Kurt A. Santarius

The protective effect of sugars on chloroplast membranes during temperature and water stress and its relationship to frost, desiccation and heat resistance.

SummaryFreezing, desiccation and high-temperature stress may under certain conditions result in inactivation of electron transport (DCIP reduction) and cyclic photophosphorylation of isolated chloroplast membranes of spinach (Spinacia oleracea L.). When sugars are present during temperature and water stress, the thylakoids may be partially or completely protected. This membrane stabilization depends on the concentration of sugars and their molecular size. The trisaccharide raffinose is, on a molar basis, more effective than the disaccharide sucrose and the latter more than the monosaccharide glucose. An uncoupling effect and a stimulation of electron transport can be observed during freezing, desiccation and heat treatment, e.g. electron transport reactions are less sensitive to temperature and water stress than is photophosphorylation. As sugars are known to accumulate in winter, unspecific membrane stabilization by sugars may help to explain the often reported parallel development of frost, drought and heat resistance in many plants during winter.

Changes in the intracellular levels of ATP, ADP, AMP and Pi and regulatory function of the adenylate system in leaf cells during photosynthesis

1. 1. Leaves were exposed to periods of light and dark, killed in liquid air and freeze-dried. The dry material was fractionated by a non-aqueous method into a chloroplastic and a residual fraction. These were analyzed for ATP, ADP, AMP, pyruvate and orthophosphate and for adenylate kinase (EC 2.7.4.3). 2. 2. The concentrations of the adenylates in chloroplasts vary between 1.5 · 10−4 and 5 · 10−4M. ATP shows a rapid increase in the light and a rapid decrease in the dark in both chloroplasts and cytoplasm. The changes in ADP are opposite to those in ATP. The changes in AMP are the same as those for ADP in Spinacia and Beta, while in Coleus an increase is observed even in the light. This may be explained by a low activity of adenylate kinase in Coleus and by high activities in Spinacia and Beta. Less than 50% of the adenylate kinase is located in the chloroplasts of the investigated species. 3. 3. The levels of inorganic phosphate in chloroplasts are relatively high (4 · 10t3 to 25 · 10−3M); they decrease rapidly in the light, sometimes by as much as 30% of the initial value, and increase slowly in a subsequent dark period. 4. 4. Pyruvate decreases in the light and increases in the dark. 5. 5. From the results it is concluded that the controlling factor in the inhibition of glycolysis and respiration by light is the increased ratio of ATP to ADP rather than a drop in the orthophosphate. 6. 6. Mass action ratios and the behaviour of AMP point to the existence of a photosynthetic reaction generating AMP.

Compartmentation and reduction of pyridine nucleotides in relation to photosynthesis

1. 1. Leaves were exposed to periods of light and dark, killed in precooled light petroleum (at approx. −120°) or liquid air and freeze-dried. The dry material was fractionated by a non-aqueous method into a chloroplastic and a residual fraction. These were analyzed for oxidized and reduced pyridine nucleotides. 2. 2. In the dark about 25% of the total chloroplastic NADP(H2) and 2–5% of the total NAD(H2) occurred in the reduced state. Upon illumination a rapid reduction of pyridine nucleotides takes place in the chloroplasts. After 15–30 sec in the light NADPH2 and NADH2 reach a maximum and then decrease, first rapidly and then slowly. Levels of chloroplastic NADP show changes opposite to those of NADPH2. Owing to the rather high content of NAD and the low content of NADH2, no light-dependent changes of NAD could be detected in chloroplasts. 3. 3. No light-dependent changes in the concentrations of pyridine nucleotides similar to those observed in the chloroplasts for NADP, NADPH2 and NADH2 have been found in the cytoplasm. Contrary to the situation in the chloroplasts, most of the cytoplasmic NADP(H2) occurred in the reduced state (70–100%). These observations strongly suggest a compartmentation of pyridine nucleotides between chloroplasts and cytoplasm. 4. 4. Aqueously isolated chloroplasts, which are suspended in an isotonic sucrose buffer, reduce exogenous NADP at a very low rate in the light. Brief ultrasonication stimulates NADP reduction by a factor of 10–20. Osmotic shock shows the same effect. Osmotically ruptured chloroplasts cannot be stimulated further by ultrasound provided that no ferrodoxin is added to the chloroplast system. These results are further evidence of a compartmentation of pyridine nucleotides between chloroplasts and cytoplasm of the leaf cell. 5. 5. The maximum observed in the chloroplastic levels of NAD(P)H2 and of ATP after 15–30 sec illumination corresponds closely to a minimum in PGA. The subsequent decrease in NAD(P)H2 and ATP in the light is accompanied by an increase in PGA. Darkening leads to a rapid rise in PGA and a concomitant fall of NAD(P)H2 and ATP levels. Thus the kinetics of PGA, NAD(P)H2 and ATP suggest a rapid reduction of PGA by reduced pyridine nucleotides and ATP in the chloroplasts of illuminated leaf cells.

