High-light stress does not impair biomass accumulation of sun-acclimated tropical tree seedlings (Calophyllum longifolium Willd. and Tectona grandis L. f.).

Studies with seedlings of tropical rainforest trees ( Calophyllum longifolium Willd.; Tectona grandis L. f.) were designed to test whether high-light stress affects photosynthetic performance and growth. Seedlings were cultivated in pots at a field site in Central Panama (9 degrees N) and separated into two groups: (1) plants exposed to full solar radiation; (2) plants subjected to automatic neutral shading (48 %) whenever visible irradiance surpassed 1000, 1200, or 1600 micromol photons m-2 s-1. After 2-4 months, chlorophyll fluorescence (Fv/Fm ratio), photosynthetic net CO2 uptake, pigment composition, alpha-tocopherol content of leaves, and plant biomass accumulation were measured. Fully sun-exposed, compared to periodically shaded plants, experienced substantial high-light stress around midday, indicated by photoinhibition of photosystem II and depressed net CO2 uptake. Higher contents of xanthophyll cycle pigments, lutein, and alpha-tocopherol showed an enhancement of photoprotection in fully sun-exposed plants. However, in all experiments, the maximum capacity of net CO2 uptake and plant dry mass did not differ significantly between the two treatments. Thus, in these experiments, high-light stress did not impair productivity of the seedlings studied. Obviously, the continuously sun-exposed plants were capable of fully compensating for any potential costs associated with photoinhibition and repair of photosystem II, reduced CO2 assimilation, and processes of high-light acclimation.

A new flux-orientated concept to derive critical levels for ozone to protect vegetation.

The current European critical levels for ozone (O3) to protect crops, natural and semi-natural vegetation and forest trees are based on a relative small number of open-top chamber experiments with a very limited number of plant species. Therefore, the working group "Effects of Ozone on Plants" of the Commission on Air Pollution Prevention of the Association of German Engineers and the German Institute of Standardization reanalysed the literature on O3 effects on European plant species published between 1989 and 1999. An exposure-response relationship for wild plant species and agricultural crops could be derived from 30 experiments with more than 30 species and 90 data points; the relationship for conifer and deciduous trees is based on 20 experiments with nine species and 50 data points. From these relationships maximum O3 concentrations for different risk stages are deduced, below which the vegetation type is protected on the basis of the respective criteria. Because it is assumed that the fumigation concentrations reflect the O3 concentrations at the top of the canopy, i.e. the upper surface boundary of the quasi-laminar layer if the micrometeorological big-leaf approach is applied, the application of these maximum O3 concentrations requires the transformation of O3 concentrations measured at a reference height above the canopy to the effective phytotoxic concentrations at the top of the canopy. Thus, the approach described in this paper is a synthesis of the classical concept of toxicology of air pollutants (critical concentrations) and the more toxicological relevant dose concept.

Physiological changes on agricultural crops induced by different ambient ozone exposure regimes: I. Effects on photosynthesis and assimilate allocation in spring wheat.

Spring wheat (Triticum aeslivum cv. Nandu) cultivated under glasshouse conditions was exposed to ozone in large fumigation chambers for 2 wk. Different exposure regimes were applied as constant concentrations as well as with ozone peaks, partly under equal dose-conditions, in times of high solar radiation during different stages of development (seedling, late tillering, anthesis). Chlorophyll fluorescence was monitored and amounts of carbohydrates (hexoses, sucrose, starch) and chlorophyll were measured in young leaves (seedling) and flag leaves (late tillering, anthesis) during and after ozone exposure. Although seedlings showed no significant response in photosynthesis, strong effects on photosynthesis and carbohydrate accumulation were measured when plants were fumigated during anthesis, especially after a heat stress period preceding ozone treatments. Under equal dose conditions chlorophyll fluorescence parameters (Fv :Fm ) and electron transport rate decreased and sucrose content of flag leaves increased significantly if ozone at a concentration of 220 µg m-3 was supplied for 4 h, indicating that peak concentrations show stronger effects than constant concentrations. The reaction of wheat plants is dependent on environmental conditions such as preceding heat stress and on the developmental stage during exposure. The results favour the hypothesis that photoinhibition and disturbance of photosynthesis are only secondary effects as a consequence of retarded sucrose export from the leaf, because of damage at the plasma membrane.

