Light-dependent inhibition of photosynthetic electron transport by zinc

The effects of zinc concentrations up to 400 μ M were examined on three photosynthetic electron transport reactions of thylakoids isolated from Pisum sativum L. cv. Meteor. Zinc (400 μ M ) had no effect on photosystem I mediated electron transport from reduced N,N,N',N'-tetramethyl- p -phenylenediamine to methyl viologen, but inhibited uncoupled electron flow from water to methyl viologen by ca 50% and to 2,6-dichlorophenol-indophenol (DCPIP) by ca 30% at saturating light levels. Zinc inhibition of DCPIP photoreduction was independent of the light intensity to which thylakoids were exposed. Decreasing the photon flux density below 400 μmol m⁻² s⁻¹ produced a logarithmic reduction in the zinc-induced inhibition of methyl viologen photoceduction; a stimulation of this reaction was observed below 80 μmol photons m⁻² s⁻¹. Increasing light intensity decreased the amount of zinc tightly bound to the thylakoid membranes, but increased the weakly associated zinc which could be removed by washing the membranes with buffer containing Mg². The results suggest that zinc acts on the photosynthetic electron transport system at two sites. Site 1 is on the oxidizing side of photosystem 2 and the inhibition by zinc is independent of the light intensity. Site 2 is between photosystems 1 and 2 and the electron flow can be positively or negatively affected by zinc depending on the light intensity.     

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.     

Chlorophyll fluorescence and photoacoustic characteristics in relationship to changes in chlorophyll and Ca2+ content of a Cu-tolerant Silene compacta ecotype under Cu treatment

The effect of Cu toxicity on photosynthetic function, chlorophyll and Ca²⁺ content of Cu-tolerant Silene compacta plants grown in nutrient solution was studied. Since, in plants grown under 8 μ M Cu, the chlorophyll and Ca²⁺ concentration as well as the photosystem II (PSII) photochemistry were increased, compared to the control, the development of an adaptive mechanism of the Cu-tolerant ecotype of S. compacta to 8 μ M Cu is suggested. Increased Cu tolerance of the S. compacta ecotype reflects modulation of the photosynthetic apparatus to optimize photosynthesis. However, exposure of plants to 160 μ M Cu resulted in a marked increase of the fraction of closed PSII centres and decreased quantum yield of PSII electron transport (Φ_PSU) which was accompanied by a significant decline of relative quantum yield for O₂ evolution (A_ox/A_pt). The concentration of chlorophyll and Ca²⁺ in leaves also decreased significantly under 160 μ M Cu treatment. Photochemical quenching (q_p) displayed a reduction as a result of perturbation of the photosynthetic electron transfer chain, while non-photochemical quenching (q_N) increased. High Cu treatment reduced photosynthetic productivity of S. compacta plants which can be attributed, in part, to pertubation of photosynthetic process and photosynthetic pigments as well as to Ca²⁺ loss.     

The composition and function of thylakoid membranes from pea plants grown under white or green light with or without far-red light

Pea plants ( Pisum sativum L. ev. Greenfeast) were grown for 2 to 3 weeks in while (˜ 50 μmol photons m⁻² s⁻¹; 400–700 nm) or green (˜ 30 μmol photons m⁻² s ⁻¹ 400–700 nm) light (16 h day/8 h night), with or without far-red light. Supplementary far-red light decreased leaf area and increased internodal length in both white and green light, demonstrating that phytochrome influenced leaf size and plant growth. However, there was no effect of far-red light on chlorophyll a /chlorophyll b ratios, chlorophyll-protein composition, the stoichiometry of electron transport complexes or photosynthetic function of isolated thylakoids. These results suggest that phytochrome is ineffective in modulating the composition and function of thylakoids in pea plants grown at low irradiance. One possible explanation of the ineffectiveness of phytochrome on thylakoids is discussed in terms of the drastic attenuation of red relative to far-red light in green tissue.     

Effects of copper on active and passive Rb+ influx in roots of winter wheat

The effects of copper (CuCl₂) on active and passive Rb⁺(⁸⁶Rb⁺) influx in roots of winter wheat grown in water culture for 1 week were studied. External copper concentrations in the range of 10–500 μ M in the uptake nutrient solution reduced active Rb⁺ influx by 20–70%, while passive influx was unaffected (ca 10% of the Rb⁺ influx in the Cu-free solution). At external Rb⁺ concentrations of up to 1 m M , Cu exposure (50 μ M decreased V_max to less than half and increased K_m to twice the value of the control. Short Cu exposure reduced the K⁺ concentration in roots of low K⁺ status. Pretreatment for 5 min in 50 μ M CuCl₂ prior to uptake experiments reduced Rb⁺ influx by 26%. After 60 min pretreatment with Cu, the corresponding reduction was 63%. Cu in the cultivation solution impeded growth, especially of the roots. The Cu concentration in the roots increased linearly with external Cu concentration (0–100 μ M ) while Cu concentration in the shoots was relatively unchanged. The K⁺ concentration in both roots and shoots decreased significantly with increased Cu in the cultivation solutions. Possible effects of Cu on membranes and ion transport mechanisms are discussed.     