Stabilization and inactivation of biological membranes during freezing in the presence of amino acids

Abstract As a contribution to the understanding of frost tolerance of cells and organisms the effect of amino acids on thylakoid membranes during freezing was investigated. Freezing inactivates photophosphorylation of thylakoids by irreversibly altering essential membrane properties. Washed thylakoids, frozen in the presence of some amino acids such as proline, threonine, γ-aminobutyric acid or lysine·HCl, were protected against inactivation by freezing. Membrane-toxic compounds such as inorganic salts reduced the protection. Other amino acids such as glycine, alanine, serine, hydroxyproline, sodium or potassium aspartate or glutamate were unable to prevent the alteration of washed membranes by freezing. In fact, membranes protected by sucrose or by cryoprotective amino acids became inactivated during freezing when an excess of these amino acids was also present. Although individually unable to provide protection, in certain combinations with one another these amino acids became protective. Even in combination of amino acid with a membrane-toxic inorganic salt such as NaCl was protection observed. For effective protection a suitable ratio between membrane-toxic compound and amino acid had to be maintained. Departure from this ratio to either side resulted in inactivation. A third group of amino acids, among them phenylalanine, tyrosine, valine, leucine, isoleucine, methionine and arginine·HCl, did not prevent freeze inactivation of thylakoid membranes either in the absence or in the presence of inorganic salts. Membranes protected against inactivation by a cryoprotectant such as sucrose became inactivated during freezing if one of these amino acids was also present. Membrane inactivation during freezing is due to the accumulation in the unfrozen part of the system of potentially membrane-toxic compounds such as inorganic salts or amino acids possessing apolar side chains, and in some instances perhaps also to eutectic solidification of the complete system. Protective compounds protect during freezing partly by colligative action, i.e. by their unspecific ability to reduce the concentration of toxic solutes below the limit of toxicity. Further, specific interaction between cryoprotectants and membranes plays an important role in membrane preservation during freezing. The results indicate that the accumulation of potentially toxic cell components, such as inorganic but also organic cell compounds, which become concentrated up to toxic levels during extracellular freezing, are the cause of injury during freezing of frost-sensitive cells. In frost-hardy cells, protection is provided by compounds that reduce non-specifically the concentration of toxic substances or that protect specifically by membrane stabilization.

Investigations on Heat Resistance of Spinach Leaves

Exposure of spinach plants to high temperature (35° C) increased the heat resistance of the leaves by about 3° C. This hardening process occurred within 4 to 6 h, whereas dehardening at 20°/15° C required 1 to 2 days. At 5° C dehardening did not take place. Hardening and dehardening occurred in both the dark and the light. The hardiness was tested by exposure of the leaves to heat stress and subsequent measurements of chlorophyll fluorescence induction and light-induced absorbance changes at 535 nm on the leaves and of the photosynthetic electron transport in thylakoids isolated after heat treatment. Heat-induced damage to both heat-hardened and non-hardened leaves seemed to consist primarily in a breakdown of the membrane potential of the thylakoids accompanied by partial inactivation of electron transport through photosystem II. The increase in heat resistance was not due to temperature-induced changes in lipid content and fatty acid composition of the thylakoids, and no conspicuous changes in the polypeptide composition of the membranes were observed. Prolonged heat treatment at 35° C up to 3 days significantly decreased the total lipid content and the degree of unsaturation of the fatty acids of membrane lipids without further increase in the thermostability of the leaves. Intact chloroplasts isolated from heat-hardened leaves retained increased heat resistance. When the stroma of the chloroplasts was removed, the thermostability of the thylakoids was decreased and was comparable to the heat resistance of chloroplast membranes obtained from non-hardened control plants. Compartmentation studies demonstrated that the content of soluble sugars within the chloroplasts and the whole leaf tissue decreased as heat hardiness increased. This indicated that in spinach leaves, sugars play no protective role in heat hardiness. The results suggest that changes in the ultrastructure of thylakoids in connection with a stabilizing effect of soluble non-sugar stroma compounds are responsible for acclimatization of the photosynthetic apparatus to high temperature conditions. Changes in the chemical composition of the chloroplast membranes did not appear to play a role in the acclimatization.

