A rapid circadian rhythm of carbon-dioxide metabolism in Bryophyllum fedtschenkoi

Leaves of Bryophyllum fedtschenkoi Hamet et Perrier maintained in a stream of normal air and at 15° C exhibit a circadian rhythm of CO₂ uptake in continuous light but not in continuous darkness. The rhythm is unusual in that it persists for at least 10 d, and has a short period of approximately 18 h. The mechanism by which this rhythm is generated is discussed.Abbreviation PEPCase phosphoenolpyruvate carboxylase     

Persistent circadian rhythms in the phosphorylation state of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi leaves and in its sensitivity to inhibition by malate

Phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPCase) from Bryophyllum fedtschenkoi leaves has previously been shown to exist in two forms in vivo. During the night the enzyme is phosphorylated and relatively insensitive to feedback inhibition by malate whereas during the day the enzyme is dephosphorylated and more sensitive to inhibition by malate. These properties of PEPCase have now been investigated in leaves maintained under constant conditions of temperature and lighting. When leaves were maintained in continuous darkness and CO₂-free air at 15°C, PEPCase exhibited a persistent circadian rhythm of interconversion between the two forms. There was a good correlation between periods during which the leaves were fixing respiratory CO₂ and periods during which PEPCase was in the form normally observed at night. When leaves were maintained in continuous light and normal air at 15°C, starting at the end of a night or the end of a day, a circadian rhythm of net uptake of CO₂ was observed. Only when these constant conditions were applied at the end of a day was a circadian rhythm of interconversions between the two forms of PEPCase observed and the rhythms of enzyme interconversion and CO₂ uptake did not correlate in phase or period.Abbreviations CAM Crassulacean acid metabolism - FW fresh weight - PEPCase phosphoenolpyruvate carboxylase - RuBPCase ribulose-1,5-bisphosphate carboxylase To whom correspondence should be addressed.     

Control of the circadian rhythm of carbon dioxide assimilation in Bryophyllum leaves by exposure to darkness and high carbon dioxide concentrations

The circadian rhythm of CO₂ assimilation in detached leaves of Bryophyllum fedtschenkoi at 15° C in normal air and continuous illumination is inhibited both by exposure to darkness, and to an atmosphere enriched with 5% CO₂. During such exposures substantial fixation of CO₂ takes place, and the malate concentration in the cell sap increases from about 20 mM to a constant value of 40–50 mM after 16 h. On transferring the darkened leaves to light, and those exposed to 5% CO₂ to normal air, a circadian rhythm of CO₂ assimilation begins again. The phase of this rhythm is determined by the time the transfer is made since the first peak occurs about 24 h afterwards. This finding indicates that the circadian oscillator is driven to, and held at, an identical, fixed phase point in its cycle after 16 h exposure to darkness or to 5% CO₂, and it is from this phase point that oscillation begins after the inhibiting condition is removed. This fixed phase point is characterised by the leaves having acquired a high malate content. The rhythm therefore begins with a period of malate decarboxylation which lasts for about 8 h, during which time the malate content of the leaf cells must be reduced to a value that allows phosphoenolpyruvate carboxylase to become active. Inhibition of the rhythm in darkness, and on exposure to 5% CO₂ in continuous illumination, appears to be due to the presence of a high concentration of CO₂ within the leaf inhibiting malic enzyme which leads to the accumulation of high concentrations of malate in the leaf cells. The malate then allosterically inhibits phosphoenolpyruvate carboxylase upon which the rhythm depends. The results give support to the view that malate synthesis and breakdown form an integral part of the circadian oscillator in this tissue.Abbreviations B. Bryophyllum - PEPCase phosphoenolpyruvate carboxylase     

Period and phase control by temperature in the circadian rhythm of carbon dioxide fixation in illuminated leaves of Bryophyllum fedtschenkoi

