Freezing of isolated thylakoid membranes in complex media. VIII. Differential cryoprotection by sucrose, proline and glycerol

The cryoprotective efficiency of sucrose, proline and glycerol for chloroplast membranes isolated from spinach leaves ( Spinacia oleracea L. cv. Monatol) was determined after freeze-thaw treatment in media containing the predominant inorganic electrolytes of the chloroplast stroma. In most cases, the protective capacity of equimolar concentrations of the cryoprotectants followed the order sucrose > proline > glycerol. The lower the freezing temperature the less cryoprotectant was necessary for comparable preservation of the capacity of photosynthetic electron transport. Likewise, the cryoprotective efficiency of sucrose for cyclic photophosphorylation and light-induced proton gradient increased with decreasing freezing temperature. In contrast, while proline effectively stabilized these membrane reactions at mild and moderate freezing temperatures, it was much less efficient at more severe freezing stress. Cryoprotection of photophosphorylation and proton gradient formation at given initial concentrations of glycerol was largely independent of the freezing temperature. While dissociation of the peripheral part of chloroplast coupling factor (CF₁) during freeze-thaw treatment cannot be prevented in the presence of lower initial concentrations of proline and glycerol and. at mild freezing temperatures, of sucrose, the latter may stabilize this protein complex at least under more severe freezing conditions. The differences in the cryoprotective efficiency of the solutes are discussed relative to their non-ideal activity-concentration profiles, solution properties and penetration behaviour across the thylakoid membrane.     

New results about structure, function and regulation of the chloroplast ATP synthase (CF0CF1)

The chloroplast ATP synthase utilises the energy of a transmembrane electrochemical proton gradient to drive the synthesis of ATP from ADP and phosphate. This multi-subunit thylakoid membrane-bound enzyme consists of a proton channel, CF₀, and an extrinsic catalytic sector, CF₁. Stimulated by the elucidation of a three-dimensional partial structure of the mitochondrial enzyme, substantial progress has been made to understand the catalytic mechanism and interesting hypotheses have been proposed about the molecular mechanism of energy coupling. The review discusses the present state of knowledge concerning the structure, molecular genetics, catalytic mechanism, energy coupling and regulation of this important enzyme involved in photophosphorylation.     

Lack of correlation between effects of tentoxin on chloroplast coupling factor and chloroplast ultrastructure

The mediation of tentoxin-induced chlorosis through inhibition of chloroplast coupling factor 1 (CF₁) ATPase activity was investigated through an examination of the effects of tentoxin on electrophoretically-separated CF₁ ATPases from sensitive and insensitive Nicotiana species. Sensitive species exhibited three major ATPases, only one of which was inhibited at some concentrations of tentoxin. Insensitive Nicotiana species showed the same three "isozymes"upon electrophoresis but none of the isozymes were tentoxin sensitive. CF₁ isolated from Zea mays L. cv. Pioneer 3541, which is insensitive to tentoxin in vivo based on lack of chlorosis, exhibited two ATPases, one of which was sensitive to tentoxin. The concentration/activity relationships between tentoxin and ATPase inhibition of the sensitive isozyme did not correlate well with the chlorosis induced at similar levels of tentoxin in vivo. Both Oenothera hookeri Torr. & Gray and the CF₁-deficient I iota mutant derived from it are sensitive to tentoxin as determined by loss of chlorophyll and ultrastructural changes typical of the tentoxin syndrome. These results support a mechanism of action different from inhibition of CF¹ for tentoxin-induced chlorosis.     

