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Landscape Architecture (E.J.S.), University ofMinnesota, St. Paul, Minnesota 55108 MATERIALS AND METHODSThis study was undertaken to quantify the effect of aluminum and calcium on membrane permeability. The influence of Ca2" (0.2-3.7 millimolar) and Al' (0-3.7 millimolar) on the permeability of root cortical cells of Quercus rubra was measured using three nonelectrolytes (urea, methyl urea, and ethyl urea) Aluminum, as Al3", can interfere with physiological processes and be toxic to plant cells (1,5,(8)(9)(10)15). Plant cell membranes may be a site of primary lesions from Al3" (1,10). Hofler (10) and Bohm-Tuchy (1) showed that Al3" changed the attachment of the plasmalemma to the cell wall causing the membrane to assume a convex shape on plasmolysis. Bohm-Tuchy (1) also suggested that Al3" solidifies the outer region of the protoplasmic layer. Vierstra and Haug (19) using EPR spectroscopy showed that Al3" decreased membrane fluidity in isolated membranes and intact cells of a thermophilic bacterium.Ca2" is another ion well known to influence the integrity and functions of membranes (13,14). Recent work (9) shows negative interactions between Al3+ and Ca2" with regard to calmodulin activity and other physiologic processes (5, 15).The capacity of Al3" and other cations to alter the biochemical and biophysical properties of both the lipid and protein portions of membranes is well reviewed by Haug (8). Changes in the lipid portion of the membrane could result in an alteration of permeability to nonelectrolytes and to water. A major objective of this study was to measure how Al3+ altered the combined permeability of the plasma and vacuolar membranes in the root cortex cells of red oak. A related objective was to determine whether the effects of Al3" vary with the concentration of Ca2". Permeability Measurements. The solute permeability of the plasma membrane and tonoplast in series was measured by the techniques described by Stadelmann (17). Immediately after sectioning, the tissue was sequentially exposed to increasing concentrations of sucrose. The sections were in 0.1 M sucrose for 1 h, in 0.2, 0.3, and 0.4 M for 30 min each, and 0.5 M for 1 h. At this osmolarity (0.59 osm) all intact cells were plasmolyzed. The tissue sections in 0.5 M sucrose were transferred in a droplet of the same solution into a perfusion chamber (21). In the chamber, the sucrose solution was replaced with isotonic solutions or urea family permeators (urea, methyl urea, or ethyl urea

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Onion (Allium cepa L.) bulbs were frozen to -4 and -11 C and kept frozen for up to 12 days. After dow thawing, a 2.5-cm square from a bulb scale was trferred to 25 ml deionized H20. After shaking for standard times, measurements were made on the effusate and on the effsed cells. The results obtained were as follows.Even when the scale tisse was completely inSltrated, and when up to 85% of the ions had diffused out, all of the cels were still alve, as revealed by cytoplasmic sa and abiliy to psmolyze. The osmotic concentration of the cell sap, as measred pamoyically, decreased in parallel to the rise in condedivity of the effuate. The K+ content of the effusate, plus its ased counterion, accounted for only 20% of the total solutes, but for 100% of the cpnductvity. A brge part of the nonelectrolytes in the remaning 80% of the solutes was gars.The increased cell injury and infiltration in the -11 C treatment, relative to the -4 C and control (unfozen) treatments, were paralleled by increases in conducdvity, K+ content, supr content, and pH of the effuste. In spite of the 100% ifltrtion of the tissue and the lage increase in conductivity of the effusate following freezing, no increase in permeability of the cells to water could be detected.The above observations may indicate that freezing or thawing involves a disruption of the active trnsport system before the cells reveal any inqju microscoplcally.In spite of the general adoption of the conductivity method, there have been no attempts to investigate the basic nature of the freeze-induced efflux of ions. In recent literature, the assumption seems to have been made that the efflux is mainly, if not solely, from dead cells, and therefore one measures the freezing injury by determining the percentage of cells killed (6,11 (see 5). This method has been used successfully, particularly for testing relative varietal resistance. The basic assumption is that the greater the injury of the living tissue, the greater the efflux of ions from the thawed cells. These authors clearly recognized that exosmosis occurs not only in dead but also in injured cells, however, without distinguishing reversible damage.

Onion (Allium cepa L.) bulbs were subjected for 12 days to either a moderate freeze (-4 C) or a severe freeze (-11 C). They were then thawed dowly over Ice. Durng 7 to 12 days following the thaw, the injury progressed with time in the severely frozen bulbs, but appeared completely repired in the moderately frozen bulbs. This was shown by the following post-thawing changes.Inftratlon of the intercellua spaces inceased from 80 to 90% to 100% after the severe freeze, and deCeaed from 30 to 50% to zero after the moderate freeze. Al of the cells were alive immediately after thawing whether the freeze was moderate or severe. Corresponding to the infiltration results 7 to 12 days later, many to most were dead folowing the severe freeze, all were alive following the moderate freeze.The conductMity of the effuate from pieces of bulb tssue inceased after the severe freezing, and decreased after the moderate freezing. The concentration of K+, totad solutes, and sgas in the effsate parleled the conductity chnges. Neither the pH of the effuste nor the perneabllty of the cells (as long as cells were livig) to water was changed following either the severe or the moderate freezes. Some treatments of the thawed tise following the severe freeze halted the progress of injury.The above results indicate that the semipermeable properties of the cell are uninjured but that the ion and suar transport mehis is damaged by freezing. Most likely the prmary injury is to the active traport me involved in their trort. It mut be conduded that the fid injouy following freezing and thawing cannot be evsduated from the degree of infltration or the conductvity of the effute immediately after thawing, since injury may progress or recede following the thawing.In agreement with earlier results obtained with other plants (6), our recent work with onion (Allium cepa L.) bulbs led us to suspect that freezing injury may occur at three moments: during freezing, during thawing, or after thawing (8). This injury results in the leakage of ions and sugars from the cells. The exosmosis was found to originate not from dead cells, since all of the cells were still alive on thawing. The damaged, but still living cells, afford us the opportunity to investigate the initial injurious events in frozen cells. The following investigation attempts to do this and to determine which treatments enhance the injury and which induce it to recede.

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This report extends research on Al-induced changes in membrane behavior of intact root cortex cells of Northern red oak (Quercus rubra). Membrane permeability was determined by the plasmometric method for individual intact cells at temperatures from 2 or 4 to 35°C. Al (0.37 millimolar) significantly increased membrane permeability to urea and monoethyl urea and decreased permeability to water. Al significantly altered the activation energy required to transport water (+32%), urea (+9%), and monoethyl urea (-7%) across cell membranes. Above 90C, Al increased the lipid partiality of the cell membranes; below 70C, Al decreased it. Al narrowed by 60C the temperature range over which plasmolysis occurred without membrane damage. These changes in membrane behavior are explainable if Al reduces membrane lipid fluidity and kink frequency and increases packing density and the occurrence of straight lipid chains.