The rhythm of CO₂ assimilation exhibited by leaves of Bryophyllum fedtschenkoi maintained in light and normal air occurs only at constant ambient temperatures between 10°C and 30°C. Over this range the period increases linearly with increasing temperature from the extremely low value of 15.7 h to 23.3 h, but shows a considerable degree of temperature compensation. Outside the range 10°C–30°C the rhythm is inhibited but re-starts on changing the temperature to 15°C. Prolonged exposure of leaves to high (40°C) and low (2°C) temperature inhibits the rhythm by driving the basic oscillator to fixed phase points in the cycle which differ by 180°, and which have been characterised in terms of the malate status of the leaf cells. At both temperatures loss of the circadian rhythm of CO₂ assimilation is due to the inhibition of phosphoenolpyruvate carboxylase (PEPCase) activity, but the inhibition is apparently achieved in different ways at 40°C and 2°C. High temperature appears to inhibit directly PEPCase activity, but not the activity of the enzymes responsible for the breakdown of malate, with the result that the leaf acquires a low malate status. In contrast, low temperature does not directly inhibit PEPCase activity, but does inhibit enzymes responsible for malate breakdown, so that the malate level in the leaf increases to a high value and PEPCase is eventually allosterically inhibited. The different malate status of leaves held at these two temperatures accounts for the phases of the rhythms being reversed on returning the leaves to 15°C. After exposure to high temperature, CO₂ fixation by PEPCase activity can begin immediately, whereas after exposure to low temperature, the large amount of malate accumulated in the leaves has to be decarboxylated before CO₂ fixation can begin.     

The role of temperature in the regulation of the circadian rhythm of CO2 fixation in Bryophyllum fedtschenkoi

Detached leaves of Bryophyllum fedtschenkoi Hamet et Perrier kept in normal air show a single period of net CO₂ fixation on transfer to constant darkness at temperatures in the range 0–25 °C. The duration of this initial fixation period is largely independent of temperature in the range 5–20 °C, but lengthens very markedly at temperatures below 4 °C, and is reduced at temperatures above 25 °C. The onset of net fixation of CO₂ on transfer of leaves to constant darkness is immediate at low temperatures, but is delayed as the temperature is increased. The ambient temperature also determines whether or not a circadian rhythm of CO₂ exchange occurs. The rhythm begins to appear at about 20 °C, is most evident at 30 °C and becomes less distinct at 35 °C. The occurrence of a distinct circadian rhythm in CO₂ output at 30° C in the absence of a detectable rhythm in PEPCase kinase activity shows that the kinase rhythm is not a mandatory requirement for the rhythm of PEPCase activity. However, when it occurs, the kinase rhythm undoubtedly amplifies the PEPCase rhythm.Abbreviation PEPCase phosphoenolpyruvate carboxylase We thank the Agricultural and Food Research Council for financial support for this work.     

Phase resetting of the circadian rhythm of carbon dioxide assimilation inBryophyllum leaves in relation to their malate content following brief exposure to high and low temperatures,darkness and 5% carbon dioxide

Leaves ofBryophyllum fedtschenkoi show a persistent circadian rhythm in CO₂ assimilation when kept in continuous illumination and normal air at 15°C. The induction of phase shifts in this rhythm by exposing the leaves for four hours at different times in the circadian cycle to 40° C, 2° C, darkness and 5% CO₂ have been investigated. Exposure to high temperature has no effect on the phase at the apex of the peak but is effective at all other times in the cycle, whereas exposure to low temperature, darkness or 5% CO₂ is without effect between the peaks and induces a phase shift at all other times. The next peak of the rhythm occurs 17 h after a 40° C treatment and 7–10 h after a 2° C, dark or 5% CO₂ treatment regardless of their position in the cycle. When these treatments are given at times in the cycle when they induce maximum phase shifts, they cause no change in the gross malate status of the leaf. The gross malate content of the leaf in continuous light and normal air at 15% shows a heavily damped circadian oscillation which virtually disappears by the time of the third cycle, but the CO₂ assimilation rhythm persists for many days. The generation of the rhythm, and the control of its phase by environmental factors are discussed in terms of mechanisms that involve the synthesis and metabolism of malate in specific localised pools in the cytoplasm of the leaf cells.     