Effects of freezing on biological membranes in vivo and in vitro.

Abstract Membrane inactivation by freezing has been investigated using intact spinach leaves and isolated thylakoid membranes from chloroplasts of leaf cells as test material. During freezing in vitro in solutions containing neutral solute and a slight excess of inorganic salts such as NaCl, electron transport is stimulated while photophosphorylation is lost. Under more drastic freezing conditions damage increases, affecting dichlorophenolindophenol reduction, the rise in variable fluorescence, ferricyanide reduction and electron transport through Photosystem I, in that order. Semipolar compounds such as phenylalanine or phenylpyruvate exhibit a much higher membrane toxicity during freezing than inorganic salts. The profile of damage caused by this class of compounds is different from that caused by salts. Damage to membranes isolated rapidly from frost-killed leaves is similar to that produced by semipolar compounds during freezing in vitro. A few sites of damage could be identified, among them the site responsible for oxidation of water during photosynthesis. The results support the view that the sensitivity of their membranes limits the ability of cells to withstand freezing and suggest that freezing sensitivity is due to the accumulation in the cells of potentially membrane-toxic organic and norganic cell constituents.

Relationship between frost tolerance and sugar concentration of various bryophytes in summer and winter

SummaryFrost resistance, measured via the photosynthetic capacity after freeze-thaw treatment, and concentrations of sucrose, glucose and fructose of thalli of seven species of Bryidae and one species of Marchantiidae were determined from January to March and June to September, respectively. A distinct increase in cold tolerance from summer to winter was found in Polytrichum formosum Hedw., Atrichum undulatum (Hedw.) P. Beauv., Plagiomnium undulatum (Hedw.) Kop., Plagiomnium affine (Funck) Kop., Mnium hornum Hedw. and Pellia epiphylla (L.) Corda. While the frost resistance of the musci differed in summer and winter by 15° to more than 25° C, the hardening capacity of the thalloid liverwort was comparably low. Except in Mnium hornum, the increase in frost hardiness was accompanied by rise of the sucrose concentration in the cells, but insignificant changes in glucose and fructose contents. In contrast, Brachythecium rutabulum (Hedw.) B.S.G. and Hypnum cupressiforme Hedw. already exhibited high frost tolerances in summer, which coincided with high sucrose levels in the tissue, comparable to those found in other musci during the winter. Highly frost-resistant musci had total sugar concentrations around 90–140 mM, of which at least 80% and often more than 90% was sucrose. Artificial degradation of sucrose during exposure of mosses to higher temperatures resulted in a decline in cold hardiness. The results signify that the concentration of sugars, mainly of sucrose, may be important for the frost tolerance of bryophytes.

[Hill reaction and photophosphorylation of isolated chloroplasts in relation to water content : I. Removal of water by means of concentrated solutions].

