Effective cryoprotection of thylakoid membranes by ATP

Freezing of isolated spinach thylakoids in the presence of NaCl uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by Mg²⁺. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaCl was saturated at about 1–2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo.Abbreviations CF₁ chloroplast coupling factor - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMS phenazine methosulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Cryoprotection of spinach chloroplast membranes by dextrans

The cryoprotective properties of dextrans have been investigated in freezing experiments with isolated spinach thylakoids (Spinacia oleracea L.). The activity of cyclic photophosphorylation was used as an assay for membrane integrity.Dextrans of average molecular weights between 10,000 and 70,000 daltons proved to be fairly nontoxic to chloroplast membranes. On a molar basis, cryoprotective action increased with increasing molecular weight; on a unit weight basis, the cryoprotective effectiveness of different dextrans was comparable. In the presence of low dextran concentrations which are not sufficient for complete membrane preservation, the effectiveness of the polymers could be considerably increased by the addition of electrolytes. This is in contrast to cryoprotection exerted by sugars. At a given dextran concentration, membrane activity is a function of the electrolyte concentration and follows an optimum curve. If membrane-toxic action of the electrolytes and salt crystallization during freezing which complicate the situation, are not taken into consideration, the increase in membrane protection during freezing by salts was dependent on the concentration of the salts and was not much influenced by the nature of the cations and anions. At 0 °C, dextrans delayed the inactivation of thylakoids suspended in NaCl solutions.From the results it is concluded that cryoprotection produced by dextrans is caused in part by specific membrane stabilization.     

Cryoprotection of mammalian cells in tissue culture with polymers; possible mechanisms

Studies were undertaken to more clearly define the mechanism of cryoprotection by polymers. Significant cryoprotection of Chinese hamster cells in tissue culture was found in the presence of hydroxyethyl starch (HES), polyvinylpyrrolidone (PVP), and dextran. The addition of PVP to the medium after thawing did not increase the survival of these cells. The presence of PVP in the medium was shown to have no effect on the transport mechanism for alanine in unfrozen cells. The source of freeze-thaw injury did not appear to be due to a direct effect on this transport mechanism. Several physical parameters of polymeric solutions were monitored at subzero temperatures. The freezing point depression was found to increase dramatically at higher polymer concentrations. Tests on the NaCl concentration in the liquid fraction of partially frozen solutions showed that the increase in salt concentration with decreasing temperature was similar in the presence of 10% PVP or 2.5% DMSO, two agents which gave similar cryoprotection at these concentrations. NMR studies showed that polymers could retain water in the liquid state at temperatures as low as −35° C, and that the remaining water was highly structured. The cryoprotective properties of polymers appear to reside in their ability to alter the physical properties of solutions during the freezing process rather than in direct effects on cell membranes.     

Cryopreservation of spinach chloroplast membranes by low-molecular-weight carbohydrates: I. Evidence for cryoprotection by a noncolligative-type mechanism

In freezing experiments with isolated spinach thylakoids (Spinacia oleracea L. cv. Monatol) the cryoprotective efficiency of various low-molecular-weight polyols was determined. The activity of cyclic photophosphorylation was used as an assay for the functional integrity of the membranes. The results were compared with the osmotic behavior of the cryoprotectants at high concentrations.Equimolal concentrations of polyols which exhibit nearly comparable freezing point depressions even at high concentrations differed considerably in their protective capacity during a freeze—thaw cycle. This was particularly distinct when glucose, galactose, and ethylene glycol monomethyl ether were compared, but was also evident when various pentoses and deoxy-hexoses were used as cryoprotectants. Even in the absence of freezing, carbohydrates exerted a stabilizing influence on biomembranes.From the data it is suggested that in addition to colligative action of the compounds, a specific noncolligative mechanism contributes to membrane protection during freezing.     