Stomatal responses of Argenteum — a mutant of Pisum sativum L. with readily detachable leaf epidermis

Epidermis is easily detached from both adaxial and abaxial surfaces of leaf four of the Argenteum mutant of Pisum sativum L. The isolated epidermis has stomata with large, easily-measured pores. Hairs and glands are absent. The density of stomata is high and contamination by mesophyll cells is low. In the light and in CO₂-free air, stomata in isolated adaxial epidermis of Argenteum mutant opened maximally after 4 h incubation at 25°C. The response of stomata to light was dependent on the concentration of KCl in the incubation medium and was maximal at 50 mol m^-3 KCl. Stomata did not respond to exogenous kinetin, but apertures were reduced by incubation of epidermis on solutions containing between 10^-5 and 10^-1 mol m^-3 abscisic acid (ABA). The responses of stomata of Argenteum mutant to light, exogenous KCl, ABA and kinetin were comparable with those described previously for stomata in isolated epidermis of Commelina communis. A method for preparing viable protoplasts of guard cells from isolated epidermis of Argenteum mutant is described. The response of guard cell protoplasts to light, exogenous KCl, ABA and kinetin were similar to those of stomata in isolated epidermis except that the increase in volume of the protoplasts in response to light was maximal at a lower concentration of KCl (10 mol m^-3) and that protoplasts responded more rapidly to light than stomata in isolated epidermis. The protoplasts did not respond to exogenous kinetin, but when incubated for 1 h in the light and in CO₂-free air on a solution containing 10^-3 mol m^-3 ABA, they decreased in volume by 30%. The advantages of using epidermis from Argenteum mutant for experiments on stomatal movements are discussed.Abbreviations ABA abscisic acid - MES 2-(N-morpholino)ethanesulfonic acid     

Stomatal movement and potassium transport in epidermal strips of Zea mays: The effect of CO2

Summary The correlation between stomatal action and potassium movement in the epidermis of Zea mays was examined in isolated epidermal strips floated on distilled water. Stomatal opening in the isolated epidermis is reversible in response to alternate periods of light or darkness, and is always correlated with a shift in the potassium content of the guard cells. K accumulates in guard cells during stomatal opening, and moves from the guard cells into the subsidiary cells during rapid stomatal closure. When epidermal strips are illuminated in normal air, as against CO₂-free air, the stomata do not open and there is a virtually complete depletion of K from the stomatal apparatus. In darkness CO₂-containing air inhibits stomatal opening and K accumulation in guard cells, but does not lead to a depletion of K from the stomata as observed in the light.     

Impairment of gas exchange and structure in birch leaves (Betula pendula) caused by low ozone concentrations

Summary Injury caused by low O₃ concentrations (0, 0.05, 0.075, 0.1 l 1^-1) was analyzed in the epidermis and mesophyll of fully developed birch leaves by gas exchange experiments and low-temperature SEM: (I) after leaf formation in O₃-free and ozonated air, and (II) after transferring control plants into ozonated air. In control leaves, autumnal senescence also was studied in O₃-free air (III). As O₃ concentration increased, leaves of (I) stayed reduced in size, but showed increased specific weight and stomatal density. The declining photosynthetic capacity, quantum yield and carboxylation efficiency lowered the light saturation of CO₂ uptake and the water-use efficiency (WUE). Carbon gain was less limited by the reduced stomatal conductance than by the declining ability of CO₂ fixation in the mesophyll. The changes in gas exchange were related to the O₃ dose and were mediated by narrowed stomatal pores (overriding the increase in stomatal density) and by progressive collapse of mesophyll cells. The air space in the mesophyll increased, preceded by exudate formation on cell walls. Ozonated leaves, which had developed in O₃-free air (II), displayed a similar but more rapid decline than the leaves from (I). In senescent leaves (III), CO₂ uptake showed a similar decrease as in leaves with O₃ injury but no changes in mesophyll structure and WUE. The nitrogen concentration declined only in senescent leaves in parallel with the rate of CO₂ uptake. A thorough understanding of O₃ injury and natural senescence requires combined structural and functional analyses of leaves.     

Environmental effects on circadian rhythms in photosynthesis and stomatal opening

Persistent circadian rhythms in photosynthesis and stomatal opening occurred in bean (Phaseolus vulgaris L.) plants transferred from a natural photoperiod to a variety of constant conditions. Photosynthesis, measured as carbon assimilation, and stomatal opening, as conductance to water vapor, oscillated with a freerunning period close to 24 h under constant moderate light, as well as under light-limiting and CO₂-limiting conditions. The rhythms damped under constant conditions conducive to high photosynthetic rates, as did rates of carbon assimilation and stomatal conductance, and this damping correlated with the accumulation of carbohydrate. No rhythm in respiration occurred in plants transferred to constant darkness, and the rhythm in stomatal opening damped rapidly in constant darkness. Damping of rhythms also occurred in leaflets exposed to constant light and CO₂-free air, demonstrating that active photosynthesis and not simply light was necessary for sustained expression of these rhythms. This is CIWDPB Publication No. 1142 This research was supported by National Science Foundation grant BSR 8717422 (C.B.F.) and a U.S. Department of Agriculture training grant to Stanford University (T.L.H.).     