The long-term responses of the photosynthetic proton circuit to drought

Proton motive force (pmf) across thylakoid membranes is not only for harnessing solar energy for photosynthetic CO₂ fixation, but also for triggering feedback regulation of photosystem II antenna. The mechanisms for balancing these two roles of the proton circuit under the long-term environmental stress, such as prolonged drought, have been poorly understood. In this study, we report on the response of wild watermelon thylakoid 'proton circuit' to drought stress using both in vivo spectroscopy and molecular analyses of the representative photosynthetic components. Although drought stress led to enhanced proton flux via a ∼34% increase in cyclic electron flow around photosystem I (PS I), an observed ∼fivefold decrease in proton conductivity, g_H⁺, across thylakoid membranes suggested that decreased ATP synthase activity was the major factor for sustaining elevated q_E. Western blotting analyses revealed that ATP synthase content decreased significantly, suggesting that quantitative control of the complex plays a pivotal role in down-regulation of g_H⁺. The expression level of cytochrome b ₆ f complex – another key control point in photosynthesis – also declined, probably to prevent excess-reduction of PS I electron acceptors. We conclude that plant acclimation to long-term environmental stress involves global changes in the photosynthetic proton circuit, in which ATP synthase represents the key control point for regulating the relationship between electron transfer and pmf.     

Membrane lipids in Ceratodon purpureus protonemata grown at high and low temperatures

Ceratodon purpureus (Hedw.) Brid. was grown at two temperatures, 20 and 4°C. The protonemata grown at 4°C fixed more CO₂ at low temperatures; but their frost tolerance, tested as the recovery of photosynthesis after frost treatment, was not better than in the protonemata grown at 20°C. The effects of the growth temperature were studied on the membrane lipids of intact protonemata and on the lipid and protein contents of isolated thylakoid membranes. A large proportion, 70 to 90%, of the thylakoid membrane lipids was lost unless precautions were taken to inhibit the lipid-degrading enzyme activities. The lipid content of the thylakoid membranes of protonemata grown at 20 and 4°C was 3.9 and 4.8 mol (mol chlorophyll)⁻¹, respectively. Only minor differences were found in the lipid class composition. Monogalactosyldi-acylglycerol constituted more than 50 mol-% of the thylakoid membrane lipids at both 20 and 4°C. However, each lipid class had a higher average number of double bonds per lipid molecule in cold growth conditions. The protein content of the thylakoid membranes was low at both 20 and 4°C. These characteristics of the thylakoid membranes may be a prerequisite for the observed ability of protonemata to photosynthesize even at subzero temperatures.     

The lower limit of photon fluence rate for phototrophic growth: the significance of 'slippage' reactions

Abstract. The role of 'slippage' reactions, in the form of passive H⁺ uniport through CF₀-CF₁, ATP synthetase and breakdown of the S₂ and S₃ intermediates of O₂ evolution, is considered in relation to the growth of phototrophic organisms at low photon fluence rates. Analysis of the limited data available suggests that adaptation (phenotypic or genotypic) to low photon fluence rates is accompanied by an increase in the ratio of light-absorbing pigments to the (potentially slippage-inducing) photosystem two units and CF₀-CF₁ ATP synthetases. Furthermore, organisms which are genotypically adapted to high photon fluence rates do not, when grown at low photon fluence rates, achieve the same low ratio of reaction centres to total light-harvesting pigments as is found in phototrophs genotypically adapted to low photon fluence rates. The limits to, and energy costs of, such a mechanism of adaptation to low photon fluence rates are also discussed.     

Effect of low growth temperature on coupling between electron transport and proton flux in Vicia faba thylakoids

Coupling between electron transport and proton flux has been compared in chloroplasts from Vicia faba (cv. Windsor) plants grown at 20 and 5°C. Proton uptake by warm-grown thylakoids was sensitive to external pH and stimulated by micromolar adenine nucleotide above pH 7.0. Electron transport was modulated by pH, adenine nucleotide and energy transfer inhibitors (triphenyltin and Hg²⁺). By contrast, proton uptake by cold-grown thylakoids was generally lower and was insensitive to micromolar ATP. The rate of non-phosphorylating electron flow in cold-grown thylakoids was relatively insensitive to pH and Hg²⁺ and was not modulated by adenine nucleotides or triphenyltin. Stimulation of electron transport by phosphorylating conditions in cold-grown thylakoids was generally lower and insensitive to pH. It is concluded that the control of proton efflux through CF₀-CF₁ differs in thylakoids of V. faba grown at warm and cold temperatures.     