Mechanical and chemical injury to thylakoid membranes during freezing in vitro

The relative contributions of membrane rupture due to osmotic stress and of chemical membrane damage due to the accumulation of cryotoxic solutes to cryoinjury was investigated using thylakoid membranes as a model system. When thylakoid suspensions were subjected to a freeze-thaw cycle in the presence of different molar ratios of NaCl as the cryotoxic solute and sucrose as the cryoprotective solute, membrane survival first increased linearly with the osmolality of the solutions used to suspend the membranes, regardless of the molar ratio of salt to sucrose. It subsequently decreased when the ratio of sucrose to salt was not sufficiently high for complete cryopreservation by sucrose. There was an optimum of cryopreservation at intermediate osmolalities (approx. 0.1 osmol/kg). This optimum of cryopreservation at a given sucrose concentration could be shifted to lower solute concentration, if mixtures of NaCl and NaBr were used instead of NaCl alone. At suboptimal initial osmolalities, damage is attributed mainly to membrane rupture. Under these conditions, cryopreservation is not influenced by the chaotropicity of the suspending medium. At supraoptimal initial solute concentrations, solute (i.e., chemical) effects determine membrane survival. Under these conditions, increased ratios of sugar to salt increased cryoprotection. In mixtures of NaCl and NaBr at constant molar ratios of salt to sucrose, chemical membrane damage was quantitatively related to the lyotropic properties of the ions used. The degree of chemical damage becomes more pronounced with rising osmolalities of the suspending media. With NaF as the cryotoxic solute, damage was more severe than should be expected from its lyotropic properties. This may reflect a specific interaction of fluoride with the membranes. Protein release from the membranes during freezing in the presence of different anions was qualitatively comparable at identical ratios of sugar to salt. However, the total amount of protein released was correlated linearly with membrane inactivation, even when different anions acted on the membranes. Gel electrophoretic analysis of proteins released from thylakoid membranes during freezing revealed discrete bands indicative of mechanical and chemical damage, respectively.     

Factors contributing to inactivation of isolated thylakoid membranes during freezing in the presence of variable amounts of glucose and NaCl.

During freezing of isolated spinach thylakoids in sugar/salt solutions, the two solutes affected membrane survival in opposite ways: membrane damage due to increased electrolyte concentration can be prevented by sugar. Calculation of the final concentrations of NaCl or glucose reached in the residual unfrozen portion of the system revealed that the effects of the solutes on membrane activity can be explained in part by colligative action. In addition, the fraction of the residual liquid in the frozen system contributes to membrane injury. During severe freezing in the presence of very low initial solute concentrations, membrane damage drastically increased with a decrease in the volume of the unfrozen solution. Freezing injury under these conditions is likely to be due to mechanical damage by the ice crystals that occupy a very high fraction of the frozen system. At higher starting concentrations of sugar plus salt, membrane damage increased with an increase in the amount of the residual unfrozen liquid. Thylakoid inactivation at these higher initial solute concentrations can be largely attributed to dilution of the membrane fraction, as freezing damage at a given sugar/salt ratio decreased with increasing the thylakoid concentration in the sample. Moreover, membrane survival in the absence of freezing decreased with lowering the temperature, indicating that the temperature affected membrane damage not only via alterations related to the ice formation. From the data it was evident that damage of thylakoid membranes was determined by various individual factors, such as the amount of ice formed, the final concentrations of solutes and membranes in the residual unfrozen solution, the final volume of this fraction, the temperature and the freezing time. The relative contribution of these factors depended on the experimental conditions, mainly the sugar/salt ratio, the initial solute concentrations, and the freezing temperature.     

Freezing of isolated thylakoid membranes in complex media: I. The effect of potassium and sodium chloride,nitrate, and sulfate

Thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were subjected to a freeze-thaw cycle in the presence of a buffered medium containing sorbitol as a cryoprotectant and various combinations of potassium and sodium chloride, nitrate, and sulfate. Above a certain total salt concentration, an increase in the concentration of a single electrolyte, or of potassium plus sodium salts with identical anions, always led to a decrease in photophosphorylation activity. A similar effect was obtained with combinations of nitrate plus chloride with identical cations and of KNO₃ plus NaCl. By contrast, in the presence of suitable combinations of NaNO₃ plus KCl, NaNO₃ plus sulfates, and chlorides plus sulfates, inactivation of photophosphorylation was diminished, sometimes dramatically, at initial molarities of nitrate or chloride which alone caused partial or complete membrane damage. When NaNO₃, KCl, and potassium or sodium sulfate were simultaneously present during freezing, thylakoids were affected very little over a wide range of concentration. Diminution or prevention of inactivation of photophosphorylation by suitable combinations of two or more cryotoxic inorganic salts can be explained by postulating that the different solutes act on different sites and that each reduces the concentration of the others by colligative action, together with specific effects of the various electrolytes on individual membrane sites.     