Effects of temperature on the activity of phosphoenolpyruvate carboxylase and on the control of CO2 fixation in Bryophyllum fedtschenkoi

The phosphorylation state and the malate sensitivity of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in Bryophyllum fedtschenkoi Hamet et Perrier are altered by changes in the ambient temperature. These effects, in turn alter the in-vivo activity of the enzyme. Low temperature (3 °C or less), stabilizes the phosphorylated form of the enzyme, while high temperature (30 °C) promotes its dephosphorylation. The catalytic activity of the phosphorylated and dephosphorylated forms of PEPCase increases with temperature, but the apparent K _i values for malate of both forms of the enzyme decrease. Results of experiments with detached leaves maintained in darkness in normal air indicate that the changes in malate sensitivity and phosphorylation state of PEPCase with temperature are of physiological significance. When the phosphorylated form of PEPCase is stabilized by reducing the temperature of leaves 9 h after transfer to constant darkness at 15 °C, a prolonged period of CO₂ fixation follows. When leaves are maintained in constant darkness at 15 °C until CO₂ output reaches a low steady-state level and the PEPCase is dephosphorylated, reducing the temperature to 3 °C results in a further period of CO₂ fixation even though the phosphorylation state of PEPCase does not change.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase We thank the Agricultural and Food Research Council for financial support for this work.     

Is the signal from the mesophyll to the guard cells a vapour‐phase ion?

Previous studies have suggested that the red light and CO₂ responses of stomata are caused by a signal from the mesophyll to the guard cells. Experiments were conducted to test the idea that this signal is a vapour‐phase ion. Stomata in isolated epidermes of Tradescantia pallida were found to respond to air ions created by an electrode that was positioned under the epidermes. Anthocyanins in the epidermes of this species were observed to change colour in response to these air ions, and this change in colour was attributed to changes in pH. A similar change in lower epidermal colour was observed in intact leaves upon illumination and with changes in CO₂ concentration. Based on the change in epidermal colour, the pH of the epidermis was estimated to be approximately 7.0 in darkness and 6.5 in the light. Stomata in isolated epidermes responded to pH when suspended over (but not in contact with) solutions of different pH. We speculate that stomatal responses to CO₂ and light are caused by vapour‐phase ions, possibly hydronium ions that change the pH of the epidermis.     

The Damping and Reinitiation of the Circadian Rhythm of CO2 Output in Bryophyllum Leaves in Relation to their Malate Content

The circadian rhythm of CO₂ output in leaves of Bryophyllumfedtschenkoi damps out after 3–4 d in continuous darknessand a CO₂-free air stream at 15°C. The rhythm is reinitiatedafter a single exposure to white light of 2, 4, 6 or 8 h duration,damps out again after a further 3–4 d and can be reinitiatedfor a second time by a further exposure to light. During the exposure to light there is a burst of CO₂ outputconsistent with the decarboxylation of malate, and the rhythmbegins afterwards with an initial high rate of CO₂ fixation.Malate gradually accumulates in the leaves in continuous darknessto attain a maximum value (35 mol m^–3) at the time whenthe circadian rhythm disappears, and decreases to a low value(19 mol m^–3) after a 4 h exposure to light which reinitiatesrhythmicity. These results support the hypothesis that damping of the rhythmof CO₂ output in continuous darkness is due to the accumulationof malate in the leaf cells, eventually reaching such a levelthat its removal from the cytoplasm into the vacuole cannottake place, with the result that PEPc activity, upon which therhythm of CO₂ output depends, remains allosterically inhibited. Key words: CAM, circadian rhythm, Bryophyllum, CO₂-fixation, malate metabolism     

Perturbations of malate accumulation and the endogenous rhythms of gas exchange in the Crassulacean acid metabolism plant<Emphasis Type="Italic"> Kalanchoë daigremontiana</Emphasis>: testing the tonoplast-as-oscillator model