Photosynthesis, growth and structural characteristics of holm oak resprouts originated from plants grown under elevated CO2

The physiological characteristics of holm oak ( Quercus ilex L.) resprouts originated from plants grown under current CO₂ concentration (350 μl l⁻¹) (A-resprouts) were compared with those of resprouts originated from plants grown under elevated CO₂ (750 μl l⁻¹) (E-resprouts). At their respective CO₂ growth concentration, no differences were observed in photosynthesis and chlorophyll fluorescence parameters between the two kinds of resprout. E-resprouts appeared earlier and showed lower stomatal conductance, higher water-use efficiency and increased growth (higher leaf, stem and root biomass and increased height). Analyses of leaf chemical composition showed the effect of elevated [CO₂] on structural polysaccharide (higher cellulose content), but no accumulation of total non-structural carbohydrate on area or dry weight basis was seen. Four months after appearance, downregulation of photosynthesis and electron transport components was observed in E-resprouts: lower photosynthetic capacity, photosystem II quantum efficiency, photochemical quenching of fluorescence and relative electron transport rate. Reduction in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) activity, deduced from the maximum carboxylation velocity of RuBisCo, accounts for the observed acclimation. Increased susceptibility of photosynthetic apparatus to increasing irradiance was detected in E-resprouts.     

Physiological and structural responses of the cyanobacterium Anabaena cylindrica to aluminium

When adding aluminium (3.7–370 μ M ) as AlCl₃–6H₂0 to cultures of the nitrogen-fixing cyanobacterium Anabaena cylindrica , strain 1403/2a (CCAP), the following responses were observed: The effects of aluminium were dependent on pH. being most drastic at pH 6.0. At this pH the growth of A. cylindrica was significantly reduced by 3.7 μ M aluminium and completely inhibited by 370 μ M . The content of chlorophyll a and phycocyanin decreased after treatment with aluminium. Also, aluminium lowered the rates of both CO₂-fixation and N₂-fixation with total inhibition of both processes by 370 μ M . At the lower concentrations used the nitrogenase activity started to recover after about 100 h. The aluminium content in the cells increased with increasing concentration and with time. At 190 μ M the aluminium concentration in the cells represented 2.4 and 3.3% of the dry weight after 6 and 24 h, respectively. Clogging of filaments and lysis of vegetative cells were apparent at higher aluminium concentrations while the frequency of heterocysts increased in all concentrations used. The most pronounced ultrastructural changes included accumulation of cyanophycin granules and degradation of the thylakoids. The ultrastructure of the heterocysts was however not affected. It is concluded that major reasons for the toxicity are interactions with membranes and phosphate deficiency.     

Kinetics of leaf oxygen uptake represent in planta activities of respiratory electron transport and terminal oxidases

We present, for the first time, the oxygen response kinetics of mitochondrial respiration measured in intact leaves (sunflower and aspen). Low O₂ concentrations in N₂ (9–1500 ppm) were preset in a flow-through gas exchange measurement system, and the decrease in O₂ concentration and the increase in CO₂ concentration as result of leaf respiration were measured by a zirconium cell O₂ analyser and infrared-absorption CO₂ analyser, respectively. The low O₂ concentrations little influenced the rate of CO₂ evolution during the 60-s exposure. The initial slope of the O₂ uptake curve on the dissolved O₂ concentration basis was relatively constant in leaves of a single species, 1.5 mm s⁻¹ in sunflower and 1.8 mm s⁻¹ in aspen. The apparent K _0.5(O₂) values ranged from 0.33 to 0.67 μ M in sunflower and from 0.33 to 1.1 μ M in aspen, mainly because of the variation of the maximum rate, V _max (leaf temperature 22°C). The initial slope of the O₂ response of respiration characterizes the catalytic efficiency of terminal oxidases, an important parameter of the respiratory machinery in leaves. The plateau of the response characterizes the activity of the mitochondrial electron transport chain and is subject to regulations in accordance with the necessity for ATP production. The relatively low oxygen conductivity of terminal oxidases means that in leaves, less than 10% of the photosynthetic oxygen can be reassimilated by mitochondria.     