Cellular compartmentalization of salt ions and protective agents with respect to freezing tolerance of leaves

Summary Evidence was given that the freezing tolerance of Halimione portulacoides, a wintergreen halophyte, can be explained by protection of sensitive cellular membranes in vivo. Experiments were done with cloned cuttings of a plant from the German North Sea coast. One series received no NaCl (O-plants) the other 3% NaCl (NaCl-plants) in the nutrient solution. During the annual course Na⁺ and Cl^- of the O-plant leaves remained on a nearly constant low level. In the leaves of the NaCl-plants Na⁺ and Cl^- concentrations changed strongly during year and reached a maximum in winter. Potassium was always on a low level. The freezing tolerance curves showed a minimum in summer and a maximum in winter. The small difference between the freezing tolerance peaks of the NaCl- and O-plants indicated that the increased salt stress did not affect freezing tolerance very much. Freezing stress in cellular membranes, like thylacoids, acts in the same way as increasing salt concentration; consequently both together must amplify the stress. For the analysis of their ion contents chloroplasts of H. portulacoides were non-aqueously isolated from leaves with different freezing tolerance during the year. In midwinter, when freezing tolerance was highest, the chloroplasts of the NaCl-plants contained about 250 mM chloride (O-plants c. 150 mM), while the non-plastidic fraction of the cell contained about 1 M (O-plants c. 400 mM) chloride. On the other hand citrate reached high concentrations in the chloroplasts in winter. Non-volatile organic acids like citrate are known to compensate colligatively the injurious action of the inorganic salt ions on thylacoids in vitro (Heber and Santarius, 1976). The molar proportion between chloride and citrate in H. portulacoides chloroplasts decreased with increasing freezing tolerance and reached values which were protective on chloroplast membranes in vitro. This relationship in vivo with H. portulacoides provides evidence supplying the concept of colligative protection of cellular membranes. Besides citrate also malate may act as a colligatively protecting agent against the amplified salt stress by freezing.     

Physical-Chemical Basis of the Protection of Slowly Frozen Human Erythrocytes by Glycerol

One theory of freezing damage suggests that slowly cooled cells are killed by being exposed to increasing concentrations of electrolytes as the suspending medium freezes. A corollary to this view is that protective additives such as glycerol protect cells by acting colligatively to reduce the electrolyte concentration at any subzero temperature. Recently published phase-diagram data for the ternary system glycerol-NaCl-water by M. L. Shepard et al. (Cryobiology, 13:9-23, 1976), in combination with the data on human red cell survival vs. subzero temperature presented here and in the companion study of Souzu and Mazur (Biophys. J., 23:89-100), permit a precise test of this theory. Appropriate liquidus phase-diagram information for the solutions used in the red cell freezing experiments was obtained by interpolation of the liquidus data of Shepard and his co-workers. The results of phase-diagram analysis of red cell survival indicate that the correlation between the temperature that yields 50% hemolysis (LT₅₀) and the electrolyte concentration attained at that temperature in various concentrations of glycerol is poor. With increasing concentrations of glycerol, the cells were killed at progressively lower concentrations of NaCl. For example, the LT₅₀ for cells frozen in the absence of glycerol corresponds to a NaCl concentration of 12 weight percent (2.4 molal), while for cells frozen in 1.75 M glycerol in buffered saline the LT₅₀ corresponds to 3.0 weight percent NaCl (1.3 molal). The data, in combination with other findings, lead to two conclusions: (a) The protection from glycerol is due to its colligative ability to reduce the concentration of sodium chloride in the external medium, but (b) the protection is less than that expected from colligative effects; apparently glycerol itself can also be a source of damage, probably because it renders the red cells susceptible to osmotic shock during thawing.     