In continuous light, leaves of the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier exhibit a circadian rhythm of CO₂ uptake, stomatal conductance and leaf-internal CO₂ pressure. According to a current quantitative model of CAM, the pacemaking mechanism involves periodic turgor-related tension and relaxation of the tonoplast, which determines the direction of the net flux of malate between the vacuole and the cytoplasm. Cytoplasmic malate, in turn, through its inhibitory effect on phosphoenolpyruvate carboxylase, controls the rate of CO₂ uptake. According to this mechanism, when the accumulation of malate is disrupted by removing CO₂ from the ambient air, the induction of a phase delay with respect to an unperturbed control plant is expected. First, using the mathematical model, such phase delays were observed in numerical simulations of three scenarios of CO₂ removal: (i) starting at a trough of CO₂ uptake, lasting for about half a cycle (ca. 12 h in vivo); (ii) with the identical starting phase, but lasting for 1.5 cycles (ca. 36 h); and (iii) starting while CO₂ increases, lasting for half a cycle again. Applying the same protocols to leaves of K. daigremontiana in vivo did not induce the predicted phase shifts, i.e. after the end of the CO₂ removal the perturbed rhythm adopted nearly the same phase as that of the control plant. Second, when leaves were exposed to a nitrogen atmosphere for three nights prior to onset of continuous light to prevent malate accumulation, a small, 4-h phase advance was observed instead of a delay, again contrary to the model-based expectations. Hence, vacuolar malic acid accumulation is ruled out as the central pacemaking process. This observation is in line with our earlier suggestion [T.P. Wyka, U. Lüttge (2003) J Exp Bot 54:1471–1479] that in extended continuous light, CO₂ uptake switches gradually from a CAM-like to a C3-like mechanism, with oscillations of the two CO₂ uptake systems being tightly coordinated. It appears that the circadian rhythm of gas exchange in this CAM plant emerges from one or several devices that are capable of generating temporal information in a robust manner, i.e. they are protected from even severe metabolic perturbations.Abbreviations CAM Crassulacean acid metabolism - c_ia Ratio of mesophyll CO₂ concentration to external CO₂ concentration - J_C Rate of carbon dioxide uptake - J_W Transpiration rate - g_W Stomatal conductance - LL Continuous light conditions - PEPC Phosphoenolpyruvate carboxylase - Rubisco d-Ribulose-1,5-bisphosphatecarboxylase/oxygenase - Effective quantum yield of photosystem II     

Effects of temperature on the gas exchange of leaves in the light and dark

Summary Evolution of CO₂ into CO₂-free air was measured in the light and in the dark over a range of temperatures from 15 to 50°. Photosynthetic rates were measured in air and O₂-free air over the same range of temperatures. Respiration in the light had a different sensitivity to temperature compared with respiration in the dark. At the lower temperatures the rate of respiration in the light was higher than respiration in the dark, whereas at temperatures above 40° the reverse was observed. For any one species the maximum rates of photosynthesis and photorespiration occur at about the same temperature. The maximum rate for dark respiration generally is found at a temperature about 10° higher. Zea mays and Atriplex nummularia showed no enhancement of photosynthesis in O₂-free air nor any evolution of CO₂ in CO₂-free air at any of the temperatures.     

Inhibition of the circadian rhythm of CO2 metabolism in Bryophyllum leaves by cycloheximide and dinitrophenol

The circadian rhythm of CO₂ output in darkened leaves of Bryophyllum fedtschenkoi R. Hamet and Perrier can be inhibited by cycloheximide (10^-6 mol) and 2,4-dinitrophenol (10^-5 mol) applied via the transpiration stream. After having been suppressed by 10^-6 M cycloheximide, the rhythm can be reinitiated with a 12-h exposure to light. Experiments using ¹⁴CO₂ show that cycloheximide abolishes the rhythm by inhibiting the dark fixation of CO₂. Cycloheximide inhibits malate accumulation and acidification of the leaves, but does not affect the amount of the CO₂-fixing enzyme phosphoenol-pyruvate carboxylase (PEP-C, EC 4.1.1.31) which can be extracted from the leaves during the 45 h of the experiment. Cycloheximide has no direct effect on the activity of the enzyme as measured in the assay. PEP-C from desalted leaf extracts was inhibited by L-malate (K_i=0.4 mmol). The most likely explanation for the inhibitory effect of cycloheximide and dinitrophenol is that they cause changes in tonoplast properties which result in a redistribution of malate from the vacuole to the cytoplasm. An increase in malate concentration in the cytoplasm will lead to inhibition of PEP-carboxylase, and hence the suppression of the rhythm of CO₂ output.Abbreviations CAM crassulacean acid metabolism - PEP-C phosphoenol-pyruvate carboxylase - MDH malate dehydrogenase - CHM cycloheximide - DNP 2,4-dinitrophenol - LD light-dark-cycle - DD continuous darkness     