Mechanism of copper-enhanced photoinhibition in thylakoid membranes

The effect of copper on photoinhibition of photosystem II (PSII) in vitro was studied in bean ( Phaseolus vulgaris L. cv. Dufrix) and pumpkin ( Cucurbita pepo L.) thylakoids. The thylakoids were illuminated at 200–2 000 μmol photons m⁻² s⁻¹ in the presence of 70–1 830 added Cu²⁺ ions per PSII. Three lines of evidence show that the irreversible damage of PSII caused by illumination of thylakoids in the presence of Cu²⁺ was mainly due to donor-side photoinhibition resulting from inhibition of the PSII donor side by Cu²⁺. First, addition of an artificial electron donor partially restored PSII activity of thylakoids that had been illuminated in the presence of Cu²⁺. Second, already moderate light was enough to cause rapid inhibition of PSII, and the inhibition could be saturated by light. Third, the extrinsic polypeptides of the oxygen-evolving complex were found to become oxidized by the combined effect of Cu²⁺ and light. The presence of oxygen was not necessary for the copper-induced enhancement of photoinhibition of PSII. When the illumination was prolonged, copper caused a gradual collapse of the thylakoid structure by increasing degradation of thylakoid proteins.     

The inhibition of photosynthesis and photorespiration in isolated mesophyll cells of Phaseolus and Lycopersicon by reduced osmotic potentials

Mesophyll cells isolated from Phaseolus vulgaris and Lycopersicon esculentum show decreasing photosynthetic rates when suspended in media containing increasing concentrations of osmoticum. The photosynthetic activity was sensitive to small changes in osmotic potential over a range of sorbitol concentrations from 0.44 M (−1.08 MPa) to 0.77 M (−1.88 MPa). Photorespiration assayed by ¹⁴CO₂ release in CO₂-free air and by ¹⁴CO₂ release from the oxidation of [1–¹⁴C] glycolate also decreased as the osmotic potential of the incubation medium was reduced. The CO₂ compensation points of the cells increased with increasing concentration of osmoticum from approximately 60 μ I⁻¹1 at −1.08 MPa to 130 μl 1⁻¹ for cells stressed at −1.88 MPa. Changes in photosynthetic and photorespiratory activities occurred at moderate osmotic potentials in these cells suggesting that in whole leaves during a reduction in water potential, non- stomatal inhibition of CO₂ assimilation and glycolate pathway metabolism occurs simultaneously with stomatal closure.     

Effects of NH+4-N and NO+3-N on growth and metabolism of Sphagnum magellanicum

Photosynthetic CO₂-fixation, chlorophyll content, growth rate and nitrate reductase activity were used to examine the influence of NH⁺₄-N and NO⁻₃-N on Sphagnum magellanicum cultivated under defined conditions in phytotrons. NO⁻₃-concentrations up to 322 μ M were found to be favourable. Increased NH⁺₄ concentrations, however, resulted in growth inhibition and decreased chlorophyll content at concentrations ≧ 255 μ M ; e.g. 600 μ M NH⁺₄ caused a 20% reduction of nitrate reductase activity and net photosynthesis. For raised bog Sphagna an improved standard nutrient solution is proposed with the following ion concentrations (μ M ): 55 Na⁺; 17 K⁺; 95 NH⁺₄; 22 Ca²⁺; 22 Mg²⁺; 2 Fe³⁺; 20 Cl⁻; 100 NO⁻₃; 57 SO^2-₄; 7.4 H₂PO⁻₄; trace elements: A-Z solution (Hoagland) 50 μl 1000 ml⁻¹; pH 5.8.     

Inhibition of electron flow around photosystem I in chloroplasts of Cd-treated maize plants is due to Cd-induced iron deficiency

Photosystem I activity of chloroplasts isolated from 21 days old maize seedlings ( Zea mays L. cv. Hidosil) cultivated in a nutrient solution containing different concentrations of Cd (10,20,30μM) was investigated. Cd markedly decreased ferredoxin(Fd)-dependent NADP⁺ photoreduction, while it had no effect on electron transport from 2. 6-dichlorophenolindophenol to methyl viologen, indicating that the metal interferred with electron transport on the reducing side of photosystem I. The decrease in electron transport correlated with a low Fd content, which in turn was correlated with a low Fe concentration, suggesting Cd-induced Fe deficiency. In in vitro experiments direct Cd inhibition of Fd-dependent NADP⁺ photoreduction required much higher Cd concentrations than those observed in Cd-treated plants.     