Colligative and non-colligative freezing damage to thylakoid membranes

When spinach thylakoid membranes were frozen in vitro in solutions containing constant molar ratios of cryotoxic to cryoprotective solute, maintenance of functional integrity strongly depended on initial osmolarities. Optimum cryopreservation of cyclic photophosphorylation was observed when the membranes were suspended in solutions of intermediate osmolarities (approx. 50–100 mM NaCl, 75–150 mM sucrose). Both higher and lower initial osmolarities were found to result in decreased cryopreservation. In the absence of added salt, more than 100 mM sucrose were needed for full cryopreservation of the membranes. When thylakoids were frozen in solutions containing low concentrations of NaCl (2 mM), the ratio of sucrose to salt necessary to give full protection was high (up to 50). When the salt concentration was about 60 mM, ratios as low as 1.5 were sufficient for maintaining membrane integrity. This ratio increased again, as the initial NaCl concentration was increased beyond 60 mM. During freezing, proteins dissociated from the membranes, and the amount of the released proteins was correlated linearly with inactivation of photophosphorylation. The gel electrophoretic pattern of proteins released at low initial osmolarities differed from that of proteins released at high initial osmolarities. Cryopreservation was also found to depend on membrane concentration. Concentrated membrane suspensions suffered less inactivation than dilute suspensions. The protective effect of high membrane concentrations was particularly pronounced at high initial solute concentrations. It is proposed that damage at low initial osmolarities is caused predominantly by mechanical stress and by osmotic contraction/expansion. Damage at high initial osmolarities is thought to be caused mainly by solute effects. Under these conditions, both the final volume of the unfrozen solution in coexistence with ice and the membrane concentration affect membrane survival by influencing the extent of the loss of membrane components through dissociation reactions. Membrane protection by sugars is caused by colligative action under these circumstances.     

Cryoprotection by glucose, sucrose, and raffinose to chloroplast thylakoids

Differential cryoprotection is afforded to chloroplast thylakoids against freeze-induced uncoupling of cyclic photophosphorylation by equimolar concentrations of glucose, sucrose, and raffinose. This differential protective effect appears to be due to nonideal activity-concentration profiles exhibited by the sugars during freezing. When cryoprotection is analyzed as a function of the mole fraction of NaCl to which the membranes are exposed during freezing, the pattern of protection to cyclic photophosphorylation and its component reactions is not dependent upon the chemical identity of the protective solute. Cryoprotective efficiency of glucose, sucrose, and raffinose can be accounted for by proposing an activity dependent alteration in the freezing environment rather than specific solute-membrane interactions.     

Boron and calcium increasePisum sativum seed germination and seedling development under salt stress

A beneficial effect of B and Ca application on symbiotic interaction between legume and rhizobia under saline conditions has recently been shown, suggesting conventional agricultural practices to increase crop salt tolerance. However, nothing is known about application of both nutrients on early events of legume development under salt stress, prior to the establishment of a symbiotic interaction. Therefore, the effects of different levels of B (from 9.3 to 93μM B) and Ca (from 0.68 to 5.44 mM Ca) on seed germination, root elongation, plant development, and mineral composition of pea (Pisum sativum L. cv. Argona) grown under 0 to 150 mM NaCl, were analysed. Development of plants previously germinated in the presence of salt was more impaired than that of plants put under salt stress once seeds were germinated. A NaCl concentration of 75 mM and 150 mM inhibited pea seed germination and seedling growth. The addition of either extra B or extra Ca to the germination solution prevented the reduction caused by 75 mM NaCl but not that of 150 mM NaCl. However, root elongation and plant development under salt stress (75 mM NaCl) was enhanced only by addition of both B and Ca. When plants were cultivated in the absence of external N, N content in roots and shoots originating from seeds was diminished by salt and enhanced by B and Ca, suggesting a role of these nutrients in remobilisation of seed nutrient stores. Salinity also led to an extremely high concentration of Na⁺ ions, and to a decrease of B and Ca concentrations. This can be overcome by addition of both nutrients, increasing salt tolerance of developing pea plants. The necessity of nutritional studies to increase crop production in saline soils is discussed and proposed.     