Kinetics of electron transport, proton transfer and photophosphorylation in chloroplasts and their relation to temperature-induced structural changes in the thylakoid membrane

Effects of various temperatures on the rates of electron transport between two photosystems, the light-induced uptake of protons, kinetics of proton efflux from the chloroplasts in the dark and photophosphorylation were studied in isolated chloroplasts. There are correlations between the physical state of thylakoid membrane and the rates of electron- and proton transport processes. The temperature dependence of "structural" parameter (fluidity of lipids in membrane) as well as the rates of electron- and proton transport processes reveal the breaks under the same temperatures. Stimulation of photophosphorylation by temperature increasing correlates with the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the protein part of CF0-CF1 complex. The rate of photophosphorylation also correlates with the physical state of membrane lipids. Thermoinduced "melting" of the thylakoid membrane inhibits the ATP formation because of a decrease in photosystem 2 photochemical activity and stimulation of membrane conductivity for protons.     

Inhibition of photosynthesis in Phaseolus vulgaris by treatment with toxic concentrations of zinc: effects on electron transport and photophosphorylation

Dwarf beans ( Phaseolus vulgaris L. cv. Limburgse Vroege) were grown on a nutrient medium containing a toxic non-lethal ZnSO₄ concentration. The electron transport and photophosphorylation activities of chloroplasts, isolated from these beans, and from control plants, grown under standard nutrient conditions, were compared. Electron transport was significantly inhibited by Zn²⁺ treatment. Photosystem 2 activity proved to be more sensitive than photosystem 1 activity.
Inhibition was dependent on electron flow rate. Activity was fully restored with semicarbazide. EDTA-washed thylakoid membranes were strongly manganese-deficient. The results suggest that photolysis of water was primarily inhibited, due to a zinc-induced deficiency in loosely bound manganese at the water-splitting site. Manganese is probably substituted by zinc, since the zinc content of thylakoids increased five-fold. Non-cyclic photophosphorylation capacity was also limited as a result of inhibition of electron transport. Phosphorylation efficiency (ATP/2e ratio) involving both energy conserving sites was hardly affected.     

Electron Transport Activities of Isolated Thylakoids from Wheat Plants Grown in Salicylic Acid

Abstract: Wheat ( Triticum aestivum cv. Sonalika) plants were grown with three different concentrations of salicylic acid (SA; 50/500/1000 μM) for 7 days and the effects on the level of thylakoid photochemical activities were examined. SA treatment stimulated photosystem II-catalyzed electron flow in all concentrations tested. Photosystem I-associated electron transport activity was stimulated at low concentrations of SA (50 μM) but at higher concentrations (500 and 1000 μM) the electron transport activity was drastically attenuated. Thylakoids isolated from the leaves of seedlings grown with high concentrations of SA (500 and 1000 μM) showed a substantial reduction in uncoupler (NH₄Cl)-mediated stimulation in electron flow. In addition, they failed to support ADP-dependent stimulation of electron transport activity and induced a significant reduction in ATPase activity. Incubation of isolated thylakoids with SA, however, had no effect on thylakoid photofunction, indicating no direct effect of SA on photoelectron transport activity. Furthermore, high concentrations of SA specifically reduce the thylakoid cytochrome f₅₅₄ level. The results suggest that SA, depending on its concentration, imparts differential effects on the photofunction of thylakoids. A low concentration of SA favours photosynthetic activity while the high concentration induces drastic attenuation of photosynthetic activity because of the decline in cytochrome f₅₅₄.     

The role of thylakoid lipids in the photodamage of photosynthetic activity

The effect of excess light at 10 or 30°C under aerobic or low O₂ condition on peroxidation of thylakoid lipids and primary photochemistry of photoinsynthesis was studied in wheat ( Triticum aestivum L. cv. HD 2329). Photoinhibitory damage to photosythesis was directly proportional to the peroxidation of thylakoid lipids. Photoinhibitory treatment given under low O₂ conditions resulted in significantly less peroxidation of the primary photochemistry of photosythesis measured using chlorophyll fluorescence and photosythetic electron trasport. Short term recovery of F_v/F_m ratio was fast while thylakoid lipids did not show much recovry. However, recovery (of F_v/F_m ratio and thylakoid lipids) was almost complete within 12 h after photoinhibition treatment. A possible relationship between peroxidation of thylakoid lipids and photodamage to photosynthesis is discussed.     