The effects of low temperature acclimation and photoinhibitory treatments on Photosystem 2 studied by thermoluminescence and fluorescence decay kinetics.

The effects of low temperature acclimation and photoinhibitory treatment on Photosystem 2 (PS 2) have been studied by thermoluminescence and chlorophyll fluorescence decay kinetics after a single turnover saturating flash. A comparison of unhardened and hardened leaves showed that, in the hardened case, a decrease in overall and B-band thermoluminescence emissions occurred, indicating the presence of fewer active PS 2 reaction centers. A modification in the form of the B-band emission was also observed and is attributed to a decrease in the apparent activation energy of recombination in the hardened leaves. The acclimated leaves also produced slower QA (-) reoxidation kinetics as judged from the chlorophyll fluorescence decay kinetics. This change was mainly seen in an increased lifetime of the slow reoxidation component with only a small increase in its amplitude. Similar changes in both thermoluminescence and fluorescence decay kinetics were observed when unhardened leaves were given a high light photoinhibitory treatment at 4°C, whereas the hardened leaves were affected to a much lesser extent by a similar treatment. These results suggest that the acclimated plants undergo photoinhibition at 4°C even at low light intensities and that a subsequent high light treatment produces only a small additive photoinhibitory effect. Furthermore, it can be seen that photoinhibition eventually gives rise to PS 2 reaction centers which are no longer functional and which do not produce thermoluminescence or variable chlorophyll fluorescence.

Oxygen dependence of photoinhibition at low temperature in intact protoplasts of Valerianella locusta L.

Photoinhibition of photosynthesis in vivo is shown to be considerably promoted by O2 under circumstances where energy turnover by photorespiration and photosynthetic carbon metabolism are low. Intact protoplasts of Valerianella locusta L. were photoinhibited by 30 min irradiation with 3000 µmol photons · m(-2) · s(-1) at 4° C in saturating [CO2] at different oxygen concentrations, corresponding to 2-40% O2 in air. The photoinhibition of light-limited CO2-dependent photosynthetic O2 evolution increased with increasing oxygen concentration. The uncoupled photochemical activity of photosystem II, measured in the presence of the electron acceptor 1,4-benzoquinone, and maximum variable fluorescence, Fv, were strongly affected and this inhibition was closely correlated to the O2 concentration. The effect of O2 did not saturate at the highest concentrations applied. An increase in photoinhibitory fluorescence quenching with [O2], although less pronounced than in protoplasts, was also observed with intact leaves irradiated at 4° C in air. Initial fluorescence, Fo, was slightly (about 10%) increased by the inhibitory treatments but not influenced by [O2]. A long-term cold acclimation of the plants did not substantially alter the O2-sensitivity of the protoplasts under the high-light treatment. From these experiments we conclude that oxygen is involved in the photoinactivation of photosystem II by excess light in vivo.

A simple model relating photoinhibitory fluorescence quenching in chloroplasts to a population of altered Photosystem II reaction centers.

A model is presented describing the relationship between chlorophyll fluorescence quenching and photoinhibition of Photosystem (PS) II-dependent electron transport in chloroplasts. The model is based on the hypothesis that excess light creates a population of inhibited PS II units in the thylakoids. Those units are supposed to posses photochemically inactive reaction centers which convert excitation energy to heat and thereby quench variable fluorescence. If predominant photoinhibition of PS IIα and cooperativity in energy transfer between inhibited and active units are presumed, a quasi-linear correlation between PS II activity and the ratio of variable to maximum fluorescence, FVFM, is obtained. However, the simulation does not result in an inherent linearity of the relationship between quantum yield of PS II and FVFM ratio. The model is used to fit experimental data on photoinhibited isolated chloroplasts. Results are discussed in view of current hypotheses of photoinhibition.