Interactions between osmoregulation and the alternative respiratory pathway in Plantago coronopus as affected by salinity

Plantago coronopus L., a species from the coastal zone, was grown in culture solution with and without 50 mM NaCl. In addition it was transferred from a non-saline solution to a solution containing 50 mM NaCl. Short term effects of NaCl on growth and various aspects of energy metabolism, including photosynthesis, shoot dark respiration, root respiration and the contribution of the SHAM-sensitive alternative pathway to root respiration were investigated. The concentrations of soluble and insoluble non-structural carbohydrates and of sorbitol a compatible osmotic solute in Plantago, in both shoots and roots were also determined. Growth of shoots and roots was largely unaffected by addition of 50 mM NaCl. Net photosynthesis, shoot dark respiration and the concentration of non-structural carbohydrates in both shoots and roots were also unaffected by salinity. The rate of root respiration immediately decreased upon addition of 50 mM NaCl. This decrease was almost exclusively attributed to a decreased activity of the SHAM-sensitive alternative pathway. The concentration of sorbitol in the roots increased quickly after addition of 50 mM NaCl, whilst the increase in sorbitol concentration in the shoots started later. The time course of the increase of sorbitol concentration was similar to that of the decrease in activity of the alternative pathway. During the first 12 h after exposure to 50 mM NaCl, the amount of carbohydrates which was saved in respiration, due to the decreased activity of the alternative pathway, was the same as that used for sorbitol synthesis in the roots. It is concluded that the activity of the alternative pathway decreased due to increased utilization of carbohydrates for sorbitol synthesis, according to a proposed ‘energy overflow model’. After 24 h, the sorbitol concentration in the cytoplasm of the root cells of plants transferred to a saline solution reached a level that was sufficient to compensate for 50 mM NaCl, assuming a cytoplasmic volume of ca. 10% of the total cell volume. The sorbitol concentration in roots of plants grown in a saline environment for several weeks was lower than that in roots of plants transferred to a saline environment for c. 24 h. It is suggested that sorbitol accumulated in roots of Plantago coronopus as an immediate reaction upon salinity, whilst other adaptations may occur thereafter.     

Ecophysiological response of Crambe maritima to airborne and soil-borne salinity

Background and Aims

There is a need to evaluate the salt tolerance of plant species that can be cultivated as crops under saline conditions. Crambe maritima is a coastal plant, usually occurring on the driftline, with potential use as a vegetable crop. The aim of this experiment was to determine the growth response of Crambe maritima to various levels of airborne and soil-borne salinity and the ecophysiological mechanisms underlying these responses.

Methods

In the greenhouse, plants were exposed to salt spray (400 mm NaCl) as well as to various levels of root-zone salinity (RZS) of 0, 50, 100, 200 and 300 mm NaCl during 40 d. The salt tolerance of Crambe maritima was assessed by the relative growth rate (RGR) and its components. To study possible salinity effects on the tissue and cellular level, the leaf succulence, tissue Na⁺ concentrations, Na⁺ : K⁺ ratio, net K⁺/Na⁺ selectivity, N, P, K⁺, Ca²⁺, Mg²⁺, proline, soluble sugar concentrations, osmotic potential, total phenolics and antioxidant capacity were measured.

Key Results

Salt spray did not affect the RGR of Crambe maritima. However, leaf thickness and leaf succulence increased with salt spray. Root zone salinities up to 100 mm NaCl did not affect growth. However, at 200 mm NaCl RZS the RGR was reduced by 41 % compared with the control and by 56 % at 300 mm NaCl RZS. The reduced RGR with increasing RZS was largely due to the reduced specific leaf area, which was caused by increased leaf succulence as well as by increased leaf dry matter content. No changes in unit leaf rate were observed but increased RZS resulted in increased Na⁺ and proline concentrations, reduced K⁺, Ca²⁺ and Mg²⁺ concentrations, lower osmotic potential and increased antioxidant capacity. Proline concentrations of the leaves correlated strongly (r = 0·95) with RZS concentrations and not with plant growth.