Depletion of Vipp1 in Synechocystis sp. PCC 6803 affects photosynthetic activity before the loss of thylakoid membranes

A vipp1 mutant of Synechocystis sp. PCC 6803 could not be completely segregated under either mixotrophic or heterotrophic conditions. A vipp1 gene with a copper-regulated promoter (P _petE -vipp1 ) was integrated into a neutral platform in the genome of the merodiploid mutant. The copper-induced expression of P _petE -vipp1 allowed a complete segregation of the vipp1 mutant and observation of the phenotype of Synechocystis 6803 with different levels of vesicle-inducing protein in plastids 1 (Vipp1). When P _petE -vipp1 was turned off by copper deprivation, Synechocystis lost Vipp1 and photosynthetic activity almost simultaneously, and at a later stage, thylakoid membranes and cell viability. The photosystem II (PSII)-mediated electron transfer was much more rapidly reduced than the PSI-mediated electron transfer. By testing a series of concentrations, we found that P _petE -vipp1 cells grown in medium with 0.025 μM Cu²⁺ showed no reduction of thylakoid membranes, but greatly reduced photosynthetic activity and viability. These results suggested that in contrast to a previous report, the loss of photosynthetic activity may not have been due to the loss of thylakoid membranes, but may have been caused more directly by the loss of Vipp1 in Synechocystis 6803.     

Freezing of isolated thylakoid membranes in complex media. VI. The effect of pH

Thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were used as a model biomembrane system for evaluating the significance of the hydrogen ion activity for cryoprotection. After freeze-thaw treatment in a buffered complex medium adjusted to various pH, light-induced photosynthetic membrane reactions were determined at optimum proton concentration. When thylakoids were suspended at hydrogen ion activities above and below the physiologically important pH range, irreversible inhibition of membrane functions was significantly less distinct after freezing at -15 degrees C than after storage for the same time at 0 degree C. It is suggested that thylakoid preservation at subfreezing temperatures could be due to temperature- and concentration-induced changes of the proton activity in the unfrozen part of the system and retardation of the temperature-dependent aging processes of the isolated membranes. In addition, the increase in the concentration of cryoprotective compounds during freezing could stabilize chloroplast membranes against the deleterious effect of unfavorable high and low proton concentrations. Thylakoid injury brought about by lowering the pH was primarily due to dissociation of the chloroplast coupling factor (CF1), which increased the proton permeability of the membranes and caused inhibition of photophosphorylation. In media adjusted to more alkaline pH, inactivation of the water oxidation system was an initial result of membrane damage. Then, noncyclic photophosphorylation was limited by photosystem II-mediated electron flow. Photosystem I-driven electron transport was substantially more stable over a wide pH range.     

Growth and development of winter rye at cold-hardening temperatures results in thylakoid membranes with increased sensitivity to low concentrations of osmoticum

Chloroplasts developed at cold-hardening (5°C) and non-hardening temperatures (20°C) were compared with respect to the stability of photosynthetic electron transport activities, the capacity to produce and maintain a H⁺ gradient and the capacity fat photophosphorylation as a function of resuspension in the presence or absence of osmoticum. The results for electron transport indicate that whole chain, photosystem I and pfaotosystem II activities in non-hardened chloroplast thyalkoids were unaffected by resuspension in the presence of high or low osmoticum. In contrast, the same electron transport activities in cold-hardened chloroplast thylakoids exhibited a 3- to 4-fold decrease in activity when resuspended in the presence of low osmoticum. Impairment of electron transport through photosystem II of cold-hardened thylakoids resuspended in the presence of low osmoticum was supported by room temperature fluorescence induction kinetics. Since the presence of Mn²⁺ partially overcame this inhibition, it is concluded that this osmotically-induced inhibition of PSII activity in cold-hardened chloroplast thylakoids may, in part, be due to damage to the H₂O-splitting side of photosystem II. Both the initial rate and the maximum capacity for cyclic photophosphorylation were significantly inhibited in cold-hardened as compared to non-hardened thylakoids upon resuspension in the presence of low concentrations of osmoticum. This was correlated with an inability of the cold-hardened chloroplast thylakoids to maintain a significant transrnembrane H⁺ gradient. The results indicate that cold-hardened thylakoid membranes required an osmotic concentration (0.8 M) twice as high as non-hardened thylakoids (0.4 M) to produce the same initial rate of H⁺ uptake. In addition, the capacity to produce a proton gradient in cold-hardened thylakoids was less stable than that in non-hardened thylakoids regardless of the osmotic concentration tested. It is concluded that development of rye thylakoid membranes at low temperature results in a differential sensitivity to low osmoticum and thus extreme caution should be exercised when comparing the structure and function of isolated thylakoids developed under contrasting thermal regimes.     