Photoinhibition at chilling temperatures and effects of freezing stress on cold acclimated spinach leaves in the field. A fluorescence study.

The role of high light stress in a natural environment was studied on spinach plants (Spinacia oleracea L. cv. Wolter) grown in the field during the winter season. Fluorescence induction (at 293 K and 77 K) of leaves was used to characterize the stress effects. Night frost with minimum temperatures between - 1.5°C and -7.5°C (i.e. above the'frost killing point'at ca. -11.5°C) led to impaired photosynthesis. This was seen as increased initial fluorescence (F(o) ), decreased ratio of variable to maximum fluorescence (F(V) /F(M) ) and lowered rates of O(2) evolution. The freezing injury was reversible within several frostless days. Exposure to high light (about 900 mol m(-2) s(-1) ) at chilling temperatures in the field caused photoinhibition, manifested as decreased variable fluorescence (F(V) ) and F(V) /F(M) ratio without changes in F(O) . The photoinhibitory fluorescence quenching was not stronger after frost than after frostless nights; synergism between light stress and preceding freezing stress was not observed. Fluorescence induction signals at 77 K showed that F(V) of photosystems I and II decreased to the same extent, indicating increased thermal deactivation of excited chlorophyll. Photoinhibition was fully reversible at +4°C within 1 h in low light, but only partially in moderate light. Preceding night frosts did not affect the recovery. The photoinhibition observed here is regarded as a protective system of thermal dissipation of excess light energy.

Reversible photoinhibition of unhardened and cold-acclimated spinach leaves at chilling temperatures.

The photoinhibition of photosynthesis at chilling temperatures was investigated in cold-acclimated and unhardened (acclimated to +18° C) spinach (Spinacia oleracea L.) leaves. In unhardened leaves, reversible photoinhibition caused by exposure to moderate light at +4° C was based on reduced activity of photosystem (PS) II. This is shown by determination of quantum yield and capacity of electron transport in thylakoids isolated subsequent to photoinhibition and recovery treatments. The activity of PSII declined to approximately the same extent as the quantum yield of photosynthesis of photoinhibited leaves whereas PSI activity was only marginally affected. Leaves from plants acclimated to cold either in the field or in a growth chamber (+1° C), were considerably less susceptible to the light treatment. Only relatively high light levels led to photoinhibition, characterized by quenching of variable chlorophyll a fluorescence (FV) and slight inhibition of PSII-driven electron transport. Fluorescence data obtained at 77 K indicated that the photoinhibition of cold-acclimated leaves (like that of the unhardened ones) was related to increased thermal energy dissipation. But in contrast to the unhardened leaves, 77 K fluorescence of cold-acclimated leaves did not reveal a relative increase of PSI excitation. High-light-treated, cold-acclimated leaves showed increased rates of dark respiration and a higher light compensation point. The photoinhibitory fluorescence quenching was fully reversible in low light levels both at +18° C and +4° C; the recovery was much faster than in unhardened leaves. Reversible photoinhibition is discussed as a protective mechanism against excess light based on transformation of PSII reaction centers to fluorescence quenchers.

Chernoff-Flury faces: a statistical means for representing multivariate response parameters of air pollution induced injury on plants.

To evaluate the effects of dry and wet deposition on forest trees (Picea abies [L.] Karst.), the LIS-Essen is operating an Open-Top Chamber Field Station within an area where novel forest decline has been prevalent since 1982. Chambers are ventilated with either ambient or charcoal-filtered air and receive either natural or artificial rain, the latter being prepared by natural rain and distilled water in ratio 1:10. Besides deposition data, acquired above and below the tree crowns as well as via lysimeters of soil percolates, various parameters describing vitality of trees are measured. To obtain a persuading representation of total parameters and their interdependencies, a multivariate graphical cluster analysis has been performed by use of Chernoff-Flury faces. Interdependencies of vitality parameters are more easily recognizable in this multivariate picture than in usually applied binary correlation diagrams.