Summary1.Water was removed by means of concentrated solutions from chloroplasts which were isolated from leaves of spinach and beets. During and after the dehydration Hill reaction and cyclic photophosphorylation with PMS as a cofactor were investigated. As osmotic amterial glucose, sucrose, lutrol and NaCl were used.2.No depression of ferricyanide reduction was obtained in 3 M sugar solution and in 2.5 M lutrol solution. These concentrations correspond to a loss of water amounting to 90% of the total water of leaf cells. In contrast, cyclic photophosphorylation was already decreased in 1–2 M solutions of sugar or lutrol, that means by much less dehydration. In 3 M solutions only 5–25% of the activity of the water saturated controls remained. However, this decrease in cyclic photophosphorylation occurred only when chloroplasts were kept dehydrated during the light reaction. When chloroplasts were permitted to return to optimal water conditions photophosphorylation was no longer inhibited. Therefore, extensive loss of water leads to reversible uncoupling of photophosphorylation from electron transport.3.Relatively low concentrations of NaCl (as compared with sugar concentrations) damage the ability of chloroplasts to perform Hill reaction and photophosphorylation. Inactivation of the reactions is partly reversible at low concentrations of NaCl and irreversible at high concentrations.4.The osmotic potential of leaves of sugar beet increased with increasing dehydration. Within a limited range the osmotic behaviour of the cell sap of leaf cells during dehydration was identical with that of NaCl solutions.5.The possibility of correlating in vitro experiments in which dehydration is simulated by exposure of chloroplasts to various solutions with in vivo experiments using intact leaves which are dehydrated to different degrees is demonstrated.Zusammenfassung1.Isolierte Chloroplasten aus Spinat- und Rübenblättern wurden mittels Lösungen verschiedener Konzentration unterschiedlich stark entwässert und während bzw. nach der Entquellung die Hill-Reaktion und die cyclische Photophosphorylierung mit PMS bestimmt. Als Osmotika hatten Glucose, Saccharose, Sorbit, Lutrol 9 und NaCl Verwendung gefunden.2.Die Ferricyanid-Reduktion wird selbst in 3 M Zuckerlösung bzw. 2,5 M Lutrollösung, also bei einem Wasserverlust von ca. 90% des Gesamtwassers der Chloroplasten, nicht wesentlich beeinflußt, und zwar unabhängig davon ob den Chloroplasten das Wasser vor oder während des Ablaufs der Lichtreaktion entzogen wird. Die cyclische Photophosphorylierung dagegen wird schon in 1–2 M Zucker- bzw. Lutrollösungen, also bei geringeren Wasserverlusten, herabgesetzt und weist in 3 M Lösungen nur noch 5–25% der Aktivität der nichtentwässerten Kontrollen auf. Dies ist allerdings nur der Fall, wenn die Chloroplasten während der Durchführung der Lichtreaktion stark entquollen waren. Chloroplasten, die vor Ablauf der Lichteaktion entwässert worden waren und danach wieder in optimale Wasserverhältnisse überführt worden sind, zeigten die gleiche Phosphorylierungs-Aktivität wie die Kontrollen; die durch die Entwässerung hervorgerufenen Veränderungen sind also reversibel. — Bei stärkerem Wasserverlust kommt es demnach zu einer mehr oder weniger starken reversiblen Entkopplung der ATP-Synthese vom Elektronentransport.3.NaCl beeinflußt im Vergleich zu den Zuckern bereits in niedrigeren Konzentrationen die Hill-Reaktion und die Photophosphorylierung beträchtlich. In Gegenwart höherer Konzentrationen an Ionen wird offenbar die Chloroplastenstruktur irreversibel verändert.4.Der osmotische Wert von Rübenblättern steigt mit zunehmendem Wasserverlust kontinuierlich an. Der Zellsaft verhält sich dabei ähnlich einer NaCl-Lösung, der in steigendem Maße Wasser entzogen wird.5.Es wird eine Möglichkeit aufgezeigt, in vitro-Untersuchungen, die in Lösungen unterschiedlicher Konzentration durchgeführt worden waren, mit in vivo-Versuchen an Blättern zu vergleichen die in verschieden starkem Ausmaß Wasser verloren haben.1. Water was removed by means of concentrated solutions from chloroplasts which were isolated from leaves of spinach and beets. During and after the dehydration Hill reaction and cyclic photophosphorylation with PMS as a cofactor were investigated. As osmotic amterial glucose, sucrose, lutrol and NaCl were used. 2. No depression of ferricyanide reduction was obtained in 3 M sugar solution and in 2.5 M lutrol solution. These concentrations correspond to a loss of water amounting to 90% of the total water of leaf cells. In contrast, cyclic photophosphorylation was already decreased in 1-2 M solutions of sugar or lutrol, that means by much less dehydration. In 3 M solutions only 5-25% of the activity of the water saturated controls remained. However, this decrease in cyclic photophosphorylation occurred only when chloroplasts were kept dehydrated during the light reaction. When chloroplasts were permitted to return to optimal water conditions photophosphorylation was no longer inhibited. Therefore, extensive loss of water leads to reversible uncoupling of photophosphorylation from electron transport. 3. Relatively low concentrations of NaCl (as compared with sugar concentrations) damage the ability of chloroplasts to perform Hill reaction and photophosphorylation. Inactivation of the reactions is partly reversible at low concentrations of NaCl and irreversible at high concentrations. 4. The osmotic potential of leaves of sugar beet increased with increasing dehydration. Within a limited range the osmotic behaviour of the cell sap of leaf cells during dehydration was identical with that of NaCl solutions. 5. The possibility of correlating in vitro experiments in which dehydration is simulated by exposure of chloroplasts to various solutions with in vivo experiments using intact leaves which are dehydrated to different degrees is demonstrated.