Detoxification of hydrogen peroxide maintains the water transport activity in figleaf gourd (Cucurbita ficifolia) root system exposed to low temperature

The figleaf gourd ( Cucurbita ficifolia Bouché) root system has the ability to take up water and nutrients at low soil temperatures, and in the present paper, we attempt to reveal some of the molecular mechanisms behind this low-temperature tolerance. Exposure of figleaf gourd root system to low temperature induced accumulation of H₂O₂ along the plasma membrane but not in the cytoplasm. H⁺-ATPase (EC 3.6.1.35) activity of isolated root plasma membranes and root hydraulic conductivity ( Lp_r ) were largely insensitive to externally applied H₂O₂. However, using bromocresol purple, it was shown that the acidification of the medium surrounding the root was strongly inhibited with low temperature- and H₂O₂-treated roots. Addition of catalase (EC 1.11.1.6) to the root medium during low-temperature exposure led to a recovery of H⁺-efflux along the root surface and increased Lp_r , demonstrating the importance of an H₂O₂ detoxification system in the root cells. Additional evidence for an increased Lp_r was obtained by the Fenton reaction wherein a warming of the solution increased the activity of the detoxification system. All available evidence suggests that the ability of figleaf gourd root system to maintain a low level of H₂O₂ in the cytoplasm and to detoxify reactive oxygen species is related to the maintenance of water transport activity at low temperatures.     

Multi-temperature effects on Hill reaction activity of barley chloroplasts

1. 1. The relationship between temperature and Hill reaction activity has been investigated in chloroplasts isolated from barley (Hordeum vulgare L. cv. Abyssinian).

2. 2. An Arrhenius plot of the photoreduction of 2,6-dichlorophenolindophenol (DCIP) showed no change in slope over the temperature range 2–38 °C. The apparent Arrhenius activation energy (E_a) for the reaction was 48.1 kJ/mol.

3. 3. In the presence of an uncoupler of photophosphorylation, methylamine, the E_a for DCIP photoreduction went through a series of changes as the temperature was increased. Changes were found at 9, 20, 29 and 36 °C. The E_a was highest below 9 °C at 63.7 kJ/mol. Between 9 and 20 °C the E_a decreased to 40.4 kJ/mol and again to 20.2 kJ/mol between 20 and 29 °C. Between 29 and 36 °C there was no further increase in activity with increasing temperature. The temperature-induced changes at 9, 20 and 29 °C were reversible. At temperatures above 36 °C (2 min) a thermal and largely irreversible inactivation of the Hill reaction occurred.

4. 4. Temperature-induced changes in E_a were also found when ferricyanide was substituted for DCIP or gramicidin D for methylamine. The addition of an uncoupler of photophosphorylation was not required to demonstrate temperature-induced changes in DCIP photoreduction following the exposure of the chloroplasts to a low concentration of cations.

5. 5. The photoreduction of the lipophilic acceptor, oxidized 2, 3, 5, 6-tetramethyl-p-phenylenediamine, also showed changes in E_a in the absence of an uncoupler.

6. 6. The temperature-induced changes in Hill activity at 9 and 29 °C coincided with temperature-induced changes in the fluidity of chloroplast thylakoid membranes as detected by measurements of electron spin resonance spectra. It is suggested that the temperature-induced changes in the properties and activity of chloroplast membranes are part of a control mechanism for regulation of chloroplast development and photosynthesis by temperature.

