An upward lateral movement of radioactivity from [(14)C]gibberellic acid (GA3) has been found to occur in geotropically stimulated coleoptiles and primary roots of intact Zea mays (L.) seedlings.

The activity of phosphoenolpyruvate carboxylase (PEPCase) kinase in leaf extracts increased markedly on dilution. This was shown to be caused by the presence of a protein that inhibits the kinase. The inhibitor protein was separated from the kinase and purified partially. It inhibited the kinase reversibly, presumably by a direct interaction; it was neither a protease nor a protein phosphatase. The amounts of kinase and inhibitor in leaves were estimated following separation by hydrophobic chromatography. The amount of inhibitor in the crassulacean acid metabolism plant Kalanchoë fedtschenkoi Hamet et Perrier was sufficient to inhibit the basal level of kinase activity present during the light period and the early stages of the dark period. Similarly, the amount of inhibitor in the C4 plant Zea mays L.was sufficient to inhibit the low amount of kinase activity present in the dark and at moderate light intensity. Analogous to the role of the protein inhibitor of mammalian cyclic AMP-dependent protein kinase, the function of the PEPCase kinase inhibitor may be to inhibit the basal level of kinase present in conditions under which rapid flux through PEPCase is not required.

Barley leaf protoplasts were incubated in light or darkness in the presence of various inhibitors, metabolites or weak acids/bases. Nitrate reductase (NR) and phosphoenolpyruvate carboxylase (PEPCase) were rapidly extracted from the protoplasts and assayed under sub-optimal conditions, i.e. in the presence of Mg2+ and malate, respectively. Under these conditions changes in activities are thought to reflect changes in the phosphorylation states of the enzymes. The NR was activated by illumination to 90% of its maximal activity within 10 min. Photosynthetic electron transport appeared necessary for light activation of NR since activation was inhibited by the photosynthetic electron-transport inhibitor 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), and, additionally, an electron acceptor (HCO 3 - ) was required. The PEPCase was also activated by light. However, this activation was not prevented by DCMU or lack of HCO 3 - . Loading of protoplasts in the dark with a weak acid resulted in activation of both NR and PEPCase. For NR, full activation was completed within 5 min, whereas for PEPCase a slower, modest activation continued for at least 40 min. Incubation of protoplasts with a weak base also gave activation of PEPCase, but not of NR. On the contrary, base loading counteracted light activation of NR. Since several treatments tested resulted in the modulation of either NR or PEPCase activity, but not both, signal transduction cascades leading to changes in activities appear to be very different for the two enzymes.

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.

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 Ki 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.

SUMMARYThis article reviews the circadian rhythm of carbon dioxide metabolism in leaves of the Crassulacean plant Bryophyllum (Kalanchoë) fedtsckenkoi which persists both in continuous darkness and a CO2‐free atmosphere, and in continuous light and normal air. Under both conditions the rhythm is due to the periodic activity of the enzyme phosphoenolpyruvate carboxylase (PEPc). The physiological characteristics of the rhythm are described in detail and, from these characteristics, hypotheses are advanced to account for both the generation of the rhythm and the regulation of its phase and period by environmental factors.The periodic activity of PEPc is ascribed to the periodic accumulation of an allosteric inhibitor, malate, in the cytoplasm and its subsequent removal either to the vacuole in continuous darkness, or by metabolism in continuous light. Also involved in the generation of the rhythm is a periodic change in the sensitivity of PEPc to malate inhibition due to the periodic phosphorylation and dephosphorylation of PEPc which changes its K1 by a factor of 10 from 30 to 0.3 mM and vice versa. This periodic phosphorylation of PEPc is apparently achieved by the periodic synthesis and breakdown of a PEPc kinase which phosphorylates the enzyme on a serine residue; dephosphorylation is achieved by a type 2A phosphatase which shows no rhythmic variation.The induction of phase shifts in the rhythm in continuous darkness and CO2‐free air has been explained in terms of light and high‐temperature activated gates or channels in the tonoplast which, when open, allow malate to diffuse between the vacuole and cytoplasm. For the rhythm in continuous light and normal air phase, control by environmental signals can be attributed to changes in the malate levels in critical cell compartments, or in particular cell populations such as the stomatal guard cells, due to regulation of the malate synthesizing enzyme system involving PEPc, and malic enzyme which is responsible for malate metabolism. The role of the stomata in the generation of the rhythm is also discussed.The biochemical events which appear to give rise to the well‐studied circadian rhythms in leaf movement in Samanea and Albizza, in luminescence in Gonyaulax polyedra and in the synthesis of the chlorophyll a/b binding protein are also reviewed in an attempt to identify similarities between these events and those involved in the Bryophyllum rhythm. Finally, the somewhat similar nature of the genes apparently responsible for circadian rhythmicity in Neurospora and Drosophila are discussed, and suggestions made for utilizing anti‐sense nucleic acid technology in the further elucidation of the critical biochemical events involved in the basic, temperature‐compensated circadian oscillator in living organisms. CONTENTS Summary 347 I. Introduction 348 II. Occurrence of circadian rhythms 348 III. Physiological characteristics of circadian rhythms 349 IV. Biochemical and molecular events involved in the circadian rhythm in Bryophyllum leaves 362 V. Biochemical and molecular events involved in the origin and control of circadian rhythmicity in other organisms 366 VI. Genetic studies 370 VII. Conclusion 371 References 372

The role of the epidermis in the generation of the endogenous circadian rhythm of CO2 exchange in leaves of Bryophyllum fedtschenkoi has been examined. At 25° C the rhythm of CO2 output exhibited by whole leaves kept in continuous darkness and an initially CO2-free air stream also occurs in isolated pieces of mesophyll. The sensitivity to light of the rhythms in whole leaves and in isolated mesophyll appears to be identical. At 15° C, however, no rhythm is observed in isolated mesophyll tissue, despite there being a conspicuous rhythm in intact leaves. The rhythm of net CO2 assimilation in whole leaves kept in continuous light and a stream of normal air at either 25° C or at 15° C is abolished by removal of the epidermis, although at 15° C and under the higher of the two light levels used, there is an indication that rhythmicity may begin to reappear after the third day of the experiment. Thus, only under certain environmental conditions is the rhythm of CO2 exchange in Bryophyllum leaves independent of the epidermis. The results indicate that the rhythm of carbon dioxide fixation in continuous darkness and CO2-free air is generated primarily in the mesophyll cells, whereas the rhythm in continuous light and normal air is generated in the stomatal guard cells or in an interaction of these cells with the mesophyll cells.

There is now good evidence that the malate sensitivity of PEPc is regulated by phosphorylation/dephosphorylation in the leaf tissue of C4 and CAM plants. This statement is based on the assessment of the phosphorylation state of PEPc in [32P]-labeled intact tissue by immunoprecipitation and the correlation between phosphorylation state and malate sensitivity that has been observed during incubation of purified PEPc in vitro with protein kinases or protein phosphatases. The phosphorylation of PEPc in the CAM plant B. fedtschenkoi is controlled by an endogenous rhythm whereas that of PEPc in the C4 plant maize is triggered directly by light. In neither case has the mechanism of signal transduction been identified. It is hoped that further work on the protein kinases and protein phosphatases involved will reveal the nature of the signalling systems. Preliminary work suggests that plant protein phosphatases are very similar to their mammalian counterparts. It is also noteworthy that higher plant genes very similar to the genes encoding the cyclic nucleotide-dependent protein kinases and the protein kinase C family have recently been identified. It is interesting to speculate that the protein kinases and phosphatases involved in signal transduction systems in plants may prove to be closely related to well-studied mammalian enzymes.

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.

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.

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.