Der Einfluß von Elektrolyten auf Chloroplasten beim Gefrieren und Trocknen

The effect of freezing, desiccation and various electrolytes on photophosphorylation, electron transport and some enzyme reactions of isolated spinach chloroplasts has been investigated. Freezing of broken chloroplasts took place at-25°C for 3 hrs; desiccation was performed at +2°C in vacuo over CaCl2 for 3 hrs. The influence of various electrolytes during freezing or drying or during incubation of thylakoids or stroma enzymes for 3 hrs at +2°C in electrolyte solutions was determined. After treatment, the activities of a number of enzymes and enzyme systems were measured under normal conditions, e. g. in the absence of elevated electrolyte levels in a reaction medium which contained only the substrates and cofactors which are necessary for the respective enzyme reactions.Only photophosphorylation and electron transport were affected by freezing, desiccation and high concentrations of electrolytes; various soluble enzymes investigated here were not inactivated under the same conditions. In general, mild dehydration and lower concentrations of electrolytes resulted in an irreversible inactivation of ATP synthesis but did not impair ferricyanide reduction. With increasing dehydration or at higher concentrations of electrolytes the Hill reaction was also inhibited. In a certain range of dehydration and electrolyte concentration uncoupling of photophosphorylation from electron transport took place. Sugar protects the sensitive structures against the deleterious effect of both dehydration and high concentration of electrolytes.Various electrolytes affected thylakoid membranes differently. Inactivation of the membranes increased with increasing ion radius and decreasing hydration envelope of univalent or divalent cations. Divalent cations were more destructive than univalent cations. Anions did not follow these rules. Within a group of similar anions (halides or organic anions) effectivity decreased with increasing hydration envelope. On a molar basis, polyvalent anions were less effective than univalent anions. Inactivation by anions followed Hofmeisters series in seversed order. However, exceptions were observed and it appears that various ions affect the membrane in a specific manner.Inactivation of photophosphorylation and electron transport due to freezing or desiccation is identical to that due to high concentrations of electrolytes. This suggests that during dehydration due to freezing or drying the concentration of electrolytes in the remaining solution is responsible for the inactivation of the sensitive membranes.: The effect of freezing, desiccation and various electrolytes on photophosphorylation, electron transport and some enzyme reactions of isolated spinach chloroplasts has been investigated. Freezing of broken chloroplasts took place at-25°C for 3 hrs; desiccation was performed at +2°C in vacuo over CaCl2 for 3 hrs. The influence of various electrolytes during freezing or drying or during incubation of thylakoids or stroma enzymes for 3 hrs at +2°C in electrolyte solutions was determined. After treatment, the activities of a number of enzymes and enzyme systems were measured under normal conditions, e. g. in the absence of elevated electrolyte levels in a reaction medium which contained only the substrates and cofactors which are necessary for the respective enzyme reactions.Only photophosphorylation and electron transport were affected by freezing, desiccation and high concentrations of electrolytes; various soluble enzymes investigated here were not inactivated under the same conditions. In general, mild dehydration and lower concentrations of electrolytes resulted in an irreversible inactivation of ATP synthesis but did not impair ferricyanide reduction. With increasing dehydration or at higher concentrations of electrolytes the Hill reaction was also inhibited. In a certain range of dehydration and electrolyte concentration uncoupling of photophosphorylation from electron transport took place. Sugar protects the sensitive structures against the deleterious effect of both dehydration and high concentration of electrolytes.Various electrolytes affected thylakoid membranes differently. Inactivation of the membranes increased with increasing ion radius and decreasing hydration envelope of univalent or divalent cations. Divalent cations were more destructive than univalent cations. Anions did not follow these rules. Within a group of similar anions (halides or organic anions) effectivity decreased with increasing hydration envelope. On a molar basis, polyvalent anions were less