Conclusions

Based on its growth response, Crambe maritima can be classified as a salt spray tolerant plant that is sensitive to root zone salinities exceeding 100 mm NaCl.     

Malic dehydrogenase from tamarix roots: effects of sodium chloride in vivo and in vitro

Soluble and mitochondrial malic dehydrogenases (MDH) were isolated from root tips of the halophyte Tamarix tetragyna L. grown in the presence and absence of NaCl. The activity of the enzymes isolated from root tips grown in the presence of NaCl was lower than that of the enzymes isolated from roots grown in absence of NaCl. The mitochondrial MDH was much more sensitive to salinity than the soluble MDH. The soluble enzyme from roots grown in NaCl had a higher Km for malate and lower Km for NAD than enzyme from the control roots. Addition of NaCl in vitro at 72 mM significantly stimulated the reductive activity of soluble MDH, while higher NaCl concentrations (240 mM and above) depressed enzyme activity. The inhibition of enzyme activity by various salts was found to be in the order MgCl₂ > NaCl = KCl > Na₂SO₄. Mannitol at equiosmotic concentrations had no effect. Substrate inhibition, typical for oxaloacetate oxidation, was not observed at high NaCl concentrations in vitro and high substrate concentrations neutralized the inhibitory effect of NaCl. Increased coenzyme concentrations had no effect. In vitro NaCl increased the Km for malate and oxaloacetate already at relatively low concentrations. At the same time NaCl decreased the Km for NAD and NADH. The inhibitory effect of NaCl on enzyme activity seems not to be due to the effect on the Km alone. Soluble and mitochondrial MDH had different responses to pH changes, mitochondrial MDH being more sensitive. Mitochondrial MDH released from the particles had a similar response to that of the entire particles. Changes of pH modified the effect of NaCl on enzyme activity. It was postulated that NaCl apparently induces conformational changes in the enzyme.     

Analysis of “solution effects” injury: Rabbit renal cortex frozen in the presence of dimethyl sulfoxide

Slices of rabbit renal cortex were frozen in 0.64 or 1.92 M dimethyl sulfoxide (Me₂SO) to various subzero temperatures, thawed, and assayed for viability. Salt and Me₂SO concentrations were calculated and correlated with the injury taking place during freezing. In separate experiments, slices were treated with NaCl or Me₂SO in concentrations sufficient to simulate the exposure brought about as a result of freezing. The effects of these treatments on cortical viability were compared with the results of freezing to equivalent concentrations of either NaCl or Me₂SO. The results show that whereas slices will tolerate exposure to at least six times the isotonic concentration of NaCl at 0 °C, they are unable to tolerate even three times the isotonic salt concentration when frozen in 1.92 M Me₂SO. They can, however, tolerate 3 × NaCl when frozen in 0.64 M Me₂SO. Freezing damage did not depend upon the amount of ice formed per se, since slices frozen in the low concentration of Me₂SO tolerated removal of about 75% of the initial fluid content of the system, whereas slices frozen in 1.92 M Me₂SO did not tolerate an identical removal of unfrozen solution. It was found that treatment of slices with high concentrations of Me₂SO at subzero temperatures in accordance with Elford's application (14) of Farrant's method (20) produced damage which correlated approximately with the damage observed when the same concentrations of Me₂SO were produced by freezing. It is concluded that most of the damage caused by freezing in 1.92 M Me₂SO is produced either directly or indirectly by Me₂SO. Possible mechanisms for this injury are discussed.     

Effect of cryoprotectants on the activity of hemolymph nucleating agents in physical solutions

The ability of 11 different organic solutes in physical solution to mask the effect of nucleating agents from hemolymph of freezing tolerant insects was tested. The masking effect was tested by measuring the supercooling points of samples with various solute concentrations, with and without hemolymph. Hemolymph was obtained from freeze-tolerant Eleodes blanchardi tenebrionid beetles.The depressive effect of the solutes on the supercooling points was nearly equivalent to the corresponding melting point depression, indicating that the depression was due only to the colligative properties of the solutes. Thus, no ability for nucleator masking was demonstrated.     

