Characterization of Solute Efflux from Dehydration Injured

Soybean (Glycine max L. Merr) seeds lose their tolerance ofdehydration between 6 and 36 hours of imbibition. Soybean axes and cotyledons were excised 6 hours (tolerant of dehydration) and 36 hours (susceptible) after commencing imbibition and subsequently dehydrated to 10% moisture. Kinetics of the efflux of potassium, phosphate, amino acid, sugar, protein, and total electrolytes were compared in the four treatments during rehydration. Only slight differences were observed in the kinetics of solute efflux between the two cotyledon treatments dehydrated at 6 and 36 hours suggesting that the cotyledons may retain their tolerance of dehydration at this stage of germination. Several symptoms of injury were observed in the axes dehydrated at 36 hours. An increase in the initial leakage of solutes during rehydration, as quantified by thep-intercept of the linear regression line for solute efflux between 2 and 8 hours suggests an increased incidence of cell rupture. An increase in the rate of solute efflux (slope of regression line between 2 and 8 hours) from fully rehydrated axes was observed in comparison to axes dehydrated at 6 hours. The Arrhenius activation energy for potassium, phosphate, and amino acid effux decreased and for protein remained unchanged. Both observations indicate an increase in membrane permeability in dehydration-injured tissue. Increasing the H+ concentration of the external solution increased K+ efflux from both control and dehydrated/rehydrated samples, increased sugar efflux from axes at 6 hours imbibition but decreased sugar efflux from axes at 36 hours imbibition, indicating changes in membrane properties during germination. The dehydration treatment did not alter the pattern of the pH response of axes dehydrated at 6 or 36 hours but did increase the quantity of potassium and sugar efflux from dehydration injured axes. These results are interpreted as indicating that dehydration of soybean axes at 36 hours of imbibition increased both the incidence of cel rupture during rehydration and altered membrane permeability of the rehydrated tissue. A seed's tolerance of dehydration is lost at a specific stage in germination (1 1). For example, soybean seeds can be imbibed for 6 h, and dehydrated to 10%7o moisture without loss of seed viability or vigor. If the seed is imbibed for 36 h, at which time the radicle is beginning to emerge from the seed coat, and then dehydrated to 10%o moisture, seed viability is lost (17). The ability of plant tissues to tolerate dehydration is thought to reflect the inherent protoplasmic properties of these tissues (2) and in seeds, the protoplasmic properties which impart tolerance are presumably lost as the seed germinates. The loss of tolerance has been asso' Supported by The Natural Sciences and Engineering Research Council of Canada and the facilities provided by The Ontario Ministry of Agriculture and Food. 2 Recipient of a graduate scholarship from International Development Research Centre, Ottawa, Canada. ciated with the initiation of cell elongation and hydration of vacuoles (11), onset of DNA replication and RNA transcription (4), and changes in cellular membranes (14). Although the loss of dehydration tolerance is coincident with the initiation of cell elongation in many systems (1 1), the relationship does not appear to be causal. Soybean axes which have been prevented from elongating by treatment with a -6 bar solution of PEG become sensitive to dehydration even though cell enlargement did not occur (17). Similarily, soybean axes imbibed in cycloheximide to inhibit protein synthesis do not elongate but become sensitive to dehydration (17). Consequently, the changes in cell volume that occur during germination are not consistently related to the loss of dehydration tolerance. Instead, the loss of tolerance may be associated with biochemical or biophysical changes in cell membranes during germination. To define these changes precisely, it is first necessary to define the nature of the metabolic lesion induced by dehydration. The effect of water loss on the structure of membranes is not well defmed. Ultrastructural studies have indicated that substantial alterations of membrane structure occur in dehydration-injured seeds (5, 6). Freeze-fracture studies on the plasmalemma of the phycobiont Trebouxia have demonstrated changes in membrane structure and distribution of intramembranous particles after prolonged drying of the thallus (16). Dehydration injury in soybean seeds occurs only in the axis, not in the cotyledons, and only after the seed has been dehydrated to less than 20%o moisture (17). The maintenance of the lipoprotein associations in membranes requires 20 to 30o hydration (10). Low angle x-ray diffraction has not confirmed Simon's (18) proposal that seed phospholipids are involved in a lamellar-hexagonal phase transition at 20o hydration (14). It remains unclear how the seed membranes change in structure during drying and also how the seed tolerates this stress at one stage in germination but is sensitive to it at another. A typical symptom of dehydration injury in seeds is an increase in the quantity of electrolytes and other cytoplasmic solutes that can be leached from the tissue during rehydration (14, 17). Solutes may leak from injured tissue more rapidly because the plasmalemma and/or tonoplast has been mechanically ruptured by the dehydration treatment or because the permeability of an intact membrane has been altered. To make this distinction, three experimental approaches were used in this study which varied time, temperature, and pH, respectively, and measured their effects on the efflux of various solutes. MATERIALS AND METHODS Soybean (Glycine max L. Merr cv Maple Arrow) seeds were imbibed and dehydrated as previously described (17). Kinetic Analysis of Solute Efflux. To quantify the rate of solute efflux from dehydration injured axes and cotyledon tissue during reimbibition, ten axes or six cotyledons, which had been previously dehydrated to 10%o moisture at 6 or 36 h of imbibition, were soaked in 10 ml distilled H20. The incubating solution was 911 www.plantphysiol.org on July 22, 2017 Published by Downloaded from Copyright © 1983 American Society of Plant Biologists. All rights reserved. SENARATNA AND McKERSIE Table I. Initial Leakage of Solutesfrom Soybean Axes and Cotyledons during Rehydration after Dehydration Treatment at 6 or 36 Hours of Imbibition Values represent the y-intercept of the linear regression line calculated using the data between 2 and 8 h and are expressed per 100 mg seed.