Circadian control of heat tolerance in stationary phase cultures of Schizosaccharomyces pombe

The capacity of stationary phase cultures of Schizosaccharomyces pombe to survive a heat treatment at 55°C is controlled by a circadian rhythm. In a synchronizing light-dark-cycle this rhythm shows a stable phase relationship to the onset of light. In continuous darkness it persists for several cycles without marked damping. The free-running period of about 27 h at 30°C is only slightly longer at 20°C, hence temperature-compensated. These results indicate that S. pombe is a suitable experimental organism for further research into both heat tolerance and circadian rhythms.     

Stomatal responses in isolated epidermis of the crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perr.

The optimal conditions for opening of stomata in detached epidermis of the Crassulacean Acid Metabolism (CAM) plant Kalanchoe daigremontiana were determined. Stomatal opening in CO₂–free air was unaffected by light so subsequently all epidermal strips were incubated in the dark and in CO₂–free air. Apertures were maximal after 3 h incubation and were significantly greater at 15° C than 25° C. Thus stomata in isolated epidermis of this species can respond directly to temperature. Stomatal opening was greatest when the incubating buffer contained 17.6 mol m^–3 K⁺, but decreased linearly with increasing K⁺ concentrations between 17.6 and 300 mol m^–3; the decrease in aperture was shown to be associated with increasing osmotic potentials of the solutions. Reasons for this behaviour, which differs from that of many C₃ and C₄ species, are discussed. Stomatal apertures declined linearly upon incubation of epidermis on buffer solutions containing between 10^–11 and 10^–5 mol m^–3 abscisic acid (ABA). Hence stomata on isolated epidermis of K. daigremontiana respond to lower concentrations of ABA than those of any species reported previously.     

The state of activation of ribulose-1,5-bisphosphate carboxylase in wheat leaves

In light and in darkness, exposure of leaf segments to CO₂-free atmospheres caused a marked reduction in extractable RuBP carboxylase activity. By contrast, darkness caused a relatively small decrease in carboxylase activity in extracts from leaf segments kept in air containing CO₂. Recovery of carboxylase activity in leaves during illumination in air after exposure to CO₂-free conditions paralleled recovery of capacity for photosynthesis; in darkness recovery of carboxylase activity in leaves was slower than in the light. Extracts from leaves exposed to CO₂-free conditions recovered activity when provided with CO₂ and Mg²⁺; there were clearly, however, substances in the extracts that modified the activity achieved and caused anomalous decreases and increases with time after extraction. Studies of the effect of orthophosphate on the activity of purified wheat carboxylase in vitro were consistent with the view that many of the effects observed on the activity of crude leaf extracts were due to orthophosphate content.     

Photosynthesis of Ivy Leaves (Hedera helix) after Heat Stress I. CO2-Gas Exchange and Diffusion Resistances

For an analysis of the inhibition of the photosynthetic CO₂-uptake after heat stress attached leaves of Hedera helix L. were heat-stressed for 30 min at various temperatures. Subsequently their photosynthetic CO₂-uptake, transpiration, respiration in light and darkness, and CO₂-compensation concentration were measured under optimal conditions. After heat stress the stomatal resistance increased only corresponding to the raised CO₂-concentration inside the leaves (due to the reduced CO₂-uptake). The physical resistance between the mesophyll cell walls and the chloroplasts remained unchanged after heat stress. A non-stomatal inhibition of the CO₂-uptake is indicated by a strong increase of the CO₂-compensation concentration after heat stress. This is hardly due to a stimulation of the respiration in light, as the CO₂-evolution into CO₂-free air in light was even reduced. Therefore, it must be concluded that the photosynthetic process itself is impaired after heat stress.