effective than univalent anions. Inactivation by anions followed Hofmeisters series in seversed order. However, exceptions were observed and it appears that various ions affect the membrane in a specific manner.Inactivation of photophosphorylation and electron transport due to freezing or desiccation is identical to that due to high concentrations of electrolytes. This suggests that during dehydration due to freezing or drying the concentration of electrolytes in the remaining solution is responsible for the inactivation of the sensitive membranes.SummaryThe effect of freezing, desiccation and various electrolytes on photophosphorylation, electron transport and some enzyme reactions of isolated spinach chloroplasts has been investigated. Freezing of broken chloroplasts took place at-25°C for 3 hrs; desiccation was performed at +2°C in vacuo over CaCl2 for 3 hrs. The influence of various electrolytes during freezing or drying or during incubation of thylakoids or stroma enzymes for 3 hrs at +2°C in electrolyte solutions was determined. After treatment, the activities of a number of enzymes and enzyme systems were measured under normal conditions, e. g. in the absence of elevated electrolyte levels in a reaction medium which contained only the substrates and cofactors which are necessary for the respective enzyme reactions.Only photophosphorylation and electron transport were affected by freezing, desiccation and high concentrations of electrolytes; various soluble enzymes investigated here were not inactivated under the same conditions. In general, mild dehydration and lower concentrations of electrolytes resulted in an irreversible inactivation of ATP synthesis but did not impair ferricyanide reduction. With increasing dehydration or at higher concentrations of electrolytes the Hill reaction was also inhibited. In a certain range of dehydration and electrolyte concentration uncoupling of photophosphorylation from electron transport took place. Sugar protects the sensitive structures against the deleterious effect of both dehydration and high concentration of electrolytes.Various electrolytes affected thylakoid membranes differently. Inactivation of the membranes increased with increasing ion radius and decreasing hydration envelope of univalent or divalent cations. Divalent cations were more destructive than univalent cations. Anions did not follow these rules. Within a group of similar anions (halides or organic anions) effectivity decreased with increasing hydration envelope. On a molar basis, polyvalent anions were less effective than univalent anions. Inactivation by anions followed Hofmeisters series in seversed order. However, exceptions were observed and it appears that various ions affect the membrane in a specific manner.Inactivation of photophosphorylation and electron transport due to freezing or desiccation is identical to that due to high concentrations of electrolytes. This suggests that during dehydration due to freezing or drying the concentration of electrolytes in the remaining solution is responsible for the inactivation of the sensitive membranes.

Short-term thermal acclimation and heat tolerance of gametophytes of mosses

Abstract The ability of gametophytes of two Bryidae, Atrichum undulatum (Hedw.) P. Beauv. and Polytrichum formosum Hedw., to rapidly acquire thermotolerance was investigated by measuring chlorophyll a fluorescence and electrolyte leakage. Short-term acclimation of turgid shoots to elevated sublethal temperatures resulted in a small but significant increase in the heat stability of the photosynthetic apparatus and of cellular membranes by around 1 K, indicating that the heat hardening capacity of hydrated mosses is very low. While thermal adaptation occurred within few hours, dehardening required several days. The pattern of rapid thermal hardening and dehardening of turgid mosses resembles that of flowering plants. However, as opposed to the low potential for short-term thermal acclimation of hydrated gametophytes, a dramatic rise in heat resistance occurred with decline of the water content of the poikilohydric shoots, which was achieved either by equilibrating of the thalli at different relative humidities or by incubation in sugar solutions of various concentrations. The lower the water potential of the tissue the higher the heat tolerance of the shoots. The data show that in contrast to homoiohydric higher plants, in desiccation-resistant mosses changes in the water status of the thalli play the dominant role in short-term variations of thermal tolerance rather than specific heat hardening and dehardening reactions.