Uptake of salicylic acid 2-O-beta-D-glucose into soybean tonoplast vesicles by an ATP-binding cassette transporter-type mechanism

In soybean ( Glycine max L.), salicylic acid (SA) is converted primarily to SA 2- O - β - d -glucose (SAG) in the cytoplasm and then accumulates exclusively in the vacuole. However, the mechanism involved in the vacuolar transport of SAG has not been investigated. The vacuolar transport of SAG was characterized by measuring the uptake of [¹⁴C]SAG into tonoplast vesicles isolated from etiolated soybean hypocotyls. The uptake of SAG was stimulated about six-fold when MgATP was included in the assay media. In contrast, the uptake of SA was only stimulated 1.25-fold by the addition of MgATP and was 2.2-fold less than the uptake of SAG providing an indication that the vacuolar uptake of SA is promoted by glucosylation. The ATP-dependent uptake of SAG was inhibited by increasing concentrations of vanadate (64% inhibition in the presence of 500 μ M ) but was not very sensitive to inhibition by bafilomycin A₁ (a specific inhibitor of vacuolar H⁺-ATPase; EC 3.6.1.3), and dissipation of the transtonoplast H⁺-electrochemical gradient. The SAG uptake exhibited Michaelis–Menten-type saturation kinetics with a K _m value of 90 μ M for SAG. SAG uptake was inhibited 60% by β ‐estradiol 17-( β - d -glucuronide), but glutathione conjugates and uncharged glucose conjugates were only slightly inhibitory. Based on the characteristics of SAG uptake into soybean tonoplast vesicles it is likely that this uptake occurs through an ATP-binding cassette transporter-type mechanism. However, this vacuolar uptake mechanism is not universal since the uptake of SAG by red beet ( Beta vulgaris L) tonoplast vesicles appears to involve an H⁺-antiport mechanism.     

Photosynthetic and respiratory electron transport in Cu2+-deficient Dunaliella

Growth inhibition of the green alga Dunalietla parva Lerche has been observed during cultivation in low Cu²⁺ media. A minimum endogenous Cu concentration for unrestricted growth of 100 to 200 nmol ml⁻¹ packed cell volume was estimated. At lower concentrations, Cu deficiency causes a decrease in photosynthesis and respiration. Assay of photosynthetic electron transport rates as well as the determination of several redox components showed that the target of Cu deprivation in the photosynthetic apparatus is the synthesis of Cu-containing plastocyanin. Consequently, inhibited formation of plastocyanin resulted in low activities of photosynthetic electron transport. A secondary, indirect effect of Cu deficiency is the reduction of thylakoid formation resulting in an additional decrease of photosynthesis compared to cultures with sufficient Cu²⁺.
The inhibitory influence of low Cu²⁺ on respiration was located at the site of cytochrome oxidase. In contrast to blue-green algae, a strong coordination of the biosynthesis of the cytochrome oxidase complex was evident. During restricted Cu²⁺ supply the formation of cytochiome aa₃ , another component besides Cu, was stalled. The resulting low activities of cytochrome oxidase are responsible for decreased respiratory electron transfer activity from NADPH to oxygen. At Cu²⁺ concentrations which exert only moderate effects on Dunalietla , the cytochrome oxidase reaction was more strongly affected than the photosystem I reaction.     

Kinetic and proteolytic identification of heat-induced conformational changes in the urea herbicide binding site of isolated Phaseolus vulgaris chloroplast thylakoids

Kinetic parameters of 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea (DCMU)-induced inhibition of electron transport in chloroplast thylakoids isolated from Phaseolus vulgaris L. cv. Oregon 1604 were determined from analysis of a convergent, parallel electrical circuit. Through this analogue, the apparent affinity of the purported binding site for DCMU (K₁) and the relative amount of DCMU-insensitive electron transport (v^max₁/v_o) were obtained using a reiterative non-linear least squares curve-fitting procedure. Exposure of thylakoids to heat caused a gradual increase in K₁ (or decrease in the affinity of the thylakoid for DCMU) with an apparent activation energy of 134 kJ mol⁻¹. Tryptic susceptibility of a protein region regulating K₁ also decreased gradually with exposure to 45°C, suggesting that the heat-induced increase in K₁ might be due to a protein conformational change. On the other hand, thylakoid exposure to 45°C resulted in a rapid (<5 min) irreversible increase in v^max_I/v_o, which was also the apparent result of a conformational change in a region of the protein which regulates this function. These results are suggestive of the existence of differential thermal sensitivities of proteins within the thylakoids and, perhaps, of different regions within a single membrane protein.     