Photoinhibition at chilling temperature : Fluorescence characteristics of unhardened and cold-acclimated spinach leaves.

The effects of moderate light at chilling temperature on the photosynthesis of unhardened (acclimated to +18° C) and hardened (cold-acclimated) spinach (Spinacea oleracea L.) leaves were studied by means of fluorescence-induction measurements at 20° C and 77K and by determination of quantum yield of O2 evolution. Exposure to 550 µmol photons·m(-2)·s(-1) at +4° C induced a strong photoinhibition in the unhardened leaves within a few hours. Photoinhibition manifested by a decline in quantum yield was characterized by an increase in initial fluorescence (F o) and a decrease in variable fluorescence (F v) and in the ratio of variable to maximum fluorescence (F V/F M), both at 77K and 20° C. The decline in quantum yield was more closely related to the decrease in the F V/F M ratio measured at 20° C, as compared with F V/F M at 77K. Quenching of the variable fluorescence of photosystem II was accompanied by a decline in photosystem-I fluorescence at 77K, indicating increased thermal de-excitation of pigments as the main consequence of the light treatment. All these changes detected in fluorescence parameters as well as in the quantum yield of O2 evolution were fully reversible within 1-3 h at a higher temperature in low light. The fast recovery led us to the view that this photoinhibition represents a regulatory mechanism protecting the photosynthetic apparatus from the adverse effects of excess light by increasing thermal energy dissipation. Long-term cold acclimation probably enforces other protective mechanisms, as the hardened leaves were insensitive to the same light treatment that induced strong inhibition of photosynthesis in unhardened leaves.

Ozone-induced nitrate formation in needles and leaves of Picea abies, Fagus sylvatica and Quercus robur.

Much attention has been paid to ozone as a major cause of novel forest decline in Europe. In combination with acidic mist, O(3) has been observed to increase ion leaching. Besides cations lake Mg(2+), Ca(2+), K(+), NH(4)(+), considerable amounts of nitrate were found to be leached by acidic mist from needles of Norway spruce. Controlled fumigation experiments, with 100, 300, and 600 microg O(3)m(-3) over 22 days continuously, have led to a nitrate accumulation of 94.1 +/- 14.8, 119.4 +/- 28.7 and 198.9 +/- 14.9 microg NO(3)(-1) g(-1) FW, respectively, in leaves of Quercus robur. Similar values were found in leaves of Fagus sylvatica and current and previous year needles of Picea abies. Nitrate levels of controls receiving charcoal filtered air were well below 40 microg NO(3)(-) g (-1) FW. Statistically significant elevated nitrate levels were observed after only 48 h of continuous fumigation with 600 microg O(3)m(-3), in all tree species tested, and after 144 h in the 100 microg O(3)m(-3) treatment. In another experiment, trees of Picea abies were kept in two charcoal (C) and two Purafil plus charcoal (P/C) ventilated chambers, and fumigated with O and 500 microg O(3)m(-3) in cabinets of each filter-type in order to eliminate NO(x) from chamber air. After 29 days of continuous ozone fumigation, NO(3)(-) accumulation in needles amounted to 102.0 +/- 37.7 and 137.4 +/- 40.5 microg g(-1) FW in P/C and C-filtered chambers, respectively. Nitrate contents of controls were below 30 microg NO(3)(-)g(-1) FW at the end of the experiment. No significant differences in NO(3)(-) accumulation between filter treatments were observed. Since NO(x) was reduced by more than 95% in the Purafil/charcoal versus the charcoal treatment, NO(3)(-) accumulation in needles can be attributed predominantly to the influence of ozone and not to direct NO(2) uptake of needles by the possible oxidation of NO to NO(2) in the presence of ozone.

Effects of freezing on plant mesophyll cells.