Enhancement of seed germination in high salinity by engineering mannitol expression in Arabidopsis thaliana

The bacterial gene mtlD, which encodes mannitol 1-phosphate dehydrogenase (E. C. 1. 1. 1. 17), was transformed into Arabidopsis thaliana and expressed under control of the CaMV 35S promoter. MtlD-transformants accumulated mannitol, a sugar alcohol that is not normally found in Arabidopsis. Amounts of soluble carbohydrates, sucrose, glucose, fructose, myo-inositol and mannitol were determined in different tissues of wild-type and transgenic plants. We estimated that less than 1& of the carbon assimilated was converted into mannitol by the transgenic plants. The establishment of individual transformed lines (after self-crossing three times) resulted in high and low mannitol-producing lines which were stably maintained. The presence of mannitol did not alter plant appearance or growth habit. When MtlD-expressing seeds and control seeds (T3 generation) were imbibed with solutions containing NaCl (range 0 to 400 mol m^?3), transgenic seeds containing mannitol germinated in medium supplemented with up to 400 mol m^?3 NaCl, while control seeds ceased germination at 100 mol m^?3 NaCl. It is doubtful whether the ability to germinate in high salt was a result of an osmotic effect exerted by elevated levels of mannitol, considering that mannitol concentrations were in the mol m^?3 range in seeds. A specific effect of polyols, for example on the integrity of subcellular membranes or enzymes, cannot be excluded.     

Effect of surface potential on P-700 reduction in chloroplasts

The effect of salt addition on the rate of reduction of P-700 oxidized by flash illumination was analyzed. In broken chloroplasts, the rate of P-700 reduction was accelerated by salts of mono-, di- and trivalent cations, with the increasing effectiveness in this order, in the presence of various artificial electron donors or acceptors. The rate was not dependent on the concentration and the valence of anions. On the other hand, in Photosystem I-enriched subchloroplast particles, added KCl did not induce the acceleration of direct reduction of P-700 by reduced DCIP.At low KCl concentrations (below 10 mM), the rate of P-700 reduction was also accelerated by added KCl in sonicated chloroplasts to which purified plastocyanin was added. The curves of dependence of the reduction rate on plastocyanin concentration were not of the Michaelis-Menten type, but sigmoidal. The maximal of P-700 reduction was higher at higher salt concentrations and the half-maximal plastocyanin concentration for P-700 reduction became lower with increasing NaCl concentrations.In broken chloroplasts treated with 50 mM glutaraldehyde, the rate of P-700 reduction was not accelerated by added KCl.The Debye-Hückel theory and the Gouy-Chapman theory were applied to our data to analyze the electrostatic interaction between electron tranfer components on thylakoid membranes. It is suggested that the major factor determining the rate of P-700 reduction is the donation of electrons from plastocyanin to P-700. Most of the observed effect is probably due to the increase in the local concentration or accessibility of plastocyanin to the site of P-700 reduction which is expected when the negative surface potential rises when salt is added.     

Salt Effects on Plastein Formation by α-Chymotrypsin

The plastein formation by α-chymotrypsin from an ovalbumin hydrolysate was affected in an order of valency of salts when the concentration of each salt was 1 m. Monovalent cations were rather effective at this concentration and enhanced the plastein yield by 10%. In the presence of NaCl, the plastein formation showed two distinct maximal rates at its concentrations of 0.1 m and 0.8 m. The first maximum was considered to be resulted from an increase in enzyme activity, since chymotryptic hydrolysis of both N-acetyl-l-tyrosine ethyl ester and benzyloxycarbonyl-l-phenylalanine p-nitrophenyl ester was activated at an NaCl concentration of 0.1 ~ 0.2 m. The second maximum was ascribed to the salting-out of the product due to the higher concentration of NaCl. A salt-tolerant protease was also used to confirm the above conclusions. It was observed that this enzyme was much effective in producing a plastein at a high NaCl concentration. This may be due to the fact that both the enzyme activation effect and the product salting-out effect participate co-operatively.