Effect of oxygen tension, salinity, temperature and organic matter concentration on the growth and nitrifying activity of an estuarine strain of Nitrosomonas

Abstract The effects of O₂ tension, temperature, salt concentration and organic matter concentration on the growth and nitrifying activity of Nitrosomonas N₃ isolated from Tay Estuary sediments have been investigated. Chemostat-grown cultures were able to grow and nitrify at dissolved O₂ concentrations as low as 0.1 mg O₂· 1⁻¹ (cell population densities were 15% of those obtained in fully aerated cultures). This bacterium was sensitive to reduced temperatures as chemostat-grown cultures washed out at growth temperatures below 15°C, at dilution rates > 0.025 · h⁻¹. Batch-grown cultures of Nitrosomonas N₃ were used to study the effects of NaCl and complex organic matter concentration on nitrifying activity. Maximum rates of NH⁺₄ oxidation were recorded at NaCl concentrations of 1% w/v, whilst tryptone soya broth (TSB), nutrient broth (NB), yeast extract broth (YEB) and peptone were inhibitory at concentrations > 10 mg · 1⁻¹.     

Cold acclimation of Ilex aquifolium under natural conditions with special regard to the photosynthetic apparatus

Frost resistance of leaves of holly ( Ilex aquifolium L.) increased from about −9°C in late summer to −24°C in mid-winter. The gradual rise in cold hardiness occurred when the minimum air temperature dropped to 0°C or below and was closely related to increase in the cellular sap concentration. Predominantly, the decrease in the osmotic potential of the cellular sap was caused by sugar accumulation, mainly of sucrose. The capacity of net photosynthesis of the leaves, as well as the total lipid and protein content and the proportion of individual lipids of the thylakoid membranes, did not significantly change during cold acclimation. The gradual shift towards desaturation in the fatty acids of the thylakoid lipids during the hardening period was neither correlated with alterations in the frost resistance nor did it affect the potential efficiency for various light-induced chloroplast membrane reactions such as linear photosynthetic electron transport, photophosphorylation and the proton gradient (ΔpH). It is suggested that in holly leaves reduction in cell volume changes during freeze-thawing and cryoprotection by sugars could play a dominant role for the increase in frost resistance. Seasonal changes in the degree of unsaturation of polar lipids of the thylakoids could contribute to maintain optimal functional efficiency of the membranes at low temperatures rather than to avoid freezing damage.     

A burst of CO2 from photosynthesizing leaves after a temperature decrease under constant light conditions

A transient CO₂ burst from seedlings of some plant species was observed after a rapid temperature decrease. The magnitude of the CO₂ release depended on initial temperature, oxygen concentration and light intensity. To obtain a maximal value of CO₂ release, the temperature had to decrease by more than 8°C. The phenomenon was detected only in the light, and was confined to C₃ species. It was inhibited by low oxygen concentration, indicating its possible connection with photorespiration.     

The Effect of Isoprene on the Properties of Spinach Thylakoids and Phosphatidyicholine Liposomes

Abstract: Isoprene is emitted from the leaves of some plants. It was recently reported that exogenous isoprene delays the onset of leaf damage during controlled increases in leaf temperature (Singsaas et al. Plant Physiology 115: 1413–1420 [1997^[17). Thylakoid membranes are presumed to be the site of action based upon isoprene's hydrophobicity, production in chloroplasts, and effect upon chlorophyll fluorescence at high temperatures. In an attempt to discern the mechanistic basis for isoprene's thermoprotective role, we studied the effect of exogenous isoprene on the peroxidation, permeability, and stability of spinach thylakoids and phosphatidylcholine liposomes. Isoprene, supplied at either 18 or 21 μ1 L¹, had no effect upon the rate of liposome peroxidation in the presence of a hydroxyl radical-generating system. Isoprene also did not affect liposome peroxidation at high temperatures. Neither the proton permeability of thylakoids nor the leakage of a fluorescent probe from liposomes was influenced by exogenous isoprene, when measured at several temperatures. Isoprene did not affect the stability of thylakoid membrane proteins during a temperature increase, as shown by differential scanning calorimetry. Therefore, despite the use of a variety of techniques to investigate fundamental membrane parameters, we were unable to demonstrate an effect of isoprene.