids, fatty acid, fatty acid methyl ester, maleimide, iodoacetamide, short chain hydrocarbon, androstane, 2,2,6,6-tetramethyl-4-aminopiperidinooxyl (TEMPAMINE) and 2,2,6,6-tetramethylpiperdinooxyl (TEMPO). All except the phosphotidylcholine spin label were found to partition throughout the cell. The phosphotidylcholine spin label may have been selectdvely retained in the plasmalemma.Changes in the plasma membranes of higher plants have been proposed as the basis of many cellular phenomena. Examples are development of cold tolerance, salt tolerance, and senescence. Also, changes in membrane structure may result from exposure to hormones, pathogens, radiation, and pressure. Usually, membrane structure has been studied with fixed tissues or with isolated membranes, precluding direct measurement of membrane changes in living cells.Spin labels have been used to study the plasma membrane of living cells. However, to selectively probe the plasmalemma of an intact protoplast, the probe must be reporting only from the plasmalemma. This condition can be met if the probe is only present in the plasmalemma or is spectroscopically inactive everywhere except in the plasmalemma. Kaplan et al. (14) claimed that by adding K3Fe(CN)6 (a spin label-activating agent) to a labeled cell suspension, only the spin-label in the plasma membrane gave an ESR signal. The assumptions that spin labels cannot move quickly between membranes and that different membranes would create different spin label spectra are also used to support the contention that fatty acid spin labels are good probes for the plasma membrane (2-4, 7, 10, 13, 24, 25). However, some investigators dispute these assumptions (12, 16), arguing that most spin labels report an average from every membrane in the cell.The objectives of this report were to: (a) evaluate whether or not fatty acid spin labels are selective for the plasmalemma of plant protoplasts; (b) develop criteria for ascertaining the selectivity of a spin label for the plasmalemma; and (c) test various classes of spin label and identify those which may have selectivity for the plasmalemma. MATERIALS AND METHODSAvena sativa L. cv. Garry and Park were grown in the laboratory at 22°C in vermiculite irrigated with White's nutrient solution (8). Fluorescent and incandescent bulbs provided a 16-h photoperiod. Primary leaves were harvested from 1-to 2-week-old seedlings.Protoplasts were isolated by peeling away the lower epidermis of the leaves with forceps and floating the leaves (peeled surface down) on a solution composed 6f 0.5% Cellulysin (CalbiochemBehring Corp.) and 0.6 M sorbitol, adjusted to pH 5.6 with KOH. The preparation was incubated at 28°C for 3 h in the light, then was swirled gently to release protoplasts. The protoplasts were fi...

Changes in Tobacco Cell Membrane Composition and Structure Caused by Cercosporin

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Studies on Freezing Injury of Plant Cells

“…Using ESR technique, Singh and Miller (17) demonstrated that isolated plant cells may undergo an irreversible reorganization of lipid bilayers to a less ordered amorphous state after freezing at a lethal temperature. However, as pointed out by Briggs et aL (6), when intact or isolated protoplasts are used for ESR studies, problems remain as to whether or not the fatty acid spin labels were selective for the plasma membranes. They showed that most of the labels were partitioned throughout the cells and, thus, the ESR signals reported an average from every membrane in the cells.…”