Freezing and thawing of leaves of herbaceous plants leads to damage when the freezing temperature falls below a certain tolerance limit, which depends on the plant species and state of acclimation. Such damage is expressed as an irreversible inhibition of photosynthesis observed after thawing. In frost-damaged leaves the capacity of photosynthetic reactions of the thylakoid membranes is impaired. Particularly, the water-oxidation system, photosystems II and I are inhibited. However, it appears that CO2 assimilation is more readily affected by freezing stress than the activity of the thylakoids. The inhibition of CO2 fixation seen in initial stages of damage seems to be independent of thylakoid inactivation. This can be shown by chlorophyll fluorescence analysis made simultaneously with measurement of CO2 assimilation. Fluorescence emission by leaves is strongly influenced by carbon assimilation activity, namely via the redox state of the photosystem II electron acceptor QA (QA-dependent quenching) and via energization of the thylakoid membranes depending on the transthylakoid proton gradient (energy-dependent quenching). Resolution of these components of fluorescence changes provides insight into alterations of the CO2 fixing capacity of the chloroplasts and properties of the thylakoids. The effects of freezing and thawing were studied in detail with isolated mesophyll protoplasts prepared from both non-hardened and cold-acclimated plants of Valerianella locusta L. Freezing damage was characterized by various parameters such as plasma membrane integrity, photosynthetic CO2 assimilation, chlorophyll fluorescence emission and activities of thylakoids isolated from the protoplasts. All tests indicated a substantially increased frost tolerance of protoplasts obtained from cold-acclimated as compared to non-hardened leaves. CO2 assimilation and related fluorescence changes were the most freezing-sensitive parameters in both types of protoplasts. Inactivation of CO2 assimilation was correlated neither to the disintegration of the plasma membrane nor to inactivation of the thylakoids. Experimental data indicate that freeze-thaw treatment affected the light-regulated enzymes of the carbon reduction cycle, such as fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-1,5-bisphosphate carboxylase. Inhibition of light-activation of these enzymes may be based on altered properties of the chloroplast envelope.

Inactivation of the photosynthetic carbon reduction cycle in isolated mesophyll protoplasts subjected to freezing stress.

Isolated mesophyll protoplasts from Valerianella locusta L. were subjected to freeze-thaw cycles. Subsequently, steady-state pool sizes of (14)C-labeled intermediates of the photosynthetic carbon reduction cycle were determined by high performance liquid chromatography. Protoplasts in which CO2 fixation was inhibited by preceding freezing stress, showed a strong increase in the proportion of fructose-1,6-bisphosphate, sedoheptulose-1,7-bisphosphate and triose phosphates. These results indicate an inhibition of the activities of stromal fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase. Furthermore, freezing stress caused a slight increase in the proportion of labeled ribulose-1,5-bisphosphate, which may be based on an inhibition or ribulose bisphosphate carboxylase activity. It was shown earlier (Rumich-Bayer and Krause 1986) that freezing-thawing readily affects photosynthetic CO2 assimilation independently of thylakoid inactivation. The present results are interpreted in terms of an inhibition of the light-activation system of the photosynthetic carbon reduction cycle, caused by freezing stress.

Simulation of in situ freezing damage of the photosynthetic apparatus by freezing in vitro of thylakoids suspended in complex media.

Chloroplast thylakoid membranes were isolated from leaves of unhardened and cold-acclimated spinach (Spinacia oleracea L.). For freezethaw treatment, the membranes were suspended in complex media composed to simulate the solute concentrations in the chloroplast stroma in the unhardened and hardened states of the leaves. In particular, high concentrations of amino acids were applied for simulating the hardened state. After frost treatment, photosynthetic activities and chlorophyll fluorescence parameters of the thylakoids were tested to determine the degree of freezing damage. The results revealed a pattern of freezing injury similar to that observed upon frost treatment of thylakoids in situ. A major manifestation of damage was the inhibition of photosynthetic electron transport. Uncoupling of photophosphorylation, which is the dominating effect of freezing of thylakoids suspended in binary solutions (e.g., containing one sugar and one inorganic salt), was also visible but less pronounced in the complex media. Thylakoids obtained from cold-acclimated leaves did not exhibit an increased frost tolerance in vitro, as compared with thylakoids from unhardened plants. The results, furthermore, indicated a strong protective effect of free amino acids at the concentrations and composition found in chloroplasts of hardened leaves. The presence of inorganic salts in the complex media slightly stabilized rather than damaged the membranes during freezing. It is concluded that inactivation of thylakoids in situ may be understood as the destabilizing action of the combined solutes surrounding the thylakoids, occurring when solute concentration is raised due to freezing of water.

Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L.

Mesophyll protoplasts were isolated from unhardened and cold-acclimated leaves of Valerianella locusta L. and subjected to freeze-thaw treatment. To evaluate the extent and course of freezing injury, photosynthetic reactions of whole protoplasts and of free thylakoid membranes, liberated from protoplasts by osmotic lysis, were measured. In addition, the integrity of the protoplasts was determined by microscopy. The results reveal an increased frost tolerance of protoplasts isolated from acclimated leaves with respect to all parameters measured. CO2-dependent O2 evolution (representing net photosynthetic CO2 fixation of protoplasts) was the most freezing-sensitive reaction; its inhibition due to freeze-thaw treatment of protoplasts was neither correlated with disintegration of the plasma membrane, nor was it initiated by inactivation of the thylakoid membranes. The frost-induced decline of protoplast integrity was not closely correlated to thylakoid damage either. Freezing injury of the thylakoid membranes was manifested by inhibition of photosynthetic electron transport and photophosphorylation. Both photosystems were affected by freezing and thawing with strongest inhibition occurring in the water-oxidation system or at the oxidizing site of photosystem II. Photophosphorylation responded more sensitively to freezing stress than electron transport, although uncoupling (increased permeability of the thylakoid membranes to protons) was not a conspicuous effect. The data are discussed in relation to freezing injury in leaves and seem to indicate that frost damage in vivo is initiated at multiple sites.

Photoinhibition of photosynthesis under anaerobic conditions studied with leaves and chloroplasts of Spinacia oleracea L.

The role of oxygen in the photoinactivation of the photosynthetic apparatus of Spinacia oleracea L. was investigated. Moderate irradiation (1200 µmol photons m(-2)s(-1)) of spinach leaves in an atmosphere of pure nitrogen caused strong inhibition of subsequently measured net CO2 assimilation, whereas considerably less photoinhibition was observed in the presence of low partial pressures (10-20 mbar) of O2. The decrease in activity caused by anaerobiosis in the light was not based on stomatal closure; the decline of assimilation represents a photoinhibition, as activity was not impaired by low irradiation (80 µmol photos m(-2)s(-1)). In contrast, gassing with pure N2 in the dark caused strong inhibition. Electron-transport rates and chlorophyll-fluorescence data of thylakoids isolated from photoinhibited leaves indicated damage to the electron-transport system, in particular to photosystem II reaction centers. In vitro, photoinhibition in isolated thylakoid membranes was also strongly promoted by anaerobiosis. Photoinhibition of electron-transport rates under anaerobic conditions was characterized by a pronounced increase in the initial fluorescence level, F0, of chlorophyll-fluorescence induction, in contrast to photoinhibition under aerobic conditions. The results are discussed in terms of two mechanisms of photoinhibition, one that is suppressed and a second that is promoted by oxygen.

Inhibition of photosynthetic reactions by light : A study with isolated spinach chloroplasts.

