Changes in Membrane Permeability of Winter Wheat Cells

Nuclear etic resoaace (NMR) relaxation times were studied in acclhnated and nonacclmated Kharkov winter wheat (Tritlum sestlvwm L.) crowns and acclimated cel aggregates to determine if membrane permeability was altered by freezing. The NMR water signal decay consisted of two exponental c ets: a short one arising from extracellular water, and a long one arising from intracelular water. A slow freezethaw treatbmet of nonacclmated and 1-week acclimated crowns decreased the long relaxation thie, suggestin m neh4ury. Similar resuts were obtained for nonacclimated and acclimated crowns kie dhrecty in lquid N2. A significant increase in plasma membrane permeabiUty to Mn2+ was observed in acclmated freeze-killed crowns and ceDl aggregates. Freezing injury to plat tissue appears to be a membrane-related phenoe, but more extensive hijury occurs to noacclimated and acclimated tissue with a high water cootent (cel aggregates) compared to acclimated tissue with a low water content (crowns). Rapid freezing of plant tissue results in intracellular ice formation which is invariably lethal to the cell (1, 2). At slow freezing rates (<10 C/hr), ice forms at extracellular sites which can accommodate the growing ice crystals (15, 18). Extracellular ice formation during slow freezing causes a dehydration stress on the protoplast and injury may be due to the secondary stress caused by the freeze-induced dehydration (16). It has been inferred by many investigators that membranes, primarily plasma membranes, are the site of freezing injury. This evidence is based upon loss of semipermeability properties of the membrane following freezing injury (7, 19). Pulse NMR3 measurements of the longitudinal relaxation time (T1) and the transverse relaxation time (T2) have been used to study water and water interactions in biological systems (5, 14, 22). NMR studies on plant tissue have shown that there are at least two populations of water, each with a different magnetic environment that causes a different relaxation time (5, 21). Stout (21) concluded that the protons with a short relaxation time were in part associated with the extracellular water and in part with the total tissue water of hydration. The protons with a long relaxation ' This work was supported by the National Research Council ofCanada Grant No. A-9661. 2Present address: Agriculture Canada, Range Research Station, 3015 Ord Road, Kamloops, B.C. Canada, V2B 8A9. 3Abbreviation: NMR: nuclear magnetic resonance. time were associated with the intracellular water. Haran and Shporer (13), working with phospholipid vesicles, also identified a short relaxation time with extracellular water and a long relaxation time with intracellular water. Intracellular and extracellular water exchange with each other across the plasma membrane, the rate of exchange depending upon the plasma membrane permeability. When the extracellular water has a short relaxation time the water exchange out of the cell results in a shortening of the intracellular relaxation time (6, 13, 22). The greater the water permeability the more the intracellular T2 is shortened. The extracellular T2 can be shortened by adding paramagnetic ions such as Mn2" which cause protons to relax in a short time. Because plant membrane permeability to divalent cations is low (20, 22), the added Mn2' does not directly decrease the intracellular water T2 for several hr (22). The objective of this study was to evaluate the effect of different freezing stresses (fast freezing versus slow freezing) on the plasma membrane of tender and hardy winter wheat (Triticum aestivum L.). Alterations to plasma water membrane permeability as a result of freezing stresses were studied by observing the effects of freezing on NMR relaxation times. Alterations to Mn2+ permeability were studied by observing the loss in the long T2 water signal as Mn2" diffused into the protoplast. MATERIALS AND METHODS