Effect of copper deficiency on photosynthetic electron transport in wheat plants

Copper deficiency in wheat ( Triticum aestivum L. cv. Nazareno Stramppeli) markedly affects photosynthetic activity. Flag leaves of copper-deficient plants showed a 50% reduction of the photosynthetic rate expressed as mg CO₂ dm⁻²h⁻¹. The activities of PSI and PSII, determined for isolated chloroplasts, as well as fluorescence measurements on intact leaves of copper-deficient plants, indicated a low activity of photosynthetic electron transport. Ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity was not affected by copper deficiency but copper deficiency affected the chloroplast ultrastructure, especially at the level of grana, where a disorganization of thylakoids is evident.     

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.     

Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris)

Sugar-beet plants ( Beta vulgaris L. cv. Monohill) were cultivated for 4 weeks in a complete nutrient solution. Indirect effects of cadmium were studied by adding 5, 10 or 20 μ M CdCl₂ to the culture medium while direct effects were determined by adding 1, 5, 20, 50 or 2 000 μ M CdCl₂ to the assay media. The photosynthetic properties were characterized by measurement of CO₂ fixation in intact plants, fluorescence emission by intact leaves and isolated chloroplasts, photosystem (PS) I and PSII mediated electron transport of isolated chloroplasts, and CO₂-dependent O₂ evolution by protoplasts. When directly applied to isolated leaves, protoplasts and chloroplasts. Cd²⁺ impeded CO₂ fixation without affecting the rates of electron transport of PSI or PSII or the rate of dark respiration. When Cd²⁺ was applied through the culture medium the capacity for, and the maximal quantum yield of CO₂ assimilation by intact plants both decreased. This was associated with: (1) decreased total as well as effective chlorophyll content (PSII antennae size), (2) decreased coupling of electron transport in isolated chloroplasts, (3) perturbed carbon reduction cycle as indicated by fluorescence measurements. Also, protoplasts isolated from leaves of Cd²⁺-cultivated plants showed an increased rate of dark respiration.     

Salt induced responses of chloroplast activities in species of differing salt tolerance. Photosynthetic electron transport in Aster tripolium and Pisum sativum

A comparative study was made of the effects of high concentrations of NaCl, KCl and MgCl₂ on two electron transport reactions of thylakoids isolated from a mesophyte, Pisum sativum and a halophyte, Aster tripolium . The rate of photosystem I mediated electron transport from reduced N, N, N', N'-tetramethyl- p -phenylenediamine (TMPD) to methyl viologen was determined polarographically, and photosystem II mediated electron flow from water to 2,6-dichlorophenolindophenol (DCPIP) was monitored spectrophotometrically. The response of photosystem II to increasing in vitro salt concentrations was similar for thylakoids isolated from both A. tripolium and P. sativum , but differences in the response of photosystem I to salinity changes were observed for the two species. Increasing NaCl, KCl and MgCl₂ concentrations produced similar patterns of response of photosystem I activity in P. sativum thylakoids, whilst for A. tripolium KCl induced a completely different response pattern compared to NaCl and MgCl₂. The salinity of the culture medium in which A. tripolium was grown also had an effect on both the absolute in vitro activities of photosystems I and II and their response to changes in salt concentration of the reaction media.     

The role of the chloroplast coupling factor in the inactivation of thylakoid membranes at low temperatures

Thylakoids isolated from spinach leaves ( Spinacia oleracea L. cv. Monatol) were exposed to variable low temperatures under non-freezing conditions. After incubation, changes in the activities of several photochemical reactions and physical properties of the membranes were measured at room temperature.
Cyclic photophosphorylation was strictly dependent on the temperature and the electrolyte concentration: decrease in temperature and increase in NaCl concentration enhanced membrane damage. Inactivation of photophosphorylation was accompanied by stimulation of non-cyclic electron transport, increase in proton permeability and decrease in δpH. When dicyclohexylcarbodiimide was added, the proton gradient became completely restored. The temperature- and salt-dependent breakdown of photophosporylation was closely related to the release of the chloroplast coupling factor (CF₁) from the membranes. The addition of Mg²⁺, very low concentrations of ATP or ADP, or higher concentrations of low-molecular-weight polyols prior to temperature treatment prevented thylakoid damage.
The data indicate that inactivation of photophosphorylation of thylakoids at low temperatures is determined to a considerable extent by the cold lability of the CF₁. As a consequence, it must be concluded that damage of biomembranes caused by freezing is not due solely to changes resulting from the ice formation but additionally by temperature-dependent alterations of cold-labile proteins. Moreover, the data explain the mechanism of non-colligative cryoprotection of isolated thylakoid membranes.