Illumination of isolated intact chloroplasts of Spinacia oleracea L. for 10 min with 850 W m(-2) red light in the absence of substrate levels of bicarbonate caused severe inhibition of subsequently measured photosynthetic activities. The capacity of CO2-dependent O2 evolution and of non-cyclic electron transport were impaired to similar degrees. This photoinactivation was prevented by addition of bicarbonate which allowed normal carbon metabolism to proceed during preillumination. Photoinhibition of electron transport was observed likewise upon illumination of intact or broken chloroplasts when efficient electron acceptors were absent. Addition of uncouplers did not influence the extent of inhibition. Studies of partial electron-transport reactions indicated that the activity of both photosystems was affected by light. In addition, the water-oxidation system or its connection to photosystem II seemed to be impaired. Preillumination did not cause uncoupling of photophosphorylation. Chlorophyll-fluorescence data obtained at room temperature and at 77 K are consistent with the view that photosystem-II reaction centers were altered. Addition of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) or 1,4-diazabicyclo(2,2,2)octane to isolated thylakoids prior to preillumination substantially diminished photoinhibition. This result shows that reactive oxygen species were involved in the damage. It is concluded that bright light, which normally does not damage the photosynthetic apparatus, may exert the described destructive effects under conditions that restrict metabolic turnover of photosynthetic energy.

Freezing injury in cold-acclimated and unhardened spinach leaves : I. Photosynthetic reactions of thylakoids isolated from frost-damaged leaves.

Spinach plants (Spinacia oleracea L.) were frost-hardened by cold-acclimation to 1° C or kept in an unhardy state at 20°/14° C in phytotrons. Detached leaves were exposed to temperatures below 0°C. Rates of photosynthetic CO2 uptake by the leaves, recorded after frost treatment, served as a measure of freezing injury. Thylakoid membranes were isolated from frost-injured leaves and their photosynthetic activities tested. Ice formation occurred at about-4° to-5° C, both in unhardened and cold-acclimated leaves. After thawing, unhardened leaves appeared severely damaged when they had been exposed to-5° to-8° C. Acclimated leaves were damaged by freezing at temperatures between-10° to-14° C. The pattern of freezing damage was complex and appeared to be identical in hardened and unhardened leaves: 1. Inactivation of photosynthesis and respiration of the leaves occurred almost simultaneously. 2. When the leaves were partly damaged, the rates of photosynthetic electron transport and noncyclic photophosphorylation and the extent of light-induced H(+) uptake by the isolated thylakoids were lowered at about the same degree. The dark decay of the proton gradient was, however, not stimulated, indicating that the permeability of the membrane to-ward protons and metal cations had not increased. 3. As shown by partial reactions of the electron transport system, freezing of leaves predominantly inhibited the oxygen evolution, but photosystem II and photosystem I-dependent electron transport were also impaired. 4. Damage of the chloroplast envelope was indicated by a decline in the percentage of intact chloroplasts found in preparations from injured leaves. The results are discussed in relation to earlier studies on freezing damage of thylakoid membranes occurring in vitro.

Freezing injury in cold-acclimated and unhardened spinach leaves : II. Effects of freezing on chlorophyll fluorescence and light scattering reactions.

Leaves from cold-acclimated and from unhardened spinach plants (Spinacia oleracea L.) were subjected to a freezing/thawing procedure in which varying minimum temperatures were reached. Subsequently, the chlorophyll fluorescence induction signal (Kautsky phenomenon) and the light-induced apparent absorbance changes at 535 nm (light-scattering changes indicative of the proton gradient, and absorbance changes induced by the membrane potential) of the leaves were studied to obtain information on the course and mechanism of frost damage to the photosynthetic apparatus. Membrane energization as indicated by these signals was related in a complex way to the inactivation of CO2 assimilation due to the progressing impact of freezing: In the absence of CO2, the maximum energization of the thylakoids was progressively decreased. According to altered fluorescence signals, the electron transport system was affected in parallel. In the presence of CO2, energization frequently appeared increased when the leaves had been partially damaged, i.e., when the CO2 assimilation rates were lowered. The results suggest that the primary frost injury in chloroplasts of intact leaves consists of an inhibition of the energy conserving photosynthetic processes and, in addition, of a partial inactivation of the carbon reduction cycle. The pattern of freezing injury was no different in frost-hardened and unhardened leaves.